Immunoconjugates and pharmaceutical uses thereof

Abstract

Disclosed are an immunoconjugate of a bispecific antibody and a small molecule drug, and use of the immunoconjugate and a pharmaceutical composition comprising the same in the preparation of a medicament for treating cancer.

Description

Technical Field

[0001] This invention relates to immunoconjugates with improved cancer targeting capabilities, comprising a bispecific antibody targeting EGFR and c-MET, and a therapeutic portion belonging to the camptothecin class of drugs. The invention further relates to the use of these immunoconjugates in the preparation of medicaments for treating cancer. Background Technology

[0002] Epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR or c-MET) have been found to be overexpressed in many tumors. For example, EGFR is overexpressed in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma. c-MET receptor is overexpressed in NSCLC and several other solid tumor types. Recently, co-expression of EGFR and c-MET has been found in many solid tumors, including head and neck squamous cell carcinoma, NSCLC, colorectal cancer, gastric cancer, renal cell carcinoma, and pancreatic ductal adenocarcinoma.

[0003] For patients with aberrant EGFR activation, EGFR-targeting tyrosine kinase inhibitors (TKIs) are the standard treatment. However, many patients develop resistance to TKIs. Among the relevant mechanisms, enhanced activation of the c-MET pathway is common in EGFR TKI-resistant patients. Furthermore, dual-receptor targeting strategies using a single-agent combination of EGFR and MET inhibitors cannot cover a wide range of resistance mechanisms. Therefore, a novel approach using bispecific antibodies that simultaneously target EGFR and c-MET has proven to be an effective way to overcome resistance and achieve better efficacy in a subgroup of NSCLC patients.

[0004] One way to improve the efficacy of antibody therapy is through antibody-drug conjugates (ADCs). ADCs are a new type of anticancer drug that combines antibodies with cytotoxins through chemical conjugation. Antibody-drug conjugation technology links small molecule drugs to biological proteins, offering the advantage of significantly improving drug efficacy while reducing side effects.

[0005] Given the common challenges of off-target toxicity and clinical resistance faced by traditional ADCs, target selection should follow these criteria: (1) Traditional guidelines involve identifying antigens that are well-endocytic, relatively poorly expressed in normal tissues, and highly expressed in tumors. (2) Considering the heterogeneity of solid tumors, it is necessary to determine the expression levels of targets in various tumor subtypes and sites to drive the optimal implementation of customized drug delivery, rather than relying on a single "magic bullet" strategy. (3) Due to the unique dual-targeting nature of bispecific ADCs (BsADCs), it is crucial to comprehensively consider the deep effects of antigen combinations. This encompasses many factors, including endocytosis, recovery, rates of turnover, lysosomal degradation, and obligate mechanisms. Thoughtful integration of these considerations is essential for the effective design of BsADCs, ensuring a deep understanding of the dynamic interactions between antigens and cellular processes (Acta Pharm Sin B. May 2024; 14(5):1965-1986).

[0006] Currently, BsADCs are still in the early stages of development, and initial clinical trials face numerous challenges. Careful selection of BsAbs, adaptors, and cytotoxic payloads is crucial for achieving the expected therapeutic outcomes of ADCs. For example, the endocytosis of BsAbs and their potential for inclusion in BsADCs are unpredictable and involve many uncertainties. Furthermore, adaptors and cytotoxic payloads also significantly impact therapeutic outcomes in ADCs.

[0007] Because EGFR and c-Met receptors are highly co-expressed and overexpressed in various types of tumor cells, ADCs that simultaneously target EGFR and c-MET (i.e., EGFR×c-MET bispecific ADCs) may have the advantage of improving therapeutic efficacy and could benefit a wider range of patients, regardless of whether abnormal EGFR and c-MET activation is a driving factor for tumorigenesis. Therefore, there is an urgent need to develop bispecific antibody-drug conjugates (bispecific ADCs) that target EGFR×c-MET, have good internalization properties, and possess strong anti-tumor effects. Summary of the Invention

[0008] The technical problem to be solved by this invention is to develop an immunoconjugate containing a bispecific antibody targeting EGFR and c-Met, as well as a therapeutic drug. This immunoconjugate exhibits good endocytosis and very strong antitumor effects in cancer. The antitumor effect can be attributed to improved antibody affinity, enhanced internalization to achieve potent endocytosis, and a higher drug-to-antibody ratio (DAR).

[0009] Specifically, the present invention covers the following aspects: This disclosure relates to an immunoconjugate, or a tautomer, mesosome, racemate, enantiomer, or diastereomer thereof, or a pharmaceutically acceptable salt or solvate thereof, the immunoconjugate being represented by general formula (A): , in: D is a cytotoxic drug; L1 and L2 are linking units; y is a number from 1 to 20, preferably from 2 to 10, more preferably from 2 to 8, even more preferably from 2 to 6 or 4 to 8, and most preferably from 2, 3, 4, 5, 6, 7, 8; Ab is an antibody or an antibody fragment thereof, comprising: a first antigen-binding domain that binds to EGFR and a second antigen-binding domain that binds to c-Met.

[0010] In some embodiments of this disclosure, the second antigen-binding domain includes a heavy chain variable region (VH) comprising the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2 HCDR3 having the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof, wherein one, two, or three amino acids of one or more of HCDR1, HCDR2, or HCDR3 are substituted with other amino acids; and a light chain variable region (VL) comprising the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO: 4. LCDR2 has the amino acid sequence shown in SEQ ID NO: 5. LCDR3 having the amino acid sequence shown in SEQ ID NO: 6, or a variant thereof, wherein one, two, or three of one or more of LCDR1, LCDR2, or LCDR3 are replaced by other amino acids.

[0011] In some embodiments of this disclosure, the second antigen-binding domain includes a heavy chain variable region (VH) comprising the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2 HCDR3 having the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region (VL) comprising the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO: 4. LCDR2 has the amino acid sequence shown in SEQ ID NO: 5. LCDR3 having the amino acid sequence shown in SEQ ID NO: 6.

[0012] In some embodiments of this disclosure, the second antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO: 13, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; and a VL region containing the amino acid sequence shown in SEQ ID NO: 14, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14.

[0013] In some embodiments of this disclosure, the second antigen-binding domain comprises: a VH region containing the amino acid sequence of SEQ ID NO: 13; and a VL region containing the amino acid sequence of SEQ ID NO: 14.

[0014] In some embodiments of this disclosure, the first antigen-binding domain includes a heavy chain variable region (VH) comprising the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7 HCDR2 has the amino acid sequence shown in SEQ ID NO: 8. HCDR3 having the amino acid sequence shown in SEQ ID NO: 9, or a variant thereof, wherein one, two, or three amino acids of one or more of HCDR1, HCDR2, or HCDR3 are substituted with other amino acids; and a light chain variable (VL) region comprising the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO: 10. LCDR2 has the amino acid sequence shown in SEQ ID NO: 11. LCDR3 having the amino acid sequence shown in SEQ ID NO: 12, or a variant thereof, wherein one, two, or three of one or more of LCDR1, LCDR2, or LCDR3 are replaced by other amino acids.

[0015] In some embodiments of this disclosure, the first antigen-binding domain includes a heavy chain variable region (VH) comprising the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7 HCDR2 has the amino acid sequence shown in SEQ ID NO: 8. HCDR3 having the amino acid sequence shown in SEQ ID NO: 9; and a light chain variable region (VL) containing the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO: 10. LCDR2 has the amino acid sequence shown in SEQ ID NO: 11. LCDR3 having the amino acid sequence shown in SEQ ID NO: 12.

[0016] In some embodiments of this disclosure, the first antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO: 15, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; and a VL region containing the amino acid sequence shown in SEQ ID NO: 16, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0017] In some embodiments of this disclosure, the first antigen-binding domain comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 15; and a VL region comprising the amino acid sequence of SEQ ID NO: 16.

[0018] In some embodiments of this disclosure, the antibody fragment is selected from: Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), diabody, triabody, scFv-Fc, disulfide bond stabilized Fv (dsFv), and CDR-containing peptides.

[0019] In some embodiments of this disclosure, the first antigen-binding domain and / or the second antigen-binding domain further include a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conserved variants, and the light chain constant region of the human antibody constant region is selected from the κ chain constant region and the λ chain constant region of the human antibody and their conserved variants. More preferably, the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 25 or 26, or has at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25 or 26; and the light chain constant region contains the amino acid sequence shown in SEQ ID NO: 27, or has at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 27. More preferably, the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 25 or 26; and the light chain constant region contains the amino acid sequence shown in SEQ ID NO: 27.

[0020] In some embodiments of this disclosure, the first antigen-binding domain and the second antigen-binding domain form a heterodimer, and the first antigen-binding domain and / or the second antigen-binding domain further include a CH1 region and a CL region, wherein the CH1 region of the second antigen-binding domain includes one or more substitutions selected from L145Q, K147E, and S181E, and the CL region of the second antigen-binding domain includes one or more substitutions selected from T129R, T178R, and T180Q; and / or the CH1 region of the first antigen-binding domain includes one or more substitutions selected from L128R and K147R; and the CL region of the first antigen-binding domain includes one or more substitutions selected from Q124E, V133Q, and T178E; wherein the constant regions are numbered according to the EU index; Preferably, the amino acid substitutions in the CH1 region of the second antigen-binding domain include L145Q, K147E, and S181E, and the amino acid substitutions in the CL region of the second antigen-binding domain include T129R, T178R, and T180Q; and / or the amino acid substitutions in the CH1 region of the first antigen-binding domain include L128R and K147R, and the amino acid substitutions in the CL region of the first antigen-binding domain include Q124E, V133Q, and T178E, wherein the constant regions are numbered according to the EU index; More preferably, the first antigen-binding domain comprises the human antibody heavy chain constant region shown in SEQ ID NO: 19 and the human light chain constant region shown in SEQ ID NO: 20; and / or the second antigen-binding domain comprises the human antibody heavy chain constant region shown in SEQ ID NO: 17 and the human light chain constant region shown in SEQ ID NO: 18.

[0021] In some embodiments of this disclosure, the first antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 23 or 30, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 30; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 24 or 31, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24 or 31; and / or The second antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 21 or 28, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or 28; and a light chain containing the amino acid sequence shown in SEQ ID NO: 22 or 29, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 or 29; In some embodiments of this disclosure, the first antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 23 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 24 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; and / or, The second antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 21 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; and a light chain containing the amino acid sequence shown in SEQ ID NO: 22 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22.

[0022] In some embodiments of this disclosure, the first antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 31 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; and / or, The second antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 28 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28; and a light chain containing the amino acid sequence shown in SEQ ID NO: 29 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.

[0023] In some embodiments of this disclosure, the first antigen-binding domain comprises: a heavy chain containing the amino acid sequence of SEQ ID NO: 23; and a light chain containing the amino acid sequence of SEQ ID NO: 24; and the second antigen-binding domain comprises: a heavy chain containing the amino acid sequence of SEQ ID NO: 21; and a light chain containing the amino acid sequence of SEQ ID NO: 22.

[0024] In some embodiments of this disclosure, the first antigen-binding domain comprises: a heavy chain containing the amino acid sequence of SEQ ID NO: 30; and a light chain containing the amino acid sequence of SEQ ID NO: 31; and the second antigen-binding domain comprises: a heavy chain containing the amino acid sequence of SEQ ID NO: 28; and a light chain containing the amino acid sequence of SEQ ID NO: 29.

[0025] In some embodiments of this disclosure, the cytotoxic agent is selected from toxins, chemotherapeutic agents, antibiotics, radioisotopes, and nucleases.

[0026] Many cytotoxic drugs are used to develop ADCs. In some embodiments of this disclosure, the cytotoxic drug is selected from microtubule inhibitors or topoisomerase inhibitors; preferably, olistatin analogs, maytansine derivatives, eribulin and its derivatives, pyrrolobenzodiazepines or camptothecin derivatives; more preferably, SN-38, MMAE, MMAF, MMAD, eribulin or eczetidine and its derivatives.

[0027] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, has one of the following structures: , , , , , , , , , ; in: L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 3, 4, 5, 6, 7, 8; Ab is the above-mentioned antibody or an antibody fragment thereof.

[0028] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is represented by general formula (D): ; in: The carbon atoms labeled p1* and p2* are each asymmetry centers, and the asymmetry centers are... S Configuration, R Configuration, or racemate; R3 and R4 are each independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, or hydroxyl; or R3 and R4 together with the atoms they are attached to form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclic group, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R5 is hydrogen, deuterium, C 1-6 Alkyl, deuterated alkyl, substituted alkyl, aryl, substituted aryl, or heteroaryl; L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 2, 3, 4, 5, 6, 7, 8; Ab is the above-mentioned antibody or an antibody fragment thereof.

[0029] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, has one of the following structures: , , , , , , , , , , , , , , , , ; in: L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 2, 3, 4, 5, 6, 7, 8; Ab is the above-mentioned antibody or an antibody fragment thereof.

[0030] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is represented by general formula (B): ; in: L1 and L2 are connecting units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, further preferably a number selected from 4 to 8, and most preferably 3, 4, 5, 6, 7, 8; Ab is the above-mentioned antibody or an antibody fragment thereof.

[0031] In some embodiments of this disclosure, -L2- is selected from -La-Lb-, and -L1- is selected from -Lc-Ld-Le-. Wherein, -La- is a covalent linking unit, -Lb- is an extension unit, -Lc- is a bond or polar hydrophilic group unit, wherein the polar hydrophilic group includes sugar residues and their derivatives, polyethylene glycol residues and their derivatives, polysarcosine residues and their derivatives or combinations thereof, -Ld- is selected from peptide residues composed of 2-8 amino acids, and -Le- is a bond or self-cleaving fragment.

[0032] In some embodiments of this disclosure, -La- is selected from: , , , , ; -Lb- is selected from: -(CH2)s 2 -C(=O)-, -CH2-C(=O)-NH-(CH2)s 1 -C(=O)-, -(CH2CH2O)s 2 -(CH2)s 1 -C(=O)-、 , ; S 1 and S 2 Independently selected as an integer from 0 to 8; -Lc- is a key. , ,or , where s3 Integers selected from 2 to 12; preferably, s 3 Choose from 6, 7, 8, 9, 10, 11, or 12; -Ld- is selected from peptide residues consisting of 2-8 amino acids, wherein the amino acids are selected from: phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine; preferably, -Ld- is selected from: , , , ; -Le- is the key. or -NH-CH2-O-(CRaRb)s 4 -CR1R2-C(O)-; where Ra and Rb may be the same or different, and are independently selected from: hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxyl, amino; s 4 The integer is selected from 0-4, preferably s 4 It is 0 or 1; R1 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; R2 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; or R1 and R2 together with the carbon atoms they are attached to form C 3-8 Cycloalkyl; preferably: R1 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; R2 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; or R1 and R2 together with the carbon atoms they are attached to form C 3-6 Cycloalkyl.

[0033] In some embodiments of this disclosure, -L2-L1- has one of the following structures: , , , , , , , , , , , , , , , , , , , , , , , .

[0034] In some embodiments of this disclosure, -L2-L1- has one of the following structures: , , , , , , , , .

[0035] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, may have the following structure: , , , , , , , , , , , , , , , , ; in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 3, 4, 5, 6, 7, 8; Ab is the above-mentioned antibody or an antibody fragment thereof.

[0036] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is represented by general formula (C): ; in: R1 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; R2 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; or R1 and R2 together with the carbon atoms they are attached to form C 3-8 Cycloalkyl.

[0037] In some embodiments of this disclosure, R1 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; R2 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; or R1 and R2 together with the carbon atoms they are attached to form C 3-6 Cycloalkyl.

[0038] In some embodiments of this disclosure, L2 is as shown in general formula (I): ; in: s 1 and s 2 Each is an integer selected independently from 0-8, preferably, s 1 and s 2 Independently selected from 1, 2, 3, 4, 5, or 6; or, s 1 It is an integer from 1 to 8, s 2 It is 0, preferably, s 1Selected from 4, 5, 6, 7 or 8, and s 2 It is 0; or, s 2 Integers selected from 2 to 8, s 1 It is 2, preferably, s 2 Choose from 2, 3, 4, 5, or 6, and s 1 It is 2.

[0039] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, may have the following structure: , , , , , , , , , , , , , ; in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 4 to 8, and most preferably 3, 4, 5, 6, 7, or 8; Ab is the antibody described above or an antibody fragment thereof.

[0040] In some embodiments of this disclosure, the immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following compounds: , , , , , , , , , , , ; in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably a number selected from 3, 4, 5, 6, 7, or 8.

[0041] On the other hand, this disclosure provides a pharmaceutical composition comprising the above-mentioned immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0042] On the other hand, this disclosure provides the use of the above-mentioned immune conjugates or their tautomers, mesosomes, racemates, enantiomers or diastereomers, or pharmaceutically acceptable salts or solvates thereof, or the above-mentioned pharmaceutical compositions in the preparation of medicaments for the treatment or prevention of diseases related to human EGFR and / or c-MET.

[0043] In some embodiments of this disclosure, the use is for preparing a medicament for treating or preventing cancers expressing EGFR and / or c-MET; preferably, the cancer is epithelial carcinoma, lung cancer, ovarian cancer, breast cancer, colorectal cancer, squamous cell carcinoma of the head and neck (SCCHN or SQHN), anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, or glioblastoma; more preferably, the lung cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, or small cell lung cancer.

[0044] The resulting immunoconjugates possess a series of superior properties: i) specific binding to EGFR and c-MET; ii) good internalization ability in cancer cells with high EGFR expression; and iii) in vitro and in vivo activity in various cancer cells with different EGFR and c-MET expression levels. Specifically, the bispecific antibody of this application, when conjugated with a cytotoxic drug, exhibits significantly better in vitro efficacy than the cytotoxic drug alone. Furthermore, it was found that other bispecific antibodies in the prior art, when conjugated with the same cytotoxic drug, showed weaker killing effects in some tumor cells compared to the drug alone. In terms of in vivo efficacy, the ADC of this application is significantly superior to the control ADC and also superior to the antibody-toxin combination group. Attached Figure Description

[0045] Figure 1 EGFRxc-MET bispecific antibody binds to SNU-1 (A) and SW620 (B) cells in vitro.

[0046] Figure 2 (A) HGF, EGF, and bispecific antibodies activate the SRE reporter gene in the HEK293 cell line. (B) Bispecific antibodies block HGF activation of reported SRE cells. (C) Bispecific antibodies block EGF activation of reported SRE cells.

[0047] Figure 3 In vitro cytotoxicity dose-response curves of EGFRxc-MET bispecific ADCs in cancer cells.

[0048] Figure 4 Tumor growth curves in mice treated with solvent (Vehicle) and ADC in the NCI-H1975 CDX model.

[0049] Figure 5 Tumor growth curves in mice treated with solvent and ADC in the NCI-H1975 CDX model.

[0050] Figure 6 Tumor growth curves in mice treated with solvent and ADC in the NCI-H1975 CDX model.

[0051] Figure 7 Tumor growth curves in mice treated with solvent and ADC in the NCI-H441 CDX model.

[0052] Figure 8 Tumor growth curves in mice treated with solvent and ADC in the SNU-1 CDX model.

[0053] Figure 9 Tumor growth curves in mice treated with solvent and ADC in the HCT-116 CDX model.

[0054] Figure 10 Tumor growth curves in mice treated with solvent and ADC in the Detroit562 CDX model. Detailed Implementation

[0055] This invention is based on the development of immune conjugates comprising bispecific antibodies and therapeutic agents capable of specifically binding to EGFR and c-Met, including, for example, eczetidine or eczetidine derivatives.

[0056] The headings used in this section are for illustrative purposes only and do not limit the invention. Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. Furthermore, unless the context specifically requires it, singular forms include plural forms, and plural forms include singular forms. Abbreviations for amino acid residues are standard three-letter and / or one-letter codes used in the art to represent one of the 20 common L-amino acids.

[0057] definition The term "antibody" is used in a broad sense to include immunoglobulin molecules, including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies), full-length antibodies, antigen-binding fragments, multispecific antibodies (such as bispecific, trispecific, tetraspecific, etc.), dimer, tetramer, or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of immunoglobulin molecules containing the antigen-binding site required for specificity.

[0058] Any antibody fragment that can bind to a relevant target molecule can be called an "antibody fragment". Examples of antibody fragments include: Fv, scFv, Fab, scFab, F(ab')2, Fab2, bisomatic antibodies, tripois antibodies, scFv-Fc, microantibodies, and single-domain antibodies (e.g., VhH).

[0059] The "antibody" or "antibody fragment" according to this disclosure comprises one or more antigen-binding domains. An "antigen-binding domain" describes a portion of a molecule that binds to all or part of a target antigen. In cases where the antigen is large, the antibody may bind only to a specific portion of the antigen, referred to as an epitope. One or more variable domains of the antibody may provide an antibody-antigen binding site. The antibody-antigen binding site preferably comprises a variable light chain region (VL) and a variable heavy chain region (VH). The VH and VL regions of the antigen-binding domain constitute the Fv region.

[0060] An antigen-binding domain typically contains six complementarity-determining regions (CDRs): three in the VH region (HCDR1, HCDR2, and HCDR3) and three in the VL region (LCDR1, LCDR2, and LCDR3). These six CDRs collectively define the antigen-binding site of the antigen-binding domain, which is the portion of the antigen-binding domain that binds to the target antigen.

[0061] The VH and VL regions contain frame regions (FRs) on both sides of each CDR, which provide support for the CDR. From the N-end to the C-end, the VH region contains the following structure: N-end - [HFR1] - [HCDR1] - [HFR2] - [HCDR2] - [HFR3] - [HCDR3] - [HFR4] - C-end; and the VL region contains the following structure: N-end - [LFR1] - [LCDR1] - [LFR2] - [LCDR2] - [LFR3] - [LCDR3] - [LFR4] - C-end.

[0062] As used herein, the term “antigen-binding fragment” for antibodies refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen (e.g., HER3). It has been shown that antigen-binding functionality of antibodies can be achieved through fragments of full-length antibodies. Examples of binding fragments covered within the term “antigen-binding fragment” for antibodies include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CHI domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341: 544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked via synthetic linkers using recombination methods, enabling the fabrication of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USÅ 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding moiety" of antibodies.

[0063] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence.

[0064] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomally modified animals (e.g., mice) or hybridomas prepared therefrom (further described in Section 1 below), (b) antibodies isolated from host cells transformed to express antibodies, e.g., from transfected tumors, (c) antibodies isolated from recombinant human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be mutagenized in vitro (or, when using animals transgenic with human Ig sequences, in vivo somatic cell mutagenization), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and associated with human germline VH and VL sequences, may not naturally exist in an in vivo germline reservoir of human antibodies.

[0065] The term "CDR" refers to one of the six hypervariable regions within the antibody variable domain, which primarily facilitates antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991), Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of the light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of the heavy chain variable domain.

[0066] Methods and techniques for identifying CDRs within amino acid sequences of HCVR and LCVR are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Examples that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art can readily identify CDRs defined by each numbering system.

[0067] The following are useful comparisons of CDR numbers: Note 1: Some of these definitions (especially for Chothia rings) vary depending on the specific publication consulted; Note 2: Any numbering scheme can be used for these CDR definitions, except for the contact definition, which uses the Chothia or Martin (enhanced Chothia) definition; Note 3: When using the Kabat numbering convention, the end of the Chothia HCDR1 ring varies between H32 and H34, depending on the ring length. This is because the Kabat numbering scheme places the insertion at H35A and H35B.

[0068] "Bispecificity" refers to the specific binding of an antibody to two different antigens or two different epitopes within the same antigen. Bispecific antibodies can be cross-reactive with other relevant antigens, such as the same antigen from other species (homologs), such as humans or monkeys (e.g., cynomolgus monkeys or pantroglodytes), or can bind to epitopes shared by two or more different antigens.

[0069] As used herein, the term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. A nucleic acid is "effectively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

[0070] The preparation method of nucleic acid is a conventional method in the art. Preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-length sequence synthesis.

[0071] Those skilled in the art will recognize that the base sequence encoding the amino acid sequence of a protein can be appropriately substituted, deleted, altered, inserted, or added to provide polynucleotide homologs. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases of a gene encoding a protein sequence within the range that maintains antibody activity.

[0072] The term "ligand" is a macromolecular compound that recognizes and binds to a target cell-associated antigen or receptor. The role of a ligand is to deliver a drug to a population of target cells that have bound to it. Ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, and other molecules capable of binding to cells. In embodiments of the invention, a ligand is designated as Ab. Linkage bonds may form between heteroatoms and linking units within the ligand.

[0073] As used herein, the term "linker unit" refers to the portion in an antibody-drug conjugate (i.e., ADC) that links an antibody to a drug, and this linker may be cleavable or non-cleavable. Cleavable linkers (i.e., breakable or biodegradable linkers) can be disrupted in or on target cells, thereby releasing the drug. In some embodiments, the linker units or linkers of the present invention exhibit very good stability, significantly reducing drug release during delivery to the target (e.g., in the bloodstream), thereby reducing side effects and toxicity. In some specific embodiments, the linker units or linkers of the present invention are selected from cleavable linkers, such as disulfide-based linkers (which are selectively disrupted in tumor cells at high thiol concentrations), peptide linkers (which are enzymatically cleaved in tumor cells), and hydrazone linkers.

[0074] The term "cytotoxic drug" refers to a chemical molecule that can strongly disrupt the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to their lack of specificity, while killing tumor cells, they also induce apoptosis in normal cells, leading to serious side effects. In embodiments of the present invention, the cytotoxic drug is referred to as "Compound C". In embodiments of the present invention, the cytotoxic drug is referred to as "Compound D", and non-limiting examples include microtubule inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, and DNA synthesis inhibitors, preferably topoisomerase inhibitors.

[0075] The term "immunoconjugate" refers to an antibody component or other targeting moiety conjugated to a therapeutic agent. In some specific embodiments, "immunoconjugate" is preferably an antibody-drug conjugate (ADC), which refers to an antibody or antibody fragment linked to a biologically active cytotoxic drug via a linker unit. As used herein, the terms "conjugate" and "immunoconjugate" are used interchangeably.

[0076] The term "drug-antibody ratio (DAR)" refers to the average amount of cytotoxic drug loaded on each ligand, and can also be expressed as the ratio of drug amount to antibody amount. It can be an integer or a non-integer. The drug loading range for each ligand (Ab) can be 1-20 cytotoxic drugs (compound C or compound D). In embodiments of the invention, the drug-antibody ratio is expressed as y, where y is a number selected from 1 to 10, preferably from 2 to 8, more preferably from 2 to 4, 2 to 6, 4 to 6, 4 to 8, 6 to 8, further preferably from 4 to 8 or 6 to 8, and most preferably from 4, 5, 6, 7 or 8. The average amount of drug in each ADC molecule after the coupling reaction can be identified by conventional methods, such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0077] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0078] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 10 carbon atoms, and most preferably an alkyl group having 1 to 6 carbon atoms (having 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers. More preferably, the alkyl group is a lower alkyl group having 1 to 6 carbon atoms, and 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, etc. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection point. The substituents are preferably selected independently from one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic thio, and oxo.

[0079] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein the alkyl or cycloalkyl group is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups may optionally be substituted or unsubstituted, and when substituted, the substituent is preferably independently selected independently from one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkoxythio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0080] The term "haloalkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0081] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (having 3, 4, 5, 6, 7, or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc. Polycyclic cycloalkyl groups include cycloalkyl groups having spirocyclic, fused, or bridged rings.

[0082] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)u heteroatoms (u being an integer from 0 to 2), excluding the -OO-, -OS-, or -SS- ring moieties, and the remaining ring atoms are carbon. In one embodiment, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include: pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. The heterocyclic ring may be fused with an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group. The heterocyclic group may optionally be substituted or unsubstituted, and when substituted, the substituent is preferably independently selected independently from one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkane thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0083] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent carbon atom pairs) group having a conjugated electron system, preferably a 6- to 10-membered ring, such as benzene, naphthyl, etc., and in one embodiment, phenyl. The aryl ring may be fused with a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring. The aryl group may be substituted or unsubstituted, and when substituted, the substituent is preferably independently selected independently from one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkanethio, alkylamino, halogen, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkanethio.

[0084] The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably a 5- to 10-membered ring, more preferably a 5- or 6-membered ring, such as furanyl, thiophene, pyridyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused with an aryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may optionally be substituted or unsubstituted; when substituted, the substituent is preferably independently selected independently from one or more of the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkane thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkyl thio, heterocyclic alkoxy, cycloalkyl thio, oxo.

[0085] As used herein, the term “transfected tumor” includes recombinant eukaryotic host cells expressing antibodies, such as CHO cells, NS / O cells, HEK293 cells, plant cells, or fungi, including yeast cells.

[0086] The sequence of the DNA molecule of the antibody or fragment thereof according to the invention can be obtained using conventional techniques (e.g., PCR amplification or genomic library screening). Furthermore, sequences encoding the light and heavy chains can be fused together to form a single-chain antibody.

[0087] Once the relevant sequence is obtained, it can be obtained in batches using recombination methods. This is typically done by cloning the sequence into a vector using conventional methods, transforming cells with the vector, and then separating the relevant sequence from the proliferating host cells.

[0088] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Typically, several small fragments are synthesized first, and then they are joined together to obtain a long sequence fragment.

[0089] Currently, the DNA sequence encoding the antibody (or a fragment thereof or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0090] Generally, host cells obtained are cultured under conditions suitable for expressing the antibodies according to the invention. The antibodies of the invention are then purified using conventional immunoglobulin purification steps, such as conventional separation and purification methods known to those skilled in the art, including protein A-agarose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0091] Monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).

[0092] The antibodies according to the invention can be expressed in cells or on cell membranes, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and any other liquid chromatography, and combinations thereof.

[0093] The term "variant" for polypeptides, such as antigen-binding fragments, proteins, or antibodies, is a polypeptide sequence in which one or more amino acid residues have been inserted, deleted, added, and / or substituted, including fusion polypeptides, compared to another polypeptide sequence. Furthermore, protein variants include protein variants that are modified by protease cleavage, phosphorylation, or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding to and specificity for HER3. Variants may be approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of the antibodies or antigen-binding fragments disclosed herein. The percentage of identity or homology (%) can be calculated with reference to the following description.

[0094] In one implementation, the homology or identity percentage can be calculated as 100 × [(identical position) / min (TGA, TGB)], where TGA and TGB are the sum of the number of residues in the compared sequences A and B and the internal vacancy positions (Russell et al., J. Mol Biol., 244: 332-350 (1994)).

[0095] In this invention, the antibodies of this invention also include conserved variants thereof, meaning that, compared to the amino acid sequence of the antibodies of this invention, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids having similar or similar properties to form a polypeptide. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table A.

[0096] Table A As used in this article, the term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. "K" D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a The Kc of an antibody is obtained and expressed as a molar concentration (M). In view of this disclosure, methods in the art can be used to determine the Kc of an antibody. D Value. For example, the K value of an antibody. D It can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g., the system) or by using biolayer interferometry (e.g., the Octet RED96 system).

[0097] The term "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen. It is determined by the characteristics of the antigen, such as its size, shape, and / or charge, and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art and can be found below.

[0098] When the dissociation constant (K) D )< l0 6 When M occurs, the antibody or its antigen-binding fragment is said to have "specifically bound" to its target (such as an antigen). When K occurs... D < l0 9 When M occurs, the antibody binds specifically to its target with "high affinity".

[0099] "Antibody endocytosis" refers to the ability of antibody molecules and immunoconjugates described herein to mediate effective endocytosis. This is particularly useful for immunoconjugates, as they ensure that the conjugate is internalized into the cell and delivered to the lysosome, where the antibody molecule is subsequently degraded, releasing the drug into the cell to exert its cellular effects, such as cytotoxicity.

[0100] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more compounds described herein, or physiologically / pharmaceutically acceptable salts thereof, or prodrugs thereof, along with other chemical components such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, which is beneficial for the absorption of the active ingredient and the exertion of its biological activity.

[0101] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "application" and "treatment" mean contacting said animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical reagent, therapeutic reagent, diagnostic reagent, or composition. "Application" and "treatment" can, for example, refer to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Cell treatment encompasses contacting cells with a reagent, as well as contacting a liquid with a reagent, wherein said liquid is in contact with cells. "Application" and "treatment" also refer to in vitro and ex vivo treatments, such as in vitro and ex vivo treatments of cells with a reagent, diagnostic agent, conjugated composition, or with another cell. When applied to humans, veterinarians, or research subjects, "treatment" means therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0102] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dosage and time. Therapeutic effective dose can depend on a variety of factors, such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment or combination therapy to elicit the desired response in the individual. Exemplary indications for effective treatment or combination therapy include, for example, improving patient well-being.

[0103] The antibody or antigen-binding fragment, immunoconjugate, or pharmaceutical composition described in this application can be used in methods of treating humans or animals. Relevant aspects of this disclosure are provided. (i) The use of the antibody or antigen-binding fragment, or immunoconjugate, or pharmaceutical composition described in this application as a medicine, (ii) The method of using the antibody or antigen-binding fragment, or immune conjugate, or pharmaceutical composition described in this application to treat a disease or condition. (iii) The use of the antibody or antigen-binding fragment, or immunoconjugate, or pharmaceutical composition described in this application in the preparation of a medicament for treating a disease or condition; and (iv) A method of treating a disease or condition in an individual, wherein the method comprises administering to the individual a therapeutically effective amount of the antibody or antigen-binding fragment, or immunoconjugate or pharmaceutical composition described in this application.

[0104] "Individual" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. The terms "individual" and "subject" are used interchangeably in this document.

[0105] Example The present invention is further illustrated by the following specific embodiments. It will be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not detailed in the following embodiments generally follow the conditions described in standard conditions, such as Sambrook J. et al., *Molecular Cloning: A Laboratory Manual* (translated by Huang Peitang et al., Beijing: Science Press, 2002), or the manufacturer's recommended conditions (such as the product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0106] The room temperature described in the examples is a conventional room temperature in the art, and is generally 10-30°C.

[0107] Example 1: Preparation of bispecific antibodies The bispecific antibody EGFRxc-MET-136 Afu (referred to as BsAb-1) was prepared according to the method of Example 6 disclosed in patent WO2023 / 186092. The patents listed herein are incorporated herein by reference in their entirety. Table 1 shows the amino acid sequences of the CDR, heavy chain variable region, light chain variable region, constant region, heavy chain, and light chain of the bispecific antibody. The amino acid residues of the CDR in VH / VL are numbered and annotated according to the Kabat & Wu definition system.

[0108] Table 1. Sequences of bispecific antibodies Example 2 Cell binding of bispecific antibodies The binding of the EGFRxc-MET bispecific antibody to EGFR on the cell surface was determined by FACS analysis using SNU-1 cancer cells expressing EGFR but not c-Met. The binding of the EGFRxc-MET bispecific antibody to c-Met on the cell surface was determined by FACS analysis using SW620 cancer cells expressing c-Met but not EGFR. SNU-1 and SW620 cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin, and streptomycin. All cell lines were cultured at 37°C under a humidified atmosphere of 5% CO2.

[0109] To determine the binding of the EGFRxc-MET bispecific antibody to EGFR and c-Met on the cell surface, cells were first harvested and then... 6 Cells were resuspended at 100 cells / mL in cell staining buffer (BioLegend). Then, the cells were treated with human Fc receptor blocking reagent (BioLegend) on ice for 10 min. The resulting cell suspension was divided into 50 μL aliquots. 50 μL of different concentrations of BsAbs were mixed with each aliquot, with the final concentration of hIgG1 mAb in the mixture ranging from 4 pM to 200 nM. The cells were incubated on ice for 1 hour, followed by washing twice with cell staining buffer. 50 μL of secondary antibody (PE-conjugated goat anti-human Fc, eBioscience) was added to each sample. TM Cells were resuspended in 1:400 dilution. Cells were then incubated on ice for 30 min. Cells were subsequently washed twice with cell staining buffer and resuspended in 4% PFA for fixation. Samples were analyzed using iQue3 to measure median fluorescence intensity using the corresponding channel. An EGFRxc-MET bispecific antibody from WO2023083846 was used as a control (referred to as Control 1 in this invention). The allotype control mAb1 was a self-made version of the antibody referenced in the literature (McLellan et al., Nat Struct Mol Biol., 2010 17:248-250).

[0110] The results were published Figure 1 As shown in Table 2, the results confirm that the EGFRxc-MET bispecific antibody binds to EGFR and c-Met primarily expressed in cancer cells.

[0111] Table 2. K values ​​of EGFRxc-MET bispecific antibodies D value Example 3: EGFRxc-MET BsAb in the SRE-HEK293 reporter cell line SRE reporter gene-HEK293 cells (BPS Bioscience) were used to evaluate the effects of the EGFRxc-MET bispecific antibody on the EGFR and c-Met signaling pathways.

[0112] To evaluate the direct effects of the EGFRxc-MET bispecific antibody on the EGFR and c-Met signaling pathways, 2 × 10⁻⁶ antibodies were used. 5 SRE-HEK293 cells were seeded on 96-well white plates and incubated overnight. Then, bispecific antibodies, recombinant human HGF, and recombinant human EGF were added to the cells, respectively. An EGFRxc-MET bispecific antibody from WO2014081954 was used as a control (referred to as Control 2 in this invention). The isotype control mAb2 was a self-made version of the antibody based on the literature (Zwick et al. JVirol., 2003, 77(10):5863-5876). After 4 hours of incubation, One-Glo luciferase assay reagent (Promega) was added to each well. Luminescence was then measured using a plate reader. Figure 2 As shown, recombinant human HGF and recombinant EGF can activate SRE-HEK293 cells, while BsAb-1 and control 2 do not activate SRE-HEK293 cells.

[0113] To evaluate whether the EGFRxc-MET bispecific antibody could block HGF or EGF-mediated activation, the bispecific antibody was first incubated with SRE-HEK293 cells in a 96-well plate. After 1 hour of incubation, HGF and EGF were added to the culture at final concentrations of 185 pm and 750 pm, respectively. After 4 hours of incubation, One-Glo luciferase assay reagent (Promega) was added to each well. Luminescence was then measured using a plate reader. Figure 2 B and Figure 2 As shown in C, BsAb-1 can effectively block HGF or EGF-mediated activation in SRE reporter cells.

[0114] Example 4: Generation of an immunoconjugate containing EGFRxc-MET bispecific antibody Purification of bispecific antibodies The bispecific antibody was purified for drug conjugation using the method described in Example 1.

[0115] Preparation of drug intermediates The following intermediate compounds are used to generate immunoconjugates of anti-EGFRxc-MET bispecific antibodies. Compound C is prepared by the method disclosed in PCT patent application (see WO2020063673 filed on September 25, 2019), and compound D is prepared by the method disclosed in PCT patent application (see WO2022161385 filed on January 26, 2022). Compound C Compound D.

[0116] Both compounds C and D depend on intracellular processes to release toxins (compound 2-A). Compound 2-A is a potent DNA topoisomerase I inhibitor disclosed in WO2021190480, which is prepared by the method disclosed in the PCT patent application (see WO2021190480, filed March 23, 2021).

[0117] .

[0118] The conjugation of bispecific antibodies with drug molecules Bispecific antibody BsAb-1 (5 mg / mL PBS solution, pH 7.4) was treated with a sufficient molar equivalent of tris(2-carboxyethyl)phosphine (TCEP, 10 mM) for 1 hour at 37°C. Isotype controls mAb1 and mAb2 were negative controls, and controls 1 and 2 were positive controls. Control 1 antibody was also conjugated with the payload SG3932, as described in patent WO2023083846.

[0119] A sufficient molar equivalent of the drug linker (e.g., a DMSO solution of compound C or compound D) was added to a PBS solution of the reducing antibody. After incubation for one hour, the reaction mixture was purified using size exclusion chromatography (SEC) to separate the ADC and the free unconjugated drug linker. The bispecific antibody conjugated with compound C has the general formula (ADC-A), and the bispecific antibody conjugated with compound D has the general formula (ADC-B).

[0120] The drug-antibody ratio (DAR) of the ADCs was determined using a TOF LC / MS system (Agilent), and the average DAR values ​​are summarized in Table 3. The average DAR values ​​for BsAb-1 conjugated with compound C were approximately 4 and 6. The average DAR values ​​for BsAb-1 conjugated with compound D were approximately 4, 6, and 8. The synthesis steps for anti-EGFRxc-MET bispecific antibodies conjugated with compound C or compound D with other DAR values ​​can be compared with the above. (ADC-A) (ADC-B) Table 3: Conjugation of Bispecific Antibodies and Linker / Payload Moment Example 5: Determining the internalization rates of bsAb and ADC BsAbs and ADCs were labeled using pHrodo™ iFL dye. pHrodo™ iFL dye exhibits minimal fluorescence at neutral pH. When a BsAb or ADC is internalized, the dye is simultaneously internalized. The fluorescence signal gradually increases as the pH decreases. The internalization activity of the BsAb or ADC is evaluated based on how the fluorescence signal increases.

[0121] In short, the density is 2×10 6 HCC827 and MDA-MB-468 cells were seeded at 50 µl per well in 96-well conical (V) plates, with 100,000 cells per well. BsAb and ADC were diluted to 40 nM or 80 nM with complete cell culture medium, and pHrodo™ iFL dye was diluted to 120 nM or 240 nM with complete cell culture medium. The ADC and pHrodo™ iFL dye dilutions were then mixed at a 1:3 molar ratio and a 1:1 v / v ratio and incubated at room temperature for 10 minutes. 50 µL of pHrodo™ iFL-labeled ADC was added to each well of the 96-well conical (V) plate. The mixture was gently mixed and incubated at 37°C. The final concentration of ADC was 10 nM, and the final concentration of pHrodo™ iFL dye was 30 nM.

[0122] Cells were removed from the 37°C incubator at 0.5 h, 2 h, and 4 h, and samples were then loaded into a flow cytometer. Data were analyzed using FlowJo analysis software. The changes in MFI for different cell types were obtained by adding MFI and pHrodo™ iFL dye to the cell wells at each time point. The data are summarized in Table 4. At all time points, ADC-12 showed slightly higher endocytosis than BsAb-1, and significantly higher endocytosis than ADC-13.

[0123] Table 4: Variation of mean fluorescence signal (MFI) at different time points Example 6: Inhibitory effect of ADC on tumor cell growth The inhibitory effect of ADCs on tumor cell growth was examined using an in vitro cell killing assay. Different tumor cells were collected during the logarithmic growth phase and distributed at 2000 cells / well in 96-well plates. After overnight incubation, ADCs were added to each well. The final concentration of ADC in the wells ranged from 0.003 nM to 200 nM. Cell viability in each well was determined by Cell Titer Glo 2.0 assay (Promega) after 4 to 7 days of incubation. IC50 was generated using sigmoid dose-response nonlinear regression fitting in GraphPad Prism. 50 Values. The data from these experiments are summarized in Table 5. Figure 3 Representative killing curves for all ADCs tested against specified cell lines are shown. The tested BsAb-1 ADCs exhibited good in vitro toxicity on tumor cells.

[0124] Table 5: Evaluation of the cytotoxicity of antibody-drug conjugates against tumor cells (IC50) 50 (nM) NA: IC 50 Unmeasurable, -: Untested.

[0125] The inhibitory effect of ADCs on tumor cell growth was detected using the CellTiter-Glo chemiluminescence assay kit. Different tumor cells were collected during the logarithmic growth phase and distributed at different densities into 96-well plates. After overnight culture, the test ADCs (maximum final concentrations of 100 nM, 300 nM, 1000 nM, or 2000 nM), 3- or 4-fold dilutions, and nine concentration gradients were added. BsAb-1 and compound 2-A served as controls. After adding the test ADCs and drugs, cells were cultured at 37°C and 5% CO2 for 6 days, followed by the addition of CellTiter-Glo reagent and shaking on a shaker to induce cell lysis and ATP release. The 96-well plates were then incubated at room temperature in the dark, and the chemiluminescence signal values ​​were measured using a microplate reader. Finally, the IC50 was calculated using sigmoid dose-response nonlinear regression fitting in GraphPad Prism. 50 value.

[0126] Data from these experiments are summarized in Table 6. The tested ADC and compound 2-A significantly inhibited tumor cells from different tissue origins expressing EGFR / cMet antigens, with ADC-12 showing superior efficacy compared to the control ADC and cytotoxic drugs.

[0127] Table 6: Evaluation of ADC cytotoxicity against tumor cells (IC50) 50 (nM) Example 7: Tumor suppression experiment of ADC on NCI-H1975 cell line subcutaneously transplanted tumor model in nude mice 1. Xenograft (CDX) model derived from NCI-H1975 cell line NCI-H1975 cells were subcutaneously transplanted into Balb / C nude mice. When the tumor volume reached approximately 150-200 mm... 3 At that time, transplanted mice were randomly divided into four groups (n=5 mice per group). These groups were treated with solvent control, ADC-08, and ADC-09, respectively. Mice were treated with intravenous ADC (3 mg / kg) QW×3. The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0128] Tumor growth in mice treated with ADC showed a statistically significant difference compared to that in the control group, leading to tumor regression.

[0129] The results of the study are shown in Table 7 and Figure 4 In mice treated with ADC-08 and ADC-09, tumor growth showed statistically significant differences compared to mice treated with solvent, with TGI values ​​of 103.2% and 104.0% on day 16, respectively.

[0130] Table 7. Therapeutic effects of ADCs in NCI-H1975 tumor-bearing mice In another experiment, NCI-H1975 tumor-bearing mice were randomly divided into 7 groups (n=5 per group). These groups were treated with solvent control, ADC-11, ADC-01, and ADC-02 at two dose levels, respectively. Tumor growth in mice treated with ADCs showed a statistically significant difference compared to the control group, leading to tumor regression. Compared to ADC-11, ADC-01 and ADC-02 significantly delayed tumor growth. ADC-02 was slightly more effective than ADC-01. The results are shown in Table 8 and... Figure 5 middle.

[0131] Table 8. Therapeutic effects of ADCs in NCI-H1975 tumor-bearing mice In another experiment, NCI-H1975 tumor-bearing mice were randomly divided into 12 groups (n=5 mice per group). These groups were treated with solvent control, BsAb-1 ADC, control 1 ADC, and BsAb-1, respectively. Compared with ADC-03, ADC-04, and BsAb-1, ADC-01 and ADC-02 significantly delayed tumor growth. The results are shown in Table 9 and... Figure 6 middle.

[0132] Table 9. Therapeutic effects of ADCs in NCI-H1975 tumor-bearing mice 2. Xenograft (CDX) model derived from NCI-H1975 (EGFR C797S) cell line NCI-H1975 (EGFR C797S) cells were subcutaneously transplanted into NOD / SCID mice. When the tumor volume reached approximately 150-200 mm... 3 At that time, transplanted mice were randomly divided into four groups (n=5 mice per group). These groups were treated with solvent control and ADC-08, respectively. Mice were treated with intravenous ADC (3 mg / kg) QW×2. The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0133] The results are shown in Table 10. Tumor growth in mice treated with ADC-08 showed a statistically significant difference compared to mice treated with the solvent, with a TGI value of 108.55% on day 28.

[0134] Table 10. The efficacy of ADCs in NCI-H1975 (EGFR C797S) tumor-bearing mice 3. Xenograft (CDX) model derived from NCI-H441 cell line NCI-H441 cells were subcutaneously transplanted into BALB / c mice. When the tumor volume reached approximately 100-200 mm... 3At that time, transplanted mice were randomly divided into four groups (n=6 per group). These groups were treated with BsAb-1 and compounds 2-A, ADC-12, and ADC-13, respectively. Mice were treated with a single intravenous dose of ADC (1, 3, 6 mg / kg) and BsAb-1 + compound 2-A (mAb + drug_6 + 0.086 mg / kg). The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0135] The results of the study are shown in Table 11 and Figure 7 In contrast to ADC-13 and the combination of BsAb-1 and compound 2-A, ADC-12 significantly delayed tumor growth.

[0136] Table 11. Therapeutic effects of ADCs in NCI-H441 tumor-bearing mice 4. Xenograft (CDX) model derived from SNU-1 cell line SNU-1 cells were subcutaneously transplanted into BALB / c mice. When the tumor volume reached approximately 150-200 mm... 3 At that time, transplanted mice were randomly divided into four groups (n=7 per group). These groups were treated with BsAb-1 and compounds 2-A, ADC-12, and ADC-13, respectively. Mice were treated with single intravenous doses of ADC (1, 3, 9 mg / kg) and BsAb-1 + compound 2-A (mAb + drug_9 + 0.128 mg / kg). The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0137] The results of the study are shown in Table 12 and Figure 8 In contrast to ADC-13 and the combination of BsAb-1 and compound 2-A, ADC-12 significantly delayed tumor growth.

[0138] Table 12. Therapeutic effects of ADCs in SNU-1 tumor-bearing mice 5. Xenograft (CDX) model derived from HCT-116 cell line HCT-116 cells were subcutaneously transplanted into BALB / c mice. When the tumor volume reached approximately 100-200 mm... 3 At that time, transplanted mice were randomly divided into four groups (n=7 per group). These groups were treated with BsAb-1 and compounds 2-A, ADC-12, and ADC-13, respectively. Mice were treated with single intravenous doses of ADC (1, 3, 8 mg / kg) and BsAb-1 + compound 2-A (mAb + drug_8 + 0.114 mg / kg). The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0139] The results of the study are shown in Table 13 and Figure 9 In contrast to ADC-13 and the combination of BsAb-1 and compound 2-A, ADC-12 significantly delayed tumor growth.

[0140] Table 13. The therapeutic effect of ADC in HCT-116 tumor-bearing mice 6. Xenograft (CDX) model derived from Detroit562 cell line Detroit562 cells were subcutaneously transplanted into BALB / c mice. When the tumor volume reached approximately 100-200 mm... 3 At that time, transplanted mice were randomly divided into four groups (n=6 mice per group). These groups were treated with BsAb-1 and compounds 2-A, ADC-12, and ADC-13, respectively. Mice were treated with a single intravenous dose of ADC (0.3, 1, 3 mg / kg) and BsAb-1 + compound 2-A (mAb + drug-3 + 0.043 mg / kg). The mean tumor growth inhibition (TGI) was calculated using the following formula: TGI = ((mean (C) - mean (C0)) - (mean (T) - mean (T0))) / (mean (C) - mean (C0)) * 100%; T is the current group value, C is the control group value, and T0 and C0 represent the tumor volume at the start of the test.

[0141] The results of the study are shown in Table 14 and Figure 10 In contrast to ADC-13 and the combination of BsAb-1 and compound 2-A, ADC-12 significantly delayed tumor growth.

[0142] Table 14. Therapeutic effects of ADCs in Detroit 562 tumor-bearing mice

Claims

1. An immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, said immunoconjugate being of general formula (A): ， in: D is a cytotoxic drug; L1 and L2 are connection units; y is a number from 1 to 20, preferably from 2 to 10, more preferably from 2 to 8, even more preferably from 2 to 6 or from 4 to 8, and most preferably from 2, 3, 4, 5, 6, 7, or 8. Ab is an antibody or an antibody fragment thereof, comprising: a first antigen-binding domain that binds to EGFR and a second antigen-binding domain that binds to c-Met; The second antigen-binding domain includes: Heavy chain variable region (VH), which contains the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2 HCDR3 having the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof, wherein one, two, or three amino acids of one or more of HCDR1, HCDR2, or HCDR3 are substituted with other amino acids; and Light chain variable region (VL), which contains the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO:

4. LCDR2 has the amino acid sequence shown in SEQ ID NO:

5. LCDR3 having the amino acid sequence shown in SEQ ID NO: 6, or a variant thereof, wherein one, two, or three of one or more of LCDR1, LCDR2, or LCDR3 are replaced by other amino acids; Preferably, the second antigen-binding domain comprises: Heavy chain variable (VH) region, which contains the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2 HCDR3 having the amino acid sequence shown in SEQ ID NO: 3; and Light chain variable (VL) region, which contains the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO:

4. LCDR2 has the amino acid sequence shown in SEQ ID NO:

5. LCDR3 having the amino acid sequence shown in SEQ ID NO:

6.

2. The immunoconjugate according to claim 1, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The second antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO: 13, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; and a VL region containing the amino acid sequence shown in SEQ ID NO: 14, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; Preferably, the second antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO:13; and a VL region containing the amino acid sequence shown in SEQ ID NO:

14.

3. The immunoconjugate according to claim 1 or 2, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The first antigen-binding domain includes: Heavy chain variable region (VH), which contains the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7 HCDR2 has the amino acid sequence shown in SEQ ID NO:

8. HCDR3 having the amino acid sequence shown in SEQ ID NO: 9, or a variant thereof, wherein one, two, or three amino acids of one or more of HCDR1, HCDR2, or HCDR3 are substituted with other amino acids; and Light chain variable region (VL), which contains the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO:

10. LCDR2 has the amino acid sequence shown in SEQ ID NO:

11. LCDR3 having the amino acid sequence shown in SEQ ID NO: 12, or a variant thereof, wherein one, two, or three of one or more of LCDR1, LCDR2, or LCDR3 are replaced by other amino acids; Preferably, The first antigen-binding domain includes: Heavy chain variable region (VH), which contains the following CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 7 HCDR2 has the amino acid sequence shown in SEQ ID NO:

8. HCDR3 having the amino acid sequence shown in SEQ ID NO: 9; and Light chain variable region (VL), which contains the following CDRs: LCDR1 has the amino acid sequence shown in SEQ ID NO:

10. LCDR2 has the amino acid sequence shown in SEQ ID NO:

11. LCDR3 having the amino acid sequence shown in SEQ ID NO:

12.

4. The immunoconjugate according to any one of claims 1 to 3, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The first antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO: 15, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; and a VL region containing the amino acid sequence shown in SEQ ID NO: 16, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; Preferably, the first antigen-binding domain comprises: a VH region containing the amino acid sequence shown in SEQ ID NO:15; and a VL region containing the amino acid sequence shown in SEQ ID NO:

16.

5. The immunoconjugate according to any one of claims 1 to 4, or its tautomer, meso compound, racemic mixture, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The antibody fragments are selected from: Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), diabody, triabody, scFv-Fc, disulfide bond stabilized Fv (dsFv), and peptides containing CDR.

6. The immunoconjugate according to any one of claims 1 to 5, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The first antigen-binding domain and / or the second antigen-binding domain further include a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conserved variants, and the light chain constant region of the human antibody constant region is selected from the κ and λ chain constant regions of human antibodies and their conserved variants. More preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 25 or 26, or has at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 25 or 26; and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 27, or has at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

27. More preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 25 or 26; and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

27.

7. The immunoconjugate according to any one of claims 1 to 6, or its tautomer, meso compound, racemic mixture, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The first antigen-binding domain and the second antigen-binding domain form a heterodimer, and the first antigen-binding domain and / or the second antigen-binding domain further include a CH1 region and a CL region. Wherein, the CH1 region of the second antigen-binding domain contains one or more substitutions selected from L145Q, K147E, and S181E, and the CL region of the second antigen-binding domain contains one or more substitutions selected from T129R, T178R, and T180Q; and / or The CH1 region of the first antigen-binding domain contains one or more substitutions selected from L128R and K147R, and the CL region of the first antigen-binding domain contains one or more substitutions selected from Q124E, V133Q, and T178E, wherein the constant regions are numbered according to the EU index. Preferably, The amino acid substitutions in the CH1 region of the second antigen-binding domain include L145Q, K147E, and S181E, and the amino acid substitutions in the CL region of the second antigen-binding domain include T129R, T178R, and T180Q; and / or The amino acid substitutions in the CH1 region of the first antigen-binding domain include L128R and K147R, and the amino acid substitutions in the CL region of the first antigen-binding domain include Q124E, V133Q, and T178E, wherein the constant regions are numbered according to the EU index. More preferably, The first antigen-binding domain comprises the human antibody heavy chain constant region shown in SEQ ID NO: 19 and the human light chain constant region shown in SEQ ID NO: 20; and / or the second antigen-binding domain comprises the human antibody heavy chain constant region shown in SEQ ID NO: 17 and the human light chain constant region shown in SEQ ID NO:

18.

8. The immunoconjugate according to any one of claims 1 to 7, or its tautomer, meso compound, racemic mixture, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The first antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 23 or 30, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 30; and a light chain containing the amino acid sequence shown in SEQ ID NO: 24 or 31, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24 or 31; and / or, The second antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 21 or 28 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21 or 28; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 22 or 29 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22 or 29; Preferably, the first antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 23, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23; and a light chain containing the amino acid sequence shown in SEQ ID NO: 24, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; and / or, The second antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 21, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 22, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; Alternatively, the first antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO: 30, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 30; and a light chain containing the amino acid sequence shown in SEQ ID NO: 31, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; and / or, The second antigen-binding domain comprises: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 28, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 29, or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 29; More preferably, The first antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO:23; and a light chain containing the amino acid sequence shown in SEQ ID NO:24; and the second antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO:21; and a light chain containing the amino acid sequence shown in SEQ ID NO:22; or The first antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO:30; and a light chain containing the amino acid sequence shown in SEQ ID NO:31; and the second antigen-binding domain comprises: a heavy chain containing the amino acid sequence shown in SEQ ID NO:28; and a light chain containing the amino acid sequence shown in SEQ ID NO:

29.

9. The immunoconjugate according to any one of claims 1 to 8, or its tautomer, meso compound, racemic mixture, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The cytotoxic drugs are selected from toxins, chemotherapy drugs, antibiotics, radioisotopes, and nucleases.

10. The immunoconjugate according to any one of claims 1 to 9, or its tautomer, meso compound, racemic mixture, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The cytotoxic drug is selected from DNA damaging agents, microtubule inhibitors, or topoisomerase inhibitors; preferably, olistatin analogs, maytansine derivatives, eribulin and its derivatives, pyrrolobenzodiazepines, or camptothecin derivatives; more preferably, SN-38, MMAE, MMAF, MMAD, eribulin, or eczema and its derivatives.

11. The immunoconjugate according to claim 10, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or its pharmaceutically acceptable salt or solvate, has one of the following structures: 、 、 、 、 、 、 、 、 , ； in L1 and L2 are connection units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably a number selected from 3, 4, 5, 6, 7, or 8. Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

12. The immunoconjugate according to claim 10, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is shown in general formula (D): ； in: The carbon atoms labeled p1* and p2* are each asymmetry centers, and the asymmetry centers are... S Configuration, R Configuration, or racemic mixture; R3 and R4 are each independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy or hydroxyl; or R3 and R4 together with the atoms they are attached to form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclic group, an unsubstituted or substituted aryl group, or an unsubstituted or substituted heteroaryl group. R5 represents hydrogen, deuterium, and C. 1-6 Alkyl, deuterated alkyl, substituted alkyl, aryl, substituted aryl, or heteroaryl; L1 and L2 are connection units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 2, 3, 4, 5, 6, 7, 8; Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

13. The immunoconjugate according to claim 12, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or its pharmaceutically acceptable salt or solvate, has one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； in L1 and L2 are connection units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 2, 3, 4, 5, 6, 7, 8; Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

14. The immunoconjugate according to claim 12, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is shown in general formula (B): ； in: L1 and L2 are connection units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 4 to 8, and most preferably 3, 4, 5, 6, 7, or 8; Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

15. The immunoconjugate according to any one of claims 11 to 14, or a tautomer, meso compound, racemic mixture, enantiomer, or diastereomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein, -L2- is selected from -La-Lb-, and -L1- is selected from -Lc-Ld-Le-, wherein, The -La- is a covalently connected unit. The -Lb- is an extension unit. The -Lc- is a bond or a polar hydrophilic group unit, wherein the polar hydrophilic group comprises sugar residues and their derivatives, polyethylene glycol residues and their derivatives, polysarcosine residues and their derivatives, or combinations thereof. The -Ld- is selected from peptide residues consisting of 2-8 amino acids. The -Le- is a bond or a self-cutting fragment.

16. The immunoconjugate according to claim 15, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The -La- is selected from: 、 、 、 、 ； The -Lb- is selected from: -(CH2)s 2 -C(=O)-、 -CH2-C(=O)-NH-(CH2)s 1 -C(=O)-、 -(CH2CH2O)s 2 -(CH2)s 1 -C(=O)-、 、 ； S 1 and S 2 Independently selected as an integer from 0 to 8; The -Lc- is a bond. , ,or , where s 3 Integers selected from 2 to 12; preferably, s 3 Choose from 6, 7, 8, 9, 10, 11, or 12; The -Ld- is selected from peptide residues composed of 2-8 amino acids, wherein the amino acids are selected from: phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine; preferably, the -Ld- is selected from: 、 、 、 ； -Le- is the key. or -NH-CH2-O-(CRaRb)s 4 -CR1R2-C(O)-; wherein Ra and Rb are the same or different, and are each independently selected from: hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxyl, amino; S 4 The integer is selected from 0-4, preferably s 4 It can be 0 or 1; R1 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; R2 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 Cycloalkyl; or, R1 and R2 together with the carbon atoms they are attached to form C 3-8 cycloalkyl; Preferably, R1 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; R2 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 Cycloalkyl; or, R1 and R2 together with the carbon atoms they are attached to form C 3-6 Cycloalkyl.

17. The immunoconjugate according to claim 16, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, -L2-L1- has one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 18. The immunoconjugate according to any one of claims 11 to 14, or a tautomer, meso compound, racemic mixture, enantiomer, or diastereomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein, -L2-L1- has one of the following structures: 、 、 、 、 、 、 、 、 。 19. The immunoconjugate according to claim 17 or 18, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or its pharmaceutically acceptable salt or solvate, has the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 3, 4, 5, 6, 7, 8; Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

20. The immunoconjugate according to claim 14, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or pharmaceutically acceptable salt or solvate thereof, is shown in general formula (C): ； in: R1 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 cycloalkyl; R2 is selected from hydrogen, C 1-6 Halogenated alkyl or C 3-8 cycloalkyl; Alternatively, R1 and R2 together with the carbon atoms they are attached to form C 3-8 cycloalkyl; Preferably, R1 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 cycloalkyl; R2 is selected from hydrogen, C 1-3 Halogenated alkyl or C 3-6 cycloalkyl; Alternatively, R1 and R2 together with the carbon atoms they are attached to form C 3-6 Cycloalkyl.

21. The immunoconjugate according to claim 20, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, L2 is shown in general formula (I): ， in: s 1 and s 2 Each is an integer selected independently from 0-8, preferably, s 1 and s 2 Select independently from 1, 2, 3, 4, 5, or 6; Or, s 1 It is an integer from 1 to 8, s 2 It is 0, preferably, s 1 Selected from 4, 5, 6, 7 or 8, and s 2 It is 0; Or, s 2 It is an integer selected from 2 to 8, s 1 It is 2, preferably, s 2 Choose from 2, 3, 4, 5, or 6, and s 1 It is 2.

22. The immunoconjugate according to claim 20 or 21, or its tautomer, meso compound, racemic mixture, enantiomer, or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or its pharmaceutically acceptable salt or solvate, has the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 ； in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 4 to 8, and most preferably 3, 4, 5, 6, 7, 8; Ab is the antibody or antibody fragment thereof as described in any one of claims 1 to 8.

23. The immunoconjugate according to any one of claims 1 to 22, or a tautomer, meso compound, racemic mixture, enantiomer, or diastereomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein, The immunoconjugate or its tautomer, meso compound, racemic compound, enantiomer or diastereomer, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following compounds: 、 、 、 、 、 、 、 、 、 、 、 ； in: y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6 or 4 to 8, even more preferably a number selected from 6 to 8, and most preferably 3, 4, 5, 6, 7, or 8.

24. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 1 to 23 or a tautomer, meso compound, racemic compound, enantiomer or diastereomer thereof, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

25. Use of any one of the immunoconjugates according to claims 1 to 23, or their tautomers, mesosomes, racemates, enantiomers or diastereomers, or pharmaceutically acceptable salts or solvates thereof, or the pharmaceutical composition according to claim 24, in the preparation of a medicament for the treatment or prevention of diseases associated with human EGFR and / or c-MET.

26. The use according to claim 25, characterized in that, It is used to prepare drugs for treating or preventing cancers expressing EGFR and / or c-MET; preferably, the cancer is epithelial carcinoma, lung cancer, ovarian cancer, breast cancer, colorectal cancer, squamous cell carcinoma of the head and neck (SCCHN or SQHN), anal cancer, prostate cancer, kidney cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, or glioblastoma; more preferably, the lung cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, or small cell lung cancer.

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