HER3 / MET binding molecules and medical uses thereof

Abstract

The present disclosure relates to HER3 / MET binding molecules and medical uses thereof. Specifically, the invention relates to an anti-HER3 / MET bispecific antibody, an anti-HER3 / MET antibody drug conjugate, and a cancer treatment method and pharmaceutical application thereof.

Description

HER3 / MET binding molecules and their medical uses Technical Field

[0001] The present disclosure relates to HER3 / MET binding molecules, such as anti-HER3 / MET bispecific antibodies, anti-HER3 / MET antibody-drug conjugates, and methods and pharmaceutical uses thereof for treating cancer. Background Art

[0002] Antibody-drug conjugates (ADCs) combine an antibody for targeting and a small-molecule drug for killing tumor cells. This perfect combination creates a "magic bullet" platform for precision therapy with minimal side effects. Since the launch of the first ADC, Mylotarg, in 2000, innovation has continued in this field, with over a dozen ADCs now available.

[0003] MET (mesenchymal-epithelial transition factor) is a membrane receptor essential for embryonic development and wound healing. Hepatocyte growth factor (HGF) is the only known ligand for the MET receptor. MET is normally expressed by cells of epithelial origin, while HGF expression is restricted to cells of mesenchymal origin. Upon HGF stimulation, MET induces several biological responses that collectively produce a program known as invasive growth. Aberrant MET activation in cancer is associated with poor prognosis, as abnormally active MET triggers tumor growth, the formation of new blood vessels that supply nutrients to the tumor (angiogenesis), and the spread of cancer to other organs (metastasis).

[0004] HER3 (epidermal growth factor receptor 3, ErbB-3 or HER3) is a member of the epidermal growth factor receptor (EGFR) family. These receptors all consist of three parts: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region contains four domains, while the intracellular region includes an intracellular tyrosine kinase domain for signal transduction and a tail with tyrosine phosphorylation residues located in the cytoplasm. When ligands bind to extracellular domains I and III, cell signaling is initiated. Under normal circumstances, these receptors mediate cell division, migration, survival, and organ development. When EGFR family members mutate, the resulting aberrant signaling stimulates cell survival and is associated with cancer progression.

[0005] c-Met and EGFR (or HER family) interact and participate in various mechanisms related to tumor growth. These proteins (targets) are typically receptor tyrosine kinases (RTKs) present on the cell surface, thereby inducing cancer cell proliferation, cancer cell penetration, angiogenesis, etc.

[0006] The present disclosure provides an anti-HER3 / MET bispecific antibody and a drug conjugate thereof. The anti-HER3 / MET bispecific antibody drug conjugate has excellent tumor growth inhibition and killing effects, good drugability, and high potential clinical safety.

[0007] Summary of the Invention

[0008] The present disclosure provides MET-binding molecules, HER3-binding molecules, and HER3 / MET-binding molecules, as well as nucleic acids encoding the same, vectors, host cells, and pharmaceutical compositions, and their use in treating cancer and related pharmaceutical applications.

[0009] MET binding molecules

[0010] The present disclosure provides MET-binding molecules comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 71; and the VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 72.

[0011] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0012] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOs: 74, 19 and 20, respectively.

[0013] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 18-20, respectively.

[0014] In some embodiments, in any of the aforementioned MET-binding molecules, the VH1 includes a K mutation at position 23 and / or a T mutation at position 78 relative to SEQ ID NO: 1; and / or the VL1 includes a T mutation at position 69 relative to SEQ ID NO: 2; preferably, any of the aforementioned MET-binding molecules comprises E23K and S78T mutations relative to SEQ ID NO: 1, and an A69T mutation relative to SEQ ID NO: 2.

[0015] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:71, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, or at least 80% identical thereto.

[0016] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or at least 80% identical thereto.

[0017] In some embodiments, the aforementioned MET-binding molecules inhibit tumor growth, or treat or ameliorate cancer.

[0018] HER3 binding molecules

[0019] The present disclosure provides a HER3-binding molecule comprising a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 13, and the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 70.

[0020] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0021] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 54, 55, and 73, respectively.

[0022] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 54-56, respectively.

[0023] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or at least 80% identical thereto.

[0024] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or at least 80% identical thereto.

[0025] In some embodiments, the aforementioned HER3 binding molecules inhibit tumor growth, or treat or alleviate cancer.

[0026] HER3 / MET binding molecules

[0027] The present disclosure provides a HER3 / MET binding molecule comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3, which can specifically bind to HER3 and MET simultaneously or separately.

[0028] Regarding the first binding domain that specifically binds to MET:

[0029] In some embodiments, the first binding domain that specifically binds to MET in the HER3 / MET binding molecule comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein: the VH1 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence of any one of SEQ ID NOs: 71, 1, 3, 5, 7, 9, and 11, and / or the VL1 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence of any one of SEQ ID NOs: 72, 2, 4, 6, 8, 10, and 12.

[0030] In some specific embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO:71, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO:72.

[0031] In some specific embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO: 1, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO: 2.

[0032] In some embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO: 3, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO: 4.

[0033] In some embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO:5, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO:6.

[0034] In some embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO:7, and the VL1 comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO:8.

[0035] In some specific embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO:9, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO:10.

[0036] In some specific embodiments, the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence of SEQ ID NO: 11, and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of SEQ ID NO: 12.

[0037] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0038] In some embodiments, the first binding domain that specifically binds to MET in the HER3 / MET binding molecule comprises a heavy chain variable region (VH1) comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 15-17, respectively, and a light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 76, 19, and 20, respectively.

[0039] In some embodiments, the first binding domain that specifically binds to MET comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any one of the following:

[0040] a-1) HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 18-20, respectively; or

[0041] a-2) HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 74, 19, and 20, respectively;

[0042] In other embodiments, the first binding domain that specifically binds to MET comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any one of the following:

[0043] i-1) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 21-23, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 24-26, respectively;

[0044] i-2) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 27-29, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 30-32, respectively;

[0045] i-3) HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 33-35, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 36-38, respectively;

[0046] i-4) HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 39-41, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 42-44, respectively; or

[0047] i-5) HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 45-47, respectively, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 48-50, respectively.

[0048] In some embodiments, the first binding domain that specifically binds to MET in the HER3 / MET binding molecule comprises VH1 and VL1, wherein: any HCDR comprised in the VH1 has 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or any LCDR comprised in the VL1 has 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned LCDRs.

[0049] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (e.g., conservative substitution of amino acids), and the mutation does not affect or does not substantially affect the function of the first binding domain that specifically binds to MET.

[0050] In some embodiments, in the first binding domain that specifically binds to MET in the HER3 / MET binding molecule, the VH1 includes a K mutation at position 23 and / or a T mutation at position 78 relative to SEQ ID NO: 1; and / or the VL1 includes a T mutation at position 69 relative to SEQ ID NO: 2; preferably, the first binding domain that specifically binds to MET comprises an E23K mutation and an S78T mutation relative to SEQ ID NO: 1, and an A69T mutation relative to SEQ ID NO: 2.

[0051] In some embodiments, in the first binding domain of the HER3 / MET binding molecule that specifically binds MET:

[0052] The VH1 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 71, 1, 3, 5, 7, 9 and 11, or having at least 80% sequence identity thereto; and / or,

[0053] The VL1 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 72, 2, 4, 6, 8, 10 and 12, or having at least 80% sequence identity thereto.

[0054] Regarding the second binding domain that specifically binds to HER3:

[0055] In some embodiments, the second binding domain that specifically binds to HER3 in the HER3 / MET binding molecule comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein: the VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 13, and the VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 70 or 14.

[0056] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0057] In some embodiments, the second binding domain that specifically binds to HER3 in the HER3 / MET binding molecule comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2, and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 51-53, respectively, and the VL2 comprises LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NOs: 54, 55, and 75, respectively.

[0058] In some embodiments, the second binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in any one of the following:

[0059] b-1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 51-56, respectively; or

[0060] b-2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 51-55 and 73, respectively;

[0061] In some embodiments, the second binding domain that specifically binds to HER3 in the HER3 / MET binding molecule comprises VH2 and VL2, wherein: any HCDR comprised in the VH2 has 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or, any LCDR comprised in the VL2 has 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned LCDRs.

[0062] In some specific embodiments, the above-mentioned amino acid mutation is an amino acid replacement, substitution, modification, deletion and / or addition (e.g., conservative substitution of amino acids), and the mutation does not affect or substantially does not affect the function of the second binding domain that specifically binds to HER3.

[0063] In some embodiments, the second binding domain of the HER3 / MET binding molecule that specifically binds to HER3:

[0064] The VH2 comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 80% sequence identity thereto; and / or,

[0065] The VL2 comprises an amino acid sequence as shown in SEQ ID NO: 70 or 14, or an amino acid sequence having at least 80% sequence identity thereto.

[0066] In some embodiments, the HER3 / MET binding molecule further comprises a human immunoglobulin Fc region. In some specific embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4, such as the Fc region of human IgG1.

[0067] In some embodiments, the Fc region is half-life extended, e.g., having M252Y / S254T / T256E mutations.

[0068] In some embodiments, the Fc region is ADCC-enhanced, e.g., having S239D / A330L / I332E mutations, or fucose removal.

[0069] In some embodiments, the Fc region can enable the binding molecule to form a dimeric molecule and simultaneously prolong the in vivo half-life of the binding molecule.

[0070] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2). In some embodiments, a mutation is introduced that causes the two subunits (Fc1, Fc2) of the Fc region to pair to form a dimer, or a mutation that reduces homodimerization. In some embodiments, the first and second subunits contain knob-into-hole mutations. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1.

[0071] In some embodiments, the Fc1 contains one or more amino acid substitutions at positions selected from the group consisting of 354, 356, 358, and 366, and the Fc2 contains one or more amino acid substitutions at positions selected from the group consisting of 349, 356, 358, 366, 368, and 407. In some specific embodiments, the Fc1 contains a mutation at position 366, and the Fc2 contains a mutation at positions 366, 368, and 407, or any combination thereof; in some specific embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349; in some specific embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains mutations at positions 349, 366, 368, and 407.

[0072] In some embodiments, the Fc1 comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the Fc2 comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, the Fc1 comprises a 366W mutation, and the Fc2 comprises a mutation selected from 366S, 368A, and 407V, or any combination thereof; in some specific embodiments, the Fc1 comprises a 354C or 356C mutation, and the Fc2 comprises a 349C mutation; or in some specific embodiments, the Fc1 comprises 354C / 366W mutations, and the Fc2 comprises 349C / 366S / 368A / 407V mutations.

[0073] In some embodiments, the Fc1 contains a T366W mutation, and the Fc2 contains a mutation selected from T366S, L368A, and Y407V, or any combination thereof; the first subunit of the Fc region contains a S354C or E356C mutation, and the second subunit contains a Y349C mutation; or the first subunit of the Fc region contains an S354C / T366W mutation, and the second subunit contains a Y349C / T366S / L368A / Y407V mutation, and the mutations are numbered according to Eu.

[0074] In some embodiments, the sequence of the Fc1 is shown in SEQ ID NO: 67, and the sequence of the Fc2 is shown in SEQ ID NO: 68.

[0075] In some embodiments, the HER3 / MET binding molecule comprises amino acid size and charge mutations to the amino acids at the interface of the heavy chain CH1 and the light chain CL, thereby reducing mispairing between the light and heavy chains. For example, Roche exchanged the CH1 and CL domains and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187–11192(2011)), MedImmune introduced disulfide bonds by mutating the heavy chain F126C and the light chain S121C (Mazor et al., mAbs, 7(2), pp.377–389(2015)), Amgen further modified the CH1-CL region with electrostatic interactions (Liu et al., Journal of Biological Chemistry, 290(12), pp.7535–7562(2015)), and Lilly (Lewis et al., Nature Biotechnology, 32(2), pp.191–198 (2014)) and Genentech (Dillon et al., mAbs, 9(2), pp.213–230 (2017)) introduced mutations in both the variable and constant domains. The constant region of the antibody was replaced with that of a TCR, as described in CN109535257A (incorporated herein by reference).

[0076] In some embodiments, the HER3 / MET binding molecule contains a linker.

[0077] In some embodiments, the linker is such as (G m S n ) h or (GGNGT (SEQ ID NO: 88)) h or (YGNGT (SEQ ID NO: 89)) h or (EPKSS (SEQ ID NO: 90)) h The amino acid sequence shown, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0078] In some embodiments, the linker is (G x S) yA linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4, or 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the linker is selected from G4S (SEQ ID NO: 91), GS, GAP, (G4S)2 (SEQ ID NO: 92), (G4S)3 (SEQ ID NO: 93), (G4S)4 (SEQ ID NO: 94), (G4S)5 (SEQ ID NO: 95), and ASGS (SEQ ID NO: 98).

[0079] In some embodiments, the HER3 / MET binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein

[0080] The first heavy chain, from N-terminus to C-terminus, is composed of: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0081] The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 2]-[Titin-T chain],

[0082] The second heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[CH1]-[second subunit of the Fc region], and

[0083] The second light chain, from N-terminus to C-terminus, is: [VL2]-[CL];

[0084] or,

[0085] The first heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0086] The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 2]-[Titin-T chain],

[0087] The second heavy chain, from N-terminus to C-terminus, consists of: [VH1]-[CH1]-[second subunit of the Fc region], and

[0088] The second light chain, from N-terminus to C-terminus, is: [VL1]-[CL];

[0089] Wherein, - represents a peptide bond, and the linker 1, linker 2 and linker 3 may be the same or different, and may exist independently or not.

[0090] In some specific embodiments, the amino acid sequence of linker 1 and linker 2 is GGGGS, and linker 3 does not exist.

[0091] In some embodiments, the titin-T chain is as shown in SEQ ID NO:96.

[0092] In some embodiments, the titin-T chain is a variant of SEQ ID NO: 96, wherein the variant has an amino acid residue substitution at one or more positions selected from positions 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 60, 64, 66, 70, 75, 77, 79, 81, 82, 83, and 84. Exemplarily, the variant has an amino acid residue substitution at one or more positions selected from 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 60S, 64T, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L. In some specific embodiments, the titin-T chain is as shown in SEQ ID NO: 64 or an amino acid sequence having at least 80% sequence identity thereto.

[0093] In some embodiments, the Obscurin-O chain is as shown in SEQ ID NO:97.

[0094] In some embodiments, the Obscurin-O chain is a variant of SEQ ID NO: 97 having an amino acid residue substitution at one or more positions selected from positions 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 48, 53, 58, 62, 66, 67, 69, 76, 82, 88, 89, 92, 93, 94, and 97. Illustratively, one or more amino acid residues are substituted from the group consisting of 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y / 13S, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P / 32F, 34E, 36T, 41K, 42L, 44I, 45T, 48V, 53L, 58V, 62E / 62K / 62H, 66C, 67Q / 67T, 69S, 76S, 82H, 88C, 89L, 92E, 93C, 94G, and 97G. In some specific embodiments, the Obscurin-O chain is as set forth in SEQ ID NO: 63, or an amino acid sequence having at least 80% sequence identity thereto.

[0095] In some embodiments, the HER3 / MET binding molecule comprises the following polypeptide chain combination:

[0096] a first heavy chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 77, 57, and 61, or having at least 90% sequence identity thereto,

[0097] a first light chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 78, 58, and 62, or having at least 90% sequence identity thereto,

[0098] a second heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90% sequence identity thereto, and / or

[0099] A second light chain comprising an amino acid sequence as set forth in either SEQ ID NO: 79 or 60, or having at least 90% sequence identity thereto.

[0100] In some embodiments, a HER3 / MET binding molecule is provided, comprising:

[0101] The first heavy chain shown in SEQ ID NO: 57,

[0102] The first light chain shown in SEQ ID NO: 58,

[0103] The second heavy chain shown in SEQ ID NO: 59, and

[0104] The second light represented by SEQ ID NO:60.

[0105] In some embodiments, a HER3 / MET binding molecule is provided, comprising:

[0106] The first heavy chain shown in SEQ ID NO: 61,

[0107] The first light chain shown in SEQ ID NO: 62,

[0108] The second heavy chain shown in SEQ ID NO: 59, and

[0109] The second light chain is represented by SEQ ID NO:60.

[0110] In some embodiments, a HER3 / MET binding molecule is provided, comprising:

[0111] The first heavy chain shown in SEQ ID NO: 77,

[0112] The first light chain shown in SEQ ID NO: 78,

[0113] The second heavy chain shown in SEQ ID NO: 59, and

[0114] The second light chain is represented by SEQ ID NO:79.

[0115] Antibody Drug Conjugates

[0116] In some embodiments, the HER3 / MET binding molecules of the present disclosure are anti-HER3 / MET antibody drug conjugates.

[0117] In some embodiments, the antibody-drug conjugate comprises an effector molecule selected from the group consisting of a radioisotope, an anti-tumor agent, an immunomodulator, a biological response modifier, a lectin, a cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, an enzyme, a metal ion, and any combination thereof.

[0118] In some embodiments, the effector molecule is a cytotoxic drug, exemplified by a microtubule aggregation inhibitor, a Topo I inhibitor, MMAE or a derivative thereof. In some embodiments, the cytotoxin is selected from MMAE or a derivative thereof, exitecan or a derivative thereof, eribulin or a derivative thereof.

[0119] Conjugates of antibody drugs (exitecan or its derivatives)

[0120] The present disclosure provides an antibody-drug conjugate having a structure shown in formula (I):

[0121] Wherein, -L- is a linker unit, which is -L 1 -L 2 -L 3 -L 4 -,

[0122] L 1 -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0123] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20;

[0124] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0125] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6;

[0126] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0127] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0128] Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0129] Ra and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0130] R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0131] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group or a heteroaryl group;

[0132] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0133] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0134] m is an integer from 0 to 4;

[0135] n is 1 to 10, and n is an integer or a decimal.

[0136] In some embodiments, the Ab is an antibody comprising a binding domain that specifically binds MET and / or a binding domain that specifically binds HER3.

[0137] In some embodiments, the antibody drug conjugate represented by formula (I) as described in any of the preceding items,

[0138] in:

[0139] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0140] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen and an alkyl group;

[0141] R 1 Selected from hydrogen atom, halogenated alkyl or C 3-6 Cycloalkyl;

[0142] R 2 selected from hydrogen atoms, halogenated alkyl groups and C 3-6 Cycloalkyl;

[0143] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0144] m is 0 or 1.

[0145] In some embodiments, the antibody drug conjugate represented by formula (I) as described in any of the preceding items,

[0146] Wherein, Y is selected from:

[0147] The O end of Y is connected to the linker unit L.

[0148] In some embodiments, the antibody drug conjugate represented by formula (I) as described in any of the preceding items,

[0149] Wherein, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0150] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0151] L 2 Selected from-NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer from 1 to 20;

[0152] L 3is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0153] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and chemical bonds, wherein t is an integer from 1 to 6;

[0154] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0155] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0156] In some embodiments, the antibody drug conjugate represented by formula (I) as described in any of the preceding items,

[0157] Wherein, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0158] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0159] L 2 Selected from-NR4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer from 1 to 20;

[0160] L 3 A peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0161] L 4 Selected from-NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - and chemical bonds, wherein t is an integer from 1 to 6, non-limiting examples being 1, 2, 3, 4, 5, and 6;

[0162] R 3 、R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0163] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0164] In some embodiments, the antibody drug conjugate represented by formula (I) as described in any of the preceding items,

[0165] Wherein, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0166] L 1 for s 1 is an integer from 2 to 8, non-limiting examples being 2, 3, 4, 5, 6, 7 and 8;

[0167] L 2 is a chemical bond;

[0168] L 3 is a tetrapeptide residue; preferably, L 3 is the tetrapeptide residue of GGFG;

[0169] L 4 -NR 5 (CR 6 R 7 )t-,R 5 、R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group, and t is 1 or 2;

[0170] The L 1 The end is connected to Ab, L 4 Connect the Y end.

[0171] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is an antibody-drug conjugate of general formula (II):

[0172] in:

[0173] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0174] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;

[0175] L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0176] R 1 is selected from hydrogen, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0177] R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;

[0178] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0179] R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0180] R 6 and R 7 are the same or different and are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

[0181] m is an integer from 0 to 4;

[0182] n is 1 to 10, and n is an integer or a decimal;

[0183] Ab is as defined in general formula (I).

[0184] In some embodiments, the antibody drug conjugate of formula (II) as described in any of the preceding items, wherein n is 1 to 10, for example, 1 to 8, 2 to 8, 2 to 7, 2 to 4, 3 to 8, 3 to 7, 3 to 6, 4 to 7 or 4 to 6, and n is a decimal or an integer. In some embodiments, n is 1 to 8, and n is a decimal or an integer. In some embodiments, n is 3 to 7, and n is a decimal or an integer. In some embodiments, n is 4 to 6, and n is a decimal or an integer. In some embodiments, n is an average value of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10. In some embodiments, n is an average of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6.

[0185] In some embodiments, the antibody drug conjugate represented by formula (II) as described in any of the preceding items,

[0186] in:

[0187] The -LY- is shown in the following structure:

[0188] s 1 is an integer from 2 to 8;

[0189] L 2 、L 3 、R 1 、R 2 、R 5 、R 6 、R 7 and m are as defined above in the general formula (II).

[0190] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is an antibody-drug conjugate of formula (III):

[0191] In some embodiments, the antibody drug conjugate represented by formula (III) as described in any of the preceding items:

[0192] in:

[0193] s 1 is an integer from 2 to 8;

[0194] Ab、R 1 、R 2 、R 5 、R 6 、R 7 , m and n are as defined in the general formula (II).

[0195] In some embodiments, the antibody drug conjugate of formula (I) as described in any of the preceding items, wherein -L- is:

[0196] In some embodiments, the antibody drug conjugate represented by the general formula (I) as described in any of the preceding items, wherein -LY- is optionally selected from:

[0197] In some embodiments, wherein -LY- is optionally selected from:

[0198] In some embodiments, wherein -LY- is:

[0199] In some embodiments, wherein -LY- is:

[0200] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0201] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0202] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0203] wherein Ab and n are as defined in the general formula (I).

[0204] Antibody-drug (eribulin or its derivatives) conjugates

[0205] The present disclosure provides an antibody drug conjugate having a structure shown in formula (IV): Ab-(L-De)k (IV)

[0206] wherein L is a linker that covalently attaches Ab to De,

[0207] k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers),

[0208] De is shown as follows:

[0209] where R 1a Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, one or more substituents are substituted; preferably R 1a It is a methyl group;

[0210] R 1b Selected from hydrogen, alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 Cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, wherein the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally selected from alkyl (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, one or more substituents are substituted; preferably R 1b is hydrogen; or

[0211] R 1a With R 1b Together with the atoms it is connected to form C 5-8 Heterocycloalkyl, the heteroalkyl is optionally replaced by an alkyl group (such as C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 Alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not hydrogen at the same time.

[0212] In some embodiments, the Ab is an antibody comprising a binding domain that specifically binds MET and / or a binding domain that specifically binds HER3.

[0213] In some embodiments, in the antibody-drug conjugate of formula (IV) as described in any of the preceding items, R1a in De is methyl.

[0214] In some embodiments, De in the antibody drug conjugate of formula (IV) as described in any of the preceding items is represented by the following formula:

[0215] In some embodiments, in the antibody drug conjugate of formula (IV) as described in any of the preceding items, k is selected from 1 to 10 and can be an integer or a decimal.

[0216] In some embodiments, the linker is extracellularly stable, such that the antibody drug conjugate of formula (IV) as described in any of the preceding items remains intact in the extracellular environment but can be cleaved upon internalization into cancer cells, for example.

[0217] In some embodiments, the linker in the antibody drug conjugate of formula (IV) as described in any of the preceding items comprises a cleavable portion; wherein the cleavable portion is positioned such that no linker and Ab remain in the drug (e.g., an eribulin derivative) after cleavage.

[0218] In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety.

[0219] In some embodiments, the addition of a cleavable moiety increases cytotoxicity and / or efficacy relative to a non-cleavable linker. In some embodiments, the cleavable peptide moiety is capable of being cleaved by an enzyme, and the linker is a linker that is cleavable by an enzyme. In some embodiments, the linker is a linker that is cleavable by a cathepsin. In certain embodiments, an enzyme-cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the above-mentioned improved properties.

[0220] In some embodiments, the linker comprises an amino acid unit (i.e., a peptide residue consisting of 2 to 7 amino acids), preferably the amino acids are selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine-lysine (Gly-Gly-lys), valine-lysine (Val-lys), valine-alanine (Val-Ala), valine-phenylalanine (Val-Phe) or glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0221] In some embodiments, the linker of the antibody drug conjugate of formula (IV) disclosed herein is selected from:

[0222] In some embodiments, the Amino Acid unit comprises Valine-Citrulline (Val-Cit).

[0223] In another aspect, some embodiments provide linkers comprising a cleavable sulfonamide moiety, wherein the linker is cleavable under reducing conditions.

[0224] In some embodiments, the linker comprises a cleavable disulfide moiety, and the linker is capable of being cleaved under reducing conditions.

[0225] In another aspect, the linker of the present disclosure comprises at least one Spacer unit that attaches De (eg, an eribulin derivative) to a cleavable moiety.

[0226] In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),

[0227] On the other hand, some embodiments provide that the antibody drug conjugate of formula (IV) is represented by the following formula:

[0228] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8;

[0229] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0230] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8;

[0231] k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0232] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0233] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0234] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0235] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0236] k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8;

[0237] k is selected from 1 to 10 and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2;

[0238] k is selected from 1 to 10 and can be an integer or a decimal; P1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2.

[0239] In some embodiments, the antibody drug conjugate of formula (IV) described in any of the preceding items is represented by the following formula:

[0240] wherein k is selected from 1 to 10 and can be an integer or a decimal; further, R in De 1a Preferably, R 1b Preferred is hydrogen.

[0241] In some embodiments, the antibody drug conjugates of the present disclosure include tautomers, mesomers, racemates, enantiomers, diastereomers, deuterated forms, or mixtures thereof.

[0242] In some embodiments, the antibody-drug conjugate of formula (I) or (IV) as described in any of the preceding items is an anti-HER3 / MET antibody-drug conjugate;

[0243] Among them, Ab is an antibody, comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3; the first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).

[0244] In some embodiments, the anti-HER3 / MET antibody drug conjugate of formula (I) or (IV), wherein Ab comprises:

[0245] The first heavy chain, from N-terminus to C-terminus, is composed of: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0246] The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 2]-[Titin-T chain],

[0247] The second heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[CH1]-[second subunit of the Fc region], and

[0248] The second light chain, from N-terminus to C-terminus, is: [VL2]-[CL];

[0249] or,

[0250] The first heavy chain, from N-terminus to C-terminus, consists of: [VH2]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region],

[0251] The first light chain, from N-terminus to C-terminus, is: [VL2]-[Linker 2]-[Titin-T chain],

[0252] The second heavy chain, from N-terminus to C-terminus, consists of: [VH1]-[CH1]-[second subunit of the Fc region], and

[0253] The second light chain, from N-terminus to C-terminus, is: [VL1]-[CL];

[0254] Wherein, - represents a peptide bond, and the linker 1, linker 2 and linker 3 may be the same or different, and may exist independently or not.

[0255] In some embodiments, the titin-T chain is as shown in SEQ ID NO:96.

[0256] In some embodiments, the titin-T chain is a variant of SEQ ID NO: 96, wherein the variant has an amino acid residue substitution at one or more positions selected from positions 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 60, 64, 66, 70, 75, 77, 79, 81, 82, 83, and 84. Exemplarily, the variant has an amino acid residue substitution at one or more positions selected from 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 60S, 64T, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L. In some specific embodiments, the titin-T chain is as shown in SEQ ID NO: 64 or an amino acid sequence having at least 80% sequence identity thereto.

[0257] In some embodiments, the Obscurin-O chain is as shown in SEQ ID NO: 97

[0258] In some embodiments, the Obscurin-O chain is a variant of SEQ ID NO: 97 having an amino acid residue substitution at one or more positions selected from positions 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 48, 53, 58, 62, 66, 67, 69, 76, 82, 88, 89, 92, 93, 94, and 97. Illustratively, one or more amino acid residues are substituted from the group consisting of 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y / 13S, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P / 32F, 34E, 36T, 41K, 42L, 44I, 45T, 48V, 53L, 58V, 62E / 62K / 62H, 66C, 67Q / 67T, 69S, 76S, 82H, 88C, 89L, 92E, 93C, 94G, and 97G. In some specific embodiments, the Obscurin-O chain is as set forth in SEQ ID NO: 63, or an amino acid sequence having at least 80% sequence identity thereto.

[0259] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to MET comprises HCDR1, HCDR2, and HCDR3 with the amino acid sequences shown in SEQ ID NOs: 15-17, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the amino acid sequences shown in SEQ ID NOs: 76, 19, and 20; the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, and HCDR3 with the amino acid sequences shown in SEQ ID NOs: 51-53, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the amino acid sequences shown in SEQ ID NOs: 54, 55, and 75.

[0260] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to MET comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 15-17, and the light chain variable region comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 18 or 74, and LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 19 and 20; the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51-53, and the light chain variable region comprises LCDR1 and LCDR2 of the amino acid sequences shown in SEQ ID NOs: 54 and 55, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 56 or 73.

[0261] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to MET comprises an amino acid sequence as shown in SEQ ID NO: 71 or 1, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 72 or 2, or at least 90% identical thereto; the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises an amino acid sequence as shown in SEQ ID NO: 13, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70 or 14, or at least 90% identical thereto.

[0262] In some embodiments, the Ab comprises a combination of polypeptide chains as set forth in SEQ ID NOs: 77, 78, 59, and 79, or comprises a combination of polypeptide chains as set forth in SEQ ID NOs: 57-60.

[0263] In some embodiments, the antibody-drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0264] in:

[0265] n is 1 to 8, n is a decimal or an integer, such as an integer or decimal of 3-7, and exemplary n is about 6; Ab comprises a polypeptide chain combination as shown in SEQ ID NOs: 77, 78, 59 and 79, or comprises a polypeptide chain combination as shown in SEQ ID NOs: 57-60.

[0266] In some embodiments, the antibody drug conjugate represented by formula (IV) as described in any of the preceding items is selected from:

[0267] in:

[0268] k is 1 to 8, k is a decimal or an integer, such as an integer or decimal of 3-7, and exemplary k is about 4 or 6; Ab comprises a polypeptide chain combination as shown in SEQ ID NOs: 77, 78, 59 and 79, or comprises a polypeptide chain combination as shown in SEQ ID NOs: 57-60.

[0269] In some embodiments, the antibody-drug conjugate of formula (I) or (IV) as described in any of the preceding items is an anti-MET antibody-drug conjugate;

[0270] Wherein, Ab is an antibody, comprising a binding domain that specifically binds to MET, and the binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1).

[0271] In some embodiments, in the anti-MET antibody drug conjugate of Formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence set forth in SEQ ID NO: 71; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence set forth in SEQ ID NO: 72.

[0272] In some embodiments, in the anti-MET antibody drug conjugate of formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 15-17, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 74, 19, and 20.

[0273] In some embodiments, the binding domain that specifically binds to MET comprises one or more amino acid mutations selected from: VH_E23K, VH_S78T, and VL_A69T in the variable region of the heavy chain.

[0274] In some embodiments, in the anti-MET antibody drug conjugate of Formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET comprises an amino acid sequence as shown in SEQ ID NO: 71, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 72, or at least 80% identical thereto.

[0275] In some embodiments, the anti-MET antibody drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0276] in:

[0277] n is 1 to 8, n is a decimal or an integer, such as an integer or decimal of 3-7, and an exemplary n is about 6;

[0278] Ab comprises a heavy chain variable region as shown in SEQ ID NO: 71 and a light chain variable region as shown in SEQ ID NO: 72.

[0279] In some embodiments, the anti-MET antibody drug conjugate represented by formula (IV) as described in any of the preceding items is selected from:

[0280] k is 1 to 8, k is a decimal or an integer, such as an integer or decimal of 3-7, and exemplary k is about 4 or 6;

[0281] Ab comprises a heavy chain variable region as shown in SEQ ID NO: 71 and a light chain variable region as shown in SEQ ID NO: 72.

[0282] In some embodiments, the binding molecule represented by formula (I) as described in any of the preceding items is an anti-HER3 antibody drug conjugate;

[0283] Wherein, Ab is an antibody, comprising a binding domain that specifically binds to HER3, wherein the binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).

[0284] In some embodiments, in the anti-HER3 antibody drug conjugate of formula (I), the heavy chain variable region of the binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 70.

[0285] In some embodiments, in the anti-HER3 antibody drug conjugate of formula (I), the heavy chain variable region of the binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 51-53, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 54, 55, and 73.

[0286] In some embodiments, in the anti-HER3 antibody drug conjugate of formula (I), the heavy chain variable region of the binding domain that specifically binds to HER3 comprises an amino acid sequence as shown in SEQ ID NO: 13, or at least 80% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or at least 80% identical thereto.

[0287] In some embodiments, the anti-HER3 antibody drug conjugate of formula (I) as described in any of the preceding items is selected from:

[0288] in:

[0289] n is 1 to 8, n is a decimal or an integer, such as an integer or decimal of 3-7, and an exemplary n is about 6;

[0290] Ab comprises a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 70.

[0291] In some embodiments, the anti-HER3 antibody drug conjugate represented by formula (IV) as described in any of the preceding items is selected from:

[0292] k is 1 to 8, k is a decimal or an integer, such as an integer or decimal of 3-7, and exemplary k is about 4 or 6;

[0293] Ab comprises a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 70.

[0294] In some embodiments, the aforementioned HER3 / MET binding molecules have at least one of the following properties:

[0295] a) specifically binds to MET or an epitope thereof; in some embodiments, the 7 K D The K D The detection method of the value is commonly used in the art, for example, the detection method in Example 2 of the present disclosure;

[0296] b) specifically binds to HER3 or an epitope thereof; in some embodiments, the 7 K D The K D The detection method of the value is commonly used in the art, for example, the detection method in Example 2 of the present disclosure;

[0297] c) Good binding activity to cells co-expressing MET and HER3; for example, EC 50 ≤5nM. For example, EC 50 ≤4nM, EC 50 ≤3nM, EC 50 ≤2nM, EC 50 ≤1nM, EC 50 ≤0.9nM, EC 50 ≤0.8nM, EC 50 ≤0.7nM, EC 50 ≤0.6nM, EC 50 ≤0.5nM, EC 50 ≤0.4nM, EC 50 ≤0.3 nM or lower; the EC 50 The detection method of the value is commonly used in the art, for example, the detection method in Example 3 of the present disclosure;

[0298] d) Good endocytic activity in cells co-expressing MET and HER3; for example, EC 50 ≤1 nM. For example, EC 50 ≤0.9nM, EC 50 ≤0.8nM, EC 50 ≤0.7nM, EC 50 ≤0.6nM, EC 50 ≤0.5nM, EC 50 ≤0.4nM, EC 50 ≤0.3nM, EC 50 ≤0.2 nM or lower; the EC 50 The detection method of the value is commonly used in the art, for example, the detection method in Example 4 of the present disclosure;

[0299] e) the endocytosis rate in cells co-expressing MET and HER3 was better than that of anti-HER3 antibody or anti-MET antibody;

[0300] f) It will not cause HER3 / MET target dimerization to activate downstream signaling pathways;

[0301] g) inhibiting tumor growth, or treating or alleviating cancer.

[0302] In some embodiments, the aforementioned HER3 / MET binding molecules of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0303] In some embodiments, the HER3 / MET binding molecules of the present disclosure are anti-HER3 / MET antibodies, such as anti-HER3 / MET bispecific antibodies or anti-HER3 / MET multispecific antibodies.

[0304] The antibodies include antigen-binding fragments, including but not limited to Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. In some specific embodiments, the antigen-binding fragment includes Fab, Fv, sFv, Fab', F(ab')2.

[0305] In some embodiments, anti-HER3 / MET antibodies are provided that bind to or compete for binding to MET and / or HER3, or bind to or compete for binding to the same epitope of MET and / or HER3, as the aforementioned HER3 / MET binding molecules of the present disclosure.

[0306] In some embodiments, anti-HER3 / MET antibodies are provided that block the aforementioned HER3 / MET binding molecules of the present disclosure from binding to MET and / or HER3.

[0307] Polynucleotides and vectors

[0308] The present disclosure provides polynucleotides encoding the HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules provided herein.

[0309] In some embodiments, the polynucleotide may be RNA, DNA, or cDNA.According to some embodiments of the present disclosure, the polynucleotide of the present disclosure is a substantially isolated nucleic acid.

[0310] The nucleic acids of the present disclosure may also be in the form of vectors, may be present in a vector and / or may be part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may be, in particular, an expression vector, i.e., a vector that provides for expression of HER3 / MET binding molecules, MET binding molecules and HER3 binding molecules in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a particular host. Regulatory elements and other elements useful or necessary for the expression of the HER3 / MET binding molecules of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0311] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0312] host cells

[0313] The present disclosure provides recombinant host cells that express or are capable of expressing one or more of the HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules of the present disclosure, and / or contain a nucleic acid or vector of the present disclosure.

[0314] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0315] Exemplary bacterial cells include, for example, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0316] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0317] Illustratively, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0318] The present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0319] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0320] Production or preparation method

[0321] The present disclosure provides methods of making the HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules of the present disclosure.

[0322] In some embodiments, the method comprises the following steps:

[0323] - culturing the host cell of the present disclosure under conditions suitable for expression of the antibody; and

[0324] - recovering the target protein expressed by the host cell from the culture; and

[0325] -Optionally, further purification and / or modification of the target protein of the present disclosure is included.

[0326] The HER3 / MET-binding molecules, MET-binding molecules, and HER3-binding molecules of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0327] Methods and reagents for recombinantly producing polypeptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for producing target proteins, such as binding molecules or antibodies, are well known to those skilled in the art. Methods for producing and purifying antibodies are well known in the art and can be found in the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture medium containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and multimers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be frozen immediately, such as at -70°C, or freeze-dried.

[0328] However, the HER3 / MET binding molecules, MET binding molecules and HER3 binding molecules of the present disclosure can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0329] The present disclosure also provides a method for preparing the aforementioned antibody-drug conjugate as shown in formula (I) or (IV), comprising the following steps:

[0330] The aforementioned Ab is coupled with a drug to obtain an antibody-drug conjugate as shown in formula (I) or (IV);

[0331] Optionally, the antibody-drug conjugate of formula (I) or (IV) is purified.

[0332] In some embodiments, a method for preparing an antibody drug conjugate as shown in formula (II) comprises the following steps:

[0333] After Ab is reduced, it is coupled with the general formula (La-Y-Dr) to obtain a compound represented by the general formula (Ab-La-Y-Dr); the reducing agent is preferably TCEP, especially preferably reducing the disulfide bonds on the antibody

[0334] wherein Ab, W, L2, L3, R1, R2, R5-R7, m and n are as defined in formula (I).

[0335] Composition

[0336] The present disclosure provides compositions comprising any one or any combination of the following: any HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule provided herein, a polynucleotide encoding a HER3 / MET binding molecule, a MET binding molecule, or a HER3 binding molecule, and a vector.

[0337] In some embodiments, the pharmaceutical composition contains an amount of the HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, encoding polynucleotide or vector described above that is effective for treating, alleviating or preventing cancer.

[0338] In some embodiments, the pharmaceutical composition comprises or consists of a mixture of HER3 / MET-binding molecules of Formula (I) having different n values, wherein at least 65% of the HER3 / MET-binding molecules of Formula (I) have n of 6. Exemplary, at least 70%, at least 75%, at least 80%, or at least 90%.

[0339] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0340] In some embodiments, the pharmaceutical composition may contain 0.01 to 99 weight percent of the HER3 / MET binding molecule, MET binding molecule, or HER3 binding molecule in a unit dose; or the amount of the HER3 / MET binding molecule, MET binding molecule, or HER3 binding molecule in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in some embodiments, 1-1000 mg.

[0341] In some embodiments, an article or product is provided comprising the aforementioned HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, polynucleotides, and / or vectors. Optionally, the article comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a composition effective for treating a condition. A label on or associated with the container indicates that the composition is used to treat the selected condition. The composition comprises the aforementioned HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, polynucleotides, and / or vectors.

[0342] Kits and Tests

[0343] The present disclosure provides kits comprising the aforementioned HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, polynucleotides, vectors, and compositions. The present disclosure also provides methods, systems, or devices for detecting MET or HER3 in vivo or in vitro, comprising treating a sample with the aforementioned binding molecules, polynucleotides, vectors, and compositions.

[0344] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0345] (1) contacting a sample with a HER3 / MET binding molecule, a MET binding molecule, a HER3 binding molecule, a polynucleotide, a vector, or a composition;

[0346] (2) detecting a complex formed between the aforementioned binding molecule, polynucleotide, carrier, and sample; and / or

[0347] (3) contacting a reference sample (e.g., a control sample) with the binding molecule, polynucleotide; and

[0348] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample or subject compared to the control sample or subject indicates the presence of MET or HER3 in the sample.

[0349] In other embodiments, the in vivo detection method, system or device may include:

[0350] (1) administering the aforementioned binding molecule, polynucleotide or vector to a subject; and

[0351] (2) Detecting the formation of a complex between the aforementioned binding molecule, polynucleotide, carrier, and subject.

[0352] Detection can include determining the position or time of complex formation. The aforementioned binding molecules and nucleic acid labels are labeled with a detectable substance, and the label is detected to detect substances (e.g., MET, HER3) that can bind to proteins and nucleic acids. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. The formation of complexes between binding molecules, nucleic acids and MET and HER3 can be detected by measuring substances that are bound or not bound to MET and HER3 or by visualizing them. Conventional detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry. For detection purposes, the binding molecules and nucleic acids of the present disclosure can be labeled with fluorophore chromophores. In some embodiments, diagnostic reagents comprising the above-mentioned nucleic acids and binding molecules are also provided, as well as related diagnostic uses.

[0353] In some embodiments, a kit is also provided, comprising the aforementioned binding molecules and polynucleotides, and further comprising instructions for use in diagnosis. The kit may further comprise at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the binding molecules may be formulated as a pharmaceutical composition.

[0354] Methods for treating diseases and pharmaceutical uses

[0355] The present disclosure provides uses and methods of HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, encoding polynucleotides, vectors or pharmaceutical compositions in preventing, treating or alleviating diseases or disorders.

[0356] In some embodiments, the present disclosure provides HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules or encoding polynucleotides, vectors, pharmaceutical compositions for treating or ameliorating cancer, or for preparing medicaments for treating or ameliorating cancer.

[0357] In some embodiments, the present disclosure provides a method for preventing, treating, or ameliorating cancer or tumors, comprising administering to a patient or subject an effective amount of a HER3 / MET binding molecule, a MET binding molecule, a HER3 binding molecule, or encoding polynucleotide, vector, or pharmaceutical composition of the present disclosure for preventing, treating, or ameliorating the disease or condition.

[0358] In some specific embodiments, the tumor or cancer is HER3 positive. In some specific embodiments, the tumor or cancer is HER3 and MET double positive.

[0359] In some specific embodiments, the tumor or cancer is resistant to EGFR-TKI (epidermal growth factor receptor tyrosinase inhibitor).

[0360] In some embodiments, the HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, or encoding polynucleotides, vectors, and pharmaceutical compositions of the present disclosure can be administered by any suitable method known in the art, and administration can be systemic or local.

[0361] In some embodiments, the dosage regimen can be adjusted to obtain the optimal intended response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation. BRIEF DESCRIPTION OF THE DRAWINGS

[0362] Figures 1A-1C show HER3 / MET dual-target IHC staining of tumor samples from patients with EGFR-TKI-resistant non-small cell lung cancer.

[0363] FIG2 shows the secondary mass spectrometry results of Antibody A and Antibody A T94V.

[0364] FIG3 shows the FACS detection of the binding of HER3 / MET bispecific antibody to MET single-positive cells.

[0365] Figure 4A shows the FACS detection of antibody binding to HER3 single-positive cells. Figure 4B shows the FACS detection of antibody binding to MET single-positive cells. Figure 4C shows the FACS detection of antibody binding to HER3 / MET double-positive cells.

[0366] Figure 5A shows the killing of antibodies in HER3 / MET double-positive cells detected by the αHFc-CL-MMAE toxin antibody system. Figure 5B shows the killing of antibodies in HER3 single-positive cells detected by the αHFc-CL-MMAE toxin antibody system. Figure 5C shows the killing of antibodies in MET single-positive cells detected by the αHFc-CL-MMAE toxin antibody system. Figure 5D shows the killing of antibodies in HER3 / MET double-positive cells detected by the αHFc-CL-MMAE toxin antibody system.

[0367] Figure 6 shows the internalization of the pHrodo-detected antibody in HER3 / MET double-positive cells.

[0368] FIG7 shows the internalization of antibodies in HER3 / MET double-positive cells detected by FACS.

[0369] Figure 8 shows the degree of ERK phosphorylation by the antibody in HER3 / MET double-positive cells.

[0370] FIG9A is a schematic diagram of a HER3 / MET antibody-drug conjugate, FIG9B is a denatured mass spectrum of the HER3 / MET antibody-drug conjugate, and FIG9C is a native mass spectrum of the HER3 / MET antibody-drug conjugate.

[0371] FIG10 is a FACS assay showing the binding of HER3 / MET bispecific antibodies and their ADCs to HER3 / MET double-positive cells.

[0372] Figure 11A shows the results of FACS analysis of HER3 / MET expression in HER3 / MET double-positive cells. Figure 11B shows the results of ADC-4 killing HER3 / MET double-positive cells. Figure 11C shows the results of ADC-1 killing HER3 / MET double-positive cells.

[0373] Figures 12A and 12B show the anti-tumor activity results of the bispecific ADC in the HCC827 Osimertinib-resistant cell xenograft model, where Figure 12A shows the changes in mouse body weight and Figure 12B shows the changes in mouse tumor volume.

[0374] Figures 13A and 13B show the anti-tumor activity results of the dual-antibody ADC in a HER3 single-positive cell xenograft model, wherein Figure 13A shows the changes in mouse body weight, and Figure 13B shows the changes in mouse tumor volume.

[0375] Figure 14 shows the killing activity of the dual-antibody ADC in HER3 / MET double-positive cells. DETAILED DESCRIPTION

[0376] Definition of terms

[0377] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0378] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0379] "MET" or "cMET" or "MET protein" or "MET polypeptide" may optionally include any such protein or variant, conjugate, or fragment thereof, including but not limited to known or wild-type MET as described herein, as well as any naturally occurring splice variants, amino acid variants, or isoforms. The complete MET sequence may be UniProt No. P08581.

[0380] "HER3" or "HER3 protein" or "HER3 polypeptide" may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type HER3 as described herein, as well as any naturally occurring splice variants, amino acid variants or isoforms. The complete HER3 sequence may be UniProt No. P21860.

[0381] The term "functional variant" includes but is not limited to homologs, fragments, truncations, mutants, modifications, etc. of the wild-type protein. The functional variant of the protein has improved, reduced or maintained protein activity compared to the wild-type protein.

[0382] The "binding protein" and "binding molecule" of the present disclosure encompass any protein, polypeptide or any molecule comprising the protein or polypeptide that can specifically bind to an antigen (e.g., MET or HER3) or a fragment or epitope thereof, including but not limited to antibodies as defined in the present disclosure.

[0383] Throughout this disclosure, "polypeptide," "peptide," or "protein" are used interchangeably to refer to a polymer of amino acid residues, or an aggregate of multiple polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptide sequences are typically described with the left-hand end of the polypeptide sequence being the amino terminus (N-terminus, N-terminus) and the right-hand end of the polypeptide sequence being the carboxyl terminus (C-terminus, C-terminus).

[0384] "Titin-T chain" or "T chain" refers to a 78-118 amino acid peptide segment of the titin protein comprising the titin Ig-like 152 domain, or a functional variant thereof. The titin-T chain is capable of binding to the obscurin Ig-like 1 domain to form a dimerization complex. Functional variants of the T chain are polypeptides in which some amino acids of the wild-type T chain are mutated, but which still bind to the obscurin Ig-like 1 domain to form a dimerization complex. For example, a suitable length of amino acids may be added or truncated at the C-terminus and / or N-terminus of the titin Ig-like 152 domain. One, two, three, four, five, six, seven, eight, nine, or ten amino acid residues may be added or truncated. For example, five amino acids, "KAGIR," located immediately adjacent to the N-terminus of the titin Ig-like 152 domain in the wild-type titin protein, may be added to the N-terminus of the titin Ig-like 152 domain, while still being able to associate with the obscurin Ig-like 1 domain to form a complex. Other mutations can also be made to the amino acids of the titin Ig-like 152 domain, for example, mutations to certain amino acids can be made to improve interchain disulfide bonds, enhance the stability of the complex, and the like.

[0385] "Obscurin-O chain" or "O chain" refers to a peptide segment of the obscurin protein containing the obscurin Ig-like 1 domain, or a functional variant thereof, that is 87-117 amino acids in length. The obscurin-O chain is capable of binding to the titin Ig-like 152 domain to form a dimerization complex. These functional obscurin-O chain variants are polypeptides that have had some amino acid mutations in the wild-type O chain but still bind to the titin Ig-like 152 domain to form a dimerization complex. For example, an appropriate length of amino acids can be added or truncated at the C-terminus and / or N-terminus of the obscurin-O domain, for example, by adding or truncating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For example, the five amino acids "DQPQF" located immediately adjacent to the N-terminus of the obscurin Ig-like 1 domain in the wild-type obscurin protein can be added to the N-terminus of the obscurin-O domain, while still functioning to bind to the titin Ig-like 152 domain to form a dimerization complex. Other amino acid mutations can also be made to portions of the obscurin Ig-like 1 domain, for example, to improve interchain disulfide bonds or enhance antibody stability.

[0386] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to any DNA or RNA molecule that is single-stranded or double-stranded and, in the case of single-stranded molecules, its complementary sequence, preferably double-stranded DNA.

[0387] "Antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, bispecific or multispecific antibodies (e.g., trispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, antigen-binding domains), as long as they exhibit the desired antigen-binding activity. Antibodies of the present disclosure include recombinant antibody forms.

[0388] Antibodies, also known as immunoglobulins, are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of immunoglobulins (Ig) heavy chains vary, resulting in different antigenicity. This classification leads to the classification of immunoglobulins into five classes, or isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same Ig class, the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds are further divided into subclasses. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on their constant regions. Each of the five Ig classes can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly, forming the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable, forming the constant region (C region). The variable region consists of three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0389] The prior art has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bivalent, trivalent, tetravalent or higher-valent bispecific antibodies. Based on whether the structure is bilaterally symmetrical, they can be divided into bispecific antibodies with symmetrical structures and bispecific antibodies with asymmetric structures. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, are formed by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, (scFv)2-Fab and other bispecific antibodies; IgG-like bispecific antibodies (for example, with Fc fragments) have a relatively large molecular weight. The Fc fragment helps in the later purification of the antibody and improves its solubility and stability. The Fc part may also bind to the receptor FcRn to increase the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019: 4516041).

[0390] "Antigen-binding fragment" or "antigen-binding domain" encompasses Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0391] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0392] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0393] Table 1. Relationships between CDR numbering systems

[0394] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0395] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation equilibrium constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of the complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and FACS, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore. TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D K value and non-target interaction D The value is evaluated.

[0396] Typically, "specific binding" refers to binding of a binding molecule (binding protein) to an epitope on an antigen. The HER3 / MET binding molecules of the present disclosure will bind to an epitope as measured in a Biacore or KinExA or Fortibio assay at a specific binding affinity of ≤10 -7 M, preferably ≤10 -8The dissociation equilibrium constant (K D ) binds to the antigen to be bound (ie, HER3 or MET) or its epitope. Any greater than 10 -4 M's K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of a binding molecule to an antigen or epitope can be determined by any suitable means known in the art, including, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry sorting (FACS) as described herein.

[0397] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0398] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two nucleic acid sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0399] The term "internalization" refers to the transport of a portion from the outside of a cell to the inside. The internalized portion can be located in an intracellular compartment. An "internalized" or "internalized" antigen or antibody refers to an antigen or antibody that is capable of being transported from the outside of a target cell to the inside. It is generally understood by those skilled in the art that the process of cellular internalization generally refers to the movement of cell surface molecules across the plasma membrane from the cell surface to the interior of the cell. After internalization, the endosome can be transported to the lysosome for degradation or recycled to the cell surface. The cellular internalization rate of a given cell surface molecule provides a measure of the kinetics of the movement of the molecule from the cell surface across the plasma membrane to the interior of the cell. Internalization activity or internalization rate of antigens and antibodies can be monitored and / or measured by various techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305–17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007). Many antibody labeling techniques, dyes, and kits for antibody labeling that can be used to quantify and monitor internalization are commercially available (e.g., pHrodo iFL antibody labeling methods, reagents, and kits sold by Thermo Fisher Scientific).

[0400] The term "antibody-drug conjugate" (ADC) refers to an antibody linked to a biologically active drug, wherein the antibody can be coupled to the drug directly or via a linker.

[0401] The term "drug loading" refers to the average number of cytotoxic drugs loaded per ligand in the ADC, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading range can be 1-20, preferably 1-10, cytotoxic drugs (D) attached per antibody (Ab). In the embodiments of the present disclosure, the drug loading is expressed as n, and is exemplified by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values ​​therebetween. The average number of drug per ADC molecule after the coupling reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, monoclonal antibody size variant assay (CE-SDS), and HPLC characterization.

[0402] While the drug-to-antibody ratio has a specific value for a particular conjugate molecule (e.g., n in Formula (I)), it will be understood that when used to describe a sample containing many molecules, this value will often be an average value due to some degree of heterogeneity typically associated with the conjugation step. The average loading of an immunoconjugate sample is referred to herein as the drug-to-antibody ratio or "DAR." In some embodiments, the DAR is between about 1 and about 10, e.g., 1-8, and is typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. Embodiments include those wherein DAR is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4 In some embodiments, a DAR of 'about x' means that the measured value of the DAR is within 20% of x.

[0403] The detection method of DAR, for example, is to extrapolate the DAR value from the LC-MS data of reduced and deglycosylated samples. LC / MS allows the quantification of the average number of payload (drug moiety) molecules connected to the antibody in the ADC. HPLC separates the antibody into light and heavy chains, and also separates the heavy chain (HC) and light chain (LC) according to the number of linker-payload groups of each chain. Mass spectrometry data can identify the types of components in the mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. Based on the average loading amount of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (payload) molecules connected to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0404] The term "camptothecin drugs" refers to cytotoxic camptothecin and its derivatives, including but not limited to 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, exitecan, irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0405] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0406] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0407] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0408] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0409] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0410] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, the ring contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls.

[0411] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group in which the monocyclic rings of 5 to 20 members share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer 0 to 2) heteroatom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of shared spiral atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group and a dispiro heterocyclic group. It is more preferably 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0412] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, wherein each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0413] The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclic groups include:

[0414] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0415] wait.

[0416] The heterocyclic group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0417] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0418] The aryl group may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0419] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0420] The heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0421] The term "amino-protecting group" is used to protect the amino group with an easily removable group in order to maintain the amino group intact while reacting other parts of the molecule. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be substituted with 1-3 substituents selected from halogen, alkoxy, or nitro. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0422] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above and wherein cycloalkyl is as defined above.

[0423] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0424] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0425] The term "hydroxy" refers to an -OH group.

[0426] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0427] The term "amino" refers to -NH2.

[0428] The term "nitro" refers to -NO2.

[0429] The term "amido" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0430] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

[0431] The present disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds of formula (I) with reference to relevant literature. When preparing deuterated forms of compounds of formula (I), commercially available deuterated starting materials can be used, or they can use conventional techniques to synthesize deuterated reagents, which include but are not limited to deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane etc.

[0432] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0433] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable vehicle" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents.

[0434] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0435] "Proliferative disease" refers to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative condition refers to cancer. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0436] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0437] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0438] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0439] "Optional" or "optionally", "optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B", "A or B", "A" (alone) and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising", "having", "including", etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to".

[0440] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0441] Example

[0442] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0443] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0444] Example 1. HER3 / MET dual-target IHC staining results of tumor samples from patients with EGFR-TKI-resistant non-small cell lung cancer

[0445] Tumor samples from patients with EGFR-TKI-resistant NSCLC were examined using HER3 / MET dual-target IHC staining. Figures 1A to 1C show that HER3 / MET is expressed in tumor tissues from patients with EGFR-TKI-resistant NSCLC, with a co-expression rate of 100%. This suggests that HER3 / MET targets are expressed and co-expressed at a high rate in patients with EGFR-TKI-resistant NSCLC. A HER3 / MET bispecific antibody ADC may achieve superior anti-tumor activity in these patients compared to either HER3 or MET monoclonal antibody ADCs.

[0446] Example 2. Design and preparation of anti-HER3 / MET bispecific antibodies

[0447] 1. Design of anti-HER3 / MET bispecific antibodies

[0448] The variable region at the HER3 end is selected from antibody A; the variable region at the MET end is selected from antibody A13, antibody S3, antibody A9, antibody P8, antibody P3, and antibody 5D5. The sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the antibodies are as follows:

[0449] >Anti-MET Antibody A13 VH

[0450] >Anti-MET Antibody A13 VL

[0451] >Anti-MET Antibody S3 VH

[0452] >Anti-MET Antibody S3 VL

[0453] >Anti-MET Antibody A9 VH

[0454] >Anti-MET Antibody A9 VL

[0455] >Anti-MET Antibody P8 VH

[0456] >Anti-MET Antibody P8 VL

[0457] >Anti-MET Antibody P3 VH

[0458] >Anti-MET Antibody P3 VL

[0459] >Anti-MET Antibody 5D5 VH

[0460] >Anti-MET Antibody 5D5 VL

[0461] >Anti-HER3 Antibody A VH

[0462] >Anti-HER3 Antibody A VL

[0463] Table 2. Antibody CDR sequences (Kabat numbering convention)

[0464] Construct a bispecific antibody with the following structure:

[0465] The first heavy chain, from N-terminus to C-terminus, is as follows: anti-MET antibody heavy chain variable region-GGGGS-[Obscurin-O chain]-[IgG1 Fc1],

[0466] The first light chain, from N-terminus to C-terminus, is: anti-MET antibody light chain variable region-GGGGS-[Titin-T chain],

[0467] The second heavy chain, from N-terminus to C-terminus, is: anti-HER3 antibody heavy chain variable region-[CH1]-[IgG1Fc2], and

[0468] The second light chain, which, from N-terminus to C-terminus, consists of: anti-HER3 antibody light chain variable region - [CL];

[0469] Wherein, -- represents a peptide bond, Obscurin-O chain is shown in SEQ ID NO: 63, Titin-T chain is shown in SEQ ID NO: 64, CH1 is shown in SEQ ID NO: 65, CL is shown in SEQ ID NO: 66, IgG1 Fc1 is shown in SEQ ID NO: 67, and IgG1 Fc2 is shown in SEQ ID NO: 68.

[0470] The heavy and light chain variable regions of the heavy chain of antibody A were operably linked to the heavy and light chain variable regions of antibodies A13, S3, A9, P8, P3, and 5D5, respectively, according to the above structure, to construct the anti-HER3 / MET bispecific antibodies 2232-01 (A13-A), 2232-02 (S3-A), A9-A, P8-A, P3-A, and 5D5-A. The heavy chain (H) and light chain (L) sequences of 2232-01 and 2232-02 are shown below:

[0471] >2232-01H1

[0472] >2232-01L1

[0473] >2232-01H2

[0474] >2232-01L2

[0475] >2232-02H1

[0476] >2232-02L1

[0477] 2232-02H2 is identical to SEQ ID NO:59;

[0478] 2232-02L2 is consistent with SEQ ID NO:60.

[0479] In the above heavy chain sequence, the italicized one is the Fc constant region of IgG1, the underlined wavy line is the Obscurin-O chain, and the underlined horizontal line is VH1; in the light chain sequence, the underlined horizontal line is the Cκ of IgG1, and the underlined wavy line is the Titin-T chain; the italicized bold one is the GGGGS (SEQ ID NO: 91) linker.

[0480] >Obscurin-O chain

[0481] >Titin-T chain

[0482] >CH1

[0483] >CL

[0484] >IgG1 Fc1(S354C / T366W)

[0485] >IgG1 Fc2(Y349C / T366S / L368A / Y407V)

[0486] 2. Antibody modification

[0487] We designed four sets of mutations, N93Q, N93T, N93S, and T94V, for the anti-HER3 antibody Antibody A (having a heavy chain as described in SEQ ID NO: 86 and a light chain as described in SEQ ID NO: 87). Antibody A N93Q is an N-to-Q mutation only at position 93 of VL relative to Antibody A, Antibody A N93T is an N-to-T mutation only at position 93 of VL relative to Antibody A, Antibody A N93S is an N-to-S mutation only at position 93 of VL relative to Antibody A, and Antibody A T94V is a T-to-V mutation only at position 94 of VL relative to Antibody A.

[0488] Experimental plan: Antibodies A, A N93Q, A N93T, A N93S, and A T94V were diluted to 1 μg / mL in HBS-EP+ buffer at a flow rate of 10 μL / min, and the capture antibody was maintained at 200 RU. His-tagged human HER3 antigen was diluted in HBS-EP+ buffer at a concentration gradient of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. Sample analysis was performed at a flow rate of 30 μL / min. The association time was 120 s, and the dissociation time was 900 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer at a flow rate of 30 μL / min for 30 s. The response signal is plotted against the analysis time on the horizontal axis and the response value on the vertical axis. The data were fitted by BIAcore 8K analysis software, and the kinetic constants such as association rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD) were determined using a 1:1 Langmuir binding model.

[0489] Experimental results: Table 3-1 and Figure 2 show that there is no significant change in the binding of the antibody to HER3 before and after the T94V mutation, but the deamidation level of the antibody is significantly reduced.

[0490] Table 3-1. Binding activity of antibody A before and after mutation

[0491] The variable region sequence of antibody A T94V is as follows:

[0492] >Anti-HER3 Antibody A VH

[0493] >Anti-HER3 Antibody A VL T94V

[0494] We designed four groups of mutations, N30Q, N30T, N30S, and T31V, for the anti-MET antibody Antibody A13 (having a heavy chain as set forth in SEQ ID NO: 84 and a light chain as set forth in SEQ ID NO: 85). Antibody A13 N30Q has an N-to-Q mutation only at position 30 of VL relative to Antibody A13, Antibody A13 N30T has an N-to-T mutation only at position 30 of VL relative to Antibody A13, Antibody A13 N30S has an N-to-S mutation only at position 30 of VL relative to Antibody A13, and Antibody A13 T31V has a T-to-V mutation only at position 31 of VL relative to Antibody A13.

[0495] Experimental protocol: Antibodies A13, A13 N30Q, A13 N30T, A13 N30S, and A13 T31V were diluted to 1 μg / mL in HBS-EP+ buffer at a flow rate of 10 μL / min, and the capture antibody was maintained at 200 RU. His-tagged human MET antigen was diluted in HBS-EP+ buffer at a concentration gradient of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. Samples were analyzed at a flow rate of 30 μL / min. Association time was 120 s, and dissociation time was 900 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer at a flow rate of 30 μL / min for 30 s. The response signal is plotted against analysis time on the horizontal axis and response value on the vertical axis. The data were fitted by BIAcore 8K analysis software, and the 1:1 Langmuir binding model was used to determine the kinetic constants such as the association rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD).

[0496] Experimental results: Table 3-2 shows that there is no significant change in the antibody's binding to HER3 before and after the N30S mutation, and the antibody's deamidation level is significantly reduced.

[0497] Table 3-2. Binding activity of antibody A13 before and after mutation

[0498] Building on the N30S construct, we further mutated the framework region (FR) of the anti-MET antibody A13. Specifically, we mutated the VH variant to E23K and S78T, and the VL variant to A69T, resulting in the antibody A13 E23K / S78T / A69T / N30S. Testing showed that the expression level of the antibody A13 E23K / S78T / A69T / N30S increased from 96 mg / L to 200 mg / L.

[0499] The variable region sequence of antibody A13 E23K / S78T / A69T / N30S is as follows:

[0500] >Anti-MET Antibody A13 E23K / S78T / A69T / N30 VH

[0501] >Anti-MET Antibody A13 E23K / S78T / A69T / N30 VL

[0502] Table 3-3. Antibody CDR (Kabat numbering convention)

[0503] That is, the antibody A of the present disclosure has the following general sequence:

[0504] HCDR1: DYAMH (SEQ ID NO: 51)

[0505] HCDR2:GISWNSGSIGYADSVKG (SEQ ID NO: 52)

[0506] HCDR3:EGLPGLDY(SEQ ID NO:53)

[0507] LCDR1: RASQHVGTYLN (SEQ ID NO: 54)

[0508] LCDR2:GAANLQS(SEQ ID NO:55)

[0509] LCDR3: QQSYX1X2PPFS (SEQ ID NO: 75), wherein X1 is selected from N, Q, T or S, and X2 is selected from T or V.

[0510] The antibody A13 of the present disclosure has the following general sequence:

[0511] HCDR1: SYGFS (SEQ ID NO: 15)

[0512] HCDR2: WISASNGNTYYAQKLQG (SEQ ID NO: 16)

[0513] HCDR3: VYADYADY (SEQ ID NO: 17)

[0514] LCDR1: RASQGIX3X4WLA (SEQ ID NO: 76), wherein X3 is selected from N, Q, T or S, and X4 is selected from T or V.

[0515] LCDR2:AASSLKS(SEQ ID NO:19)

[0516] LCDR3: QQANSFPLT (SEQ ID NO: 20).

[0517] The above mutated sequence was used to construct the anti-HER3 / MET bispecific antibody 2232-06, and the full-length amino acid sequence is as follows:

[0518] >2232-06(A13 E23K / S78T / A69T / N30S,AT94V)H1 heavy chain

[0519] >2232-06(A13 E23K / S78T / A69T / N30S,AT94V)L1 light chain

[0520] >2232-06 (A13 E23K / S78T / A69T / N30S, A T94V) H2 heavy chain is identical to SEQ ID NO: 59;

[0521] >2232-06(A13 E23K / S78T / A69T / N30S,AT94V)L2 light chain

[0522] 3. Control Antibodies

[0523] The full-length sequences of the heavy chain (HC) and light chain (LC) of the naked antibody of the control antibody U3-1402 (U3-1402 Ab), antibody S3, antibody A13, and antibody A are as follows:

[0524] >U3-1402 Ab HC

[0525] >U3-1402 Ab LC

[0526] >Antibody S3 HC

[0527] >Antibody S3 LC

[0528] >Antibody A13 HC

[0529] >Antibody A13 LC

[0530] >Antibody A HC

[0531] >Antibody A LC

[0532] 4. Antibody Preparation

[0533] The nucleotide sequences encoding the heavy and light chains of the antibody were cloned into the pTT5 vector and transfected into ExpiCHO cells. After 8 days, the cells were removed by centrifugation, and the cell culture fluid was collected and filtered. The harvested cell culture fluid was purified using a Protein A affinity column (MabSelect SuRe, GE). The bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris and then desalted. After testing, the target antibody was obtained.

[0534] Example 3. Antigen Binding Activity Detection of Anti-HER3 / MET Bispecific Antibodies

[0535] In this example, FACS experiments were used to detect the binding activity of the anti-HER3 / MET bispecific antibody to the human MET protein and HER3 / MET protein on the cell surface.

[0536] 1. Detection of binding activity to cell surface MET protein

[0537] Experimental Methods: NCI-H1975 (lung cancer cell line) is a MET single-positive cell line. The cell culture medium was RPMI 1640 medium (ATCC modification) (Gibco, Cat# A1049101) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffered saline, pH 7.40) containing 2% fetal bovine serum (the same below). NCI-H1975 cells were washed twice with experimental medium and 1×10 cells were plated per well. 5 Cells were seeded in 96-well U-bottom plates, and the test samples were added at different concentrations. After incubation at 4°C for 1 hour, the cells were washed twice with experimental culture medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was then added. After washing twice, the fluorescence signal value was read by flow cytometry.

[0538] Experimental results: The FACS results in Figure 3 show that different anti-HER3 / MET bispecific antibodies (same anti-HER3 antibody sequence, different anti-MET antibody sequences) have different binding abilities to MET single-positive cells. Among them, A13-A has the strongest binding ability and is an anti-HER3 / MET bispecific antibody with high affinity for MET; P8-A has the weakest binding ability and is an anti-HER3 / MET bispecific antibody with low affinity for MET; S3-A binds to EC 50 The affinity of the anti-HER3 / MET bispecific antibody was 10-fold different from that of A13-A, indicating a low-to-medium affinity for MET. 2232-01 (A13-A), with high affinity for MET, and 2232-02 (S3-A), with medium-to-low affinity for MET, were selected for the next screening step.

[0539] 2. Detection of binding activity with human HER3 / MET protein on cell surface

[0540] Experimental Methods: MDA-MB-453 (breast cancer cell line) is a HER3 single-positive cell line. The cell culture medium is Leibovitz's L-15 (Gibco, Cat#11415064) supplemented with 10% fetal bovine serum and cultured in air. NCI-H1703 (lung cancer cell line) is a MET single-positive cell line. The cell culture medium is RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) supplemented with 10% fetal bovine serum. The HCC827 osimertinib-resistant cell line (lung cancer cell line) is an in-house constructed HER3 / MET double-positive cell line. HCC827 cells were generated by gradient resistance induction with osimertinib. The cell culture medium is RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) supplemented with 10% fetal bovine serum. Wash MDA-MB-453, NCI-H1703 or HCC827 Osimertinib-resistant cells twice with experimental medium and add 1 × 10 cells per well. 5 Cells were seeded in 96-well U-bottom plates and tested at various concentrations. After incubation at 4°C for 1 hour, the cells were washed twice with experimental culture medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat# 209-605-098) was then added. After two washes, the fluorescence signal was measured by flow cytometry. IgG1 was used as an isotype control antibody.

[0541] Experimental Results: The FACS results in Tables 4-1 and 4-2, and Figures 4A and 4B, demonstrate that the anti-HER3 / MET bispecific antibodies bind to cell surface HER3 / MET antigens. In MET single-positive cells, the high-affinity MET binding of 2232-01 was superior to the medium-low-affinity MET binding of 2232-02. In HER3 single-positive cells, the binding abilities of both molecules to HER3 were similar, though slightly weaker than those of Antibody A.

[0542] The results in Table 4-3 and Figure 4C show that in HER3 / MET double-positive cells, the maximum fluorescence value of the anti-HER3 / MET bispecific antibody is higher than that of the anti-HER3 antibody (antibody A and U3-1402 Ab) and the anti-MET antibody (antibody S3), indicating that more anti-HER3 / MET bispecific antibodies are bound to the double-positive cells.

[0543] Table 4-1. Results of anti-HER3 / MET bispecific antibodies binding to HER3 single-positive cells

[0544] Table 4-2. Results of anti-HER3 / MET bispecific antibodies binding to MET single-positive cells

[0545] Table 4-3. Results of anti-HER3 / MET bispecific antibodies binding to HER3 / MET double-positive cells

[0546] Example 4. Detection of endocytic activity of anti-HER3 / MET bispecific antibodies

[0547] 1. Evaluation of the Internalization Activity of Antibodies to αHFc-CL-MMAE Toxin

[0548] The αHFc-CL-MMAE toxin antibody internalization activity evaluation system was used to assess antibody internalization and toxin cytotoxicity. αHFc-CL-MMAE is an anti-human Fc antibody conjugated to the MMAE toxin, which has a stable, cleavable linker that specifically binds to the Fc portion of human IgG. MMAE is a toxic small molecule that inhibits cell division by blocking tubulin polymerization. The linker is stable in the extracellular matrix and, upon endocytosis, is cleaved by lysosomal cathepsins, releasing the toxin. Therefore, the cytotoxicity of an antibody can be used to evaluate its endocytic activity.

[0549] Experimental Methods: NCI-H441 (lung cancer cell line) was used as a MET / HER3 double-positive cell line, MDA-MB-453 (breast cancer cell line) was used as a HER3 single-positive cell line, NCI-H1703 (lung cancer cell line) was used as a MET single-positive cell line, and the HCC827 osimertinib-resistant cell line (lung cancer cell line, constructed in-house) was used as a MET / HER3 double-positive cell line. HCC827 cells were induced with osimertinib gradient resistance. The culture medium and culture conditions were the same as in Example 3. αHFc-CL-MMAE (Moradec, Cat# AH-102AE-50) and the test antibody were mixed at an equal molar concentration of 1:1 by volume, incubated at 37°C for 30 minutes, and then diluted three-fold in complete culture medium. The cells were added to cells plated one day earlier (600 cells / well) and incubated in a 5% CO2 incubator at 37°C for 6 days. After the incubation, CellTiter-Glo (Promega, Cat#G7570) was added and incubated at room temperature in the dark for 10 min. Chemiluminescence was read on a PerkinElmer ENVISION and EC was calculated. 50 Values ​​and Emax values ​​(relative to the values ​​of the group without antibody).

[0550] Experimental results: Table 5-1 and Figure 5A show that different anti-HER3 / MET bispecific antibodies (same anti-HER3 antibody sequence, different anti-MET antibody sequences) have different killing activities against HER3 / MET double-positive cells, among which A13-A has the strongest killing ability and P3-A has the weakest killing ability. 2232-01 (A13-A) and 2232-02 (S3-A) were selected for the next step of screening to explore the anti-tumor activity of bispecific antibody ADCs with different MET affinities.

[0551] The cell viability assay results in Tables 5-2 and 5-3, Figures 5B and 5C show that in HER3 single-positive cells, the cytotoxicity of the αHFc-CL-MMAE toxin antibody of the anti-HER3 / MET bispecific antibody was similar to that of Antibody A. In MET single-positive cells, the cytotoxicity of the αHFc-CL-MMAE toxin antibody of 2232-01 was superior to that of 2232-02.

[0552] The cell activity test results in Table 5-4 and Figure 5D show that the killing activity of the αHFc-CL-MMAE toxin antibody of the anti-HER3 / MET bispecific antibody in double-positive cells is stronger than that of antibody S3 or antibody A and U3-1402 Ab, indicating that the endocytic activity of the bispecific antibody is stronger in double-positive cells.

[0553] Table 5-1. Results of endocytic activity assay of anti-HER3 / MET bispecific antibodies in HER3 / MET double-positive cells (NCI-H441)

[0554] Table 5-2. Results of endocytic activity detection of anti-HER3 / MET bispecific antibodies in HER3 single-positive cells (MDA-MB-453)

[0555] Table 5-3. Results of endocytic activity assay of anti-HER3 / MET bispecific antibodies in MET single-positive cells (NCI-H1703)

[0556] Table 5-4. Results of endocytic activity assay of anti-HER3 / MET bispecific antibody in HER3 / MET double-positive cells (HCC827 Osimertinib)

[0557] 2. Zenon TM pHrodo TM iFL IgG evaluation

[0558] Zenon TM pHrodo TM iFL IgG Indicator Reagent (Invitrogen, Cat# Z25612) provides a rapid, real-time, and reliable method for assessing antibody internalization. The pHrodo iFL Red-labeled Fab fragment binds to the Fc portion of intact IgG antibodies, forming a labeled complex within 5 minutes. Once the complex enters the cell via endocytosis, fluorescence increases dramatically as the acidity of the surrounding environment increases. The fluorescence intensity can be recorded in real time using the Incucyte instrument, thereby determining the degree of antibody internalization.

[0559] Experimental Methods: 5000 osimertinib-resistant HCC827 cells were plated overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Each well was incubated with 50 μL of the pre-incubated antibody and labeled complex. The next day, 50 μL of the pre-incubated antibody and labeled complex were added to each well. The plates were placed in an Incucyte instrument (IncuCyte S3 Live-Cell Analysis System, Sartorius). Reading conditions and intervals were set according to the instrument's protocol. After the plate was read, data were analyzed using the instrument's included analysis software.

[0560] As shown in Figure 6, the endocytic activity of the anti-HER3 / MET bispecific antibody is significantly better than that of the monoclonal antibody, indicating that more bispecific antibody molecules enter the cell through endocytosis.

[0561] 3. FACS Endocytosis Assessment

[0562] Antibodies undergo endocytosis at 37°C. By comparing the antibody incubated at 4°C, the endocytosis rate of the antibody at each time point can be obtained and the endocytic activity of the antibody can be evaluated.

[0563] Experimental Methods: HCC827 osimertinib-resistant cell line (lung cancer cell line) was constructed in-house as a HER3 / MET double-positive cell line. HCC827 cells were induced to resist osimertinib by gradient induction. The cell culture medium was RPMI 1640 medium (ATCC modification) (Gibco, Cat# A1049101) containing 10% fetal bovine serum. HCC827 osimertinib-resistant cells were washed twice with experimental culture medium and 1×10 cells were plated per well. 5 Cells were seeded in 96-well U-bottom plates. At each time point, a 96-well U-bottom plate was added with a saturating concentration of the test sample, incubated at 4°C for 1 hour, and then washed twice with experimental medium. The samples were incubated at 37°C and 4°C, respectively, and removed for analysis at each time point. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat# 209-605-098) was then added, washed twice, and fluorescence signals were read by flow cytometry. The internalization rate was calculated as follows: (MFI of the antibody at 4°C incubation - MFI of the antibody at 37°C incubation) - MFI of the antibody at 37°C incubation.

[0564] As shown in Table 6 and Figure 7, the endocytosis rate of anti-HER3 antibody A is better than that of anti-MET antibody A13, and the endocytosis rate of anti-HER3 / MET bispecific antibody 2203-01 is better than that of anti-HER3 antibody A and anti-MET antibody A13.

[0565] Table 6. Endocytosis rate from 0 to 6 hours

[0566] Example 5. Detection of ERK phosphorylation activation activity

[0567] HER3 / MET dimerization leads to activation of downstream signaling pathways (Tanizaki et al., British Journal of Cancer, 105(6), pp.807-813 (2011)), promoting tumor cell proliferation and survival. To test whether the disclosed antibodies have agonist activity, ERK phosphorylation was evaluated.

[0568] Experimental method: 4×10 HCC827 osimertinib-resistant cells were cultured in 96-well flat-bottom plates. 4Plate cells overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The next day, replace the low-serum medium (50 μL / well) with RPMI 1640 medium supplemented with 0.05% FBS and starve the cells for 5 hours. Dilute the anti-HER3 / MET bispecific antibody and control antibody in serum-free medium, add 50 μL / well to the 96-well plate, and incubate at 37°C for 5 minutes. Following incubation, rapidly measure ERK phosphorylation levels within the tumor using the Advanced Phospho-ERK1 / 29 (THR202 / TYR204) Kits (PerkinElmer, Cat# 64ERKPEG).

[0569] Experimental results: Figure 8 shows that anti-HER3 antibody A does not induce ERK phosphorylation, and anti-HER3 / MET bispecific antibody 2232-01 does not significantly induce ERK phosphorylation. This indicates that anti-HER3 / MET bispecific antibodies do not induce HER3 / MET target dimerization and activate downstream signaling pathways. In other words, the bispecific antibody 2232-01 of the present disclosure does not promote tumor cell proliferation or survival.

[0570] Example 6. Design and Preparation of Anti-HER3 / MET Antibody Drug Conjugates (ADCs)

[0571] 1. Preparation of Compounds

[0572] 1.1 Preparation of Compounds 9A and 9B

[0573] N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0574] N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0575] first step

[0576] Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0577] 2a (1.3 g, 11.2 mmol; prepared using the method disclosed in patent application WO2013 / 106717) was dissolved in 50 mL of acetonitrile. Potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 hours, filtered through celite, and the filter cake was rinsed with ethyl acetate (10 mL). The combined filtrates were concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography using solvent System C to obtain the title product 9a (2 g, 86.9% yield).

[0578] Step 2

[0579] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid benzyl ester 9b

[0580] 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol, prepared using the method disclosed in patent application "CN105829346A") were added to a reaction flask, and 4 mL of tetrahydrofuran was added. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. Potassium tert-butoxide (109 mg, 0.98 mmol) was added. The ice bath was removed, and the mixture was warmed to room temperature and stirred for 40 minutes. 10 mL of ice water was added, and the mixture was extracted with ethyl acetate (20 mL x 2) and chloroform (10 mL x 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, and sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added. The mixture was stirred at room temperature for 2 hours. 20 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent System C to obtain the title product 9b (48 mg, yield: 19%).

[0581] MS m / z(ESI):515.0[M+1].

[0582] Step 3

[0583] 10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid 9c

[0584] 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (v:v = 2:1). Palladium on carbon (12 mg, 10% content, dry form) was added, and the atmosphere was replaced with hydrogen three times. The reaction was stirred at room temperature for 1 hour. The reaction solution was filtered through celite, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude title product 9c (13 mg). This product was directly used in the next reaction without purification.

[0585] MS m / z(ESI):424.9[M+1].

[0586] Step 4

[0587] (9H-fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

[0588] 1b (10 mg, 18.8 μmol) was added to a reaction flask, followed by 1 mL of N,N-dimethylformamide. The atmosphere was replaced with argon three times, and the temperature was cooled to 0-5°C in an ice-water bath. One drop of triethylamine was added dropwise, followed by the addition of crude product 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol). The reaction was stirred in an ice-water bath for 40 minutes. 10 mL of water was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure. The resulting residue was purified by thin-layer chromatography using developing solvent System B to obtain the title product 9d (19 mg, 73.6% yield).

[0589] MS m / z(ESI):842.1[M+1].

[0590] Step 5

[0591] 2-((2-Aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide 9e

[0592] 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, 1 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1 mL of toluene was added, and the mixture was concentrated under reduced pressure twice. 3 mL of n-hexane was added to the residue, and the supernatant was poured out after standing to retain the solid. The solid residue was concentrated under reduced pressure and pumped dry to obtain the crude title product 9e (17 mg), which was used directly in the next reaction without purification.

[0593] MS m / z(ESI):638.0[M+18].

[0594] Step 6

[0595] N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0596] N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0597] The crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, replaced with argon three times, and cooled to 0-5°C in an ice-water bath. 8 g (21.2 mg, 44.8 μmol, prepared by the method disclosed in patent application "EP2907824") in 0.3 mL of N,N-dimethylformamide solution was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes. The ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title product (9-A: 2.4mg, 9-B: 1.7mg).

[0598] MS m / z(ESI):1074.4[M+1].

[0599] Single configuration compound 9-A (shorter retention time):

[0600] UPLC analysis: retention time 1.14 minutes, purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0601] 1 H NMR (400MHz, DMSO-d6): δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H) ,7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,1 H),4.54-4.40(m,2H),3.76-3.64(m,4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,1H),2.80-2.62(m,1H),2.45-2.30(m, 3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H).

[0602] Single configuration compound 9-B (longer retention time):

[0603] UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).

[0604] 1H NMR (400MHz, DMSO-d6): δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H), 7.31(s,1H),7.26-7.13(m,3H),6.99(s,1H),6.55-6.48(m,1H),5.60- 5.50(m,1H),5.41(s,2H),5.35-5.15(m,2H),4.78-4.68(m,1H),4.60- 4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H) ,3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13( m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52- 1.39(m,3H),1.34-1.12(m,4H),0.91-0.79(m,3H),0.53-0.34(m,4H).

[0605] 1.2 Preparation of compound L-1

[0606] Compound L-1 was synthesized according to the method provided in "Example 1 on page 28 of the specification of CN117460540A".

[0607] 1.3 Preparation of Compound 1

[0608] Compound 1 was synthesized according to the method provided in "Example 58 on page 163 of the specification of patent CN104755494A".

[0609] 2. Anti-HER3 / MET Antibody-Drug Conjugates

[0610] 2.1 The following antibody-drug conjugates (ADCs) were prepared, wherein the Ab in ADC-1 was 2232-01, the Ab in ADC-2 was 2232-02, and the Ab in ADC-5 was 2232-06. The target DAR was 6.

[0611] Preparation method:

[0612] ADC-1: To a solution of antibody 2232-01 (5.0 mg / mL, 2.0 mL, 70 nmol) in PBS (pH 7.2), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) aqueous solution (10 mM, 35 μL, 350 nmol, 5.0 eq) was added and placed on a temperature-controlled shaker for 3 hours at 37°C. The reaction system was then cooled to 25°C. A solution of compound 9-A (1.13 mg, 1050 nmol, 15.0 eq) in dimethyl sulfoxide was added to the reaction system and placed on a temperature-controlled shaker for 3 hours at 25°C. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer: PBS pH 7.2, flow rate 10 mL / min) to obtain ADC-1 in PBS buffer (2.0 mg / mL, 4.7 mL) with a yield of 94%. The solution was stored refrigerated at 4°C. The target DAR was 6. The drug loading was calculated by RP-HPLC, and the measured DAR was 5.90. As shown in Figure 9B , the main peaks were LC1, LC2 + 1 drug, HC1 + 2 drugs, and HC2 + 3 drugs.

[0613] ADC-2: 2232-02 was coupled with the aforementioned compound 9-A, and ADC-2 was obtained by referring to the preparation method of ADC-1.

[0614] ADC-5: 2232-06 was conjugated with the aforementioned compound 9-A, following the preparation method of ADC-1, to obtain ADC-5. Native mass spectrometry analysis, as shown in Figure 9C, revealed an average DAR value of 5.87, with DAR6 accounting for 94.9%, DAR2 and DAR4 accounting for 1.3% and 3.8%, respectively, and DAR8 undetectable. This indicates that this anti-HER3 / MET antibody-drug conjugate (ADC) exhibits high homogeneity, with a high proportion of DAR6 and low proportions of other DAR values.

[0615] 2.2 Preparation Antibody drug conjugates (ADCs) with the following structures were obtained. The Ab in ADC-6 and ADC-7 was 2232-06. The target DAR value for ADC-6 was 4, and the target DAR value for ADC-7 was 6.

[0616] Preparation method:

[0617] ADC-6: To a solution of antibody 2232-06 (7.0 mg / mL, 1.0 mL, 48 nmol) in PBS (pH 7.2) was added an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 17 μL, 173 nmol, 3.6 eq). The mixture was placed on a temperature-controlled shaker and incubated at 37°C for 2 hours. The reaction system was then cooled to 25°C. A solution of prepared compound L-1 (0.53 mg, 384 nmol, 8.0 eq) in dimethyl sulfoxide was added to the reaction system and incubated on a temperature-controlled shaker at 25°C for 2 hours. The reaction mixture was desalted using a desalting column (HiPrep 26 / 10, GE) and exchanged for PBS buffer (pH 7.2, flow rate 10 mL / min) to obtain ADC-6 in PBS buffer (3.44 mg / mL, 1.71 mL) with a yield of 84.1%. The product was stored at 4°C. The target DAR was 4, and the drug loading was calculated by mass spectrometry, resulting in a measured DAR of 3.90.

[0618] ADC-7: To a solution of antibody 2232-06 (7.0 mg / mL, 1.0 mL, 48 nmol) in PBS (pH 7.2), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) aqueous solution (10 mM, 48 μL, 480 nmol, 10 eq) was added and placed on a temperature-controlled shaker for 2 hours at 37°C. The reaction system was then cooled to 25°C. A solution of prepared compound L-1 (0.99 mg, 720 nmol, 15 eq) in dimethyl sulfoxide was added to the above reaction system and placed on a temperature-controlled shaker for 2 hours at 25°C. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer: PBS pH 7.2, flow rate 10 mL / min) to obtain ADC-7 in PBS buffer (3.21 mg / mL, 1.83 mL) with a yield of 84.0%. The solution was stored refrigerated at 4°C. The target DAR was 6, and the drug loading was calculated by mass spectrometry, and the measured DAR was 5.69.

[0619] 2.3 An antibody-drug conjugate (ADC) with the following structure was prepared, wherein Ab in the ADC is 2232-06 and the target DAR value is 6.

[0620] ADC-10: 2232-06 was coupled with compound 1, and the anti-HER3 / MET antibody drug conjugate ADC-10 of DAR6 was obtained by referring to the preparation method described in step 2.1 above.

[0621] Example 7. Preparation of anti-HER3 ADC or anti-MET ADC

[0622] 1) Prepare an antibody-drug conjugate (ADC) with the following structure, wherein the Ab in anti-MET ADC-3 is antibody S3 and the Ab in anti-HER3 ADC-4 is antibody A. The target DAR value is 4

[0623] Preparation method:

[0624] Anti-MET ADC-3: To a solution of antibody S3 (10.0 mg / mL, 1.0 mL, 70 nmol) in PBS (pH 7.2) was added a solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 17.5 μL, 175 nmol, 2.5 eq). The mixture was placed on a temperature-controlled shaker and shaken at 37°C for 3 hours. The reaction system was then cooled to 25°C. A solution of compound 9-A (0.60 mg, 560 nmol, 8.0 eq) in dimethyl sulfoxide was added to the reaction system and placed on a temperature-controlled shaker and shaken at 25°C for 3 hours. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) and the buffer was exchanged (buffer: PBS pH 7.2, flow rate 10 mL / min) to obtain ADC-3 in PBS buffer (2.0 mg / mL, 4.7 mL). The drug loading was calculated by RP-HPLC, and the measured DAR was 4.7.

[0625] Anti-HER3 ADC-4: Antibody A was conjugated with compound 9-A, following the preparation method of ADC-3, to obtain ADC-4. The drug loading was calculated by RP-HPLC, and the measured DAR was 4.0.

[0626] 2) The ADC structure of U3-1402 is shown in the figure below:

[0627] Preparation method of U3-1402:

[0628] To a solution of naked antibody against U3-1402 (5.76 mg / mL, 552 nmol) in PBS (pH 7.2), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) aqueous solution (10 mM, 828 μL, 8280 nmol, 15.0 eq) was added, and the mixture was placed on a temperature-controlled shaker and shaken at 37°C for 2 hours. The reaction system was then cooled to 25°C. A solution of compound 1 (5.7 mg, 5520 nmol, 10.0 eq) in dimethyl sulfoxide was added to the reaction system, and the mixture was placed on a temperature-controlled shaker and shaken at 25°C for 2 hours. The reaction mixture was desalted using a desalting column (HiPrep 26 / 10, GE) and exchanged with PBS pH 7.2 buffer (10 mL / min) to obtain U3-1402 in PBS buffer (4.18 mg / mL, 18 mL) with a yield of 94%. The product was stored at 4°C. The drug loading was calculated by mass spectrometry, yielding a DAR of 7.88.

[0629] Example 8. Anti-HER3 / MET Antibody Drug Conjugate Binding Detection

[0630] 1. Flow cytometry (FACS) detection

[0631] FACS experiments were used to detect the binding activity of anti-HER3 / MET bispecific antibodies and their ADCs to human HER3 / MET proteins on the cell surface.

[0632] Experimental method: HCC827 Osimertinib-resistant cell line was used, cells were washed twice with experimental culture medium, and 1×10 5 Cells were seeded in 96-well U-bottom plates, and the test samples were added at different concentrations. After incubation at 4°C for 1 hour, the cells were washed twice with experimental culture medium. Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was then added, and after two washes, the fluorescence signal value was read by flow cytometry.

[0633] Experimental Results: The FACS results in Figure 10 show that both the anti-HER3 / MET bispecific antibody and its conjugate can bind to the cell surface HER3 / MET antigen, with comparable binding abilities before and after conjugation (the dashed and solid lines overlap in Figure 10). This indicates that toxin conjugation did not affect the antibody's target binding.

[0634] 2. Surface Plasmon Resonance (SPR) Detection

[0635] SPR detection was performed using a BIAcore 8K system (Cityva). Protein A sensor chips and related reagents were purchased from Cytiva.

[0636] Experimental method: ADC-1, ADC-2 and ADC-5 were diluted to 1 μg / mL with HBS-EP+ buffer, the flow rate was set to 10 μL / min, and the capture antibody was adjusted to 200RU level. The HER3 / MET antigen with His tag was diluted with HBS-EP+ buffer in a certain proportion to a concentration gradient of 6.25nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, and 400nM, respectively. The flow rate was set to 30 μL / min during sample analysis. The binding time was 120s and the dissociation time was 900s. Regeneration was then performed, using pH 1.5Gly-HCl buffer as the regeneration buffer, the regeneration flow rate was set to 30 μL / min, and the regeneration was 30s. The response signal was plotted with the analysis time as the horizontal axis and the response value as the vertical axis. The obtained data were fitted by BIAcore 8K analysis software, and the 1:1 Langmuir binding model was used to determine its binding rate constant (K a ), dissociation rate constant (K d ) and the dissociation equilibrium constant (K D ) and other kinetic constants.

[0637] Experimental results: Table 8 shows that the anti-HER3 / MET antibody drug conjugate can bind to human HER3 antigen and human MET antigen.

[0638] Table 8-1. Binding kinetic parameters of anti-HER3 / MET antibody drug conjugates

[0639] Table 8-2. Binding kinetic parameters of anti-HER3 / MET antibody drug conjugates

[0640] Example 9. Anti-HER3 / MET Antibody Drug Conjugates Inhibit Tumor Cell Proliferation in Vitro

[0641] Experimental Methods: Double-positive NCI-H441, NCI-H226, and PC-9 osimertinib-resistant cell lines (created in-house, using PC-9 cells induced with osimertinib gradient resistance) were cultured in RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) supplemented with 10% fetal bovine serum. Cells were grown overnight in 96-well plates at a density of 600 cells / well. The next day, equal volumes of serially diluted ADC were added. Six days later, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Cat#G7570) as described in the product instructions. Cell viability was assessed as a percentage of untreated control cells.

[0642] Experimental Results: As shown in the FACS results in Figure 11A, HER3 expression was similar among the three osimertinib-resistant cell lines, NCI-H441, NCI-H226, and PC-9. NCI-H441 expressed the highest MET expression, while the PC-9 osimertinib-resistant cell line expressed the least MET. The ADC cytotoxicity results in Figures 11B and 11C show that ADC-4 had similar cytotoxicity against the three cell lines, consistent with HER3 expression. ADC-1 had the strongest cytotoxicity against NCI-H441 and the weakest cytotoxicity against the PC-9 osimertinib-resistant cell line, consistent with MET expression, indicating that the MET arm enhances the cytotoxicity of the anti-HER3 / MET antibody-drug conjugate.

[0643] Example 10. Evaluation of anti-HER3 / MET antibody drug conjugates in mouse anti-tumor models

[0644] The calculation formula used in this disclosure is as follows:

[0645] The tumor volume (TV) was calculated using the formula TV = 1 / 2 × a × b2, where a and b represent the long and short diameters of the tumor, respectively.

[0646] Relative tumor growth rate T / C% = (T-T0) / (C-C0) × 100%; tumor inhibition rate TGI% = 1-T / C%.

[0647] Experimental methods: The anti-tumor activity was evaluated using a mouse xenograft model of the HCC827 osimertinib-resistant cell line (constructed in-house, using HCC827 cells induced by osimertinib gradient resistance). HCC827 osimertinib-resistant cells were cultured at a rate of 1×10 7100 μL / mouse was inoculated subcutaneously into NCG mice (provided by Jicui Yaokang) and the tumor was grown to about 120 mm. 3 Patients were randomly divided into groups according to tumor volume at the time of treatment and received anti-HER3 / MET antibody-drug conjugate administration. Tumor volume was measured twice weekly during the treatment and observation period, and the measured values ​​were recorded.

[0648] Experimental Results: As shown in Figure 12A, the experimental animals showed no significant weight loss during the high and low dose periods, indicating that the test drug did not cause significant toxic side effects in the experimental animals and showed good toxin tolerance and safety. As shown in Figure 12B and Table 9, the high-dose dual-antibody ADC demonstrated good anti-tumor activity. At equivalent doses, ADC-5 exhibited superior anti-tumor activity to ADC-3 and ADC-4. At equivalent toxin administration, ADC-5 also exhibited superior anti-tumor activity to ADC-3 and ADC-4.

[0649] Table 9. Antitumor effects of different HER3xMET dual-antibody ADCs on mouse transplanted tumors

[0650] Example 11. Evaluation of anti-HER3xMET antibody drug conjugates in a HER3 single-positive anti-tumor model in mice

[0651] The anti-tumor activity was evaluated using the MDA-MB-453 mouse xenograft model. MDA-MB-453 cells were plated at 5 × 10 6 The cells / 100 μL / mouse were inoculated subcutaneously into Nude mice (provided by Shanghai Bikeway Co., Ltd.) and the tumors were grown to about 120 mm. 3 Forty-two mice were randomly divided into seven groups (six mice per group) based on tumor volume: vehicle, ADC-1 (10 mg / kg), ADC-1 (3 mg / kg), ADC-2 (10 mg / kg), and ADC-2 (3 mg / kg). Dosing was once weekly, and twice weekly. Tumor volume was measured twice weekly during the dosing and observation period, and the measured values ​​were recorded.

[0652] The tumor volume (TV) was calculated using the formula TV = 1 / 2 × a × b2, where a and b represent the long and short diameters of the tumor, respectively.

[0653] Relative tumor growth rate T / C% = (T-T0) / (C-C0) × 100%; tumor inhibition rate TGI% = 1-T / C%.

[0654] CR% (complete tumor regression ratio) = complete tumor regression (<150 mm 3 ) of mice / number of mice enrolled.

[0655] As shown in Figure 13A, the experimental animals showed no significant weight loss during the high and low dose periods, indicating that the test drug did not cause significant toxic side effects in the experimental animals and that the toxin was well tolerated and safe. As shown in Figure 13B and Table 10, the high dose of the dual-antibody ADC showed good anti-tumor activity.

[0656] Table 10. Antitumor effects of different HER3xMET dual-antibody ADCs on mouse transplanted tumors

[0657] Example 12. Anti-HER3 / MET Antibody Drug Conjugate ADC-6 Inhibits Tumor Cell Proliferation in Vitro

[0658] Experimental Methods: Double-positive NCI-H441 cells were cultured in RPMI 1640 medium (ATCC modification) (Gibco, Cat# A1049101) supplemented with 10% fetal bovine serum. Cells were grown overnight in 96-well plates at a density of 600 cells / well. The following day, equal volumes of serially diluted ADC were added. Six days later, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Cat# G7570) as described in the product manual. Cell viability was assessed as a percentage of untreated control cells.

[0659] As shown in FIG14 , ADC-6 and ADC-7 had killing activity against double-positive cells.

Claims

1. A HER3 / MET binding molecule comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3; The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71 or 1; the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72 or 2; and / or, The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 70 or 14, The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

2. A HER3 / MET binding molecule comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3; The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the VL1 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 76, 19 and 20, respectively; and / or, The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 55 and 75, respectively; Preferably, The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the VL1 comprises LCDR1 as shown in SEQ ID NOs: 18 or 74, and LCDR2 and LCDR3 as shown in SEQ ID NOs: 19 and 20, respectively; and / or, The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the VL2 comprises LCDR1 and LCDR2 as shown in SEQ ID NOs: 54 and 55, respectively, and LCDR3 as shown in SEQ ID NOs: 56 or 73; More preferably, The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 18-20, respectively; and The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54-56, respectively; or The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 74, 19, and 20, respectively; and The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the VL2 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 54, 55 and 73, respectively.

3. The HER3 / MET binding molecule according to claim 1 or 2, wherein The VH1 of the first binding domain that specifically binds to MET includes a mutation of K at position 23 and / or a mutation of T at position 78 relative to SEQ ID NO: 1; and / or the VL1 of the first binding domain that specifically binds to MET includes a mutation of T at position 69 relative to SEQ ID NO: 2; Preferably, the first binding domain that specifically binds to MET comprises mutations of 23K and 78T relative to the natural number of SEQ ID NO:1, and mutation of 69T relative to the natural number of SEQ ID NO:

2.

4. The HER3 / MET binding molecule according to any one of claims 1 to 3, wherein The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises an amino acid sequence as shown in SEQ ID NO: 71 or 1, or having at least 90% identity thereto, and the VL1 comprises an amino acid sequence as shown in SEQ ID NO: 72 or 2, or having at least 90% identity thereto; and / or, The second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises an amino acid sequence as shown in SEQ ID NO: 13 or having at least 90% identity thereto, and the VL2 comprises an amino acid sequence as shown in SEQ ID NO: 70 or 14 or having at least 90% identity thereto; Preferably, The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the VH1 and VL1 comprise the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively; and the second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), and the VH2 and VL2 comprise the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 70, respectively; or The first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 and VL1 comprise the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and, the second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein VH2 and VL2 comprise the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; More preferably, the HER3 / MET binding molecule is an anti-HER3 / MET antibody.

5. The HER3 / MET binding molecule according to any one of claims 1 to 4, further comprising a human immunoglobulin Fc region; Preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4.

6. The HER3 / MET binding molecule of claim 5, wherein The Fc region comprises a first subunit and a second subunit, wherein the first subunit and the second subunit have a knob-into-hole structure; Preferably, the first subunit of the Fc region is a knob chain, and the second subunit of the Fc region is a hole chain; Preferably, The first subunit of the Fc region contains a mutation at position 366, and the second subunit contains a mutation selected from positions 366, 368 and 407 or any combination thereof; The first subunit of the Fc region contains a mutation at position 354 or 356, and the second subunit contains a mutation at position 349; or The first subunit of the Fc region contains mutations at positions 354 or 356, and the second subunit contains mutations at positions 349, 366, 368 and 407; More preferably, The first subunit of the Fc region contains a 366W mutation, and the second subunit contains a mutation selected from 366S, 368A and 407V or any combination thereof; The first subunit of the Fc region contains a 354C or 356C mutation, and the second subunit contains a 349C mutation; or The first subunit of the Fc region contains 354C / 366W mutations, and the second subunit contains 349C / 366S / 368A / 407V mutations, and the mutations are numbered according to Eu.

7. The HER3 / MET binding molecule according to any one of claims 1 to 6, further comprising a linker, preferably, the linker is such as (G m S n ) h or (G m Q n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, m and n are each independently selected from integers of 1-8, and h is independently selected from integers of 1-20.

8. The HER3 / MET binding molecule of any one of claims 1 to 7, comprising: The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region], The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 2]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[CH1]-[second subunit of the Fc region], and The second light chain, from N-terminus to C-terminus, is: [VL2]-[CL]; in, - represents a peptide bond, The amino acid sequence of the Obscurin-O chain is shown in SEQ ID NO: 63, The amino acid sequence of the Titin-T chain is shown in SEQ ID NO: 64, The linker 1, linker 2 and linker 3 may be the same or different, and may exist independently or not; Preferably, the amino acid sequences of linker 1 and linker 2 are GGGGS (SEQ ID NO: 91), and linker 3 does not exist.

9. The HER3 / MET binding molecule according to any one of claims 1 to 8, comprising the following polypeptide chain combination: A first heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 77 or 57, or having at least 90% sequence identity thereto, A first light chain comprising an amino acid sequence as shown in SEQ ID NO: 78 or 58, or having at least 90% sequence identity thereto, a second heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 59, or having at least 90% sequence identity thereto, and a second light chain comprising an amino acid sequence as set forth in SEQ ID NO: 79 or 60, or having at least 90% sequence identity thereto; Preferably, The HER3 / MET binding molecule comprises: The first heavy chain shown in SEQ ID NO: 77, The first light chain shown in SEQ ID NO: 78, The second heavy chain shown in SEQ ID NO: 59, and The second light chain shown in SEQ ID NO: 79; or The HER3 / MET binding molecule comprises: The first heavy chain shown in SEQ ID NO: 57, The first light chain shown in SEQ ID NO: 58, The second heavy chain shown in SEQ ID NO: 59, and The second light chain represented by SEQ ID NO:

60.

10. An antibody-drug conjugate comprising an antibody and an effector molecule, wherein the antibody comprises a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3; The heavy chain variable region in the first binding domain that specifically binds to MET comprises SEQ ID NO:15-17, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO:76, 19, 20; and, the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO:51-53, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NO:54, 55, 75; Preferably, the heavy chain variable region in the first binding domain that specifically binds to MET comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 15-17, and the light chain variable region comprises LCDR1 of the amino acid sequence shown in SEQ ID NOs: 18 or 74, and LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 19 and 20; and the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51-53, and the light chain variable region comprises LCDR1 and LCDR2 of the amino acid sequences shown in SEQ ID NOs: 54 and 55, and LCDR3 of the amino acid sequence shown in SEQ ID NOs: 56 or 73; More preferably, the heavy chain variable region in the first binding domain that specifically binds to MET comprises an amino acid sequence as shown in SEQ ID NO: 71 or 1, or having at least 90% identity thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 72 or 2, or having at least 90% identity thereto; and the heavy chain variable region in the second binding domain that specifically binds to HER3 comprises an amino acid sequence as shown in SEQ ID NO: 13, or having at least 90% identity thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70 or 14, or having at least 90% identity thereto. More preferably, the antibody comprises: The first heavy chain shown in SEQ ID NO: 77, The first light chain shown in SEQ ID NO: 78, The second heavy chain shown in SEQ ID NO: 59, and The second light chain shown in SEQ ID NO: 79; or The antibody comprises: The first heavy chain shown in SEQ ID NO: 57, The first light chain shown in SEQ ID NO: 58, The second heavy chain shown in SEQ ID NO: 59, and The second light chain represented by SEQ ID NO:

60.

11. The antibody-drug conjugate drug according to claim 10, wherein the effector molecule is a cytotoxin; Preferably, the cytotoxin is selected from: microtubule aggregation inhibitors, Topo I inhibitors, MMAE or derivatives thereof; More preferably, the cytotoxin is selected from MMAE or its derivatives, exitecan or its derivatives, eribulin or its derivatives.

12. The antibody-drug conjugate according to claim 10, which has a structure shown in formula (I): in, -L- is a linker unit, which is -L 1 -L 2 -L 3 -L 4 -, L 1 -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10, and n is an integer or a decimal; Ab is an antibody as defined in claim 10.

13. The antibody drug conjugate according to any one of claims 10 to 12, which has a structure shown in formula II in: W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 1 is selected from a hydrogen atom, a halogen, a cycloalkylalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; m is an integer from 0 to 4; n as defined in claim 12; Ab is an antibody as defined in claim 10.

14. The antibody-drug conjugate according to any one of claims 10 to 13, which has a structure represented by formula (III): in: s 1 is an integer from 2 to 8; preferably 5; R 1 , R 2 , R 5 ~R 7 , m as defined in claim 13; n as defined in claim 12; Ab is an antibody as defined in claim 10.

15. The antibody drug conjugate of claim 12, wherein -LY- is selected from: Preferably 16. The antibody drug conjugate according to any one of claims 10 to 15, It is selected from the following structural formula: in: n is 1 to 10, which can be an integer or a decimal, preferably n is an integer or a decimal from 1 to 6; Ab Ab is an antibody as defined in claim 10.

17. A method for preparing an antibody-drug conjugate as claimed in any one of claims 13 to 16, comprising reacting the Ab with a drug Preferably, it comprises the following steps:

18. The antibody-drug conjugate according to claim 10, which has a structure shown in formula (IV): Ab-(L-De)k (IV) -L- is a linker unit that covalently attaches Ab to De, k is an integer or decimal from 1 to 20, preferably n is an integer or decimal from 1 to 6; De is shown as follows: in, R 1a is selected from hydrogen, alkyl, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl. The aryl group is substituted by one or more substituents, preferably R 1a It is methyl; R 1b is selected from hydrogen, alkyl, alkoxy, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, preferably R 1b is hydrogen; or R 1a With R 1b Together with the atoms it is attached to, it forms C 5-8 Heterocycloalkyl; optionally, the heteroalkyl is substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not at the same time hydrogen; Ab is an antibody as defined in claim 10.

19. The antibody drug conjugate of claim 18, wherein the linker unit comprises a cleavable peptide portion; Preferably, the cleavable peptide portion is cleavable by an enzyme, More preferably, the enzyme is a cathepsin.

20. The antibody drug conjugate of claim 18 or 19, wherein: The linker unit comprises a peptide residue consisting of 2 to 7 amino acids, The amino acids are selected from the group consisting of: phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; More preferably, the peptide residues are selected from the group consisting of: valine-citrulline, alanine-alanine-asparagine, glycine-glycine-lysine, valine-lysine, valine-alanine, valine-phenylalanine, glycine-glycine-phenylalanine-glycine (GGFG, SEQ ID NO: 69).

21. The antibody drug conjugate of claim 18, wherein the linker unit comprises a cleavable sulfonamide moiety or a cleavable disulfide moiety; Preferably, the linker unit is cleavable under reducing conditions.

22. The antibody drug conjugate of any one of claims 18 to 21, wherein the Linker unit comprises a Spacer unit attached to De; Preferably, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB).

23. The antibody drug conjugate of any one of claims 18 to 22, which is represented by any one of the following structures: k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p2 is selected from 2, 4, 6 or 8; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; k is selected from 1 to 10 and can be an integer or a decimal; p1 is selected from 2, 4, 6 or 8; P3 is selected from 0, 1 or 2; Preferably, k is selected from 1 to 10 and is an integer or a decimal. 24.Antibody drug conjugate, the structure of which is as follows: n is 1 to 10, and n is an integer or a decimal; or k is selected from 1 to 10 and is an integer or a decimal; The Ab comprises a first binding domain that specifically binds to MET as described in claims 1-9 and a second binding domain that specifically binds to HER3.

25. A MET-binding molecule comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71; and the VL1 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 15-17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 74, 19 and 20, respectively.

26. The MET-binding molecule of claim 25, wherein The VH1 comprises a mutation of K at position 23 and / or a mutation of T at position 78 relative to SEQ ID NO: 1; and / or the VL1 comprises a mutation of T at position 69 relative to SEQ ID NO: 2; Preferably, the MET binding molecule comprises E23K and S78T mutations relative to the natural numbering of SEQ ID NO:1, and A69T mutation relative to the natural numbering of SEQ ID NO:

2.

27. The MET-binding molecule of claim 25 or 26, wherein The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:71, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:72, or at least 90% identical thereto.

28. A HER3 binding molecule comprising a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 70; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 51-53, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOs: 54, 55 and 73, respectively.

29. The HER3 binding molecule of claim 28, wherein The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or at least 90% identical thereto.

30. A polynucleotide encoding the HER3 / MET binding molecule of any one of claims 1 to 9, the MET binding molecule of any one of claims 25 to 27, or the HER3 binding molecule of claim 28 or 29.

31. A vector comprising the polynucleotide according to claim 30.

32. A host cell comprising the polynucleotide of claim 30, or the vector of claim 31.

33. A pharmaceutical composition comprising the HER3 / MET binding molecule of any one of claims 1 to 9, the antibody drug conjugate of any one of claims 10 to 16 and 18 to 24, the antibody drug conjugate prepared by the method of claim 17, the MET binding molecule of any one of claims 25 to 27, the HER3 binding molecule of claim 28 or 29, the polynucleotide of claim 30, or the vector of claim 31; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants.

34. A method for preparing the HER3 / MET binding molecule of any one of claims 1 to 9, the MET binding molecule of any one of claims 25 to 27, or the HER3 binding molecule of claim 28 or 29, comprising culturing the host cell of claim 32, and expressing the HER3 / MET binding molecule of any one of claims 1 to 9, the MET binding molecule of any one of claims 25 to 27, or the HER3 binding molecule of claim 28 or 29, Optionally, it comprises isolating and / or purifying the HER3 / MET binding molecule, MET binding molecule or HER3 binding molecule.

35. The HER3 / MET binding molecule of any one of claims 1 to 9, the antibody drug conjugate of any one of claims 10 to 16 and 18 to 24, the antibody drug conjugate prepared by the method of claim 17, the MET binding molecule of any one of claims 25 to 27, the HER3 binding molecule of claim 28 or 29, the polynucleotide of claim 30, or the vector of claim 31, or the pharmaceutical composition of claim 33 for use in treating or alleviating cancer, or in preparing a medicament for treating or alleviating cancer; Preferably, the cancer is breast cancer or lung cancer, preferably, the cancer is HER3 positive, preferably HER3 positive breast cancer or lung cancer; Preferably, the cancer is HER3 and MET double positive, preferably HER3 and MET double positive breast cancer or lung cancer.

36. A method for treating or alleviating cancer, comprising the steps of: Administering to a subject a therapeutically effective amount of the HER3 / MET binding molecule of any one of claims 1 to 9, the antibody drug conjugate of any one of claims 10 to 16 and 18 to 24, the antibody drug conjugate prepared by the method of claim 17, the MET binding molecule of any one of claims 25 to 27, the HER3 binding molecule of claim 28 or 29, the polynucleotide of claim 30, or the vector of claim 31, or the pharmaceutical composition of claim 33; Preferably, the cancer is breast cancer or lung cancer, preferably, the cancer is HER3 positive; preferably, the cancer is HER3 and MET double positive.

37. Antibody-drug conjugates, The Ab is an antibody, comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3; the first binding domain that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), and the second binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2); The Ab comprises: The first heavy chain, from N-terminus to C-terminus, is: [VH1]-[Linker 1]-[Obscurin-O chain]-[Linker 3]-[First subunit of the Fc region], The first light chain, from N-terminus to C-terminus, is: [VL1]-[Linker 2]-[Titin-T chain], The second heavy chain, from N-terminus to C-terminus, is: [VH2]-[CH1]-[second subunit of the Fc region], and The second light chain, from N-terminus to C-terminus, is: [VL2]-[CL]; in, - represents a peptide bond, and the linker 1, linker 2 and linker 3 may be the same or different, Can exist or not exist independently Preferably, i) the antibody-drug conjugate is represented by the general formula (I): Wherein, -L- is a linker unit, which is -L 1 -L 2 -L 3 -L 4 -, L 1 -(succinimidyl-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms, the heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 、-C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6; R 3 , R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group; Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group or a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; or, ii) The antibody-drug conjugate has a structure shown in the general formula Ab-(L-De)k: L is a linker that covalently attaches Ab to De, De is represented by formula (III): in, R 1a is selected from hydrogen, alkyl, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, preferably R 1a It is methyl; R 1b is selected from hydrogen, alkyl, alkoxy, cycloalkyl, aryl and heteroaryl, Optionally, the alkyl, cycloalkyl, aryl and heteroaryl groups are each independently substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, preferably R 1b is hydrogen; or R 1a With R 1b Together with the atoms it is attached to, it forms C 5-8 Heterocycloalkyl; optionally, the heteroalkyl is substituted with one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and R 1a and R 1b Not hydrogen at the same time.

Images