Anti-EGFR×cmet bispecific antibody-drug conjugate and use thereof

Abstract

A bispecific antibody-drug conjugate targeting tumor-associated antigens EGFR and cMet, a preparation method therefor, a corresponding pharmaceutical composition, and a use of the antibody-drug conjugate in tumor treatment. The anti-EGFR×cMet bispecific antibody-drug conjugate targets tumor cells by specifically recognizing EGFR and cMet, and kills tumor cells more effectively by means of mechanisms such as toxin killing, bystander effects, signal pathway inhibition, and receptor degradation.

Description

Drug conjugates of anti-EGFR×cMet bispecific antibodies and their uses

[0001] This disclosure claims priority to Chinese Patent Application No. 202410701939.4, filed on May 31, 2024, entitled "Drug Conjugate of Anti-EGFR×cMet Bispecific Antibody and Its Use Thereof", and Chinese Patent Application No. 202510295625.3, filed on March 12, 2025, entitled "Drug Conjugate of Anti-EGFR×cMet Bispecific Antibody and Its Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedicine, and more specifically, to a class of bispecific antibodies or antigen-binding fragments capable of specifically binding to EGFR and cMet, the corresponding antibody-drug conjugates, their preparation methods, pharmaceutical compositions, and their use as antitumor drugs. Background Technology

[0003] Epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) tyrosine kinase family. EGFR overexpression is primarily found in gastric cancer, renal cell carcinoma, glial cell carcinoma, and thymic carcinoma. Many targeted therapies against EGFR have been approved; however, intrinsic alterations in the signaling pathway and acquired resistance mechanisms have led to clinical limitations, and these therapies have not yet met the needs of cancer patients.

[0004] Mesenchymal-epidermal transition factor (cMet) is a tyrosine kinase receptor, and hepatocyte growth factor (HGF) is the only known cMet ligand. Aberrant activation of cMet is frequently associated with tumor drug resistance, metastasis, and invasion. Furthermore, cMet overexpression is negatively correlated with patient prognosis and survival. Aberrant activation of cMet can occur through HGF-independent mechanisms, such as mutations in the Met gene, gene amplification, and upregulation at the transcriptional level.

[0005] The combined use of EGFR- and cMet-targeted drugs can demonstrate clinical activity, but it also faces relatively serious adverse reactions. Clinically, EGFR-related adverse reactions are mainly skin rashes, while cMet-related adverse reactions are mainly peripheral edema and hypoalbuminemia.

[0006] Antibody-drug conjugates (ADCs), as novel targeted therapies, combine the tumor-targeting properties of antibodies with the highly effective killing effects of toxins by linking monoclonal antibodies that specifically bind to tumor cell surface antigens to biologically active toxin molecules. This approach avoids the drawbacks of lower efficacy associated with the former and excessive toxicity and poor drug-likeness associated with the latter. Therefore, the development of EGFR×cMet bispecific antibody ADCs has significant potential clinical demand. Summary of the Invention

[0007] In the development of antibody-drug conjugates (ADCs), the characteristics of each component are crucial to their targeted therapeutic effect, and the selection of each component can also affect the safety and efficacy of the ADC. For bispecific antibody ADCs, the structural and sequence characteristics of the bispecific antibody molecules, such as the affinity of the binding arm, antibody molecular weight, frame region size, antibody backbone characteristics, and charge composition, all influence the tumor cell targeting, target-mediated endocytosis, efficacy, and toxicity of the ADC molecule.

[0008] Through extensive experimentation and screening, the applicant discovered a bispecific antibody or antigen-binding fragment, constructed with a specific format and sequence, capable of specifically binding to EGFR and cMet, as described in this disclosure, which is particularly suitable for the preparation of ADC drugs.

[0009] This disclosure further provides nucleic acids, vectors, and cells encoding bispecific antibodies or antigen-binding fragments that specifically bind to EGFR and cMet; antibody-drug conjugates based on the antibodies; pharmaceutical compositions containing the antibodies or antigen-binding fragments, or antibody-drug conjugates based on the antibodies; and related uses of the pharmaceutical compositions in the treatment of tumors.

[0010] In a first aspect, this disclosure provides a bispecific antibody or antigen-binding fragment capable of specifically binding EGFR and cMet, said antibody or antigen-binding fragment comprising a first antigen-binding domain specifically binding EGFR and a second antigen-binding domain specifically binding cMet.

[0011] In some specific embodiments, the first antigen-binding domain that specifically binds to EGFR includes the heavy chain variable region VH. EGFR and / or light chain variable region VL EGFR The second antigen-binding domain that specifically binds to cMet includes the heavy chain variable region VH. cMet and / or light chain variable region VL cMet .

[0012] In some specific embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region. Optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment includes CH1, a hinge region, CH2, CH3, CL, or Fc region. Optionally, the heavy chain constant region is selected from the human or mouse IgG1, IgG2, IgG3, or IgG4 constant region, and the light chain constant region is selected from the human or mouse kappa constant region or lambda constant region.

[0013] In some specific embodiments, the antibody or antigen-binding fragment comprises a first peptide chain, a second peptide chain, and a third peptide chain, wherein the first peptide chain comprises VL cMet -CL, the second peptide chain contains VH cMet -CH1-Fc, the third peptide chain contains a single-chain unit VL EGFR -VH EGFR -Fc.

[0014] In some specific embodiments, the VL in the single-chain unit of the third peptide chain EGFR and VH EGFR It may also contain a cysteine ​​mutation to introduce a disulfide bond, preferably, the cysteine ​​mutation is VL. EGFR The 100th amino acid is mutated from G to C, and / or the cysteine ​​is mutated to VH. EGFR The 44th amino acid is mutated from G to C, thereby introducing a disulfide bond.

[0015] In some specific embodiments, the antibody or antigen-binding fragment comprises a first peptide chain, a second peptide chain, and a third peptide chain, wherein the first peptide chain comprises VL EGFR -CL, the second peptide chain contains VH EGFR -CH1-Fc, the third peptide chain contains a single-chain unit VL cMet -VH cMet -Fc.

[0016] In some specific embodiments, the VL in the single-chain unit of the third peptide chain cMet and VH cMet It may also contain a cysteine ​​mutation to introduce a disulfide bond, preferably, the cysteine ​​mutation is VL. cMet The 100th amino acid is mutated from G to C, and / or the cysteine ​​is mutated to VH. cMet The 44th amino acid is mutated from G to C, thereby introducing a disulfide bond.

[0017] In some specific embodiments, the antibody or antigen-binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise VL cMet -VL EGFR -CL, the second peptide chain and the third peptide chain respectively contain VH cMet -VH EGFR -CH1-Fc.

[0018] In some specific embodiments, the antibody or antigen-binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise VL cMet -CL, the second peptide chain contains VH cMet -CH1-Fc, the third peptide chain contains VH cMet -CH1-Fc-VL EGFR -VH EGFR .

[0019] In some specific embodiments, the antibody or antigen-binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise VL cMet -CL, the second peptide chain contains VH cMet -CH1-Fc, the third peptide chain contains VH cMet -CH1-VL EGFR -VH EGFR -Fc.

[0020] In some specific embodiments, the antibody or antigen-binding fragment comprises a "protrusion chain" and a "cave chain";

[0021] In some preferred embodiments, the antibody or antigen-binding fragment comprises a T366W mutation in the CH3 domain of the "protrusion chain" and a T366S, L368A, or / and Y407V mutation in the CH3 domain of the "cave chain".

[0022] In some specific implementations, the VH EGFR Includes HCDR1 EGFR HCDR2 EGFR and HCDR3 EGFR , and / or the VL EGFR Includes LCDR1 EGFR LCDR2 EGFR and LCDR3 EGFR .

[0023] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFRHCDR1-HCDR3, respectively, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:9, and / or the LCDR1. EGFR -LCDR3 EGFR LCDR1-LCDR3 are respectively SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8.

[0024] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:38-40 respectively, and / or the LCDR1 EGFR -LCDR3 EGFR As shown in SEQ ID NO:55-57 respectively.

[0025] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:41-43 respectively, and / or the LCDR1 EGFR -LCDR3 EGFR As shown in SEQ ID NO:58-59 and 57 respectively.

[0026] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:44-46 respectively, and / or the LCDR1 EGFR -LCDR3 EGFR As shown in SEQ ID NO:55-57 respectively.

[0027] In some specific implementations, the VH cMet Includes HCDR1 cMet HCDR2 cMet and HCDR3 cMet , and / or the VL cMet Includes LCDR1 cMet LCDR2 cMet and LCDR3 cMet .

[0028] In some specific implementations, the HCDR1 cMet -HCDR3 cMet HCDR1-HCDR3 of SEQ ID NO:13, and / or LCDR1, respectively. cMet -LCDR3 cMetThese are LCDR1-LCDR3, which are SEQ ID NO:12 respectively.

[0029] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:47-49 respectively, and / or the LCDR1 cMet -LCDR3 cMet As shown in SEQ ID NO:60-62 respectively.

[0030] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:50-52 respectively, and / or the LCDR1 cMet -LCDR3 cMet As shown in SEQ ID NO:63-64 and 62 respectively.

[0031] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:53-54, 49 respectively, and / or the LCDR1 cMet -LCDR3 cMet As shown in SEQ ID NO:60-62 respectively.

[0032] In some specific implementations, the VH EGFR Contains an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:9, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or the VL EGFR It contains an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0033] In some specific embodiments, the VH cMet Contains an amino acid sequence as shown in SEQ ID NO. 13, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or the VL cMet It contains an amino acid sequence as shown in SEQ ID NO:12, or a sequence that has at least 70% identity or up to 15 mutations compared to it.

[0034] In some specific embodiments, the Fc region has an amino acid sequence as shown in SEQ ID NO:18 and / or SEQ ID NO:19, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0035] In some specific embodiments, the CL region has an amino acid sequence as shown in SEQ ID NO:20.

[0036] In some specific embodiments, the CH1 region has an amino acid sequence as shown in SEQ ID NO:21.

[0037] In some specific embodiments, the antibody or antigen-binding fragment comprises a heavy chain HC and a light chain LC, wherein,

[0038] The heavy chain HC has an amino acid sequence as shown in SEQ ID NO:23, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0039] The light chain LC has an amino acid sequence as shown in SEQ ID NO:22, or a sequence that has at least 70% identity or up to 15 mutations compared to it.

[0040] In some specific embodiments, the antibody or antigen-binding fragment comprises a first heavy chain HC-1, a second heavy chain HC-2, and a light chain LC, wherein,

[0041] The first heavy chain HC-1 has an amino acid sequence as shown in SEQ ID NO:25 or SEQ ID NO:30, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0042] The second heavy chain HC-2 has an amino acid sequence as shown in any of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:31, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0043] The light chain LC has an amino acid sequence as shown in SEQ ID NO:24 or SEQ ID NO:29, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0044] In some implementations, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 15 mutations is at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations.

[0045] In some implementations, the mutation is an insertion, deletion, or substitution, the substitution is preferably a conserved amino acid substitution, and the mutation is preferably a reversion mutation or a hotspot mutation.

[0046] In some specific embodiments, the antibody or antigen-binding fragments described in this disclosure include:

[0047] (1) a chimeric antibody or a fragment thereof; and / or

[0048] (2) Humanized antibodies or fragments thereof; and / or,

[0049] (3) Fully human antibodies or fragments thereof;

[0050] Preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0051] In some specific embodiments, the antigen-binding fragments described herein are selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

[0052] In a second aspect, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD)n.

[0053] in,

[0054] D is a cytotoxic drug;

[0055] L represents the connecting subunit;

[0056] Pc is a bispecific antibody or its antigen-binding fragment that specifically binds to EGFR and cMet;

[0057] Furthermore, n is a real number from 1 to 16.

[0058] In some specific embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or its pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment Pc is the antibody or antigen-binding fragment described in the first aspect of this disclosure.

[0059] In some specific embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or its pharmaceutically acceptable salt thereof, wherein the cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics.

[0060] In some specific implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt thereof, wherein the cytotoxic drug D is selected from tubulin inhibitors, DNA damaging agents, or DNA topoisomerase inhibitors, wherein the tubulin inhibitors include, but are not limited to, dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins, wherein the DNA damaging agents include, but are not limited to, PBD drugs, and wherein the DNA topoisomerase inhibitors include, but are not limited to, camptothecin drugs.

[0061] In some specific embodiments, the cytotoxic drug D is selected from auristatin drugs or DNA topoisomerase inhibitors.

[0062] In some specific embodiments, the cytotoxic drug D is selected from MMAE (monomethyl auristatin E) or a compound represented by the following formula (DI).

[0063] in,

[0064] R 1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more D atoms;

[0065] R 4 Selected from H or C1-C3 alkyl groups;

[0066] R 5 Selected from H, halogens, CN, OH, NH2, or C1-C3 alkyl groups;

[0067] R 6 Selected from H or C1-C3 alkyl groups;

[0068] R7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

[0069] In some specific implementations, the R 1 R 2 The atoms connected to them together form

[0070] In some specific implementations, R 4 Selected from H.

[0071] In some specific implementations, R 5 It is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl.

[0072] In some specific implementations, R 5 Selected from H.

[0073] In some specific implementations, R 6 Selected from H.

[0074] In some specific implementations, R 7 Selected from cyclopropyl.

[0075] In some specific embodiments, the compound represented by formula (DI) is selected from one of the following compounds:

[0076] In some specific embodiments, the cytotoxic drug D is selected from compounds represented by formula (DI) as defined above.

[0077] In some specific embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or its pharmaceutically acceptable salt, wherein the linker unit L is selected from... Its a-terminus is covalently linked to the aforementioned bispecific antibody that specifically binds to EGFR and cMet, or its antigen-binding fragment Pc, and its b-terminus is covalently linked to the cytotoxic drug D, wherein:

[0078] Ring A is selected from R b1 R b2 Each is independently selected from H, halogen, CN, C1-C6 alkyl or C3-C6 cycloalkyl, or R b1 R b2The carbon atoms connected to them together form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, which is optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic group;

[0079] m1 is selected from integers 2 to 8;

[0080] L a Selected from chemical bonds or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl groups, m2 is selected from integers 1 to 8, R b4 R b5 Each is independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl,

[0081] L b The peptide residues are selected from 1 to 8 amino acids, and the peptide residues are optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic groups.

[0082] In some specific implementations, ring A is selected from... R b1 R b2 All are selected from H or R b1 R b2 The carbon atoms connected to them together form a 4-7 membered heterocyclic group, which is optionally substituted by one or more substituents among halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclic group.

[0083] In some specific implementations, ring A is selected from... R b1 R b2 All are selected from H or R b1 R b2 The carbon atoms connected to them together form 4-7 membered heterocyclic groups.

[0084] In some specific implementations, ring A is selected from...

[0085] In some specific implementations, L a Selected from chemical bonds or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl, R b4 R b5 One is selected from H, and the other is selected from

[0086] In some specific implementations, L a Selected from chemical bonds or Its * end and L b connect.

[0087] In some specific implementations, the L b It consists of Gly-Gly-Phe-Gly tetrapeptide residues.

[0088] In some specific implementations, m1 is selected from integers 2 to 8.

[0089] In some specific implementations, m1 is an integer from 2 to 6.

[0090] In some specific implementations, m1 is 2, 3, 4 or 5.

[0091] In some specific implementations, the connecting subunit L is

[0092] Its a-terminus is covalently linked to the aforementioned bispecific antibody that specifically binds to EGFR and cMet or its antigen-binding fragment Pc, and its b-terminus is covalently linked to the cytotoxic drug D.

[0093] In some specific embodiments, the aforementioned antibody-drug conjugate or its pharmaceutically acceptable salt, wherein n is selected from real numbers from 1 to 16, for example, n is selected from real numbers from 2 to 12, for example, n is selected from real numbers from 4 to 10, for example, n is selected from real numbers from 3 to 9, for example, n is selected from real numbers from 4 to 8, for example, n is selected from real numbers from 6 to 8.

[0094] In some specific implementations, n is selected from real numbers from 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 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, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0095] In some specific implementations, n is selected from real numbers from 4 to 8, for example, n is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 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, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0096] In some specific implementations, n is selected from real numbers from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0097] In some specific embodiments, the antibody-drug conjugates or pharmaceutically acceptable salts thereof disclosed herein are selected from the following compounds or pharmaceutically acceptable salts thereof:

[0098] Where Pc and n are as defined in any of the preceding definitions.

[0099] In a third aspect, this disclosure provides an isolated nucleic acid molecule that encodes a bispecific antibody or antigen-binding fragment that specifically binds EGFR and cMet as described in this disclosure.

[0100] In some specific embodiments, the antibody or antigen-binding fragment includes a first antigen-binding domain that specifically binds to EGFR and a second antigen-binding domain that specifically binds to cMet, wherein the first antigen-binding domain that specifically binds to EGFR includes the heavy chain variable region VH. EGFR and / or light chain variable region VLEGFR The second antigen-binding domain that specifically binds to cMet includes the heavy chain variable region VH. cMet and / or light chain variable region VL cMet .

[0101] In some specific implementations, the VH EGFR Includes HCDR1 EGFR HCDR2 EGFR and HCDR3 EGFR , and / or the VL EGFR Includes LCDR1 EGFR LCDR2 EGFR and LCDR3 EGFR .

[0102] In some specific implementations, the VH EGFR Includes HCDR1 EGFR HCDR2 EGFR and HCDR3 EGFR , and / or the VL EGFR Includes LCDR1 EGFR LCDR2 EGFR and LCDR3 EGFR .

[0103] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR HCDR1-HCDR3, respectively, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:9, and / or the LCDR1. EGFR -LCDR3 EGFR LCDR1-LCDR3 are respectively SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8.

[0104] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:38-40 respectively, and / or the LCDR1 EGFR -LCDR3 EGFR As shown in SEQ ID NO:55-57 respectively.

[0105] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:41-43 respectively, and / or the LCDR1 EGFR -LCDR3 EGFRAs shown in SEQ ID NO:58-59 and 57 respectively.

[0106] In some specific implementations, the HCDR1 EGFR -HCDR3 EGFR As shown in SEQ ID NO:44-46 respectively, and / or the LCDR1 EGFR -LCDR3 EGFR As shown in SEQ ID NO:55-57 respectively.

[0107] In some specific implementations, the VH cMet Includes HCDR1 cMet HCDR2 cMet and HCDR3 cMet , and / or the VL cMet Includes LCDR1 cMet LCDR2 cMet and LCDR3 cMet .

[0108] In some specific implementations, the HCDR1 cMet -HCDR3 cMet HCDR1-HCDR3 of SEQ ID NO:13, and / or LCDR1, respectively. cMet -LCDR3 cMet LCDR1-3 are SEQ ID NO:12, respectively.

[0109] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:47-49 respectively, and / or the LCDR1 cMet -LCDR3 cMet As shown in SEQ ID NO:60-62 respectively.

[0110] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:50-52 respectively, and / or the LCDR1 cMet -LCDR3 cMet As shown in SEQ ID NO:63-64 and 62 respectively.

[0111] In some specific implementations, the HCDR1 cMet -HCDR3 cMet As shown in SEQ ID NO:53-54, 49 respectively, and / or the LCDR1 cMet-LCDR3 cMet As shown in SEQ ID NO:60-62 respectively.

[0112] In some specific implementations, the VH EGFR Contains an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:9, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or the VL EGFR It contains an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:8, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0113] In some specific implementations, the VH cMet Contains an amino acid sequence as shown in SEQ ID NO:13, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or the VL cMet It contains an amino acid sequence as shown in SEQ ID NO:12, or a sequence that has at least 70% identity or up to 15 mutations compared to it.

[0114] In some specific embodiments, the Fc region has an amino acid sequence as shown in SEQ ID NO:18 and / or SEQ ID NO:19, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0115] In some specific embodiments, the CL region has an amino acid sequence as shown in SEQ ID NO:20.

[0116] In some specific embodiments, the CH1 region has an amino acid sequence as shown in SEQ ID NO:21.

[0117] In some specific embodiments, the antibody or antigen-binding fragment comprises a heavy chain HC and a light chain LC, wherein,

[0118] The heavy chain HC has an amino acid sequence as shown in SEQ ID NO:23, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0119] The light chain LC has an amino acid sequence as shown in SEQ ID NO:22, or a sequence that has at least 70% identity or up to 15 mutations compared to it.

[0120] In some specific embodiments, the antibody or antigen-binding fragment comprises a first heavy chain HC-1, a second heavy chain HC-2, and a light chain LC, wherein,

[0121] The first heavy chain HC-1 has an amino acid sequence as shown in SEQ ID NO:25 or SEQ ID NO:30, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0122] The second heavy chain HC-2 has an amino acid sequence as shown in any of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:31, or a sequence having at least 70% identity or up to 15 mutations compared to it; and / or

[0123] The light chain LC has an amino acid sequence as shown in SEQ ID NO:24 or SEQ ID NO:29, or a sequence having at least 70% identity or up to 15 mutations compared to it.

[0124] In some implementations, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 15 mutations is at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations.

[0125] In some implementations, the mutation is an insertion, deletion, or substitution, the substitution is preferably a conserved amino acid substitution, and the mutation is preferably a reversion mutation or a hotspot mutation.

[0126] In some specific embodiments, the antibody or antigen-binding fragment described in this disclosure is:

[0127] (1) Chimeric antibodies or fragments thereof;

[0128] (2) Humanized antibodies or fragments thereof; and / or,

[0129] (3) Fully human antibodies or fragments thereof;

[0130] Preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0131] In some specific embodiments, the antigen-binding fragments described herein are selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

[0132] In a fourth aspect, this disclosure provides an expression vector comprising the isolated nucleic acid molecules described in the third aspect of this disclosure.

[0133] In a fifth aspect, this disclosure provides a host cell comprising the isolated nucleic acid molecules described in the third aspect of this disclosure, and / or the expression vector described in the fourth part.

[0134] In some specific embodiments, the host cell is a eukaryotic cell or a prokaryotic cell.

[0135] Preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0136] In a sixth aspect, this disclosure provides a pharmaceutical composition comprising a bispecific antibody or antigen-binding fragment that specifically binds EGFR and cMet as described in this disclosure, or an antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0137] In a seventh aspect, this disclosure provides a method for treating cancer or tumors in mammals, comprising administering to a mammal, preferably a human, a therapeutically effective amount of the aforementioned bispecific antibody or antigen-binding fragment that specifically binds to EGFR and cMet, or an antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a corresponding pharmaceutical composition.

[0138] In an eighth aspect, this disclosure provides the use of the aforementioned bispecific antibody or antigen-binding fragment that specifically binds to EGFR and cMet, or antibody-drug conjugates of the general formula Pc-(LD)n or pharmaceutically acceptable salts thereof, or corresponding pharmaceutical compositions, in the preparation of medicaments for treating cancer or tumors.

[0139] In a ninth aspect, this disclosure provides a bispecific antibody or antigen-binding fragment that specifically binds to EGFR and cMet, or an antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a corresponding pharmaceutical composition, for the treatment of cancer or tumors.

[0140] In a tenth aspect, this disclosure provides the aforementioned bispecific antibody or antigen-binding fragment that specifically binds EGFR and cMet for the treatment of cancer or tumors, or an antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a corresponding pharmaceutical composition.

[0141] In some specific embodiments of aspects seven to ten above, the cancer or tumor includes solid tumors.

[0142] In some specific embodiments, the cancer or tumor includes one or more of the following: lung cancer, pancreatic cancer, liver cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, stomach cancer, esophageal cancer, nasopharyngeal carcinoma, kidney cancer, cervical cancer, prostate cancer, uterine cancer, melanoma, squamous cell carcinoma of the head and neck, bile duct cancer, thyroid cancer, ovarian cancer, or glioblastoma.

[0143] Terminology Definitions and Explanations

[0144] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0145] Furthermore, unless otherwise stated herein, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0146] The terms “comprising,” “including,” and “having” are used interchangeably in this document to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” in this document also provides for schemes “consisting of…”.

[0147] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0148] The term “and / or” as used herein includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0149] The term "c-Met" used in this article, also known as "Met" or "HGFR," is a member of the tyrosine kinase receptor superfamily. The ligand for c-Met is hepatocyte growth factor (HGF). "c-Met" in this article includes mature or immature full-length wild-type c-Met proteins or their mutants (e.g., point mutations, insertion mutations, or deletion mutations), splice variants, orthologs, and fragments of the aforementioned c-Met. "c-Met" in this article can be derived from humans, primates such as monkeys (e.g., cynomolgus monkeys, rhesus monkeys), and rodents (e.g., mice and rats). For example, the amino acid sequence of human c-Met can be found in UniProt: P08581, and the amino acid sequence of green monkey c-Met can be found in UniProt: Q2IBA6.

[0150] The term "EGFR" in this article, also known as "ErbB-1" or "HER1," is a member of the epidermal growth factor receptor (HER) family. Currently, six ligands for EGFR have been identified, primarily epidermal growth factor (EGF) and transforming growth factor α (TGF-α), along with bidirectional regulatory protein (AR), β-cytokinin (BTC), heparin-binding EGF-like growth factor (HBEGF), and epidermal regulatory factor (EPR). In this article, "EGFR" includes mature or immature full-length wild-type EGFR proteins or their mutants (e.g., point mutations, insertion mutations, or deletion mutations), splice variants, orthologs, and fragments of the aforementioned EGFRs. "EGFR" in this article can be derived from humans, primates such as monkeys (e.g., cynomolgus monkeys and rhesus monkeys), and rodents (e.g., mice and rats). For example, the amino acid sequence of human EGFR can be found in UniProt: P00533, and the amino acid sequence of macaque EGFR can be found in UniProt: P55245.

[0151] The term "antibody" in this document, used in the broadest sense, refers to a polypeptide or combination of polypeptides containing sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain to specifically bind to an antigen. The term "antibody" in this document encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity. The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document and refer to structures that are substantially similar to those of natural antibodies.

[0152] The terms "antibody fragment" or "antigen-binding fragment" used herein refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, etc., regardless of its structure, which binds to the same antigen recognized by the intact antibody. "Antibody fragment" or "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen and forming a complex. For example, antibody fragments include isolated fragments consisting of light chain variable regions, "Fv" fragments consisting of heavy and light chain variable regions, recombinant single-chain polypeptide molecules (scFv) in which the light and heavy chain variable regions are linked by peptide linkers, and the smallest recognizing unit consisting of amino acid residues mimicking hypervariable regions. The terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and refer to a fragment that does not possess the full structure of a complete antibody but only contains a portion or a partial variant of the complete antibody capable of binding antigens. "Antigen-binding fragment" or "antibody fragment" includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0153] The term "antibody" in this article also includes alternative protein scaffolds or artificial scaffolds having a transplantable complementarity-determining region (CDR) or a CDR derivative. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004); the contents of which are incorporated herein by reference. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for transplanting CDRs, including but not limited to tendinins, fibronectin, peptide aptamers, etc.

[0154] Basic antibody structures include typical "quadruple-chain antibodies," which belong to immunoglobulins composed of two heavy chains (HC) and two light chains (LC). The heavy chain is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0155] The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0156] In this article, "antibody" can be derived from any animal, including but not limited to humans and non-human animals. Non-human animals can be selected from primates, mammals, rodents, and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0157] In this article, "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0158] The term "monoclonal antibody" as used herein refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that, apart from possible variants (e.g., containing naturally occurring mutations or generated during the manufacturing process of the formulation, such variants are typically present in small amounts), the individual antibodies comprising said population are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. The modifier "monoclonal" herein should not be construed as requiring the production of said antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0159] The term "natural antibody" in this article refers to antibodies produced and paired by the immune system of multicellular organisms. The term "engineered antibody" in this article refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (such as scFv), bispecific antibodies, etc.

[0160] The term "monospecific antibody" in this article refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen.

[0161] The term "multispecific antibody" in this article refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0162] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing variable domains of the heavy and light chains, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" in this paper refers to the light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. The "Fv fragment" is the smallest fragment produced by IgG and IgM, containing an intact antigen-binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as the Fab fragment; the VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0163] The term "scFv" (single-chain variable fragment) in this paper refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); the contents of which are incorporated herein by reference). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:30) amino acid sequence or a variant thereof. For example, a adapter having the amino acid sequence (GGGGS)4 (SEQ ID NO:31) may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448, the contents of which are incorporated herein by reference). Other adapters that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. (the contents of which are incorporated herein by reference). In some cases, disulfide bonds can also exist between VH and VL of scFv, forming a disulfide-bonded Fv (dsFv).

[0164] The term "nanobody" in this article refers to a naturally occurring heavy chain antibody in camels that lacks a light chain. Cloning its variable region can yield a single-domain antibody consisting only of the heavy chain variable region.

[0165] The term "single-domain antibody" in this article refers to a single-domain antibody consisting only of the heavy chain variable region obtained from the variable region of a naturally occurring heavy chain antibody lacking the light chain in a cloned camel. It is also called VHH (Variable domain of heavy chain antibody), and it is the smallest functional antigen-binding fragment.

[0166] The term "diabody" used in this paper refers to a single polypeptide chain in which the VH and VL domains are expressed, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994), the contents of which are incorporated herein by reference).

[0167] The term "chimeric antibody" in this paper refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). For example, the term "chimeric antibody" can include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0168] The term "humanized antibody" in this article refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0169] The term "fully human antibody" in this document refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" in this document does not include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0170] The term "smallest unit of antibody recognition" in this article refers to the smallest unit of antigen that an antibody can recognize in an antigen-antibody binding reaction.

[0171] The term "naked antibody" as used herein refers to an antibody that is not linked, fused to, or conjugated with another agent or molecule (e.g., a label or drug), peptide, or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells may be glycosylated by the host cell's glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are not glycosylated when expressed by host cells that do not possess their own glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0172] The term "variable region" in this article refers to the region in the antibody heavy or light chain involved in enabling antibody binding to antigens. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably in this article. They typically refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form a precise complementarity with the antigen epitope in its spatial structure. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" and "FR region" are used interchangeably, referring to the amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. A typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0173] In this paper, "CDR" may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tools and websites used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abysis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0174] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. A "heavy chain constant region" contains at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from the CH1, CH2, CH3 domains, or the Fc region.

[0175] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0176] The term "Fc" in this document refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically containing the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447.

[0177] One approach to circumventing the mismatch byproduct problem (called “knob-into-hole”) aims to modify the contact interface by introducing mutations into the CH3 domain, thereby forcing two different antibody heavy chains to pair. On one chain, a bulky amino acid is replaced with an amino acid with a shorter side chain to create a “hole”. Conversely, an amino acid with a larger side chain is introduced into the other CH3 domain to create a “protrusion”. By co-expressing these two heavy chains (and two identical light chains, which must be adapted to both heavy chains), a higher yield of heterodimer formation (“knob-hole”) relative to homodimer formation (“hole-hole” or “protrusion-protrusion”) was observed (Ridgway, JB, Protein Eng. 9 (1996) 617-621; and WO96 / 027011). The percentage of heterodimers can be further increased by using phage display to reconstruct the interaction surface of the two CH3 domains and introducing disulfide bridges to stabilize the heterodimers (Merchant A.M. et al., Nature Biotech 16(1998)677-681; Atwell, S. et al., J. Mol. Biol. 270(1997)26-35). For example, the antibody or antigen-binding fragment may contain the T366W mutation in the CH3 domain of the “protrusion chain” and the T366S, L368A, or / and Y407V mutations in the CH3 domain of the “hole chain”. Additional interchain disulfide bridges between the CH3 domains may also be used (Merchant, AM et al., Nature Biotech. 16(1998)677-681).

[0178] In some practical technical solutions, the interaction surface of the two CH3 domains of the antibody or antigen-binding fragment can be altered using the knock-in-hole technique to enhance the heterodimerization of the two heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "protrusion," and the other a "hole." The introduction of disulfide bridges further stabilizes the heterodimer and improves the yield.

[0179] In some practical technical solutions, the antibody or antigen-binding fragment contains the T366W mutation in the CH3 domain of the "protrusion chain" and the T366S, L368A, or / and Y407V mutations in the CH3 domain of the "cave chain". Additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, AM et al., Nature Biotech. 16(1998) 677-681), which is achieved, for example, by introducing the Y349C mutation into the CH3 domain of the "protrusion" or "cave" chain and introducing the E356C or S354C mutation into the CH3 domain of the other chain. Thus, in another preferred embodiment, the bispecific, tetravalent antigen-binding protein comprises an S354C or / and T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A or / and Y407V mutation in the other of the two CH3 domains; or the bispecific, tetravalent antigen-binding protein comprises an E356C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A or / and Y407V mutation in the other of the two CH3 domains; or the... Bispecific, tetravalent antigen-binding proteins contain a Y349C or / and T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A or / and Y407V mutation in the other of the two CH3 domains, or the bispecific, tetravalent antigen-binding protein contains a Y349C or / and T366W mutation in one of the two CH3 domains and an S354C, T366S, L368A or / and Y407V mutation in the other of the two CH3 domains (numbering is always in accordance with Kabat's EU index).

[0180] The term "epitope" in this document refers to any protein determinant capable of specifically binding to immunoglobulins, scFv, or T-cell receptors. Epitope determinants typically consist of chemically active surface clusters of molecules such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, antibodies can target the N-terminus or C-terminus of a polypeptide.

[0181] The term "valence" in this article refers to the presence of a specified number of binding sites in an antibody / antigen binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" represent the presence of one, two, four, and six binding sites in an antibody / antigen binding molecule, respectively, while "multivalent" represents the presence of multiple binding sites in an antibody / antigen binding molecule.

[0182] The terms "specific binding," "immunobinding," and "immunobinding properties" used in this document refer to the type of non-covalent interaction formed between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed as the dissociation constant (KD) of the interaction, where a smaller KD indicates a greater affinity. The immunobinding properties of a selected peptide can be quantitatively determined using methods well-known in the art. One such method requires determining the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex's partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Therefore, the "binding rate constant" (KD) is used. on ) and "dissociation rate constant" (K off It can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87(1993)). K off / K on The ratio allows for the elimination of all parameters unrelated to affinity, and equals the dissociation constant KD. KD is preferably determined using surface plasmon resonance assays (BIAcore, GE-Healthcare Uppsala, Sweden). When the equilibrium binding constant (KD) is ≤1 μM, for example ≤200 nM in some embodiments, ≤100 nM in some embodiments, ≤10 nM in some embodiments, and ≤100 pM to about 1 pM in some embodiments, the antibody or antigen-binding fragment of this disclosure is considered to specifically bind to the target antigen EGFR and / or cMet.

[0183] The term "mutation" in this article includes gene mutations and amino acid mutations. Gene mutations refer to deletions, insertions, inversions, or substitutions of heterologous nucleic acids, which may lead to changes in the amino acid sequence of the corresponding protein product. Amino acid mutations, also known as nonsynonymous single nucleotide mutations, are caused by changes in a few single bases, resulting in changes in the amino acid sequence of the protein product. Changes in amino acids can affect protein stability, interactions, and enzyme activity, thereby leading to disease.

[0184] The term "amino acid substitution" in this article refers to the removal of at least one amino acid residue from the natural or starting sequence, and the insertion of different amino acids at the same position. Substitutions can be single, where only one amino acid in the molecule is substituted, or they can be multiple, where two or more amino acids in the same molecule are substituted.

[0185] The term "conservative amino acid substitution" in this document refers to the substitution of an amino acid normally present in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitution include replacing a nonpolar (hydrophobic) residue such as isoleucine, valine, and leucine with another nonpolar residue. Similarly, examples of conservative substitution include replacing a polar (hydrophilic) residue with another residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, replacing a basic residue such as lysine, arginine, or histidine with another residue, or replacing an acidic residue such as aspartic acid or glutamic acid with another acidic residue, are additional examples of conservative substitution. Examples of nonconservative substitution include replacing a nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or replacing a polar residue with a nonpolar residue.

[0186] The term "mutant" in this document refers to a "variant" of the protein or peptide that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity with the amino acid sequence of the protein or peptide.

[0187] The term "identity" in this document can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0188] Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the common positions shared by the sequences.

[0189] The term "antibody-drug conjugate" (ADC) refers to an antibody or its antigen-binding fragment linked to a biologically active drug via a stable linker unit. This link can be a covalent bond or a non-covalent interaction, such as through electrostatic forces. Various linkers known in the art can be used to form the immunoconjugate. The biologically active drugs described herein include cytotoxic drugs, such as chemotherapeutic agents or antibiotics. Exemplarily, the cytotoxic drugs are selected from tubulin inhibitors, DNA damaging agents, or topoisomerase inhibitors. The tubulin inhibitors include, but are not limited to, dolastatin, auristatin, maytansine, tubulolysins, and cryptomycins. The DNA damaging agents include, but are not limited to, PBD drugs. The topoisomerase inhibitors include, but are not limited to, camptothecin-based drugs.

[0190] The term "DAR" or "drug-antibody ratio" refers to the average number of small molecule cytotoxic drugs linked to each antibody molecule. In the antibody-drug conjugates disclosed herein, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0191] The term "nucleic acid" in this document includes any compound and / or substance comprising a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for the direct expression of the antibodies disclosed herein in vitro and / or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0192] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of this disclosure contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express the antibodies and antibody fragments of this disclosure include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. The expression vectors of this disclosure may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norsocrine.

[0193] The term "host cell" in this article refers to a cell in which foreign nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0194] In this article, "n is a real number from 1 to 16" means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0195] In this article Indicates the connection site.

[0196] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0197] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0198] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0199] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0200] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0201] C in this article m -C n , refers to having an integer number of carbon atoms in the range mn.

[0202] The term "alkyl" refers to the general formula CnH. 2n+1The alkyl group can be straight-chain or branched. The term "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0203] The “C1-C6 alkyl” mentioned in this article may further include “C1-C3 alkyl”.

[0204] The term "cycloalkyl" refers to a fully saturated carbon ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0205] The term "heterocyclic group" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridged ring group containing 1-5 heteroatoms or heteroatom groups (i.e., atomic groups containing heteroatoms). These "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, containing 1-3 independently selected heteroatoms or heteroatom groups as described above. The term "5-6 membered heterocyclic group" refers to a heterocyclic group with 5 or 6 ring atoms, and whose ring atoms contain 1-3 independent heteroatoms or heterogroups selected from those described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrocyclobutane and oxacyclobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. "4-7 membered heterocyclic group" can encompass the ranges of "4-7 membered heterocyclic alkyl," "5-6 membered heterocyclic group," and "5-6 membered heterocyclic alkyl."

[0206] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0207] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0208] The term "effective dose" refers to the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0209] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals that receive treatment for a disease or condition.

[0210] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0211] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0212] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0213] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the disclosed compounds to an organism.

[0214] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0215] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0216] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0217] Certain isotope-labeled compounds of this disclosure (e.g., using...) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0218] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0219] In all methods of administration of the compounds described herein, the daily dose is from 0.001 mg / kg to 600 mg / kg body weight, preferably from 0.05 mg / kg to 200 mg / kg body weight, more preferably from 0.1 mg / kg to 100 mg / kg body weight, in the form of single or separate doses.

[0220] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0221] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0222] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety.

[0223] The term "tumor" or "carcinoma" in this document refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The term "cancer" in this document refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The terms "cancer" and "tumor" are not mutually exclusive when used herein. Exemplarily, tumors or cancers described in this disclosure include, but are not limited to, solid tumors such as lung cancer, pancreatic cancer, liver cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal carcinoma, kidney cancer, cervical cancer, prostate cancer, uterine cancer, melanoma, squamous cell carcinoma of the head and neck, cholangiocarcinoma, thyroid cancer, ovarian cancer, or glioblastoma, etc.

[0224] The term "EC50" in this document refers to the half-maximal effective concentration, which includes the antibody concentration that induces a half-range response between baseline and maximum value after a specified exposure time. EC50 essentially represents the antibody concentration at which 50% of its maximum effect is observed, and can be measured by methods known in the art. The term "IC" in this document... 50 "Half-maximum inhibitory concentration" refers to the amount of inhibitor that can reduce the reaction rate by half when the inhibitor concentration reaches half. Attached Figure Description

[0225] Figure 1 shows an exemplary format of the EGFR×cMet bispecific antibody, where Format-1 to 21 are exemplary format diagrams of the EGFR×cMet bispecific antibody of this disclosure, and Format-22 is an antibody format diagram of the α-glucan molecule AZD9592.

[0226] Figure 2. ELISA detection of the binding of EGFR×cMet double antibody to human EGFR (A) and human cMet (B);

[0227] Figure 3. FACS detection of the binding of EGFR×cMet double antibody to NCI-H1975 cells (A) and MKN45 cells (B);

[0228] Figure 4. ELISA detection of the binding of ADC-1 to 5 with human EGFR (A) and human cMet (B);

[0229] Figure 5. FACS detection of the binding of ADC-1 to ADC-5 with NCI-H1975 cells (A) and MKN45 cells (B);

[0230] Figure 6. In vitro proliferation inhibition curves of ADC-1 to 5 on NCI-H1975 cells (A) and MKN45 cells (B);

[0231] Figure 7. Tumor growth curves of NCI-H1975 subcutaneous tumor model with ADC-1 to 5 (A); (B) Body weight change curve of subcutaneous tumor model mice;

[0232] Figure 8. Pharmacokinetic curves of ADC-1 to ADC-5 in mice;

[0233] Figure 9. In vitro proliferation inhibition curves of ADC-6 to 10 (using drug linker L3-1) on NCI-H1975 cells (A) and MKN45 cells (B);

[0234] Figure 10. (A) Tumor growth curve of NCI-H1975 subcutaneous tumor model with ADC-9; (B) Body weight change curve of NCI-H1975 subcutaneous tumor model mice with ADC-9; (C) Effect of administration of ADC-9 to large tumors on tumor volume of NCI-H1975 tumor-bearing mice.

[0235] Figure 11. (A) Tumor growth curve of HCT116 subcutaneous tumor model of ADC-9; (B) Body weight change curve of mouse HCT116 subcutaneous tumor model of ADC-9.

[0236] Figure 12. (A) Tumor growth curve of ADC-9 IM95#206 subcutaneous tumor model; (B) Body weight change curve of ADC-9 IM95#206 subcutaneous tumor model mice.

[0237] Figure 13. In vitro proliferation inhibition curves of ADC-16-20 on NCI-H1975 cells (A) and MKN45 cells (B). Detailed Implementation

[0238] This disclosure relates to bispecific antibodies or antigen-binding fragments constructed from specific formats and sequences, particularly suitable for the preparation of ADC drugs, capable of specifically binding to EGFR and cMet, and corresponding antibody-drug conjugates.

[0239] The present disclosure will be further described below with reference to specific embodiments, and the advantages and features of the present disclosure will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0240] The embodiments disclosed herein are merely exemplary and do not constitute any limitation on the scope of this disclosure. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions disclosed herein without departing from the spirit and scope of this disclosure, but all such modifications and substitutions fall within the protection scope of this disclosure.

[0241] Experimental methods

[0242] 1. SEC purity analysis of bispecific antibodies and ADC molecules

[0243] SEC-HPLC was used to analyze antibody samples, characterize antibody molecular size uniformity, and determine antibody purity. The HPLC system used was an Agilent 1260, with a TSKgel G3000SWXL column from Tosoh Bioscience. The mobile phase was 200 mM phosphate buffer (pH 7.0), the detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 minutes. For the SEC-HPLC data, the chromatograms were analyzed using manual integration, and protein purity was calculated using the area normalization method. The main peak was considered the monomer, the peaks before the main peak were called aggregates, and the peaks after the main peak were called fragments.

[0244] 2. ADC yield (%) calculation: ADC amount / antibody amount added * 100%

[0245] 3. Homogeneity analysis of ADC molecules

[0246] The homogeneity of the ADC sample was characterized by analysis using HIC-HPLC. The HPLC system used was an Agilent 1260, with a TSKgel Butyl-NPR column from Tosoh Bioscience. Mobile phase A consisted of 1.5M ammonium sulfate and 50mM phosphate buffer (pH 7.0), while mobile phase B consisted of 25mM phosphate buffer (pH 7.0) containing 20% ​​isopropanol. The detection temperature was 30℃, the flow rate was 0.5 mL / min, and the detection wavelengths were 280 nm and 390 nm. The sample was first concentrated to 1 mg / mL, then 2M ammonium sulfate and 100mM phosphate buffer (pH 7.0) were added to bring the ammonium sulfate concentration to 0.8 M. After thorough mixing, the supernatant was collected, and the target protein was loaded at a rate of 40 μg. The mobile phase gradient was achieved by increasing mobile phase B from 0% to 70% over 35 minutes, with an analysis time of 50 minutes.

[0247] For HIC-HPLC data, the chromatograms were analyzed using the manual integration method, and the percentage of peak area of ​​each chromatographic peak at the detection wavelength of 280 nm was calculated.

[0248] Example 1: Preparation of EGFR×cMet bispecific antibody

[0249] 1.1 Antibody Sequence

[0250] The anti-EGFR antibodies used in this disclosure for constructing EGFR×cMet bispecific antibodies include: panitumumab (EP0979246B1), cetuximab (US7060808B1), and humanized cetuximab hCe01, hCe02, and hCe10; the anti-cMet antibodies used for constructing EGFR×cMet bispecific antibodies include onartuzumab (CN101035808B) and CE355621 (US7498420B2), and the corresponding antibody sequences are shown below.

[0251] The variable region sequence of the anti-EGFR antibody:

[0252] Panitumumab_VL

[0253] Panitumumab_VH

[0254] >cetuximab_VL

[0255] >cetuximab_VH

[0256] hCe01_VL

[0257] hCe01_VH

[0258] hCe02_VL

[0259] hCe02_VH

[0260] hCe10_VL

[0261] hCe10_VH

[0262] Variable region sequence of anti-cMet antibody:

[0263] >Onartuzumab_VL

[0264] >Onartuzumab_VH

[0265] >CE355621_VL

[0266] >CE355621_VH

[0267] 1.2 Construction and Production of EGFR×cMet Bispecific Antibodies

[0268] The method for constructing the EGFR×cMet bispecific antibody molecule is as follows: the nucleic acid sequences encoding the antibodies VH and VL are recombined into the expression vector pTT5 (purchased from UBO Biotechnology, catalog number: VT2202) containing the signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 14) and the heavy chain constant region / light chain constant region sequence, to obtain the recombinant plasmid expressing VH-CH / VL-CL. For specific methods, please refer to Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).

[0269] This disclosure selects different antibody sequences from the above-mentioned anti-EGFR antibody and anti-cMet antibody sequences for cross-combination, and selects 21 bispecific antibody formats to construct a total of 61 bispecific antibodies (related composition information is shown in Table 1). The format diagram is shown in Figure 1.

[0270] Table 1 Summary of Bispecific Antibiotics

[0271] Based on the aforementioned experimental methods 1-3 and the DAR value determination method in Example 3.2 below, the purity of the 61 bispecific antibody molecules obtained, as well as the purity, yield, DAR value, and uniformity of the corresponding ADC molecules after ADC construction, were characterized. Five bispecific antibodies (numbered 3, 5, 8, 13, and 28 in Table 1) with better overall performance in terms of ADC purity, yield, and uniformity were selected for subsequent experiments.

[0272] The five bispecific antibodies selected are numbered 3, 5, 8, 13, and 28 in Table 1, and are sequentially numbered EGFR×cMet-BsAb-1-5. Their basic structural composition is shown below:

[0273] EGFR×cMet-BsAb-1: Constructed from humanized cetuximab hCe01 and anti-cMet antibody CE355621 based on Format 1 in Figure 1, comprising a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each contain VL cMet -VL EGFR -CL, the second peptide chain and the third peptide chain respectively contain VH cMet -VH EGFR -CH1-Fc* (i.e., antibody number 3 in Table 1);

[0274] EGFR×cMet-BsAb-2: Constructed from humanized cetuximab hCe10 and anti-cMet antibody CE355621 based on Format 2 in Figure 1, it comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each contain VL cMet -CL, the second peptide chain contains VH cMet -CH1-Fc, the third peptide chain contains VH cMet -CH1-Fc-VL EGFR -VH EGFR (i.e., antibody number 5 in Table 1);

[0275] EGFR×cMet-BsAb-3: Constructed from humanized cetuximab hCe01 and anti-cMet antibody CE355621 based on Format 3 in Figure 1, comprising a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each contain VL cMet -CL, the second peptide chain contains VH cMet-CH1-Fc, the third peptide chain contains VH cMet -CH1-VL EGFR -VH EGFR -Fc (i.e., antibody number 8 in Table 1);

[0276] EGFR×cMet-BsAb-4: Constructed from humanized cetuximab hCe01 and anti-cMet antibody CE355621 based on Format 4 in Figure 1, wherein the first peptide chain contains VL cMet -CL, the second peptide chain contains VH cMet -CH1-Fc, the third peptide chain contains a single-chain unit VL EGFR -VH EGFR -Fc (i.e., antibody number 13 in Table 1);

[0277] EGFR×cMet-BsAb-5: Constructed from humanized cetuximab hCe01 and anti-cMet antibody CE355621 based on Format 5 in Figure 1, comprising a first peptide chain, a second peptide chain, and a third peptide chain, wherein the first peptide chain contains VL EGFR -CL, the second peptide chain contains VH EGFR -CH1-Fc, the third peptide chain contains a single-chain unit VL cMet -VH cMet -Fc (i.e., antibody number 28 in Table 1).

[0278] The constituent elements and heavy and light chain sequences of the above-mentioned EGFR×cMet bispecific antibody EGFR×cMet-BsAb-1-5, Yangshen antibody AZD9592 BsAb (WO2023083846, structure shown in Figure 1 Format-22) and anti-FITC isotype control antibody are shown in Table 2. The CDR sequences classified according to Kabat, IMGT and Chothia are shown in Table 3.

[0279] Table 2. Sequence information of EGFR×cMet bispecific antibody and control antibody

[0280] Table 3. CDR sequence information of EGFR×cMet bispecific antibody

[0281] Note: EGFR×cMet-BsAb-1-5 uses the same CDR.

[0282] The plasmid and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed well, and incubated for 15 min. The mixture was then added to Expi293F cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C with 5% CO2 at 120 rpm. On the second day after transfection, OPM-293ProFeed (Shanghai AOPMai, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected.

[0283] Protein A column (Mabselect SuRe™, Cytiva) was used for initial purification. The Protein A column was equilibrated with 3–5 column volumes of equilibration buffer (PBS buffer, pH 7.4), and then the clear culture supernatant was loaded at a flow rate of 8 mL / min. After loading, 3–5 column volumes of high-salt eluent (20 mM phosphate buffer, 1 M NaCl, pH 7.4) were used for elution. The protein bound to the Protein A column was eluted with elution buffer (50 mM sodium acetate buffer, pH 3.5), and the elution was monitored by the A280 UV absorption peak. The eluted protein was collected and neutralized to pH 5–6 with 1 M pH 8.0 Tris-HCl. Then, the protein was purified using Capto SP ImpRes (Cytiva). The column was equilibrated with 3–5 column volumes of equilibration buffer (Buffer A: 50 mM HAC, pH 5.5), and then the purified protein solution was loaded at a flow rate of 5 mL / min. After sample loading, wash with 3-5 column volumes of Buffer A, followed by elution with a salt gradient: Buffer B: Buffer A + 1M NaCl, pH 5.5, 0% B-40% B gradient elution. After concentration, dialyze to 55g buffer (10mM HAC, 9% sucrose, pH 5.5). Filter aseptically through a 0.22μm filter and store aseptically. The purification method for Yangshen antibody AZD9592 BsAb is based on patent WO2023083846.

[0284] As described in Experimental Method 1 above, the obtained antibody molecules were subjected to SEC purity analysis. The SEC purity of the EGFR×cMet bispecific antibody and the control antibody is shown in Table 4.

[0285] Table 4. SEC purity of EGFR×cMet bispecific antibody and control antibody

[0286] Example 2: Detection of binding activity of EGFR×cMet bispecific antibody and control antibody

[0287] 2.1 Preparation of EGFR antigen and cMet antigen

[0288] The full-length amino acid sequence of human EGFR (Uniprot ID: P00533, SEQ ID NO: 65) and the full-length amino acid sequence of monkey EGFR (Uniprot ID: P55245, SEQ ID NO: 66) were synthesized by Anhui General Biotechnology Co., Ltd., and the sequence information is shown in Table 5. The full-length amino acid sequence of human cMet (purchased from Sinocare, catalog number: HG10692-CH) and the full-length amino acid sequence of monkey cMet (purchased from Sinocare, catalog number: CG90304-CH) were also synthesized. The extracellular domain (ECD) of the above proteins was sequentially linked to the human His tag and the tag from the N-terminus to the C-terminus to obtain antigen sequences for immunoassay and antibody screening and identification. General Biosystems (Anhui) Co., Ltd. was commissioned to synthesize the nucleotide sequences corresponding to the amino acid sequences and clone them into the pTT5 vector containing the signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 14). Cellular expression, purification, and subsequent SEC purity analysis of the obtained antibody molecules as described in Experimental Method 1 above are shown in Table 6.

[0289] Table 5. Full-length amino acid sequence of human / cynomolgus monkey EGFR

[0290] Table 6. SEC purity of EGFR / cMet protein

[0291] 2.2 Enzyme-linked immunosorbent assay (ELISA) to detect the binding of EGFR×cMet double antibody to human EGFR and cMet proteins.

[0292] Human EGFR-His and human cMet-his were diluted to a final concentration of 4 μg / mL with PBS, and 50 μL was added to each well of a 96-well ELISA plate. The plates were incubated overnight at 4°C. The next day, the plates were washed twice with PBST (PBS + 0.5% Tween 20). After blocking with PBS + 5% (w / w) skim milk at room temperature for 2 hours, the plates were washed three times with PBST and then blotted dry. The test antibody and control antibody were diluted with 1% BSA-PBS, starting at 20 nM, and serially diluted 5-fold. 50 μL / well of the diluted antibody was added to the coated plates, and the plates were incubated at 37°C for 1 hour, followed by washing three times with PBST. HRP (horseradish peroxidase)-labeled secondary antibody (109-035-098, purchased from Jackson Immune) was added, and the plates were incubated at 37°C for 1 hour, followed by washing five times with PBST. Add 50 μl of TMB substrate to each well and incubate at room temperature for 5–10 minutes. Then add 50 μl of stop solution (1.0 M HCl) to each well. Read the OD450 nm value using an ELISA reader (PowerWaveHT, Biotek). As shown in Table 7, all five EGFR×cMet bispecific antibodies showed good binding activity to both human EGFR protein (Figure 2, A) and human cMet protein (Figure 2, B).

[0293] Table 7. ELISA detection of binding between EGFR×cMet double antibody and human EGFR and cMet proteins

[0294] 2.3 Flow cytometry (FACS) assay to detect the binding of EGFR×cMet bispecific antibody to cells

[0295] The desired cell types (lung cancer NCI-H1975 and gastric cancer MKN45 cells) were cultured in T-175 cell culture flasks until the logarithmic growth phase. For adherent cells, the culture medium was aspirated, and the cells were washed twice with PBS buffer. Cells were then digested with trypsin, and after digestion was terminated, the cells were washed twice with PBS buffer. After cell counting, the cell pellet was resuspended in [PBS + 2% (w / w) FBS] blocking buffer to a concentration of 2 × 10⁻⁶. 6Cells were added at a rate of 50 μL per well to a 96-well FACS plate, along with 50 μL of the test sample containing the bispecific antibody per well. The plate was incubated at 4°C for 1 hour. After washing three times with PBS buffer, 50 μL of Alexa Flour647-labeled secondary antibody (109-605-098, purchased from Jackson Immuno) was added to each well, and the plate was incubated at 4°C for 45 minutes. After washing five times with PBS buffer, the results were detected and analyzed using FACS (FACS Canto™, purchased from BD). Data analysis was performed using CellQuest software to obtain the mean fluorescence density (MFI) of the cells. Further analysis was performed using GraphPad Prism software to perform data fitting and calculate EC50. As shown in Table 8 and Figure 3, all five EGFR×cMet bispecific antibodies could bind to the NCI-H1975 and MKN45 cell lines, respectively.

[0296] Table 8. FACS detection of EGFR×cMet bispecific antibody binding activity

[0297] Example 3: Preparation of EGFR×cMet dual antibody ADC

[0298] 3.1 The structure of the drug-linker L1-1 is shown below, and its preparation is based on patent document WO2023217227A1 (Example 39).

[0299] 3.2 Conjugation of EGFR×cMet bispecific antibodies and drug-linker L1-1

[0300] The antibody prepared in Example 1 was dialyzed and the buffer was changed to 20 mM PB, 1 mM EDTA solution (pH 6.5). 10-15 molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution, and the mixture was incubated on a constant-temperature metal shaker at 4°C for 16-18 h to reduce the antibody. 10-15 molar equivalents of the above drug-linker L1-1 compound were dissolved in DMSO and added to the reaction system. The reaction solution was coupled at 25°C for 6 h. The reaction product was desalted using a G25 column and the buffer was changed to 20 Mm NaCl-HAC, 9% sucrose, pH 5.5 buffer to remove unreacted free small molecule toxins, yielding the antibody-drug conjugate. The conjugate of the positive molecule AZD9592 was obtained using the same method, and its conjugate SG3932 (US20200306243A1) was purchased from MCE, catalog number HY-145399. For the SEC purity analysis of the bispecific antibody ADC, please refer to Experimental Method 1.

[0301] DAR value determination: The DAR value of ADC molecules was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-sugar modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37℃ for 1 h to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected into a Waters ACQUITY Protein BEH size-exclusion column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min, and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were: spray voltage 3.8 kV, capillary heating temperature 300℃, sheath gas flow rate 35 arb, and precursor ion scan range 800-3000. Finally, the mass spectrometry data was analyzed using the Biopharma Finder software. 4.1 The Respect algorithm was used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component, thereby calculating the DAR value of the ADC sample to be tested. Simultaneously, the SEC purity of the antibody-drug conjugate was analyzed according to the aforementioned experimental method 1. The DAR value and SEC purity of the prepared antibody-drug conjugate are shown in Table 9.

[0302] Table 9. Preparation, DAR value, and SEC purity of antibody-drug conjugates

[0303] Example 4: Detection of binding activity of EGFR×cMet bispecific antibody ADC

[0304] 4.1 Enzyme-linked immunosorbent assay (ELISA) to detect the binding of EGFR×cMet bispecific antibody ADC to human EGFR and cMet proteins.

[0305] For the specific method of ELISA detection of the binding of EGFR×cMet bispecific antibody ADC to human EGFR and cMet proteins, please refer to Example 2.2. As shown in Table 10, ADC-1 to ADC-5 have good binding activity to both human EGFR protein (Figure 4A) and human cMet protein (Figure 4B).

[0306] Table 10. ELISA detection of binding between EGFR×cMet bispecific antibody ADC and human EGFR and cMet proteins.

[0307] 4.2 Flow cytometry (FACS) assay to detect the binding of EGFR×cMet bispecific antibody ADC to cells

[0308] The specific method for detecting the binding of EGFR×cMet bispecific antibody ADCs to cells using FACS is described in Example 2.3. The results are shown in Table 11 and Figure 5. All five EGFR×cMet bispecific antibody ADCs (ADC-1 to ADC-5) bound to the NCI-H1975 and MKN45 cell lines. Furthermore, the endocytic ability of ADC-1 to ADC-5 was verified to have good endocytosis by conventional methods in the art.

[0309] Table 11. FACS detection of EGFR×cMet bispecific antibody ADC binding activity

[0310] 4.3 Affinity determination of EGFR×cMet bispecific antibody ADC

[0311] To determine the affinity of EGFR×cMet bispecific antibody ADCs for human EGFR and human cMet proteins, five EGFR×cMet bispecific antibody ADCs (ADC-1–5) and the anolyte molecule AZD9592 were captured using a Protein A chip (Cytiva; 29-127-558). Samples and run buffer were prepared using HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P2O) (Cytiva; BR-1006-69). The flow cell was set to 25°C, and the sample block was set to 16°C. Both were pretreated with run buffer. In each cycle, the antibody was first captured using the Protein A chip, followed by the injection of a single concentration of human EGFR or human cMet antigen protein. The binding and dissociation of the antibody and antigen protein were recorded, and the chip was regenerated using Glycine pH 1.5 (Cytiva; BR-1003-54). Binding was measured by injecting different concentrations of human EGFR or human cMet antigen protein into the solution for 240 seconds at a flow rate of 30 μL / min, starting from 200 nM (actual concentrations tested are detailed in the results), diluted 1:1, for a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds, triggered by switching from the sample solution to the run buffer. The surface was regenerated by washing with 10 mM glycine solution (pH 1.5) at a flow rate of 30 μL / min for 30 seconds. Bulk refractive index differences were corrected by subtracting the response value obtained from the reference channel. Blank injections (=dual reference) were also subtracted. The Langmuir 1:1 model was used to calculate the apparent KD and other kinetic parameters. The binding affinity (KD) of ADC-1–5 to human antigen proteins is shown in Table 12.

[0312] Table 12. Biacore detection of the binding of EGFR×cMet bispecific antibody ADC to human protein

[0313] 4.4 Species Cross-Binding Activity of EGFR×cMet Bispecific Antibodies

[0314] Following the method described in Example 4.3, the affinity of the EGFR×cMet bispecific antibody ADC for cynomolgus monkey EGFR protein and cynomolgus monkey cMet protein was tested. The results are shown in Table 13. ADC-1 to 5 showed good binding activity to both cynomolgus monkey EGFR and cynomolgus monkey cMet.

[0315] Table 13. Biacore detection of the binding of EGFR×cMet bispecific antibody ADC to cynomolgus monkey protein

[0316] Example 5: In vitro assay of EGFR×cMet bispecific antibody ADC inhibiting tumor cell proliferation

[0317] MKN45 and NCI-H1975 cells were digested with trypsin (Gibco, catalog number 25200072), then resuspended in RPMI 1640 + 10% FBS medium to adjust cell density. MKN45 cells were cultured at 2.5 × 10⁻⁶ cells / year. 3 Cells / 90 μL, NCI-H1975 at 1.05 × 10⁻⁶ 3 Cells / 90 μL were seeded into 96-well plates (purchased from Corning, catalog number 3610) and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the antibody-drug conjugate was serially diluted with complete culture medium according to the experimental design, and 10 μL of the diluted antibody-drug conjugate was transferred to the corresponding well. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 5 days. After 5 days, 50 μL of Key was added to each well. The Luminescent Cell Viability Detection Kit (purchased from vkey-bio, catalog number A2010003N, usage instructions follow the product manual) was used to detect cell viability by reading fluorescence values ​​on an Envision instrument (purchased from PerkinElmer, model Envision 2105).

[0318] A positive control group and a negative control group were set up as controls for 0% and 100% cell killing, respectively. The positive control group received no test drug, but all other procedures were the same as the experimental group. The negative control group received no cells, only the same volume of culture medium, and all other procedures were the same as the experimental group. The cell proliferation inhibition rate was calculated using the formula: Inhibition rate = ((Positive control - Sample well reading) / (Positive control - Negative well reading)) × 100%. This formula yielded the inhibition rate of the corresponding drug in different cell types, with the maximum inhibition rate denoted by Emax. GraphPad Prism 9.0 software was used for graphing and IC50 calculation. 50 .

[0319] The results are shown in Table 14 and Figure 6. ADC-1 to ADC-5 all showed good inhibitory activity against tumor cells expressing EGFR and cMet, and their in vitro inhibitory activity was no weaker than that of the control molecule AZD9592.

[0320] Table 14. Inhibitory effect of EGFR×cMet bispecific antibody ADC on tumor cell proliferation in vitro

[0321] Example 6. Efficacy evaluation of NCI-H1975 subcutaneous tumor model

[0322] Cells and reagents: Lung cancer NCI-H1975 cells; RPMI-1640 medium: Gbico; Cat No.: A104910; Fetal bovine serum: Gibco; Cat No.: 10099-141C; 0.25% trypsin-EDTA: Gibco, Cat No.: 25200-072; D-PBS (calcium- and magnesium-free phosphate buffer): Hyclone, Cat No.: SH30256.01; Matrigel: Corning, Cat No.: 356237

[0323] Animal Information: Balb / c nude mice, female, 6-7 weeks old, weighing approximately 16-22 grams. The animals were purchased from Beijing Vital River Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0324] Cell culture: The lung cancer NCI-H1975 cell line was cultured in vitro under the following conditions: RPMI-1640 (cell culture medium) with 10% fetal bovine serum and 1% Pen Strep, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using a routine digestion process with 0.25% trypsin-EDTA digestion solution. When the cell saturation reached 80%-90% and the required number was achieved, the cells were harvested and counted.

[0325] Cell seeding: Add 0.1 ml of (containing 5 × 10⁻⁶ cells) to the cell line. 6 NCI-H1975 cell suspension (RPMI-1640: Matrigel, volume ratio 1:1) was subcutaneously inoculated into the axilla of each mouse. On day 8 post-inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day being Day 0.

[0326] Administration: The antibody-drug conjugates ADC-1, ADC-2, ADC-3, ADC-4, and ADC-5, the positivist molecule AZD9592, and the negative control molecule ADC-ISO-L1 were administered at a dose of 5 mg / kg via a single tail vein injection. Six mice were used in each group.

[0327] Tumor measurement and experimental indicators: Tumor diameter was measured twice weekly using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly. The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0328] As shown in Table 15 and Figure 7, in the mouse subcutaneous xenograft NCI-H1975 model, ADC-1, ADC-2, ADC-3, ADC-4, and ADC-5, when administered via a single tail vein dose of 5 mg / kg, all significantly inhibited tumor growth (P<0.0001). Furthermore, compared to the ginseng molecule AZD9592, ADC-2, ADC-4, and ADC-5 were significantly superior to AZD9592 (P<0.0001), and ADC-1 was also significantly superior to AZD9592 (P<0.001). In this embodiment, no effect on mouse body weight or death was observed at the tested doses; the mice tolerated the medication well.

[0329] Table 15. Tumor volume in the NCI-H1975 subcutaneous tumor model

[0330] Example 7. Pharmacokinetic Study of EGFR×cMet ADC in Mice

[0331] 7.1 Screening of monoclonal antibodies against drug 001

[0332] Monoclonal antibodies against drug 001 were generated by immunizing mice. The immunogen was drug 001 (structure shown below, preparation method as described in Example 39 of WO2023020605A1). Hybridoma cells were prepared from splenic lymphocytes of mice with high antibody titers in serum that tended to plateau. Positive hybridoma clones were obtained by screening using conventional methods in the art, such as ELISA. These clones could specifically recognize antibody-drug conjugates (ADCs) coupled with drug 001. The hybridoma clones were cultured using serum-free cell culture to further prepare antibodies, which were then purified to obtain the anti-drug 001 antibody.

[0333] 7.2 Mouse PK Experiment of EGFR×cMet ADC

[0334] Experimental design: Three Balb / c mice were selected to receive each drug molecule via intravenous injection at a dose of 5 mg / kg. Blood collection points were set at 0 hr before administration, and 15 min, 2 hr, 8 hr, 24 hr (Day 1), 72 hr (Day 3), 120 hr (Day 5), 168 hr (Day 7), 240 hr (Day 10), 336 hr (Day 14), and 504 hr (Day 21) after administration. The total antibody and ADC concentrations in mouse plasma were detected using ELISA.

[0335] The detection method for total antibody (TA): Goat Anti-Human IgG Fc antibody (Cat: 109-006-098, Jackson) was prepared to a concentration of 0.25 μg / mL using phosphate-buffered saline (Cat: AR0030, Boster Biologics). 100 μL was added to each well of a high-affinity 96-well plate, and the plate was sealed with a membrane and incubated overnight at 4°C. After coating, the 96-well plate was removed, the membrane was peeled off, and the liquid in the wells was shaken dry. 300 μL of PBST was added to each well, the plate was gently shaken, and the liquid was shaken off. This washing process was repeated twice. Then, 250 μL of 1% BSA-PBST solution was added to each well, and the plate was incubated at 37°C for 1 hour for blocking. A standard curve with a concentration range of 1000-15.625 ng / mL was prepared using blank mouse plasma (purchased from IPHASE, catalog number: 032E13.12). After sealing the 96-well plate, wash it twice. Dilute the standard curve sample 100-fold with 1% BSA-PBST solution. Dilute the test sample 100-fold with 1% BSA-PBST solution, and then further dilute with 1% BSA-PBST solution containing 1% blank mouse plasma to ensure the estimated concentration of the sample is within the curve range. After washing the 96-well plate twice, add 100 μL of standard curve sample and test sample to each well. Incubate the 96-well plate on a Thermo microplate shaker (model: 88880024) at 500 rpm for 2 hours at room temperature. After incubation, remove the 96-well plate and wash it four times. Add 100 μL of Goat Anti-Human IgG Fc HRP (Cat: 109-036-170, Jackson) diluted 10,000 times with 1% BSA-PBST solution to each well. Incubate at 500 rpm for 1 hour at room temperature. After incubation, remove the 96-well plate and wash it four times. Add 100 μL of TMB chromogenic solution (Cat: 34029, Thermo) to each well. Incubate at room temperature in the dark for 7-10 minutes. Terminate the reaction with 1M H2SO4. Read the data at 450nm-630nm using a microplate reader (Model: Multiskan FC, Brand: Thermo). Analyze the collected data using a four-parameter fitting method.

[0336] ADC detection method: Goat Anti-Human IgG Fc antibody (Cat: 109-006-098, Jackson) was prepared to a concentration of 0.25 μg / mL using phosphate-buffered saline (Cat: AR0030, Boster Biologics). 100 μL was added to each well of a high-affinity 96-well plate, and the plate was sealed with a membrane and incubated overnight at 4°C. After coating, the 96-well plate was removed, the membrane was peeled off, and the liquid in the wells was shaken dry. 300 μL of PBST was added to each well, the plate was gently shaken, and the liquid was shaken off. This washing process was repeated twice. Then, 250 μL of 1% BSA-PBST solution was added to each well, and the plate was incubated at 37°C for 1 hour for blocking. A standard curve with a concentration range of 1000-15.625 ng / mL was prepared using blank mouse plasma. The standard curve sample was diluted 100-fold with 1% BSA-PBST solution. The test sample was also diluted 100-fold with 1% BSA-PBST solution, and then appropriately diluted using 1% BSA-PBST solution containing 1% blank mouse plasma to ensure the estimated concentration was within the curve range. After sealing the 96-well plate, it was washed twice, and 100 μL of the standard curve sample and test sample were added to each well. The 96-well plate was then incubated at 500 rpm at room temperature for 2 hours using a Thermo microplate shaker (model: 88880024). After incubation, the 96-well plate was washed four times, and 100 μL of a Biotion-labeled monoclonal antibody solution diluted to 0.25 μg / mL with 1% BSA-PBST solution was added to each well. The plate was then incubated at 500 rpm at room temperature for 1 hour. After incubation, remove the 96-well plate, wash it four times, and add SA-HRP (Cat: 21126, Thermo) diluted 10,000 times with 1% BSA-PBST solution. Incubate at room temperature for 1 hour with the plate shaker at 500 rpm for 50 minutes. After incubation, wash the 96-well plate four times, add 100 μL of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 7-10 minutes. Terminate the reaction with 1M H₂SO₄. Read the values ​​using a microplate reader at wavelengths of 450 nm-630 nm. The collected data were analyzed using a four-parameter fitting method.

[0337] As shown in Figure 8, the total anti-inflammatory and anti-inflammatory blood drug exposure of ADC-1 to ADC-5 are similar to those of ADC, which is significantly better than that of Yangshen molecule AZD9592. Furthermore, calculations of the half-life of the above molecules show that the half-life of ADC-1 to ADC-5 is also significantly better than that of Yangshen molecule AZD9592.

[0338] Example 8: Preparation method of drug linker

[0339] 8.1 Synthesis of drug-linker L3-1

[0340] Synthesis route:

[0341] Step 1: Synthesis of tert-butyl 3-(2-cyclopentathio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionate (intermediate 3-2)

[0342] At room temperature, cyclopentylthiol (367 mg) and 2 M sodium hydroxide aqueous solution (1.65 mL) were added sequentially to a solution of compound 3-1 (1.066 g) dissolved in DCM (10 mL). The resulting mixture was stirred at room temperature for 30 min. The mixture was then separated by column chromatography (EA / PE, 0-50%) to obtain the crude title compound, which was used directly in the next reaction.

[0343] Step 2: Synthesis of tert-butyl 3-(2'-(cyclopentylthio)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-3)

[0344] Intermediate 3-2 (200 mg) was dissolved in N,N-dimethylformamide (2 mL) at 0 °C under a nitrogen atmosphere. NaH (127.15 mg, 60% effective content) and 1-iodo-2-(2-iodoethoxy)ethane (518.01 mg) were added. The reaction mixture was stirred at 0 °C for 1 h under nitrogen protection. Ice water (2 mL) was then added to the reaction mixture, followed by extraction with ethyl acetate (1 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (ethyl acetate / petroleum ether, 0-60%) to give the title compound (110 mg).

[0345] MS m / z (ESI): 448.2 [M+H] + .

[0346] Step 3: Synthesis of tert-butyl 3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyrano-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionate (intermediate 3-4)

[0347] Intermediate 3-3 (60 mg) was dissolved in anhydrous dichloromethane (1 mL) at 0 °C, and m-chloroperoxybenzoic acid (108.86 mg, 85% purity) was added. The reaction mixture was stirred at 25 °C for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure and separated by column chromatography (ethyl acetate / petroleum ether, 0-55%) to give the title compound (9.8 mg).

[0348] MS m / z (ESI): 480.3 [M+H]+ .

[0349] 1 H NMR (400MHz, Chloroform-d) δ = 9.40 (s, 1H), 4.35-4.18 (m, 1H), 4.00 (m, 2H), 3.86-3.72 (m, 6H), 2.68 (t, J= 6.3Hz,2H),2.31-2.23(m,2H),2.18(m,2H),2.06(m,2H),1.93-1.81(m,2H),1.77-1.65(m,4H),1.45(s,9H)

[0350] Step 4: Synthesis of 3-(2'-(cyclopentanyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionic acid (intermediate 3-5)

[0351] Intermediate 3-4 (680 mg) was dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (0.68 mL) was added to the reaction solution. The reaction solution was stirred at 25 °C for 1 hour. After the reaction was complete, the solvent was removed by direct concentration under reduced pressure. The residue was purified by rapid silica gel column chromatography. 12g The title compound (200 mg) was obtained by rapid silica column chromatography with a gradient of 0–53% tetrahydrofuran / petroleum ether at a flow rate of 40 mL / min.

[0352] MS m / z (ESI): 423.8 [M+H] +

[0353] Step 5: Synthesis of (S)-5-allyloxycarbonyl-1-(9H-fluorene-9-yl)-8,11,14,17,20,23,26,29,32-nonamethyl-3,7,10,13,16,19,22,25,28,31-decaoxo-2-oxa-4,8,11,14,17,20,23,26,29,32-decaazatritetradecane-34-carboxylic acid (intermediates 3-6)

[0354] This compound was synthesized using a peptide solid-phase synthesis method, as follows:

[0355] 1) Dichloromethane was added to a mixture of CTC resin (0.64 mmol / g, 12.5 g) and N-(((9H-fluorene-9-yl)methoxy)carbonyl)-N-methylglycine (2.50 g) under nitrogen protection;

[0356] 2) Add diisopropylethylamine (DIEA) dropwise and mix and stir for 2 hours;

[0357] 3) Add methanol (13 mL) and mix and stir for 0.5 h;

[0358] 4) Filter and wash three times with DMF;

[0359] 5) Add 20% piperidine / DMF solution and react for 30 min;

[0360] 6) Filter and wash five times with DMF;

[0361] 7) Add the substances listed in the "Ingredients" column of the table below and mix for 30 seconds, then add the substances listed in the "Reagents" column of the table below. The reaction is carried out under nitrogen bubbling conditions for 1 hour, where Fmoc-Sar-OH represents N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine, and Fmoc-Asp-OAll-OH represents (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(allyloxy)-4-oxobutyric acid;

[0362] 8) Repeat steps 4-7 to complete the desired peptide synthesis.

[0363] Note:

[0364] After solid-phase preparation, a 20% dichloromethane solution of 1,1,1,3,3,3-hexafluoroisopropanol was added to the resulting mixture and the mixture was stirred for 1.5 h. The mixture was then filtered, the filtrate was collected and concentrated, and the resulting mixture was purified by preparative liquid chromatography (A: 0.075% aqueous trifluoroacetic acid, B: acetonitrile) to give the title compound (2.15 g).

[0365] MS m / z (ESI): 1035.6 [M+H] + .

[0366] Step 6: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid allyl ester (intermediate 3-7)

[0367] Intermediate 3-6 (1 g), ammonium chloride (155.09 mg), diisopropylethylamine (499.44 mg), and HATU (728.95 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 3 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (800 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0368] MS m / z (ESI): 1034.6 [M+H] + .

[0369] Step 7: Synthesis of (S)-30-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-1-amino-3,6,9,12,15,18,21,24,27-nonamethyl-1,4,7,10,13,16,19,22,25,28-decaoxo-3,6,9,12,15,18,21,24,27-nonazatrione-31-carboxylic acid (intermediates 3-8)

[0370] Intermediate 3-7 (800 mg), tetraphenylphosphine palladium (201.84 mg), and 1,3-dimethylbarbituric acid (108.71 mg) were added to DMF (5 mL), purged three times with nitrogen, and stirred at 25 °C for 4 h. After the reaction was complete, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The title compound (700 mg) was obtained by mixing 80 g of C 18 column with a mobile phase gradient of 0–40% acetonitrile / water at a flow rate of 40 mL / min.

[0371] MS m / z (ESI): 994.9 [M+H] + .

[0372] Step 8: Synthesis of resin-supported (2S,10S,19S)-19-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-48-amino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediates 3-9)

[0373] Intermediate 3-8 (700 mg) was added to DMF (10 mL), followed by intermediate L1-25 (total weight of resin and substrate 400 mg, content approximately 0.18 mmol), prepared according to Example 22 of PCT / CN2024 / 131800, approximately 0.18 mmol, O-benzotriazole-tetramethylurea hexafluorophosphate (336.58 mg), and diisopropylethylamine (205.62 mg). The reaction mixture was purged with nitrogen three times and then reacted at 25 °C with shaking for 1 h. After the reaction was complete, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated three times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (420 mg).

[0374] MS m / z (ESI): 1461.4 [M+Na] + .

[0375] Step 9: Synthesis of resin-supported (2S,10S,19S)-19,48-diamino-10-benzyl-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid (intermediate 3-10)

[0376] Intermediate 3-9 (420 mg) was dissolved in DMF (5 mL), and piperidine (1.25 mL) was added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 h. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (400 mg).

[0377] MS m / z (ESI): 1217.4 [M+H] + .

[0378] Step 10: Resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34 Synthesis of 37,40,43,46-Nonmethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-Decadecyloxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-Tetraazaoctadecane-1-carboxylic acid (intermediate 3-11)

[0379] Intermediate 3-10 (880 mg) and intermediate 3-5 (393.14 mg) were dissolved in N,N-dimethylformamide (15 mL), and O-benzotriazole-tetramethylurea hexafluorophosphate (453.65 mg) and N,N-diisopropylethylamine (313.82 μL) were added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 hour. After the reaction was completed, the resin was washed sequentially with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (920 mg).

[0380] Step 11: (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyrido[4,3-d]pyrimidine]-6'(7'H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34,37 Synthesis of 40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoic acid-1-carboxylic acid (intermediate 3-12)

[0381] Intermediate 3-11 (920 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25 °C for 0.5 hr. After the reaction was complete, the reaction solution was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (65 mg).

[0382] Step 12: (S)-N 4 -(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosane)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxopentano[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxa-2,7,10,13,16-pentazaoctadecane-18-yl)-2-(3-(2'-(cyclopentanesulfonyl)-5'-oxo-2,3,5,6-tetrahydro-5'H-spiro[pyran-4,8'-pyridino[4,3-d]pyrimidinyl]-6'(7'H)-yl)propionylamino)-N 4 Synthesis of methylbutyramide (drug linker L3-1)

[0383] Intermediate 3-12 (65 mg), intermediate 2-11 (22.10 mg, preparation method described in section 8.2 below), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.36 mg), pyridine (9.51 mg), and 1-hydroxybenzotriazole (10.82 mg) were dissolved in N,N-dimethylformamide (3 mL). The mixture was purged with nitrogen three times, and the reaction solution was stirred for 2 hours at 25°C under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [phase A: water (0.225% formic acid), phase B: acetonitrile]; B%: 21%-51%, 11 min) to obtain the title compound (35 mg).

[0384] 1H NMR (400MHz, DMSO-d6) δ = 9.28 (s, 1H), 8.69-8.55 (m, 2H), 8.30-8.21 (m, 2H), 8.01 (m, 2H), 7.79 (s, 1H), 7.52 (s, 1H), 7.39-7.27 (m, 1H), 7. 26-7.21(m,5H),7.15(s,1H),7.11-7.00(m,1H),6.49(s,1H),5.43(d,J=5.8Hz,4H),4.86-4.70(m,2H),4.70-4.63(m,1H),4.62-4.53(m,1 H),4.52-4.44(m,1H),4.40-4.17(m,10H),4.12-3.89(m,9H),3.86-3.76(m,5H),3.75-3.65(m,9H),3.63-3.52(m,2H),3.51-3.43(m,2H) ,3.00-2.68(m,32H),2.07-1.92(m,6H),1.91-1.80(m,2H),1.71-1.59(m,6H),1.01-0.92(m,1H),0.88(t,J=7.3Hz,3H),0.38-0.27(m,4H)

[0385] MS m / z(ESI): 1014.8 [(M+2) / 2] + .

[0386] 8.2 Synthesis of drug-linker L2-1

[0387] Synthesis route:

[0388] Synthesis of intermediates 2-4

[0389] Step 1: Synthesis of tert-butyl 3-(2-cyclopentathio-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionate (intermediate 2-2)

[0390] At room temperature, cyclopentylthiol (367 mg) and 2M sodium hydroxide solution (1.65 mL) were added sequentially to a dichloromethane (1.066 g) solution of compound 2-1. The mixture was stirred at room temperature for 30 min. The mixture was then separated by column chromatography (EA / PE, 0-50%) to obtain the crude title compound, which was used directly in the next reaction.

[0391] LC-MS: m / z (ESI): 378.4 [M+H] + .

[0392] Step 2: Synthesis of tert-butyl 3-(2-cyclopentanyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionate (intermediate 2-3)

[0393] At 0°C, mCPBA (2.43 g, 85% purity) was added to a mixture of crude intermediate 2-2 obtained in the previous step and DCM (50 mL). The mixture was reacted at room temperature for 30 min. The mixture was then concentrated under reduced pressure, and the residue was separated by column chromatography (EA / PE, 0-100%) to give the title compound (890 mg).

[0394] LC-MS: m / z (ESI): 410.5 [M+H] + .

[0395] 1 H NMR (400MHz, DMSO-d6) δ9.23 (s, 1H), 4.30-4.17 (m, 1H), 3.76 (t, J = 6.7Hz, 2H), 3.70 (t, J = 7.0Hz, 2H ),3.24(t,J=6.7Hz,2H),2.56(t,J=7.0Hz,2H),2.04-1.85(m,4H),1.74-1.57(m,4H),1.39(s,9H).

[0396] Step 3: Synthesis of 3-(2-cyclopentanyl-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionic acid (intermediates 2-4)

[0397] Trifluoroacetic acid (2 mL) was added to a mixture of intermediate 2-3 (135 mg) and DCM (10 mL) at 0 °C, and the mixture was reacted at room temperature for 2 h. The solvent was then removed under reduced pressure, and the mixture was separated by column chromatography (EA / PE, 0-100%) to give the title compound (80 mg).

[0398] LC-MS: m / z (ESI): 354.4 [M+H] + .

[0399] Synthesis of intermediate 2-11:

[0400] Step 1: Synthesis of 1-(benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 2-5)

[0401] 3',4'-Dihydroxyacetophenone (3 g) was dissolved in anhydrous DMF (25 mL), and deuterated dichloromethane (8.57 g) and potassium carbonate (8.18 g) were added. After the addition was complete, the mixture was heated to 90 °C and stirred for 16 h. The reaction mixture was then added to water (100 mL) and extracted with ethyl acetate (200 mL x 2). The combined organic phases were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (2.4 g).

[0402] MS m / z (ESI): 167.1 [M+H] + .

[0403] Step 2: Synthesis of 1-(6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)ethane-1-one (intermediate 2-6)

[0404] Intermediate 2-5 (2.4 g) was dissolved in anhydrous acetic acid (10 mL), and concentrated nitric acid (32.50 g, 70% purity) was added dropwise at 0 °C, followed by stirring at 0 °C for 10 min. The mixture was then heated to room temperature and stirred for 1 h. After the reaction was complete, the reaction solution was added dropwise to ice water (200 mL), filtered, and the filter cake was dried to give the title compound (1.9 g).

[0405] MS m / z(ESI): 212.0 [M+H] + .

[0406] 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H),7.30(s,1H),2.49(s,3H).

[0407] Step 3: Synthesis of N-(6-acetylbenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 2-7)

[0408] Intermediate 2-6 (1.8 g) was dissolved in acetic acid (25 mL), and acetic anhydride (1.84 g) and reduced iron powder (4.76 g) were added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.5 g).

[0409] MS m / z(ESI): 224.1 [M+H] + .

[0410] Step 4: Synthesis of N-(6-(2-bromoacetyl)benzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)acetamide (intermediate 2-8)

[0411] A solution of HBr in acetic acid (2.39 g, 33% purity) was added dropwise to a solution of intermediate 2-7 (1.45 g) in anhydrous acetic acid (25 mL), and then Br was added dropwise. 2( 1.07 g was added dropwise, and the mixture was stirred at room temperature for 1 h. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure. The residue was added to water (50 mL), extracted with ethyl acetate (50 mL * 2), the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:1) to give the title compound (1.3 g).

[0412] MS m / z (ESI): 302.1 [M+H] + .

[0413] Step 5: Synthesis of 1-(6-aminobenzo[d][1,3]dioxacyclopenten-5-yl-2,2-d2)-2-chloroethane-1-one (intermediate 2-9)

[0414] Intermediate 2-8 (1.2 g) and concentrated hydrochloric acid (144.82 mg) were dissolved in ethanol (15 mL), and the reaction mixture was stirred at 60 °C for 16 h. After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high performance liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; water (containing 0.05% NH4HCO3) and a mixture of acetonitrile with decreasing polarity as the eluent (acetonitrile gradient ratio 40%-50%), to give the title compound (577 mg).

[0415] MS m / z(ESI): 216.0 [M+H] + .

[0416] Step 6: Synthesis of (S)-14-(chloromethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 2-10)

[0417] Intermediate 2-9 (100.0 mg) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazine-3,6,10(4H)-trione (109.87 mg) were dissolved in toluene (1 mL) and acetic acid (1 mL), and pyridinium p-toluenesulfonic acid salt (5.24 mg) was added. The reaction mixture was stirred at 100 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature and concentrated to dryness under reduced pressure. Ethanol (5 mL) was added, and the mixture was stirred at 25 °C for 0.5 h. The mixture was filtered, and the filter cake was washed with ethanol (5 mL * 2) to obtain the title compound (100.0 mg).

[0418] MS m / z(ESI): 443.0 [M+H] + .

[0419] Step 7: Synthesis of (S)-14-(aminomethyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxacyclopenteno[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione-2,2-d2 (intermediate 2-11)

[0420] Intermediate 2-10 (100.00 mg) was dissolved in anhydrous ethanol (1.5 mL) and anhydrous DMF (1.5 mL), and hexamethylenetetramine (94.97 mg) was added. The reaction mixture was stirred at 50 °C for 6 h. After the reaction was completed, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was purified by high performance liquid chromatography (column: Boston Green ODS150*30 mm*5 μm; mobile phase: [A: water (formic acid), B: acetonitrile]; B%: 0%-30%, 12 min) to give the title compound (25.0 mg).

[0421] MS m / z(ESI): 424.0 [M+H] + .

[0422] Synthesis of drug-linker L2-1

[0423] Step 1: Synthesis of resin-supported (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoate-1-carboxylic acid (intermediate 2-12)

[0424] Intermediate 3-10 (535 mg) and intermediate 2-4 (187.41 mg) were dissolved in N,N-dimethylformamide (10 mL), and O-benzotriazole-tetramethylurea hexafluorophosphate (270 mg) and diisopropylethylamine (309 μL) were added. The reaction solution was placed on a shaker at 25 °C and shaken for 1 hour. After the reaction was completed, the resin was washed successively with methanol (10 mL) and dichloromethane (10 mL), repeated 3 times. The resin was then filtered to dryness and dried under vacuum to obtain the title compound (550 mg).

[0425] Step 2: Synthesis of (2S,10S,19S)-48-amino-10-benzyl-19-(3-(2-(cyclopentanesulfonyl)-5-oxo-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propionylamino)-2-cyclopropyl-22,25,28,31,34,37,40,43,46-nonamethyl-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-pentadecaoxo-3-oxa-5,8,11,14,17,22,25,28,31,34,37,40,43,46-tetradecanoate-1-carboxylic acid (intermediate 2-13)

[0426] Intermediate 2-12 (550 mg) was added to a mixed solvent of dichloromethane (8 mL) and 1,1,1,3,3,3-hexafluoroisopropanol (2 mL) and shaken at 25 °C for 0.5 hr. After the reaction was complete, the reaction solution was filtered to remove the resin, and the filtrate was concentrated to dryness under reduced pressure. The residue was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 18%-38%, 20 min) to obtain the title compound (35 mg).

[0427] Step 3: (S)-N 4-(26-amino-3,6,9,12,15,18,21,24-octamethyl-2,5,8,11,14,17,20,23,26-nonaoxo-3,6,9,12,15,18,21,24-octaazahexacosane-1-yl)-N 1 -((4S,12S)-12-benzyl-4-cyclopropyl-1-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxadideuteropentano[4,5-g]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-14-yl-2,2-d2)-3,8,11,14,17-pentoxo-5-oxa-2,7,10,13,16-pentazaoctadecane-18-yl)-2-(3-(2-(cyclopentanylsulfonyl)-5-oxo-7,8-dihydropyridino[4,3-d]pyrimidin-6(5H)-yl)propionylamino)-N 4 Synthesis of methylbutyramide (drug linker L2-1)

[0428] Intermediate 2-13 (35 mg), intermediate 2-11 (9.54 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.64 mg), pyridine (5.35 mg), and 1-hydroxybenzotriazole (6.09 mg) were dissolved in N,N-dimethylformamide (1 mL). The mixture was purged with nitrogen three times, and the reaction solution was stirred for 2 hours at 25°C under nitrogen protection. After the reaction was completed, the reaction solution was directly purified by high performance liquid chromatography (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [A: water (0.225% formic acid), B: acetonitrile]; B%: 25%-45%, 20 min) to obtain the title compound (8.2 mg).

[0429] 1H NMR (400MHz, DMSO-d6) δ = 9.22 (s, 1H), 8.72-8.64 (m, 1H), 8.59 (d, J = 4.9Hz, 1H), 8.35-8.21 (m, 2H), 8.09-7.91 (m, 2H),7.80(s,1H),7.52(s,1H),7.35-7.19(m,5H),7.18-7.15(m,1H),7.11-7.04(m,1H),6.53-6.49(m,1H),5.44( d,J=9.8Hz,3H),4.83-4.30(m,9H),4.28-3.82(m,13H),3.78-3.48(m,18H),3.08-2.64(m,30H),2.04-1.80(m,6H ),1.73-1.56(m,4H),1.53-1.39(m,1H),1.24(s,3H),1.01-0.92(m,1H),0.87(t,J=7.2Hz,3H),0.43-0.26(m,4H)

[0430] MS m / z(ESI): 979.6 [(M+2H) / 2] + .

[0431] 8.3 Conjugation of EGFR×cMet bispecific antibodies with drug linkers L2-1 and L3-1

[0432] The specific methods for conjugation and analysis are described in Example 3. The DAR value and SEC purity of the prepared antibody-drug conjugates are shown in Table 16.

[0433] Table 16. Preparation, DAR value, and SEC purity of antibody-drug conjugates

[0434] Example 9: In vitro assay of EGFR×cMet bispecific antibody ADC inhibiting tumor cell proliferation

[0435] The specific experimental methods are described in Example 5. The results are shown in Table 17 and Figure 9. ADC-6 to 10 all exhibited good inhibitory activity against the proliferation of two tumor cells expressing EGFR and cMet, and their in vitro inhibitory activity was no weaker than that of the Yangshen molecule AZD9592. Comparison with Table 14 shows that the killing activity of EGFR×cMet dual antibodies coupled with different compounds was comparable.

[0436] Table 17. Inhibitory effect of EGFR×cMet bispecific antibody ADC on tumor cell proliferation in vitro

[0437] Example 10: Efficacy Evaluation of the NCI-H1975 Subcutaneous Tumor Model

[0438] Cells: Lung cancer NCI-H1975 cells.

[0439] Animal information: Balb / c nude mouse, female, 6-7 weeks old, weighing approximately 16-22 grams.

[0440] Administration: The dosage of the antibody-drug conjugate ADC-9 and the ginseng molecule AZD9592 disclosed herein was 0.3, 1.5, and 5 mg / kg; the dosage of the control ADC-ISO-L3 was 5 mg / kg; administration was via a single tail vein injection. Six mice were used in each group.

[0441] The specific experimental method is described in Example 6, where the average tumor volume (mm³) was measured at specific time points (day 0, day 2, day 5, day 8, day 12, day 15, day 19, day 21, and day 25). 3 The results are shown in Table 18 and Figure 10. In the mouse subcutaneous xenograft NCI-H1975 model, ADC-9 significantly inhibited tumor growth at doses of 1.5 mg / kg and 5 mg / kg (P<0.0001), which was significantly superior to the same dose of Yangshen AZD9592 (P<0.0001). Furthermore, at the 5 mg / kg dose, tumor recurrence began in the AZD9592 group from day 16, while the efficacy of ADC-9 was maintained at least until day 25 post-administration. In this example, no effect on mouse body weight or death was observed at the tested doses, and the mice tolerated the medication well.

[0442] Table 18. Tumor volume in the NCI-H1975 subcutaneous tumor model

[0443] Example 11: Efficacy Evaluation of HCT116 Subcutaneous Tumor Model

[0444] Cells and experimental reagents: HCT116 colorectal cancer cells; McCoy'5A culture medium: Gbico; Cat No.: 16600-082; cultured and expanded using conventional methods in the art.

[0445] Animal information: Balb / c nude mouse, female, 6-7 weeks old, weighing approximately 16-22 grams.

[0446] Drug administration: In the seeded cells (0.1 ml / (containing 2.5 × 10⁻⁶ cells), administer the drug. 6 On day 7 after subcutaneous inoculation (under the armpit), mice were randomly assigned to groups based on tumor volume, with the grouping day designated as Day 0. The antibody-drug conjugate ADC-9 and the ginseng molecule AZD9592 were administered at doses of 0.3, 1.5, and 5 mg / kg; the control ADC-ISO-L3 was administered at doses of 1.5 and 5 mg / kg; administration was a single tail vein injection. Six mice were in each group.

[0447] The remaining experimental methods were the same as in Example 6, with the average tumor volume (mm²) measured at specific time points (day 0, day 5, day 8, day 13, day 16, day 19, day 22, and day 26). 3 The results are shown in Table 19 and Figure 11. In the mouse subcutaneous xenograft HCT116 model, ADC-9 significantly inhibited tumor growth at doses of 1.5 and 5 mg / kg (P<0.0001); and ADC-9 was significantly superior to the same dose of Yangshen AZD9592 at medium and high doses (P<0.0001, P<0.001). In this example, no effect on mouse body weight or death was observed at the tested doses, and the mice tolerated the treatment well.

[0448] Table 19. Tumor volume in the HCT116 subcutaneous tumor model

[0449] Example 12: Efficacy Evaluation of IM95#206 Subcutaneous Tumor Model

[0450] Cells and experimental reagents: Gastric cancer IM95#206 cells: Primary extracted cells, obtained by inoculating mice with IM95 cell line to form tumors and expanding them according to conventional tumor primary cell preparation methods in the art; DMEM culture medium: Gbico; Cat No.: 10569044; cultured and expanded using conventional methods in the art.

[0451] Animal information: Balb / c nude mouse, female, 6-7 weeks old, weighing approximately 16-22 grams.

[0452] Administration: In the seeded cells (0.1 ml / (containing 1×10⁻⁶ cells)... 7 On day 13 after subcutaneous inoculation (in the axilla), mice were randomly assigned to groups based on tumor volume, with the grouping day designated as Day 0. The antibody-drug conjugate ADC-9 and the ginseng molecule AZD9592 were administered at doses of 0.3, 1.5, and 5 mg / kg via a single tail vein dose; the isotype control ADC-ISO-L3 was administered at doses of 1.5 and 5 mg / kg via a single tail vein dose. Six mice were in each group.

[0453] The remaining experimental methods were the same as in Example 6, with the average tumor volume (mm²) measured at specific time points (day 0, day 2, day 6, day 9, day 13, day 16, day 20, day 23, day 27, day 36, and day 43). 3The results are shown in Table 20 and Figure 12. In the mouse subcutaneous xenograft tumor IM95#206 model, ADC-9 significantly inhibited tumor growth at doses of 1.5 and 5 mg / kg (P<0.0001), and was significantly superior to the same dose of Yangshen AZD9592 (P<0.0001). Furthermore, the inhibitory effect of ADC-9 on tumor growth at a dose of 1.5 mg / kg was better than that of Yangshen AZD9592 at a dose of 5 mg / kg. In this example, no effect on mouse body weight or death was observed at the tested doses, and the mice could tolerate the treatment.

[0454] Table 20. Tumor volume in the IM95#206 subcutaneous tumor model

[0455] Example 13: Conjugation of EGFR×cMet bispecific antibody and vcMMAE

[0456] The antibody prepared in Example 1 was dialyzed to a 20 mM PB, 1 mM EDTA solution (pH 6.5). Two to eight molar equivalents of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) were added to the antibody solution, and the mixture was incubated on a metal shaker at 4°C for 16-18 hours to reduce the antibody. Eight to ten molar equivalents of the drug-linker L4-2 were dissolved in DMSO and added to the reaction system. The reaction mixture was coupled at 25°C for 2 hours. The reaction product was desalted using a G25 column and the buffer was changed to a 10 mM NaAC-HAC, 9% sucrose pH 5.5 buffer to remove unreacted free small molecule toxins, yielding the antibody-drug conjugate. Purity and DAR value analysis were performed according to Experimental Method 1 and Example 3.2. The DAR value and SEC purity of the prepared antibody-drug conjugate are shown in Table 22.

[0457] The structure of the drug-linker L4-2 is shown below. It was purchased from MedChemExpress (MCE) under catalog number HY-15575.

[0458] Table 22. Preparation, DAR value, and SEC purity of antibody-drug conjugates

[0459] Example 14: In vitro assay of EGFR×cMet bispecific antibody ADC inhibiting tumor cell proliferation

[0460] The specific experimental methods are described in Example 5. The results are shown in Table 23 and Figure 13. ADC-16-20 exhibited good inhibitory activity against the proliferation of both EGFR and cMet-expressing tumor cells, and its in vitro inhibitory activity was comparable to that of the Yangshen molecule AZD9592. Comparison with Tables 14 and 17 shows that the killing activity of EGFR×cMet dual antibodies coupled with different compounds was comparable.

[0461] Table 23. Inhibitory effect of EGFR×cMet bispecific antibody ADC on tumor cell proliferation in vitro

[0462] All teachings of the patents, publications and references cited in this article are incorporated herein by reference in their entirety.

[0463] While exemplary embodiments have been specifically shown and described, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the embodiments covered by the appended claims.

Claims

1. A bispecific antibody or antigen-binding fragment capable of specifically binding EGFR and cMet, wherein, The antibody or antigen binding fragment comprises a first antigen binding domain that specifically binds EGFR comprising a heavy chain variable region VH EGFR and / or a light chain variable region VL EGFR , and a second antigen binding domain that specifically binds cMet comprising a heavy chain variable region VH cMet and / or a light chain variable region VL cMet ; wherein, (1) the antibody or antigen binding fragment comprises a first peptide chain, a second peptide chain, and a third peptide chain, wherein the first peptide chain comprises VL cMet -CL, the second peptide chain comprises VH cMet -CH1-Fc, and the third peptide chain comprises a single chain unit VL EGFR -VH EGFR -Fc; or (2) the antibody or antigen binding fragment comprises a first peptide chain, a second peptide chain, and a third peptide chain, wherein the first peptide chain comprises VL EGFR -CL, the second peptide chain comprises VH EGFR -CH1-Fc, and the third peptide chain comprises a single chain unit VL cMet -VH cMet -Fc; or (3) the antibody or antigen binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise a VL cMet - VL EGFR - CL, the second peptide chain and the third peptide chain each comprise a VH cMet - VH EGFR - CH1-Fc; or (4) the antibody or antigen binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise VL cMet -CL, the second peptide chain comprises VH cMet -CH1-Fc, the third peptide chain comprises VH cMet -CH1-Fc-VL EGFR -VH EGFR ; or (5) the antibody or antigen binding fragment comprises a first peptide chain, a second peptide chain, a third peptide chain, and a fourth peptide chain, wherein the first peptide chain and the fourth peptide chain each comprise VL cMet -CL, the second peptide chain comprises VH cMet -CH1-Fc, the third peptide chain comprises VH cMet -CH1-VL EGFR -VH EGFR -Fc.

2. The antibody or antigen-binding fragment of claim 1, wherein, the VH EGFR comprises HCDR1 EGFR , HCDR2 EGFR , and HCDR3 EGFR , and / or the VL EGFR comprises LCDR1 EGFR , LCDR2 EGFR , and LCDR3 EGFR ; wherein (1) the HCDR1 EGFR -HCDR3 EGFR HCDR1-HCDR3 of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 9, respectively, and / or the LCDR1 EGFR -LCDR3 EGFR LCDR1-LCDR3 of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 8, respectively; or (2) the HCDR1 EGFR -HCDR3 EGFR are as set forth in SEQ ID NOs: 38-40, respectively, and / or the LCDR1 EGFR -LCDR3 EGFR are as set forth in SEQ ID NOs: 55-57, respectively; or (3) the HCDR1 EGFR -HCDR3 EGFR respectively as shown in SEQ ID NOs: 41-43, and / or the LCDR1 EGFR -LCDR3 EGFR respectively as shown in SEQ ID NOs: 58-59, 57; or (4) the HCDR1 EGFR -HCDR3 EGFR respectively as set forth in SEQ ID NOs: 44-46, and / or the LCDR1 EGFR -LCDR3 EGFR respectively as set forth in SEQ ID NOs: 55-57.

3. The antibody or antigen-binding fragment of claim 1, wherein, the VH cMet comprises HCDR1 cMet , HCDR2 cMet , and HCDR3 cMet , and / or the VL cMet comprises LCDR1 cMet , LCDR2 cMet , and LCDR3 cMet ; wherein (1) the HCDR1 cMet -HCDR3 cMet are HCDR1-HCDR3 of SEQ ID NO: 13, respectively, and / or the LCDR1 cMet -LCDR3 cMet are LCDR1-LCDR3 of SEQ ID NO: 12, respectively; or (2) the HCDR1 cMet -HCDR3 cMet are as set forth in SEQ ID NOs: 47-49, respectively, and / or the LCDR1 cMet -LCDR3 cMet are as set forth in SEQ ID NOs: 60-62, respectively; or (3) the HCDR1 cMet - HCDR3 cMet as set forth in SEQ ID NOs: 50-52, respectively, and / or the LCDR1 cMet - LCDR3 cMet as set forth in SEQ ID NOs: 63-64, 62, respectively; or (4) the HCDR1 cMet - HCDR3 cMet are as set forth in SEQ ID NOs: 53-54, 49, respectively, and / or the LCDR1 cMet - LCDR3 cMet are as set forth in SEQ ID NOs: 60-62, respectively.

4. The antibody or antigen binding fragment of claim 1 or 2, wherein the VH EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 9, or a sequence having at least 70% identity or at most 15 mutations thereto; and / or The VL EGFR comprises an amino acid sequence as set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 8, or a sequence having at least 70% identity or at most 15 mutations thereto.

5. The antibody or antigen binding fragment of claim 1 or 3, wherein the VH cMet comprises an amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having at least 70% identity or at most 15 mutations thereto; and / or the VL cMet comprises an amino acid sequence as set forth in SEQ ID NO: 12, or a sequence having at least 70% identity or up to 15 mutations thereto.

6. The antibody or antigen-binding fragment of any one of claims 1-5, wherein, The antibody or antigen binding fragment comprises a heavy chain HC and a light chain LC, wherein The heavy chain HC has an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence having at least 70% identity or at most 15 mutations thereto; and / or The light chain LC has an amino acid sequence as set forth in SEQ ID NO: 22, or a sequence having at least 70% identity or at most 15 mutations thereto.

7. The antibody or antigen-binding fragment of any one of claims 1-5, wherein, The antibody or antigen binding fragment comprises a first heavy chain HC-1, a second heavy chain HC-2, and a light chain LC, wherein The first heavy chain HC-1 has an amino acid sequence as set forth in SEQ ID NO: 25 or SEQ ID NO: 30, or a sequence having at least 70% identity or at most 15 mutations thereto; and / or The second heavy chain HC-2 has an amino acid sequence as set forth in any one of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 31, or a sequence having at least 70% identity or at most 15 mutations thereto; and / or The light chain LC has an amino acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 29, or a sequence having at least 70% identity or at most 15 mutations thereto.

8. The antibody or antigen binding fragment of any one of claims 4-7, wherein The at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; The at most 15 mutations are at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 mutations; The mutations are insertions, deletions, or substitutions, preferably back mutations or hot spot mutations; The substitutions are preferably conservative amino acid substitutions.

9. The antibody or antigen-binding fragment of any one of claims 1-8, wherein, The antibody or antigen binding fragment further comprises a "knob chain" and a "hole chain".

10. The antibody or antigen-binding fragment of claim 9, wherein, The antibody or antigen binding fragment comprises a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, or / and Y407V mutation in the CH3 domain of the "hole chain".

11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein, VL in the single chain unit EGFR and VH EGFR or VL cMet and VH cMet Cysteine mutations to introduce disulfide bonds can also be included.

12. The antibody or antigen-binding fragment of claim 11, wherein, the cysteine mutation is VL EGFR or VL cMet the amino acid at position 100 is mutated from G to C, and / or the cysteine mutation is VH EGFR or VH cMet the amino acid at position 44 is mutated from G to C, thereby introducing a pair of disulfide bonds.

13. The antibody or antigen-binding fragment of any one of claims 1-12, wherein, The antibody or antigen binding fragment comprises: (1) a chimeric antibody or fragment thereof; and / or (2) a humanized antibody or fragment thereof; and / or, (3) a fully human antibody or fragment thereof; Preferably, the antibody or antigen binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv, a diabody, or a single domain antibody.

14. The antibody or antigen-binding fragment of any one of claims 1-13, wherein, The antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody, and antibody minimal recognition unit.

15. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having a general structure of Pc-(L-D)n, wherein, D is a cytotoxic drug; L is a linker unit; Pc is the bispecific antibody or antigen-binding fragment capable of specifically binding to EGFR and cMet according to any one of claims 1-14; and n is a real number from 1 to 16.

16. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 15, wherein, The cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics; Preferably, the cytotoxic drug D is selected from microtubulin inhibitors including dolastatin, auristatin, maytansine, Tubulysins, and cryptomycins, DNA damaging agents including PBDs, and DNA topoisomerase inhibitors including camptothecins. Preferably, the cytotoxic drug D is selected from auristatin or DNA topoisomerase inhibitors.

17. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 15 or 16, wherein, the cytotoxic drug D is selected from MMAE or a compound of formula (D-I), wherein, R 1 , R 2 with the atom to which they are attached form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, which is optionally substituted by one or more D atoms; R 4 selected from H or C1-C3alkyl; R 5 selected from H, halogen, CN, OH, NH2or Ci-C3alkyl; R 6 selected from H or C1-C3alkyl; R 7 is selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, said C1-C3 alkyl or C3-C6 cycloalkyl being optionally substituted by D, halogen, CN, =0, OH, NH2or C1-C3 alkyl.

18. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 17, wherein, R 1 R 2 with the atom to which they are attached 19. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 17 or 18, wherein, R 4 , R 5 , R 6 are each selected from H.

20. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 17-19, wherein, said R 7 selected from cyclopropyl.

21. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 17-20, wherein, The compound of formula (D-I) is selected from one of the following compounds:

22. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 15 or 16, wherein, The cytotoxic drug D is selected from one of the following compounds:

23. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 15-22, wherein, said linker unit L is selected from covalently attached at the a end to Pc as described in claim 15 and covalently attached at the b end to a cytotoxic drug D, wherein: Ring A is selected from R b1 , R b2 each independently is selected from H, halogen, CN, C1-C6 alkyl or C3-C6 cycloalkyl, or R b1 , R b2 together with the carbon atom to which they are attached form a C3-C6 cycloalkyl or 4-7 membered heterocyclyl, said C3-C6 cycloalkyl or 4-7 membered heterocyclyl being optionally substituted with one or more substituents selected from halogen, CN, =0, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl; m1 is selected from an integer from 2 to 8; L a selected from a chemical bond or Its * end and L b Connection, R b3 Selected from H or C1-C6 alkyl groups, m2 is selected from integers 1 to 8, R b4 R b5 Each is independently selected from H, halogen, CN, C1-C6 alkyl, C3-C6 cycloalkyl, L b a peptide residue consisting of 1 to 8 amino acids, which is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, and 4-7 membered heterocyclyl.

24. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 23, wherein, said ring A is selected from R b1 , R b2 are each selected from H, or R b1 , R b2 together with the carbon atom to which they are attached form a 4-7 membered heterocyclyl group, optionally substituted with one or more halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl, and 4-7 membered heterocyclyl substituents; Preferably, said ring A is selected from R b1 , R b2 are each selected from H, or R b1 , R b2 together with the carbon atom to which they are attached form a 4-7 membered heterocyclyl group; More preferably, said ring A is selected from 25. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 23 or 24, wherein, L a is selected from a chemical bond or is selected from a chemical bond or b is selected from H or C1-C6 alkyl, R b3 is selected from H or C1-C6 alkyl, R b4 , R b5 one is selected from H and the other is selected from Preferably, said L a is selected from a chemical bond or Its end with L b connected.

26. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 23-25, wherein, The L b a peptide residue consisting of 1 to 8 amino acids, which is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6alkyl, OH, 0(C1-C6alkyl), NH2, NH(C1-C6alkyl), N(C1-C6alkyl)2, C3-C6cycloalkyl and 4-7 membered heterocyclyl; Preferably, said L b is a Gly-Gly-Phe-Gly tetrapeptide residue.

27. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 23-26, wherein, m1 is selected from an integer from 2 to 8; preferably, m1 is an integer from 2 to 6; more preferably, m1 is 2, 3, 4, or 5.

28. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 15-27, wherein, The linker unit L is which is covalently attached at the a end to the Pc described in claim 15 and at the b end to the cytotoxic drug D.

29. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 15, wherein, The antibody-drug conjugate or pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

30. An isolated nucleic acid molecule, wherein, The nucleic acid molecule encodes the antibody or antigen-binding fragment according to any one of claims 1-14.

31. An expression vector, wherein, The expression vector comprises the nucleic acid molecule according to claim 30.

32. A host cell, wherein, The host cell comprises the nucleic acid molecule according to claim 30, and / or the expression vector according to claim 31; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, E. coli, and / or B. subtilis; more preferably, the host cell is selected from Expi293 or CHO cell.

33. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the antibody or antigen-binding fragment according to any one of claims 1-14, or the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 15-29, and a pharmaceutically acceptable excipient.

34. The pharmaceutical composition of claim 33, wherein, The pharmaceutical composition further comprises other therapeutic agents; preferably, each part in the pharmaceutical composition, such as the antibody-drug conjugate and other therapeutic agents, can be packaged independently or in combination.

35. A method of treating cancer or a tumor in a mammal, wherein, comprising administering to a mammal, preferably a human, in need of such therapy, a therapeutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-14, or the antibody-drug conjugate of any one of claims 15-29, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 33 or 34; optionally, the cancer or tumor comprises one or more of lung cancer, pancreatic cancer, liver cancer, hepatocellular liver cancer, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer, kidney cancer, cervical cancer, prostate cancer, uterine cancer, melanoma, head and neck squamous cell carcinoma, cholangiocarcinoma, thyroid cancer, ovarian cancer, or glioblastoma.

36. The method of treating a tumor in a mammal according to claim 33, wherein, The antibody or antigen-binding fragment of any one of claims 1-14, or the antibody-drug conjugate of any one of claims 15-29, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 33 or 34 can be used in combination with other therapeutic agents.

37. Use of the antibody or antigen-binding fragment of any one of claims 1-14, or the antibody-drug conjugate of any one of claims 15-29, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 33 or 34 in the manufacture of a medicament for treating a cancer or tumor; optionally, the cancer or tumor comprises one or more of lung cancer, pancreatic cancer, liver cancer, hepatocellular liver cancer, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer, kidney cancer, cervical cancer, prostate cancer, uterine cancer, melanoma, head and neck squamous cell carcinoma, cholangiocarcinoma, thyroid cancer, ovarian cancer, or glioblastoma.

38. A product produced by a process of any one of claims 1-14, or a product produced by a process of any one of claims 15-29, or a pharmaceutical composition of claim 33 or 34, wherein, The product is for treating a cancer or tumor; optionally, the cancer or tumor comprises one or more of lung cancer, pancreatic cancer, liver cancer, hepatocellular liver cancer, breast cancer, colorectal cancer, gastric cancer, esophageal cancer, nasopharyngeal cancer, kidney cancer, cervical cancer, prostate cancer, uterine cancer, melanoma, head and neck squamous cell carcinoma, cholangiocarcinoma, thyroid cancer, ovarian cancer, or glioblastoma.

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