Conjugate combined with DDX5 protein and connected with camptothecin drug and acid-sensitive linker and immunoconjugate using conjugate

Abstract

The present invention relates to a conjugate in which a camptothecin-based drug (e.g., epatan and dxd) of formula 1, which is designed so as to bind to a DDX5 protein, is linked to an acid-sensitive linker, and to a carrier-drug conjugate using the conjugate. The present invention relates to: a conjugate in which a camptothecin-based drug (e.g., epatan and dxd) of formula 1, which is designed so as to bind to a DDX5 protein, is linked to an acid-sensitive linker; the camptothecin-based drug of formula 1 designed in a manner of binding to DDX5 protein according to the present invention not only can bind to DDX5 protein in cells and induce cell death by degradation of DDX5 protein, but also can preferably solve the problem of aggregation of a carrier-drug conjugate together with an acid-sensitive linker when used as a payload. Furthermore, the carrier-drug conjugate according to the present invention, when linked to a camptothecin-based drug of formula 1 by an acid-sensitive linker, prevents phagocytosis by macrophages in in vivo administration, distribution and elimination pathways after in vivo injection, and, due to the phagocytosis of the macrophages on reticuloendothelial system organs, has a significant therapeutic effect on the reticuloendothelial system organs. The composition cannot be absorbed by liver, spleen, bone marrow or lymph nodes, and can provide desired blood circulation characteristics.

Description

Technical Field

[0001] The present invention relates to a conjugate of a camptothecin drug (e.g., exatecan and dxd) of formula 1 designed in a manner to bind to a DDX5 protein and connected to an acid-sensitive linker, and a carrier-drug conjugate using the conjugate.

[0002] According to the present invention, the camptothecin drug of formula 1 designed in a manner to bind to the DDX5 protein can not only bind to the DDX5 protein in the cell and induce cell death through the degradation of the DDX5 protein, but also, preferably, when used as a payload, can solve the problem of aggregation of its carrier-drug conjugate and the acid-sensitive linker. Background Art

[0003] Cancer genes are genes that originally exist to control the normal functions of cells, but if they mutate or become abnormally regulated, they can lead to cancer. One of the normal cell functions is apoptosis, and related to this, there are apoptosis-promoting factors and apoptosis-inhibiting factors.

[0004] For example, cancer genes include (i) oncogenes that actively promote cell division when mutated and (ii) tumor suppressor genes that cannot suppress cell division when mutated. Examples of oncogenes include Src, EGFR, HER2, RAS, APC, BCL-2, etc., and examples of tumor suppressor genes include p53, BRCA, Rb, PTEN, BAX, etc.

[0005] Cancer is a group of diseases in which the cell cycle of growth and division is dysregulated. Fig.11 As shown, cancer occurs when the programmed cell death (e.g., apoptosis) machinery is impaired.

[0006] Cancer is caused by mutations in genes whose protein products are involved in cell cycle regulation. Many cancers are associated with the overexpression of specific genes or the abnormal activity of their mutant protein products (Oncogenes).

[0007] Proteins encoded by viral oncogenes are similar to cellular proteins with important regulatory functions. These cellular homologs are called proto-oncogenes or normal cellular oncogenes. Mutations in proto-oncogenes actively promote cell proliferation. Mutant cH-ras proteins contain mutations that impair the ability to hydrolyze GTP. This allows the mutant protein to maintain an active signaling mode and stimulate cell division. Mutated versions of c-ras have been found in multiple types of tumors.

[0008] A single mutation does not usually cause cancer. Often, several genes that regulate cell growth mutate before a cell becomes cancerous.

[0009] HER2-positive breast cancer, which has a high level of abnormal HER2 expression in breast epithelial cells, accounts for about 1 / 4 of all breast cancer patients. Compared with female hormone-related breast cancer, it has a higher recurrence rate during chemotherapy and a poorer prognosis. Herceptin (ingredient name: Trastuzumab), a HER2-targeted therapeutic drug, treats breast cancer by reducing the amount of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.

[0010] HER2-positive breast cancer is a breast cancer that retains amplification or overexpression of the HER2 (ErbB2) oncogene, a member of the ErbB receptor. Trastuzumab (Herceptin) has improved the treatment of HER2-positive breast cancer, but compared with other breast cancers, HER2-positive breast cancer is more aggressive, and more than half of patients develop resistance to existing targeted therapies or develop resistance during treatment.

[0011] As ligands of ErbB receptors attached to the cell membrane, NRG1, HB-EGF, activated ErbB ligands bind to their receptors (EGFR, HER3) and catalyze phosphorylation (phosphorylation, P) through the binding between receptors (EGFR-HER2, HER2-HER3) to induce intracellular growth and proliferation signals. Excessive activation of these ErbB receptors can lead to resistance to anti-HER2 therapeutic drugs.

[0012] The STAT (abbreviation for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6. When stimulated by growth factors, cytokines, interferons or oncogenes, JAK kinases activate STAT proteins to regulate interferon signaling. The DNA binding domain of STAT is an immunoglobulin-like structure that mediates the binding of specific DNA target sequences. During activation, phosphorylation of STAT3 by JAK kinase activates dimerization. The dimer then moves to the nuclear compartment and binds to the sequence of the target promoter to induce the expression of the target gene. STAT3 target genes include survivin, bcl-xl, mcl-1, Cyclin D1, MMP2, MMP9, VEGF, Myc, Sox2, etc. STAT proteins regulate many biological processes, including cell growth, apoptosis, differentiation and immunity. Among the STAT family members, STAT3 and STAT5 are most strongly associated with cancer progression.

[0013] Dysregulated STAT3 and activated STAT5 are considered oncogenes that increase angiogenesis and improve the survival of cancer cells. In a study using a large number of samples from the cBioPortal database, STAT3 was found to be more frequently overexpressed in lung cancer, ovarian cancer, gastric cancer, hematopoietic cancer, and brain cancer compared with normal tissues, and STAT3 overexpression was associated with poor overall survival. Phosphorylation of STAT3 by JAK kinase is the first step in STAT3 activation. In another study of 90 glioblastoma patients, high p-STAT3 values ​​were found to be significantly associated with decreased progression-free survival and overall survival. Multivariate survival analysis showed that high p-STAT3 levels can have an important prognostic role for poor progression-free survival and overall survival.

[0014] Type 1 topoisomerase (Topoisomerase I) inhibitors (irinotecan, topotecan, etc.) are anticancer mechanisms whose efficacy and safety have been clinically verified, and their excellent anticancer efficacy against various refractory solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer has been verified in clinical trials.

[0015] In this regard, camptothecin derivatives are known as low-molecular compounds that exhibit antitumor effects by inhibiting type 1 topoisomerase (topoisomerase-1).

[0016] Camptothecin is a selective inhibitor of topoisomerase-1 (an isomerase involved in DNA replication and recombination, etc.). It is a natural antitumor alkaloid isolated by Wall et al. in the United States in 1966 from Camptotheca acuminata, which is native to China. Since it was found to have strong cytotoxicity in vitro, the National Cancer Research Center (NCI) of the United States and others began to develop it through clinical trials, but due to the limitation of extremely poor solubility, the development was stopped due to various side effects such as bone marrow suppression and hemorrhagic cystitis. However, since 1990, it has been confirmed that camptothecin has a unique mechanism of action, that is, unlike the inhibition mechanism of DNA type 2 topoisomerase (topoisomerase-2), it selectively inhibits DNA type 1 topoisomerase to show antitumor effects.

[0017] DNA topoisomerases are members of the gyrase family of enzymes. These are enzymes in the cell nucleus that temporarily cut DNA or unwind the double helix when the cell needs to access genetic material for replication or transcription. They are also involved in a variety of intracellular activities, including chromosome condensation and recombination, DNA repair, etc. The genetic code of topoisomerases is highly conserved between species.

[0018] Type 1 topoisomerase, the drug target of camptothecin, has been found to be elevated in a variety of malignancies. The drug does not inhibit the free enzyme, but rather stabilizes the covalent bonds of the topoisomerase-DNA complex, preventing the cleaved DNA fragments from rejoining. Therefore, the sensitivity of cells to these topoisomerase-targeted drugs is related to the level of enzyme present in the cell nucleus. The drugs work by interfering with the recombination of DNA, thereby preventing transcription from taking place. The greater the amount of type 1 topoisomerase, the more cleavable complexes are formed, which means a high sensitivity to the drug. This has important clinical relevance, because type 1 topoisomerase inhibitors serve to increase the expression of type 2 topoisomerase, making it more sensitive to type 2 topoisomerase inhibitors. The antagonistic relationship between type 1 and type 2 topoisomerases supports this result. Unlike type 2 topoisomerase, these type 1 topoisomerases are not closely related to proliferation in normal tissues. They are present in solid cancers such as colon cancer, ovarian cancer, esophageal cancer, etc., which are in the "S" phase when cell division is active, including lymphoma, and are present in larger quantities compared to the surrounding normal tissues. In fact, it is known that the "S" phase in the cell cycle may cause cell death by blocking gene replication and transcription in tumor cells.

[0019] All camptothecin derivatives identified so far contain a five-ring parent structure that is required for cytotoxicity ( Figure 1). In terms of molecular structure, the E ring, A ring, and B ring sites are confirmed as important sites. Camptothecin contains a five-ring lactone structure in the E ring required for cytotoxicity. Among them, the lactone group and α hydroxyl group at the C20 position of the E ring are important for the stability of type 1 topoisomerase-DNA byproducts, and it has been confirmed that modifications of the A ring and the B ring can increase water solubility and activity. Modifications on the first ring have been confirmed to increase solubility in water and receive greater tolerance, for example, in the case of the drugs mentioned above.

[0020] CKD-602 also attempts to replace the B-ring site of carbon 7 to increase water solubility and anticancer effects. Lee et al. reported that CKD-602 has excellent anticancer effects in a variety of cancer cell lines compared to camptothecin and topotecan. In addition, it was confirmed to be a relatively safe drug in the L1210 leukemia nude mouse model, with a maximum tolerated dose (MTD) of 25 mg / kg. Generally, the side effects of known camptothecin drugs can be roughly divided into hematological side effects and non-hematological side effects. As hematological side effects, there are neutropenia, sepsis, bleeding, etc. accompanied by fever, and as non-hematological side effects, skin side effects such as nausea, vomiting, alopecia, etc., as well as toxicity to the gastrointestinal tract, kidneys, and nervous systems can be cited. In the CKD-602 animal study, Kim et al. found that among the side effects of these camptothecin drugs, no abnormal drug reactions other than increased gastric secretion were found in the case of high-volume administration more than 10 times higher than the clinically applicable dose. In addition, its relative safety has been demonstrated in recent clinical studies in South Korea, and only reversible and controllable side effects of neutropenia and leukopenia have been reported, with no reports of severe systemic toxicity. However, so far, its main indications are patients who have failed or cannot undergo standard therapy, that is, patients who have relapsed or worsened after standard therapy and are judged to be difficult to achieve results through further anticancer chemotherapy or surgery, and the treatment of refractory or recurrent ovarian cancer and colon cancer, refractory or recurrent limited-stage small cell lung cancer, and extensive-stage small cell lung cancer after initial chemotherapy failure, and its use is restricted.

[0021] Antibody-drug conjugates (ADCs) are a new drug platform that utilizes the high tissue selectivity of antibodies to selectively deliver payloads with strong anti-cancer efficacy only to cancer tissues. ADCs can selectively deliver strong effective carriers (payloads) that can kill cancer cells at low concentrations of pM levels to cancer tissues and minimize systemic drug exposure, thereby ensuring both anti-cancer efficacy and safety.

[0022] ADC consists of three components, including drugs, monoclonal antibodies, and linkers that connect antibodies and drugs. ADC technology is a method of delivering drugs to tumor cells using antibodies that specifically bind to specific antigens expressed on the surface of cancer cells. Most ADCs' intracellular transduction is carried out through the function of clathrin-coated pits. ADCs that migrate into cells fall off clathrin, fuse with other vesicles in the cell, and then proceed through the endosome-lysosome pathway. Next, proteases present in the acidic environment of endosomes cleave the linker, and the activated "free" drug passes through the lysosomal membrane, migrates to the cytoplasm, and then binds to the molecular target of the drug, thereby stopping the cell cycle of tumor cells and causing cancer cell death due to apoptosis. A predetermined amount of drug is passively diffused from the cell, actively transported, or leaked out of the cell through dead cells. At this time, if the leaked drug has cell membrane permeability, it also enters the surrounding cells to cause the so-called bystander cell-killing phenomenon.

[0023] The linker needs to be stable in the bloodstream, preventing the drug from separating from the antibody, keeping it in a prodrug state until it reaches the target, and minimizing damage to normal tissues. In addition, the ADC needs to maintain the same affinity as the antibody before the antibody binds to the drug. That is, interference with the antibody-antigen binding caused by the drug bound to the antibody should not occur.

[0024] After binding to target cells, ADCs are internalized into cells through a process called receptor-mediated endocytosis. At this point, sufficient concentrations of active drugs are required to enter the cells, but the internalization process through the antigen-antibody complex is usually inefficient, and the number of antigens on the cell surface is usually limited to <1×10 5Therefore, it is necessary to use a sufficiently potent drug to be able to fully kill cancer cells even at low drug concentrations. Therefore, drugs combined with antibodies for use as ADCs need to use drugs with cytotoxicity that is 100-1000 times higher than conventional anticancer drugs.

[0025] In order to deliver potent cytotoxic drugs only to specific cancer cells, determining the target antigen is the first major step in ADC development. By using antibodies, the high specificity and long half-life of the target can make long-term systemic circulation, so that cytotoxic drugs can be selectively accumulated only to tumor cells and the exposure of normal tissues can be minimized, thereby reducing damage, reducing side effects, and increasing the therapeutic effect. To this end, it is necessary to find a target antigen that can specific tumor cells, which requires the following conditions. First, the target antigen needs to be evenly overexpressed on the surface of tumor cells, and the expression in normal cells should be relatively low or not expressed. A representative example has human epidermal growth factor receptor 2 (HER2), which is known to be expressed more than 100 times as much as normal cells in HER2-positive breast cancer. Therefore, before making antibodies, the tumor expression of the target antigen is analyzed by various analyses, and if the overexpression of a specific antigen is confirmed, a monoclonal antibody that recognizes the antigen is generated. Second, the binding force to the antigen is that due to the characteristic of antibodies that internalize through receptors, the stronger the binding force to the epitope of the antigen, the more internalization may occur, thereby increasing the therapeutic effect. In addition, it has low immunogenicity.

[0026] Antibodies that recognize cancer cell antigens need to be internalized into cells together with drugs. In order to improve the internalization of cancer cells, bispecific antibodies are being developed.

[0027] SN-38 has an IC in the nanomolar range in various cell lines 50 It is a potent topoisomerase-I inhibitor with high value. It is the active form of the prodrug irinotecan used for the treatment of colorectal cancer, and is also active in lung cancer, breast cancer, and brain cancer. Trop-2-SN-38ADC has been successfully developed in various cancer types such as TNBC, bladder cancer, and gastric cancer, but there are still drug resistance problems caused by overexpression of drug efflux transporters, epigenetic silencing of Top1, and increase in anti-apoptotic proteins.

[0028] According to US 9629926 B, the in vivo distribution of hRS7-CL2A-SN-38 is similar to that of the parental hRS7 IgG in tumor uptake, but due to the hydrophobicity of SN-38, it is quickly eliminated by a 2-fold higher liver uptake. Since the ADC is eliminated by the liver, the dose to the liver and gastrointestinal tract is expected to be limited.

[0029] On the other hand, Daiichi Sankyo Co., Ltd. The development of DXD (exatecan class), a cytotoxic drug that is approximately 10 times more active in cancer cells than SN-38, was used. DXD has good solubility, is relatively safe, and has good surrounding cell killing effects, so it has advantages in the treatment of heterogeneous tumors. However, the half-life that can reduce off-target effects is very short. DXD was bioconjugated to the cysteine ​​residues of the anti-HER2 antibody via a maleimide linker, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD is very stable, with only 2.1% released into plasma within 21 days (Ogitani et al., 2016). Uherde was approved by the US Food and Drug Administration (US FDA) in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have received at least two HER2-targeted therapies in the past.

[0030] Antibody-drug conjugates (ADCs) such as Trodelvy and Enhertu that successfully use camptothecin derivatives as payloads can deliver camptothecin compounds that show strong anti-cancer efficacy only selectively to cancer cells, and can also bring excellent anti-cancer efficacy without serious systemic side effects, although there are still many shortcomings, the most representative example is the drug resistance caused by overexpression of the ABCG2 drug efflux pump. Many camptothecin derivatives are rapidly exported to the extracellular space due to ABCG2, so compared with cancer cells that do not express ABCG2 or express it at a low normal level, cancer cells that overexpress ABCG2 show a problem of severely reduced therapeutic efficacy. Overexpression of ABCG2 is not only the main reason for the reduced therapeutic efficacy of low-molecular-weight camptothecin compounds such as irinotecan and topotecan, but also the main reason for the reduced therapeutic efficacy of ADCs such as Trodelvy and Enhertu that use camptothecin compounds as payloads.

[0031] On the other hand, there are also research results reporting that DXd of Euheride has high membrane permeability compared to DM1 of Herceptin, and as a result, the payload released inside the cell is delivered to cells that do not express HER2 near the adjacent target cells. Euheride has a potential bystander effect due to the characteristics of the payload, and may also provide clinical advantages for patients refractory to Herceptin or Herceptin. Summary of the invention

[0032] Technical issues

[0033] The present inventors synthesized the FL118 compound, which is a dual MoA camptothecin drug with excellent type 1 topoisomerase inhibition ability and degradation of tumor protein (oncoprotein) DDX5 (p68), and continued to conduct various studies on its physicochemical properties. However, due to the limitations of its physicochemical properties, the FL118 compound is limited in the development of dosage forms.

[0034] In this regard, based on the structure of the FL118 compound which is different from the SN38 drug, the present inventors designed a camptothecin derivative having a dual mechanism of action (dual MoA) in terms of type 1 topoisomerase inhibition and DDX5 degradation, which are the advantages of the FL118 compound. During the design process, it was found that camptothecin derivatives such as Exitecan or Dxd also play a mechanism of action (MoA) of degrading DDX5 protein ( Figure 4 and Figure 5 ).

[0035] Therefore, the present invention aims to design and provide a novel antibody-drug conjugate (ADC) as a prodrug connected by an acid-sensitive linker in the absence of aggregation problems in body fluids, so that the camptothecin drug of formula 1 having a dual mechanism of action (dual MoA) of type 1 topoisomerase inhibition and degradation of tumor protein (oncoprotein) DDX5 is mainly released only at target sites such as tumor tissues.

[0036] In addition, the present invention aims to solve the aggregation problem of ADC (e.g., hRS7-CL2A-SN-38) with SN-38 as a payload and the dose limitation problem due to liver toxicity, and / or to improve the relatively weak (inadequate) efficacy of SN-38, when one end of the acid-sensitive linker is connected to the α-hydroxyl group at the C20 position of the E ring of the camptothecin derivative, as a candidate drug that not only has a more enhanced hydrogen bond strength and orientation than SN-38 in the molecular structure, but also can cause apoptosis of target cells, using a camptothecin drug of Formula 1 (e.g., exitecan or Dxd) designed in a manner to bind to the DDX5 protein as a payload, thereby connecting the camptothecin drug of Formula 1 to an acid-sensitive linker to improve the water dispersibility of the conjugate.

[0037] In addition, the present invention aims to design a conjugate of a camptothecin drug of Formula 1 (e.g., exotecan or Dxd) connected to an acid-sensitive linker with various payload-acid-sensitive linker combinations so as to (i) provide the desired hepatic clearance and blood circulation characteristics and / or (ii) penetrate deep into the tumor tissue while exerting a bystander effect or controlling the extent of its effect.

[0038] Furthermore, the object of the present invention is to provide an anti-cancer mechanism, which is a new therapy (New Modality) that kills cancer cells and / or surrounding cells before intrinsic or acquired drug resistance, so that the camptothecin derivative of Formula 1 with exitecan as the mother core has a bystander effect and binds to DDX5 protein in the cell to induce cell apoptosis (cell death) through degradation of DDX5 protein.

[0039] Solutions to the problem

[0040] The first embodiment of the present invention provides an immunoconjugate or a pharmaceutically acceptable salt thereof, which is directed to release the camptothecin drug of Formula 1 at a target site in vivo by comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment containing its antigen binding site], characterized in that the camptothecin drug of Formula 1 is a payload designed in a manner to bind to DDX5 protein and / or E3 ligase, at least one camptothecin drug of Formula 1 is connected to the antibody or a fragment containing its antigen binding site through an acid-sensitive linker, after targeting cancer cells through the antigen binding site of an antigen targeting cancer cells, the acid-sensitive linker is degraded in the acidic environment (pH≤7) surrounding the cancer, at least a portion of the camptothecin drug of Formula 1 is freed, the free camptothecin drug of Formula 1 penetrates the cell membrane and migrates into the cell, and optionally, the immunoconjugate connected to the camptothecin drug of Formula 1 is internalized into the cell, and the camptothecin drug of Formula 1 is freed from lysosomes.

[0041] Formula 1

[0042]

[0043] X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur,

[0044] X1 and X3 can be the same or different.

[0045] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0046] X1, (X2)n and X3 can form a pentagonal ring, a hexagonal ring or a septagonal ring (n = 0 to 2),

[0047] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0048] A second aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate of the first aspect or a pharmaceutically acceptable salt thereof as an active ingredient.

[0049] For example, it can be administered to treat HER2-positive cancer, breast cancer, lung cancer, and colon cancer.

[0050] Preferably, the pharmaceutical composition for preventing or treating cancer can be used as a primary treatment drug after cancer diagnosis or administered to a subject (over)expressing DDX5 in cancer tissue.

[0051] The third embodiment of the present invention provides a drug-linker conjugate in which a camptothecin drug of Formula 1-1 or Formula 1-2 is connected to an acid-sensitive linker of Formula 3, or a pharmaceutically acceptable salt thereof.

[0052] Formula 1-1

[0053]

[0054] Formula 1-2

[0055]

[0056] Formula 3

[0057]

[0058] Wherein, X1 and X2 are each independently -H or -halogen;

[0059] Y is -NH-, -NR A -, or nothing (null);

[0060] 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)-;

[0061] W is -R B -、-M--R B -M-, -MR B -or-R B -MR C -;

[0062] R A To R C are each independently a C1-C4 alkyl group,

[0063] M is as well as

[0064] n is an integer of 5-9.

[0065] The fourth embodiment of the present invention provides an immunoconjugate or a pharmaceutically acceptable salt thereof, characterized in that it comprises (a) a drug-linker conjugate of the third formula or a pharmaceutically acceptable salt thereof; and (b) an antibody or a fragment containing its antigen binding site, wherein at least one camptothecin drug of Formula 1-1 or Formula 1-2 is connected to the antibody or a fragment containing its antigen binding site via an acid-sensitive linker of Formula 3.

[0066] The fifth embodiment of the present invention provides a method for preparing a carrier-drug conjugate, characterized in that the drug-linker conjugate of the third formula or a pharmaceutically acceptable salt thereof is used to connect at least one camptothecin drug of Formula 1-1 or Formula 1-2 to a carrier via an acid-sensitive linker of Formula 3.

[0067] Hereinafter, the present invention will be described.

[0068] In the present specification, the camptothecin drugs of Formula 1-1 or Formula 1-2 are mixed and used by exatecan or Dxd, respectively.

[0069] In this specification, cancer and tumor are used interchangeably.

[0070] In the present invention, the term "drug-linker conjugate" is a substance used to prepare an immunoconjugate or a carrier-drug conjugate, and refers to a substance that is not connected to an antibody, a fragment containing its antigen binding site, or a carrier, and can also be arbitrarily combined with an antibody, a fragment containing its antigen binding site, or a carrier according to the purpose to be used as an immunoconjugate or a carrier-drug conjugate.

[0071] In this specification, the camptothecin drug of Formula 1 can be provided by the following steps:

[0072] (1) selecting an active camptothecin derivative designed to have a mechanism of action (MoA) for inhibiting type 1 topoisomerase and / or a mechanism of action (MoA) for degrading oncoprotein DDX5 from a library of compounds comprising a camptothecin skeleton represented by Formula 1 as a core, and / or

[0073] (2) A step of confirming whether a compound comprising a camptothecin skeleton represented by Formula 1 as a core inhibits type 1 topoisomerase and / or degrades oncoprotein DDX5 by in vitro experiments and / or in vivo experiments.

[0074] Only by understanding the characteristics of drugs can we use them correctly. The pharmacodynamic and pharmacokinetic parameters of drugs help us understand the characteristics of drugs.

[0075] In pharmacodynamics, the magnitude and pattern of changes (effects) (cell viability, clinical effect) (therapeutic action, toxic effect, adverse effect) that occur in cells or in vivo after a drug binds to its receptor are described by their relationship to drug concentration.

[0076] Pharmacokinetics (PK) shows how the drug concentration changes as it moves through different compartments of the body through metabolism and excretion (ADME), depending on the drug or drug's mode of action.

[0077] From a pharmacokinetic perspective, failures in the development of new drugs are due to the possibility that toxic drugs may accumulate, useful drugs may not have an advantage because doses are too small to establish treatment, and drugs may be rapidly metabolized.

[0078] Therefore, in order to use drugs correctly, it is necessary to select appropriate drugs and apply appropriate doses-dosages.

[0079] Dose refers to the amount of a drug given at one time. The dose of a drug is usually prescribed by a healthcare provider based on factors such as the patient's age, weight, and health status. Conversely, Dosage refers to the frequency and duration of drug administration. It measures the total amount of a drug given over a predetermined period, usually expressed as a daily or weekly amount. Dose and Dosage are important considerations for the safe and effective use of a drug.

[0080] On the other hand, the in vivo efficacy and in vivo side effects of anticancer drugs are closely related to the absorption, distribution, metabolism and excretion (ADME) properties of anticancer drugs. The ADME of a drug determines its pharmacokinetics, which describes how a drug moves through the body and interacts with tissues and organs.

[0081] Drug absorption affects efficacy and side effects. Poorly absorbed drugs do not reach therapeutic concentrations in target tissues, which may reduce efficacy. On the other hand, well-absorbed drugs may cause off-target side effects due to increased systemic exposure.

[0082] The distribution of drugs can also affect efficacy and side effects. Drugs that are poorly distributed in target tissues may be ineffective, while drugs that are widely distributed in non-target tissues may induce toxicity in the corresponding tissues. In addition, drugs with high binding capacity to plasma proteins may have reduced distribution to target tissues, which may lead to reduced efficacy.

[0083] The distribution of a drug in the body may be affected by its physicochemical properties, including size, charge, and lipophilicity. The ability of a drug to penetrate into tumor tissue may also be affected by factors such as the tumor microenvironment, such as blood flow and cell density. Some anticancer drugs accumulate in specific tissues, which may cause toxic effects.

[0084] Therefore, it is necessary to analyze the ADME properties of anticancer drugs to optimize efficacy and minimize side effects in vivo. That is, when ADME characteristics are evaluated and optimized, the therapeutic index of these drugs is improved, thereby leading to better outcomes for cancer patients.

[0085] Surprisingly, as described below, the present inventors designed a camptothecin derivative having a dual mechanism of action (dual MoA) based on the structure of the FL118 drug of formula 2 which is different from the SN38 drug, from the perspective of inhibiting type 1 topoisomerase and degrading DDX5, which are the advantages of the FL118 drug ( Figure 1 ), and found that ixetine or Dxd also played a role in degrading DDX5 protein (MoA) ( Figure 4 and Figure 5 ).

[0086] Formula 2

[0087]

[0088] Surprisingly, it was found that, like FL118, exatecan not only strongly inhibited the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another major cause of anticancer drug resistance, at low concentrations, but also blocked the resistance mechanism ( Figure 4 and Figure 5 ).

[0089] The present invention is based on these findings.

[0090] Therefore, in the present invention, the camptothecin drug of Formula 1 used as a payload by connecting with [acid-sensitive linker] is designed in the same manner as the compound of Formula 1-1 (exitecan) and the compound of Formula 1-2 (dxd) in that R1 and R2 (A group) on the A ring in Formula 1 are similar to the FL118 compound of Formula 2 ( Figure 4 ), characterized in that it exerts an anti-cancer mechanism of degrading the intracellular oncoprotein DDX5 (Example 1).

[0091] Formula 1

[0092]

[0093] The synthetic design concept of active camptothecin derivatives with dual MoA of type 1 topoisomerase inhibition and oncoprotein DDX5 degradation is as follows: Figure 1 As shown, and can be designed into the following structure based on the structure-activity relationship (SAR), that is, the structure of group C as the type 1 topoisomerase inhibition region is maintained, and group A as the binding site for DDX5 degradation is also similar to FL118, while maintaining the same structure as the compound of formula 1-1 (ixitencan) and the compound of formula 1-2 (dxd), and adjusting the structure (R3 and R4) of group B in general formula 1 to be as shown in formula 1, so that it can be designed into the following structure, that is, not only the physicochemical properties of the drug can be improved to solve the aggregation problem of the drug, but also the cytotoxicity of the drug and / or the tumor tissue penetration and / or cell membrane permeability of the drug can be adjusted to be able to accurately adjust the bystander killing effect.

[0094] In Formula 1 or Formula 2, the C group site binds to type 1 topoisomerase and the A group site binds to DNA to stabilize the covalent bonds of the topoisomerase-DNA complex to hinder reconnection of the cut DNA fragments, and / or, in Formula 1 or Formula 2, the A group site binds to DDX5 and the C group site binds to E3 ligase to induce DDX5 degradation (refer to PCT / KR2023 / 005380, incorporated herein by reference).

[0095] In the present invention, it is preferred to design a camptothecin drug of Formula 1, and by modifying R3 and / or R4 in Formula 1, the cell membrane permeability can be adjusted as needed, so that an appropriate bystander effect can be exerted in tumor tissues.

[0096] Therefore, the main feature of the present invention is that, taking into account various side effects and reduced therapeutic coefficient, the selection range of the effective load of the ADC that exerts appropriate anti-cancer efficacy can be extended to various candidate groups including the active camptothecin derivatives of Formula 1, wherein the active camptothecin derivatives of Formula 1 are combined with the DDX5 protein and the E3 ligase using a molecular glue degrader.

[0097] Formula 1

[0098]

[0099] X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, and X1 and X3 may be the same or different,

[0100] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0101] X1, (X2)n and X3 can form a pentagonal ring, a hexagonal ring or a septagonal ring (n = 0 to 2),

[0102] Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0103] Among them, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may 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 , 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 Hm , 0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl or Br), - COSH, - COOCO -, - CORCO - (R = C l 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, epoxy, -hydrazone, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C≡C- and a functional group in the group consisting of hydrocarbons of two or more carbons.

[0104] According to the present invention, the camptothecin drugs of Formula 1 designed in a manner of binding to the DDX5 protein and / or the E3 ligase can kill target cells expressing the DDX5 protein through a molecular glue degrader mechanism (MoA).

[0105] The target cell can be a cancer cell or a senescent cell. Senescent cells also include cells that do not perform long-term intrinsic characteristic functions.

[0106] Preferably, according to the present invention, the camptothecin drugs of Formula 1 designed in a manner to bind to DDX5 protein and / or E3 ligase may have multiple mechanisms of action (MoA) of type 1 topoisomerase inhibition and degradation of oncoprotein DDX5.

[0107] According to the present invention, the camptothecin derivative represented by Formula 1, such as Formula 1-1 and 1-2, is Figure 1Like the camptothecin shown in , it has a five-ring structure with a lactone in the E ring required for cytotoxicity and is designed to maintain the lactone group and α-hydroxyl group at the C20 position of the E ring, which are important for the stability of type 1 topoisomerase-DNA byproducts. In general formula 1, the structural characteristics of exatecan drugs, namely, (1) the structural characteristics of R1 and R2 (CH3-C=CF) are maintained, which bind to the DDX5 protein and are similar to the orientation structure of the FL118 drug (-OCH2O-(methylenedioxo) 5-membered ring), and (2) through R3 and R4, compared with SN-38 that induces aggregation by π-π stacking of the aromatic ring formed by the A ring and the B ring, various orientations of continuous carbon-carbon single bonds of the 6- or 7-angled ring extending from the A ring and the B ring are in dynamic equilibrium, resulting in that the stacking of the aromatic ring formed by the A ring and the B ring can be weakened or inhibited.

[0108] In addition, for the compound represented by Formula 1-1, the molecular bond can be rotated relative to the carbon-carbon single bond in the hexagonal ring extending from the A ring and the B ring, so that the -NH2 with a large degree of freedom is exposed to water (H2O) to carry a (+) charge or form a hydrogen bond with water to increase water dispersibility.

[0109] In addition, for the compound represented by Formula 1-2, the molecular bond can be rotated relative to the carbon-carbon single bond in the hexagonal ring extending from the A ring and the B ring, so that the functional group CH2(OH)CONH- in the form of lactic acid (Lactic acid) of -NH2 with a large degree of freedom is exposed to water (H2O), and as it rotates like a propeller, it forms hydrogen bonds with water to increase water dispersibility.

[0110] On the other hand, the DXd payload used in Enhertu was originally made from the exatecan compound, which is hardly affected by ABCG2, but is strongly affected by ABCG2 due to the presence of the glycolic acid (alpha-hydroxy acetic acid) functional group used to convert exatecan into DXd. However, the glycolic acid functional group plays a very important role in the excellent safety / efficacy characteristics of Enhertu, and when it is eliminated, it will bring difficulties to ADC preparation and will also lead to the problem of worsening the characteristics of ADC in animal models and clinical trials (safety issues or reduced efficacy).

[0111] like Figures 4 to 7As shown, various camptothecin drugs were treated at different concentrations in A549 cells, which are cancer cells that overexpress FaDu and ABCG2, and FaDu and ABCG2 are Her2-low / mid cancer cells that do not express ABCG2, so that the degree of degradation of intracellular DDX5 protein and the resulting expression inhibitory activity of cancer-associated survival genes such as survivin, Mcl-1, XIAP, and cIAP2 could be confirmed, and the cell membrane permeability could be indirectly compared and confirmed.

[0112] In addition, the present invention is characterized in that it provides an immunoconjugate comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment containing its antigen binding site]; or a carrier-drug conjugate comprising [camptothecin drug of Formula 1]-[acid-sensitive linker], wherein when a compound (e.g., exitecan or Dxd) comprising a camptothecin skeleton represented by Formula 1 as a core is arbitrarily combined with an antibody, a fragment containing its antigen binding site, or a carrier to be used as an effective load of the immunoconjugate or carrier-drug conjugate, the following characteristics are optimized or maximized: (1) an excellent apoptotic effect through the mechanism of action (MoA) of intracellular degradation of DDX5 protein; (2) a bystander effect optimized as it penetrates deep into tumor tissue; and (3) excellent physicochemical properties suitable for the generation of high DAR ADC.

[0113] According to the present invention, an immunoconjugate comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment containing its antigen binding site]; or a carrier-drug conjugate comprising [camptothecin drug of Formula 1]-[acid-sensitive linker], the acid-sensitive linker is degraded in the acidic environment (pH≤7) surrounding the cancer, and at least a portion of the camptothecin drug of Formula 1 is free, so in order to exert a suitable surrounding cell killing effect (bystander effect) or control the extent of its effect while penetrating deep into the tumor tissue, the relative hydrophilicity / hydrophobicity of the camptothecin drug of Formula 1 has an important influence on the solubility, absorption, distribution, metabolism and excretion (ADME) of the drug. In particular, the ease with which the drug passes through the cell membrane and the interaction between it and the drug target DDX5 protein and / or E3 ligase are important.

[0114] like Figure 2 As shown in Figure 2, the hydrophobicity of various camptothecin drugs can be expressed by the partition coefficient (p).

[0115] In the case of hydrophobicity, the p value is large, and in the case of hydrophilicity (polarity), the p value is small. In practice, its log P value is used as a measure of hydrophobicity. Clog P refers to the log P value of a given compound calculated by appropriate software. The smaller the cLogP value, the higher the polarity.

[0116] The polar surface area (PSA) or topological polar surface area (TPSA) of a molecule is defined as the sum of the surface areas of all polar atoms or molecules, primarily oxygen and nitrogen, including attached hydrogen atoms. tPSA is a commonly used medicinal chemistry assay to optimize the cell penetration ability of a drug. Molecules smaller than Only tPSA can cross the blood-brain barrier and act on receptors in the central nervous system.

[0117] The camptothecin drugs of formula 1 used as effective loads in the present invention are hydrophobic low molecules, and even if R3 and R4 of general formula 1 are modified, they can penetrate the cell membrane, so when delivered to tumor tissue, they can penetrate deep into the tumor tissue and accumulate at high concentrations. After the camptothecin drugs of formula 1 or their prodrug ADC penetrate the cell membrane, the camptothecin drugs of formula 1 bind to DDX5 inside the cell as molecular glue degraders, causing cell apoptosis and release into the extracellular fluid, and continuously penetrate the cell membrane in the surrounding cells and move into the cell to exert the apoptosis mechanism. Therefore, the camptothecin drugs of formula 1 that are advantageous from the immunoconjugate or carrier-drug coupling of the present invention can convert solid cancers that aggregate with high cell density into cancers that disperse with low cell density, and / or, can convert cold tumors with low immune activity into hot tumors with high immune activity. In addition, the surrounding cell killing effect is high, so it is advantageous for the treatment of heterogeneous tumors.

[0118] Drug efficacy refers to the ability to remain in the body within the expected time to show effect on the target indication without degradation.

[0119] The in vivo efficacy of anticancer drugs can be affected by their absorption, distribution, metabolism, and excretion (ADME) properties. The ADME characteristics of a drug can affect its ability to reach and act on cancer cells. That is, the ADME properties of anticancer drugs can have a significant impact on their in vivo efficacy.

[0120] The ability of a drug to penetrate into tumor tissue may also be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0121] Therefore, understanding the ADME properties of anticancer drugs and optimizing the selection of drugs / drug modalities as well as dosage / administration regimens can improve the therapeutic index of these anticancer drugs, thereby leading to better outcomes for cancer patients.

[0122] Antibody-drug conjugates (ADCs) have different life times and ADME characteristics from antibodies (Abs) due to their different structures and mechanisms of action, which depend not only on the drug itself as its payload but also on the various combinations with linkers.

[0123] Antibodies are large proteins that react to common foreign substances (antigens) and are naturally produced by the immune system. Due to their size and complex structure, they have a long circulation half-life (weeks to months), which protects them from degradation and elimination. Antibodies are distributed throughout the body, including tissues, and are able to interact with target antigens with high specificity and affinity. Antibodies are mainly eliminated through degradation metabolism in the reticuloendothelial system (RES), including the liver and spleen, and in the kidneys and other organs.

[0124] ADCs consist of an antibody (usually 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, in turn, provides cytotoxic activity that kills target cells. ADCs have a shorter half-life than antibodies, typically a few days to a week. This is because ADCs are internalized into target cells, resulting in lysosomal degradation of the ADC and release of the drug payload ( Figure 3 ADCs are primarily eliminated by the RES, but the drug payload can also be metabolized and excreted by the liver and kidneys.

[0125] Antibodies are usually administered by subcutaneous or intravenous injection and absorbed into the bloodstream. They can be distributed throughout the body (including tissues), but are usually limited to the extracellular space due to their size. ADCs are also administered by injection and absorbed into the bloodstream, but due to the antibody component, the drug payload released from the ADC can penetrate into cells and tissues after targeting tumors or diseased tissues (in some cases, receptor-mediated endocytosis). Furthermore, the metabolism and excretion of ADCs vary depending on the specific structure and the specific drug, linker or antibody used.

[0126] In the present invention, an acid-sensitive linker refers to a linker that is stable in the neutral environment of blood at pH 7.3 to 7.5, but causes internalization around tumor cells (pH 6.5 to 7.2) or in cells and hydrolyzes in weakly acidic environments such as endosomes (pH 5.0 to 6.5) and lysosomes (pH 4.5 to 5.0) to release the drug. Therefore, in the present invention, the acid-sensitive linker has a hydrophilic molecular structure to produce a hydrolysis environment. To this end, the acid-sensitive linker may include, for example, a polyethylene glycol (PEG) spacer.

[0127] The ADC of trastuzumab (Trastuzumab), a kind of HER2 antibody, connected to the camptothecin drug of Formula 1-1 or Formula 1-2 through the CL2A linker, which is a kind of acid-sensitive linker of Formula 3, i.e., Trastuzumab-CL2A-exadecane (Preparation Example 3) and Trastuzumab-CL2A-dxd (Preparation Example 4) (DAR 7-8) is easy to synthesize and has no aggregation problem.

[0128] like Figure 8 As shown, in the Her2-low / mid cancer cells FaDu that do not express ABCG2 and the cancer cells A549 that overexpress 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) of Trastuzumab-CL2A-exadecane (Preparation Example 3) were shown in a concentration-dependent manner.

[0129] like Fig. 9 and Fig.10As shown, Trastuzumab-CL2A-exadecane (Preparation Example 3) exhibited cytotoxicity in Her2-high cell lines (MDA-MB-453) and HER2-positive breast cancer-derived cell lines (SK-BR-3).

[0130] Therefore, the present invention provides various camptothecin drugs of Formula 1 connected to DDX5 protein and E3 ligase by molecular glue degrader as various effective load candidates, so that more than one suitable drug can be selected and its dosage-administration can be appropriately applied to accurately control the desired efficacy (e.g., anti-cancer, combination therapy) and side effects (aggregation problem of camptothecin drugs).

[0131] For example, for the camptothecin drug of Formula 1, depending on the drug mode (small molecule, ADC), exceeding a specific drug concentration (high) in tumor tissue may be important, and depending on the situation, the time for which the specific drug concentration (low) is maintained in the tumor tissue may be important.

[0132] [Distribution and Excretion Control of Carrier-Drug Conjugates by Various Combinations of Camptothecin Drugs of Formula 1 and Acid-Sensitive Linkers of Formula 3]

[0133] Once a drug enters the body, the excretion process begins. The main routes of drug excretion are (1) liver metabolism, (2) bile excretion, and (3) urine excretion.

[0134] Carrier-drug conjugates such as antibody-drug conjugates (ADCs) have the potential to selectively deliver drugs to disease sites or to deliver larger amounts of drugs to disease sites. However, the limitation of most drug nanoformulations such as artificial nanocarriers, liposomes, and polymer nanoparticles is that they are engulfed by the reticuloendothelial system of the human immune system before reaching the disease site, thereby having a rapid elimination from the blood circulation system.

[0135] The reticuloendothelial system is also called the macrophage system or the mononuclear phagocyte system (MPS). It is a cell that absorbs specific substances from various parts of the human body. These cells form part of the body's defense mechanism.

[0136] Reticuloendothelial cells are produced by progenitor cells in the bone marrow. The progenitor cells develop into phagocytes that are released into the blood, called monocytes. Some monocytes remain in the circulation, but most enter the body's tissues and become larger phagocytes called macrophages. Most macrophages are stationary cells that remain in the tissues, filtering foreign matter and destroying it. However, some break off and wander in the circulation or in the spaces between cells.

[0137] Tissue macrophages have different shapes and names depending on where they are located. Reticular cells are found in lymph nodes, spleen, and bone marrow, whereas histiocytes are found in large numbers in subcutaneous tissue. Microglia are found in nervous tissue, alveolar macrophages in the alveoli of the lungs, and Kupffer cells in the liver. Through phagocytosis, macrophages form the first line of defense against harmful particles that enter the body.

[0138] Nanoparticles accumulated in the body vary in size, the organs into which they are taken up, and the resulting distribution in the body. Nanoparticles larger than 50 nm are engulfed by Kupffer cells in the liver, an organ with a well-developed reticuloendothelial system, and rapidly accumulate in the liver.

[0139] When the carrier-drug conjugate is aggregated with the hydrophobic camptothecin drugs connected by the linker and accumulated in the liver through phagocytosis of macrophages, a part of the camptothecin drugs connected with the acid-sensitive linker is released as free hydrophobic camptothecin drugs in the liver, which may cause cytotoxic hepatotoxicity problems after penetrating the cell membrane of normal cells.

[0140] The present invention is characterized in that, in order to make the carrier-drug conjugate aggregated by the hydrophobic camptothecin drug connected via an acid-sensitive linker and not be accumulated in the liver through macrophage phagocytosis, (1) as a payload of the camptothecin drug connected to the acid-sensitive linker, instead of SN-38, which induces aggregation by π-π stacking of the aromatic ring formed by the A ring and the B ring, a compound (e.g., exitecan or Dxd) containing the skeleton of the camptothecin represented by Formula 1 as a parent core is used, and various orientations of the continuous carbon-carbon single bonds of the hexagonal ring or the heptagonal ring extending from the A ring and the B ring form a dynamic equilibrium to be able to inhibit the aggregation of the A ring. and stacking of aromatic rings formed by ring A and ring B, (2) preferably, one end of the linker is connected to the α-hydroxyl group at the C20 position of the E ring of the camptothecin drug of formula 1, so that it is designed to be able to rotate the molecular bond relative to the carbon-carbon single bond on the hexagonal ring or the heptagonal ring extending from the A ring and the B ring, and the -NH2 or -OH with large freedom is exposed to the aqueous environment (aqueous environment), that is, exposed to water (H2O), carries a (+) charge or forms a hydrogen bond with water, so as to improve the water dispersibility of the camptothecin drug site of formula 1 directed by the acid-sensitive linker in the carrier-drug conjugate.

[0141] That is, the present invention is designed such that the carrier-drug conjugate connected to the camptothecin drug of Formula 1 is aggregated by the camptothecin drug and does not accumulate in the liver through macrophage phagocytosis, thereby solving the aggregation problem of ADC with SN-38 as a payload (e.g., hRS7-CL2A-SN-38) and the dose limitation problem caused by the hepatotoxicity induced thereby.

[0142] In addition, the present invention improves the water dispersibility of the camptothecin drug site of formula 1 directed by the acid-sensitive linker in the carrier-drug conjugate, thereby reducing the phagocytosis caused by the reticuloendothelial system in the liver, thereby increasing the retention time in the blood, and being able to maintain the blood drug concentration for a long time at a high concentration. In addition, when the hydrophobicity of the linker and the drug increases, the cohesion of the ADC increases, resulting in a corresponding decrease in the therapeutic coefficient of the drug. Therefore, by adjusting the hydrophilicity of various combinations of the camptothecin drug of formula 1 and the acid-sensitive linker of formula 3, the carrier-drug conjugate can be provided with the desired hepatic clearance characteristics and blood circulation characteristics.

[0143] [Antitumor mechanism of FL118 as a molecular glue degrader binding to DDX5]

[0144] FL118 of Formula 2 can act as a molecular glue degrader that degrades DDX5 by directly conjugating DDX5 and ubiquitination regulatory factors.

[0145] FL118 acts as a molecular glue. Without reducing DDX5 mRNA, it directly connects to the tumor protein DDX5, which is a multifunctional masterregulator, through the proteasome degradation pathway and has the function of dephosphorylating and degrading it. DDX5 silencing indicates that DDX5 is a master regulator for regulating the expression of multiple oncogenic proteins including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras.

[0146] In addition, drug resistance can be avoided by fundamentally blocking the overexpression of anti-apoptotic proteins, which is a common mechanism for the development of drug resistance in most cancers. Biomarkers (DDX5, K-ras, p53) and companion diagnostic technologies that can predict patients' anti-cancer responses have been established. Since customized biomarkers are ensured, customized treatment through companion diagnostics can be achieved, especially in p53 / K-ras mutant cancer cells with poor prognosis, showing strong efficacy.

[0147] FL118 effectively inhibits cancer initiation, development, metastasis, recurrence and treatment resistance by indirectly controlling DDX5 downstream targets, as demonstrated in studies using human colorectal cancer / pancreatic cancer cells and tumor models.

[0148] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Studies in human tumor animal models have shown that FL118 shows high efficiency in eliminating human PDAC and CRC tumors with high DDX5 expression, while FL118 is more effective in PDAC and CRC tumors with low DDX5 expression.

[0149] DDX5 protein is the direct target of FL118 drug and can be used as a biomarker to measure PDAC and CRC tumor sensitivity to FL118.

[0150] On the other hand, FL118 has the same level of Top1 inhibition efficacy as SN-38 in cancer cells and is 5-20 times more potent than SN-38 in various cancer cell lines, i.e., at a low level of IC 50 The numerical values ​​show cytotoxicity. The evaluation results of 140 atoms in various cancer cell lines showed very strong anti-cancer efficacy, with IC values ​​of 0.1% for most cancer cells. 50 <100nM. The FL118 drug ensures excellent safety through GLP-toxicity tests in rats and beagles, and shows superior efficacy than SN-38 in various cancer cell line Xenograft models. On the other hand, for Camptothecin anticancer drugs, when used in patients, they initially show excellent anticancer responses, but through epigenetic silencing of the Top1 gene and Top2 dependence of cancer cells, they show strong resistance to these drugs. Conversely, the FL118 drug shows strong efficacy even in xenograft models of cancer cell lines that do not express Top1 through epigenetic silencing or knockout.

[0151] In addition, FL118 directly targets the established anticancer target type 1 topoisomerase, and is a triple-target anticancer drug that simultaneously inhibits Bcl family members (Bcl family) such as the resistance protein Survivin involved in the drug resistance mechanism, and inhibits the single-acting efflux pump.

[0152] Specifically, SN-38 and other camptothecin anticancer drugs show drug resistance in the way they release drugs outside cells due to overexpression of ABCG2 Transporter, but FL118 drug is not affected by ABCG2 transporter, so it can overcome drug resistance. FL118 drug can block the expression of drug resistance by strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), which is another major cause of anticancer drug resistance, at low concentrations.

[0153] Therefore, FL118 will not be excreted from cells by the efflux pump ABCG2, and can block drug resistance caused by various anti-apoptotic proteins. As a result, FL118 can overcome various drug resistance mechanisms of SN-38 / exatecan.

[0154] When FL118 was administered in vivo at the same amount as SN-38, it showed a more potent (tumor regression) effect than SN-38 in colon cancer, head and neck cancer, pancreatic cancer, etc.

[0155] After FL118 induced SN-38 resistance in tumor xenografts, FL118 retained its strong anticancer efficacy when administered.

[0156] Furthermore, the FL118 drug has an optimal PK / safety profile for targeted drug delivery (e.g., carrier-drug conjugate applications). When the FL118 drug is administered alone systemically, it is rapidly metabolized and metabolized / excreted from the blood, but it rapidly accumulates in cancer tissues immediately after administration and maintains high concentrations for a long time. It only shows a low concentration, but it rapidly accumulates in cancer tissues immediately after administration and maintains high concentrations for a long time. For example, when ADC is applied, maximum selectivity between tumor tissues and normal tissues can be ensured in the system.

[0157] Based on this, according to the present invention, the camptothecin drugs of Formula 1 are designed in various ways to be used as effective loads, so as to be able to exert various advantages as anticancer drugs exemplified in the FL118 drug.

[0158] [Antitumor mechanism as a molecular glue degrader binding to DDX5]

[0159] The ubiquitin-proteasome system (UPS) is an important pathway for degrading intracellular proteins, which are used to regulate a wide range of cellular processes. Ubiquitin is a small protein covalently linked to lysine residues of substrate proteins through a series of enzymatic reactions including ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process is called ubiquitination and is an important mechanism for regulating protein degradation, signal transduction, and transport.

[0160] Ubiquitin ligases are responsible for substrate specificity in the ubiquitination pathway.

[0161] In relation to this, the camptothecin drug of Formula 1 according to the present invention is designed in such a manner as to be linked to the DDX5 protein and the E3 ligase.

[0162] The transformation of normal cells into cancerous cells occurs due to the deregulation of a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cell metabolism, but when they are deregulated, they can accelerate neoplastic transformation. DDX5 can interact with hundreds of other cellular proteins, and both proteins can act as tumor suppressors or oncogenes, depending on the specific pathways involved.

[0163] The DEAD-box family of RNA helicases participates in a variety of metabolic pathways, from transcription and translation to cell proliferation, innate immunity, and stress responses. Given their diverse roles, their deregulation or their mutations are associated with a variety of pathological conditions, including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation to behave as a tumor suppressor, while in other cases, overexpression of the same enzyme favors cancer progression and acts as a classic oncogene.

[0164] DDX5 (also known as p68) is a multifunctional master regulator that acts on the following mechanisms: (1) the biological process of co-activation of transcription of a large number of tumor genes through direct interaction with various transcription factors (e.g., c-Myc) in 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).

[0165] Camptothecin drugs of Formula 1 according to the present invention bind to DDX5 protein in the absence of DDX5 mRNA reduction and are degraded through functional dephosphorylation and proteasome degradation pathways, which means that camptothecin drugs of Formula 1 can attach to both DDX5 and ubiquitin-involved protein stability / degradation regulators and act as "molecular glue degraders".

[0166] As is well known, DDX5 downstream protein targets are all related to the occurrence, development, metastasis, recurrence and treatment resistance of cancer. Therefore, when the DDX5 downstream targets are indirectly blocked by the degradation of the DDX5 protein of the camptothecin drug of Formula 1 according to the present invention, the camptothecin drug of Formula 1 can show high anti-tumor efficacy.

[0167] DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and a transcription cofactor. Therefore, when cancer occurs due to transcription factor deregulation caused by the physiological state of DDX5, DDX5 (p68) as a transcription cofactor can be selectively degraded to treat cancer. Similarly, cancer can be prevented by selectively degrading DDX5 (p68) as a transcription cofactor.

[0168] According to the camptothecin drug of Formula 1 of the present invention, the DDX5 protein is a drug target, so drug resistance, resistance to targeted therapy and / or drug resistance during treatment can be avoided. In addition, the transcriptional induction of anti-apoptotic genes can be blocked (off) by the camptothecin drug of Formula 1. In addition, the DDX5 protein as a transcription cofactor is degraded by the camptothecin drug of Formula 1, so the sensitivity of cancer cells to chemotherapy or radiotherapy can be maintained or improved.

[0169] [Mechanism of Action (MoA) of the Immunoconjugate of the Present Invention]

[0170] One embodiment of the present invention relates to an immunoconjugate comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment thereof containing an antigen-binding site], characterized in that:

[0171] (i) the camptothecin drug of formula 1 is a payload designed to bind to the DDX5 protein and / or the E3 ligase,

[0172] (ii) at least one camptothecin drug of Formula 1 is linked to an antibody or a fragment thereof containing an antigen binding site via an acid-sensitive linker,

[0173] (iii) after targeting cancer cells by targeting the antigen binding site of the antigen of the cancer cell, the acid-sensitive linker is degraded in the acidic environment (pH ≤ 7) surrounding the cancer, and at least a portion of the camptothecin drug of Formula 1 is freed, and the free camptothecin drug of Formula 1 penetrates the cell membrane and migrates into the cell,

[0174] (iv) Optionally, the immunoconjugate linked to the camptothecin drug of Formula 1 is internalized into cells, and the camptothecin drug of Formula 1 is released from lysosomes.

[0175] The immunoconjugate of the present invention uses an acid-sensitive linker, so that after antigen binding, the camptothecin drug of Formula 1 can be effectively released not only inside the cancer cell but also around the cancer tissue, thereby overcoming the drug resistance mechanism associated with ADC treatment ( Figure 3 ).

[0176] In order for ADCs to work effectively, it is not enough to maximize potent antigen-selective cytotoxicity against cells in vitro; they also need to be able to penetrate cancer tissues well and deliver drugs effectively in vivo or in actual patients.

[0177] The immunoconjugate of the present invention targets cancer cells by targeting the antigen binding site of the antigen of the cancer cell, and then the acid-sensitive linker is degraded in the acidic environment (pH≤7) around the cancer, so that at least part of the camptothecin drug of Formula 1 is freed, and the free camptothecin drug of Formula 1 is a hydrophobic low molecular weight, but compared with SN-38, it is a drug with improved water dispersibility to prevent aggregation in body fluids such as blood and interstitial fluid, so as to penetrate into the deep tumor tissue and move into the cell through the cell membrane.

[0178] Therefore, the immunoconjugate of the present invention uses an acid-sensitive linker that degrades in the acidic environment (pH≤7) surrounding the cancer, so that the drug can be quickly released in the tumor microenvironment surrounding the cancer. The free camptothecin drug of Formula 1 has a low molecular weight and, unlike antibodies, has a high ability to penetrate cancer tissues. Therefore, the problem of antibodies in existing ADCs that cannot penetrate deep into cancer tissues can be solved.

[0179] Therefore, the free camptothecin drug of Formula 1 moves into cells and can target cancer cells and / or their surrounding cells.

[0180] In addition, the camptothecin drugs of Formula 1 are hydrophobic low molecules that can penetrate cell membranes. Therefore, the camptothecin drugs of Formula 1 released from the immunoconjugate of the present invention through the degradation of the extracellular linker around cancer cells are rapidly accumulated in tumor tissues and can maintain high concentrations for a long time, penetrate the cell membrane, exert cytotoxicity inside the cell, and are released after causing cell apoptosis, thereby continuously penetrating the cell membrane of surrounding cells and moving into the cell to work.

[0181] In short, the [camptothecin drug of Formula 1]-[acid-sensitive linker] conjugate of the present invention, which can be used in the carrier-drug conjugate of the present invention, is characterized in that:

[0182] (i) in the acidic environment (pH ≤ 7) surrounding the tumor microenvironment of the cancer, a hydrophobic low molecular weight drug that can penetrate the cell membrane and act inside the cell is released, and then an acid-sensitive linker such as the CL2A linker of Formula 3 is used to introduce a large amount of free hydrophobic low molecular weight drug into the cell and / or penetrate deep into the tissue; and

[0183] (ii) In the tumor microenvironment surrounding the cancer in an acidic environment (pH ≤ 7), a large amount of free drugs released by degradation of the acid-sensitive linker can penetrate deep into the tumor tissue and penetrate the cell membrane to move into the cell for intracellular enrichment. Although it is a hydrophobic low-molecular drug that can penetrate the cell membrane, the camptothecin drug of formula 1 with improved water dispersibility compared to SN-38 is used to prevent aggregation in body fluids such as blood and interstitial fluid.

[0184] Another feature is that the organic mechanism of action of the camptothecin drug-acid-sensitive linker of Formula 1 is utilized in combination with anticancer efficacy and aggregate toxicity.

[0185] Preferably, the camptothecin drug of Formula 1 and the acid-sensitive linker are linked via a carbonate or ester bond, so that when degraded in an acidic environment (pH≤7), the free camptothecin drug of Formula 1 is released when the acid-sensitive linker is degraded.

[0186] In the present invention, the connection between [acid-sensitive linker]-[antibody or a fragment containing its antigen-binding site] can be a "click" reaction through reaction formula 1, wherein the thiol group contained in the antibody or its antigen-binding fragment is connected to the maleimide group (maleimide) or maleic hydrazide (maleic hydrazide) of the acid-sensitive linker for binding.

[0187] Reaction 1

[0188]

[0189] In addition, the present invention provides a drug-linker conjugate formed by connecting a camptothecin drug of Formula 1 with various acid-sensitive linkers, and uses the drug-linker conjugate to provide a carrier-drug conjugate, wherein the carrier-drug conjugate is formed by connecting the camptothecin drug of Formula 1 with various carriers through various acid-sensitive linkers.

[0190] In addition, the present invention also provides a method for preparing a carrier-drug conjugate, characterized in that at least one camptothecin drug of Formula 1 is connected to a carrier via an acid-sensitive linker of Formula 3 using the drug-linker conjugate of the present invention as described above or a pharmaceutically acceptable salt thereof.

[0191] [Payload for various carrier-drug conjugates]

[0192] Inhibitors of topoisomerase I (irinotecan, topotecan, etc.) are clinically proven anticancer mechanisms with proven efficacy and safety, and have been clinically proven to have excellent anticancer efficacy in various refractory solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer. Camptothecin drugs such as exatecan and SN-38 have been developed as payloads for ADCs.

[0193] Exatecan of Formula 1-1 is a camptothecin derivative and is an antitumor low molecular weight compound that inhibits topoisomerase type 1. Exatecan is a substance that has been proven to have a potent cytotoxicity that is 5 to 10 times higher than that of SN-38.

[0194] Unlike irinotecan, ixotecan does not require enzyme activation. It has stronger topoisomerase type 1 inhibitory activity than SN-38, the main drug of irinotecan, or topotecan, which is used in the same clinic, and has stronger cytotoxic activity against various cancer cells in vitro. In particular, it is also effective against cancer cells that show drug resistance to SN-38 and other drugs due to the expression of P-glycoprotein. In addition, it has shown strong anti-tumor effects in a subcutaneous transplantation model of human tumors in mice, and clinical trials have been conducted.

[0195] Dxd (exatecan derivative for ADC) of formula 1-2 is a potent DNA topoisomerase I inhibitor and is used as an IC conjugate drug for HER2-targeted ADC (DS-8201a). 50 It is 0.31μM.

[0196] Although not limited to this, even if a simple functional group such as C 1-3 Alkyl, hydroxyl, halogen, amine, etc. are added to the compound of Formula 1, or existing hydroxyl, ethyl, oxo functional groups are replaced or eliminated with other functional groups, but it is obvious that they are included in the equivalent scope of the present invention that shows the same efficacy as the immunoconjugate or carrier-drug conjugate of the present invention.

[0197] [Linker technology for effective drug delivery]

[0198] For ADCs using existing super toxins such as MMAE, calicheamicin, and PBD, the Stable Linker system (a characteristic of second-generation ADCs) is used to minimize the separation of the drug from the blood before it reaches cancer tissue.

[0199] The principle of how most second-generation ADCs work in cancer cells is that, in the first step, the antibody part that makes up the ADC binds to the antigen overexpressed in the cancer cell. In the second step, the ADC that binds to the antigen on the surface of the cancer cell through the antigen-antibody reaction passes through the endosome and lysosome and is transported into the cancer cell. In the third step, the antigen-antibody part is degraded from the lysosome and the enzyme (cathepsin B) drug is released. In the last step, the cancer cell undergoes apoptosis through the drug released from the inside of the cancer cell.

[0200] In order to develop new ADCs that can be selectively used for various solid cancers, it is necessary to use a linker system that exceeds the drug delivery efficiency of existing ADCs. In order to develop ADCs targeting new antigens such as Trop-2, Her2, and folate receptors, which are established drug targets, it is necessary to develop ADCs targeting slow internalizing antigens such as CEACAM-5 and / or cancer-specific antigens such as NY-ESO-1 / HLA Complex, but it is difficult to deliver sufficient amounts of drugs using the limited drug delivery efficiency of existing Val-Cit or MAC-glucuronide linker systems.

[0201] In particular, for effective drug delivery of antigens such as CEACAM-5, the antibody-drug conjugate needs to maintain stability in the surrounding environment of blood and / or normal tissues, but needs to have the property of rapid drug release in the surrounding environment of cancer cells such as the tumor microenvironment.

[0202] When the drug can be released rapidly in the tumor microenvironment surrounding the cancer, it can also be advantageously used to target various antigens with limited ADC uptake rate (representatively CEACAM-5, various cancer specific antigen-HLA complexes, etc.).

[0203] Therefore, the camptothecin drug of Formula 1 is a drug with improved water dispersibility compared to SN-38, so that it will not aggregate in body fluids such as blood and interstitial fluid. In order to fully utilize the ability of hydrophobic low molecules to penetrate deep into tumor tissues and penetrate cell membranes to move into cells, the present invention selects an acid-sensitive linker that degrades in the acidic environment (pH≤7) surrounding cancer as a release profile linker for rapid and effective drug delivery after reaching cancer tissues.

[0204] According to the present invention, the antibody connected to the camptothecin drug of Formula 1 through an acid-sensitive linker binds to the antigen overexpressed on the surface of the cancer cell in the same manner as the first step of the second-generation ADC in cancer cells, but some undergo intracellular processing steps in the same manner as the second-generation ADC, and a considerable portion of the rest can release the drug through the low pH on the cancer cell line. Then, the drug released in the cancer tissue moves to the inside of the cancer cell by diffusion, not only directly acting on the cancer cell to induce apoptosis without passing through the endosome or lysosome, but also without reacting with the enzyme (cathepsin B). Therefore, compared with the second-generation ADC that can only release drugs through enzymatic reactions, although the antigen selectivity of cancer cells is the same, the pH-sensitive linker can maximize the efficiency of drug release and delivery into cancer cells, which is the main feature of the immunoconjugate of the present invention.

[0205] In addition, the linker is stable in the bloodstream, preventing the drug from being separated from the antibody, keeping it in a prodrug state until it reaches the target, and minimizing the damage to normal tissues. Nevertheless, the present invention uses an acid-sensitive linker with a hydrophilic molecular structure to create a hydrolytic environment, which is combined with a hydrophobic drug to alleviate the problem of ADC aggregation.

[0206] Depending on the type of linker, different metabolites are formed. In this regard, preferably, the [camptothecin drug of Formula 1]-[acid-sensitive linker] of the present invention is formed by linking the camptothecin drug of Formula 1 and the acid-sensitive linker via a carbonate or ester bond, so that the free camptothecin drug of Formula 1 is released when the acid-sensitive linker is degraded.

[0207] Generally, carbamate bonds provide excellent drug linker stability compared to ester bonds and carbonate bonds with respect to hydrolysis. However, the present invention is characterized in that unstable ester bonds or carbonate bonds are used instead of carbamate bonds in order to enable the camptothecin drug designed as Formula 1 to be separated from the drug linker both outside and inside the cancer cell under the acidic environment (pH) around the cancer cell.

[0208] The pH value of blood is constant at 7.3 to 7.4. Therefore, the camptothecin drug of Formula 1 will not be cleaved from the acid-sensitive linker in the blood. Even if it is cleaved, the release rate of the camptothecin drug of Formula 1 from ADC at the neutral pH of serum is much lower than that of tumor tissue in the acidic environment.

[0209] Furthermore, in order to reduce the aggregation of ADC in plasma caused by hydrophobic drugs, the present invention is characterized in that the α-hydroxyl group at the C20 position of the E ring in the camptothecin drug of Formula 1 is connected to an acid-sensitive linker.

[0210] In this case, in camptothecin drugs, the molecular bonds can be rotated relative to, for example, the carbon-carbon single bonds in the hexagonal or heptagonal rings extending from the A ring and the B ring, and the -NH2 or -OH with a large degree of freedom is exposed to water (H2O) in an aqueous environment to carry a (+) charge or form a hydrogen bond with water, thereby improving the water dispersibility of the ixotecan or Dxd drug site directed by the acid-sensitive linker.

[0211] Tetrapeptide linkers are conjugated to hydrophobic drugs to display restrictions on possible ADC aggregation.

[0212] The linker CL2A used in the current ADC Trodelvy approved by the U.S. Food and Drug Administration (FDA) is a linker that meets the following three points: (i) storage stability after preparation; (ii) stability in the blood during administration (almost no free payload is exposed in plasma); and (iii) rapid release of the effective carrier (payload) in cancer tissues.

[0213] The Phe-Lys peptide inserted in the original CL2 derivative is cleavable by cathepsin B. As part of simplifying the synthesis process, phenylalanine was eliminated from CL2A, thereby eliminating the cleavage site for cathepsin B. These changes had no effect on conjugate binding, stability, or efficacy. This suggests that SN-38 is primarily released from the conjugate by cleavage of the pH-sensitive benzyl carbonate bond on the lactone ring, rather than the cathepsin B cleavage site in CL2.

[0214] The acid-sensitive linker used in the present invention can utilize the CL2A linker and can be designed as shown in Formula 3, so as to selectively and effectively deliver the camptothecin drug of Formula 1 to cancer tissue. That is, in the present invention, the acid-sensitive linker can be a compound derived from the following Formula 3:

[0215] Formula 3

[0216]

[0217] Wherein, X1 and X2 are each independently -H or -halogen;

[0218] Y is -NH-, -NRA -, or nothing (null);

[0219] 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)-;

[0220] W is -R B -、-M--R B -M-, -MR B -or-R B -MR C -;

[0221] R A To R C are each independently a C1-C4 alkyl group,

[0222] M is as well as

[0223] n is an integer of 5-9.

[0224] Preferably, in Formula 3,

[0225] X1 and X2 may each independently be -H or -halogen;

[0226] Y can be -NR A -, or nothing (null);

[0227] Z may be -C1-C4 alkyl-, -(C1-C2 alkyl)-(C3-C6 cycloalkyl)-, or -(C3-C6 cycloalkyl)-(C1-C2 alkyl)-;

[0228] W can be -R B -or-R B -MR C -;

[0229] R A To R C Can each independently be a C1-C4 alkyl;

[0230] M can be as well as

[0231] N may be an integer from 5 to 9.

[0232] In the present invention, the length of the linker, that is, in Formula 3, n 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 deviates from the above range, if there is no significant difference in the effect caused by the change in the linker length, it should all be included in the equivalent scope of the present invention.

[0233] The water dispersibility of the drug can be improved by placing a polyethylene glycol (PEG) spacer between the drug and the carrier, so the linker of Formula 3 contains a low molecular weight PEG portion containing a limited number (n=5 to 9) of PEG monomers.

[0234] The acid-sensitive linker of Formula 3 is a custom linker that can be optimized according to the characteristics of the targeting target, payload, and carrier.

[0235] The camptothecin drugs of Formula 1 have sites that are easily attached to various linkers to prepare carrier-drug conjugates. For example, the alcohol group site of the camptothecin drugs of Formula 1 can be used as the attachment site between the linker.

[0236] Therefore, in the present invention, [camptothecin drug of Formula 1]-[acid-sensitive linker] can be formed by connecting the alcohol site of the camptothecin drug of Formula 1 with the alcohol site of the acid-sensitive linker of Formula 3.

[0237] In the present invention, the [camptothecin drug of Formula 1]-[acid-sensitive linker] conjugate may be a compound represented by Formula 4 or 5 below.

[0238] Formula 4

[0239]

[0240] Formula 5

[0241]

[0242] (wherein n is each independently an integer from 5 to 9).

[0243] In order to improve the drug delivery efficiency, for the acid-sensitive linker of the present invention, a multivalent linker system that can overcome the problem of low DAR (which is considered to be a disadvantage of site-specific conjugation) is developed and applied, and a method for ensuring the attachment of 2-3 payloads at one attachment site can be used to prepare a high DAR ADC of DAR4-12 even in site-specific conjugation.

[0244] To this end, in the present invention, a Bridgeable Linker system can be used as a linker, as shown in Formulas 4 and 5.

[0245] This bridgeable linker system can improve the efficiency of CMC and simplify the process, so that the ADC of DAR 4 can be easily prepared while maintaining the rapid release characteristics of the effective carrier (payload) in cancer tissues, which is the biggest advantage of the CL2A linker system. The site-specific antibody-drug complex of DAR 4 can be prepared without additional antibody engineering. When the disulfide (-SS-) present in the antibody is reduced, two thiols (-SH) are formed, and the thiol (Thiol) thus generated forms a conjugate with the camptothecin drug of the new bridgeable linker-formula 1 at a ratio of 2:1. Therefore, the antibody-drug complex of DAR 4 can be easily prepared as a single product by reacting all four disulfides in the conventional antibody.

[0246] [Advantages of the drug model of molecular glue degrader]

[0247] Proteins in cells in the body are naturally degraded within a few hours to a few days after they have fulfilled their functions. In all cells in the body, there is a purification system called the Ubiquitin proteasome system (UPS) that degrades proteins. In this process, ubiquitin plays a marker role to inform proteins that need to be degraded. The proteasome has the function of recognizing ubiquitin tags and destroying the corresponding proteins. That is, next to the protein that has fulfilled its function, multiple substances called ubiquitin are attached like tags. The so-called proteasome only picks up proteins with the tags and then degrades them like a shredder. E3 ligases are enzymes that cause protein degradation systems in the body and are responsible for substrate specificity in the ubiquitination pathway.

[0248] Molecular glue degraders or molecular glues are compounds with adhesive functions that attach target proteins to specific enzymes (E3 ligases) in our body. One of the advantages of molecular glue is the catalytic effect, which degrades the target protein and then detaches again to degrade another target protein.

[0249] When the E3 ligase is attached to the tumor protein through the molecular glue, the tumor protein is degraded, and other tumor proteins are continuously degraded until the targeted tumor protein disappears, thereby preventing the proliferation of cancer cells. Therefore, the molecular glue of tumor protein overcomes the drug resistance that is a problem of targeted anticancer drugs, and has a high therapeutic effect even at a low dosage.

[0250] The camptothecin drug of formula 1 used as an effective load according to the present invention is a molecular glue degrader that binds to the DDX5 protein and the E3 ligase, that is, a molecular glue degrader that activates the degradation of the tumor protein DDX5 or its phosphorylated DDX5 protein (p-DDX5). Figure 1 , Figures 4 to 8 ).

[0251] Molecular glue degrader is not only a ligand (warhead) that binds to the target protein, but also can act as a ligand (binder) for E3 ligase.

[0252] Therefore, the camptothecin drug of Formula 1 according to the present invention is a molecular glue degrading agent that activates the degradation of tumor protein DDX5, and thus can be used as a ligand targeting DDX5 protein or a ligand binding to DDX5 protein.

[0253] Unlike kinase inhibitors, molecular glue degraders are "proximity-driven" and their ability to induce degradation is "event-driven" and depends on the formation of a temporary "target protein-molecular glue-E3 ligase" ternary complex. After degradation occurs, the separated molecular glue forms additional ternary complexes with the target protein, which can undergo multiple degradation processes until the target protein disappears.

[0254] Most proteins that are drug targets develop drug resistance that evolves in response to the drugs. However, molecular glue, a low-molecular compound that functions as an adhesive to bind tumor proteins and E3 ligases, is a suitable model for cancer therapeutic drugs because it is more resistant to drug resistance.

[0255] Every time a gene mutation occurs, the shape of the drug target protein changes slightly. It would be great to block the activity of all variants with one drug, but this is impossible due to selectivity issues. In order to block the activity of all mutations with one drug, the drug target protein is degraded and completely eliminated.

[0256] Therefore, the camptothecin drugs of Formula 1 according to the present invention are drugs that can accurately bind to the DDX5 tumor protein and can avoid the drug resistance problem of targeted therapeutic drugs by a molecular glue approach method that selectively degrades the protein.

[0257] The camptothecin drug of Formula 1 according to the present invention determines the binding strength with the target protein DDX5 tumor protein according to its physicochemical properties, and is preferably an irreversible drug, which binds so strongly that it cannot return to its previous state.

[0258] Unlike the existing SN38 drug, the camptothecin drug of formula 1 according to the present invention has a high affinity for DDX5, is not easy to fall off, and degrades DDX5 through the molecular glue function, thereby not only irreversibly inhibiting DDX5-related signal transduction in cancer cells, but also adjusting the cell membrane permeability of the drug as needed according to its physicochemical properties to exert a bystander effect or control the extent of its effect, thereby having a high effect on killing surrounding cells, which is beneficial to the treatment of heterogeneous tumors, and can inhibit long-term cancer progression, reduce the risk of drug resistance expression, and improve the treatment response rate.

[0259] The camptothecin drugs of Formula 1 according to the present invention can bind to DDX5, which acts as an oncoprotein in cells, so as to induce cell death by degradation of DDX5 protein ( Fig.11 ).

[0260] In cancer treatment, intrinsic drug resistance can be caused by abnormally expressed transcription factors, which are key regulators of cell proliferation and apoptosis. Therefore, the camptothecin drugs of Formula 1 according to the present invention can induce DDX5 protein degradation and the resulting cell death by acting as a transcription factor cofactor and a molecular glue degrader of an oncoprotein that binds to DDX5. At this time, DDX5 protein degradation can downregulate the transcription of anti-apoptotic genes ( Figures 4 to 8 ). Therefore, unlike other targeted therapeutic drugs, drug resistance caused by abnormally expressed transcription factors related to cell proliferation and / or apoptosis does not occur, and / or acquired drug resistance induced by activation of transcription factors that are deregulated during chemotherapy and / or radiotherapy can be suppressed.

[0261] That is, the anti-cancer mechanism of the camptothecin drugs of Formula 1 according to the present invention can bypass the molecular mechanism of cancer treatment resistance, so the problem of drug resistance can be avoided. When drug resistance occurs, the treatment effect is lower than before, so the camptothecin drugs of Formula 1 according to the present invention can be preferably used as standard treatment drugs or primary treatment drugs after cancer diagnosis.

[0262] In short, the camptothecin drugs of Formula 1 used as a payload according to the present invention can be targeted anticancer drugs acting on DDX5 protein, which plays an important role in the growth, survival (cell survivor), proliferation, metastasis and / or metabolism of cancer cells.

[0263] [Immunoconjugate]

[0264] In the present invention, the term "immunoconjugate" refers to a complex in which a cytotoxic drug-linker conjugate is linked to an antibody or an antigen-binding fragment thereof.

[0265] Antibody-drug conjugate (ADC) is an example of an immunoconjugate, and thus, in the present invention, descriptions of ADC and immunoconjugate are used interchangeably.

[0266] When the immunoconjugate is administered in vivo, the antibody or a fragment containing its antigen binding site as one of its components binds to the targeted antigen and then releases the drug, allowing the drug to act on target cells and / or surrounding cells, thereby expecting excellent efficacy and reduced side effects as a targeted drug.

[0267] Factors that have a significant impact on the effect of immunoconjugates include, in particular, (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. Since many factors have a complex impact on the effect, it is very difficult to predict the effect of a combined immunoconjugate based only on the known facts of each element.

[0268] For immunoconjugates designed to release drugs after internalization, when the internalization process is inefficient, sufficient concentrations of active drugs cannot be delivered to the interior of cells. Even if hydrophobic drugs are used as cytotoxic drugs, there is a disadvantage that it is difficult to anticipate the by-stander cell-killing phenomenon of surrounding cells.

[0269] In order to solve the above-mentioned problems, the immunoconjugate of the present invention is characterized in that:

[0270] (i) In the tumor microenvironment (pH ≤ 7) surrounding cancer, hydrophobic drugs that can penetrate cell membranes and act inside cells are released, and then acid-sensitive linkers are used to introduce a large amount of free hydrophobic drugs into cells;

[0271] (ii) In the acidic environment (pH ≤ 7) surrounding cancer cells, a camptothecin drug of Formula 1 is used as a hydrophobic drug capable of penetrating the cell membrane, so that the acid-sensitive linker is degraded and the drug is released, and a large amount of free drug penetrates the cell membrane to aggregate inside the cell ( Figure 4 ).

[0272] Camptothecin drugs of Formula 1 are topoisomerase I inhibitors that can exhibit cytotoxicity against cancer cells, but examples of using them as payloads and preparing immunoconjugates through acid-sensitive linkers have not been reported so far.

[0273] The camptothecin drugs of Formula 1 are hydrophobic low molecules that can penetrate cell membranes, accumulate rapidly in tumor tissues, and can rapidly accumulate at high concentrations for a long time, diffuse inside cells and exert cytotoxicity, killing cells, and then are released and continuously penetrate cell membranes to surrounding cells and move into cells to exert their effects.

[0274] As described above, the immunoconjugate of the present invention has the technical feature of combining the camptothecin drug of Formula 1 with an acid-sensitive linker, so the immunoconjugate can be designed according to the desired purpose to bind any antibody or a fragment containing its antigen binding site, which are all included in the scope of the present invention. For example, for excellent anti-cancer effects, the camptothecin drug-acid-sensitive linker conjugate of Formula 1 of the present invention can be combined with trastuzumab, cetuximab and sacituzumab to prepare an immunoconjugate.

[0275] In the present invention, the average drug-to-antibody ratio (DAR) of the immunoconjugate may be 2 to 12, and preferably DAR 4 to 12.

[0276] [Target antigen_drug target]

[0277] When designing the antibody-drug conjugate (ADC) or immunoconjugate of the present invention, the targeting targets can be extended not only to cancer cells, but also to senescent cells, infectious disease organisms and / or cells associated with autoimmune diseases.

[0278] Thus, cells targeted by antibodies or fragments thereof containing antigen binding sites may be cancer cells, senescent cells, infectious disease organisms, and / or cells associated with autoimmune diseases.

[0279] Non-limiting examples of target antigens include antigens selectively distributed on the surface of cancer, such as Her2, FolR, PSMA, etc., and cancer cell overexpressed antigens that are also distributed in small amounts on normal tissues, such as Trop2.

[0280] Cancer cell target antigens can be, for example, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, aromatase, ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), otein)), B4GALNT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1(MDR15), BIyS, BMP1, B MP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (clathrin), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPR IN-1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCLI1 (chemokine), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22 (MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / chemokine-2), C CL25 (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-IRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6),CCR7(CKR7 / EBI1), CCR8 or CDw198(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), CD13, CD164, CD19, CDH6, CDIC, CD2, CD20, CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, C D69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH 1 (E-cadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wa p1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB, chitinase, CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN6, CLDN7 (Claudin-7), CLDN18, CLEC5A, CLEC6A, CL EC11A, CLEC14A, CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, C-MET, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTAG1B (NY-ESO-1), CTLA4, CTL8, CTNNB1 (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO),CXCLI1(1-TAC / IP-9), CXCL12(SDF1), CXCL13, CXCL14, CXCL16, CXCL2(GRO2), CXCL3(GRO3), CX CL5(ENA-78 / LIX), CXCL6(GCP-2), CXCL9(MIG), CXCR3(GPR9 / CKR-L2), CXCR4, CXCR6(TYMSTR / S). TRL33 / Bonzo) CYB5 CYC1 CYSLTR1 DAB2IP DES DKFZp451J0118 DLK1 DNCL1 DPP4 E2F1 En gel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA 2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB 5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EP HRIN-B2 EPHRIN-B3 EPHB4 EPG ERBB2(HER-2) ERBB3 ERBB4 EREG ERK8 F3(TF), FADD, FAP, FAP, FASL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), FGF10, and FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3( int-2, FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF(VEGFD), FI L1(EPSILON), FBL1(ZETA), FLJ12584, FLJ25530, FLRT1 (protein transcription factor), FLT1, FLT-3, FOLR1, FOS, FOSL1(FRA-1), FR-α FY(DARC) GABRP(MARKET) GAGEB1 GAGEC1 GALNAC4S-6ST GATA3 GD2 GD3 GDF5 GFI1 GFRA1 GGT1 GM-CSFGNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, histamine and histamine receptors, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNγ, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1(CD121a), IL1R2(CD121b), IL-IRA, IL-2, IL2RA(CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1(IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R(C D129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHY I, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27,IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (type II hair-specific keratin), L1CAM, LAG3, LAMA5, LAMP1, LEP (leptin), Lewis Y antigen ("LeY"), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​LY75, LYPD3, MACMARCKS, MAG or OMgp, MAGEA3, MAGEA6, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MLSN, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, neuronal protein Glycan (neurocan), NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOTCH3, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H 2. NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3 C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI,P2RX7, PAP, PART1, PATE, PAWR, P-cadherin, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-β, P PBP(CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2(COX-2), PT N, PVRIG, RAC2(P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O(ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (cytokine that activates endothelial monocytes), SDF2, SERPENA1, SERPINA3, SERPINB5 (mammaglobin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor (tissuefactor), TLR1, TLR2, TL R3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8,TNFRSF9, TNFSF1O(TRAIL), TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRSF17, TNFSF1 1(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNFSF14(HVEM-L), TNFRSF14(HVEM), TNFSF15(VEGI), TNFSF18, TNFSF4(OX40 ligand), TNFSF5(CD40 ligand), TNFSF6(FasL), TNFSF7(CD27 ligand), TNFSF8(CD30 ligand), TNFSF9(4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A(Topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (lymphotexin), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85 I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 ( CD337, NKp30), OSCAR, TARM1, CD30, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D),<h2 style=";text-align:left;direction:ltr">NCR2(CD336、NKp44)、PILRB、SIGLEC1(CD169、SN)、SIGLEC5、SIGLEC6、SIGLEC7、SIGLEC8、SIGLEC9、SIGLEC10、SIGLEC11、SIGLEC12、SIGLEC14、SIGLEC 15(CD33L3)、SIGLEC16、SIRPA、SIRPB1(CD172B)、TREM1(CD354)、TREM2、KL RF1(NKp80)、17-1A、SLAM7、MSLN、CTAG1B / NY-ESO-1、MAGEA3 / A6、ATP5I(Q06) 185)、OAT(P29758)、AIFM1(Q9Z0X1)、AOFA(Q64133)、MTDC(P18155)、CMC1( Q8BH59)、PREP(Q8K411)、YMEL1(O88967)、LPPRC(Q6PB66)、LONM(Q8CGK3)、A CON(Q99KI0)、ODO1(Q60597)、IDHP(P54071)、ALDH2(P47738)、ATPB(P56480)、AATM(P05202)、TMM93(Q9CQW0)、ERGI3(Q9CQE7)、RTN4(Q99P72)、CL041( Q8BQR4)、ERLN2(Q8BFZ9)、TERA(Q01853)、DAD1(P61804)、CALX(P35564)、C ALU(O35887)、VAPA(Q9WV55)、MOGS(Q80UM7)、GANAB(Q8BHN3)、ERO1A(Q8R18) 0)、UGGG1(Q6P5E4)、P4HA1(Q60715)、HYEP(Q9D379)、CALR(P14211)、AT2A2 (O55143)、PDIA4(P08003)、PDIA1(P09103)、PDIA3(P27773)、PDIA6(Q922R8) )、CLH(Q68FD5)、PPIB(P24369)、TCPG(P80318)、MOT4(P57787)、NICA(P57716)、BASI(P18572)、VAPA(Q9WV55)、ENV2(P11370)、VAT1(Q62465)、4F2(P10 852)、ENOA(P17182)、ILK(O55222)、GPNMB(Q99P91)、ENV1(P10404)、ERO1A(Q8R180)、CLH(Q68FD5)、DSG1A(Q61495)、AT1A1(Q8VDN2)、HYOU1(Q9JKR6)、TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433), but not limited thereto.

[0281] The target antigen may be an antigen that is distributed 10 times more in cancer cells than in normal cells.

[0282] [Antibodies or fragments thereof containing antigen-binding sites]

[0283] According to the immunoconjugates of the present invention comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment containing its antigen-binding site], as long as the antibody or the fragment containing its antigen-binding site targets the antigen of the cancer cell, regardless of its type, the organic mechanism of action according to the combined use of [camptothecin drug of Formula 1]-[acid-sensitive linker] as described above can be exerted. In particular, free exitecan or Dxd drug has an anticancer efficacy that is 5-20 times stronger than that of SN-38 in various cancer cell lines, that is, at a low level of IC 50 The numerical value shares very strong anti-cancer efficacy. In addition, Irinotecan or Dxd drugs use the anti-cancer mechanism whose efficacy / safety has been clinically verified, namely, inhibitors of topoisomerase I. Topoisomerase I inhibitors (Irinotecan, Topotecan, etc.) have been clinically proven to have excellent anti-cancer efficacy in various refractory solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer.

[0284] In this specification, the term "antibody" refers to a protein molecule that acts as a ligand for specific antigen recognition, including immunoglobulin molecules that react with specific antigens, including polyclonal antibodies, monoclonal antibodies, and whole antibodies. In addition, the term includes chimeric antibodies and bivalent or bispecific molecules, double antibodies, three antibodies, and four antibodies. The term also includes single-chain antibodies, scaps, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that retain the function of binding to FcRn. A whole antibody is a structure with two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0285] In the present specification, the term "fragment containing an antigen binding site" may be 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. The Fd refers to the heavy chain portion contained in the Fab fragment. The Fab has a structure of variable regions of light and heavy chains, a constant region of the light chain and a first constant region (CH1 domain) of the heavy chain, and has an antigen binding site. Fab' differs from Fab in that the C-terminus of the heavy chain CH1 domain has a hinge region containing at least one cysteine ​​residue. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment containing only heavy chain variable sites and light chain variable sites. In the case of a disulfide Fv (dsFv), the heavy chain variable site and the light chain variable site are linked by a disulfide bond, and in the case of a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are usually covalently linked by a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (e.g., Fab can be obtained by restricted cleavage of whole antibodies by papain, and F(ab')2 fragments can be obtained by cleavage with pepsin), preferably, can be prepared by genetic recombination technology.

[0286] [Pharmaceutically acceptable salt]

[0287] In this specification, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, such as: salts of inorganic ions, sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc.; salts of inorganic acids, hydrochloric acid, phosphoric acid and sulfuric acid, etc.; salts of organic acids, ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, acetylsalicylic acid, etc.; amino acid salts, lysine, arginine, guanidine, etc. And salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, benzethonium, etc. that may be used in drug reactions, purification and separation processes. However, the types of salts referred to in the present invention are not limited to the types of these salts listed.

[0288] [Various Carrier-Drug Conjugates]

[0289] In the present specification, prodrugs include but are not limited to prodrugs that are biologically activated or further activated after being placed in a predetermined physiological environment.

[0290] For example, it refers to a compound designed to chemically modify a physiologically active substance or a therapeutically active organic compound and to free or release the parent compound under enzymatic or other conditions in vivo. Prodrugs are converted into target compounds in vivo after administration. Although they are useful drugs, they have unsuitable characteristics in terms of side effects, stability, solubility, absorbability, duration of action, etc., and are chemically modified to enable them to be used clinically.

[0291] Carrier-Drug Conjugate is also a type of prodrug.

[0292] The [camptothecin drug of Formula 1]-[acid-sensitive linker] conjugate according to the present invention can be applied to various types of drug carriers other than antibodies. Unlike antibodies, it can penetrate deep into cancer tissues well and can be well used for various purposes using a carrier with CMC-friendly properties.

[0293] Therefore, the method for preparing the carrier-drug conjugate of the present invention is characterized in that:

[0294] Using the [camptothecin drug of Formula 1]-[acid-sensitive linker of Formula 2] conjugate according to the present invention or a pharmaceutically acceptable salt thereof, at least one camptothecin drug of Formula 1 is connected to a carrier via a linker of Formula 3.

[0295] In this case, the carrier may be an antibody, a peptide, a repeat, and / or an aptamer.

[0296]

[0297] For example, antibody-drug conjugates (ADCs) are new drug platforms that utilize the high tissue selectivity of antibodies that specifically bind to specific antigens expressed on the surface of cancer cells to selectively deliver payloads with strong anti-cancer efficacy only to cancer tissues. ADCs can selectively deliver strong payloads that kill cancer cells at low concentrations at low pM levels only to cancer tissues, and minimize systemic exposure of the drug, so as to ensure both anti-cancer efficacy and safety.

[0298] For HER2-positive cancers, according to the present invention, the prodrug ADC of the camptothecin drug of Formula 1 using the DDX5 protein as a drug target can become a new treatment method that can address the drug resistance of anti-HER2 therapy.

[0299] The prodrug of the present invention may be an immunoconjugate comprising an antibody or a fragment containing an antigen binding site thereof, or a pharmaceutically acceptable salt thereof, which is designed to release the camptothecin drug of Formula 1 in vivo.

[0300] The immunoconjugate of the present invention specifically binds to an antigen of a cancer cell and exhibits cytotoxicity by releasing the drug inside and outside the cancer cell, and thus can be effectively used for treating or preventing cancer.

[0301] Aptamer-Drug Conjugate (ApDC) is an aptamer introduced into ADC to replace the antibody of ADC. Aptamer is a single-stranded nucleic acid with a three-dimensional structure. It is discovered through the "SELEX" (Systematic Evolution of Ligands by Exponential enrichment) process. SELEX is a technology that adds target protein molecules to the compound library to obtain functional nucleic acids that bind to it.

[0302] Aptamers can bind to targets very strongly and selectively, so they are also called chemical antibodies. Aptamers are known to be about 20 kDa in size and have excellent cell permeability and low immunogenicity compared to antibodies.

[0303] Aptamers can be chemically synthesized, so when preparing aptamer-drug conjugates, the conjugation position and amount of the drug can be precisely designed. The production cost is lower than that of ADC.

[0304] Aptamers are generally made of natural nucleic acids and are therefore degraded by nucleic acid-degrading enzymes in the body, making them less stable in the body. However, the convenience of chemical modification of aptamers can be used to overcome the limitations of modified aptamer stability.

[0305] Peptide-Drug Conjugate (PDC) is a form of ADC that introduces peptides to replace antibodies. Peptides are composed of amino acids and range in size from 500 to 5000 Da (Daltons). This is a very small size compared to antibodies above 150 kDa (kilodaltons). Therefore, peptide-based PDCs have excellent cell penetration compared to ADCs and are less likely to produce immunogenicity. In addition, peptides can be chemically synthesized. Therefore, PDCs not only have very low production costs, but also can precisely control the conjugation position and ratio of peptides to drugs.

[0306] In general, peptides are easily degraded by proteolytic enzymes and thus have a short biological half-life. To overcome these limitations of peptide-based drug conjugates, strategies such as the use of modified peptides, such as cyclic peptides, the introduction of unnatural amino acids, etc. have been proposed.

[0307] Repebodies are artificial antibodies that do not have an antibody skeleton but have the ability to recognize antigens like antibodies. Repebodies specific to target proteins can be discovered through phage display technology.

[0308] Phage display is a technology for expressing desired proteins on the surface of phages. The size of repetitive bodies is about 30kDa, which is about 20% of antibodies. Therefore, it is known that compared with antibodies, it has relatively low immunogenicity and improved cell permeability. In addition, it is expected that the structural stability can be improved by controlling the thermal stability and pH stability of repetitive bodies. Compared with antibodies, the production cost is also evaluated as relatively low. Due to these advantages of repetitive bodies, people are increasing their attention to the development of repetitive body-drug conjugates (Repebody-DC) as a strategy for replacing antibodies with repetitive bodies.

[0309] [About ADC, its role as an antibody therapeutic drug]

[0310] The ADC of the present invention can also be used as a basic therapeutic antibody, which has played an important role in the treatment of cancer, autoimmune diseases and the like in the past decade.

[0311] Antibodies have the ability to distinguish between antigens and cells in our body, so they have excellent screening functions that can selectively act on antigens. Antibody therapeutic drugs are drugs that utilize these antigen-selective binding properties of antibodies in disease treatment drugs. Antibodies are in the shape of a "Y", with an antigen binding site (Antigen binding site) on each arm, and complementary determining regions (CDR) enable antibodies to selectively recognize target antigens. In addition, there are roughly five types of antibodies (IgM, IgD, IgG, IgA, and IgE / Immunoglobulin (Ig)), among which IgG is mainly used in antibody therapeutic drugs.

[0312] Antibody therapeutics can selectively react with antigens and remove them. To do this, various mechanisms are used, and the characteristics of the representative mechanisms used in antibody therapeutics are as follows.

[0313] The first mechanism is blocking. Antibodies selectively bind to target cell receptors and block the physiological functions of target cells. Antibodies bind to ligands or receptors expressed on the cell surface, blocking the target signal transduction pathway. Reduced signal transduction leads to loss of cell activity, proliferation inhibition, and cell apoptosis.

[0314] When antibodies target receptors on the cell surface and bind to them, they change the structure of the receptors or prevent factors that should bind to the receptors from binding, thereby inhibiting intracellular signal transduction and thus controlling cell growth and differentiation. In addition, when antibodies bind to receptors, the receptors enter the cell through an intracellular influx mechanism, thereby reducing the number of receptors on the cell surface, thereby regulating the physiological mechanism of the cell. A representative antibody therapeutic drug that shows excellent effects through this mechanism is trastuzumab (Trastuzumab, which targets HER2-positive breast cancer). ), which is one of the human epidermal growth factor receptors (HER / EGFR / ERBB).

[0315] The second mechanism is antibody-dependent cell-mediated cytotoxicity (ADCC). For cancer cells, they have specific receptors or overexpressed receptors that are different from those of ordinary cells. The immune system recognizes them, produces antibodies and binds to the cell surface. When NK cells recognize Fc receptors (FcγRIII), they induce cell apoptosis when activated.

[0316] The third mechanism is complement-dependent cytotoxicity (CDC). It binds to the surface of cancer cells and activates complement protein (C1q), triggering the classical pathway and attacking the cell membrane, creating pores and causing the lysis of target cells. In addition, it has mechanisms such as opsonization, neutralization, and inflammatory response.

[0317] Therapeutic antibodies are usually made into optimized forms by redesigning antibodies from different angles to achieve specific therapeutic purposes. And the antibody engineering technology used to develop these therapeutic monoclonal antibodies is being expanded and applied to optimize the development of ADC. For example, most of the current therapeutic antibodies or ADCs in clinical development are IgG1, and only a few use IgG2 and IgG4. The selection of antibody isotype is equally important for therapeutic antibodies and ADCs. The reason is that the isotype of the antibody affects the functions of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). In addition, IgG4 is functionally monovalent through in vivo Fab arm exchange. In addition, the in vivo efficacy of ADC can be improved by isotype ADC efficacy and Fc domain engineering of applied antibodies.

[0318] [Pharmaceutical composition for preventing or treating cancer]

[0319] The present invention provides a pharmaceutical composition for preventing or treating cancer, the composition comprising the immunoconjugate according to the present invention as described above or a pharmaceutically acceptable salt thereof as an effective ingredient.

[0320] In addition, according to one embodiment of the present invention, a method for treating or preventing cancer is provided, the method comprising the step of administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof. The subject may be a mammal including a human.

[0321] The immunoconjugate of the present invention specifically binds to the antigen of cancer cells and releases drugs inside and outside the cancer cells to show cytotoxicity, and thus can be effectively used for the treatment or prevention of cancer. The anticancer activity of the immunoconjugate of the present invention is as described above.

[0322] In the present invention, the cancer includes all cancers that can be treated by inhibiting topoisomerase I, and may be solid cancer or blood cancer. For example, it can be selected from pseudomyxoma, intrahepatic biliary cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney At least one of the group consisting of cancer, heart disease, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric cancer lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal cancer, 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 cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer and thymic cancer, but not limited thereto. In addition, the cancer includes not only primary cancer but also metastatic cancer.

[0323] The term "therapeutically effective amount" used in the present invention means the amount of the immunoconjugate that is effective for the treatment or prevention of cancer. Specifically, "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio that can be applied to medical treatment. The effective dose level can be determined according to the type and severity of the subject, age, sex, disease type, drug activity, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, factors including drugs used simultaneously, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic drug or in combination with other therapeutic drugs, or can be administered sequentially or simultaneously with commercially available therapeutic drugs. In addition, it can be administered once or multiple times. Taking into account all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects. The immunoconjugate of the present invention exhibits a dose-dependent effect, so the ordinary technicians in the technical field of the present invention can determine the dosage according to various factors such as patient status, age, sex, and complications. The active ingredient of the pharmaceutical composition of the present invention has excellent safety, so it can be used above the determined dosage.

[0324] In addition, according to a specific example of the present invention, the present invention provides the use of the immunoconjugate in the preparation of a medicament for treating or preventing cancer. The immunoconjugate used to prepare the medicament can be mixed with an acceptable adjuvant, diluent, carrier, etc., and can be prepared into a composite preparation together with other active preparations to have a synergistic effect of the active ingredients.

[0325] Matters mentioned in the uses, compositions and methods of treatment of the present invention are equally applicable unless they conflict with each other.

[0326] Effects of the Invention

[0327] According to the present invention, the camptothecin drug of formula 1 designed in a manner to bind to the DDX5 protein can not only bind to the DDX5 protein in the cell and induce cell death through the degradation of the DDX5 protein, but also, preferably, when used as a payload, can solve the problem of aggregation of its carrier-drug conjugate and the acid-sensitive linker.

[0328] According to the carrier-drug conjugate of the present invention, when the camptothecin drug of formula 1 is connected through the acid-sensitive linker of formula 3, after in vivo injection, it is prevented from being phagocytosed by macrophages in the in vivo administration, distribution and elimination pathways. Due to the phagocytosis of macrophages on the organs of the reticuloendothelial system, it will not be absorbed by the liver, spleen, bone marrow or lymph nodes, etc., and can provide the desired blood circulation characteristics. Therefore, the immunoconjugate of the present invention can be administered at a dose of 11 mg / kg or more.

[0329] In addition, the immunoconjugate of the present invention is a hydrophobic low molecular weight drug. Due to the combined use of the camptothecin drug of formula 1 with cell membrane permeability and the acid-sensitive linker, the internalization process of the antigen-antibody complex is inefficient, which can solve the problem that the drug of sufficient concentration does not enter the cell and the problem that the antibody cannot penetrate well into the deep part of the cancer tissue, and can maximize the by-stander effect of entering not only the targeted cells but also the surrounding cells.

[0330] Among the problems identified during the development of the first to third generation ADCs, the immunoconjugates of the present invention can overcome the problems of drug resistance to ADCs and a narrow therapeutic window due to the relatively weak efficacy of the payloads used in the third generation ADCs. That is, due to the combined use of the camptothecin drug of Formula 1 and the acid-sensitive linker, the immunoconjugates of the present invention can exhibit an optimized therapeutic window by balancing strong anticancer efficacy and in vivo safety compared to existing ADC payloads. BRIEF DESCRIPTION OF THE DRAWINGS

[0331] Figure 1 It is the structural formula of various camptothecin anticancer drugs (SN-38, exatecan, Dxd, FL118).

[0332] Figure 2 The structural formula of camptothecin (CPT) and its binding to topoisomerase-1 are shown.

[0333] Figure 3 Schematic diagram showing the effective drug release mechanism of the immunoconjugate using an acid-sensitive linker according to the present invention.

[0334] Figures 4 to 7The results of western blot show whether the DDX5 and p-DDX5 proteins of various camptothecin drugs (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016) are degraded / degraded, and the inhibition degree of other various anti-apoptotic proteins in FaDu cell lines and A549 cell lines. Figure 5 and Figure 7 Results of western blot experiments in FaDu and A549 cell lines ( Figure 4 and Figure 6 ) to quantify the concentration graphically.

[0335] Figure 8 The western blot results of Tra-CL2A-FL118 / Tra-CL2A-exatecan are shown in FIG.

[0336] Fig. 9 and Fig.10 The results of in vitro cell viability comparison and evaluation of various camptothecin drugs and ADCs using them as payloads, namely Tra-CL2A-FL118 / Tra-CL2A-exatecan, were performed in MDA-MB-453 (HER2++) cell lines and SK-BR-3 cell lines, respectively.

[0337] Fig.11 A detailed classification diagram of apoptosis associated with programmed cell death. DETAILED DESCRIPTION

[0338] The present invention is further described in detail below through examples. However, the following examples are only used to illustrate the present invention, rather than to limit the scope of protection of the present invention.

[0339] Preparation Example 1: Synthesis of CL2A-exatecan

[0340]

[0341] 1-1: Synthesis of compound 2

[0342]

[0343] Triethylamine (0.098 mL, 0.705 mmol) was added to a suspension of exetidecan mesylate dihydrate (100 mg, 0.176 mmol) in N,N-dimethylformamide (dry) (3 mL) at room temperature under argon atmosphere. Then, MMTrCl (109 mg, 0.352 mmol) was added. The reaction mixture was diluted with dimethyl sulfoxide (DMSO) and purified by basic preparative medium pressure chromatography (MPLC) (XSelect40-80) to obtain, after lyophilization, (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((4-methoxyphenyl)diphenylmethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (103 mg, 83%) as an off-white solid.

[0344] SC_BASE: m / z 708.4[M+H] +

[0345] 1 H NMR(400MHz,DMSO-d6)δ7.73(d,J=10.9Hz,1H),7.58-7.49(m,4H),7.45-7.38(m,2H),7.37-7.27(m,5 H),7.27-7.20(m,2H),6.88(d,J=8.8Hz,2H),6.49(s,1H),5.42(s,2H),5.17(d,J=19.2Hz,1H),4.91( d,J=19.2Hz,1H),4.00-3.90(m,1H),3.74(s,3H),3.63(d,J=7.4Hz,1H),3.23-3.11(m,1H),2.80-2.6 8(m,1H),2.33(s,3H),1.96-1.80(m,2H),1.67-1.55(m,1H),1.31-1.19(m,1H),0.88(t,J=7.3Hz,3H).

[0346] 1-2: Synthesis of compound 4

[0347]

[0348] Under nitrogen atmosphere, (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((4-methoxyphenyl)diphenylmethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (85 mg, 0.120 mmol) and DMAP (55 mg, 0.456 mmol) were added to a dry flask and dissolved in dichloromethane (6 mL). After stirring for 5 minutes, a solution of triphosgene (14.2 mg, 0.048 mmol) in dichloromethane (0.750 mL) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. After 10 minutes, according to liquid chromatography (LCMS) analysis (sample in methanol (MeOH)), good conversion to methyl carbonate was observed. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azatriacontamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (140 mg, 0.132 mmol) was added to dichloromethane (1.5 mL), and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, dissolved in dimethyl sulfoxide (DMSO), and purified by basic preparative medium pressure chromatography (MPLC) (XSelect50-100), and after freeze-drying, the product (153 mg, 71%) was obtained as an off-white solid.

[0349] SC_BASE_M1800: m / z 1793.5[M+H] +

[0350] 1H NMR (400MHz, CDCl3) δ8.60(s,1H),7.69(d,J=10.6Hz,1H),7.59-7.49(m,6H),7.44(d,J=8.4Hz,6H),7.38-7.28(m,8 H),7.24-7.09(m,6H),6.89-6.74(m,4H),5.65(d,J=17.3Hz,1H),5.36(d,J=17.2Hz,1H),5.11(d,J=12.0Hz,1H),4. 97(d,J=12.1Hz,1H),4.69-4.44(m,3H),4.21-3.97(m,5H),3.84-3.74(m,6H),3.68-3.51(m,33H),3.50-3.41(m,2H ),3.40-3.27(m,3H),2.96-2.85(m,1H),2.43(s,3H),2.33-1.79(m,7H),1.78-1.34(m,14H),0.97(t,J=7.4Hz,3H).

[0351] 1-3. Synthesis of Compound 5

[0352]

[0353] In dichloromethane (9 mL), 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontamide)benzyl((1S,9S)-9-ethyl-5-fluoro-1-(((4-methoxyphenyl)diphenylmethyl)amino)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (0.070 g, 0.256 mmol), copper(I) bromide (7.3 mg, 0.051 mmol) and DIPEA (0.045 mL, 0.256 mmol) were added to a solution of 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (0.070 g, 0.256 mmol), copper(I) bromide (7.3 mg, 0.051 mmol) and DIPEA (0.045 mL, 0.256 mmol). The reaction mixture was stirred at room temperature overnight. An additional amount of copper(I) bromide (7.3 mg, 0.051 mmol) was added. After stirring for 3 hours, the product in sample-2 on liquid chromatograph (LCMS) increased. The reaction mixture was stirred for another 5 hours and concentrated under reduced pressure. The residue was purified by basic preparative medium pressure chromatography (MPLC) (XSelect 50-100) to give the product as an off-white solid after lyophilization (133 mg, 75%).

[0354] SC_BASE_M1800:m / z 1795.5[M-MMT+H] + ,1523.5[M-2xMMT+H] +

[0355] 1H NMR (400MHz, DMSO-d6) δ10.16(s,1H),8.24-8.11(m,2H),8.11-8.02(m,1H),7.81(s,1H),7.78-7.69(m,1H),7.63-7. 49(m,6H),7.44-7.08(m,27H),7.03-6.96(m,3H),6.89(d,J=8.5Hz,2H),6.85-6.78(m,2H),5.60-5.45(m,2H),5.30- 4.97(m,4H),4.51-4.39(m,3H),4.24(d,J=5.6Hz,2H),4.08-3.93(m,5H),3.83-3.59(m,10H),3.53-3.36(m,36H),3. 27-3.19(m,6H),2.22-1.86(m,7H),1.81-1.38(m,13H),1.38-1.11(m,7H),0.90(t,J=7.3Hz,4H),0.87-0.80(m,1H).

[0356] 1-4. Synthesis of CL2A-exatecan compound

[0357]

[0358] In dichloromethane (anhydrous, 1.5 mL), 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontamide)benzyl((1S,9S)-9- To a solution of ethyl-5-fluoro-1-(((4-methoxyphenyl)diphenylmethyl)amino)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (100 mg, 0.048 mmol) was added anisole (0.528 mL, 4.83 mmol) at room temperature under an inert atmosphere, followed by dropwise addition of dichloroacetic acid (0.160 mL, 1.934 mmol). Subsequently, methyl tert-butyl ether (MTBE) (~3 mL) was added. The reaction mixture became a fine suspension. Heptane (~3 mL) was added. The solvent was removed as much as possible using a pipette. The residue was washed several times with a mixture of methyl tert-butyl ether (MTBE) / heptane (1:1, ~4 mL). The wet residue was dried under reduced pressure overnight to obtain a light green solid (85 mg, 89%).

[0359] AN_ACID:m / z 763.0[M+2H] 2+ / 2

[0360] 1H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.26-8.14(m,2H),8.08(t,J=5.7Hz,1H),7.89(d,J=10.8Hz,1H),7.81(s,1H),7 .78-7.50(m,5H),7.31(d,J=8.3Hz,2H),7.07(s,1H),7.01(s,2H),6.17(s,3H),5.79-5.41(m,4H),5.19-4.99(m,3H),4 .52-4.42(m,3H),4.25(d,J=5.6Hz,2H),4.09-3.94(m,4H),3.78(t,J=5.3Hz,2H),3.55-3.41(m,33H),3.25-3.19(m,4 H),2.83-2.72(m,2H),2.44-2.42(m,3H),2.26-2.00(m,5H),1.80-1.46(m,10H),1.44-1.18(m,6H),0.95-0.85(m,5H).

[0361] Preparation Example 2: Synthesis of CL2A-Dxd

[0362]

[0363] 2-1: Synthesis of Compound 1

[0364]

[0365] 2-hydroxyacetic acid (133mg, 1.749mmol) and triethylamine (0.729ml, 5.25mmol) are dissolved in dichloromethane (4ml). The reaction mixture is cooled to 0 ° C. Then, under an argon atmosphere, a solution of MMTrCl (702mg, 2.273mmol) is added to dichloromethane (4.00ml). The reaction mixture is slowly brought to room temperature and stirred for 2 days. The reaction mixture is concentrated under reduced pressure. The residue is purified by flash chromatography (silicon dioxide, 0% to 10% methanol + 1% TEA (triethylamine) in dichloromethane) to obtain a white solid product, which is contaminated by residual triethylamine. Yield: 500mg, 30% (corrected for residual triethylamine). The product can be used without further purification.

[0366] SC_BASE: m / z 347.2[M+H] +

[0367] 1H NMR (400MHz, CDCl3) δ7.59-7.50(m,4H),7.45-7.38(m,2H),7.28-7.13(m,6H),6.83-6.76(m,2H),3.77(s,3H),3.59(s,2H).

[0368] 2-2: Synthesis of Compound 2

[0369]

[0370] A solution of 2-((4-methoxyphenyl)diphenylmethoxy)acetic acid (126 mg, 0.254 mmol), NHS (29.2 mg, 0.254 mmol) and EDCI·HCl (48.7 mg, 0.254 mmol) were added to N,N-dimethylformamide (dry) (1.2 mL). The reaction mixture was stirred at room temperature for 1 hour. Then, it was added to a suspension of exitecan mesylate (90 mg, 0.169 mmol) and triethylamine (0.026 mL, 0.186 mmol) in N,N-dimethylformamide (dry) (1.2 mL), and stirred at room temperature overnight. The reaction mixture was diluted with dimethyl sulfoxide (DMSO), purified by basic preparative medium pressure chromatography (MPLC) (XSelect 30-70), concentrated from a mixture of acetonitrile and water (1:1, 10 mL), and lyophilized to afford N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-((4-methoxyphenyl)diphenylmethoxy)acetamide as an off-white solid. Yield: 89 mg, 68%.

[0371] SC_BASE: m / z 766.4[M+H] +

[0372] 1H NMR (400MHz, CDCl3) δ7.74 (d, J = 10.6Hz, 1H), 7.61 (s, 1H), 7.26-7.09 (m, 13H), 6. 90(d,J=9.1Hz,1H),6.75-6.69(m,2H),5.76(d,J=16.4Hz,1H),5.61-5.52(m,1H), 5.35-5.20(m,3H),4.03-3.93(m,2H),3.74(s,3H),3.22-3.12(m,1H),3.10-2.99 (m,1H),2.44(s,3H),2.27-2.19(m,2H),1.98-1.84(m,2H),1.06(t,J=7.4Hz,3H).

[0373] 2-3: Synthesis of compound 4

[0374]

[0375] In a dry flask under nitrogen atmosphere, N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-((4-methoxyphenyl)diphenylmethoxy)acetamide (80 mg, 0.104 mmol) and DMAP (48.5 mg, 0.397 mmol) were dissolved in dichloromethane (6 mL). After stirring for 5 minutes, a solution of triphosgene (12.4 mg, 0.042 mmol) was added in one portion in dichloromethane (0.750 mL), and the reaction mixture was stirred at room temperature for 15 minutes. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azatriacontamide)-N-(4-(hydroxymethyl))phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (122 mg, 0.115 mmol) was added to dichloromethane (1.5 mL) and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure and dissolved in dimethyl sulfoxide (DMSO), purified by basic preparative medium pressure chromatography (MPLC) (XSelect50-100), and freeze-dried to obtain a colorless solid. Yield: 0.129 g, 66%.

[0376] SC_BASE_M1900:m / z 1580.0[M-MMT+H] +

[0377] 1H NMR (400MHz, DMSO-d6) δ10.14(s,1H),8.32(d,J=8.7Hz,1H),8.13(d,J=8.0Hz ,1H),8.10-8.01(m,1H),7.80(d,J=10.9Hz,1H),7.58(d,J=8.5Hz,2H),7.45- 7.33(m,8H),7.33-7.11(m,19H),7.02(s,1H),6.89-6.78(m,4H),5.56(s,1H) ,5.50(s,2H),5.33-5.15(m,2H),5.09(q,J=12.2Hz,2H),4.47-4.40(m,1H),4. 00(s,2H),3.96(d,J=4.2Hz,2H),3.71(s,3H),3.70-3.68(m,3H),3.64-3.57( m,4H),3.56-3.52(m,4H),3.52-3.47(m,24H),3.43-3.36(m,5H),3.28-3.21(m ,3H),3.13(s,2H),2.42-2.36(m,4H),2.24-2.10(m,4H),2.00-1.88(m,2H),1 .73-1.53(m,2H),1.53-1.42(m,2H),1.41-1.22(m,3H),0.90(t,J=7.4Hz,3H).

[0378] 2-4: Synthesis of compound 5

[0379]

[0380] In dichloromethane (7 mL), 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontamide)benzyl((1S,9R)-9-ethyl-5-fluoro-1-(2-((4-methoxyphenyl)diphenylmethoxy)acetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (57 mg, 0.209 mmol), copper(I) bromide (6 mg, 0.042 mmol) and DIPEA (0.036 mL, 0.209 mmol) were added to a solution of H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-9-yl)carbonate (0.129 g, 0.070 mmol). The reaction mixture was stirred at room temperature overnight. An additional amount of copper(I) bromide (6 mg, 0.042 mmol) was added. Stir for another 3 hours, then the reaction mixture was concentrated under reduced pressure. The residue was purified by basic preparative medium pressure preparative chromatography (MPLC) (XSelect50-100). The product fractions were lyophilized to obtain an off-white solid. Yield: 106 mg, 71%.

[0381] SC_BASE: m / z 1854.2[M-MMT+H] + ,1582.0[M-2xMMT+H] +

[0382] 1H NMR (400MHz, DMSO-d6) δ10.15(s,1H),8.34(d,J=8.6Hz,1H),8.24-8.03(m,3H),7.87-7.73(m,2H),7.67-7.53(m,2H),7.44-7 .34(m,9H),7.30-7.10(m,19H),7.07-6.99(m,2H),6.90-6.77(m,5H),5.61-5.39(m,2H),5.29-4.99(m,4H),4.53-4.38(m,4H) ,4.32-4.20(m,2H),4.02-3.93(m,5H),3.78-3.66(m,10H),3.62(d,J=9.7Hz,2H),3.52-3.38(m,38H),3.25-3.20(m,3H),2.42 -2.36(m,3H),2.24-2.10(m,3H),1.99-1.87(m,3H),1.76-1.55(m,6H),1.55-1.41(m,4H),1.29(s,6H),0.90(t,J=7.0Hz,4H).

[0383] 2-5: Synthesis of compound CL2A-Dxd

[0384]

[0385] 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontamide)benzyl ((1 ... To a solution of (4-((4-methoxyphenyl)diphenylmethoxy)acetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (80 mg, 0.038 mmol) was added anisole (0.411 ml, 3.76 mmol) followed by dropwise addition of dichloroacetic acid (0.047 ml, 0.564 mmol). After stirring for 1 hour, MTBE (~3 mL) was added. The reaction mixture became a fine suspension. Heptane (~3 mL) was added to induce precipitation. The solvent was removed as much as possible using a pipette. The residue was washed several times with a mixture of MTBE / heptane (1:1, ~4 mL). The wet residue was dried under reduced pressure, transferred to a vial, and dried under vacuum overnight to obtain an off-white solid. Yield: 57 mg, 96%. Purity (LC-UV: 78%).

[0386] AN_ACID:792.0[M+2H] 2+ / 2

[0387] 1 H NMR analysis contains the expected signals.

[0388] 1H NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.43(d,J=9.0Hz,1H),8.26-8.15(m,2H),8.12-8.03(m,1H),7.86-7.53(m,8H) ,7.45-7.18(m,6H),7.05-6.97(m,3H),6.19(s,2H),5.66-5.54(m,1H),5.50(s,3H),5.23(s,2H),5.17-5.00(m,2H),4 .52-4.44(m,3H),4.25(d,J=5.7Hz,2H),4.06-3.92(m,7H),3.81-3.74(m,3H),3.74-3.68(m,1H),3.58-3.46(m,33H), 3.23(d,J=7.1Hz,7H),2.40(s,3H),2.27-1.98(m,7H),1.82-1.44(m,11H),1.28(d,J=12.2Hz,6H),0.95-0.81(m,6H).

[0389] Preparation Example 3: Preparation of anti-HER2 antibody-CL2A linker-exatecan / Dxd immunoconjugate (DAR 8)

[0390] 2mg / ml of anti-HER2 antibody (trastuzumab) in reaction buffer (20mM histidine, 150mM NaCl, pH6.0) was reduced with 825uM TCEP at 25°C for 2 hours (hrs), and the thiol site required for the reaction was generated by the disulfide of the antibody. After reduction, TCEP was removed by a spin desalting column (PD-10).

[0391] Dimethyl sulfoxide (DMSO), compound solutions of Preparation Example 1 and Preparation Example 2 (5 mM stock solution in DMSO) were added to the reduced antibody solution, and the final DMSO concentration was 10%, which was 12 equivalents (eq.) relative to the antibody. The reaction mixture was appropriately mixed and the reaction bottle was placed at 25°C for 1 hour.

[0392] After conjugation, the reaction mixture was passed through a spin desalting column (PD-10) to remove unreacted compounds and isolate only the ADC. The product was then sterile filtered through a 0.2 μm polyvinylidene fluoride (PVDF) disposable filter. The immunoconjugate as the resulting product was characterized and 0.07 mM PS80 and 20 mM trehalose dehydrate were added.

[0393] The DAR of the immunoconjugate of Preparation Example 3 was determined using hydrophobic interaction chromatography (HIC) and mass spectrometry (MS), and it was confirmed that the average MS-DAR value was 7.14 and the HIC-DAR value was 8.00.

[0394] Example 1. Confirmation of the expression inhibitory activity of cancer-associated survival genes DDX5, p-DDX5, survivin, Mcl-1, XIAP, and cIAP2 by western blot

[0395] For FL118, exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38, and Tra-CL2A-Exatecan (Preparation Example 3), the following anti-apoptotic protein expression inhibition analysis was performed.

[0396] Tra-CL2A-FL118 and Tra-CL2A-SN-38 were prepared in the same manner as in Preparation Example 3, except that FL118 drug and SN-38 were used instead of Exatecan drug, respectively.

[0397] (1) Protein extraction

[0398] FaDu cell line was seeded at 200,000 cells / well in a 6-well plate and incubated at a constant temperature (37°C, 5% CO2). After 24 hours, drugs (FL118, Exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38, and Tra-CL2A-Exatecan) were treated in the wells at concentrations of 0 nM, 10 nM, and 100 nM, respectively. Incubate at a constant temperature (37°C, 5% CO2) for 24 hours. 100 ul of RIPA buffer dissolved with protease inhibitor cocktail was treated in the wells. The plate was placed on ice and incubated at a constant temperature on an orbital shaker for 2 hours. The RIPA buffer containing lysed cells was transferred to an eptube and centrifuged (16000 rcf, 20 min, 4°C). Then, only the supernatant was transferred to a new eptube. The protein concentration was confirmed by protein assay.

[0399] (2) Protein separation by electrophoresis

[0400] Protein sample and 4x SDS-PAGE loading buffer were mixed at a ratio of 3:1, boiled at 95°C for 10 minutes, and then cooled. The samples were loaded into the gel wells so that the amount of protein was the same. Electrophoresis was performed at 60V in the gel.

[0401] (3) Transfer of proteins from gel to membrane

[0402] Turbo TM Transfer system( Turbo TM Into the cassettes of the Transwell Transfer System, 7 activated filter papers, PVDF membrane, gel, and 7 filter papers were placed in sequence, then the lid was closed, inserted into the instrument, and the experimental protocol was started.

[0403] (4) Antibody incubation

[0404] Immerse the transferred membrane in blocking buffer and incubate at a constant temperature (RT, 1h). Then, incubate at a constant temperature (4°C, overnight) in the primary antibody solution. Rinse with hybridization membrane cleaning solution (TBST buffer) for 3 minutes (min) (repeat 3 times). Incubate at a constant temperature (RT, 1h) in the secondary antibody solution conjugated with HRP (horseradish peroxidase). Rinse with hybridization membrane cleaning solution (TBST buffer) for 3 minutes (min) (repeat 3 times).

[0405] (5) Imaging and result analysis

[0406] After the membrane is immersed in ECL (enhanced chemiluminescence) substrate for about 3 to 5 minutes, use ChemiDoc TM MP Imaging System (ChemiDoc TM MP Imaging System) to confirm the signal. HRP (horseradish peroxidase) bound to the secondary antibody oxidizes the luminol of ECL, and the released light is detected to display as an image. The thickness of the band is proportional to the amount of protein, so the amount of protein can be compared by the thickness of the band.

[0407] like Figure 4 and Figure 5 As shown, it was confirmed how the protein expression level changes due to drug (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 and Tra-CL2A-exatecan) treatment. Glyceraldehyde phosphate dehydrogenase (GAPDH) is an enzyme involved in glycolysis, which is an essential metabolic process in cells. GAPDH is a gene that is always expressed in cells and its expression level is not easily changed, which can indicate whether the sample is loaded with the same amount of protein on the gel.

[0408] In the same way, A549 cells overexpressing ABCG2 were seeded to replace the FaDu cell line not expressing ABCG2, 4ug protein was loaded, and western blot was performed ( Figure 6 , Figure 7 as well as Figure 8 ).

[0409] It has been confirmed that various camptothecin compounds degrade DDX5 or phosphorylate DDX5 (p-DDX5), among which DDX5 is a factor directly involved in the expression of anti-apoptotic proteins. In addition, the expression of anti-apoptotic proteins such as Survivin, Mcl-1, cIAP2, and XIAP was also confirmed to be partially inhibited, thus confirming that various camptothecin compounds are compounds with dual mechanisms, which can not only inhibit topoisomerase I, but also act as anti-apoptotic protein inhibitors.

[0410] Example 2. In vitro cell viability test of Tra-CL2A-FL118 and Tra-CL2A-Exatecan

[0411] Trastuzumab (molecular weight 148 kDa, purity 97%) provided by BCD in a solution state stored at -80°C was dissolved in a buffer solution (20 mM histidine, 150 mM NaCl, pH 6.0) (concentration 10.09 mg / mL) and used as a control group.

[0412] Her2-high cell line (MDA-MB-453) and HER2-positive breast cancer derived cell line (SK-BR-3) were seeded at 3000 cells / well in a 96-well plate and incubated at a constant temperature (37°C, 5% CO2). After 24 hours, the cells were treated with 100ul of 9 concentrations of drugs (serial dilution starting from 1000nM in 1 / 5 steps). At this time, trastuzumab, Tra-CL2A-FL118 and Tra-CL2A-exatecan were treated. Cell viability was observed by constant temperature incubation (37°C, 5% CO2) for 3 or 6 days. 100ul of CellTiter-Glo reagent (using Luminescent Cell Viability Assay Kit ( Luminescent cell viability Assay kit (Promega, G7571))) was used, and then pipetted. After incubation at constant temperature (RT) for 10 minutes, the luminescence was measured. When the luminescence value when the drug concentration was 0 was regarded as 100%, the concentration representing the luminescence value of 50% was the IC 50 value.

[0413] like Fig. 9 and Fig.10 As shown, Trastuzumab-CL2A-exadecane (Preparation Example 3) showed cytotoxicity in Her2-high cell line (MDA-MB-453) and HER2-positive breast cancer-derived cell line (SK-BR-3).

[0414] Thus, it was confirmed that ADCs connected to an acid-sensitive linker (CL2A) with various camptothecin compounds of Formula 1 (eg, FL118 to exotecan) as payloads showed excellent cytotoxicity and operated with high efficiency.

Claims

1. An immunoconjugate or a pharmaceutically acceptable salt thereof, designed to release the camptothecin drug of Formula 1 at a target site in vivo by comprising [camptothecin drug of Formula 1]-[acid-sensitive linker]-[antibody or a fragment containing its antigen-binding site], characterized in that: The camptothecin drug of Formula 1 is a payload designed to bind to the DDX5 protein and / or E3 ligase. At least one camptothecin drug of Formula 1 is linked to an antibody or a fragment containing an antigen binding site thereof via an acid-sensitive linker, After targeting cancer cells by targeting the antigen binding site of the antigen of the cancer cell, the acid-sensitive linker is degraded in the acidic environment surrounding the cancer, i.e., pH ≤ 7, and at least a portion of the camptothecin drug of Formula 1 is freed, and the free camptothecin drug of Formula 1 penetrates the cell membrane and migrates into the cell. Optionally, the immunoconjugate linked to the camptothecin drug of Formula 1 is internalized into the cell, and the camptothecin drug of Formula 1 is released from the lysosome: Formula 1 X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a pentagonal ring, a hexagonal ring or a septagonal ring, and n=a value of 0 to 2, Y1, Y2 and Y3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

2. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: Used to administer to a patient population in which DDX5 acts as a tumor protein, or oncoprotein, within cells.

3. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The camptothecin drugs of Formula 1 are designed to adjust the cell membrane permeability as needed through the modification of X1, (X2)n, X3, Y1, Y2 and / or Y3, so as to exert a bystander effect in tumor tissues or control the extent of its effect.

4. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The camptothecin drug of Formula 1 is a camptothecin drug of Formula 1-1 or Formula 1-2: Formula 1-1 Formula 1-2 5. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The acid-sensitive linker is stable in the neutral environment of blood, i.e., pH 7.3-7.5, but is hydrolyzed in the surroundings of tumor cells, i.e., pH 6.5-7.2, or in endosomes, i.e., pH 5.0-6.5, or lysosomes, i.e., pH 4.5-5.0, after being internalized into cells, to release the active camptothecin drug of formula 1.

6. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: After targeting cancer cells by targeting the antigen binding site of the cancer cell antigen, the acid-sensitive linker degrades in the acidic environment surrounding the cancer, i.e., pH ≤ 7, so that at least part of the camptothecin drug of Formula 1 is freed, and the free camptothecin drug of Formula 1 penetrates deep into the tumor tissue and penetrates the cell membrane to migrate into the cell.

7. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The camptothecin drug of Formula 1 and the acid-sensitive linker are linked via a carbonate or ester bond, so that the free camptothecin drug of Formula 1 is released when the acid-sensitive linker is degraded.

8. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The free camptothecin drugs of Formula 1 migrate into cells to target cancer cells and / or their surrounding cells.

9. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody or its antigen-binding site binds to a receptor on the cell surface.

10. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody or its antigen-binding site will target antigens that are selectively distributed on the surface of cancer or antigens overexpressed by cancer cells in small amounts on normal tissues.

11. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The antibody or its antigen binding site targets HER2, FolR, PSMA or Trop-2, one of the human epidermal growth factor receptors, HER / EGFR / ERBB.

12. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The acid-sensitive linker is derived from the compound of the following formula 3: Formula 3 Wherein, X1 and X2 are each independently -H or -halogen; Y is -NH-, -NR A -, or nothing, i.e. 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 -; R A To R C Each is independently a C1-C4 alkyl group; M is as well as n is an integer of 5-9.

13. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 12, characterized in that: The alcohol group site at the C20 position of the camptothecin drug of Formula 1 is connected to the alcohol group site of the acid-sensitive linker of Formula 3.

14. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: [Camptothecin drug of Formula 1]-[acid-sensitive linker] is derived from a compound represented by the following Formula 4 or Formula 5: Formula 4 Formula 5 wherein n is an integer from 5 to 9 independently.

15. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The connection between [acid-sensitive linker]-[antibody or a fragment containing its antigen-binding site] is formed by the binding of the thiol group contained in the antibody or its antigen-binding fragment to the maleimide group or maleic hydrazide of the acid-sensitive linker.

16. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The camptothecin drugs of Formula 1 designed in a manner of linking to the DDX5 protein and the E3 ligase kill target cells expressing the DDX5 protein through a molecular glue degrader mechanism (MoA).

17. The immunoconjugate according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The camptothecin drug of formula 1, which is designed in a manner of connecting to the DDX5 protein and the E3 ligase, has a mechanism of action (MoA) of degrading the tumor protein, ie, oncoprotein DDX5, while having type 1 topoisomerase inhibitory ability.

18. The immunoconjugate or a pharmaceutically acceptable salt thereof according to claim 17, characterized in that: (1) In Formula 1, the C group site selectively binds to type 1 topoisomerase and the A group site binds to DNA to stabilize the covalent bonds of the topoisomerase-DNA complex to prevent religation of the cut DNA fragments, and / or, (2) In Formula 1, the A group site binds to the DDX5 protein and, selectively, the C group site binds to the E3 ligase to induce degradation of the DDX5 protein: Formula 1 19. A pharmaceutical composition for preventing or treating cancer, wherein: The composition comprises the immunoconjugate according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof as an active ingredient.

20. A drug-linker conjugate or a pharmaceutically acceptable salt thereof, wherein: The camptothecin drug of Formula 1-1 or Formula 1-2 is connected with the acid-sensitive linker of Formula 3: Formula 1-1 Formula 1-2 Formula 3 Wherein, X1 and X2 are each independently -H or -halogen; Y is -NH-, -NR A -, or nothing, i.e. 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 -; R A To R C Each is independently a C1-C4 alkyl group; M is as well as n is an integer of 5-9.

21. The drug-linker conjugate or a pharmaceutically acceptable salt thereof according to claim 20, characterized in that: The camptothecin drug of Formula 1-1 or Formula 1-2 and the acid-sensitive linker are connected via a carbonate or ester bond, so that the free camptothecin drug of Formula 1-1 or Formula 1-2 is released when the acid-sensitive linker is degraded.

22. The drug-linker conjugate or a pharmaceutically acceptable salt thereof according to claim 20, characterized in that: The alcohol group site at the C20 position of the camptothecin drug of Formula 1-1 or Formula 1-2 is connected to the alcohol group site of the acid-sensitive linker of Formula 3.

23. The drug-linker conjugate or a pharmaceutically acceptable salt thereof according to claim 20, characterized in that: The drug-linker conjugate is a compound represented by the following Formula 4 or Formula 5: Formula 4 wherein n is an integer from 5 to 9 independently.

24. The drug-linker conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 23, characterized in that: The acid-sensitive linker of Formula 3 is degraded in an acidic environment, i.e., pH ≤ 7, and the camptothecin drug of Formula 1-1 or Formula 1-2 is free.

25. An immunoconjugate or a pharmaceutically acceptable salt thereof, characterized in that: Include: (a) the drug-linker conjugate according to any one of claims 20 to 23, or a pharmaceutically acceptable salt thereof; and (b) an antibody or a fragment thereof containing an antigen-binding site, At least one camptothecin drug of Formula 1-1 or Formula 1-2 is linked to an antibody or a fragment containing an antigen binding site thereof via an acid-sensitive linker of Formula 3.

26. The immunoconjugate according to claim 25 or a pharmaceutically acceptable salt thereof, characterized in that: The antibody is trastuzumab, cetuximab, or certolizumab pegol.

27. A method for preparing a carrier-drug conjugate, characterized in that: Using the drug-linker conjugate according to any one of claims 20 to 23 or a pharmaceutically acceptable salt thereof, at least one camptothecin drug of Formula 1-1 or Formula 1-2 is linked to a carrier via an acid-sensitive linker of Formula 3.

28. The method for preparing a carrier-drug conjugate according to claim 27, characterized in that: The carrier is an antibody or a fragment, repeat, aptamer, and / or peptide containing the antigen binding site thereof.

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