EGFR × her3 bispecific antibody-drug conjugate, preparation method therefor, and use thereof

Abstract

Disclosed in the present invention are an EGFR x HER3 bispecific antibody-drug conjugate, a preparation method therefor, and use thereof. The structure of the antibody-drug conjugate is A-(L1-L2-L3-T)n, wherein A refers to an antibody; L1 refers to a coupling linker; L2 refers to x PEG spacers; L3 refers to an enzymatically cleavable or cleavable linker, or a non-cleavable linker; and T refers to an active small molecule. The antibody-drug conjugate provided in the present invention can bind to an HER3 protein of a human EGFR protein, has cytotoxicity to cancer cells expressing EGFR and / or HER3, and exhibits better efficacy and safety in in vivo tumor treatment.

Description

An EGFR×HER3 bispecific antibody-drug conjugate, its preparation method and application

[0001] This application claims priority to Chinese Patent Application No. 2024115933096, filed on November 8, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] In the field of biotechnology, the invention specifically relates to an EGFR×HER3 bispecific antibody-drug conjugate, its preparation method, and its application. Background Technology

[0003] EGFR, also known as HER1, is a member of the human epidermal growth factor receptor (HER) family. It is a receptor tyrosine kinase that, when activated by its ligands such as EGF and TGF-α, initiates a series of signaling pathways that regulate cell proliferation, survival, and differentiation. Aberrant activation of EGFR is frequently observed in various cancers, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and head and neck cancer. Mutations and overexpression of EGFR are associated with disease progression and poor prognosis due to their role in promoting metastatic behavior and resistance to treatment [1][3]. Numerous studies have established a strong association between EGFR expression and various malignancies. High levels of EGFR are associated with aggressive tumor behavior and poor clinical outcomes. For example, EGFR mutations are prevalent in NSCLC and often lead to the development of targeted therapies, although resistance mechanisms, such as secondary mutations, frequently occur [1][4].

[0004] HER3 is another member of the HER family, characterized by impaired kinase activity, but plays a crucial role as a heterodimerization partner for other HER family members, particularly EGFR and HER2. HER3 is frequently upregulated in cancer, activating downstream signaling pathways such as PI3K / AKT and MAPK / ERK, which are essential for tumor progression. Recent studies have highlighted the importance of HER3 in mediating treatment resistance, sparking interest in therapeutic targeting of this receptor [2][4]. Clinical data suggest that targeting HER3 can improve the efficacy of existing therapies, especially in cases of failure of EGFR inhibitors [2][4].

[0005] The development of antibody-drug conjugates (ADCs) targeting EGFR and HER3 is progressing rapidly. MRG003 and patritumab deruxtecan (U3-1402) are the most advanced representatives in their respective fields. In addition, CPO-301 (targeting EGFR), YL202 (targeting HER3) and SHR-A2009 (targeting HER3) are also in early clinical stages.

[0006] MRG003 is an antibody-drug conjugate (ADC) targeting EGFR, composed of a humanized IgG1 monoclonal antibody and MMAE conjugated via a cleavable valine-citrulline linker (vc). MRG003 has demonstrated significant efficacy and manageable safety in clinical studies for the treatment of recurrent / metastatic nasopharyngeal carcinoma (NPC) and head and neck squamous cell carcinoma (SCCHN). Patritumab deruxtecan is an antibody-drug conjugate (ADC) targeting HER3, developed using Daiichi Sankyo's proprietary DXd technology. In the recently announced results of the HERTHENA-Lung01 Phase II clinical trial, this drug achieved an objective response rate (ORR) of 29.8% in patients with EGFR-mutant non-small cell lung cancer (NSCLC) who had previously received EGFR TKIs and platinum-based chemotherapy. The median duration of response was 6.4 months. These findings support pertuzumab deruxtecan as a potential treatment option for heavily pre-treated patients.

[0007] Although the above experimental therapies have shown promise in clinical studies, EGFR ADCs have shown common problems of skin and mucosal toxicity; while HER3 ADCs have shown poor efficacy against tumors with low expression. At present, the field tends to address these problems through dual targeting. Bispecific antibodies (bsAbs) have become a promising cancer treatment method because they can target two different antigens at the same time. Compared with monospecific antibodies, this dual targeting ability can improve efficacy and reduce toxicity. In the field of antibody-drug conjugates (ADCs), bispecific antibodies offer more selective tumor targeting and have the potential to improve internalization, thereby better clearing tumor cells. EGFR and HER3 are usually co-expressed in solid tumors, and studies have shown that their expression levels are significantly correlated. In non-small cell lung cancer (NSCLC), EGFR and HER3 are expressed in a large proportion of tumors. One study found that 52.3% of primary tumors were EGFR positive, while the proportion of HER3 positive was 82.7% [2]. In colorectal cancer, EGFR and HER3 are highly expressed (2+ and 3+) in 64.2% and 85.0% of primary tumors, respectively [3]. This co-expression pattern is particularly pronounced in breast cancer, where HER3 overexpression is often associated with EGFR and / or HER2 overexpression [1]. The co-expression of these receptors provides a strong theoretical basis for the development of bispecific antibodies that simultaneously target EGFR and HER3.

[0008] Duligotuzumab, also known as MEHD7945A, is a bispecific human IgG1 monoclonal antibody designed specifically for the treatment of cancer. As a monoclonal antibody, duligotuzumab targets both the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 3 (HER3). A phase II study in patients with metastatic colorectal cancer (mCRC) with KRAS exon 2 wild-type tumors found that duligotuzumab did not provide a benefit in progression-free survival (PFS) or overall survival (OS) compared to cetuximab, although it did cause fewer rash events but more cases of diarrhea [6][8]. In head and neck cancers, duligotuzumab has been evaluated in combination with cisplatin and 5-fluorouracil (5-FU) or paclitaxel and carboplatin in patients with recurrent / metastatic squamous cell carcinoma of the head and neck (r / m SCCHN). Although there were partial response rates in patients resistant to cetuximab and in patients who had previously undergone radiotherapy and chemotherapy, the overall outcome was not significantly improved compared to existing treatments [9]. The development of duligotuzumab highlights the complexity of simultaneously targeting EGFR and HER3. Despite its promising performance in preclinical models and certain patient subgroups, limited clinical benefits have led to the discontinuation of some of its development programs targeting specific cancer types[5][7]. Nevertheless, the dual-targeting strategy of duligotuzumab continues to provide valuable insights into the potential and challenges of bispecific antibodies in cancer therapy.

[0009] Recent clinical data have demonstrated the potential of EGFRxHER3 bispecific ADCs. BL-B01D1, an EGFRxHER3 bispecific ADC, has shown potential antitumor activity in a phase 1 trial across various solid tumors, including NSCLC. This approach combines the targeting specificity of a bispecific antibody with the potent cytotoxicity of an ADC payload.

[0010] In summary, compared with single-target ADCs, bispecific ADCs (such as BL-B01D1) have several potential advantages: enhanced tumor selectivity, improved internalization, reduced drug resistance, and a broader patient population. EGFRxHER3 bispecific ADCs combine the advantages of bispecific antibodies with the potent cytotoxicity of ADCs, representing a promising new direction for targeted cancer therapy. The co-expression of EGFR and HER3 in solid tumors provides strong biological evidence for this approach. This invention preferentially uses ADC molecules with low EGFR affinity and high HER3 affinity, which will result in better therapeutic efficacy and safety.

[0011] References:

[0012] [1]https: / / jeccr.biomedcentral.com / articles / 10.1186 / s13046-022-02515-x

[0013] [2]https: / / www.nature.com / articles / s41598-019-43678-6

[0014] [3]https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9334602 /

[0015] [4]https: / / www.med.kindai.ac.jp / life / files / r03 / Nishio_01.pdf

[0016] [5]https: / / en.wikipedia.org / wiki / Duligotuzumab

[0017] [6]https: / / aacrjournals.org / clincancerres / article / 24 / 10 / 2276 / 80758 / Phase-II-Study-of-the-Dual-EGFR-HER3-Inhibitor

[0018] [7]https: / / www.nature.com / articles / s41598-018-27454-6

[0019] [8]https: / / pubmed.ncbi.nlm.nih.gov / 29506988 /

[0020] [9]https: / / www.creativebiolabs.net / duligotuzumab-overview.htm。 Summary of the Invention

[0021] Bispecific antibodies commonly used in bispecific antibody-drug conjugates generally fall into the following categories: (1) Mab+sFv or Mab+VHH; (2) hybridization of two half-antibodies; and (3) bispecific antibodies based on a common light chain. These bispecific antibodies suffer from narrow antibody selectivity, steric hindrance, and issues related to large-scale production or pharmacokinetics. To address the shortcomings of existing EGFR×HER3 bispecific ADCs in terms of in vivo efficacy, pharmacokinetics, and safety, this invention utilizes a simple IgG(k) antibody that binds to both EGFR and HER3, providing an EGFR×HER3 bispecific antibody-drug conjugate, its preparation method, and its application. The antibody-drug conjugate is a simple IgG(k) EGFR×HER3 bispecific antibody-drug conjugate.

[0022] The present invention solves the above-mentioned technical problems through the following technical means:

[0023] The first aspect of the present invention provides an antibody-drug conjugate, a tautomer, an enantiomer, a diastereomer, or a mixture of isomers thereof, or a pharmaceutically usable salt thereof, wherein the antibody-drug conjugate has the structure A-(L1-L2-L3-T)n.

[0024] Where A represents the antibody;

[0025] L1 is the coupling connector;

[0026] L2 consists of x PEG spacers;

[0027] L3 is a cleavable or cleavable linker, or L3 is a non-cleavable linker;

[0028] T represents a small, reactive molecule;

[0029] n is any integer or decimal from 1 to 20; for example, 4, 6.06, 8.10, 9, 10.11, 13.05, 17.99, 16, or 18.

[0030] x is any integer from 0 to 20; for example, 0, 2, 4, 6, 8, 9, 10, 13, 16, 17, or 18.

[0031] The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:23, respectively. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:24, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0032] In some embodiments of the present invention, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or,

[0033] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0034] In some preferred embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4; and / or, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:9 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:9; or,

[0035] The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:14; and / or, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:17 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17.

[0036] In some embodiments of the present invention, the antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region and / or the light chain constant region are derived from human antibodies.

[0037] In some embodiments of the present invention, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region.

[0038] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:15, or SEQ ID NO:19; and / or, the amino acid sequence of the light chain constant region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18.

[0039] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:5, and / or, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:10; or,

[0040] The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:15, and / or, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:18; or,

[0041] The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:19, and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO:18.

[0042] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31; and / or, the amino acid sequence of the light chain of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.

[0043] In some embodiments of the present invention, the amino acid sequences of the heavy and light chains of the antibody are selected from any one of the following groups:

[0044] (i) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively;

[0045] (ii) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:31 and SEQ ID NO:28, respectively;

[0046] (iii) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively;

[0047] (iv) The amino acid sequences of the heavy and light chains of the antibody are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively.

[0048] In some embodiments of the present invention, L1 is maleimide or methanesulfonylpyrimidine.

[0049] In some embodiments of the present invention, L3 is a degradable linker peptide, such as one or more selected from VA, VC and GGFG (SEQ ID NO:21); preferably VC or GGFG.

[0050] In some embodiments of the present invention, L3 is a cleavable linker, such as glucuronide phenol.

[0051] In this invention, the non-splittable connector is conventionally chosen in the art.

[0052] In some embodiments of the present invention, T is a cytotoxic agent, such as a microtubule inhibitor cytotoxic agent or a DNA topoisomerase I inhibitor.

[0053] In some embodiments of the present invention, the antibody is (i) or (iii) above, and n is any integer or decimal from 1 to 8; for example, 1, 2.06, 3, 4.9 or 8, preferably 8; or, the antibody is (ii) or (iv) above, and n is any integer or decimal from 1 to 4; for example, 1, 2.06, 3 or 4, preferably 4.

[0054] In some preferred embodiments of the present invention, the microtubule inhibitor cytotoxicity is selected from one or more of Exd, MMAE, MMAF, DM1, DM4, ducarmycin and eribulin, such as Exd, DM1, MMAE or eribulin; the DNA topoisomerase I inhibitor is Dxd and / or SN38.

[0055] In a specific embodiment of the present invention, the antibody-drug conjugate is:

[0056] The antibody is as described in (i); L1 is mesylate pyrimidine; L3 is GGFG; T is Dxd; n is 4-6; x is 0 or 2; or,

[0057] The antibody is as described in (i); L1 is methanesulfonylpyrimidine; L3 is GGFG or glucuronide phenol; T is SN38; n is 4-6; x is 0-8; or,

[0058] The antibody is as described in (i); L1 is maleimide; L3 is VC; T is MMAE; n is 2-4; x is 2 or 3; or,

[0059] The antibody is as described in (ii); L1 is mesylate pyrimidine; L3 is VC or a non-cleavable linker; T is DM1; n is 2-4; x is 0; or,

[0060] The antibody is as described in (ii); L1 is maleimide; L3 is VC; T is MMAE; n is 4; x is 4-8; or,

[0061] The antibody is as described in (ii); L1 is maleimide; L3 is VC; T is eribulin; n is 4; x is 2-8, for example 2, 4 or 8; or,

[0062] The antibody is as described in (ii); L1 is maleimide; L3 is GGFG; T is Exd; n is 4; x is 0; or,

[0063] The antibody is as described in (ii); L1 is mesylate pyrimidine; L3 is VC; T is Exd; n is 4; x is 4-8; or,

[0064] The antibody is as described in (iii); L1 is mesylate pyrimidine; L3 is GGFG; T is Dxd; n is 8; x is 0 or 2; or,

[0065] The antibody is as described in (iii); L1 is mesylate pyrimidine; L3 is GGFG or glucuronide phenol; T is SN38; n is 8; x is 0-8; or,

[0066] The antibody is as described in (iii); L1 is maleimide; L3 is VC; T is MMAE; n is 2-4; x is 2 or 3; or,

[0067] The antibody is as described in (iv); L1 is mesylate pyrimidine; L3 is a non-cleavable linker; T is DM1; n is 2-4; x is 0; or,

[0068] The antibody is as described above (iv); L1 is maleimide; L3 is VC; T is MMAE; n is 4; x is 4-8; or,

[0069] The antibody is as described in (iv); L1 is maleimide; L3 is VC; T is eribulin; n is 4; x is 2-8, for example 2, 4 or 8; or,

[0070] The antibody is as described above (iv); L1 is maleimide; L3 is GGFG; T is Exd; n is 4; x is 0; or,

[0071] The antibody is as described above (iv); L1 is mesylate pyrimidine; L3 is VC; T is Exd; n is 4; x is 4-8.

[0072] A second aspect of the present invention provides a method for preparing an antibody-drug conjugate as described in the first aspect of the present invention, the method comprising mixing the antibody with L1-L2-L3-T. Wherein, L1, L2, L3, and T are defined as previously stated.

[0073] In some preferred embodiments of the present invention, the step further includes using a reducing agent.

[0074] In some preferred embodiments of the present invention, the reducing agent is tris(2-carboxyethyl)phosphine.

[0075] A third aspect of the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0076] In this invention, pharmaceutical compositions may comprise suitable pharmaceutically acceptable carriers, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextran and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing the antibody-drug conjugate of the invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of lyophilized formulations or aqueous solutions. Sustained-release formulations can also be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody-drug conjugate of the invention, said matrix being in the form of shaped articles, such as films or microcapsules.

[0077] A fourth aspect of the present invention provides a complex comprising an antibody-drug conjugate as described in the first aspect of the present invention and / or a pharmaceutical composition as described in the second aspect of the present invention.

[0078] In this invention, a complex refers to an antibody-drug conjugate as described in the first aspect of the invention and / or a pharmaceutical composition as described in the second aspect of the invention, with the addition of active and inactive substances known in the art to promote formulation stability and / or enhance efficacy. Preferably, these active substances have complementary activities that do not adversely affect each other, and are suitably combined in an amount effective for the intended use.

[0079] The fifth aspect of the present invention provides the use of one or more of the antibody-drug conjugates as described in the first aspect of the present invention, the pharmaceutical compositions as described in the third aspect of the present invention, and the complexes as described in the fourth aspect of the present invention in the preparation of a medicament for treating cancer.

[0080] In this invention, the cancer is a cancer that is positive for EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma.

[0081] In some preferred embodiments of the present invention, the cancer is selected from one or more of lung cancer, skin cancer, and colorectal cancer.

[0082] The sixth aspect of the present invention provides a method for treating cancer, comprising administering to a subject in need an effective amount of one or more of the antibody-drug conjugate as described in the first aspect of the present invention, the pharmaceutical composition as described in the third aspect of the present invention, and the complex as described in the fourth aspect of the present invention.

[0083] In this invention, the cancer is a cancer that is positive for EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma.

[0084] In some preferred embodiments of the present invention, the cancer is selected from one or more of lung cancer, skin cancer, and colorectal cancer.

[0085] The seventh aspect of the present invention provides an antibody-drug conjugate as described in the first aspect of the present invention, a pharmaceutical composition as described in the third aspect of the present invention, or a complex as described in the fourth aspect of the present invention for treating cancer.

[0086] In this invention, the cancer is a cancer that is positive for EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma.

[0087] In some preferred embodiments of the present invention, the cancer is selected from one or more of lung cancer, skin cancer, and colorectal cancer.

[0088] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0089] The reagents and raw materials used in this invention are all commercially available.

[0090] The present invention achieves the following technical effects:

[0091] The antibody-drug conjugates provided by this invention can bind to human EGFR and HER3 proteins, and the conjugation of small molecules does not affect the binding activity of the antibody to human EGFR protein. They are cytotoxic to cancer cells expressing HER3 and EGFR, and show superior efficacy and safety in in vivo tumor treatment. Attached Figure Description

[0092] Figure 1 shows the format of the antibodies involved in the patent; BB-0850-2 and BB-0856-3 are ordinary IgG1.

[0093] Figure 2 shows the affinity of antibodies BB0850-2 and BB0856-3 with different concentrations of HER3.

[0094] Figure 3 shows the affinity of antibodies BB0850-2 and BB0856-3 with different concentrations of EGFR.

[0095] Figure 4 shows a comparison of the efficacy of Exd-ADC with reference ADCs at various clinical stages. Detailed Implementation

[0096] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0097] Unless otherwise specified, all materials and reagents used in the embodiments of the present invention are commonly used materials and reagents in the art and can be obtained through conventional commercial channels.

[0098] In this invention, the abbreviations are as follows:

[0099] VC (Valine-Citrulline);

[0100] VA (valine-alanine);

[0101] GGFG (glycine-glycine-phenylalanine-glycine);

[0102] Exd (Exatecan);

[0103] MMAE(Monomethyl auristatin E);

[0104] MMAF (Monomethyl auristatin F);

[0105] DM1 (Medane DM1);

[0106] DM4 (Maytansin DM4);

[0107] Dxd(Exatecan);

[0108] MC (maleimide);

[0109] PEG (polyethylene glycol).

[0110] The format of the antibodies involved in the patent is shown in Figure 1; BB-0850-2 and BB-0856-3 are ordinary IgG1.

[0111] Example 1: Design and production of fusion proteins BB0850 and BB0856

[0112] The plasmids (pcDNA3.4 plasmid) of fusion proteins BB0850-2 (SEQ ID NO:15, SEQ ID NO:16) and BB0856-3 (SEQ ID NO:17, SEQ ID NO:18) were constructed and expressed, and HEK293 cells (Gibco) were transiently transfected with the plasmids. Sufficient quantities of fusion proteins BB0850-2 and BB0856-3 were obtained through one-step purification using a Protein A affinity column. The antibodies prepared in this form showed good expression and high yield, and can be purified to high purity using a one-step Protein A purification process. The purity and yield of fusion proteins BB0850-2 and BB0856-3 were detected and identified by SEC-HPLC (Thermo Fisher, Vanquish F) and NanoDrop (Merinton, SMA4000), and the results are shown in Table 1. It can be seen that the fusion proteins have high stability, and stable and homogeneous fusion proteins can be obtained according to conventional methods.

[0113] Table 1 Purity and yield of fusion proteins BB0850-2 and BB0856-3

[0114] Example 2: Coupling to Generate ADC

[0115] 1. Production of DAR4 and DAR8 ADCs

[0116] The antibodies BB0850-2 and BB0856-3 were subjected to ultrafiltration replacement. The buffer was replaced with EDTA / DPBS, and TCEP reducing agent (Sigma) was added. The mixture was reduced at 37°C for 1-2 hours. DM1 (Lianning), MMAE (Lianning), or Exd (MCE, GGFG linker) containing MC-(PEG)x-VC or mesylate-pyrimidine-(PEG)x-VC linkers were added. The mixture was reacted at room temperature for 1-2 hours. The buffer was replaced 5 times using a 30KD ultrafiltration tube (Amicon) to remove excess small molecules. The mixture was then removed to obtain ADC (DAR4).

[0117] The antibodies BB0850 (SEQ ID NO:13, SEQ ID NO:14) and BB0856 (SEQ ID NO:11, SEQ ID NO:12) were subjected to ultrafiltration replacement. The buffer was replaced with EDTA / DPBS, and TCEP reducing agent (Sigma) was added. The mixture was reduced at 37°C for 1-2 hours. Dxd (MCE) or SN38 (MCE) containing MC-(PEG)x-GGFG or mesylate-pyrimidine-(PEG)x-GGFG linkers was added, and the mixture was reacted at room temperature for 1-2 hours. The buffer was replaced 5 times using a 30KD ultrafiltration tube (Amicon) to remove excess small molecules. The mixture was then removed to obtain the ADC (DAR8).

[0118] The prepared ADCs are shown in Table 2.

[0119] Table 2 ADC Note: "-" indicates a non-cleavable linker without an enzymatically cleavable portion, which is the conventional choice in this field.

[0120] 2. Coupling Result Analysis

[0121] The coupling results were analyzed using HIC (Thermo Fisher, model Q Exactive Plus).

[0122] After BB0850, BB0856, BB0850-2 and BB0856-3 were conjugated with small molecule toxins, the peak times of the antibodies and ADCs were different. The peak times of the ADCs were all later than the peak times of the corresponding antibodies, which proves that the antibodies were conjugated with small molecule toxins respectively.

[0123] Example 3: Combining Activity Detection

[0124] Human EGFR or HER3 protein (1000 ng / mL) (Acro) was coated onto 96-well plates and incubated overnight at 4°C. BB0850 and BB0856 and their ADC samples (Dxd or SN38 ADC (DAR8) prepared in Example 2) were serially diluted 5-fold starting at 5000 ng / mL, for a total of 8 dilutions. The diluted samples were then transferred to EGFR-coated plates and incubated at room temperature for 2 hours. A colorimetric reaction was performed using HRP-labeled anti-human IgG Fc (Sigma) as the detection antibody and TMB (Sino Biological Inc). The absorbance at 450 / 650 nm was read using a microplate reader (Molecular Devices, SpectraMax i3X), and the dose-response curve was fitted using a four-parameter equation for data analysis.

[0125] The results showed that the BB0850 and BB0856 antibodies and their ADCs could bind to the human EGFR protein HER3, and that conjugation with small molecules did not affect the binding activity of the antibodies to the human EGFR protein.

[0126] Example 4 Affinity Detection

[0127] The antibodies of BB0850-2 and BB0856-3 prepared in Example 2 were diluted to 5 μg / mL and immobilized onto the AHC chip. When the detection signal reached or approached 1.5 nm, the sample was then detected with different concentrations of EGFR and HER3 (200 nM initial 2-fold dilution) using an instrument (Octet RED96).

[0128] Protein binding and dissociation analysis was performed. The binding and dissociation constants (KD) were then calculated using instrumental analysis software, as shown in Figure 2 (binding and dissociation analysis with different concentrations of HER3) and Figure 3 (binding and dissociation analysis with different concentrations of EGFR).

[0129] Example 5 Cytotoxicity Detection

[0130] Human lung cancer cells H1975 and H441, and human skin cancer cells A431 were seeded into 96-well cell culture plates, and serially diluted BB0856-3 ADC or BB0850-2 ADC (Exd conjugate prepared in Example 2) were added. The cells were cultured at 37°C and 5% CO2 for 5 days. Then, CCK-8 reagent (Baiying Biotechnology) was added, and the absorbance at 450 / 650 nm was measured using a SpectraMax microplate reader. The cytotoxicity of BB0850-2 ADC and BB0856-3 ADC to tumor cells was evaluated by detecting the number of viable cells.

[0131] The results showed that BB0850-2 ADC and BB0856-3 ADC were cytotoxic to all three cancer cell types.

[0132] Example 6: In vivo efficacy

[0133] Female nude mice (Balb / c nude, purchased from Jicui Yaokang) aged 6-7 weeks and weighing 18-22g were injected subcutaneously with 10 x ... 6 Cells / Human lung cancer H441 cell line only, with an average subcutaneous tumor volume of approximately 200 mmHg. 3 Mice were randomly assigned to groups (n=5) based on average tumor volume and administered the drug (tail vein injection, once). The mice were divided into a solvent control group (Vehicle, PBS), BB0850-2-Exd (DAR4, 6 mg / kg), BB0856-3-Exd (DAR4, 6 mg / kg), and Isotype Control IgG-Exd (DAR4, 6 mg / kg). The long axis (a, mm) and short axis (b, mm) of the tumor were measured three times a week, and the tumor volume was calculated using the formula: V(mm²) 3 ) = 1 / 2 * a * b 2 Figure 4 shows the changes in tumor volume over time after administration of various Exd-coupled ADCs.

[0134] After administration, the various Exd-conjugated ADCs exhibited different efficacies. Isotype Control-Exd showed better safety (TGI 10.65%), while BB-0856-3-Exd showed the best efficacy (TGI 87.48%). BB-0856-3-Exd showed better in vivo efficacy than BB0850-2-Exd, which has a stronger EGFR binding affinity. The efficacy of each ADC was measured by TGI (see Table 3).

[0135] Table 3. Efficacy of H441 in vivo model (mean tumor growth inhibition rate TGI%)

[0136] The weakened EGFR binding ability, ease of binding, and ease of dissociation endow BB0856-3-Exd with optimal efficacy and safety.

[0137] The sequence of the present invention is as follows:

[0138] BB0850 Heavy Chain CDR:

[0139] CDR1: GDWIH (SEQ ID NO:1)

[0140] CDR2:EISAAGGYTDYADSVKG(SEQ ID NO:2)

[0141] CDR3:ESRVSFEAAMDY(SEQ ID NO:3)

[0142] BB0850 Heavy Chain Variable Region:

[0143] BB0850 Heavy Chain Constant Region:

[0144] BB0850 Light Chain CDR:

[0145] CDR1:RASQNIATDVA(SEQ ID NO:6)

[0146] CDR2: SASFLYS (SEQ ID NO:7)

[0147] CDR3: QQSEPEPYT (SEQ ID NO:8)

[0148] BB0850 Light Chain Variable Zone:

[0149] BB0850 light chain constant region:

[0150] BB0856 Heavy Chain CDR:

[0151] CDR1: GNWIH (SEQ ID NO:11)

[0152] CDR2: EISPSGGYTDYADSVKG(SEQ ID NO:12)

[0153] CDR3:ESRVSYEAAMDY(SEQ ID NO:13)

[0154] BB0856 Heavy Chain Variable Region:

[0155] BB0856 Heavy Chain Constant Region:

[0156] BB0856 Light Chain CDR:

[0157] CDR1:RASQDLATDVA(SEQ ID NO:16)

[0158] CDR2: (SEQ ID NO:7)

[0159] CDR3: (SEQ ID NO:8)

[0160] BB0856 Light Chain Variable Region:

[0161] BB0856 Light Chain Constant Region:

[0162] BB0850-2 Heavy Chain CDR:

[0163] CDR1: (SEQ ID NO:1)

[0164] CDR2: (SEQ ID NO:2)

[0165] CDR3: (SEQ ID NO:3)

[0166] BB0850-2 Heavy chain variable region: (SEQ ID NO:4)

[0167] BB0850-2 Heavy Chain Constant Region:

[0168] BB0850-2 Light Chain CDR:

[0169] CDR1: (SEQ ID NO:6)

[0170] CDR2: (SEQ ID NO:7)

[0171] CDR3: (SEQ ID NO:8)

[0172] BB0850-2 Light Chain Variable Region: (SEQ ID NO:9)

[0173] BB0850-2 Light Chain Constant Region: (SEQ ID NO:18)

[0174] BB0856-3 Heavy Chain CDR:

[0175] CDR1: (SEQ ID NO:11)

[0176] CDR2: (SEQ ID NO:12)

[0177] CDR3: (SEQ ID NO:13)

[0178] BB0856-3 Heavy chain variable region: (SEQ ID NO:14)

[0179] BB0856-3 Heavy chain constant region: (SEQ ID NO:19)

[0180] BB0856-3 Light Chain CDR:

[0181] CDR1: (SEQ ID NO:16)

[0182] CDR2: (SEQ ID NO:7)

[0183] CDR3: (SEQ ID NO:8)

[0184] BB0856-3 Light chain variable region: (SEQ ID NO:17)

[0185] BB0856-3 Light chain constant region: (SEQ ID NO:18)

[0186] SEQ ID NO:20: GXWIH, where X is D or N.

[0187] SEQ ID NO:21: GGFG

[0188] SEQ ID NO:22: EISX1X2GGYTDYADSVKG, where X1 is A or P, and X2 is A or S.

[0189] SEQ ID NO:23: ESRVSX3EAAMDY, where X3 is F or Y.

[0190] SEQ ID NO:24: RASQX4X5ATDVA, where X4 is N or D, and X5 is I or L.

[0191] BB0850 heavy chain (SEQ ID NO:25):

[0192] BB0850 light chain (SEQ ID NO:26):

[0193] BB0856 heavy chain (SEQ ID NO:27):

[0194] BB0856 light chain (SEQ ID NO:28):

[0195] BB0850-2 heavy chain (SEQ ID NO:29):

[0196] BB0850-2 Light Chain (SEQ ID NO:30):

[0197] BB0856-3 heavy chain (SEQ ID NO:31):

[0198] BB0856-3 Light Chain (SEQ ID NO:28)

[0199] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody-drug conjugate, its tautomers, enantiomers, diastereomers, or mixtures of isomers, or a pharmaceutically usable salt thereof, characterized in that, The structure of the antibody-drug conjugate is A-(L1-L2-L3-T)n; Where A represents the antibody; L1 is the coupling connector; L2 consists of x PEG spacers; L3 is a cleavable or cleavable linker, or a non-cleavable linker; T represents a small, reactive molecule; n is any integer or decimal from 1 to 20; for example, 4, 6.06, 8.10, 9, 10.11, 13.05, 17.99, 16, or 18. x is any integer from 0 to 20; for example, 0, 2, 4, 6, 8, 9, 10, 13, 16, 17, or 18. The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:23, respectively. The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:24, SEQ ID NO:7, and SEQ ID NO:8, respectively.

2. The antibody-drug conjugate as described in claim 1, characterized in that, The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or, The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:16, SEQ ID NO:7 and SEQ ID NO:8 respectively. Preferably, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4; and / or, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:9 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:9; or, The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:14; and / or, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:17 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

17.

3. The antibody-drug conjugate as described in claim 1 or 2, characterized in that, The antibody further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region and / or the light chain constant region are derived from human antibodies; Preferably, the heavy chain constant region is derived from the human heavy chain IgG1 constant region; and / or, the light chain constant region is derived from the human light chain κ chain constant region; More preferably, the amino acid sequence of the heavy chain constant region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:15, or SEQ ID NO:19; and / or, the amino acid sequence of the light chain constant region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:18; More preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:5, and / or, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:10; or, The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:15, and / or, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:18; or, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:19, and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

18.

4. The antibody-drug conjugate according to any one of claims 1-3, characterized in that, The amino acid sequence of the heavy chain of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:31; and / or, the amino acid sequence of the light chain of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:

30.

5. The antibody-drug conjugate as described in claim 4, characterized in that, The amino acid sequences of the heavy and light chains of the antibody are selected from any of the following groups: (i) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively; (ii) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:31 and SEQ ID NO:28, respectively; (iii) The amino acid sequences of the heavy chain and light chain of the antibody are shown in SEQ ID NO:25 and SEQ ID NO:26, respectively; (iv) The amino acid sequences of the heavy and light chains of the antibody are shown in SEQ ID NO:29 and SEQ ID NO:30, respectively.

6. The antibody-drug conjugate according to any one of claims 1-5, characterized in that, The antibody-drug conjugate meets one or more of the following conditions: (1) L1 is maleimide or methanesulfonylpyrimidine; (2) L3 is a degradable linker, such as one or more selected from VA, VC and GGFG; preferably VC or GGFG; or L3 is a cleavable linker, such as glucuronide phenol; (3) T is a cytotoxic agent, such as a microtubule inhibitor cytotoxic agent or a DNA topoisomerase I inhibitor; and, (4) The antibody is (i) or (iii) of the antibody-drug conjugate as described in claim 5, where n is any integer or decimal from 1 to 8; for example, 1, 2.06, 3, 4.9 or 8, preferably 8; or, the antibody is (ii) or (iv) of the antibody-drug conjugate as described in claim 5, where n is any integer or decimal from 1 to 4; for example, 1, 2.06, 3 or 4, preferably 4; Preferably, the microtubule inhibitor cytotoxic agent is selected from one or more of MMAE, MMAF, DM1, DM4, ducarmycin, and eribulin, such as DM1, MMAE, or eribulin; the DNA topoisomerase I inhibitor is Exd, Dxd, and / or SN38. More preferably, the antibody-drug conjugate is: The antibody is (i) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is GGFG; T is Dxd; n is 4-6; x is 0 or 2; or, The antibody is (i) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is GGFG or glucuronide phenol; T is SN38; n is 4-6; x is 0-8; or, The antibody is (i) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is VC; T is MMAE; n is 2-4; x is 2 or 3; or, The antibody is (ii) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is VC or L3 is a non-cleavable linker; T is DM1; n is 2-4; x is 0; or, The antibody is (ii) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is VC; T is MMAE; n is 4; x is 4-8; or, The antibody is (ii) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is VC; T is eribulin; n is 4; x is 2-8, for example 2, 4 or 8; or, The antibody is (ii) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is GGFG; T is Exd; n is 4; x is 0; or, The antibody is (ii) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is VC; T is Exd; n is 4; x is 4-8; or, The antibody is (iii) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is GGFG; T is Dxd; n is 8; x is 0 or 2; or, The antibody is (iii) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is GGFG or glucuronide phenol; T is SN38; n is 8; x is 0-8; or, The antibody is (iii) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is VC; T is MMAE; n is 2-4; x is 2 or 3; or, The antibody is (iv) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is a non-cleavable linker; T is DM1; n is 2-4; x is 0; or, The antibody is (iv) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is VC; T is MMAE; n is 4; x is 4-8; or, The antibody is as described in (iv) of the antibody-drug conjugate as claimed in claim 5; L1 is maleimide; L3 is VC; T is eribulin; n is 4; x is 2-8, for example 2, 4 or 8; or, The antibody is (iv) of the antibody-drug conjugate as described in claim 5; L1 is maleimide; L3 is GGFG; T is Exd; n is 4; x is 0; or, The antibody is (iv) of the antibody-drug conjugate as described in claim 5; L1 is mesylate pyrimidine; L3 is VC; T is Exd; n is 4; x is 4-8.

7. A method for preparing an antibody-drug conjugate according to any one of claims 1-6, the method comprising mixing the antibody with L1-L2-L3-T; Preferably, the step further includes using a reducing agent; More preferably, the reducing agent is tris(2-carboxyethyl)phosphine.

8. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 1-6, and a pharmaceutically acceptable carrier.

9. A complex, characterized in that, The complex comprises an antibody-drug conjugate as described in any one of claims 1-6 and / or a pharmaceutical composition as described in claim 8.

10. Use of one or more of the antibody-drug conjugates of any one of claims 1-6, the pharmaceutical composition of claim 8, and the complex of claim 9 in the preparation of a medicament for treating cancer; Preferably, the cancer is a cancer expressing EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma; More preferably, the cancer is selected from one or more of lung cancer, skin cancer, head and neck cancer, and colorectal cancer.

11. A method of treating cancer, comprising administering to a subject in need an effective amount of one or more of the antibody-drug conjugate as claimed in any one of claims 1-6, the pharmaceutical composition as claimed in claim 8, and the complex as claimed in claim 9; Preferably, the cancer is a cancer expressing EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma; More preferably, the cancer is one or more of lung cancer, skin cancer, head and neck cancer, and colorectal cancer.

12. An antibody-drug conjugate as described in any one of claims 1-6, a pharmaceutical composition as described in claim 8, or a complex as described in claim 9 for the treatment of cancer; Preferably, the cancer is a cancer expressing EGFR and / or Her3, for example, the cancer is selected from one or more of the following: skin cancer, breast cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma; More preferably, the cancer is one or more of lung cancer, skin cancer, head and neck cancer, and colorectal cancer.

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