Antigen-binding molecule specifically binding to EGFR and cmet, drug conjugate thereof, and medical use thereof

Abstract

The present invention relates to an antigen-binding molecule specifically binding to EGFR and cMET, a drug conjugate thereof, and medical use thereof. In particular, the present invention relates to a bispecific antibody-drug conjugate specifically binding to EGFR and cMET, and use thereof in treating a tumor.

Description

Antigen-binding molecules that specifically bind to EGFR and cMET, their drug conjugates, and their pharmaceutical uses Technical Field

[0001] This disclosure pertains to the field of biotechnology and relates to antigen-binding molecules that specifically bind to EGFR and cMET, their drug conjugates, and their pharmaceutical uses. Background Technology

[0002] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0003] Epidermal growth factor receptor (EGFR, also known as HER1 or ERBB1) is a receptor-type tyrosine kinase. It is overexpressed in various epithelial tumors and is associated with tumor proliferation, angiogenesis, and metastasis; it is also expressed in many normal tissues, such as epithelial tissues like skin and hair follicles.

[0004] EGFR is a crucial drug target in the clinical treatment of NSCLC, and several small molecule inhibitors targeting EGFR have been approved for marketing. However, with the clinical use of these small molecule inhibitors, various resistance mechanisms have emerged. For example, with osimertinib, the world's best-selling third-generation EGFR small molecule inhibitor, nearly 30-50% of patients currently lack corresponding targeted clinical treatment options after developing resistance (Osimertinib Resistance: Molecular Mechanisms and Emerging Treatment Options, Georgia Gomatou, et al., Cancers, 2023, 15, 841). This has created opportunities for the development of large molecule drugs targeting EGFR.

[0005] Hepatocyte growth factor receptor (HGFR, also known as cMET) is a receptor-type tyrosine kinase. In cancer, gene mutations, overexpression, and amplification of cMET can lead to abnormal activation of the HGF-cMET signaling pathway, promoting tumor development and progression. In normal tissues, cMET expression is limited and at low levels. cMET amplification is one of the important reasons for osimertinib resistance, and a high proportion (62%) of patients with osimertinib resistance have intermediate to high expression of cMET (MET Biomarker-based Preliminary Efficacy Analysis in SAVANNAH: savolitinib+osimertinib in EGFRm NSCLC Post-Osimertinib, Mj. Ahn, et al., Journal of Thoracic Oncology, 17(9S), S470).

[0006] Several EGFR-targeting ADCs have entered clinical trials and shown promising efficacy, such as Sichuan Baili Pharmaceutical's EGFR-HER3 bispecific antibody ADC BL-B01D1. However, clinical data indicate that this molecule has significant hematologic toxicity (BL-B01D1, a first-in-class EGFRxHER3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic solid tumor: Results from a first-in-human phase 1 study, Li Zhang et al., Journal of Clinical Oncology, 41(16)). Therefore, there is still a need in clinical practice to develop EGFR-targeting ADCs. Summary of the Invention

[0007] <Antigen-binding molecules that specifically bind to EGFR and cMET>

[0008] This disclosure provides an antigen-binding molecule that specifically binds to EGFR and cMET, comprising at least one antigen-binding module specifically binding to EGFR and at least one antigen-binding module specifically binding to cMET. The EGFR-binding module comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the cMET-binding module comprises a heavy chain variable region cMET-VH and a light chain variable region cMET-VL.

[0009] (1) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0010] The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25; or

[0011] (2) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0012] The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25.

[0013] In some embodiments, the antigen-binding molecules that specifically bind to EGFR and cMET as described above, wherein the heavy chain variable regions HCDR1, HCDR2, and HCDR3 and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to a numbering rule selected from Kabat, IMGT, Chothia, AbM, and Contact.

[0014] In some implementations, the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the light chain variable regions LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering rules.

[0015] In some embodiments, the antigen-binding molecule that specifically binds EGFR and cMET as described in any of the preceding embodiments, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 14 or 15, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 16; and the cMET-VH comprises the amino acid sequence of SEQ ID NO: 26; and the cMET-VL comprises the amino acid sequence of SEQ ID NO: 27.

[0016] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments, wherein the antigen-binding module that specifically binds to EGFR or the antigen-binding module that specifically binds to cMET comprises a Titin chain and an Obscurin chain capable of forming a dimer (the Titin chain and Obscurin chain in WO2022237882A1 are incorporated by reference).

[0017] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments includes an Fc region, which is preferably an IgG Fc region, more preferably an IgG1 Fc region; more preferably, the Fc region includes one or more amino acid substitutions that can reduce the binding of the Fc region to the Fcγ receptor.

[0018] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0019] In some embodiments, Fc1, as described in any of the preceding embodiments, has a protruding structure according to the pestle and mortar technique, and Fc2 has a hole structure according to the pestle and mortar technique; or, Fc2 has a protruding structure according to the pestle and mortar technique, and Fc1 has a hole structure according to the pestle and mortar technique.

[0020] In some embodiments, Fc1, as described in any of the preceding embodiments, has a raised structure according to the pestle and mortar technique, and Fc2 has a hole structure according to the pestle and mortar technique.

[0021] In some embodiments, as described in any of the preceding embodiments, Fc1 has amino acid C at position 354 and amino acid W at position 366; and Fc2 has amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index; or, Fc2 has amino acid C at position 354 and amino acid W at position 366; and Fc1 has amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0022] In some implementations, Fc1 and Fc2, as described in the preceding one, further include amino acid A at position 234 and amino acid A at position 235, numbered according to the EU index.

[0023] In some embodiments, as described in the preceding one, Fc1 has amino acid A at position 234, amino acid A at position 235, amino acid C at position 354, and amino acid W at position 366; and Fc2 has amino acid A at position 234, amino acid A at position 235, amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0024] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 34 and Fc2 comprises the amino acid sequence of SEQ ID NO: 35.

[0025] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET, as described in any of the preceding embodiments, wherein the antigen-binding molecule comprises an antigen-binding module that specifically binds to EGFR and an antigen-binding module that specifically binds to cMET.

[0026] In some embodiments, the antigen-binding molecule that specifically binds EGFR and cMET as described in any of the preceding embodiments comprises an antigen-binding module that specifically binds EGFR and an antigen-binding module that specifically binds cMET, wherein the antigen-binding module that specifically binds cMET is a Fab, and the antigen-binding module that specifically binds EGFR is a replaced Fab comprising a dimeric Titin chain and an Obscurin chain; or, the antigen-binding module that specifically binds cMET is a replaced Fab comprising a dimeric Titin chain and an Obscurin chain, and the antigen-binding module that specifically binds EGFR is a Fab.

[0027] In some embodiments, an antigen-binding molecule that specifically binds EGFR and cMET, as described in any of the preceding embodiments, comprises an antigen-binding module that specifically binds EGFR and an antigen-binding module that specifically binds cMET, wherein the antigen-binding module that specifically binds cMET is a Fab, and the antigen-binding module that specifically binds EGFR is a replaced Fab comprising a Titin chain and an Obscurin chain capable of forming a dimer.

[0028] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET, as described in any of the preceding embodiments, comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d).

[0029] (a)[cMET-VH]-[CH1]-[Fc1],

[0030] (b)[cMET-VL]-[CL],

[0031] (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0032] (d)[EGFR-VL]-[connector 2]-[Obscurin],

[0033] The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0034] The structures shown in equations (a), (b), (c) and (d) are arranged from the N end to the C end.

[0035] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET, as described in any of the preceding embodiments, comprises a first chain having the structure shown in formula (e), a second chain having the structure shown in formula (f), a third chain having the structure shown in formula (g), and a fourth chain having the structure shown in formula (h).

[0036] (e)[EGFR-VH]-[CH1]-[Fc1],

[0037] (f)[EGFR-VL]-[CL],

[0038] (g)[cMET-VH]-[connector 3]-[Titin]-[Fc2],

[0039] (h)[cMET-VL]-[connector 4]-[Obscurin],

[0040] The linker 3 and linker 4 may be the same or different, and are peptide linkers; or linker 3 or linker 4 may not exist.

[0041] The structures shown in equations (e), (f), (g), and (h) are arranged from the N end to the C end.

[0042] In some embodiments, the antigen-binding molecules that specifically bind to EGFR and cMET as described in any of the preceding claims, wherein the linkers 1-4 are peptide linkers known in the art, provided that the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide having 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, or GGGGS (SEQ ID NO: 33), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 48), and the peptide linker is not a bond. In some embodiments, linker 1 and linker 2 are identical, and their amino acid sequences are as shown in SEQ ID NO: 33.

[0043] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 32.

[0044] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments, wherein the CL is a light chain constant region of the antibody. In some embodiments, the antigen-binding molecule that describes in any of the preceding embodiments, wherein the CL is a light chain constant region of kappa or lambda. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 13.

[0045] In some embodiments, an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 30 and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 31.

[0046] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET, as described in any of the preceding embodiments, is a bispecific antibody that specifically binds to EGFR and cMET (also known as an anti-EGFR-cMET bispecific antibody).

[0047] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments has:

[0048] (1) A first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 38, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39; or

[0049] (2) A first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 40 and a fourth chain containing the amino acid sequence of SEQ ID NO: 39.

[0050] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 38, and a fourth chain as shown in SEQ ID NO: 39.

[0051] In some embodiments, the antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding embodiments has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 40, and a fourth chain as shown in SEQ ID NO: 39.

[0052] <Couple>

[0053] On the other hand, this disclosure provides a conjugate comprising an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims and a payload, wherein the payload is conjugated to the antigen-binding molecule; preferably, the payload is a topoisomerase inhibitor; more preferably, the payload is eczema or a derivative thereof.

[0054] On the other hand, this disclosure provides a compound or a pharmaceutically acceptable salt thereof, which is a compound or a pharmaceutically acceptable salt of general formula (I):

[0055] Ab-(LYD) n (I)

[0056] Wherein Ab is an antigen-binding molecule that specifically binds to EGFR and cMET; preferably, Ab is an anti-EGFR-cMET bispecific antibody, which comprises:

[0057] (1) A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d).

[0058] (a)[cMET-VH]-[CH1]-[Fc1],

[0059] (b)[cMET-VL]-[CL],

[0060] (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0061] (d)[EGFR-VL]-[connector 2]-[Obscurin],

[0062] The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0063] The structures shown in equations (a), (b), (c), and (d) are arranged from the N-end to the C-end; or

[0064] (2) A first chain having the structure shown in equation (e), a second chain having the structure shown in equation (f), a third chain having the structure shown in equation (g), and a fourth chain having the structure shown in equation (h).

[0065] (e)[EGFR-VH]-[CH1]-[Fc1],

[0066] (f)[EGFR-VL]-[CL],

[0067] (g)[cMET-VH]-[connector 3]-[Titin]-[Fc2],

[0068] (h)[cMET-VL]-[connector 4]-[Obscurin],

[0069] The linker 3 and linker 4 may be the same or different, and are peptide linkers; or linker 3 or linker 4 may not exist.

[0070] The structures shown in equations (e), (f), (g), and (h) are arranged from the N end to the C end;

[0071] L represents the connector unit;

[0072] Y is selected from -O-(CR) a R b ) m -CR 1 R 2 -C(O)-、-O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0073] R a and R b They may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

[0074] Or, R a and Rb Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, or heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino.

[0075] R 1 The group is selected from halogen, alkyl, haloalkyl, hydroxy, hydroxyalkyl, alkoxy, cyano, amino, carboxyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, heterocyclic alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl, wherein each of the cycloalkyl, heterocyclic, aryl, and heteroaryl groups is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino.

[0076] R 2 Selected from hydrogen atom, halogen, alkyl, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, cyano, amino, -(CR) c R d ) p -NR e R f 、-(CR c R d ) p -COOH, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, heterocyclic alkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl, wherein each of the cycloalkyl, heterocyclic, aryl and heteroaryl groups is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0077] Or, R 1 and R 2 Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, and heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino groups.

[0078] Or, R a and R 2 Together with the carbon atom attached thereto, they form cycloalkyl, heterocyclic, aryl, and heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano, and amino groups.

[0079] R c and R dThey may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl;

[0080] Or, R c and R d Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, or heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino.

[0081] R e and R f The same or different, and each independently selected from hydrogen atoms, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein each cycloalkyl, heterocyclic, aryl and heteroaryl is independently optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino;

[0082] Or R e and R f Together with the nitrogen atoms attached to them, they form a heterocyclic group, which is optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino.

[0083] m can be 0, 1, 2, 3, or 4;

[0084] p is 0, 1, 2, 3, 4, 5 or 6;

[0085] D is ixotecan or its derivatives;

[0086] n is 1 to 10; preferably, n is 1 to 8; more preferably, n is 4 to 8; most preferably, n is about 6.

[0087] In some embodiments, the compound represented by the general formula (I) as described above, or a pharmaceutically acceptable salt thereof, wherein Fc1 has a raised structure according to the mortar and pestle technique, and Fc2 has a porous structure according to the mortar and pestle technique; or, Fc2 has a raised structure according to the mortar and pestle technique, and Fc1 has a porous structure according to the mortar and pestle technique.

[0088] In some embodiments, the compound represented by general formula (I) as described above, or a pharmaceutically acceptable salt thereof, is a compound represented by general formula (II) or a pharmaceutically acceptable salt thereof:

[0089] Ab, L, Y and n are defined as in general formula (I).

[0090] In some embodiments, the compound or a pharmaceutically acceptable salt thereof as described in any of the preceding embodiments, wherein:

[0091] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0092] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 alkoxy, hydroxy and C 1-6 Hydroxyalkyl;

[0093] R 1 Halogen, C 1-6 Halogenated alkyl, 3- to 6-membered cycloalkyl and 3- to 6-membered cycloalkyl C 1-6 alkyl;

[0094] R 2 Selected from hydrogen atoms, halogens, C 1-6 Halogenated alkyl, 3- to 6-membered cycloalkyl and 3- to 6-membered cycloalkyl C 1-6 alkyl;

[0095] Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups;

[0096] m can be 0, 1, 2, 3, or 4.

[0097] In some embodiments, the compound represented by general formula (I) or general formula (II) as described in any of the preceding embodiments, wherein:

[0098] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0099] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 alkyl;

[0100] R 1 It is a 3- to 6-membered cycloalkyl or a 3- to 6-membered cycloalkyl-C 1-6 alkyl;

[0101] R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and 3- to 6-membered cycloalkyl groups;

[0102] Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups;

[0103] m can be 0, 1, 2, 3, or 4.

[0104] In some embodiments, the compound represented by general formula (I) or general formula (II) as described in any of the preceding embodiments, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0105] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkylene and C 1-8 alkylene-3 to 6-membered cycloalkyl, wherein the C 1-8 Alkylene or C 1-8 The alkylene group (3 to 6-membered cycloalkyl group) is independently selected from halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group;

[0106] L 2 Selected from -NR 4 (CH2CH2O) p1 CH2CH2C(O)-、-NR 4 (CH2CH2O) p1 CH2C(O)-、-S(CH2) p1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0107] L 3 A peptide consisting of 2 to 7 amino acid residues, selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally selected from halogens, hydroxyl groups, cyano groups, amino groups, C groups, and C groups. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group;

[0108] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -、-C(O)NR 5 (CH2) t - and chemical bonds, where t is 1, 2, 3, 4, 5 or 6;

[0109] R 3 R 4 and R 5 Whether the atoms are the same or different, and each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl;

[0110] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl, hydroxyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups and C 1-6 Hydroxyalkyl;

[0111] The L of -L- 1 The terminal is connected to Ab, L 4 The end is connected to Y.

[0112] In some embodiments, the compound represented by general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof, wherein L 1 It is -(succinimide-3-yl-N)-WC(O)-, where W is as defined in general formula (I) or general formula (II).

[0113] In some embodiments, the compound represented by general formula (I) or general formula (II) or a pharmaceutically acceptable salt thereof, wherein L 4 For -NR 5 (CR 6 R 7 ) t -, where t is an integer from 1 to 6; R 5 R 6 and R 7 As defined in general formula (I) or general formula (II).

[0114] In some embodiments, the compound of general formula (I), general formula (II), or a pharmaceutically acceptable salt thereof, as described in any of the preceding embodiments, is a compound of general formula (III) or a pharmaceutically acceptable salt thereof:

[0115] Where R 1 It is a 3- to 6-membered cycloalkyl or a 3- to 6-membered cycloalkyl-alkyl;

[0116] R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and 3- to 6-membered cycloalkyl groups;

[0117] Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups;

[0118] W is selected from C 1-8 Alkylene and C 1-8 alkylene-3 to 6-membered cycloalkyl, wherein the C 1-8 Alkylene and C 1-8 The alkylene group (3 to 6-membered cycloalkyl group) is independently selected from halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 It is substituted with one or more substituents of alkoxy and 3 to 6-membered cycloalkyl groups;

[0119] L 2 Selected from -NR 4 (CH2CH2O) p1 CH2CH2C(O)-、-NR 4 (CH2CH2O) p1 CH2C(O)-、-S(CH2) p1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0120] L 3 A peptide consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally selected from halogens, hydroxyl groups, cyano groups, amino groups, C groups, etc. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group;

[0121] R 4 and R 5 Selected from hydrogen atoms, C 1-6 Alkyl, C1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl;

[0122] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl;

[0123] m can be 0, 1, 2, 3, or 4;

[0124] Where Ab and n are defined as in general formula (I) or general formula (II).

[0125] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein m is 0 or 1; preferably, m is 0.

[0126] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein R 1 It is a 3- to 6-membered cycloalkyl group; preferably, R 1 It is cyclopropyl.

[0127] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein R 2 It is a hydrogen atom.

[0128] In some embodiments, the compound of general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 Together with the carbon atoms attached to it, they form 3 to 6-membered cycloalkyl groups.

[0129] In some embodiments, the compound represented by general formula (I), general formula (II), or general formula (III), or a pharmaceutically acceptable salt thereof, wherein W is selected from C 1-8 Alkylene.

[0130] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein L 2 It is a chemical bond.

[0131] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein L 3A peptide consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally selected from halogens, hydroxyl groups, cyano groups, amino groups, C groups, etc. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents from a 3- to 6-membered cycloalkyl group; preferably, L 3 It is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid; more preferably, L 3 It is a tetrapeptide residue; most preferably, L 3 It is a tetrapeptide residue represented by GGFG (SEQ ID NO: 47).

[0132] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein R 5 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl; preferably, R 5 It is a hydrogen atom or a carbon atom. 1-6 Alkyl; more preferably, R 5 It is a hydrogen atom.

[0133] In some embodiments, the compound represented by general formula (I), general formula (II) or general formula (III) or a pharmaceutically acceptable salt thereof, wherein R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl; preferably, R 6 and R 7 They may be the same or different, and each is independently a hydrogen atom or a carbon atom. 1-6 Alkyl; more preferably, R 6 and R 7 All are hydrogen atoms.

[0134] In some embodiments, the compound represented by general formula (I), general formula (II), or general formula (III), or a pharmaceutically acceptable salt thereof, wherein the connector unit-L 1 -L 2 -L 3 -L 4 -as follows:

[0135] L 1 for Where s 1 It can be 1, 2, 3, 4, 5, 6, 7, or 8;

[0136] L 2 It is a chemical bond;

[0137] L 3 It is a tetrapeptide residue; preferably, L 3 It is the tetrapeptide residue shown in GGFG (SEQ ID NO: 47);

[0138] L 4 For -NR 5 (CR 6 R 7 )t-, where R 5 R 6 and R 7 They may be the same or different, and each is independently a hydrogen atom or a carbon atom. 1-6 Alkyl group, where t is 1 or 2;

[0139] The L of -L- 1 The terminal is connected to Ab, L 4 The end is connected to Y.

[0140] In some embodiments, the compound or a pharmaceutically acceptable salt thereof represented by general formula (I), general formula (II) or general formula (III) as described in any of the preceding claims is a compound or a pharmaceutically acceptable salt thereof with the following structure:

[0141] Where Ab and n are defined as in general formula (I).

[0142] In some embodiments, a compound of formula (I), formula (II), or formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-EGFR-cMET bispecific antibody, comprising a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d).

[0143] (a)[cMET-VH]-[CH1]-[Fc1],

[0144] (b)[cMET-VL]-[CL],

[0145] (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0146] (d)[EGFR-VL]-[connector 2]-[Obscurin],

[0147] The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0148] The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end; Fc1 has a protruding structure according to the pestle and mortar technique, and Fc2 has a hole structure according to the pestle and mortar technique; wherein:

[0149] (1) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0150] The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25; or

[0151] (2) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0152] The cMET-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; and the cMET-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25.

[0153] Preferably, EGFR-VH contains the amino acid sequence of SEQ ID NO: 14 or 15, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 16; and cMET-VH contains the amino acid sequence of SEQ ID NO: 26; and cMET-VL contains the amino acid sequence of SEQ ID NO: 27.

[0154] In some embodiments, the compound or a pharmaceutically acceptable salt thereof of formula (I), formula (II) or formula (III) as described in any of the preceding claims, wherein Fc1 has amino acid A at position 234, amino acid A at position 235, amino acid C at position 354, and amino acid W at position 366; and Fc2 has amino acid A at position 234, amino acid A at position 235, amino acid C at position 349, amino acid S at position 366, amino acid A at position 368, and amino acid V at position 407, numbered according to the EU index.

[0155] In some embodiments, a compound of formula (I), formula (II) or formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 34 and Fc2 comprises the amino acid sequence of SEQ ID NO: 35.

[0156] In some embodiments, the linkers 1-4 are peptide linkers known in the art, provided that the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide having 1-50 or 3-20 amino acid residues. In some embodiments, each of the peptide linkers independently has an L1-(GGGGS)n-L2 structure, wherein L1 is a bond, A, G, GS, GGG, GGS, or GGGGS (SEQ ID NO: 33), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 48), and the peptide linker is not a bond. In some embodiments, linkers 1 and 2 are identical, having the amino acid sequence shown in SEQ ID NO: 33.

[0157] In some embodiments, the compound represented by formula (I), formula (II), or formula (III) as described in any of the preceding embodiments, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 32.

[0158] In some embodiments, the compound represented by formula (I), formula (II), or formula (III) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein the CL is a light chain constant region of an antibody. In some embodiments, the antigen-binding molecule as described in any of the preceding embodiments, wherein the CL is a light chain constant region of kappa or lambda. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 13.

[0159] In some embodiments, a compound of formula (I), formula (II) or formula (III) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein the Titin chain comprises the amino acid sequence of SEQ ID NO: 30, and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 31.

[0160] In some embodiments, a compound of formula (I), formula (II), or formula (III) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-EGFR-cMET bispecific antibody having:

[0161] (1) A first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 38, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39; or

[0162] (2) A first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 40 and a fourth chain containing the amino acid sequence of SEQ ID NO: 39.

[0163] In some embodiments, a compound of formula (I), formula (II) or formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the Ab is an anti-EGFR-cMET bispecific antibody, wherein the Ab has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 38 and a fourth chain as shown in SEQ ID NO: 39.

[0164] In some embodiments, a compound of formula (I), formula (II) or formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the Ab is an anti-EGFR-cMET bispecific antibody, wherein the Ab has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 40 and a fourth chain as shown in SEQ ID NO: 39.

[0165] In some embodiments, the compound or its pharmaceutically acceptable salt represented by formula (I), formula (II) or formula (III) as described in any of the preceding claims, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the average of any two values; preferably, n is 1-10; more preferably, n is 1-8, or 2-8, or 2-7, or 2-6, or 2-5, or 2-4, or 3-8, or 3-7, or 3-6, or 4-8, or 4-7, or 4-6, or the average of 4-5.

[0166] In some embodiments, the compound or a pharmaceutically acceptable salt thereof of formula (I), formula (II) or formula (III) as described in any of the preceding embodiments is used, wherein n is 1 to 10; preferably, n is 1 to 8; more preferably, n is 4 to 8; and most preferably, n is about 6.

[0167] On the other hand, this disclosure provides a method for preparing a compound as shown in general formula (III) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0168] After reduction, Ab undergoes a coupling reaction with a compound of general formula (IIIa) or its salt to give a compound of general formula (III) or its pharmaceutically acceptable salt.

[0169] Among them, Ab, W, L 2 L 3 R 1 R 2 R 5 To R 7 m and n are as defined in general formula (III).

[0170] In some implementations, n is 1 to 10 (inclusive of decimals or integers, the same below), for example, calculated as an average of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0171] In some implementations, n is 1 to 8; preferably, n is 4 to 8; more preferably, n is 6.

[0172] On the other hand, this disclosure provides a pharmaceutical composition comprising an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of formula (I), formula (II), or formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0173] In some embodiments, based on the total weight of the pharmaceutical composition, the pharmaceutical composition contains 0.01-99.99% of an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0174] In some embodiments, the pharmaceutical composition contains 0.1-99.9% of an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0175] In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, the pharmaceutical composition contains 1% to 99% of an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0177] In some embodiments, the pharmaceutical composition contains 2% to 98% of an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof.

[0178] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable diluents or excipients based on the total weight of the pharmaceutical composition.

[0179] In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable diluent or excipient.

[0180] In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients.

[0181] In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable diluents or excipients.

[0182] In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable diluents or excipients.

[0183] On the other hand, this disclosure provides isolated nucleic acids that encode antigen-binding molecules that specifically bind to EGFR and cMET as described in the previous one.

[0184] On the other hand, this disclosure provides a host cell containing isolated nucleic acids as described in any of the preceding claims.

[0185] On the other hand, this disclosure provides a method for preventing or treating a disease, the method comprising administering to a subject an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound represented by general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; preferably, the disease is a tumor; more preferably, the disease is selected from lung cancer, breast cancer, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, ovarian cancer, salivary gland cancer, head and neck cancer, and glioblastoma; most preferably, the disease is non-small cell lung cancer.

[0186] On the other hand, this disclosure provides a use in the preparation of a medicament for the prevention or treatment of a disease, comprising administering to a subject an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0187] On the other hand, this disclosure provides an antigen-binding molecule that specifically binds to EGFR and cMET as described in any of the preceding claims, a conjugate as described in any of the preceding claims, or a compound of general formula (I), general formula (II), or general formula (III) as described in any of the preceding claims, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In some embodiments, the medicament is used for the prevention or treatment of a disease.

[0188] In some embodiments, the disease described in any of the preceding embodiments is a tumor; in some embodiments, the disease is selected from lung cancer, breast cancer, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, ovarian cancer, salivary gland cancer, head and neck cancer, and glioblastoma; most preferably, the disease is non-small cell lung cancer.

[0189] In some embodiments, the compounds represented by general formulas (I), (II), and (III) of this disclosure, or their pharmaceutically acceptable salts, as well as all anti-EGFR-cMET bispecific antibody ADCs disclosed herein, have a high DAR6 content (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%), while other DAR values ​​(e.g., DAR2, DAR4, and DAR8) have a low content. That is, ADCs containing the anti-EGFR-cMET bispecific antibody structures disclosed herein can achieve stable conjugation with high DAR values ​​and high homogeneity; specifically, DAR6 exhibits high homogeneity. Attached Figure Description

[0190] Figure 1 is a schematic diagram of the molecular structure of the anti-EGFR-cMET bispecific antibody;

[0191] Figure 2 shows the DAR distribution of ADC-1;

[0192] Figure 3 shows the DAR distribution of ADC-2;

[0193] Figure 4 shows the selective binding experiment of anti-EGFR-cMET bispecific antibody to tumor cells. Detailed Implementation

[0194] the term

[0195] To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0196] The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

[0197] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0198] The term "cytokine" is a general term for proteins released by a population of cells that act as intercellular mediators in other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include mIL-2, IFNγ, TNFα, CCL-2, and IL-6.

[0199] The term "and / or" implies both "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0200] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0201] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0202] The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In one embodiment, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and / or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this paper, the amino acid residue at a specific site can be represented by the format of position + amino acid residue. For example, 82aR indicates that the amino acid residue at the 82a site is R. S82aR indicates that the amino acid residue at the 82a (also known as 82A) site has changed from S to R.

[0203] The term "antigen-binding molecule" is used in the broadest sense to encompass molecules that specifically bind antigens, including but not limited to antibodies, other peptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two, as well as any molecule containing the aforementioned antibodies, peptides, or antibody fusion proteins, provided they exhibit the desired antigen-binding activity. The antigen-binding molecules described herein comprise a variable region (VH) and a variable region (VL), which together constitute the antigen-binding domain. Exemplarily, the antigen-binding molecules described herein are bispecific antigen-binding molecules (e.g., bispecific antibodies).

[0204] The term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0205] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies; and based on whether the structure is symmetrical, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc part may also bind to the receptor FcRn, increasing the antibody serum half-life.

[0206] "Natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL). The terms "full-length antibody," "complete antibody," and "all antibody" are used interchangeably herein to refer to antibodies with a structure substantially similar to that of natural antibodies or heavy chains with an Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL). VL is located at the amino terminus of the light chain, and the constant region includes the κ chain and the λ chain. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3). VH is located at the amino terminus of the heavy chain, and the constant region is located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0207] The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0208] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between various numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0209] Table 1. Relationship between CDR numbering systems

[0210] Unless otherwise stated, the variable areas and CDRs in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0211] The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody is bound. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0212] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0213] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remaining portion of the heavy and / or light chain is derived from another different source or species.

[0214] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0215] The terms "human antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine ​​residues or glycosylation sites that might cause undesirable folding. This term also encompasses antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies that have been fed to transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The meaning of "human antibody" explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0216] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

[0217] As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

[0218] The term “surface plasmon resonance” refers to the optical phenomenon of analyzing real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore™ system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

[0219] The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0220] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibody formulations typically contain multiple different antibodies with different amino acid sequences in their variable structural domains, and they generally specifically target different epitopes. "Monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

[0221] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder, such as an antigen-binding protein (including, for example, an antibody), and is further capable of being used in animals to produce antibodies that can bind to that antigen. An antigen may have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0222] The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the antibody loses binding to the conformational epitope. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0223] The terms "specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous) (such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset)).

[0224] The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

[0225] The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

[0226] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0227] The term “nucleic acid” is used interchangeably with the term “polynucleotide” herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). “Isolated” nucleic acid refers to a nucleic acid molecule that has been separated from its components in its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain such molecules but are located extrachromosomally or at chromosomal locations other than their natural chromosomal locations. Isolated nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including one or more such nucleic acid molecules in a single or separate vector, and one or more such nucleic acid molecules present at one or more locations in a host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues.

[0228] The terms “polypeptide” and “protein” are used interchangeably in this document and refer to polymers of amino acid residues.

[0229] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when two sequences are optimally aligned, with gaps introduced where necessary to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0230] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and / or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

[0231] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous 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 derived progeny, regardless of passage number. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. This includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *Candida polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*. Pichia, any Saccharomyces, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0232] As used in this application, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny will have identical DNA contents. This includes mutant progeny that have the same function or biological activity as the original transformed cells.

[0233] The term "alkyl" refers to a saturated, straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C2). 1-20 Alkyl group). The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C12). 1-12 Alkyl groups, more preferably alkyl groups having 1 to 6 carbon atoms (i.e., C14-C6 ... 1-6 Alkyl groups). Non-limiting examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. Alkyl groups can be substituted or unsubstituted. When substituted, they can be substituted at any usable connection point. The substituents are preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0234] The term "alkenyl" refers to an alkyl group in which the molecule contains at least one carbon-carbon double bond, wherein the alkyl group is defined as described above and has 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms (i.e., C atoms). 2-12 Alkenyl). The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (i.e., C). 2-6 Alkenyl). Non-limiting examples include vinyl, propenyl, isopropenyl, butenyl, etc. Alkenyl groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable connection point. Substituents are preferably selected from one or more of the following: D atom, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0235] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy, etc. Alkoxy groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable linker. The substituent is preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0236] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic carbocyclic ring (i.e., monocyclic cycloalkyl) or polycyclic system (i.e., polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 3 to 20 membered cycloalkyl). The cycloalkyl is preferably a cycloalkyl having 3 to 12 ring atoms (i.e., 3 to 12 membered cycloalkyl), more preferably a cycloalkyl having 3 to 8 ring atoms (i.e., 3 to 8 membered cycloalkyl), and most preferably a cycloalkyl having 3 to 6 ring atoms (i.e., 3 to 6 membered cycloalkyl).

[0237] Non-limiting examples of the monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl.

[0238] The polycyclic alkyl groups include: spirocyclic alkyl groups, fused cyclic alkyl groups, and bridged cyclic alkyl groups.

[0239] The term "spirocycloalkyl" refers to a polycyclic system in which rings share a single carbon atom (called a spiro atom), and the ring may contain one or more double bonds, or one or more heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-), provided that it contains at least one full carbon ring with a bonding point on that full carbon ring, having 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20-membered spirocycloalkyl). The spirocycloalkyl is preferably a spirocycloalkyl having 6 to 14 ring atoms (i.e., 6 to 14-membered spirocycloalkyl), more preferably a spirocycloalkyl having 7 to 10 ring atoms (i.e., 7 to 10-membered spirocycloalkyl). The spirocyclic alkyl group includes monospirocyclic alkyl and polyspirocyclic alkyl (such as bispirocyclic alkyl, etc.), preferably monospirocyclic alkyl or bispirocyclic alkyl, more preferably 3 / 4, 3 / 5, 3 / 6, 4 / 4, 4 / 5, 4 / 6, 5 / 3, 5 / 4, 5 / 5, 5 / 6, 5 / 7, 6 / 3, 6 / 4, 6 / 5, 6 / 6, 6 / 7, 7 / 5 or 7 / 6 monospirocyclic alkyl. Non-limiting examples include:

[0240] Its connection point can be anywhere;

[0241] wait.

[0242] The term "fused cycloalkyl" refers to a polycyclic system in which two adjacent carbon atoms are shared between rings. This system is a monocyclic cycloalkyl group fused with one or more monocyclic cycloalkyl groups, or a monocyclic cycloalkyl group fused with one or more heterocyclic, aryl, or heteroaryl groups, wherein the bonding point is on the monocyclic cycloalkyl group. The ring may contain one or more double bonds and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20-membered fused cycloalkyl groups). The fused cycloalkyl group is preferably a fused cycloalkyl group having 6 to 14 ring atoms (i.e., 6 to 14-membered fused cycloalkyl groups), and more preferably a fused cycloalkyl group having 7 to 10 ring atoms (i.e., 7 to 10-membered fused cycloalkyl groups). The fused cyclic alkyl group includes bicyclic fused cyclic alkyl groups and polycyclic fused cyclic alkyl groups (such as tricyclic fused cyclic alkyl groups, tetracyclic fused cyclic alkyl groups, etc.), preferably bicyclic fused cyclic alkyl groups or tricyclic fused cyclic alkyl groups, more preferably ternary / quadrivalent, ternary / pentary, ternary / hexavalent, quadrivalent / quadrivalent, quadrivalent / pentary, quadrivalent / hexavalent, pentary / pentary ... or pentary / pentary bicyclic fused cyclic alkyl groups. Non-limiting examples include:

[0243] Its connection point can be anywhere;

[0244] wait.

[0245] The term "bridged cycloalkyl" refers to a fully carbon polycyclic system in which two non-directly connected carbon atoms are shared between rings, and the ring may contain one or more double bonds and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., 5 to 20-membered bridged cycloalkyl). The bridged cycloalkyl is preferably a bridged cycloalkyl having 6 to 14 carbon atoms (i.e., 6 to 14-membered bridged cycloalkyl), more preferably a bridged cycloalkyl having 7 to 10 carbon atoms (i.e., 7 to 10-membered bridged cycloalkyl). The bridged cycloalkyl includes bicyclic bridged cycloalkyl and polycyclic bridged cycloalkyl (e.g., tricyclic bridged cycloalkyl, tetracyclic bridged cycloalkyl, etc.), preferably bicyclic or tricyclic bridged cycloalkyl. Non-limiting examples include:

[0246] Its connection point can be anywhere.

[0247] The cycloalkyl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0248] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic heterocycle (i.e., monocyclic heterocyclic group) or polycyclic heterocyclic system (i.e., polycyclic heterocyclic group) containing at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), and having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) ring atoms (i.e., 3 to 20 membered heterocyclic groups). The heterocyclic group is preferably a heterocyclic group having 3 to 12 ring atoms (i.e., a 3 to 12-membered heterocyclic group), for example, a 4 to 12-membered heterocyclic group containing at least one nitrogen atom; more preferably a heterocyclic group having 3 to 8 ring atoms (i.e., a 3 to 8-membered heterocyclic group); more preferably a heterocyclic group having 3 to 6 ring atoms (i.e., a 3 to 6-membered heterocyclic group); and most preferably a heterocyclic group having 5 or 6 ring atoms (i.e., a 5 or 6-membered heterocyclic group).

[0249] Non-limiting examples of the monocyclic heterocyclic group include: pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl, etc.

[0250] The polycyclic heterocyclic groups include spirocyclic heterocyclic groups, fused heterocyclic groups, and bridged heterocyclic groups.

[0251] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic system in which rings share a single atom (called a spiro atom), which may contain one or more double bonds and at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-), provided that at least one monocyclic heterocyclic group is present and the bonding point is on the monocyclic heterocyclic group, which has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20-membered spiroheterocyclic groups). The spiroheterocyclic group is preferably a spiroheterocyclic group having 6 to 14 ring atoms (i.e., a 6 to 14-membered spiroheterocyclic group), more preferably a spiroheterocyclic group having 7 to 10 ring atoms (i.e., a 7 to 10-membered spiroheterocyclic group). The spiroheterocyclic group includes monospirocyclic and polyspirocyclic groups (such as bispirocyclic groups), preferably monospirocyclic or bispirocyclic, more preferably 3 / 4, 3 / 5, 3 / 6, 4 / 4, 4 / 5, 4 / 6, 5 / 3, 5 / 4, 5 / 5, 5 / 6, 5 / 7, 6 / 3, 6 / 4, 6 / 5, 6 / 6, 6 / 7, 7 / 5, or 7 / 6 monospirocyclic. Non-limiting examples include:

[0252] wait.

[0253] The term "fused heterocyclic group" refers to a polycyclic heterocyclic system in which two adjacent atoms are shared between rings. The ring may contain one or more double bonds and at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-). It is a monocyclic heterocyclic group fused with one or more monocyclic heterocyclic groups, or a monocyclic heterocyclic group fused with one or more cycloalkyl, aryl, or heteroaryl groups, wherein the bonding point is on the monocyclic heterocyclic group and has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 5 to 20 membered fused heterocyclic groups). The fused heterocyclic group is preferably a fused heterocyclic group having 6 to 14 ring atoms (i.e., a 6 to 14-membered fused heterocyclic group), more preferably a fused heterocyclic group having 7 to 10 ring atoms (i.e., a 7 to 10-membered fused heterocyclic group). The fused heterocyclic group includes bicyclic and polycyclic fused heterocyclic groups (such as tricyclic fused heterocyclic groups, tetracyclic fused heterocyclic groups, etc.), preferably bicyclic or tricyclic fused heterocyclic groups, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 3-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 5-membered / 7-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, 6-membered / 6-membered, 6-membered / 7-membered, 7-membered / 5-membered, or 7-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples include:

[0254] wait.

[0255] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic system in which two non-directly connected atoms are shared between the rings. The rings may contain one or more double bonds, and the rings contain at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized to form nitrogen oxides; the sulfur may optionally be oxidized to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-). The system has 5 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., a 5- to 20-membered bridged heterocyclic group). The bridged heterocyclic group is preferably a bridged heterocyclic group with 6 to 14 ring atoms (i.e., a 6- to 14-membered bridged heterocyclic group), and more preferably a bridged heterocyclic group with 7 to 10 ring atoms (i.e., a 7- to 10-membered bridged heterocyclic group). Based on the number of constituent rings, heterocyclic groups can be classified into bicyclic bridged heterocyclic groups and multicyclic bridged heterocyclic groups (such as tricyclic bridged heterocyclic groups, tetracyclic bridged heterocyclic groups, etc.), with bicyclic bridged heterocyclic groups or tricyclic bridged heterocyclic groups being preferred. Non-limiting examples include:

[0256] wait.

[0257] The heterocyclic group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0258] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aryl) or a polycyclic aromatic ring system (i.e., polycyclic aryl) having a conjugated π-electron system, having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 6 to 14-membered aryl). The aryl is preferably an aryl having 6 to 10 ring atoms (i.e., 6 to 10-membered aryl). The monocyclic aryl is, for example, phenyl. Non-limiting examples of the polycyclic aryl include naphthyl, anthraceneyl, phenanthrene, etc. The polycyclic aryl further includes fusion of the phenyl with one or more heterocyclic groups or cycloalkyl groups, or fusion of the naphthyl with one or more heterocyclic groups or cycloalkyl groups, wherein the bonding point is on the phenyl or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples including:

[0259] wait.

[0260] The aryl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0261] The term "heteroaryl" refers to a monocyclic heteroaryl ring (i.e., monocyclic heteroaryl) or a polycyclic heteroaryl ring system (i.e., polycyclic heteroaryl) having a conjugated π-electron system, containing at least one (e.g., 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS- or -SS-), having 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14) ring atoms (i.e., 5 to 14-membered heteroaryl). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5 to 10-membered heteroaryl group), more preferably a monocyclic heteroaryl group having 5 or 6 ring atoms (i.e., a 5 or 6-membered monocyclic heteroaryl group) or a bicyclic heteroaryl group having 8 to 10 ring atoms (i.e., an 8 to 10-membered bicyclic heteroaryl group), and most preferably a 5 or 6-membered monocyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur or an 8 to 10-membered bicyclic heteroaryl group containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and sulfur.

[0262] Non-limiting examples of the aforementioned monocyclic heteroaryl groups include: furanyl, thiopheneyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furazonyl, pyrroleyl, N-alkylpyrroleyl, pyridyl, pyrimidinyl, pyridoneyl, N-alkylpyridone (e.g.) (etc.), pyrazinyl, pyridazinyl, etc.

[0263] Non-limiting examples of the polycyclic heteroaryl groups include: indolyl, indazole, quinolinyl, isoquinolinyl, quinoxalinyl, phthalazinyl, benzimidazolyl, benzothiophene, quinazolinyl, benzothiazolyl, carbazole, etc. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused with one or more aryl groups, wherein the connecting point is on the aromatic ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaryl ring system. The polycyclic heteroaryl groups also include monocyclic heteroaryl groups fused with one or more cycloalkyl or heterocyclic groups, wherein the connecting point is on the monocyclic heteroaryl ring, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic heteroaryl ring system. Non-limiting examples include:

[0264] wait.

[0265] The heteroaryl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0266] The aforementioned cycloalkyl, heterocyclic, aryl, and heteroaryl residues include residues derived from removing one hydrogen atom from a parent ring atom, or residues derived from removing two hydrogen atoms from the same ring atom or two different ring atoms of the parent, namely "divalent cycloalkyl", "divalent heterocyclic", "aryl", and "heteroaryl".

[0267] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations.

[0268] The compounds disclosed herein include all suitable isotopic derivatives thereof. The term "isotopic derivative" refers to a compound in which at least one atom is replaced by an atom having the same atomic number but a different atomic mass. Examples of isotopes that may be introduced into the compounds of this disclosure include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, for example, […]. 2 H (deuterium, D) 3 H (tritium, T) 11 C 13 C 14 C 15 N、 17 O、 18 O、 32 p、 33 p、 33 S, 34 S, 35 S, 36 S, 18 F, 36 Cl、 82 Br、 123 I, 124 I, 125 I, 129 I and 131 Grade I, with deuterium as the preferred grade.

[0269] Compared to undeuterated drugs, deuterated drugs offer advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations of the compounds disclosed herein, regardless of radioactivity, are included within the scope of this disclosure. Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom, wherein the deuterium substitution can be partial or complete; partial deuterium substitution refers to the replacement of at least one hydrogen atom with at least one deuterium atom.

[0270] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0271] The term "pharmaceutical composition" means a mixture containing one or more antigen-binding molecules that specifically bind EGFR and cMET as described herein, as well as conjugates or compounds thereof or pharmaceutically acceptable salts thereof, and other chemical components such as physiological / pharmaceutical carriers and excipients.

[0272] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is distinct from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0273] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably herein. In some embodiments, the individual or subject is a human being.

[0274] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0275] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within the subject's body. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

[0276] "Treatment" and "treatment" (and their grammatical variations) refer to clinical interventions that attempt to alter the natural processes of the individual being treated, and can be implemented for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, antibodies disclosed herein are used to delay disease onset or slow disease progression.

[0277] An "effective dose" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate such symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, an effective dose is a therapeutically effective dose or a preventatively effective dose. A "therapeuticly effective dose" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A "preventatively effective dose" is an amount that, when administered to a subject, will have a predetermined preventative effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. A complete therapeutic or preventative effect may not occur after the administration of a single dose, but may occur after the administration of a series of doses. Therefore, a therapeutically or preventatively effective dose may be administered in a single or multiple-dose manner. "Therapeutic effective dose" and "preventive effective dose" can vary depending on a number of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination of treatments to elicit the desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved health status in the patient.

[0278] Exemplary antigen-binding molecules that specifically bind to EGFR and cMET

[0279] The embodiments disclosed herein reveal antigen-binding molecules that specifically bind to EGFR and cMET.

[0280] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein is an anti-EGFR-cMET bispecific antibody.

[0281] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein comprises a first strand having the structure shown in formula (a), a second strand having the structure shown in formula (b), a third strand having the structure shown in formula (c), and a fourth strand having the structure shown in formula (d).

[0282] (a)[cMET-VH]-[CH1]-[Fc1],

[0283] (b)[cMET-VL]-[CL],

[0284] (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0285] (d)[EGFR-VL]-[connector 2]-[Obscurin],

[0286] The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0287] The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end;

[0288] The Fc1 has a protruding structure according to the pestle and mortar technique, and the Fc2 has a hole structure according to the pestle and mortar technique;

[0289] in:

[0290] (1) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0291] The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25; or

[0292] (2) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6.

[0293] The cMET-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; and the cMET-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25.

[0294] Preferably, the EGFR-VH contains the amino acid sequence of SEQ ID NO: 14 or 15, and the EGFR-VL contains the amino acid sequence of SEQ ID NO: 16; the cMET-VH contains the amino acid sequence of SEQ ID NO: 26; and the cMET-VL contains the amino acid sequence of SEQ ID NO: 27.

[0295] For example, the antigen-binding molecules that specifically bind to EGFR and cMET disclosed herein, wherein linker 1 and linker 2 are identical and their amino acid sequences are shown in SEQ ID NO: 33.

[0296] For example, the antigen-binding molecule disclosed herein specifically binds to EGFR and cMET, wherein CH1 contains the amino acid sequence of SEQ ID NO: 32.

[0297] For example, the antigen-binding molecule disclosed herein specifically binds to EGFR and cMET, wherein CL contains the amino acid sequence of SEQ ID NO: 13.

[0298] For example, the antigen-binding molecule disclosed herein specifically binds to EGFR and cMET, wherein the Titin chain contains the amino acid sequence of SEQ ID NO: 30 and the Obscurin chain contains the amino acid sequence of SEQ ID NO: 31.

[0299] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein has a first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 38, and a fourth chain containing the amino acid sequence of SEQ ID NO: 39.

[0300] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 38, and a fourth chain as shown in SEQ ID NO: 39.

[0301] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein has a first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 40, and a fourth chain containing the amino acid sequence of SEQ ID NO: 39.

[0302] For example, the antigen-binding molecule that specifically binds to EGFR and cMET disclosed herein has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 40, and a fourth chain as shown in SEQ ID NO: 39.

[0303] Exemplary compounds or their pharmaceutically acceptable salts

[0304] Examples of this disclosure disclose a compound of general formula (II) or a pharmaceutically acceptable salt thereof:

[0305] in:

[0306] Ab is a bispecific antibody against EGFR-cMET, comprising a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d).

[0307] (a)[cMET-VH]-[CH1]-[Fc1],

[0308] (b)[cMET-VL]-[CL],

[0309] (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0310] (d)[EGFR-VL]-[connector 2]-[Obscurin],

[0311] The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0312] The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end;

[0313] The Fc1 has a protruding structure according to the pestle and mortar technique, and the Fc2 has a hole structure according to the pestle and mortar technique;

[0314] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0315] Among them, R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 alkyl;

[0316] R 1 It is a 3- to 6-membered cycloalkyl or a 3- to 6-membered cycloalkyl-C 1-6 alkyl;

[0317] R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and 3- to 6-membered cycloalkyl groups;

[0318] Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups;

[0319] m can be 0, 1, 2, 3, or 4;

[0320] L represents the connector unit;

[0321] n is approximately 6.

[0322] For example, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, are compounds represented by general formula (III) or pharmaceutically acceptable salts thereof:

[0323] in:

[0324] W is selected from C 1-8 Alkylene; L 2 For chemical bonds; L3 It is a tetrapeptide residue (preferably, L) 3 (The tetrapeptide residues shown in GGFG (SEQ ID NO: 47)); R 1 It is a 3- to 6-membered cycloalkyl group; R 2 For hydrogen atoms; R 5 It is a hydrogen atom or a carbon atom. 1-6 Alkyl; R 6 and R 7 They may be the same or different, and each is independently a hydrogen atom or a carbon atom. 1-6 Alkyl; m is 0 or 1; n is approximately 6; wherein Ab is as defined in general formula (I) or general formula (II).

[0325] For example, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, are compounds or pharmaceutically acceptable salts thereof with the following structures:

[0326] Where n is approximately 6; Ab is defined as in general formula (I), general formula (II) or general formula (III).

[0327] For example, in the compounds described above or their pharmaceutically acceptable salts, Ab is an anti-EGFR-cMET bispecific antibody, wherein the HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9, and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6;

[0328] The cMET-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; and the cMET-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25.

[0329] Preferably, the EGFR-VH contains the amino acid sequence of SEQ ID NO: 14, and the EGFR-VL contains the amino acid sequence of SEQ ID NO: 16; the cMET-VH contains the amino acid sequence of SEQ ID NO: 26; and the cMET-VL contains the amino acid sequence of SEQ ID NO: 27.

[0330] More preferably, Ab has a first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 38, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39.

[0331] Most preferably, Ab has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 38, and a fourth chain as shown in SEQ ID NO: 39.

[0332] For example, in the compounds described above or their pharmaceutically acceptable salts, Ab is an anti-EGFR-cMET bispecific antibody, wherein the HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11, and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 6;

[0333] The cMET-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; and the cMET-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25.

[0334] Preferably, the EGFR-VH contains the amino acid sequence of SEQ ID NO: 15, and the EGFR-VL contains the amino acid sequence of SEQ ID NO: 16; the cMET-VH contains the amino acid sequence of SEQ ID NO: 26; and the cMET-VL contains the amino acid sequence of SEQ ID NO: 27.

[0335] More preferably, Ab has a first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 40, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39.

[0336] Most preferably, Ab has a first chain as shown in SEQ ID NO: 36, a second chain as shown in SEQ ID NO: 37, a third chain as shown in SEQ ID NO: 40, and a fourth chain as shown in SEQ ID NO: 39.

[0337] Measurement

[0338] The antigen-binding molecules that specifically bind to EGFR and cMET, as well as their conjugates or pharmaceutically acceptable salts, provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities using a variety of assays known in the art. In one aspect, activity is tested, for example, by known methods such as ELISA, Western blotting, etc.

[0339] Detailed Implementation

[0340] The present disclosure is further described below with reference to embodiments and test examples, but these embodiments and test examples are not intended to limit the scope of the present disclosure. Experimental methods in the embodiments or test examples of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer; reagents and materials whose specific sources are not specified are commercially available.

[0341] Example

[0342] Example 1: Modification of anti-EGFR antibody

[0343] Molecules that specifically bind to EGFR can be derived from any suitable antibody, such as zalutumumab or its variants, the sequence of which is shown below.

[0344] Table 2. CDR sequences of Zalutumumab

[0345] The heavy chain variable region sequence of Zalututumumab:

[0346] >Zalutumumab light chain variable region sequence:

[0347] By mutating amino acids at positions 31 and / or 97 of the heavy chain variable region and / or position 1 of the light chain variable region of zalutumumab, and linking them to the human IgG1 heavy chain constant region (containing L234A and L235A mutations) and the kappa light chain constant region, respectively, two anti-EGFR antibodies were obtained: ZalH4 and ZalH10, with the specific sequences as follows:

[0348] Table 3. Amino acid sequences of the replaced CDRs

[0349] Human IgG1 heavy chain constant region sequence (containing L234A and L235A mutations):

[0350] Human light chain constant region sequence:

[0351] The heavy chain variable region sequence of ZalH4 (abbreviated as "ZalVH4"):

[0352] The heavy chain variable region sequence of ZalH10 (abbreviated as "ZalVH10"):

[0353] Light chain variable region sequences of ZalH4 and ZalH10 (hereinafter referred to as "ZalVL"):

[0354] The heavy chain sequence of ZalH4:

[0355] Heavy chain sequence of ZalH10:

[0356] The light chain sequences of ZalH4 and ZalH10:

[0357] Note: In the antibody sequences above, the underlined part is the antibody variable region sequence, the double underlined part is the antibody CDR sequence, the ununderlined part is the antibody constant region sequence, and the bolded letters are mutant amino acids.

[0358] Example 2: Anti-cMET antibody

[0359] The molecule that specifically binds to cMET can be derived from any suitable antibody, such as Omab (where the VH / VL sequence of Omab is derived from Onartuzumab), whose sequence is shown below.

[0360] Table 4. CDR sequences of Omab

[0361] The heavy chain variable region sequence of Omab (abbreviated as "Omab VH"):

[0362] >Omab light chain variable region sequence (abbreviated as "Omab VL"):

[0363] Human IgG1 heavy chain constant region sequence (containing L234A and L235A mutations): SEQ ID NO: 12

[0364] Human light chain constant region sequence: SEQ ID NO: 13

[0365] Omab's heavy chain sequence:

[0366] Omab's light chain sequence:

[0367] Example 3: Construction of anti-EGFR-cMET bispecific antibody

[0368] The EGFR-cMET bispecific antibody is in a 1:1 molecular form. Omab is selected as the cMET arm and assembled with EGFR antibodies ZalH4 and ZalH10, respectively. The VH of the EGFR antibody is combined with Titin, and the VL is combined with Obscurin. Furthermore, S354C, T366W mutations (knob) and L234A, L235A mutations are introduced into the Omab antibody heavy chain, while Y349C, T366S, L368A, Y407V mutations (hole) and L234A, L235A mutations are introduced into the EGFR antibody heavy chain. The format is an asymmetric molecule containing four chains.

[0369] Chain 1: [VH(anti-cMET)]-[IgG1(CH1)]-[Fc(Knob)];

[0370] Chain 2: [VL(anti-cMET)]-[CL];

[0371] Chain 3: [VH(anti-EGFR)]-[connector 1]-[Titin]-[Fc(Hole)];

[0372] Chain 4: [VL(anti-EGFR)]-[connector 2]-[Obscurin]; its schematic diagram is shown in Figure 1 (where T represents Titin and O represents Obscurin).

[0373] Table 5. Anti-EGFR-cMET bispecific antibodies disclosed herein Note: For example, Omab-ZalH4 means that the molecule uses the variable region of Omab as the cMET binding domain, the variable region of ZalH4 as the EGFR binding domain, and adopts the Format shown in Figure 1 as the molecular structure, and so on.

[0374] Titin Chain:

[0375] Obscurin chain:

[0376] CH1:

[0377] >CL:SEQ ID NO:13

[0378] Connector 1 and connector 2: GGGGS (SEQ ID NO:33)

[0379] >Fc(knob):

[0380] >Fc(hole):

[0381] The full-length sequence of the bispecific antibody is as follows:

[0382] Omab-ZalH4 sequence:

[0383] Chain 1 (Omab VH-CH1-Fc(Knob)):

[0384] Chain 2 (Omab VL-CL):

[0385] Chain 3 (ZalVH4-connector 1-Titin-Fc(Hole)):

[0386] Chain 4 (ZalVL-connector 2-Obscurin):

[0387] Omab-ZalH10 sequence:

[0388] Chain 1 (Omab VH-CH1-Fc(Knob)): SEQ ID NO:36

[0389] Chain 2 (Omab VL-CL): SEQ ID NO:37

[0390] Chain 3 (ZalVH10-connector 1-Titin-Fc(Hole)):

[0391] Chain 4 (ZalVL-connector 2-Obscurin): SEQ ID NO:39

[0392] Note: In the antibody sequences above, the underlined part is the antibody variable region sequence, the ununderlined part is the antibody constant region sequence, and the wavy line is the linker sequence.

[0393] In addition, the positive control antibody T antibody in this embodiment (constructed with reference to sequence 59 / 60 / 61 / 62 of WO2023083846A1) has the following sequence.

[0394] Chain 1:

[0395] Chain 2:

[0396] Chain 3:

[0397] Chain 4:

[0398] The VH / VL sequence of the negative control antibody Isotype used in this disclosure is derived from patent US6114143A. The heavy chain constant region and light chain constant region sequences are SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and their full-length sequences are shown below.

[0399] Isotype relinks:

[0400] Isotype Light Chain:

[0401] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0402] Example 4: Preparation of anti-EGFR-cMET bispecific antibody ADC

[0403] The above general reaction formula applies to ADC-1, ADC-2 and ADC-3.

[0404] ADC-1

[0405] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 15.4 μL, 154 nmol) was added to the PBS buffered aqueous solution of antibody Omab-ZalH4 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.46 mL, 30.7 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0406] Compound 9-A (0.39 mg, 368.4 nmol) was dissolved in 23 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a constant temperature shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.03 M His buffer aqueous solution at pH = 5.0) to obtain His buffer of the title product ADC-1, which was stored at 4 °C.

[0407] Natural mass spectrometry analysis (results shown in Figure 2) revealed an average DAR value of 5.87; DAR6 accounted for 95.46%, DAR2 and DAR4 for 1.95% and 2.59% respectively, and DAR8 was undetectable. Clearly, this anti-EGFR-cMET bispecific antibody ADC exhibits high DAR6 content and low proportions of other DAR values, demonstrating high homogeneity.

[0408] ADC-2

[0409] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 61.1 μL, 0.611 μmol) was added to the PBS buffered aqueous solution of antibody Omab-ZalH10 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.31 mL, 87.3 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0410] Compound 9-A (1.13 mg, 1.05 μmol) was dissolved in 65 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a constant temperature shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.03 M His buffer aqueous solution at pH = 5.0) to obtain His buffer of the title product ADC-2, which was stored at 4 °C.

[0411] Natural mass spectrometry analysis (results shown in Figure 3) revealed an average DAR value of 5.85; DAR6 accounted for 95.64%, DAR2 and DAR4 for 3.20% and 1.16% respectively, and DAR8 was undetectable. This indicates that this anti-EGFR-cMET bispecific antibody ADC exhibits high DAR6 content and low proportions of other DAR values, demonstrating high homogeneity.

[0412] ADC-3

[0413] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 178.4 μL, 1784 nmol) was added to the PBS buffered aqueous solution of antibody Isotype (0.05 M PBS buffered aqueous solution of pH = 6.3; 10.0 mg / mL, 7.84 mL, 540.7 nmol). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours. The reaction was then stopped, and the reaction solution was cooled to 25°C.

[0414] Compound 9-A (6.97 mg, 6.49 μmol) was dissolved in 390 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a constant-temperature shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.03 M His buffer aqueous solution at pH 5.0) to obtain His buffer of the title product ADC-3, which was stored at 4 °C.

[0415] The average number of drugs bound per antibody molecule was calculated by RP-HPLC: n = 6.17.

[0416] ADC-4

[0417] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 416.0 μL, 4160 nmol) was added to a PBS-buffered aqueous solution of T antibody (ab replaced with T antibody in the synthetic route) (pH = 6.3, 0.05 M PBS buffer; 10.0 mg / mL, 13.0 mL, 866.7 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C, and then the solution was transferred to a pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0418] Compound SG3932 (synthesized according to Example 1 of WO2020200880A1) (11.94 mg, 10.4 μmol) was dissolved in 650 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a constant temperature shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.03 M His buffer aqueous solution at pH = 5.0) to obtain His buffer of the title product ADC-4, which was stored at 4 °C. The average drug binding number per antibody molecule was calculated by MS: n = 6.17.

[0419] Test case

[0420] Test Example 1: Biacore Affinity Assay for Anti-EGFR Antibody

[0421] 1. Test instruments: Biacore 8K, Cytiva.

[0422] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva).

[0423] 3. Test reagents:

[0424] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva);

[0425] 2) 10mM Glycine-HCl (pH 1.5) (Cat.#BR-1003-54, Cytiva);

[0426] 3) EGFR-His(ACRO,EGR-H5222);

[0427] 4) The sample to be tested;

[0428] The affinity of the disclosed antibody for the human EGFR-His antigen was tested using a Biacore 8K instrument.

[0429] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then a certain concentration of antigen molecules was flowed through the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The reaction signal was monitored in real-time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The results are shown in Table 6.

[0430] Table 6. Affinity test results of anti-EGFR antibodies to human EGFR antigen

[0431] The results showed that the disclosed anti-EGFR antibody had good affinity for both human EGFR antigen.

[0432] Test Example 2: Biacore Affinity Test for Anti-EGFR-cMET Bispecific Antibody and its ADC

[0433] 1. Test instruments: Biacore T200 or Biacore 8K, Cytiva.

[0434] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva).

[0435] 3. Test reagents:

[0436] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva);

[0437] 2) 10mM Glycine-HCl (pH 1.5) (Cat.#BR-1003-54, Cytiva);

[0438] 3) EGFR-His(ACRO,EGR-H5222);

[0439] 4) cMET-His(ACRO,HGF-H5227);

[0440] 5) Antibody sample to be tested.

[0441] 2.1 The affinity of the disclosed anti-EGFR-cMET bispecific antibody and its ADC for human EGFR-His antigen was tested using a Biacore T200 instrument.

[0442] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then an antigen of a certain concentration was passed through the chip surface for 180 s, followed by dissociation for 600 s. The reaction signal was monitored in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Gly-HCl pH 1.5. A 1:1 model was used for data fitting. The results are shown in Table 7.

[0443] Table 7. Affinity test results of anti-EGFR-cMET bispecific antibodies and their ADCs to human EGFR antigen

[0444] The results showed that the disclosed anti-EGFR-cMET bispecific antibody and its ADC had comparable affinity to human EGFR antigen.

[0445] 2.2 The affinity of the disclosed anti-EGFR-cMET bispecific antibody and its ADC for human cMET-His antigen was tested using a Biacore T200 instrument.

[0446] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then an antigen of a certain concentration was passed through the chip surface for 180 s, followed by dissociation for 600 s. The reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Gly-HCl pH 1.5. A 1:1 model was used for data fitting. The results are shown in Table 8.

[0447] Table 8. Affinity test results of anti-EGFR-cMET bispecific antibodies and their ADCs to human cMET antigen

[0448] The results showed that the disclosed anti-EGFR-cMET bispecific antibody and its ADC had comparable affinity to the human cMET antigen.

[0449] 2.3 The affinity of the disclosed anti-EGFR-cMET bispecific antibody to bind to both targets simultaneously was tested using a Biacore 8K instrument.

[0450] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip. A high concentration of the first antigen was then passed through the chip surface for 150 seconds, followed by the injection of a second antigen (dissolved in the first antigen) for 180 seconds. Dissociation was then performed in the high concentration of the first antigen for 420 seconds. The reaction signal was monitored in real-time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated by injecting 10 mM Gly-HCl at pH 1.5 for 60 seconds. A 1:1 model was used for data fitting. The results are shown in Table 9.

[0451] Table 9. Affinity test results of anti-EGFR-cMET bispecific antibodies binding to both targets simultaneously.

[0452] The results showed that the anti-EGFR-cMET bispecific antibody disclosed herein had essentially no impact on its binding ability to the other antigen when binding to one antigen.

[0453] Test Example 3: FACS Detection of Cell Binding to Anti-EGFR-cMET Bispecific Antibody

[0454] Discard the culture medium from the cell culture flask, wash once with PBS, and then add an appropriate amount of trypsin (Invitrogen, catalog number 25200072) to digest the cells. Add HCC827 (EGFR+++, cMET+), H441 (EGFR+, cMET+), and EBC-1 (EGFR+, cMET+++) cells at 100,000 cells / well to a 96-well plate. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells once with PBS buffer containing 2% FBS. Use antibody at a starting concentration of 20 μg / mL, serially dilute 5-fold in PBS buffer containing 2% FBS, for a total of 8 concentration gradients. Add 100 μL of antibody sample to each well, resuspend the cells, and incubate at 4°C for 1 hour. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells twice with PBS buffer containing 2% FBS. Add secondary antibody (Alexa). 488goat anti-human IgG (H+L) 1:1000 (Invitrogen, A11013) was incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and then read. The results are shown in Table 10.

[0455] Table 10. FACS detection of cell binding of anti-EGFR-cMET bispecific antibodies and their ADCs

[0456] The results showed that the disclosed anti-EGFR-cMET bispecific antibody and its ADC had good affinity for HCC827, H441 and EBC-1 tumor cell lines.

[0457] Test Example 4: Anti-EGFR-cMET Dual Anti-tumor Cell Endocytosis Assay

[0458] Discard the culture medium from the HCC827 and EBC-1 tumor cell culture flasks and wash once with PBS. Then, add an appropriate amount of trypsin (Invitrogen, catalog number: 25200072) to digest the cells, centrifuge, and resuspend the cells in 2 mL of culture medium (medium: RPMI-1640 + 20% Low IgG FBS for HCC827, MEM + 20% Low IgG FBS + 2% NEAA + 2 mM PNa for EBC-1), count the cells, and adjust the cell density with the appropriate culture medium. Add 50 μL of the above cells to columns 2-10 of rows 2 to 7 of a 96-well plate (CORNING, 3610), add only 50 μL of seed medium to column 11, and add 100 μL of culture medium to the remaining wells. Incubate the cell plate overnight at 37°C with 5% CO2. Prepare 800 nM (4×) antibody (RPMI-1640 for HCC827, MEM for EBC-1) using FBS-free medium. Prepare 4800 nM (4×) DT3C (Diphtheria toxin & spg 3C domain) using FBS-free medium. Mix 75 μL of DT3C and 75 μL of antibody at a 1:1 volume ratio and incubate at room temperature for 30 minutes. Then, serially dilute the mixture 5-fold with FBS-free medium, resulting in 8 dilutions, with the 9th and 10th spots being pure medium. Add 50 μL of the diluted mixture to the wells of the cell plate, with two replicates for each antibody concentration. Incubate the cell plate in a cell culture incubator for 3 days. After 3 days, remove the cell plate and prepare according to the instructions. Following the instructions for the Luminescent Cell Viability Assay (Promega, G7573), prepare the CTG working solution, then add 50 μL of CTG working solution to each well. Gently vortex to mix and incubate at room temperature in the dark for 10 minutes. Read the luminescence signal values ​​using a VICTOR3 microplate reader. The experimental data were analyzed using Graphpad Prism 10.0.1 software, and the results are shown in Table 11.

[0459] Table 11. Endocytotic activity of EGFR-cMET bispecific antibodies

[0460] The results showed that the EGFR-cMET bispecific antibodies Omab-ZalH4 and Omab-ZalH10 disclosed herein exhibited good endocytic activity in HCC827 and EBC-1 cells.

[0461] Test Example 5: Anti-EGFR-cMET Bispecific Anti-ADC Tumor Cell Killing Experiment

[0462] Tumor cells (HCC827, H441, EBC-1) were grown in suitable culture media. The culture medium in the cell culture flasks was discarded, the cells were washed once with PBS, and then an appropriate amount of trypsin (Invitrogen, catalog number: 25200072) was added to digest the cells. The cells were centrifuged, resuspended in their respective culture media, counted, and the cell density was adjusted. 135 μL of cells were added to the wells of a 96-well plate (CORNING, 3610) (1000 cells per well for HCC827, 2000 cells per well for H441, and 1500 cells per well for EBC-1). Only 150 μL of cell growth medium was added to the edge wells. The cell culture plates were incubated overnight. A 10×ADC initial concentration was prepared with PBS, and then serially diluted 5-fold with PBS to nine concentration points. 15 μL of the diluted drug was added to the wells of the cell culture plate, with two replicates for each ADC concentration point. The cell culture plates were incubated for 6 days. The cell plate was removed 6 days later, according to the instructions. Following the instructions for the Luminescent Cell Viability Assay (Promega, G7573), prepare the CTG working solution by adding 50 μL of CTG working solution to each well, gently vortexing to mix, and incubating at room temperature in the dark for 10 minutes. Then, read the luminescence values ​​using a VICTOR3 microplate reader. The experimental data were analyzed using Graphpad Prism 10.0.1 data processing software, and the results are shown in Table 12.

[0463] Table 12. Data on tumor cell killing

[0464] The results showed that ADC-1 and ADC-2 exhibited superior cytotoxic activity compared to ADC-4 in this study. Particularly in HCC827 and H441 cells, the cytotoxic activity of ADC-1 and ADC-2 was significantly better than that of ADC-4.

[0465] Test Example 6: Anti-EGFR-cMET Bilateral Antitumor Selectivity Experiment

[0466] Tumor cells HCC827, H441, EBC-1, and EGFR-expressing cells CHO-K1 / EGFR were grown in suitable culture media. The culture medium in the cell culture flasks was discarded, and the cells were washed once with PBS. Then, an appropriate amount of trypsin (Invitrogen, catalog number: 25200072) was added to digest the cells. After centrifugation, the cells were washed twice with PBS and resuspended in Facs buffer (PBS containing 2% FBS). Cell density was adjusted, and CHO-K1 / EGFR and HCC827, CHO-K1 / EGFR and H441, and CHO-K1 / EGFR and EBC-1 were seeded at a 1:1 ratio (200,000:200,000 cells) into the upper and lower chambers of a transwell (CORNING, 3378). EGFR / CHOK1 was seeded into the upper chamber at 200 μL / well. HCC827, H441, and EBC-1 tumor cells were seeded into the lower chamber at 800 μL / well. The antibody to be tested was adjusted to 330 nM (11X) with Facs buffer, and then 100 μL was added to the lower chamber of the transwell plate. The mixture was incubated at 4°C on ice for 2 hours. After 2 hours, cells from the upper chamber of each well were collected into a U-shaped 96-well plate (CORNING, 3788), and cells from the lower chamber were collected into 1.5 mL centrifuge tubes. After centrifuging the 1.5 mL tubes containing cells from the lower chamber, the enriched cells were transferred to the remaining wells of the U-shaped 96-well plate containing cells from the upper chamber. Finally, the cells were washed twice with Facs buffer. 100 μL of fluorescent secondary antibody (Goat anti-Human IgG (H+L) Secondary Antibody, diluted 200 times with Facs buffer) was added to each well of the U-shaped 96-well plate. The sample was incubated with 488 conjugate (Life Technologies, A-11013) on ice for 30 minutes. It was then washed three times with FACS buffer and analyzed by flow cytometry. The mean fluorescence intensity (MFI) was calculated using Flowjo flow cytometry software. The selective tumor cell binding activity of the analyte was characterized by the ratio of the MFI value of each molecule on tumor cells to the MFI value on CHO-K1 / EGFR cells. The results are shown in Figure 4.

[0467] The results showed that, on HCC827, H441, and EBC-1 cells, the anti-EGFR-cMET bispecific antibodies Omab-ZalH10 and Omab-ZalH4 disclosed in this study exhibited significantly improved binding selectivity on all tumor cells compared to their respective EGFR monoclonal antibodies. This indicates that the EGFR-cMET bispecific antibodies can accumulate on tumor cells more effectively than EGFR monoclonal antibodies, thus enhancing the safety of the molecules.

[0468] Test Example 7: Efficacy Experiment of Anti-EGFR-cMET Bispecific Anti-ADC HCC827 CDX Model

[0469] 1. Experimental objective: This experiment aims to evaluate the in vivo efficacy and toxic side effects of different doses of bispecific antibody ADC administered intraperitoneally to human non-small cell lung cancer cells HCC827 tumor-bearing mice.

[0470] 2. Experimental principle: This experiment constructs an HCC827 tumor-bearing mouse model on Balb / c nude mice, administers the test molecules intraperitoneally, evaluates the pharmacodynamics of different molecules, and compares the efficacy of bispecific antibody ADCs.

[0471] 3. Laboratory animals: Balb / c nude rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0472] 4. Experimental apparatus:

[0473] CO2 incubator: HERACELL 150i GP INCUBATOR, Thermo Fisher Scientific, USA;

[0474] Biosafety cabinet: HFsafe-1800LC, Likang Company;

[0475] Inverted microscope: DMi1, LEICA Corporation;

[0476] Digital Vernier Caliper: 1195-200C, INSIZE Company;

[0477] Electronic balance: MP6001, Shanghai Sunny Hengping Scientific Instruments Co., Ltd.;

[0478] Centrifuge: L500-A, Hunan Xiangyi Instrument Equipment Co., Ltd.;

[0479] Electric thermostatic water bath: DK-S22, Shanghai Jinghong Company;

[0480] Electronic balance: ML204T-02, Mettler Company;

[0481] Microplate reader: Spark, TECAN;

[0482] Biochemical incubator: BSP-150, Shanghai Boxun Medical Bio-Instrument Co., Ltd.

[0483] 5. Experimental Procedure: 100 μL of HCC827 human non-small cell lung cancer cells (5 × 10⁶ cells) per mouse were inoculated subcutaneously into the right rib area of ​​160 female nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 15 days, the tumor grew to approximately 150 mm. 3Afterwards, mice were removed based on body weight and the size of tumors (too large or too small). Mice were then randomly divided into eight groups (D0) according to tumor volume and body weight: a Vehicle (PBS) control group, a negative control ADC-3 group, a positive control ADC-4 group, the disclosed EGFR-cMET bispecific antibody ADC-1 group, and an ADC-2 group. Each group consisted of eight mice. The ADCs were administered intraperitoneally on D0 mice as a single dose. Tumor volume and animal body weight were measured twice weekly, and data were recorded.

[0484] 6. Data Processing: The tumor volume of each group of animals was expressed as mean ± standard error (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two / one way ANOVA was used for statistical analysis to calculate the tumor inhibition rate. The formula is: Tumor proliferation rate (T / C%) = (T - T0 / C - C0) × 100%, Tumor inhibition rate % = 1 - T / C.

[0485] 7. The experimental results are shown in Table 13.

[0486] Table 13. Efficacy of ADC in the HCC827 CDX model in vivo (analysis using data from day 35) Note: D0 represents the grouping day, D1 represents the first day, and D9 represents the ninth day; / represents that it cannot be calculated; ns represents that it is not statistically significant, and the same applies below.

[0487] The results showed that ADC-1 and ADC-2 disclosed herein had good tumor-suppressing effects in the HCC827 CDX model, and their tumor-suppressing effects were better than those of ADC-4; especially at 1.5 mpk, the tumor-suppressing effects of ADC-1 and ADC-2 were significantly better than those of ADC-4.

[0488] Test Example 8: Efficacy Experiment of Anti-EGFR-cMET Bispecific Anti-ADC EBC-1CDX Model

[0489] 1. Experimental objective: This experiment aims to evaluate the in vivo efficacy and toxic side effects of different doses of bispecific antibody ADC administered intraperitoneally in mice bearing human lung cancer squamous cell carcinoma EBC-1 tumor cells.

[0490] 2. Experimental principle: This experiment constructs an EBC-1 tumor-bearing mouse model in B-NDG mice, administers the test molecules intraperitoneally, evaluates the pharmacodynamics of different molecules, and compares the efficacy of bispecific antibody ADCs.

[0491] 3. Experimental animals: B-NDG mice, purchased from Biocytogen Pharmaceuticals Co., Ltd.

[0492] 4. Experimental apparatus:

[0493] CO2 incubator: HERACELL 150i GP INCUBATOR, Thermo Fisher Scientific, USA;

[0494] Biosafety cabinet: HFsafe-1800LC, Likang Company;

[0495] Inverted microscope: DMi1, LEICA Corporation;

[0496] Digital Vernier Caliper: 1195-200C, INSIZE Company;

[0497] Electronic balance: MP6001, Shanghai Sunny Hengping Scientific Instruments Co., Ltd.;

[0498] Centrifuge: L500-A, Hunan Xiangyi Instrument Equipment Co., Ltd.;

[0499] Electric thermostatic water bath: DK-S22, Shanghai Jinghong Company;

[0500] Electronic balance: ML204T-02, Mettler Company;

[0501] Microplate reader: Spark, TECAN;

[0502] Biochemical incubator: BSP-150, Shanghai Boxun Medical Bio-Instrument Co., Ltd.

[0503] 5. Experimental Procedure: EBC-1 human lung cancer squamous cell carcinoma cells (5×10⁻¹¹) 6 100 μL of 1 / +gel (per mouse) was injected subcutaneously into the right rib area of ​​155 female B-NDG mice (purchased from Biocytogen Pharmaceuticals Co., Ltd.). After 10 days, the tumor grew to approximately 200 mm. 3 Afterwards, mice were removed based on body weight and the size of tumors (too large or too small). Mice were then randomly divided into seven groups (7 groups, 10 mice per group, D0) according to tumor volume and body weight: a Vehicle (PBS) control group, a negative control ADC-3 group, a positive control ADC-4 group, the disclosed EGFR-cMET bispecific antibody ADC-1 group, and an ADC-2 group. The ADCs were administered intraperitoneally on D1 as a single dose. Tumor volume and animal body weight were measured twice weekly, and data were recorded.

[0504] 6. Data Processing: The tumor volume of each group of animals was expressed as mean ± standard error (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two / one way ANOVA was used for statistical analysis to calculate the tumor inhibition rate. The formula is: Tumor proliferation rate (T / C%) = (T - T0 / C - C0) × 100%, Tumor inhibition rate % = 1 - T / C.

[0505] 7. The experimental results are shown in Table 14.

[0506] Table 14. Efficacy of ADCs in the EBC-1CDX model (analysis using data from day 20) Note: D0 represents the grouping day, D1 represents the first day, and D9 represents the ninth day; / represents that it cannot be calculated; ns represents that it is not statistically significant, and the same applies below.

[0507] The results showed that ADC-1 and ADC-2 disclosed herein had good tumor-suppressing effects in the EBC-1CDX model. Furthermore, at 1.5 mpk, the tumor-suppressing effects of ADC-1 and ADC-2 were superior to those of ADC-4.

[0508] Test Example 9: Killing Experiment of Osimertinib-Resistant Cell Lines by Anti-EGFR-cMET Bispecific Antibody ADC

[0509] Osimertinib-resistant EGFR mutant cell lines H1975-L858R-T790M-C797S (H975-LTC, constructed by Shanghai Hengrui) and Ba / F3-L858R-T790M-C797S (Ba / F3-LTC, (KYinno, KC-0122)) were grown in suitable culture media. The culture media in the cell culture flasks were discarded, the cells were washed once with PBS, and then an appropriate amount of trypsin (Invitrogen, catalog number: 25200072) was added to digest the cells. The cells were centrifuged, resuspended in their respective culture media, counted, and their densities were adjusted. Add 135 μL of cells to the wells of a 96-well plate (CORNING, 3610) (1000 cells per well for H1975-LTC, 1500 cells per well for BaF3-LTC), and add only 150 μL of cell growth medium to the edge wells. Incubate the plate overnight in a cell culture incubator. Prepare a 10× ADC initial concentration with PBS, and then perform 5-fold serial dilutions with PBS to nine concentration points. Add 15 μL of the diluted drug to the wells of the cell plate, with two replicates for each ADC concentration point. Dissolve Osimertinib in DMSO, and further perform 5-fold serial dilutions with DMSO to nine concentration points. Then, dilute the above series of concentrations 50-fold in PBS, and add 15 μL to the wells of the cell plate, with two replicates for each concentration point. Incubate the cell plate in a cell culture incubator for 6 days. After 6 days, remove the cell plate and prepare according to the instructions. Following the instructions for the Luminescent Cell Viability Assay (Promega, G7573), prepare the CTG working solution by adding 50 μL of CTG working solution to each well, gently vortexing to mix, and incubating at room temperature in the dark for 10 minutes. Then, read the luminescence values ​​using a VICTOR3 microplate reader. The experimental data were analyzed using Graphpad Prism 10.0.1 data processing software, and the results are shown in Table 15.

[0510] Table 15. Killing data of Osimertinib-resistant cell lines

[0511] The results showed that in the two Osimertinib-resistant cell lines H1975-LTC and Ba / F3-LTC, ADC-1 and ADC-2 had comparable activities and significantly stronger cell-killing activity compared to Osimertinib.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, which is a compound or a pharmaceutically acceptable salt of general formula (I): Ab-(LYD) n (I) Wherein Ab is an antigen-binding molecule that specifically binds to EGFR and cMET; preferably, Ab is an anti-EGFR-cMET bispecific antibody, which comprises: (1) A first chain having the structure shown in equation (a), a second chain having the structure shown in equation (b), a third chain having the structure shown in equation (c), and a fourth chain having the structure shown in equation (d). (a)[cMET-VH]-[CH1]-[Fc1], (b)[cMET-VL]-[CL], (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2], (d)[EGFR-VL]-[connector 2]-[Obscurin], The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist. The structures shown in equations (a), (b), (c), and (d) are arranged from the N-end to the C-end; or (2) A first chain having the structure shown in equation (e), a second chain having the structure shown in equation (f), a third chain having the structure shown in equation (g), and a fourth chain having the structure shown in equation (h). (e)[EGFR-VH]-[CH1]-[Fc1], (f)[EGFR-VL]-[CL], (g)[cMET-VH]-[connector 3]-[Titin]-[Fc2], (h)[cMET-VL]-[connector 4]-[Obscurin], The linker 3 and linker 4 may be the same or different, and are peptide linkers; or linker 3 or linker 4 may not exist. The structures shown in equations (e), (f), (g), and (h) are arranged from the N end to the C end; L represents the connector unit; Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b They may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl; Or, R a and R b Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, or heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino. R 1 The group is selected from halogen, alkyl, haloalkyl, hydroxy, hydroxyalkyl, alkoxy, cyano, amino, carboxyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, heterocyclic alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl, wherein each of the cycloalkyl, heterocyclic, aryl, and heteroaryl groups is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino. R 2 Selected from hydrogen atom, halogen, alkyl, haloalkyl, hydroxyl, hydroxyalkyl, alkoxy, cyano, amino, -(CR) c R d ) p -NR e R f 、-(CR c R d ) p -COOH, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, heterocyclic alkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl, wherein each of the cycloalkyl, heterocyclic, aryl and heteroaryl groups is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; Or, R 1 and R 2 Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, and heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino groups. Or, R a and R 2 Together with the carbon atom attached thereto, they form cycloalkyl, heterocyclic, aryl, and heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxyl, cyano, and amino groups. R c and R d They may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl; Or, R c and R d Together with the carbon atoms connected thereto, they form cycloalkyl, heterocyclic, aryl, or heteroaryl groups, each of which is independently and optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano, and amino. R e and R f The same or different, and each independently selected from hydrogen atoms, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein each cycloalkyl, heterocyclic, aryl and heteroaryl is independently optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino; Or R e and R f Together with the nitrogen atoms attached to them, they form a heterocyclic group, which is optionally substituted by one or more substituents selected from oxo, halogen, alkyl, haloalkyl, alkoxy, hydroxyalkyl, hydroxy, cyano and amino. m can be 0, 1, 2, 3, or 4; p is 0, 1, 2, 3, 4, 5 or 6; D is ixotecan or its derivatives; n is 1 to 10; preferably, n is 1 to 8; more preferably, n is 4 to 8; most preferably, n is about 6.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by general formula (II) or a pharmaceutically acceptable salt thereof: Where Ab, L, Y and n are defined as in claim 1.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein: Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 alkoxy, hydroxy and C 1-6 Hydroxyalkyl; R 1 Halogen, C 1-6 Halogenated alkyl, 3- to 6-membered cycloalkyl and 3- to 6-membered cycloalkyl C 1-6 alkyl; R 2 Selected from hydrogen atoms, halogens, C 1-6 Halogenated alkyl, 3- to 6-membered cycloalkyl and 3- to 6-membered cycloalkyl C 1-6 alkyl; Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups; m can be 0, 1, 2, 3, or 4; Preferably, Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-; Among them, R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 alkyl; R 1 It is a 3- to 6-membered cycloalkyl or a 3- to 6-membered cycloalkyl-C 1-6 alkyl; R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and 3- to 6-membered cycloalkyl groups; Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups; m can be 0, 1, 2, 3, or 4.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -, in: L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkylene and C 1-8 alkylene-3 to 6-membered cycloalkyl, wherein the C 1-8 Alkylene or C 1-8 The alkylene group (3 to 6-membered cycloalkyl group) is independently selected from halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group; L 2 Selected from -NR 4 (CH2CH2O) p1 CH2CH2C(O)-、-NR 4 (CH2CH2O) p1 CH2C(O)-、-S(CH2) p1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20; L 3 A peptide consisting of 2 to 7 amino acid residues, selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally selected from halogens, hydroxyl groups, cyano groups, amino groups, C groups, and C groups. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -、-C(O)NR 5 (CH2) t - and chemical bonds, where t is 1, 2, 3, 4, 5 or 6; R 3 R 4 and R 5 Whether the atoms are the same or different, and each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 1-6 Halogenated alkyl and C 1-6 Hydroxyalkyl; R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl, hydroxyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups and C 1-6 Hydroxyalkyl; The L of -L- 1 The terminal is connected to Ab, L 4 The end is connected to Y.

5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by general formula (III) or a pharmaceutically acceptable salt thereof: Where R 1 It is a 3- to 6-membered cycloalkyl or a 3- to 6-membered cycloalkyl-C 1-6 alkyl; R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and 3- to 6-membered cycloalkyl groups; Or, R 1 and R 2 Together with the carbon atoms attached thereto, they form 3- to 6-membered cycloalkyl groups; W is selected from C 1-8 Alkylene and C 1-8 alkylene-3 to 6-membered cycloalkyl, wherein the C 1-8 Alkylene and C 1-8 The alkylene group (3 to 6-membered cycloalkyl group) is independently selected from halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 It is substituted with one or more substituents of alkoxy and 3 to 6-membered cycloalkyl groups; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20; L 3 A peptide consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, alanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid, and optionally selected from halogens, hydroxyl groups, cyano groups, amino groups, C groups, etc. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 The alkoxy group is substituted with one or more substituents in a 3- to 6-membered cycloalkyl group; R 4 and R 5 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; m can be 0, 1, 2, 3, or 4; Where Ab and n are defined as in claim 1.

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the compound is a pharmaceutically acceptable salt thereof as follows: Where Ab and n are defined as in claim 1.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-EGFR-cMET bispecific antibody comprising a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d). (a)[cMET-VH]-[CH1]-[Fc1], (b)[cMET-VL]-[CL], (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2], (d)[EGFR-VL]-[connector 2]-[Obscurin], The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist. The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end; Fc1 has a protruding structure according to the pestle and mortar technique, and Fc2 has a hole structure according to the pestle and mortar technique; wherein: (1) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

6. The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25; or (2) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

6. The cMET-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; and the cMET-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO:

25. Preferably, EGFR-VH contains the amino acid sequence of SEQ ID NO: 14 or 15, and EGFR-VL contains the amino acid sequence of SEQ ID NO: 16; and cMET-VH contains the amino acid sequence of SEQ ID NO: 26; and cMET-VL contains the amino acid sequence of SEQ ID NO: 27; More preferably, Ab has: (1) A first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 38, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39; or (2) A first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 40 and a fourth chain containing the amino acid sequence of SEQ ID NO:

39.

8. A method for preparing a compound of formula (III) or a pharmaceutically acceptable salt thereof, comprising the steps of: After reduction, Ab undergoes a coupling reaction with a compound of general formula (IIIa) or its salt to give a compound of general formula (III) or its pharmaceutically acceptable salt. Among them, Ab, W, L 2 L 3 R 1 R 2 R 5 To R 7 m and n are as defined in claim 5.

9. An antigen-binding molecule that specifically binds to EGFR and cMET, comprising at least one antigen-binding module specifically binding to EGFR and at least one antigen-binding module specifically binding to cMET, wherein the antigen-binding module specifically binding to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module specifically binding to cMET comprises a heavy chain variable region cMET-VH and a light chain variable region cMET-VL; wherein: (1) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 1, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 9; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

6. The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO: 25; or (2) The HCDR1 of the EGFR-VH contains the amino acid sequence of SEQ ID NO: 10, the HCDR2 contains the amino acid sequence of SEQ ID NO: 2, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 11; and the LCDR1 of the EGFR-VL contains the amino acid sequence of SEQ ID NO: 4, the LCDR2 contains the amino acid sequence of SEQ ID NO: 5, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

6. The cMET-VH's HCDR1 contains the amino acid sequence of SEQ ID NO: 20, HCDR2 contains the amino acid sequence of SEQ ID NO: 21, and HCDR3 contains the amino acid sequence of SEQ ID NO: 22; the cMET-VL's LCDR1 contains the amino acid sequence of SEQ ID NO: 23, LCDR2 contains the amino acid sequence of SEQ ID NO: 24, and LCDR3 contains the amino acid sequence of SEQ ID NO:

25.

10. The antigen-binding molecule that specifically binds EGFR and cMET according to claim 9, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 14 or 15, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 16; and the cMET-VH comprises the amino acid sequence of SEQ ID NO: 26; and the cMET-VL comprises the amino acid sequence of SEQ ID NO:

27.

11. The antigen-binding molecule that specifically binds EGFR and cMET according to claim 9 or 10, wherein the antigen-binding module that specifically binds EGFR or the antigen-binding module that specifically binds cMET comprises a Titin chain and an Obscurin chain capable of forming a dimer. Preferably, the titin chain contains the amino acid sequence of SEQ ID NO: 30, and the obscurin chain contains the amino acid sequence of SEQ ID NO:

31.

12. The antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 11, comprising an Fc region, wherein the Fc region is preferably an IgG Fc region, more preferably an IgG1 Fc region; more preferably, the Fc region comprises one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor.

13. The antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 12, comprising an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 and Fc2 each independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; Preferably, Fc1 has a protruding structure according to the pestle and mortar technique, and Fc2 has a hole structure according to the pestle and mortar technique; or, Fc2 has a protruding structure according to the pestle and mortar technique, and Fc1 has a hole structure according to the pestle and mortar technique. More preferably, the amino acid at position 354 of Fc1 is C, and the amino acid at position 366 is W; and the amino acid at position 349 of Fc2 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index; or, the amino acid at position 354 of Fc2 is C, and the amino acid at position 366 is W; and the amino acid at position 349 of Fc1 is C, the amino acid at position 366 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index; Most preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 34, and Fc2 contains the amino acid sequence of SEQ ID NO:

35.

14. The antigen-binding molecule that specifically binds EGFR and cMET according to any one of claims 9 to 13, comprising an antigen-binding module that specifically binds EGFR and an antigen-binding module that specifically binds cMET, wherein the antigen-binding module that specifically binds cMET is a Fab, and the antigen-binding module that specifically binds EGFR is a replaced Fab comprising a dimeric Titin chain and an Obscurin chain; or, the antigen-binding module that specifically binds cMET is a replaced Fab comprising a dimeric Titin chain and an Obscurin chain, and the antigen-binding module that specifically binds EGFR is a Fab; Preferably, (1) The antigen-binding molecule that specifically binds to EGFR and cMET comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d). (a)[cMET-VH]-[CH1]-[Fc1], (b)[cMET-VL]-[CL], (c)[EGFR-VH]-[connector 1]-[Titin]-[Fc2], (d)[EGFR-VL]-[connector 2]-[Obscurin], The linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist. The structures shown in equations (a), (b), (c), and (d) are arranged from the N-end to the C-end; or (2) The antigen-binding molecule that specifically binds to EGFR and cMET comprises a first chain having the structure shown in formula (e), a second chain having the structure shown in formula (f), a third chain having the structure shown in formula (g), and a fourth chain having the structure shown in formula (h). (e)[EGFR-VH]-[CH1]-[Fc1], (f)[EGFR-VL]-[CL], (g)[cMET-VH]-[connector 3]-[Titin]-[Fc2], (h)[cMET-VL]-[connector 4]-[Obscurin], The linker 3 and linker 4 may be the same or different, and are peptide linkers; or linker 3 or linker 4 may not exist. The structures shown in equations (e), (f), (g), and (h) are arranged from the N end to the C end.

15. The antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 14, wherein it is an anti-EGFR-cMET bispecific antibody; preferably, it comprises: (1) A first strand containing the amino acid sequence of SEQ ID NO: 36, a second strand containing the amino acid sequence of SEQ ID NO: 37, a third strand containing the amino acid sequence of SEQ ID NO: 38, and a fourth strand containing the amino acid sequence of SEQ ID NO: 39; or (2) A first chain containing the amino acid sequence of SEQ ID NO: 36, a second chain containing the amino acid sequence of SEQ ID NO: 37, a third chain containing the amino acid sequence of SEQ ID NO: 40 and a fourth chain containing the amino acid sequence of SEQ ID NO:

39.

16. A conjugate comprising an antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 15 and a payload, wherein the payload is conjugated to the antigen-binding molecule; preferably, the payload is a topoisomerase inhibitor; more preferably, the payload is eczema or a derivative thereof.

17. A pharmaceutical composition comprising an antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 15, a conjugate according to claim 16, or a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

18. An isolated nucleic acid encoding an antigen-binding molecule that specifically binds to EGFR and cMET according to any one of claims 9 to 15.

19. A host cell comprising the isolated nucleic acid as described in claim 18.

20. A method for preventing or treating a disease, the method comprising administering to a subject an antigen-binding molecule that specifically binds to EGFR and cMET as claimed in any one of claims 9 to 15, a conjugate as claimed in claim 16, or a compound as claimed in any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 17; preferably, the disease is a tumor; more preferably, the disease is selected from lung cancer, breast cancer, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, ovarian cancer, salivary gland cancer, head and neck cancer, and glioblastoma; most preferably, the disease is non-small cell lung cancer.

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