Antibody drug conjugate platform using bispecific antibodies

Abstract

The present invention relates to an antibody drug conjugate using a bispecific antibody and use thereof. According to the antibody drug conjugate of the present invention, by binding a conjugate of a bivalent calicheamicin peptide and a drug to an anti-calicheamicin single chain variable fragment, a complex of an antibody and a drug can be easily formed without a multi-step synthetic process. In addition, the antibody drug conjugate of the present invention can effectively deliver a drug to a target to which an antibody specifically binds, and can enhance a therapeutic effect by increasing a half-life of a drug in vivo.

Description

Technical Field

[0001] This invention relates to antibody-drug conjugates using bispecific antibodies and their uses.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 352804, filed June 21, 2016, the entire contents of which are disclosed in the specification and drawings of which are incorporated herein by reference. Background Technology

[0004] Antibody-drug conjugates (ADCs) are a novel class of anticancer agents and have been developed for the selective delivery of cytotoxic agents to tumor cells expressing antigens. Traditional ADCs offer many advantages, including high applicability and the use of site-specific binding methods. However, the multi-step binding process required to conjugate cytotoxic agents to antibodies, along with the need to optimize the process for individual antibodies, makes ADCs difficult to use.

[0005] Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the ErbB family. EGFR overexpression is frequently observed in various human cancers, including head and neck cancer, colorectal cancer, lung cancer, breast cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, and bladder cancer. In cancer treatment, two pharmacological approaches have been used to block EGFR signaling: monoclonal antibodies (cetuximab (Erbitux) and panitumumab (Vetibix)) and small molecule tyrosine kinase inhibitors (gefitinib (Iressa) and erlotinib (Tarceva)). However, mutations in EGFR and its downstream signaling molecules have led to poor clinical responses and difficulties in EGFR-targeted therapy. For example, KRAS mutations affect primary resistance to EGFR-targeted therapy in patients with EGFR-positive cancers. KRAS mutations have been found in 25% of non-small cell lung cancer (NSCLC) and 39% of colorectal cancer cases. Although KRAS has been identified as one of the common oncogene aberrations in human cancers, there is currently no effective treatment.

[0006] On the other hand, ADCs can be effective against EGFR-positive cancers with KRAS mutations. ADCs can be prepared by crosslinking cytotoxic agents to monoclonal antibodies using linkers. ADCs can overcome the therapeutic limitations of traditional antibodies or nonspecific cytotoxic agents. For example, two ADCs (Bentuximab (Adcetris) and Trastuzumab-Kadcyla) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of lymphoma and human epidermal growth factor receptor-2 (Her2)-positive metastatic breast cancer, respectively.

[0007] Traditional conjugation of small molecules to antibodies uses ε-amino acid chains on lysine residues or deoxy disulfide bonds on cysteine ​​residues. Nonspecific conjugation results in heterogeneous ADC mixtures with variable drug-antibody ratios (DARs) and conjugation site positions. This heterogeneity can affect the solubility, stability, pharmacokinetics, and batch-to-batch variability of ADCs.

[0008] To avoid the heterogeneity of ADCs as described above, site-specific coupling methods using intentionally inserted cysteine ​​residues or non-natural amino acid residues, or enzymatic coupling, have been investigated. Furthermore, the binding activity, stability, and coupling efficiency of ADCs vary with different coupling sites. Therefore, to maximize the properties of ADCs, methods are needed to find suitable coupling residues and optimize them for each antibody. Typically, ADCs require multi-step, complex coupling processes and sophisticated analyses to validate the final product. As a result, there are many difficulties in developing conventional ADCs using site-specific coupling. Summary of the Invention

[0009] Technical issues

[0010] The problem this invention aims to solve is to provide a new antibody-drug conjugate platform that overcomes the shortcomings of traditional antibody-drug conjugates.

[0011] In addition, the present invention aims to provide a pharmaceutical composition comprising the antibody-drug conjugate platform and a method for treating diseases using it.

[0012] Technical solution

[0013] The present invention provides an antibody-drug conjugate platform comprising a bispecific antibody containing an anti-cotinine single-chain variable fragment (scFv); and a conjugate of divalent cotinine and a drug crosslinked with a peptide.

[0014] The inventors of this invention conducted in-depth research to address the drawbacks of traditional antibody-drug conjugates (ADCs) requiring multi-step binding processes and antibody optimization. As a result, they developed a new antibody-drug conjugate platform that uses a tetravalent bispecific antibody that simultaneously binds to a cytotoxic agent conjugated to human EGFR and a hapten.

[0015] First, the inventors selected a previously reported tetravalent bispecific antibody form and developed a bispecific antibody that reacts with human EGFR and cotinine. The bispecific antibody of this invention uses an anti-cotinine single-chain variable fragment (scFv) to form a complex with cotinine. Cotinine is a major metabolite of nicotine and, due to its exogenous nature, physiological inertness, and non-toxicity, can be used as an ideal hapten in clinical applications.

[0016] In one example of the invention, the bispecific antibody (ERC6) of the present invention is formed comprising a human EGFR-binding antibody (cetuximab). Then, a cross-linked divalent cotinine-binding peptide (cotinine-cetuximab) is prepared using duocarmycin, and an antibody-drug conjugate is formed by mixing it with the bispecific antibody. As a result, the inventors of the present invention have developed an antibody-drug conjugate platform consisting of a tetravalent conjugate comprising bispecific cetuximab × anti-cotinine antibody and a divalent cotinine-binding peptide cross-linked with duocarmycin (cotinine-cetuximab). Figure 1a The antibody-drug conjugate platform of this invention demonstrated significant antitumor activity against EGFR-positive cetuximab-refractory lung adenocarcinoma with KRAS mutations in both in vitro and in vivo models. These experimental results suggest that the ADC platform of this invention, using bispecific antibodies, could be a useful delivery tool for treating diseases such as cancer.

[0017] The anti-cotinine single-chain variable fragment (scFv) of the bispecific antibody of the present invention can form an antibody-drug conjugate by specifically binding to the divalent cotinine of a drug-conjugated divalent cotinine peptide. The nucleotide sequence of the anti-cotinine single-chain variable fragment can consist of the heavy chain of SEQ ID NO:1 and the light chain of SEQ ID NO:2. Furthermore, the amino acid sequence of the anti-cotinine single-chain variable fragment can consist of the heavy chain of SEQ ID NO:3 and the light chain of SEQ ID NO:4. Additionally, the amino acid sequence (Gly-Gly-Gly-Gly-Ser) n (n is an integer from 1 to 5), preferably (Gly-Gly-Gly-Gly-Ser)4 can be inserted between the heavy chain and light chain sequences of the anti-cotinine single-chain variable fragment. Thus, it can help the expressed anti-cotinine single-chain variable fragment to properly perform antigen-antibody reactions.

[0018] Preferably, the bispecific antibody of the present invention can be used in C H A glycine- and serine-rich peptide linker (Gly-Gly-Gly-Gly-Ser) is inserted between the 3-domain and the anti-cotinine single-chain variable fragment (scFv). n (n is an integer from 1 to 5), preferably (Gly-Gly-Gly-Gly-Ser)3. Therefore, the bispecific antibody of the present invention can improve flexibility.

[0019] In this article, divalent cotinine can be two cotinines that are crosslinked to the N-terminus and C-terminus of a 6-18 peptide, respectively. Preferably, when cotinine is coupled to a peptide, the cotinine can be carboxycotinine (trans-4-cotinine carboxylic acid), and crosslinking with the peptide can be effectively achieved through its carboxyl group.

[0020] In this document, peptides of 6 to 18 lengths can be used as crosslinking peptides with two cotinines. The peptides can consist of one or more peptides selected from the group consisting of glycine (G), serine (S), and lysine (K). Preferably, GSKGSK, GGGGSKGGGGSK, GSKGSKGSKGSKK, or GGGSGGGSKGGGSGGGSK can be used as peptides. More preferably, GSKGSKGSKGSKK can be used as a peptide, and it can be stably conjugated to the drug of the present invention (e.g., tebuconazole).

[0021] In this paper, the conjugation of divalent cotinine and the drug can be achieved by conjugating the drug to the ε-amino group of the lysine residue in the 6-18 peptide crosslinked with divalent cotinine.

[0022] The drug used in the antibody-drug conjugate of the present invention can be any drug selected from the group consisting of: auristatin, colchicine, anthracycline, caricimonic acid, maytansinoid, pyrrolobenzodiazepine, salivarius toxin, tubulolysin, maytansin, doxorubicin, novozymin, epothilone, safranin, deacetylated colchicine, maytansin, vedotin, mafodotin, metansine, ravtansine, soravtansine, talirine, tesirine, indoline benzodiazepine, irinotecan prodrug, eczetine derivatives, and tubulin inhibitors, etc., which are cytotoxic agents. Preferably, the antibody-drug conjugate of the present invention may contain auristatin or metansin. Due to its strong DNA alkylation activity, pyruvic acid can be used as an effective cytotoxic drug to kill cancer cells. For example, when pyruvic acid forms a drug conjugate by binding to a peptide, it can be used in the form of dimethylaminoethyl pyruvic acid linked to four valine-citrulline PAB-. Alternatively, for example, when pyruvic acid forms a drug conjugate by binding to a peptide, it can bind via an MCC (cyclohexane-1-carboxylic acid maleimide methyl ester) linker. In an experimental example of the invention, experiments were conducted using pyruvic acid (… Figure 3e ) or Metannew ( Figure 6b The antibody-drug conjugates of this invention exhibited cytotoxicity against cancer cells and, in both cases, showed significant inhibitory activity against A549 cells. These results demonstrate the versatility of the antibody-drug conjugate platform of this invention in binding to the aforementioned drugs.

[0023] Furthermore, the drug used in the antibody-drug conjugate of the present invention can be siRNA that inhibits the expression of genes involved in cancer. For example, the siRNA can be selected from siRNAs that inhibit the expression of one or more genes selected from the group consisting of: Mcl-1, Wnt-1, Hec1, Survivin, Livin, Bcl-2, XIAP, Mdm2, EGF, EGFR, VEGF, VEGFR, GASC1, IGF1R, Akt1, Grp78, STAT3, STAT5a, β-catenin, WISP1, c-myc, RRM2, KSP, PKN3, PLK1, KRAS, MYC, and EPHA2, etc. Here, the siRNA inhibits the expression of specific genes expressed in cancer cells through ribonucleic acid-mediated interference (RNA-mediated interference, RNAi) to kill cancer cells; therefore, antibody-drug conjugates containing siRNA can be used as excellent anticancer agents.

[0024] In this paper, the conjugation of siRNA and cotinine-peptide can be achieved through a linker. Therefore, the antibody-drug conjugate of the present invention has the versatility to bind various types of siRNA to the conjugate. For example, the linker can be an SMCC (succinimide-trans-4-(maleimide-methyl)cyclohexane-1-carboxylate) linker or a valine-citrulline-PAB linker. Specifically, when siRNA binds to cotinine-peptide via the SMCC linker, the 5' or 3' end of the siRNA can be modified and cross-linked with the SMCC linker, and it can be conjugated to cotinine-GSCGSCGSCGSCK-cotinine (C=cysteine). Additionally, when siRNA binds to cotinine-peptide via the valine-citrulline-PAB linker, the siRNA can be conjugated to cotinine-GSKGSKGSKGSKK-cotinine via this linker.

[0025] The bispecific antibody included in the antibody-drug conjugate of the present invention may include any one selected from the group consisting of: cetuximab, trastuzumab, ovovacumab, ezetoxumab, alemtuzumab, labetuzumab, bevacizumab, tiimomab, oflamumab, panitumumab, rituximab, tosimob, ipilimumab, gemtruzumab, brentuximab, vadastuximab, and glembat. umumab), depatuxizumab, polatuzumab, denintuzumab, enfortumab, telisotuzumab, tisotumab, pinatuzumab, lidostuzumab, indusatumab, vantosu Vandortuzumab, Sofituzumab, Vorsetuzumab, Trastuzumab, Mirvetuximab, Coltuximab, Naratuximab, Indatuximab, Anetumab, Lovotuzumab, Cantuzumab The antibody-drug conjugates of the present invention may include cetuximab, laprituximab, bivatuzumab, vataximab, lovatuzumab, inotuzumab, sacituzumab, labetuzumab, milatuzumab, lupartumab, and aprutumab. Preferably, the antibody-drug conjugates of the present invention may include human EGFR-binding antibody (cetuximab). Cetuximab is an FDA-approved monoclonal antibody for the clinical treatment of non-small cell carcinoma, metastatic colorectal cancer, or head and neck squamous cell carcinoma (HNSCC). The nucleotide sequence of cetuximab may consist of the heavy chain of SEQ ID NO:5 and the light chain of SEQ ID NO:6. In addition, the amino acid sequence of cetuximab can be composed of the heavy chain of SEQ ID NO:7 and the light chain of SEQ ID NO:8.

[0026] Furthermore, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate for treating cancer. The cancers for which the antibody-drug conjugate of the present invention exhibits therapeutic efficacy can be one or more selected from the group consisting of: head and neck cancer, colon cancer, lung cancer, breast cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, brain cancer, or bladder cancer, etc. Preferably, the antibody-drug conjugate of the present invention, comprising a tetravalent conjugate containing bispecific cetuximab × anti-cotinine and a divalent cotinine-binding peptide crosslinked with pyruvicin (cotinine-pyruvicin), exhibits significant therapeutic efficacy against KRAS-mutant lung adenocarcinoma with primary resistance to EGFR-targeted cetuximab therapy. Preferably, the total amino acid sequence of the antibody-drug conjugate comprising a tetravalent conjugate containing bispecific cetuximab × anti-cotinine antibody and cotinine-pyruvicin can consist of the light chain sequence of SEQ ID NO:9 and the heavy chain sequence of SEQ ID NO:10.

[0027] The pharmaceutical compositions of the present invention can be administered by various methods known in the art. The route or method of administration may vary depending on the desired outcome and may be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or near the target site. The antibody-drug conjugates of the present invention can be formulated into pharmaceutically acceptable forms of administration using conventional methods known to those skilled in the art.

[0028] Furthermore, the present invention provides a method for preparing antibody-drug conjugates, the method comprising (s1) preparing a bispecific antibody comprising an anti-cotinine single-chain variable fragment (scFv); (s2) preparing a conjugate of divalent cotinine and a drug crosslinked with a peptide; and (s3) mixing the bispecific antibody prepared in step (s1) and the conjugate prepared in step (s2). In step (s3), the anti-cotinine single-chain variable fragment and divalent cotinine specifically bind to prepare the antibody-drug conjugate. Alternatively, divalent cotinine crosslinked with the N-terminus and C-terminus of a 6-18 peptide can be used. Additionally, the preparation method may include a) inserting a nucleic acid molecule encoding an anti-cotinine scFv-binding bispecific antibody into a vector; b) introducing the vector into a host cell; and c) culturing the host cell.

[0029] Furthermore, the present invention provides a method for treating cancer, comprising the step of administering an effective dose of the antibody-drug conjugate to an animal. Additionally, the present invention provides the use of the antibody-drug conjugate of the present invention in the preparation of pharmaceutical compositions for treating cancer.

[0030] The method of treating cancer according to the present invention may include administering a composition comprising the antibody-drug conjugate of the present invention at a therapeutically effective dose. Hereinafter, the term "therapeutically effective dose" refers to the amount of the antibody-drug conjugate of the present invention that is effective in preventing or treating cancer-related diseases, or the amount of a composition comprising the antibody-drug conjugate.

[0031] The actual dose level of the antibody-drug conjugate in the pharmaceutical composition of the present invention can vary in order to achieve an effective amount of active ingredient that is non-toxic to the patient and achieves the desired therapeutic response for a specific patient, composition, and method of administration. The dose level can vary depending on various pharmacokinetic factors, such as the activity of the antibody-drug conjugate used, route of administration, time of administration, elimination rate of the conjugate used, duration of treatment, other drugs, compounds, or materials used with the conjugate, age, sex, weight, disease condition, overall health status and medical history of the patient to be treated, or other factors.

[0032] In this document, the therapeutic dose of the pharmaceutical composition can be titrated to optimize stability and efficacy. When the antibody-drug conjugate of the present invention is administered systemically, the dose range can be from about 0.0001 mg to 100 mg per kg of host body weight, and more typically from 0.01 mg to 15 mg. For example, the treatment method can be accompanied by systemic administration once every two weeks, once a month, or once every 3 to 6 months.

[0033] Furthermore, this invention provides a method for improving the half-life of a drug using antibody-drug conjugates. In Experimental Example 3 of this invention, pharmacokinetic analysis was performed to evaluate the stability of the cotinine payload when a conjugate with ERC6 was formed. The results confirmed that the cotinine payload was rapidly removed from the bloodstream due to its low molecular weight, but on the other hand, the half-life was prolonged when it bound to ERC6. Moreover, by using divalent cotinine crosslinked with a peptide, the antibody-drug conjugate of this invention significantly enhances the stability of the conjugate compared to a single cotinine-drug conjugate.

[0034] In this document, "antibody" refers to a substance produced by antigen stimulation in the immune system, and there is no particular limitation on its type. In this document, antibodies may include animal antibodies, chimeric antibodies, humanized antibodies, or complete human antibodies. Additionally, in this document, antibodies may include antibody fragments with antigen-binding capabilities, such as Fab. A chimeric antibody is an antibody in which the variable region or its complementarity-determining region (CDR) is derived from a different animal or from other parts of the antibody. This antibody may be one in which the variable region is derived from an animal other than a human (e.g., mouse, rabbit, poultry, etc.), and the invariant region is derived from a human. This chimeric antibody can be prepared by methods known in the art (e.g., genetic recombination).

[0035] In this article, "heavy chain" refers to a chain containing a variable structural domain V. H and 3 invariant structural domains C H 1. C H 2 and C H 3 and its fragments, all full-length heavy chains, the variable structural domain V H It contains an amino acid sequence that provides a variable region sufficient to provide specificity for the antigen. Additionally, "light chain" refers to a chain containing a variable domain V. L and invariant structural domain C L All the full-length light chains, the variable structural domain V L It contains an amino acid sequence that provides a variable region specific to the antigen.

[0036] In this document, a "conjugate" refers to a heterologous molecule that can be generated by covalently linking one or more polypeptides (usually a polypeptide) to one or more non-polypeptide moieties (particularly polymeric moieties, such as polymer molecules, lipophilic compounds, carbohydrate moieties, and organic derivatives). Additionally, conjugates can be linked to one or more carbohydrate moieties, particularly using N-glycosylation or O-glycosylation. Covalent linkage means the direct covalent linking of polypeptide and non-polypeptide moieties to each other, or the indirect covalent linking of polypeptide and non-polypeptide moieties to each other through cross-linking bridges, spaces, linking moieties, or mediating moieties. This definition includes, for example, conjugates linking the drugs disclosed herein and cotinine.

[0037] Furthermore, this invention provides an in vitro bioanalytical method in which a conjugate of cotinine and the drug is used as the analytical tool. The in vitro bioanalytical method can be selected from the group consisting of: cell counting, Western blotting, immunoprecipitation, and enzyme-linked immunochemical analysis, etc.

[0038] The conjugates of anti-cotinine antibodies with the drug and cotinine according to the invention retain all the unique characteristics of the drug and antibody by using cotinine as a hapten. Specifically, the antibody-drug conjugates of the present invention can retain the specific reactivity and function of the molecules, as well as complement-mediated cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), and long in vivo half-life characteristic of antibodies.

[0039] Beneficial effects

[0040] The antibody-drug conjugates of the present invention can effectively deliver drugs to targets that are specifically bound by antibodies and increase the half-life of drugs in vivo, thereby improving therapeutic efficacy. In particular, the bispecific conjugate of a divalent cotinine-peptide conjugated with cetuximab × anti-cotinine antibody and pyruvicin showed significant antitumor activity against EGFR-positive cetuximab-refractory lung adenocarcinoma with KRAS mutations.

[0041] Furthermore, the antibody-drug conjugate platform of the present invention does not require antibody optimization and conjugates cytotoxic agents with antibodies of interest through simple culture, thereby significantly reducing the complex multi-step conjugation process. In other words, the antibody-drug conjugate platform of the present invention can be used to overcome the limitations of traditional ADCs and ultimately develop more effective therapeutic agents as described below. Attached Figure Description

[0042] Figures 1a to 1d This study demonstrates the preparation and characterization of a bispecific (cetuximab × anti-cotinine) antibody (ERC6). Figure 1a This is a schematic diagram of an antibody-drug conjugate (ADC) using a bispecific antibody. The bispecific (cetuximab × anti-cotinine) antibody (ERC6) is designed to bind specifically to both human EGFR and cotinine loading. Figure 1b Displaying SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results. Purified ERC6 was inserted into 4% to 12% (w / v) SDS-PAGE gels. The gels were stained with Coomassie Brilliant Blue to visualize the bands. Lane 1 or Lane 2 represents the sample with or without the reducing agent, respectively. Figure 1c It is size exclusion chromatography (SEC). The purified ERC6 was analyzed by SEC using high-performance liquid chromatography (SEC-HPLC). Figure 1d The pharmacokinetic analysis results of ERC6 are presented. 200 μg of ERC6 was injected intravenously into Balb / c mice (n=4), and blood samples were collected via the orbital vein. Circulating serum levels of ERC6 were analyzed by enzyme-linked immunosorbent assay (ELISA). Results are shown as mean ± SD obtained from three experiments.

[0043] Figures 2a to 2d The binding reactivity of ERC6 with human EGFR and cotinine was demonstrated. Figure 2a The results of the enzyme immunoassay were displayed. Cetuximab, anti-cotinine-IgG, and ERC6 were added to the wells of a microtiter plate coated with human EGFR or cotinine. The wells were probed with HRP-conjugated anti-human IgG (Fab-specific) antibody. Figure 2b The chemical structure of cross-linked dPEG6 biotin and divalent cotinine-coupled peptide (cotinine-biotin) is shown. Figure 2c Cetuximab, anti-cotinine-IgG, and ERC6 were added to the remaining wells of a microtiter plate coated with human EGFR, followed by the addition of cotinine-biotin and detection using HRP-conjugated streptavidin. Background signal was measured in control wells coated with BSA. Absorbance was measured at 650 nm. Figure 2dPharmacokinetic analysis of ERC6-complex cotinine-biotin was performed. 200 μg of ERC6, pre-cultured with 1.85 μg of cotinine-biotin dissolved in 100 μL sterile PBS, was intravenously injected into Balb / c mice (n = 4). Blood samples were collected via the orbital vein, and circulating serum levels of ERC6-complex cotinine-biotin were measured by ELISA. Results are shown as mean ± SD from three experiments performed.

[0044] Figures 3a to 3e This study demonstrated the enhanced antiproliferative effect of the ERC6 complex of cotinine-benzamycin in EGFR-positive lung adenocarcinoma cell lines. Figure 3a These are flow cytometry results. A549 cells were cultured with cetuximab, anti-cotinine-IgG, or ERC6, with or without cotinine-biotin. For analysis, cells were detected using PE-conjugated streptavidin and FITC-conjugated anti-human Fc. Figure 3b The structure of free betamethasone is shown. Figure 3c The chemical structure of the divalently coupled peptide (cotinine-cotinine) crosslinked with four pyruvicides is shown. R represents dimethylaminoethyl pyruvicides linked by a valine-citrulline PAB. Figure 3d This is the result of cell viability analysis of free betaine. Figure 3e This is the result of cell viability analysis for cotinine-carcinomycin. A549 cell lines were treated with palizumab and DMSO (●); cetuximab and DMSO (■); ERC6 and DMSO (▲); palizumab and carcinomycin (○); cetuximab and carcinomycin (□); and ERC6 and carcinomycin (△). Palizumab was used as an isotype control for the bispecific antibody. DMSO was used as a carrier control for cotinine-carcinomycin. Relative cell viability was measured by measuring cellular ATP content using Cell Titer-Glo reagent after 72 hours of cell culture. Results are shown as mean ± SD from three experiments performed.

[0045] Figures 4a to 4e This study demonstrated the enhanced antiproliferative effect of the ERC6-complex cotinine-benzamine in a mouse xenograft tumor model. A549 cells were subcutaneously injected into the left and right sides of each Balb / c nude mouse. The effect was assessed when the tumor volume reached 150 mm². 3Mice were randomly divided into three groups (n=4 per group) and treated for 5 weeks. Each group received intraperitoneal injections of palizumab and cotinine-based cytosine, ERC6 and a carrier, or a combination of ERC6 and cotinine-based cytosine. Palizumab was used as an isotype control for the bispecific antibody. DMSO was used as a carrier control for cotinine-based cytosine. Figure 4a This is the result of measuring tumor volume 32 days ago. Figure 4b It is the result of observing weight during treatment. Figure 4c This is the average tumor volume on day 32. Figure 4d It is the average mass of tumors dissected when mice are euthanized. Figure 4e Tumor images of the three treatment groups at the endpoint are shown. Results are presented as mean ± SD; *P<0.05, **P<0.01, Student's t-test compared to the group.

[0046] Figure 5 shows the pharmacokinetic analysis results of ERC6-complexed cotinine-biotin. (a) 200 μg of ERC6 pre-cultured with 964 picomol cotinine-biotin dissolved in 100 μL sterile PBS at a 1:1 molar ratio was intravenously injected into Balb / c mice (n = 4). The peptide has various lengths of 6, 12, or 18 amino acids between the two cotinine molecules. Blood samples were collected via the orbital vein, and the circulating serum levels of ERC6-complexed cotinine-biotin were determined by ELISA. (b) The circulating serum levels of total ERC6 were measured by ELISA. Results are shown as mean ± SD; *P < 0.05, **P < 0.01, Student t-test.

[0047] Figure 6 shows the antiproliferative effect of ERC6-complex cotinine-DM1 (Metancin) in EGFR-positive lung adenocarcinoma cell lines. Figure 6a The chemical structure of a divalent cotinine-coupled peptide (cotinine-DM1) crosslinked with four DM1 molecules is shown. R represents MCC-linked DM1. Figure 6bThis is the result of cell viability analysis using cotinine-DM1 (Metancin). A549 cell lines were treated with palizumab and DMSO (●); cetuximab and DMSO (■); ERC6 and DMSO (▲); palizumab and cotinine-DM1 (○); cetuximab and cotinine-DM1 (□); and ERC6 and cotinine-DM1 (△). Palizumab was used as an isotype control for the bispecific antibody. DMSO was used as a carrier control for cotinine-duocarmycin. Relative cell viability was measured by measuring cellular ATP content using Cell Titer-Glo reagent after 72 hours of cell culture. Results are shown as mean ± SD from three experiments performed. Detailed Implementation

[0048] Examples will be described in detail below to facilitate understanding of the invention. However, the examples according to the invention can be modified into various other forms, and the scope of the invention should not be construed as limited to the following examples. Examples of the invention are provided to enable those skilled in the art to gain a more complete understanding of the invention.

[0049] Example 1: Cell Culture

[0050] A549 (human lung adenocarcinoma) cells were obtained from a Korean cell line bank and grown in RPMI-1640 medium (Welgene, Seoul, South Korea) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin under a humidified atmosphere containing 5% CO2. HEK293F cells (Invitrogen, Carlsbad, CA, USA) were grown in conical tissue culture flasks with vent caps (Corning Inc., NY, USA) in FreeStyle medium containing 100 U / mL penicillin and 100 μg / mL streptomycin. TM It was grown in 293 Expression medium (GIBCO) in a rotary shaker incubator at 135 rpm (Minitron, INFORS HT, Botmingen, Switzerland) at 37°C in a 70% humid atmosphere containing 7% CO2.

[0051] Example 2: Construction and purification of bispecific cetuximab × anti-cotinine antibody

[0052] To construct a bispecific cetuximab × anti-cotinine antibody expression vector, genes encoding the cetuximab light chain and the cetuximab heavy chain linker (Gly-Gly-Gly-Gly-Ser)3-anti-cotinine single-chain variable fragment (scFv) were chemically synthesized (Genscript, Piscatave, New Jersey, USA). Restriction sites AgeI and XbaI were inserted into the 5' and 3' ends of the gene encoding the cetuximab light chain, respectively. Additional restriction sites NheI and BsiWI were inserted into the 5' end of the gene encoding the cetuximab heavy chain and the 3' end of the anti-cotinine scFv gene. Light chain and heavy chain-linker-anti-cotinine-scFv subcloning into mammalian expression vectors designed for secretion of recombinant proteins, as described in the following method: Park S, Lee DH, Park JG, Lee YT, Chung JA. Sensitive enzyme immunoassay for measuring cottonine in passive smokers. Clin Chim Acta 2010;411(17-18):1238-42.

[0053] As described below: Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B et al., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci US A 1995; 92(16): 7297-301, transfected the expression vector encoding ERC6 into HEK293F (Invitrogen) with 25-kDa linear polyethyleneimine (Polyscience, Warrington, PAN, USA). As described below: Kim H, Park S, Lee HK, Chung J. Application of bispecific antibody against antigen and happened for immunodetection and immunopurification. Exp Mol Med 2013; 45:e43, purified ERC6 from culture supernatant by affinity chromatography using protein A agarose beads (RepliGen, Waltham, MD, USA).

[0054] Example 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0055] Analysis was performed according to the manufacturer's instructions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a NuPage 4% to 12% Bis-Tris gel (Invitrogen). The gel was stained with Coomassie Brilliant Blue R-250 (Amresco, Cologne, Oregon, USA).

[0056] Example 4: Size Exclusion Chromatography (SEC)

[0057] Use equipment containing * Purified ERC6 was analyzed by SEC-HPLC using an Agilent 1260 Infinity high-performance liquid chromatography (HPLC) system (Agilent 1260, Agilent Technologies Inc., California, USA) with 3 μm pore size and a Bio SEC-3 column (7.8 mm × 300 mm). The mobile phase consisted of 50 mM sodium phosphate and 150 mM sodium chloride at pH 7.0. 20 μL of ERC6 (1 mg / mL) was injected and eluted isocratically at a flow rate of 1 mL / min for 30 min. The column elution was monitored at 280 nm using a UV detector and expressed as mAU. The percentages of monomers, aggregates, and fragments were quantified based on peak area.

[0058] Example 5: Synthesis of cotinine conjugates

[0059] All peptides used in this experiment were synthesized using Fmoc-solid-phase peptide synthesis (Peptron, Korea). Two trans-4'-cotinine carboxylic acids (Sigma-Aldrich, St. Louis, Mississippi, USA) were crosslinked with free amino groups at the N-terminus and C-terminus of the GGGGSKGGGGSK and GSKGSKGSKGSKK peptides. After removing the alloc group on the lysine residue in the middle of the peptide with triphenylphosphine(tetra)palladium, dPEG6 (Peptide international Inc., Louisville, Kentucky, USA) was coupled with the free amino group of the GGGGSKGGGGSK peptide. Biotin was then crosslinked with the free amino group on the dPEG6-coupled peptide (Peptron). For simplicity, the biotin-coupled divalent cotinine peptide (cotinine-GGGGSK[(dPEG6)-biotin]GGGGSK-cotinine) is abbreviated as cotinine-biotin.

[0060] Four valine-citrulline PAB-linked dimethylaminoethyl pyruvicin are coupled to the free amino acid groups of four lysine residues present in the divalent cotinine-crosslinked GSKGSKGSKGSKK peptide (Concortis, San Diego, California, USA). For simplicity, the divalent cotinine-coupled peptide (cotinine-[GSK(pyruvicin)]4K-cotinine) crosslinked with four pyruvicin residues is abbreviated as cotinine-pyruvicin.

[0061] Cotinine-Biotin and Cotinine-Betaine were purified by reverse-phase extraction using a C18 column. After purification, the samples were then processed using a Capcell Pak C18 column (4.6 mm × 50 mm). They were analyzed and validated using an Agilent 1100 capillary LC and HPLC system (Shimadzu Corp., Kyoto, Japan) for mass spectrometry.

[0062] Example 6: Composite of ERC6 and cotinine loaded

[0063] To generate a complex of ERC6 and cotinine-conjugated loaded compound, the cotinine load and ERC6 were mixed at a 1:1 molar ratio by aspiration. The mixture was then incubated at room temperature for 30 minutes. The compound was then used in vitro or in vivo without further modification.

[0064] Example 7: Enzyme-linked immunosorbent assay (ELISA)

[0065] The wells of a 96-well microtiter plate (Corning) were coated overnight at 4°C with human EGFR (Sigma) or BSA-conjugated cotinine in coating buffer (0.1M sodium bicarbonate in distilled water, pH 8.6) and blocked at 37°C for 1 hour with 3% bovine serum albumin (BSA) in PBS. 50 μL of antibody at a concentration of 1 μg / mL was added to each well, and the plate was incubated at 37°C for 2 hours. After washing with 0.05% Tween 20 in PBS (PBST), HRP-conjugated anti-human IgG (Fab-specific) antibody (Sigma) diluted in blocking buffer was added, and the plate was incubated at 37°C for 1 hour. Then, after washing the plate again with 0.05% PBST, 50 μL of 3,3',5,5'-tetramethylbenzidine substrate solution (TMB) (GenDEPOT, Barker, Texas, USA) was added to each well, and the absorbance was measured at 650 nm using a Multiskan Ascent microplate reader (Labsystems, Helsinki, Finland).

[0066] To confirm the bispecificity of ERC6, wells coated with human EGFR were cultured using the antibody described above. After washing with 0.05% PBST, cotinine-biotin was added to each well, and the mixture was incubated at 37°C for 1 hour. Then, HRP-conjugated streptavidin (Thermo Fishers Scientific, Waltham, MA) diluted in blocking buffer was added, and the mixture was incubated at 37°C for 1 hour. After washing, TMB was added to each well, and the absorbance was measured at 650 nm.

[0067] Example 8: Pharmacokinetic Analysis

[0068] All animals used in this experiment were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center Research Institute of Korea (License No.: NCC-15-267). The animals were kept in the National Cancer Center animal facility in accordance with the AAALAC International Animal Care Policy.

[0069] Eight-week-old male Balb / c mice were intravenously injected with a complex of ERC6 (200 μg) and cotinine-biotin (1.85 μg) dissolved in 100 μL of sterile PBS at a 1:1 molar ratio (n = 4 mice / group). Blood samples were collected from the intraorbital vein at 0, 1, 24, 48, 72, 96, and 168 hours post-injection. Samples were incubated at room temperature for 2 hours until blood clotted. Serum was then obtained by centrifugation at 3500 rpm for 15 minutes at 4°C. Circulating serum levels of total ERC6 and the ERC6-cotinine complex were determined by enzyme-linked immunosorbent assay (ELISA).

[0070] Total ERC6 in serum samples was measured as follows: Wells of a 96-well microtiter plate (Corning) were coated overnight at 4°C with goat anti-human IgG (Fc-specific) capture antibody (EMD Millipore, Darmstadt, Germany) in coating buffer, and blocked at 37°C for 1 hour with 3% BSA in PBS. Serum samples diluted in blocking buffer and standard solutions were added to each well and incubated at 37°C for 2 hours. After washing with 0.05% PBST, HRP-conjugated anti-human C6 antibody diluted in blocking buffer was added. κ Antibody (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added and incubated at 37°C for 1 hour. After washing, TMB (GenDEPOT) was added to each well, and absorbance was measured at 650 nm.

[0071] The ERC6 and cotinine-biotin complex in serum samples was measured as follows: Wells coated with human EGFR (Sigma) were cultured using serum samples as described above. After washing with 0.05% PBST, HRP-conjugated streptavidin (ThermoFisher Scientific Pierce) was added, and the mixture was incubated at 37°C for 1 hour. After washing, TMB was added to each well, and the absorbance was measured at 650 nm.

[0072] Example 9: Flow Cytometry

[0073] A549 cells were seeded in V-bottom 96-well plates (Corning) to a final concentration of 4 × 10⁻⁶ cells per sample. 5 Cells were treated with 0 or 100 nM ERC6 and cotinine-biotin diluted in flow cytometry buffer (PBS solution containing 1% BSA in sodium azide, 0.1% [w / v]) at 37°C for 30 min. For control experiments, palizumab (Synagis, Boehringer Ingelheim Pharma, Biberlach-sur-Ries, Germany), anti-cotinine-IgG, or cetuximab (Erbitux, Merck K GaA, Darmstadt, Germany) were used instead of ERC6. After washing with flow cytometry buffer, cells were cultured in the dark at 37°C for 1 h with phycoerythrin (PE)-conjugated streptavidin (BD Biosciences Pharmanogen, San Diego, California, USA) and FITC-conjugated anti-human IgG (Fc-specific) antibody (Thermo Fisher Scientific Pierce). After washing again with the same buffer, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry using a FACS Canto II instrument equipped with a 488 nm laser (BD Bioscience, San Diego, California, USA). Each measurement consisted of 10,000 cells, and the data were analyzed using FlowJo software (TreeStar, Ashland, Oregon, USA).

[0074] Example 10: Cell viability assay

[0075] The effects of ERC6 and cotinine-betamethasone on tumor cell viability were evaluated using Cell Titer-Glo reagent (Promega Corp., Madison, Wisconsin, USA). A549 cells were seeded in 50 μL of RPMI-1640 medium in 96-well black-walled plates (4000 cells per well) and allowed to adhere overnight at 37°C, 5% CO2, and a humidified atmosphere. The ERC6 and cotinine-betamethasone complex was then serially diluted 10-fold (0.02 nM to 2000 nM) in fresh medium. In control experiments, palizumab (Boehringer Ingelheim Pharma) or cetuximab (Merck KGaA) was used instead of ERC6. Cotinine-betamethasone diluted in 50 μL of medium and the antibody were added to each well and incubated for 72 hours. After adding Cell Titer-Glo reagent (Promega Corp.) to each well, the luminescence signal was measured using a microplate luminometer (PerkinElmer, Waltham, Massachusetts, USA) according to the manufacturer's instructions. All experiments were performed in triplicate. Relative cell viability was calculated by dividing by the control luminescence signal [viability % = (test - background) / (control - background) × 100].

[0076] Example 11: Xenotransplantation

[0077] Six-week-old female Balb / c nude mice were subcutaneously injected with A549 (1x10) on both the left and right sides of each mouse. 7 (cells). When the tumor volume reaches approximately 150 mm... 3 All animals were randomly assigned to three groups (n = 4 mice / group) and treated for five weeks. For the first two weeks, mice were administered appropriate controls via intraperitoneal injection twice weekly: Group I received palizumab (2.15 mg / kg) and cotinine-betamethasone (95 μg / kg); Group II received ERC6 (3 mg / kg) and dimethyl sulfoxide (DMSO); and Group III received a complex of ERC6 (3 mg / kg) and cotinine-betamethasone (95 μg / kg). Then, for the next three weeks, mice were injected with three times the recommended dose of the drug three times weekly. Palizumab was used as an isotype control for the bispecific antibody. DMSO was used as a carrier control for cotinine-betamethasone. Tumor volume was measured twice weekly using digital calipers for 32 days post-injection. Tumor volume was calculated as length × (width). 2 ×0.5, where length is the longest axis and width is the distance perpendicular to the length. Systemic toxicity was assessed by measuring body weight twice weekly. Mice were sacrificed on day 35 post-injection, and tumors were dissected and weighed.

[0078] Example 12: Immunofluorescence

[0079] Balb / c nude mice were subcutaneously injected with A549 (1x10) on both the left and right sides. 7 (cells). When the tumor volume reaches 500 mm... 3 Mice with tumors were administered a single intraperitoneal (ip) injection as follows: Group I received 144 μg palizumab and 1.85 μg cotinine-biotin; Group II received 200 μg ERC6 and a carrier (distilled water); Group III received a complex of ERC6 (200 μg) and cotinine-biotin (1.85 μg). Within 24 hours post-injection, mice were anesthetized with isoflurane and euthanized by cardiac perfusion of 10 ml of 4% [w / v] paraformaldehyde PBS. Dissected tumors were cryopreserved in PBS containing 30% [w / v] sucrose, equilibrated at 4°C for 24 hours, frozen in liquid nitrogen in optimal cutting temperature embedding medium (Sakura Finetek, Torrance, CAR, USA), and stored at -80°C until cutting. For immunofluorescence staining, 4 μm thick frozen sections were prepared and fixed at room temperature with 4% paraformaldehyde PBS for 10 minutes. After washing with PBS, the sections were blocked for 1 hour at room temperature with 10% [v / v] IHC-Tek antibody dilution solution (pH 7.4) in standard goat serum (CST, Denver, MD, USA) (IHC WORLD, Woodstock, MD, USA). These tissue sections were then incubated for 8 hours with streptavidin conjugated with Alexa Fluor 488 (Molecular Probes Inc., Eugene, Oregon, USA) and stained in the dark at 4°C in a humidified chamber with anti-human IgG antibody conjugated with Alexa Fluor 594 (Molecular Probes Inc.). After washing with PBS, the cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI; Pierce, Rockford, Illinois, USA) according to the manufacturer's instructions. The sections were fixed onto glass slides containing fluorescent fixation medium (DAKO, Grosstup, Denmark), and 40x magnified images were obtained using an FV1000 laser scanning microscope (Olympus, Tokyo, Japan) with FV10ASW software. Emission and excitation filters were arranged to enable imaging in three colors.

[0080] Statistical analysis

[0081] Statistics used in the experiments were performed using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, California, USA). Results are expressed as the mean ± standard deviation (SD) of a specified number of individual measures. Statistical significance was determined using a two-tailed unpaired Student t-test, and a p-value less than 0.05 was considered statistically significant. P-values ​​are represented in the graphs and their legends.

[0082] Experimental Example 1: Design of an antibody-drug conjugate platform using a tetravalent bispecific antibody.

[0083] By fusing two single-stranded variable fragments (scFv) into the heavy chain C H In the 3-domain structure, a tetravalent bispecific antibody based on IgG was designed. Figure 1a For flexibility, a peptide linker rich in glycine and serine [(Gly-Gly-Gly-Gly-Ser)3] is inserted into C. H Between the 3-domain and the scFv. This results in a tetravalent antibody containing two bispecific antibody Fab arms and two heavy chain C-terminals of the scFv, thereby simultaneously targeting epidermal growth factor receptor (EGFR) and cotinine, respectively. To develop the bispecific cetuximab × anti-cotinine scFv antibody (ERC6), the gene structures encoding cetuximab IgG, the linker, and the anti-cotinine scFv were cloned into a eukaryotic expression vector, and ERC6 was purified from temporarily transfected HEK393F culture supernatant using protein A affinity column chromatography.

[0084] Traditional antibody-drug conjugates require a multi-step process to conjugate the drug with the antibody, and process optimization is often necessary. On the other hand, targeted drug delivery using bispecific antibodies conjugated with cotinine requires conjugation of cotinine and the drug. Therefore, if a chemical cross-linking process of carboxycotinine (trans-4-cotinine carboxylic acid) is established, this platform will have advantages over traditional ADCs. Two carboxycotinine groups are cross-linked at the N-terminus and C-terminus of a 13-amino acid peptide. The binding affinity of the bispecific antibody to the divalent cotinine-crosslinked peptide is more stable due to the titer effect (Figure 5). Depending on the purpose, various loadings can be conjugated to the ε-amino acid chain of lysine in the peptide. In this invention, biotin or pyruvic acid is conjugated to the divalent cotinine-conjugated peptide (…). Figure 2b , Figure 3c ).

[0085] Experimental Example 2: Properties of Bispecific Cetuximab × Anti-Cotinine scFv Antibody (ERC6)

[0086] To analyze the purity and molecular weight of purified ERC6, it was visualized in a sodium dodecyl sulfate (SDS) polyacrylamide gel stained with Coomassie brilliant blue. A major band with a molecular weight of 206 kDa was observed under non-reducing conditions, and two major bands with molecular weights of 78 kDa and 25 kDa were also observed under the same conditions. Figure 1b The recombinant protein with a molecular weight of 206 kDa corresponds to the fully assembled ERC6 as predicted by the ProtParam tool (ExPASy). This is achieved by reducing disulfide bonds to connect the 25 kDa unmodified light chain and the 75 kDa C-linked protein. H Visualization of a heavy chain fused to an scFv on the 3-domain. Therefore, the data demonstrate that the bispecific antibody is pure and prepared without damage.

[0087] The physicochemical properties of the purified recombinant protein were analyzed using size exclusion chromatography (SEC-HPLC) with high performance liquid chromatography. ERC6 appeared as a single main peak with a clear molecular weight corresponding to the correct assembly form. These data indicate that ERC6 does not produce fragments, it does not aggregate, and it produces multimers. Figure 1c ).

[0088] In addition, pharmacokinetic analyses were performed to measure the stability of ERC6 in vivo. The serum half-life of ERC6 was determined in Balb / c mice (n=4). Circulating serum levels of ERC6 were measured using enzyme-linked immunosorbent assay (ELISA) using blood samples collected from the orbital vein. Intravenously administered ERC6 was stable in mouse serum until day 5, similar to the results for cetuximab IgG.

[0089] Experimental Example 3: ERC6's binding responsiveness to human EGFR and cotinine

[0090] To test the responsiveness of ERC6 to EGFR and cotinine, an enzyme immunoassay will be performed. Figure 2a The confirmed binding activity of ERC6 to EGFR demonstrated that EGFR reactivity is independent of other scFvs. Furthermore, it was confirmed that the affinity of the anti-cotinine scFv module for cotinine was maintained. Figure 2a ).

[0091] To determine whether ERC6 simultaneously binds to human EGFR and cotinine, an additional enzyme immunoassay was performed using a streptavidin-biotin assay system. After culturing human EGFR-coated microtiter plates with ERC6, a biotin-crosslinked divalent cotinine-conjugated peptide (cotinine-biotin) was added to each well. Figure 2b Then, HRP-conjugated streptavidin was added. ERC6 is simultaneously conjugated with EGFR and cotinine ( Figure 2c ).

[0092] To evaluate the stability of the cotinine loading when forming a complex with ERC6, pharmacokinetic analyses were performed. Mice (n=4) were intravenously injected with ERC6 pre-cultured with cotinine-biotin, and circulating serum levels were measured by ELISA. Due to its low molecular weight, the cotinine loading was rapidly cleared from the mouse bloodstream (t0.05). 1 / 2 =0.557h). On the other hand, by binding to ERC6, the circulating half-life of cotinine-biotin is prolonged (t 1 / 2 =18 hours)( Figure 2d The binary nature of ERC6 and cotinine enhances the stability of the cotinine-biotin conjugate through increased binding affinity (Figure 6). Furthermore, the clearance pattern of cotinine-biotin matches the degradation pattern of ERC6, implying that the half-life of the conjugate is primarily dependent on the pharmacokinetics of ERC6. Figure 2d ).

[0093] Experimental Example 4: ERC6-based cotinine-benzamycin induces strong cytotoxicity against lung adenocarcinoma cells with KRAS mutations.

[0094] A549 is a lung adenocarcinoma cell line expressing wild-type EGFR and possessing a KRAS mutation, exhibiting primary resistance to EGFR-targeted therapies (e.g., cetuximab). Therefore, this cell line was used to test the efficacy of an ERC6-conjugated cotinine cytotoxic agent against cancer cells with wild-type EGFR and KRAS mutations.

[0095] Prior to cytotoxicity analysis, flow cytometry analysis confirmed the expression of EGFR on the surface of A549 cells. Figure 3a ERC6 exhibited binding activity similar to that of EGFR expressed in the plasma membrane, comparable to levels seen with cetuximab. As predicted, cotinine-biotin showed binding activity against A549 cells in the presence of ERC6. A mixture of cotinine-biotin and anti-cotinine IgG or cetuximab did not produce any significant signal from streptavidin-PE. Furthermore, cotinine-biotin did not affect the binding affinity of ERC6 to EGFR. These data demonstrate that ERC6 mediates cotinine-biotin delivery to EGFR-positive cells in a target-specific manner through simultaneous binding activity.

[0096] The cytotoxic activity against A549 cells was studied by cell viability analysis. Figure 3e Relative cell viability was determined by measuring cellular ATP content, which is directly related to the number of viable cells. Unloaded betaine (free betaine) showed stronger antitumor activity than cotinine-betaine. Figure 3d , Figure 3e If the toxicity of cotinine-betamethrin is lower than that of free betamethrin, its penetration through the cell membrane will be less effective.

[0097] Neither ERC6 nor cetuximab significantly inhibited the proliferation of A549 cells, due to the primary resistance of A549 cells to EGFR-targeted therapy. Figure 3e However, when ERC6 and cotinine-betamethasone were combined, the cytotoxicity was significantly enhanced compared to cetuximab or the negative control antibody.

[0098] Half of the maximum inhibitory concentration (IC50) 50 The concentration was 0.3 nM. Experimental results showed that ERC6 effectively promoted the internalization of cotinine-benzamycin into EGFR-positive cells via receptor-mediated endocytosis. This demonstrated that KRAS-mediated primary resistance to the ERC6-complex cotinine-benzamycin was overcome.

[0099] In addition, the cytotoxicity of the antibody-drug conjugate containing ERC6 and cotinine-metazidine (Cot-DM1) was investigated in another experiment. This antibody-drug conjugate also showed significantly high cytotoxicity against the A549 cell line, similar to the cotinine-metazidine conjugate. Figure 6b ).

[0100] Experimental Example 5: ERC6-complex cotinine-pyramidin inhibits tumor growth in an animal model of lung adenocarcinoma.

[0101] To evaluate the in vivo efficacy of the ERC6-combined cotinine-benzamycin, cetuximab-refractory A549 cells were transplanted into mice (n=4 mice / group). When the tumor volume reached 150 mmHg... 3 Mice were administered palizumab and cotinine-carcinomycin, ERC6 and its carrier, or a combination of ERC6 and cotinomycin, via intraperitoneal injection for five weeks. The drugs were administered twice weekly for two weeks, followed by three weekly administrations for three weeks.

[0102] Compared with animals treated with cotinine-betamethasone or ERC6 alone, mice receiving cotinine-betamethasone in combination with ERC6 showed inhibition of tumor growth. Figure 4aAlthough cotinine-betamycin showed potent antiproliferative activity against A549 in vitro, the isotype control of the bispecific antibody did not inhibit tumor growth in vivo. This observation suggests that only cotinine-betamycin could not selectively deliver to the tumor site and inhibit tumor growth. This insufficient efficacy can be explained by its nonspecificity and low molecular weight, leading to rapid clearance from the mouse bloodstream. On the other hand, ERC6 allows cotinine-betamycin to have a prolonged circulating half-life and be delivered to EGFR-expressing tumor tissues. Therefore, ERC6 and cotinine-betamycin synergistically enhance the antiproliferative efficacy against EGFR-positive cetuximab-refractory tumors in vivo.

[0103] Furthermore, during the 5-week treatment period, the mice did not experience significant weight loss. Figure 4b This observation indirectly implies that ERC6 and cotinine-benzamine have no systemic toxicity. Therefore, these data suggest that ERC6 can function as a drug delivery system, selectively delivering cotinine-bound cytotoxic drugs to EGFR-expressing tumor tissues in a targeted and specific manner without systemic toxicity.

[0104] Experimental Example 6: Tissue distribution of ERC6-complexed cotinine loading in a mouse xenograft tumor model

[0105] Pharmacokinetic studies demonstrated that the circulating half-life of the ERC6 complex loading was prolonged by binding to ERC6. To investigate the specific delivery of cotinine loading to tumor tissues expressing the antigen, immunofluorescence analysis was performed in an A549 xenograft mouse model. A549 cells were subcutaneously injected into the left side of each Balb / c nude mouse. When the tumor reached 500 mm... 3 In this study, palizumab and cotinine-biotin, cetuximab and a loading agent, or cotinine-biotin complexed with ERC6 were injected intraperitoneally into mice with tumors. Twenty-four hours after injection, the animals were sacrificed, and the dissected tumor tissue was imaged ex vivo. Cotinine loading and antibodies in the tumor tissue were detected by fluorescently labeled secondary antibodies. Antibodies were detected by Alexa 594-labeled anti-human Fc (red), and cotinine-biotin was detected by Alexa 488-labeled streptavidin (green). For reference, cell nuclei were stained with DAPI (blue), and images were magnified x40. The tissue distribution of ERC6-complexed cotinine-biotin was observed using confocal microscopy.

[0106] ERC6 accumulation was observed in human EGFR-positive tumor tissue compared to palizumab, which was used as an isotype control for bispecific antibodies. Furthermore, cotinine-biotin was observed in tumors only upon ERC6 injection. Due to its low molecular weight, cotinine-biotin without ERC6 was rapidly cleared from the bloodstream. These observations suggest that ERC6 selectively delivers cotinine loading to the desired tumor site in vivo in a target-specific manner. <110> Seoul National University Industry-Academia Collaboration Group <120> Antibody-drug conjugate platform using bispecific antibodies <130> PCT17‑057, SNU‑2016‑0600‑US‑PC <150> US 62 / 352,804 <151> 2016-06-21 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Heavy chain of anticotinine scFv <400> 1 gaggtgcagc tggtggagag cgggggcggc ctggtgcagc ctggaggaag cctgcgactg 60 tcctgtgcag cttctggaca cctgcggaga agggactgga tgaactgggt gcggcaggca 120 ccaggaaaag gcctggagtg ggtcgcagcc atcggacgat ccggcgacac ctactatgct 180 acatgggcaa aaggcaggtt cacaattagt gctgatactt caaagaacac cgcatacctg 240 cagatgaata gtctgagggc cgaagacact gctgtgtact attgctcccg catcccttat 300 tttgggtgga acaatggaga tatttggggg cagggaacac tggtgactgt cagctcc 357 <210> 2 <211> 119 <212> PRT <213> Synthetic Sequence <220> <223> Heavy chain of cotinine-specific scFv <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Leu Arg Arg Arg Asp 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Gly Arg Ser Gly Asp Thr Tyr Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Ile Pro Tyr Phe Gly Trp Asn Asn Gly Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 3 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Light chain of anticotinine scFv <400> 3 gacatccaga tgacacagtc tccatctagt ctgagtgcat cagtgggcga tagagtcacc 60 attacatgtc agtcaagcca gagtccctac tcaaacgagt ggctgagctg gtatcagcag 120 aagccccggaa aagcccctaa gctgctgatc tacaggatta gcacactggc ttccggcgtg 180 ccttctcggt tcagcggctc cagatctggg actgacttta ctctgaccat ctcctctctg 240 cagccagagg atttcgcaac ctactattgc gccggcgggt ataattttgg cctgttcctg 300 tttggccagg ggaccaaagt ggaaattaag 330 <210> 4 <211> 110 <212> PRT <213> Artificial sequence <220> <223> Light chain of anticotinine scFv <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Pro Tyr Ser Asn 20 25 30 Glu Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Arg Ile Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Asn Phe 85 90 95 Gly Leu Phe Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 5 <211> 1347 <212> DNA <213> Artificial Sequence <220> <223> Heavy Chain of Cetuximab <400> 5 caggtgcagc tgaagcagag cggccccgga ctggtgcagc cctcacagag cctgtccatc 60 acttgcaccg tgagtggctt ctcactgaca aactacggag tccactgggt gcgacagagc 120 cctggcaagg ggctggagtg gctgggcgtg atctggtccg gagggaacac tgactataat 180 actcccttca ccagccggct gtccattaac aaggataact ctaagagtca ggtgttcttt 240 aagatgaact ctctgcagag taatgacaca gctatctact attgcgctcg cgcactgact 300 tactatgatt acgagttcgc atattgggga cagggcacac tggtcaccgt gagcgccgcc 360 agcaccaagg gacccagcgt gtttccactg gcccccagct ccaaatcaac cagcggagga 420 acagcagccc tgggatgtct ggtgaaggac tacttcccag aacccgtcac agtgtcctgg 480 aactctgggg cactgacatc tggagtccat acttttccag ccgtgctgca gtctagtggg 540 ctgtacagcc tgtcaagcgt ggtcactgtc ccctcctcta gtctgggaac acagacttat 600 atctgcaacg tgaatcacaa gccaagtaat accaaggtcg acaaaagagt ggagcccaag 660 agctgtgata aaacccatac atgcccccct tgtcctgcac cagaactgct ggggggaccc 720 tccgtgttcc tgtttccacc caagcctaaa gacaccctga tgatttctag gactcccgag 780 gtcacctgcg tggtcgtgga cgtgagccac gaggatcctg aagtcaagtt caactggtac 840 gtggatggcg tcgaagtgca taatgctaag acaaaacctc gggaggaaca gtacaacagc 900 acttatagag tcgtgtccgt cctgaccgtg ctgcaccagg attggctgaa cgggaaagag 960 tataagtgca aagtgagcaa taaggccctg cccgctccta tcgagaaaac catttccaag 1020 gccaaaggcc agcctaggga accacaggtg tacacactgc ctccatcccg cgaggaaatg 1080 accaagaacc aggtctctct gacatgtctg gtgaaaggat tctatccttc agacatcgct 1140 gtggagtggg aaagcaatgg ccagccagag aacaattaca agaccacacc ccctgtgctg 1200 gacagtgatg gctcattctt tctgtattct aagctgaccg tggataaaag tcgatggcag 1260 caggggaatg tcttttcctg ttctgtgatg cacgaagccc tgcacaacca ttacacccag 1320 aagagcctga gcctgtcccc cggcaaa 1347 <210> 6 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain of Cetuximab <400> 6 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lights <210> 7 <211> 642 <212> DNA <213> The snowstorm <220> <223> Thanks for watching <400> 7 gatattctgc tgactcagag ccccgtgatt ctgtctgtca gccccggcga gcgggtgtct ttcagttgca gagcatcga gagcatcgga acaaatattc actggtacca gcagaggact aacggctccc cacgcctgct gatcaagtat gcttccgat ctatcagtgg gattccctct cggttctcag gcagcgggtc cggaacagac tttactctgt ctatcaatag tgtggagtca 240 gaagacattg ccgattacta ttgccagcag aacaataact ggcctaccac attcggcgct 300 gggaccaagc tggagctgaa acgaacagtg gccgctccaa gtgtcttcat ttttccccct 360 agcgacgaac agctgaaatc cgggaccgcc tctgtggtct gtctgctgaa taacttttac 420 cctagagagg caaaggtgca gtggaaagtc gataatgccc tgcagagcgg aaactcccag 480 gagtctgtga ctgaacagga cagtaaggat tcaacctata gcctgagctc cactctgacc 540 ctgtccaaag ctgattacga aaagcataaa gtctatgcat gtgaggtcac tcatcagggg 600 ctgtccagtc cagtcaccaa gtccttcaat cggggggaat gc 642 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of cetuximab <400> 8 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 9 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Light chain of a tetravalent complex containing bispecific cetuximab x anti-cotinine antibody <400> 9 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 10 <211> 715 <212> PRT <213> Artificial sequence <220> <223> Heavy chain containing a tetravalent complex of bispecific cetuximab x anti-cotinine antibody <400> 10 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 465 470 475 480 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly His Leu Arg Arg 485 490 495 Arg Asp Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 500 505 510 Trp Val Ala Ala Ile Gly Arg Ser Gly Asp Thr Tyr Tyr Ala Thr Trp 515 520 525 Ala Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 530 535 540 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 545 550 555 560 Cys Ser Arg Ile Pro Tyr Phe Gly Trp Asn Asn Gly Asp Ile Trp Gly 565 570 575 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 580 585 590 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 595 600 605 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 610 615 620 Thr Ile Thr Cys Gln Ser Ser Gln Ser Pro Tyr Ser Asn Glu Trp Leu 625 630 635 640 Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 645 650 655 Arg Ile Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 660 665 670 Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 675 680 685 Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Asn Phe Gly Leu Phe 690 695 700 Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 705 710 715

Claims

1. An antibody drug conjugate comprising a bispecific antibody comprising an anti-azirin single chain variable fragment (scFv) consisting of a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 4; and a conjugate of a divalent azirin and a drug crosslinked with a peptide, wherein the peptide crosslinked with the divalent azirin consists of an amino acid sequence GSKGSKGSKGSKK, wherein the anti-azirin single chain variable fragment binds to the divalent azirin, wherein the bispecific antibody comprises cetuximab, wherein the drug is duocarmycin or maytansine. wherein the bispecific antibody is a bispecific antibody with a peptide linker (Gly-Gly-Gly-Gly-Ser)3 inserted between the C H 3 domain and the anti-colistin single chain variable fragment scFv. 2.The antibody drug conjugate of claim 1, wherein, the divalent azirin is a divalent azirin in which two azirins are crosslinked with the N-terminal and C-terminal of the amino acid sequence GSKGSKGSKGSKK, respectively. 3.The antibody drug conjugate of claim 1, wherein the drug is conjugated with a lysine residue in the amino acid sequence GSKGSKGSKGSKK crosslinked with the divalent azirin. 4.A pharmaceutical composition for treating cancer, comprising the antibody drug conjugate of claim 1.

5. The pharmaceutical composition for use in the treatment of cancer according to claim 4, wherein, The cancer is lung adenocarcinoma with KRAS mutation. 6.A method for preparing an antibody drug conjugate, comprising: (s1) a step of preparing a bispecific antibody comprising an anti-azirin single chain variable fragment (scFv) consisting of a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO: 4; (s2) a step of preparing a conjugate of a divalent azirin and a drug crosslinked with a peptide; and (s3) a step of mixing the bispecific antibody prepared in the step (s1) and the conjugate prepared in the step (s2), wherein the peptide crosslinked with the divalent azirin consists of an amino acid sequence GSKGSKGSKGSKK, wherein the anti-azirin single chain variable fragment binds to the divalent azirin, wherein the bispecific antibody comprises cetuximab, wherein the drug is duocarmycin or maytansine, wherein the bispecific antibody is a bispecific antibody with a peptide linker (Gly-Gly-Gly-Gly-Ser)3 inserted between the C H 3 domain and the anti-colistin single chain variable fragment scFv.

7. The method for preparing an antibody drug conjugate according to claim 6, wherein, the divalent azirin is a divalent azirin in which two azirins are crosslinked with the N-terminal and C-terminal of the amino acid sequence GSKGSKGSKGSKK, respectively.

8. The method for preparing an antibody drug conjugate according to claim 6, wherein, the drug is conjugated with a lysine residue in the amino acid sequence GSKGSKGSKGSKK crosslinked with the divalent azirin. 9.Use of the antibody drug conjugate of claim 1 for the preparation of a medicament for treating cancer. 10.Use of the antibody drug conjugate of claim 1 for the preparation of a medicament for improving the half-life of a drug.

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