Antibody-drug conjugate in which one or more drug linker conjugates are bound to an antibody, and method for producing the same.

Abstract

The present invention relates to an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to a single antibody, and to an efficient method for producing the same. Specifically, at least one drug linker conjugate selected from the group consisting of a drug linker conjugate (A) which is a combination of a camptothecin-based drug and an acid-sensitive linker; a drug linker conjugate (B) which is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; a drug linker conjugate (C) which is a combination of a camptothecin-based drug and an enzyme-sensitive linker; and a drug linker conjugate (D) which is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker may be bound to the cysteine ​​and lysine residues of the antibody.

Description

[Technical Field]

[0001] The present invention relates to an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are bound (linked) to an antibody, and to an efficient method for producing the same.

[0002] Specifically, at least one drug linker complex selected from the group consisting of (A) a combination of a camptothecin-based drug and an acid-sensitive linker; (B) a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; (C) a combination of a camptothecin-based drug and an enzyme-sensitive linker; and (D) an anti-apopototic protein inhibitor drug and an enzyme-sensitive linker may be bound to an amino acid residue of the antibody. [Background technology]

[0003] Antibody-drug conjugates (ADCs) are a novel drug platform that utilizes the high tissue selectivity of antibodies to selectively deliver a payload with strong anti-cancer efficacy only to cancer tissue. ADCs can selectively deliver a potent payload that kills cancer cells even at low pM levels only to cancer tissue, minimizing systemic drug exposure and ensuring both anti-cancer efficacy and safety simultaneously.

[0004] ADC technology is a method of delivering drugs to tumor cells using antibodies that specifically bind to certain antigens expressed on the surface of cancer cells. Most ADCs are introduced into cells via clathrin-coated pits. Once inside the cell, the ADC detaches from clathrin, fuses with other intracellular vesicles, and then proceeds into the endosomal-lysosomal pathway. Next, proteases in the acidic environment of the endosome cleave the linker, and the activated "free" drug crosses the lysosomal membrane and moves into the cytoplasm. Thereafter, it binds to the drug's molecular target, halting the tumor cell cycle and causing cancer cell death by apoptosis. Of these, a certain amount of the drug is either passively diffused within the cell, actively transported, or leached out of the cell via dead cells. If the leached drug is permeable to the cell membrane, it can enter surrounding cells, leading to what is known as bystander cell killing.

[0005] Antibody-drug conjugates (ADCs) are broadly composed of three elements. (i) An antibody that specifically acts against a target antigen that is restrictively expressed in normal cells. (ii) A cytotoxic drug (payload) designed to kill target cancer cells. (iii) A chemical linker that attaches cytotoxic drugs to antibodies.

[0006] In other words, ADCs are antibody drugs that combine the targeting ability of monoclonal antibodies with the potent cell-killing ability of cytotoxic drugs.

[0007] The potency of a cytotoxic drug (payload) bound to an ADC is typically 100 to 1000 times greater than the potency of the toxic drug used alone. Thus, the goal of ADC development is to develop drugs that act very specifically on target cancer cells without causing serious side effects in normal tissue.

[0008] Developing ADCs requires an understanding of target antigen selection, the encapsulation effect of ADCs by tumor cells, drug titer, and the stability of linkers between drugs and antibodies. Furthermore, the conjugation method of cytotoxic drugs and antibodies, the drug-antibody ratio (DAR), the effects of antibody properties and linker type on drug binding affinity are crucial for developing safe and effective ADCs.

[0009] The inhibitory factors for ADCs include competition from conventional therapies and other innovative antibody drugs, relatively difficult manufacturing processes and high costs compared to conventional therapies, linker stability, and heterogeneous drug-antibody ratio (DAR) profiles.

[0010] In particular, the stability of the linker that connects antibodies and cytotoxic drugs is closely related to toxicity in clinical trials.

[0011] In other words, the biggest potential problem with ADCs is that undesirable toxicity can occur if the linker connecting the monoclonal antibody and the cytotoxic drug is cleaved prematurely in normal tissues other than the target cancer cells. Mylotarg, the first ADC in the US market, was withdrawn in 2010, reportedly due to the instability of the linker attached to the antibody and the resulting potent toxicity of the cytotoxic drug. Other toxicity-related issues arise when ADCs target antigens also found in normal tissues.

[0012] To date, approved ADCs and those in the clinical pipeline are manufactured by linking small molecule cytotoxic drugs to lysine or cysteine ​​residues of antibodies via linkers. Conventional manufacturing processes, up to Adcetris, Kadcyla, and Mylotarg (re-approved in 2017), only allow for limited control of the number of cytotoxic drugs attached to the antibody. For example, studies on the drug-antibody ratio (DAR) profile of Kadcyla show that an average of 4 drugs are bound to the antibody, but with approximately 80 usable binding sites (lysine residues) on a monoclonal antibody, and around 8-10 highly reactive surface lysine residues, the DAR profile varies widely from 0 to 8. Several companies are developing platforms for appropriately binding drug-antibody ratios (DAR), including Pfizer's Besponsa (Inotuzumab ozogamicin), which was newly approved in 2017.

[0013] However, ADCs, including T-DM1 (trade name: Cadcyla®), have been plagued by heterogeneity since their initial development. Specifically, because small molecule drugs are randomly reacted with approximately 70-80 Lys residues in the antibody, the drug / antibody ratio (DAR) and conjugation site are not constant. Typically, when ADCs are produced using such random conjugation methods, the DAR ranges from 0 to 8, and it is known that multiple drugs with different numbers of drug bindings are produced. Recently, it has been reported that changing the number and binding site of drugs in an ADC alters its pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control over the number and location of conjugated drugs. If the number and location are constant, it is possible to achieve the expected efficacy, address variations in conjugated drugs, and resolve issues related to lot-to-lot differences and so-called regulation.

[0014] Antibody regioselective modification methods include genetic engineering techniques and enzyme-based modification methods. Genetic engineering modification methods allow for control over regioselectivity and antibody quantification.

[0015] Recently, the CCAP (Chemical Conjugation by Affinity Peptide) method has been developed (US10227383B2, US2021 / 0139548A1, ACS Omega (2019) Vol.4, pp.20564-20570, all of which are incorporated herein). The CCAP method successfully achieves regioselective modification of antibodies by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide (i.e., a method for producing ADCs via a linker containing the peptide portion). The CCAP method is the first in the world to successfully achieve regioselective modification of the antibody Fc region with a drug using a chemical synthesis method, and furthermore, good practical results have been confirmed [reaction time 30 minutes, yield 70% (for DAR1), regioselectivity 100%). It has been demonstrated that the DAR can be controlled to 2 by adding approximately 5 equivalents of the peptide reagent, and this is groundbreaking because the modification position can also be controlled.

[0016] The drug-antibody ratio (DAR) is one of the most important properties that influences the pharmacokinetic properties and in vivo distribution of ADCs. Furthermore, ADCs with higher DARs show greater efficacy in in vitro experiments. However, it has been reported that ADCs with high DARs have lower in vivo efficacy, because the plasma washing rate of ADCs with more bound drugs is higher than that of ADCs with fewer bound drugs. Based on these observations, current ADCs under development, including approved ADCs, have DARs adjusted to 2-4, thereby primarily using the same technology used for drug binding to cysteine ​​or lysine residues of antibodies. Many attempts have been made to increase the DAR, but most have failed, mainly because the cytotoxic drugs and linkers are all hydrophobic, or for other reasons, such as ADC aggregation, loss of affinity to target antigens, or high plasma washing rates. Recently, homogeneous ADCs with a DAR value of 8 have been produced via cysteine ​​binding, but these ADCs also exhibit high plasma washing rates due to their unique drug-linker complex and significant deformation of the antibody.

[0017] The concept of an "ideal" ADC is to deliver the most cytotoxic drug to target cancer cells. Developing high-DAR ADCs that remain in plasma for extended periods requires careful study and establishment of a delicate balance between the number of drugs bound to the antibody and the degree of antibody deformation. Furthermore, technological development is needed for hydrophilic or branched linkers, such as PEG linkers, that overcome the limitations of conventional ADC linker technologies.

[0018] Furthermore, as the field of ADC technology develops, it has become important not only to selectively deliver potent anticancer chemicals or radionuclides (radionuclides) to cancer tissue, but also to efficiently deliver payloads with various pharmacological effects, or combinations thereof, to cancer tissue to maximize anticancer efficacy. In particular, even when using potent payloads such as MMAE, hemiasterlin, and PBD for the manufacture of ADCs, unlike in preclinical animal studies, the potent anticancer efficacy sufficient to completely eliminate cancer is not obtained in humans. Therefore, rather than simply using large amounts of payloads such as MMAE, hemiasterlin, and PBD to improve the efficacy of ADCs, methods to achieve maximum efficiency through combinations (e.g., anti-apoptotic protein inhibitors; (combinations of Bcl-XL inhibitors (such as Navitoclax) and MMAE or Top1 inhibitors, combinations of CHEK1 inhibitors and Top1 inhibitors)) are being attempted. However, to date, there is a problem in that methods for efficiently manufacturing dual-payload ADCs are limited. Current clinical attempts mostly use potent anticancer drugs such as MMAE and Top1 inhibitors in the form of ADCs, and anti-apoptotic protein inhibitors It is common to use combination drugs such as inhibitors for systemic administration. AbbVie is developing an ADC that uses a Bcl-XL inhibitor like ABBV-155 as the payload, but since only a limited number of antigens are present on the surface of cancer cells, there is a high risk that the efficiency of drug delivery will be limited by using two different ADCs. Furthermore, it is predicted that its application will be limited due to the difficulty of using two or more ADCs, which are more expensive than typical anticancer drugs. Debiopharm is delivering two drugs using a linker-payload system that combines two payloads in a 1:1 ratio, but it still has limitations, such as a relatively high aggregation rate and the requirement to deliver only two drugs at a specific ratio.That is, there is still no ADC with a new structure that can efficiently deliver one or more payloads at various ratios while reducing the concern of aggregation.

Summary of the Invention

Problems to be Solved by the Invention

[0019] In order to solve the above-mentioned problems, the present invention provides an antibody-drug conjugate (ADC) in which one or more drug-linker conjugates are linked to one antibody via a linker, and the pharmacokinetic properties and in vivo distribution are adjusted as desired.

Means for Solving the Problems

[0020] A first aspect of the present invention is an antibody-drug conjugate (ADC) in which one or more drug-linker conjugates are linked to one antibody, and the drug-linker conjugate (A) is a combination of a camptothecin-based drug and an acid-sensitive linker; the drug-linker conjugate (B) is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; the drug-linker conjugate (C) is a combination of a camptothecin-based drug and an enzyme-sensitive linker; and the drug-linker conjugate (D) is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker. An antibody-drug conjugate (ADC) is provided, characterized in that at least one or more drug-linker conjugates selected from the group consisting of are each bound to the cysteine and lysine residues of the antibody.

[0021] A second aspect of the present invention, according to the first aspect, is a method for producing an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to one antibody, the drug linker conjugate (A) being a combination of a camptothecin-based drug and an acid-sensitive linker; the drug linker conjugate (B) being a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; the drug linker conjugate (C) being a combination of a camptothecin-based drug and an enzyme-sensitive linker; and the drug linker conjugate (D) being a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker, the method comprising a first step of selecting at least one drug linker conjugate from the group consisting of; and a second step of using (i) the amino acid residues at the binding positions with the antibody, (ii) the binding order and / or (iii) the binding method to be different from each other by the drug linker conjugate, and binding each drug linker conjugate to a cysteine or lysine residue of the antibody, and providing an ADC characterized by including the above.

[0022] Hereinafter, the present invention will be described. As used herein, the term "antibody" means a protein molecule that serves as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that is reactive with an immunologically specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and whole antibodies. The term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that possess a binding function for FcRn. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The whole antibodies include IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes IgG1, IgG2, IgG3, and IgG4.

[0023] Preferably, the antibodies of the present invention are urelumab, utomilumab, bebtelovimab, aducanumab, bapineuzumab, crenezumab, donanemab, gantenerumab, lecanemab, solanezumab, nesvacumab, evinacumab, and Noblitzuzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Tepliz Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, U blituximab), veltuzumab, epratuzumab, basiliximab, daclizumab, varlilumab, lulizumab, iratumumab, lintuzumab, daratumumab, felzartamab, isatuximab, mezagitamab,Bleserumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, In Inclacumab, Cusatuzumab, Oleclumab, Miratuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab nartuzumab, eculizumab, pozelimab, ravolizumab, lacnotuzumab, axatilimab, cabiralizumab, emactuzumab, ipilimumab, quavonlimab, tremelimumab, zalifrelimab, cetuximab imab), Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab,Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Kodori Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Aniflorumab olumab), Emapalumab, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab, Canakinumab, Gevokizumab, Briakinumab ), ustekinumab, anrukinzumab, sendakimab, lebrikizumab, tralokinumab, brodalumab, bimekizumab, ixekizumab, secukinumab, brazikumab, guselkumab, mirikizumab,Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levirimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Favezelimab, Fianlimab, Ieramilimab, Relamilimab (atlimab), Simtuzumab, Abagobomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bococizumab, Ebronucimab ), Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab,Pidilizumab, Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab Lizumab), Avelumab, Cosibelimab, Sugemalimab, Durvalumab, Envafolimab, Suvratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociperlima b) Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Gatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab,Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Obrindatamab, Elranatamab, Linvoseltamab, Teclistamab, Epcolitamab (Epcoritamab), Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naptumomab, Belantamab, Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibuprofen Ibritumomab, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorvotuzumab, Polatuzumab, Tusamitamab, Telisotuzumab, Rovalpituzumab umab), Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab, Vobarilizumab, Cadonilimab, Vudalimab, Tebotelimagne, Ivonescimab,At least one drug selected from the group consisting of erfonrilimab, ozoralizumab, faricimab, vanucizumab, and navigationixizumab.

[0024] In this specification, the term "drug linker complex" means a substance for the manufacture of antibody-drug conjugates (ADCs) that does not contain an antibody.

[0025] In this specification, the term "drug linker complex linked" means that the drug / antibody ratio (DAR) and / or conjugation site are constant.

[0026] The drug linker complex of the present invention may have one or more (preferably two) amino acid residues containing cysteine ​​or lysine of an antibody, unnatural amino acid residues, or glycans, each of which may be bound.

[0027] Even linkers attached to the same antibody amino acid sequence can influence in vivo transformation through linker decoupling from a steric and electromagnetic perspective, depending on the type and length of the linker, and the position of the antibody to which the linker is conjugated. Furthermore, ADCs must maintain the same affinity as the antibody before it bound to the drug. In other words, the drug bound to the antibody must not interfere with antibody-antigen binding.

[0028] Therefore, various drug linker complex combinations can be designed taking these points into consideration.

[0029] In this invention, drug linker complexes are classified into (A) a combination of a camptothecin-based drug and an acid-sensitive linker; (B) a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; (C) a combination of a camptothecin-based drug and an enzyme-sensitive linker; and (D) an anti-apopototic protein inhibitor drug and an enzyme-sensitive linker, in order to select one or more drug linker complexes that homogeneously and symmetrically bind to the cysteine ​​and lysine residues of an antibody using (i) amino acid residues at the binding site with the antibody, (ii) binding sequence, and / or (iii) binding method. However, using different enzymatically cleavable linkers or binding two drugs of different classes also falls within the scope of this invention.

[0030] As long as the two drugs have different cytotoxicities, they each belong to the categories of camptothecin-based drugs and non-camptothecin-based supertoxin drugs of the present invention.

[0031] For example, the antibody-drug conjugate of the present invention may be a compound to which two compounds corresponding to the drug linker conjugates (B) and (C) described above are linked.

[0032] In this case, the antibody-drug conjugate can be manufactured by preparing a mixture by mixing selected drug linker conjugates, and then reacting the mixture with a reduced antibody to perform conjugation.

[0033] In yet another specific example, an antibody-drug conjugate can be produced by adding the drug-linker conjugate of (B) to the reduced antibody in an amount 2 to 4 times relative to the molar equivalent of the antibody to produce a primary reaction product, and then adding the drug-linker of (C) to the primary reaction product to produce a secondary reaction product.

[0034] As long as the two drugs have different cytotoxicities, they belong to the categories of camptothecin-based drugs and non-camptothecin-based supertoxin drugs of the present invention, respectively.

[0035] The dual-payload ADC (antibody-drug conjugate in which two drug-linkers are bound to one antibody; hereinafter also referred to as Dual-payload ADC) according to the present invention has the characteristic of being an ADC with a homogeneous structure in which two types of payloads are bound in a predetermined ratio, and has the characteristic of being able to freely introduce two or more types of payloads to obtain various pharmacological effects.

[0036] In one specific example of the present invention, the antibody-drug conjugate (ADC) is an antibody to which a first drug linker conjugate and a second drug linker conjugate are bound. The first drug linker conjugate and the second drug linker conjugate are each one type of conjugate selected from four groups of drug linker conjugates (A, B, C, D). In the present invention, the linkers contained in the first drug linker conjugate and the second drug linker conjugate may be the same or different from each other. For example, the linker contained in the first drug linker conjugate or the second drug linker conjugate may contain at least one structure selected from the group consisting of GGFG, val-cit, and derivatives thereof.

[0037] In one specific example of the present invention, an antibody-drug conjugate (ADC) is an antibody to which a first drug linker conjugate and a second drug linker conjugate are bound. The first drug linker conjugate is a drug linker conjugate (B) which is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker, and the second drug linker conjugate is a drug linker conjugate (C) which is a combination of a camptothecin-based drug and an enzyme-sensitive linker. In the present invention, the DAR of the antibody-drug conjugate can be 4 to 8. When the total DAR is 8, the drugs contained in the first drug conjugate and the drugs contained in the second drug conjugate may be bound to the antibody in a ratio of 1:1 to 1:7 (e.g., 1:7, 2:6, 3:4, or 4:4). When the total DAR is 6, they may be bound to the antibody in a ratio of 1:1 to 1:5 (e.g., 1:5, 2:4, 3:3), and when the total DAR is 4, they may be bound to the antibody in a ratio of 1:1 to 1:3 (e.g., 1:3, 2:2).

[0038] The linkers contained in the first drug linker complex and the second drug linker complex, respectively, may be the same or different from each other.

[0039] In a preferred example of the present invention, the drug contained in the first drug linker complex is an auristatin compound, and the drug contained in the second drug linker complex is a topoisomerase I inhibitor. Here, the topoisomerase I inhibitor may be at least one compound selected from the group consisting of SN-38, exatecan, Dxd, FL118, and 7-aminoalkyl-substituted camptothecin compounds, or a derivative thereof, but is not limited to these. Any compound that can potently inhibit the topoisomerase I enzyme inside cancer cells and produce a cancer cell death effect can be used without limitation. The drug-to-antibody ratio of the first drug linker complex can be adjusted in various ways as needed.

[0040] The drug linker complex containing the topoisomerase I inhibitor as a drug may be represented by any one of the following chemical formulas 2 to 5. [Chemical formula 2] [ka] [Chemical formula 3] [ka] [Chemical formula 4] [ka] [Chemical formula 5] [ka]

[0041] The drug linker complex containing the auristatin compound as a drug may be represented by either one of the following chemical formulas 6 or 7. [Chemical formula 6] [ka] [Chemical formula 7] [ka]

[0042] In the present invention, the linker can be one which is readily cleaved by an enzyme selectively activated inside cancer cells or in the tumor microenvironment to release a drug. Specifically, GGFG linkers, AAA linkers, Val-Cit linkers, Val-Ala linkers, and glucuronidase-cleaved linkers and regmine-cleaved linkers that are cleaved by these enzymes can also be readily used. Furthermore, tandem linkers that release a drug after cleavage by two enzymes, rather than just one, can also be used.

[0043] [Cytotoxic drugs] After binding to target cells, ADCs are internalized into the cells through a receptor-mediated endocytosis process. This requires a sufficient concentration of the active drug to enter the cell; however, the internalization process via antigen-antibody complexes is generally inefficient, and the number of antigens on the cell surface is typically <1 × 10⁻⁶. 5 Because it is limited to receptors / cells, it must be possible to kill tumor cells effectively even at low concentrations using very potent drugs. Therefore, the drugs used in ADCs (antibody-drug conjugates) that bind to antibodies are typically 100 to 1000 times more cytotoxic than commonly used anticancer drugs.

[0044] Most potent cytotoxic drugs introduced into ADCs are highly toxic and affect normal cells through the bystander effect. Furthermore, the amount of payload that can be delivered is limited because the drug must be conjugated while minimizing its impact on antibodies. This indicates that cytotoxic drugs for application to ADCs must be able to kill most tumor cells at low concentrations (nM or pM) and exhibit therapeutic effects through regulation of drug release. Therefore, the cytotoxic drugs that can be applied as payloads are highly diverse. They must have low immunogenicity and stable properties during circulation in the body. This is because unstable cytotoxic payloads can lead to drug deformation during conjugation or storage. Currently, cytotoxic drugs used in approved or clinically active ADCs are broadly divided into two categories: microtubule-destroying drugs and DNA-modifying drugs. Recently, with the approval of topoisomerase inhibitors, interest in drugs with various mechanisms of action has increased.

[0045] The two main categories of cytotoxic drugs used in ADCs are microtubule-destroying agents and DNA-modifying agents, and anti-apoptotic protein inhibitors are also used.

[0046] Microtubules play a crucial role in the cell cycle, but abnormalities in microtubules can prevent cell division. Most known microtubule disruptors are derived from natural products and exhibit high cytotoxicity. Unlike drugs that affect microtubules and act only at specific stages of the cell cycle, DNA-modifying agents can kill cells at any point in the cycle.

[0047] Monomethyl auristatin E (MMAE, also known as "vedotin"), a cytotoxic drug, binds to microtubules, halts the cell cycle, and induces cell death.

[0048] Table 1 classifies cytotoxic drugs primarily used in clinical-stage ADCs according to their mechanism of action.

[0049] [Table 1]

[0050] A commonly used microtubule-destroying drug is the auristatin series from Seattle Genetics. Monomethyl auristatin E (MMAE), or vedotin, is a cytotoxic drug derived from drastatin 10, a naturally occurring cytotoxic peptide isolated from the Indian Ocean sea scallop (Dolabella auricularia), and is a potent microtubule polymerization inhibitor. While this drug was designed to be cleaved and released from the antibody by the cathepsin B enzyme, its secondary derivative, MMAF, is being developed in a form conjugated to a non-degradable linker, limiting its cellular release.

[0051] Another microtubule inhibitor, the ansamycin antibiotic developed by Immunogen, is derived from maytansine, a natural product isolated from the Ethiopian shrub Maytenus serrata. Maytansine binds to tubulin, inhibiting microtubule aggregation and inducing microtubule degradation, thereby interfering with cell division. Maytansine exhibits cytotoxicity in many tumor cell lines and can suppress tumor growth. Such derivatives are designed to be conjugated to a linker via a thiol group. In the case of DM1, it is readily cleaved under reducing conditions inside the cell. On the other hand, in the case of DM4, a methyl group is substituted for the disulfide bond, making reduction less likely.

[0052] Cancer cells, which divide rapidly, are particularly sensitive to the effects of cytotoxic substances compared to slow-dividing normal cells, and therefore can be eliminated using DNA-modified preparations. While DNA damage is the mechanism of action for many of the most commonly used chemotherapy agents, clinically used therapeutics are difficult to employ due to the risk of toxicity as their therapeutic window narrows as their potency increases. However, by binding such potent drugs to highly targeted antibodies, both the safety and efficacy of the drug can be enhanced simultaneously.

[0053] Most DNA-modified drugs are derived from natural products. Bristol-Myers Squibb and Medarex have developed various natural product-derived drug sources that induce mutagenesis through DNA alkylation mechanisms. Synthon (formerly Syntarga), a Dutch-based company, also uses a synthetic toxin called duocarmycin analog to bind to DNA in this way. Currently, SYD985 (Trastuzumab duocarmazine), developed by Synthon and in Phase 3 clinical trials, is known to use duocarmycin.

[0054] Other drugs used as DNA modification preparations include calicheamicin, pyrrolobenzodiazempine (PBD), and SN-38 (an active metabolite of irinotecan). Calicheamicin, isolated by Pfizer from bacteria, binds to the DNA minor groove and then cleaves the DNA strand. Calicheamicin is used in both of Pfizer's ADCs, Mylotarg and Besponsa. In the case of the PBD dimer, Seattle Genetics entered clinical trials applying it to CD-70-targeting ADCs, a new cytotoxic drug developed since the auristatin-based ADC platform, but development is currently suspended. SN-38 is an active metabolite of irinotecan and inhibits DNA topoisomerase. It is known to be a less potent drug compared to conventionally approved cytotoxic drugs such as auristatin, maytansinoids, and calicheamicin. This makes it possible to develop ADCs with reduced side effects and a higher drug-antibody ratio. Currently, IMMU-130 and IMMU-132, developed by Immunomedics and in clinical trials, are representative ADCs that apply SN-38 to cytotoxic drugs.

[0055] [Camptothecin-based drugs] Cancer is a disease characterized by undiscriminate cell proliferation and uncontrolled cell division. Normal cells have cell cycle checkpoints that check for genetic and cellular damage during cell division. If DNA damage is detected, repair occurs, or, in severe cases, cell death occurs. However, if DNA damage persists without repair, cancer develops. The most representative example is the BRCA gene, which plays a crucial role in cancer suppression and is essential for repairing double-strand DNA breaks through homologous recombination. When DNA damage occurs while a mutation exists in the BRCA gene, proper DNA repair fails, increasing the risk of mutations in other genes. Indeed, mutations in the BRCA gene significantly increase the incidence of female cancers such as breast and ovarian cancer. It is known that women with BRCA gene mutations have a 5-6 times higher incidence of breast cancer and a 10-fold higher incidence of ovarian cancer compared to the general population. This reveals that the failure to properly repair DNA damage can induce cancer by causing genetic instability in cells.

[0056] A common characteristic of cancer is genetic instability. While we don't know specifically which defects in DNA damage reactions cause cancer cells to develop in most cancers, the link between defects in DNA damage reactions and cancer is undeniable.

[0057] For example, 15% of sporadic colorectal tumors have abnormally short or long dinucleotide repeat sequences. Such DNA mutations, known as microsatellite instability, are thought to arise from the accumulation of DNA replication errors due to dysfunctional mismatch repair. Microsatellite instability appears not only in sporadic colorectal tumors but also in hereditary non-polyposis colorectal cancer. Hereditary non-polyposis colorectal cancer is associated with loss-of-function mutations in mismatch repair genes such as MSH2 and MLH1.

[0058] Similarly, there are reports of defects in homologous recombination repair leading to sporadic or hereditary tumors. According to one study, when 500 specimens of high-grade serous ovarian adenocarcinoma were analyzed, 50% of the specimens showed defects in homologous recombination repair.

[0059] Defects in homologous recombination repair are mainly due to mutations in BRCA genes or the occurrence of epigenetic silencing. Other reports indicate that loss-of-function mutations in genes involved in homologous recombination repair, such as BRCA1, BRCA2, ATM, RAD51C, and RAD51D, have been found in hereditary breast cancer, uterine cancer, and pancreatic cancer.

[0060] Cancer cells have a defect in their DNA damage response. They are unable to properly cope with DNA damage. Therefore, the goal is to induce cell death in cancer cells by using substances that can cause DNA damage as anticancer agents.

[0061] A recent strategy in anticancer drugs involves targeting the activity of specific proteins that participate in DNA damage reactions; such anticancer drugs are called targeted therapies. An example of a targeted therapy is a topoisomerase inhibitor. The function of topoisomerase is to cleave and rejoin the phosphodiester backbone of DNA to untwist the DNA structure, thereby facilitating transcription and DNA replication. By inhibiting this function and leaving the DNA in a cleaved state, topoisomerase inhibitors are used as effective anticancer drugs in several types of cancer, including colorectal cancer and lung cancer.

[0062] Inhibitors of type 1 topoisomerase (Topoisomerase I) (such as Irinotecan and Topotecan) are anticancer agents with clinically validated efficacy and safety, demonstrating excellent anticancer effects in various refractory solid tumors, including colorectal cancer, lung cancer, breast cancer, and ovarian cancer. Camptothecin-based drugs such as Exatecan and SN-38 are being developed as ADC payloads.

[0063] Camptothecin is a selective inhibitor of topoisomerase-1, an isomerase involved in DNA replication and recombination. It is a natural antitumor alkaloid that was isolated in 1966 by Wall et al. in the United States from Camptotheca acuminata, a plant native to China. After being found to exhibit potent cytotoxicity in vitro, development through clinical trials was initiated at the National Cancer Institute (NCI) and other institutions. However, due to its extremely poor solubility, development was halted due to various side effects such as myelosuppression and hemorrhagic cystitis associated with it. However, since 1990, it has been confirmed that camptothecin exhibits an antitumor effect by selectively inhibiting DNA topoisomerase-1, which differs from the DNA topoisomerase-2 inhibitory mechanism.

[0064] DNA topoisomerases are a group of gyrase enzymes. These are nuclear enzymes that temporarily cleave DNA or unwind the double helix when cells require access to genetic material for replication and transcription. They also participate in various intracellular activities such as chromosome condensation and recombination, and DNA repair. The genetic code of topoisomerase enzymes is fairly conserved across species.

[0065] Camptothecin's drug target, type 1 topoisomerase, has been observed to be elevated in various malignancies. This drug does not inhibit the free enzyme but stabilizes the covalent bonds of the topo-DNA complex, preventing further linking of cleaved DNA fragments. Therefore, the cellular sensitivity to such topoisomerase-target drugs is related to the level of the enzyme present in the nucleus. This drug makes transcription impossible by interfering with DNA recombination. Higher levels of type 1 topoisomerase lead to the formation of more cleavable complexes, which in turn results in greater drug sensitivity. This has significant clinical relevance, and type 1 topoisomerase inhibitors are used to increase the expression of type 2 topoisomerase, thereby leading to greater sensitivity to type 2 topoisomerase inhibitors. Such results are supported by the antagonistic relationship between type 1 and type 2 topoisomerase. Unlike type 2 topoisomerase, type 1 topoisomerase is not closely associated with proliferation in normal tissues. However, it is found in large quantities in rapidly dividing "S" phase solid tumors such as colorectal cancer, ovarian cancer, and esophageal cancer, which are included in lymphomas, compared to surrounding normal tissues. It is known that type 1 topoisomerase can actually induce cell death by blocking gene replication and transcription within tumor cells during the "S" phase of the cell cycle.

[0066] Camptothecin (CPT) has low water solubility, and to prepare for clinical trials, the National Cancer Institute (NCI) manufactured a water-soluble sodium salt (NSC100880). Phase I and II clinical trials were not completed due to the high toxicity observed by the compound (hemorrhagic cystitis, gastrointestinal toxicity, e.g., nausea, vomiting, diarrhea, and myelosuppression, particularly leukopenia and hypothrombocytosis).

[0067] Subsequently, numerous CPT congeners were synthesized to obtain compounds with lower toxicity and higher water solubility. Two such drugs are irinotecan (CPT-11) and topotecan.

[0068] Irinotecan (CPT-11), jointly developed by Dallchi and Yakult in Japan, was the world's first camptothecin-based anticancer drug in 1994. Its effectiveness against lung cancer (small cell and non-small cell lung cancer) was proven, and it was launched in Europe and Japan. In 1995, its effectiveness against colorectal cancer and breast cancer was further demonstrated. Topotecan, developed by Glaxo Smith Kline, was approved by the US FDA in April 1995 for its effectiveness against metastatic ovarian cancer and was launched. Recently, CKD-602 (belotecan), a new camptothecin derivative developed in Korea, has successfully overcome the toxicity caused by the poor solubility of conventional camptothecin, in addition to its potent type 1 topoisomerase inhibitory effect, by being water-soluble.

[0069] All camptothecin derivatives identified to date contain a parent structure with five rings essential for cytotoxicity. Structurally, the E-ring and A- and B-ring sites were identified as important. Camptothecin consists of a pentacyclic structure with a lactone on the E-ring, which is essential for cytotoxicity. Of these, the lactone group and alpha-hydroxyl group located at carbon 20 of the E-ring are important for the stability of the type 1 topoisomerase-DNA byproduct, and it has been proven that modifications to the A- and B-rings can increase water solubility and activity. Modifications on the first ring, for example, in the case of the aforementioned drug, have been shown to increase water solubility and allow for greater tolerance of the drug.

[0070] CKD-602 also underwent a substitution of the B-ring site at the 7th carbon to increase its water solubility and anticancer effect. Lee et al. stated that CKD-602 has superior anticancer effects compared to camptothecin and topotecan in a wide range of cancer cell lines. They also confirmed that it is a relatively safe drug with a maximum tolerated dose (MTD) of 25 mg / kg in an L1210 leukemia nude mouse model. The side effects of commonly known camptothecin-based drugs can be broadly classified into hematological and non-hematological side effects. Hematological side effects include neutropenia with fever, sepsis, and bleeding, while non-hematological side effects include skin side effects such as nausea, vomiting, and hair loss, as well as toxicity to the gastrointestinal tract, kidneys, and nervous system. Kim et al., in their animal studies of CKD-602, found no abnormal drug reactions other than increased gastric acid secretion, even when administering doses more than 10 times higher than the clinically applicable dose of such camptothecin-based drugs. Furthermore, recent clinical studies in Korea have demonstrated relatively stable drug use, reporting only reversible and controllable levels of side effects such as neutropenia and leukopenia, rather than serious systemic toxicity. However, to date, its use is restricted, primarily indicated for patients who have failed or cannot undergo standard chemotherapy—that is, refractory or recurrent ovarian and colorectal cancers that have relapsed or worsened after standard treatment and are deemed unlikely to respond to further anti-cancer chemotherapy or surgery; refractory or recurrent limited-stage small cell lung cancer that has failed first-line chemotherapy; and extensive-stage small cell lung cancer.

[0071] Exatecan is a camptothecin derivative and an antitumor small molecule compound that inhibits type 1 topoisomerase. Exatecan has been shown to exhibit 5 to 10 times stronger cytotoxicity than SN-38.

[0072] Dxd (Exatecan derivative for ADCs) is an IC used as a conjugate drug for HER2-targeted ADCs (DS-8201a). 50 It is a potent DNA topoisomerase I inhibitor with a concentration of 0.31 μM.

[0073] FL118 is a potent topoisomerase I inhibitor that shares the same camptothecin nucleus structure as conventionally commercialized Top 1 inhibitors such as SN-38, topotecan, and exatecan. However, even when administered alone, it exhibits differentiated anticancer efficacy and superior safety compared to SN-38 in various cancer cell and animal models, ensuring a broad therapeutic window.

[0074] Specifically, the FL118 drug exhibits Top1 inhibitory efficacy equivalent to or greater than SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., it induces low IC levels. 50 It numerically indicates cytotoxicity, and in evaluations of 140 cell lines derived from various cancers, it showed <100 nM IC for the vast majority of cancer cells. 50 It demonstrated extremely powerful anti-cancer efficacy.

[0075] [Non-camptothecin supertoxin drugs] Conventionally, in the case of ADCs utilizing super toxins such as MMAE, hemiastalin, calicheamicin, and PBD, a stable linker system was used (a characteristic of second-generation ADCs) with the aim of minimizing the separation of the drug from the blood before it reaches cancer tissue.

[0076] The principle by which second-generation ADCs such as Hoto work in cancer cells is as follows: In the first stage, the antibody portion of the ADC binds to an antigen that is overexpressed in the cancer cell; in the second stage, the ADC bound to the antigen on the surface of the cancer cell is transported into the interior of the cancer cell via endosomes and lysosomes through an antigen-antibody reaction; in the third stage, the antigen-antibody portion is broken down in the lysosome, and an enzyme (cathepsin B) drug is released; and in the final stage, the drug released from inside the cancer cell kills the cancer cell.

[0077] Examples of microtubule-destroying drugs include MMAE (monomethyl auristatin E) or MMAF, which are similar peptide-based microtubule polymerization inhibitors.

[0078] PBD (Pyrrolebenzodiazepine) is a DNA alkylating agent, and includes SG3199 and SG2057.

[0079] Cytotoxic drugs like PBD (pyrrolebenzodiazepine) are far more effective than conventional cytotoxic drugs, but they also pose toxicity problems when exposed to systemic exposure.

[0080] [Anti-apoptotic protein inhibitor drugs] Anti-apoptotic proteins that are the main cause of resistance to anticancer drugs include sulubivin, cIAP2, and XIAP.

[0081] Examples of anti-apoptotic protein inhibitors include Bcl-XL inhibitors, sulfibine inhibitors, MCL-1 inhibitors, and CHK inhibitors.

[0082] Generally, the apoptosis mechanism is a series of processes in which intracellular proteins are broken down by proteolytic enzymes called caspases, and signals are transmitted. Various types of caspases are involved in apoptosis. Caspase-8 is activated by cell death-inducing substances such as TNF-α or Fas ligand, and induces cell death by activating a series of other caspases. During the cell death process, cytochrome c is released through a pathway located in the mitochondrial membrane and is regulated by BCL-2 system proteins that constitute this pathway. It has been reported that released cytochrome c binds to Apaf-1, caspase-9, and dATP, activating caspase-9, which in turn activates caspase-3, thereby inducing cell death.

[0083] Two factors determine tumor growth: first, cell proliferation, and second, cell death. When the cell cycle of tumor cells is stopped by cytotoxic substances, the tumor cells die through apoptosis.

[0084] B-cell lymphoma (BCL)-2 is apoptosis-mediated.

[0085] While various cancer therapies induce different types of cell death, the activation of cell death pathways regulated by BCL-2 is the most crucial factor in the therapeutic efficacy of oncogenic kinase inhibitors and cytotoxic agents. However, defects in the mitochondrial cell death pathway lead to resistance to cytotoxic drugs in various cancers.

[0086] BCL-2 overexpression has been demonstrated in chronic lymphocytic leukemia (CLL) cells, mediating tumor cell survival and associated with resistance to chemotherapy agents.

[0087] Venetoclax is a potent and selective small molecule inhibitor of the anti-apoptotic protein BCL-2. In other words, venetoclax is an apoptosis-inducing anticancer agent. Venetoclax directly binds to the BH3-binding groove of BCL-2, replacing BH3 motif-containing pro-apoptotic proteins such as BIM. This allows BIM, which does not bind to BCL-2, to exhibit mitochondrial membrane permeabilization (MOMP), caspase activity, and apoptosis. In non-clinical studies, this drug showed cytotoxicity in tumor cells overexpressing BCL-2.

[0088] Since sulvivin is mainly distributed in cancer cells, in the development of anticancer drugs, sulvivin inhibitors induce apoptosis by binding to sulvivin and suppressing its activity. This allows them to act selectively only on cancer cells, greatly minimizing side effects on the human body.

[0089] [Linker] Among the components that make up an ADC (antibody-drug converter), the linker is what binds the antibody and the cytotoxic drug together.

[0090] The linker must be stable in the bloodstream, prevent the drug from separating from the antibody, maintain a prodrug state until it reaches the target, and minimize damage to healthy tissue.

[0091] The most ideal linker is one that remains stable when the ADC circulates systemically, yet is cleaved within the target cell, allowing for the proper release of the cytotoxic drug, thus ensuring both efficacy and safety in the ADC. One of the main challenges in developing safe and effective ADCs is developing a suitable chemical linker between the cytotoxic drug and the monoclonal antibody. Linker synthesis is highly complex, and the type of linker used significantly impacts the efficient release of the cytotoxic drug.

[0092] (1) Linker stability Linker development is crucial because it reflects the long half-life advantage of monoclonal antibodies (mAbs), ensuring the mAbs remain stable in systemic circulation, and that the binding of the linker to the cytotoxic drug does not affect antibody stability or pharmacokinetics. Many ADCs that showed promising preclinical data early on have failed in clinical development despite their potential due to the inability to use appropriate linkers. It has been revealed that the linkers used in these failed ADCs released the antibody-bound cytotoxic drug prematurely. Therefore, the importance of linker manufacturing strategies in the ADC development process is further highlighted. Novel ADC clinical pipelines with improved linker technology demonstrate greater safety and superior efficacy potential compared to conventional ADCs.

[0093] Generally, the catabolic effects that can occur in the systemic circulation of linker-cytotoxic drugs are as follows, and there are also various other catabolic effects that have not yet been clarified.

[0094] Hydrazone cleavage, protease-mediated dipeptide cleavage, esterase-mediated carbamate cleavage, hydrolysis of acetate ester, disulfide cleavage, succinimide ring opening.

[0095] Linkers—catabolism occurring at specific sites on cytotoxic drugs—may sometimes maintain cytotoxic effects and exhibit activity at the target, but may cause toxicity in systemic blood circulation. Conversely, if cytotoxic effects are lost, the drug may not exhibit pharmacological effects even if it reaches the target. For example, tylanstatin, developed as a payload for spliceosome inhibitors, maintains activity even if ester group hydrolysis occurs, while cryptophycin and tubulin inhibitors lose activity if ester group hydrolysis occurs.

[0096] Most ADCs currently in clinical development use a limited range of chemical linkers, including hydrazones, disulfide, or thioether bonds.

[0097] [Table 2]

[0098] In principle, such chemical linkers induce cytotoxic drug release within cancer cells by utilizing differences in intracellular pH and enzyme concentration. Ensuring the stability of drug-linkers in the body after administration is the biggest challenge in ADC development, but chemically unstable hydrazones and disulfide linkers are not sufficiently stable in plasma.

[0099] Peptide-based linkers possess excellent plasma stability while maintaining well-regulated drug-linker stability and drug release capabilities. Since cleavable dipeptide linkers such as valine-Alanine (Val-Ala) and valine-Citrulline (Val-Cit) undergo rapid hydrolysis in the presence of lysosomal extracts or purified human cathepsin B, the cleavage principle of the linker depends on the intracellular environment.

[0100] Furthermore, the limited drug delivery efficiency of conventional Val-Cit or MAC-glucuronide linker systems makes it difficult to deliver a sufficient amount of drug.

[0101] (2) Non-cutting linkers and cutting linkers Linkers currently in general use are broadly classified into cleavable and non-cleavable linkers. Representative types of cleavable linkers include acid-labile, oxidation-reduction reaction, and enzyme-labile linkers, while non-cleavable linkers include thioether linkers (Table 3).

[0102] [Table 3]

[0103] Acid-labile linkers are chemically unstable linkers developed in the early stages of ADC development. Although they have low stability, they are still used today. A typical linker is the hydrazone linker. While stable in the neutral environment of blood (pH 7.3-7.5), it undergoes hydrolysis in weakly acidic environments such as endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) after internalization around tumor cells (pH 6.5-7.2) and within cells, releasing the drug. However, acidic conditions are not limited to the tumor microenvironment and are frequently found outside cells, potentially leading to nonspecific drug release. Mylotarg®, the first ADC approved by the FDA, is a prime example of a hydrazone linker. Its low stability in plasma led to its withdrawal from the US market in 2010, but it was re-approved in 2017. More recently, silyl ether linkers have been studied. They exhibit high stability in plasma, with a plasma half-life of over 7 days, a significant improvement over hydrazone's 2-3 days.

[0104] Related to oxidation-reduction reaction linkers, disulfide linkers are also a type of chemically unstable linker that is based on oxidation-reduction reactions. In endogenous ADCs, drug release occurs when the linker is broken down via disulfide exchange or reducing agents such as glutathione after the antibody is broken down by enzymes. Glutathione is a low molecular weight thiol known as an antioxidant that regulates cell proliferation and death and protects cells from inflammation and oxidative stress. Glutathione is present in cells at concentrations of 0.5–10 mM, but in hypoxic tumors, it can be up to 1000 times higher. In plasma, it is present at low concentrations (2–20 μM), and disulfide linkers, due to their high plasma stability, reduce nonspecific drug release, making them relatively safe and tumor-specific linkers.

[0105] In relation to enzyme-labile linkers, peptide linkers consist of dipeptides or tetrapeptides that, once the ADC is internalized, are recognized and cleaved by lysosomal proteolytic enzymes. Tetrapeptides were used in the early stages of development, but limitations arose, such as relatively slow drug release and the possibility of aggregation when binding to hydrophobic drugs. These problems were solved by the development of dipeptide linkers such as Val-Cit, Phe-Lys, Val-Lys, and Val-Ala, which have been successfully applied to several ADCs such as Adcetris® and Vedotin®.

[0106] The β-glucuronide linker is a linker introduced by Seattle Genetics in 2006 that releases cytotoxic drugs when hydrolyzed by β-glucuronidase, a glycosulfating enzyme in lysosomes. β-glucuronidase is abundant within lysosomes and is known to be overexpressed in some tumors. This enzyme is highly active at low pH but decreases to 10% at neutral pH. This characteristic improves the plasma stability of ADCs with β-glucuronide linkers, preventing drug release outside the target area. To confirm the plasma stability of the β-glucuronide linker, experiments were conducted in mouse plasma with the Val-Cit linker. After 7 days, the half-lives were less than 89% and 50%, respectively, demonstrating the exceptional stability of the β-glucuronide linker, with half-lives measured at approximately 81 days and 6 days, respectively. Furthermore, ADCs containing β-glucuronide linkers demonstrated high stability and efficacy despite binding to high doses (up to 8) of cytotoxic drugs.

[0107] Non-cleavable linkers, such as thioether linkers, exhibit even higher plasma stability compared to cleavable linkers. Unlike cleavable linkers, non-cleavable linkers do not degrade themselves, and therefore, if antibody degradation occurs after intracellular delivery in ADC form, drug release is possible.

[0108] Drugs released in this manner are charged and less likely to diffuse to surrounding cells (bystander effect). Although there is no bystander effect indicating toxicity to surrounding cells, the effect appears only in target cells after internalization within the target cells, meaning it is relatively safe. This indicates that ADCs produced with non-cleavage linkers are even more dependent on the biological mechanisms within the target cells. Many studies have shown that ADCs with non-cleavage linkers have high stability and efficacy, and they are being used as linkers for ADC development. Currently, Kadcycla® is an ADC that utilizes this technology.

[0109] In the case of Brentuximab vedotin (Adcetris), it is composed of a cleavable Val-Cit dipeptide linker that is selectively degraded by cathepsin B present in the lysosomes of target cancer cells, releasing MMAE, and is stable when circulating in the body. ADCs using cleavable dipeptide linkers such as Val-Ala and Val-Cit bind the antibody domain of the ADC to the antigen of the target cancer cell, forming an ADC-antigen complex, which is then encapsulated inside the cancer cell via the endosomal-lysosome pathway. In this case, the intracellular release of cytotoxic drugs is regulated by the internal environment of the endosome / lysosome. Specifically, hydrazone linkers are acidic and unstable, releasing the drug upon degradation, while Val-Cit dipeptide linkers release MMAE by cathepsin B, a proteolytic enzyme in lysosomes.

[0110] On the other hand, ado-trastuzumab emtansine (Kadcyla) has a non-cleaving SMCC linker. The non-cleaving linker allows the ADC, once it has entered the target cell, to be degraded by lysosomes, thereby releasing the cytotoxic drug. This not only avoids unwanted drug release in the body but can also alter the chemical properties of the bound drug, adjusting its affinity to the carrier or improving its efficacy.

[0111] ADC stability during target migration is crucial for achieving the desired therapeutic objective, regardless of the type of linker used, such as cleavage or non-cleavage linkers.

[0112] [click response] In the present invention, the linking of the [linker]-[antibody] may be achieved by the bonding of a thiol group contained in the antibody to a maleimide group or maleic hydrazide group of an acid-sensitive linker through the "click" reaction of reaction formula 1. [Reaction Equation 1] [ka]

[0113] [Hydrophilic spacer] Hydrophilic linkers containing sulfonates or PEG, which exhibit high solubility in both organic solvents and aqueous solutions, solve various problems observed with hydrophobic linkers. PEG linkers have advantages such as water solubility, low toxicity, low immunogenicity, and controlled linker chain length. In connection with this, studies have reported that using PEG linkers significantly improves the in vivo pharmacokinetic profile, increases half-life and plasma concentration, and increases the plasma concentration-time curve (AUC).

[0114] [Enzyme-sensitive linker] An enzyme-sensitive linker according to one specific example of the present invention may be -S-maleimide-spacer-enzyme cleavage site-self-sacrificing spacer-(payload) or -S-dipromaleimide-spacer-enzyme cleavage site-self-sacrificing spacer(payload).

[0115] In vivo transformations through linker deconjugation can occur through chemical separation or enzymatic cleavage.

[0116] In the case of ADCs in which a linker is linked to a lysine residue via an amide bond, the linker-cytotoxic drug can be eliminated in the form of a linker-cytotoxic drug by enzymatic amide hydrolysis. In the case of ADCs in which a linker containing a maleimide group or a disulfide group is linked to a cysteine ​​residue, the sulfur (S) of the cysteine ​​residue is reduced, and the linked linker-cytotoxic drug can be eliminated via exchange. In the eliminated state, the cysteine ​​residue of the monoclonal antibody can exist in a state of disulfide bonding with other cysteine ​​amino acids or endogenous or exogenous substances containing sulfur (S), such as glutathione (GSH). Therefore, while the ADC loses its mechanism of activity at the target by the cytotoxic drug, the eliminated linker-cytotoxic drug can also form adducts to other proteins or enzymes, or be metabolized to become active and cause toxicity.

[0117] [Acid-sensitive linker] In this invention, an acid-sensitive linker is a linker that is stable in the neutral environment of blood (pH 7.3-7.5), but undergoes hydrolysis in weakly acidic environments such as around tumor cells (pH 6.5-7.2) or after intracellular internalization, such as endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0), releasing a drug. Therefore, in this invention, the acid-sensitive linker has a hydrophilic molecular structure to create a hydrolysis environment. For this purpose, the acid-sensitive linker may contain, for example, a polyethylene glycol (PEG) spacer.

[0118] It is preferable that the camptothecin-based drug and the acid-sensitive linker are linked by a carbonate or ester bond so that they decompose in an acidic atmosphere (pH ≤ 7) and free camptothecin-based drugs are released when the acid-sensitive linker decomposes.

[0119] The tetrapeptide linker has reached a limit in which it can bind to hydrophobic drugs and cause ADC aggregation.

[0120] CL2A, the linker used in Troderbi, a conventional FDA-approved ADC, is a linker that satisfies all of the following characteristics: (i) storage stability after manufacturing, (ii) stability in the blood at the time of administration (almost no exposure of the free payload to the plasma), and (iii) rapid release of the payload in cancer tissue.

[0121] The Phe-Lys peptide initially inserted into the CL2 derivative allows for cleavage via cathepsin B. As part of simplifying the synthetic process, phenylalanine was removed from CL2A, thereby eliminating the cathepsin B cleavage site. Such changes did not affect complex binding, stability, or efficacy. This suggests that the release from the complex was primarily due to the cleavage of the pH-sensitive benzyl carbonate bond to the lactone ring of SN-38, rather than the cathepsin B cleavage site in CL2.

[0122] The acid-sensitive linker used in the present invention can utilize a CL2A linker and may be designed as shown in Chemical Formula 1 below to selectively and efficiently deliver camptothecin-based drugs to cancer tissue. That is, the acid-sensitive linker in the present invention may be derived from the compound of Chemical Formula 1 below: [Chemical formula 1] [ka]

[0123] Here, X1 and X2 are independently -H or -halogen; Y is -NH-, -NR A -, or direct combination; Z is -C1-C4 alkylene-, -C3-C6 cycloalkylene-, -(C1-C2 alkylene)-(C3-C6 cycloalkylene)-, -(C3-C6 cycloalkylene)-(C1-C2 alkylene)-, or -(C1-C2 alkylene)-(C3-C6 cycloalkylene)-(C1-C2 alkylene)-; W is -RB -, -M--R B -M-, -M-R B - or -R B -M-R C - is; R A ~R C each is independently C1-C4 alkylene; M is

Chemical formula

[0124] Preferably, in the chemical formula 1, X1 and X2 are each independently -H or -halogen; Y is -NR A -, or a direct connection; Z is -C1-C4 alkylene-, -(C1-C2 alkylene)-(C3-C6 cycloalkylene)-, or -(C3-C6 cycloalkylene)-(C1-C2 alkylene)-; W is -R B - or -R B -M-R C - is;<000​​​​​​​​​​​​​​​​​​​​In the present invention, the linker length, i.e., n in the chemical formula 1, may be an integer from 5 to 9, more specifically, n may be an integer from 6 to 8, and more specifically, n may be 7, but is not limited to these. Even if it falls outside the above range, if there is no difference in special effects due to the change in linker length, it is naturally included within the equivalent range of the present invention.

[0126] The water-to-acidity of a drug can be improved by placing a polyethylene glycol (PEG) spacer between the drug and the carrier; therefore, the linker of chemical formula 1 contains a low molecular weight PEG monomer containing a limited number (n=5-9) of PEG monomers.

[0127] The acid-sensitive linker of chemical formula 1 is a custom linker that can be optimized according to the characteristics of the target, payload, and carrier.

[0128] Camptothecin-based drugs have attachment sites that readily adhere to various linkers for the production of carrier-drug complexes. For example, the alcohol group portion of a camptothecin-based drug can be used as an attachment site to a linker.

[0129] Therefore, in the present invention, the [camptothecin-based drug]-[acid-sensitive linker] may be formed by linking the alcohol group portion of the camptothecin-based drug with the alcohol group portion of the acid-sensitive linker of chemical formula 1.

[0130] [Mechanism of action of ADCs equipped with a drug linker complex (A), which is a combination of a camptothecin-based drug and an acid-sensitive linker] In this specification, the term "immune complex" means a complex in which a cytotoxic drug linker complex is linked to an antibody or its antigen-binding fragment, and falls within the category of ADCs of the present invention.

[0131] Since antibody-drug conjugates (ADCs) are an example of immune conjugates, the descriptions of ADCs and immune conjugates in this invention can be used interchangeably.

[0132] When administered in vivo, the aforementioned immune complex, after its constituent antibody or a fragment containing its antigen-binding site binds to the target antigen, releases the drug, allowing it to act on target cells and / or surrounding cells. This allows for superior efficacy and reduced side effects as a targeted drug.

[0133] Factors that significantly influence the effectiveness of immune complexes include, in particular, (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. Since various factors interact to influence the effect, it is extremely difficult to predict the effect of immune complexes, which are combinations of these factors, based solely on known facts about each individual factor.

[0134] Immune complexes designed to release drugs after internalization have a drawback: if the internalization process is inefficient, sufficient concentrations of the active drug may not be delivered into the cell. Furthermore, even if hydrophobic drugs are adopted as cytotoxic agents, bystander cell-killing of surrounding cells is unlikely to be expected.

[0135] To solve these problems, the immune complex of the present invention is (i) After releasing hydrophobic drugs that can penetrate the cell membrane and perform their intended role inside the cell in the tumor microenvironment surrounding the cancer (pH ≤ 7), an acid-sensitive linker is used to introduce a large amount of the released hydrophobic drug into the cell; (ii) The method is characterized by using a camptothecin-based drug, which is a hydrophobic drug that can penetrate the cell membrane, in order to release the drug by degrading the acid-sensitive linker in an acidic atmosphere (pH ≤ 7) surrounding cancer cells, and to concentrate a large amount of the released drug inside the cell by penetrating the cell membrane.

[0136] Camptothecin-based drugs are hydrophobic low-molecular-weight substances that can penetrate cell membranes. Therefore, they accumulate rapidly in cancer tissue, can maintain high concentrations for extended periods, diffuse into the cell interior, exert cytotoxicity, kill cells, and are then released, allowing them to continuously penetrate cell membranes and act on surrounding cells.

[0137] As described above, the immune complexes of the present invention are technically characterized by combining a camptothecin-based drug with an acid-sensitive linker, so that any antibody or a fragment containing its antigen-binding site can be attached to design an immune complex depending on the desired purpose, and this is all within the scope of the present invention. For example, for excellent anticancer effects, trastuzumab, cetuximab, and sacituzumab can be attached to the camptothecin-based drug-acid-sensitive linker complex of the present invention to produce an immune complex.

[0138] The antibody linked to a camptothecin-based drug via an acid-sensitive linker, according to the present invention, binds to antigens overexpressed on the surface of cancer cells in the same initial step as when second-generation ADCs act on cancer cells. However, a portion of the antibody undergoes an intracellular processing step, also similar to second-generation ADCs, while a substantial portion is released by the low pH surrounding the cancer cells. Subsequently, the drug released from the cancer tissue migrates into the interior of the cancer cells by diffusion and acts immediately on the cancer cells without passing through endosomes or lysosomes, and without enzymatic (cathepsin B) reactions, inducing cell death. Thus, compared to second-generation ADCs, which can release drugs solely by enzymatic reactions, the antigen selectivity of cancer cells is the same, but the efficiency of drug release and delivery into cancer cells can be maximized by using a pH-sensitive linker, which is a key feature of the immune complex of the present invention.

[0139] Furthermore, although the linker must be stable in the bloodstream, prevent the drug from separating from the antibody, maintain it in a prodrug state until it reaches the target, and minimize damage to normal tissue, the present invention can mitigate the problem of ADC aggregation by using an acid-sensitive linker having a hydrophilic molecular structure to create a hydrolysis environment, which binds to hydrophobic drugs.

[0140] Different metabolites (catabolites) are formed depending on the type of linker. In this regard, in the present invention, the camptothecin-based drug and the acid-sensitive linker are preferably linked by a carbonate or ester bond so that the free camptothecin-based drug is released when the acid-sensitive linker is degraded.

[0141] Generally, in relation to hydrolysis, carbamate bonds offer superior drug linker stability compared to ester and carbonate bonds. However, the present invention is characterized by the use of unstable ester or carbonate bonds instead of carbamate bonds in order to design camptothecin-based drugs to be completely separable from the drug linker both extracellularly and intracellularly in the acidic atmosphere (pH) surrounding cancer cells.

[0142] Blood maintains a constant pH of 7.3-7.4. Therefore, camptothecin-based drugs are not cleaved from acid-sensitive linkers in the blood, and even if cleavage occurs, the release rate of camptothecin-based drugs from ADCs at the neutral pH of serum is further reduced than in tumor tissue in an acidic atmosphere.

[0143] In the present invention, the immune complex may have a mean drug-to-antibody ratio (DAR) of 4 to 8, preferably 4 or 8.

[0144] [Mechanism of action of ADCs comprising a drug linker complex (A), which is a combination of a camptothecin-based drug and an acid-sensitive linker; and a drug linker complex (B), which is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker] Drug release proceeds rapidly from Group A, primarily attacking cancer cells, while Group B releases a supertoxin with slower but potent anticancer efficacy, leading to a secondary attack and subsequent cancer cell death. For example, in the case of supertoxins such as MMAE and hemiasterlin, DAR 4 or higher cannot be used during ADC manufacturing due to toxicity, but MMAE can be used at DAR 4 or lower, and the resulting toxicity can be compensated for with a TOP1 inhibitor such as a camptothecin-based drug.

[0145] All disulfide bonds exposed outside a single antibody can be conjugated with drug-linkers of group A and group B, in which case DAR8 (group A: 4-7, group B: 1-4) is possible, with (MMAE-Cit-Val)2-Trastuzumab-(CL2A-FL118)6 being a non-restrictive example.

[0146] [Mechanism of action of ADCs equipped with a drug linker complex (A), which is a combination of a camptothecin-based drug and an acid-sensitive linker; and a drug linker complex (C), which is a combination of a camptothecin-based drug and an enzyme-sensitive linker] The same payload (or a payload with similar performance) can be configured with different drug release rates using two different linker systems.

[0147] By allowing drug release to proceed rapidly from Group A, where drug release is highly efficient in the tumor microenvironment, and then relatively slowly from Group B, cancer cell death can be promoted with a time lag.

[0148] Drug-linkers of group A and group C can be conjugated to all disulfide bonds exposed outside a single antibody, in which case DAR8 (group A: 1-7, group C: 1-7) is possible, with (Dxd-GFGG)4-Trastuzumab-(CL2A-Dxd)4 being a non-restrictive example.

[0149] [Mechanism of action of ADCs equipped with a drug linker complex (B), which is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; and a drug linker complex (C), which is a combination of a camptothecin-based drug and an enzyme-sensitive linker]

[0150] Both Group B and Group C can be delivered to cancer cells at a relatively slow rate (continuously), and TOP1 inhibitors can compensate for the disadvantage of supertoxins, which generally cannot be used above DAR4 due to toxicity. Group B and Group C can be delivered competitively into cancer cells and induce cancer cell death.

[0151] Drug-linkers of group B and group C can be conjugated to all disulfide bonds exposed outside a single antibody, in which case the antibody-drug conjugate may be DAR8 (group A: 4-7, group B: 1-4), with (MMAE-Cit-Val)2-Trastuzumab-(GGFG-Dxd)6 being a non-restrictive example.

[0152] [Mechanism of action of ADCs comprising a drug linker complex (B), which is a combination of a non-camptothecin supertoxin drug and an enzyme-sensitive linker; and a drug linker complex (D), which is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker.] Both Group B and Group D can be delivered to cancer cells at a relatively slow rate (continuously), and the disadvantage of supertoxins, which generally cannot be used above DAR4 due to toxicity, can be compensated for by anti-apoptotic protein inhibitors. Group B and Group D can be competitively delivered into cancer cells and induce cancer cell death.

[0153] Furthermore, a potent anticancer effect can be achieved by using Group D, which can enhance the effects of Group B drugs. Groups B and D can be adjusted in a 1:2 or 1:4 ratio, and the drug-linker can be conjugated to all disulfide bonds exposed outside the single antibody. A non-restrictive example is (MMAE-Cit-Val)2-Mirzotamab-(Val-Ala-Clezutoclax)4.

[0154] [Target antigen] To deliver potent cytotoxic drugs only to specific cancer cells, determining the target antigen is the first major step in ADC development. Using antibodies allows for high specificity to the target and long half-lives, enabling long-term systemic circulation. This allows for the selective accumulation of cytotoxic drugs only in tumor cells, minimizing exposure to normal tissue, reducing damage, decreasing side effects, and increasing therapeutic efficacy. To achieve this, target antigens that can identify tumor cells must be sought, and the following conditions are necessary: ​​Firstly, the target antigen must be uniformly overexpressed on the surface of tumor cells, preferably with relatively low or absent expression in normal cells. A typical example is the human epidermal growth factor receptor 2 (HER2) receptor, which is known to be expressed more than 100 times more in HER2-positive breast cancer than in normal cells. Therefore, before creating antibodies, tumor expression of the target antigen is analyzed through various profiling methods. Once the overexpression of a specific antigen is confirmed, a monoclonal antibody that recognizes this antigen is generated. Secondly, due to the antibody's binding affinity to the antigen, which is a characteristic of antibodies that undergo internalization via receptors, a stronger binding affinity to the antigen's epitope allows for more internalization and thus increases the therapeutic effect. Furthermore, it exhibits low immunogenicity. Initially, first-generation ADCs were produced using antibodies produced via mice. In first-generation ADCs, mouse antibodies were injected into humans, but side effects and antibody neutralization occurred due to the human immune response to the administered mouse antibodies, making it difficult to observe anticancer effects. The problems with the immune response have been largely improved with the development of genetic engineering technology, through the production of chimeric antibodies, humanized antibodies, and fully human antibodies.

[0155] Antibodies that recognize antigens on cancer cells must be internalized along with the drug. Bispecific antibodies are being developed to enhance internalization in cancer cells. MEDI4267 (Trastuzumab-META), under development by Medimmune and Astrazeneca, demonstrates that a biparatopic antibody targeting two non-overlapping epitopes in HER2 induces HER2 receptor clustering, thereby promoting intracellular integration, lysosomal trafficking, and degradation.

[0156] Furthermore, bispecific antibodies incorporating lysosomal markers such as CD63 or APLP2, or the prolactin receptor, along with tumor-targeting antibodies, demonstrated improved intracellular relocation compared to single antibodies, along with enhanced tumor antigen recognition. A bispecific ADC (HER2xCD63-duostatin-3, Creative Biolabs) targeting HER2 and CD63 was also found, demonstrating potent anticancer effects by reducing delivery to normal tissues and ensuring specific delivery to cancer cells.

[0157] The targeting of antibody-drug conjugates (ADCs) or immunoconjugates in the design of the present invention can be extended to include not only cancer cells but also infectious disease organisms and / or cells associated with autoimmune diseases.

[0158] Therefore, the cells targeted by antibodies or their antigen-binding site-containing fragments may be cancer cells, infectious disease organisms, and / or cells associated with autoimmune diseases.

[0159] Non-restrictive examples of target antigens include antigens that are selectively distributed on the surface of cancer, such as Her2, FolR, and PSMA, as well as cancer cell overexpression antigens that are also distributed in small amounts in normal tissues, such as Trop2.

[0160] がん cell-targeted antigens は, えば, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, アグリカン, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AM H, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, アロマターゼ(aromatase), ASPH, ATX, AX1, AXL, AZGP1(zinc-a-glycoprotein), B4GAL NT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1(MDR15), BIyS, BMP1, BMP2, BMP3B(GD FIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1(プレクチン), BRCA1, C19orflO(IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CASP1 , CASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCLI1(エオタキシン), CCL13(MCP-4), CCL15(MIP-Id), CCL16(HCC-4), CCL17(TARC), CCL18(PARC), CCL19( MIP-3b), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MEP-2), SLC, exodus-2, CCL22(MDC / STC-I), CCL23(MPIF-I), CCL24(MPIF-2 / エオタキシン-2), CCL25 (TECK), CCL26(エオタキシン-3), CCL27(CTACK / ILC), CCL28, CCL3(MIP-Ia), CCL4(MIPIb), CCL5(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND 1. CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-IRB / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6),CCR7(CKR7 / EBI1), CCR8, CDw198(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6). )、CCRL1(VSHK1),CCRL2(L-CCR),CD13,CD164,CD19,CDH6,CDIC,CD2,CD20 CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37C D38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, CD 69 CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD9 9, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH1( E-カドヘリ) CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CD H8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A(p21Wap1 / Cip1) 、CDKN1B(p27Kip1) 、CDKN1C 、CDKN2A(p16INK4a) 、CDKN2B(CDKN2C)C DKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB (Chit inase) CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLF SF7, CKLFSF8, CLDN3, CLDN6, CLDN7(Cluster-7), CLDN18, CLEC5A, CLEC6A, CLE C11A, CLEC14A, CLN3, CLU(ツテン), CMKLR1, CMKOR1(RDC1), CNR1, C-MET, C.S OL18A1, COLIA1, COL4A3, COL6A1, CR2, Crypto, CRP, CSF1(M-CSF), CSF2(GM). -CSF), CSF3(GCSF), CTAG1B(NY-ESO-1), CTLA4, CTL8, CTNNB1(b-テニ), CT SB(テシB), CX3CL1(SCYD1), CX3CR1(V28), CXCL1(GRO1), CXCL1O(IP-IO).CXCLI1(1-TAC / IP-9), CXCL12(SDF1), CXCL13, CXCL14, CXCL16, CXCL2(GRO2), CXCL3(GRO3), CXC L5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR4, CXCR6 (TYMSTR / STRL 33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA2, EPHA 3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRIN-A3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2 (HER-2), ERBB3, ERBB4, EREG, ERK8, estrogen receptor, Earl, ESR2, F3 (TF), FADD, FAP, farnesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FG F3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF(VEG FD), FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (Fibronectin), FLT1, FLT-3, FOLR1, FOS, FOSL1 (FRA-1), FR-Alpha, FY (DARC), GABRPGABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1,GFRA1, GGT1, GMCSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, Histamine and Histamine Receptor, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFN gamma, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1 (CD121a), IL1R2 (CD121b), ILIRA, IL-2, IL2RA (CD25), IL2RB (CD122), IL2RG (CD132), IL-4, IL-4R (CD123), IL-5, IL5RA (CD125), IL3RB (CD131), IL-6, IL6RA, (CD126), IR6RB (CD130), IL-7, IL7RA (CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R (CD129), IL-10, IL10RA (CD210), IL10RB (CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL3 0, IL3RA, IL4, 1L4, IL6ST (Symbol 130), ILK, INHA, INHBA, INSL3, INSL4, IRA K1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6(α6イテグリン), ITGAV, ITGB3, ITGB4(α 4. JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5(GC Box BP)、KLF6、KLK10、KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK6、KLK9、KRT1、KRT19(ケラチン19) KRT2A, KRTHB6 (hair)-derivatives II )、L1CAM、LAG3、LAMA5、LAMP1、LEP(レプチン) Lewis Y (LeY), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA(TNFb), LTB. LTB4R(GPR16), LTB4R2, LTBR, ​​LY75, LYPD3, MACMARCKS, MAG, OMgp, MAGEA3, MAGEA6, MA P2K7(c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MLSN, MK, MKI67(Ki-67) MMP2, MMP9, MS4A1, MSMB, MT3 (mucin-UI), mTOR, MTSS1, MUC1(mucin), MUC16, MYC, and M.S YD88, NCK2, NCR3LG1, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, N.K gRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI(NM23A), NOTCH, NOTCH1, NOTCH3, NOX5, and NPP B. NROB1, NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1 NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1NRP1、NRP2、NT5E、NTN4、NY-ESO1、ODZI、OPRDI、P2RX7、PAP、PART1、PATE、PAWR、P-カドヘリン、PCA3、PCD1、PD-L1、PCDGF、PCNA、PDGFA、PDGFB、PDGFRA、PDGFRB、PECAMI、L1、 -CAM, peg-clinic chain PF4(CXCL4), PGF, PGR, glucose (phosphacan), PIAS2, PI3, PIK3CG, P.S LAU(uPA), PLG, PLXDCI, PKC, PKC-ータ, PPBP(CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PRO C. PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2(COX-2), PTN, PVRIG, RAC2(P21Rac2). ANK, RANK, RARB, RGS1, RGS13, RGS3, RNFI1O(ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB 1D2(Shell B) SCGB2A1(Shell2) SCGB2A2(Shell1) SCYE1 (Small-scale sludge-processing agents) SDF2, SERPENA1, SERPINA3, SERPINB5 (Sarpena). )、SERPINEI(PAI-I)、SERPINFI、SHIP-1、SHIP-2、SHB1、SHB2、SHB G, SfcAZ, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, S.K LIT2, SPP1, SPRR1B(Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, and TGFB1I 1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1(トロTHBS-1), THBS2, THBS4THPO, TIE(Tie-1), TIMP3, activator (tissue factor) (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10 TLR11, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFR SF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF1O(TRAIL), TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRSF17, TNFSF1 1(TRANCE)、TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEML), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), T OLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6 A (tectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (tectin-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G) , ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), N CR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD30, CD300C, CD300E, CD300L B (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD 314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC5, SIGLEC6, SIGLEC7,SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPA, SIRPB1 (CD172B), TREM1 (CD354), TREM2, KLRF1 (NKp80), 17-1A, SLAM7, MSLN, CTAG1B / NY-ESO-1, MAGEA3 / A6, ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q 8BH59)、PREP(Q8K411)、YMEL1(O88967)、LPPRC(Q6PB66)、LONM(Q8CGK3)、 ACON(Q99KI0)、ODO1(Q60597)、IDHP(P54071)、ALDH2(P47738)、ATPB(P564) 80)、AATM(P05202)、TMM93(Q9CQW0)、ERGI3(Q9CQE7)、RTN4(Q99P72)、CL04 1(Q8BQR4)、ERLN2(Q8BFZ9)、TERA(Q01853)、DAD1(P61804)、CALX(P35564) CALU(O35887), VAPA(Q9WV55), MOGS(Q80UM7), GANAB(Q8BHN3), ERO1A(Q8R180), UGGG1(Q6P5E4), P4HA1(Q60715), HYEP(Q9D379), CALR(P14211), A T2A2(O55143), PDIA4(P08003), PDIA1(P09103), PDIA3(P27773), PDIA6(Q922R8), CLH(Q68FD5), PPIB(P24369), TCPG(P80318), MOT4(P57787), NICA (P57716)、BASI(P18572)、VAPA(Q9WV55)、ENV2(P11370)、VAT1(Q62465)、 4F2(P10852)、ENOA(P17182)、ILK(O55222)、GPNMB(Q99P91)、ENV1(P10404) (、ERO1A(Q8R180)、CLH(Q68FD5)、DSG1A(Q61495)、AT1A1(Q8VDN2)、HYOU1) Q9JKR6)、TRAP1(Q9CQN1)、GRP75(P38647)、ENPL(P08113)、CH60(P63038)、It could also be CH10(Q64433), but is not limited to these.

[0161] The target antigen may be an antigen that is distributed more than 10 times more abundantly in cancer cells than in normal cells.

[0162] [Pharmacologically acceptable salts] In this specification, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, including, for example, salts of inorganic ions such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, and iron, and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid. Other examples include 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, and acetylsalicylic acid, as well as amino acid salts such as lysine, arginine, and guanidine. Additionally, there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium, which may be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts as defined in this invention are not limited by these listed salts.

[0163] [Various Carrier-Drug Conjugates] The two drug linker conjugates selected by this invention can be applied to various types of drug carriers other than antibodies. Unlike antibodies, they can penetrate deep into cancer tissue and have the characteristic of being easily converted into cancer cells (CMC), making them suitable for a wide range of applications.

[0164] The carrier may be the antigen-binding site, repibody, and / or aptamer of the antibody.

[0165] Aptamers are DNA / RNA-based biomolecules that, like antibodies, possess antigen-binding affinity at the nM level and can be used as cancer tissue-selective carriers for drugs.

[0166] Repebodies are a new class of immunoproteins discovered in fish. Like antibodies and nanobodies (single-chain antibodies derived from mammals such as llamas), they can recognize antigens and possess antigen-binding affinity at the several nM level, making them potentially useful as selective carriers of drugs for cancer tissue. In particular, their lack of immunogenicity makes them suitable for use as drug carriers, while their low molecular weight enhances cancer cell penetration. They are also easy to produce consistently, resulting in repebodies of uniform quality, making them ideal for the production of drug conjugates with enhanced cancer tissue penetration.

[0167] [Pharmaceutical compositions for the prevention or treatment of cancer] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt thereof according to the present invention as described above, as an active ingredient.

[0168] Furthermore, according to one specific example of the present invention, a method for treating or preventing cancer is provided, comprising the step of administering a therapeutically effective amount of the antibody-drug conjugate (ADC) to a subject in need. The subject refers to all animals, such as mice, livestock, and humans, that are likely to develop or have developed the disease for which treatment is the objective, but may exclude humans.

[0169] The antibody-drug conjugate (ADC) of the present invention specifically binds to antigens on cancer cells and releases drugs inside and outside the cancer cells, exhibiting cytotoxicity; therefore, it can be usefully used for the treatment or prevention of cancer. The anticancer activity of the immune conjugate of the present invention is as described above.

[0170] In the present invention, the cancer may be a solid tumor or a hematological cancer. For example, 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, epithelial ovarian cancer, ovarian germ cell tumor, male breast cancer, brain tumor, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, nasal cavity cancer / paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain tumor, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, One or more of the following cancers may be selected from the group consisting of renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue tumor, malignant bone tumor, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary origin, gastric lymphoma, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal tumor, Wilms' tumor, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone tumor, metastatic brain tumor, mediastinal tumor, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's 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, hematological cancer, and thymic cancer, but are not limited to these. Furthermore, the aforementioned cancers include not only primary cancers but also metastatic cancers.

[0171] The term "therapeutably effective amount" as used in this invention refers to the amount of the immune complex effective for the treatment or prevention of cancer. Specifically, "therapeutably effective amount" means an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the individual's species and severity, age, sex, type of disease, drug activity, drug sensitivity, administration time, route of administration and elimination rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of this invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. It can also be administered single or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and since the immune complex of this invention exhibits a dose-dependent effect, the dose can be easily determined by those skilled in the art based on various factors such as the patient's condition, age, sex, and comorbidities. The active ingredient of the pharmaceutical composition of this invention is highly safe and can therefore be used at doses higher than the determined dose.

[0172] Furthermore, according to one specific example of the present invention, the present invention provides a use for the immune complex for use in the manufacture of a medicine for use in the treatment or prevention of cancer. The immune complex for the manufacture of the medicine can be mixed with acceptable adjuvants, diluents, carriers, etc., and can be manufactured into a complex formulation together with other active ingredients, which can have a synergistic effect of the active ingredients.

[0173] The matters referred to in the uses, compositions, and therapeutic methods of the present invention shall apply equally to each other, insofar as they do not contradict each other. [Effects of the Invention]

[0174] The present invention provides an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to a single antibody via a linker, allowing for desired adjustment of pharmacokinetic properties and in vivo distribution. This enables, for example, increased residence time in the bloodstream, maintaining high blood drug concentrations for extended periods, and providing the ADC with a desired hepatic clearance profile and blood circulation profile.

[0175] The present invention provides an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to a single antibody via a linker, allowing for desired adjustment of pharmacokinetic properties and in vivo distribution. This enables, for example, increased residence time in the bloodstream, maintaining high blood drug concentrations for extended periods, and providing the ADC with a desired hepatic clearance profile and blood circulation profile. [Brief explanation of the drawing]

[0176] [Figure 1] Figure 1 shows the results of size exclusion chromatography (SEC) analysis of dual-payload ADCs obtained by simultaneous reaction according to one embodiment of the present invention. [Figure 2] Figure 2 shows the results of hydrophobic interaction chromatography (HIC) analysis of dual-payload ADCs produced by simultaneous reaction according to one embodiment of the present invention. [Figure 3] Figure 3 shows the SDS-PAGE analysis results of dual-payload ADCs produced by simultaneous reaction according to one embodiment of the present invention. [Figure 4] Figure 4 shows the results of confirming the correlation between the mixing ratio and DAR of dual-payload ADCs produced by simultaneous reaction according to one embodiment of the present invention. [Figure 5] Figure 5 shows the results of an analysis of cell viability after treatment with dual-payload ADCs produced according to one embodiment of the present invention. [Figure 6] Figure 6 shows the results of an analysis of cell viability after treatment with dual-payload ADCs produced according to one embodiment of the present invention. [Best Mode for Carrying Out the Invention]

[0177] The present invention will be described more specifically below based on examples. However, the following examples are merely for the purpose of clearly illustrating the technical features of the present invention and do not limit the scope of protection of the present invention.

[0178] [Examples] Example 1: Manufacturing of a comparative single-payload ADC The antibody was buffer-exchanged using a PD-10 desalting column with reducing buffer (150 mM NaCl, 50 mM histidine, pH 6.0). Then, 4 mg / ml of antibody was treated with 9 times the molar equivalent of TCEP at 25°C for 2 hours to reduce the disulfide bonds of the antibody.

[0179] The reduced antibody was desalted using a PD-10 desalting column to remove excess TCEP, and the conjugation reaction was carried out by reacting 2 mg / ml of reduced antibody with 12 times the molar equivalent of 25-6 (chemical formula 3 below) linker-payload in a reaction buffer containing 10% DMSO (25 mM histidine, pH 6.0) at 25°C for 1 hour.

[0180] After the conjugation reaction, the excess linker-payload mixture was removed using a PD-10 desalting column to obtain the final ADC0 (DAR8; ADC with 25-6 bound to DAR8).

[0181] Example 2: Method for manufacturing a dual-payload ADC when payloads 1 and 2 are different 2-1. Method for manufacturing a dual-payload ADC through the simultaneous reaction of two linker-payloads

[0182] The antibody was buffer-exchanged using a PD-10 desalting column with reducing buffer (150 mM NaCl, 50 mM histidine, pH 6.0). Then, 4 mg / ml of antibody was treated with 9 times the molar equivalent of TCEP at 25°C for 2 hours to reduce the disulfide bonds of the antibody.

[0183] Three types of mixtures were prepared by mixing mc-val-cit-PAB-MMAE (Maleimide-Caproyl amide-Val-Cit-PAB-MMAE; hereafter referred to as vcMMME) dissolved in DMSO with 25-6 (chemical formula 3 below) linker-payload in molar ratios of 2:6, 3:5, and 4:4.

[0184] The reduced antibody was desalted using a PD-10 desalting column to remove excess TCEP, and the conjugation reaction was carried out by reacting 2 mg / ml of the reduced antibody with 12 times the molar equivalent of the linker-payload mixture in a reaction buffer (25 mM histidine, pH 6.0) containing 10% DMSO at 25°C for 1 hour.

[0185] After the conjugation reaction, the excess linker-payload mixture was removed using a PD-10 desalting column to obtain the final dual-payload ADC1-7. [Chemical formula 3] [ka]

[0186] The size exclusion chromatography (SEC) analysis results of the dual-payload ADC produced by this embodiment are shown in Figure 1, the hydrophobic interaction chromatography (HIC) analysis results are shown in Figure 2, the SDS-PAGE analysis results of the dual-payload ADC are shown in Figure 3, the DAR analysis results are shown in Table 4 below, and the correlation between the reaction ratio of the dual-payload ADC and the DAR is shown in Figure 4. [Table 4] [Table 5]

[0187] 2-2. Method for manufacturing a dual-payload ADC through a sequential reaction of two linker-payloads The antibody was buffer-exchanged using a PD-10 desalting column with reducing buffer (150 mM NaCl, 50 mM histidine, pH 6.0). Then, 4 mg / ml of antibody was treated with 9 times the molar equivalent of TCEP at 25°C for 2 hours to reduce the disulfide bonds of the antibody.

[0188] The reduced antibody was desalted using a PD-10 desalting column to remove excess TCEP, and the primary conjugation reaction was carried out by reacting 2 mg / ml of reduced antibody with 2.0, 2.1, 2.2 or 4.0, 4.1, 4.2 times the molar equivalent of the mc-vc-MMAE linker-payload in a reaction buffer (25 mM histidine, pH 6.0) containing 10% DMSO at 25°C for 1 hour.

[0189] The primary reaction mixture was mixed with a 12-fold molar equivalent of 25-6 linker payload, and the reaction was carried out at 25°C for 1 hour to advance the secondary conjugation reaction.

[0190] After all reactions, the excess linker-payload mixture was removed using a PD-10 desalting column to obtain the final dual-payload ADC8-13. [Table 6]

[0191] 2-3. Cell viability assay Cell lines of various cancer types were seeded at a rate of 3000 cells per well in 96-well plates (KPL4, FaDu, BT474, MDA-MB-468) and cultured at constant temperature (37°C, 5% CO2). After 24 hours, the cells were treated with 100 µl of ADC samples at nine different concentrations (serially diluted from 1000 nM in 1 / 5 increments). This treatment included single-payload ADC (Tra-25-6 (DAR8)) and dual-payload ADC (Tra-vcMMAE-25-6 (DAR1+7)). A control group (ADC concentration 0) was also prepared without ADC treatment. After 6 days of constant temperature incubation (37°C, 5% CO2), 100 μl of CellTiter-Glo reagent (CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)) was added to each well, pipetted, and incubated at room temperature (RT) for 10 minutes before measuring luminescence. If the luminescence value at an ADC concentration of 0 is considered 100%, the concentration that yields 50% of the luminescence value is the IC. 50 It is a value.

[0192] 2-4. Results of cell viability assay To evaluate the in vitro efficacy of dual payload ADCs compared to single payload ADCs, which are targeted by HER2 expression, various cancer cell lines showing differences in HER2 expression levels were treated with Tra-25-6 (DAR8) as a single payload ADC and Tra-vcMMAE-25-6 (DAR1+7) as a dual payload ADC, respectively, and cell viability was observed over a 6-day incubation period.

[0193] Referring to Figures 5 and 6, in HER2-positive cell lines, ADC4 and ADC6, acting as dual-payload ADCs, showed lower IC50 values ​​compared to ADC0 (Tra-25-6(DAR(0+8))), confirming their potent cytotoxic efficacy. Furthermore, depending on the HER2 expression level, in cell lines showing high expression (KPL4, BT474) and cell lines showing relatively mild expression (FaDu), ADC4 and ADC6 as dual-payload ADCs showed even lower IC50 values ​​than ADC0 as a single-payload ADC. 50 The values ​​indicate that the cell killing effect of a dual-payload ADC is stronger than that of a single-payload ADC.

[0194] This confirms that two payloads with different mechanisms of action (a topoisomerase I inhibitor and a microtubule inhibitor) can work together to show a synergistic effect from an efficacy standpoint.

[0195] On the other hand, in HER2-negative cell lines, Tra-25-6(DAR8) and Tra-vcMMAE-25-6(DAR1+7)ADC showed similar levels of IC. 50 The observed values ​​confirm that the cell death effect was not significantly induced, and that it exhibited a comparable level of cytotoxicity.

[0196] Furthermore, it was observed that differences in the degree of cytotoxicity to the drug payload appeared depending on the degree of sensitivity to the drug payload among various cancer cell lines.

[0197] In other words, according to this embodiment, the antibody-drug conjugate (ADC) may contain one or more drug linker conjugates, and when the drugs contained in each drug linker conjugate are of different types, a significant synergistic effect is observed in killing cancer cells.

[0198] The present invention has been described above, focusing on preferred embodiments.

[0199] In the examples, it was shown that when manufacturing a Dual payload ADC of DAR8 consisting of two different payloads 1 and 2, the respective DARs of payloads 1 and 2 can be freely adjusted to be 1 and 7, 2 and 6, 3 and 5, or 4 and 4. This is possible by adjusting the molar equivalent ratio of the reactants through simultaneous or sequential reactions, and the method of the present invention makes it possible to manufacture Dual payload ADCs of DAR4 or DAR6. That is, when manufacturing a Dual payload ADC of DAR4, the respective DARs of the payloads can be freely adjusted to be 1 and 3, or 2 and 2, and in the case of DAR6, they can be adjusted to be 1 and 5, 2 and 4, or 3 and 3.

[0200] Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be embodied in modified forms that do not depart from the essential characteristics of the invention. Accordingly, the disclosed embodiments should be considered in an explanatory rather than restrictive view. The scope of the present invention is shown in the claims, not in the above description, and all differences within an equivalent scope should be construed as being included in the present invention.

Claims

1. An antibody-drug conjugate (ADC) is an antibody to which one or more drug linker conjugates are bound, A drug linker complex (A) is a combination of a camptothecin-based drug and an acid-sensitive linker; Drug linker complexes (B) are combinations of non-camptothecin supertoxin drugs and enzyme-sensitive linkers; A drug linker complex (C), which is a combination of a camptothecin-based drug and an enzyme-sensitive linker; and An antibody-drug conjugate (ADC) characterized in that at least one drug linker conjugate selected from the group consisting of a drug linker conjugate (D) which is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker, is bound to an amino acid residue containing cysteine ​​or lysine, a non-natural amino acid residue, or a sugar chain of an antibody.

2. An antibody-drug conjugate (ADC) in which a first drug linker conjugate and a second drug linker conjugate are bound to one antibody, The first drug linker complex is a drug linker complex (B) which is a combination of a non-camptothecin-based supertoxin drug and an enzyme-sensitive linker; The second drug linker complex is an antibody-drug conjugate, which is a drug linker complex (C) consisting of a combination of a camptothecin-based drug and an enzyme-sensitive linker.

3. The antibody-drug conjugate according to claim 2, characterized in that the antibody-drug conjugate has a drug-antibody ratio (DAR) of 4 to 8.

4. The antibody-drug conjugate according to claim 2, wherein the first drug-linker conjugate is a compound of the following chemical formula 6 or 7, and the second drug-linker conjugate is one compound selected from the following chemical formulas 2 to 5; [Chemical formula 2] 【Chemistry 1】 [Chemical formula 3] 【Chemistry 2】 [Chemical formula 4] 【Transformation 3】 [Chemical formula 5] 【Chemistry 4】 [Chemical formula 6] 【Transformation 5】 [Chemical formula 7] 【Transformation 6】

5. The antibody-drug conjugate according to claim 2, wherein the drug contained in the first drug-linker conjugate and the drug contained in the second drug-linker conjugate are bound to the antibody in a ratio of 1:1 to 1:

7.

6. The antibody-drug conjugate according to claim 2, characterized in that the antibody-drug conjugate is produced by sequential or simultaneous reactions.

7. The drug contained in the first drug linker complex is an auristatin compound. The antibody-drug conjugate according to claim 2, wherein the drug contained in the second drug-linker conjugate is a camptothecin derivative that inhibits topoisomerase I.

8. The drug contained in the first drug linker complex is an auristatin compound, and is at least one compound selected from the group consisting of MMAE, MMAU, Aur0101, Duostatin 5, and tubulin inhibitors. The antibody-drug conjugate according to claim 7, wherein the drug contained in the second drug-linker conjugate is a topoisomerase I inhibitor, and the topoisomerase I inhibitor is at least one compound or derivative thereof selected from the group consisting of SN-38, exatecan, Dxd, FL118, and 7-aminoalkyl-substituted camptothecin compounds.

9. The antibodies mentioned above are urelumab, utomirumab, bebuterovimab, aducanumab, bapineuzumab, crenezumab, donanemab, gantenerumab, lecanemab, solanezumab, nesvacumab, evinacumab, and enobrituzumab. blituzumab, omburtamab, belimumab, ianalumab, tabalumab, bertilimumab, mogamulizumab, leronlimab, ciplizumab, foralumab, muromonab-CD3, otelixizumab, teplizumab ), ibalizumab, tregalizumab, zanolimmab, itolizumab, efalizumab, inebilizumab, tafasitamab, tositumomab, ocrelizumab, ofatumumab, rituximab, ublituximab, beltz Veltuzumab, Epratuzumab, Basiliximab, Daclizumab, Vallilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab, Mezagitamab, BleselumabDacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab b) Cusatuzumab, Oleculumab, Miratuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab ), eculizumab, pozelimab, ravulizumab, lacnotuzumab, axatilimab, cabilalizumab, emactuzumab, ipilimumab, quavonlimab, tremelimumab, zalifrelimab, cetuximab Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab,Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Pronmarlimab, Kodori Tuzumab (Codrituzumab), Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Dunogotuzumab, Ficlatuzumab, Rilotumumab, Aromfilimab, Aniflorumab olumab, emapalumab, ligerizumab, omalizumab, cyclostomab, dalotuzumab, figitumumab, ganitumab, teprotumumab, bermekimab, canakinumab, gevokizumab, briakinumab ), ustekinumab, anlukinzumab, sendakimab, lebrikizumab, tralokinumab, brodalumab, bimekizumab, ixekizumab, secukinumab, brazikumab, guselkumab, mirikizumab,Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab ), olokizumab, siltuximab, silukumab, ziltivekimab, levirimab, sarilumab, satralizumab, tocilizumab, abituzumab, favezerimab, fianlimab, ieramilimab, relatrimab (Rel) atlimab, simtuzumab, avagovomab, oregovomab, tanezumab, ivuxolimab, locatinlimab, tavolimab, telazololimab, vonlerolizumab, alirocumab, bococizumab, ebronucimab ), evolocumab, flubocimab, ongericimab, taforecimab, dostarlimab, valstilimab, camrelizumab, cemiplimab, geptanolimab, nivolumab, pembrolizumab, penplimab,Pidilizumab, Progolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislerizumab, Tripalimab, Ezabenlizumab, Zimberelimab, Atezolizumab lizumab, avelumab, cosibelimab, sugemalimab, durvalumab, envafolimab, subratoxumab, denosumab, zirovertamab, elotuzumab, domvanalimab, etigilimab, osperirumab b) Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Gatuzumab, Tezeperumab, Gatipotuzumab, Cabilizumab, Bevacizumab, Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab,Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Obrindatamab, Elranatamab, Limboseltamab, Teclistamab, Epcoritamab amab), Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab, Amibanta Amivantamab, Emicizumab, Xenocutuzumab, Zanidatamab, Tibrizumab, Naptumomab, Belantamab, Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumoma Ibritumob, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorbotuzumab, Polatuzumab, Tusamitamab, Terisotuzumab, Robalpituzumab ab), Depatuxizumab, Farletuzumab, Mirbetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab, Vobarilizumab, Cadonilimab, Vudalimab, Teboterimab, Ivonescimab,The antibody-drug conjugate according to claim 2, wherein the conjugate is at least one selected from the group consisting of erfonrilimab, ozoralizumab, faricimab, vanucizumab, and navicixizumab.

10. A method for producing an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to one antibody, A drug linker complex (A) is a combination of a camptothecin-based drug and an acid-sensitive linker; Drug linker complexes (B) are combinations of non-camptothecin supertoxin drugs and enzyme-sensitive linkers; A drug linker complex (C), which is a combination of a camptothecin-based drug and an enzyme-sensitive linker; and A first step of selecting at least one drug linker complex (D) from the group consisting of a drug linker complex (D) which is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker; and A method for producing an antibody-drug conjugate (ADC), comprising a second step of using drug linker complexes in a manner that differs from one another: (i) amino acid residues at the binding site with the antibody, (ii) binding sequence, and / or (iii) binding method, thereby binding each drug linker complex to an amino acid residue of the antibody.

11. The method for producing an antibody-drug conjugate according to claim 10, characterized in that (B) is a compound of chemical formula 6 or 7 below, and (C) is one compound selected from chemical formulas 2 to 5. [Chemical formula 2] 【Transformation 7】 [Chemical formula 3] 【Transformation 8】 [Chemical formula 4] 【Chemistry 9】 [Chemical formula 5] 【Chemistry 10】 [Chemical formula 6] 【Chemistry 11】 [Chemical formula 7] 【Chemistry 12】

12. The method for producing an antibody-drug conjugate according to claim 10, wherein the second step is to mix the drug-linker conjugates of (B) and (C) to prepare a mixture; and to react the mixture with a reduced antibody to cause conjugation.

13. The method for producing an antibody-drug conjugate according to claim 10, wherein the second step includes the steps of: adding the drug-linker conjugate of (B) to the reduced antibody in an amount of 2 to 4 times the molar equivalent of the antibody to produce a primary reaction product; and adding the drug-linker of (C) to the primary reaction product to produce a secondary reaction product.

14. A pharmaceutical composition for the prevention or treatment of cancer comprising an antibody-drug conjugate (ADC) in which one or more drug linker conjugates are linked to an antibody, The antibody-drug conjugate (ADC) is A drug linker complex (A) is a combination of a camptothecin-based drug and an acid-sensitive linker; Drug linker complexes (B) are combinations of non-camptothecin supertoxin drugs and enzyme-sensitive linkers; A drug linker complex (C), which is a combination of a camptothecin-based drug and an enzyme-sensitive linker; and A pharmaceutical composition characterized in that at least one drug linker complex selected from the group consisting of a drug linker complex (D) which is a combination of an anti-apoptotic protein inhibitor drug and an enzyme-sensitive linker, is bound to an amino acid residue containing cysteine ​​or lysine of an antibody, a non-natural amino acid residue, or a sugar chain.

15. The aforementioned cancers include 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, epithelial ovarian cancer, ovarian germ cell tumor, male breast cancer, brain tumor, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, nasal cavity cancer / paranasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain tumor, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, Renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue tumors, malignant bone tumors, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary origin, gastric lymphoma, stomach cancer, gastric carcinoid tumor, gastrointestinal stromal tumor, Wilms' tumor, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic osteoma The pharmaceutical composition according to claim 14, which is at least one selected from the group consisting of ulcer, metastatic brain tumor, mediastinal tumor, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's 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, hematological cancer, and thymic cancer.

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