Anti-EGFR antibody drug conjugate and use thereof

Abstract

A new anti-EGFR antibody drug conjugate and the use thereof are disclosed in the present disclosure. The anti-EGFR antibody drug conjugate has a reduced ADCC effect, excellent therapeutic efficacy and excellent safety.

Description

Anti-EGFR antibody drug conjugates and their uses Technical Field

[0001] This invention relates to the field of antibody-drug conjugate technology, specifically to an anti-EGFR antibody-drug conjugate and its uses. Background Technology

[0002] EGFR (Epidermal Growth Factor Receptor) is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. It belongs to the ErbB receptor family, which includes EGFR (HER1, ErbB-1), HER2 (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). EGFR is involved in the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. Blocking the EGFR signaling pathway, or by blocking the EGFR binding site in the extracellular region of the receptor, or by inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumors and improve the patient's condition. Currently approved anti-EGFR antibodies include cetuximab, panitumumab, necitumumab, nimotuzumab, and amivantamab. While these monoclonal antibodies have shown some efficacy in their respective indications, there is still room for improvement in efficacy, and drug resistance is easily developed after a period of treatment. Currently, only one anti-EGFR antibody-drug conjugate (ADC) has been approved—cetuximab Sarotalocan, marketed as Akalux. This drug consists of cetuximab and the payload IRDye700DX, a photoreactive dye that undergoes a photochemical reaction upon exposure to infrared light to exert its therapeutic effect. Based on this ADC's mechanism of action, it is only suitable for superficial tumors such as skin tumors, but it is difficult to treat intracranial or internal (organ) tumors. Furthermore, it requires infrared light irradiation after administration, causing inconvenience for patients.

[0003] Therefore, there is still a need to develop new anti-EGFR antibody-drug conjugates with lower off-target toxicity, better efficacy, and not limited to superficial tumors. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-EGFR antibody-drug conjugate with excellent efficacy and safety.

[0005] The first aspect of the present invention discloses an anti-EGFR antibody-drug conjugate.

[0006] An anti-EGFR antibody-drug conjugate having the structure shown in formula (Ⅰ):

[0007] Formula (Ⅰ), where mAb represents an anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4; R is H or halogen.

[0008] As one example, in the aforementioned anti-EGFR antibody-drug conjugate, R is H or F.

[0009] As a further example, the aforementioned anti-EGFR antibody-drug conjugate has the structure shown in formula (II):

[0010] Formula (II), where mAb represents anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4.

[0011] As a further embodiment, the aforementioned anti-EGFR antibody drug conjugate has the structure shown in formula (Ⅲ):

[0012] Formula (Ⅲ), where mAb represents anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4.

[0013] An anti-EGFR antibody-drug conjugate having the following structure (Ⅳ):

[0014] Formula (Ⅳ), where mAb represents an anti-EGFR antibody; m is 1-8, preferably m is 4 or 8, and more preferably m is 8.

[0015] As a further example, the aforementioned anti-EGFR antibody-drug conjugate contains anti-EGFR antibodies such as cetuximab, panitumumab, necitumumab, nimotuzumab, matuzumab, amivantamab, demupitamab, depatuxizumab, futuximab, imatrozumab, and latuximab. The monoclonal antibody may be selected from the following: prituximab, rosatuxizumab, zatuximab, pimurutamab, serclutamab, tomuzotuximab, or zalutumumab or their biosimilars, preferably cetuximab or its biosimilars, and preferably necitumumab or its biosimilars; the choice of monoclonal antibody is not limited, as long as the structure employs the conjugate of this invention is within the scope of protection of this invention.

[0016] As a further example, the anti-EGFR antibody drug conjugate described above has a DAR of 2-8, preferably 4±0.5, 6±0.5 or 8±0.5.

[0017] As one embodiment, the anti-EGFR antibody-drug conjugate described above has the following anti-EGFR antibody: heavy chain CDR1 as described in SEQ ID NO:1, heavy chain CDR2 as described in SEQ ID NO:2, heavy chain CDR3 as described in SEQ ID NO:3, light chain CDR1 as described in SEQ ID NO:4, light chain CDR2 as described in SEQ ID NO:5, and light chain CDR3 as described in SEQ ID NO:6.

[0018] As one embodiment, the anti-EGFR antibody-drug conjugate described above has an anti-EGFR antibody having a heavy chain variable region as described in SEQ ID NO:7 and a light chain variable region as described in SEQ ID NO:8. The monoclonal antibody used in this invention is not limited; it can be an antibody with an Fc segment mutation or an antibody without an Fc segment mutation.

[0019] As one embodiment, the anti-EGFR antibody-drug conjugate described above has the anti-EGFR antibody having the heavy chain CDR1 as described in SEQ ID NO:14, the heavy chain CDR2 as described in SEQ ID NO:15, the heavy chain CDR3 as described in SEQ ID NO:16, the light chain CDR1 as described in SEQ ID NO:17, the light chain CDR2 as described in SEQ ID NO:18, and the light chain CDR3 as described in SEQ ID NO:19.

[0020] As one embodiment, the anti-EGFR antibody-drug conjugate described above has an anti-EGFR antibody having a heavy chain variable region as described in SEQ ID NO:20 and a light chain variable region as described in SEQ ID NO:21.

[0021] In some implementations, the Fc fragment of the anti-EGFR antibody has a reduced ADCC effect.

[0022] Furthermore, the anti-EGFR antibody is an IgG1 subtype antibody, and it has a reduced ADCC effect compared to wild-type IgG1.

[0023] In some implementations, the reduced ADCC effect is achieved through Fc mutations, which can be single-point mutations, two-site mutations, or multi-site mutations, with mutation sites selected from P232-S239, D265-D270, Y296-T299, and K322-I332.

[0024] Furthermore, Fc mutations include N297A mutation, L234A and L235A (LALA) mutation, L234A, L235A and P329G (LALAPG) mutation, L234A, L235A and K322A (LALAKA) mutation, L234F, L235E and D265A (LFLEDA) mutation, or L234F, L235E and P331S (LFLEPS) mutation.

[0025] In some embodiments, the anti-EGFR antibody has the heavy chain CDR1 of SEQ ID NO:1, the heavy chain CDR2 of SEQ ID NO:2, the heavy chain CDR3 of SEQ ID NO:3, the light chain CDR1 of SEQ ID NO:4, the light chain CDR2 of SEQ ID NO:5, and the light chain CDR3 of SEQ ID NO:6.

[0026] Furthermore, the anti-EGFR antibody has a heavy chain variable region, as shown in SEQ ID NO:7, and a light chain variable region, as shown in SEQ ID NO:8.

[0027] Furthermore, the anti-EGFR antibody has a heavy chain as in SEQ ID NO:11 and a light chain as in SEQ ID NO:13.

[0028] Furthermore, the anti-EGFR antibody has a heavy chain as in SEQ ID NO:12 and a light chain as in SEQ ID NO:13.

[0029] In some embodiments, the anti-EGFR antibody has, for example, the heavy chain CDR1 of SEQ ID NO:14, the heavy chain CDR2 of SEQ ID NO:15, the heavy chain CDR3 of SEQ ID NO:16, the light chain CDR1 of SEQ ID NO:17, the light chain CDR2 of SEQ ID NO:18, and the light chain CDR3 of SEQ ID NO:19.

[0030] Furthermore, the anti-EGFR antibody has a heavy chain variable region as in SEQ ID NO:20 and a light chain variable region as in SEQ ID NO:21.

[0031] Furthermore, the anti-EGFR antibody has a heavy chain as described in SEQ ID NO:24 and a light chain as described in SEQ ID NO:26.

[0032] Furthermore, the anti-EGFR antibody has a heavy chain as described in SEQ ID NO:25 and a light chain as described in SEQ ID NO:26.

[0033] This invention engineered antibodies to mutate the Fc segment of anti-EGFR antibodies, reducing the binding of antibody Fc to FcR, thereby reducing off-target adverse reactions caused by ADC drugs being internalized into FcR-expressing cells.

[0034] A second aspect of the present invention discloses the use of anti-EGFR antibody-drug conjugates in the preparation of medicaments for treating diseases.

[0035] In some embodiments, the anti-EGFR antibody-drug conjugate of the present invention can be used to prepare drugs for treating diseases such as colorectal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, pancreatic cancer, glioma, breast cancer, and esophageal cancer.

[0036] Furthermore, the disease is lung adenocarcinoma.

[0037] Furthermore, the disease is non-small cell lung cancer.

[0038] Furthermore, the disease is glioma.

[0039] Furthermore, the disease is metastatic colorectal cancer.

[0040] A third aspect of the present invention discloses a method for treating a disease.

[0041] The method of treating a disease according to the present invention comprises administering to a subject an effective dose of the anti-EGFR antibody-drug conjugate of the first aspect of the present invention.

[0042] In some implementation schemes, the diseases include colorectal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, pancreatic cancer, glioma, breast cancer, and esophageal cancer.

[0043] Furthermore, the disease is lung adenocarcinoma.

[0044] Furthermore, the disease is non-small cell lung cancer.

[0045] Furthermore, the disease is glioma.

[0046] Furthermore, the disease is metastatic colorectal cancer.

[0047] The anti-EGFR antibody-drug conjugate of this invention can be administered by any suitable means, including gastrointestinal, parenteral, intrapulmonary, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0048] For the prevention or treatment of disease, a generally suitable approach for the anti-EGFR antibody-drug conjugate of the present invention is to administer the drug to the patient in a single or multiple doses. Depending on the type and severity of the disease, the initial candidate dose of the anti-EGFR antibody-drug conjugate administered to the patient can be from about 1 μg / kg to 100 mg / kg, for example, 0.1 mg / kg to 20 mg / kg. For repeated administrations lasting several days or longer, treatment is typically continued until the desired suppression of disease symptoms occurs, depending on the condition. Example doses of the anti-EGFR antibody-drug conjugate can range from about 0.05 mg / kg to about 10 mg / kg.

[0049] The present invention relates to an anti-EGFR antibody drug conjugate that involves multiple administrations of the drug to the patient, with a dosing frequency of once every 1-6 weeks, for example, once every 2 weeks, 3 weeks, or 4 weeks, until the subject is cured, the disease progresses, or the subject dies.

[0050] A fourth aspect of the present invention discloses a non-therapeutic method for inhibiting tumor cells.

[0051] The non-therapeutic method for inhibiting tumor cells of the present invention comprises adding an effective dose of the anti-EGFR antibody-drug conjugate described in the first aspect of the present invention to a tumor cell system.

[0052] In some implementations, tumor cells include colorectal cancer cells, head and neck cancer cells, lung cancer cells, nasopharyngeal cancer cells, pancreatic cancer cells, glioma cells, breast cancer cells, and esophageal cancer cells.

[0053] Furthermore, the disease is lung adenocarcinoma.

[0054] Furthermore, the disease is non-small cell lung cancer.

[0055] Furthermore, the disease is glioma.

[0056] It should be understood that one, some, or all of the various embodiments described herein can be combined to form other embodiments of the invention. These and other embodiments of the invention are further described in the following detailed description. Attached Figure Description

[0057] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0058] Figure 1: Effect of test substance ERH001-CPT01 on tumor growth of human lung adenocarcinoma cell line NCI-H292 xenograft tumor;

[0059] Figure 2: Effect of test substance ERH001-CPT01 on body weight of nude mice with human lung adenocarcinoma cell line NCI-H292 xenograft tumor;

[0060] Figure 3: Effects of the test substances ERH001-CPT01, ERH001-DXd-1 and ERH001-DXd-2 on tumor growth of human glioma LN-229-4A7-2H8 xenograft.

[0061] Figure 4: Effects of the test substances ERH001-CPT01, ERH001-DXd-1 and ERH001-DXd-2 on the body weight of nude mice with human glioma LN-229-4A7-2H8 xenograft tumors.

[0062] Figure 5: Effects of test substances ERH001-CPT01, ERH001-DXd-1 and ERH001-DXd-2 on tumor growth of human non-small cell lung cancer HCC827 xenograft tumors;

[0063] Figure 6: Effects of test substances ERH001-CPT01, ERH001-DXd-1 and ERH001-DXd-2 on body weight in nude mice with human non-small cell lung cancer HCC827 xenograft tumors.

[0064] Figure 7: Effect of test substance ERH001-CPT02 on tumor growth of human glioma LN-229-4A7-2H8 xenograft;

[0065] Figure 8: Effect of test substance ERH001-CPT02 on body weight in nude mice with human glioma LN-229-4A7-2H8 xenograft tumor;

[0066] Figure 9: Effects of test substances ERH001-CPT02 and ERH001-DXd on tumor growth of human lung cancer cell NCI-H292 xenograft tumors;

[0067] Figure 10: Effects of test substances ERH001-CPT02 and ERH001-DXd on body weight in nude mice with human lung cancer cell NCI-H292 xenograft tumors. Detailed Implementation

[0068] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0070] In this invention, the term "antibody" refers to a full-length immunoglobulin molecule or the immunoactive portion of a full-length immunoglobulin molecule, such as a molecule containing an antigen-binding site that specifically binds to a target antigen or a portion of a target antigen. Targets include, but are not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins in this invention can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulins.

[0071] The term "effective dose" refers to the amount of medication that effectively achieves the desired therapeutic outcome at the required dose and time period. Effective doses can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the treatment agent or combination of treatment agents to elicit the desired response in the individual.

[0072] The term "cytotoxic activity" refers to the cytotoxic effect of a drug, a camptothecin conjugate, or a camptothecin conjugate's intracellular metabolite. Cytotoxic activity can be expressed as the IC50 value, which is the concentration (moles or mass / unit volume) at which half of the cells survive.

[0073] The terms “administration” or “dosage” refer to the transfer, delivery, introduction, or transport of a drug or other pharmaceutical agent to a subject in any manner. These methods include oral, local contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, or intrathecal administration.

[0074] The term "halogen" refers to the elements in Group VIIA of the periodic table; including fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At). (Ts) etc.

[0075] Example 1: Engineering of anti-EGFR antibodies

[0076] This invention employs conventional antibody engineering methods to mutate the Fc terminus of anti-EGFR antibodies. Exemplary mutations include (1) two-site mutations at L234A and L235A (LALA) and (2) three-site mutations at L234A, L235A, and P329G (LALAPG). The resulting antibodies are exemplary ERH001, ERH001A, ERH001B, and ERH001C, whose sequences are shown in Tables 1-4 below.

[0077] Table 1 ERH001 sequence

[0078] Table 2 ERH001A sequence

[0079] Table 3 ERH001B sequence

[0080] Table 4 ERH001C sequence

[0081] Example 2: In vitro evaluation of anti-EGFR antibodies

[0082] 1. Antibody affinity flow cytometry detection

[0083] The antibody affinity was detected by flow cytometry using MDA MB 468 cells. As shown in Table 5, the flow cytometry results indicated that the antibodies Erbitux (original cetuximab), ERH000 (self-made cetuximab biosimilar), and ERH001 had similar affinities, and the LALA mutation did not alter antibody affinity.

[0084] Table 5. Results of Antibody Affinity Flow Cytometry Detection

[0085] 2. Antibody-Fc Receptor Affinity - Fortebio BLI Assay

[0086] Referring to Table 6, the affinity evaluation results for Fc receptors show that Erbitux (the original cetuximab) and ERH001 have the same affinity for Human FcRn, and the LALA-mutated antibody can maintain the same in vivo half-life as the original drug. ERH001 has a weaker affinity for Human FcγRI and Human FcγRIIA than Erbitux (the original cetuximab), and the binding of the antibody to the Fc receptor is reduced, thus reducing the side effects caused by Fc receptor binding.

[0087] Table 6 Results of antibody-Fc receptor affinity assay (Kd(M): antibody, Fc receptor affinity)

[0088] 3. Antibody endocytosis efficiency - flow cytometry detection (MDA MB 468 cells)

[0089] The antibody endocytosis efficiency was detected by flow cytometry using MDA MB 468 cells (see Table 7). The antibody endocytosis results showed that the endocytosis efficiencies of Erbitux (the original cetuximab) and ERH001 antibodies were similar, and the LALA mutation did not change the antibody endocytosis performance.

[0090] Table 7 Results of antibody endocytosis efficiency assay

[0091] Example 3: Exemplary ADC Fabrication

[0092] 3.1 Synthesis of CPT01 (Compound 8)

[0093] Preparation of (S)-(2-((4,11-diethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]inzizo[1,2-b]quinoline-9-yl)oxy)ethyl)tert-butyl carbamate (compound 2):

[0094] At 0 °C, triphenylphosphine (0.86 g, 3.3 mmol), (2-hydroxyethyl)carbamate (0.70 g, 4.4 mmol), and ERHD (0.57 g, 3.3 mmol) were added to a solution of (S)-4,11-diethyl-8-fluoro-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazazino[1,2-b]quinoline-3,14(4H)-dione (compound 1, 0.45 g, 1.1 mmol) tetrahydrofuran (40 mL). After addition, the reaction was stirred at room temperature for 3 h, concentrated, and purified by silica gel column chromatography to give compound 2. MS-ESI (m / z): 554.22 [M+H] + .

[0095] Preparation of (S)-9-(2-aminoethoxy)-4,11-diethyl-8-fluoro-4-hydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indazano[1,2-b]quinoline-3,14(4H)-dione (compound 3):

[0096] Compound 2 (0.37 g, 0.67 mmol) was dissolved in 3 mL of dichloromethane, and 6 mL of trifluoroacetic acid was added with stirring. The mixture was stirred at room temperature for 1 h, and then concentrated to give a yellow solid, compound 3 (0.64 g). MS-ESI (m / z): 454.17 [M+H] + .

[0097] Preparation of compound 6:

[0098] 4-({4-[(3-carboxypropyl)amino]-2,3-bis(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-1,4-dioxobutyl}amino)butyric acid (compound 4, 1.5 g, 3.1 mmol), ((S)-35-amino-24-methyl-1-(11-oxo)-29-oxo3,6,9,12,15,18,21,24,27-nonoxy-30-azahexahexadecyl-36-acyl)-L-valine -L-alanine tert-butyl ester (compound 5, 5.5 g, 6.6 mmol) was dissolved in DMF (100 ml), then HATU (4.8 g, 12.5 mmol) and NMM (1.3 g, 12.5 mmol) were added. The reaction was allowed to proceed to completion at room temperature. The solution was concentrated and poured into 200 ml of water. The mixture was extracted with dichloromethane (3 × 100 ml), and the organic layers were combined. The mixture was washed with water (50 ml) and brine (50 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 6. MS-ESI (m / z): 1063.78 [M + 2H] 2+ .

[0099] Preparation of compound 7:

[0100] Compound 6 (6.5 g, 3.1 mmol) was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (50 mL) was added. The mixture was stirred for 2 h, concentrated, and purified by HPLC to obtain compound 7. MS-ESI (m / z): 1006.13 [M+2H] 2+ .

[0101] Preparation of compound 8:

[0102] Compound 7 (280 mg, 0.139 mmol) and compound 3 (0.278 mmol) were dissolved in DMF (5 mL), and HATU (158 mg, 0.417 mmol) and DIPEA (92 μL, 0.557 mmol) were added. The mixture was stirred at room temperature until the reaction was complete. After concentration, compound 8 was purified by HPLC. MS-ESI (m / z): 1442.45 [M+2H] 2+ .

[0103] 3.2 Synthesis of CPT02 (Compound 10)

[0104] Following the synthetic route of CPT01 (compound 8), the synthesis process is the same except that the toxin molecule is replaced by 7-ethyl-10-hydroxycamptothecin from compound 1.

[0105] The structure of 7-ethyl-10-hydroxycamptothecin is as follows:

[0106] The structure of the synthesized CPT02 (compound 10) is as follows:

[0107] 3.3 Synthesis of ERH001-CPT01 / ERH001-CPT02

[0108] The antibody (ERH001) was prepared to a concentration of 7-10 mg / ml with phosphate buffer at pH 6.8-7.2. 3.8 eq of TCEP (10 mM) was added, and the mixture was reduced at 4°C for 15-16 h. CPT01 (compound 8) / CPT02 (compound 10) (7.0 eq, 100 mM) was added for a coupling reaction for 2-3 h. Then, N-acetylcysteine ​​(14 eq, 100 mM) was added to quench the reaction. Finally, the mixture was purified using a Zeba Spin Desalt Column desalting column to obtain ERH001-CPT01 / ERH001-CPT02.

[0109] 3.4 Synthesis of ERH001-DXd-1(DAR4)

[0110] The toxin linker for ERH001-DXd-1 is a commercially available product (compound 9), with the following structure:

[0111] The antibody (ERH001) was prepared to a concentration of 7-10 mg / ml with phosphate buffer at pH 6.8-7.2. 3.8 eq of TCEP (10 mM) was added, and the mixture was reduced at 4°C for 15-16 h. Compound 9 (7.0 eq, 100 mM) was added for a coupling reaction for 2-3 h. Then, N-acetylcysteine ​​(14 eq, 100 mM) was added to quench the reaction. Finally, the mixture was purified using a Zeba Spin Desalt Colum desalting column to obtain ERH001-DXd-1.

[0112] 3.4 Synthesis of ERH001-DXd-2(DAR8)

[0113] The antibody (ERH001) was prepared to 10 mg / ml with phosphate buffer at pH 6.8-7.2. 6.0 eq of TCEP (10 mM) was added, and the mixture was reduced at 37 °C for 2-3 h. The mixture was then cooled to 25 °C and compound 9 (12.0 eq, 100 mM) was added for coupling reaction for 2-3 h. The reaction was then quenched by adding N-acetylcysteine ​​(14 eq, 100 mM). Finally, the mixture was purified using a Zeba Spin Desalt Colum desalting column to obtain ERH001-DXd-2.

[0114] Example 4: In vitro antitumor efficacy study of anti-EGFR antibody-drug conjugates

[0115] 1. Test substance

[0116] Test sample 1: ERH001-CPT01, DAR4.39(RP)

[0117] Test sample 2: ERH001-DXd-1, DAR 4.05 (RP);

[0118] Test sample 3: ERH001-CPT02, DAR4.27(RP);

[0119] Positive control: Paclitaxel injection, concentration 6.0 mg / mL.

[0120] 2. Cells

[0121] Four types of EGFR-positive target cells and human colorectal adenocarcinoma cells (SW620) were selected: human lung adenocarcinoma cells (NCI-H292), human non-small cell lung cancer cells (HCC827), human glioblastoma cells (LN-229-EGFRvⅢ-4A7-2H8), and human pharyngeal carcinoma pleural effusion metastatic cells (Detroit526).

[0122] 3. Experimental Procedure

[0123] 3.1 Cell preparation: Collect cells in good growth condition, centrifuge at 1000 rpm for 5 min, resuspend in complete culture medium, count the cells, and dilute the cell density to 1×10⁻⁶. 6 per mL.

[0124] 3.2 Cell Plating: 180 μL / well of cell suspension was seeded into the drug-treated wells and control wells of a 96-well plate (Experiment 1: 3000 cells / well for NCI-H292 and HCC827, 2000 cells / well for LN229-EGFRvⅢ-4A7-2H8 and SW620 cells; Experiment 2: 6000 cells / well for NCI-H292 and HCC827, 6000 cells / well for Detroit526 cells). 200 μL / well of complete culture medium was added to the zeroing wells, and 300 μL / well of PBS was added to the edge 36 wells. The plates were incubated statically at 37°C in a 5% CO2 incubator for 12–24 h.

[0125] 3.3 Sample Dilution: Eight concentration gradients of samples were prepared using complete culture medium. In Experiment 1, for all test cells, the initial concentration of the test substances ERH001-CPT01 and ERH001-DXd-1 was 100 μg / mL, with a dilution factor of 5; the initial concentration of the test substance Paclitaxel was 6 μg / mL, with a dilution factor of 5. In Experiment 2, for HCC827 and Detroit526 cells, the initial concentrations of the test substances ERH001-CPT02 and ERH001-DXd-1 were 100 μg / mL, with a dilution factor of 5. The initial concentration of H001-DXd-1 was 100 μg / mL, with a dilution factor of 5. The initial concentration of the test substance Paclitaxel was 6 μg / mL, with a dilution factor of 5. In NCI-H292 cells, the initial concentration of the test substances ERH001-CPT02 and ERH001-DXd-1 was 20 μg / mL, with a dilution factor of 5. The initial concentration of the test substance Paclitaxel was 6 μg / mL, with a dilution factor of 5.

[0126] 3.4 Sample incubation: Add 20 μL of sample diluent to each well, with 3 replicates per dose, and add 20 μL of complete culture medium to each control well; Experiment 1: Incubate at 37℃ in a 5% or 0% CO2 incubator for 144±6 h; Experiment 2: Incubate at 37℃ in a CO2 incubator for 96 h.

[0127] 3.5 Color development: Add 20 μL / well of CCK8 to the drug addition well, control well and zeroing well; incubate at 37℃ in a carbon dioxide incubator for 1.5 h.

[0128] 3.6 Reading: Remove the cell culture plate and place it in a microplate reader. Read the OD value at 450nm / 650nm (absorption wavelength / reference wavelength).

[0129] 3.7 Data Analysis: The cell survival rate (Survival%) and proliferation inhibition rate (Inhibition%) at each sample concentration were calculated; the dose-response relationship of the samples was analyzed using GraphPad Prism software according to log(inhibitor) vs. response – Variable slope (four parameters). Inhibition%=100%-Survival%

[0130] 4. Test Results

[0131] Results of Experiment 1:

[0132] The inhibitory effects of ERH001-CPT01, ERH001-DXd-1, and Paclitaxel on the proliferation of EGFR-positive cells NCI-H292, HCC827, LN-229-EGFRvⅢ-4A7-2H8, and EGFR-negative cells SW620 are shown in Table 8.

[0133] Table 8. Proliferation inhibition results of ERH001-CPT01, ERH001-DXd, and Paclitaxel in various cell lines.

[0134] The experimental results showed that the IC50 values ​​of ERH001-CPT01 and ERH001-DXd-1 against the HCC827 cell line were 0.10 nM and 0.04 nM, respectively, which were superior to Paclitaxel's 1.04 nM. The IC50 values ​​of ERH001-CPT01 and ERH001-DXd-1 against the LN-229-EGFR-4A7-2H8 cell line were 0.19 nM and 0.04 nM, respectively, which were superior to Paclitaxel's 1.60 nM. In other words, the in vitro killing effects of ERH001-CPT01 and ERH001-DXd-1 on HCC827 and LN-229-EGFR-4A7-2H8 cells were similar, and superior to Paclitaxel's in vitro killing effects on HCC827 and LN-229-EGFR-4A7-2H8 cells.

[0135] In EGFR-negative target cells SW620, neither ERH001-CPT01 nor ERH001-DXd-1 had a significant effect on cell proliferation inhibition, indicating that the inhibitory effects of ERH001-CPT01 and ERH001-DXd on cell proliferation are target-dependent.

[0136] Results of Experiment 2:

[0137] The inhibitory effects of ERH001-CPT02, ERH001-DXd-1, and Paclitaxel on the proliferation of EGFR-positive cells NCI-H292, HCC827, and Detroit526 are shown in Table 9.

[0138] Table 9. Proliferation inhibition results of ERH001-CPT02, ERH001-DXd, and Paclitaxel in various cell lines.

[0139] The experimental results showed that the IC50 values ​​of ERH001-CPT02 and ERH001-DXd-1 against the NCI-H292 cell line were 0.068 nM and 0.070 nM, respectively, which were superior to Paclitaxel's 1.991 nM. The IC50 values ​​of ERH001-CPT02 and ERH001-DXd-1 against the HCC827 cell line were both 0.012 nM, which were superior to Paclitaxel's 0.095 nM. The in vitro killing effects of ERH001-CPT02 and ERH001-DXd-1 on NCI-H292 and HCC827 cells were superior to Paclitaxel.

[0140] Combining the results of Experiment 1 and Experiment 2, ERH001-CPT01, ERH001-CPT02, and ERH001-DXd-1 showed similar in vitro killing effects, all demonstrating excellent in vitro tumor cell killing activity.

[0141] Example 5: Intra-in vivo pharmacodynamic study of anti-EGFR antibody drug conjugates

[0142] Experiment 1: Efficacy study of anti-EGFR antibody-drug conjugate against human lung adenocarcinoma cell line NCI-H292 xenograft tumor

[0143] The experiment used a human lung adenocarcinoma cell line NCI-H292 BALB / cNude mouse xenograft model. NCI-H292 tumor cells were subcutaneously inoculated into the right side of nude mice at a concentration of 5 × 10⁻⁶ cells / mL. 6 Cells / mouse, tumors grew to an average size of 150 mm 3 Animals were randomly divided into 5 groups of 5 animals each. Each group received a single intravenous injection of sodium chloride injection (Saline), or ERH001-CPT01 (DAR3.8) at doses of 1 mg / kg, 2 mg / kg, 4 mg / kg, and 8 mg / kg, respectively. The day of grouping was considered Day 1. During the experiment, animal weight and tumor size were measured twice weekly, and clinical symptoms were observed and recorded. When the average tumor volume in the negative control group exceeded 1000 mmHg... 3 The experiment was terminated by euthanizing the mice at the designated time.

[0144] Data Analysis: Data from each group on the day of euthanasia in the negative control group were used to calculate relevant indicators. The evaluation index for antitumor activity was the relative tumor proliferation rate (T / C%). A T / C% > 40% was considered ineffective, and a T / C% ≤ 40% was considered effective after statistical processing (p < 0.05). The formula for calculating T / C% is:

[0145] T / C(%)=(TRTV / CRTV)×100%.

[0146] T RTV For the treatment group RTV, C RTV RTV was used as the negative control group.

[0147] RTV: Relative Tumor Volume. The RTV of each tumor is the ratio of the tumor volume at the time of measurement to the tumor volume at the initial state of the experiment.

[0148] TGI tumor inhibition rate (%) = (mean tumor volume or weight in negative control group - mean tumor volume or weight in treatment group) / mean tumor volume or weight in negative control group × 100%.

[0149] The weight change (%) of tumor-bearing animals was calculated as follows: (weight at measurement - weight at grouping) / weight at grouping × 100%.

[0150] Experiment 2: Efficacy study of anti-EGFR antibody-drug conjugate against human glioma LN-229-4A7-2H8 xenograft tumor

[0151] The experiment used a human glioma LN-229-4A7-2H8 BALB / cNude mouse subcutaneous xenograft model. LN-229-4A7-2H8 tumor cells were subcutaneously inoculated into the right side of nude mice at a concentration of 5 × 10⁶ cells / mL. 6 Cells / mouse, tumor growth to 100-300 mm 3 Animals were randomly divided into 6 groups of 5 animals each. Each group received a single intravenous injection of sodium chloride injection (Saline), at doses of 1.5 mg / kg, 3 mg / kg, 6 mg / kg ERH001-CPT01 (DAR 4.39), 6 mg / kg ERH001-DXd-1 (DAR 4.05), and 6 mg / kg ERH001-DXd-2 (DAR 7.65). The day of group assignment was considered Day 1. During the experiment, animal weight and tumor size were measured twice weekly, and clinical symptoms were observed and recorded. When the average tumor volume in the negative control group exceeded 1000 mmHg... 3 At that point, the mice were euthanized to end the experiment. Data analysis was the same as in Experiment 1.

[0152] Experiment 3: Efficacy study of anti-EGFR antibody-drug conjugate against human non-small cell lung cancer HCC827 xenograft tumor

[0153] The experiment used a human non-small cell lung cancer HCC827 BALB / cNude mouse subcutaneous xenograft model. HCC827 tumor cells were subcutaneously inoculated into the right side of nude mice at a concentration of 3 × 10⁻⁶ cells / mL. 6 Cells / mouse, tumor growth to a size of 100-300 mm 3 Suitable tumors were selected and grouped into 6 groups, with 5 animals in each group. Animals received a single intravenous injection of PBS, 2 mg / kg, 4 mg / kg, 8 mg / kg ERH001-CPT01 (DAR 4.39), 8 mg / kg ERH001-DXd-1 (DAR 4.05), and 8 mg / kg ERH001-DXd-2 (DAR 7.22), respectively. The day of grouping was considered Day 1. During the experiment, animal weight and tumor size were measured twice weekly, and clinical symptoms were observed and recorded. When the average tumor volume in the negative control group exceeded 1000 mmHg... 3 At that point, the mice were euthanized to end the experiment. Data analysis was the same as in Experiment 1.

[0154] Experiment 4: Efficacy study of anti-EGFR antibody-drug conjugate ERH001-CPT02 against human glioma LN-229-4A7-2H8 xenograft tumor

[0155] The experiment used a human glioma LN-229-4A7-2H8 BALB / cNude mouse subcutaneous xenograft model. LN-229-4A7-2H8 tumor cells were subcutaneously inoculated into the right side of nude mice at a concentration of 1×10⁻⁶. 7 Cells / mouse, tumor growth to a size of 100-300 mm 3 Suitable tumors were selected and randomly divided into 4 groups of 4 animals each. Each group received a single intravenous injection of PBS at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg ERH001-CPT02 (DAR4.27). The day of grouping was considered Day 1. During the experiment, animal weight and tumor size were measured twice weekly, and clinical symptoms were observed and recorded. When the average tumor volume in the negative control group exceeded 2000 mmHg... 3 At that point, the mice were euthanized to end the experiment. Data analysis was the same as in Experiment 1.

[0156] Experiment 5: Efficacy study of anti-EGFR antibody-drug conjugates ERH001-CPT02 and ERH001-DXd against human lung cancer cell NCI-H292 xenograft tumors.

[0157] The experiment used a subcutaneous xenograft tumor model of human lung cancer cells NCI-H292 BALB / cNude mice. NCI-H292 tumor cells were subcutaneously inoculated into the right side of nude mice at a concentration of 1×10⁻⁶ cells. 7 Cells / mouse, tumor volume reaches 100-300 mm 3 Suitable tumors were selected and randomly divided into 3 groups of 5 animals each. Animals were administered PBS, 3 mg / kg ERH001-CPT02 (DAR 4.27), or 3 mg / kg ERH001-DXd (DAR 6.82) via single intravenous injection. The day of grouping was considered Day 1. Animal weight and tumor size were measured twice weekly during the experiment. Animals were euthanized at the end of the experiment. Data analysis was the same as in Experiment 1.

[0158] Experimental results:

[0159] 1. Effects of the test substance on animal body weight

[0160] Referring to Figures 2, 4, 6, 8, and 10, in this in vivo study, the mice in all experiments tolerated the anti-EGFR antibody-drug conjugate of this invention well. No significant weight loss or death in the animals due to drug toxicity was observed.

[0161] 2. Effects of the test substance on tumor growth in animals

[0162] In Experiment 1, as shown in Figure 1, the average tumor volume of mice in the solvent control group on Day 22 was 1334.27 ± 61.08 mm. 3 The mean tumor volume on Day 22 in the treatment groups of the test substance ERH001-CPT01 (1 mg / kg, 2 mg / kg, 4 mg / kg and 8 mg / kg) was 967.27 ± 26.03 mm. 3 452.88±71.58mm 3 20.5±5.96mm 3 4.26±1.95mm 3 The T / C (%) values ​​were 76.0, 34.5, 1.2, and 0.3, respectively, all of which were statistically significant compared to the control group (p<0.01, p<0.001, p<0.001, and p<0.001, respectively). The tumor inhibition rates (TGI) were 34.5%, 66.06%, 98.46%, and 99.68%, respectively. The tumor growth curves showed that the inhibition of tumor growth by each dose group was dose-dependent. The 4 and 8 mg / kg ERH001-CPT01 showed significant anti-tumor efficacy, with tumors in all animals nearly disappearing or disappearing.

[0163] In Experiment 2, as shown in Figure 3, the mean tumor volume of the negative control group Saline mice on Day 25 was 1196.94 ± 288.74 mm.3 The mean tumor volume on Day 25 was 556.51 ± 47.77 mm in the test substance ERH001-CPT01 groups at concentrations of 1.5 mg / kg, 3 mg / kg, and 6 mg / kg. 3 240.37±41.91mm 3 and 39.69±13.62mm 3 The relative tumor proliferation rates (T / C%) of the low, medium, and high dose groups of ERH001-CPT01 were 46.1%, 19.8%, and 3.0%, respectively, showing a significant dose-dependent relationship (p<0.05 between groups). At the end of the experiment, the TGI (tumor genicity percentage) calculated based on tumor weight were 35.3%, 78.6%, and 97.4%, respectively. The mean tumor volume on Day 25 in the 6 mg / kg ERH001-DXd-1 and ERH001-DXd-2 groups was 15.4 ± 4.32 mm. 3 and 32±26.54mm 3 The relative tumor proliferation rates (T / C%) were 3.5% and 5.5%, respectively. After the experiment, the total tumor growth index (TGI) calculated based on tumor weight was 98.08% and 96.02%, respectively. Compared with ERH001-DXd-1 and ERH001-DXd-2, at a dose of 6 mg / kg, the antitumor effects of ERH001-CPT01, ERH001-DXd-1, and ERH001-DXd-2 were comparable, with no significant difference observed.

[0164] In Experiment 3, as shown in Figure 5, the average tumor volume of the negative control group PBS mice on Day 29 was 1024.78 ± 102.59 mm. 3 The mean tumor volume on Day 29 was 384.42 ± 87.81 mm in the test substance ERH001-CPT01 groups at 2 mg / kg, 4 mg / kg, and 8 mg / kg. 3 111.72±44.86mm 3 and 35.26±5.85mm 3 The differences between the two groups and the negative control group were statistically significant (p<0.01). On Day 29, the relative tumor proliferation rates (T / C%) of the low, medium, and high dose groups of ERH001-CPT01 were 39.08%, 12.46%, and 3.69%, respectively, showing a clear dose-dependent relationship. After the experiment, the TGI (%) calculated based on tumor weight were 65.55%, 89.69%, and 97.52%, respectively. The mean tumor volume of ERH001-DXd-1 and ERH001-DXd-2 on Day 29 was 32.69±1.78 mm. 3 and 41.69±15.72mm 3The relative tumor proliferation rates (T / C%) were 3.54% and 4.00%, respectively. After the experiment, the total tumor growth index (TGI) calculated based on tumor weight was 97.42% for both. Compared with ERH001-DXd at different DAR values, at a dose of 8 mg / kg, the antitumor effects of ERH001-CPT01 and ERH001-DXd were comparable, with no significant difference observed.

[0165] In Experiment 4, as shown in Figure 7, the average tumor volume of the negative control mice on Day 25 was 1212.3 ± 425.3 mm. 3 The mean tumor volume on Day 25 in the treatment groups of the test substance ERH001-CPT02 at 1 mg / kg, 2 mg / kg, and 4 mg / kg was 661.0 ± 206.9 mm. 3 501.7±182.2mm 3 and 109.8±83.5mm 3 The relative tumor proliferation rates (T / C%) of the low, medium, and high dose groups of ERH001-CPT02 were 52.98%, 40.25%, and 9.50%, respectively, showing a significant dose-dependent relationship (p<0.05 between groups). At the end of the experiment, the TGI (Transcription Gibbs Intake) calculated based on tumor weight were 39.06%, 57.10%, and 89.33%, respectively. The ERH001-CPT02 at 4 mg / kg showed a highly significant difference compared to the control group.

[0166] In Experiment 5, as shown in Figure 9, Day 19, the average tumor volume in the negative control group mice was 1596.4 ± 492.9 mm. 3 The mean tumor volume in the 3 mg / kg ERH001-CPT02 and 3 mg / kg ERH001-DXd treatment groups was 9.8 ± 7.5 mm. 3 238.2±354.6mm 3 The relative tumor proliferation rates (T / C%) were 0.61% and 16.74%, respectively. After the experiment, the tumor growth index (TGI) calculated based on tumor volume was 99.39% and 85.08%, respectively. The results showed that both ERH001-CPT02 and ERH001-DXd at 3 mg / kg could effectively inhibit tumor growth, and ERH001-CPT02 had a better antitumor effect than ERH001-DXd.

[0167] The T / C and TGI for each experimental drug group are shown in Table 10-12:

[0168] Table 10. T / C and TGI in each treatment group of the NCI-H292 model.

[0169] Table 11. T / C and TGI of each treatment group in the LN-229-4A7-2H8 model

[0170] Table 12. T / C and TGI of each treatment group in the HCC827 model.

[0171] In summary, the anti-EGFR antibody-drug conjugates ERH001-CPT01, ERH001-CPT02, ERH001-DXd-1, and ERH001-DXd-2 of this invention all exhibit excellent in vivo antitumor activity, with similar antitumor effects. The antitumor effects of ERH001-CPT01 and ERH001-CPT02 are dose-dependent.

[0172] Example 6: Acute toxicity study of anti-EGFR antibody-drug conjugate in mice

[0173] Experiment 1: Acute toxicity of ICR mice was evaluated by single intravenous injection of 200 mg / kg ERH001-CPT01, ERH001-DXd-1, and ERH001-DXd-2.

[0174] Six-week-old female ICR mice, weighing 25-28.6g, were used in the experiment. The test substances were ERH001-CPT01, ERH001-DXd-1, and ERH001-DXd-2, with DAR values ​​of 4.01, 4.07, and 7.72, respectively. PBS was used as a control. The dosage was 200mg / kg. The experiment lasted for 12 days (including the day of administration). The body weight was measured daily. Blood was collected on days 5 and 12, and mice 1-3 and 4-6 in each group were dissected to detect serum biochemical indicators and blood cell counts. Heart, liver, spleen, lung, kidney, and brain were collected, and the liver-to-brain ratio was calculated.

[0175] Experiment 2: Acute toxicity of ICR mice was evaluated by single intravenous injection of 300 mg / kg ERH001-CPT02 and ERH001-DXd.

[0176] The experiment used 5-8 week old female ICR mice, weighing 23-27g. The test substances included ERH001-CPT02 and ERH001-DXd with DAR values ​​of 3.79 and 6.45, respectively. PBS was used as a control. The dosage was 300mg / kg. Serum biochemical indicators and blood cell counts were detected on DAY5 and DAY15. Heart, liver, spleen, lung, kidney and brain were collected and the liver-brain ratio was calculated.

[0177] Experimental results:

[0178] 1. Effects of the test substance on animal body weight

[0179] The weight loss in each treatment group was small, with the maximum decrease being less than 5%, and no significant effects related to the test substance were observed.

[0180] 2. Effects of the test substance on blood biochemical parameters

[0181] In Experiment 1, no significant differences were observed in blood biochemical indicators between the drug-treated groups and the negative control group.

[0182] In Experiment 2, on day 15, the GGT level of DEA001-DXd was 2.5 times that of the negative control group, which was statistically significant. The GGT level of ERH001-CPT02 was 1.0 times that of the negative control group, which was not statistically significant. There were no statistically significant differences in other serum biochemical indicators among all treatment groups.

[0183] 3. Effect of the test substance on blood cell count

[0184] In Experiment 1, on day 12, 200 mg / kg ERH001-CPT01 showed lower inhibition of RET than DEA001-DXd-2; it had almost no inhibitory effect on HCT%, which was superior to DEA001-DXd-1 and DEA001-DXd-2; ERH001-CPT01 showed less inhibition of WBC, LYMPH, and EO than DEA001-DXd-1 and DEA001-DXd-2. No statistically significant differences were found in other indicators.

[0185] In Experiment 2, no significant differences in blood cell counts were observed between the drug-treated groups and the negative control group.

[0186] In summary, the maximum tolerated dose of ERH001-CPT01, ERH001-DXd-1, and ERH001-DXd-2 is not less than 200 mg / kg, and the maximum tolerated dose of ERH001-CPT02 is not less than 300 mg / kg.

[0187] At the same dosage, ERH001-CPT01 (DAR = 4.07) is less toxic than ERH001-DXd-1 (DAR = 4.01) and less toxic than ERH001-DXd-2 (DAR = 7.72). The toxicity response at a dose of 200 mg / kg is sufficient for clinical application; therefore, ERH001-CPT01 has a better safety profile than DEA001-DXd-1 and DEA001-DXd-2.

[0188] At the same dosage, ERH001-CPT02 (DAR = 3.79) is less toxic than ERH001-DXd (DAR = 6.45). The toxicity response at a dose of 300 mg / kg is sufficient for clinical application; therefore, ERH001-CPT02 is safer than DEA001-DXd.

[0189] The main change indicators are shown in Table 13:

[0190] Table 13 Key Changes in Acute Poisoning Tests

[0191] The acute toxicity test showed that the anti-EGFR antibody-drug conjugate of the present invention has excellent safety, and the safety of ERH001-CPT01 and ERH001-CPT02 is better than that of the ERH001-DXd control group.

[0192] In summary, this invention provides a novel anti-EGFR antibody-drug conjugate that reduces the ADCC effect without affecting antibody affinity through antibody engineering.

[0193] In vitro and in vivo efficacy tests and acute toxicity tests show that the anti-EGFR antibody drug conjugate of the present invention has excellent efficacy and safety. In particular, ERH001-CPT01 has low toxicity and optimal safety, and has excellent drug development potential.

[0194] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

Claims

1. An anti-EGFR antibody-drug conjugate, characterized in that, The anti-EGFR antibody drug conjugate has the following formula (I) structure: Formula (Ⅰ), where mAb represents an anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4; R is H or halogen.

2. The anti-EGFR antibody drug conjugate of claim 1, wherein, R is either H or F.

3. The anti-EGFR antibody-drug conjugate according to claim 1, characterized in that, The anti-EGFR antibody drug conjugate has the following formula (II) structure: Formula (II), where mAb represents anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4.

4. The anti-EGFR antibody-drug conjugate according to claim 1, characterized in that, The anti-EGFR antibody drug conjugate has the following formula (III) structure: Formula (Ⅲ), where mAb represents anti-EGFR antibody, n is 1-4, preferably n is 2-4, and even more preferably n is 4.

5. An anti-EGFR antibody-drug conjugate, characterized in that, The anti-EGFR antibody drug conjugate has the structure (IV) as shown below: Formula (Ⅳ), where mAb represents an anti-EGFR antibody; m is 1-8, preferably m is 4 or 8, and more preferably m is 8.

6. The anti-EGFR antibody drug conjugate of any one of claims 1-5, wherein, The anti-EGFR antibodies mentioned are cetuximab, panitumumab, necitumumab, nimotuzumab, matuzumab, amivantamab, demupitamab, depatuxizumab, futuximab, imatrozumab, and latoxa. Laprituximab, Losatuxizumab, Zatuximab, Pimurutamab, Serclutamab, Tomuzotuximab, Zalutumumab or their biosimilars, preferably Cetuximab or its biosimilars, and preferably Necitumumab or its biosimilars.

7. The anti-EGFR antibody drug conjugate of any one of claims 1-5, wherein, The DAR of the anti-EGFR antibody-drug conjugate is 2-8, preferably 4±0.5, 6±0.5 or 8±0.

5.

8. The anti-EGFR antibody drug conjugate of claim 1 or 5, wherein, The anti-EGFR antibody has the heavy chain CDR1 as described in SEQ ID NO:1, the heavy chain CDR2 as described in SEQ ID NO:2, the heavy chain CDR3 as described in SEQ ID NO:3, the light chain CDR1 as described in SEQ ID NO:4, the light chain CDR2 as described in SEQ ID NO:5, and the light chain CDR3 as described in SEQ ID NO:

6.

9. The anti-EGFR antibody drug conjugate of claim 8, wherein, The anti-EGFR antibody has a heavy chain variable region as described in SEQ ID NO:7 and a light chain variable region as described in SEQ ID NO:

8.

10. The anti-EGFR antibody drug conjugate of claim 1 or 5, wherein, The anti-EGFR antibody has the heavy chain CDR1 as described in SEQ ID NO:14, the heavy chain CDR2 as described in SEQ ID NO:15, the heavy chain CDR3 as described in SEQ ID NO:16, the light chain CDR1 as described in SEQ ID NO:17, the light chain CDR2 as described in SEQ ID NO:18, and the light chain CDR3 as described in SEQ ID NO:

19.

11. The anti-EGFR antibody drug conjugate of claim 10, wherein, The anti-EGFR antibody has a heavy chain variable region as described in SEQ ID NO:20 and a light chain variable region as described in SEQ ID NO:

21.

12. The anti-EGFR antibody drug conjugate of any one of claims 1-5, wherein, The Fc fragment of the anti-EGFR antibody has a reduced ADCC effect.

13. The anti-EGFR antibody drug conjugate of claim 12, wherein, The anti-EGFR antibody is an IgG1 subtype antibody, and has a reduced ADCC effect compared to wild-type IgG1.

14. The anti-EGFR antibody-drug conjugate as described in claim 13, characterized in that, The reduced ADCC effect is obtained through Fc mutation, which is a single point mutation, a two-site mutation, or a multi-site mutation, and the mutation sites are selected from P232-S239, D265-D270, Y296-T299, and K322-I332.

15. The anti-EGFR antibody-drug conjugate as described in claim 14, characterized in that, The Fc mutation is N297A mutation, L234A and L235A mutation, L234A, L235A and P329G mutation, L234A, L235A and K322A mutation, L234F, L235E and D265A mutation or L234F, L235E and P331S mutation.

16. The anti-EGFR antibody-drug conjugate as described in claim 15, characterized in that, The anti-EGFR antibody has the heavy chain CDR1 as described in SEQ ID NO:1, the heavy chain CDR2 as described in SEQ ID NO:2, the heavy chain CDR3 as described in SEQ ID NO:3, the light chain CDR1 as described in SEQ ID NO:4, the light chain CDR2 as described in SEQ ID NO:5, and the light chain CDR3 as described in SEQ ID NO:

6.

17. The anti-EGFR antibody-drug conjugate as described in claim 16, characterized in that, The anti-EGFR antibody has a heavy chain variable region as described in SEQ ID NO:7 and a light chain variable region as described in SEQ ID NO:

8.

18. The anti-EGFR antibody-drug conjugate as described in claim 17, characterized in that, The anti-EGFR antibody has a heavy chain as described in SEQ ID NO:11 and a light chain as described in SEQ ID NO:

13.

19. The anti-EGFR antibody-drug conjugate as described in claim 17, characterized in that, The anti-EGFR antibody has a heavy chain as described in SEQ ID NO:12 and a light chain as described in SEQ ID NO:

13.

20. The anti-EGFR antibody-drug conjugate as described in claim 15, characterized in that, The anti-EGFR antibody has the heavy chain CDR1 as described in SEQ ID NO:14, the heavy chain CDR2 as described in SEQ ID NO:15, the heavy chain CDR3 as described in SEQ ID NO:16, the light chain CDR1 as described in SEQ ID NO:17, the light chain CDR2 as described in SEQ ID NO:18, and the light chain CDR3 as described in SEQ ID NO:

19.

21. The anti-EGFR antibody-drug conjugate as described in claim 20, characterized in that, The anti-EGFR antibody has a heavy chain variable region as described in SEQ ID NO:20 and a light chain variable region as described in SEQ ID NO:

21.

22. The anti-EGFR antibody-drug conjugate as described in claim 20, characterized in that, The anti-EGFR antibody has a heavy chain as described in SEQ ID NO:24 and a light chain as described in SEQ ID NO:

26.

23. The anti-EGFR antibody-drug conjugate as described in claim 20, characterized in that, The anti-EGFR antibody has a heavy chain as described in SEQ ID NO:25 and a light chain as described in SEQ ID NO:

26.

24. The use of an anti-EGFR antibody-drug conjugate as described in any one of claims 1-23 in the preparation of a medicament for treating diseases, characterized in that, The diseases mentioned include colorectal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, pancreatic cancer, glioma, breast cancer, and esophageal cancer.

25. The use as described in claim 24, characterized in that, The disease in question is lung adenocarcinoma.

26. The use as described in claim 24, characterized in that, The disease in question is non-small cell lung cancer.

27. The use as described in claim 24, characterized in that, The disease in question is glioma.

28. The use as described in claim 24, characterized in that, The disease is metastatic colorectal cancer.

29. A method for treating a disease, characterized in that, Administering an effective dose of the anti-EGFR antibody-drug conjugate as described in claim 1 or 5 to a subject, wherein the disease includes colorectal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, pancreatic cancer, glioma, breast cancer, and esophageal cancer.

30. The method as described in claim 29, characterized in that, The disease in question is lung adenocarcinoma.

31. The method as described in claim 29, characterized in that, The disease in question is non-small cell lung cancer.

32. The method as described in claim 29, characterized in that, The disease in question is glioma.

33. The method as described in claim 29, characterized in that, The disease is metastatic colorectal cancer.

34. A non-therapeutic method for inhibiting tumor cells, characterized in that, Add an effective dose of the anti-EGFR antibody-drug conjugate as described in claim 1 or 5 to a tumor cell system, wherein the tumor cells include colorectal cancer cells, head and neck cancer cells, lung cancer cells, nasopharyngeal cancer cells, pancreatic cancer cells, glioma cells, breast cancer cells, and esophageal cancer cells.

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