Anti-epidermal growth factor receptor (EGFR) antibody-drug conjugates and their applications

Abstract

This invention relates to antibody-drug conjugates (ADCs) and their applications. Specifically, it concerns novel anti-epidermal growth factor receptor antibody-drug conjugates, and methods and applications for treating diseases or conditions related to EGFR.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. CN202310519224.2, filed on 9 May 2023, and Chinese Patent Application No. CN202311314455.6, filed on 10 October 2023.

[0002] This application belongs to the pharmaceutical field and relates to antibody-drug conjugates (ADCs) and their uses. Specifically, this application relates to novel anti-epidermal growth factor receptor antibody-drug conjugates and methods and uses for treating diseases or conditions related to EGFR. [Background technology]

[0003] The human epidermal growth factor receptor (also known as HER-1 or Erb-B1, and referred to herein as "EGFR") is a 170 kDa transmembrane receptor encoded by the c-erbB oncogene, exhibiting intrinsic tyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996), Herbst and Shin, Cancer 94:1593-1611 (2002)). EGFR is a member of the epidermal growth factor receptor family and plays a crucial role in cell growth, development, and differentiation. EGFR regulates various cellular processes through tyrosine kinase-mediated signaling pathways, including, but not limited to, the activation of signaling pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitosis, and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003), Tsao and Herbst, Signal 4:4-9 (2003), Herbst and Shin, Cancer 94:1593-1611 (2002), Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)).

[0004] EGFR is highly expressed in various solid tumors, and many well-known drugs targeting EGFR, such as osimertinib, have been developed over the past thirty years. However, small molecule therapies often cause mutations in the target gene, leading to treatment failure. Therefore, there is a constant need for the development of next-generation EGFR inhibitor small molecule drugs that can counteract mutations. Among antibody drugs, cetuximab was approved in 2004 and is effective against colorectal cancer, nasopharyngeal cancer, and other cancers. However, cetuximab also needs to be used in combination with chemotherapy to enhance its therapeutic effect.

[0005] Therefore, in order to further improve therapeutic effects and reduce side effects, this field urgently needs humanized anti-epidermal growth factor receptor drugs with higher biological activity, particularly drugs that are effective in treating patients with KRAS mutations or TKI resistance, such as antibody-drug conjugates. In the development of macromolecular drugs targeting EGFR, an important kinase target, novel drug molecules with different anticancer mechanisms of action have been demonstrated. Antibody-drug conjugates targeting EGFR are one of the hot spots for the development of next-generation targeted drugs. Antibody-drug conjugates (ADCs) consist of a monoclonal antibody (Antibody) that targets a tumor-specific antigen or tumor-associated antigen and a small molecule toxin (Payload) with high anticancer activity, conjugated via a linker. Antibody-drug conjugates combine the advantages of both the high targetability of monoclonal antibody drugs and the high activity of cytotoxins in tumor tissue, synergistically leveraging the benefits of antibody drugs and small molecule drug loading, thereby making it easier to kill tumor cells more efficiently and specifically. Compared to monotherapy drugs, antibody-drug conjugates have lower toxic side effects and exhibit better therapeutic efficacy compared to conventional antibody-based oncosuria drugs. [Overview of the Initiative]

[0006] This application relates to an antibody-drug conjugate of formula (I) as defined herein and a pharmaceutical composition comprising the antibody-drug conjugate. A feature of this application is a method for treating or preventing an EGFR-related disease or condition, comprising administering to a subject in need a therapeutically effective amount of an antibody-drug conjugate of formula (I) as defined herein, its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt. The method of this application can be used to treat or prevent an EGFR-related disease or condition.

[0007] A first aspect of this application relates to an antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, wherein the antibody-drug conjugate has the structure of formula (I), TP-[L1-L2-L3-D] k (I) Here, TP is a target region that selectively binds to or recognizes EGFR. L1 is an extension unit that connects TP and L2. L2 is a short-chain peptide consisting of an optional amino acid residue or 2 to 10 amino acid residues. L3 is a spacer element, D is a bioactive molecule, k represents any number between approximately 0.1 and approximately 10.0.

[0008] Another aspect of this application relates to a pharmaceutical composition comprising an antibody-drug conjugate of formula (I), a prodrug thereof, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0009] Another aspect of this application relates to a method for treating EGFR-related diseases or conditions. The method comprises administering to a subject in need an antibody-drug conjugate of formula (I), its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt or a pharmaceutical composition containing the same. The EGFR-related diseases or conditions are selected from epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma.

[0010] Another aspect of this application relates to the use of an antibody-drug conjugate of formula (I), its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition containing the same, in the manufacture of a drug.

[0011] This application further provides antibody-drug conjugates and compositions having improved efficacy and / or antitumor activity compared to known prior art anti-EGFR-ADCs.

[0012] Details of this application are described in the attached description below. Similar or equivalent methods and materials described herein may be used in the practice or testing of this application, but only exemplary methods and materials are described here. Other features, purposes and advantages of this application will become apparent from the specification and claims. In the specification and the attached claims, singular nouns include plural nouns unless otherwise explicitly specified in the context. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. All patents and publications incorporated herein by reference are incorporated herein by reference in their entirety.

[0013] The contents of all references invoked throughout this application (including references to references to references to references to references to references to references to patents, granted patents, disclosed patent applications and co-pending patent applications) are incorporated herein by express reference as a whole. Unless otherwise defined, all technical and scientific terms used herein are to have the meaning commonly known to those skilled in the art. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the affinity of anti-EGFR antibody-drug conjugates to EGFR-positive cells A549 and SW480. (A) Affinity of anti-EGFR antibody-drug conjugate MAB07-LP62 (DAR4 or DAR8) to A549 cells. (B) Affinity of anti-EGFR antibody-drug conjugate MAB07-LP62 (DAR8) to SW480 cells. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, DAR represents the drug-to-antibody ratio, and IgG1 represents human IgG1, which is the κ isotype control. [Figure 2] This figure shows that the anti-EGFR antibody-drug conjugate is internalized by EGFR-positive cells A549 as incubation time is extended. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, DAR represents the loading drug:antibody ratio, and IgG1 represents human IgG1, which is the κ isotype control. [Figure 3]This figure shows the cytotoxic activity of the anti-EGFR antibody-drug conjugate MAB07-LP62 (DAR8). (A) Cytotoxic activity of MAB07-LP62 (DAR8) against the NCI-H1993 cell line. (B) Cytotoxic activity of MAB07-LP62 (DAR8) against the EBC-1 cell line. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, and DAR represents the loading drug:antibody ratio. [Figure 4] This figure shows the cross-binding activity of anti-EGFR antibody-drug conjugates against EGFR from different species. (A) Binding of MAB07-LP62 (DAR8) to human EGFR ECD. (B) Binding of MAB07-LP62 (DAR8) to cynomolgus monkey EGFR ECD. (C) Binding of MAB07-LP62 (DAR8) to rat EGFR ECD. (D) Binding of MAB07-LP62 (DAR8) to mouse EGFR ECD. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, DAR represents the loading drug:antibody ratio, and IgG1 represents human IgG1, which is the κ isotype control. [Figure 5] This figure shows the antitumor activity of anti-EGFR antibody-drug conjugates in a subcutaneous xenograft BALB / c nude mouse model of human epidermal carcinoma cell line A431. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing; MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62; cetuximab-LP62 represents the antibody-drug conjugate formed by the conjugation of cetuximab and LP62; IgG1-LP62 represents the antibody-drug conjugate formed by the conjugation of IgG1 and LP62; and DAR represents the loading drug:antibody ratio. [Figure 6]This figure shows the antitumor activity of anti-EGFR antibody-drug conjugates in a subcutaneous xenograft BALB / c nude mouse model of the human non-small cell lung cancer cell line NCI-H1993. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing; MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62; MAB07-VC-MMAE represents the antibody-drug conjugate formed by the conjugation of MAB07 and the linker-toxin MC-VC-PABC-MMAE; MAB07-GGFG-DXD represents the antibody-drug conjugate formed by the conjugation of MAB07 and the linker-toxin GGFG-DXD; A 1.3 represents the toxin compound A 1.3 in Example 1, which is the free loading drug, and DAR represents the loading drug:antibody ratio. [Figure 7] This figure shows the antitumor activity of anti-EGFR antibody-drug conjugates in a subcutaneous xenograft BALB / c nude mouse model of human non-small cell lung cancer cell line EBC-1. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing; MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62; MAB07-VC-MMAE represents the antibody-drug conjugate formed by the conjugation of MAB07 and the linker-toxin MC-VC-PABC-MMAE; MAB07-GGFG-DXD represents the antibody-drug conjugate formed by the conjugation of MAB07 and the linker-toxin GGFG-DXD; A 1.3 represents the toxin compound A 1.3 in Example 1, which is the free loading drug, and DAR represents the loading drug:antibody ratio. [Figure 8]This figure shows the antitumor effect of an anti-EGFR antibody-drug conjugate in a HuPrime® lung cancer LU3075 subcutaneous xenograft female BALB / c nude mouse model. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, and DAR represents the drug-to-antibody ratio. [Figure 9] This figure shows the antitumor effect of an anti-EGFR antibody-drug conjugate in an NCG mouse model of human lung cancer LD1-0025-200717 PDX transplanted tumor. Note: MAB07 represents the anti-EGFR antibody, and its amino acid sequence is listed in the sequence listing. MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62. Serplulimab is an anti-PD-1 antibody, and its amino acid sequence is listed in the sequence listing. IgG1 represents human IgG1, which is a κ isotype control. IgG1-LP62 represents the antibody-drug conjugate formed by the conjugation of IgG1 and LP62. The DAR value is 8, and DAR represents the drug-to-antibody load ratio. [Figure 10] This figure shows the antitumor activity of an anti-EGFR antibody-drug conjugate in a human colon cancer HT-29 cell allograft NCG mouse model using a human-brain-mediated immune system (PBMC). Note: MAB07 represents the anti-EGFR antibody, and its amino acid sequence is listed in the sequence listing. MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62. Serplulimab is an anti-PD-1 antibody, and its amino acid sequence is listed in the sequence listing. DAR represents the drug-to-antibody ratio. [Figure 11]This figure shows the antitumor effect of an anti-EGFR antibody-drug conjugate in a human head and neck squamous cell carcinoma LD1-2023-411020 PDX transplanted tumor NCG mouse model. Note: MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, and DAR represents the drug-to-antibody ratio. [Figure 12] This figure shows the antitumor activity of an anti-EGFR antibody-drug conjugate in a human gastric cancer LD1-0017-411335PDX transplanted tumor NU / NU mouse model. Note: MAB07 represents the anti-EGFR antibody, its amino acid sequence is listed in the sequence listing, MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62, A 1.3 represents the toxin compound A 1.3 in Example 1, which is the free loading drug, and DAR represents the loading drug:antibody ratio. [Figure 13] This figure shows the antitumor activity of an anti-EGFR antibody-drug conjugate in a subcutaneous xenograft NOD / SCID mouse model using human-derived esophageal cancer KYSE-150 cell lines. Note: IgG1 represents human IgG1, which is the κ isotype control; MAB07 represents the anti-EGFR antibody, whose amino acid sequence is listed in the sequence listing; MAB07-LP62 represents the antibody-drug conjugate formed by the conjugation of MAB07 and LP62; and DAR represents the drug-to-antibody ratio. [Modes for carrying out the invention]

[0015] This disclosure provides a novel anti-EGFR antibody-drug conjugate. The anti-EGFR antibody-drug conjugate exhibits good affinity for EGFR-positive cells and can be effectively internalized into EGFR-positive cells, thereby exhibiting good cytotoxic activity even against tumor cells that are not sensitive to anti-EGFR antibodies or small molecule EGFR inhibitors. Furthermore, the anti-EGFR antibody-drug conjugate shows excellent tumor inhibitory activity in various tumor models (including osimertinib and / or Enhertu-resistant tumor models).

[0016] Rather than being limiting, the forms for carrying out the invention can be categorized as follows, for clarity.

[0017] 1. Definition, 2. Antibody-drug conjugates, 3. How to use; 4. Pharmaceutical formulations, and 5. Products and kits.

[0018] 1. Definition In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, the terms and laboratory procedures used herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are terms and procedures that are widely used in the corresponding fields. In addition, to better understand this invention, definitions and interpretations of the relevant terms are provided below.

[0019] The term "EGFR" refers to the human epidermal growth factor receptor (also known as HER-1 or Erb-B1), a 170kDa transmembrane receptor encoded by the c-erbB oncogene, which exhibits intrinsic tyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996), Herbst and Shin, Cancer 94:1593-1611 (2002)). Entry P00533 in the SwissProt database provides the sequence of human EGFR. EGFR regulates various cellular processes through tyrosine kinase-mediated signaling pathways, including, but not limited to, the activation of signaling pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitosis, and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003), Tsao and Herbst, Signal 4:4-9 (2003), Herbst and Shin, Cancer 94:1593-1611 (2002), Modjtahedi et al., Br.J. Cancer 73:228-235 (1996)).

[0020] The term "antibody" is used in its broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to the full-length antibody or its antigen-binding fragment.

[0021] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the heavy and light chains are responsible for antigen binding. The variable domains of the heavy and light chains are each called "V H " and "V L The variable region in the two strands typically contains three highly variable loops, which are called complementarity-determining regions (CDRs) (the light chain (LC) CDR, which includes LC-CDR1, LC-CDR2, and LC-CDR3; and the heavy chain (HC) CDR, which includes HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody-antigen binding fragments disclosed herein may be defined or identified by the convention of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997, Chothia 1985, Chothia 1987, Chothia 1989, Kabat 1987, Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are even more conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of the constant region of the antibody heavy chain. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgGA1 (α1 heavy chain), or IgGA2 (α2 heavy chain).

[0022] As used herein, the term “antigen-binding fragment” refers to an antibody fragment, which includes, for example, bispecific antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-bonded Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-bonded bispecific antibodies (ds bispecific antibodies), single-chain Fv (scFv), scFv dimers (bivalent diaboves), multispecific antibodies formed from antibody moieties containing one or more CDRs, camelized single-domain antibodies, nanoantibodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that bind to an antigen but do not contain a complete antibody structure. Antigen-binding fragments can bind to antigens with the same parental antibody or parental antibody fragment (e.g., parental scFv) binding. In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody, the CDRs being grafted onto framework regions from one or more different human antibodies.

[0023] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions are discussed in Kabat et al., J. Biol. Chem., 252:6609-6616 (1977), Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), and Lefranc MP et al., Dev. Comp.Immunol., 27:55-77. (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where overlaps or subsets containing amino acid residues are defined for comparison purposes. However, any CDR referring to an antibody or transplanted antibody or its variant, applied to any one of these definitions, is intended to be within the scope of the terminology defined and used herein. The amino acid residues covering the CDRs as defined in each of the above references are included in Table 1 below for comparison. The CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references referenced in this chapter and section are incorporated herein by reference as a whole, used in this application, and may be included in one or more claims herein.

[0024] [Table 1]

[0025] 1 Residue numbering follows the nomenclature of Kabat et al. (ibid.). 2 Residue numbering follows the nomenclature of Chothia et al. (ibid.). 3 Residue numbering follows the nomenclature of MacCallum et al. (ibid.). 4 Residue numbering follows the nomenclature of Lefranc et al. (ibid.). 5 Residue numbering follows the nomenclature of Honegger and Pluckthun (ibid.).

[0026] The expressions “e.g., Kabat variable domain residue numbering” or “e.g., Kabat amino acid position numbering” and their variants refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the Kabat et al. antibody assembler described above. Using this numbering system, the actual linear amino acid sequence may contain fewer or different amino acids corresponding to the shortening or insertion of the FR or highly variable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after H2 residue 52 (based on Kabat residue 52a) and an insertion residue after heavy chain FR residue 82 (e.g., based on Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by alignment of the antibody sequence with the homology region of the “standard” Kabat numbered sequence.

[0027] Unless otherwise specified herein, amino acid residues covering the CDR of a full-length antibody are defined according to the Kabat nomenclature of Kabat et al., and the residue numbering in the immunoglobulin heavy chain, for example, the Fc region, is the EU index numbering described by Kabat et al., and all amino acid residues except those covering the CDR of any shared sequence are defined according to the Kabat nomenclature, where modifications are based on experimental conditions. "Kabat EU index" refers to the residue numbering of human IgG1 EU antibody.

[0028] "Framework" or "FR" residues are variable domain residues other than the CDR residues as defined herein.

[0029] Humanized non-human (e.g., rodent) antibodies are chimeric antibodies that contain a minimal sequence derived from the non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody), where residues from the receptor hypervariability region (HVR) are replaced with residues from a hypervariability region (donor antibody) of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) that have the desired antigen specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. The humanized antibody may also contain residues not found in the receptor antibody or donor antibody. These modifications are made to further improve antibody performance. Typically, the humanized antibody contains substantially all of at least one and usually two variable domains, where all or substantially all of the hypervariability loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies further optionally contain at least a portion of the constant region (Fc) of immunoglobulins, typically at least a portion of human immunoglobulins. For further details, see Jones et al., Nature, 321: 522-525, (1986), Riechmann et al., Nature, 332: 323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0030] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or is prepared by any technique for the preparation of human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). It is also possible to use the methods described by Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991), which can also be used for the preparation of monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared, for example, by administering an antigen to a transgenic animal (e.g., immunized xenomice) in which the endogenous locus has been rendered ineffective but which has been modified to produce such antibodies in response to antigen challenge (e.g., XENOMOUSE TM see U.S. Pat. Nos. 6,075,181 and 6,150,584 related to the technology). Human antibodies produced by human B cell hybridoma technology are further described, for example, in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006).

[0031] "Percent amino acid sequence identity (%)" or "homology" relating to the polypeptide identified herein and the antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the polypeptide being compared, after sequence alignment (considering any conservative substitutions as part of sequence identity). For the purpose of determining the percentage amino acid sequence identity, alignment can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters to be used for measurement alignment, including any algorithm that achieves maximum alignment within the full-length range of the sequences being compared. However, for the purposes of this specification, the sequence comparison computer program MUSCLE is used to produce the value of amino acid sequence identity % (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0032] "Homologie" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. Two comparison sequences are homologous at a given position when one position is occupied by the same base or amino acid monomer subunit; for example, when one position in each of two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences match or are homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Comparisons are typically performed when the two sequences are aligned to give maximum homology.

[0033] The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence than another part of the immunoglobulin, namely the variable domain, and it contains the antigen-binding site. The constant domain is the C of the heavy chain. H 1, C H 2 and C H 3 domains (C H (collectively referred to as) and the light chain CHL (or C L ) Includes the domain.

[0034] The "light chain" of any mammalian antibody (immunoglobulin) can be designated as one of two distinctly different types, depending on the amino acid sequence of its constant domain, and these are called kappa ("κ") and lambda ("λ"), respectively.

[0035] The "CH1 domain" (also known as the "C1" in the "H1" domain) typically consists of approximately 118 to 215 amino acids (according to the EU numbering system).

[0036] The "hinge region" is usually defined as the region in IgG corresponding to Glu216 to Pro230 in human IgG1 (Burton, Molec. Immunol., 22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the disulfide bond between the heavy chains in the same position.

[0037] The "CH2 domain" of the human IgG Fc region (also called the "C2" domain) is typically between approximately 231 and 340 amino acids. The CH2 domain is unique because it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete, natural IgG molecule. It is speculated that the carbohydrates may provide a domain-domain pairing substitute, contributing to the stabilization of the CH2 domain. (Burton, Molec Immunol., 22:161-206 (1985)).

[0038] The "CH3 domain" (also called the "C2" domain) includes the residue region in the Fc region that is close to the C-terminus of the CH2 domain (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence (typically located at amino acid residues 446 or 447 of IgG)).

[0039] In this specification, “Fc region” or “fragment crystallizable region” is used to define the C-terminal region of an immunoglobulin heavy chain and includes both native sequence Fc regions and mutant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain are variable, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl group terminus. For example, the C-terminal lysine of the Fc region (based on residue 447 in the EU numbering system) can be removed during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, a complete antibody composition may include antibody groups with all K447 residues removed, antibody groups with and without K447 residues, and antibody mixtures with or without K447 residues. Suitable native sequence Fc regions used in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0040] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is the natural human FcR. The preferred FcR is an FcR that binds to an IgG antibody (γ receptor) and includes the receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, with the main distinction being in their cytoplasmic domain. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcR is outlined in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991), Capel et al., Immunomethods 4: 25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term “FcR” as used herein covers other FcRs and includes FcRs identified in the future.

[0041] As used herein, the terms “specifically bind,” “specifically recognize,” and “specific to…” refer to measurable and reproducible interactions, such as the binding of a target to an antibody or antibody moiety, which determines the presence of the target in the presence of heteromolecules (including biomolecules). For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) is an antibody or antibody moiety that binds to the target, and its affinity, binding, readiness, and / or duration are longer than that of binding to other targets. In some embodiments, the degree of binding to an antibody-unrelated target is about 10% less than the degree of binding to an antibody-target as measured, for example, by radioimmunoassay (RIA). In some embodiments, the dissociation constant (K) of an antibody that specifically binds to a target isD )≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤10 -12 M is the key factor. In some embodiments, the antibody specifically binds to an epitope for a conserved protein from a different species of protein. In some embodiments, the specific binding may include, but is not limited to, exclusive binding. The binding specificity of the antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include Western blotting, ELISA, RIA, ECL, IRMA, EIA, and BIACORE. TM - This includes, but is not limited to, assays and peptide scanning.

[0042] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term also includes radioactive isotopes, e.g., At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 , C 60 It is intended to include low-molecular-weight toxins or toxins such as enzyme-active toxins of bacterial, fungal, plant, or animal origin, including radioactive isotopes of Lu, chemotherapeutic agents, and their synthetic analogs and derivatives.

[0043] A "linker," "linker unit," or "linker" refers to a chemical module containing covalent bonds or atomic chains that covalently bond an antibody or target portion to a drug module.

[0044] As used herein, the terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are interchangeable and refer to compounds or derivatives thereof that are conjugated to antibodies (e.g., anti-EGFR antibodies), and are defined by the general formula Ab-(LD)k, where Ab = antibody portion (i.e., antibody or antigen-binding fragment), L = linker portion, D = drug portion, and k = number of drug portions conjugated to each antibody portion.

[0045] Currently, there are two main methods of conjugation between antibodies and linkers in commercially available antibody-drug conjugates: (1) Lysine is the most common linking site in antibodies, and its ε-amino group can react with the activated carboxyl group of the linker to form an amide bond. Currently, there are technologies that can achieve site-directed conjugation, namely, activating the carboxyl group of the linker with an activating group, and then forming an amide bond with a specific lysine ε-amino group in the antibody to complete the conjugation. (2) The mercapto groups (SH) of antibody cysteine ​​all exist in the form of disulfide bonds. Opening the disulfide bond in the antibody can provide multiple free mercapto groups as conjugation sites. One method for conjugating with antibody mercapto groups is for the free mercapto groups on the antibody to undergo a Michael addition reaction with maleimide, or a specific substrate may undergo two Michael addition reactions with the free mercapto groups on the antibody to form a unique sulfur crosslink bond.

[0046] The three-letter and one-letter amino acid codes used herein are as defined in J. boil. Chem. 1968, 243, 3558.

[0047] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0048] As used herein, the term “alkyl group” refers to a saturated aliphatic hydrocarbon group which is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl This includes groups such as 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof.More preferably, the lower alkyl group contains 1 to 6 carbon atoms, and non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl group may be substituted or unsubstituted, and if substituted, the substituent may be substituted at any available linking site, and the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, amino groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0049] As used herein, the term “haloalkyl group” refers to an alkyl group substituted with one or more halogens, where the alkyl group is as defined above.

[0050] As used herein, the term "cyano group" refers to -CN.

[0051] As used herein, the term "sulfonic acid group" refers to the group -SO3H.

[0052] As used herein, the term "carboxyl group" refers to -C(O)OH.

[0053] As used herein, the term “alkoxy group” refers to -O-(alkyl) and -O-(cycloalkyl), where the definitions of alkyl or cycloalkyl groups are as set forth above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy groups. Alkoxy groups may be optionally substituted or unsubstituted, and if substituted, the substituent is preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio groups.

[0054] The term "alkenyl group" refers to an alkyl compound containing at least one carbon-carbon double bond in the molecule, where the definition of an alkyl group is as described above, and it has 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) carbon atoms (i.e., C 2-10 (Alkenyl group). The alkenyl group is preferably an alkenyl group having 2 to 6 carbon atoms (i.e., C 2-6 The alkenyl group is an alkenyl group. The alkenyl group may be substituted or unsubstituted, and if substituted, the substituent is preferably one or more selected from an alkoxy group, halogen, haloalkyl group, haloalkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0055] The term "alkynyl group" refers to an alkyl compound containing at least one carbon-carbon triple bond in the molecule, where the definition of an alkyl group is as described above, and it has 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) carbon atoms (i.e., C 2-10 Alkynyl group). The alkynyl group is preferably an alkynyl group having 2 to 6 carbon atoms (i.e., C 2-6 The alkynyl group is substituted or unsubstituted. If substituted, the substituent is preferably one or more selected from alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclyloxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups.

[0056] As used herein, the term “deuterated alkyl group” refers to an alkyl group substituted with one or more deuterium atoms, where alkyl group is as defined above.

[0057] The present invention further includes various deuterated forms of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently substituted with a deuterium atom. Those skilled in the art can synthesize the deuterated forms of formula (I) by referring to relevant literature. When producing the deuterated forms of formula (I), commercially available deuterated starting materials may be used, or they may be synthesized using conventional techniques with deuterating reagents, non-limiting examples of deuterating reagents include borane deuterated, borane tetrahydrofuran trihydrogenated solution, lithium aluminum deuterated, iodoethane deuterated, and iodomethane deuterated.

[0058] As used herein, the term “DAR” refers to the drug-to-antibody ratio, which represents the average number of cytotoxic drugs loaded onto each antibody, and can also be expressed as a ratio of drug amount to antibody amount, where the drug loading range may be 0 to 12, preferably 1 to 10, cytotoxic drugs (D) linked to each antibody (Ab). In embodiments of the present invention, DAR is represented as k, and as an example that is not limiting, it may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Conventional methods, such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC, can be used to identify the average number of drugs on each ADC molecule after the conjugation reaction.

[0059] In one embodiment of the present invention, a cytotoxic drug is conjugated via a linker unit to an open interchain cysteine ​​mercapto-SH group of an antibody and / or to a site-specifically mutated cysteine ​​mercapto-SH group. Generally, in a conjugation reaction, the number of drug molecules that can be conjugated with an antibody is less than or equal to the theoretical maximum value.

[0060] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, (1) Controlling the molar ratio of the linker-loading drug, reducing agent reagent, and monoclonal antibody, (2) Controlling the reaction time, pH value and temperature, (3) This includes selecting different reaction reagents and their quantities.

[0061] As used herein, the terms “solvate” or “solvate” refer to the ligand (or target moiety)-drug conjugate of the present invention forming a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, and dimethylacetamide (DMAC).

[0062] When used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an antibody-drug conjugate. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bistartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugarates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthylates)). A pharmaceutically acceptable salt may contain other molecules, such as acetate ions, succinate ions, or other counterions. The counterion may be any organic or inorganic module that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If multiple charged atoms constitute a pharmaceutically acceptable salt, it may also have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0063] A "pharmaceutically acceptable solvate" refers to the bonding of one or more solvent molecules with an ADC or a salt thereof. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0064] As used herein, “treatment” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, reducing one or more symptoms caused by the disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying disease exacerbation), preventing or delaying the transmission of the disease (e.g., metastasis), preventing or delaying disease recurrence, slowing or mitigating the rate of disease progression, improving the disease state, providing some or all relief of the disease, reducing the dosage of one or more other drugs necessary for the treatment of the disease, delaying disease progression, increasing or improving quality of life, increasing weight gain, and / or extending survival. “Treatment” further covers reducing the pathological outcomes of cancer (e.g., tumor volume). The methods of this application consider one or more of these therapeutic embodiments.

[0065] In the context of cancer, the term “treatment” includes any or all of the following: inhibition of cancer cell growth, inhibition of cancer cell replication, reduction of the overall tumor burden, and improvement of one or more symptoms associated with the disease.

[0066] The term “inhibition” or “inhibit” refers to a reduction or cessation of any phenotypic feature, or a reduction or cessation of the incidence, degree, or possibility of such feature. Compared to the reference, “reduction” or “inhibition” refers to reducing, reducing, or blocking activity, function, and / or quantity. In some embodiments, “reduction” or “inhibition” refers to the ability to cause a general reduction of 20% or more. In another embodiment, “reduction” or “inhibition” refers to the ability to cause a general reduction of 50% or more. Also in one embodiment, “reduction” or “inhibition” refers to the ability to cause a general reduction of 75%, 85%, 90%, 95%, or more.

[0067] As used herein, “reference” refers to any sample, standard, or level used for comparison. References may be obtained from healthy and / or uninfected samples. In some embodiments, references may be obtained from untreated samples. In some embodiments, references may be obtained from uninfected or untreated samples of individuals. In some examples, references may be obtained from one or more healthy individuals that are not the aforementioned individual or patient.

[0068] As used herein, “delaying disease progression” means slowing, inhibiting, slowing, stabilizing, inhibiting, and / or delaying the progression of a disease (e.g., cancer). This delay may have different durations depending on the disease history and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay may practically include prevention, since the individual is not suffering from the disease. For example, it may be possible to delay the progression of advanced cancer (e.g., metastatic progression).

[0069] As used herein, “prevention” includes providing prevention against the onset or recurrence of the disease in an individual who may be susceptible to the disease but has not been diagnosed with the disease.

[0070] As used herein, “inhibiting” a function or activity means reducing its function or activity compared to other identical conditions or parameters other than those of interest, or alternatively, compared to other conditions. For example, compared to the tumor growth rate in the absence of the antibody, an antibody that inhibits tumor growth reduced the tumor growth rate.

[0071] The terms “subject,” “individual,” and “patient” are interchangeable herein and refer to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0072] The “effective dose” of a drug refers to the amount that effectively achieves the required therapeutic or prophylactic effect within the required dosage and duration. The specific dose may vary depending on one or more of the following: the chosen drug, the administration regimen to be followed, whether or not it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the accompanying physical delivery system.

[0073] The "therapeutic effective dose" of the substance / molecule, agonist, or antagonist of this application may vary depending on factors such as disease state, age, sex, and body weight of the individual, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. The therapeutic effective dose is further defined as the amount at which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are offset by the therapeutically beneficial effects. The therapeutic effective dose can be delivered in one or more doses.

[0074] The "prophylactic effective dose" refers to the effective dose and duration required to achieve the desired preventive outcome. Typically, since prophylactic doses are not essential and are used in subjects before or early in the course of the disease, such prophylactic effective doses are smaller than therapeutic effective doses.

[0075] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a formulation in which the biological activity of one or more active ingredients is effective, and which does not contain other ingredients that are unacceptably toxic to the individual to whom the formulation is administered. Such formulations may be sterile.

[0076] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, compounding adjuvant, or carrier of the art used in conjunction with a therapeutic agent, both of which together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used.

[0077] In this specification, the KRAS gene has the same meaning as the K-RAS gene, which is a member of the RAS gene family, codes for the K-ras protein, and is involved in the generation, proliferation, migration, diffusion, and angiogenesis of several types of tumors. Common mutation sites are codons 12 and 13 of exon 2 and codon 61 of exon 3 of the K-RAS gene, where there are seven mutation hotspots: G12C, G12R, G12S, G12V, G12D, G12A, and G13V / D, which account for more than 90% of mutations. In one embodiment of the present invention, the tumor is a tumor with a KRAS gene mutation associated with EGFR expression.

[0078] In this specification, the BRAF (v-raf murine sarcoma viral oncogene homolog B1) gene is a proto-oncogene and a member of the RAF family. Approximately 8% of human tumors are BRAF mutations, and the majority of BRAF gene mutations are BRAF V600E mutations, which persistently activate the downstream MEK / ERK signaling pathway and play a crucial role in tumor growth, proliferation, invasion, and metastasis. In one embodiment of the present invention, the tumor is a BRAF gene mutation associated with EGFR expression.

[0079] In this application, if the chemical name and chemical structure do not match (or do not match), it should be understood that the chemical structure shall prevail.

[0080] It should be understood that the embodiments of the application described herein include "...consisting of..." and / or "substantially consisting of...".

[0081] In this specification, a “approximately” value or parameter reference includes (and describes) a change in the value or parameter itself. For example, a description of “approximately X” includes a description of “X”.

[0082] As used herein, references to a value or parameter that is "not" usually mean and describe a value or parameter that is "different." For example, "not a method for treating type X cancer" means that the method is used to treat cancers other than type X.

[0083] As used herein, the term "approximately X to Y" has the same meaning as "from approximately X to approximately Y".

[0084] As used herein, when the term “about” modifies the number of components or reactants of the present invention, it refers to variations in numerical amounts, which may occur, for example, due to typical measurements and the preparation of concentrates or liquid handling procedures in which the solution is actually used, accidental errors in these procedures, differences in the manufacturer, raw material source, or purity of components for the preparation of the composition or implementation of the method. The term “about” also includes amounts that differ due to different equilibrium conditions for the composition obtained from a particular starting mixture. Whether modified by the term “about” or not, claims include equivalent amounts of the quantity. In one embodiment, the term “about” refers to a range of 10% of the reported numerical value, preferably a range of 5% of the reported numerical value.

[0085] Those skilled in the art should understand that when a numerical value is stated in a claim, regardless of whether or not the prefix "approximately" is used, the actual value of the numerical value may vary by 10% (±10%) above or below the stated value, and preferably by 5% (±5%) above or below.

[0086] When used in this specification and the accompanying claims, the singular forms “a,” “an,” “or,” and “the” refer to multiple objects unless the context clearly indicates otherwise.

[0087] 2. Antibody-drug conjugates Anti-EGFR antibodies can be conjugated with cytotoxic or cell-inhibiting moieties (including their pharmaceutically compatible salts) to form antibody-drug conjugates (ADCs). Particularly suitable moieties for conjugation with antibodies are cytotoxic agents (e.g., chemotherapeutic agents), prodrug convertases, radioisotopes or compounds, or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-EGFR antibodies can be conjugated with cytotoxic agents, such as chemotherapeutic agents or toxins (e.g., cell inhibitors or cytotoxic agents, e.g., abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin).

[0088] Anti-EGFR antibodies can be conjugated with prodrug convertases. Prodrug convertases can be recombinantly fused or chemically conjugated with the antibody by known methods. Exemplary prodrug convertases include carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0089] The techniques for conjugating therapeutic agents with proteins, particularly with antibodies, are well-known. (For example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy (edited by Reisfeld et al., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (edited by Robinson et al., Marcel Dekker, Inc., 2nd edition 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," Monoclonal Antibodies '84: Biological And Clinical Applications (edited by Pinchera et al., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy (edited by Baldwin et al., Academic) See Press, 1985, and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, for example, PCT disclosure number WO 89 / 12624.

[0090] The therapeutic agent can be conjugated in a manner that reduces its activity, except when it is cleaved by the antibody (e.g., hydrolysis, degradation by the antibody, or cleavage by a cleavage agent). Such a therapeutic agent is conjugated to an antibody with a cleavable linker, and the cleavable linker is sensitive to cleavage in the intracellular environment or tumor microenvironment of EGFR-expressing cancer cells, and when the conjugate is in such an environment, it is cleaved by the antibody (e.g., in endosomes, or, for example, in a pH-sensitive or protease-sensitive lysosomal environment, caveolae, or tumor microenvironment).

[0091] Typically, an ADC includes a linker region between the therapeutic agent and an anti-EGFR antibody. As described above, typically, the linker is cleavable under intracellular conditions or in the tumor microenvironment, and cleavage of the linker causes the therapeutic agent to be released from the antibody in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae) or in the tumor microenvironment. The linker may be a peptide linker that is cleaved by, for example, an intracellular peptidase or protease (including lysosomal or endosomal proteases). Typically, the peptide linker is at least two amino acid lengths or at least three amino acid lengths. The cleavage agents may include cathepsins B and D and plasmin, etc. (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). Most typically, the linker is an enzyme present in EGFR-expressing cells or a peptide linker that is cleavable in the tumor microenvironment. For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, may be used (e.g., a linker containing the Phe-Leu or Gly-Phe-Leu-Gly peptide). Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In specific embodiments, the peptide linker cleavable by intracellular proteases includes the Val-Cit linker or the Phe-Lys dipeptide (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker). One advantage of releasing therapeutic agents by hydrolysis by intracellular proteins is that the drug is typically attenuated during conjugation, and the blood stability of the conjugate is usually high.

[0092] The cleavable linker may also be pH-sensitive, i.e., susceptible to hydrolysis at certain pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) may be used. (See, for example, U.S. Patents 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (e.g., blood) but unstable below pH 5.5 or 5.0 (approximate lysosomal pH). In some embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether bonded to the therapeutic agent via an acylhydrazone linkage) (see, for example, U.S. Patent No. 5,622,929).

[0093] Other linkers (e.g., disulfide linkers) can be cleaved under reducing conditions. Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. For example, see Thorpe et al., 1987, Cancer Res. 47:5924-5931, and Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (edited by CW Vogel, Oxford U. Press, 1987; see also U.S. patent number 4,880,935).

[0094] The linker may be a malonic acid ester linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimide benzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3 (10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3 (10): 1305-12). The linker may be a malonic acid ester linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimide benzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0095] The linker may be an inclementable linker, such as a maleimide-alkylene- or maleimide-aryl linker that directly binds to a therapeutic agent (e.g., a drug). The active drug-linker is released upon degradation of the antibody.

[0096] Typically, the linkers are largely insensitive to the circulatory environment, meaning that when ADCs are present in plasma, approximately 20% or less, typically 15% or less, more typically 10% or less, and more typically 5% or less, 3% or less, or 1% or less of the linkers in the ADC sample will be cleaved.

[0097] For example, by independently culturing both (a) an ADC ("ADC sample") and (b) an equimolar amount of an unconjugated antibody or therapeutic agent ("control sample") in plasma for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of the unconjugated antibody or therapeutic agent present in the ADC sample, measured, for example by high-performance liquid chromatography, with the amount present in the control sample, it is possible to determine whether the linker is largely unsensitive to the circulating environment.

[0098] The anti-EGFR antibody can be conjugated with a linker via heteroatoms of the antibody. These heteroatoms may be present on the antibody in its natural state or may be introduced into the antibody. In some embodiments, the anti-EGFR antibody conjugates with the linker via the nitrogen atom of a lysine residue. In other embodiments, the anti-EGFR antibody conjugates with the linker via the sulfur atom of a cysteine ​​residue. The cysteine ​​residue may be naturally occurring or an antibody-engineered residue. Methods for conjugating linkers and drug-linkers with antibodies via lysine and cysteine ​​residues are known in the art.

[0099] An exemplary antibody-drug conjugate further comprises a camptothecin-based antibody-drug conjugate (i.e., the drug component is the camptothecin drug). Camptothecin is a topoisomerase inhibitor that has been demonstrated to have anticancer activity. Typically, a camptothecin-based antibody-drug conjugate includes a linker between the camptothecin drug and an anti-EGFR antibody. The linker may be, for example, a cleavable linker (e.g., a peptide linker, a carbohydrate linker) or an incleavable linker (e.g., a linker released by the degradation of the antibody). The synthesis and structure of an exemplary camptothecin drug linker are described in PCT / US19 / 025968, which is incorporated herein by reference in whole for all purposes.

[0100] Other exemplary antibody-drug conjugates include meitansinoid antibody-drug conjugates (i.e., the drug component is a meitansinoid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is a benzodiazepine (e.g., pyrrolo[l,4]benzodiazepine dimer (PBD dimer), indole benzodiazepine dimer, and oxazolidino benzodiazepine dimer)).

[0101] Useful categories of cytotoxic agents that conjugate with anti-EGFR antibodies include, for example, antitubulins, DNA minor groove conjugates, DNA replication inhibitors, and chemotherapeutic sensitizers. Other exemplary categories of cytotoxic agents include anthracyclines, auristatins, camptothecines, duocalmycin, etoposide, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include camptothecin derivatives (e.g., irinotecan, DXD, etc.), auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., enediyne and lexitropsin), duocalmycin, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulisin M, doxorubicin, morpholino-doxorubicin, and cyclomorpholino-doxorubicin.

[0102] The cytotoxic agent may be a chemotherapeutic agent, such as doxorubicin, paclitaxel, melphalan, vinca alkaloid, methotrexate, mitomycin C, or etoposide. The agent may also be a CC-1065 analog, calichemycin, meitansin, a dorastatin 10 analog, rhizoxin, or palytoxin.

[0103] The cytotoxic agent may be auristatin. Auristatin may also be an auristatin E derivative, such as an ester formed between auristatin E and a keto acid. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include auristatin T, AFP, MMAF, and MMAE. The synthesis and structures of several auristatins are described, for example, in US 2005-0238649 and US2006-0074008.

[0104] This invention relates to a novel antibody-drug conjugate (ADC) targeting EGFR, which mainly consists of three parts: a monoclonal antibody targeting EGFR, a cytotoxic small molecule drug (payload), and a linker sensitive to the tumor microenvironment. Here, the loading drug is a novel topoisomerase (TOP) I inhibitor with higher sensitivity to tumors. After the anti-EGFR antibody-drug conjugate of this invention enters the body, its antibody portion specifically binds to EGFR highly expressed on tumor cells, is internalized by an antigen-mediated drug, and then enters cancer cells. Inside tumor cells, during the process of capture and transport of the anti-EGFR antibody-drug conjugate of this invention by lysosomes, the linker of the anti-EGFR antibody-drug conjugate cleaves under the special environment inside the tumor, efficiently releasing the payload. The payload small molecule drug exerts a cytotoxic effect and can further kill cancer cells. Because a portion of the payload has high lipophilicity, it exhibits a "bystander effect," entering and acting in cells surrounding cancer cells. This anticancer mechanism is extremely effective even against tumor cells with low antigen expression or heterogeneity.

[0105] In some embodiments, the present disclosure provides an antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, wherein the antibody-drug conjugate has the structure of formula (I), TP-[L1-L2-L3-D] k (I) Here, TP is a target region that selectively binds to or recognizes EGFR. L1 is an extension unit that links TP and L2, where TP is linked to L1 via an active group (e.g., a mercapto group or an amino group), preferably where L1 is conjugated to an open interchain cysteine ​​mercapto group-SH and / or a site-specifically mutated cysteine ​​mercapto group-SH in TP. L2 is a short-chain peptide consisting of an optional amino acid residue or 2 to 10 amino acid residues. L3 is a spacer element, D is a bioactive molecule, k represents any number between 0.1 and 10.0.

[0106] For antibody-drug conjugates, the subscript k represents the drug load and, depending on the context, can be an integer value, as it can represent the number of drug-linker molecules bound to a single antibody molecule, or it can be an integer or a non-integer value, as it can represent the average drug load. The average drug load represents the average number of drug-linker molecules for each antibody in the population. Usually, but not always, when referring to antibodies, such as monoclonal antibodies, we are referring to the antibody molecule population. In a composition containing an antibody-drug conjugate molecule population, the average drug load is an important mass attribute as it determines the amount of drug that can be delivered to target cells. The percentage of unconjugated antibody molecules in the composition is included in the average drug load value.

[0107] In preferred embodiments of the present invention, when referring to a composition comprising a population of antibody-drug conjugate compounds, the average drug load is 1 to about 16, preferably about 2 to about 14, and more preferably about 2 to about 10.

[0108] For MMAE and camptothecin ADCs, for example, the exemplary ones herein, the preferred average drug load is about 2, 4, or 8, and the particularly preferred average drug load is about 8. In one embodiment, the preferred average drug load for MMAE ADC is 2 or 4. In one embodiment, the preferred average drug load for camptothecin ADC is 4 or 8. In exemplary embodiments, the drug-linker conjugates with a cysteine ​​residue of the reduced interchain disulfide. In some embodiments, the actual drug load of a single antibody molecule in the antibody-drug conjugate compound population is from 1 to 10 (or 6 to 10 or 6 to 8), where the primary drug load is 8.

[0109] In some embodiments of formula (I), the TP is an antibody or its antigen-binding fragment.

[0110] In some embodiments of formula (I), the antigen-binding fragment is selected from full-length immunoglobulin, single-stranded Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2, Fv fragment, Fv fragment with stable disulfide bond (dsFv), (dsFv)2, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, tetrabody, or any combination thereof.

[0111] In some embodiments of formula (I), the TP is a humanized antibody or its antigen-binding fragment.

[0112] In some embodiments of formula (I), the TP includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) includes a heavy chain complementarity determination region 1 (HCDR1), HCDR2, and HCDR3 included in SEQ ID NO:7, and the light chain variable region (VL) includes a light chain complementarity determination region 1 (LCDR1), LCDR2, and LCDR3 included in SEQ ID NO:8.

[0113] In some embodiments of formula (I), the amino acid sequence of HCDR1 is SEQ ID NO:1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3, and the amino acid sequence of LCDR1 is SEQ ID NO:4, the amino acid sequence of LCDR2 is SEQ ID NO:5, and the amino acid sequence of LCDR3 is SEQ ID NO:6.

[0114] In some embodiments of formula (I), the TP comprises the Fc sequence of human IgG.

[0115] In some embodiments of formula (I), the IgG is selected from IgG1, IgG2, IgG3, and IgG4.

[0116] In some embodiments of formula (I), the TP comprises a heavy chain variable region (VH), and the amino acid sequence of the heavy chain variable region (VH) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:7.

[0117] In some embodiments of formula (I), the TP includes a light chain variable region (VL), and the amino acid sequence of the light chain variable region (VL) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:8.

[0118] In some embodiments of formula (I), the TP comprises a heavy chain (HC), and the amino acid sequence of the heavy chain (HC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:9.

[0119] In some embodiments of formula (I), the TP comprises a light chain (LC), and the amino acid sequence of the light chain (LC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:10.

[0120] In some embodiments of formula (I), the TP is a multispecific antibody.

[0121] In some embodiments of formula (I), the TP is an afucosylated antibody.

[0122] In some embodiments of formula (I), the Fc region includes a C-terminal lysine.

[0123] In some embodiments of formula (I), the Fc region includes a deletion of the C-terminal lysine.

[0124] In some embodiments of formula (I), L1 is selected from the following: [ka] Here, L1 is linked to TP via an S atom at position a and to L2 at position b, m is any integer between 1 and 10, n is any integer between 1 and 10, and X and Y are independently C, O, S, or N.

[0125] In some embodiments of formula (I), L1 is as follows: [ka] Here, L1 is linked to TP via an S atom at position a and to L2 at position b, and m is selected from 1, 2, 3, 4, and 5.

[0126] In some embodiments of formula (I), L1 is as follows: [ka] Here, L1 is linked to TP via an S atom at position a, and to L2 at position b.

[0127] In some embodiments, L1 is linked at the α position to the cysteine ​​of TP (more specifically, to the mercapto group of cysteine).

[0128] In some embodiments of formula (I), L2 is selected from the following: [ka] Here, L2 is connected to L1 at position c and to L3 at position d.

[0129] In some embodiments of formula (I), L2 is as follows: [ka] Here, L2 is connected to L1 at position c and to L3 at position d.

[0130] In some embodiments of formula (I), L3 is a single bond or selected from the following groups: [ka] Here, L3 is connected to L2 at position e and to D at position f. p and q are independent integers between 0 and 10. W is C, O, S, or N. Z is hydrogen, halogen, C1-C 20 Alkan, C1-C 20 Haloalkane, cyano group, sulfonic acid, carboxylic acid, C1-C 20 Alkoxy group, C2-C 10 The selection is made from unsaturated alkanes, natural amino acids, unnatural amino acids, and short-chain peptides composed of the above amino acids.

[0131] In some embodiments of formula (I), Z is selected from the following: [ka] Here, Z is connected to the remainder of L3 at position g, z1 to z9 are each independent integers between 0 and 10, and T is either NH2 or OH.

[0132] In some embodiments of formula (I), D is a cytotoxic agent.

[0133] In some embodiments of formula (I), D is selected from the following: [ka] Here, D is connected to L3 at position h.

[0134] In some embodiments of formula (I), the antibody-drug conjugate is

[0135] [ka]

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] Selected from. Here, Ab represents an antibody or its antigen-binding fragment as defined in the embodiments described above.

[0140] In some embodiments of formula (I), k is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 The range is 0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 or any range between the two. In a further embodiment, k is a range of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any range between the two. In a further embodiment, k is 4.0, 6.0, or 8.0.

[0141] 3. How to use The anti-EGFR antibody-drug conjugate of the present invention can be used to treat cancer. Some such cancers exhibit detectable EGFR levels, measured at the protein (e.g., by immunoassay using one of the exemplary antibodies) or mRNA level. Some such cancers exhibit elevated EGFR levels in the same type of non-cancerous tissue (preferably from the same patient). Optionally, the level of EGFR in the cancer is measured before treatment.

[0142] Examples of cancers associated with EGFR expression and suitable for treatment include epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat epidermal cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat colon cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat rectal cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat head and neck cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat lung cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat osimertinib-resistant lung cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat non-small cell lung cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat small cell lung cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat ovarian cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat cervical cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat bladder cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat esophageal cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat breast cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat kidney cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat breast cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat prostate cancer.In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat gastric cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat Enhertu-resistant gastric cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat pancreatic cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used to treat glioma. The treatment can be applied to patients suffering from these types of primary or metastatic tumors. The treatment can also be applied to patients who are refractory to conventional treatment or to patients who have relapsed after responding to such treatment.

[0143] The antibody-drug conjugate (ADC) of the present invention can be used in combination with other TKI inhibitors or PD1 / PDL1 inhibitors and can be used to treat a variety of diseases or conditions, such as diseases or conditions characterized by EGFR overexpression. Typical diseases or hyperproliferative conditions include benign or malignant tumors, including epidermal carcinoma, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma. In one embodiment, the tumor or cancer is a tumor or cancer of KRAS gene mutation and / or BRAF gene mutation. In a further embodiment, the tumor or cancer of KRAS gene mutation and / or BRAF gene mutation is colon cancer, rectal cancer, lung cancer, or pancreatic cancer of KRAS gene mutation and / or BRAF gene mutation. In one embodiment, the PD1 inhibitor is an anti-PD1 antibody. In a further embodiment, the anti-PD1 antibody is serplulimab.

[0144] The antibody-drug conjugate of the present invention is administered in an effective regimen, where the effective regimen means a dose, route of administration, and frequency of administration that can delay the onset, reduce the severity, prevent further progression, and / or improve at least one sign or symptom of cancer. If the patient already has cancer, the regimen may be called a therapeutic effective regimen. If the patient is at higher risk of developing cancer compared to the general population but has not yet experienced symptoms, the regimen may be called a prophylactic effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in individual patients compared to historical controls or past experiences in the same patient. In other cases, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials compared to a control group of untreated patients.

[0145] The exemplary doses of the antibody-drug conjugate may be 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 20 mg / kg), e.g., 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg or 3 mg / kg to 7.5 mg / kg of subject body weight, or 0.1 to 20 mg / kg of body weight or 0.5 to 5 mg / kg of body weight (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg), or a fixed dose of 10 to 1500 mg or 200 to 1500 mg. In some cases, the patient is administered a dose of at least 1 mg / kg, at least 1.5 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 8 mg / kg, or at least 10 mg / kg once weekly, every three weeks, or at intervals of more than one week. The dose is determined by factors such as the frequency of administration, the patient's condition and response to previous treatments (if any), whether the treatment is prophylactic or therapeutic, and whether the symptoms are acute or chronic.

[0146] Administration may be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. Direct administration into the tumor is also possible. Preferably, it is administered into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration may be performed, for example, by intravenous drip infusion over a set period of time (e.g., 30-90 min) or by a single bolus intravenous injection.

[0147] The frequency of administration is determined by factors such as the half-life of the circulating conjugate, the patient's condition, and the route of administration. The frequency may be set daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of cancer during treatment. In a continuous course of treatment, exemplary frequencies of intravenous administration range from twice weekly to once quarterly, but higher or lower frequencies are also possible. Other exemplary frequencies of intravenous administration in a continuous course of treatment range from once weekly to three times every four weeks, but higher or lower frequencies are also possible. For subcutaneous administration, exemplary frequencies range from daily to once monthly, but higher or lower frequencies are also possible.

[0148] The number of doses depends on the nature of the cancer (e.g., whether it is acute or chronic) and the response to the treatment of the symptoms. For acute symptoms or acute exacerbations of chronic symptoms, doses of 1 to 10 are generally sufficient. In some cases, a single bolus dose (optionally in divided form) is sufficient for acute symptoms or acute exacerbations of chronic symptoms. For relapses or acute exacerbations of acute symptoms, treatment can be repeated. For chronic symptoms, antibodies may be administered at regular intervals, e.g., once a week, once every two weeks, once a month, once a quarter, or once every six months, and may be continued for at least 1, 5, or 10 years, or for the patient's lifetime.

[0149] Pharmaceutical compositions for parenteral administration are preferably sterile, basically isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for single administration). The pharmaceutical composition can be prepared with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The preparation depends on the chosen route of administration. For injection, the antibody can be prepared in an aqueous solution, preferably formulated in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline or acetate buffer (to reduce injection site discomfort). The solution may contain the prepared drug, such as a suspension, stabilizer, and / or dispersant. Alternatively, before use, the antibody may be in lyophilized form and reconstituted in a suitable medium (e.g., sterile, pyrogenic water). The concentration of the antibody in the liquid formulation may be, for example, 1 to 100 mg / ml, for example, 10 mg / ml.

[0150] Therapies using the antibody-drug conjugate of the present invention can be combined with immunotherapy, chemotherapy, radiation, stem cell therapy, surgery, and treatments effective for symptoms during treatment. Other useful categories of drugs that can be used in combination with the EGFR-targeting antibody-drug conjugates described herein include, for example, antibodies that target other receptors expressed on cancer cells (e.g., anti-PDL1 antibodies), immunotherapies (e.g., anti-PD1 antibodies), antitubulins (e.g., auristatin), DNA minor groove conjugates, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono(platinum), bis(platinum) and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolic acids, antimetabolites, chemotherapeutic sensitizers, duocalmycin, etoposide, pyrimidine fluorophosphates, ionophores, lexitropsin, nitrosourea, cisplatin, preformed compounds, purine antimetabolites, promycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0151] Compared to the same treatment without the antibody-drug conjugate (e.g., chemotherapy), treatment with the antibody-drug conjugate, when optionally combined with any other drug or regimen, can increase the median progression-free survival or overall survival of patients with tumors (e.g., epidermal carcinoma, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma) by at least 30% or 40%, preferably by 50%, 60%, to 70%, or even 100% or more. Additionally or alternatively, compared to the same treatment without the antibody-drug conjugate (e.g., chemotherapy), a treatment including the antibody-drug conjugate (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) in patients with tumors by at least 30% or 40%, preferably by 50%, 60% to 70%, or even 100%.

[0152] 4. Pharmaceutical formulations The antibody-drug conjugates described herein can be used alone (e.g., administered), but generally, they are preferably present in the form of a composition or compound.

[0153] In one embodiment, the composition is a pharmaceutical composition (e.g., a formulation, preparation, or drug) comprising an antibody-drug conjugate and a pharmaceutically acceptable carrier, diluent, or excipient as described herein.

[0154] In one embodiment, the composition is a pharmaceutical composition comprising at least one antibody-drug conjugate described herein and one or more other pharmaceutically acceptable components well known to those skilled in the art, the components including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents and sweeteners.

[0155] In one embodiment, the composition further comprises other activators, such as other therapeutic or prophylactic agents.

[0156] Appropriate carriers, diluents, and excipients can be found in standard drug literature. For example, see Handbook of Pharmaceutical Additives, 2nd edition (edited by M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, Lippincott, Williams & Wilkins, 2000, and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0157] Another aspect of the present invention relates to a method for producing a pharmaceutical composition, which comprises mixing at least one [11C]-radiolabeled antibody-drug conjugate or antibody-drug conjugate-like compound as defined herein with one or more other pharmaceutically acceptable components well known to those skilled in the art (e.g., carriers, diluents, excipients, etc.). When prepared in discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dose) of the active compound.

[0158] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions, dosage forms, etc., that are suitable, within reasonable medical judgment, to come into contact with the tissues of the subject under consideration (e.g., human) without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that have a reasonable benefit-risk ratio. Each carrier, diluent, excipient, etc., must also be “acceptable” in terms of compatibility with other components of the formulation.

[0159] The formulation may be prepared by any method well known in the pharmaceutical field. Such a method involves associating the active compound with a carrier constituting one or more auxiliary components. Generally, the formulation is produced by homogeneously and tightly associating the active compound with a carrier (e.g., a liquid carrier, a fragmented solid carrier, etc.) and then forming the product as needed.

[0160] The aforementioned formulations may be prepared to achieve rapid release, slow release, immediate release, delayed release, time-controlled release, sustained release, or a combination thereof.

[0161] Formulations applied to parenteral administration (e.g., injection) comprise an aqueous or non-aqueous, isotonic, nonpyrogenic sterile liquid (e.g., solution, suspension) in which the active ingredient is provided in dissolution, suspension, or other manner (e.g., within liposomes or other microparticles). Such liquids may further contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspension aids, thickeners, and solutes, which can be made isotonic with the blood (or other relevant body fluids) of the target recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerin, and vegetable oils. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Generally, the concentration of the active ingredient in the liquid is about 1 ng / ml to about 10 μg / ml, for example, about 10 ng / ml to about 1 μg / ml. The formulation may be present in unit or multi-dose sealed containers (e.g., ampoules and vials), and may be stored under lyophilization conditions, and may be used for injection only immediately before use by adding a sterile liquid carrier (e.g., water). Temporary solutions and suspensions for injection may be prepared from sterile powders, granules, and tablets.

[0162] 5. Products and Kits In another embodiment, a product or kit is provided, the product or kit comprising the anti-EGFR antibody-drug conjugate described herein. The product or kit may further include instructions for using the anti-EGFR antibody-drug conjugate described herein in a method of the present invention. Thus, in some embodiments, the product or kit includes instructions for using the anti-EGFR antibody-drug conjugate described herein in a method for treating a subject's cancer (e.g., epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma), the method comprising administering an effective amount of the anti-EGFR antibody-drug conjugate described herein to the subject. In some embodiments, the subject is a human.

[0163] The product or kit may further include a container. Suitable containers include, for example, bottles, vials (e.g., two-chamber vials), syringes (e.g., single-chamber or two-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container may be made of various materials (e.g., glass or plastic). The container holds the formulation.

[0164] The product or kit may further include a label or packaging insert on or associated with the container that can indicate guide instructions regarding the use of the reconstitution and / or formulation. The label or packaging insert may further indicate that the formulation is available for use or will be used to treat cancer (e.g., epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma) in a subject when administered subcutaneously, intravenously (e.g., intravenous infusion), or by other means of administration. The container containing the formulation may be a single-use vial or a multi-use vial, which allows for repeated administration of the reconstituted formulation. The product or kit may further include a second container containing a suitable diluent. The product or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and packaging inserts printed with instructions for use.

[0165] The products or kits of this specification optionally further comprise a container containing a second drug, wherein the anti-EGFR antibody-drug conjugate is the first drug, and the products or kit further comprise instructions on a label or packaging insert for treating a subject with an effective amount of the second drug. In some embodiments, the second drug is used to eliminate or reduce the severity of one or more adverse events.

[0166] In some embodiments, the anti-EGFR antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in an airtight container (e.g., a vial, ampoule, or pouch) which indicates the amount of activator. When the drug is administered by injection, an ampoule of sterile water for injection or saline may be provided, for example, optionally, as part of the kit to allow the components to be mixed before administration. If necessary, such a kit may further include one or more containers with various common drug components, e.g., one or more pharmaceutically acceptable carriers, another container, etc., as will be apparent to those skilled in the art. The kit may further include printed instructions as an insert or label which indicate the amount of component to be administered, administration guide and / or guide for mixing components.

[0167] [Table 2-1]

[0168] [Table 2-2]

[0169] [Table 2-3]

[0170] [Table 2-4]

[0171] [Table 2-5]

[0172] Examples The following embodiments are for illustrative purposes only and should not be considered as limiting in any way.

[0173] Reagents and materials: Organic solvents such as dimethyl sulfoxide were purchased from Spectrum Chemical Mfg Corp, and the TCEP-HCl reagent was purchased from Thermo Fisher and other sources.

[0174] IgG1 is human IgG1, a κ isotype control, and was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0175] pH value sensitive Zenon TM pHrodo TM The iFL IgG labeling reagent was purchased from Thermo Fisher.

[0176] CellTiter-Glo solution was purchased from Promega.

[0177] Cell lines A549 and NCI-H1993 were purchased from ATCC, cell line SW480 was purchased from the Chinese Academy of Sciences Cell Bank, and EBC-1 cell line was purchased from the JCRB Cell Bank. Each PDX transplant tumor was established and maintained by Shanghai Lidi Biotechnology Co., Ltd.

[0178] Manufacturing scheme: The structures of the compounds described in the following examples have nuclear magnetic resonance spectra ( 1 Confirmed by 1HNMR or liquid chromatography-mass spectrometry (LC-MS).

[0179] Nuclear magnetic resonance spectrum ( 1 A Bruker 400 MHz nuclear magnetic resonance spectrometer was used as the instrument for measuring 1H NMR. The measurement solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexaduterodimethyl sulfoxide (DMSO-d6), and the internal standard was tetramethylsilane (TMS).

[0180] The abbreviations used in the nuclear magnetic resonance (NMR) patterns in the examples are shown below.

[0181] s: single peak, d: double peak, t: triple peak, q: quartet, dd: double double peak, qd: quarter double peak, ddd: double double double peak, ddt: double double triple peak, dddd: double double double double peak, m: multiple peak, br: broad peak, J: bond constant, Hz: Hertz, DMSO-d6: deuterated dimethyl sulfoxide. The Δ value was expressed in ppm.

[0182] Liquid chromatography-mass spectrometry (LC-MS) detection was performed using an Agilent (ESI) analyzer, model number Agilent 6120B.

[0183] Example 1. Production of Linker-Toxin (LP62) [ka]

[0184] Example 1.1: Synthesis of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4',6,7]indolidino[1,2-b]quinoline-8,11(7H)-dione (A1.3) The synthesis route for compound A1.3 is as shown in Scheme 1 below. [ka]

[0185] The specific steps are as follows: [ka]

[0186] Step 1: Under ice bath conditions, ferrous sulfate heptahydrate (570 mg of ferrous sulfate heptahydrate dissolved in 1 mL of water) and 4,4-dimethoxychlorobutane (3.89 g) were added to a 75% sulfuric acid solution (5 mL) of (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4',6,7]indolidino[1,2-b]quinoline-8,11(7H)-dione (A1.1, 500 mg). After stirring the reaction mixture for 3 minutes, hydrogen peroxide solution (29%, 2.5 mL) was continuously added dropwise under ice bath conditions. After the dropwise addition was complete, the reaction mixture was stirred at 0°C for 5 minutes, then the temperature was raised to room temperature and stirred for 3 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (80 mL x 2). The organic phase was washed with saturated brine and then dried over anhydrous sodium sulfate. The organic phase was evaporated under reduced pressure and then concentrated to obtain the crude product. The crude product was purified by preparative liquid chromatography with a C18 column (5%-60% aqueous solution of acetonitrile / 0.05% formic acid) to finally obtain the target compound (A1.2, yellow solid, 400 mg, yield: 67%).

[0187] LC-MS (ESI) [M+H] + :468.9: 1 HNMR (400Hz,DMSO-d6) δ7.65 (s,1H),7.51 (s,1H),7.24 (s,1H),6.50 (s,1H),6.30 (s,2H),5.42 (s,2H),5.26 (s,2H),3.81(d,J=5.9Hz,2H),3.22 (s,2H),1.98 (d,J=6.7Hz,4H),0.88(t,J=7.2Hz,3H).

[0188] [ka]

[0189] Step 2: (S)-7-ethyl-7-hydroxy-14-(3-chloropropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4',6,7]indolidino[1,2-b]quinoline-8,11(7H)-dione (A1.2, 100 mg, 0.213 mmol) was dissolved in 10% sulfuric acid (5 mL) solution, and the reaction mixture was allowed to react at 110°C for 48 hours. Saturated sodium bicarbonate (30 mL) solution was added to the reaction mixture, extracted with dichloromethane (10 mL x 5), dried over anhydrous sodium sulfate, filtered by suction, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by high-performance liquid preparative fractionation (acetonitrile / water, containing 0.05% formic acid) to obtain (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4',6,7]indolidino[1,2-b]quinoline-8,11(7H)-dione (A1.3, 1.78 mg).

[0190] LC-MS (ESI) [M+H] + :451.0: 1HNMR (400Hz,DMSO-d6) δ7.63 (s,1H),7.50 (s,1H),7.24 (s,1H),6.48 (s,1H),6.28 (s,2H),5.47-5.37 (m,2H),5.32-5.19 (m,2H),3.51-3.46 (m,2H),3.17-3.13 (m,2H),1.92-1.76 (m,4H),0.90-0.84 (m,3H).

[0191] Example 1.2: Synthesis of Compound A2.3 The synthesis route for compound A2.3 is as shown in Scheme 2 below. [ka]

[0192] The specific steps for synthesizing compound A2.3 are as follows: [ka]

[0193] Step 1: Compound A2.1 (368 mg), compound A1.3 (440 mg), and pyridinium p-toluenesulfonate (PPTS, 25 mg) were refluxed in dichloromethane (20 mL) at 40°C for 20 hours. The mixtures were then washed with aqueous sodium bicarbonate and aqueous hydrochloric acid, respectively, and the organic solvent was removed under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the target compound A2.2 (240 mg). LC-MS (ESI) [M+H] + :747:

[0194] [ka]

[0195] Step 2: Dissolve A2.2 (240 mg) in DMF (5 mL), add piperidine (1 mL), stir at room temperature for 20 minutes, remove low-boiling components under pressure, and use the residue directly in the next step of synthesis. A small amount of crude product was purified by reverse-phase chromatography (aqueous solution of acetonitrile / 0.05% FA: 5%~50%) to obtain the target compound A2.3.

[0196] LC-MS (ESI) [M+H] + :525: 1 H NMR (400Hz,DMSO-d6) δ9.13 (t,1H),8.04 (br,2H),7.58 (s,1H),7.51 (s,1H),7.25 (s,1H),6.29 (s,2H),5.43 (s,2H),5.21 (s,2H),4.65 (d,2H),3.63 (m,2H),3.53 (m,2H),3.11 (m,2H),1.87 (m,4H),0.88 (t,3H).

[0197] Example 1.3: Synthesis of Compound A3.5 The synthesis route for compound A3.5 is as shown in Scheme 3 below.

Chem.

[0198] The specific synthesis steps of compound A3.5 are as follows.

Chem.

[0199] Step 1: A solution of hydrogen chloride - dioxane (dioxone, C4H8O2) (100 mL) was added to compound A3.1 (10.0 g), and the reaction was carried out at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the target compound A3.2 (8.0 g), which is a white solid.

[0200] LC-MS (ESI) [Μ+Η] + : 380.1 1 H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 7.4 Hz, 1H), 7.40 - 7.30 (m, 5H), 7.26 (d, J = 8.8 Hz, 1H), 5.08 - 4.99 (m, 2H), 4.23 - 4.11 (m, 1H), 3.94 - 3.88 (m, 1H), 2.78 - 2.73 (m, 2H), 2.03 - 1.94 (m, 1H), 1.77 - 1.51 (m, 4H), 1.44 - 1.31 (m, 2H), 0.87 (dd, J = 17.3, 6.6 Hz, 6H).

[0201]

Chem.

[0202] Step 2: Compound A3.2 (5.0 g, 12 mmol) was dissolved in dichloromethane (100 mL), and n-propionaldehyde (4.2 g, 72.3 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 10 minutes. Then, sodium triacetoxyborohydride (12.8 g, 60.25 mmol) was added to the reaction mixture, and the reaction was stirred at room temperature for 1 hour. LC-MS indicated completion of the reaction. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was stirred for 1 hour. The mixture was dried by rotation, filtered, and the filtrate was separated and purified using a C18 reversed-phase column to obtain the target compound A3.3 (4.57 g, yield 82.0%), which was a white solid.

[0203] LC-MS (ESI) [M+H] + :464.0: 1 H NMR(400MHz,DMSO-d6) δ 7.81 (d,J= 7.3Hz,IH),7.38-7.28 (m,5H),5.04 (d,J =1.7Hz,2H),4.12- 4.02 (m,IH),3.94-3.82 (m,1H),2.65-2.52 (m,6H),2.06-1.94 (m,1H),1.76-1.64 (m,1H),1.64-1.53 ​​(m,1H),1.52-1.40 (m,6H),1.34-1.18 (m,2H),0.92-0.80 (m,12H).

[0204] [ka]

[0205] Step 3: At room temperature, compound 3.3 (2.0 g, 4.32 mmol) was dissolved in methanol (80 mL), and Pd / C (0.16 g) was added to the reaction mixture. The mixture was stirred at room temperature in the presence of hydrogen gas for 12 hours to allow the reaction to proceed. LC-MS confirmed completion of the reaction. The reaction mixture was filtered and concentrated under reduced pressure to obtain the target compound A3.4 (1.2 g, yield 85.5%), which was a white solid.

[0206] [ka]

[0207] Step 4: Dissolve 6-(2-(methylsulfonyl)pyrimidine-5-yl)hexa-5-ic acid (100 mg, 0.373 mmol) in DMF (1 mL), then add HATU (142 mg, 0.373 mmol) and N,N-diisopropylethylamine (120 mg, 0.93 mmol), stir the system for 30 minutes, then add compound A3.4 (122 mg, 0.371 mmol), and stir the reaction mixture at room temperature for 1 hour. After detecting completion of the reaction by LC-MS, the reaction mixture was directly purified using a C18 reversed-phase column (acetonitrile and 0.05% formic acid aqueous solution system) to obtain the target compound N 6 ,N 6 -Dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidine-5-yl)hexa-5-inoyl)-L-valine)-L-lysine (compound A3.5, 50 mg, yield 28%) was obtained, and it was a pale yellow solid.

[0208] LC-MS (ESI) [M+H] + :580.0: 1HNMR (400MHz,DMSO-d6) δ 8.24 (s,2Η),7.98-7.93 (m,2Η),4.24-4.16 (m,1H),4.10 (d,J=5.2Hz,1H),3.41 (s,3H),2.79-2.64 (m,6H),2.55 (t,J=7.1Hz,2H),2.45-2.26 (m,2H),2.06-1.91 (m,1H),1.89-1.78 (m,2H),1.76-1.66 (m,1H),1.64-1.57 (m,1H),1.57-1.42 (m,6H). 1.37-1.24 (m,2H),0.93-0.78 (m,12H).

[0209] Example 1.4: Synthesis of linker-toxin (LP62) [ka]

[0210] Step 1: Compound A3.5 (43 mg, 0.074 mmol) and Compound A2.3 (40 mg, 0.074 mmol) were dissolved in 1 mL of DMF, and then HBTU (28 mg, 0.075 mmol) and N,N-diisopropylethylamine (24 mg, 0.187 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 1 hour. After detecting completion of the reaction by LC-MS, the reaction mixture was directly purified by preparative chromatography (0.01% aqueous trifluoroacetic acid solution, acetonitrile) to obtain the trifluoroacetate salt of the target compound LP62 (8.5 mg, yield 10%), which was a yellow solid.

[0211] LC-MS (ESI) [M+H] + :1098.6: 1 H NMR (400MHz,DMSO-d6): δ 9.10 (s,2H),9.03 (s,1H),8.64 (t,J=6.4Hz,1H),8.19 (m,J=5.9Hz,1H),8.08 (d,J=7.4Hz,1H),7.92 (d,J=8.5Hz,1H),7.59 (s,1H),7.51 (s,1H),7.24 (s,1H),6.49 (s,1H),6.29 (s,2H),5.42 (s,2H),5.24 (s,2H),4.66-4.52 (m,2H),4.31-4.21 (m,1H),4.19-4.10 (m,1H),3.74 (d,J=5.5Hz,2H),3.49-3.48 (m,2H),3.40 (s,3H),3.15-3.06 (m,2H),3.02-2.95 (m,6H),2.59-2.52 (m,3H),2.41-2.29 (m,2H),2.50-1.90 (m,2H),1.91-1.77 (m,6H),1.63-1.57 (m,6H),1.31-1.29 (m,2H),0.92-0.80 (m,15H).

[0212] Example 2. Antibody preparation Unless otherwise specified, the anti-EGFR antibody used in this invention is MAB07, and its heavy and light chain amino acid sequences are described in the sequence listing.

[0213] Generally, the antibody was prepared as follows. A stable CHO cell line secreting and expressing MAB07 was constructed. The stable CHO cell line was cultured in an appropriate medium. The culture supernatant was harvested, and processes such as depth filtration, protein A column affinity chromatography, virus inactivation, filtration, anion chromatography, cation chromatography, and nanofiltration / ultrafiltration were performed in sequence. Finally, the target antibody was obtained.

[0214] Example 3. Production and Detection of Antibody-Drug Conjugate Example 3.1: Production of MAB07-LP62 [Chemical formula]

[0215] Here, n = 8 represents the molar ratio of the linker-loaded drug to the antibody.

[0216] Take 2 - 5 mL of anti-EGFR antibody MAB07 (Anti-EGFR_mAb, 15 - 25 mg / mL) and place it in a reaction tube. Add EDTA with a final concentration of 10 - 20 mM, and then adjust the pH to 6.0 - 7.5 with a 0.5 - 1 M disodium hydrogen phosphate solution. Slowly add 10 - 20 mM TCEP (tris(2-carboxyethyl)phosphine, 4 - 10 equivalents, 0.1 - 0.4 mL), mix uniformly, and react at room temperature for 50 - 80 minutes. After the MAB07 reduction reaction is completed, add a linker-toxin LP62 solution (4 - 12-fold equivalents, 1 - 8 μmol) dissolved in advance in dimethyl sulfoxide DMSO or DMAC solvent to the above solution system, stir, mix uniformly, and react at room temperature for 100 - 150 minutes. After purification by cation chromatography, the buffer of the system was replaced with 10 - 50 mM PB or histidine-hydrochloric acid histidine with Amicon Ultra-15, and the pH was adjusted to 5.5 - 7.5 to obtain the conjugate product MAB07-LP62 of MAB07 and linker-toxin LP62. Its DAR value and monomer content were verified by LC-MS, HIC, and HPLC-SEC analysis and met the standards.

[0217] Example 3.2: Manufacturing of the reference ADC Cetuximab-LP62. [ka]

[0218] Here, n=8 represents the molar ratio of the linker-loading drug to the antibody.

[0219] 2-5 mL of cetuximab (15-25 mg / mL) was placed in a reaction tube. EDTA with a final concentration of 10-20 mM was added, and then the pH was adjusted to 6.0-7.5 with a 0.5-1 M disodium hydrogen phosphate solution. 10-20 mM tris(2-carboxyethyl)phosphine (TCEP, 4-10 equivalents, 0.1-0.4 mL) was slowly added and mixed uniformly, and the reaction was allowed to proceed at room temperature for 50-80 minutes. After the cetuximab reduction reaction was complete, linker-toxin LP62 solution (4-12 equivalents, 1-8 μmol) pre-dissolved in dimethyl sulfoxide (DMSO) or DMAC solvent was added to the above solution system, stirred and mixed uniformly, and the reaction was allowed to proceed at room temperature for 100-150 minutes. After purification by cationic chromatography, the buffer solution in the system was replaced with 10-50 mM PB or histidine hydrochloride using Amicon Ultra-15 to adjust the pH to 5.5-7.5, yielding the conjugate product Cetuximab-LP62 of Cetuximab and the linker-toxin LP62. The DAR value and monomer content were verified by LC-MS, HIC, and HPLC-SEC analysis and met the standards.

[0220] Example 3.3: Manufacturing of Reference ADC MAB07-VC-MMAE [ka]

[0221] MAB07-VC-MMAE was prepared using a conventional, general-purpose method. Specifically, 2-5 mL of anti-EGFR antibody MAB07 (Anti-EGFR mAb, 15-25 mg / mL) was taken and placed in a reaction tube. 10-20 mM EDTA was added, and then the pH was adjusted to 6.0-7.5 with 0.5-1 M disodium hydrogen phosphate solution. 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 4-10 equivalents, 0.1-0.4 mL) was slowly added and mixed uniformly, and the reaction was allowed to proceed at room temperature for 50-80 minutes. After the MAB07 reduction reaction was completed, a solution of linker toxin MC-VC-PABC-MMAE (whose structure is as follows), pre-dissolved in dimethyl sulfoxide DMSO or DMAC solvent (4-12 equivalents, 1-8 μmol), was added to the above solution system, stirred, and mixed uniformly. The reaction was then allowed to proceed at room temperature for 100-150 minutes. The buffer solution of the system was replaced with 10-50 mM PB or histidine-histidine hydrochloride using Amicon Ultra-15 to adjust the pH to 5.5-7.5, yielding the conjugate product MAB07-VC-MMAE of MAB07 and linker toxin MC-VC-PABC-MMAE. Its DAR value and monomer content were verified by LC-MS, HIC, and HPLC-SEC analysis and met the standards.

[0222] [ka]

[0223] Example 3.4: Manufacturing of Reference ADC MAB07-GGFG-DXD [ka]

[0224] MAB07-GGFG-DXD was prepared using a conventional method. Specifically, 2-5 mL of anti-EGFR antibody MAB07 (Anti-EGFR mAb, 15-25 mg / mL) was taken and placed in a reaction tube. EDTA with a final concentration of 10-20 mM was added, and then the pH was adjusted to 6.0-7.5 with a 0.5-1 M disodium hydrogen phosphate solution. 10-20 mM TCEP (tris(2-carboxyethyl)phosphine, 4-10 equivalents, 0.1-0.4 mL) was slowly added and mixed uniformly, and the reaction was allowed to proceed at room temperature for 50-80 minutes. After the MAB07 reduction reaction was completed, a solution of linker-toxin MC-GGFG-DXD (whose structure is as follows), pre-dissolved in dimethyl sulfoxide DMSO or DMAC solvent (8-12 equivalents, 2-10 μmol), was added to the above solution system, stirred, and mixed uniformly. The mixture was then reacted at room temperature for 100-150 minutes. The buffer solution of the system was replaced with 10-50 mM PB or histidine-histidine hydrochloride using Amicon Ultra-15 to adjust the pH to 5.5-7.5, yielding the conjugate product MAB07-GGFG-DXD of MAB07 and linker-toxin MC-GGFG-DXD. The DAR value and monomer content were verified by LC-MS, HIC, and HPLC-SEC analysis and met the standards.

[0225] [ka]

[0226] Example 3.5: Size exclusion chromatography (HPLC-SEC) for monomer and conjugate aggregation analysis Size exclusion chromatography analysis was performed using an Agilent 1260 liquid chromatography system equipped with a TSKgel® G3000SWxL column (Tosoh Bioscience LLC, 7.8 mm × 30 cm, 5 μm pore size). Bare antibody or conjugate samples were eluted from the column for approximately 30–60 minutes at a flow rate of 0.5–1.0 mL / min using 0.1 M sodium phosphate containing 0.15 M sodium chloride and 10–20% isopropanol (pH 7.4). All data were analyzed using Agilent ChemStation software. The agglutination percentages of bare antibodies or conjugates were calculated as follows:

number

[0227] The calculated monomer purities of the conjugates are listed below.

[0228] [Table 2]

[0229] As the results show, the obtained anti-EGFR antibody-drug conjugates existed mainly in monomeric form and exhibited excellent anti-aggregation properties.

[0230] Example 3.6: Analysis of the drug-to-antibody ratio (DAR) of antibody-drug conjugates by LC-MS Antibody-drug conjugate samples were reduced by deconvolutional therapy (DTT) at 37°C for 10 minutes, and the DAR value of the conjugate was analyzed by LC-MS. Specifically, the reduced samples were injected into LC-MS systems (Agilent 1290 Infinity II LC system and Agilent 6545XT AdvanceBio Q-TOF) equipped with Acquity UPLC BEH C4 reversed-phase columns (Waters Corporation, 2.1 mm × 50 mm, 1.7 μm, 300 Å). The drug-to-antibody ratio (DAR) was calculated using the provided deconvolution LC-MS results.

[0231] [Table 3]

[0232] Example 4. Measurement of the affinity of anti-EGFR antibody-drug conjugate to EGFR-positive cells. Normal passaged A549 and SW480 tumor cells were collected separately, washed in PBS, resuspended, and divided into 5 × 10⁶ cells. 5 Cells were inoculated into 96-well plates at 50 μl / well. The test samples were then serially diluted equally with FACS buffer (PBS + 2% FBS). The diluted test samples and control samples were sequentially added to the 96-well plates inoculated with cells, where the control sample IgG1 was an isotype control (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.). The sample plates were incubated at 4°C for 0.5 hours, centrifuged, the supernatant removed, and washed with 300 μl / well of FACS buffer. Fluorescently labeled sheep anti-human IgG antibody detection solution was then sequentially added to the sample plates (100 μl / well) and incubated with the cells. The samples were incubated at 4°C for 0.5 hours, washed with 300 μl / well of FACS buffer, and finally the cells were resuspended. Fluorescence values ​​for each sample were detected by flow cytometry. Data were analyzed using Graphpad prism 8.1, and fitted with a four-parameter equation.

[0233] As shown in Figure 1A, regardless of whether the DAR was high (DAR8) or low (DAR4), the anti-EGFR antibody-drug conjugate MAB07-LP62 showed good affinity to EGFR-positive cells A549, and this affinity was equivalent to that of the unconjugated antibody. Similarly, as shown in Figure 1B, MAB07-LP62 (DAR8) also showed equivalent affinity to EGFR-positive cells SW480 compared to the unconjugated antibody.

[0234] Example 5. Measurement of internalization of anti-EGFR antibody-drug conjugate into EGFR-positive cells A certain volume of normally passaged A549 cells was taken, centrifuged, the supernatant was removed, and the cells were resuspended in cell medium (RPMI-1640 medium + 10% FBS). 50 μl of the cell suspension was inoculated into a 96-well plate and prepared for use (1 × 10⁶). 5 (Individual cells / well). pH value sensitive in cell culture medium. Zenon TM pHrodo TM iFL IgG labeling reagent (purchased from ThermoFisher) was prepared in 4x activator 1 (480 nM). The conjugate of this pH-sensitive dye and IgG does not fluoresce extracellularly but fluoresces brightly in an acidic environment (including lysosomes). The test material was prepared in cell medium in 4x activator 2 (160 nM). 25 μl each of activator 1 and activator 2 were placed in a 96-well plate, mixed, and incubated. The mixed liquid was aspirated into 50 μl of cell suspension, mixed uniformly, placed in an incubator, and cultured. Samples were taken at 0, 2, 16, and 24 h, centrifuged, and the supernatant was removed. The samples were then washed with 300 μl / well of FACS buffer, resuspended, and the fluorescence value of each sample was measured by flow cytometry.

[0235] As shown in Figure 2, compared to IgG1 isotype control data, the rate of internalization of MAB07-LP62 and MAB07 increased with increasing incubation time, reaching a peak at 16 hours, demonstrating that the anti-EGFR antibody-drug conjugate MAB07-LP62 can effectively bind to EGFR antigens on the cell surface and be internalized into the intracellular lysosomal pathway.

[0236] Example 6. Measurement of the cytotoxic activity of the anti-EGFR antibody-drug conjugate. Cells in the logarithmic growth phase were harvested, and the number of viable cells was counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to the appropriate concentration with culture medium. The cell suspension was added to a cell culture plate at a rate of 80 μl per well. The test sample (5-fold dilution) was serially diluted, and 20 μl of each diluted test sample was taken and added to the cell suspension in the cell culture plate. The cell culture plate was then placed in an incubator and cultured for 72 hours. After 72 hours, 50 μl of CellTiter-Glo solution (purchased from Promega), which had been pre-dissolved and equilibrated to room temperature according to the CellTiter-Glo instruction manual, was added to each well. The mixture was then homogenized for 2 minutes using a microwell plate shaker, left at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision2104 plate reader.

[0237] As shown in Figure 3, MAB07-LP62 showed strong cytotoxic activity against EGFR-positive tumor cells NCI-H1993 and EBC-1, which are not sensitive to MAB07.

[0238] Example 7. Comparison of cytotoxic activity of anti-EGFR antibody-drug conjugates. Human colon adenocarcinoma cell line SW480 in the logarithmic growth phase was harvested, and the number of viable cells was counted using a Vi-Cell XR cell counter. The cell suspension was adjusted to the appropriate concentration with culture medium. The cell suspension was added to a cell culture plate at a rate of 80 μl per well. The test sample (5-fold dilution) was serially diluted, and 20 μl of each diluted test sample was taken and added to the cell suspension in the cell culture plate. The cell culture plate was then placed in an incubator and cultured for 72 hours. After 72 hours, 50 μl of CellTiter-Glo solution (purchased from Promega), which had been pre-dissolved and equilibrated to room temperature according to the CellTiter-Glo instructions, was added to each well. The mixture was then homogenized for 2 minutes using a microwell plate shaker, left at room temperature for 10 minutes, and the fluorescence signal was measured using an Envision2104 plate reader.

[0239] As shown in Table 4, the in vitro cell-killing activity of MAB07-LP62 was superior to that of Cetuximab-LP62.

[0240] [Table 4]

[0241] Example 8. Cross-binding activity of anti-EGFR antibody-drug conjugates against EGFR derived from different species. The extracellular domain (ECD) of EGFR derived from humans, cynomolgus monkeys, rats, and mice was diluted in 1×PBS to a working concentration of 2 μg / mL and used as the ELISA coating solution. The test plates were washed three times with PBST (PBS containing 0.05% Tween20). The plates were incubated at room temperature in the blocking solution SuperBlock T20 (PBS) Blocking Buffer (60 μL / well), and the test samples were added to the test plates after being serially diluted 1:1 ratio, and incubated at room temperature. The test plates were washed, and HRP enzyme-labeled goat anti-human IgG, used as the secondary antibody, was added and incubated. The test plates were washed, stopped with stop solution (30 μL / well), centrifuged, and the light absorption at 450 nm wavelength was read. The raw data were analyzed using Graphpad prism 8.1 and fitted with a four-parameter equation.

[0242] As shown in Figure 4, the binding affinity of MAB07-LP62 to cynomolgus monkey EGFR ECD was consistent with that of human EGFR ECD, with EC50 values ​​of 0.074 nM and 0.033 nM, respectively. MAB07-LP62 (DAR8) showed weak specific binding to both rat and mouse EGFR ECD.

[0243] Example 9. Evaluation of the antitumor activity of anti-EGFR antibody-drug conjugate in a BALB / c nude mouse model of human epidermal carcinoma cell line A431 subcutaneous xenograft. A431 cells were cultured in growth medium (DMEM 1640 + 10% FBS). Cells in the logarithmic growth phase were harvested, resuspended in PBS and Matrixgel matrix, and used for subcutaneous inoculation of mice. Mice to be inoculated were 7-8 weeks old, and A431 cells (5 × 10⁶) were placed in the right dorsal region. 6 ( / animal) was subcutaneously inoculated. The average volume was 152.78 mm³. 3When the tumors had grown to a certain size, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three doses. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0244] As shown in Figure 5, on day 21 after administration, cetuximab-LP62 did not show tumor inhibitory activity (TGI -15.47%) compared to the control group (PBS). MAB07-LP62 showed tumor inhibitory activity (TGI 42.66%). This demonstrated that the in vivo antitumor activity of MAB07-LP62 was superior to that of cetuximab-LP62.

[0245] Example 10. Evaluation of the antitumor activity of anti-EGFR antibody-drug conjugate in a subcutaneous xenograft BALB / c nude mouse model of human non-small cell lung cancer cell line NCI-H1993. NCI-H1993 cells were cultured in growth medium (RPMI 1640 + 10% FBS). Cells in the logarithmic growth phase were harvested, resuspended in PBS and Matrixgel matrix, and used for subcutaneous inoculation of mice. Mice to be inoculated were 6-7 weeks old, and NCI-H1993 cells (1 × 10⁶) were placed in the right dorsal region. 7 ( / animal) was subcutaneously inoculated. The average volume was 199.56 mm³. 3 When the tumors had grown to a certain size, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three doses. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0246] As shown in Figure 6, on day 24 after administration, MAB07-LP62 (DAR4) (3 mg / kg and 8 mg / kg) and MAB07-LP62 (DAR8) (3 mg / kg and 8 mg / kg) both showed significant inhibition of tumor growth compared to the control group (MAB07), with TGI% of 47.0% (p=0.18), 81.5% (p<0.001), 53.4% ​​(p=0.0106), and 84.7% (p<0.001), respectively. Here, both MAB07-LP62 (DAR8) and MAB07-LP62 (DAR4) showed significant dose-dependent antitumor effects. Compared to conventional antibody-drug conjugates constructed using conventional conjugation techniques, MAB07-LP62 (DAR8) showed significantly superior antitumor activity compared to MAB07-VC-MMAE (DAR4) (day 24, 3 mg / kg TGI% 38.6%) and MAB07-GGFG-DXD (DAR8) (day 24, 3 mg / kg, TGI% 28.4%, 8 mg / kg 73.3%). None of the animals experienced severe weight loss throughout the entire treatment period.

[0247] Example 11. Evaluation of the antitumor activity of an anti-EGFR antibody-drug conjugate in a subcutaneous xenograft BALB / c nude mouse model of human non-small cell lung cancer cell line EBC-1. EBC-1 cells were cultured in growth medium (MEM + 10% FBS + 1% NEAA). Cells in the logarithmic growth phase were harvested, resuspended in PBS and Matrixgel matrix, and used for subcutaneous inoculation of mice. Mice to be inoculated were 7-8 weeks old, and EBC-1 cells (3 × 10⁴) were placed in the right dorsal region. 6 ( / animal) was subcutaneously inoculated. The average volume was 200.41 mm³. 3 When the tumors had grown to a certain size, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three doses. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3) = 1 / 2 × (a × b²) (where a represents the major axis and b represents the minor axis).

[0248] As shown in Figure 7, on day 17 after administration, MAB07-LP62 (DAR4) (3 mg / kg and 8 mg / kg), MAB07-LP62 (DAR8) (3 mg / kg and 8 mg / kg), MAB07-GGFG-DXd (DAR8) (8 mg / kg), and MAB07-VC-MMAE (DAR4) (3 mg / kg) all showed significant tumor inhibitory effects compared to the control group (MAB07). Their TGI% were 67.06% (p=0.00126), 96.58% (p<0.001), 97.83% (p<0.001), 100.00% (p<0.001), 82.12% (p<0.001), and 99.21% (p<0.001), respectively. All of the test drugs in this experiment showed dose-dependent antitumor effects. Compared to other investigational drugs, MAB07-LP62 (DAR8) achieved a complete response (CR) in the tumor model. In contrast, the other investigational drugs all resulted in varying degrees of tumor growth during the 4-week observation period after the end of administration. This demonstrated that MAB07-LP62 (DAR8) has an extremely strong tumor inhibitory effect. None of the animals experienced significant weight loss throughout the entire treatment period.

[0249] Example 12. Evaluation of the antitumor effect of an anti-EGFR antibody-drug conjugate in a HuPrime® lung cancer LU3075 subcutaneous xenograft female BALB / c nude mouse model. Tumor tissue was collected from HuPrime® lung cancer xenograft model LU3075 tumor-bearing mice and cut into tumor masses with a diameter of 2-3 mm. The obtained tumor masses were subcutaneously inoculated into the right anterior scapula of 8-9 week old BALB / c nude mice. The average volume was 181.60 mm³. 3When the tumors had grown to a certain size, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for three doses. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0250] As shown in Figure 8, on day 28 after administration, MAB07-LP62 (DAR8) showed strong tumor inhibitory effects at doses of 1 mg / kg (TGI 79.88%), 3 mg / kg (TGI 76.55%), and 8 mg / kg (TGI 91.47%). Furthermore, the combination group of 3 mg / kg MAB07-LP62 (DAR8) and 20 mg / kg osimertinib (day 28, TGI 93.80%) showed superior efficacy compared to MAB07-LP62 (DAR8) alone (day 28, TGI 76.55%) or osimertinib alone (day 28, TGI 61.00%). The TGI on day 28 after administration of 8 mg / kg MAB07 was 35.20%. This example demonstrated the superiority of MAB07-LP62 in combination with a small molecule inhibitor (e.g., osimertinib). None of the animals experienced significant weight loss throughout the entire treatment period.

[0251] Example 13. Evaluation of the antitumor effect of anti-EGFR antibody-drug conjugate in a human lung cancer PDX (patient-derived xenograft tumor) transplant tumor NCG mouse model. A human lung cancer LD1-0025-200717 PDX model with osimertinib resistance and EGFR exon19del / T790M / C797S mutation was resuscitated in NCG mice, with tumors measuring 500-800 mm. 3When the tumor tissue had grown to a certain extent, it was surgically removed aseptically, and non-tumor tissue and necrotic tissue were removed. The tumor tissue was dissected and uniformly cut into tissue pieces of approximately 3 mm x 3 mm x 3 mm, and then small pieces of tumor tissue were inoculated subcutaneously into the right side of the back of NCG mice. Six days after inoculation, PBMC 5 x 10 per mouse 6 Each dose was administered by intraperitoneal injection. The average volume was 110 mm³. 3 When the tumors had grown to a certain extent, the mice were randomly divided into groups according to tumor size and administered the treatment to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the treatment was administered either as a single dose or for three weeks. Clinical symptoms observed during the experiment were recorded. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0252] As shown in Figure 9, in the osimertinib-resistant lung cancer PDX model LD1-0025-200717, the IgG1-LP62 TGI% was 58.1% (p<0.0001) compared to the PBS group. Osimertinib and serplulimab did not have a significant tumor inhibitory effect, while MAB07-LP62 (DAR8) caused sustained tumor reduction in both single doses and combination therapy (in combination with osimertinib or serplulimab) at doses of 1 mg / kg, 3 mg / kg, and 8 mg / kg. None of the animals experienced significant weight loss throughout the treatment period.

[0253] Example 14. Evaluation of the antitumor activity of anti-EGFR antibody-drug conjugate in a PBMC immune system humanized mouse NCG mouse model with human colon cancer HT-29 cell allograft. HT-29 cells were cultured in a growth medium (McCoy’s 5A + 10% FBS). Human PBMCs were inoculated into NCG mice to complete the reconstruction of the mouse immune system. After 3 weeks, HT-29 cells in the logarithmic growth phase were collected, resuspended in PBS and Matrixgel matrix, and used for subcutaneous inoculation of mice. The mice to be inoculated were 6 - 8 weeks old and were subcutaneously inoculated with HT-29 cells (3×10 6 / mouse) on the right back. When the tumor grew until the average volume reached 140.5 mm 3 , the mice were randomly grouped according to the tumor size for administration, ensuring that the tumor volumes between different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, administered once a week, and administered three times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated by the following formula. Tumor volume (mm 3 ) = 1 / 2×(a×b 2 ) (where a represents the long diameter and b represents the short diameter).

[0254] As shown in Figure 10, on the 17th day after administration, compared with the control group (PBS), both MAB07-LP62 (DAR8) (1 mg / kg and 3 mg / kg) showed significant tumor growth inhibition and dose-dependent antitumor effects, and their TGI% were 49.3% (p = 0.0017) and 94.6% (p < 0.001), respectively. In addition, the combination group of 10 mg / kg serplulimab + 1 mg / kg MAB07-LP62 (DAR8) showed better tumor growth inhibition than 10 mg / kg serplulimab or 1 mg / kg MAB07-LP62 (DAR8), and their TGI% were 82.1%, 18.5% and 49.3%, respectively. It was shown that the combination of serplulimab and MAB07-LP62 (DAR8) had a significant synergistic effect. All animals did not show a significant decrease in body weight throughout the treatment period.

[0255] Example 15. Evaluation of the antitumor effect of an anti-EGFR antibody-drug conjugate in a human head and neck squamous cell carcinoma LD1-2023-411020 PDX transplanted tumor NCG mouse model. NCG mice were used to resuscitate a human head and neck squamous cell carcinoma (PDX) model, with tumors measuring 500-800 mm. 3 When the tumor tissue had grown to a certain size, it was dissected and uniformly cut into tissue clumps of approximately 3 mm x 3 mm x 3 mm. These small clumps of tumor tissue were then inoculated subcutaneously into the right dorsal side of NCG mice. The average volume was 138.88 mm³. 3 When the tumors had grown to a certain extent, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for a total of three doses. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0256] As shown in Figure 11, on day 21, MAB07-LP62 (DAR8) at a dose of 8 mg / kg showed a strong tumor inhibitory effect, with a TGI of 121.62% (p<0.001). This demonstrated that MAB07-LP62 (DAR8) has an extremely strong tumor inhibitory effect. None of the animals experienced a significant decrease in body weight throughout the entire treatment period.

[0257] Example 16. Evaluation of the antitumor effect of anti-EGFR antibody-drug conjugate in a human gastric cancer LD1-0017-411335 PDX transplanted tumor NU / NU mouse model. Human gastric cancer PDX model resistant to DS-8201 (fam-trastuzumab deruxtecan-nxki, trade name ENHERTU®) was resuscitated in NU / NU mice, with tumors measuring 500-800 mm. 3 When the tumor tissue had grown to a certain size, it was dissected and uniformly cut into tissue clumps of approximately 3 mm x 3 mm x 3 mm. These small clumps of tumor tissue were then inoculated subcutaneously into the right dorsal side of NU / NU mice. The average volume was 176.58 mm³. 3When the tumor grew to a certain extent, it was randomly grouped according to tumor size and administered to ensure that the tumor volumes among different groups were similar. The day of grouping was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, administered once a week, and administered 4 times. The clinical symptoms observed during the experiment were recorded. The tumor volume was calculated by the following formula. Tumor volume (mm 3 ) = 1 / 2×(a×b 2 )(where a represents the long diameter and b represents the short diameter).

[0258] As shown in Figure 12, on day 28, MAB07-LP62 (DAR8) showed strong tumor inhibitory effects at both doses of 3 mg / kg and 8 mg / kg, and the TGI was 87.04% (p = 0.0003) and 98.01% (p < 0.0001), respectively. In contrast, naked anti-MAB07 did not show significant antitumor activity at 8 mg / kg, and the TGI was 27.59% (p = 0.1976), while the TGI of the free-loaded drug A1.3 was 30.68%. It was shown that MAB07-LP62 (DAR8) had an extremely strong tumor inhibitory effect. All animals did not show severe weight loss throughout the treatment period.

[0259] Example 17. Evaluation of the antitumor effect of an anti-EGFR antibody-drug conjugate in a subcutaneous xenograft NOD / SCID mouse model of human-derived esophageal cancer KYSE-150 cell line KYSE-150 cells were cultured in a growth medium (RPMI 1640 / F12 + 2% FBS). Cells in the logarithmic growth phase were collected, resuspended in PBS and Matrixgel matrix, and used for subcutaneous inoculation of mice. The mice to be inoculated were 7 - 8 weeks old, and KYSE-150 cells (1×10 7 / mouse) were subcutaneously inoculated on the right back. The average volume was 199.66 mm 3When the tumors had grown to a certain size, the mice were randomly divided into groups according to tumor size and administered the drug to ensure similar tumor volumes between different groups. The day of group division was defined as day 0. After the start of administration, the body weight and tumor size of the mice were measured twice a week, and the drug was administered once a week for a total of three doses. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the major axis and b represents the minor axis.)

[0260] As shown in Figure 13, on day 21, MAB07-LP62 (DAR8) showed dose-dependent tumor inhibitory effects in the 1 mg / kg, 3 mg / kg, and 8 mg / kg dose groups, with TGIs of 28.6% (p=0.482), 94.5% (p<0.001), and 97.9% (p<0.001), respectively. Naked anti-MAB07 showed only partial antitumor activity at 8 mg / kg, with a TGI of 66.4% (p<0.001). This demonstrated that MAB07-LP62 (DAR8) has an extremely strong tumor inhibitory effect. None of the animals experienced severe weight loss throughout the entire treatment period.

[0261] In addition to the illustrated and claimed embodiments, the disclosed themes further cover other embodiments having other combinations of the features disclosed and claimed herein. Thus, certain features presented herein may be combined with each other in other ways within the scope of the disclosed themes, such that the disclosed themes include any suitable combination of the features disclosed herein. The above description of specific embodiments of the disclosed themes is provided for illustrative and explanatory purposes only. The above description is not intended to be exhaustive or to limit the disclosed themes to those disclosed embodiments.

[0262] It will be apparent to those skilled in the art that various modifications and changes can be made to the structure and methods of the disclosed theme without departing from the spirit or scope of the disclosed theme. Accordingly, the disclosed theme is intended to include modifications and changes that fall within the scope of the appended claims and their equivalents.

[0263] Various publications, patents, and patent applications are incorporated herein by reference, and their contents are incorporated herein by reference as a whole.

Claims

1. An antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a pharmaceutically acceptable salt solvate thereof, wherein the antibody-drug conjugate has the structure shown in formula (I), TP-[L1-L2-L3-D] ｋ (I) Here, TP is a target region that selectively binds to or recognizes EGFR. L1 is an extension unit that connects TP and L2. L2 is an optional amino acid residue or a short-chain peptide consisting of 2 to 10 amino acid residues. L3 is a spacer element, D is a bioactive molecule, k represents any numerical value between 0.1 and 10.0, and the antibody-drug conjugate, its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

2. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to claim 1, wherein the TP is an antibody or an antigen-binding fragment thereof.

3. The aforementioned antigen-binding fragments include full-length immunoglobulin, single-stranded Fv (scFv) fragment, Fab fragment, Fab' fragment, and F(ab'). ２ Fv fragment, Fv fragment with stable disulfide bond (dsFv), (dsFv) ２ An antibody-drug conjugate according to claim 2, selected from Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, triabody, tetrabody or any combination thereof, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

4. The TP is a humanized antibody or an antigen-binding fragment thereof, the antibody-drug, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

5. The TP comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 included in SEQ ID NO: 7, and the light chain variable region (VL) comprises a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 included in SEQ ID NO: 8, wherein the antibody-drug conjugate, its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims.

6. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to claim 5, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO: 2, the amino acid sequence of HCDR3 is SEQ ID NO: 3, and the amino acid sequence of LCDR1 is SEQ ID NO: 4, the amino acid sequence of LCDR2 is SEQ ID NO: 5, and the amino acid sequence of LCDR3 is SEQ ID NO:

6.

7. The TP comprises the Fc sequence of human IgG, the antibody-drug conjugate according to any one of the claims, the prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

8. The IgG is selected from IgG1, IgG2, IgG3 and IgG4, and is an antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims.

9. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims, wherein the TP comprises a heavy chain variable region (VH), and the amino acid sequence of the heavy chain variable region (VH) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

7.

10. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate of a pharmaceutically acceptable salt, or a solvate of a pharmaceutically acceptable salt according to any one of the claims, wherein the TP comprises a light chain variable region (VL), and the amino acid sequence of the light chain variable region (VL) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

8.

11. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims, wherein the TP comprises a heavy chain (HC), and the amino acid sequence of the heavy chain (HC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

9.

12. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims, wherein the TP comprises a light chain (LC), and the amino acid sequence of the light chain (LC) is at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:

10.

13. The TP is a multispecific antibody, the antibody-drug conjugate according to any one of the claims, the prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

14. The TP is an afucosylated antibody, the antibody-drug conjugate according to any one of the claims, the prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

15. The Fc region comprises a C-terminal lysine-containing antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims.

16. The Fc region comprises a deletion of C-terminal lysine, the antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

17. The aforementioned L1 is selected from the following: 【Chemistry 1】 Herein, L1 is linked to TP via an S atom at position a and linked to L2 at position b, m is any integer between 1 and 10, n is any integer between 1 and 10, and X and Y are independently C, O, S, or N, an antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims.

18. The aforementioned L1 is as follows: 【Chemistry 2】 Herein, L1 is linked to TP via an S atom at the a position and to L2 at the b position, and m is selected from 1, 2, 3, 4, 5, the antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

19. The aforementioned L1 is as follows: 【Transformation 3】 Herein, L1 is linked to TP via an S atom at the a position and to L2 at the b position, the antibody-drug, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

20. The aforementioned L2 is selected from the following: 【Chemistry 4】 Herein, L2 is linked to L1 at the c position and to L3 at the d position, the antibody-drug, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

21. The aforementioned L2 is as follows: 【Transformation 5】 Herein, L2 is linked to L1 at the c position and to L3 at the d position, the antibody-drug, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims.

22. The aforementioned L3 is a single bond, or is selected from the following groups: 【Transformation 6】 Here, L3 is connected to L2 at position e and to D at position f. p and q are independent integers between 0 and 10. W is C, O, S, or N. Z is hydrogen, halogen, C １ -C ２０ alkane, C １ -C ２０ haloalkane, cyano group, sulfonic acid, carboxylic acid, C １ -C ２０ alkoxy group, C ２ -C １０ an unsaturated alkane, a natural amino acid, an unnatural amino acid, and a short-chain peptide composed of the above amino acids, the antibody-drug conjugate according to any one of the preceding claims, its prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

23. Z is selected from the following: 【Transformation 7】 Here, Z is connected to the remainder of L3 at position g, z1 to z9 are each independent integers between 0 and 10, and T is NH ２ An antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, according to any one of the claims, or an OH.

24. The D is a cytotoxic agent, an antibody-drug conjugate according to any one of the claims, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt.

25. The aforementioned D is selected from the following: 【Transformation 8】 Here, D is an antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims, linked to L3 at the h position.

26. The antibody-drug conjugate is 【Chemistry 9】 【Chemistry 10】 Selected from, Here, Ab is an antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt according to any one of the claims, representing the antibody or its antigen-binding fragment.

27. An antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt according to any one of the claims, wherein k is about 4.0 to about 8.0, preferably k is about 4.0, about 6.0, or about 8.

0.

28. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

29. An antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28, used as a drug.

30. An antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28, used for treating or preventing a tumor or cancer.

31. The tumor or cancer is an EGFR-related tumor or cancer, the antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing the tumor or cancer according to claim 30.

32. The tumor or cancer is selected from epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, gastric cancer, pancreatic cancer, and glioma, and the antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing the tumor or cancer according to claim 30.

33. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing the tumor or cancer according to claim 32, wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

34. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing the tumor or cancer according to claim 32, wherein the lung cancer is osimertinib-resistant lung cancer.

35. The antibody-drug conjugate, prodrug, pharmaceutically acceptable salt, solvate, or solvate of a pharmaceutically acceptable salt or pharmaceutical composition for treating or preventing the tumor or cancer according to claim 32, wherein the gastric cancer is ENHERTU-resistant gastric cancer.

36. An antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt or a pharmaceutical composition for treating or preventing the tumor or cancer according to any one of claims 30 to 35, wherein the tumor or cancer is a tumor or cancer of a KRAS gene mutation and / or a BRAF gene mutation.

37. An antibody-drug conjugate, a prodrug, a pharmaceutically acceptable salt, a solvate or a solvate of a pharmaceutically acceptable salt or a pharmaceutical composition for treating or preventing the tumor or cancer according to claim 36, wherein the tumor or cancer of the KRAS gene mutation and / or BRAF gene mutation is colon cancer, rectal cancer, lung cancer or pancreatic cancer of the KRAS gene mutation and / or BRAF gene mutation.

38. A product or kit comprising an antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28 and instructions for use.

39. A method for preventing or treating a tumor or cancer, comprising administering to a subject in need thereof a prophylactic or therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28.

40. The method according to claim 39, wherein the tumor or cancer is a tumor or cancer related to EGFR.

41. The method according to claim 39, wherein the tumor or cancer is selected from epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, and glioma.

42. The method according to claim 41, wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

43. The method according to claim 41, wherein the lung cancer is osimertinib-resistant lung cancer.

44. The method according to claim 41, wherein the gastric cancer is ENHERTU-resistant gastric cancer.

45. The method according to any one of claims 39 to 44, wherein the tumor or cancer is a tumor or cancer of a KRAS gene mutation and / or a BRAF gene mutation.

46. The method according to claim 45, wherein the tumor or cancer of the KRAS gene mutation and / or BRAF gene mutation is colon cancer, rectal cancer, lung cancer, or pancreatic cancer of the KRAS gene mutation and / or BRAF gene mutation.

47. The method according to any one of claims 39 to 46, wherein the method comprises administering an additional therapeutic agent to the subject, the therapeutic agent being selected from antitumor agents, chemotherapeutic agents, proliferation inhibitors and cytotoxic agents.

48. The method according to claim 47, wherein the additional therapeutic agent is osimertinib.

49. The method according to claim 47, wherein the additional therapeutic agent is a TKI inhibitor or a PD1 / PDL1 inhibitor.

50. The method according to claim 47, wherein the additional therapeutic agent is an anti-PD1 antibody.

51. The method according to claim 50, wherein the anti-PD1 antibody is celpullimab.

52. The method according to any one of claims 39 to 51, further comprising administering radiotherapy to the subject.

53. Uses of an antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate or solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28 in the manufacture of a drug for treating or preventing tumors or cancer.

54. Uses in the manufacture of drugs for treating or preventing EGFR-related diseases or symptoms or osimertinib-resistant tumors or cancers, of an antibody-drug conjugate according to any one of claims 1 to 27, its prodrug, a pharmaceutically acceptable salt, a solvate or solvate of a pharmaceutically acceptable salt, or a combination of the pharmaceutical composition according to claim 28 and an additional therapeutic agent.

55. The use according to claim 53 or 54, wherein the additional therapeutic agent is osimertinib or an anti-PD1 antibody.

56. The use according to claim 55, wherein the anti-PD1 antibody is celpullimab.

57. A method for preventing or treating osimertinib-resistant tumors or cancers, comprising administering to a subject in need thereof a prophylactic or therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 27, a prodrug thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a pharmaceutically acceptable salt, or a pharmaceutical composition according to claim 28.

58. The method according to claim 57, wherein the tumor or cancer is selected from epidermal cancer, colon cancer, rectal cancer, head and neck cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, and glioma.

59. The method according to claim 58, wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

60. The method according to any one of claims 57 to 59, wherein the method comprises administering an additional therapeutic agent to the subject, the additional therapeutic agent being selected from an antitumor agent, a chemotherapeutic agent, a proliferation inhibitor, and a cytotoxic agent.

61. The method according to claim 60, wherein the additional therapeutic agent is osimertinib.

62. The method according to claim 60, wherein the additional therapeutic agent is a TKI inhibitor or a PD1 / PDL1 inhibitor.

63. The method according to claim 60, wherein the additional therapeutic agent is an anti-PD1 antibody.

64. The method according to claim 63, wherein the anti-PD1 antibody is celpullimab.

65. The method according to any one of claims 57 to 64, further comprising administering radiotherapy to the subject.

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