HER3 / MET binding molecules and their pharmaceutical use

Abstract

This disclosure relates to HER3 / MET binding molecules and their pharmaceutically acceptable uses. Specifically, it relates to anti-HER3 / MET bispecific antibodies, anti-HER3 / MET antibody-drug conjugates, and methods and pharmaceutically acceptable uses thereof for treating cancer.

Description

[Technical Field]

[0001] This 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 technology]

[0002] Antibody-drug conjugates (ADCs) provide targeting through antibodies, while the coupled small molecule chemotherapeutic plays a role in killing tumor cells. This perfect combination forms a "magic bullet" platform for precise treatment with minimal side effects. Since the first ADC, Mylotarg, was commercialized in 2000, there has been continuous innovation in this field, and more than 10 different ADCs have been introduced to date.

[0003] MET (Mesenchymal-Epithelial Metastasis 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 epithelial-derived cells, while HGF expression is limited to mesenchymal-derived cells. HGF stimulation induces various biological responses in MET, which together generate a program known as invasive growth. Abnormal MET activation in cancer is associated with a poor prognosis, as this abnormal MET activity induces tumor growth, the formation of new blood vessels supplying 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 regions: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains four domains, while the intracellular domain includes one intracellular tyrosine kinase domain for signal transduction and one cytoplasmic tail with a tyrosine phosphorylation residue. Cellular signaling is initiated when a ligand binds to extracellular domains I and III. Under normal circumstances, these receptors mediate cell division, migration, survival, and organ development. Mutations in EGFR family members lead to abnormal signal transduction, stimulating cell survival and contributing to cancer progression.

[0005] c-Met interacts with EGFR (or the HER family) and is involved 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 invasion, and angiogenesis.

[0006] This disclosure provides an anti-HER3 / MET bispecific antibody and its coupling with a drug. Anti-HER3 / MET bispecific antibody-drug coupling exhibits excellent tumor growth inhibition and killing activity, good drug discovery potential, and high potential for clinical safety. [Overview of the project]

[0007] This disclosure provides MET-binding molecules, HER3-binding molecules, and HER3 / MET-binding molecules, as well as their coding nucleic acids, vectors, host cells, pharmaceutical compositions, and their therapeutic and related pharmaceutical uses for cancer.

[0008] MET binding molecule This disclosure provides a MET-binding molecule comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72.

[0009] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the above CDR is defined according to the Kabat numbering system.

[0010] In some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as shown in SEQ ID NOs: 15-17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as shown in SEQ ID NOs: 74, 19, and 20.

[0011] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, respectively, as shown in SEQ ID NOs: 15-17, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, respectively, as shown in SEQ ID NOs: 18-20.

[0012] In some embodiments, in any one of the MET-binding molecules described above, VH1 includes a mutation in which the 23rd position of the natural count for SEQ ID NO: 1 is K and / or a mutation in which the 78th position is T, and / or VL1 includes a mutation in which the 69th position of the natural count for SEQ ID NO: 2 is T, and preferably, any one of the MET-binding molecules described above includes the natural count E23K and S78T mutations for SEQ ID NO: 1, and the natural count A69T mutation for SEQ ID NO: 2.

[0013] In some embodiments, the heavy chain variable region includes an amino acid sequence represented by SEQ ID NO: 71 or having at least 80% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 72 or having at least 80% identity thereto.

[0014] In some embodiments, the heavy chain variable region includes an amino acid sequence shown in SEQ ID NO: 1 or having at least 80% identity thereto, and the light chain variable region includes an amino acid sequence shown in SEQ ID NO: 2 or having at least 80% identity thereto.

[0015] In some embodiments, the MET-binding molecule inhibits tumor growth or treats or alleviates cancer.

[0016] HER3 binding molecule This 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 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.

[0017] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the above CDR is defined according to the Kabat numbering system.

[0018] In some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, indicated by SEQ ID NOs. 51 to 53, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, indicated by SEQ ID NOs. 54, 55, and 73.

[0019] In some embodiments, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by sequence numbers 51 to 53, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by sequence numbers 54 to 56.

[0020] In some embodiments, the heavy chain variable region includes an amino acid sequence represented by SEQ ID NO: 13 or having at least 80% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 70 or having at least 80% identity thereto.

[0021] In some embodiments, the heavy chain variable region includes an amino acid sequence shown in SEQ ID NO: 13 or having at least 80% identity thereto, and the light chain variable region includes an amino acid sequence shown in SEQ ID NO: 14 or having at least 80% identity thereto.

[0022] In some embodiments, the HER3-binding molecule inhibits tumor growth or treats or alleviates cancer.

[0023] HER3 / MET binding molecule This 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 bind to HER3 and MET simultaneously or specifically to each.

[0024] Regarding the first binding domain that specifically binds to MET, In some embodiments, the first binding domain in the HER3 / MET binding molecule that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs. 71, 1, 3, 5, 7, 9, and 11, and / or VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs. 72, 2, 4, 6, 8, 10, and 12.

[0025] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72.

[0026] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 1, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 2.

[0027] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 3, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 4.

[0028] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 5, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 6.

[0029] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 7, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 8.

[0030] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 9, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 10.

[0031] In some specific embodiments, VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 11, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 12.

[0032] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the above CDR is defined according to the Kabat numbering system.

[0033] In some embodiments, the first binding domain in the HER3 / MET binding molecule that specifically binds to MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, indicated by SEQ ID NOs. 15-17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, indicated by SEQ ID NOs. 76, 19, and 20, respectively.

[0034] In some specific embodiments, the first binding domain that specifically binds to the MET includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which are represented by one of the following: a-1) HCDR1, HCDR2, HCDR3 shown in sequence numbers 15-17, respectively, and LCDR1, LCDR2, and LCDR3 shown in sequence numbers 18-20, respectively, or a-2) HCDR1, HCDR2, and HCDR3, represented by sequence numbers 15 to 17, respectively, and LCDR1, LCDR2, and LCDR3, represented by sequence numbers 74, 19, and 20, respectively.

[0035] In some other embodiments, the first binding domain that specifically binds to the MET includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which are represented by one of the following: i-1) HCDR1, HCDR2, and HCDR3 shown in sequence numbers 21 to 23, respectively, and LCDR1, LCDR2, and LCDR3 shown in sequence numbers 24 to 26, respectively. i-2) HCDR1, HCDR2, and HCDR3 shown in sequence numbers 27-29, respectively, and LCDR1, LCDR2, and LCDR3 shown in sequence numbers 30-32, respectively. i-3) HCDR1, HCDR2, and HCDR3, respectively, indicated by sequence numbers 33 to 35, and LCDR1, LCDR2, and LCDR3, respectively, indicated by sequence numbers 36 to 38. i-4) HCDR1, HCDR2, HCDR3 shown in sequence numbers 39-41 respectively, and LCDR1, LCDR2, and LCDR3 shown in sequence numbers 42-44 respectively, or i-5) HCDR1, HCDR2, and HCDR3, represented by sequence numbers 45-47, respectively, and LCDR1, LCDR2, and LCDR3, represented by sequence numbers 48-50, respectively.

[0036] In some embodiments, the first binding domain in the HER3 / MET binding molecule that specifically binds to MET comprises VH1 and VL1, wherein any one HCDR contained in VH1 has 1, 2, 3, 4, or 5 amino acid mutations compared to any one HCDR, and / or any LCDR contained in the indicated VL1 has 1, 2, 3, 4, or 5 amino acid mutations compared to any one LCDR.

[0037] In some specific embodiments, the amino acid mutation is an exchange, substitution, modification, deletion, and / or addition of an amino acid (e.g., a conservative substitution of an amino acid), and the mutation does not affect, or essentially does not affect, the function of the first binding domain that specifically binds to MET.

[0038] In some embodiments, in the first binding domain that specifically binds to MET in the HER3 / MET binding molecule, VH1 includes a mutation in which the 23rd position of the natural count for SEQ ID NO: 1 is K and / or a mutation in which the 78th position is T, and / or VL1 includes a mutation in which the 69th position of the natural count for SEQ ID NO: 2 is T. Preferably, the first binding domain that specifically binds to MET includes the natural count E23K mutation and S78T mutation for SEQ ID NO: 1, and the natural count A69T mutation for SEQ ID NO: 2.

[0039] In some embodiments, in the first binding domain that specifically binds to MET in the HER3 / MET binding molecule, The above VH1 comprises an amino acid sequence represented by any one of sequence numbers 71, 1, 3, 5, 7, 9, and 11, or having at least 80% sequence identity thereto, and / or The above VL1 includes an amino acid sequence represented by any one of sequence numbers 72, 2, 4, 6, 8, 10, and 12, or having at least 80% sequence identity with them.

[0040] Regarding the second binding domain that specifically binds to HER3, In some embodiments, the second binding domain in the HER3 / MET binding molecule that specifically binds to HER3 includes a heavy chain variable region (VH2) and a light chain variable region (VL2), of which VH2 includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and VL2 includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 70 or 14.

[0041] The above CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the above CDR is defined according to the Kabat numbering system.

[0042] In some embodiments, the second binding domain in the HER3 / MET binding molecule 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 of the amino acid sequences shown in SEQ ID NOs. 51 to 53, respectively, and the VL2 comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs. 54, 55, and 75, respectively.

[0043] In some specific embodiments, the second binding domain that specifically binds to the above HER3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which are any one of the following: b-1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, indicated by sequence numbers 51-56, or b-2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, indicated by sequence numbers 51-55 and 73.

[0044] In some embodiments, the second binding domain in the HER3 / MET binding molecule that specifically binds to HER3 comprises VH2 and VL2, wherein any one HCDR contained in VH2 has 1, 2, 3, 4, or 5 amino acid mutations compared to any one HCDR, and / or any LCDR contained in the indicated VL2 has 1, 2, 3, 4, or 5 amino acid mutations compared to any one LCDR.

[0045] In some specific embodiments, the above amino acid mutations are replacements, substitutions, modifications, deletions and / or additions of amino acids (e.g., conservative substitutions of amino acids), and the mutations do not affect, or essentially do not affect, the function of the second binding domain that specifically binds to HER3.

[0046] In some embodiments, in the second binding domain that specifically binds to HER3 in the HER3 / MET binding molecule, The above VH2 includes an amino acid sequence shown in SEQ ID NO: 13 or having at least 80% sequence identity thereto, and / or, The above VL2 includes an amino acid sequence represented by SEQ ID NO: 70 or 14, or having at least 80% sequence identity thereto.

[0047] 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, for example, the Fc region of human IgG1.

[0048] In some specific embodiments, the Fc region has an extended half-life and, for example, has an M252Y / S254T / T256E mutation.

[0049] In some specific embodiments, the Fc region is enhanced with ADCC, for example, by having the S239D / A330L / I332E mutation, or by removing fucosyl.

[0050] In some embodiments, the Fc region allows the binding molecule to form a dimer molecule while simultaneously extending the in vivo half-life of the binding molecule.

[0051] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2). In some embodiments, mutations are introduced that pair the two subunits (Fc1, Fc2) of the Fc region to form a dimer, or mutations that reduce homodimerization. In some embodiments, the first and second subunits include knob-into-hole mutations. For example, a protrusion (or knob) is created on the surface of the CH3 domain of Fc1 by mutating one, two, or more amino acid residues in the CH3 domain of Fc1 with amino acid residues having a larger side chain volume within the CH3 / CH3 interface, and a depression (or hole) is created on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1 by mutating one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 with amino acid residues having a smaller side chain volume.

[0052] In some embodiments, Fc1 comprises one or more amino acid substitutions at a site selected from 354, 356, 358, and 366, and Fc2 comprises one or more amino acid substitutions at a site selected from 349, 356, 358, 366, 368, and 407. In some specific embodiments, Fc1 comprises a mutation at position 366, and Fc2 comprises a mutation selected from positions 366, 368, and 407 or any combination thereof. In some specific embodiments, Fc1 comprises a mutation at position 354 or 356, and Fc2 comprises a mutation at position 349. In some specific embodiments, Fc1 comprises a mutation at position 354 or 356, and Fc2 comprises mutations at positions 349, 366, 368, and 407.

[0053] In some embodiments, Fc1 comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and Fc2 comprises one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, Fc1 comprises a 366W mutation, and Fc2 comprises a mutation selected from 366S, 368A, and 407V or any combination thereof; in some specific embodiments, Fc1 comprises a 354C or 356C mutation, and Fc2 comprises a 349C mutation; or in some specific embodiments, Fc1 comprises a 354C / 366W mutation, and Fc2 comprises a 349C / 366S / 368A / 407V mutation.

[0054] In some specific embodiments, Fc1 comprises the T366W mutation, Fc2 comprises a mutation selected from T366S, L368A, and Y407V or any combination thereof, the first subunit of the Fc region comprises the S354C or E356C mutation and the second subunit comprises the Y349C mutation, or the first subunit of the Fc region comprises the S354C / T366W mutation and the second subunit comprises the Y349C / T366S / L368A / Y407V mutation, and the mutations are numbered according to Eu.

[0055] In some embodiments, the sequence of Fc1 is shown in sequence number 67, and the sequence of Fc2 is shown in sequence number 68.

[0056] In some embodiments, the HER3 / MET binding molecule reduces the mismatch between the light chain and the heavy chain by inducing mutations in the amino acid size and charge of the heavy chain CH1 and the light chain CL interface amino acids. For example, Roche swapped 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 mutated the heavy chain F126C and light chain S121C to introduce a disulfide bond (Mazor et al., mAbs, 7(2), pp.377-389 (2015)), Amgen further modified the CH1-CL region with electrostatic action (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. Yakumei Seizo replaced the constant region of the antibody with the constant region of the TCR, as described in CN109535257A (referenced and incorporated into the full text).

[0057] In some embodiments, the HER3 / MET binding molecule includes a linker.

[0058] In some embodiments, the linker is, for example, (G m S n ) h Or (GGNGT(Sequence No. 88)) h Or (YGNGT(Sequence No. 89)) h Or (EPKSS (SEQ ID NO: 90)) hThe amino acid sequence is shown as follows: m and n are integers independently selected from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is an integer independently selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0059] In some embodiments, the linker is (G x S) y The linker is an integer selected from 1 to 5 (e.g., 1, 2, 3, 4, or 5) and y is an integer selected from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the linker is selected from G4S (sequence number 91), GS, GAP, (G4S)2 (sequence number 92), (G4S)3 (sequence number 93), (G4S)4 (sequence number 94), (G4S)5 (sequence number 95), and ASGS (sequence number 98).

[0060] 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, of which, The first heavy chain consists of [VH1]-[linker 1]-[Obscurin-O chain]-[linker 3]-[first subunit of the Fc region] in order from the N-terminus to the C-terminus. The first light chain is [VL1]-[linker2]-[Titin-T chain] in order from the N-terminus to the C-terminus. The second heavy chain consists of [VH2]-[CH1]-[the second subunit of the Fc region] in order from the N-terminus to the C-terminus, and The second light chain is [VL2]-[CL] from the N-terminus to the C-terminus, Or, The first heavy chain consists of [VH2]-[linker 1]-[Obscurin-O chain]-[linker 3]-[first subunit of the Fc region] in order from the N-terminus to the C-terminus. The first light chain is [VL2]-[linker2]-[Titin-T chain] in order from the N-terminus to the C-terminus. The second heavy chain consists of [VH1]-[CH1]-[second subunit of the Fc region] in order from the N-terminus to the C-terminus, and The second light chain is [VL1]-[CL] from the N-terminus to the C-terminus, Among these, hyphens represent peptide bonds, and linker 1, linker 2, and linker 3 may be the same or different, and may be present or absent independently.

[0061] In some specific embodiments, the amino acid sequences of linker 1 and linker 2 are GGGGS, and linker 3 is absent.

[0062] In some embodiments, the titin-T chain is represented by SEQ ID NO: 96.

[0063] [ka]

[0064] In some embodiments, the titin-T chain is a variant of SEQ ID NO: 96, and the variant has one or more amino acid residue substitutions 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 one or more amino acid residue substitutions 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 several specific examples, the titin-T chain is an amino acid sequence shown in SEQ ID NO: 64 or having at least 80% sequence identity thereto.

[0065] In some embodiments, the Obscurin-O chain is represented by Sequence ID No. 97.

[0066] [ka]

[0067] In some embodiments, the Obscurin-O chain is a variant of Sequence ID No. 97, which has an amino acid residue substitution at one or more sites 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. Exemplary examples include substitutions of one or more amino acid residues selected from 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 examples, the Obscurin-O chain is an amino acid sequence shown in SEQ ID NO: 63 or having at least 80% sequence identity thereto.

[0068] In some embodiments, the above HER3 / MET binding molecule is a combination of the following polypeptide chains, namely, A first heavy chain comprising an amino acid sequence represented by any one of sequence numbers 77, 57, and 61, or having at least 90% sequence identity thereto, A first light chain comprising an amino acid sequence represented by any one of sequence numbers 78, 58, and 62, or having at least 90% sequence identity thereto, A second heavy chain comprising an amino acid sequence shown in Sequence ID No. 59 or having at least 90% sequence identity thereto, and / or It includes a second light chain containing an amino acid sequence represented by either SEQ ID NO: 79 or 60, or having at least 90% sequence identity thereto.

[0069] In some embodiments, The first heavy chain, shown in Sequence ID No. 57, The first light chain, shown in Sequence ID 58, The second heavy chain, shown in sequence number 59, This provides a HER3 / MET binding molecule containing a second light chain, as indicated by Sequence ID No. 60.

[0070] In some embodiments, The first heavy chain shown in Sequence ID No. 61, The first light chain, shown in Sequence ID 62, The second heavy chain, shown in sequence number 59, This provides a HER3 / MET binding molecule containing a second light chain, as indicated by Sequence ID No. 60.

[0071] In some embodiments, The first heavy chain, shown in sequence number 77, The first light chain, indicated by sequence number 78, The second heavy chain, shown in sequence number 59, This provides a HER3 / MET binding molecule containing a second light chain, as indicated by Sequence ID No. 79.

[0072] Antibody-drug conjugates In some embodiments, the HER3 / MET binding molecule of this disclosure is an anti-HER3 / MET antibody-drug conjugate.

[0073] In some embodiments, the antibody-drug conjugate comprises an effector molecule, which is selected from radioisotopes, antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0074] In some specific embodiments, the effector molecule is a cytotoxic drug, exemplified by tubulin polymerization inhibitors, Topo I inhibitors, MMAEs, or derivatives thereof. In some specific embodiments, the cytotoxin is selected from MMAEs or derivatives thereof, exatecans or derivatives thereof, eribulins or derivatives thereof.

[0075] Conjugate of antibody drug (exatecan or derivative thereof) The present disclosure provides an antibody-drug conjugate having the structure represented by formula (I),

Chemical structure

[0076] In some embodiments, Ab is an antibody comprising a binding domain that specifically binds to MET and / or a binding domain that specifically binds to HER3.

[0077] In some embodiments, an antibody-drug conjugate represented by formula (I) described in any one of the above, Eventually, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b These are identical or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and alkyl groups. R 1 is a hydrogen atom, a haloalkyl group, or C 3-6 Selected from cycloalkyl groups, R 2 is selected from a hydrogen atom, a haloalkyl group and C 3-6 cycloalkyl groups, or alternatively, R 1 and R 2 together with the carbon atoms connected thereto form a C 3-6 cycloalkyl group, m is 0 or 1.

[0078] In some embodiments, the antibody-drug conjugate represented by formula (I) according to any one of the above, wherein Y is, JPEG2026531614000004.jpg47163wherein the O-terminus of Y is linked to the linker unit L.

[0079] In some embodiments, the antibody-drug conjugate represented by formula (I) according to any one of the above, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 is selected from -(succinimide-3-yl-N)-W-C(O)-, -CH2-C(O)-NR 3 -W-C(O)- and -C(O)-W-C(O)-, wherein W is C 1-8 alkyl, C 1-8 alkyl-cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 atoms, said heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the above C 1-8 alkyl group, cycloalkyl group and straight-chain heteroalkyl group are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group, L 2 is -NR 4 (CH2CH2O)pCH2CH2C(O)-, -NR 4is selected from (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer of 1 to 20, L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein said amino acid residues are selected from amino acid residues consisting of amino acids 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, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl, L 4 is -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer of 1 to 6, R 3 , R 4 and R 5 are the same or different and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group, R 6 and R 7 are the same or different and each is independently selected from a hydrogen atom, halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0080] In some embodiments, the antibody-drug conjugate represented by formula (I) according to any one of the above, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 is -(succinimide-3-yl-N)-W-C(O)-, -CH2-C(O)-NR 3-WC(O)- and -C(O)-WC(O)- are selected, of which W is C 1-8 Alkyl alkyl group, C 1-8 Selected from alkyl-cycloalkyl groups and linear heteroalkyl groups of 1 to 8 chain atoms, the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, and S, among which C 1-8 Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)pCH2CH2C(O)-, -NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)-, or a chemical bond, where p is an integer from 1 to 20. L 3 This is a peptide residue consisting of 2 to 7 amino acid residues, of which the above amino acid residues are selected from amino acids among phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), and aspartic acid (D), and are further optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 4 -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -C(O)NR 5 (CH2) t - and selected from chemical bonds, where t is an integer from 1 to 6, and non-limiting examples are 1, 2, 3, 4, 5 and 6. R 3 , R 4 and R 5They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R 6 and R 7 These elements may be identical or different, and each may be independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

[0081] In some embodiments, an antibody-drug conjugate represented by formula (I) described in any one of the above, Of these, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -and, JPEG2026531614000005.jpg28163L 2 It is a chemical bond, L 3 is a tetrapeptide residue, preferably L 3 This is the tetrapeptide residue of GGFG, L 4 -NR 5 (CR 6 R 7 ) t - and R 5 , R 6 or R 7 are the same or different and each is independently a hydrogen atom or an alkyl group, and t is 1 or 2. Of these, the above L 1 The end is connected to Ab, L 4 The ends are connected to a Y.

[0082] In some embodiments, the antibody-drug conjugate represented by formula (I) described in any one of the above is an antibody-drug conjugate represented by general formula (II), [ka] Eventually, W is C 1-8 Alkyl alkyl group, C 1-8Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, and of which, C 1-8 Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, selected from p 1 is an integer between 1 and 20, L 3 It is a peptide residue consisting of 2 to 7 amino acids, of which the amino acids are further optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R 1 This is selected from a hydrogen atom, halogen, cycloalkylalkyl group, deuterated alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group. R 2 This is selected from a hydrogen atom, halogen, haloalkyl group, deuterated alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group. Alternatively, R 1 and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. R 4 and R 5 They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R 6 and R7 They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. m is an integer between 0 and 4. n is between 1 and 10, and n can be an integer or a decimal. Ab is defined as shown in general formula (I).

[0083] In some embodiments, the antibody-drug conjugate is represented by formula (II) described in any one of the above, where 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 the mean 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.

[0084] In some embodiments, an antibody-drug conjugate represented by formula (II) described in any one of the above, Eventually, The above -LY- is represented by the following structure: [ka] s 1 These are integers from 2 to 8, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 And m are as defined in the general formula (II) above.

[0085] In some embodiments, the antibody-drug conjugate represented by general formula (I) described above is an antibody-drug conjugate represented by general formula (III), [ka]

[0086] In some embodiments, an antibody-drug conjugate represented by the general formula (III) described in any one of the above, Eventually, s 1 These are integers from 2 to 8, Ab, R 1 , R 2 , R 5 , R 6 , R 7 m and n are as defined in general formula (II).

[0087] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein -L- is JPEG2026531614000009.jpg29143

[0088] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein -LY- is optionally, JPEG2026531614000010.jpg51148

[0089] In some embodiments, -LY- is optionally, JPEG2026531614000011.jpg53148

[0090] In some embodiments, -LY- is, JPEG2026531614000012.jpg28148

[0091] In some embodiments, -LY- is, JPEG2026531614000013.jpg29148

[0092] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the antibody-drug conjugate is JPEG2026531614000014.jpg107163

[0093] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the antibody-drug conjugate is JPEG2026531614000015.jpg59163

[0094] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the antibody-drug conjugate is JPEG2026531614000016.jpg112167 Of these, Ab and n are as defined by general formula (I).

[0095] Antibody drugs (Eribulin or its derivatives) complex This disclosure provides an antibody-drug conjugate having the structure represented by formula (IV), Ab-(L-De)k (IV) Among them, L is a linker that covalently bonds Ab to De, 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), De is shown by the following formula: [ka] Eventually, R 1a is hydrogen, alkyl (for example, C 1-6 Alkyl alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and cycloalkyl groups (e.g., C 3-8A cycloalkyl group is selected from a cyclopropyl group, a cyclopentyl group, or a cyclohexyl group (but is not limited to these), an aryl group, and a heteroaryl group, and each of the alkyl group, cycloalkyl group, aryl group, and heteroaryl group is independently and optionally selected as an alkyl group (for example, C 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (e.g., C 1-6 The alkoxy group is substituted with one or more substituents selected from methoxy, ethoxy, propoxy, and isopropoxy groups (but not limited to these), halogens (e.g., fluorine, chlorine, bromine), deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R 1a It is a methyl group, R 1b is hydrogen, alkyl (for example, C 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups), alkoxy groups, cycloalkyl groups (e.g., C 3-8 A cycloalkyl group is selected from a cyclopropyl group, a cyclopentyl group, or a cyclohexyl group (but is not limited to these), an aryl group, and a heteroaryl group, and each of the alkyl group, cycloalkyl group, aryl group, and heteroaryl group is independently and optionally selected as an alkyl group (for example, C 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (e.g., C 1-6 The alkoxy group is substituted with one or more substituents selected from methoxy, ethoxy, propoxy, and isopropoxy groups (but not limited to these), halogens (e.g., fluorine, chlorine, bromine), deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R 1b It is hydrogen, or R 1a and R 1bC 5-8 A heterocycloalkyl group is formed, and the heteroalkyl group can optionally be an alkyl group (e.g., C 1-6 Alkyl groups, including but not limited to methyl, ethyl, and isopropyl groups, and alkoxy groups (e.g., C 1-6 Alkoxy groups, including but not limited to methoxy, ethoxy, propoxy, and isopropoxy groups), halogens (e.g., fluorine, chlorine, bromine), deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups (e.g., C 3-8 A cycloalkyl group, which includes but is not limited to a cyclopropyl group, a cyclopentyl group, or a cyclohexyl group, is substituted with one or more substituents from a heterocyclyl group, an aryl group, and a heteroaryl group, and R 1a and R 1b It is not hydrogen at the same time.

[0096] In some embodiments, Ab is an antibody comprising a binding domain that specifically binds to MET and / or a binding domain that specifically binds to HER3.

[0097] In some embodiments, in the antibody-drug conjugate represented by formula (IV) described in any one of the above, R1a in De is a methyl group.

[0098] In some embodiments, in the antibody-drug conjugate represented by formula (IV) described in any one of the above, De is represented by the following formula: [ka]

[0099] In some embodiments, in the antibody-drug conjugate represented by formula (IV) described in any one of the above, k is selected from 1 to 10 and may be an integer or a decimal.

[0100] In some embodiments, the linker is stable extracellularly and, in the antibody-drug conjugate represented by formula (IV) described in any one of the above, is fully maintained in the extracellular environment but can be cleaved when internalized, for example, in cancer cells.

[0101] In some embodiments, the linker in the antibody-drug conjugate represented by formula (IV) described in any one of the above includes a cleavable portion, and depending on the location of the cleavable portion, there is no linker and Ab remaining in the drug (e.g., eribulin derivative) after cleavage.

[0102] In some specific embodiments, the cleavable portion in the linker is a cleavable peptide portion (moiety).

[0103] In some embodiments, cytotoxicity and / or effects are increased by adding a cleavable portion to a non-cleavable linker. In some embodiments, the cleavable peptide portion can be cleaved enzymatically, and the linker is also enzymatically cleavable. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the enzymatically cleavable linker (e.g., a cathepsin-cleavable linker) exhibits one or more of the improved properties described above.

[0104] In some embodiments, the linker comprises an amino acid unit (i.e., a peptide residue consisting of 2 to 7 amino acids), the amino acids being preferably selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and 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).

[0105] In some embodiments, the linker of the antibody-drug conjugate represented by formula (IV) of this disclosure is [ka] They are selected from among them.

[0106] In some embodiments, the amino acid unit includes valine-citrulline (Val-Cit).

[0107] On the other hand, linkers provided by some embodiments include a cleavable sulfonamide portion, and the linker is cleavable under reducing conditions.

[0108] In some embodiments, the linker includes a cleavable disulfide portion, and the linker is cleavable under reducing conditions.

[0109] On the other hand, the linker according to the present disclosure includes at least one spacer unit that bonds De (e.g., an eribulin derivative) to a cleavable portion.

[0110] In some embodiments, the spacer unit is made of p-aminobenzyloxycarbonyl (PAB), JPEG2026531614000020.jpg2754

[0111] On the other hand, the antibody-drug conjugate represented by formula (IV) above, provided in several embodiments, is represented by the following formula: JPEG2026531614000021.jpg233167JPEG2026531614000022.jpg182161

[0112] In some embodiments, the antibody-drug conjugate represented by formula (IV) described in any one of the above is represented by the following formula: [ka] Among them, k is selected from 1 to 10 and may be an integer or a decimal, and furthermore, R in De 1a The group is preferably selected from methyl groups, R 1b It is preferably selected from hydrogen.

[0113] In some specific embodiments, the antibody-drug conjugates of the present disclosure include tautomers, meso-compounds, racemics, enantiomers, diastereomers, deuterated compounds, or mixtures thereof.

[0114] In some embodiments, the antibody-drug conjugate represented by general formula (I) or (IV) described above is an anti-HER3 / MET antibody-drug conjugate, Among these, Ab is an antibody comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3, wherein the first binding domain that specifically binds to MET includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the second binding domain that specifically binds to HER3 includes a heavy chain variable region (VH2) and a light chain variable region (VL2).

[0115] In some embodiments, an anti-HER3 / MET antibody-drug conjugate represented by formula (I) or (IV), wherein Ab is The first heavy chain consists of [VH1]-[linker 1]-[Obscurin-O chain]-[linker 3]-[first subunit of the Fc region] in order from the N-terminus to the C-terminus, The first light chain consists of [VL1]-[linker2]-[Titin-T chain] in order from the N-terminus to the C-terminus, From the N-terminus to the C-terminus, in order, the second heavy chain is [VH2]-[CH1]-[the second subunit of the Fc region], It includes a second light chain that is [VL2]-[CL] in order from the N-terminus to the C-terminus, Or, The first heavy chain consists of [VH2]-[linker 1]-[Obscurin-O chain]-[linker 3]-[first subunit of the Fc region] in order from the N-terminus to the C-terminus, The first light chain is [VL2]-[linker 2]-[Titin-T chain] in order from the N-terminus to the C-terminus, From the N-terminus to the C-terminus, in order, the second heavy chain is [VH1]-[CH1]-[the second subunit of the Fc region], It includes a second light chain that is [VL1]-[CL] in order from the N-terminus to the C-terminus, Among these, hyphens represent peptide bonds, and linker 1, linker 2, and linker 3 may be the same or different, and may be present or absent independently.

[0116] In some embodiments, the titin-T chain is represented by SEQ ID NO: 96.

[0117] [ka]

[0118] In some embodiments, the titin-T chain is a variant of SEQ ID NO: 96, and the variant has one or more amino acid residue substitutions 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 one or more amino acid residue substitutions 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 several specific examples, the titin-T chain is an amino acid sequence shown in SEQ ID NO: 64 or having at least 80% sequence identity thereto.

[0119] In some embodiments, the Obscurin-O chain is represented by Sequence ID No. 97.

[0120] [ka]

[0121] In some embodiments, the Obscurin-O chain is a variant of Sequence ID No. 97, which has an amino acid residue substitution at one or more sites 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. Exemplary examples include substitutions of one or more amino acid residues selected from 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 examples, the Obscurin-O chain is an amino acid sequence shown in SEQ ID NO: 63 or having at least 80% sequence identity thereto.

[0122] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to the MET includes the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 15 to 17, and the light chain variable region includes the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs. 76, 19, and 20. The heavy chain variable region in the second binding domain that specifically binds to the HER3 includes the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 51 to 53, and the light chain variable region includes the amino acid sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs. 54, 55, and 75.

[0123] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to the MET includes the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 15 to 17, and the light chain variable region includes the amino acid sequence LCDR1 shown in SEQ ID NOs. 18 or 74 and the amino acid sequences LCDR2 and LCDR3 shown in SEQ ID NOs. 19 and 20. The heavy chain variable region in the second binding domain that specifically binds to the HER3 includes the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs. 51 to 53, and the light chain variable region includes the amino acid sequences LCDR1 and LCDR2 shown in SEQ ID NOs. 54 and 55 and the amino acid sequence LCDR3 shown in SEQ ID NOs. 56 or 73.

[0124] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds to the MET includes an amino acid sequence represented by SEQ ID NO: 71 or 71, or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 72 or 72, or having at least 90% identity thereto, and the heavy chain variable region in the second binding domain that specifically binds to the HER3 includes an amino acid sequence represented by SEQ ID NO: 13, or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 70 or 14, or having at least 90% identity thereto.

[0125] In some embodiments, Ab includes a combination of polypeptide chains represented by SEQ ID NOs. 77, 78, 59, and 79, or a combination of polypeptide chains represented by SEQ ID NOs. 57 to 60.

[0126] In some embodiments, an antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the antibody-drug conjugate is Selected from JPEG2026531614000026.jpg57164, of which, n is between 1 and 8, and n is a decimal or integer, for example, an integer or decimal between 3 and 7, an exemplary n is about 6, and Ab includes combinations of polypeptide chains shown in SEQ ID NOs. 77, 78, 59 and 79, or combinations of polypeptide chains shown in SEQ ID NOs. 57 to 60.

[0127] In some embodiments, an antibody-drug conjugate represented by the general formula (IV) described in any one of the above, wherein the antibody-drug conjugate is selected from: [ka] Eventually, k is 1 to 8, k is a decimal or integer, for example, an integer or decimal from 3 to 7, exemplary k is about 4 or 6, and Ab includes combinations of polypeptide chains shown in SEQ ID NOs. 77, 78, 59 and 79, or combinations of polypeptide chains shown in SEQ ID NOs. 57 to 60.

[0128] In some embodiments, the antibody-drug conjugate represented by general formula (I) or (IV) described in any one of the above is an anti-MET antibody-drug conjugate, Of these, Ab is an antibody containing a binding domain that specifically binds to MET, and the binding domain that specifically binds to MET includes a heavy chain variable region (VH1) and a light chain variable region (VL1).

[0129] In some embodiments, in an anti-MET antibody-drug conjugate represented by formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET includes HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72.

[0130] In some embodiments, in an anti-MET antibody-drug conjugate represented by formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs. 15-17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs. 74, 19, and 20.

[0131] In some embodiments, the binding domain that specifically binds to the MET comprises one or more amino acid mutations selected from the heavy chain variable region 23K, the heavy chain variable region 78T, or the light chain variable region 69T. Exemplary examples include the VH_E23K, VH_S78T, and VL_A69T mutations.

[0132] In some embodiments, in an anti-MET antibody-drug conjugate represented by formula (I) or (IV), the heavy chain variable region of the binding domain that specifically binds to MET comprises an amino acid sequence represented by SEQ ID NO: 71 or having at least 80% identity thereto, and the light chain variable region comprises an amino acid sequence represented by SEQ ID NO: 72 or having at least 80% identity thereto.

[0133] In some embodiments, an anti-MET antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the anti-MET antibody-drug conjugate is Selected from JPEG2026531614000028.jpg53151, of which, n is between 1 and 8, and n can be a decimal or an integer, for example, an integer or decimal between 3 and 7, with an exemplary n being approximately 6. Ab includes the heavy chain variable region shown in Sequence ID 71 and the light chain variable region shown in Sequence ID 72.

[0134] In some embodiments, an anti-MET antibody-drug conjugate represented by the general formula (IV) described in any one of the above, wherein the anti-MET antibody-drug conjugate is selected from: [ka] k is 1 to 8, and k is a decimal or an integer, for example, an integer or decimal from 3 to 7, and exemplary k is about 4 or 6, Ab comprises a heavy chain variable region shown in SEQ ID NO: 71 and a light chain variable region shown in SEQ ID NO: 72.

[0135] In some embodiments, the binding molecule represented by general formula (I) according to any one of the above is an anti-HER3 antibody-drug conjugate, wherein Ab is an antibody comprising a binding domain that specifically binds to HER3, and said binding domain that specifically binds to HER3 comprises a heavy chain variable region (VH2) and a light chain variable region (VL2).

[0136] In some embodiments, in the anti-HER3 antibody-drug conjugate represented by 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.

[0137] In some embodiments, in the anti-HER3 antibody-drug conjugate represented by formula (I), the heavy chain variable region of the binding domain that specifically binds to HER3 comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51 to 53, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 54, 55 and 73.

[0138] In some embodiments, in the anti-HER3 antibody-drug conjugate represented by formula (I), the heavy chain variable region of the binding domain that specifically binds to HER3 comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70 or an amino acid sequence having at least 80% identity thereto.

[0139] In some embodiments, an anti-HER3 antibody-drug conjugate represented by the general formula (I) described in any one of the above, wherein the anti-HER3 antibody-drug conjugate is Selected from JPEG2026531614000030.jpg58153, of which, n is between 1 and 8, and n can be a decimal or an integer, for example, an integer or decimal between 3 and 7, with an exemplary n being approximately 6. Ab includes the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 70.

[0140] In some embodiments, an anti-HER3 antibody-drug conjugate represented by the general formula (IV) described in any one of the above, wherein the anti-HER3 antibody-drug conjugate is selected from: [ka] k is between 1 and 8, and k can be a decimal or an integer, for example, an integer or decimal between 3 and 7, with an exemplary k being approximately 4 or 6. Ab includes the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 70.

[0141] In some embodiments, the HER3 / MET binding molecule has at least one of the following properties.

[0142] a) Specifically binds to MET or its epitope, and in some embodiments, ≤10 7 K D To bind to human MET by value, the above K D A method for detecting values ​​is commonly used in this field, for example, the detection method in Example 2 of this disclosure.

[0143] b) Specifically binds to HER3 or its epitope, and in some embodiments, ≤10 7 K D To bind to human HER3 at a certain value, the above K D A method for detecting values ​​is commonly used in this field, for example, the detection method in Example 2 of this disclosure.

[0144] c) It exhibits good binding activity to cells that co-express 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 ≤0.3nM or lower, the above EC 50 A method for detecting values ​​is commonly used in this field, for example, the detection method in Example 3 of this disclosure.

[0145] d) Endocytosis activity is good in cells co-expressing MET and HER3, for example, EC 50 ≤ 1nM. 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 ≤0.2nM or lower, the above EC 50 A method for detecting values ​​is commonly used in this field, for example, the detection method in Example 4 of this disclosure.

[0146] e) Endocytosis rates in cells co-expressing MET and HER3 are superior to those of anti-HER3 antibodies or anti-MET antibodies.

[0147] f) It does not cause activation of downstream signaling pathways through HER3 / MET target dimerization.

[0148] g) To inhibit tumor growth, or to treat or alleviate cancer.

[0149] In some embodiments, the HER3 / MET binding molecule of the present disclosure can inhibit tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0150] In some embodiments, the HER3 / MET binding molecule of the present disclosure is an anti-HER3 / MET antibody, for example, an anti-HER3 / MET bispecific antibody or an anti-HER3 / MET multispecific antibody.

[0151] The antibody comprises an antigen-binding fragment, and the antigen-binding fragment includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. In some specific embodiments, the antigen-binding fragment comprises Fab, Fv, sFv, Fab', and F(ab')2.

[0152] In some embodiments, there is provided a HER3 / MET antibody that binds to the HER3 / MET binding molecule of the present disclosure, competitively binds to MET and / or HER3, or binds to or competitively binds to the same epitope of MET and / or HER3.

[0153] In some embodiments, there is provided an anti-HER3 / MET antibody that blocks the binding of the HER3 / MET binding molecule of the present disclosure to MET and / or HER3.

[0154] Polynucleotides and vectors The present disclosure provides polynucleotides encoding the HER3 / MET binding molecules, MET binding molecules and HER3 binding molecules provided herein.

[0155] 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 essentially an isolated nucleic acid.

[0156] The nucleic acids of this disclosure may be in the form of a vector, present in a vector, and / or part of a vector, such vector being, for example, a plasmid, cosmid, YAC, or viral vector. The vector may be an expression vector in particular, i.e., a vector capable of providing in vitro and / or in vivo expression (i.e., in a suitable host cell, host organism, and / or expression system) of a HER3 / MET binding molecule, a MET binding molecule, and a HER3 binding molecule. Such an expression vector typically comprises at least one nucleic acid of this disclosure, which is operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selecting the above elements and their sequences for expression in a particular host is common sense for those skilled in the art. Regulatory elements and other elements useful or essential for the expression of the HER3 / MET binding molecule of this disclosure include, for example, promoters, enhancers, terminators, embedded factors, selection markers, reader sequences, and reporter genes.

[0157] The nucleic acids of this disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA technology) and / or isolated from suitable natural sources, based on information regarding the amino acid sequences of the polypeptides of this disclosure.

[0158] host cell This disclosure provides recombinant host cells that express or can express one or more of the HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules of this disclosure, and / or that include the nucleic acids or vectors of this disclosure.

[0159] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0160] Exemplary examples include bacterial cells, such as Gram-negative strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0161] Exemplary examples include fungal cells of species belonging to the genera Trichoderma, Neurospora, and Aspergillus, or cells belonging to species belonging to the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0162] Examples of mammalian cells include, for instance, HEK293 cells, CHO cells, BHK cells, HeLa cells, and COS cells.

[0163] This disclosure may also use amphibian cells, insect cells, plant cells, and any other cells for expressing heterologous proteins in the art.

[0164] The cells described herein cannot develop into a complete plant or animal organism.

[0165] Production or preparation method This disclosure provides a method for preparing the HER3 / MET binding molecule, the MET binding molecule, and the HER3 binding molecule as described herein.

[0166] In some embodiments, the above method is - A step of culturing the host cells of this disclosure under conditions suitable for the expression of the above antibody, - A step of recovering the target protein expressed in the host cells from the culture, -The step of selectively further purifying and / or modifying the target protein of the present disclosure.

[0167] The HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules of this disclosure may be produced intracellularly in the above-mentioned cells (e.g., in the cytoplasm, periplasm, or in inclusion bodies), then isolated from the host cells, and optionally further purified; or they may be produced extracellularly (e.g., in the culture medium for host cells), then isolated from the medium, and optionally further purified.

[0168] Methods and reagents for recombinant polypeptide production, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, techniques for isolating and purifying target proteins applied to the production of the binding molecules or antibodies of this disclosure are known to those skilled in the art. Methods for producing and purifying antibodies are well known in the prior art and can be found, for example, in the Cold Spring Harbor Manual of Antibody Experimental Techniques (Chapters 5-8 and 15). The engineered antibodies of this disclosure may be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains may be cloned and recombinant into an expression vector. The recombinant immunoglobulin expression vector can stably transfect cells. Mammalian expression systems will induce antibody glycosylation, particularly at the highly conserved N-terminus of the Fc region. Stable clones can be obtained by expressing antibodies that specifically bind to human antigens. Positive clones can be expanded and cultured in serum-free medium in a bioreactor to produce antibodies. The culture medium containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving or ion exchange. The resulting product must be immediately frozen at -70°C or similar temperature, or freeze-dried.

[0169] However, the HER3 / MET binding molecules, MET binding molecules, and HER3 binding molecules of this 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.

[0170] This disclosure relates to a method for preparing an antibody-drug conjugate represented by formula (I) or (IV) above, The steps include: coupling the above Ab with a drug to obtain an antibody-drug conjugate represented by formula (I) or (IV); The present invention further provides a method comprising the step of selectively purifying an antibody-drug conjugate represented by formula (I) or (IV) above.

[0171] In some embodiments, a method for preparing an antibody-drug conjugate represented by formula (II) is: [ka] The process includes reducing Ab and then coupling it with a compound represented by the general formula (Ab-La-Y-Dr) to obtain a compound represented by the general formula (Ab-La-Y-Dr). The reducing agent is preferably TCEP, and particularly preferably a disulfide bond on a reducing antibody. Of these, Ab, W, L2, L3, R1, R2, R5~R7, m, and n are defined as shown in equation (I).

[0172] composition This disclosure provides compositions comprising any one or combination of polynucleotides, vectors, etc., that encode any HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, HER3 / MET binding molecule, MET binding molecule, or any HER3 binding molecule provided herein.

[0173] In some embodiments, the pharmaceutical composition comprises an effective amount of the above-mentioned HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, coding polynucleotide, or vector for treating, mitigating, or preventing cancer.

[0174] In some embodiments, the pharmaceutical composition comprises or consists of a mixture of HER3 / MET binding molecules of different n values ​​represented by formula (I), of which at least 65% of the HER3 / MET binding molecules represented by formula (I) have an n of 6. Exemplarily, this can be at least 70%, at least 75%, at least 80%, or at least 90%.

[0175] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0176] In some specific embodiments, the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of HER3 / MET binding molecules, MET binding molecules, or HER3 binding molecules, or the amount of HER3 / MET binding molecules, MET binding molecules, or HER3 binding molecules in the unit dose of the pharmaceutical composition may be 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0177] In several embodiments, products or manufactured goods comprising the above-mentioned HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, polynucleotide and / or vector are provided. Selectively, the product comprises a container and a label. The container is, for example, a vial, syringe and test tube. The container contains a composition effective for the treatment of a medical condition. A label on or attached to the container indicates that the composition is used for the treatment of a selected medical condition. The composition comprises the above-mentioned HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, polynucleotide and / or vector.

[0178] Reagent kits and detection This disclosure provides a reagent kit comprising the above-mentioned HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, polynucleotide, vector, and composition. This disclosure further provides a method, system, or apparatus for detecting MET, HER3 in vivo or in vitro, which includes treating a sample with the above-mentioned binding molecule, polynucleotide, vector, and composition of this disclosure.

[0179] In some embodiments, the in vitro detection method, system, or apparatus is, for example, (1) Contact the sample with a HER3 / MET binding molecule, a MET binding molecule, a HER3 binding molecule, a polynucleotide, a vector, or a composition. (2) To detect complexes formed between the above-mentioned binding molecules, polynucleotides, vectors and the sample, and / or (3) Contacting a reference sample (e.g., a control sample) with the binding molecule, polynucleotide, and (4) This may include determining the degree of composite formation by comparing it with a reference sample. For example, a change in composite formation in the sample or subject compared to that in a control sample or subject (e.g., a statistically significant change) indicates that the sample contains MET and HER3.

[0180] In some other embodiments, an in vivo detection method, system, or apparatus is: (1) Administering the above-mentioned binding molecule, polynucleotide, or vector to the subject, (2) This may also include detecting the formation of a complex between the binding molecule, polynucleotide, vector and the target.

[0181] Detection may include determining the location or time of complex formation. Detection of the binding molecule, nucleic acid (e.g., MET, HER3) is achieved by labeling the binding molecule, nucleic acid with a detectable substance and detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. The formation of complexes between the binding molecule, nucleic acid and MET, HER3 can be detected by measuring or visualizing substances that bind to or do not bind to MET, HER3. For example, conventional detection assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry can be used. For detection purposes, the binding molecule, nucleic acid of this disclosure can be labeled with a fluorophore chromophore. In some embodiments, diagnostic reagents comprising the nucleic acid, binding molecule are further provided, and related diagnostic uses are provided.

[0182] In some embodiments, a reagent kit is further provided, comprising the binding molecule, polynucleotide, and possibly a diagnostic manual. The reagent kit may further include at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the binding molecule may be prepared as a pharmaceutical composition.

[0183] Methods of treating diseases and pharmaceutical uses This disclosure provides the use and methods of HER3 / MET-binding molecules, MET-binding molecules, HER3-binding molecules, coding polynucleotides, vectors, or pharmaceutical compositions in the prevention, treatment, or mitigation of diseases or medical conditions.

[0184] In some embodiments, the present disclosure provides HER3 / MET-binding molecules, MET-binding molecules, HER3-binding molecules, or coding polynucleotides, vectors, and pharmaceutical compositions for treating or mitigating cancer, or for preparing agents for treating or mitigating cancer.

[0185] In some embodiments, the Disclosure provides a method for preventing, treating or alleviating cancer or tumor, comprising administering to a patient or subject an effective amount of the HER3 / MET binding molecule, MET binding molecule, HER3 binding molecule, or coding polynucleotide, vector, or pharmaceutical composition of the Disclosure that prevents, treats or alleviates the disease or condition.

[0186] 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.

[0187] In some specific embodiments, the tumor or cancer is drug-resistant to EGFR-TKIs (epidermal growth factor receptor tyrosinase inhibitors).

[0188] In some embodiments, the HER3 / MET binding molecules, MET binding molecules, HER3 binding molecules, or coding polynucleotides, vectors, and pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and the administration may be systemic or topical.

[0189] In some embodiments, the administration regimen can be adjusted to obtain the optimal target response (e.g., a therapeutic or prophylactic response). For example, it may be administered as a single dose, multiple doses within a certain time period, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. [Brief explanation of the drawing]

[0190] [Figure 1] This is HER3 / MET dual-target IHC staining of tumor samples from EGFR-TKI-resistant non-small cell lung cancer patients. [Figure 2] These are the results of secondary mass spectrometry of antibody A and antibody A T94V. [Figure 3] This involves detecting the binding of HER3 / MET bispecific antibodies to MET monopositive cells using FACS. [Figure 4A] This involves detecting the binding of antibodies to HER3 monopositive cells using FACS. [Figure 4B] This involves detecting antibody binding to MET-positive single cells using FACS. [Figure 4C] This involves detecting antibody binding to HER3 / MET bipositive cells using FACS. [Figure 5A] This involves detecting antibody toxicity in HER3 / MET bipositive cells using an αHFc-CL-MMAE toxin antibody system. [Figure 5B] This involves detecting antibody killing in HER3 monopositive cells using the αHFc-CL-MMAE toxin antibody system. [Figure 5C] This involves detecting antibody killing in MET monopositive cells using an αHFc-CL-MMAE toxin antibody system. [Figure 5D] This involves detecting antibody toxicity in HER3 / MET bipositive cells using an αHFc-CL-MMAE toxin antibody system. [Figure 6] This involves the detection of antibody endocytosis in HER3 / MET bipositive cells using pHrodo. [Figure 7] This involves the detection of antibody endocytosis in HER3 / MET bipositive cells using FACS. [Figure 8] This represents the degree of ERK phosphorylation of antibodies in HER3 / MET bipositive cells. [Figure 9A] This is a schematic diagram of the HER3 / MET antibody-drug conjugate. [Figure 9B]This is the denatured mass spectrometry spectrum of the HER3 / MET antibody-drug conjugate. [Figure 9C] This is the natural mass spectrometry spectrum of the HER3 / MET antibody-drug conjugate. [Figure 10] This involves detecting the binding of HER3 / MET bispecific antibodies and their ADCs to HER3 / MET bipositive cells using FACS. [Figure 11A] This is the result of detecting HER3 / MET expression in HER3 / MET bipositive cells using FACS. [Figure 11B] This shows the results of ADC-4 killing in HER3 / MET double-positive cells. [Figure 11C] This shows the results of ADC-1 chemoscopy in HER3 / MET bipositive cells. [Figure 12] The results of the antitumor activity of the biantibody ADC in an HCC827 osimertinib-resistant cell xenograft model are shown, with Figure 12A illustrating the change in mouse body weight and Figure 12B illustrating the change in mouse tumor volume. [Figure 13] The results show the antitumor activity of the dual antibody ADC in a HER3 mono-positive cell xenograft model. Figure 13A shows the change in mouse body weight, and Figure 13B shows the change in mouse tumor volume. [Figure 14] This describes the killing activity of the biantibody ADC in HER3 / MET bipositive cells. [Modes for carrying out the invention]

[0191] Definition of Terms To facilitate understanding of this disclosure, several technical and scientific terms are defined below. Unless otherwise explicitly defined in this disclosure, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0192] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0193] "MET," "cMET," "MET protein," or "MET polypeptide" may optionally include, but not limited to, known or wild-type MET as described herein, and any naturally occurring splice variants, amino acid variants, or isoforms, or any such protein or its variants, conjugates, or fragments. The complete MET sequence may be UniProt number P08581.

[0194] "HER3," "HER3 protein," or "HER3 polypeptide" may optionally include, but is not limited to, any known or wild-type HER3 as described herein, and any naturally occurring splice variants, amino acid variants, or isoforms, or any such protein or its variants, conjugates, or fragments. The complete HER3 sequence may be UniProt number P21860.

[0195] The term "functional variant" includes, but is not limited to, congeners, fragments, cleavage, mutants, and modifications of wild-type proteins. Functional variants of proteins have increased, decreased, or maintained protein activity compared to wild-type proteins.

[0196] The “binding protein” and “binding molecule” in this disclosure cover any protein, polypeptide, or any molecule containing such protein or polypeptide that can specifically bind to an antigen (e.g., MET or HER3) or a fragment thereof or its epitope, and include, but are not limited to, antibodies as defined in this disclosure.

[0197] In this disclosure, “polypeptide,” “peptide,” or “protein” may be used interchangeably and refer to polymers of amino acid residues or aggregates of amino acid residue polymers. These terms are used for amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, and for naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A polypeptide sequence is typically described as having an amino group terminus (N-terminus, N-end) at its left end and a carboxyl group terminus (C-terminus, C-end) at its right end.

[0198] "Titin-T chain" or "T chain" refers to a peptide fragment containing the Titin Ig-Like 152 domain, having a length of 78 to 118 amino acids, in the Titin protein, or a functional variant thereof. The Titin-T chain can bind to the Obscurin Ig-Like 1 domain to form a dimerization complex. The functional variant of the T chain has a polypeptide that can bind to the Obscurin Ig-Like 1 domain and form a dimerization complex even if some amino acids of the wild-type T chain are mutated. For example, the C-terminus and / or N-terminus of the Titin Ig-Like 152 domain can be increased or cleaved by an appropriate length of amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. For instance, increasing the five amino acids "KAGIR" adjacent to the N-terminus of the Titin Ig-Like 152 domain in the wild-type Titin protein to the N-terminus of the Titin Ig-Like 152 domain still allows it to associate with the Obscurin Ig-Like 1 domain and form a complex. Other mutations may also be introduced into the amino acids of the Titin Ig-Like 152 domain, for example, by mutating certain amino acids to improve interchain disulfide bonds and enhance the stability of the complex.

[0199] "Obscurin-O chain" or "O chain" refers to a peptide fragment containing the Obscurin Ig-Like 1 domain, which has a length of 87 to 117 amino acids in the Obscurin protein, or a functional variant thereof. The Obscurin-O chain can bind to the Titin Ig-Like 152 domain to form a dimerization complex. The functional variant of the Obscurin-O chain has a polypeptide that can bind to the Titin Ig-Like 152 domain and form a dimerization complex even if some amino acids in the wild-type O chain are mutated. For example, even if an appropriate length of amino acids is added or cleaved at the C-terminus and / or N-terminus of the Obscurin-O domain, for example, if 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are added or cleaved, or if five amino acids labeled "DQPQF" adjacent to the N-terminus of the Obscurin Ig-Like 1 domain in the wild-type Obscurin protein are added to the N-terminus of the Obscurin-O domain, it will still function to bind to the Titin Ig-Like 152 domain and form a dimerization complex. Other mutations may be introduced to some amino acids in the Obscurin Ig-Like 1 domain, for example, by mutating certain amino acids to improve interchain disulfide bonds or to improve antibody stability.

[0200] "Nucleic acid" or "polynucleotide" may be used interchangeably herein and refers to any single-stranded or double-stranded DNA or RNA molecule, and, in the case of single-stranded, a molecule of its complementary sequence, preferably double-stranded DNA.

[0201] The term "antibody" covers a variety of antibody structures as long as they exhibit the desired antigen-binding activity, and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, bispecific or multispecific antibodies (e.g., tripspecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, antigen-binding domains). The antibodies of this disclosure include recombinant antibody forms.

[0202] Antibodies may also refer to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Because the amino acid composition and sequence order of the constant region of the immunoglobulin heavy chain differ, their antigenicity also differs. As a result, immunoglobulins can be divided into five types, or immunoglobulin isotypes, called IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Identical Ig cells can be further divided into different subclasses based on differences in the amino acid composition of their hinge region and the number and position of disulfide bonds in the heavy chain; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains can be divided into κ or λ chains based on differences in the constant region. Each of the five types of Ig cells may have either a κ or λ chain. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is significantly altered, forming a variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable, forming a constant region (C region). The variable region includes 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 from the amino end to the carboxyl end in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0203] The prior art has disclosed bispecific antibodies of various structures, which may be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies based on the integrity of the IgG molecule; they may be classified into bivalent, trivalent, tetravalent, or more multivalent bispecific antibodies based on the number of antigen-binding regions; and they may be classified into symmetrical bispecific antibodies and asymmetrical bispecific antibodies based on whether the structure is bilaterally symmetrical. Among these, bispecific antibodies based on antibody fragments include, for example, Fab fragments lacking Fc fragments. By binding two or more Fab fragments to a single molecule, a bispecific antibody is formed. These antibodies have relatively low immunogenicity, a small molecular weight, and relatively high tumor tissue penetration. Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies like IgG-like bispecific antibodies (e.g., those with Fc fragments) have a relatively large relative molecular weight. The Fc fragment contributes to the later purification of the antibody and improves its solubility and stability. The Fc portion can also bind to the receptor FcRn, increasing the half-life of the antibody serum. Typical structural models of bispecific antibodies include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. These are bispecific antibodies such as 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, and F(ab)4-CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019), and Chen S1 et al., J Immunol Res. 2019 Feb 11, 2019:4516041).

[0204] "Antigen-binding fragments" or "antigen-binding domains" cover Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and any one of the above epitope-binding fragments (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136, Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for producing and preparing these antigen-binding fragments are known in this art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0205] The definitive determination or definition of a CDR can be achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex, thereby achieving a reliable description of the CDR and identification of residues containing the antibody binding site. This can be achieved by any one of the various techniques known to those skilled in the art, for example, by X-ray crystallography. Multiple analytical methods can be used for the identification of CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformational definition.

[0206] The Kabat numbering system is a standard for numbering residues in antibodies and is typically 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 the Chothia numbering system takes into account the location 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 from Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272, "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd). The AbM numbering system uses a combination of a knowledge database and the ab initio method to model the tertiary structure of antibodies from their basic sequences (see Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach"). Contact definitions are based on the analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J.Mol.Biol., 5:732-45). In conformational definitions, the position of the CDR can be identified as a residue that makes an enthalpy contribution to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Furthermore, while the definitions of other CDR boundaries may not strictly follow one of the methods described above, they still overlap with at least a portion of the Kabat CDR, although they may be shortened or extended, based on predictions or experimental results that certain residues or groups of residues do not significantly affect antigen binding. As used in this disclosure, CDR can refer to a CDR defined by any method (including combinations of methods) known in the art. Correspondences between various numbering systems are well known to those skilled in the art and are shown exemplarily in Table 1 below.

[0207] [Table 1]

[0208] The CDR amino acid residues in the VL and VH regions of the antibodies disclosed herein correspond in number and position to known Kabat numbering systems.

[0209] In this disclosure, "binding affinity" or "affinity" is used as an indicator of the strength of the non-covalent interaction between two molecules (e.g., an antibody or a portion thereof and an antigen). The binding affinity between two molecules is determined by the dissociation equilibrium constant (K). D This can be quantified by determining the K. For example, by measuring the dynamics of composite formation and dissociation using the surface plasmon resonance (SPR) method (Biacore). D This can be determined. The rate constants corresponding to the bonding and dissociation of the composite are called the bonding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D is, K DThe dissociation constant is related to ka and kd by the equation =kd / ka. The value of the dissociation constant can be directly determined by well known methods, and can also be calculated for complex mixtures by methods such as those described by Caceci et al. (1984, Byte 9:340-362). For example, by a double-filtration nitrocellulose filter binding assay disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432), K D This can be determined. Other standard assays for evaluating the antibody's binding ability to the target antigen are known in the art and include, for example, ELISA, Western blotting, RIA and FACS, as well as other assays cited elsewhere in this disclosure. Antibody binding kinetics and binding affinity can be determined using standard assays known in the art, such as surface plasmon resonance (SPR), for example, Biacore. TM The K of each antibody / antigen complex can be evaluated by the system or KinExA. D By comparing values, we can compare binding affinities related to interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of the interaction is the K of the target interaction (e.g., a specific interaction between an antibody and an antigen). D Value and non-objective interaction K D The value can be determined and compared to evaluate it.

[0210] Typically, "specific binding" refers to the binding of a binding molecule (binding protein) to an epitope on an antigen. The HER3 / MET binding molecule in this disclosure is measured in Biacore, KinExA, or Fortibio assays with an epitope of ≤10. -7 M, preferably ≤10 -8 The dissociation equilibrium constant (K) of M D ) binds to the antigen (i.e., HER3 or MET) or its epitope. -4 K is larger than M. DThe values ​​are generally considered to indicate nonspecific binding. Specific binding of the binding molecule to an antigen or epitope can be measured by any known suitable method, including, for example, surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry (FACS) as described herein.

[0211] A "conservative substitution" refers to the substitution of an original amino acid residue with another amino acid residue that has similar properties. 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. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have polar side chains with no charge, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Also, 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 when amino acid residues in the group exhibiting the above-mentioned similar properties are substituted, it does not result in any particular change in properties.

[0212] "Homologousity," "identity," or "sequence identity" refers to the sequence similarity between two nucleic acid sequences or two polypeptides. Two sequences being compared are homologous at a given position if all positions in those sequences are occupied by the same nucleotide or amino acid monomer—for example, if the positions in two DNA molecules are occupied by the same nucleotide. The percentage of homology between two sequences is a function of dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and multiplying by 100%. For example, if the sequences are optimally aligned, and six out of ten positions in the two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, two sequences are compared when they are aligned to obtain the highest possible percentage of homology.

[0213] The term "internalization" refers to the transport of a portion of a substance from the outside of a cell to the inside. The internalized portion may be located in an intracellular septum. An "internalized" or "internalizing" antigen or antibody refers to an antigen or antibody that can be transported from the outside of a target cell to the inside. As is generally understood by those skilled in the art, the process of intracellular internalization usually refers to the movement of cell surface molecules from the cell surface to the cell interior across the quality membrane. After internalization, endosomes can be transported to lysosomes for degradation or recycled back to the cell surface. Given the rate of intracellular internalization of cell surface molecules, a measurement of the dynamics of the movement of these molecules from the cell surface across the quality membrane into the cell interior is provided. The internalization activity or rate of antigens and antibodies can be monitored and / or measured by various techniques known in this art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305-17, 2008) and toxin virulence measurement (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 antibody labeling reagent kits are commercially available for the quantification and monitoring of internalization (e.g., pHrodo iFL antibody labeling method, reagents, and reagent kits sold by Thermo Fisher Scientific).

[0214] The term "antibody-drug conjugate" (ADC) refers to a device in which an antibody is linked to a biologically active drug. The antibody can be coupled to the drug either directly or via a linking unit.

[0215] The term "drug load" refers to the average amount of cytotoxic drug loaded onto each ligand in the ADC, and may be expressed as a ratio of drug load to antibody load. The drug load range may be such that each antibody (Ab) is linked to 1 to 20, preferably 1 to 10, cytotoxic drugs (D). In embodiments of this disclosure, the drug load is denoted by n, which may be exemplary 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average value between any two of these numbers. The average drug load on each ADC molecule after the coupling reaction can be characterized and identified by conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA, monoclonal antibody molecular size variant assay (CE-SDS), and HPLC.

[0216] While the drug-to-antibody ratio has a precise value (e.g., n in formula (I)) for a particular complex molecule, it should be understood that when used to describe a sample containing many molecules, this value is always an average value, which is due to a certain degree of heterogeneity typically associated with the complexing step. The average loading amount of an immunocomplex sample is referred to herein as the drug-to-antibody ratio or "DAR". In some examples, the DAR is between approximately 1 and approximately 10, for example, 1 to 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, and 8.0. The examples include those with DAR values ​​of approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0. The immunocomplexes include approximately 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0. In some examples, a DAR of "approximately x" means that the measured value of DAR is within 20% of x.

[0217] One method for detecting DAR is to estimate the DAR value from LC-MS data of reduced and deglycosylated samples. LC / MS allows for the quantification of the average number of payload (drug portion) molecules linked to the antibody in the ADC. HPLC isolates the antibody into light and heavy chains, and further isolates the heavy chain (HC) and light chain (LC) based on the number of linker-payload groups in 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. The average DAR of the ADC can be calculated from the average loading of the LC and HC chains. The DAR of a given immunocomplex sample indicates the average number of drug (payload) molecules linked to a tetrameric antibody containing two light chains and two heavy chains. For example, this is the DAR detection method in WO2018142322.

[0218] The term "camptothecin-based drugs" refers to cytotoxic camptothecin and its derivatives, and is not limited to 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, exatecan, irinotecan, or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0219] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a linear or branched 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 group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl This includes n-octyl groups, 2,3-dimethylpentyl groups, 2,4-dimethylpentyl groups, 2,2-dimethylpentyl groups, 3,3-dimethylpentyl groups, 2-ethylpentyl groups, 3-ethylpentyl groups, n-octyl groups, 2,3-dimethylhexyl groups, 2,4-dimethylhexyl groups, 2,5-dimethylhexyl groups, 2,2-dimethylhexyl groups, 3,3-dimethylhexyl groups, 4,4-dimethylhexyl groups, 2-ethylhexyl groups, 3-ethylhexyl groups, 4-ethylhexyl groups, 2-methyl-2-ethylpentyl groups, 2-methyl-3-ethylpentyl groups, n-nonyl groups, 2-methyl-2-ethylhexyl groups, 2-methyl-3-ethylhexyl groups, 2,2-diethylpentyl groups, n-decyl groups, 3,3-diethylhexyl groups, 2,2-diethylhexyl groups, and various branched isomers thereof.More preferably, it is a lower alkyl group containing 1 to 6 carbon atoms, and non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl group may be substituted or not, and if substituted, the substituent may be substituted at any available linking point, and preferably the substituent is independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0220] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, where the alkyl group is as defined above.

[0221] The term "alkylene group" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and is a linear or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2)-, 1,1-propyridene (-CH(CH2CH3)-), 1,2-propyridene (-CH2CH(CH3)-), 1,3-propyridene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), and 1,5-butylidene (-CH2CH2CH2CH2CH2-). The alkylene group may be substituted or not, and if substituted, the substituent may be substituted at any available linking point, and it is preferable that the substituent is independently and optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0222] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), with the definitions of alkyl group and cycloalkyl group being as described above. Non-exclusive examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, and cyclohexyloxy group. Alkoxy groups may be optionally substituted or unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxyl group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0223] The term "cycloalkyl group" refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituents, where a 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 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, while polycyclic cycloalkyl groups include cycloalkyl groups of spiro rings, fused rings, and crosslinked rings.

[0224] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more ring atoms are nitrogen, oxygen, or S(O). mThe heteroatoms are selected from (where m is an integer from 0 to 2), but do not contain the -OO-, -OS-, or -SS- ring portion, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro rings, fused rings, and bridging rings.

[0225] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclyl group with 5 to 20 members, in which monocyclic rings share one atom (called a spiro atom), and one or more of these ring atoms are nitrogen, oxygen, or S(O). m The heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. It may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably it is 6 to 14 members, more preferably 7 to 10 members. The spiroheterocyclyl group is divided into a monospiroheterocyclyl group, a bisspiroheterocyclyl group, or a polyspiroheterocyclyl group depending on the number of spiroatoms shared between the rings, preferably a monospiroheterocyclyl group and a bisspiroheterocyclyl group. More preferably it is a 4-member / 4-member, 4-member / 5-member, 4-member / 6-member, 5-member / 5-member, or 5-member / 6-member monospiroheterocyclyl group. Non-limiting examples of spiroheterocyclyl groups are: JPEG2026531614000034.jpg32156

[0226] The term "condensed heterocyclyl group" refers to a polycyclic heterocyclyl group with 5 to 20 members, in which each ring in the system shares one pair of adjacent atoms with the other rings in the system, and one or more rings may contain one or more double bonds, but none of the rings have a fully conjugated π-electron system, and one or more of the ring atoms are nitrogen, oxygen, or S(O). mThe heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. Preferably, it has 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings that make up the group, it can be classified into bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclyl groups, preferably bicyclic or tricyclic, and more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of fused heterocyclyl groups are: JPEG2026531614000035.jpg55161

[0227] The term "bridged heterocyclyl group" refers to a polycyclic heterocyclyl group having 5 to 14 members, in which any two rings share two atoms that are not directly linked, and which may contain one or more double bonds, but none of the rings have a fully conjugated π-electron system, and of which one or more ring atoms are nitrogen, oxygen, or S(O) m The heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. Preferably, it has 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings that make up the group, it can be divided into bicyclic, tricyclic, tetracyclic, or polycyclic bridging heterocyclyl groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridging heterocyclyl groups are: [ka] Includes.

[0228] The above heterocyclyl ring may be condensed with an aryl group, a heteroaryl group, or a cycloalkyl ring, of which the ring linked to the parent structure is a heterocyclyl group, and a non-limiting example thereof is: JPEG2026531614000037.jpg24161

[0229] The heterocyclyl group may be optionally substituted or left unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0230] The term "aryl group" refers to a 6-14 member all-carbon monocyclic or condensed polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6-10 membered, such as a phenyl group and a naphthyl group, preferably a phenyl group. The above aryl ring may be condensed with a heteroaryl group, a heterocyclyl group, or a cycloalkyl ring, of which the ring linked to the parent structure is an aryl ring, and non-limiting examples include: JPEG2026531614000038.jpg47161

[0231] The aryl group may be substituted or not. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0232] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, of which the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 10 membered, more preferably 5 or 6 membered, and include, for example, furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidine, pyrazinyl, imidazolyl, and tetrazolyl groups. The above heteroaryl ring may be condensed with an aryl group, a heterocyclyl group, or a cycloalkyl ring, of which the ring linked to the parent structure is a heteroaryl ring, and non-limiting examples include: JPEG2026531614000039.jpg40154

[0233] The heteroaryl group may be optionally substituted or left unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0234] The term "amino protecting group" refers to a group that protects an amino group with an easily detachable group so that the amino group is not altered when other parts of the molecule react. Non-limiting examples include the 9-fluorenylmethyloxycarbonyl group, the tert-butoxycarbonyl group, the acetyl group, the benzyl group, the allyl group, and the p-methoxybenzyl group. These groups can be optionally substituted with 1 to 3 substituents selected from halogens, alkoxy groups, or nitro groups. The 9-fluorenylmethyloxycarbonyl group is preferred as the amino protecting group.

[0235] The term "cycloalkylalkyl group" refers to a group in which an alkyl group is substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, where the alkyl group is as defined above, and the cycloalkyl group is as defined above.

[0236] The term "haloalkyl group" refers to a group in which an alkyl group is substituted with one or more halogens, where the alkyl group is as defined above.

[0237] The term "deuterated alkyl group" refers to a group in which an alkyl group is substituted with one or more deuterium atoms, where the alkyl group is as defined above.

[0238] The term "hydroxyl group" refers to the -OH group.

[0239] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0240] The term "amino group" refers to -NH2.

[0241] The term "nitro group" refers to -NO2.

[0242] The term "amide group" refers to -C(O)N(alkyl group) or (cycloalkyl group), of which alkyl groups and cycloalkyl groups are defined above.

[0243] The term "carboxylic acid ester group" refers to -C(O)O(alkyl group) or (cycloalkyl group), of which alkyl groups and cycloalkyl groups are defined above.

[0244] This disclosure further includes compounds of formula (I) in various deuterated forms. Each available hydrogen atom bonded to a carbon atom may be independently substituted with a deuterium atom. Those skilled in the art can synthesize compounds of formula (I) in deuterated forms by referring to relevant literature. When preparing compounds of formula (I) in deuterated forms, commercially available deuterated starting materials may be used, or they may be synthesized using deuterating reagents by general techniques, including, but not limited to, borane deuterated, borane tetrahydrofuran trihydrogenated solution, lithium aluminum hydride deuterated, iodoethane deuterated, and iodomethane deuterated.

[0245] "Substituting" means that one or more hydrogen atoms in a group, preferably five or fewer, more preferably one to three hydrogen atoms, are substituted by a number of substituents that correspond to each other independently. Of course, substituents can only be located in their chemically possible sites, and those skilled in the art can determine possible or impossible substitutions with little effort (by experiment or theory). For example, an amino group or hydroxyl group with free hydrogen can become unstable when bonded to a carbon atom with an unsaturated (e.g., olefin) bond.

[0246] The terms "pharmaceutically acceptable additive" or "pharmaceutically acceptable excipient" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the target immune system. Examples include, but are not limited to, any standard drug carriers, such as buffered saline solutions, water, emulsions such as oil / water emulsions, and various wetting agents.

[0247] "Inhibition" and "blockage" may be used interchangeably and cover both partial and complete inhibition / blockage. "Inhibition of growth" (e.g., for cells) is intended to also include any measurable reduction of cell growth.

[0248] "Proliferative disorder" refers to a medical condition associated with a certain degree of abnormal cell proliferation. In one embodiment, proliferative disorder refers to cancer. "Tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive as they appear in this disclosure.

[0249] "Give," "administer," and "process" refer to the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids, e.g., for therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell processing includes contact between reagents and cells, and contact between reagents and fluids, where the fluid comes into contact with the cells. "Give," "administer," and "process" also mean in vitro and ex vivo processing of cells, e.g., with reagents, diagnostics, conjugated compositions, or with other types of cells. When applied to humans, veterinary medicine, or research subjects, they also mean therapeutic processing, preventive or precautionary measures, research, and diagnostic use.

[0250] "Treatment" means administering an internal or external therapeutic agent, for example, one of the binding proteins or a pharmaceutical composition thereof of the Disclosure, to a subject who has, is at risk of, or is prone to having one or more proliferative disorders or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the subject or population being treated is given the therapeutic agent in a dose that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable degree. The dose of the therapeutic agent that effectively alleviates any specific disease symptom (also called the "therapeutic dose") can vary depending on various factors, such as the subject's disease state, age and weight, and the agent's ability to produce the necessary therapeutic effect on the subject. Whether or not the disease symptoms are reduced can be assessed by any clinical detection method commonly used by a physician or other professional healthcare provider to assess the severity or progression of the symptoms. Embodiments of the present disclosure (e.g., a treatment method or product) may be ineffective in alleviating a target disease symptom in a given subject, but any statistical test known in the art, such as the Student t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, may determine that they should alleviate a statistically significant number of subjects.

[0251] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also refers to an amount sufficient to enable or facilitate a diagnosis. The effective dose used for a subject may vary depending on factors such as the medical condition being treated, the subject's overall health, the method, route, and dosage of administration, and the severity of side effects. An effective dose may also be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0252] "Optional" or "optionally," "selective" or "selectively" means that the event or situation described thereafter may occur, but does not necessarily have to occur, and such description includes both cases in which the event or situation occurs and cases in which it does not. "And / or" should be considered as specifically indicating whether each of the two specified features or components has or does not have the other. Accordingly, the term "and / or" as used in the phrase "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless otherwise specifically stated in the context, words such as "contains," "has," and "includes" throughout the specification and claims should be understood to have a comprehensive meaning, i.e., "includes, but not limited to," rather than an exclusive or exhaustive meaning.

[0253] In this disclosure, “subjects” and “patients” refer to mammals, especially primates, and especially humans.

[0254] Examples The present disclosure will be further described below in accordance with the examples, but these examples are not intended to limit the scope of the present disclosure.

[0255] Experimental methods in the examples or test cases of this disclosure that do not specify concrete conditions generally follow normal conditions or conditions recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Molecular Biology Methods, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents whose specific source is not specified are commonly available commercially.

[0256] Example 1. Results of HER3 / MET dual-target IHC staining of tumor samples from EGFR-TKI-resistant non-small cell lung cancer patients. Tumor samples from EGFR-TKI-resistant non-small cell lung cancer patients were detected using HER3 / MET bitargeted IHC staining. As shown in Figures 1A to 1C, both HER3 and MET were expressed in the tumor tissue of EGFR-TKI-resistant non-small cell lung cancer patients, with a co-expression rate of 100%. This demonstrates that EGFR-TKI-resistant non-small cell lung cancer patients exhibit HER3 / MET target expression and a high co-expression rate, suggesting that HER3 / MET bispecific antibody ADCs can achieve superior antitumor activity compared to HER3 monoclonal antibody ADCs or MET monoclonal antibody ADCs in this segment of patients.

[0257] Example 2. Design and preparation of anti-HER3 / MET bispecific antibodies 1. Design of anti-HER3 / MET bispecific antibodies The variable region at the HER3 terminus was selected from antibody A, and the variable region at the MET terminus was 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 above antibodies were as follows.

[0258] >Anti-MET antibody A13 VH [ka]

[0259] >Anti-MET antibody A13 VL [ka]

[0260] >Anti-MET antibody S3 VH [ka]

[0261] >Anti-MET antibody S3 VL [ka]

[0262] Anti-MET antibody A9 VH

change

[0263] Anti-MET antibody A9 VL

change

[0264] Anti-MET antibody P8 VH

change

[0265] Anti-MET antibody P8 VL

change

[0266] >Anti-MET antibody P3 VH

change

[0267] >Anti-MET antibody P3 VL

change

[0268] Anti-MET antibody 5D5 VH

change

[0269] Anti-MET antibody 5D5 VL

change

[0270] Anti-HER3 antibody A VH [ka]

[0271] >Anti-HER3 antibody A VL [ka]

[0272] [Table 2-1] [Table 2-2] JPEG2026531614000056.jpg154157

[0273] A bispecific antibody with the following structure was constructed.

[0274] The first heavy chain consists of the anti-MET antibody heavy chain variable region -GGGGS-[Obscurin-O chain]-[IgG1 Fc1] in order from the N-terminus to the C-terminus. From the N-terminus to the C-terminus, the first light chain is the anti-MET antibody light chain variable region -GGGGS-[Titin-T chain]. The second heavy chain consists of the anti-HER3 antibody heavy chain variable region -[CH1]-[IgG1 Fc2], in order from the N-terminus to the C-terminus, and From the N-terminus to the C-terminus, the second light chain is the variable region of the anti-HER3 antibody light chain -[CL]. Of these, -- represents a peptide bond, Obscurin-O chain refers to SEQ ID NO: 63, Titin-T chain refers to SEQ ID NO: 64, CH1 refers to SEQ ID NO: 65, CL refers to SEQ ID NO: 66, IgG1 Fc1 refers to SEQ ID NO: 67, and IgG1 Fc2 refers to SEQ ID NO: 68.

[0275] The heavy / light chain variable region of the antibody A heavy chain was operably linked to the heavy / light chain variable regions selected from antibody A13, antibody S3, antibody A9, antibody P8, antibody P3, and antibody 5D5, respectively, according to the above structure, and anti-HER3 / MET bispecific antibodies 2232-01(A13-A), 2232-02(S3-A), A9-A, P8-A, P3-A, and 5D5-A were constructed and obtained. Of these, the heavy chain (H) and light chain (L) sequences of 2232-01 and 2232-02 are shown below.

[0276] >2232-01 H1 [ka]

[0277] >2232-01 L1 [ka]

[0278] >2232-01 H2 [ka]

[0279] >2232-01 L2 [ka]

[0280] >2232-02 H1 [ka]

[0281] >2232-02 L1 [ka]

[0282] 2232-02 H2 matches sequence number 59. 2232-02 L2 matched sequence number 60.

[0283] In the heavy chain sequence shown above, the italicized part is the Fc constant region of IgG1, the underlined tilde is the Obscurin-O chain, and the underlined transverse is VH1. In the light chain sequence, the underlined transverse is the Cκ of IgG1, the underlined tilde is the Titin-T chain, and the italicized bold part is the GGGGS (sequence number 91) linker.

[0284] >Obscurin-O chain [ka]

[0285] >Titin-T chain [ka]

[0286] >CH1 [ka]

[0287] >CL [ka]

[0288] >IgG1 Fc1(S354C / T366W) [ka]

[0289] >IgG1 Fc2(Y349C / T366S / L368A / Y407V) [ka]

[0290] 2. Antibody modification Four groups of mutants, N93Q, N93T, N93S, and T94V, were designed for the anti-HER3 antibody-antibody A (which has the heavy chain described in SEQ ID NO: 86 and the light chain shown in SEQ ID NO: 87). Antibody A N93Q had a mutation from N to Q only at position 93 of the VL compared to antibody A; antibody A N93T had a mutation from N to T only at position 93 of the VL compared to antibody A; antibody A N93S had a mutation from N to S only at position 93 of the VL compared to antibody A; and antibody A T94V had a mutation from T to V only at position 94 of the VL compared to antibody A.

[0291] Experimental Design: Antibodies A, A N93Q, A N93T, A N93S, and A T94V were diluted to 1 μg / mL with HBS-EP+ buffer, and the flow rate was set to 10 μL / min to capture antibodies at the 200 RU level. His-tagged human HER3 antigens were diluted at a constant ratio with HBS-EP+ buffer, resulting in concentration gradients of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The flow rate was set to 30 μL / min during sample analysis. The binding time was 120 s and the dissociation time was 900 s. Next, regeneration was performed using Gly-HCl buffer at pH 1.5 as the regeneration buffer, with a regeneration flow rate of 30 μL / min, for 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data was fitted using BIAcore 8K analysis software, and dynamical constants such as the coupling rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD) were determined using a 1:1 Langmuir coupling model.

[0292] Experimental results: As is clear from the results in Table 3-1 and Figure 2, the binding of the antibody to HER3 before and after the T94V mutation did not change significantly, but the level of antibody deamidation decreased dramatically.

[0293] [Table 3]

[0294] The variable region sequence of antibody A T94V was as follows:

[0295] >Anti-HER3 antibody A VH [ka]

[0296] >Anti-HER3 antibody A VL T94V [ka]

[0297] Four groups of mutations, N30Q, N30T, N30S, and T31V, were designed for the anti-MET antibody A13 (which has a heavy chain indicated by SEQ ID NO: 84 and a light chain indicated by SEQ ID NO: 85). In antibody A13 N30Q, the mutation from N to Q is present only at position 30 of the VL compared to antibody A13. In antibody A13 N30T, the mutation from N to T is present only at position 30 of the VL compared to antibody A13. In antibody A13 N30S, the mutation from N to S is present only at position 30 of the VL compared to antibody A13. In antibody A13 T31V, the mutation from T to V is present only at position 31 of the VL compared to antibody A13.

[0298] Experimental Design: Antibodies A13, A13 N30Q, A13 N30T, A13 N30S, and A13 T31V were diluted to 1 μg / mL with HBS-EP+ buffer, and the flow rate was set to 10 μL / min to capture the antibodies at the 200 RU level. His-tagged human MET antigen was diluted at a constant ratio with HBS-EP+ buffer, and the concentration gradients were 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The flow rate was set to 30 μL / min during sample analysis. The binding time was 120 s and the dissociation time was 900 s. Next, regeneration was performed using Gly-HCl buffer at pH 1.5 as the regeneration buffer, with the regeneration flow rate set to 30 μL / min and regeneration performed for 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data was fitted using BIAcore 8K analysis software, and dynamical constants such as the coupling rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD) were determined using a 1:1 Langmuir coupling model.

[0299] Experimental results: As is clear from the results in Table 3-2, the binding of the antibody to HER3 before and after the N30S mutation did not change significantly. Furthermore, the level of antibody deamidation decreased significantly.

[0300] [Table 4]

[0301] Based on N30S, the inventors further mutated the framework region (FR) of the anti-MET antibody A13, specifically by performing E23K and S78T mutations on VH and A69T mutations on VL, thereby obtaining antibody A13 E23K / S78T / A69T / N30S. Detection revealed an increase in the expression level of antibody A13 E23K / S78T / A69T / N30S from 96 mg / L to 200 mg / L.

[0302] The variable region sequence of antibody A13 E23K / S78T / A69T / N30S was as follows:

[0303] >Anti-MET antibody A13 E23K / S78T / A69T / N30 VH [ka]

[0304] >Anti-MET antibody A13 E23K / S78T / A69T / N30 VL [ka]

[0305] [Table 5]

[0306] In other words, antibody A of this disclosure had the following general formula sequence.

[0307] HCDR1:DYAMH (Sequence ID 51) HCDR2:GISWNSGSIGYADSVKG (Sequence ID 52) HCDR3:EGLPGLDY (Sequence ID 53) LCDR1:RASQHVGTYLN (Sequence ID 54) LCDR2:GAANLQS(Sequence ID 55) LCDR3:QQSYX1X2PPFS (Sequence ID 75), where X1 is selected from N, Q, T, or S, and X2 is selected from T or V.

[0308] The antibody A13 of this disclosure had the following general formula sequence.

[0309] HCDR1:SYGFS (Sequence ID 15) HCDR2:WISASNGNTYYAQKLQG (Sequence ID 16) HCDR3:VYADYADY (Sequence ID 17) LCDR1:RASQGIX3X4WLA (Sequence ID 76), where X3 is selected from N, Q, T, or S, and X4 is selected from T or V.

[0310] LCDR2:AASSLKS(Sequence ID 19) LCDR3:QQANSFPLT (SEQ ID NO: 20).

[0311] The anti-HER3 / MET bispecific antibody 2232-06 was constructed using the sequence after the above mutation, and its full-length amino acid sequence was as follows.

[0312] >2232-06 (A13 E23K / S78T / A69T / N30S, A T94V) H1 heavy chain [ka]

[0313] >2232-06 (A13 E23K / S78T / A69T / N30S, A T94V) L1 light chain [ka]

[0314] >2232-06(A13 E23K / S78T / A69T / N30S, A T94V)H2 double chain matches sequence number 59. >2232-06 (A13 E23K / S78T / A69T / N30S, A T94V) L2 light chain [ka]

[0315] 3. Control antibody The full-length sequences of the naked antibody (U3-1402 Ab) of the control antibody U3-1402, antibody S3, antibody A13, and the heavy chain (HC) and light chain (LC) of antibody A were as follows.

[0316] >U3-1402 Ab HC [ka]

[0317] >U3-1402 Ab LC

change

[0318] Antibody S3 HC

change

[0319] Antibody S3 LC

change

[0320] Antibody A13 HC

change

[0321] Antibody A13 LC

change

[0322] Antibody A HC

change

[0323] Antibody A LC

change

[0324] 4. Modulation of antibodies The nucleotide sequences encoding the heavy and light chains of the antibody were cloned into pTT5 vectors, respectively. ExpiCHO cells were then transfected. After 8 days, the cells were removed by centrifugation, the cell culture medium was collected and filtered, and the resulting cell culture medium was purified using a Protein A affinity column (MabSelect SuRe, GE). The bound antibody was eluted with glycine, the eluent was neutralized with 1M Tris, and then desalted. The target antibody was obtained by detection.

[0325] Example 3. Detection of antigen-binding activity of anti-HER3 / MET bispecific antibody In this example, the binding activity of an anti-HER3 / MET bispecific antibody to human MET protein and HER3 / MET protein on the cell surface was detected by FACS experiment.

[0326] 1. Detection of binding activity to cell surface MET proteins Experimental Method: NCI-H1975 (lung cancer cell lineage) was a MET monopositive cell lineage. The cell medium was RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. The experimental medium was sterile PBS (phosphate buffer, pH 7.40) containing 2% fetal bovine serum (the same applies below). NCI-H1975 cells were washed twice with the experimental medium, and 1 × 10⁶ cells were prepared. 5 Cells were inoculated into a 96-well U-bottom plate at a rate of one cell per well, samples of different concentrations awaiting measurement were added, and the cells were incubated at 4°C for 1 hour. After that, the cells were washed twice with experimental medium, then Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added, and after two washes, the fluorescence signal values ​​were read using a flow cytometer.

[0327] Experimental Results: As is clear from the FACS results in Figure 3, different anti-HER3 / MET bispecific antibodies (same anti-HER3 antibody sequence, different anti-MET antibody sequence) showed different binding affinities to MET monopositive cells. Of these, A13-A showed the strongest binding affinity and is a high-affinity anti-HER3 / MET bispecific antibody, while P8-A showed the weakest binding affinity and is a low-affinity anti-HER3 / MET bispecific antibody, and S3-A binding EC 50 This antibody differed tenfold from A13-A and was a medium-to-low affinity anti-HER3 / MET bispecific antibody. High-affinity MET antibody 2232-01 (A13-A) and medium-to-low affinity MET antibody 2232-02 (S3-A) were selected and the next screening was performed.

[0328] 2. Detection of binding activity to human HER3 / MET protein on the cell surface Experimental methods: MDA-MB-453 (breast cancer cell line) was a HER3 monopositive cell line, cultured in air using Leibovitz's L-15 (Gibco, Cat#11415064) containing 10% fetal bovine serum. NCI-H1703 (lung cancer cell line) was a MET monopositive cell line, cultured in RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. The HCC827 Osimertinib drug-resistant cell line (lung cancer cell line) was obtained by constructing HER3 / MET double-positive cells internally and inducing Osimertinib gradient drug resistance using HCC827 cells, cultured in RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. Wash MDA-MB-453, NCI-H1703, or HCC827 osimertinib-resistant cells twice with experimental medium, and then 1 × 10⁶ 5Cells were inoculated into a 96-well U-bottom plate at a rate of one cell per well, samples of different concentrations awaiting measurement were added, and the cells were incubated at 4°C for 1 hour. Afterward, the cells were washed twice with experimental medium, then Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added, followed by two more washes. Fluorescence signal values ​​were then read using a flow cytometer. IgG1 was the isotype control antibody, and the process was similar for subsequent steps.

[0329] Experimental Results: As is clear from the FACS detection results in Tables 4-1 and 4-2, and Figures 4A and 4B, the anti-HER3 / MET bispecific antibodies were able to bind to the cell surface HER3 / MET antigen. In MET monopositive cells, the binding ability of MET-high affinity 2232-01 was superior to that of MET-medium affinity 2232-02. In HER3 monopositive cells, the two molecules had similar binding abilities to HER3, both being slightly weaker than antibody A.

[0330] As is clear from the results in Table 4-3 and Figure 4C, in HER3 / MET bispecific cells, the maximum fluorescence value of anti-HER3 / MET bispecific antibodies was higher than that of anti-HER3 antibodies (antibody A and U3-1402 Ab) and anti-MET antibodies (antibody S3), indicating that more anti-HER3 / MET bispecific antibodies bind to bispecific cells.

[0331] [Table 6]

[0332] [Table 7]

[0333] [Table 8]

[0334] Example 4. Detection of endocytosis activity of anti-HER3 / MET bispecific antibody 1. Evaluation of the internalization activity of αHFc-CL-MMAE toxin antibodies The internalization and toxicogenic activity of the αHFc-CL-MMAE toxin antibody were evaluated using an internalization activity evaluation system. αHFc-CL-MMAE is a coupling of an anti-human Fc antibody to the MMAE toxin, possessing a stable, cleavable linker capable of specifically binding 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, after being taken up by cells via endocytosis, can be cleaved by lysosomal cathepsin to release the toxin. Therefore, the endocytic activity of the antibody can be evaluated by its cellular toxicity.

[0335] Experimental method: NCI-H441 (lung cancer cell line) was used as a MET / HER3 bipositive cell line, MDA-MB-453 (breast cancer cell line) as a HER3 monopositive cell line, NCI-H1703 (lung cancer cell line) as a MET monopositive cell line, and the HCC827 Osimertinib drug-resistant cell line (lung cancer cell line, internally constructed) was used as a MET / HER3 bipositive cell line. Osimertinib gradient drug resistance was obtained using HCC827 cells, and the culture medium and conditions were the same as in Example 3. αHFc-CL-MMAE (Moradec, Cat#AH-102AE-50) and the antibody awaiting measurement were uniformly mixed in an equimolar ratio of 1:1 volume, incubated at 37°C for 30 minutes, then gradient diluted 3-fold with complete medium and added to cells seeded 1 day prior (600 cells / well). The mixture was incubated in a 5% CO2 incubator at 37°C for 6 days. After incubation is complete, CellTiter-Glo (Promega, Cat#G7570) is added and incubated at room temperature in the dark for 10 minutes. Chemiluminescence is read using PerkinElmer ENVISION and EC 50 The values ​​and Emax values ​​(readings relative to the antibody-free group) were calculated.

[0336] Experimental results: As is clear from Table 5-1 and Figure 5A, different anti-HER3 / MET bispecific antibodies (same anti-HER3 antibody sequence, different anti-MET antibody sequence) exhibited different killing activities against HER3 / MET bipositive cells. Of these, A13-A showed the strongest killing activity, and P3-A showed the weakest. Therefore, 2232-01 (A13-A) and 2232-02 (S3-A) were selected to proceed to the next step of screening, where the antitumor activity of bispecific antibody ADCs with different MET affinity was explored.

[0337] As is clear from the cell activity detection results in Tables 5-2 and 5-3, and Figures 5B and 5C, the killing activity of the anti-HER3 / MET bispecific antibody αHFc-CL-MMAE toxin antibody was similar to that of antibody A in HER3 monopositive cells. In MET monopositive cells, the killing activity of αHFc-CL-MMAE toxin antibody 2232-01 was superior to that of antibody 2232-02.

[0338] As is clear from the cell activity detection results in Table 5-4 and Figure 5D, the killing activity of the αHFc-CL-MMAE toxin antibody, an anti-HER3 / MET bispecific antibody, was stronger in bipositive cells than that of antibody S3 or antibody A and U3-1402 Ab, indicating that the endocytosis activity of the bispecific antibody is stronger in bipositive cells.

[0339] [Table 9]

[0340] [Table 10]

[0341] [Table 11]

[0342] [Table 12]

[0343] 2. Zenon TM pHrodo TM iFL IgG Rating Zenon TM pHrodo TM The iFL IgG indicator reagent (Invitrogen, Cat#Z25612) provided a rapid, real-time, and reliable method for evaluating antibody internalization. pHrodo iFL Red-labeled Fab fragments could bind to the Fc portion of complete IgG antibodies, forming a labeled complex within 5 minutes. After the complex was taken up by cells via endocytosis, fluorescence rapidly increased with increasing acidity in the surrounding environment. The fluorescence intensity could be recorded in real time using an Incucyte instrument, thereby determining the degree of antibody internalization.

[0344] Experimental Method: 5000 HCC827 osimertinib-resistant cells per well were pre-seed overnight in 50 μL / well of RPMI 1640 medium containing 10% fetal bovine serum. The following day, 50 μL of pre-incubated antibody and labeled complex was added to each well. The plates were placed in an IncuCyte instrument (IncuCyte S3 Live-Cell Analysis System, Sartorius), and the plate reading conditions and time intervals were set according to the instrument's operating steps. After plate reading was complete, the data were analyzed according to the analysis software steps provided with the instrument.

[0345] As is clear from the results in Figure 6, the endocytosis activity of the anti-HER3 / MET bispecific antibody was significantly superior to that of the monoclonal antibody, indicating that more bispecific antibody molecules were taken up by cells via endocytosis.

[0346] 3. FACS Endocytosis Assessment The antibodies underwent endocytosis at 37°C, and by comparing this with antibodies incubated at 4°C, the endocytosis rate of the antibodies at each time point was obtained, allowing for the evaluation of the antibody's endocytic activity.

[0347] Experimental method: The HCC827 osimertinib-resistant cell line (lung cancer cell line) was constructed by constructing HER3 / MET double-positive cells within it and obtaining osimertinib gradient resistance induction using HCC827 cells. The cell medium was RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. The HCC827 osimertinib-resistant cells were washed twice with the experimental medium and 1 × 10⁶ cells were obtained. 5 Cells were inoculated into 96-well U-bottom plates at a rate of cells / well. At each time point, one 96-well U-bottom plate containing the 0h point was used (incubated at 4°C). Samples awaiting saturation concentration measurement were added, and the cells were incubated at 4°C for 1 hour. After incubation, the cells were washed twice with experimental medium, and the samples were incubated at 37°C and 4°C respectively. The samples were removed and detected at each time point. Then, Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added, and after two washes, the fluorescence signal value was read by flow cytometry. Endocytosis rate calculation: (Antibody incubation at 4°C MFI - Antibody incubation at 37°C MFI) - Antibody incubation at 37°C MFI.

[0348] As is clear from the results in Table 6 and Figure 7, the endocytosis rate of anti-HER3 antibody A was superior to that of anti-MET antibody A13, and the endocytosis rate of anti-HER3 / MET bispecific antibody 2203-01 was superior to that of both anti-HER3 antibody A and anti-MET antibody A13.

[0349] [Table 13]

[0350] Example 5. Detection of activation activity by ERK phosphorylation HER3 / MET dimerization triggered 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 detect whether the antibodies disclosed herein possess agonist activity, they were evaluated with ERK phosphate.

[0351] Experimental method: HCC827 osimertinib-resistant cells were placed in a 96-well flat-bottom plate in 4 × 10⁶ wells. 4 Cells were seeded overnight in one well, and the cell medium was RPMI 1640 medium containing 10% fetal bovine serum. The following day, the medium was changed to low-serum medium (50 μL / well), and the cell medium was RPMI 1640 medium containing 0.05% fetal bovine serum, and the cells were starved for 5 hours. Anti-HER3 / MET bispecific antibody and control antibody were diluted in serum-free medium and added to a 96-well plate at 50 μL / well, and incubated at 37°C for 5 minutes. After incubation, ERK phosphorylation levels in the tumor were detected using Advanced Phospho-ERK1 / 29 (THR202 / TYR204) Kits (PerkinElmer, Cat#64ERKPEG) rapidly.

[0352] Experimental results: As is clear from Figure 8, anti-HER3 antibody A did not induce ERK phosphorylation, and anti-HER3 / MET bispecific antibody 2232-01 clearly did not induce ERK phosphorylation. This indicates that anti-HER3 / MET bispecific antibodies do not induce activation of downstream signaling pathways through HER3 / MET target dimerization. In other words, the bispecific antibody 2232-01 of this disclosure did not induce tumor cell proliferation or survival.

[0353] Example 6. Design and preparation of anti-HER3 / MET antibody-drug conjugates (ADCs) 1. Preparation of the compound 1.1 Preparation of Compound 9A and Compound 9B 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]indolidino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide 9-A 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]indolidino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide 9-B [ka]

[0354] Step 1 2-Cyclopropyl-2-hydroxyacetate benzyl 9a 2a (1.3 g, 11.2 mmol, prepared by the method disclosed in patent application "WO2013 / 106717") was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol), and ammonium tetrabutyliodide (413 mg, 1.1 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 48 hours, filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate (10 mL), the filtrates were combined and 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, yield: 86.9%).

[0355] Step 2 10-Cyclopropyl-1-(9H-Fluoren-9-yl)-3,6-Dioxo-2,9-Dioxa-4,7-Diazoundecane-11-ate benzyl 9b 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol, prepared by the method disclosed in patent application "CN105829346A") were added to a reaction flask, 4 mL of tetrahydrofuran was added, the mixture was purged three times with argon gas, the temperature was lowered to 0-5°C in an ice bath, potassium tert-butoxide (109 mg, 0.98 mmol) was added, the ice bath was removed, the temperature was raised 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), and the organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and 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 using silica gel column chromatography with solvent system C to obtain the title product 9b (48 mg, yield: 19%).

[0356] MS m / z (ESI): 515.0 [M+1].

[0357] Step 3 10-Cyclopropyl-1-(9H-Fluoren-9-yl)-3,6-Dioxo-2,9-Dioxa-4,7-Diazoundecane-11-acid 9c 9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V=2:1), palladium carbon (12 mg, 10% content, dry form) was added, and the mixture was purged three times with hydrogen gas. The reaction was carried out at room temperature for 1 hour with stirring. The reaction mixture was filtered through diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product, the title product 9c (13 mg). The product was then carried out in the next step without purification.

[0358] MS m / z (ESI): 424.9 [M+1].

[0359] Step 4 (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]indolidino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d Add 1b (10 mg, 18.8 μmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon gas, cool to 0-5°C in an ice bath, add 1 drop of triethylamine, add crude product 9c (13 mg, 30.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol), and react with stirring in an ice bath for 40 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL x 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL x 2), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The resulting residue was purified by thin-layer chromatography using solvent system B to obtain the title product 9d (19 mg, yield: 73.6%).

[0360] MS m / z (ESI): 842.1[M+1].

[0361] Step 5 2-((2-aminoacetylamino)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]indolidino[1,2-b]quinoline-1-yl)acetamide 9e 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 mixture was concentrated under reduced pressure, and the concentration under reduced pressure was repeated twice with the addition of 1 mL of toluene. 3 mL of n-hexane was added to the residue to form a slurry, and after standing, the supernatant was poured off, retaining the solid. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product, the title product 9e (17 mg), which was used directly in the next step without purification.

[0362] MS m / z (ESI): 638.0[M+18].

[0363] Step 6 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]indolidino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide 9-A 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]indolidino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide 9-B Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon gas, cooled to 0-5°C in an ice bath, 0.3 mL of 8 g (21.2 mg, 44.8 μmol, prepared by the method disclosed in patent application "EP2907824") N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol) was added, and the mixture was reacted with stirring in an ice bath for 10 minutes. The ice bath was removed, the temperature was raised to room temperature, and the mixture was stirred for 1 hour to produce compound 9. The reaction mixture was purified by high-performance liquid chromatography (isolation conditions: column: XBridge Prep C18 OBD 5μm 19×250mm, mobile phase: A-water (10 mmol NH4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).

[0364] MS m / z (ESI): 1074.4 [M+1].

[0365] Single-configuration compound 9-A (relatively short retention time): UPLC analysis: Retention time: 1.14 minutes, Purity: 85% (Column: ACQUITY UPLC BEHC18 1.7μm 2.1×50mm, Mobile phase: A-Water (5 mmol NH4OAc), B-Acetonitrile).

[0366] 1H NMR (400 MHz, DMSO-d6): δ 8.60 (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, 1H), 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).

[0367] Compound 9-B in a single configuration (comparatively long retention time): UPLC analysis: retention time: 1.16 minutes, purity: 89% (Kurarum: ACQUITY UPLC BEHC18 1.7μm 2.1×50mm, mobile phase: A-water (5mmol NH4OAc), B-アセトニトリル).

[0368] 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (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).

[0369] 1.2 Preparation of Compound L-1 [ka] Compound L-1 was synthesized by referring to the method provided in "Example 1 on page 28 of the specification of patent CN117460540A".

[0370] 1.3 Preparation of Compound 1 [ka] Compound 1 was synthesized by referring to the method provided in "Example 58 on page 163 of the specification of patent CN104755494A".

[0371] 2. Anti-HER3 / MET antibody-drug conjugate 2.1 Antibody-drug conjugates (ADCs) with the following structure were prepared and obtained. Of these, Ab in ADC-1 was 2232-01, Ab in ADC-2 was 2232-02, and Ab in ADC-5 was 2232-06. The target DAR was 6.

[0372] [ka] Preparation method: A PBS aqueous solution (pH=7.2) of antibody 2232-01 (5.0 mg / mL, 2.0 mL, 70 nmol) of ADC-1 was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 35 μL, 350 nmol, 5.0 eq) and the mixture was placed in a temperature-controlled oscillator and reacted at 37°C for 3 hours with shaking. The reaction system was then cooled to 25°C. A dimethyl sulfoxide solution of the prepared compound 9-A (1.13 mg, 1050 nmol, 15.0 eq) was added to the reaction system and the mixture was placed in a temperature-controlled oscillator and reacted at 25°C for 3 hours with shaking. 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%, which was stored refrigerated at 4°C. The target DAR was 6, and the drug load was calculated by RP-HPLC, with an actual measured DAR of 5.90. Denatured mass spectrometry detection revealed that the main peaks were LC1, LC2+1 drug, HC1+2 drug, and HC2+3 drug, as shown in Figure 9B.

[0373] ADC-2:2232-02 was coupled with compound 9-A, and ADC-2 was obtained by referring to the preparation method for ADC-1.

[0374] ADC-5:2232-06 was coupled with compound 9-A, and ADC-5 was obtained by following the preparation method for ADC-1. Natural mass spectrometry detection, as shown in Figure 9C, revealed an average DAR value of 5.87, of which DAR6 accounted for 94.9%, DAR2 and DAR4 accounted for 1.3% and 3.8%, respectively, and DAR8 was not detected. This structure of anti-HER3 / MET antibody-drug conjugate (ADC) showed a high proportion of DAR6 and a low proportion of other DAR values, demonstrating a high degree of uniformity.

[0375] 2.2 Antibody-drug conjugates (ADCs) with the following structure were prepared and obtained, and the Ab values ​​for ADC-6 and ADC-7 were 2232-06. The target DAR value for ADC-6 was 4, and the target DAR value for ADC-7 was 6.

[0376] [ka] Preparation method: A PBS aqueous solution (pH=7.2) of antibody 2232-06 (7.0 mg / mL, 1.0 mL, 48 nmol) of ADC-6 was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 17 μL, 173 nmol, 3.6 eq) and the mixture was placed in a temperature-controlled oscillator and reacted at 37°C for 2 hours with shaking. The reaction system was then cooled to 25°C. A dimethyl sulfoxide solution of the prepared compound L-1 (0.53 mg, 384 nmol, 8.0 eq) was added to the reaction system and the mixture was placed in a temperature-controlled oscillator and reacted at 25°C for 2 hours with shaking. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer solution: PBS 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%, and stored refrigerated at 4°C. The target DAR was 4, and the drug load was calculated by mass spectrometry, with an actual measured DAR of 3.90.

[0377] A PBS aqueous solution (pH=7.2) of antibody 2232-06 (7.0 mg / mL, 1.0 mL, 48 nmol) of ADC-7 was mixed with an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 48 μL, 480 nmol, 10 eq) and the mixture was placed in a temperature-controlled oscillator and reacted at 37°C for 2 hours with shaking. The reaction system was then cooled to 25°C. A dimethyl sulfoxide solution of the prepared compound L-1 (0.99 mg, 720 nmol, 15 eq) was added to the reaction system and reacted at 25°C for 2 hours with shaking in a temperature-controlled oscillator. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer solution: 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%, and stored refrigerated at 4°C. The target DAR was 6, and the drug load was calculated by mass spectrometry, with an actual measured DAR of 5.69.

[0378] 2.3 Antibody-drug conjugates (ADCs) with the following structure were prepared and obtained, with Ab being 2232-06 in the ADC, and the target DAR value being 6. [ka] ADC-10:2232-06 was coupled with compound 1, and the DAR6 anti-HER3 / MET antibody-drug conjugate ADC-10 was obtained by referring to the preparation method described in step 2.1 above.

[0379] Example 7. Preparation of anti-HER3 ADC or anti-MET ADC 1) Antibody-drug conjugates (ADCs) with the following structure were prepared and obtained. In anti-MET ADC-3, the Ab was antibody S3, and in anti-HER3 ADC-4, the Ab was antibody A. The target DAR value was 4.

[0380] [ka] Preparation method: Anti-MET ADC-3: An aqueous solution of antibody S3 (10.0 mg / mL, 1.0 mL, 70 nmol) was added to a PBS aqueous solution (pH=7.2) containing tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 17.5 μL, 175 nmol, 2.5 eq). The mixture was placed in a temperature-controlled oscillator and reacted at 37°C for 3 hours with shaking. The reaction system was then cooled to 25°C. A dimethyl sulfoxide solution of compound 9-A (0.60 mg, 560 nmol, 8.0 eq) was added to the reaction system and reacted at 25°C for 3 hours with shaking. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer solution: 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 load was calculated by RP-HPLC, and the measured DAR was 4.7.

[0381] Anti-HER3 ADC-4: Antibody A was coupled with compound 9-A, and ADC-4 was obtained by following the preparation method for ADC-3. The drug load was calculated by RP-HPLC, and the measured DAR was 4.0.

[0382] 2) The ADC structure of U3-1402 is shown in the figure below.

[0383] [ka] Preparation method for U3-1402: To an aqueous PBS solution (pH=7.2) containing the naked antibody of U3-1402 (U3-1402 Ab) (5.76 mg / mL, 552 nmol), an aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) (10 mM, 828 μL, 8280 nmol, 15.0 eq) was added, and the mixture was placed in a temperature-controlled oscillator and reacted with shaking for 2 hours at 37°C. The reaction system was then cooled to 25°C. A dimethyl sulfoxide solution of compound 1 (5.7 mg, 5520 nmol, 10.0 eq) was added to the above reaction system, and the mixture was placed in a temperature-controlled oscillator and reacted with shaking for 2 hours at 25°C. Finally, the reaction solution was desalted using a desalting column (HiPrep 26 / 10, GE) (buffer solution: PBS pH 7.2, flow rate 10 mL / min) to obtain PBS buffer for U3-1402 (4.18 mg / mL, 18 mL), with a yield of 94%, and was stored refrigerated at 4°C. The drug load was calculated by mass spectrometry, and a DAR of 7.88 was obtained.

[0384] Example 8. Detection of binding of anti-HER3 / MET antibody-drug conjugate to antigen 1. Detection by flow cytometry (FACS) 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.

[0385] Experimental method: Using the HCC827 osimertinib drug-resistant cell line, the cells were washed twice with experimental medium and 1 × 10⁶ cells were obtained. 5 Cells were inoculated into a 96-well U-bottom plate at a rate of one cell per well, samples of different concentrations awaiting measurement were added, and the cells were incubated at 4°C for 1 hour. After that, the cells were washed twice with experimental medium, then Alexa Fluor 647-mouse anti-human (IgG, Fcγ fragment specific) antibody (Jackson, Cat#209-605-098) was added, and after two washes, the fluorescence signal values ​​were read using a flow cytometer.

[0386] Experimental results: As is clear from the FACS detection results in Figure 10, both the anti-HER3 / MET bispecific antibody and its complex were able to bind to the cell surface HER3 / MET antigen, and their binding ability before and after coupling was considerable (overlap of dashed and solid lines in Figure 10). This indicates that toxin coupling did not affect the target binding of the antibody.

[0387] 2. Detection of surface plasmon resonance (SPR) SPR detection was performed using the BIAcore 8K (Cityva) system. The detection sensor chip protein A and related reagents were purchased from Cytiva.

[0388] Experimental method: ADC-1, ADC-2, and ADC-5 were each diluted to 1 μg / mL with HBS-EP+ buffer, and the flow rate was set to 10 μL / min to capture the antibodies at the 200 RU level. His-tagged HER3 / MET antigens were diluted in HBS-EP+ buffer at a constant ratio to create concentration gradients of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively. The flow rate was set to 30 μL / min during sample analysis. The binding time was 120 s and the dissociation time was 900 s. Next, regeneration was performed using Gly-HCl buffer at pH 1.5 as the regeneration buffer, with the regeneration flow rate set to 30 μL / min and regeneration performed for 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data was fitted using BIAcore 8K analysis software, and the binding rate constant (K) was determined using a 1:1 Langmuir binding model. a ), dissociation rate constant (K d ) and the dissociation equilibrium constant (K D Dynamical constants such as ) were determined.

[0389] Experimental results: As is clear from Table 8, the anti-HER3 / MET antibody-drug conjugate was able to bind to the human HER3 antigen and the human MET antigen.

[0390] [Table 14]

[0391] [Table 15]

[0392] Example 9. In vitro inhibition of tumor cell proliferation by anti-HER3 / MET antibody-drug conjugate. Experimental Methods: Double-positive cell lines NCI-H441, NCI-H226, and PC-9 osimertinib-resistant cell lines (internal construction, obtained by osimertinib gradient resistance induction using PC-9 cells) were cultured in RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. Cells were grown overnight in 96-well culture plates, with a yield of 600 cells / well. The following day, isovolume gradient-diluted ADC was added. After 6 days, cell viability was determined using the CellTiter-Glo luminescent cell viability measurement reagent kit (Promega, Cat#G7570) as described in the product instructions. Cell viability was evaluated as a percentage of untreated cells.

[0393] Experimental Results: As is clear from the FACS results in Figure 11A, HER3 expression was similar in the three cell lines NCI-H441, NCI-H226, and PC-9 Osimertinib-resistant cell line, with MET expression being highest in NCI-H441 and lowest in the PC-9 Osimertinib-resistant cell line. As is clear from the ADC killing results in Figures 11B and 11C, ADC-4 showed similar killing ability in the three cell lines, consistent with HER3 expression. ADC-1 had the strongest killing ability against NCI-H441 and the weakest killing ability against the PC-9 Osimertinib-resistant cell line, consistent with MET expression, indicating that the MET arm contributes to the enhancement of killing by the anti-HER3 / MET antibody-drug conjugate.

[0394] Example 10. Evaluation of anti-HER3 / MET antibody-drug conjugates in a mouse antitumor model. The calculation formula used in this disclosure was as follows:

[0395] 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 measured tumor, respectively.

[0396] Relative tumor growth rate T / C%=(T-T0) / (C-C0)×100%, tumor inhibition rate TGI%=1-T / C%.

[0397] Experimental Method: Antitumor activity was evaluated using a mouse xenograft model of HCC827 osimertinib-resistant cell lines (internal construction, obtained by osimertinib gradient drug resistance induction using HCC827 cells). 1 × 10⁶ HCC827 osimertinib-resistant cells were used. 7 The drug was inoculated subcutaneously into NCG mice (provided by Jiucui Yaokang) at a dose of 100 μL per mouse, and the tumor was approximately 120 mm in size. 3 Once the tumors grew, they were randomly divided into groups based on tumor volume and administered an anti-HER3 / MET antibody-drug conjugate. Tumor volume was measured twice a week during the administration and observation period, and the measurements were recorded.

[0398] Experimental Results: As is clear from Figure 12A, the experimental animals did not experience any significant decrease in body weight during the high and low dose administration periods, indicating that the test drug did not cause any significant toxicity or side effects in the experimental animals, demonstrating good tolerance and safety to the toxin. As is clear from Figure 12B and Table 9, the biantibody ADC showed good antitumor activity at high doses, and at equid doses, the antitumor activity of ADC-5 was superior to that of ADC-3 and ADC-4. At equitoxin administration, the antitumor activity of ADC-5 was also superior to that of ADC-3 and ADC-4.

[0399] [Table 16]

[0400] Example 11. Evaluation of anti-HER3 × MET antibody drug conjugates in a HER3 mono-positive antitumor model in mice. The antitumor activity of MDA-MB-453 was evaluated using a mouse xenograft model. 5 × 10⁶ cells of MDA-MB-453 were used. 6 Nudified mice (provided by Shanghai Bikai Keyi Co., Ltd.) were subcutaneously inoculated at a dose of 100 μL per mouse, and the tumor was approximately 120 mm in size. 3 When the tumors grew, 42 individuals were selected based on tumor volume and randomly divided into 7 groups of 6 individuals each. Each group received one of the following: solvent, ADC-1 (10 mg / kg), ADC-1 (3 mg / kg), ADC-2 (10 mg / kg), and ADC-2 (3 mg / kg). The treatment was administered once a week for two weeks. Tumor volume was measured twice a week during the administration and observation period, and the measured values ​​were recorded.

[0401] 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 measured tumor, respectively.

[0402] Relative tumor growth rate T / C%=(T-T0) / (C-C0)×100%, tumor inhibition rate TGI%=1-T / C%.

[0403] CR% (tumor complete regression ratio) = complete tumor regression (<150mm 3 ) Number of mice / Number of mice included in the group.

[0404] As is clear from Figure 13A, the experimental animals did not experience any significant decrease in body weight during the high and low dose administration periods, indicating that the test drug did not cause any significant toxicity or side effects in the experimental animals, demonstrating good tolerance and safety to toxins. As is clear from Figure 13B and Table 10, the biantibody ADC showed good antitumor activity at high doses.

[0405] [Table 17]

[0406] Example 12. In vitro inhibition of tumor cell proliferation by anti-HER3 / MET antibody-drug conjugate ADC-6. Experimental Method: The NCI-H441 double-positive cell medium was RPMI 1640 medium (ATCC modification) (Gibco, Cat#A1049101) containing 10% fetal bovine serum. Cells were grown overnight in 96-well culture plates, with a yield of 600 cells / well. The following day, isovolume gradient diluted ADC was added. After 6 days, 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 evaluated as a percentage of untreated cells.

[0407] As is clear from Figure 14, ADC-6 and ADC-7 exhibited 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 the MET comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71 or 71, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 72 or 72, 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 VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13, and VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO: 70 or 14. The CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. HER3 / MET binding molecule.

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

3. The first binding domain VH1 that specifically binds to MET includes a mutation in sequence number 1 where the 23rd position of the natural count is K, and / or a mutation where the 78th position is T, and / or the first binding domain VL1 that specifically binds to MET includes a mutation in sequence number 2 where the 69th position of the natural count is T. Preferably, the first binding domain that specifically binds to MET includes mutations at natural counts of 23K and 78T for SEQ ID NO: 1, and a mutation at natural count of 69T for SEQ ID NO:

2. The HER3 / MET binding molecule according to claim 1 or 2.

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

5. It further contains the human immunoglobulin Fc region, Preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3, or IgG4. A HER3 / MET-binding molecule according to any one of claims 1 to 4.

6. The Fc region includes a first subunit and a second subunit having 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 includes a mutation at position 366, and the second subunit includes a mutation selected from positions 366, 368, and 407, or any combination thereof. The first subunit of the Fc region includes a mutation at position 354 or 356, and the second subunit includes a mutation at position 349, or The first subunit of the Fc region includes a mutation at position 354 or 356, and the second subunit includes mutations at positions 349, 366, 368 and 407. more, The first subunit of the Fc region includes a mutation at position 366W, and the second subunit includes a mutation selected from 366S, 368A, and 407V, or any combination thereof. The first subunit of the Fc region includes a 354C or 356C mutation, and the second subunit includes a 349C mutation, or The first subunit of the Fc region contains the 354C / 366W mutation, and the second subunit contains the 349C / 366S / 368A / 407V mutation, and the mutations are numbered according to Eu. The HER3 / MET binding molecule according to claim 5.

7. further comprising a linker, preferably, the linker is (G ｍ S ｎ ) ｈ or (G ｍ Q ｎ ) ｈ or (GGNGT) ｈ or (YNGT) ｈ or (EPKSS) ｈ represented by, wherein m and n are each independently an integer selected from 1 to 8, and h is independently an integer selected from 1 to 20. A HER3 / MET-binding molecule according to any one of claims 1 to 6.

8. The first heavy chain consists of [VH1] - [linker 1] - [Obscurin-O chain] - [linker 3] - [first subunit of the Fc region] in order from the N-terminus to the C-terminus, The first light chain is [VL1]-[linker2]-[Titin-T chain] in order from the N-terminus to the C-terminus, From the N-terminus to the C-terminus, in order, the second heavy chain is [VH2]-[CH1]-[the second subunit of the Fc region], It includes a second light chain that is [VL2]-[CL] in order from the N-terminus to the C-terminus, Among them, the hyphen represents a peptide bond. The amino acid sequence of the Obscurin-O chain is shown in Sequence ID No.

63. The amino acid sequence of the titin-T chain is shown in SEQ ID NO:

64. The linkers 1, 2, and 3 may be the same or different, and may exist independently or not exist independently. Preferably, the amino acid sequences of linker 1 and linker 2 are GGGGS (SEQ ID NO: 91), and linker 3 is absent. A HER3 / MET-binding molecule according to any one of claims 1 to 7.

9. The following combinations of polypeptide chains, namely, A first heavy chain comprising an amino acid sequence represented by Sequence ID No. 77 or 57, or having at least 90% sequence identity thereto, A first light chain comprising an amino acid sequence represented by Sequence ID No. 78 or 58, or having at least 90% sequence identity thereto, A second heavy chain comprising an amino acid sequence shown in Sequence ID No. 59 or having at least 90% sequence identity thereto, A second light chain comprising an amino acid sequence represented by Sequence ID No. 79 or 60, or having at least 90% sequence identity thereto, Preferably, The aforementioned HER3 / MET binding molecule is The first heavy chain shown in Sequence ID 77, The first light chain, indicated by sequence number 78, The second heavy chain, shown in Sequence ID 59, The second light chain, as shown in Sequence ID No. 79, and, or The aforementioned HER3 / MET binding molecule is The first heavy chain shown in Sequence ID No. 57, The first light chain, indicated by Sequence ID No. 58, The second heavy chain, shown in Sequence ID 59, The second light chain, indicated by Sequence ID No. 60, A HER3 / MET-binding molecule according to any one of claims 1 to 8.

10. An antibody-drug conjugate comprising an antibody and an effector molecule, the antibody comprising 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 includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs. 15 to 17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of 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 includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs. 51 to 53, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs. 54, 55, and 75. Preferably, the heavy chain variable region in the first binding domain that specifically binds to MET includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 15-17, and the light chain variable region includes 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 includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51-53, and the light chain variable region includes LCDR1, 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 includes an amino acid sequence represented by SEQ ID NO: 71 or 71 or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 72 or 72 or having at least 90% identity thereto, and the heavy chain variable region in the second binding domain that specifically binds to HER3 includes an amino acid sequence represented by SEQ ID NO: 13 or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO: 70 or 14 or having at least 90% identity thereto. More preferably, the antibody is The first heavy chain shown in Sequence ID 77, The first light chain, indicated by sequence number 78, The second heavy chain, shown in Sequence ID 59, The second light chain, as shown in Sequence ID No. 79, and, or The aforementioned antibody is The first heavy chain shown in Sequence ID No. 57, The first light chain, indicated by Sequence ID No. 58, The second heavy chain, shown in Sequence ID 59, The second light chain, indicated by Sequence ID No. 60, Antibody-drug conjugate.

11. Effector molecules are cytotoxins, Preferably, the cytotoxin is selected from tubulin polymerization inhibitors, Topo I inhibitors, MMAEs, or derivatives thereof. More preferably, the cytotoxin is selected from MMAE or its derivatives, exatecan or its derivatives, eribulin or its derivatives, The antibody-drug conjugate drug according to claim 10.

12. Having the structure shown in formula (I), 【Chemistry 1】 Eventually, -L- is a linker unit, and it is -L １ -L ２ -L ３ -L ４ - and L １ is -(succinimido-3-yl-N)-W-C(O)-,-CH ２ -C(O)-NR ３ -W-C(O)- or -C(O)-W-C(O)-, where W is C １－８ alkyl group, C １－８ Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, and of which C １－８ Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ C(O)- or chemical bond is selected, and among them, p １ is an integer between 1 and 20, L ３ It is a peptide residue consisting of 2 to 7 amino acids, of which the amino acids are further optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ４ -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ , -C(O)NR ５ (CH ２ ) ｔ - Or selected from chemical bonds, where t is an integer from 1 to 6, R ３ , R ４ and R ５ They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. Y is -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 Selected from -C(O)- R ａ and R ｂ They are the same or different, and each is 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 heterocyclyl group, or R ａ and R ｂ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. R １ This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. R ２ This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. Or, R ａ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. m is an integer between 0 and 4. n is between 1 and 10, and n can be an integer or a decimal. Ab is an antibody as defined in claim 10. The antibody-drug conjugate according to claim 10.

13. The structure is shown by equation II, 【Chemistry 2】 Eventually, W is C １－８ alkyl group, C １－８ Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, and of which C １－８ Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ is selected from -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ )p １ C(O)- or a chemical bond, p １ is an integer from 1 to 20, L ３ It is a peptide residue consisting of 2 to 7 amino acids, of which the amino acids are further optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. R １ This is selected from a hydrogen atom, halogen, cycloalkylalkyl group, deuterated alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group. R ２ This is selected from a hydrogen atom, halogen, haloalkyl group, deuterated alkyl group, cycloalkyl group, heterocyclyl group, aryl group, or heteroaryl group. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. R ４ and R ５ They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. m is an integer between 0 and 4. n is as defined in claim 12, Ab is an antibody as defined in claim 10. The antibody-drug conjugate according to any one of claims 10 to 12.

14. The structure is represented by equation (III), 【Transformation 3】 Eventually, s １ is an integer between 2 and 8, preferably 5. R １ , R ２ , R ５ ~R ７ ,m is as defined in claim 13, n is as defined in claim 12, Ab is an antibody as defined in claim 10. The antibody-drug conjugate according to any one of claims 10 to 13.

15. -L-Y- is optional. The antibody-drug conjugate according to claim 12.

16. The following structural formula, that is, 【Chemistry 4】 Selected from, Eventually, n is between 1 and 10, and may be an integer or a decimal, preferably an integer or decimal between 1 and 6. Ab Ab is an antibody as defined in claim 10. The antibody-drug conjugate according to any one of claims 10 to 15.

17. A method for preparing an antibody-drug conjugate according to any one of claims 13 to 16, comprising a coupling reaction between Ab and a drug, Preferably, the following steps are included: 【Transformation 5】 method.

18. Having the structure shown by formula (IV), Ab-(L-De)k (IV) -L- is a linker unit that covalently bonds Ab to De, k is an integer or decimal number between 1 and 20, and preferably n is an integer or decimal number between 1 and 6. De is given by the following formula: 【Transformation 6】 Eventually, R １ａ This is selected from hydrogen, alkyl groups, cycloalkyl groups, aryl groups and heteroaryl groups. Optionally, the alkyl group, cycloalkyl group, aryl group, and heteroaryl group are each independently substituted with one or more substituents selected from alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R １ａ It is a methyl group, R １ｂ This is selected from hydrogen, alkyl groups, alkoxy groups, cycloalkyl groups, aryl groups and heteroaryl groups. Optionally, the alkyl group, cycloalkyl group, aryl group, and heteroaryl group are each independently substituted with one or more substituents selected from alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R １ｂ is hydrogen, or R １ａ and R １ｂ C ５－８ A heterocycloalkyl group is formed, and optionally the heteroalkyl group is substituted with one or more substituents from among alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, and R １ａ and R １ｂ It is not hydrogen at the same time, Ab is an antibody as defined in claim 10. The antibody-drug conjugate according to claim 10.

19. The linker unit includes a cleavable peptide portion, Preferably, the cleavable peptide portion is cleavable by an enzyme, More preferably, the enzyme is a cathepsin. The antibody-drug conjugate according to claim 18.

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

21. The linker unit includes a cleavable sulfonamide portion or a cleavable disulfide portion, Preferably, the linker unit is detachable under reducing conditions. The antibody-drug conjugate according to claim 18.

22. The linker unit includes a spacer unit coupled to De, Preferably, the spacer unit contains p-aminobenzyloxycarbonyl (PAB), The antibody-drug conjugate according to any one of claims 18 to 21.

23. It is represented by one of the following structures: 【Transformation 7】 k is selected from 1 to 10 and may be an integer or a decimal, and p1 is selected from 2, 4, 6, or 8. 【Transformation 8】 k is selected from 1 to 10 and may be an integer or a decimal; p1 is selected from 2, 4, 6, or 8; and P3 is selected from 0, 1, or 2. 【Chemistry 9】 k is selected from 1 to 10 and may be an integer or a decimal, and p1 is selected from 2, 4, 6, or 8. 【Chemistry 10】 k is selected from 1 to 10 and may be an integer or a decimal; p1 is selected from 2, 4, 6, or 8; and P3 is selected from 0, 1, or 2. 【Chemistry 11】 k is selected from 1 to 10 and may be an integer or a decimal, and p2 is selected from 2, 4, 6, or 8. 【Chemistry 12】 k is selected from 1 to 10 and may be an integer or a decimal, and p2 is selected from 2, 4, 6, or 8. 【Chemistry 13】 k is selected from 1 to 10 and may be an integer or a decimal, and p2 is selected from 2, 4, 6, or 8. 【Chemistry 14】 k is selected from 1 to 10 and may be an integer or a decimal, and p2 is selected from 2, 4, 6, or 8. 【Chemistry 15】 k is selected from 1 to 10 and may be an integer or a decimal, and p2 is selected from 2, 4, 6, or 8. 【Chemistry 16】 k is selected from 1 to 10 and may be an integer or a decimal; p1 is selected from 2, 4, 6, or 8; and P3 is selected from 0, 1, or 2. 【Chemistry 17】 k is selected from 1 to 10 and may be an integer or a decimal; p1 is selected from 2, 4, 6, or 8; and P3 is selected from 0, 1, or 2. Preferably, [Chemistry 18] k is selected from 1 to 10 and can be an integer or a decimal. The antibody-drug conjugate according to any one of claims 18 to 22.

24. An antibody-drug conjugate having the following structure, 【Chemistry 19】 n is between 1 and 10, and n can be an integer or a decimal. or 【Chemistry 20】 k is selected from 1 to 10 and can be an integer or a decimal. The Ab comprises a first binding domain that specifically binds to MET as described in claims 1 to 9, and a second binding domain that specifically binds to HER3. Antibody-drug conjugate.

25. A MET-binding molecule comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 71, and VL1 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:

72. The aforementioned CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. Preferably, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NOs: 15 to 17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NOs: 74, 19, and 20. MET binding molecule.

26. The VH1 includes a mutation in sequence number 1 where the 23rd position of the natural count is K and / or a mutation where the 78th position is T, and / or the VL1 includes a mutation in sequence number 2 where the 69th position of the natural count is T. Preferably, the MET-binding molecule includes naturally occurring E23K and S78T mutations for SEQ ID NO: 1, and a naturally occurring A69T mutation for SEQ ID NO:

2. The MET-binding molecule according to claim 25.

27. The heavy chain variable region includes an amino acid sequence shown in SEQ ID NO: 71 or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence shown in SEQ ID NO: 72 or having at least 90% identity thereto. The MET-binding molecule according to claim 25 or 26.

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 aforementioned CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. Preferably, the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as shown in SEQ ID NOs: 51 to 53, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as shown in SEQ ID NOs: 54, 55, and 73. HER3 binding molecule.

29. The heavy chain variable region includes an amino acid sequence shown in Sequence ID No. 13 or having at least 90% identity thereto, and the light chain variable region includes an amino acid sequence shown in Sequence ID No. 70 or having at least 90% identity thereto. The HER3-binding molecule according to claim 28.

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

31. A polynucleotide comprising the polynucleotide described in claim 30, vector.

32. A polynucleotide according to claim 30, or a vector according to claim 31, host cell.

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

34. A method for preparing a HER3 / MET binding molecule according to any one of claims 1 to 9, a MET binding molecule according to any one of claims 25 to 27, or a HER3 binding molecule according to claim 28 or 29, comprising: culturing a host cell according to claim 32; and expressing the HER3 / MET binding molecule according to any one of claims 1 to 9, a MET binding molecule according to any one of claims 25 to 27, or a HER3 binding molecule according to claim 28 or 29, This includes selectively isolating and / or purifying the HER3 / MET binding molecule, the MET binding molecule, or the HER3 binding molecule. Preparation method.

35. The use of a HER3 / MET binding molecule according to any one of claims 1 to 9, an antibody-drug conjugate according to any one of claims 10 to 16 and 18 to 24, an antibody-drug conjugate prepared by the method of claim 17, a MET binding molecule according to any one of claims 25 to 27, a HER3 binding molecule according to claim 28 or 29, a polynucleotide according to claim 30, or a vector according to claim 31, or a pharmaceutical composition according to claim 33, in order to treat or alleviate cancer, or to prepare a drug 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, and more preferably, HER3 and MET double-positive breast cancer or lung cancer. use.

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

37. An antibody-drug conjugate, The aforementioned Ab is an antibody comprising a first binding domain that specifically binds to MET and a second binding domain that specifically binds to HER3, wherein 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 aforementioned Ab is, The first heavy chain consists of [VH1] - [linker 1] - [Obscurin-O chain] - [linker 3] - [first subunit of the Fc region] in order from the N-terminus to the C-terminus, The first light chain is [VL1]-[linker2]-[Titin-T chain] in order from the N-terminus to the C-terminus, From the N-terminus to the C-terminus, in order, the second heavy chain is [VH2]-[CH1]-[the second subunit of the Fc region], It includes a second light chain that is [VL2]-[CL] in order from the N-terminus to the C-terminus, Among these, the hyphen (-) represents a peptide bond, and linker 1, linker 2, and linker 3 may be the same or different, and may be present or absent independently. Preferably, i) the antibody-drug conjugate is represented by general formula (I), 【Chemistry 21】 Among them, -L- is the linker unit, and that is -L １ -L ２ -L ３ -L ４ - and L １ is -(succinimido-3-yl-N)-W-C(O)-,-CH ２ -C(O)-NR ３ -W-C(O)- or -C(O)-W-C(O)-, where W is C １－８ alkyl group, C １－８ Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups of 1 to 8 atoms, wherein the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, and of which C １－８ Alkyl groups, cycloalkyl groups, and linear heteroalkyl groups are each independently and optionally further substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ２ -NR ４ (CH ２ CH ２ O)p １ CH ２ CH ２ C(O)-, -NR ４ (CH ２ CH ２ O)p １ CH ２ C(O)-, -S(CH ２ ) p １ C(O)- or chemical bond is selected, and among them, p １ is an integer between 1 and 20, L ３ It is a peptide residue consisting of 2 to 7 amino acids, of which the amino acids are further optionally substituted with one or more substituents selected from halogens, hydroxyl groups, cyano groups, amino groups, alkyl groups, chloroalkyl groups, deuterated alkyl groups, alkoxy groups, and cycloalkyl groups. L ４ -NR ５ (CR ６ R ７ ) ｔ -, -C(O)NR ５ , -C(O)NR ５ (CH ２ ) ｔ - Or selected from chemical bonds, where t is an integer from 1 to 6, R ３ , R ４ and R ５ They are the same or different, and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group. R ６ and R ７ They are the same or different, and each is independently selected from a hydrogen atom, halogen, alkyl group, haloalkyl group, deuterated alkyl group, and hydroxyalkyl group. Y is -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 Selected from -C(O)- R ａ and R ｂ They are the same or different, and each is 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 heterocyclyl group, or R ａ and R ｂ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. R １ This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. R ２ This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, or heteroaryl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. Or, R ａ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocycline group. m is an integer between 0 and 4. Or, ii) The antibody-drug conjugate has a structure represented by the general formula Ab-(L-De)k, L is a linker that covalently bonds Ab to De, De is shown by equation (III), 【Chemistry 22】 Eventually, R １ａ This is selected from hydrogen, alkyl groups, cycloalkyl groups, aryl groups and heteroaryl groups. Optionally, the alkyl group, cycloalkyl group, aryl group, and heteroaryl group are each independently substituted with one or more substituents selected from alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R １ａ It is a methyl group, R １ｂ This is selected from hydrogen, alkyl groups, alkoxy groups, cycloalkyl groups, aryl groups and heteroaryl groups. Optionally, the alkyl group, cycloalkyl group, aryl group, and heteroaryl group are each independently substituted with one or more substituents selected from alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, preferably R １ｂ is hydrogen, or R １ａ and R １ｂ C ５－８ A heterocycloalkyl group is formed, and optionally the heteroalkyl group is substituted with one or more substituents from among alkyl groups, alkoxy groups, halogens, deuterium, amino groups, cyano groups, nitro groups, hydroxyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, and R １ａ and R １ｂ It is not hydrogen at the same time. Antibody-drug conjugate.

Images