Antibody-dual drug conjugate comprising asymmetric antibody and method for preparing same

Abstract

The present invention relates to an antibody-drug conjugate (ADC) using an asymmetric antibody having a knob-in-hole (KIH) structure. Specifically, the antibody according to the present invention comprises an IgG1-derived Fab-Fc arm specific to a first epitope and an scFv-Fc arm specific to a second epitope, and forms a disulfide bond in a hinge region. In addition, the antibody is designed such that a specific amino acid position is substituted with cysteine and a drug can be conjugated to the antibody at a DAR of 1, thereby enabling stable and precise drug conjugation. Therefore, the present invention provides a novel antibody-drug conjugate capable of realizing efficient drug delivery while maintaining the structural stability and target specificity of the antibody.

Description

Antibody-double drug conjugate containing an asymmetric antibody and method for preparing the same

[0001] The present invention relates to an antibody-drug conjugate (ADC). More specifically, the present invention relates to an antibody-double drug conjugate comprising an asymmetric antibody having a knob-in-hole structure and a method for manufacturing the same, wherein the asymmetric antibody comprises arms that recognize identical or different epitopes and enables stable and efficient drug delivery by achieving a uniform drug-antibody binding ratio (DAR).

[0002] Antibody-drug conjugates (ADCs) are precision therapies that combine the target specificity of antibodies against tumor cells with the potent cytotoxic effects of cytotoxic drugs; to date, several products have been approved, and numerous candidates are currently under development in clinical trials. However, despite their excellent antitumor efficacy, conventional ADCs have consistently faced safety concerns due to serious side effects and inherent toxicity, which limits their clinical application. Therefore, there is a need to develop next-generation ADCs that possess an improved safety profile while maintaining superior antitumor activity.

[0003] More than 80% of approved ADCs utilize linkers that can be cleaved by intracellular enzymes. However, cleaved linkers present a problem of premature drug release into the bloodstream before reaching tumor tissue. This premature release can lead to the delivery of the drug to non-specific tissues during circulation, potentially causing severe off-target toxicity, while simultaneously reducing the efficacy of the residual ADC and acting as a dose-limiting factor. This is known to be a major obstacle that not only complicates the evaluation of anti-tumor efficacy in preclinical animal models but also hinders therapeutic potential during clinical application.

[0004] Furthermore, most existing ADCs are based on a symmetric IgG structure; in this case, the two arms of the antibody are identical, allowing them to recognize only a single target, and the random drug binding sites result in heterogeneity of the drug-antibody ratio (DAR). This heterogeneity not only reduces formulation stability but also negatively impacts the reproducibility of efficacy and safety. Moreover, due to their single-specificity structure, there are limitations in effectively targeting the complex tumor microenvironment.

[0005] Therefore, there is a need to develop a new type of antibody-drug conjugate platform that can overcome the limitations of existing symmetric antibody-based ADCs, achieve bispecificity, and simultaneously ensure drug binding site selectivity and DAR uniformity.

[0006] The present invention was devised to solve the above-mentioned problems and address the aforementioned needs, and the objective of the present invention is to provide an antibody-drug conjugate comprising an asymmetric antibody having a knob-in-hole structure.

[0007] Another objective of the present invention is to provide an asymmetric antibody comprising a Fab-Fc arm specific to a first epitope and a scFv-Fc arm specific to a second epitope, thereby realizing dual specificity capable of recognizing two targets that are the same or different from each other.

[0008] In addition, another objective of the present invention is to improve drug delivery efficiency and the safety of the formulation by substituting a specific amino acid with cysteine ​​to secure a uniform drug-antibody ratio (DAR) and stably binding the linker and the drug.

[0009] Furthermore, according to one embodiment of the present invention, the antibody-drug conjugate can simultaneously achieve target specificity for tumor cells and selective release of the drug, thereby overcoming the limitations of conventional symmetric antibody-based ADCs and being usefully applied as a more precise and efficient next-generation anticancer therapeutic agent.

[0010] Methods and materials similar or equivalent to those disclosed herein may be used in the practice or experimentation of the embodiments disclosed herein, although some preferred methods, configurations, apparatuses, and materials are disclosed herein. However, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols disclosed herein, as materials and methods may vary depending on routine experimentation and optimization prior to disclosure. It should be understood that the terms used in the description are used solely for the purpose of describing specific versions or embodiments and are not intended to limit the scope of the embodiments disclosed herein.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. However, in the event of a conflict, this specification, including definitions, shall prevail. Accordingly, the following definitions apply in the context of the embodiments disclosed herein.

[0012] The total DAR values ​​used in this specification do not necessarily mean integers. The DAR values ​​include values ​​with an error range of approximately 15% of the defined integer values. For example, a total DAR 8 may mean an average value obtained by rounding to include an error range, and may, without limitation, mean 7.6 to 8.4, 7.7 to 8.3, 7.8 to 8.2, or 7.9 to 8.1. Also, DAR 1 does not necessarily mean an integer. As a non-limiting example, DAR 1 may mean 0.8 to 1.2, 0.9 to 1.1, 0.92 to 1.08, or 0.95 to 1.05.

[0013] Unless otherwise indicated, all numbers expressing the size, quantity, and physical properties of a feature used in this specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, the numerical parameters disclosed in this specification and appended claims are approximations that may vary depending on the desired properties to be obtained by a person skilled in the art using the teachings disclosed in this specification. Preferably, "approximately" may mean ±15% of the stated values.

[0014] As used herein, the term “comprise” and its linguistic variations mean the presence of the cited features(s), elements(s), method steps(s), etc., without excluding the presence of additional features(s), elements(s), method steps(s), etc. Conversely, the term “consisting of” and its linguistic variations mean the presence of the cited features(s), elements(s), method steps(s), etc., while generally excluding unmentioned features(s), elements(s), method steps(s), etc., with the exception of related impurities. The phrase “consisting essentially of” refers to the mentioned features(s), elements(s), method steps(s), etc., and features(s), elements(s), method steps(s), etc., that do not substantially affect the fundamental characteristics of the composition, system, or method. Many embodiments of the present invention are described using the open “comprising” language. These embodiments include a number of closed "consisting of" and / or "consisting essentially of" embodiments that may alternatively be claimed or described using such language.

[0015] As used herein, the terms “bonded” or “linked” refer to a state in which two components are operably joined, either directly or indirectly, by chemical, physical, or biological interactions. Specifically, these terms may include chemical bonds such as covalent bonds, disulfide bonds, amide bonds, ester bonds, etc., and, in some cases, forms of indirect joining through linkers, spacers, or other intermediates. Accordingly, the terms “bonded” or “linked” are used to encompass a functional joining relationship that is consistent with the purpose of the invention, going beyond a mere state of contact.

[0016]

[0017] To solve the above-mentioned problem, the present invention provides an antibody for an antibody-drug conjugate to which one or more drugs are bound. The antibody is an asymmetric antibody having a knob-in-hole structure, comprising: a) a first arm consisting of a Fab and Fc region derived from an IgG1 antibody specific to a first epitope; and b) a second arm consisting of a ScFv and Fc region fusion protein specific to a second epitope, wherein the Fc region of the second arm comprises a sequence derived from an IgG1 antibody, and the first epitope and the second epitope are identical or different from each other.

[0018] In the present invention, when the Fc region molecules of the first and second arms are bonded, at least two disulfide bonds can be formed in the hinge region.

[0019] In addition, any one of the amino acids, including lysine at position 183 of the first female heavy chain, lysine at position 149 of the first female light chain, lysine at positions 290, 326, and 392 of the first female or second female Fc region, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328, may be substituted with cysteine.

[0020] The first drug may be bound to the antibody at a ratio of about 1 DAR, and if a second drug is additionally bound, the second drug may be bound to the antibody at a ratio of 5 to 7 DAR. The DAR ratio of the first drug and the second drug is preferably about 1:5 to 7, and most preferably about 1:6.

[0021] In this specification, the term "arm" refers to a structural unit constituting one side of an antibody. Specifically, the arm may include a form in which a Fab region that binds to an antigen and an Fc region are combined, and in some cases, may include a protein in which a single-chain variable fragment (scFv) and an Fc region are fused. Accordingly, in the present invention, the first arm may include a Fab-Fc structure derived from an IgG1 antibody, and the second arm may include a scFv-Fc fusion protein structure.

[0022] In this specification, "scFv" refers to a single-chain variable fragment, which includes a form in which the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody are connected into a single continuous polypeptide.

[0023]

[0024] The present invention also provides an antibody-drug conjugate in which a drug is bound to the asymmetric antibody in a ratio of DAR 1.

[0025] An antibody-drug conjugate according to one embodiment of the present invention comprises a form in which a specific amino acid of an asymmetric antibody is substituted with cysteine ​​and a drug is bound. Specifically, any one of i) lysine at position 183 of the first arm heavy chain, ii) lysine at position 149 of the first arm light chain, or iii) lysine at positions 290, 326, and 392 located in the Fc region of the first or second arm, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328 may be substituted with cysteine. The cysteine ​​substituted as described above can form a bond with the drug (or the drug via a linker), thereby inducing binding at a ratio of approximately DAR 1.

[0026]

[0027] The present invention also provides an antibody-double drug conjugate in which a first drug and a second drug are bound to the asymmetric antibody. In the present invention, the antibody-double drug conjugate in which the first drug and the second drug are bound is also referred to as an "antibody-double drug conjugate" or "AD2C". In the case of an antibody-double drug conjugate (AD2C) in which a double drug is bound to an antibody, the first drug and the second drug may be different from each other. The ratio of the DAR of the first drug and the second drug is 1:5 to 7, and the total DAR of the antibody-double drug conjugate is 6 to 8. The first drug and the second drug may each be connected to the antibody through a linker.

[0028]

[0029] The first drug of the present invention may be a microtubule polymerization inhibitor or a microtubule inhibitor, and may include auristatin compounds (or derivatives thereof), and any compounds having a bystander effect may be used as the first drug of the present invention. Specifically, MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F), MMAU (Monomethyl auristatin U), auristatin E, DM1 (Emtansine), DM4, Eribulin, Chemical Formula 1, Aur0101, Duostatin 5, etc. may be used without limitation, and compounds having a structure in which the bystander effect is controlled to an optimal range by controlling a specific position of these compounds may also be used in the present invention.

[0030] [Chemical Formula 1]

[0031]

[0032] The second drug of the present invention is a compound that inhibits topoisomerase I activity, and may be used without limitation as long as it is a compound that includes a topoisomerase I inhibitor and can strongly inhibit the topoisomerase I enzyme inside cancer cells to bring about a death effect on cancer cells.

[0033] Specific examples of the second drug of the present invention may include DXd, Exatecan (Chemical Formula 6), FL118, SN-38, Camptothecin-based compounds (e.g., compounds of Chemical Formula 2 and Chemical Formula 3), Chemical Formulas 4 to 15 and their derivatives, Belotecan and its derivatives, Topotecan and its derivatives, etc. A more preferred example may be a compound represented by Chemical Formulas 3 to 5. Since the compound represented by Chemical Formula 3 or Chemical Formula 5 is selected as the second drug and conjugated to an antibody together with the aforementioned first drug, the first drug and the second drug may each provide improved anticancer efficacy compared to the combined administration of an antibody-linked antibody-drug conjugate.

[0034] [Chemical Formula 2]

[0035] ,

[0036] [Chemical Formula 3]

[0037] ,

[0038] [Chemical Formula 4]

[0039] ,

[0040] [Chemical Formula 5]

[0041] ,

[0042] [Chemical Formula 6]

[0043] ,

[0044] [Chemical Formula 7]

[0045] ,

[0046] [Chemical Formula 8]

[0047] ,

[0048] [Chemical Formula 9]

[0049] ,

[0050] [Chemical Formula 10]

[0051] ,

[0052] [Chemical Formula 11]

[0053] ,

[0054] [Chemical Formula 12]

[0055] ,

[0056] [Chemical Formula 13]

[0057] ,

[0058] [Chemical Formula 14]

[0059] ,

[0060] [Chemical Formula 15]

[0061]

[0062] The compounds corresponding to the first and second drugs according to the present invention include not only the aforementioned compounds or isomers thereof, but also their pharmaceutically acceptable salts, their solvates, and their prodrugs.

[0063] In this specification, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, such as, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., and salts of inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, etc., as well as salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, acetylsalicylic acid, etc., and salts of amino acids such as lysine, arginine, and guanidine. Additionally, there are, but are not limited to, salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium that can be used in pharmaceutical reactions, purification and separation processes.

[0064] The term "prodrug" carries the meaning used in the relevant technical field. For instance, it refers to an inactive compound that is converted into an active state through drug metabolism within the body. Specifically, it refers to a compound designed to chemically modify a physiologically active substance or a therapeutically active organic compound to release or release the parent compound under enzymatic or other conditions within the body. After administration, the prodrug transforms into the target compound within the body. It enables clinical use by chemically modifying substances that, despite being useful, possess properties unsuitable in terms of side effects, safety, solubility, absorption, or duration of action.

[0065] "Solvent" means a compound or its isomer and its pharmaceutically acceptable salt, which additionally contain stoichiometric or non-stoichiometric amounts of solvent bound by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

[0066]

[0067] In the antibody-drug conjugate of the present invention, the drug can be linked to a linker at an appropriate site as long as its characteristics, such as anticancer activity, are not altered.

[0068] The linkers used to attach the first and second drugs of the present invention to the antibody may be linkers that are easily cleaved by enzymes selectively activated inside cancer cells or in the tumor microenvironment to release the drug (payload). Specifically, GGFG linkers, AAA linkers, Val-Cit linkers, Val-Ala linkers, glucuronidase cleavage linkers cleaved by other enzymes, legumarin cleavage linkers, etc., may be used. In addition, tandem linkers that release the drug only after cleaving by two enzymes rather than one may also be used.

[0069] In the present invention, the "linker" must be stable in the bloodstream to prevent the drug from separating from antibodies, etc., and maintain its structure until it reaches a target such as an antigen, thereby minimizing damage to normal tissues. Ideally, the antibody-drug conjugate, etc., is stable when circulating systemically, but is cleaved at target cells to appropriately release cytotoxic drugs, thereby safely delivering the drug to the target and ensuring that the antibody-drug conjugate, etc., possesses both efficacy and safety.

[0070] In the present invention, the linker may be in a form that can be cleaved under specific intracellular environments and / or conditions, that is, a form that allows a drug from an antibody to be released through the cleavage of the linker in an intracellular environment.

[0071] For example, the linker may be a peptide linker that can be cleaved by a cleaving agent present in the intracellular environment, e.g., lysosomes or endosomes, and cleaved by intracellular peptidase or protease enzymes, e.g., lysosome or endosome proteases. Generally, the peptide linker has a length of at least two amino acids. The cleaving agent may include cathepsin B and cathepsin D, plasmin, and hydrolyze the peptide to enable the release of the drug into the target cell. The peptide linker may be cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, and may be used, for example, but is not limited to, Gly-Gly-Phe-Gly (GGFG), Phe-Leu, or Gly-Phe-Leu-Gly linkers. In addition, the above peptide linker can be cleaved by, for example, an intracellular protease, and may be a Val-Cit linker or a Phe-Lys linker.

[0072] In the present invention, the cleavable linker is pH sensitive and may be sensitive to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, acid-unstable linkers that can be hydrolyzed in lysosomes may be, for example, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.

[0073] In addition, in the present invention, the linker may be cleaved under reducing conditions, for example, a disulfide linker may be used. Various disulfide bonds may 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-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0074] The above linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or overexpressed in some tumor cells. For example, a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, such as a beta-glucuronide linker containing a self-immolative group, may be used.

[0075] Additionally, the linker may be, for example, a non-cleavable linker, and the drug is released through an antibody hydrolysis step to produce, for example, an amino acid-linker-drug complex. This type of linker may be a thioether group or a maleimidocaproyl group and can maintain stability in the blood.

[0076] According to a preferred embodiment, the linker according to the present invention may include GGFG. According to one embodiment of the present invention, a portion of at least one amino acid side chain forming the linker may be substituted with a hydrophilic functional group that can be separated under specific conditions. The hydrophilic functional group is preferably a monovalent hydrophilic functional group, and examples may include, but are not limited to, β-glucuronide or an ester or carbonate having a PEG (Polyethylene Glycol) group having 3 to 100 ethylene glycol repeating units.

[0077] More preferably, the linker may have a structure of Formula 16 to Formula 18, but is not limited thereto.

[0078] [Chemical Formula 16]

[0079]

[0080] [Chemical Formula 17]

[0081]

[0082] [Chemical Formula 18]

[0083]

[0084] In the above chemical formula 18, n can be an integer from 3 to 10.

[0085]

[0086] In one embodiment of the present invention, the linker has the structure of maleimidocaproyl (MC)-valine (Val)-citrulline (Cit)-p-aminobenzyl carbamate (PABC), GGFG, or GGYG, and when the linker is GGYG, β-glucuronic acid is attached to the hydroxyl group of tyrosine.

[0087] In one embodiment of the present invention, the first drug is a linker-drug conjugate of the form MC-Val-Cit-PABC-MMAE, and the second drug is bound to an antibody in the form of a linker-drug conjugate of MC-GGFG-DXd, MC-GGFG-Formula 3, MC-GGYG-Formula 3, MC-GGFG-Formula 5, or MC-GGYG-Formula 5, wherein if the linker has a GGYG structure, β-glucuronic acid is bound to the hydroxyl group of tyrosine (Y). In this specification, a hyphen ('-') in notations such as 'MC-Val-Cit-PABC-MMAE' indicates a covalent linkage or chemical connection between each component. For example, 'MC-Val-Cit-PABC-MMAE' implies that it has a modular structure and that these components are connected to each other by covalent linkage or other chemical methods.

[0088]

[0089] For example, specific examples of linker-drug (payload) combinations that can be used to conjugate the first drug according to the present invention to a carrier are as shown in the following chemical formulas 19 to 26, but are not limited thereto.

[0090] [Chemical Formula 19]

[0091]

[0092] [Chemical Formula 20]

[0093]

[0094] [Chemical Formula 21]

[0095]

[0096] [Chemical Formula 22]

[0097]

[0098] [Chemical Formula 23]

[0099]

[0100] [Chemical Formula 24]

[0101]

[0102] [Chemical Formula 25]

[0103]

[0104] [Chemical Formula 26]

[0105]

[0106]

[0107] The present invention also provides a method for manufacturing an antibody-double drug conjugate to which a heterogeneous drug is combined.

[0108] The above antibody-drug conjugate is prepared by comprising the following steps: i) adding an excess amount of reducing agent to the antibody to expose reducible free thiol groups (-SH) within the antibody; ii) adding an excess amount of oxidizing agent to the antibody obtained according to step i) to reform disulfide bonds and expose free thiol groups (-SH) in monomeric form; iii) conjugating a first drug to the free thiol groups (-SH) of step ii) to form an antibody-first drug conjugate of DAR 1; iv) adding an excess amount of reducing agent to the antibody-first drug conjugate to expose reducible free thiol groups (-SH); and v) conjugating a second drug to the antibody obtained in step iv) to obtain an antibody-drug conjugate conjugated with a heterogeneous drug.

[0109] Herein, the antibody is an asymmetric antibody having a knob-in-hole structure, comprising: a) a first arm consisting of Fab and Fc regions derived from an IgG1 antibody specific to the first epitope; and b) comprising a second arm composed of a ScFv and Fc region fusion protein specific to the second epitope, wherein the Fc region of the second arm comprises a sequence derived from an IgG1 antibody, wherein the first epitope and the second epitope are identical or different from each other, and at least two disulfide bonds are formed in the hinge region upon intermolecular binding of the Fc regions of the first arm and the second arm, and any one of the amino acids including lysine at position 183 of the first arm heavy chain, lysine at position 149 of the first arm light chain, lysine at positions 290, 326, and 392 of the first arm or second arm Fc region, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328 It may be substituted with cysteine.

[0110]

[0111] The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer comprising the antibody-drug conjugate (or antibody-double drug conjugate). The description of the antibody-drug conjugate applies equally to the extent that it does not limit the inherent characteristics of the pharmaceutical composition.

[0112] The cancer of the present invention includes all cancers treatable with inhibitors of topoisomerase I and / or microtubulin-targeted therapies (inhibitors), and may be solid tumors or blood cancers. Specifically, pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial carcinoma, ovarian germ cell carcinoma, ovarian cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampullary carcinoma, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, rhinosinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, Includes, but is not limited to, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, triple-negative breast cancer (TNBC), sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer. In addition, the above cancer includes not only primary cancer but also metastatic cancer.

[0113] The antibody-drug conjugate of the present invention or a pharmaceutical composition containing the same can be administered to a subject in need of the same in a therapeutically effective amount.

[0114] In this specification, “patient,” “subject,” and “object” refer to animals such as mammals. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal, such as a dog, cat, livestock (e.g., horse, pig, or donkey), chimpanzee, or monkey.

[0115] The term "therapeutically effective amount" as used in the present invention refers to an amount of the antibody-drug conjugate or a pharmaceutical composition containing the same that is effective for the treatment or prevention of cancer. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, gender, type of disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and since the antibody-drug conjugate of the present invention exhibits dose-dependent effects, the dosage can be easily determined by a person skilled in the art based on various factors such as the patient's condition, age, gender, and complications. Since the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it can be used even at doses greater than the determined dosage.

[0116] In addition, the present invention provides a use of the antibody-drug conjugate for use in the manufacture of a medicament for the treatment or prevention of cancer.

[0117] The antibody-drug conjugate for the manufacture of the drug may be mixed with pharmaceutically acceptable adjuvants, diluents, carriers, etc., and may be manufactured as a complex formulation with other active agents to have a synergistic effect of the active ingredients.

[0118] In this specification, the anticancer effect or therapeutic effect by an anticancer agent may refer to an action that reduces the severity of cancer, reduces the size of a tumor, or delays or slows down the progression of cancer, which occurs while a patient is suffering from a specific cancer.

[0119] For example, the anticancer effect of an anticancer drug can be the cell viability (degree of cytotoxicity or change in cell number) of cancer cells after treating them with the drug in vitro and / or in vivo. For instance, it can be indirectly confirmed through drug response tests using cell lines or xenografts. Additionally, the anticancer effect of the drug can be directly verified in cancer patients to derive related data, which can then be used as a database. Furthermore, animal model PK parameters and / or toxicity profiles can be considered in parallel when designing guidelines for anticancer drug administration.

[0120] The anticancer effect of an anticancer drug can be inferred from in-vitro data, such as the % Maximum effect of the anticancer drug (e.g., IC50, IC60, IC70, IC80, and IC90), and can also be confirmed in non-clinical animal models and clinical cancer patients through in-vivo data, such as the maximum blood concentration (Cmax) and / or the area under the blood drug concentration-time curve (AUC).

[0121] The responsiveness of an anticancer drug refers to clinical sensitivity in terms of anticancer effect.

[0122] When referring to treatment using anticancer drugs, "sensitivity" and "sensitive" are relative terms describing the degree of effect of a compound in alleviating or reducing the progression of the tumor or disease being treated.

[0123] "Effective anticancer effect / response in patients" may be inhibition of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more in patient response, as measured by any suitable means, such as gene expression, cell counting, analysis results, etc.

[0124] In this specification, the dosage is the dose at which a therapeutic effect is expected. In the present invention, the therapeutic effect may be an anticancer effect. The responsiveness (anticancer effect) of the anticancer agent is the degree of response and may be the % maximum effect of the anticancer agent, such as IC50, IC60, IC70, IC80, and IC90, or the value exhibiting toxicity to normal cells (LC50).

[0125] For example, oral formulations may be formulated using various formulation technologies known in the art. For example, they may include a biodisintegrable (hydrolyzable) polymeric carrier used for attachment to the oral mucosa. They are manufactured to gradually erode over a set period, wherein drug delivery is essentially provided entirely.

[0126] Drug delivery in oral formulations avoids the weaknesses encountered in oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract, and / or first-pass inactivation in the liver. Regarding biodisintegrable (hydrolyzable) polymeric carriers, virtually any such carrier may be used as long as the desired drug release profile is not compromised, and the carrier is compatible with any other component present in the oral dosage unit. Generally, the polymeric carrier comprises a hydrophilic (water-soluble and water-swellable) polymer that adheres to the moist surface of the oral mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers (e.g., carbomers). In some embodiments, non-limiting examples of other components that may be incorporated into the oral formulation include disintegrants, diluents, binders, lubricants, flavorings, colorings, preservatives, etc. In some embodiments, for oral or sublingual administration, it may be in the form of a tablet, lozenge, or gel formulated in a conventional manner.

[0127] In some embodiments, if the patient's condition improves, administration of the compound is continued at the discretion of the physician; alternatively, the dose of the drug to be administered may be temporarily reduced or temporarily discontinued for a certain length of time (i.e., "drug break"). The length of the drug break may vary between 2 days and 1 year and includes, by example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dose reduction during the drug withdrawal is 10%-100% and includes, by example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0128] If the patient's condition improves, a maintenance dose is administered if necessary. Subsequently, the dosage, frequency of administration, or both may be reduced as a function of symptoms to a level where the improved disease, disorder, or condition is maintained. However, the patient requires intermittent treatment over an extended period in the event of any recurrence of symptoms.

[0129] The amount of a given formulation corresponding to such an amount will vary depending on factors of the subject requiring treatment, such as the specific compound, the severity of the disease, and characteristics (e.g., body weight), but nevertheless may be routinely determined in the manner known in the art, for example, depending on the formulation to be administered, the route of administration, and specific circumstances surrounding the subject to be treated. Generally, however, the dose used for the treatment of adult humans will typically be in the range of 0.02–5000 mg / day, or about 1–1500 mg / day.

[0130] In this specification, a single dose may be provided as a single dose or as a divided dose administered simultaneously, for example, as 2, 3, 4 or more sub-dose portions.

[0131] In some embodiments, the oral formulation is a unit dose form suitable for a single administration of an accurate dosage. In the unit dose form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. In some embodiments, the unit dose is in the form of a pack containing separate amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powder vials or ampoules. The aqueous suspension composition may be packaged in a single-dose non-resealable container. Alternatively, a multi-dose resealable container may be used, in which case it is typical to include a preservative in the composition.

[0132] In some embodiments, the parenteral injectable formulation is provided with an added preservative in a unit dosage form, including, but not limited to, an ampoule, or in a multi-dose container.

[0133] It is typically manufactured for parenteral administration, i.e., bolus, intravenous, and intratumoral injection, in a unit-dose injectable form with a pharmaceutically acceptable parenteral vehicle. It is optionally mixed in the form of a lyophilized preparation or an aqueous solution with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).

[0134] The matters mentioned in the antibody-drug conjugate, pharmaceutical composition containing the same, use, method of preparation, and method of treatment presented in the present invention apply equally unless they are contradictory.

[0135] According to the present invention, an antibody-double drug conjugate (AD2C) can be stably produced by selectively conjugating a first drug and a second drug at different locations to an asymmetric antibody having a knop-in-hole structure.

[0136] The antibody and manufacturing method of the present invention have the advantage of enabling the realization of an antibody-drug conjugate (AD2C) in which the binding ratio of the two drugs is approximately 1:6, by allowing the first drug to selectively bind to a specific site of the antibody to exhibit a DAR value corresponding to an average drug-to-antibody ratio of 1, and the second drug to selectively bind to engineered cysteine ​​residues of the antibody to exhibit a DAR value corresponding to an average drug-to-antibody ratio of 6.

[0137] When using the asymmetric antibody according to the present invention, the first drug and the second drug are stably conjugated through different binding sites, thereby enabling control of the overall DAR and providing the advantage of securing a uniform drug distribution. Accordingly, the AD2C of the present invention can simultaneously improve drug delivery efficiency and therapeutic effect while ensuring structural stability and reproducibility.

[0138] Furthermore, the AD2C according to the present invention exhibited a single peak and high purity in SEC and HIC analyses, and maintained stability even under repeated freeze / thaw conditions. Unlike conventional random conjugation methods, this enables the production of uniform and reproducible ADC / AD2C, thereby improving drug delivery efficiency and reducing non-specific toxicity.

[0139] Therefore, the antibody-double drug conjugate of the present invention can achieve synergy in therapeutic effects and suppress resistance by simultaneously delivering drugs with different mechanisms, and can be usefully utilized as a therapeutic platform applicable to various types of cancer and intractable diseases.

[0140] Figure 1 is a schematic diagram showing the structure of an asymmetric antibody including a knop-in-hole structure.

[0141] Figures 2a to 2i show the results of analyzing changes in molecular weight by LC-MS for the light chain (LC), knob structure heavy chain (HC2), and hole structure heavy chain (HC1) after treating the asymmetric antibody with mc-vc-MMAE in untreated, 6-fold overdose, and 12-fold overdose.

[0142] Figure 2a shows the LC-MS analysis results for the light chain (LC) of the asymmetric antibody under mc-vc-MMAE untreated conditions. Figure 2b shows the LC-MS analysis results for the knob structure heavy chain (HC2) under mc-vc-MMAE untreated conditions. Figure 2c shows the LC-MS analysis results for the hole structure heavy chain (HC1) under mc-vc-MMAE untreated conditions. Figure 2d shows the LC-MS analysis results for the light chain (LC) of the asymmetric antibody under mc-vc-MMAE 6-fold overdose conditions. Figure 2e shows the LC-MS analysis results for the knob structure heavy chain (HC2) under mc-vc-MMAE 6-fold overdose conditions. Figure 2f shows the LC-MS analysis results for the hole structure heavy chain (HC1) under mc-vc-MMAE 6-fold overdose conditions. Figure 2g shows the LC-MS analysis results for the light chain (LC) of the asymmetric antibody under 12-fold overdose conditions of mc-vc-MMAE. Figure 2h shows the LC-MS analysis results for the Knob structure heavy chain (HC2) under 12-fold overdose conditions of mc-vc-MMAE. Figure 2i shows the LC-MS analysis results for the Hall structure heavy chain (HC1) under 12-fold overdose conditions of mc-vc-MMAE.

[0143] FIGS. 3a to 3c are drawings showing the molecular weight distribution results obtained through LC-MS analysis for a light chain (LC), a knob structure heavy chain (HC1), and a hole structure heavy chain (HC2), respectively, according to an embodiment of the present invention. FIG. 3a is a drawing showing the molecular weight distribution measured by LC-MS for the light chain (LC: SEQ ID NO. 1) of an asymmetric antibody. FIG. 3b is a drawing showing the molecular weight distribution measured by LC-MS for the knob structure heavy chain (HC1: SEQ ID NO. 2) of an asymmetric antibody. FIG. 3c is a drawing showing the molecular weight distribution measured by LC-MS for the hole structure heavy chain (HC2: SEQ ID NO. 3) of an asymmetric antibody.

[0144] Figure 4 is an image showing the DAR values ​​calculated by LC-MS analysis after reacting mc-vc-MMAE with an asymmetric antibody for 16 hours under untreated (Intact), 6x overdose (6x), and 12x overdose (12x) conditions according to one embodiment of the present invention.

[0145] Figure 5 is a figure showing the results of SDS-PAGE analysis of samples and antibody-drug conjugates (PE1-VM-109H06-1, PE1-VM-109H06-2) that underwent reduction (Red) and oxidation (Oxi) processes for asymmetric antibodies prepared according to the present invention, under reduced and non-reduced conditions.

[0146] Figures 6a to 6e show the results of SEC and HIC analysis of the antibody-drug conjugate (AD2C) and the unbound antibody (control, naked), confirming that AD2C maintains high purity and stability while increasing hydrophobicity due to the binding of the two drugs. Figure 6a shows the SEC analysis results of the control antibody. Figure 6b shows the SEC analysis results of the manufactured antibody-drug conjugate (AD2C). Figure 6c shows the SEC analysis results of the manufactured antibody-drug conjugate (AD2C) after two freeze / thaw treatments. Figure 6d shows the HIC analysis results of the control antibody. Figure 6e shows the HIC analysis results of the manufactured antibody-drug conjugate (AD2C).

[0147] Figure 7 is a figure showing the results of SDS-PAGE analysis under reducing and non-reducing conditions of a sample that underwent reduction, oxidation, binding of the first drug (LP1), re-reduction, and binding of the second drug (LP2) in the process of preparing an antibody-double drug conjugate (AD2C) from an asymmetric antibody.

[0148] Figures 8a to 8f show the LC-MS analysis results for the light chain (LC), hole structure heavy chain (HC1), and knob structure heavy chain (HC2) of a naked antibody and an antibody-drug conjugate (AD2C), demonstrating that the target drug binding ratio was achieved in AD2C by LP1 selectively binding to HC1 and LP2 binding to both the LC and the two heavy chains. Figure 8a shows the LC-MS analysis results for the light chain (LC) of the naked antibody. Figure 8b shows the LC-MS analysis results for the hole structure heavy chain (HC1) of the naked antibody. Figure 8c shows the LC-MS analysis results for the knob structure heavy chain (HC2) of the naked antibody. Figure 8d shows the LC-MS analysis results for the light chain (LC) of the antibody-drug conjugate (AD2C), where a peak bound to LP2 is identified. Figure 8e shows the LC-MS analysis results for the hole structure heavy chain (HC1) of the antibody-drug conjugate (AD2C), where a peak bound to LP1 and LP2 is identified. Figure 8f shows the LC-MS analysis results for the knob structure heavy chain (HC2) of the antibody-drug conjugate (AD2C), where a peak bound to LP2 is identified.

[0149] Figure 9 is a table showing the DAR values ​​of LP1 and LP2 and the total DAR calculated based on the LC-MS analysis results of the control (naked) antibody and the antibody-drug conjugate (AD2C).

[0150] The present invention will be described in detail below. The advantages and features of the present invention and the embodiments described below for achieving them will become clear. However, the present invention is not limited to the embodiments disclosed below but can be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Throughout the specification, the same reference numerals refer to the same components.

[0151] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise. The terms used herein are for describing embodiments and are not intended to limit the present invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text.

[0152]

[0153] In the present invention, an antibody-drug conjugate (AD2C) capable of simultaneously conjugating two different types of drugs was prepared using an asymmetric antibody having a knob-in-hole structure. It was confirmed that the antibody of the present invention stably binds the first drug to an average DAR level of 1 and the second drug to an average DAR level of 6 through designed engineered cysteine ​​residues, thereby enabling the realization of an AD2C in which the DAR ratio of the two drugs is approximately 1:6.

[0154] AD2C prepared according to the present invention exhibited a single peak and high purity in SEC and HIC analyses, and demonstrated that structural stability was maintained even under repeated freeze-thaw conditions. In addition, LC-MS analysis confirmed that the first and second drugs were selectively conjugated to different binding sites, and DAR analysis verified that the drugs were bound at the target ratio.

[0155] AD2C according to the present invention can simultaneously deliver two drugs with different mechanisms of action via an antibody, thereby achieving synergy in therapeutic effects, suppression of drug resistance, and reduction of non-specific toxicity. Therefore, the antibody and manufacturing method of the present invention serve as a next-generation antibody-drug conjugate platform and can be usefully utilized for the treatment of various types of cancer and intractable diseases.

[0156]

[0157] [Preparation Example]

[0158] 1. Ingredients

[0159] mc-vc-MMAE was purchased from MedChemExpress. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Thermo Fischer Scientific. Dehydroascorbic acid (dhAA) was purchased from Sigma Aldrich.

[0160]

[0161] 2. Preparation of Chemical Formula 3

[0162]

[0163] (R)-2-hydroxypropanoic acid (37 mg, 0.41 mmol) was dissolved in 2.0 mL of N,N-dimethylformamide. HOSu (48 mg, 0.41 mmol) and EDCI.HCl (79 mg, 0.41 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. 0.7 mL of this solution was added to a suspension of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]diosolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione methanesulfonate (75 mg, 0.14 mmol) and diisopropylethylamine (96 μL, 0.55 mmol) dissolved in N,N-dimethylformamide (3.0 mL). The mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted in DMSO and heated to obtain a clear solution. The mixture was purified twice by acid preparative MPLC (5-40), and after freeze-drying the product fraction, a grayish-white solid was obtained. Yield: 45 mg, 58%.

[0164] U_AN_ACID: m / z 520.2 [M+H] +

[0165] 1 H NMR (400 MHz, DMSO) δ 8.39 (d,J= 9.1 Hz, 1H), 7.40 (s, 1H), 7.23 (s, 1H), 6.47 (s, 1H), 6.28 (d,J= 2.3 Hz, 2H), 5.61 - 5.49 (m, 2H), 5.40 (s, 2H), 5.22 - 5.00 (m, 2H), 4.17 - 4.07 (m, 1H), 3.13 - 2.95 (m, 2H), 2.19 - 2.03 (m, 2H), 1.94 - 1.77 (m, 2H), 1.39 (d,J= 6.8 Hz, 3H), 0.87 (t,J= 7.3 Hz, 3H).

[0166]

[0167] 3. Preparation of a linker-drug conjugate (LP2) containing Chemical Formula 3

[0168]

[0169] Manufacturing method

[0170] 3-1. Step 1

[0171]

[0172] Fmoc-Gly-Gly-OH (5.0 g, 14.1 mmol) was partially dissolved in tetrahydrofuran (125 mL), toluene (42.6 mL), and pyridine (2.15 mL). Then, lead (IV) acetate (7.8 g, 17.6 mmol) was added, and the reaction mixture turned orange. The mixture was heated to reflux temperature. After stirring for 3 hours, the reaction mixture was cooled to room temperature, filtered through a Celite layer, and washed with ethyl acetate. It was then concentrated under reduced pressure. The residue was purified by flash chromatography (10-100% ethyl acetate in silica and heptane) to obtain (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)-methyl acetate (3.0 g, 57% yield) as a white solid.

[0173] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d,J= 7.4 Hz, 2H), 7.59 (d,J= 7.5 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.36 - 7.29 (m, 2H), 6.98 (s, 1H), 5.34 (s, 1H), 5.26 (d,J= 7.3 Hz, 2H), 4.46 (d,J= 6.8 Hz, 2H), 4.23 (t,J= 6.8 Hz, 1H), 3.94 - 3.84 (m, 2H), 2.06 (s, 3H). m / z 391.2 [M+Na] +

[0174]

[0175] 3-2. Step 2

[0176]

[0177] (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)-methyl acetate (0.3 g, 0.814 mmol) was dissolved in dichloromethane (4.35 mL), and then benzyl (R)-2-hydroxypropanoate (1.46 g, 8.14 mmol) was added. Subsequently, pyridinium p-toluenesulfonate (0.020 g, 0.081 mmol) was added, and the mixture was stirred overnight at reflux temperature. After cooling the reaction mixture to room temperature, it was diluted with ethyl acetate (20 mL), washed with water (3 x 20 mL), and dried with Na2SO4. It was then filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (ethyl acetate 10-70% in silica and heptane). Yield: 0.35 g, 88%.

[0178] 1 H NMR (400 MHz, CDCl3) δ 7.77 (d,J= 7.5 Hz, 2H), 7.59 (d,J= 7.5 Hz, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.28 (m, 7H), 6.66 - 6.55 (m, 1H), 5.21 - 5.12 (m, 3H), 4.93 - 4.73 (m, 2H), 4.46 (d,J= 6.7 Hz, 2H), 4.26 - 4.19 (m, 2H), 3.85 - 3.69 (m, 2H), 1.42 (d,J= 6.8 Hz, 3H). m / z 511.2 [M+Na] +

[0179]

[0180] 3-3. Step 3

[0181]

[0182] Diethylamine (1.7 mL) was added at room temperature to a suspension of benzyl (R)-1-(9H-fluorene-9-yl)-10-methyl-3,6-dioxo-2,9-diosa-4,7-diazaundecane-11-oate (0.35 g, 0.716 mmol) in dichloromethane (3.4 mL). After stirring for 2 hours, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain benzyl (R)-2-((2-aminoacetamido)-methoxy)propanoate as a white solid. The product was used as is. m / z 267.0 [M+H] +

[0183]

[0184] 3-4. Step 4

[0185]

[0186] Fmoc-Phe-OSu (1.041 g, 2.148 mmol) was added to a 3.35 mL solution of N,N-dimethylformamide (dry) containing benzyl (R)-2-((2-aminoacetamido)methoxy)propanoate (0.191 g, 0.716 mmol) under an argon atmosphere at room temperature. After stirring for 50 minutes, the reaction mixture was diluted with ethyl acetate and washed with water and brine. It was then dried with Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (10-100% ethyl acetate in silica and heptane) to obtain benzyl (5S,13R)-5-benzyl-1-(9H-fluorene-9-yl)-13-methyl-3,6,9-trioxo-2,12-diosa-4,7,10-triazatetradecane-14-oate (0.295 g, 64% yield) as a colorless oil.

[0187] 1H NMR (400 MHz, CDCl3) δ 7.76 (d,J= 7.6 Hz, 2H), 7.56 - 7.49 (m, 2H), 7.44 - 7.37 (m, 2H), 7.37 - 7.27 (m, 10H), 7.21 - 7.14 (m, 2H), 6.93 - 6.73 (m, 1H), 6.30 - 6.09 (m, 1H), 5.25 - 5.16 (m, 1H), 5.16 - 5.08 (m, 2H), 4.81 - 4.77 (m, 1H), 4.71 - 4.67 (m, 1H), 4.51 - 4.25 (m, 3H), 4.22 - 4.15 (m, 2H), 3.89 - 3.69 (m, 2H), 3.19 - 2.98 (m, 2H), 1.37 (d,J= 6.9 Hz, 3H). m / z 658.4 [M+Na] +

[0188]

[0189] 3-5. Step 5

[0190]

[0191] Diethylamine (1.1 mL) was added at room temperature to a suspension of benzyl (5S,13R)-5-benzyl-1-(9H-fluorene-9-yl)-13-methyl-3,6,9-trioxo-2,12-diosa-4,7,10-triazatetradecane-14-oate (0.295 g, 0.464 mmol) in dichloromethane (2.2 mL). After stirring for 2 hours, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain benzyl (R)-2-((2-((S)-2-amino-3-phenylpropanamido)acetamido)methoxy)propanoate as a colorless residue, which solidified upon standing. The product was used as is. m / z 414.2 [M+H] +

[0192]

[0193] 3-6. Step 6

[0194]

[0195] DIPEA (0.324 mL, 1.856 mmol) was added to a solution of benzyl (R)-2-((2-((S)-2-amino-3-phenylpropanamido)acetamido)methoxy)propanoate (0.192 g, 0.464 mmol) in N,N-dimethylformamide (dry) (1.482 mL), followed by the addition of Fmoc-Gly-Gly-OH (0.329 g, 0.928 mmol) and HATU (0.353 g, 0.928 mmol). The reaction mixture was stirred at room temperature for 45 minutes. Subsequently, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with 10% water-soluble citric acid (10 mL). The aqueous layer was extracted with ethyl acetate (2 x 10 mL). The combined organic layer was washed with water (5 mL), saturated water-soluble NaHCO3 (5 mL), and brine (5 mL), respectively, and then dried with Na2SO4. Subsequently, it was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (0-8% methanol in silica and dichloromethane) to obtain benzyl (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-diosa-4,7,10,13,16-pentazaicosan-20-oate (0.259 g, 74% yield) as a white solid.

[0196] 1H NMR (400 MHz, DMSO-d6) δ 8.63 - 8.54 (m, 1H), 8.39 - 8.29 (m, 1H), 8.16 (d,J= 8.2 Hz, 1H), 8.07 - 7.97 (m, 1H), 7.89 (d,J= 7.4 Hz, 2H), 7.70 (d,J= 7.6 Hz, 2H), 7.64 - 7.55 (m, 1H), 7.44 - 7.29 (m, 9H), 7.26 - 7.13 (m, 5H), 5.21 - 5.07 (m, 2H), 4.66 - 4.45 (m, 3H), 4.33 - 4.17 (m, 4H), 3.82 - 3.56 (m, 6H), 3.05 (dd,J= 13.7, 4.5 Hz, 1H), 2.84 - 2.73 (m, 1H), 1.26 (d,J= 6.8 Hz, 3H). m / z 772.4 [M+Na] +

[0197]

[0198] 3-7. Step 7

[0199]

[0200] A solution of benzyl (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-diosa-4,7,10,13,16-pentazaicosan-20-oate (259 mg, 0.345 mmol) dissolved in ethanol (4.1 mL) and ethyl acetate (2.0 mL) was purged with argon for 10 minutes. Then, Pd / C (10%, 50% wet) (73.5 mg, 0.035 mmol) was added and stirred for 2 hours in a hydrogen atmosphere (balloon). The reaction mixture was filtered through a celite layer, washed with methanol, and concentrated under reduced pressure to obtain (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-diosa-4,7,10,13,16-pentazaicosan-20-oxan (0.204 g, 90% yield) as a pale white solid.

[0201] 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 7.89 (d,J= 7.6 Hz, 2H), 7.72 (d,J= 7.4 Hz, 2H), 7.59 (d,J= 7.2 Hz, 0H), 7.46 - 7.28 (m, 4H), 7.24 (d,J= 4.4 Hz, 4H), 7.18 (t,J= 4.6 Hz, 1H), 4.69 (s, 1H), 4.58 - 4.46 (m, 2H), 4.35 (s, 1H), 4.31 - 4.18 (m, 3H), 3.97 (d,J= 7.7 Hz, 1H), 3.84 - 3.70 (m, 2H), 3.67 - 3.54 (m, 4H), 3.07 (dd,J= 13.9, 4.5 Hz, 1H), 2.89 - 2.76 (m, 1H), 1.23 (d,J= 6.8 Hz, 3H). m / z 682.2 [M+Na] +

[0202]

[0203] 3-8. Step 8

[0204]

[0205] Diethylamine (0.25 mL) was added at room temperature to a suspension of (11S,19R)-11-benzyl-1-(9H-fluorene-9-yl)-19-methyl-3,6,9,12,15-pentaoxo-2,18-diosa-4,7,10,13,16-pentazaicosan-20-oxan (50 mg, 0.076 mmol) in dichloromethane (0.5 mL). After stirring for 3 hours, the reaction mixture was concentrated under reduced pressure and co-evaporated three times with dichloromethane to obtain (2R,10S)-16-amino-10-benzyl-2-methyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanosan as a white solid. The product was used as is. m / z 438.2 [M+H] +

[0206]

[0207] 3-9. Step 9

[0208]

[0209] (2R,10S)-16-amino-10-benzyl-2-methyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecanosane (33.2 mg, 0.076 mmol) was dissolved in N,N-dimethylformamide (dry) (1 mL), and DIPEA (0.086 mL, 0.494 mmol) was added at room temperature under an argon atmosphere. Subsequently, 6-maleimidohexanoic acid N-hydroxysuccinimide ester (35.1 mg, 0.114 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with 1 mL of DMF and purified using an acidic preparative MPLC (Luna 5-40). Subsequently, (2R,10S)-10-benzyl-23-(2,5-dioso-2,5-dihydro-1H-pyrrole-1-yl)-2-methyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentazatricosanoic acid (24 mg, 50% yield) was obtained as a white solid by freeze-drying.

[0210] 1 H NMR (400 MHz, DMSO-d6) δ 12.60 (s, 1H), 8.61 (s, 1H), 8.32 - 7.93 (m, 4H), 7.28 - 7.21 (m, 4H), 7.20 - 7.15 (m, 1H), 7.00 (s, 2H), 4.68 - 4.60 (m, 1H), 4.59 - 4.44 (m, 2H), 4.04 - 3.96 (m, 1H), 3.80 - 3.55 (m, 6H), 3.40 - 3.34 (m, 2H), 3.06 (dd,J= 13.9, 4.6 Hz, 1H), 2.81 (dd,J= 13.9, 9.7 Hz, 1H), 2.10 (t,J= 7.3 Hz, 2H), 1.52 - 1.41 (m, 4H), 1.26 - 1.14 (m, 5H). m / z 653.4 [M+Na] +

[0211]

[0212] 3-10. Step 10

[0213]

[0214] (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione methanesulfonate (410 mg, 1 eq, 754 μmol) and (2R,10S)-10-benzyl-23-(2,5-dioso-2,5-dihydro-1H-pyrrole-1-yl)-2-methyl-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentazatricosanoic acid (466 mg, 0.98 eq, 739 μmol) was suspended in N,N-dimethylformamide (10.0 mL). Subsequently, DIPEA (439 mg, 591 μL, 4.5 eq, 3.39 mmol) was added, followed by HATU (430 mg, 1.5 eq, 1.13 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified by acidic preparative MPLC (Luna 10-50) three times. The resulting product fractions were combined and freeze-dried to obtain a pale yellow solid. Yield: 510 mg, 63%.

[0215] 1H NMR (400 MHz, DMSO-d6) δ 8.62 (t,J= 6.6 Hz, 1H), 8.50 (d,J= 9.1 Hz, 1H), 8.29 (t,J= 5.9 Hz, 1H), 8.13 - 7.96 (m, 3H), 7.39 (s, 1H), 7.27 - 7.12 (m, 6H), 6.99 (s, 2H), 6.48 (s, 1H), 6.26 (d,J= 6.0 Hz, 2H), 5.61 - 5.51 (m, 1H), 5.45 - 5.33 (m, 2H), 5.20 - 4.99 (m, 2H), 4.72 - 4.63 (m, 1H), 4.57 - 4.41 (m, 2H), 4.11 (q,J= 6.7 Hz, 1H), 3.78 - 3.53 (m, 6H), 3.40 - 3.34 (m, 2H), 3.16 - 2.95 (m, 3H), 2.74 (dd,J= 13.8, 9.5 Hz, 1H), 2.17 - 2.03 (m, 4H), 1.91 - 1.78 (m, 2H), 1.52 - 1.35 (m, 7H), 1.25 - 1.12 (m, 2H), 0.87 (t,J= 7.3 Hz, 3H). m / z 1060.02 [M+H] +

[0216]

[0217] [Example 1]

[0218] 1-1. LC-MS for DAR 1 Condition Analysis of Asymmetric Antibodies

[0219] In this embodiment, an asymmetric antibody having a knob-in-hole structure was used. Specifically, the knob structure arm was composed of a light chain (SEQ No. 1) and a heavy chain (SEQ No. 2), and the hole structure arm was constructed with a heavy chain containing scFv (SEQ No. 3). The antibody contains a total of three interchain disulfide bonds, and one free cysteine ​​is introduced into the knob structure heavy chain (HC1: SEQ No. 2).

[0220] A reduction reaction was performed on 4 mg / ml of antibody using 30 molar equivalents of the reducing agent tris(2-carboxyethyl)phosphine (TCEP, Thermo Fisher Scientific) at 25°C for 2 hours. Residual TCEP was removed using a PD-10 column (Cytiva), and interchain disulfide bonds were reformed using the oxidizing agent dehydroascorbic acid (dhAA, Sigma Aldrich). Subsequently, residual dhAA was removed using a PD-10 column.

[0221] 6 and 12 times the molar amount of mc-vc-MMAE (MedChemExpress) were added to the 2 mg / mL antibody obtained after a series of reductive oxidation processes. The mixed solution was reacted at 25°C for 16 hours. After the reaction, the remaining mc-vc-MMAE was removed using a PD-10 column.

[0222] The purified antibody-drug conjugate was analyzed using liquid chromatography-mass spectrometry (LC-MS, ACQUITY Arc Bio System, Waters). For the analysis, a sample (1 μg) was used with ACQUITY TM After separation on a Primer Protein BEH C4 column (2.1 Х 50 mm, 1.7 μm, Waters), the process was performed under ESI (positive ion mode) conditions, and the molecular weight distributions of LC, HC1, and HC2 were determined using UNIFI software.

[0223] LC-MS analysis revealed no molecular weight change corresponding to the binding of mc-vc-MMAE in the light chain (LC: SEQ ID NO. 1) and the hole structure heavy chain (HC2: SEQ ID NO. 3). In contrast, a distinct new mass peak corresponding to the binding of mc-vc-MMAE was observed in the knop structure heavy chain (HC1: SEQ ID NO. 2), and drug binding was confirmed under both 6-fold and 12-fold excess doses of mc-vc-MMAE relative to the antibody (see Figs. 2a to 2i, Figs. 3a to 3c).

[0224] Through the above results, it was confirmed that a single free cysteine ​​residue introduced into the heavy chain (HC1) of the knob structure acts as a reaction site for drug binding. Accordingly, it can be seen that the asymmetric antibody of the present invention can induce a drug to selectively bind to a specific heavy chain (HC1), thereby enabling the stable implementation of the DAR 1 condition per antibody.

[0225]

[0226] 1-2. Confirmation of DAR of Asymmetric Antibody-Based ADCs

[0227] The DAR calculated based on the LC-MS analysis results of Example 1-1 above is shown in Fig. 4. Referring to Fig. 4, no drug binding was observed in the light chain (LC) and the hole structure heavy chain (HC2), whereas selective binding of mc-vc-MMAE was confirmed only in the knob structure heavy chain (HC1). As a result, the average DAR value per antibody was approximately 0.93 when treated with a 6-fold overdose of mc-vc-MMAE and approximately 0.95 when treated with a 12-fold overdose, indicating that the asymmetric antibody of the present invention selectively bound to the drug in accordance with the DAR 1 condition.

[0228]

[0229] 1-3. SDS-PAGE Analysis of Antibody Reduction and Oxidation Processes

[0230] Intact, Reduced, and Oxidized antibodies, along with ADC samples (PE1-VM-109H06-1, PE1-VM-109H06-2), were prepared and subjected to electrophoresis on a 4–15% gradient SDS-PAGE gel. After treating the samples under reduced and non-reduced conditions, the bands were visualized by staining with Coomassie Brilliant Blue (see Fig. 5).

[0231] Under non-reduced conditions, the antibody showed a distinct single band around 150 kDa. In contrast, under red conditions, the antibody separated into heavy and light chains, and separate bands were observed. Subsequently, under oxidized conditions, a single band formed again around 150 kDa, confirming that the reduced antibody was successfully reoxidized. Additionally, the ADC samples (PE1-VM-109H06-1 and PE1-VM-109H06-2) also showed a band around 150 kDa under non-reduced conditions, confirming that the antibody structure was maintained stably.

[0232] The above results demonstrate that the reduction and oxidation steps performed during the preparation of the antibody-drug conjugate of the present invention proceeded normally. In other words, it can be confirmed that the reduction and reformation of the disulfide bond were stably carried out, thereby preserving the structural integrity of the antibody.

[0233]

[0234] [Example 2]

[0235] Antibody double drug conjugate (AD2C)

[0236] 2-1. Preparation of Antibody-Double Drug Conjugates (AD2C) and SEC and HIC Analysis

[0237] In this embodiment, an asymmetric antibody with a knob-in-hole structure was used. In the antibody, a specific amino acid is substituted with cysteine ​​(Cys) in the heavy chain containing the knob, and the heavy chain containing the hole contains scFv. The antibody contains three interchain disulfide bonds and has one free cysteine ​​residue introduced into the heavy chain containing the knob. Hereinafter, LP (Linker-payload) refers to a linker-drug conjugate in which a linker is attached to a drug.

[0238] First, according to the DAR 1 preparation conditions established in Example 1-1, an antibody-drug conjugate (ADC) of DAR 1 was prepared by conjugating mc-vc-MMAE (LP1) to the free cysteine ​​of the antibody. Subsequently, the ADC was treated with 30 equivalents of TCEP relative to the molar amount of the ADC, and a reduction process was performed at 25°C for 2 hours. The remaining TCEP was removed using a PD-10 column. Then, a linker-drug conjugate (LP2, see Preparation Example 3) containing 12 equivalents of Formula 3 relative to the molar amount of the ADC was reacted with the ADC at 25°C for 1 hour. The remaining LP2 was removed using a PD-10 column to finally obtain an antibody-double drug conjugate (AD2C) in which the binding ratio of the first drug (LP1) to the second drug (LP2) was approximately 1:6.

[0239] The prepared AD2C was analyzed by size exclusion chromatography (SEC, TSKgel G3000S Wxl, Tosoh) and hydrophobic interaction chromatography (HIC, MabPAC butyl HIC column, Thermo Fisher Scientific) (see Figs. 6a to 6e).

[0240] According to SEC results, high purity of approximately 94-95% and a single peak maintained even after freezing and thawing were confirmed, supporting the structural stability and process suitability of the manufactured AD2C (see Figs. 6a-6c). Furthermore, the increase in RT of approximately 1.2 minutes and the detection of a single peak in HIC indicate that the hydrophobicity of the antibody increased due to the binding of LP1 (including MMAE, DAR 1) and LP2 (including Formula 3, DAR 6), which implies that the binding of the dual drugs was successfully achieved (see Figs. 6d, 6f).

[0241]

[0242] 2-2. SDS-PAGE Analysis of Antibody-Double Drug Conjugate Manufacturing Process

[0243] Samples were prepared by performing reduction, oxidation, and drug conjugation processes step by step on an asymmetric antibody (PE1). Specifically, an unmodified antibody (Intact PE1), a reduced antibody (Reduced PE1, R1), an oxidized antibody (Oxidized PE1, O), an ADC conjugated with a first drug (mc-vc-MMAE, LP1) (PE1-VM-109H19, C1), a reduced version of the ADC conjugated with LP1 (Reduced PE1-VM-109H19, R2), and an AD2C conjugated with a second drug (Formula 3, LP2) (PE1-DP1-1609H20, C2) were prepared, respectively.

[0244] After performing 4–15% gradient SDS-PAGE gel electrophoresis on the above samples under reduced and non-reduced conditions, protein bands were identified by Coomassie Brilliant Blue staining (see Fig. 7).

[0245] Through SDS-PAGE, it can be confirmed that the antibody's reduction, oxidation, LP1 binding, re-reduction, and LP2 binding reactions occurred normally. Looking at the non-reduced gel in Figure 7, it can be seen that the antibody is separated into heavy and light chains during the reduction process (R1), and that a band appears again around 150 kDa during the oxidation process (O). Similarly, when the LP1-bound ADC was reduced (R2), it was confirmed that it was separated into heavy and light chains again and could be used for the conjugation reaction.

[0246] According to the results, it can be seen that the reduction, oxidation, first drug binding, re-reduction, and second drug binding steps performed during the preparation of the antibody-double drug conjugate of the present invention proceeded normally. In addition, it can be confirmed that the structural integrity of the antibody is maintained at each step (see Fig. 7).

[0247]

[0248] 2-3. LC-MS and DAR Analysis of Antibody-Drug Conjugates (AD2C)

[0249] According to the method of the preceding example, a first drug (mc-vc-MMAE, LP1) was conjugated to a non-asymmetric antibody with a knob-in-hole structure at a DAR ratio of 1, and then a second drug (Chemical Formula 3, LP2) was added in an excess amount of 12 times relative to the moles of the antibody through a reduction process to prepare an antibody-double drug conjugate (AD2C) targeting a ratio of LP1:LP2 = 1:6.

[0250] Changes in molecular weight for the light chain (LC), knot structure heavy chain (HC1), and hole structure heavy chain (HC2) of the prepared samples and control (naked) antibodies were measured using LC-MS (ACQUITY Arc Bio System, Waters). Deconvolution analysis was performed on the LC-MS data using UNIFI software (see Figs. 8a to 8f, Fig. 9).

[0251] As a result of this experiment, no drug binding was observed in the LC, HC1, and HC2 of the control (naked) antibody (Figs. 8a to 8c). On the other hand, in AD2C, a peak with one LP2 molecule bound was detected in the light chain (LC) (Fig. 8d), a peak with one LP1 molecule and two LP2 molecule bound was confirmed in the knot structure heavy chain (HC1) (Fig. 8e), and a peak with two LP2 molecule bound was confirmed in the hole structure heavy chain (HC2) (Fig. 8f). Based on this, the calculated DAR values ​​were an average of 0.96 for LP1 and 5.70 for LP2, with a total DAR value of 6.65. Therefore, the binding ratio of the two drugs was 1:5.96, which is consistent with the target of 1:6 (see Fig. 9).

[0252] That is, it was confirmed that in the asymmetric antibody of the present invention, one first drug (LP1) selectively binds to the knop structure heavy chain (HC1), and a total of 5 to 6 second drugs (LP2) bind to the cysteine ​​residues derived from the interchain disulfide bond between the light chain and the two heavy chains (HC1, HC2). Therefore, it can be seen that an antibody-drug conjugate (AD2C) corresponding to the target DAR ratio (1:6) can be stably realized by the manufacturing method of the present invention.

[0253]

[0254] Through the results of these examples, it was confirmed that free cysteine ​​residues designed in asymmetric antibodies having a knob-in-hole structure can be selectively utilized for drug conjugation. SDS-PAGE analysis demonstrated that antibodies that underwent a reduction-oxidation process could be used in drug conjugation reactions while maintaining structural stability (see Figs. 5 and 7), and SEC and HIC analysis confirmed that the prepared antibody-double drug conjugate (AD2C) maintained high purity and stability (see Figs. 6a-6e).

[0255] In addition, through LC-MS analysis, it was confirmed that the first drug (LP1) binds to a specific site of the antibody at a DAR level of 1, and the second drug (LP2) binds to the cysteine ​​residue at a DAR level of 6, thereby enabling the realization of AD2C with a drug ratio of approximately 1:6 (see Figs. 8a-8f, Fig. 9).

[0256] Accordingly, it can be concluded that the present invention enables the production of not only single-drug conjugates (ADCs) but also antibody-double-drug conjugates (AD2Cs) in which two different drugs are combined, by fabricating antibodies with asymmetric structures. This platform allows for precise control of the drug binding site and ratio, enabling the production of uniform and reproducible ADCs / AD2Cs, which can contribute to the maximization of therapeutic effects and the minimization of side effects.

[0257]

[0258] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0259] Sequence No. 1

[0260] Light chain 1_LC

[0261] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0262]

[0263] Sequence No. 2

[0264] Heavy chain 1_knob structure_HC1

[0265] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0266]

[0267] In sequence number 2, italics represent the Fab sequence, bold C represents S239C, and bold W represents the Knob sequence.

[0268]

[0269] Sequence No. 3

[0270] Heavy chain 2_hole structure_HC2

[0271] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFG QGTKVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0272]

[0273] In sequence number 3, the underline represents the linker sequence, the italicized text before the underline represents the VH sequence, and the italicized text after the underline represents the VL sequence. The bold text represents the Hole sequence.

Claims

1. An antibody for an antibody-drug conjugate to which one or more drugs are bound, The above antibody is an asymmetric antibody having a knob-in-hole structure, a) a first arm consisting of Fab and Fc regions derived from an IgG1 antibody specific to the first epitope; and b) comprising a second arm composed of a ScFv and Fc region fusion protein specific to the second epitope, and The Fc region of the second cancer above includes an IgG1 antibody-derived sequence, wherein The first epitope and the second epitope are identical or different from each other, When the Fc regions of the first and second arms are bonded intermolecularly, at least two disulfide bonds are formed in the hinge region, and i) lysine at position 183 of the first female heavy chain, ii) lysine at position 149 of the first female light chain, and iii) any one of the amino acids among lysine at positions 290, 326, and 392, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328 of the first female or second female Fc region, which is substituted with cysteine; Antibody for antibody-drug conjugates, in which the first drug is bound at a ratio of DAR 1.

2. An antibody; and an antibody-drug conjugate comprising a drug bound to the antibody, The above antibody is an asymmetric antibody having a knob-in-hole structure, a) a first arm consisting of Fab and Fc regions derived from an IgG1 antibody specific to the first epitope; and b) comprising a second arm composed of a ScFv and Fc region fusion protein specific to the second epitope, and The Fc region of the second cancer above includes an IgG1 antibody-derived sequence, wherein The first epitope and the second epitope are identical or different from each other, When the Fc regions of the first and second arms are bonded intermolecularly, at least two disulfide bonds are formed in the hinge region, and i) lysine at position 183 of the first female heavy chain, ii) lysine at position 149 of the first female light chain, and iii) any one of the amino acids among lysine at positions 290, 326, and 392, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328 of the first female or second female Fc region, which is substituted with cysteine; The above drug is an antibody-drug conjugate bound to an antibody at a ratio of DAR 1.

3. In Paragraph 2, An antibody-drug conjugate in which the first epitope and the second epitope are identical epitopes.

4. In Paragraph 2, An antibody-drug conjugate in which the first epitope and the second epitope are different epitopes.

5. Antibody; an antibody-double drug conjugate comprising a first drug and a second drug bound to the antibody, wherein The above antibody is an asymmetric antibody having a knob-in-hole structure, a) a first arm consisting of Fab and Fc regions derived from an IgG1 antibody specific to the first epitope; and b) comprising a second arm composed of a ScFv and Fc region fusion protein specific to the second epitope, and The Fc region of the second cancer above includes an IgG1 antibody-derived sequence, wherein The first epitope and the second epitope are identical or different from each other, When the Fc regions of the first and second arms are bonded intermolecularly, at least two disulfide bonds are formed in the hinge region, and Any one of the amino acids—lysine at position 183 of the first female heavy chain, lysine at position 149 of the first female light chain, lysine at positions 290, 326, and 392 of the first female or second female Fc region, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328—is substituted with cysteine, The first drug is an antibody-double drug conjugate bound to an antibody at a ratio of DAR 1.

6. In Paragraph 5, The second drug is an antibody-double drug conjugate bound to the antibody at a ratio of DAR 5 to 7.

7. In Paragraph 5, The above-mentioned first drug and second drug are different from each other, and The above-mentioned first drug is an auristatin-based drug and the second drug is a topoisomerase I inhibitor, an antibody-double drug conjugate.

8. In Paragraph 7, The first drug is an antibody-double drug conjugate, wherein the first drug is any one compound selected from the group consisting of monomethyl auristatin E, monomethyl auristatin F, monomethyl auristatin U, auristatin E, emtansine, eribulin, formula 1, duostatin 5, and derivatives thereof: [Chemical Formula 1] 9. In Paragraph 7, The above-mentioned second drug is an antibody-double drug conjugate, wherein the second drug is any one compound selected from the group consisting of the following chemical formulas 2 to 15 and their derivatives: [Chemical Formula 2] , [Chemical Formula 3] , [Chemical Formula 4] , [Chemical Formula 5] , [Chemical Formula 6] , [Chemical Formula 7] , [Chemical Formula 8] , [Chemical Formula 9] , [Chemical Formula 10] , [Chemical Formula 11] , [Chemical Formula 12] , [Chemical Formula 13] , [Chemical Formula 14] , [Chemical Formula 15] .

10. In Paragraph 5, The above-mentioned first drug and second drug are each bound to an antibody through a linker, forming an antibody-double drug conjugate.

11. In Paragraph 5, The first and second drugs are each bound to an antibody through a linker, and The above linker has the structure of maleimidocaproyl (MC)-valine (Val)-citrulline (Cit)-p-aminobenzyl carbamate (PABC), GGFG, or GGYG, and An antibody-drug conjugate in which β-glucuronic acid is attached to the hydroxyl group of tyrosine when the above linker is GGYG.

12. In Paragraph 5, The first drug above is a linker-drug conjugate of the form MC-Val-Cit-PABC-MMAE, The second drug is bound to the antibody in the form of a linker-drug conjugate of MC-GGFG-DXd, MC-GGFG-Formula 3, MC-GGYG-Formula 3, MC-GGFG-Formula 5, or MC-GGYG-Formula 5, and An antibody-double drug conjugate in which, when the above linker has an GGYG structure, β-glucuronic acid is bound to the hydroxyl group of tyrosine (Y): [Chemical Formula 3] , [Chemical Formula 5] .

13. A method for preparing an antibody-double drug conjugate to which a heterogeneous drug is combined, i) a step of adding an excess amount of reducing agent to the antibody to expose reducible free thiol groups (-SH) within the antibody; ii) a step of adding an excess amount of oxidizing agent to the antibody obtained according to step i) above to reform disulfide bonds and expose free thiol groups (-SH) in monomeric form; iii) a step of forming a DAR 1 antibody-first drug conjugate by binding a first drug to the free thiol group (-SH) of step ii); iv) adding an excess amount of reducing agent to the antibody-first drug conjugate to expose reducible free thiol groups (-SH); and v) a step of binding a second drug to the antibody obtained in step iv) above to obtain an antibody-double drug conjugate bound to a heterologous drug, and The above antibody is an asymmetric antibody having a knob-in-hole structure, a) a first arm consisting of Fab and Fc regions derived from an IgG1 antibody specific to the first epitope; and b) comprising a second arm composed of a ScFv and Fc region fusion protein specific to the second epitope, and The Fc region of the second cancer above includes an IgG1 antibody-derived sequence, wherein The first epitope and the second epitope are identical or different from each other, When the Fc regions of the first and second arms are bonded intermolecularly, at least two disulfide bonds are formed in the hinge region, and A method for preparing an antibody-drug conjugate in which any one of the amino acids—lysine at position 183 of the first cancer heavy chain, lysine at position 149 of the first cancer light chain, lysine at positions 290, 326, and 392 of the first or second cancer Fc region, alanine at position 339, serine at positions 239, 440, and 442, aspartic acid at position 265, and leucine at position 328—is substituted with cysteine.

14. In Paragraph 13, A method for preparing an antibody-double drug conjugate in which the second drug is bound to the antibody at a ratio of DAR 5 to 7.

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