An antibody-drug conjugate in which a camptothecin-based drug is linked via a linker to an antibody with low antigen-binding affinity

Abstract

The present invention relates to a method for treating a cell surface protein epitope in which a camptothecin-based drug (A) has a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 The present invention relates to an immune complex or a pharmaceutically acceptable salt thereof, characterized in that it is bound, via a linker (C), to an antibody or a fragment thereof containing its antigen-binding site (B-1) of less than M; or an antibody or a fragment thereof containing its antigen-binding site (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than when binding monovalently.

Description

[Technical Field]

[0001] The present invention relates to a method for degrading DDX5 protein, in which a camptothecin-based drug (A) has a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 The present invention relates to an immune complex or a pharmaceutically acceptable salt thereof, characterized in that it is bound, via a linker (C), to an antibody (B-1) having a molecular weight of less than M or a fragment thereof containing its antigen-binding site; or an antibody (B-2) having a molecular weight of less than M or a fragment thereof containing its antigen-binding site, which binds to a cell surface protein expressed in both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein in a bivalent manner than when binding to the epitope in a monovalent manner. [Background technology]

[0002] Organs are created through evolutionary arrangements of various cell types and matrix components to form specific microarchitectures that optimize structure, efficacy, and function. The epithelial cell membrane of the small intestine, muscle cells, and nerve cells in muscles form specific microanatomical structures during development, and even in adults, these cells maintain homeostasis of the tissue or organ.

[0003] As cancer grows, the surrounding normal tissue and cancer tissue undergo dynamic and continuous structural transformations, disrupting homeostasis. Excessive cancer cell division, proteolytic enzyme activity, and matrix production can all alter cell-cell or cell-matrix relationships, affecting morphogenesis. The well-known epithelial-mesenchymal transition (EMT) can be considered an example of a morphogenetic change in which cells transform from an epithelial state, influenced only by a two-dimensional surface, to a permeable mesenchymal state, surrounded by a three-dimensional extracellular matrix.

[0004] Camptothecin is a selective inhibitor of type I topoisomerase-1, an isomerase involved in DNA replication and recombination. It is a natural antitumor alkaloid isolated in 1966 by Wall et al. in the United States from Camptotheca acuminata, a plant native to China. After its potent in vitro cytotoxicity was demonstrated, clinical development was initiated at the National Cancer Institute (NCI) and other institutions. However, due to its extremely poor solubility and associated side effects, such as bone marrow suppression and hemorrhagic cystitis, development was halted. However, since 1990, camptothecin's unique mechanism of action, namely, its selective inhibition of type I DNA topoisomerase, distinct from its inhibition of type II DNA topoisomerase-2, has been confirmed to exert its antitumor effects.

[0005] DNA topoisomerases are members of the gyrase family of enzymes. They are nuclear enzymes that temporarily break DNA or unwind the double helix when cells require access to genetic material for replication or transcription. They also participate in various cellular activities, such as chromosome condensation and recombination, and DNA repair. The genetic code for topoisomerase enzymes is fairly conserved across species.

[0006] The drug target of camptothecin, type I topoisomerase, has been observed to be elevated in various malignant tumors. This drug does not inhibit the free enzyme, but rather stabilizes the covalent bond of the topo-DNA complex, preventing further ligation of the broken DNA fragments. Therefore, the sensitivity of cells to such topoisomerase-targeting drugs is related to the level of the enzyme present in the nucleus. This drug inhibits DNA recombination. This prevents transcription from proceeding. The greater the amount of type I topoisomerase, the more cleavable complexes formed, which means greater drug sensitivity. This has important clinical relevance, as type I topoisomerase inhibitors are used to increase the expression of type II topoisomerase, thereby increasing sensitivity to type II topoisomerase inhibitors. This result is supported by the antagonistic relationship between type I and type II topoisomerases. Unlike type II topoisomerases, type I topoisomerases are not closely related to proliferation in normal tissues. They are present in greater amounts in solid tumors in the "S" phase, where cell division is active, such as lymphoma, colorectal cancer, ovarian cancer, and esophageal cancer, than in surrounding normal tissues. They are known to actually induce cell death by blocking gene replication and transcription in tumor cells during the "S" phase of the cell cycle.

[0007] Camptothecin (CPT) has low aqueous solubility, and in preparation for clinical trials, the National Cancer Institute (NCI) produced a water-soluble sodium salt (NSC100880). Phase I and II clinical trials were not completed due to the high toxicity exhibited by the compound (hemorrhagic cystitis, gastrointestinal toxicity such as nausea, vomiting, and diarrhea, and bone marrow suppression, particularly leukopenia and hypothrombocytosis).

[0008] Subsequently, numerous CPT congeners have been synthesized to obtain compounds with lower toxicity and higher aqueous solubility. Two drugs are irinotecan (CPT-11) and topotecan.

[0009] Irinotecan (CPT-11), jointly developed by Dallchi and Yakult in Japan, was the world's first camptothecin-based anticancer drug in 1994, and its effectiveness against lung cancer (small cell and non-small cell lung cancer) was proven. It was launched in Europe and Japan, and in 1995, its effectiveness against colon cancer and breast cancer was further proven. Furthermore, topotecan, developed by GlaxoSmithKline, was approved by the US FDA in April 1995 for its effectiveness against metastatic ovarian cancer and launched. CKD-602 (belotecan), a new camptothecin derivative recently developed in Korea, is a water-soluble compound that not only exhibits the effects of a potent type I topoisomerase inhibitor, but also successfully overcomes the toxicity caused by existing poor solubility.

[0010] All camptothecin derivatives identified to date contain the parent structure with five rings essential for cytotoxicity (Figure 1). The E-ring and the A- and B-rings have been identified as key sites in the molecular structure. Camptothecin has a pentacyclic structure with a lactone group in the E-ring, which is essential for cytotoxicity. The lactone group and alpha-hydroxyl group at carbon 20 of the E-ring are important for stabilizing type I topoisomerase-DNA byproducts. Modifications to the A- and B-rings have been shown to increase water solubility and activity. Modifications to the first ring, for example, have been shown to increase the aqueous solubility of the aforementioned drugs, allowing for greater tolerability.

[0011] CKD-602 also attempted to substitute the B-ring moiety at carbon 7 to improve water solubility and anticancer efficacy. Lee et al. reported that CKD-602 had superior anticancer efficacy compared to camptothecin and topotecan in a wide range of cancer cell lines. Furthermore, in an L1210 leukemia nude mouse model, the maximum tolerated dose (MTD) was 25 mg / kg, demonstrating its relative safety. The commonly known side effects of camptothecin-based drugs can be broadly categorized into hematologic and non-hematologic side effects. Hematologic side effects include neutropenia accompanied by fever, sepsis, and bleeding. Non-hematologic side effects include cutaneous side effects such as nausea, vomiting, and hair loss, as well as toxicity to the gastrointestinal tract, kidneys, and nervous system. Kim et al. demonstrated the effectiveness of camptothecin in animal studies of CKD-602. No abnormal drug reactions other than increased gastric secretion were observed, even when administered at doses 10 times higher than the clinically applicable dose. Furthermore, a recent clinical study in Korea also reported only reversible and manageable side effects such as neutropenia and leukopenia, rather than severe systemic toxicity, demonstrating the relatively stable side effects. However, to date, its use has been limited to patients who have failed or cannot undergo standard chemotherapy, i.e., those who have relapsed or worsened after standard chemotherapy and are unlikely to benefit from further anti-cancer chemotherapy or surgery, those with refractory or recurrent ovarian or colorectal cancer who have failed first-line chemotherapy, and those with extensive disease who have advanced-stage small cell lung cancer.

[0012] SN-38 has an IC in the nanomolar range in a variety of cell lines. 50Trop-2-SN-38 is a potent topoisomerase I inhibitor with therapeutic value. It is the active form of irinotecan, a precursor drug used in the treatment of colorectal cancer, and also shows activity in lung, breast, and brain cancers. Trop-2-SN-38 ADCs have been successfully developed for a number of cancer types, including TNBC, bladder, and gastric cancers, but resistance issues to SN-38 remain (e.g., overexpression of drug efflux transporters, epigenetic silencing of Top1, and increased levels of anti-apoptotic proteins).

[0013] According to US9629926B, the biodistribution of hRS7-CL2A-SN-38 is similar to that of the parent hRS7 IgG, with tumor uptake by the tumor, but the hydrophobicity of SN-38 results in rapid clearance with two-fold higher hepatic uptake. Because the ADC is eliminated via the liver, hepatic and gastrointestinal toxicity is expected to be dose-limiting.

[0014] Camptothecin derivatives, such as SN-38 and Dxd (a derivative of the clinical-stage topoisomerase I inhibitor exatecan), have recently been successfully developed as novel payloads for antibody-drug conjugates (ADCs). As a result, two ADCs using a camptothecin payload have been developed: Trodelvy (sacituzumab govitecan) with an SN-38 payload. and Enhertz (trastuzumab deruxtecan), which has a Dxd payload, was recently approved by the FDA. This payload has intermediate cytotoxicity compared to existing highly toxic ADC payloads such as MMAE, MMAF, or PBD, but its excellent safety profile allows for the use of a higher drug-to-antibody ratio (DAR) of 8 rather than 2 or 4 as in existing ADCs. Furthermore, linker systems with faster drug release profiles, such as the CL-2A or GGFG linker systems, can be used with this payload. Combining these components has led to the development of new ADCs with a broader therapeutic range.

[0015] Antibody-drug conjugates (ADCs) are a new drug development modality in which a potent anticancer drug (payload) that can efficiently inhibit cancer cell growth even in small doses is attached to an antibody that can selectively recognize cancer cells via a specially structured linker. ADCs selectively deliver drugs with strong anticancer efficacy to cancer cells via antibodies, demonstrating strong anticancer efficacy without systemic side effects, but efficacy can be limited by the amount of drug delivered.

[0016] Generally, when a hydrophilic antibody is linked to a mostly hydrophobic linker-payload, no major problems occur when the DAR, which indicates the number of drugs attached per antibody, is in the range of 1 to 8. However, if this is exceeded, problems may arise in production / transport / use due to the occurrence of aggregation, and safety issues may arise due to the formation of aggregates in the blood. Furthermore, due to high lipid solubility, drug absorption and payload release do not depend on the antibody's drug target, but may occur non-selectively in macrophages, etc., resulting in unexpected side effects. Furthermore, it is possible to increase the antibody dosage while fixing the DAR to a specific value, but in this case, (1)A There are problems with DCs, such as non-selective absorption and subsequent off-target toxicity, and (2) the antibodies that make up ADCs bind with strong affinity to drug targets on the surface of cancer cells. However, in this case, the drug targets around blood vessels are first saturated, and after a certain amount of antibody has bound, additional antibodies (ADCs) cannot penetrate into the cancer tissue, which can result in the administration of ADCs being wasted.

[0017] To address the issue of off-target toxicity that can occur after non-selective uptake, many ADCs using camptothecin compounds that are relatively safe in normal tissues have been developed, and the effectiveness of this approach has been demonstrated through the success of Daiichi-Sankyo's Enhertu and Gilead's Trodelvy. Part of Enhertu's success comes from the use of a safe payload, which increases the ADC dose from the commonly used 2.7mpk level to 4.8-5.4mpk, allowing the payload released from the ADC to be distributed uniformly throughout the cancer tissue. Summary of the Invention [Problem to be solved by the invention]

[0018] It is very important to easily deliver ADC or other targeted drug conjugates into cancer tissues. In fact, the results of a peptide drug conjugate targeting Nectin-IV recently published by Bicycle showed that a peptide drug conjugate with a low DAR (DAR1) had superior efficacy compared to Padcev (DAR4), a commercial ADC with a high DAR, demonstrating the importance of efficiently distributing the carrier into cancer tissues. Nevertheless, until recently, antibodies used in ADC development were mostly K d The value is 1*10 -10 M or more 1*10 -8 These drugs use antibodies that bind strongly to drug targets of less than M, which means that the ADC and the payload released from the ADC mainly accumulate around the blood vessels inside the cancer, limiting their efficacy.

[0019] From this, the present inventors have concluded that (1) for smooth penetration of antibodies into cancer tissues, d The value is 1*10 -8 M or more 1*10 -6(2) They derived an ADC that uses a camptothecin compound as a payload to ensure sufficient ADC dosage, thereby simultaneously ensuring sufficient ADC dosage and strong efficacy through penetration into cancer tissues.

[0020] Furthermore, due to the characteristic of antibodies that undergo receptor-mediated internalization, the stronger the binding strength to the antigen epitope, the greater the internalization that can occur. Therefore, by utilizing the binding strength to the antigen to increase the therapeutic effect, the present invention aims to design an ADC that solves the problem of on-target toxicity and exhibits anti-cancer effects by using an antibody that targets a cell surface protein expressed in both normal and cancer cells and has a higher binding affinity when binding to the cell surface protein epitope in a bivalent manner than in a monovalent manner. [Means for solving the problem]

[0021] In the first aspect of the present invention, a camptothecin-based drug (A) that degrades DDX5 protein has a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 an antibody or a fragment thereof containing an antigen-binding site (B-1) of less than M; or an antibody or a fragment thereof containing an antigen-binding site (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when bivalently binding to an epitope of the cell surface protein than when monovalently binding to the epitope of the cell surface protein, bound via a linker (C); or a pharmaceutically acceptable salt thereof.

[0022] A second aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the immunoconjugate of the first aspect or a pharmaceutically acceptable salt thereof as an active ingredient.

[0023] The present invention will be described below. As used herein, cancer and tumor may be used interchangeably.

[0024] The therapeutic index of a drug is a measure of the safety and efficacy of a drug in medical treatment. It is defined as the ratio between the dose that produces a therapeutic effect and the dose that produces toxicity or adverse effects. In other words, it indicates the range between the therapeutic and toxic doses of a drug.

[0025] A high therapeutic index indicates a wide margin of safety, where the effective dose is significantly lower than the toxic dose. This means that a drug can be administered at therapeutic levels without inducing serious side effects or toxicity. Drugs with a high therapeutic index are generally considered safer and more desirable for clinical use.

[0026] On the other hand, a low therapeutic index means a narrow margin of safety. In such cases, the effective dose and toxic dose are relatively close, increasing the risk of side effects and toxicity when using the drug. Drugs with a low therapeutic index require careful monitoring and accurate dosing to avoid harming the patient.

[0027] The therapeutic index is an important consideration in drug development because it helps determine the dose range that will provide the desired therapeutic effect while minimizing the risk of side effects. It provides useful information when prescribing drugs and allows one to assess the drug's overall benefit-to-risk ratio.

[0028] For a drug to work effectively in the body, its concentration must be maintained within the therapeutic range for a certain period of time. If too much drug is present in the body, it will be toxic, and if too little, it will not have any therapeutic effect.

[0029] Drug efficacy means that the drug remains in the body without being broken down for the expected time it will be effective for the target indication. The slower the metabolic rate, the longer the drug will remain in the bloodstream, and therefore the longer the duration of efficacy.

[0030] As used herein, the term "antibody" includes immunoglobulin molecules immunologically reactive with a specific antigen, including, for example, protein molecules that act as ligands specifically recognizing cell membrane receptors that are antigens, and includes polyclonal antibodies, monoclonal antibodies, and whole antibodies. The term also includes chimeric antibodies, 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 retain FcRn-binding function. A whole antibody has a structure consisting of two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. The whole antibodies include IgA, IgD, IgE, IgM, and IgG, and IgG is divided into subtypes: IgG1, IgG2, IgG3, and Ig4. Includes G4.

[0031] As used herein, the term "antigen-binding site-containing fragment" refers to any fragment of an antibody that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single-chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv, or scFv. Fd refers to the heavy chain portion contained in the Fab fragment. Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1 domain), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are formed by disulfide bond formation between cysteine ​​residues in the hinge region of Fab'. Fv (variable fragment) refers to the minimum antibody fragment containing only the heavy chain variable region and the light chain variable region. In double-disulfide Fvs (dsFvs), the heavy-chain variable region and the light-chain variable region are linked by a disulfide bond, while in single-chain Fvs (scFvs), the heavy-chain variable region and the light-chain variable region are generally covalently linked via a peptide linker. Such antibody fragments can be obtained using protease hydrolases (for example, Fab fragments can be obtained by limited digestion of a whole antibody with papain, or F(ab') fragments can be obtained by digestion with pepsin), or preferably, can be produced using genetic recombination techniques.

[0032] In the present invention, the term "drug linker conjugate" refers to a substance for producing an immune conjugate or a carrier-drug conjugate, and does not only mean a substance to which an antibody, an antigen-binding site-containing fragment thereof, or a carrier is not linked, but also can be used as an immune conjugate or a carrier-drug conjugate by binding to any antibody, an antigen-binding site-containing fragment thereof, or a carrier depending on the purpose.

[0033] In the present invention, the term "immunoconjugate" refers to a conjugate in which a cytotoxic drug linker conjugate is linked to an antibody or an antigen-binding fragment thereof.

[0034] An antibody-drug conjugate (ADC) is an example of an immunoconjugate, and therefore, in the present invention, the description of an ADC and the description of an immunoconjugate may be used interchangeably.

[0035] When the immune complex is administered in vivo, one of its components, an antibody or a fragment thereof containing the antigen-binding site, binds to a target antigen and then releases a drug, allowing the drug to act on the target cell and / or surrounding cells. This makes it possible to expect excellent efficacy and reduced side effects as a targeted drug.

[0036] Factors that have a significant impact on the efficacy of immunoconjugates include (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. Because various factors have a complex effect on efficacy, it is extremely difficult to predict the efficacy of an immunoconjugate, which is a combination of these factors, based solely on the known facts about each factor.

[0037] In the present invention, the immune complex may have an average drug-to-antibody ratio (DAR) of 2 to 12, and preferably, the DAR, which indicates the number of drugs attached to one antibody based on the antibody, may be 4 to 8, or the DAR, which indicates the number of drugs attached to one antibody based on the antigen-binding site-containing fragment, may be 1 to 4.

[0038] If you know the characteristics of a drug, you can use it correctly. Pharmacodynamic and pharmacokinetic parameters are useful.

[0039] Pharmacodynamics describes the magnitude and nature of the changes (drug efficacy, effects) (cell viability, clinical effects) (therapeutic effects, toxic effects, side effects) that occur in cells and the body after a drug binds to a receptor, in relation to drug concentration.

[0040] Pharmacokinetics (PK) describes how a drug or drug modality changes its concentration as it moves through ADME and into other compartments of the body.

[0041] The in vivo efficacy and side effects of anticancer drugs are closely related to their absorption, distribution, metabolism, and excretion (ADME) properties. A drug's ADME determines its pharmacokinetics, which describes how it moves through the body and interacts with tissues and organs.

[0042] The immunoconjugate or a pharmaceutically acceptable salt thereof according to the present invention comprises: Camptothecin-based drugs (A), which degrade DDX5 protein, bind to epitopes on cell surface proteins with a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 or an antibody or a fragment thereof containing its antigen-binding site (B-2) that binds to a cell surface protein expressed in both normal and cancer cells and has a higher binding affinity when binding to an epitope of the cell surface protein in a bivalent manner than when binding monovalently, and that is linked via a linker (C).

[0043] In the present invention, the camptothecin-based drug that degrades DDX5 protein may be an activated camptothecin derivative represented by the following Chemical Formula 1, Chemical Formula 2, or Chemical Formula 1-1, which is designed to bind to DDX5 protein and E3 ligase.

[0044] [ka]

[0045] [ka]

[0046] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different; X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond; X1, (X2)n and X3 can form a 5-, 6- or 7-membered ring (n = a value from 0 to 2); Y1, Y2 and Y3 can each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0047] [ka]

[0048] Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur include -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR 1 )(OR 2 )(R 1 , R 2 =C s H t N u O w S x P y X z , X=-F, -Cl, -Br or -I, 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), -SO3H, -OSO3H, -NO2, -N3, -NR3OH(R=C n H 2n+1 , 0≦n≦16), -NR3 + X - (R=C n H m , 0≦n≦16, 0≦m≦34, X=OH, Cl or Br), NR4 + X - (R=C n H m , 0≦n≦16, 0≦m≦34, X=OH, Cl or Br), -COSH, -COOCO-, -CORCO-(R=Cl H m , 0≦l≦3, 0≦m≦2l+1), -COOR, -CN, -N3, - N2, -NROH (R=C s H t N u O w S x P y X z , X=-F, -Cl, -Br or -I, 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), -NR 1 NR 2 R 3 (R 1 , R 2 , R 3 =C s H t N u O w S x P y X z , X=-F, -Cl, -Br or -I, 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), -CONHNR 1 R 2 (R 1 , R 2 =C s H t N u O w S x P y X z , X=-F, -Cl, -Br or -I, 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), -NR 1 R 2 R 3 X ′ (R 1 , R 2 , R 3 =C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, X ′ =F- , Cl - , Br - , or I - , 0≦s≦20, 0≦t≦2(s+u)+1, 0≦u≦2s, 0≦w≦2s, 0≦x≦2s, 0≦y≦2s, 0≦z≦2s), —OH, —O—, >C═O, —SS—, —SO—, —NO2, —COX (X═F, Cl, Br or I), —COOCO—, —CONH—, —CN, —SCOCH3, —SCN, —NCS, —NCO, —OCN, —CN, —F, —Cl, —I, —Br, an epoxy group, a hydrazone, —ONO2, —PO(OH)2, —C═NNH2, —HC═CH—, —C═C—, —C≡C—, and a hydrocarbon having two or more carbon atoms.

[0049] Non-limiting examples of active camptothecin derivatives represented by Chemical Formula 1 are as follows:

[0050] [ka]

[0051] [ka]

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] [ka]

[0057] Considering the design intent of ADCs—to deliver cytotoxic molecules to targets by linking them to antibodies targeting tumor-specific antigens—ADCs are expected to be less toxic than existing chemotherapeutics. However, most ADCs still suffer from on-target toxicities and other less well-known, potentially life-threatening side effects, as well as off-target toxicities similar to those of cytotoxic payloads. As the clinical indications for ADCs rapidly expand, including their use in various therapeutic settings and combinations, extensive efforts are underway to improve safety. Current approaches include clinical trials optimizing doses and treatment schedules, modifying individual ADC components, identifying predictive biomarkers for toxicity, and developing innovative diagnostic tools.

[0058] Taking these points into consideration, the dosage of the immunoconjugate of the present invention or a pharmaceutically acceptable salt thereof, which is an activated camptothecin derivative designed to bind to DDX5 protein and E3 ligase according to the present invention, can be 4.8 mpk or less, preferably 2 mpk to 4.5 mpk.

[0059] Furthermore, the immunoconjugate or a pharmaceutically acceptable salt thereof of the present invention may have a DAR, which indicates the number of drugs attached per antibody based on the antibody, of 4 to 8, or a DAR, which indicates the number of drugs attached per antibody based on the antigen-binding site-containing fragment, of 1 to 4.

[0060] When the antibody constituting the ADC binds to the drug target on the surface of cancer cells with strong affinity, it first saturates the drug target around the blood vessels, and after a certain amount of antibody binds, additional antibody (ADC) does not penetrate into the cancer tissue, resulting in wasted ADC. In order to solve this problem and to ensure that the payload released from the ADC is uniformly distributed throughout the cancer tissue, the immunoconjugate of the present invention has a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 The antibody or its antigen-binding site-containing fragment (B-1) is characterized by the use of an antibody or its antigen-binding site-containing fragment (B-2) that binds to a cell surface protein expressed in both normal cells and cancer cells and has a higher binding affinity when binding to an epitope of the cell surface protein in a bivalent manner than when binding monovalently (Figure 22).

[0061] The aforementioned antibody or its antigen-binding site-containing fragment (B-2) enables the immune complex of the present invention and / or the payload released from the immune complex to penetrate into the interior of the cancer tissue without accumulating only around the blood vessels inside the cancer.

[0062] Furthermore, due to the presence of the aforementioned antibody or its antigen-binding site-containing fragment (B-2), the immunoconjugate of the present invention not only (i) weakens internalization via receptor-mediated endocytosis, thereby increasing its therapeutic effect, but also (ii) resolves the problem of on-target toxicity of the immunoconjugate and exerts an anti-cancer effect, and (iii) the therapeutic effect of the immunoconjugate that has penetrated into the cancer tissue alleviates the solid pressure within the cancer microenvironment formed as cancer cells proliferate, and optionally thereby increases the accessibility of anti-cancer drugs and / or immunotherapeutic agents.

[0063] In this case, the anticancer drug may contain the immunoconjugate of the present invention and / or a payload excreted from the immunoconjugate, and therefore, the payload excreted from the immunoconjugate of the present invention can effectively exert a bystander effect.

[0064] Furthermore, in the immune complex of the present invention, the camptothecin-based drug (A), which decomposes DDX5 protein, is converted from its active lactone form, which acts as a TOP1 inhibitor, to its inactive carboxylate form at a pH of 7.4, which is the pH of blood or extracellular fluid (Figures 24 and 25), and the antibody or its antigen-binding site-containing fragment can selectively recognize cancer cells that are to be killed by the camptothecin-based drug.

[0065] Therefore, the present invention not only exhibits potent anticancer efficacy without systemic side effects by selectively delivering a drug with potent anticancer efficacy to cancer cells via an antibody or its antigen-binding site-containing fragment, but also solves the problem of efficacy being limited by the amount of payload drug delivered by using a camptothecin-based drug (A) that degrades DDX5 protein as the payload.

[0066] [Antibody-drug conjugates (ADCs) and their side effects] Antibody-drug conjugates (ADCs) can be used as a type of targeted therapy in cancer treatment. For example, a monoclonal antibody is conjugated to a cytotoxic drug to deliver the drug specifically to cancer cells. ADCs are antibodies that can selectively recognize cancer cells by binding to specific antigens expressed on the surface of cancer cells, and a linker is used that maintains the bound state without releasing the drug until the ADC is internalized inside the cell, and then releases the drug immediately after internalization, allowing them to potently attack cancer cells even at low concentrations of a few pM to a few nM. This is a new anti-cancer therapeutic modality that combines anti-cancer drugs that exhibit anti-cancer efficacy.

[0067] After binding to the target cell, ADCs are internalized into the cell through a process called receptor-mediated endocytosis. For this to occur, a sufficient concentration of the active drug must enter the cell. However, the internalization process via antigen-antibody complexes is generally inefficient, and the number of antigens present on the cell surface is generally <1 × 10. 5 Because ADCs are limited by receptors / cells, they require highly potent drugs to kill tumor cells even at low concentrations. Furthermore, because the drug must bind to the antibody while minimizing its effect, the amount of payload that can be delivered is limited. Therefore, the drugs used in ADCs that are bound to antibodies are 100 to 1,000 times more cytotoxic than commonly used anticancer drugs.

[0068] Most of the potent cytotoxic drugs incorporated into ADCs were highly toxic and affected normal cells through bystander effects. This indicates that cytotoxic drugs for application in ADCs must be able to kill most tumor cells at low concentrations (nM or pM) and have a regulated drug release to exert therapeutic effects.

[0069] ADCs combine the selectivity of antibodies with the potent cytotoxicity of anticancer drugs, offering new possibilities for demonstrating potent anticancer therapeutic efficacy while reducing the risk of systemic side effects in many cancer patients. However, many existing ADCs have various limitations in their practical application. When the DAR exceeds a certain number, the ADC is absorbed indiscriminately into normal cells / tissues other than cancer cells (non-selective absorption) due to the hydrophobicity of the drug and linker used. There were problems with this drug releasing, resulting in poor PK profiles and unexpected toxicity.

[0070] Daiichi-Sankyo's Enhertz is a high-DAR ADC that uses a combination of a relatively nontoxic payload and a hydrophilic linker. To conjugate the drug and linker to the antibody, all interchain disulfide bonds in the IgG1 format are cleaved, resulting in eight free thiol functional groups, which are then reacted with the drug-linker combination to create a pseudo-homogeneous ADC with a DAR of ~8. The linker and payload used here are more hydrophilic than previously used linkers and payloads, and despite the high DAR, they have experienced little aggregation issues and maintained a stable PK profile. Nevertheless, non-selective absorption remains an issue, resulting in severe inflammatory side effects in 10-15% of patients.

[0071] The term "non-selective uptake" generally refers to the uptake of any substance by cells without a specific target or preference. In the context of ADCs, non-selective uptake refers to the uptake of the ADC or its payload by cells that do not express the target antigen.

[0072] It is estimated that only approximately 0.1% of an ADC injected dose is delivered to the targeted diseased cell population, and most of the administered dose is metabolized "off-site" within healthy cells, potentially inducing undesirable toxicity. Off-site ADC toxicity can be classified as "on-target" or "off-target." On-target toxicity occurs via ADC binding to target cell surface proteins in healthy cells. Each component of an ADC, including the antibody, linker, and payload, can affect the degree of ADC-induced toxicity.

[0073] The mechanism of ADC toxicity is illustrated in Figure 26. Uptake of intact ADCs into normal cells can occur via nonspecific intracellular uptake or internalization upon binding to target antigens or Fc / C-type lectin receptors. Payloads released by ADC deconjugation or other target / non-target cell killing in the extracellular fluid can also enter normal cells via passive diffusion in the case of membrane-permeable payloads.

[0074] Conceptually, ADCs can enhance the selectivity of chemotherapy by facilitating targeted delivery of cytotoxic payload molecules to desired cell populations (on-target, on-site toxicity), while simultaneously reducing payload delivery to non-target healthy tissues, broadening the therapeutic index.

[0075] In the early stages of technology development, a predicted safety issue for anticancer ADCs was target (i.e., target-mediated) toxicity in tissues where the target antigen was expressed to some extent, and differential expression of the target between cancer cells and healthy tissues was predicted to be an important factor determining the therapeutic index of the ADC. However, subsequent clinical experience with ADCs has demonstrated that dose-limiting toxicities (DLTs) caused by target expression in healthy tissues are rare.

[0076] Preclinical and clinical data from 20 ADC Investigational New Drug (IND) applications submitted between 2012 and 2013 show that ADCs with the same type of linker / payload generally share very similar toxicity profiles, DLTs, and maximum tolerated doses (MTDs), regardless of the target antigen and the degree of antigen expression in healthy tissues.

[0077] Lipophilic payloads have high plasma membrane permeability, allowing the released payload to efficiently enter non-target cells (e.g., via membrane diffusion) and potentially induce undesirable cytotoxicity.

[0078] Therefore, in order to control the bystander effect and / or ADME profile of the activated camptothecin derivative so that it is removed quickly or slowly via lymphatic drainage by providing a desired degree of hydrophobicity and / or controlling aggregation, the camptothecin drug that degrades DDX5 protein in the immunoconjugate of the present invention can be selected from activated camptothecin derivatives represented by the following Chemical Formula 1, Chemical Formula 2, or Chemical Formula 1-1, which are designed to bind to DDX5 protein and E3 ligase. Anticancer drug formulations containing such immunoconjugates of the present invention, as well as their doses and dosages, can be designed in a variety of ways.

[0079] Reducing the DAR is a well-established method for reducing non-selective uptake of ADCs. However, for immunoconjugates of the present invention that use drugs with relatively low cytotoxicity, such as camptothecin-based payloads, it is also important to maintain a high DAR of greater than 4 to ensure sufficient efficacy.

[0080] The IC was 0.01 in MDA-MB-468, a Her2-negative cell line that corresponds to "non-selective uptake" of ADCs. 50 The results are as follows: for trastuzumab-25-6 (DAR6) (IC 50 =97.61 nM) for trastuzumab-25-6 (DAR4) (IC 50 = 240.7 nM), which was 2.5 times lower.

[0081] This is due to the fact that DAR modulation of camptothecin-based payloads enhances their therapeutic index in terms of off-target toxicity (by reducing payload delivery to non-target healthy tissues). In other words, even if a higher dose of ADC is administered when DAR=4 than when DAR=6, the by-stander cell-killing phenomenon, which is a type of "non-selective uptake" of the payload, can be kept low.

[0082] [Clinical Results and Side Effects of ADCs with Camptothecin Payloads] Trastuzumab deruxtecan (Enhertz) is a humanized anti-HER2 antibody linked via a stable linker to the camptothecin derivative deruxtecan. In the first phase 1 dose-escalation study, 22 patients with HER-2-positive advanced or metastatic breast cancer, gastric cancer, or other HER-2-expressing solid tumors were treated with trastuzumab deruxtecan administered every 3 weeks. Patients were treated with luxtecan 0.8 mg / kg to 8.0 mg / kg. No dose-limiting toxicities were observed, and the MTD was not reached. Target drug exposure was achieved at a dose of 6.4 mg / kg, which was selected as the recommended biphasic dose.

[0083] The pivotal, single-arm, phase 2 DESTINY-Breast01 clinical trial consisted of two parts. In the first part, patients with advanced / metastatic breast cancer who had previously been treated with two or more anti-HER2 therapies were randomly assigned to receive trastuzumab delta-3000 or trastuzumab delta-3000. In the second part, 134 patients were treated with trastuzumab deruxtecan at a dose of 5.4 mg / kg (n=50), 6.4 mg / kg (n=48), or 7.4 mg / kg (n=21) once every 3 weeks. Based on the efficacy and toxicity data from Part 1, 134 patients were treated with trastuzumab deruxtecan at a dose of 5.4 mg / kg. Among 184 patients, the most frequent adverse reactions of any grade (≥20%) were nausea (77.5%), fatigue (49.8%), alopecia (49.8%), vomiting (44.3%), neutropenia (40.3%), constipation (37.5%), anemia (33.6%), decreased appetite (33.2%), diarrhea (29.2%), leukopenia (26.9%), and thrombocytopenia (24.9%). Grade 3 or higher adverse reactions occurred in 57.1% of patients, with neutropenia (20.7%), anemia (8.7%), nausea (7.6%), leukopenia (6.5%), lymphopenia (6.5%), and fatigue (6.0%) being the most frequent. Dose interruptions, dose reductions, and treatment interruptions due to adverse reactions occurred in 35.3%, 23.4%, and 15.2% of patients, respectively, with pneumonia (11 patients) and interstitial lung disease (5 patients) being the most frequent reasons. These included interleukin-11 (ILD) and pneumonia. Treatment-related interstitial lung disease and fatal outcomes occurred in 9% and 2.6% of patients, respectively. As with other HER-2-targeted ADCs, patients treated with trastuzumab deruxtecan-1 had Patients also had an increased risk of embryo-fetal toxicity and left ventricular dysfunction.

[0084] Sacituzumab govitecan (Trodelv) is linked via a pH-sensitive linker. It is a humanized anti-TOP-2 IgG linked to the active metabolite of rinotecan (SN-38). In a first-in-human, dose-escalation phase 1 / 2 study, 25 patients with a variety of metastatic solid tumors received sacituzumab govitecan at 8 mg / kg on days 1 and 8 of a 21-day cycle. The MTD for the first cycle was determined to be 12 mg / kg, with neutropenia being the dose-limiting toxicity. However, this dose level was highly toxic in subsequent cycles, and doses of 8 mg / kg and 10 mg / kg were selected for the phase 2 clinical trial. In this phase 2 clinical trial, sacituzumab govitecan was administered at 8 mg / kg (n=81) or 10 mg / kg in patients with a variety of metastatic epithelial cancers who had received various prior therapies. g (n=97). The most frequent adverse reactions of any grade (≥25%) reported in the 8 mg / kg and 10 mg / kg cohorts were nausea (59% vs. 63%), diarrhea (53% vs. 62%), neutropenia (42% vs. 58%), fatigue (61% vs. 52%), vomiting (36% vs. 43%), anemia (38% vs. 42%), alopecia (46% vs. 37%), and constipation (33% vs. 43%). The most frequent grade 3 or higher (≥10%) adverse reactions reported in the 8 mg / kg and 10 mg / kg cohorts were neutropenia (30% vs. 36%), anemia (13% vs. 12%), diarrhea (4% vs. 10%), and leukopenia (6% vs. 12%). Dose reductions occurred in 19% and 28% of patients in the 8 mg / kg and 10 mg / kg cohorts, respectively. Neutropenia was the most frequent adverse reaction leading to dose delay or reduction. Significantly more patients in the 10 mg / kg cohort experienced grade 3 or higher neutropenia after the first dose than in the 8 mg / kg cohort (47% vs. 21%).

[0085] Black box warnings for severe or life-threatening neutropenia and severe diarrhea were added to the sacituzumab govitecan label. These side effects were reported in the This is likely due to its SN-38-mediated toxicity and is associated with the same toxicities as the SN-38 precursor drug, irinotecan.

[0086] All grades and grade 3 or higher among all patients treated with sacituzumab govitecan Febrile neutropenia occurred in 61% and 47%, respectively. Febrile neutropenia occurred in 7% of patients. It occurred in 65% and 12% of patients overall treated with sacituzumab govitecan. Diarrhea of ​​any grade and grade ≥ 3 occurred in all patients. Neutropenic colitis occurred in 0.5% of patients.

[0087] [Camptothecin-based drugs (A) that degrade DDX5 protein and their anticancer mechanisms] Targeted therapies differ in their therapeutic mechanism from previously used cytotoxic anticancer drugs. Cytotoxic anticancer drugs act on the DNA and microtubules of rapidly dividing cells, so toxicity to normal cells is unavoidable. However, targeted therapies use molecular targets to selectively attack only cancer cells. Molecular targets of targeted therapies include angiogenesis, cell death, cell signaling pathways, and cell cycle regulators, and targeted therapies targeting a variety of targets have been developed. The therapeutic effect of targeted therapies usually involves suppressing cell proliferation, so they are generally used alone for long periods of time or in combination with other cytotoxic anticancer drugs.

[0088] While antibodies are considered to be the key component in determining the efficacy of ADCs, it is cytotoxic drugs that carry out tumor cell killing. Cytotoxic drugs are small molecule drugs that induce tumor cell killing. In ADCs, cytotoxic drugs, which are the payload, play a key role in killing cancer cells and are a very important component in demonstrating drug efficacy.

[0089] While more potent cytotoxic drugs have been incorporated into ADCs, most of them are highly toxic and affect normal cells through bystander effects. Furthermore, the amount of payload that can be delivered is limited because the drug must be bound to the antibody while minimizing its impact. This means that cytotoxic drugs for use in ADCs must be able to kill most tumor cells at low concentrations (nM or pM) and demonstrate therapeutic efficacy through regulated drug release. Therefore, the cytotoxic drugs that can be used as payloads are highly diverse, but above all, they must have low immunogenicity and be stable during circulation. This is because unstable cytotoxin payloads can lead to drug deformation during conjugation or storage.

[0090] Anticancer drugs used as payloads in immunoconjugates such as ADCs can inhibit tumor-promoting oncoproteins associated with promoting cell growth, cell survival, and cell motility, or restore, reactivate, or stabilize tumor suppressor proteins (e.g., p53) associated with suppressing cell growth, cell death, and cell motility.

[0091] Cell death plays a vital role in maintaining tissue homeostasis and morphology, regulating cell number, eliminating damaged or abnormal cells, and defending against infection. Living organisms maintain normal life by precisely regulating cell growth, differentiation, and death. Cell death can be classified as either necrosis or programmed cell death (apoptosis). Apoptosis is a complex process that induces cell death through the expression of various genes and the activation of their protein products. Apoptosis is a physiological, active suicide mechanism within cells that plays an essential role in maintaining the development, differentiation, and homeostasis of multicellular organisms. Apoptosis occurs in various cell types due to various factors. Unlike necrosis, apoptosis is characterized by the absence of lysosomal enzyme release, the formation of apoptotic bodies, cell shrinkage, nuclear condensation, and the formation of characteristic ladder-like DNA fragments.

[0092] As shown in Figures 7 and 8, which show the degree of inhibition of anti-apoptotic proteins, camptothecin-based drugs that bind to and degrade the tumor protein DDX5 can control apoptosis-related proteins selected from the group consisting of c-Myc, survivin, cIAP2, Bcl-2, XIAP, and mutant Kras as molecular targets.

[0093] Therefore, camptothecin-based drugs that bind to and degrade the tumor protein DDX5 are suitable payloads for ADCs and can kill most tumor cells at low concentrations (nM or pM).

[0094] In consideration of this, the payload in the immunoconjugate of the present invention is characterized by using a camptothecin-based drug that binds to DDX5 protein and E3 ligase to degrade DDX5 protein, and can be selected from activated camptothecin derivatives represented by Chemical Formula 1, Chemical Formula 2, or Chemical Formula 1-1 that are designed to bind to DDX5 protein and E3 ligase.

[0095] The camptothecin-based drug (A), which degrades the DDX5 protein, the payload of the immunoconjugate of the present invention, is a hydrophobic small molecule that can permeate cell membranes, and therefore can penetrate deep into cancer tissues and accumulate at high concentrations.It penetrates the cell membrane and exerts cytotoxicity inside the cell, killing the cell, and then is released, and can continuously penetrate the cell membranes of surrounding cells to move into the cell and act there.

[0096] The camptothecin-based drug (A) that degrades the DDX5 protein, which is the payload of the immunoconjugate of the present invention, can be selected from activated camptothecin derivatives represented by Chemical Formula 1, Chemical Formula 2, or Chemical Formula 1-1 below, which are designed to bind to DDX5 protein and E3 ligase in order to control the bystander effect and / or ADME profile of the activated camptothecin derivative so that it is removed quickly or slowly via lymphatic drainage by providing a desired degree of hydrophobicity and / or controlling aggregation. Anticancer drug formulations containing such immunoconjugates of the present invention, as well as their doses and dosages, can be designed in a variety of ways.

[0097] Anti-apoptotic proteins that are the main cause of anti-cancer drug resistance include survivin, cIAP2, and XIAP (Figure 6).

[0098] Unlike SN-38, B, such as survivin, which is a resistance protein involved in the resistance mechanism, Non-limiting examples of camptothecin-based cytotoxic drugs that inhibit the cl family and bind to and degrade the tumor protein DDX5 (p68), which regulates c-Myc, survivin, and mutant Kras, include FL118 (chemical formula 1-1), and activated camptothecin derivatives represented by chemical formula 1 or chemical formula 2 (exatecan, Dxd) (Figure 7).

[0099] Targeting the apoptosis pathway to induce cell death is also an effective cancer treatment. Furthermore, camptothecin-based cytotoxic drugs, which inhibit type I topoisomerase, are DNA-altering agents and can kill cells regardless of the cell cycle. Therefore, camptothecin-based cytotoxic drugs that degrade DDX5 protein are designed to bind to DDX5 protein and E3 ligase. In addition to their ability to inhibit type I topoisomerase, they can kill cells through their MoA, which degrades the oncoprotein DDX5, and they also have excellent cell killing effects regardless of the cell cycle.

[0100] FL118 (chemical formula 1-1) can act as a molecular glue degrader that directly binds DDX5 to ubiquitination regulators and degrades DDX5.

[0101] [ka]

[0102] FL118, acting as a molecular glue, directly binds to the oncoprotein DDX5, a multifunctional master regulator, and dephosphorylates and degrades it via the proteasomal degradation pathway without reducing DDX5 mRNA. Silencing of DDX5 indicates that DDX5 is a master regulator that regulates the expression of various oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras.

[0103] In addition, it is possible to avoid resistance by essentially blocking the overexpression of anti-apoptotic proteins, which is a common mechanism of resistance development in most cancers; biomarkers (DDX5, K-ras, p53) that can predict anti-cancer responses in patients and companion diagnostic techniques have already been established, and securing custom-made biomarkers makes it possible to provide personalized treatment through companion diagnostics, and it shows particularly strong efficacy in p53 / K-ras mutant cancer cells, which have a poor prognosis.

[0104] FL118 indirectly regulates DDX5 downstream targets and has high potency in preventing cancer initiation, development, metastasis, recurrence, and treatment resistance, as demonstrated in studies using human colorectal cancer / pancreatic ductal adenocarcinoma cells and tumor models. suppression, metastasis, recurrence and treatment resistance.

[0105] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. KO PDAC cells are resistant to FL118 treatment. Studies in human tumor animal models showed that FL118 was highly effective in eliminating human PDAC and CRC tumors with high DDX5 expression, whereas FL118 was less effective in PDAC and CRC tumors with low DDX5 expression.

[0106] DDX5 protein is a direct target of the FL118 drug and can serve as a biomarker for predicting PDAC and CRC tumor sensitivity to FL118.

[0107] Furthermore, the FL118 drug has a similar or higher level of Top1 inhibitory efficacy to SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low IC 50It showed cytotoxicity with IC values ​​of <100 nM against the majority of cancer cells, as assessed against 140 cell lines derived from various cancer types. 50 FL118 demonstrated extremely potent anti-cancer efficacy, demonstrating excellent safety profiles through GLP toxicity studies in mice and beagle dogs, and demonstrated superior efficacy compared to SN-38 in various cancer cell line xenograft models. Camptothecin-based anti-cancer drugs initially demonstrate excellent anti-cancer responses when administered to patients, but strong resistance to these drugs develops due to epigenetic silencing of the Top1 gene and cancer cell dependency on Top2. Meanwhile, FL118 also demonstrated potent efficacy in cancer cell line xenograft models in which Top1 is not expressed due to epigenetic silencing or knockout.

[0108] In addition, the FL118 drug is a triple-targeted anticancer drug that directly targets type I topoisomerase, a well-established anticancer target, while simultaneously inhibiting Bcl family members such as survivin, a resistance protein involved in the resistance mechanism, and suppressing the action of efflux pumps.

[0109] Specifically, while camptothecin-based anticancer drugs such as SN-38 exhibit resistance due to the overexpression of the ABCG2 transporter, which causes the drug to be effluxed from the cell, the FL118 drug is not affected by the ABCG2 transporter, making it possible to overcome this resistance. FL118 drug can block the development of resistance by strongly suppressing the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.), which are the main cause of resistance to other anticancer drugs, at low concentrations.

[0110] Therefore, FL118 is not exported outside the cell by the efflux pump ABCG2 and can block resistance mediated by various anti-apoptotic proteins, thereby overcoming various resistance mechanisms of SN-38 / exatecan.

[0111] When administered in vivo at the same dose as SN-38, the drug FL118 showed stronger tumor regression efficacy than SN-38 in colon cancer, head and neck cancer, pancreatic cancer, etc.

[0112] The FL118 drug retained potent anticancer efficacy even when administered after induction of SN-38 resistance in tumor xenografts.

[0113] Furthermore, FL118 drugs are ideal for targeted drug delivery applications (e.g., drug-carrier complexes). It has a favorable PK / safety profile. When administered alone, FL118 is rapidly metabolized and excreted from the blood, resulting in low concentrations. However, it accumulates rapidly in cancer tissues immediately after administration and maintains high concentrations for a long time. For example, when used as an ADC, it ensures maximum selectivity between tumor and normal tissues.

[0114] Exatecan is a camptothecin derivative and an antitumor small molecule compound that inhibits type I topoisomerase. Exatecan has been shown to have cellular cytotoxicity that is 5 to 10 times more potent than SN-38.

[0115] Unlike irinotecan, exatecan does not require enzymatic activation. Furthermore, it has stronger type I topoisomerase inhibitory activity than SN-38, the active ingredient of irinotecan, and topotecan, the compound used in clinical trials. It has also demonstrated stronger cytotoxic activity against various cancer cells in vitro. It was particularly effective against cancer cells resistant to SN-38 and other drugs due to the expression of P-glycoprotein. Furthermore, exatecan demonstrated strong antitumor activity in a mouse model of human tumors subcutaneously transplanted into the body, leading to clinical trials.

[0116] Dxd (exatecan derivative for ADC) is an IC used in the conjugate drug of HER2-targeting ADC (DS-8201a). 50 It is a potent DNA topoisomerase I inhibitor with a potency of 0.31 μM.

[0117] Surprisingly, while designing camptothecin derivatives with a structure that exhibits a dual mechanism of action (MoA) based on the structure of the FL118 drug, which is differentiated from the SN38 drug (see below), and that takes advantage of the FL118 drug's advantages of type I topoisomerase inhibition and DDX5 degradation (Figure 1), the inventors discovered that exatecan or Dxd also exhibits a mechanism of action (MoA) that degrades the DDX5 protein (Figures 7 to 10).

[0118] Based on this, another feature of the present invention is that camptothecin-based drugs of Chemical Formula 1 or Chemical Formula 2 are designed in various ways to exhibit various advantages as anticancer drugs, as exemplified by the FL118 drug, and are used as the payload of the immunoconjugate of the present invention.

[0119] [ka]

[0120] [ka]

[0121] X1 and X3 are each independently carbon, oxygen, nitrogen, or sulfur, and X1 and X3 may be the same or different; X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond; X1, (X2)n and X3 can form a 5-, 6- or 7-membered ring (n = a value from 0 to 2); Y1, Y2 and Y3 can each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0122] The camptothecin-based drug of Chemical Formula 1 or Chemical Formula 2 used as a payload in the present invention is characterized by exerting an anti-cancer mechanism of degrading the intracellular oncoprotein DDX5 (Figures 7 to 10) by designing R1 and R2 (Group A) on the A ring of General Formula 1 to be identical or structurally very similar to those of the FL118 compound or exatecan / dxd (Figure 1).

[0123] [ka]

[0124] The synthetic design concept for active camptothecin derivatives with a dual mechanism of action (MoA) that degrades the oncoprotein DDX5 as well as type I topoisomerase inhibitory activity is to maintain the structure of group C, which is the type I topoisomerase inhibitory region, based on the structure-activity relationship (SAR), and to maintain the same structure as FL118 or exatecan and dxd for group A, which is the binding site for DDX5 degradation. By adjusting the structure (R3 and R4) of group B in general formula 1 to formula 1 or formula 2, the physicochemical properties of the drug can be improved, so as to solve the aggregation problem of ADCs that use this as a payload. This not only improves the cytotoxicity of the payload (active camptothecin-based drug) released from the ADC, but also regulates the drug's tumor tissue penetration and / or cell membrane permeability, thereby enabling precise control of the bystander effect.

[0125] In general formula 1, the group C site may bind to type I topoisomerase and the group A site may bind to DNA and stabilize the covalent bond of the topoisomerase-DNA complex, preventing / preventing further ligation of the cleaved DNA fragments, or the group A site in general formula 1 may bind to DDX5 and the group C site may bind to E3 ligase and induce DDX5 degradation (see PCT / KR2023 / 005380, incorporated herein in its entirety).

[0126] In the present invention, it is preferable to design a camptothecin-based drug of Chemical Formula 1 or Chemical Formula 2 so that cell membrane permeability can be adjusted as desired through modification of R3 and / or R4 in General Formula 1, thereby exerting an appropriate bystander effect in tumor tissue.

[0127] Therefore, taking into consideration not only various side effects but also issues such as a reduced therapeutic index, the selection range of ADC payloads that exhibit appropriate anti-cancer efficacy can be expanded to a variety of candidate groups including activated camptothecin derivatives of Chemical Formula 1 or Chemical Formula 2, which are designed to bind to DDX5 protein and E3 ligase via a molecular glue degrader, which is another major feature of the present invention.

[0128] Camptothecin-based drugs of Formula 1 or 2, which are designed to bind to DDX5 protein and / or E3 ligase according to the present invention, can kill target cells expressing DDX5 protein via a molecular glue degrader mechanism (MoA).

[0129] The target cells can be cancer cells or senescent cells, including cells that do not perform the characteristic functions of an organ.

[0130] Preferably, the camptothecin-based drug of Formula 1 or 2 designed to bind to DDX5 protein and / or E3 ligase according to the present invention may have multiple mechanisms of action (MoA) that degrade the oncoprotein DDX5, along with type I topoisomerase inhibitory ability.

[0131] The camptothecin derivatives represented by chemical formula 1, e.g., PBX-7011 and PBX-7012, according to the present invention, like camptothecin shown in FIG. 1, have a pentacyclic structure with a lactone in the E-ring, which is essential for cytotoxicity, and are designed to maintain the lactone group and alpha hydroxyl group at carbon 20 of the E-ring, which are important for stabilizing type I topoisomerase-DNA by-products. They also have the structural features of exatecan-based drugs in general formula 1, i.e., (1) the FL118 drug (-OCHO- While maintaining the structural features of R1 and R2 (CH3-C=CF) that are similar in orientation to the (methylenedioxo) pentagonal ring, (2) compared to SN-38, in which aggregation is induced by the π-π stacking of the aromatic rings formed by the A- and B-rings via R3 and R4, various orientations of the consecutive carbon-carbon single bonds of the 6- or 7-membered rings extended from the A- and B-rings can form a dynamic equilibrium, weakening or suppressing the stacking of the aromatic rings formed by the A- and B-rings.

[0132] In addition, the PBX-7011 compound is capable of molecular bond rotation relative to the carbon-carbon single bond in the six-membered ring extended from the A- and B-rings, and the -NH2 with a large degree of freedom is exposed to water (HO) and can take on a (+) charge or hydrogen bond with water, increasing water dispersibility.

[0133] In addition, the PBX-7014 compound is capable of molecular bond rotation relative to the carbon-carbon single bond in the six-membered ring extended from the A- and B-rings, and the functional group CH2(OH)CONH- in the lactic acid form of -NH2, which has a large degree of freedom, is exposed to water (H2O) and can hydrogen bond with water while rotating like a propeller, increasing its water dispersibility.

[0134] In addition, the PBX-7016 compound is a PBX-7014 compound with a metabolically unstable functional group, a methyl group, introduced to reduce the drug's lifespan. Drugs that are extremely metabolically stable and metabolized very slowly are necessary to ensure an appropriate residence time, as accumulation can lead to toxicity and serious side effects.

[0135] The DXd payload used in Enhertz was originally derived from the exatecan compound, which is largely unaffected by ABCG2. However, the glycolic acid (α-hydroxyacetic acid) functional group used to convert exatecan to DXd renders it highly susceptible to ABCG2. However, the glycolic acid functional group plays a crucial role in Enhertz's excellent safety / efficacy profile. Removing this functional group not only poses challenges in ADC manufacturing, but also leads to poor ADC performance in animal models and clinical trials (emergence of safety issues or reduced efficacy). Therefore, there is a significant need for new camptothecin derivatives that can be easily used in ADC manufacturing and are unaffected by ABCG2. The PBX-7024 compound, derived from the PBX-7011 compound, is a new camptothecin compound that is unaffected by ABCG2 and can be easily used in ADC manufacturing.

[0136] To confirm the anticancer efficacy of PBX-7024, we evaluated its efficacy in FaDu, a cancer cell line that does not express ABCG2, and A549, a cancer cell line that overexpresses ABCG2. As shown in Figure 9, in the A549 cancer cell line that overexpresses ABCG2, camptothecin compounds such as DXd showed high IC values. 50It can be seen that PBX-7016 and PBX-7024 still maintain strong efficacy.

[0137] As shown in Figure 9, various camptothecin-based drugs were treated at various concentrations in FaDu, a Her2-low / mid cancer cell line that does not express ABCG2, and A549, a cancer cell line that overexpresses ABCG2. The degree of degradation of intracellular DDX5 protein and the resulting inhibitory activity on the expression of cancer-associated survival genes, survivin, Mcl-1, XIAP, and cIAP2, were confirmed, and cell membrane permeability was indirectly confirmed by comparison.

[0138] [Anti-cancer mechanism as a molecular glue degrader that binds to DDX5] The ubiquitin-proteasome system (UPS) is a key pathway for intracellular protein degradation that regulates a wide range of cellular processes. Ubiquitin is a small protein that is covalently attached to lysine residues on substrate proteins through a series of enzymatic reactions involving ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process, called ubiquitination, is an important mechanism regulating protein degradation, signaling, and trafficking.

[0139] Ubiquitin ligases are responsible for substrate specificity in the ubiquitination pathway. In this regard, the camptothecin-based drug of Formula 1 or 2 according to the present invention is designed to bind to DDX5 protein and E3 ligase.

[0140] The transformation of normal cells into cancerous cells occurs through the deregulation of different metabolic pathways that involve a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cellular metabolism, but their deregulation can accelerate tumor transformation. DDX5 interacts with hundreds of different cellular proteins, and along related specific pathways, both proteins can act as tumor suppressors or oncogenes.

[0141] DDX5 (also known as p68) is a multifunctional master regulator that acts on the following mechanisms: (1) the biological process of co-activating the transcription of many oncogenes through direct interaction with various transcription factors (e.g., c-Myc) at oncogenic gene promoters; (2) the biological process of regulating miRNA and pre-RNA splicing (e.g., U1, U2, U3, ... snRNP); and (3) ribosome biogenesis (e.g., 32S rRNA, pre-ribosome).

[0142] The camptothecin-based drugs of Chemical Formula 1 or 2 according to the present invention bind to DDX5 protein without reducing DDX5 mRNA and functionally degrade it via dephosphorylation and the proteasome degradation pathway. This suggests that the camptothecin-based drugs of Chemical Formula 1 or 2 can bind to both DDX5 and ubiquitin-involved protein stability / degradation regulators, and thus act as "molecular adhesive degraders."

[0143] DDX5 downstream protein targets are all known to be involved in cancer initiation, progression, metastasis, recurrence, and treatment resistance. Therefore, when DDX5 downstream targets are indirectly blocked through degradation of DDX5 protein by the camptothecin-based drug of Chemical Formula 1 or Chemical Formula 2 according to the present invention, the camptothecin-based drug of Chemical Formula 1 or Chemical Formula 2 can exhibit high antitumor efficacy.

[0144] DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and transcription cofactor. Therefore, if the physiological state of DDX5 leads to deregulation of transcription factors and the development of cancer, selective degradation of DDX5 (p68) as a transcription cofactor can be used to treat cancer. Similarly, selective degradation of DDX5 (p68) as a transcription cofactor can be used to prevent cancer.

[0145] The camptothecin drugs of Formula 1 or 2 according to the present invention target the DDX5 protein, thereby avoiding drug resistance, refractory to targeted therapy, and / or resistance during treatment. Furthermore, the camptothecin drugs of Formula 1 or 2 can turn off the transcriptional induction of anti-apoptotic genes. Furthermore, the camptothecin drugs of Formula 1 or 2 can degrade the transcription cofactor DDX5 protein, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy and radiotherapy.

[0146] [Advantages of molecular glue degrader drug modalities] After completing their function, intracellular proteins in the body are naturally degraded within hours to days. Every cell in the body has a cleansing mechanism known as the ubiquitin proteasome system (UPS) that degrades proteins. During this process, ubiquitin acts as a marker, indicating proteins that should be degraded, and the proteasome recognizes the ubiquitin tag and acts as a grinder to destroy the protein. That is, multiple ubiquitin molecules are attached to the side of proteins that have completed their function, and the proteasome selects and breaks down only proteins that have this tag. E3 ligases are enzymes that initiate the body's protein degradation system and are responsible for substrate specificity in the ubiquitination pathway.

[0147] Molecular glue degraders, or molecular glues, are compounds that act as glue to bond target proteins to specific enzymes (E3 ligases) in the human body. One of the advantages of molecular glues is their catalytic role: after degrading a target protein, they can be further separated and degrade other target proteins.

[0148] When E3 ligase enzymes attach to tumor proteins via molecular glue, the tumor proteins are degraded, and other tumor proteins are successively degraded until the target tumor protein is gone, thereby preventing the proliferation of cancer cells. Therefore, molecular glues for tumor proteins overcome the problem of drug resistance, which is a problem with targeted anticancer drugs, and are highly effective even at low doses.

[0149] The camptothecin-based drug of Formula 1 or 2 used as a payload in the present invention is a molecular glue degrader that binds to DDX5 protein and E3 ligase, i.e., activates the degradation of the tumor protein DDX5 or its phosphorylated DDX5 protein (p-DDX5) (Figures 3, 26, and 27).

[0150] Molecular glue degraders can act not only as warheads that bind to target proteins, but also as binders for E3 ligases.

[0151] Therefore, the camptothecin-based drug of Formula 1 or 2 according to the present invention is a molecular adhesive that activates the degradation of the tumor protein DDX5, and therefore can be used as a ligand that targets or binds to the DDX5 protein.

[0152] Molecular glue degraders are kinase inhibitors Unlike the previous methods, they are "proximity-driven" and "event-driven," in that their ability to induce degradation depends on the formation of a transient "target protein-molecular glue-E3 ligase" ternary complex. After degradation occurs, the dissociated molecular glue forms another ternary complex with the target protein, allowing several rounds of degradation to proceed until the target protein is depleted.

[0153] While most drug target proteins evolve resistance to the drug, molecular glues, small molecules that act as adhesives to bind tumor proteins and E3 ligases together, are resistant to resistance and are therefore a suitable modality for cancer treatment.

[0154] Each time a genetic mutation occurs, the shape of the drug target protein changes slightly. While it would be ideal if a single drug could block the activity of all mutations, this is not possible due to selectivity issues. Therefore, in order to block the activity of all mutations with a single drug, the drug target protein is degraded and removed from the beginning.

[0155] Therefore, the camptothecin-based drug of Formula 1 or 2 according to the present invention is a drug that can precisely bind to the DDX5 tumor protein and can avoid the resistance problem of targeted therapeutic agents through a molecular glue approach that quickly and selectively degrades the protein.

[0156] The camptothecin-based drug of Formula 1 or 2 according to the present invention is preferably an irreversible drug whose binding affinity to the target protein, DDX5 oncoprotein, is determined by its physicochemical properties, and which binds so strongly to the target protein that it cannot return to its original state.

[0157] Unlike existing SN38 drugs, the camptothecin-based drugs of Formula 1 or 2 according to the present invention have high affinity with DDX5 and do not easily dissociate. They degrade DDX5 through their molecular glue function, thereby irreversibly inhibiting DDX5-related cancer cell signaling. Furthermore, their physicochemical properties allow them to regulate the cell membrane permeability of the drug as desired, exerting a bystander effect or controlling the degree of this effect, resulting in a high killing effect on surrounding cells. This not only provides advantages in the treatment of heterogeneous tumors, but also inhibits cancer progression for a long period of time, reduces the risk of resistance development, and increases the therapeutic response rate.

[0158] The camptothecin-based drugs of Formula 1 or 2 according to the present invention can bind to DDX5, which acts as an oncoprotein in cells, and induce cell death through DDX5 proteolysis (Figures 9 and 10).

[0159] Inherent drug resistance in cancer treatment can be caused by abnormally expressed transcription factors, which are crucial regulators of cell proliferation and cell death. Therefore, the camptothecin-based drugs of Chemical Formula 1 or Chemical Formula 2 according to the present invention can induce DDX5 proteolysis and subsequent cell death through their mechanism of action as molecular glue degraders that bind to DDX5, a transcription factor cofactor and oncoprotein. In this case, DDX5 proteolysis can downregulate the transcription of anti-apoptotic genes (Figures 26 and 27). Therefore, unlike other targeted therapeutic agents, drug resistance caused by abnormally expressed cell proliferation and / or cell death-related transcription factors is not common or does not occur, and the drug is effective against chemotherapy and / or chemotherapy-associated cancers. It can suppress acquired drug resistance induced by radiation therapy through activation of transcription factors that are not regulated by radiation therapy.

[0160] That is, the anti-cancer mechanism of the camptothecin-based drugs of Chemical Formula 1 or 2 according to the present invention can bypass the molecular mechanism of cancer treatment resistance, thereby eliminating the problem of drug resistance. Since the occurrence of resistance reduces the therapeutic effect, the camptothecin-based drugs of Chemical Formula 1 or 2 according to the present invention are preferred as standard or first-line therapeutic agents after cancer diagnosis.

[0161] In summary, the camptothecin-based drugs of Chemical Formula 1 or Chemical Formula 2 used as payloads in the present invention can be targeted anticancer drugs that act on the DDX5 protein, which plays an important role in the growth, survival, proliferation, metastasis, and / or metabolism of cancer cells.

[0162] [ADME profile of antibody-drug conjugates (ADCs)] The in vivo efficacy and in vivo side effects of anticancer drugs can be influenced by the absorption, distribution, metabolism, and excretion (ADME) properties of the ADC and the payload released from it. The ADME profile of a drug can affect its ability to reach and act on cancer cells. Thus, the ADME properties of the ADC and the payload released from it can significantly affect in vivo efficacy and in vivo side effects.

[0163] The ability of ADCs and the payloads they release to penetrate tumor tissue can also be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0164] Therefore, understanding the ADME properties of ADCs and their released payloads and optimizing drug / drug modality selection and dose / administration can improve the therapeutic index of such ADCs and their released payloads, leading to better outcomes for cancer patients.

[0165] Antibodies (Abs) and antibody-drug conjugates (ADCs) have different lifetimes and ADME profiles due to their different structures and mechanisms of action.

[0166] Antibodies are large proteins naturally produced by the immune system in response to foreign substances (antigens). Their size and complex structure give them a long circulating half-life (weeks to months) and protect them from degradation and clearance. Antibodies are distributed throughout the body, including tissues, and can interact with target antigens with high specificity and affinity. Antibodies are primarily cleared by the reticuloendothelial system (RES), including the liver and spleen, and by catabolism in the kidneys and other organs.

[0167] ADCs consist of an antibody (typically a monoclonal antibody) conjugated to a cytotoxic drug molecule. The antibody component provides specificity and targeting to tumors or diseased tissues, while the drug component (the payload released from the ADC) provides the cytotoxic activity that kills the target cells. ADCs have a shorter half-life than antibodies, typically ranging from several days to a week. This is because ADCs are internalized into target cells, leading to lysosomal degradation of the ADC and release of the drug payload. ADCs are primarily eliminated via the RES, but the drug payload released from the ADC can also undergo metabolism and excretion in the liver and kidney via lymphatic drainage and subsequently via the circulatory system (Example 2).

[0168] As predicted by the pharmacokinetics and biodistribution of monoclonal antibodies, in ADCs, high-affinity mAb binding to cell membrane proteins can localize a significant portion of the mAb to the target cell population, and chemical conjugation of the payload to the anti-cancer mAb increases the selectivity with which the payload is delivered to cancer cells, thereby increasing the therapeutic index of the payload.

[0169] Antibodies are typically administered subcutaneously or intravenously and absorbed into the bloodstream. They can distribute throughout the body, including tissues, but are generally restricted to the extracellular space due to their size. ADCs are also administered by injection and absorbed into the bloodstream. However, after targeting to tumors or diseased tissues via the antibody component and, in some cases, receptor-mediated endocytosis, the drug payload released from the ADC can penetrate cells and tissues due to its physicochemical properties (e.g., hydrophobicity, size, presence or absence of aggregation) and exert a bystander effect on surrounding cells through non-selective uptake (Figure 26). Furthermore, the metabolism and excretion of ADCs vary depending on their specific structure and the specific drug or antibody used.

[0170] The drug-to-antibody ratio (DAR) is a very important characteristic in ADC development, determining its pharmacokinetic properties and biodistribution.

[0171] The higher the DAR of an ADC, the higher its efficacy in in vitro tests. However, ADCs with inherently high DARs often exhibit lower in vivo efficacy than expected. This is presumably because the higher the conjugated drug, the higher the plasma washout rate. For some time, the DAR of ADC formulations has been limited to approximately 2-4. As a result, technologies that use drug conjugation to antibody cysteine ​​or lysine residues are primarily used.

[0172] The hydrophobicity of many commonly used cytotoxic drugs and linkers leads to problems such as ADC aggregation, loss of affinity for target antigens, and high plasma washout rates. Hydrophilic linkers containing sulfonates or polyethylene glycol (PEG) overcome the problems associated with hydrophobic linkers. PEG linkers have the advantages of water solubility, low toxicity, and low immunogenicity.

[0173] A DAR of around 4 has long been considered optimal, but in fact this applies to second-generation linkers using drugs such as MMAE or DM1; for third-generation linkers, a higher DAR is better. Many ADCs approved in recent years have DAR values ​​approaching 8, and new ADCs currently undergoing clinical trials have DAR values ​​ranging from 1 to 15.

[0174] Antibody-drug conjugates (ADCs) using camptothecin-based payloads have attracted considerable attention as a novel approach to treat various cancers, especially solid tumors. Among these ADCs, new ADCs (e.g., Enherz) containing newly synthesized camptothecin-based payloads (e.g., DXd) optimized for ADC modality have recently attracted particular attention due to successful clinical results.

[0175] The success of these ADCs is due in part to the fact that the new camptothecins possess characteristics such as (1) superior cell growth inhibition; (2) a better safety profile than existing ADC payloads; (3) optimized bystander effects; and (4) excellent physicochemical properties suitable for generating high DAR ADCs. Despite the remarkable success of ADCs utilizing camptothecin-based payloads, such as 1062a, there are clear unmet needs, as previously mentioned, including improving the safety profile (minimizing interstitial lung disease (ILD) and neutropenia) and developing new ADCs with multiple MoA payloads to address cancer heterogeneity.

[0176] To address these unmet needs, the present invention provides active camptothecin derivatives of Chemical Formula 1 or Chemical Formula 2, which are designed to bind to DDX5 protein and E3 ligase as molecular glue degraders, as candidate drugs offering various physicochemical properties. Furthermore, new camptothecin-based payloads (PBX series compounds) represented by Chemical Formula 1 or Chemical Formula 2 were synthesized (Preparation Examples 1 to 4) and evaluated (Figures 18 to 23).

[0177] The new PBX series compounds, represented by Chemical Formula 1, are potent dual Top1 / anti-cell death pathway inhibitors with an excellent safety profile. These PBX series derivatives, represented by Chemical Formula 1-1, are FL118-based nucleus-based compounds that exhibit potent ex vivo cytotoxicity comparable to that of Dxd, represented by Chemical Formula 2. They also exhibit an excellent safety profile in preliminary toxicity studies in mice and rapid elimination in PK studies (Example 2, Tables 3 and 4). This minimizes systemic side effects when the linker is cleaved early during use as an ADC payload.

[0178] As shown in Figure 24 (Example 3), which shows the results of the lactone-to-carboxylate conversion experiment at pH 7.4, it was confirmed that PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 were converted from the lactone form (active form) to the carboxylate form (inactive form), which is inactive as a TOP1 inhibitor, at a higher rate and faster than other reference compounds (exatecan, DXd, SN-38, FL118) at pH 7.4, which is the pH of blood or extracellular fluid.

[0179] In particular, the extent (63–86%) and rate (0.4–0.8% / min) of formation of the carboxylate form, which is inactive as a TOP1 inhibitor at blood pH 7.4, varied significantly among camptothecin derivatives (Figure 24). PBX-7014 and PBX-7016 formed over 86% of the carboxylate form, and all were converted from the lactone form to the carboxylate form relatively quickly at a level of 0.8% per min. The competing drug, DXd, formed 76.5% of the carboxylate form and was converted from the lactone form to the carboxylate form at a level of 0.6% per min.

[0180] The rate of formation of the lactone form (measured as the carboxylate form) active as a TOP1 inhibitor at a pH of 6.0, the pH of the tumor microenvironment (TME), was confirmed to be similar for all evaluated drugs (20% of the carboxylate form was formed after approximately 800 minutes) (Figure 25).

[0181] As a result, compounds represented by Chemical Formula 1, such as PBX-7014, PBX-7016, and PBX-7024, are predicted to exhibit relatively low toxicity, i.e., high safety in normal cells, by forming an inactive carboxylate form rather than an active lactone form that exhibits cytotoxicity relatively quickly and at a high rate in blood or extracellular fluid (normal tissues other than tumor tissue) compared to reference compounds. However, in the tumor microenvironment, PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 all form lactone forms that have activity at the same level as other TOP1 inhibitors, which means there is no factor that reduces efficacy.

[0182] The immunoanticancer agent of the present invention is designed to have excellent dose-dependent anticancer efficacy (conc. vs. effect) and / or time-dependent anticancer efficacy (effect vs. time). However, in some cases, the excellent anti-cancer efficacy over time may be accompanied by side effects (sustained T cell activation and accumulation of inhibitory signals due to chronic antigen exposure, resulting in T cell exhaustion, and T cell damage due to tumor tissue and connective tissue surrounding the tumor). Side effects due to the killing of peripheral fibroblasts (ILD, neutropenia) may also be significant. For example, depending on the drug modality (small molecule vs. ADC) of the active camptothecin derivative of Formula 1 or Formula 2, it is important to exceed a certain drug concentration (high), and in some cases, the time for which the drug concentration is maintained at or above a certain drug concentration (low) may be important. It may also be important to achieve fast or slow removal through lymphatic drainage by providing a desired degree of hydrophobicity and / or controlling aggregation.

[0183] Therefore, taking into consideration not only various side effects but also issues such as a reduced therapeutic index, the selection range of ADC payloads that exhibit appropriate anti-cancer efficacy can be expanded to a variety of candidate groups including activated camptothecin derivatives of Chemical Formula 1 or Chemical Formula 2, which are designed to bind to DDX5 protein and E3 ligase via a molecular glue degrader, which is another major feature of the present invention.

[0184] Despite the remarkable success of ADCs utilizing camptothecin-based payloads, such as Enhertz or DS-1062a, the present invention provides a variety of activated camptothecin derivatives represented by Chemical Formula 1 or Chemical Formula 2 that are designed to bind to DDX5 protein and E3 ligase as candidates for multiple MoA payloads to improve safety profiles (minimize ILD and neutropenia) and address cancer heterogeneity. This allows for precise control of desired efficacy (e.g., anticancer, combination therapy) and side effects (carcinogenic inflammation, drug resistance, ILD, neutropenia) by selecting one or more appropriate active camptothecin derivatives of Chemical Formula 1 or Chemical Formula 2, selecting an appropriate linker, and applying an appropriate dose and dosage.

[0185] [ADC toxicity profile, non-selective uptake, and bystander effect] The therapeutic index of a drug is a measure of the safety and efficacy of a drug in medical treatment. It is defined as the ratio between the dose that produces a therapeutic effect and the dose that produces toxicity or adverse effects. In other words, it indicates the range between the therapeutic and toxic doses of a drug.

[0186] A high therapeutic index indicates a wide margin of safety, where the effective dose is significantly lower than the toxic dose. This means that a drug can be administered at therapeutic levels without inducing serious side effects or toxicity. Drugs with a high therapeutic index are generally considered safer and more desirable for clinical use.

[0187] On the other hand, a low therapeutic index means a narrow margin of safety. In such cases, the effective dose and toxic dose are relatively close, increasing the risk of side effects and toxicity when the drug is used. Drugs with a low therapeutic index require careful monitoring and accurate dosing to avoid harming the patient.

[0188] The therapeutic index is an important consideration in drug development because it helps determine the dose range that can provide the desired therapeutic effect while minimizing the risk of side effects. It provides useful information when prescribing drugs and helps determine the overall benefit-to-risk ratio of a drug. sk ratio) can be evaluated.

[0189] Antibody-drug conjugates (ADCs) are a rapidly growing class of anti-cancer therapeutics, with over 100 ADCs currently in clinical studies. , brentuximab vedotin (Adcetris), inotuzumab ozogamicin (Besponsa), trastuzumab emtansine (Kadcyla), polatuzumab vedotin (Polivy), enfortumab vedotin (Padcev), trastuzumab deruxtecan (Enhertu), sacituzumab govitecan (Trodelvy), Twelve ADCs have been approved by the US Food and Drug Administration (FDA), including belantamab mafodotin (Blenrep), loncastuximab tesirine (Zynlonta), tisotumab vedotin (Tivdak), and mirvetuximab soravtansine (Elahere). Furthermore, a relatively small number of payload molecules (e.g., MMAE, MMAF, DM1, DM4, calisemysin, SN38, Dxd, PBD) are used in many approved and investigational ADCs.

[0190] Although some ADCs have demonstrated sufficient efficacy and safety to receive FDA approval, clinical use of all ADCs induces significant toxicity in treated patients, and many ADCs have failed during clinical development due to unacceptable toxicity profiles. This is because off-site toxicity remains a problem, limiting tolerable ADC doses below those required for substantial anti-cancer efficacy. Even with FDA-approved ADCs, a significant number of treated patients require adjuvant therapy to reduce the severity of ADC-associated toxicity, with many patients requiring dose reductions, treatment delays, or treatment interruptions.

[0191] Analysis of clinical data has demonstrated that dose-limiting toxicities (DLTs) are often shared by various ADCs delivering the same cytotoxic payload, regardless of the target antigen and / or cancer type being treated. DLTs are generally associated with cells and tissues that do not express the target antigen (i.e., off-target toxicity) and often limit ADC doses below the levels required for optimal anticancer efficacy.

[0192] ADCs have the potential to safely improve the efficacy of cytotoxic drugs compared to when used as a single drug. ADCs bind drugs to antibodies so that the antibodies specifically target the lesion site, ensuring that the drug is delivered only to the lesion site and not to normal tissue. In the case of cancer cells, antibodies that specifically bind to specific antigens expressed on the surface of cancer cells are used to deliver highly toxic drugs specifically to cancer cells, killing only the cancer cells.

[0193] For ADCs to work, they must enter target cells. After the ADC's antibodies specifically bind to specific antigens expressed on the surface of target cells, such as cancer cells, they enter the target cells via clathrin-coated pits in the cell membrane.

[0194] Once inside the cell, the ADC dissociates from clathrin, fuses with other vesicles within the cell, and then travels through the endosome-lysosome pathway. After reaching the endosome, the drug is separated from the antibody by specific factors in the specific tumor cell environment. The free cytotoxic drug, now free from the antibody, penetrates the lysosomal membrane and enters the cytoplasm. The activated drug exerts its pharmacological effect by binding to its molecular target in the periphery, inducing cell death and killing the cancer cell.

[0195] During this process, some cytotoxic drugs are passively diffused, actively transported, or exit the cell through dead cells. If drugs that have spread to the surrounding area penetrate the cell membrane, they may enter neighboring cells and kill them as well (a phenomenon known as bystander cell-killing).

[0196] Most of the potent cytotoxic drugs loaded into ADCs are highly toxic, and the drug payload released from ADCs can also affect normal cells through bystander effects (Figure 26).

[0197] Therefore, ADCs must be developed that act highly specifically on target cancer cells without inducing serious side effects in normal cells.

[0198] Fibroblasts, members of connective tissue, are the predominant cells in the stroma, particularly in breast, prostate, and pancreatic cancers.

[0199] Stromal fibroblasts (stromal fibroblasts), also known as cancer-associated fibroblasts (CAFs), are a specific type of fibroblast found within the tumor microenvironment. Stromal fibroblasts have attracted considerable attention due to their important role in tumor growth, invasion, and metastasis. CAFs are responsible for the production of paracrine growth factors, proteolytic enzymes, and ECM components.

[0200] The camptothecin-based drug represented by Chemical Formula 1 or Chemical Formula 2 according to the present invention is a hydrophobic small molecule that can permeate cell membranes, and therefore can accumulate at high concentrations while penetrating deep into cancer tissues. It penetrates the cell membrane to exert cytotoxicity inside the cell, killing the cell, and then is released, and subsequently penetrates the cell membrane of surrounding cells to move into the cells and act there. In this case, the camptothecin-based drug has a high killing effect on surrounding cells, which can be advantageous for the treatment of heterogeneous tumors. However, in some cases, to provide a modality for mitigating or preventing camptothecin-based payload-based ADC toxicity in ILD, a type of autoimmune disease, the camptothecin-based payload released from the ADC may be designed to penetrate the interior of cancer tissues, not permeate the cell membrane of fibroblasts, or be released via IC, so as not to exert its killing effect on fibroblasts surrounding cancer cells. 50Payloads that have high activity or are rapidly eliminated via lymphatic drainage and do not accumulate at high concentrations can be designed / selected from various candidate groups, including active camptothecin derivatives of Chemical Formula 1 or Chemical Formula 2. Furthermore, by appropriately applying the dosage and administration method, the desired efficacy (e.g., anticancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD) can be precisely controlled.

[0201] Furthermore, in order to increase the therapeutic index of the camptothecin drug payload and suppress non-selective uptake of the camptothecin drug and / or ADC released from dead cells, the present invention allows for the selection of an ADC payload from a group of candidate camptothecin drugs represented by Chemical Formula 1 or Chemical Formula 2. For example, by selecting a camptothecin drug with a high killing effect on target cancer cells from among low-concentration drugs (payloads) from the candidate drugs represented by Chemical Formula 1 or Chemical Formula 2, the ADC dose can be adjusted to reduce the total concentration of the ADC payload, as shown in Figure 26, thereby suppressing the by-stander cell-killing effect on normal cells of the free drug (payload) released from dead cells or overcoming the off-target toxicity of the free camptothecin drug (payload) released from target / non-target apoptotic cells.

[0202] [FL118 drug (Chemical Formula 1-1) - ADC with acid-sensitive linker] The present inventors synthesized a new ADC using FL118, a new camptothecin payload represented by Chemical Formula 1-1, which exhibits excellent in vitro / in vivo antitumor efficacy and an excellent safety profile (Example 4). The FL118 payload exhibited a similar safety profile to that of Trodelvy's payload, SN-38. Mouse and monkey models confirmed that the CL2A linker system, which has a highly efficient release profile in the tumor microenvironment, can be utilized without causing serious toxicity. Furthermore, the use of the hydrophilic CL2A linker system ensured minimal aggregation of the ADC, despite the extreme hydrophobicity of FL118 itself.

[0203] Specifically, as shown in Figure 3, PBX-001 is an ADC composed of the humanized anti-Trop2 monoclonal antibody hRS7, the novel topoisomerase I inhibitor FL118, and the CL2A linker system. PBX-001 has a high DAR of approximately 7-8, enabling highly efficient release of the FL118 payload in the low pH tumor microenvironment. As shown in Figures 11-13, in vitro and in vivo evaluations of PBX-001 demonstrated superior efficacy compared to Trodelvy. Furthermore, non-human primate toxicity studies demonstrated the superior safety of PBX-001.

[0204] Based on this, the present invention proposes an FL118-based ADC platform that can be adapted to other targets using alternative monoclonal antibodies. In this study, we newly designed nimotuzumab FL118 (an ADC composed of the topoisomerase I inhibitor FL118 and the CL2A linker system (an ADC comprising nimotuzumab-CL2A-FL118) by substituting the humanized anti-EGFR monoclonal antibody h-R3 for the humanized anti-Trop2 monoclonal antibody hRS7 in sacituzumab FL118 (PBX-001), an ADC composed of the humanized anti-Trop2 monoclonal antibody hRS7 (Example 4).

[0205] In other words, the company hopes to develop an EGFR-targeted ADC with reduced on-target toxicity by manufacturing an ADC using nimotuzumab, an antibody drug with low binding affinity, instead of cetuximab, an existing antibody drug, for EGFR, one of the targets that is difficult to develop as an ADC because it is widely distributed in normal cells.

[0206] Cetuximab K d The value is 1.8*10 -9 M, whereas K for nimotuzumab d The value is 2.9*10 -8 It's M.

[0207] Example 4 and Figure 22 show the synthesis method of nimotuzumab-CL2A-FL118, a new ADC targeting EGFR, and the results of in vivo evaluation of the ADC using two types of ADC: cetuximab and nimotuzumab.

[0208] Although nimotuzumab-CL2A-FL118 was designed to address toxicity issues, it was surprisingly found to exhibit superior in vivo efficacy compared to the reference cetuximab-CL2A-FL118 (Figure 22). The ADC in vitro assays in Figures 18 to 20 predicted that the two ADCs would exhibit similar efficacy, with cetuximab-CL2A-FL118 exhibiting slightly superior efficacy, but the in vivo assay yielded unexpected results (Figure 22).

[0209] By using an acid-sensitive linker, the immunoconjugate of the present invention can efficiently release camptothecin-based drugs not only inside cancer cells but also around cancer tissues after antigen binding, thereby overcoming resistance mechanisms associated with ADC processing (Figure 3).

[0210] Thus, one embodiment of the present invention provides a compound comprising [FL118 drug of Formula 1]-[acid-sensitive (ac an immunoconjugate comprising a nimotuzumab or an antigen-binding site-containing fragment thereof; (i) one or more FL118 drugs of Chemical Formula 1 are linked to nimotuzumab or an antigen-binding site-containing fragment thereof via an acid-sensitive linker; (ii) after being targeted to cancer cells by the antigen-binding moiety that targets the EGFR antigen on the cancer cells, the acid-sensitive linker is decomposed in the acidic environment (pH≦7) around the cancer, at least a portion of the FL118 drug of Chemical Formula 1 is liberated, and the free FL118 drug of Chemical Formula 1 penetrates the cell membrane and moves into the cells; (iii) the FL118 drug of Chemical Formula 1 inhibits the action of an efflux pump, resulting in enrichment of the intracellular free FL118 drug of Chemical Formula 1; (iv) Optionally, the immunoconjugate to which the FL118 drug of Chemical Formula 1 is linked may be internalized into cells, and the FL118 drug of Chemical Formula 1 may be released from lysosomes.

[0211] [Receptor-ligand binding and dissociation constant K d ] The present invention provides a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 An antibody or a fragment thereof containing an antigen-binding site (B-1) of less than M; or an antibody or a fragment thereof containing an antigen-binding site (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has a higher binding affinity when binding to an epitope on the cell surface protein bivalently than monovalently, and the binding between the epitope on the cell surface protein and the antibody or a fragment thereof containing an antigen-binding site corresponds to a type of receptor-ligand binding.

[0212] where the dissociation constant K d is a quantitative measure of the strength of ligand binding (corresponding to antibody binding).

[0213] All cells in a multicellular organism must receive and respond to signals from other cells to know when to grow, divide, differentiate, join, disperse, or die. Cells must receive molecular signals through membrane-bound or intracellular receptors and convert the received signals into meaningful cellular responses, such as cell division or differentiation.

[0214] After the binding interaction between a receptor and its cognate (matching) ligand, the responding cell must transmit knowledge of this receptor-ligand interaction to members of a molecular signaling pathway that can trigger an appropriate cellular response. This process is called signal transduction. The end result of a signaling pathway is a change in the behavior of the responding cell. These changes collectively represent the result of (or response to) the receptor's encounter with the ligand and can include a combination of cell division, differentiation, migration, changes in metabolic state, changes in the expression of surface or cytoplasmic molecules, and the secretion of new compounds such as chemokines or cytokines.

[0215] Enzyme-substrate binding interactions are generally monovalent (one enzyme molecule binds one substrate molecule) and have intermediate affinity, with only a small number of enzymes able to interact with any one substrate. In contrast, interactions between antigens and receptor molecules can have very high affinity, and the diversity of receptors capable of recognizing a particular antigen is astounding.

[0216] The receptor molecule is attached to the ligand through non-covalent chemical bonds of the same type that enzymes use to bind to their substrates. These include hydrogen and ionic bonds, hydrophobic and van der Vals interactions. The essence of a meaningful receptor-ligand interaction is that the sum of the binding interactions maintains the two interacting surfaces together with sufficient binding energy and for sufficient time for the receptor to bind. The goal of the receptor-ligand interaction is to allow the cell to receive a molecular signal indicating that the receptor has bound the matching ligand. Because these noncovalent interactions are individually weak, many such interactions are required to form a biologically relevant receptor-ligand linkage. Furthermore, each such noncovalent interaction occurs over a very short distance (typically about 1 angstrom (1 Å = 10 -10 Because receptors operate solely on the surface of the ligand, high affinity receptor-ligand interactions depend on a very close "fit" or degree of complementarity between the receptor and the ligand. In an aqueous environment, non-covalent interactions are weaker and depend on close complementarity between the receptor and the ligand.

[0217] In essence, multiple non-covalent bonds allow receptor-ligand interactions to achieve sufficient binding energy and interaction time to activate a molecular reaction.

[0218] When the immunoconjugate of the present invention binds to an epitope on a surface protein of a target cell via its antigen-binding site, and is then internalized into the cell along with the payload drug through a process known as receptor-mediated endocytosis, the aforementioned rules of chemistry related to receptor-ligand interaction / binding are also followed.

[0219] Antibodies that recognize antigens on cancer cells must be internalized together with drugs via receptor-mediated endocytosis. To enhance internalization in cancer cells, bispecific antibodies have been developed.

[0220] MEDI4267 (trastuzumab-META), a biparatopic antibody targeting two non-overlapping epitopes on HER2, under development by Medimmune and Astrazeneca, has been shown to induce HER2 receptor clustering, thereby promoting cellular internalization, lysosomal trafficking, and degradation.

[0221] The strength of the binding interaction between a receptor binding site (S) and a ligand (L) can be described using the following equation: (formula) Kd = [S][L] / [SL] Here, the dissociation constant, Kd, ​​of the reaction defines the relationship between the concentration of the receptor-ligand pair [SL], the concentration of free receptor sites [S], and the concentration of free ligand [L]. The unit of the dissociation constant is molar (M). K d The lower the value, the higher the affinity of the interaction. When 50% of the binding sites are occupied, [SL] = [S], K d = concentration of free ligand.

[0222] Dissociation constant K d is a quantitative measure of the strength of ligand binding. d The lower the K, the higher the affinity of the interaction. d If the same, 50% of the ligand will bind to the receptor. For example, in Figure 17, BC 50 means the concentration bound at 50% maximum. For comparison, the K for many enzyme-substrate interactions d K values ​​for antigen-antibody interactions with somewhat lower affinity early in the immune response d Value and similar 10 -3 ~10 -5 It is in the range of M. However, antibody genes expressed in the initial antigen stimulation undergo mutation and selection during the progression of the immune response, so antigen-antibody interactions in the later stages of the immune response are K d is 10 -12This is a very strong interaction, and the antigen concentration can be as low as 10 -12 Even if the K value is as low as M, half of the antigen molecules bind completely to the receptor. Recent studies on innate Toll-like receptors have shown that the K value of the interaction of these receptors with their respective ligands is d is 10 -7 ~10 -8 It is in the range of M.

[0223] The affinity of the receptor-ligand interaction can be measured by equilibrium dialysis or surface plasmon resonance (SPR).

[0224] The interaction between receptor and ligand can be multivalent. Many biological receptors, including B cell receptors, are characterized as multivalent because they have more than one ligand-binding site per molecule. When the receptors and ligands are all bivalent (e.g., a bivalent B cell receptor binds two identical ligands on the surface of bacteria), the overall binding interaction is identical but significantly stronger than the interaction between a monovalent receptor and ligand. (However, binding to two identical ligands on the same cell via two identical receptor sites on a single molecule may be slightly less than twice as strong as binding via a single receptor site because bivalent binding can slightly distort the geometry of the receptor or ligand, slightly disrupting the "fit" of the individual interactions.)

[0225] The many advantages of bivalent binding begin with the fact that noncovalent interactions are inherently reversible. A ligand spends part of its time bound to the receptor and part of its time in an unbound, or "off," state. When two or more binding sites are involved, the likelihood of all receptor sites being simultaneously in the "off" state is low, making it less likely that the receptor will release the ligand. Figure 28 compares monovalent (a) and bivalent (b) interactions. In Figure 28(a), when a monovalent receptor approaches a multivalent antigen, the receptor exists in equilibrium with the ligand (represented by the red circle), spending part of its time bound (the "on" state) and part of its time unbound (the "off" state). The proportion of time spent in the "on" and "off" states determines the affinity of the receptor-ligand interaction, which is related to the strength of the sum of the noncovalent interactions between the receptor and the ligand. In Figure 28(b), bivalent or multivalent binding occurs when one When a site releases a ligand instantaneously, it serves to ensure that the interaction between the two molecules (or indeed two cells) is not lost, just as it would be with a monovalent bond.

[0226] The term avidity is used to describe the overall strength of the collective binding interactions that occur during multivalent binding.

[0227] The fluid nature of cell membranes allows many membrane-bound antigen receptors to function in a multivalent manner, even though individual receptor molecules are essentially monovalent.

[0228] Figure 29 is a conceptual diagram of how cell surface receptors bind to multivalent antigens to form clusters. (a) When a cell surface receptor encounters a multivalent array of ligands, for example, on the surface of a bacterial cell, (b) the receptors move in the plane of the membrane to form occupied receptor clusters. That is, Figure 29 shows how multivalent antigens interacting with membrane-bound receptors in a reversible manner gradually stabilize larger and larger receptor clusters on the cell membrane.

[0229] This is because, once one or two stable connections are made, other receptors that diffuse randomly in the fluid environment of the plasma membrane are captured into the receptor cluster, thereby enlarging it. Such receptor clusters then facilitate intramolecular interactions in the cytoplasm and transmit cell activation signals to the nucleus.

[0230] In summary, multivalent binding interactions have increased preference over monovalent interactions, resulting in longer ligand occupancy times at the receptor. Monovalent receptors can also form multivalent clusters when interacting with membrane-bound multivalent ligands. Even if the topes are monovalent, multivalent clusters can be formed in the case of immune complexes of the present invention that use bivalent antibodies.

[0231] When cells are activated, such as cancer cells, their receptor expression patterns can change, making them more or less responsive to specific signals. When considering the strength of interactions between receptors and ligands, it is important to take into account the anatomical environment in which such interactions occur (e.g., pH of tumor tissue, solid tumors, cold tumors / hot tumors).

[0232] Immune antigen receptors can be transmembrane, cytoplasmic, or secreted (eg, antibodies).

[0233] To most biologists, the word "receptor" conjures up images of transmembrane proteins, dutifully waiting for water-soluble ligands to diffuse into the periphery so that they can respond to the ligand's signal.

[0234] In this context, antibodies with low affinity and low concentrations cannot bind to antigens. Therefore, the dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 The immunoanticancer agent of the present invention, which comprises an antibody of less than M or a fragment thereof containing its antigen-binding site (B-1); or an antibody or a fragment thereof containing its antigen-binding site (B-2) which binds to a cell surface protein that is expressed in both normal cells and cancer cells but has higher binding affinity when binding to an epitope of the cell surface protein in a bivalent manner than when binding monovalently, can penetrate deep into cancer tissue.

[0235] In relation to the antigen binding affinity of an antibody, the immunoglobulin fold, including antibodies, is composed of a pair of β-sheets formed by β-strands connected by loops that define the protein binding specificity.

[0236] In antibodies, each immunoglobulin domain forms an amino acid domain ring with a characteristic spacing of disulfide bonds (approximately 67 amino acids separating two cysteine ​​residues). Most immunoglobulin domains contain approximately 110 amino acids, and each beta sheet is composed of three to six strands. Each pair of intradomain beta sheets is stabilized by intrastrand disulfide bonds. Adjacent domains are connected to each other by relatively unstructured polypeptide chains.

[0237] Within each immunoglobulin (Ig) domain, multiple β-strands are arranged in parallel to form a pair of β-sheets. Hydrophobic and hydrophilic amino acids alternate along the amino acid sequence of each β-strand, with the hydrophobic amino acids of one sheet pointing toward the amino acids of the opposite sheet. The hydrophilic residues interact with the environment. Thus, the two β-sheets form a highly stable "hydrophobic sandwich," with each domain stabilized by the hydrophobic interactions between the sheets, resulting in significant solubility of the entire protein.

[0238] Immunoglobulin domains consist of amino acid residues arranged in beta sheets connected by variable loops. The two beta wrinkled sheets are shown in two shades of blue. They are held together by hydrophobic interactions and conserved disulfide bonds (not shown). The three loops of each variable domain, shown in red, are highly variable in length and amino acid sequence and comprise the antigen-binding site; they are therefore called complementarity-determining regions (CDRs).

[0239] Antibody heavy chains contain four or five domains, whereas light chains contain only two. In each case, the antigen-binding function is located within the amino (N)-terminal domain of the protein.

[0240] [EGFR targeting antibody-drug conjugate] The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, plays a central role in tumorigenesis in various types of solid tumors.

[0241] EGFR mutations and / or upregulation have been frequently identified in patients with non-small cell lung cancer (NSCLC), colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. Inhibition of EGFR activation with tyrosine kinase inhibitors (TKIs) or monoclonal antibodies (mAbs) has achieved great clinical success in suppressing tumor growth and improving patient survival.

[0242] However, even after initial response, disease progression appears inevitable, which may be due to primary or acquired resistance mechanisms, such as EGFR secondary or tertiary mutations, mutations in downstream mediators of EGFR signaling, or bypassing of RTK signaling. Overcoming resistance to EGFR-targeted therapeutic agents is currently a clinical challenge in cancer treatment and represents a major unmet medical need.

[0243] In this regard, camptothecin-based drugs (A), which degrade DDX5 protein, have a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 The immune complex of the present invention, which is bound via a linker (C) to an antibody or antigen-binding site-containing fragment thereof (B-1) of less than M; or an antibody or antigen-binding site-containing fragment thereof (B-2) that has higher binding affinity when binding to an epitope of an EGFR protein bivalently than when binding monovalently, can provide an EGFR-targeted therapeutic agent that can solve the above-mentioned difficult problems.

[0244] Antibody-drug conjugates (ADCs) are a new class of drugs that use antibodies to selectively deliver cytotoxic payloads to tumors expressing high levels of tumor-associated antigens. Localized accumulation of toxins in tumors minimizes systemic toxicity, increases cancer cell death, and offers a higher therapeutic index than chemotherapy or parent antibodies. ADCs can also circumvent some resistance mechanisms by selectively delivering anti-mitotic or DNA-damaging toxins to cancer cells to induce cell death, a mechanism independent of oncogenic signal suppression. Recently, two ADCs targeting EGFR variant III (AMG-595 and ABT-414) demonstrated promising monotherapy efficacy in patients with recurrent glioblastoma. Such responses correlated with EGFR amplification or the presence of EGFR variant III, providing early clinical validation of EGFR-targeted ADCs in patients with EGFR-amplified or variant cancers.

[0245] Nimotuzumab (humanized IgG1, also known as hR3) is an EGFR-targeting antibody approved for the treatment of non-small cell lung cancer and glioma in many developing countries. The outstanding selectivity of hR3 for EGFR-positive cancer cells enhances cancer cell killing while minimizing toxicity to normal tissues, providing a strong rationale for designing hR3-based ADCs that can treat patients who have failed anti-EGFR therapy.

[0246] Nimotuzumab is also undergoing clinical trials for the treatment of various solid tumors, including non-small cell lung cancer, CRC, gastric cancer, and pancreatic cancer. Due to its unique binding kinetics with the EGFR receptor, hR3-related side effects in humans have been mild to moderate in severity. In particular, the incidence of severe acne rash and gastrointestinal mucosal toxicity in patients was remarkably low, and these side effects were at levels that limit the toxicity typically observed with EGFR-targeted therapeutics.

[0247] Nimotuzumab binds to an epitope on the EGFR protein with a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 or antibodies that have higher binding affinity when binding to an epitope of the EGFR protein bivalently than monovalently (B-2).

[0248] While inhibiting EGFR-mediated signaling has proven effective in treating certain types of cancer, the efficacy and usefulness of approaches that inhibit EGFR function are limited by the rapidly evolving mechanisms that restore EGFR signaling or activate alternative pathways that promote the proliferation and survival of malignant cells. Given the fact that EGFR overexpression is commonly observed in many cancers, EGFR-targeted antibody-drug conjugates (ADCs) can selectively kill cancer cells without blocking EGFR-mediated signaling.

[0249] [K d The value is 1*10 -8 M or more 1*10 -6 (B) an antibody or antigen-binding site-containing fragment thereof that has a binding affinity of less than M or higher when binding bivalently than when binding monovalently The immune complex of the present invention has a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6It is preferable to use an antibody or a fragment thereof containing its antigen-binding site (B-1) that is less than M; or an antibody or a fragment thereof containing its antigen-binding site (B-2) that binds to a cell surface protein that is expressed on both normal and cancer cells and has a higher binding affinity when binding to an epitope of the cell surface protein in a bivalent manner than when binding monovalently.

[0250] Non-limiting examples of antibodies or antigen-binding site-containing fragments (B-2) that bind to cell surface proteins that are expressed in both normal cells and cancer cells and have higher binding affinity when binding to epitopes of cell surface proteins bivalently rather than monovalently include antibodies that bind to cell surface proteins that are expressed in both normal cells and cancer cells when they pair up, particularly in cancer cells, such as the growth factors illustrated in Figure 2.

[0251] Thus, antibodies with higher binding affinity for cell surface protein epitopes in a bivalent rather than monovalent manner can target cell surface proteins whose expression patterns change upon cell activation, such as cancer cells.

[0252] [Antibodies that bind to cell surface proteins expressed on both normal and cancer cells and that have higher binding affinity when bivalently binding to an epitope on a cell surface protein than when monovalently binding] can be [antibodies that bind bivalently to EGFR epitopes], such as nimotuzumab, or can have lower binding affinity.

[0253] The reduced side effects of nimotuzumab (mAb) are related to its inherent binding affinity and density dependence. The low affinity between nimotuzumab and EGFR allows for optimal drug doses below toxic doses.

[0254] Mathematical models predict that a binding affinity (Kd) of 10 for anti-EGFR mAbs is required to maximize tumor cell targeting while minimizing toxicity to normal cells. -8M~10 -9 M must be within the range [Crombet T, Osorio M, Cruz T, et al. Use of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients. J Clin Oncol 2004;22(9):1646-54. Nimotuzumab falls into this category, as do cetuximab and panitumumab. nimotuzumab (a mAb) has over 10 times stronger binding avidity. According to a recently proposed density model, nimotuzumab's transient monovalent and strong bivalent binding properties to the EGFR epitope further explain its reduced side effects. EGFR expression in normal cells (e.g., skin epithelial cells) is too low for nimotuzumab bivalent binding, limiting the efficacy of the mAb and avoiding unwanted toxicity. On the other hand, overexpressing tumor cells have sufficient receptor density for nimotuzumab to bind bivalently and potently inhibit the receptor. This theory is supported by comparative dose-binding curves of nimotuzumab and cetuximab to normal skin and kidney cells, where nimotuzumab shows much less binding than cetuximab [Garrido G, Rabasa A, Gracia E, et al. Binding properties of the anti-EGFR monoclonal antibody,nimotuzumab,limit its interaction with the EGFR in renal and epidermal cells.AACR 100th Annual Meeting, 2009(abstract 2763). Available from:http: / / www.ymbiosciences.com / upload_files / nimo_poster_AACR2009.pdf[Last accessed 22 June 2009]].

[0255] [K d The value is 1*10 -8 M or more 1*10 -6 Advantages of immunological anticancer agents having antibodies or antigen-binding site-containing fragments (B) that are less than M or have higher binding affinity when binding bivalently than when binding monovalently] By this invention, d The value is 1*10 -8 M or more 1*10 -6 An immunoanticancer agent having an antibody or its antigen-binding site-containing fragment (B) with a binding affinity less than M or higher when binding bivalently than monovalently can relieve solid pressure within the cancer microenvironment, thereby increasing the accessibility of anticancer agents and / or immunotherapeutic agents, thereby enhancing the therapeutic effect.

[0256] Traditionally, cancer has been considered a cytogenetic disease characterized by mutations in genes that regulate cell division, differentiation, and death. However, recently, the cancer microenvironment has also attracted significant attention due to its role in cancer initiation, progression, immune surveillance evasion, and therapeutic response. As cancer cells proliferate, they modify the structure and function of their surrounding environment biochemically and physically / mechanically. Consequently, these physical modifications affect both the cancer cells and the microenvironment, potentially promoting cancer progression or conferring resistance to treatment.

[0257] The eight typical characteristics of cancer are: 1. persistent cell division signals, 2. evasion of growth inhibitory signals, 3. resistance to cell death, 4. unlimited cell division, 5. promotion of angiogenesis, 6. activation of tissue invasion and metastasis, 7. alteration of energy metabolic activity, and 8. evasion of immune destruction.

[0258] As cancer grows, it biochemically and physically transforms surrounding tissues, and these transformations can induce physical deformities that can affect cancer cells and the surrounding microenvironment.

[0259] Four additional characteristics based on the physical deformity of cancer are: (i) solid compression and elastic energy, (ii) interstitial fluid compression, (iii) firmness (elasticity), and (iv) stromal structure. Although conceptually distinct, these four hallmarks can synergistically interact with each other and exacerbate eight existing cancer biological hallmarks, promoting cancer cell division, metastasis, evasion of immune surveillance, and resistance to treatment.

[0260] In relation to solid stress and elastic energy, solid stress, also known as "residual stress," is a compressive force. This refers to mechanical forces, such as resilient, tensile, and shear, inherent in and transmitted between solid elastic elements of cells and the extracellular matrix. The magnitude of solid compression is expressed in Pascals (Pa) or millimeters of mercury (mmHg) and varies widely, ranging from approximately 0.7 mmHg for glioblastoma to 75 mmHg for pancreatic ductal adenocarcinoma.

[0261] The various mechanisms that induce solid compression in cancer are as follows:

[0262] (1) Increase in tissue volume due to cell division, cell penetration, and matrix deposition This increased volume pushes out viscoelastic structures inside and outside the cancer tissue, causing solid pressure on the cancer and surrounding tissue. Therefore, when the number of cancer cells decreases due to anti-cancer treatment, the solid pressure decreases, and the pressure exerted on the blood vessels surrounding the cancer tissue also decreases.

[0263] (2) Deformation of normal tissue surrounding cancer tissue Some cancers grow into well-controlled nodular masses, which coexist with the surrounding normal tissue and push out the surrounding normal tissue, increasing mechanical stress. Some cancers seek out weak areas in the surrounding normal tissue and infiltrate there, growing by intermixing with the normal tissue, resulting in weaker solid compression than the former.

[0264] (3) Swelling due to water absorption Complex polysaccharides in the matrix, such as hyaluronic acid, increase the water content through osmotic pressure, and this increased water content in cancer tissue induces a form of solid compression that differs from hydraulic pressure.

[0265] (4) Actomyosin-mediated cell contraction Fibroblasts, immune cells, and cancer cells can contract the matrix when they move within cancer tissue or attempt to repair damaged tissue. Cell contraction generates tensile forces that contract the extracellular matrix, inducing tension in one part of the cancer tissue, which is soon balanced by compression in other parts.

[0266] It was first reported in 1997 that solid compression can inhibit cancer growth, but this physical force is strong enough to compress surrounding blood vessels and lymphatic vessels, and a reduction in blood flow can induce hypoxia, promoting the metastasis of cancer cells and preventing the access of anticancer drugs and immunotherapy agents.Cancer cells and normal cells have mechanosensing systems that can sense externally applied physical forces, and they can respond directly to cell-extracellular matrix or cell-cell forces, or indirectly to changes in the extracellular matrix that are deformed by solid compression.

[0267] In animal cancer models, relieving solid pressure reduces vascular pressure, which increases the accessibility of anticancer drugs and leads to increased survival rates. This approach of relieving solid pressure can also enhance the efficacy of other treatments, such as immunotherapy. Furthermore, the payloads released from the immunoconjugates of the present invention can effectively exert a bystander effect.

[0268] In most organs, blood enters through arteries and exits through veins, and some of the remaining tissue fluid is discharged through lymphatic vessels, maintaining the homeostasis of interstitial fluid. In normal organs, the interstitial fluid pressure is close to zero. When a tumor develops, this balance is disrupted due to increased permeability of blood vessels and pressure on blood / lymphatic vessels. Slack blood vessels and narrowed drainage channels lead to an increase in interstitial fluid within the tissue, which in turn leads to an increase in interstitial fluid pressure. The interstitial fluid pressure, which remains constant within the cancer tissue, suddenly decreases in the areas surrounding the tumor, which causes tissue fluid to flow from the center of the tumor to the surrounding normal tissue where lymphatic drainage channels are located.

[0269] This flow of interstitial fluid exerts shear stress on the extravascular cells, which induces the following changes in cancer cells and surrounding cells: fibrocyte activation, regulation of blood and lymphatic vessel formation, regulation of matrix metalloproteinase (MMP) activity, regulation of cancer cell metastasis and migration, and cell cycle inhibition. Furthermore, because immune cells can sense the flow of interstitial fluid, this flow of tissue fluid may also be involved in regulating immunity.

[0270] In addition to directly regulating mechanotransduction mechanisms, tissue fluid flow due to differences in interstitial fluid pressure is also involved in cancer progression and response to anticancer therapy. High interstitial fluid pressure can inhibit the diffusion of anticancer drugs delivered via the blood vessels into the cancer tissue, and interstitial fluid flow from the cancer tissue toward the blood / lymphatic vessels can also help cancer cells spread to other tissues via the blood vessels and lymphatic vessels.

[0271] Accumulated solid pressure compresses blood vessels, obstructing blood flow and increasing interstitial fluid pressure. Therefore, the method of relieving solid pressure using the immunoconjugate of the present invention can also remove pressure applied to blood / lymphatic vessels and improve blood flow, thereby maintaining a normal level of interstitial fluid pressure.

[0272] Linker Among the components that make up ADCs, the linker connects the antibody to the cytotoxic drug.

[0273] When designing ADCs, it is important to adopt efficient drugs to enhance tumor cell killing efficacy while enhancing specificity for target tumor cells, ensuring stability in plasma, and reducing toxicity to normal cells.

[0274] The linker must be stable in the bloodstream, prevent the drug from separating from the antibody, maintain the drug in a prodrug state until it reaches the target, and minimize damage to normal tissues. The ideal linker is one that is stable when the ADC is circulating throughout the body, yet is cleaved in the target cells, appropriately releasing the cytotoxic drug and safely delivering the drug to the target, ensuring that the ADC is both effective and safe.

[0275] When a drug is attached to an antibody via a linker, the drug must not affect the structural stability, substrate binding properties, or pharmacokinetics of the antibody.

[0276] In particular, ADCs must maintain the same affinity as the antibody before it was conjugated with the drug, i.e., the drug conjugated to the antibody must not interfere with antibody-antigen binding.

[0277] Because the camptothecin-based drug (A), which degrades the DDX5 protein, is highly hydrophobic, drug absorption and payload release do not depend on the antibody's drug target, but rather occur non-selectively in macrophages, etc., resulting in unexpected side effects. To reduce such side effects, it is preferable that the linker (C) be hydrophilic.

[0278] Linkers used in ADCs can be divided into non-cleavable and cleavable linkers based on their cleavage ability. Non-cleavable linkers have relatively high plasma stability and are resistant to proteolysis. A typical example of a non-cleavable linker is a thioether linker. Unlike cleavable linkers, non-cleavable linkers do not decompose themselves, and therefore have the property of allowing the drug to be released when the antibody is degraded after intracellular introduction in the form of an ADC. For example, when the antibody is degraded after intracellular introduction, the drug is released in the form of a conjugate with the linker, and this conjugate has pharmacological activity. The released drug carries a charge and is less likely to diffuse into surrounding cells (bystander effect). While there is no bystander effect, which indicates toxicity to surrounding cells, the drug only exerts its effect on the target cells after internalization, meaning that it is relatively safe. ADCs manufactured with non-cleavable linkers are more dependent on the biological mechanisms within the target cells.

[0279] The cleavable linker is cleaved in response to specific environmental factors, releasing the drug into the cytoplasm. Cleavable types include enzyme-cleavable and non-enzyme-cleavable types. Non-enzymatically cleavable linkers include acid-labile linkers and oxidation-reduction reaction linkers.

[0280] An antibody-drug conjugate (ADC) according to one embodiment of the present invention uses (i) an acid-sensitive linker or (ii) an enzyme-sensitive linker (FIGS. 21 to 23). Since cancer cells have high proteolytic enzyme activity, it is better to use an enzyme-sensitive linker. The enzymatically cleaved forms are cleaved by enzymes such as cathepsin B, β-glucuronidase, phosphatase, pyrophosphatase, and sulfatase.

[0281] Peptide linkers are degraded by proteases, and protease inhibitors are present in plasma, making them highly stable. Cathepsin B is a typical protease used in ADCs. Cathepsin B is present at high levels in tumor tissue, conferring tumor selectivity to ADCs. Peptide linkers consist of dipeptides or tetrapeptides that are recognized and cleaved by lysosomal proteases upon internalization of the ADC. Tetrapeptides were used early in development but presented limitations, such as relatively slow drug release and potential aggregation when combined with hydrophobic drugs. These issues were resolved with the development of dipeptide linkers such as Val-Cit, Phe-Lys, Val-Lys, and Val-Ala.

[0282] The β-glucuronide linker is degraded by β-glucuronidase, a glycolytic enzyme present in lysosomes, which is overexpressed in some tumor cells and confers tumor specificity. β-Glucuronidase is abundantly present inside 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. These properties enhance plasma stability of ADCs bearing β-glucuronide linkers, preventing off-target drug release. To confirm the plasma stability of β-glucuronide linkers, experiments were performed in mouse plasma with Val-Cit linkers. The β-glucuronide linkers demonstrated exceptional stability, with 89% and less than 50% activity after 7 days, respectively, and half-lives of approximately 81 and 6 days, respectively. Furthermore, ADCs bearing β-glucuronide linkers demonstrated high stability and efficacy despite the conjugation of high doses (up to 8 molecules) of cytotoxic drugs.

[0283] According to one embodiment of the present invention, the enzyme-sensitive linker is -S-maleimide-spacer-enzyme The linker may be cleavage site-self-immolative spacer-(payload) or -S-dibromomaleimide-spacer-enzyme cleavage site-self-immolative spacer-(payload). Figure 23 analyzes the in vivo efficacy of ADCs of the invention (DAR8 or DAR4) that use enzyme-sensitive linkers. Here, non-limiting examples of self-immolative spacers are illustrated in FIG.

[0284] Biotransformation by linker deconjugation can occur by chemical separation or enzymatic cleavage. In the case of ADCs in which a linker is attached to a lysine residue via an amide bond, the linker-cytotoxic drug can be released by enzymatic amide hydrolysis. In the case of ADCs in which a maleimide- or disulfide-containing linker is attached to a cysteine ​​residue, the S group of the cysteine ​​residue is reduced, allowing the conjugated linker-cytotoxic drug to be released via exchange. In the released state, the cysteine ​​residue of a monoclonal antibody can form a disulfide bond with other cysteine ​​amino acids or endogenous or exogenous substances containing S, such as glutathione (GSH). Therefore, while the ADC loses its mechanism of action via the cytotoxic drug at the target, the released linker-cytotoxic drug can further form adducts with other proteins or enzymes, or become metabolized and become active, causing toxicity.

[0285] A typical example of a redox-reactive linker is a disulfide linker. Disulfide linkers are also a type of chemically unstable linker that rely on redox reactions. After internalization, the linker is degraded by disulfide exchange or by reducing agents such as glutathione, releasing the cytotoxic drug.

[0286] 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 at concentrations of 0.5-10 mM intracellularly, but is present at concentrations up to 1,000-fold higher in hypoxic tumors. It is present at low concentrations (2-20 μM) in plasma. The disulfide linker provides high plasma stability, reducing nonspecific drug release and providing a relatively safe, tumor-specific linker.

[0287] In the present invention, an acid-sensitive linker refers to a linker that is stable in the neutral environment of blood (pH 7.3-7.5) but is hydrolyzed to release a drug in a weakly acidic environment, such as the periphery of tumor cells (pH 6.5-7.2) or in endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) after internalization within cells. Therefore, in the present invention, the acid-sensitive linker has a hydrophilic molecular structure that creates a hydrolysis-prone environment. For this purpose, the acid-sensitive linker may contain, for example, a polyethylene glycol (PEG) spacer.

[0288] However, acidic conditions are not limited to the tumor microenvironment and are often found outside the cell, which can lead to nonspecific drug release. Typical linkers include hydrazone linkers, and more recently, silyl ether linkers have been studied. These linkers have high plasma stability, with a plasma half-life of over 7 days, a significant improvement over the 2-3 days of hydrazone linkers.

[0289] The camptothecin drug (A) and the acid-sensitive linker (C) are preferably linked by a carbonate or ester bond so that they are decomposed in an acidic environment (pH ≦ 7) and the free camptothecin drug is released upon decomposition of the acid-sensitive linker.

[0290] The tetrapeptide linker has limitations, as it can cause ADC aggregation when conjugated with hydrophobic drugs. CL2A, the linker used in Trodelvy, an existing FDA-approved ADC, is a linker that meets all of the following characteristics: (i) storage stability after manufacturing, (ii) stability in the blood upon administration (almost no exposure of free payload in plasma), and (iii) rapid release of the payload in cancer tissue.

[0291] The Phe-Lys peptide originally inserted into the CL2 derivative allows cleavage via cathepsin B. As part of a synthetic streamlining effort, phenylalanine was removed from CL2A, thereby eliminating the cathepsin B cleavage site. This change had no effect on complex binding, stability, or potency, suggesting that release from the complex was primarily via cleavage of the pH-sensitive benzyl carbonate bond to the lactone ring of SN-38, rather than the cathepsin B cleavage site in CL2.

[0292] The acid-sensitive linker used in the present invention can utilize a CL2A linker and can be designed as shown in the following chemical formula 7 to selectively and efficiently deliver camptothecin-based drugs to cancer tissues. That is, the acid-sensitive linker in the present invention can be derived from a compound of the following chemical formula 7:

[0293] [ka]

[0294] wherein X1 and X2 are each independently -H or -halogen; Y is -NH-, -NR A - or nothing(null); Z is -C1-C4 alkyl-, -C3-C6 cycloalkyl-, -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-, -(C3-C6 cycloalkyl)-(C1-C2 alkyl)-, or -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-(C1-C2 alkyl)-; W is -R B -,-M--R B -M-, -MR B -or-R B -MR C - and; R A ~R C are each independently C1-C4 alkyl; M is

[0295] [ka]

[0296] and n is an integer of 5 to 9. Preferably, in the above Chemical Formula 7, X1 and X2 are each independently -H or -halogen; Y is -NR A - or nothing(null); Z is -C1-C4 alkyl-, -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-, or -(C3-C6 cycloalkyl)-(C1-C2 alkyl)-; W is -R B -or-R B -MR C - and; R A ~R C are each independently C1-C4 alkyl; M is

[0297] [ka]

[0298] and n can be an integer from 5 to 9.

[0299] In the present invention, the length of the linker, for example, n in Formula 7 above, may be an integer of 5 to 9, specifically, n may be an integer of 6 to 8, more specifically, n may be 7, but is not limited thereto. Even if it is outside the above range, as long as there is no difference in other effects due to the change in linker length, it is naturally included in the equivalent scope of the present invention.

[0300] The hydrophilicity of the drug can be improved by placing a polyethylene glycol (PEG) spacer between the drug and the antibody. Therefore, the linker in Formula 7 contains a low molecular weight PEG moiety containing a limited number (n = 5-9) of PEG monomers.

[0301] The acid-sensitive linker of Chemical Formula 7 is a custom-made linker that can be optimized depending on the target, payload, and carrier properties.

[0302] Camptothecin-based drugs have attachment sites that facilitate attachment to various linkers for the preparation of antibody-drug conjugates. For example, the alcohol group of camptothecin-based drugs can be used as an attachment site for the linker.

[0303] Therefore, in the present invention, the [camptothecin drug]-[acid-sensitive linker] may be a combination of the alcohol group site of the camptothecin drug and the alcohol group site of the acid-sensitive linker of Chemical Formula 7.

[0304] To develop new ADCs that can be used selectively for various solid cancers, it is essential to utilize linker systems that exceed the drug delivery efficiency of existing ADCs. To develop ADCs targeting new antigens beyond established drug targets such as Trop-2, Her2, and folate receptors, Development of ADCs against slow-internalizing antigens such as CEACAM-5 and / or cancer-specific antigens such as the NY-ESO-1 / HLA complex is necessary, but the limited drug delivery efficiency of existing Val-Cit or MAC-glucuronide linker systems makes it difficult to deliver sufficient amounts of drug.

[0305] For efficient drug delivery, particularly to antigens such as CEACAM-5, antibody-drug bonds must remain stable in the blood and / or surrounding environment of normal tissues, but must rapidly release the drug in the environment surrounding cancer cells, such as the tumor microenvironment.

[0306] If the drug can be rapidly released even in the tumor microenvironment surrounding the cancer, it can also be advantageously used to target various antigens (typically CEACAM-5, various cancer-specific antigen-HLA complexes, etc.) that have limited ADC absorption rates.

[0307] Therefore, camptothecin-based drugs that bind to and decompose DDX5 have improved water dispersibility compared to regular SN-38, preventing them from aggregating in body fluids such as blood and interstitial fluid. Taking full advantage of this fact, the present invention is characterized by the selection of an acid-sensitive linker that decomposes in the acidic environment around the cancer (pH ≦ 7) as a linker with a release profile that delivers the drug quickly and efficiently after reaching the cancer tissue.

[0308] Furthermore, the linker must be stable in the bloodstream, prevent the drug from separating from the antibody, maintain the drug in a prodrug state until it reaches the target, and minimize damage to normal tissues. However, the present invention uses an acid-sensitive linker with a hydrophilic molecular structure to create a hydrolysis environment, thereby alleviating the problem of ADC aggregation when linked to a hydrophobic drug.

[0309] Different catabolites are formed depending on the type of linker. In this regard, in the camptothecin drug-acid-sensitive linker of the present invention, the camptothecin drug and the acid-sensitive linker are preferably linked via a carbonate or ester bond so that the free camptothecin drug is released upon decomposition of the acid-sensitive linker.

[0310] Generally, carbamate bonds provide superior drug linker stability relative to ester and carbonate bonds in relation to hydrolysis. However, the present invention is characterized by the use of labile ester or carbonate bonds instead of carbamate bonds to design a camptothecin-based drug that can be cleaved from the drug linker both extracellularly and intracellularly in the acidic environment (pH) surrounding cancer cells.

[0311] The pH of blood is maintained at a constant level of 7.3 to 7.4. Therefore, camptothecin-based drugs are not cleaved from the acid-sensitive linker in blood, and even if cleaved, the release rate of camptothecin-based drugs from ADCs at the neutral pH of serum is even slower than that in the acidic environment of tumor tissue.

[0312] [FL118 drug of Chemical Formula 1-1]-[acid-sensitive linker] may be derived from any one selected from the group consisting of compounds represented by the following Chemical Formulas 7-1 to 7-3.

[0313] [ka]

[0314] [ka]

[0315] [ka]

[0316] (wherein each n is independently an integer of 5 to 9).

[0317] For an ADC to work effectively, it is not enough to simply maximize potent, antigen-selective cytotoxicity against cells in vitro; it must also be able to penetrate well into cancer tissues in vivo or in actual patients and deliver the drug efficiently.

[0318] The immunoconjugate of the present invention is targeted to cancer cells via the antigen-binding site that targets an antigen on the cancer cells. After that, the acid-sensitive linker is decomposed in the acidic environment (pH≦7) around the cancer, liberating at least a portion of the camptothecin drug. The free camptothecin drug is a hydrophobic small molecule, but has improved water dispersibility compared to SN-38 so that it does not aggregate in body fluids such as blood and interstitial fluid. Therefore, it can penetrate deep into the cancer tissue, penetrate the cell membrane, and migrate into the cells.

[0319] Therefore, the immunoconjugate of the present invention can rapidly release drugs in the tumor microenvironment surrounding the cancer by using an acid-sensitive linker that is degraded in the acidic environment (pH ≦ 7) surrounding the cancer. Free camptothecin-based drugs have a low molecular weight and, unlike antibodies, have high penetration ability into cancer tissues, thereby solving the problem of existing ADCs, which have the problem of antibodies not being able to penetrate deep into cancer tissues.

[0320] Therefore, the cells into which the free camptothecin-based drug is transported may be the targeted cancer cells and / or their surrounding cells.

[0321] In the present invention, the linker-antibody or antigen-binding site-containing fragment thereof may be linked by binding a thiol group contained in the antibody or antigen-binding fragment thereof to a maleimide group or maleic hydrazide group of the linker via a "click" reaction as shown in Reaction Scheme 1.

[0322] [ka]

[0323] The synthesis of linkers is very complicated, and the type of linker used affects the efficient release of cytotoxic drugs. Furthermore, even if linkers are attached to the same antibody amino acid sequence, the type and length of the linker and the position of the antibody to which the linker is attached can affect the biotransformation due to deconjugation of the linker from the perspective of the steric and electromagnetic environments.

[0324] The development of linkers must reflect the long half-life of monoclonal antibodies (mAbs), which is an advantage of mAbs, and ensure that mAbs are stable during systemic circulation. It is also important that the conjugation of the linker with the cytotoxic drug does not affect the stability and pharmacokinetics of the antibody.

[0325] In general, the catabolic events that can occur during systemic circulation of linker-cytotoxic drugs include the following, with various other events remaining to be defined: hydrazone cleavage, protease-mediated dipeptide cleavage, esterase-mediated carbamate cleavage, and acetate ester hydrolysis. of acetate ester, disulfide cleavage, succinimide ring opening.

[0326] Catabolism occurring at a specific site of the linker-cytotoxic drug may maintain its cytotoxic effect and be active at the target, but may cause toxicity in the systemic blood circulation. Conversely, if the cytotoxic effect is lost, the drug may not have a pharmacological effect even if it reaches the target.

[0327] A significant number of ADCs currently undergoing clinical trials employ chemical linkers such as hydrazone, disulfide, peptide, or thioether bonds. The process of drug release from the linker utilizes specific differences in pH or enzyme concentration within cancer cells. Hydrazone and disulfide linkers typically have limited stability in plasma. In contrast, peptide-based linkers have excellent plasma stability and allow for easy control of drug release.

[0328] Peptide-based valine-citrulline linkers are cleaved by cathepsin enzymes. Cleavable dipeptide linkers such as valine-alanine (Val-Ala) and valine-citrulline (Val-Cit) are cleaved by lysosomal extracts or purified human cathepsins. Since it undergoes rapid hydrolysis in the presence of B, the cleavage principle of the linker depends on the environment inside the cell.

[0329] Some chemical linkers balance the hydrophobicity of the antibody and drug, preventing aggregation of the ADC in the hydrophilic environment of the bloodstream. Hydrophilic linkers and spacers, such as cyclodextrin, polyethylene glycol (PEG), or other polymers, play a role in bloodstream stability and pharmacokinetic properties.

[0330] Hydrophilic spacer-containing linkers containing sulfonate or PEG, which exhibit high solubility in both organic solvents and aqueous solutions, overcome the various problems observed with hydrophobic linkers. PEG linkers offer advantages such as water solubility, low toxicity, low immunogenicity, and controlled linker chain length. In relation to this, studies have shown that the use of PEG linkers significantly improves in vivo pharmacokinetic profiles, increasing half-life and plasma concentration, and increasing the area under the plasma concentration-time curve (AUC).

[0331] The linker used in the present invention may be any known linker in the art, including, for example, CL-2A, MC-VC-PABC, MC-GGFG, MC-AAA, MC-VA-PABC, and Mac-glucuronide linker.

[0332] [Antibodies and their modifications] Therapeutic antibodies are used to treat a variety of diseases, including cancer, autoimmune and inflammatory diseases, and infectious diseases, and in some cases have revolutionized the treatment of serious illnesses. A variety of therapeutic antibodies are currently being used in three clinical settings: cancer, autoimmune and inflammatory diseases, and infectious diseases. Therapeutic antibodies used in cancer treatment target specific molecules expressed on tumor cells. Numerous studies have shown that these antibodies kill tumor cells through two mechanisms. IgG1 antibodies bound to tumor cells are recognized by the Fcγ receptors of macrophages, activating phagocytosis (referred to in cancer literature as antibody-dependent cellular phagocytosis, or ADCP). Furthermore, FcγRIII on NK cells binds to tumor cell-bound antibodies, inducing apoptosis of tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC). Another therapeutic antibody with a specific target, trastuzumab (Herceptin), reacts with the epidermal growth factor receptor (EGFR, also known as HER-2), which is found in some advanced breast cancers. EGF is necessary for tumor growth, and blocking the receptor deprives cells of this essential activator. Herceptin has also been shown to induce both ADCP and ADCC, providing protective functions.

[0333] Antibodies that react with two co-inhibitory receptors (CTLA-4 and PD-1) or the co-inhibitory ligand PD-L1 on tumor cells cooperate with the patient's immune system to eliminate tumors. Antibody blockade of inhibitory proteins such as CTLA-4, PD-1, and PD-L1 allows existing tumor-specific T cells to destroy highly difficult-to-treat cancers.

[0334] Monoclonal antibody therapeutic agents have many advantages over small molecule therapeutic agents. Their high target specificity allows them to act on off-target sites, resulting in fewer side effects. Their large molecular weight prevents them from passing through the blood-cerebrospinal wall, resulting in fewer side effects in the central nervous system. Furthermore, they are broken down into amino acids and metabolized by the reticuloendothelial system, bypassing hepatic metabolism and renal excretion. They have almost no drug interactions with other drugs, making them relatively safe for use in patients with liver or kidney disease. Their long half-life is an advantage, requiring only one dose every few weeks to months. However, because they are proteins, their large molecular weight means they are easily broken down in the digestive tract by protease enzymes, making them unsuitable for oral administration. Another disadvantage is that their long half-life means they cannot be quickly and non-invasively removed when unexpected side effects occur. Unlike small molecule drugs, This can lead to the production of neutralizing antibodies and hypersensitivity at the injection site. Furthermore, monoclonal antibodies cross the placenta during the second half of pregnancy, which may affect the development of the fetus and placenta.

[0335] To reduce immunogenicity in vivo, chimeric, humanized or fully humanized monoclonal antibodies have been produced, the sequences of which are partially or completely human.

[0336] Therapeutic antibodies have been engineered to optimize in vivo functions such as complement activation, Fc receptor binding to activate phagocytosis, ADCC, inhibitory signaling, and extended life span in the blood.

[0337] Human epidermal growth factor receptors (HER / EGFR / ERBB) include EGFR, HER2, FolR, PSMA, and Trop-2.

[0338] EGFR, the first identified growth factor receptor among receptor tyrosine kinases, is known to be overexpressed in over 70% of non-small cell lung cancers, and EGFR overexpression is associated with a poor prognosis. When EGFR binds to its ligand, it undergoes receptor dimerization, leading to autophosphorylation of the activation loop of the intracellular tyrosine kinase domain, activating key oncogene signaling pathways, including cell proliferation, cell survival, angiogenesis, and metastasis, via pathways such as mitogen-activated protein kinase (MAPK) and Akt. EGFR can be blocked using two approaches: 1) the use of EGFR monoclonal antibodies, which block ligand binding to EGFR, and 2) the use of EGFR-TKIs, small molecules that competitively interfere with ATP during the phosphorylation process of the EGFR tyrosine kinase domain intracellularly.

[0339] The use of EGFR-TKIs has been shown to induce apoptosis and have rapid effects.

[0340] Erlotinib and gefitinib were developed as tyrosine kinase inhibitors and are used as therapeutic agents for non-small cell lung cancer, and EGFR mutations are known as biomarkers.

[0341] EGFR mutations are known to selectively activate the Akt and STAT pathways, which promote cell proliferation and anti-apoptotic effects, but do not affect the ERK pathway, which is associated with cell proliferation.

[0342] Additionally, gefitinib and erlotinib are used as drugs for EGFR-mutant late-stage non-small cell lung cancer. Panitumumab and cetuximab, antibody drugs targeting EGFR, have been developed and are used to treat EGFR-wildtype metastatic colorectal cancer. Cetuximab, an anti-EGFR antibody targeting the ectodomain of EGFR, is approved for the treatment of late-stage colorectal cancer and squamous cell carcinoma, while panitumumab is approved for the treatment of metastatic colorectal cancer. Anti-EGFR therapeutic agents, for which KRAS mutations are known as biomarkers, have recently been shown to provide even higher survival rates in p16-negative squamous cell carcinoma.

[0343] Cetuximab, a monoclonal antibody against EGFR, has shown activity in cell lines resistant to EGFR-TKIs in vitro. ABX-EGF has been developed, and trastuzumab, which targets Her-2 / neu (erbB2), has been developed.

[0344] Cetuximab (C225, Erbitux) is a mouse-human chimeric monoclonal antibody that binds to domain III of the extracellular soluble portion of EGFR and blocks EGFR dimerization. Cetuximab is known to be more effective in combination with existing cytotoxic anticancer therapies than as a monotherapy. In one study, cetuximab was administered at a dose of 200-400 mg / m in combination with cisplatin. 2 It was confirmed that there was no cumulative toxicity at this dose, and in a phase 2 study of recurrent non-small cell lung cancer patients with EGFR expression of +1 or higher by immunohistochemistry, when it was treated in combination with docetaxel, it showed encouraging results, with a response rate of 25% and a disease control rate of over 60%. Based on this, a phase 3 study of combination treatment with docetaxel or pemetrexed is currently underway. Cetuximab is currently approved for colorectal cancer, and has been reported to have an increased effect when combined with radiation therapy for locally advanced head and neck cancer.

[0345] Unlike cetuximab, ABX-EGF is a fully humanized IgG2 monoclonal antibody known to have a higher affinity for EGFR than cetuximab. Although skin toxicity was reported as the most common toxicity in a phase 1 study, it was not a serious problem, and a phase 3 study of combination therapy with paclitaxel / carboplatin was conducted.

[0346] It is predicted that molecularly targeted anticancer drugs will be effective in treating cancer because they focus on individual cancer-specific cells or genes.

[0347] HER2 (human epidermal growth factor receptor-2), a receptor tyrosine kinase, is known to be overexpressed in breast cancer, and therefore has been proposed as a biomarker to predict the efficacy of drugs targeting HER2. In fact, three drugs developed to target HER2, trastuzumab, pertuzumab, and trastuzumab emtansine, have been shown to inhibit HER-2- It has been approved for the treatment of breast cancer.

[0348] Trastuzumab (Herceptin), a chimeric monoclonal antibody against HER2 / neu (erbB2), is an FDA-approved drug for metastatic breast cancer. To date, no notable results have been reported for non-small cell lung cancer, likely because Her2 expression is not observed significantly in patients with this cancer.

[0349] HER2 overexpression in breast cancer is known to promote cell survival, proliferation, angiogenesis, invasion, and metastasis. It has also been shown to be resistant to traditional systemic anticancer chemotherapy and predict poor prognosis in breast cancer, making it a target for tumor therapy. Therefore, HER2 receptor testing is essential before deciding on anticancer chemotherapy for metastatic breast cancer patients. HER2 overexpression can be assessed using immunohistochemical (IHC) staining (3+) or fluorescence in situ hybridization (FISH) or silver in situ hybridization (SISH) for HER2 IHC (2+).

[0350] Trastuzumab was developed in 1998 as a humanized monoclonal antibody that targets the extracellular domain of the HER2 protein and was the first to receive FDA approval.

[0351] Trastuzumab acts on the HER2 protein by first reducing the amount of overexpressed HER2, thereby suppressing cancer cell growth, resulting in cell death in only a small number of cancer cells. However, when administered in combination with cytotoxic anticancer drugs, cell death is known to be significantly increased. Second, it assists cancer-killing cells such as NK cells and macrophages in killing HER2-positive breast cancer cells.

[0352] Since the formation of new blood vessels is involved in tumor growth, tumor cells promote angiogenesis. Vascular endothelial growth factor (VEGF) is a substance required for this process, and VEGF receptors are overexpressed in tumor blood vessels compared to normal blood vessels. Bevacizumab is a recombinant humanized mouse monoclonal antibody that binds to VEGF and prevents it from binding to the receptor, thereby inhibiting the formation of new blood vessels. It exhibits anti-cancer effects by suppressing

[0353] The dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 To provide an EGFR ADC using antibodies less than K d The value is 1.8*10 -9Instead of cetuximab in M, K d The value is 1*10 -8 M or more 1*10 -6 Less than M, for example, 2.9*10 -8 Nimotuzumab M can be used.

[0354] The dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 To provide Trop-2 ADCs using antibodies less than K d The value is 1.7*10 -9 The Fv sequence was changed one amino acid at a time from sacituzumab M to K d The value is 1*10 -8 M or more 1*10 -6 Less than M, e.g., 4*10 -8 An antibody against M (sacituzumab-1) can be produced.

[0355] The dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 To provide Her2 ADCs using antibodies less than K d The value is 2.7*10 -9 The Fv sequence was changed one amino acid at a time from M trastuzumab to K d The value is 1*10 -8 M or more 1*10 -6 Less than M, for example, 3*10 -8 An antibody against M (trastuzumab-1) can be produced.

[0356] Antibodies are essential components of the adaptive immune response. Immunoglobulin G (IgG) is the most common type of antibody found in the circulation and extracellular fluids. While IgG alone can directly protect the body from infection through the activity of its antigen-binding region, most IgG immune functions are mediated through proteins and receptors expressed by specialized cell subsets that bind the fragment crystallinity (Fc) region of IgG. Fc gamma (γ) receptors (FcγRs) belong to a broad protein family that now includes not only classical membrane-bound surface receptors but also atypical intracellular receptors and cytoplasmic glycoproteins. Among the atypical FcγRs, the neonatal Fc receptor (FcRn) has gained increasing notoriety due to its intimate influence on IgG biology and its ability to also bind albumin. FcRn functions as a recycling or transcytosis receptor, responsible for maintaining IgG and albumin in the circulation and transporting these two ligands in both directions across polarized cell barriers. More recently, FcRn has been recognized to act as an immune receptor by interacting with peptides derived from IgG immune complexes (ICs) and promoting antigen presentation.

[0357] Antibodies bind to targets in the bloodstream and are taken up by cells. Once in the endosomal compartment, antibodies are protected from degradation by FcRn and recycled. In this manner, the long half-life of therapeutic antibodies is preserved, making them more effective even at substoichiometric levels.

[0358] Alterations to the hinge and Fc sequences by mutation have been made to improve desired functional properties of the antibody.

[0359] For example, the Fc region was engineered to produce an antibody with a 50-fold higher binding affinity for the C1q complement component, allowing a single IgG antibody to activate complement-mediated tumor cell killing.

[0360] Other amino acid substitutions in the Fc region, which is recognized by FcRn in vascular endothelial cells, can increase the half-life of IgG in the circulation. For many therapeutic antibodies, extending their lifespan in vivo can significantly benefit patients in terms of antibody efficacy, reduced injection frequency, and reduced treatment costs.

[0361] Therapeutic immunoglobulin G (IgG) antibodies are endogenous to the neonatal Fc receptor (FcRn). It has a relatively long half-life because it binds to IgG Fc at the acidic pH of the blood, protecting IgG from degradation. To further extend the half-life, amino acid-substituted antibodies with high affinity for FcRn have been developed, and one such therapeutic antibody (ravulizumab) has been approved.

[0362] As the use of monoclonal antibodies (mAbs) expands as a treatment for various human diseases, including chronic inflammation, infection, cancer, autoimmune diseases, cardiovascular disease, and transplantation medicine, FcRn has emerged as a key regulator of mAb efficacy. This directly correlates with the persistence of therapeutic antibodies in the bloodstream, which ultimately increases their localization to target sites. pH-dependent binding and FcRn-dependent recycling are crucial to ensure a long circulating half-life of IgG. Importantly, limited binding at neutral pH is necessary for proper release of IgG from cells, while increased mAb affinity for FcRn at acidic pH correlates with prolonged half-life. Therefore, IgG Fc engineering to optimize pH-dependent binding to FcRn has been developed to tune pharmacokinetics and increase mAb half-life. For example, MST mutations (Met252Tyr / Ser254Thr / Thr256Glu) increased IgG persistence by up to five-fold in humans and monkeys. In a phase 2 clinical trial, the IgG MST variant demonstrated a half-life of 80–120 days. Similarly, the MN (Met428Leu / Asn434Ser) mutation adjacent to the critical FcRn binding site of the IgG Fc shows potential for extending IgG half-life for therapeutic antibodies.

[0363] For example, a library containing random amino acid mutations in specific regions of the constant domain of human IgG molecules can be screened to collect and characterize mutations that increase the affinity of the IgG molecule for FcRn, thereby preparing antibodies with extended serum half-lives.

[0364] The K d The value is 1*10 -8 M or more 1*10 -6 The various antibodies and various engineered antibodies described above can be utilized to provide antibodies or antigen-binding site-containing fragments (B) that have a binding affinity less than M or that are higher when binding bivalently than monovalently.

[0365] [Pharmaceutically acceptable salts] As used herein, "pharmaceutically acceptable salts" refers to 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; 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; and salts of amino acids such as lysine, arginine, and guanidine. Also included are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, and benzethonium, which can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts referred to in the present invention are not limited to these listed salts.

[0366] [Various Carrier-Drug Conjugates] It is very important to easily deliver ADC or other targeted drug conjugate substances into cancer tissues. The [camptothecin-based drug]-[linker] conjugate of the present invention can be applied to various types of drug carriers other than antibodies, and unlike antibodies, it can penetrate deep into cancer tissues and, by using carriers with easy CMC properties, can be effectively used in various applications. The carrier can be an antigen-binding portion of an antibody, a peptide, a repibody, and / or an aptamer.

[0367] [Table 1]

[0368] Aptamer-drug conjugates (ApDCs) are ADCs that use an aptamer instead of the antibody. Aptamers are single-stranded nucleic acids with a three-dimensional structure. They are discovered through the "SELEX" (Systematic Evolution of Ligands by Exponential Enrichment) process. SELEX is a technology that involves adding target protein molecules to a compound library and obtaining functional nucleic acids that bind to them.

[0369] Aptamers, also known as chemical antibodies, can bind to targets with very strong and selective binding. Aptamers are approximately 20 kDa in size and are known to have superior cell permeability and low immunogenicity compared to antibodies. Aptamers can be chemically synthesized, allowing precise design of the conjugation position and number of drugs when producing aptamer-drug conjugates. Production costs are lower than ADCs. Aptamers are generally composed of natural nucleic acids and are easily degraded by nucleases in the body, resulting in reduced in vivo stability. However, the ease with which aptamers can be chemically modified can be used to overcome the limitations on the stability of modified aptamers.

[0370] Peptide-drug conjugates (PDCs) are ADCs that incorporate peptides instead of antibodies. Peptides are composed of amino acids and range in size from 500 to 5,000 Da (Daltons). This is significantly smaller than antibodies, which are over 150 kDa (kilodaltons). Therefore, peptide-based PDCs have superior cell penetration ability compared to ADCs and are less likely to cause immunogenicity. Furthermore, peptides can be chemically synthesized. This not only reduces production costs, but also allows precise control of the binding site and ratio of peptides and drugs.

[0371] In general, peptides have a short biological half-life due to their susceptibility to proteolytic degradation. To overcome this limitation of peptide-based drug conjugates, strategies have been proposed that utilize modified peptides, such as cyclic peptides and peptides incorporating unnatural amino acids.

[0372] Repebodies are a type of artificial antibody that does not have an antibody skeleton but has the ability to recognize antigens like antibodies. Repebodies specific to target proteins are , can be discovered through phage display.

[0373] Phage display is a technology that expresses desired proteins on the surface of bacteriophages. Repebodies are approximately 30 kDa in size, 20% the size of antibody drugs. Therefore, they are known to have relatively low immunogenicity and improved cell permeability compared to antibodies. In addition, the thermal and pH stability of Repebodies can be adjusted, which is expected to enhance their structural stability. Their production costs are also considered to be relatively low compared to antibodies. Due to these advantages of Repebodies, there is growing interest in the development of Repebody-drug conjugates (Repebody-DCs) as a strategy to convert antibodies into Repebodies.

[0374] Therefore, the method for producing the carrier-drug conjugate of the present invention includes the following steps:

[0375] The present invention is characterized in that the [camptothecin drug]-[acid-sensitive linker of Chemical Formula 2] conjugate or a pharmaceutically acceptable salt thereof according to the present invention is used to link one or more camptothecin drugs to a carrier via the linker of Chemical Formula 2.

[0376] [Pharmaceutical composition for preventing or treating cancer] The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-described immunoconjugate according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

[0377] In addition, one embodiment of the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof. The subject may be a mammal, including a human.

[0378] The immunoconjugate of the present invention specifically binds to an antigen on a cancer cell, releases a drug inside or outside the cancer cell, and exhibits cytotoxicity, and therefore can be useful for treating or preventing cancer. The anticancer activity of the immunoconjugate of the present invention is as described above.

[0379] In the present invention, the cancer may be a solid cancer or a blood cancer, such as myxoma, Intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cavity cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, epithelial ovarian cancer, ovarian germ cell tumor, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus and nasal cavity cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, childhood brain cancer, childhood lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, Renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, The cancer may be one or more selected from the group consisting of, but not limited to, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid, 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 cancer, metastatic brain cancer, mediastinal tumor, rectal cancer, rectal carcinoid, vaginal cancer, spinal cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsillar cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer. Furthermore, the cancer includes not only primary cancers but also metastatic cancers.

[0380] The term "therapeutically effective amount" as used herein refers to an amount of the immunoconjugate that is effective in treating or preventing cancer. Specifically, a "therapeutically effective amount" refers to a medically "Effective dose" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to that treatment, and the effective dose level can be determined based on factors including the type and severity of the individual, age, sex, type of disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present 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 singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect at the minimum dose without side effects. Since the immunoconjugate of the present invention exhibits dose-dependent effects, the administration dose can be determined depending on various factors such as the patient's condition, age, sex, and comorbidities. Therefore, it can be easily determined by a person skilled in the art. The active ingredient of the pharmaceutical composition of the present invention has excellent safety, so it can be used even at a dose higher than the determined dose.

[0381] According to one embodiment of the present invention, there is also provided a use of the immunoconjugate for the manufacture of a medicament for use in the treatment or prevention of cancer. The immunoconjugate for the manufacture of the medicament can be prepared by adding an acceptable adjuvant, diluent, carrier, or the like to the immunoconjugate. It can be mixed with other active ingredients to produce a complex preparation, which can have a synergistic effect of the active ingredients.

[0382] The matters mentioned in the uses, compositions, and treatment methods of the present invention are all applicable to the same unless they contradict each other. [Effects of the Invention]

[0383] The present invention provides a K d The value is 1*10 -8 M or more 1*10 -6 It uses an antibody less than M and a camptothecin-based topoisomerase I inhibitor that degrades the DDX5 protein as a payload, which has stronger potency than existing ADCs. It is possible to provide an antibody-drug conjugate (ADC) that can exhibit high efficacy while ensuring safety.

[0384] The [antibody or antigen-binding site-containing fragment thereof that exhibits higher binding affinity when bivalently binding to a cell surface protein epitope than monovalently binding] used in the present invention is designed to target [cell surface proteins expressed on both normal and cancer cells]. Due to the characteristic of antibodies that undergo receptor-mediated internalization, the stronger the binding affinity to the antigen epitope, the greater the degree of internalization that can occur. By utilizing this binding affinity to the antigen, it is possible to solve the problem of on-target toxicity. In addition, when used in combination with [camptothecin-based drugs that degrade DDX5 protein], it is possible to increase the therapeutic effect through the mechanism of apoptosis.

[0385] The present invention provides a dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 Immunoconjugates using an antibody or its antigen-binding site-containing fragment (B-1) of less than M; or an antibody or its antigen-binding site-containing fragment (B-2) that binds to a cell surface protein expressed on both normal and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than monovalently can improve target tissue penetration and maximize the targeted therapeutic effect. [Brief explanation of the drawings]

[0386] [Figure 1] FIG. 1 shows the synthetic design concept of a new active camptothecin derivative (a) that has a dual mechanism of action (MoA) that degrades the oncoprotein DDX5 as well as type I topoisomerase inhibitory activity, based on an improved FL118 structure. [Figure 2]FIG. 2 is a diagram showing the mechanism of action of cell membrane receptors (HER2, EGFR, and other receptor tyrosine kinases) to which growth factors bind. [Figure 3] Figure 3 is a schematic diagram showing the efficient drug release mechanism and anticancer mechanism of action of ADCs (e.g., PBX-001) using an FL118-acid-sensitive linker. [Figure 4] Figure 4 shows the analytical results confirming that the DAR of FL118-CL2A linker-sacituzumab (PBX-001) was 8. [Figure 5] FIG. 5 shows the results showing that FL118 and exatecan drugs have better efficacy against in vitro cytotoxicity than SN-38. [Figure 6] Figure 6 shows Western blot results showing the degree of inhibition of anti-apoptotic proteins, indicating that FL118 and exatecan drugs have better efficacy in down-regulating tumor proteins than SN-38 drug in HCT-8 and FaDu cell lines. [Figure 7] Figure 7 shows Western blot results showing the presence / extent of DDX5 and p-DDX5 protein degradation by various camptothecin-based drugs (FL118, SN-38, exatecan, PBX-7011, PBX-7014, and PBX-7016) in the A549 cell line, as well as the degree of inhibition of various other anti-apoptotic proteins. [Figure 8] FIG. 8 shows the results of a Western blot experiment (FIG. 7) performed on the A549 cell line, where the concentrations were quantified in a graph. [Figure 9] FIG. 9 shows the results of a comparative evaluation of in vitro cell viability in FaDu or A549 cell lines in response to various camptothecin-based drugs. [Figure 10] FIG. 10 shows the results of a comparative evaluation of in vitro cell viability in MDA-MB-453 (HER2++) and FaDu (HER2+) cell lines in response to various camptothecin-based drugs. [Figure 11]Figure 11 shows the mechanism by which FL118-CL2A linker-sacituzumab (PBX-001) overcomes the ABCG2 efflux pump: cells with the ABCG2 efflux pump are resistant to Trodelvy but are unaffected by PBX-001. [Figure 12] Figures 12 and 13 show the evaluation results of FL118-CL2A linker-sacituzumab (PBX-001, DAR8) in the FaDu xenograft model, demonstrating excellent anti-cancer efficacy without weight loss in the in vivo xenograft model. [Figure 13] Figures 12 and 13 show the evaluation results of FL118-CL2A linker-sacituzumab (PBX-001, DAR8) in the FaDu xenograft model, demonstrating excellent anti-cancer efficacy without weight loss in the in vivo xenograft model. [Figure 14] Figure 14 shows the results of confirming the production of ADC of cetuximab-CL2A-FL118 (DAR8) using LC-MS. [Figure 15] Figure 15 shows the results of confirming the production of ADC of nimotuzumab-CL2A-FL118 (DAR8) using LC-MS. [Figure 16] FIG. 16 confirms EGFR expression in various cell lines and shows the cell line selection process for in vitro and in vivo experiments targeting EGFR. [Figure 17] Figure 17 shows the results of a comparison of the binding avidity to EGFR of nimotuzumab and nimotuzumab modified to extend its serum half-life (YTE) with ADCs conjugated with the FL118 drug-CL2A linker or the PBX-7016 drug-GGFG linker. [Figure 18] Figure 18 shows the results of in vitro assays of various nimotuzumab ADCs in EGFR-positive and -negative cells. [Figure 19] FIG. 19 shows the results of in vitro assays of nimotuzumab ADC and cetuximab ADC in EGFR-positive and -negative cells. [Figure 20]FIG. 20 shows the results of in vitro assays of nimotuzumab ADC and nimotuzumab (YTE) ADC in EGFR-positive and -negative cells. [Figure 21] Figure 21 shows the results of in vivo evaluation of the EGFR-targeted ADC cetuximab-CL2A-FL118 in the MDA-MB-468 model. [Figure 22] Figure 22 shows the results of in vivo evaluation of the EGFR-targeted ADCs cetuximab-CL2A-FL118 and nimotuzumab-CL2A-FL118 in the MDA-MB-468 model. [Figure 23] Figure 23 shows the results of in vivo evaluation of EGFR-targeted ADCs nimotuzumab-GGFG-FL118 / PBX-7016 and nimotuzumab(YTE)-GGFG-FL118 / PBX-7016 prepared in Example 5 in the MDA-MB-468 model. [Figure 24] FIG. 24 shows the results of carboxylate to lactone form conversion at pH 7.4. [Figure 25] FIG. 25 shows the results of carboxylate to lactone form conversion at pH 6.0. [Figure 26] Figure 26 shows the mechanism of ADC toxicity (Source: Cancers 2023, 15(3), 713; https: / / doi.org / 10.3390 / cancers15030713) [Figure 27] FIG. 27 illustrates the mechanism of action of various self-immolative spacers. [Figure 28] FIG. 28 is a conceptual diagram of monovalent and divalent or polyvalent bonds. [Figure 29] FIG. 29 is a conceptual diagram showing how cell surface receptors bind to multivalent antigens to form clusters. DETAILED DESCRIPTION OF THE INVENTION

[0387] The present invention will be described in more detail below with reference to examples, which are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of protection of the present invention.

[0388] Preparation Example 1: Synthesis of active camptothecin derivatives (PBX-7011 and PBX-7012) of formula 3 or formula 3-1

[0389] [ka]

[0390] 1-1. Synthesis of Compound 2 [ka]

[0391] To a solution of 5-nitrobenzo[d][1,3]dioxole (25 g, 150 mmol) in dichloromethane (748 mL) was added silver triflate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol). The solution was stirred in a dark room at room temperature under a N atmosphere. The AgI was removed by filtration, and the solid was washed with dichloromethane (100 mL). The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc (250 mL) and 5% (v / v) NH4OH / H2O. The mixture was partitioned between HCl and HCl (200 mL). The organic layer was separated, washed with 1 M NaSO (5 x 200 mL) and brine (200 mL), dried over NaSO, treated with activated carbon, and filtered through celite. The solvent was evaporated under reduced pressure to provide the crude product as a brown solid. This was triturated with ice-cold EtOH (400 mL), filtered, and the solid was washed with ice-cold EtOH (100 mL) to provide the product as a light brown-gray solid (14.3 g). The solvent was removed from the filtrate, and the crude product was triturated a second time with ice-cold EtOH (300 mL). The solid was collected by filtration and washed with EtOH (50 mL) to provide an additional amount of product (10.2 g) as a dark brown-gray solid. Both batches were used directly without further purification. SC_ACID: m / z 294.2[M+H] +

[0392] 1-2. Synthesis of Compound 3 [ka]

[0393] To a suspension of 4-iodo-6-nitrobenzo[d][1,3]dioxole (8.75 g, 29.9 mmol) in a mixture of water (80 mL), methanol (40.0 mL), and tetrahydrofuran (40.0 mL) was added iron powder (6.67 g, 119 mmol) and ammonium chloride (6.39 g, 119 mmol). The suspension was heated to 75 °C and stirred for 16.5 h. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure. The resulting black solid was suspended in ethyl acetate (100 mL) and the solution was poured off. The residue was washed with EtOAc (3 × 50 mL). The mixture was transferred to a separatory funnel, water was added, and the aqueous layer was removed. The organic layer was washed with saturated aqueous NaHCO solution (150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and the solvent removed under reduced pressure to give a brown solid (4.75 g, 60% yield). The iron residue was washed thoroughly with ethyl acetate to recover additional product. The organic fraction was then washed with saturated aqueous NaHCO3 solution (150 mL), brine (150 mL), and dried over Na2SO4. The solvent was removed under reduced pressure to give a brown solid (1.74 g, 22% yield). SC_ACID: m / z 264.0[M+H] +

[0394] 1-3. Synthesis of Compound 4 [ka]

[0395] To a solution of 7-iodobenzo[d][1,3]dioxol-5-amine (6.39 g, 24.29 mmol) in dichloromethane (49 mL) was added acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol). The reaction mixture was stirred at room temperature overnight. After several hours, additional DCM (10 mL) was added. The suspension was filtered through a sintered funnel and washed with ice-cold DCM (10 mL). The product was further dried under reduced pressure to give a light gray solid (4.46 g). The filtrate was concentrated under reduced pressure, and the residue was dissolved in EtOAc, washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to give a brown solid (~3 g). The brown solid was purified by flash column chromatography (80 g Si, 0–100% ethyl acetate in heptane). All batches of product were triturated from ice-cold EtOAc (5-10 mL). The two batches were combined and further dried to give an off-white solid. Total yield: 5.0 g, 66% SC_ACID: m / z 306.0[M+H] + 1 H NMR (400MHz, DMSO-d6) δ9.88(s, 1H), 7.39(d, J=1.9Hz, 1H), 7.17(d, J=1.9Hz, 1H), 6.04(s, 2H), 1.99(s, 3H).

[0396] 1-4. Synthesis of Compound 5 [ka]

[0397] A slurry of N-(7-iodobenzo[d][1,3]dioxol-5-yl)acetamide (5.06 g, 16.59 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol), and potassium carbonate (2.98 g, 21.56 mmol) in acetonitrile (40 mL) was cooled to 0-5 °C in a three-necked flask equipped with a condenser. Water (13.33 mL) was slowly added to evolve gas. When gas evolution ceased, the mixture was degassed with Ar for 30 min. Tri-o-tolylphosphine (0.505 g, 1.659 mmol) and palladium acetate (0.186 g, 0.829 mmol) were added, and the mixture was degassed for an additional 30 min before being heated to reflux under Ar. The reaction was cooled to room temperature and filtered through celite. The filter cake was washed with HO and EtOAc. The organic solvent was removed from the filtrate under vacuum, and the aqueous material was acidified with concentrated HCl solution. The aqueous layer was extracted with EtOAc until pH=1-2, and the combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was then cooled and concentrated in ice. Trituration from EtOAc (30 mL) and collection of the solid by filtration afforded the product as a brown solid. The mother liquor was concentrated under reduced pressure and purified via flash chromatography (80 g Si, 0 to 100% EtOAc in heptane). The product fractions were concentrated under reduced pressure to afford a light brown foam. Total yield: 3.46 g, 75%. A mixture of E / Z isomers was obtained. SC_ACID: m / z 264.4[M+H] +

[0398] 1-5. Synthesis of Compound 6 [ka]

[0399] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) in tetrahydrofuran (50 mL) / water (50 mL) was degassed with N for 15 min. Pd / C (2.97 g, 1.397 mmol) was added and the suspension degassed with H for 5 min before heating under H (balloon) at 40 °C. After 24 h, nitrogen and additional Pd / C (2.97 g, 1.397 mmol) were added to the reaction mixture. Hydrogen was bubbled through the reaction mixture for 10 min and stirred under hydrogen at 45 °C overnight. The reaction was filtered through celite. The filter cake was washed with H2O (50 mL). EtOAc (50 mL) and organic solvents were removed from the filtrate under vacuum. The aqueous solution was acidified with concentrated HCl to pH = 1, and the dark brown / green precipitate was collected by filtration through a sintered funnel. The aqueous filtrate was extracted. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent removed in vacuo to give a brown oil. Due to low product recovery, the filter cake was added to a flask containing EtOAc (50 mL), water (200 ml), and MeOH (400 ml), the water / EtOAc flush, and the solid from the MeOH flush. The aqueous layer was acidified with concentrated HCl until pH = 1, and an off-white precipitate formed, which was collected by filtration through a sintered funnel. After extracting the aqueous filtrate with EtOAc (3 x 200 mL), LCMS indicated that all the product had been removed aqueously. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent removed in vacuo to give a light brown solid. All product batches were combined and purified via flash column chromatography (40 g Si, 0-10% MeOH in DCM). The product fractions were concentrated to give a light brown solid. Yield: 2.52 g, 72%. SC_ACID:266.2[M+H] + 1H NMR (400MHz, DMSO-d6) δ12.07(s, 1H), 9.78(s, 1H), 7.14(d, J=2.0Hz, 1H), 6.80(d, J=2.2Hz, 1H), 5.95(s, 2H), 2.50-2.46(m, 2H), 2.23(t, J=7.4Hz, 2H), 1.98(s, 3H), 1.84-1.70(m, 2H).

[0400] 1-6. Synthesis of Compound 7 [ka]

[0401] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)butanoic acid (150 mg, 0.565 mmol) in TFA (433 μL, 5.65 mmol) was cooled on ice. TFAA (157 μL, 1.131 mmol) was added. The mixture was stirred at 0–5°C for 1 h, over which time it turned to a dark solution. The reaction mixture was added dropwise to ice-cold saturated aqueous NaHCO3 solution (10 mL), and the aqueous solution was extracted with ethyl acetate (3 × 25 mL). The combined organic layers were dried over saturated NaHCO3 and brine, filtered, and the solvent was removed in vacuo to give a pale pink solid. Yield: 140 mg, 100%. SC_ACID: m / z 248.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ12.34(s, 1H), 8.11(s, 1H), 6.13(s, 2H), 2.80(t, J=6.2Hz, 2H), 2.66-2.58(m, 2H), 2.12(s, 3H), 2.01-1.91(m, 2H).

[0402] 1-7. Synthesis of Compound 8 [ka]

[0403] A suspension of N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (50 mg, 0.202 mmol) in tetrahydrofuran (1.2 mL) was cooled to 0 °C, and potassium tert-butoxide (27.2 mg, 0.243 mmol) and isoamyl nitrite (35.0 μL, 0.263 mmol) were added. The dark green mixture was stirred on ice (<5 °C) for 1.5 h. Acetic acid (170 μL, 2.94 mmol), acetic anhydride (170 μL, 1.810 mmol), and zinc dust (66.1 mg, 1.011 mmol) were added to the reaction mixture. The suspension was stirred at 0 °C for 2 h. The reaction mixture was filtered through celite and flushed with DCM. The filtrate was concentrated under reduced pressure to give a black oil. The crude product was purified using flash column chromatography (4 g Si, 0-4% MeOH in DCM). The product fractions were concentrated to give a grey solid (32 mg, 52%) in 80-90% purity. C, a sample of even higher purity can be obtained. SC_ACID: m / z 305.4[M+H] + 1 H NMR (400MHz, DMSO-d6) δ12.10(s, 1H), 8.21(d, J=8.0Hz, 1H), 8.12(s, 1H), 6.15(d, J=9.3Hz, 2H ), 4.66-4.56(m, 1H), 2.93(dd, J=8.9, 4.0Hz, 2H), 2.14(s, 3H), 2.13-1.93(m, 2H), 1.91(s, 3H).

[0404] 1-8. Synthesis of Compound 9 [ka]

[0405] N,N'-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diacetamide (244 mg, 0.802 mmol) was suspended in 2 M hydrochloric acid (4.69 mL, 9.38 mmol) in ethanol / water (5 / 1). The mixture was heated at 55 °C for 4 h. The black mixture was allowed to cool to 0-5 °C. Triethylamine (1.4 mL, 10.04 mmol) was added dropwise with stirring. The mixture was then diluted with EtOH and evaporated to dryness. The residue was partitioned between water and DCM. The layers were separated, and the aqueous layer was extracted once with DCM. The combined organic layers were dried over Na2SO4 and concentrated to give the product as a brown solid (178 mg, 73%) in 86% purity. SC_ACID: m / z 263.0[M+H] + 1 H NMR (400MHz, DMSO) δ8.04(d, J=8.0Hz, 1H), 6.20(s, 1H), 5.94(d, J=6.0Hz, 2H), 4.48-4.41( m, 1H), 3.08(s, 2H), 2.88-2.69(m, 2H), 2.15-2.03(m, 1H), 1.88(s, 3H), 1.86-1.75(m, 1H).

[0406] 1-9. Synthesis of Compound 10 [ka]

[0407] (4S)-4-Ethyl-7,8-dihydro-4-hydroxy-1H-pyrano[3,4- f] Indolizine-3,6,10(4H)-trione (146 mg, 0.555 mmol) and N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (112 mg, 0.427 mmol) were dissolved in dry toluene (4.5 ml), PPTS (21 mg, 0.085 mmol) was added, and the reaction mixture was stirred at 115°C for 40 hours. The reaction mixture was allowed to cool to room temperature. The suspension was diluted with 2 mL DCM and filtered to give a black residue (210 mg). The crude product was purified by column chromatography (12 g Si, 0-7% methanol in DCM) to afford the product (45 mg, 21%) as a brown solid. LCMS analysis showed two partial stereoisomers. SC_ACID: m / z 490.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.47(t, J=9.3Hz, 1H), 7.42(s, 1H), 7.24(s, 1H), 6.49(s, 1H), 6.29(d, J=5.2Hz, 2H), 5.57-5.49(m, 1H) , 5.41(s, 2H), 5.23-5.07(m, 2H), 3.09-3.00(m, 2H), 2.11-2.01(m, 2H), 1.91(s, 3H), 1.89-1.79(m, 2H), 0.87(t, J=7.1Hz, 3H).

[0408] 1-10.Synthesis of PBX-7011 and PBX-7012 [ka]

[0409] N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]ky (Norin-1-yl)acetamide (73.5 mg, 0.150 mmol) was dissolved in 1.0 mL of 6 N HCl and stirred at 90 °C for 8 hours, followed by overnight at room temperature. The reaction mixture was concentrated under reduced pressure and purified by two runs (Luna 2-30) using acidic preparative MPLC. The fractions containing the separated partial stereoisomers were acidified with 5 drops of 3 N HCl and lyophilized to give the product as a yellow solid. First eluting isomer: PBX-7011, 23 mg, 34% yield U_AN_ACID: m / z 448.4[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.54(s, 3H), 7.50(s, 1H), 7.27(s, 1H), 6.52(s, 1H), 6.34(d, J=13.3Hz, 2H), 5.77(d, J=19.3Hz, 1H), 5.44(s, 2 H), 5.37(d, J=19.3Hz, 1H), 5.05(s, 1H), 3.19-3.02(m, 2H), 2.46(s, 1H), 2.20-2.03(m, 1H), 1.94-1.81(m, 2H), 0.88(t, J=7.3Hz, 3H). Second eluting isomer: PBX-7012, 28 mg, 41% yield U_AN_ACID: m / z 448.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.57(d, J=4.7Hz, 3 H), 7.51(s, 1H), 7.27(s, 1H), 6.52(s, 1H), 6.34(d, J=12.2Hz, 2H), 5.76(d, J=19.4Hz, 1H), 5.44(s, 2H), 5.37(d, J=19) .4Hz, 1H), 5.08(s, 1H), 3.16-2.98(m, 2H), 2.46(s, 1H), 2.18-2.06(m, 1H), 1.94-1.80(m, 2H), 0.87(t, J=7.3Hz, 3H).

[0410] Preparation Example 2: Synthesis of active camptothecin derivatives (PBX-7014 and PBX-7015) of formula 4 or formula 4-1 This example describes the synthesis of compounds PBX-7014 and PBX-7015 starting from two separated partial stereoisomers of exatecan-hybrid compounds (PBX-7011 and PBX-7012).

[0411] [ka]

[0412] 2-1: Synthesis of PBX-7014 [ka]

[0413] A stock solution of activated glycolic acid was prepared by the following procedure: Glycolic acid (17 mg, 0.224 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. Next, 0.4 mL of activated acid solution was added to a suspension of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (40 mg, 0.089 mmol) and triethylamine (0.025 mL, 0.179 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 3 h. 0.05 mL of freshly prepared activated acid solution was added. The reaction mixture was then stirred at room temperature for an additional 2 h. The reaction mixture was evaporated to dryness. The crude product was purified by column chromatography (0-8% methanol in chloroform). This provided a yellow solid containing the PBX-7014 product and residual succinimide. The product was further purified by acidic preparative MPLC (Luna 5-40) to give a bright yellow solid after lyophilization of the product fractions. Yield: 20 mg, 50% U_AN_ACID: m / z 506.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.40(d, J=8.9Hz, 1H), 7.40(s, 1H), 7.23(s, 1H), 6.49(s, 1H), 6.28(d, J=4.6Hz, 2H), 5.61-5.45(m, 2H), 5 .45-5.35(m, 2H), 5.19-5.06(m, 2H), 3.95(s, 2H), 3.13-2.96(m, 2H), 2.21-2.02(m, 2H), 1.94-1.77(m, 2H), 0.87(t, J=7.3Hz, 3H).

[0414] 2-2: Synthesis of PBX-7015 [ka]

[0415] A stock solution of activated acid was prepared by the following procedure: Glycolic acid (17.00 mg, 0.223 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour. Next, (1R,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3', To a suspension of 4':6,7]indolizino[1,2-b]quinoline-11,14-dione (25 mg, 0.056 mmol) and triethylamine (0.016 mL, 0.112 mmol) was added 0.25 mL of activated acid solution. The mixture was stirred overnight at room temperature. 0.03 mL of freshly prepared activated acid solution was added. The reaction mixture was then stirred at room temperature for 2 hours. The reaction mixture was combined with a previous smaller batch and evaporated to dryness. The crude product was purified by column chromatography. This provided a yellow solid containing the PBX-7015 product and residual succinimide. The product was purified by acidic preparative MPLC (Luna 5-40). The product fractions were lyophilized to give a bright yellow solid. Yield: 15 mg, 38% U_AN_ACID:506.2[M+H] + 1H NMR (400MHz, DMSO) δ8.44(d, J=9.0Hz, 1H), 7.41(s, 1H), 7.24(s, 1H), 6.48(s, 1H), 6.29(d, J=2.3Hz, 2H), 5.61-5.44(m, 2H), 5.44-5. 36(m, 2H), 5.20-5.07(m, 2H), 3.96(s, 2H), 3.10-2.96(m, 2H), 2.20-2.06(m, 2H), 1.95-1.79(m, J=7.3Hz, 2H), 0.87(t, J=7.3Hz, 3H).

[0416] Preparation Example 3: Synthesis of active camptothecin derivative (PBX-7016) of formula 5 This example describes the synthesis of compound PBX-7016 starting from an exatecan-hybrid compound (PBX-7011). [ka]

[0417] A stock solution of activated D-lactic acid was prepared by the following procedure. D-Lactic acid (22 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (27 mg, 0.235 mmol) and EDC (38.6 mg, 0.201 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Next, 0.3 mL of activated acid solution was added to a solution of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (36 mg, 0.080 mmol) and triethylamine (0.022 mL, 0.161 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature for 6 hours. 0.05 mL of the prepared activated acid solution was added. The reaction mixture was then stirred overnight at room temperature. The reaction mixture was directly purified by acidic preparative MPLC (Luna 10-50), yielding a bright yellow solid after lyophilization of the product fraction. Yield: 22 mg, 52% U_AN_ACID: m / z 520.2[M+H] + 1 H NMR (400MHz, DMSO) δ8.43(d, J=9.1Hz, 1H), 7.41(s, 1H), 7.23(s, 1H), 6.50 (s, 1H), 6.29(d, J=2.1Hz, 2H), 5.62-5.58(m, 1H), 5.58-5.51(m, 1H), 5.41 (s, 2H), 5.21-5.01(m, 2H), 4.17-4.07(m, 1H), 3.14-2.95(m, 2H), 2.19-2. 04(m, 2H), 1.92-1.78(m, 2H), 1.39(d, J=6.8Hz, 3H), 0.87(t, J=7.3Hz, 3H). As described above, PBX-7016 of Chemical Formula 5 can be synthesized from PBX-7011 of Chemical Formula 3, and PBX-7017 of Chemical Formula 5-1 can be synthesized from PBX-7012 of Chemical Formula 3-1 in the same manner.

[0418] Preparation Example 4: Synthesis of active camptothecin derivative (PBX-7024) of formula 9 [ka]

[0419] PBX-7024 was produced in high yield by coupling (2S)-2-cyclopropyl-2-hydroxyacetic acid to the PBX-7011 compound. (2S)-2-Cyclopropyl-2-hydroxyacetic acid (26 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (26 mg, 0.226 mmol) and EDC (42 mg, 0.219 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Next, (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14 in N,N-dimethylformamide (2.5 mL) was added. To a solution of 40 mg (0.089 mmol) of 2-dione and 0.047 mL (0.268 mmol) of DIPEA was added 0.6 mL of the activated acid solution. The mixture was stirred overnight at room temperature. The reaction mixture was directly purified by acidic preparative MPLC (Luna 10-50) to give a bright white solid after lyophilization of the product fraction. Yield: 32 mg, 65% U_AN_ACID: m / z 520.2[M+H] + 1 H NMR (400MHz, DMSO-d6) δ8.33(d, J=8.7Hz, 1H), 7.39(s, 1H), 7.23(s, 1H), 6.4 8(s, 1H), 6.28(d, J=4.9Hz, 2H), 5.50(q, J=6.7Hz, 1H), 5.40(s, 3H), 5.23-5.0 6(m, 2H), 3.62(d, J=6.3Hz, 1H), 3.03(q, J=6.2Hz, 2H), 2.21-2.02(m, 2H), 1. 92-1.79(m, 2H), 1.18-1.08(m, 1H), 0.87(t, J=7.3Hz, 3H), 0.47-0.30(m, 4H).

[0420] Example 1 FL118, exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, and PBX-7024 were subjected to an anti-apoptotic protein expression inhibition assay and an in vitro cell viability assay as described below.

[0421] 1-1: Western blot analysis of DDX5, survivin, Mcl-1, XIAP, and cIAP2 to confirm their inhibitory activity against cancer-associated survival genes (1) Protein extraction FaDu cells were seeded at 200,000 cells per well in a 6-well plate and incubated at 37°C and 5% CO2. After 24 hours, the wells were treated with drugs (FL118, SN-38, exatecan, PBX-7011, PBX-7014, and PBX-7016) at concentrations of 0 nM, 10 nM, and 100 nM. The plates were incubated at 37°C and 5% CO2 for 24 hours. 100 μl of RIPA buffer containing a protease inhibitor cocktail was added to each well. The plate was placed on ice and incubated on an orbital shaker for 2 hours. The RIPA buffer containing the lysed cells was transferred to an EP tube and centrifuged at 16,000 rcf for 20 minutes at 4°C. The supernatant was then transferred to a new EP tube. Protein concentration was confirmed by protein assay. (2) Protein separation through electrophoresis Protein samples were mixed with 4x SDS-PAGE loading buffer in a 3:1 ratio, boiled at 95°C for 10 minutes, and then cooled. Samples were loaded into gel wells to ensure identical protein amounts. Gel electrophoresis was performed at 60V. (3) Transferring proteins from the gel to a membrane Trans-Blot® Turbo TM Seven sheets of activated filter paper, a PVDF membrane, a gel, and seven more sheets of filter paper were placed in the cassette of the transfer system in that order, the lid was closed, and the cassette was inserted into the machine, and the protocol was then run. (4) Antibody incubation The transferred membrane was immersed in blocking buffer and incubated at room temperature for 1 hour. Then, it was incubated at room temperature with primary antibody solution (4°C overnight). It was washed with TBST buffer for 3 minutes (repeated 3 times). It was incubated at room temperature with HRP-conjugated secondary antibody solution (1 hour). It was washed with TBST buffer for 3 minutes (repeated 3 times). (5) Imaging and analysis of results The membrane was immersed in the ECL substrate for 3-5 minutes, and then the ChemiDoc TMThe signal was confirmed using the MP imaging system. The HRP conjugated to the secondary antibody oxidized the luminol in the ECL, detecting the light emitted and displaying it in an image. The band thickness is proportional to the amount of protein, so the amount of protein can be compared based on the band thickness. Using the same method, A549 cells overexpressing ABCG2 were seeded instead of the FaDu cell line that does not express ABCG2, and 4 μg of protein was loaded and subjected to Western blotting. As shown in Figures 6 to 8, we investigated how protein expression levels change with drug treatment (FL118, SN-38, exatecan, PBX-7011, PBX-7014, and PBX-7016). GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells. It is a gene that is constantly expressed in cells but whose expression level does not change significantly. This gene is an indicator of whether samples have the same amount of protein loaded onto the gel.

[0422] Discussion: Evaluation of PBX-7011, PBX-7014, and PBX-7016 The degree of inhibition of anti-apoptotic protein expression in the FaDu cell line was confirmed by Western blot analysis. PBX-7011, PBX-7014, and PBX-7016 showed a decrease in the expression of survivin, cIAP2, Mcl-1, and XIAP as the concentration increased. Compared with the existing drugs FL118, SN-38, and exatecan, the levels were similar to those of FL118 and exatecan, but better than SN-38. In Example 1-1, it was confirmed that the compound exhibited a dual inhibitory effect of suppressing type I topoisomerase 1 while also degrading DDX5, and that it had the unique characteristic of degrading DDX5 and the resulting anti-cancer effect. Specifically, two cell lines (FaDu and A549) were treated with drugs at a fixed concentration (0, 10, 100 nM) and each protein (DDX, survivin, Mcl-1, XIAP) was expressed. ) expression levels were confirmed by Western blot analysis. DDX5 degradation by FL118 was superior to that by other substances, followed by 7011, 7016, and 7014. This trend was similar not only for DDX5 but also for other downstream anti-apoptotic proteins. In summary, PBX-7014 and PBX-7016 exhibit a dual mechanism of action (MoA) that not only inhibits type I topoisomerases but also acts as a degrader of DDX5 (p68), an oncoprotein that regulates survivin, Mcl-1, XIAP, and others. As a result of the evaluation, we observed that PBX-7016, out of PBX-7014 and PBX-7016, inhibited DDX5 in a concentration-dependent manner, and as a result, inhibited survivin, Mcl-1, and XIAP. This not only demonstrated the role of PBX-7016 as a topoisomerase I inhibitor, but also as a DDX5 degrader of FL118, and confirmed that PBX-7016 acted more effectively than PBX-7014 (Figures 7 and 8).

[0423] 1-2: Cell viability assay for FaDu cell line / A549 cell line FaDu cell line (cancer cells that do not express ABCG2) or A549 cell line (cancer cells that overexpress ABCG2) were seeded at 3,000 cells per well in a 96-well plate and incubated at 37°C and 5% CO2. After 24 hours, the cells were treated with 100 μL of drugs at nine concentrations (serial dilutions of 1 / 5 starting from 1,000 nM). These included Dxd drugs, PBX-7014, PBX-7016, and PBX-7024. A control group (drug concentration 0) was also prepared without drug treatment. The cells were incubated at 37°C and 5% CO2 for 3 or 6 days. 100 μL of CellTiter-Glo reagent (using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega, G7571)) was added to each well and then pipetted. After 10 minutes of incubation at room temperature (RT), luminescence was measured. The luminescence value at 0 drug concentration was taken as 100%, and the concentration at which 50% of the luminescence value was obtained was the IC 50value. Two cell lines (FaDu, A549) were treated with two compounds (PBX-7016, PBX-7024) and reference compounds (Dxd, SN-38, exatecan, FL118), and cell viability was monitored over a 3-day incubation period. As shown in Figure 9, PBX-7024 exhibits potent cell killing efficacy not only in the FaDu cell line, which does not express ABCG2, but also in the A549 cancer cell line, which overexpresses ABCG2. That is, it exhibits IC at levels equal to or higher than Dxd, the payload used in existing enhancers. 50 It was confirmed that it has the following. In the cancer cell line A549, which overexpresses ABCG2, camptothecin compounds including Dxd showed high IC 50 It can be seen that, whereas PBX-7016 and PBX-7024 still maintain strong efficacy. In other words, when the new camptothecin compounds PBX-7016 and PBX-7024 according to the present invention are used, they have the effect of overcoming the resistance mechanism caused by overexpression of ABCG2, unlike ADCs that use existing camptothecin compounds such as SN-38 and DXd.

[0424] 1-3: New PBX series compounds as potential ADC payloads: In vitro cell viability studies Cell viability assays were performed on the MDA-MB-453 (HER2++) and FaDu (HER2+) cell lines in the same manner as in Example 1-2. Two cell lines (MDA-MB-453 and FaDu) were treated with two compounds (PBX-7014, PBX-7016, PBX-7018, PBX-7020, and PBX-7022) and one reference compound (Dxd), and cell viability was monitored over a 3-day incubation period. The results are shown in Figure 10. As shown in Figure 10, the evaluation results show that PBX-7016, PBX-7018, PBX-7020, and PBX-7022 are ICs with the same or higher level than Dxd.50 Furthermore, in the FaDu cell line, which does not express ABCG2, IC was confirmed to be equal to or greater than that of Dxd. 50 It was confirmed that it had a value.

[0425] Example 2. PBX-7016 IV and IP Pharmacokinetic Profile in OD SCID Mice The experimental method is as shown in Table 2 below. PBX-7016 IV plasma concentration-time data is shown in Table 3, and PBX-7016 IP plasma concentration-time data is shown in Table 4.

[0426] [Table 2]

[0427] [Table 3]

[0428] [Table 4]

[0429] The results in Tables 3 and 4 confirm that PBX-7016 compound is cleared from the blood very quickly when administered intravenously or intraperitoneally to mice, suggesting that PBX-7016 compound alone, rather than in the form of a prodrug or ADC, is highly safe when administered in vivo. Because PBX-7016 compound is released from the body very quickly when administered IV, it can act as a safety device that reduces systemic exposure to the drug even when the drug is released prematurely after entering the body. Furthermore, this experiment suggests that the PK of the PBX-7016 compound used as the payload alone is a safe payload, with clearance of the PBX-7016 drug occurring in the blood within a short period of time. This suggests that after exerting its bystander killing effect at the tumor site, the PBX-7016 compound is safely released from the body within a very short period of time while circulating in the blood.

[0430] Example 3. Carboxylate to Lactone Conversion Rate of PBX-7016 at pH 6.0 Example 3 (FIGS. 24 and 25) is an experiment related to the activation of the lacto portion of camptothecin, a known active agent in Top1 inhibitors.

[0431] 3-1. Conversion of carboxylate to lactone form at pH 6.0 PBX-7016 powder was prepared by dissolving in DMSO at 5 mM. PBX-7016 dissolved in DMSO exists in the lactone form, so it was diluted 1 / 10 with 0.1 N NaOH and incubated in a 25°C water bath for 30 minutes to convert it to the carboxylate form. The carboxylate form of PBX-7016 was diluted to 3 μM with 10% DMSO in PBS (pH 6.0) at 4°C. An equivalent volume of HCl was added to the 0.1 N NaOH used to minimize pH changes. After dilution, the solution was centrifuged at 13,000 RCF for 2 minutes at 4°C, and the supernatant was used as the sample. The lactone form of PBX-7016 was separated by RP-HPLC using two solvents: Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6 x 50 mm column, and the analytical conditions were as follows: 1) 20 min: 5% B, 2) sample injection, 3) 2 min: 5% B, 4) 10 min: 5% B → 50% B, 5) 1 min: 50% B. To measure changes over time, the sample chamber was maintained at 37°C and samples were injected at each measurement time. The measurement times were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720, 840, 960, and 1000. The times were 1200 and 1440 minutes. The lactone and carboxylate contents were calculated from their peak areas. Because the absorption coefficients of lactone and carboxylate are different, the lactone area was calculated by multiplying the carboxylate area by the following formula: Lactone EC 390 / Carboxylate EC 390 =0.918 The same experiment as in Example 3-1 was carried out, except that FL118, SN38, DxD, exatecan, PBX-7014, and PBX-7024 were used instead of PBX-7016. The results of the carboxylate-to-lactone conversion experiment at pH 6.0 are shown in Figure 25. As shown in Figure 25, various camptothecin-based compounds were confirmed to convert from the carboxylate to the active lactone form, which inhibits topoisomerase I, at similar rates and in similar amounts at pH 6.0, and are therefore expected to exhibit similar levels of activity in a tumor microenvironment with a pH of 6.0.

[0432] 3-2.Conversion of lactone to carboxylate form at pH 7.4 PBX-7016 powder was dissolved in DMSO at 5 mM and diluted with 10% DMSO in PBS (pH 7.4) at 4°C to prepare 3 μM PBX-7016. This was then centrifuged at 13,000 RCF for 2 minutes at 4°C, and the supernatant was used as the sample. PBX-7016 dissolved in DMSO existed in the lactone form. When the PBX-7016 carboxylate form was produced, it was separated by RP-HPLC using two solvents: Solvent A: 100 mM acetate buffer pH 5.5 and Solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6 x 50 mm column, and the analytical conditions were as follows: 1) 20 min: 5% B, 2) sample injection, 3) 2 min: 5% B, 4) 10 min: 5% B → 50% B, 5) 1 min: 50% B. To measure changes over time, the sample chamber was maintained at 37°C and samples were injected at the following measurement times: 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, and 720 minutes. The lactone and carboxylate contents were calculated from their peak areas. Because the absorption coefficients of lactone and carboxylate are different, the lactone area was calculated by multiplying the carboxylate area by the following formula: Lactone EC 390 / Carboxylate EC 390 =0.918 The same experiment as in Example 3-2 was carried out, except that FL118, SN38, DxD, exatecan, PBX-7014, and PBX-7024 were used instead of PBX-7016. As shown in Figure 24, which shows the results of the lactone-to-carboxylate conversion experiment at pH 7.4, it was confirmed that at 7.4, the pH of blood or extracellular fluid, PBX-7014, PBX-7016, and PBX-7024 compounds represented by Chemical Formula 1 were converted from the lactone form (active form) to the carboxylate form (inactive form), which is inactive as a TOP1 inhibitor, not only more quickly but also at a higher rate than other reference compounds (exatecan, DXd, SN-38, FL118). In particular, the extent (63–86%) and rate (0.4–0.8% / min) of formation of the carboxylate form, which is inactive as a TOP1 inhibitor at blood pH 7.4, varied significantly among camptothecin derivatives. PBX-7014 and PBX-7016 formed over 86% of the carboxylate form, and all were converted from the lactone form to the carboxylate form relatively quickly at a level of 0.8% per min. The competing drug, DXd, formed 76.5% of the carboxylate form and was converted from the lactone form to the carboxylate form at a level of 0.6% per min. Through this, PBX-7014 and PBX-7016 compounds are not active lactones that show rapid and high cytotoxicity in the blood compared to the reference compounds, but inactive lactones. It exists as a carboxylate and is predicted to be highly safe in normal cells.

[0433] Production Example 5: Synthesis of CL2A-FL118 The CL2A-FL118 conjugate (chemical formula 7-1) was synthesized by the method described in Example 1 of WO2022-015110A1. [ka]

[0434] Preparation Example 6: Synthesis of 25-4 and 25-6 from PBX-7014 and PBX-7016 Linker-payloads containing two compounds (PBX-7014 and PBX-7016) and incorporating the enzyme-cleavable linker system GGFG, 25-4 (Chemical Formula 9) and 25-6 (Chemical Formula 10), were synthesized. [ka]

[0435] [ka]

[0436] The molecular structure of 25-4 is GGFG-PBX-7014, and the molecular structure of 25-6 is GGFG-PBX-7016. That is, each uses the same GGFG linker as Enhertu (registered trademark).

[0437] Comparative Example 1: Preparation of cetuximab-CL2A-FL118 immunoconjugate (DAR8) Cetuximab-CL2A-FL118 immunoconjugate (DAR8) was produced by the method described in Example 8 of WO2022-015110A1. The anti-EGFR antibody (cetuximab) in the original buffer (20 mM histidine, 150 mM NaCl, pH 6.0) was reduced with 5 mM TCEP, and the reaction vial was placed in an incubator shaker at 22 °C with a rotation speed of 60 rpm for 3 h. After reduction, the TCEP was removed through a spin desalting column (40K, 2 mL x 2). DMSO and CL2A-FL118 compound solution (10 mg / mL stock in DMSO, 12 eq. relative to antibody) were added to the reduced antibody solution for a final DMSO concentration of 10%. The reaction mixture was mixed properly and the reaction vial was left at 4°C for 2 hours. After conjugation, the reaction mixture was passed through a spin desalting column (40K, 10 mL) into 18 mM MES (pH 6.5) exchange buffer. The product was then sterile filtered through a 0.2 μm PVDF disposable filter. The resulting immunoconjugate was characterized and lyophilized after adding 0.07 mM PS80 and 20 mM trehalulose dehydrate. The DAR of the cetuximab-CL2A-FL118 immunoconjugate of Comparative Example 1 was determined using LCMS, and the average MS-DAR value was confirmed to be 7.99 (FIG. 14).

[0438] Example 4: Preparation of nimotuzumab-CL2A-FL118 immunoconjugate (DAR8) Nimotuzumab-CL2A-FL118 immunoconjugate (DAR8) was prepared in a similar manner to Comparative Example 1, except that nimotuzumab was used instead of cetuximab. The DAR of the nimotuzumab-CL2A-FL118 immunoconjugate of Example 4 was determined using LCMS, and the average MS-DAR value was determined to be 7.94 (FIG. 15).

[0439] Example 5: Preparation of ADCs with 19-1 (CL2A-FL118, DAR 8), 25-6 (GGFG-PBX-7016, DAR 8), and 25-6-a (GGFG-PBX-7016, DAR 4) 5-1. Manufacturing method of nimotuzumab (YTE) Nimotuzumab (YTE) is an antibody with an improved half-life of nimotuzumab. Nimotuzumab (YTE) is a modified IgG comprising a human IgG normal domain or an FcRn-binding fragment thereof, which contains substitutions at amino acid residues 252, 254, and 256 based on the EU numbering of Kabat relative to the wild-type human IgG normal domain, and the modified IgG has an increased half-life compared to that of an IgG having the wild-type human IgG normal domain, and in nimotuzumab, the substitution at amino acid residue 252 is with tyrosine, the substitution at amino acid residue 254 is with threonine, and the substitution at amino acid residue 256 is with glutamic acid.

[0440] 5-2. Manufacturing method of nimotuzumab (YTE)-19-1 Nimotuzumab (YTE) antibody was buffer exchanged into reduction buffer (150 mM NaCl, 50 mM histidine, pH 6.0) using a PD-10 desalting column, and then 27.5 μM of antibody was treated with 825 μM TCEP at 25°C for 2 hours to reduce the antibody disulfide bonds. After that, excess TCEP was removed using a PD-10 desalting column, and the conjugation reaction was carried out by reacting 165 μM 19-1 linker payload with 13.8 μM reduced antibody in a reaction buffer (25 mM histidine, pH 6.0) containing 10% DMSO at 25° C. for 1 hour. After the conjugation reaction, excess linker payload was removed using PD-10 to obtain the final product.

[0441] 5-3. Manufacturing method of nimotuzumab-25-6 and nimotuzumab(YTE)-25-6 (DAR8) Nimotuzumab and nimotuzumab (YTE) antibodies were each buffer-exchanged into a reduction buffer (150 mM NaCl, 50 mM histidine, pH 6.0) using a PD-10 desalting column, and then 7.5 μM of antibody was treated with 825 μM TCEP at 25°C for 2 hours to reduce the antibody disulfide bonds. After that, excess TCEP was removed using a PD-10 desalting column, and the conjugation reaction was carried out by reacting 165 μM 25-6 linker payload (DAR8) with 13.8 μM of reduced antibody in a reaction buffer (25 mM histidine, pH 6.0) containing 15% DMSO at 25° C. for 1 hour. After the conjugation reaction, excess linker payload was removed using PD-10 to obtain the final product.

[0442] 5-4. Manufacturing method of nimotuzumab-25-6-a and nimotuzumab(YTE)-25-6-a (DAR4) Nimotuzumab and nimotuzumab (YTE) antibodies were each buffer-exchanged into a reduction buffer (150 mM NaCl, 50 mM histidine, pH 6.0) using a PD-10 desalting column, and then 27.5 μM of antibody was treated with 825 μM TCEP at 25°C for 2 hours to reduce the disulfide bonds of the antibody. After that, excess TCEP was removed using a PD-10 desalting column, and the conjugation reaction was carried out by reacting 82.5 μM 25-6-a linker payload (DAR4) with 13.8 μM of reduced antibody in a reaction buffer (25 mM histidine, pH 6.0) containing 10% DMSO at 25°C for 1 hour. After the conjugation reaction, excess linker payload was removed using PD-10 to obtain the final product.

[0443] Example 6. Anti-HER2 ADC with PBX-payload: Binding affinity analysis / Nimotuzumab ADC-EGFR binding For in vitro and in vivo experiments targeting EGFR, the background for the selection of cell lines is shown in Figure 16. The experiments were then performed using MDA-MD-468, which overexpresses EGFR, as a positive cell line, and SW620, which does not express EGFR (low expression), as a negative cell line. Nimotuzumab ADC-EGFR binding affinity analysis was performed using a modified method described in "Example 6. Anti-HER2 ADC with a PBX-payload: Binding affinity analysis" in PCT / KR2023 / 009854 (Figure 17). A total of six ADCs using nimotuzumab and nimotuzumab (YTE), a derivative of nimotuzumab with an improved half-life, as the antibody were synthesized and evaluated. When analyzed by ELISA, each of the ADCs created was confirmed to have binding affinity that was not significantly different from that of the antibody alone (nimotuzumab and nimotuzumab (YTE)). B.C. 50 : Maximum 50% binding concentration 19-1:CL2A-FL118, 25-6:GGFG-PBX-7016(DAR8), 25-6-a:GGFG-PBX-7016(DAR4)

[0444] Example 7: In vitro assay of nimotuzumab and cetuximab ADCs Various evaluations were performed on EGFR-positive and -negative cells, and the results are shown in Figures 18, 19, and 20. In Figure 18, the payload is released extracellularly (extracellular payload With the exception of the ADC 19-1 (CL2A-FL118) which is releasable, the ADC 25-6 (GGFG-PBX-7016) which uses an enzyme-cleavable linker showed no cytotoxicity in any cell line. d Nimotuzumab and nimotuzumab (YTE) ADC, which have 1 / 10 the binding affinity of cetuximab, have very high IC 50 showed. In Figure 19, cetuximab, which has approximately 10 times the binding affinity to EGFR compared to nimotuzumab, showed excellent IC 50Not only did they demonstrate significant efficacy, but they also distinguished between positive and negative cell lines very well. In vitro experiments showed that the cetuximab ADC performed very well, while the nimotuzumab ADC did not perform as well as an ADC. In Figure 20, the payload is released extracellularly (extracellular payload With the exception of the ADC 19-1 (CL2A-FL118) which is releasable, all enzyme-cleavable ADCs showed very high IC in both positive and negative cell lines. 50 showed.

[0445] Example 8: In vivo efficacy study in the MDA-MB-468 model 8-1. Animal model and material preparation All procedures related to animal handling, care, and treatment in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Model Organisms Center, Inc. in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The experiment was carried out in accordance with the approved guidelines. Twenty-one female mice were placed in individual cages (200 mm 3 The animals were housed three per cage, maintained at constant temperature (18–26°C) and humidity (40–70%), and given free access to radiation-sterilized dry granular feed and sterile drinking water in individually ventilated cages. The specific method for evaluating in vivo efficacy is shown in Table 5 below.

[0446] [Table 5]

[0447] 8-2.Cell culture MDA-MB-468 breast cancer cells were cultured in RPMI containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C with 5% CO and routinely subcultured twice weekly by trypsin-EDTA treatment. Exponentially growing cells were harvested and counted for tumor inoculation.

[0448] 8-3.Tumor inoculation MDA-MB-468 cells (1 x 10) in 0.2 mL of DPBS with Matrigel 7 ) was inoculated subcutaneously into the right anterior flank of each mouse. The average tumor volume was approximately 215 mm 3 When this was reached, the animals were randomly grouped and treatment for the drug efficacy study was initiated.

[0449] 8-4. Observation During routine monitoring, animals were checked daily for normal behavior such as mobility, food and water consumption (observation only), weight change (weighed twice weekly), eye / hair matting, tumor growth, and treatment effects. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0450] 8-5. Tumor Measurement and Endpoints The primary endpoint was the time point at which tumor growth could be delayed or mice could be treated. The bidimensional size of the tumor was measured twice weekly using a caliper, and the volume was calculated using the formula (V = 0.5×b 2 , where a and b are the long and short diameters of the tumor, respectively) in mm 3 It was displayed as. Tumor growth inhibition (TGI) was calculated for each group. TGI(%)=[1-(T i -T0) / (V i -V0)] × 100 (T i is the mean tumor volume of the treatment group on a particular day, T0 is the mean tumor volume of the treatment group on the first day of treatment, V i is T i and the mean tumor volume of the vehicle control group on the same day, V0 is the mean tumor volume of the vehicle group on the first day of treatment). Body weight loss of 15% or more or tumor volume of 3000mm 3 Animals above this age were euthanized at a humane endpoint.

[0451] 8-6.Statistical analysis Results are expressed as mean and standard error (mean ± SEM). Data were analyzed using two-way RM ANOVA Dunnett's multiple comparison test using Graphpad Prism 6.0 software, and p<0.05 was considered statistically significant.

[0452] Body weight of the MDA-MB-468 tumor model The body weight of the MDA-MB-468 tumor model mice was monitored regularly. As shown in Figure 13, no obvious weight loss was observed in any of the treatment groups. Unlike humans, mouse plasma contains high levels of esterase activity. Since the immunoconjugate of the present invention has a cleavage activity and can easily cleave acid-sensitive bonds such as ester and carbonate functional groups, we expected that side effects such as weight loss would occur when the immunoconjugate of the present invention was administered to tumor model mice. However, surprisingly, no such side effects were observed. The above results demonstrate that the immunoconjugate of the present invention does not exhibit toxicity to normal cells even when it releases FL118 in the blood.

[0453] 8-8. Tumor growth inhibition analysis in MDA-MB-468 tumor model mice (Figures 21 to 23) Figures 21 to 23 show the results of three in vivo experiments using EGFR-targeting ADCs. The average tumor volume of MDA-MB-468 tumor model mice (n=3) in each group is shown. The TGI values ​​of the MDA-MB-468 tumor model mice (n=3) were then calculated based on tumor size (n=3) on day 21 after treatment. In Figure 21, the cetuximab-CL2A-FL118 ADC demonstrated tumor regression in a mouse MDA-MB-468 tumor model. In the case of Figure 22, K d Compared to the cetuximab ADC, which has a lower K value (i.e., a higher binding affinity), dNimotuzumab ADCs with higher values ​​(i.e., lower binding affinity) showed relatively better tumor regression, i.e., were superior ADCs. FIG. 22 shows the dissociation constant K d The value is 1*10 -8 M or more 1*10 -6 This demonstrates that immunoconjugates using antibodies less than M have superior efficacy compared to cetuximab, which is known to exhibit excellent antitumor activity on its own. Nimotuzumab-CL2A-FL118 was designed to address toxicity issues, but surprisingly, it was found to exhibit superior efficacy compared to the reference cetuximab-CL2A-FL118. While it was expected that the two ADCs would exhibit similar efficacy, or that cetuximab-CL2A-FL118 would exhibit slightly superior efficacy, unexpected results were obtained. In Figure 23, the following results were obtained: (1) The ADC of nimotuzumab-25-6 showed superior tumor regression compared to nimotuzumab-19-1. (2) A comparison between nimotuzumab and nimotuzumab (YTE), i.e., an ADC with an altered antibody half-life, showed no significant difference. From this, it can be inferred that the ADC of the present invention penetrates well into cancer tissues and exerts a cytotoxic effect early in ADC administration. (3) When comparing the doses (5mpk, 10mpk), the 5mpk group already showed sufficient tumor regression. (4) When comparing DAR4 and DAR8 ADCs, DAR4 alone showed sufficient tumor regression. is doing. (5) Only in the case of Nim(YTE) 5mpk, DAR 4, an exceptionally small tumor regression was observed. It is unclear whether this was due to the antibody (Nim(YTE)), the administered dose (5mpk), the DAR (4), or the sum of all factors. 21 to 23, the dissociation constant K d The value is 1*10 -8 M or more 1*10 -6In the case of ADCs that use an antibody of less than M or a fragment thereof containing its antigen-binding site (B-1); or an antibody or a fragment thereof containing its antigen-binding site (B-2) that binds to a cell surface protein expressed on both normal and cancer cells and has a higher binding affinity when binding to the epitope of the cell surface protein bivalently than monovalently, the antibody that constitutes the ADC indirectly binds to the drug target on the surface of cancer cells with strong affinity. In this case, it is possible to solve the problem of the drug target around the blood vessels first being saturated, and after a certain amount of antibody has bound, additional antibody (ADC) cannot penetrate into the cancer tissue, resulting in wasted ADC. It has also been demonstrated that the payload released from the ADC can be distributed uniformly throughout the cancer tissue. The immunoconjugates of the present invention have been shown to exhibit significantly superior anti-cancer activity at all administered doses, without causing side effects such as weight loss. Furthermore, since the immunoconjugates of the present invention have been shown to exhibit dose-dependent responses, they can be administered in a customized dosage regimen to maximize efficacy and minimize toxicity for each patient. Therefore, the immunoconjugate of the present invention can be very useful for preventing or treating cancer, and the FL118-linker conjugate of the present invention can be very useful for producing the immunoconjugate.

[0454] Example 9 Antibodies that can be used in the ADC of the present invention include antibodies that can target common drug targets for developing ADCs for solid cancers. d The value is 1*10 -8 M or more 1*10 -6 It has the characteristic that it is less than M. d Measurement of the values ​​can be measured using SPR methods or via quantitative Western blot analysis. Drug targets for ADCs of the present invention include various examples such as Her2, EGFR, Trop2, c-Met, CEACAM-5, B7H3, B7H4, Muc1, CDH6, and CLDN18.2. However, any surface antigen that has been identified as being overexpressed in cancer cells and that can be used as a drug target for ADCs can also be a suitable drug target for the ADCs of the present invention. The method for obtaining the antibody of the present invention includes: (1) generating a K antibody from the process of producing an initial antibody against an antigen; d The value is 1*10 -8 M or more 1*10 -6 (2) A method for obtaining antibodies in the range of less than M, and (3) a method for affinity maturation by changing the amino acids in the Fv region of an already known antibody (e.g., trastuzumab, sacituzumab, etc.), without increasing affinity, but with increasing affinity, d The value is 1*10 -8 M or more 1*10 -6 It can be obtained by making the affinity low in the range of less than M. The target antibodies in this example obtained by method (1) include nimotuzumab and Fc region mutations obtained to improve the PK profile of nimotuzumab, and in this example, new target antibodies such as trastuzumab-1 and sacituzumab-1 were produced by method (2). Representative camptothecin-based compounds that can be used in the ADCs of the present invention include camptothecin, potecan, and exatecan, as well as SN-38, which has already been used in Trodelvy, various exatecan derivatives including deruxtecan (DXd), which has been used in Daiichi-Sankyo, 10,11-methylenedioxy-camptothecin (FL118) and its derivatives (PBX-7011) and its various derivatives (PBX-7016, etc.). In the examples of the present invention, FL118 and PBX-7016 were used as the payload. However, as anyone skilled in the art can infer, the use of FL118 and PBX-7016 is not essential for practicing the present invention, and the same effects as those in the examples of the present invention can be achieved by using an appropriate camptothecin-based compound. PBX-7011 is a compound for which the present inventors filed a Korean patent application dated April 20, 2022. PBX-7016 is a camptothecin derivative for which patent application was filed under Korean Patent Application No. 0-2022-0049158. In addition to its potent topoisomerase I inhibitory activity, PBX-7016 can also inhibit the expression of various anti-apoptotic proteins that cause cancer cell resistance to existing anticancer drugs. PBX-7016 is described in Korean Patent Application No. 10-2022-0103591, filed by the present inventors on August 18, 2022. The most representative examples of linker-payloads used in the present invention include the following compounds 19-1 (CL2A-FL118) and 25-6 (MC-GGFG-NHCH2-PBX7016). In addition, CL2A-SN38 used in the development of Trodelvy, CL2A-belotecan used in the development of SKB264, MC-GGFG-NHCH2-DXd used in Enhertz, and various other derivatives can be used as needed. In addition, to adjust the DAR to a lower value other than 8, the antibody binding portion of the linker compound can be changed from maleimide to dibromomaleimide or a bridge compound that functions equivalently thereto, and a specific example is shown in 25-6-a below.

[0455] [ka]

[0456] In the case of 19-1 (CL2A-FL118), n=7 in chemical formula 7-1. [ka]

[0457] [ka]

[0458] The ADC of this example can be synthesized using the method described in the examples of Korean Patent Registration No. 10-2349925 for the appropriate antibody and linker-payload compound of the present invention. That is, all interchain disulfide bonds of the antibody used in the present invention are cleaved using a reducing agent to expose free SH groups, and then an excess amount of linker-payload compound is added to the antibody to proceed with the conjugation reaction. The resulting antibody-drug conjugate can then be purified by an appropriate method to prepare the ADC compound of the present invention.

[0459] 9-1. EGFR ADC Production K as a control antibody d The value is 1.8*10 -9 Cetuximab M was used, and K was used as an antibody suitable for use in the present invention. d The value is 2.9*10 -8 Nimotuzumab (M) was used. As exemplified in Examples 4 and 5, first, ADCs with DAR8 (nimotuzumab / 19-1, cetuximab / 19-1) were produced using the 19-1 compound, and simultaneously, ADCs with DAR8 (nimotuzumab / 25-6, cetuximab / 25-6) were produced using the 25-6 compound.

[0460] 9-2. Production of Trop-2 ADC K d The value is 1.7*10 -9 Using sacituzumab M as a control antibody, the Fv sequence was changed one amino acid at a time from sacituzumab to produce antibodies suitable for use in the present invention. d The value is 4*10 -8 We produced an antibody (sacituzumab-1) for M. Sacituzumab-1 / 19-1(DAR8), sacituzumab-1 / 25-6(DAR8), sacituzumab / 19-1(DAR8), and sacituzumab / 25-6(DAR8) were produced by the method described in Example 9-1.

[0461] 9-3.Her2 ADC Production K d The value is 2.7*10 -9Trastuzumab M was used as a control antibody, and to produce antibodies suitable for use in the present invention, amino acids in the Fv sequence were changed one by one from trastuzumab to K d The value is 3*10 -8 The antibody (trastuzumab-1) of M was produced. Trastuzumab-1 / 19-1(DAR8), trastuzumab-1 / 25-6(DAR8), trastuzumab / 19-1(DAR8), and trastuzumab / 25-6(DAR8) were produced by the method described in Example 9-1.

[0462] 9-4. In vivo evaluation of EGFR ADCs In a xenograft model of the MDA-MB-468 cell line, a TNBC model overexpressing EGFR, tumor volumes reached 200 mm 3 After reaching this level, nimotuzumab / 19-1 and cetuximab / 19-1 were administered at 1 mpk, 5 mpk, and 10 mpk, respectively, and as shown in Figure 22, it was confirmed that the nimotuzumab-based ADC administration group showed even better efficacy. Similar experiments were conducted using nimotuzumab / 25-6 and cetuximab / 25-6 in the same animal model, and as shown in Figure 22, the nimotuzumab / 25-6 group showed a TGI improvement effect of approximately 25 to 40% or more compared to the cetuximab / 25-6 group.

[0463] 9-5. In vivo evaluation of Trop-2 ADC In a xenograft model of MDA-MB-468 cell line, a TNBC model overexpressing Trop-2, tumor volume reached 200 mm 3 After reaching this target, sacituzumab-1 and two types of sacituzumab-based ADCs were administered at 5mpk, and it was confirmed that the sacituzumab-1-based ADC showed a TGI that was improved by more than 30% compared to the original sacituzumab-based ADC.

[0464] 9-6. In vivo evaluation of Her2 ADC In a xenograft model of JIMT-1 cell line, a breast cancer model overexpressing Her2, tumor volume reached 200 mm 3After reaching this level, 2 types of trastuzumab-1 and 2 types of trastuzumab-based ADC were administered at 5mpk, and as can be seen from the figure, it was confirmed that the trastuzumab-1-based ADC showed a TGI that was improved by approximately 20% or more compared to the original trastuzumab-based ADC.

[0465] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. In this regard, the above-described embodiments should be understood to be illustrative in all respects and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below and their equivalents, rather than the above detailed description.

Claims

1. The camptothecin-based drug (A) binds to an epitope of a cell surface protein with a dissociation constant K ｄ The value is 1*10 －８ M or more 1*10 －６ an antibody or antigen-binding site-containing fragment thereof (B-1) having a molecular weight of less than M; or an antibody or antigen-binding site-containing fragment thereof (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than when binding monovalently;

2. The immune complex or a pharmaceutically acceptable salt thereof according to claim 1, wherein the camptothecin-based drug is an activated camptothecin derivative represented by the following Chemical Formula 1, Chemical Formula 2, or Chemical Formula 1-1, which is designed to bind to DDX5 protein and E3 ligase. 【Chemical 1】 【Chemistry 2】 X １ and X ３ are each independently carbon, oxygen, nitrogen, or sulfur; X １ and X ３ may be the same or different, X ２ is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X １ , (X ２ ) n and X ３ can form a 5-, 6-, or 7-membered ring (n=values ​​from 0 to 2); Y １ , Y ２ and Y ３ may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus, or sulfur. 【Chemistry 3】

3. Dissociation constant K for cell surface protein epitopes ｄ The value is 1*10 －８ M or more 1*10 －６ The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the immune complex and / or payload excreted from the immune complex do not accumulate only around blood vessels inside the cancer but penetrate into the interior of the cancer tissue, due to the antibody (B-1) or antigen-binding site-containing fragment thereof that binds to a cell surface protein expressed on both normal cells and cancer cells and that has higher binding affinity when binding to an epitope of the cell surface protein bivalently than when binding monovalently, or the antibody (B-2) or antigen-binding site-containing fragment thereof.

4. Dissociation constant K for cell surface protein epitopes ｄ The value is 1*10 －８ M or more 1*10 －６ M or a fragment thereof containing the antigen-binding site thereof (B-1); or an antibody or a fragment thereof containing the antigen-binding site thereof (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than when binding monovalently, thereby weakening the internalization of the immune complex via receptor-mediated endocytosis, thereby increasing the therapeutic effect.

5. Dissociation constant K for cell surface protein epitopes ｄ The value is 1*10 －８ M or more 1*10 －６ The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, characterized in that the therapeutic effect of the immunoconjugate penetrated into the interior of cancer tissue by an antibody or antigen-binding site-containing fragment thereof (B-1) of less than M; or an antibody or antigen-binding site-containing fragment thereof (B-2) that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than monovalently is to relieve solid pressure within the cancer microenvironment formed as cancer cells proliferate, and optionally, thereby increasing the accessibility of anticancer drugs and / or immunotherapeutic agents.

6. Dissociation constant K for cell surface protein epitopes ｄ The value is 1*10 －８ M or more 1*10 －６ an antibody or its antigen-binding site-containing fragment (B-1) of less than M; or an antibody or its antigen that binds to a cell surface protein expressed on both normal cells and cancer cells and has higher binding affinity when binding to an epitope of the cell surface protein bivalently than when binding to an epitope of the cell surface protein monovalently. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the immunoconjugate exerts an anti-cancer effect while solving the problem of on-target toxicity of the immunoconjugate due to the binding site-containing fragment (B-2).

7. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the dosage of the immunoconjugate or pharmaceutically acceptable salt thereof is at a level of 4.8 mpk or less, preferably at a level of 2 mpk to 4.5 mpk.

8. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the DAR, which indicates the number of drugs attached per antibody based on the antibody, is 4 to 8, or the DAR, which indicates the number of drugs attached per antibody based on the antigen-binding site-containing fragment, is 1 to 4.

9. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the linker (C) is (i) an acid-sensitive linker or (ii) an enzyme-sensitive linker.

10. 2. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody or antigen-binding site-containing fragment thereof (B) targets an EGFR antigen distributed on the surface of cancer and / or an EGFR antigen also distributed in small amounts in normal tissues.

11. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody or its antigen-binding site-containing fragment (B) is nimotuzumab, a modified antibody having the antigen-binding site of nimotuzumab, or a fragment thereof having the antigen-binding site of nimotuzumab.

12. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that (B) an antibody or an antigen-binding site-containing fragment thereof that binds to a cell surface protein selected from the group consisting of Her2, EGFR, Trop2, c-Met, CEACAM-5, B7H3, B7H4, Mucl, CDH6, CLDN18.2, FolR, and PSMA is used.

13. 10. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein after being targeted to cancer cells by the antigen-binding site that targets the EGFR epitope of the cancer cells, the acid-sensitive linker is degraded in the acidic environment (pH≦7) around the cancer, at least a portion of the camptothecin drug (A) that degrades the DDX5 protein is liberated, and the free camptothecin drug (A) penetrates deep into the cancer tissue, penetrates the cell membrane, and moves into the cells.

14. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein the acid-sensitive linker is stable in the neutral environment of the bloodstream, that is, pH 7.3 to 7.5, but is hydrolyzed around tumor cells (pH 6.5 to 7.2) or in endosomes (pH 5.0 to 6.5) or lysosomes (pH 4.5 to 5.0) after being internalized into the cells, thereby releasing the active drug.

15. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 9, characterized in that after being targeted to cancer cells by the antigen-binding site that targets an antigen on the cancer cells, the acid-sensitive linker is decomposed in the acidic environment (pH≦7) around the cancer, at least a portion of the camptothecin drug is liberated, and the drug is released into both the inside and outside of the cells.

16. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein the camptothecin drug and the acid-sensitive linker are linked via a carbonate or ester bond so that the free camptothecin drug is released upon decomposition of the acid-sensitive linker.

17. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 9, wherein the acid-sensitive linker is derived from the following Chemical Formula 7: 【Chemistry 4】 Here, X １ and X ２ are each independently —H or -halogen; Y is -NH-, -NR Ａ - or nothing (null); Z is -C １ -C ４ Alkyl-, -C ３ -C ６ Cycloalkyl-, -(C １ -C ２ alkyl)-(C ３ -C ６ cycloalkyl)-, -(C ３ -C ６ cycloalkyl)-(C １ -C ２ alkyl)-, or -(C １ -C ２ alkyl)-(C ３ -C ６ cycloalkyl)-(C １ -C ２ alkyl)-; W is -R Ｂ --, --M--R Ｂ -M-, -M-R Ｂ -or-R Ｂ -M-R Ｃ - and; R Ａ ~R Ｃ are each independently C １ -C ４ is alkyl, M is, 【Chemistry 5】 and n is an integer from 5 to 9.

18. 2. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the camptothecin-based drug (A) that degrades DDX5 protein kills target cells, then passes through the cell membrane and kills surrounding cells; and / or penetrates deep into cancer tissue; and / or aggregates with itself to be absorbed by macrophages and removed from the body.

19. A pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18 as an active ingredient.

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