WO2023214849A1 - Conjugate of ddx5 protein-binding camptothecin-based drug linked to acid-sensitive linker and immunoconjugate using same - Google Patents

Conjugate of ddx5 protein-binding camptothecin-based drug linked to acid-sensitive linker and immunoconjugate using same Download PDF

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WO2023214849A1
WO2023214849A1 PCT/KR2023/006170 KR2023006170W WO2023214849A1 WO 2023214849 A1 WO2023214849 A1 WO 2023214849A1 KR 2023006170 W KR2023006170 W KR 2023006170W WO 2023214849 A1 WO2023214849 A1 WO 2023214849A1
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formula
drug
camptothecin
acid
cancer
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Korean (ko)
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정두영
이진수
조현용
이병성
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주식회사 피노바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a conjugate in which a camptothecin drug of formula 1 (e.g., Exatecane and dxd) designed to bind to DDX5 protein is linked to an acid-sensitive linker, and a carrier-drug conjugate using the same.
  • a camptothecin drug of formula 1 e.g., Exatecane and dxd
  • the camptothecin-based drug of Formula 1 designed to bind to the DDX5 protein according to the present invention, not only binds to the DDX5 protein within cells and induces cell death through DDX5 protein degradation, but is preferably used as a payload. When used as a payload, it can solve the problem of aggregation of its carrier-drug conjugate with an acid-sensitive linker.
  • Cancer genes are genes that originally exist to control the normal functions of cells, but can cause cancer if mutated or regulated abnormally.
  • One of the normal cell functions is apoptosis, and related to this, there are death promoting factors and death suppressing factors.
  • cancer genes include (i) oncogenes that actively promote cell division when mutated, and (ii) tumor suppressor genes that do not suppress cell division when mutated. ).
  • oncogenes include Src, EGFR, HER2, RAS, APC, and BCL-2
  • tumor suppressor genes include p53, BRCA, Rb, PTEN, and BAX.
  • Cancer is a group of diseases in which the cell cycle of growth and division is dysregulated. As shown in Figure 11, cancer can occur when programmed cell death (e.g., apoptosis) mechanisms are impaired.
  • programmed cell death e.g., apoptosis
  • 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).
  • Proteins encoded by viral oncogenes are similar to cellular proteins that have important regulatory functions. These cellular homologs are called proto-oncogenes or normal cellular oncogenes. Mutations in proto-oncogenes actively promote cell proliferation. The mutant cH-ras protein contains mutations that impair its ability to hydrolyze GTP. This keeps the mutant protein in an active signaling mode and stimulates cell division. Mutant versions of c- ras have been found in many types of tumors.
  • HER2-positive breast cancer in which HER2 is expressed abnormally in breast epithelial cells, accounts for about 1 ⁇ 4 of all breast cancer patients, and has a higher recurrence rate and worse prognosis when treated with chemotherapy than female hormone-related breast cancer.
  • Herceptin (generic name: Trastuzumab), a HER2-targeted treatment, treats breast cancer by reducing the number of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.
  • HER2-positive breast cancer is breast cancer that has amplification or overexpression of the HER2 (ErbB2) oncogene, a member of the ErbB receptor.
  • Trastuzumab Herceptin
  • HER2-positive breast cancer shows an aggressive pattern compared to other breast cancers, and more than half of patients are resistant to existing targeted treatments or develop resistance during treatment.
  • Ligands of the ErbB receptor attached to the cell membrane include NRG1 and HB-EGF, and activated ErbB ligands bind to their receptors (EGFR, HER3) and undergo phosphorylation by binding between receptors (EGFR-HER2, HER2-HER3). It catalyzes phosphorylation (P) and induces growth and proliferation signals within cells. This excessive activation of the ErbB receptor results in resistance to anti-HER2 treatment.
  • the STAT (short for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6.
  • STAT proteins can be activated by JAK kinases upon stimulation with growth factors, cytokines, interferons, or oncogenes to regulate interferon signaling.
  • the DNA binding domain of STAT is an immunoglobin-like structure that mediates binding to specific DNA target sequences.
  • phosphorylation of STAT3 by JAK kinases activates dimerization. The dimer then moves to the nuclear compartment and binds to the sequence of the target promoter, leading to expression of the target gene.
  • STAT3 target genes include survivin, bcl-xl, mcl-1, cyclin D1, MMP2, MMP9, VEGF, Myc, and Sox2.
  • STAT proteins regulate many biological processes, including cell growth, apoptosis, differentiation, and immunity. Within the STAT family, STAT3 and STAT5 have been implicated in cancer progression.
  • Dysregulated STAT3 and activated STAT5 are considered oncogenes, increasing angiogenesis and improving cancer cell survival.
  • STAT3 was found to be more frequently overexpressed in lung, ovarian, gastric, hematopoietic, and brain cancers compared to normal tissues, and overexpression of STAT3 was associated with poor overall survival.
  • Phosphorylation of STAT3 by JAK kinases is the first step in STAT3 activation.
  • Another study of 90 patients with glioblastoma found that high p-STAT3 levels were significantly associated with poorer progression-free and overall survival. Multivariate survival analysis showed that high p-STAT3 levels could serve as an important prognostic indicator for poor progression-free survival and overall survival.
  • Inhibitors of type 1 topoisomerase I are anticancer mechanisms whose efficacy/safety has been proven clinically, and are excellent anticancer agents for various intractable solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer in clinical trials. Efficacy has been proven.
  • camptothecin derivatives which are low-molecular-weight compounds that exhibit anti-tumor activity by inhibiting type 1 topoisomerase (topoisomerase-1), are known.
  • Camptothecin is a selective inhibitor of type 1 topoisomerase-1, an isomerase involved in DNA replication and recombination. It is a natural anti-tumor alkaloid isolated from Camptotheca acuminata, native to China, by Wall et al. in the United States in 1966. Since it was found to exhibit strong cytotoxicity in vitro, development through clinical trials was initiated at the National Cancer Research Center (NCI), etc., but due to the limitation of extremely poor solubility, various side effects such as bone marrow suppression and hemorrhagic cystitis were associated with it. As a result, development was halted.
  • NCI National Cancer Research Center
  • camptothecin has a unique mechanism of action, that is, unlike the DNA type 2 topoisomerase (topoisomerase-2) inhibition mechanism, it selectively inhibits DNA type 1 topoisomerase, showing an antitumor effect. This has been confirmed.
  • DNA topoisomerase is a member of the gyrase enzyme family. These are enzymes in the nucleus that temporarily cut DNA or unwind the double helix when the cell needs access to genetic material for replication or transcription. They also participate in a variety of intracellular activities, including chromosome condensation and recombination, and DNA repair. The genetic code of topoisomerase is highly conserved among species.
  • Type 1 topoisomerase the drug target of camptothecin, has been observed to have increased levels in various malignant tumors. This drug does not inhibit free enzymes, but stabilizes the covalent bond of the topoisomerase-DNA complex, preventing the reconnection of cut DNA fragments. Therefore, the sensitivity of cells to these topoisomerase-targeting drugs is related to the level of the enzyme present in the nucleus. This drug prevents transcription from proceeding by interfering with DNA recombination. The higher the amount of type 1 topoisomerase, the more cleavable complexes are formed, which means higher drug sensitivity.
  • type 1 topoisomerase inhibitors are used to increase the expression of type 2 topoisomerases, making them more susceptible to type 2 topoisomerase inhibitors.
  • type 1 topoisomerase is not closely related to proliferation in normal tissues, and is an “S” stage solid cancer with active cell division, such as colon cancer, ovarian cancer, and esophageal cancer, including lymphoma. It is present in large quantities compared to surrounding normal tissues, and is known to actually cause cell death by blocking the replication and transcription of genes in tumor cells during the “S” phase of the cell cycle.
  • camptothecin derivatives identified to date contain a parent structure with five rings essential for cytotoxicity ( Figure 1).
  • the E-ring and A- and B-ring regions were identified as important regions.
  • Camptothecin contains a pentacyclic lactone structure in the E-ring, which is essential for cytotoxicity.
  • the lactone group and alpha hydroxyl group located at carbon 20 of the E-ring are important for the stability of type 1 topoisomerase-DNA by-products, and the fact that modification of the A- and B-rings can increase water solubility and activity. This has been proven. Modifications on the first ring have been proven to increase the solubility in water and allow greater tolerability, for example in the case of the drugs mentioned above.
  • CKD-602 also attempted to substitute the B-ring portion at carbon 7 to increase water solubility and anticancer effect.
  • Lee et al. reported that CKD-602 has superior anticancer effects compared to camptothecin and topotecan in a wide range of cancer cell lines.
  • MTD maximum tolerated dose
  • the generally known side effects of camptothecin-based drugs can be broadly classified into hematological side effects and non-hematological side effects.
  • Hematological side effects include neutropenia with fever, sepsis, and bleeding, while non-hematological side effects include skin side effects such as nausea, vomiting, and hair loss, and toxicity to the gastrointestinal tract, kidneys, and nervous system.
  • Kim et al. found no adverse drug reactions other than increased gastric secretion, even when administered at doses 10 times higher than the clinically applicable dose, among the side effects of these camptothecin drugs.
  • recent domestic clinical studies have proven its relative safety, with only reversible and controllable side effects such as neutropenia and leukopenia reported rather than serious systemic toxicity.
  • Antibody-drug conjugate is a new drug platform that utilizes the high tissue selectivity of antibodies to selectively deliver a payload with strong anticancer efficacy only to cancerous tissues.
  • ADC selectively delivers a powerful payload that kills cancer cells even at concentrations as low as pM, and minimizes systemic exposure to the drug, ensuring anticancer efficacy and safety at the same time.
  • ADC consists of three components, including a drug, a monoclonal antibody, and a linker that connects the antibody and the drug.
  • ADC technology uses antibodies that specifically bind to specific antigens expressed on the surface of cancer cells. This is a method of delivering drugs to tumor cells. In most cases, intracellular transduction of ADC proceeds through the clathrin-coated pit function. ADC moved into the cell detaches from clathrin, fuses with other vesicles within the cell, and then proceeds to the endosome-lysosome pathway.
  • proteases in the acidic environment of endosomes cleave the linker, and the activated “free” drug passes through the lysosomal membrane, moves to the cytoplasm, and binds to the drug’s molecular target, thereby arresting the cell cycle of tumor cells and causing apoptosis.
  • a certain 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 may enter surrounding cells and cause so-called by-stander cell-killing.
  • the linker must be stable in the bloodstream, preventing the drug from separating from the antibody, maintaining it in a prodrug state until it reaches the target, and minimizing damage to normal tissues. Additionally, the ADC must maintain the same affinity as the antibody before it is combined with the drug. In other words, the drug bound to the antibody should not interfere with antibody-antigen binding.
  • ADC After binding to the target cell, ADC is internalized into the cell by a process called receptor-mediated endocytosis. At this time, a sufficient concentration of active drug must enter the cell, but the internalization process by the antigen-antibody complex is generally inefficient and the number of antigens on the cell surface is generally limited to ⁇ 1 ⁇ 10 5 receptors/cell, making it a very powerful drug. It must be possible to sufficiently kill tumor cells even at low concentrations of the drug. Therefore, drugs bound to antibodies and used as ADCs are drugs that are 100 to 1,000 times more cytotoxic than commonly used anticancer drugs.
  • determining the antigen to be targeted is the first major step in ADC development.
  • high specificity for the target and long half-life enable long-term systemic circulation. This allows cytotoxic drugs to selectively accumulate only in tumor cells and minimizes exposure to normal tissues, thereby reducing damage, reducing side effects and providing treatment. The effect can be increased.
  • a target antigen that can identify tumor cells must be found, and the following conditions are required. First, the target antigen must be uniformly overexpressed on the surface of tumor cells and have relatively low or no expression on normal cells.
  • a representative example is Human epidermal growth factor receptor 2 (HER2), and it is known to be expressed more than 100 times more in HER2-positive breast cancer than in normal cells. Therefore, before making an antibody, the tumor expression of the target antigen is analyzed through various profiling, and if overexpression of a specific antigen is confirmed, a monoclonal antibody that recognizes this antigen is generated. The second is the binding ability to the antigen. Due to the characteristic of antibodies that internalization occurs through receptors, the stronger the binding force to the epitope of the antigen, the more internalization can occur, which can increase the therapeutic effect. Additionally, there is low immunogenicity.
  • HER2 Human epidermal growth factor receptor 2
  • Antibodies that recognize antigens on cancer cells must be internalized into cells together with the drug. Bispecific antibodies are being developed to increase cell internalization in cancer cells.
  • SN-38 is a potent topoisomerase-I inhibitor with IC 50 values in the nanomolar range in several cell lines. It is the active form of irinotecan, a prodrug used to treat colorectal cancer, and is also active in lung, breast, and brain cancer.
  • Trop-2-SN-38 ADC it is currently being successfully developed in a number of cancer types such as TNBC, bladder cancer, and stomach cancer, but resistance problems due to drug efflux transporter overexpression, epigenetic silencing of Top1, and increased anti-apoptotic protein still remain. there is.
  • DXD a cytotoxic drug that is 10 times more active in cancer cells than SN-38
  • DXD has good solubility, is relatively safe, and has a high killing effect on surrounding cells, making it advantageous in the treatment of heterogeneous tumors.
  • the half-life to reduce off-target effects is short.
  • DXD was bioconjugated to the cysteine residue of the anti-HER2 antibody with a maleimide linker, and the homogeneous DAR value reached 8.
  • DXD is highly stable, with only 2.1% released in plasma over 21 days (Ogitani et al., 2016).
  • Enhertu was approved by the US FDA in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have received HER2-targeted therapy at least twice in the past.
  • ADCs Antibody-drug conjugates
  • camptothecin derivatives as payload, such as Trodelvy and Enhertu
  • Many camptothecin derivatives are quickly exported out of cells by ABCG2, and as a result, cancer cells that overexpress ABCG2 show a problem of seriously reduced therapeutic efficacy compared to cancer cells that do not express ABCG2 or express ABCG2 at a low normal level.
  • Enhertu's DXd had high membrane permeability, and as a result, research results showed that the payload released inside the cell was delivered to cells that do not express HER2 adjacent to the target cell. Due to the nature of Enhertu's payload, this could provide clinical benefit to Herceptin or Kaesyla refractory patients as well, with a potential bystander effect.
  • the present inventors synthesized compound FL118, a camptothecin-based drug with excellent type 1 topoisomerase inhibition ability and dual MoA that decomposes the oncoprotein DDX5 (p68), and conducted various studies on its physicochemical properties. was continuing to be performed.
  • the FL118 compound had limitations in formulation development due to limitations in physicochemical properties.
  • camptothecin derivatives with a structure that exhibits 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.
  • dual MoA dual mechanism of action
  • camptothecin derivatives such as exatecan or Dxd also exert a mechanism of action (MoA) to degrade DDX5 protein ( Figures 4 and 5).
  • the present invention provides a camptothecin-based drug of Formula 1, which has a type 1 topoisomerase inhibitory ability and a dual MoA to decompose the oncoprotein DDX5, mainly only at target sites such as tumor tissue.
  • ADC antibody-drug conjugate
  • the present invention is intended to solve the aggregation problem of ADC (e.g., hRS7-CL2A-SN-38) using SN-38 as a payload and the capacity limitation problem due to liver toxicity and/or the relatively weak SN-38.
  • ADC e.g., hRS7-CL2A-SN-38
  • SN-38 as a payload
  • capacity limitation problem due to liver toxicity and/or the relatively weak SN-38 e.g., hRS7-CL2A-SN-38
  • the camptothecin-based drug of Formula 1 (e.g., exatecan or Dxd) designed to bind to the DDX5 protein is intended to be used as a payload, resulting in the drug of Formula 1
  • the present invention (i) provides a desired hepatic clearance profile and blood circulation profile and/or (ii) penetrates deep into the tumor tissue to exert a bystander effect or the extent of the effect.
  • a camptothecin drug of Formula 1 e.g., exatecan or Dxd
  • the present invention provides that the camptothecin derivative of Formula 1, which has exatecan as its parent nucleus, has a bystander effect and binds to DDX5 protein within cells to induce cell death through DDX5 protein degradation.
  • the camptothecin derivative of Formula 1 which has exatecan as its parent nucleus, has a bystander effect and binds to DDX5 protein within cells to induce cell death through DDX5 protein degradation.
  • the first aspect of the present invention is a camptothecin-based drug of Formula 1, including [camptothecin-based drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof].
  • the camptothecin-based drug of Formula 1 is a payload designed to bind to DDX5 protein and/or E3 ligase,
  • At least one camptothecin-based drug of Formula 1 is linked to the antibody or its antigen-binding site-containing fragment through an acid-sensitive linker,
  • the acid-sensitive linker After being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ⁇ 7) surrounding the cancer, and at least part of the camptothecin-based drug of Formula 1 is released, and the free form of Formula 1 Camptothecin drugs penetrate the cell membrane and move into the cell.
  • the immunoconjugate to which the camptothecin drug of Formula 1 is linked is internalized into cells and the camptothecin drug of Formula 1 is released from lysosomes.
  • An immunoconjugate or pharmaceutical thereof Provides an acceptable salt.
  • X 1 and X 3 are each independently carbon, oxygen, nitrogen, or sulfur, and X 1 and X 3 may be the same or different,
  • X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond
  • Y 1 , Y 2 and Y 3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.
  • 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.
  • it may be administered to treat HER2-positive cancer, breast cancer, lung cancer, and colon cancer.
  • the pharmaceutical composition for preventing or treating cancer can be used as a primary treatment after cancer diagnosis or administered to an individual that (over)expresses DDX5 in cancer tissue.
  • the third aspect of the present invention is a camptothecin system of Formula 1-1 or Formula 1-2.
  • X 1 and X 2 are each independently -H or -halogen
  • Y is -NH-, -NR A -, or nothing (null);
  • Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
  • W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
  • R A to R C are each independently C 1 -C 4 alkyl
  • n is an integer from 5 to 9.
  • a fourth aspect of the present invention provides (a) the drug-linker conjugate of the third aspect or a pharmaceutically acceptable salt thereof; and (b) an antibody or an antigen-binding site-containing fragment thereof, wherein the antibody or an antigen-binding site-containing fragment contains at least one camptothecin-based drug of Formula 1-1 or Formula 1-2 through an acid-sensitive linker of Formula 3.
  • An immunoconjugate characterized by being linked or a pharmaceutically acceptable salt thereof is provided.
  • the fifth aspect of the present invention uses the drug-linker conjugate of the third aspect or a pharmaceutically acceptable salt thereof to form a drug of Formula 1-1 or Formula 1-2 through an acid-sensitive linker of Formula 3 in a carrier.
  • a method for manufacturing a carrier-drug conjugate characterized by linking one or more camptothecin drugs is provided.
  • camptothecin-based drugs of Formula 1-1 or Formula 1-2 are used interchangeably as exatecan or Dxd, respectively.
  • cancer and tumor may be used interchangeably.
  • drug-linker conjugate refers to a material for the production of an immunoconjugate or carrier-drug conjugate, which not only refers to an antibody, an antigen-binding site-containing fragment thereof, or a carrier that is not linked. Depending on the purpose, it can be used as an immunoconjugate or carrier-drug conjugate by combining with any antibody, antigen-binding site-containing fragment thereof, or carrier.
  • camptothecin-based drug of Formula 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)
  • Pharmacodynamics explains the size and pattern of changes (cell viability, clinical effect) (therapeutic action, toxic effect, adverse effect) that occur in cells or the body after a drug binds to a receptor in terms of their relationship with drug concentration. .
  • PK Pharmaco-kinetics
  • the appropriate drug in order to use the drug properly, the appropriate drug must be selected and the appropriate dose-administration must be applied.
  • Dose refers to the amount of drug administered at one time.
  • the dosage of the drug is usually prescribed by a health care provider based on factors such as the patient's age, weight, and health status.
  • dosage refers to the frequency and duration of drug administration.
  • ADME absorption, distribution, metabolism and excretion
  • Absorption of a drug can affect its efficacy and side effects. Drugs that are poorly absorbed may not reach therapeutic concentrations in target tissues, resulting in reduced efficacy. On the other hand, drugs that are well absorbed may increase systemic exposure and cause off-target side effects.
  • Distribution of a drug can also affect efficacy and side effects. Drugs that are poorly distributed in target tissues may be ineffective, and drugs that are widely distributed in non-target tissues may cause toxicity in those tissues. Additionally, drugs with high binding affinity to plasma proteins may have reduced distribution to target tissues, resulting in reduced efficacy.
  • Drug distribution in the body can be influenced by its physicochemical properties, including size, charge, and lipophilicity.
  • the ability of a drug to penetrate tumor tissue may also be affected by factors such as the tumor's microenvironment, including blood flow and cell density.
  • Some anticancer drugs can accumulate in certain tissues and cause toxic effects.
  • ADME ADME properties of anticancer drugs
  • the present inventors based on the structure of the FL118 drug of Chemical Formula 2, which is differentiated from the SN38 drug, developed a dual mechanism of action (dual MoA) in terms of type 1 topoisomerase inhibition and DDX5 degradation, which are the advantages of the FL118 drug. While designing a camptothecin derivative with a structure that exerts a structure (Figure 1), it was discovered that exatecan or Dxd also exerts a mechanism of action (MoA) to degrade the DDX5 protein ( Figures 4 and 5).
  • Exatecan drug like the FL118 drug, not only strongly inhibits the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another main cause of anticancer drug resistance, at low concentrations. Rather, it was discovered that this could block the drug resistance mechanism ( Figures 4 and 5).
  • the present invention is based on these findings.
  • the camptothecin-based drug of Formula 1 used as a payload by connecting to an [acid-sensitive linker] has R 1 and R 2 (Group A) on ring A in General Formula 1 similar to the FL118 compound of Formula 2. Meanwhile, by designing it in the same way as the compound of Formula 1-1 (exadecane) and the compound of Formula 1-2 (dxd) ( Figure 4), it is characterized by exerting an anti-cancer mechanism that decomposes the intracellular oncoprotein DDX5. (Example 1).
  • the Group C site binds to type 1 topoisomerase and the Group A site binds to DNA to stabilize the covalent bond of the topoisomerase-DNA complex, allowing the cut DNA fragments to be reconnected. and/or, in General Formula 1 or Formula 2, the Group A site binds to DDX5 and the Group C site binds to E3 ligase to induce DDX5 degradation (PCT/KR2023/005380, incorporated herein by reference) reference).
  • camptothecin-based drug of Formula 1 it is desirable to design the camptothecin-based drug of Formula 1 to exert an appropriate bystander effect in tumor tissue by controlling the cell membrane permeability as desired through modification of R 3 and/or R 4 in Formula 1. .
  • the selection range of ADC payload that exhibits appropriate anticancer efficacy is designed to bind to the DDX5 protein and E3 ligase as a molecular glue degrader.
  • Another key feature of the present invention is that it can be expanded to a variety of candidates including active camptothecin derivatives of Formula 1.
  • X 1 and X 3 are each independently carbon, oxygen, nitrogen, or sulfur, and X 1 and X 3 may be the same or different,
  • X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond
  • Y 1 , Y 2 and Y 3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.
  • the camptothecin-based drug of Formula 1 which is designed to bind to DDX5 protein and/or E3 ligase according to the present invention, can kill target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).
  • MoA molecular glue degrader mechanism
  • Target cells may be cancer cells or senescent cells. Senescent cells also include cells that do not perform organ-specific functions.
  • the camptothecin-based drug of Formula 1 designed to bind to DDX5 protein and/or E3 ligase according to the present invention has the ability to inhibit type 1 topoisomerase and has multiple actions to degrade the oncoprotein DDX5. It may have a mechanism (MoA).
  • camptothecin derivatives represented by Formula 1, such as Formulas 1-1 and 1-2 have a pentacyclic structure having a lactone in the E-ring essential for cytotoxicity, like camptothecin shown in Figure 1,
  • the compound represented by Formula 1-1 is capable of rotating the molecular bond with respect to the carbon-carbon single bond in the hexagonal ring extended from the A- and B-rings, so that -NH 2 with a large degree of freedom is water ( When exposed to H 2 O), water dispersibility can be increased by taking on a (+) charge or by hydrogen bonding with water.
  • the compound represented by Formula 1-2 is a Lactic acid of -NH 2 with a large degree of freedom as the molecular bond can be rotated about the carbon-carbon single bond in the hexagonal ring extending from the A- and B-rings.
  • the functional group in the form of CH 2 (OH)CONH- is exposed to water (H 2 O) and rotates like a propeller, forming a hydrogen bond with water to increase water dispersibility.
  • the DXd payload used in Enhertu was originally made from the compound Exatecan, which is almost unaffected by ABCG2, but due to the presence of the glycolic acid (alpha-hydroxy acetic acid) functional group used to convert Exatecan to DXd, it is converted to DXd by ABCG2. was strongly influenced.
  • the glycolic acid functional group plays a very important role in Enhertu's excellent safety/efficacy profile, and if it is removed, it will cause difficulties in ADC manufacturing and at the same time deteriorate the performance of ADC in animal models and clinical trials (resulting in safety issues or efficacy). decrease) brings problems.
  • FaDu a Her2-low/mid cancer cell that does not express ABCG2, and A549, a cancer cell that overexpresses ABCG2, were treated with various camptothecin-based drugs at various concentrations to increase intracellular DDX5.
  • the present invention relates to a compound comprising the camptothecin-based skeleton represented by Formula 1 (e.g., exatecan or Dxd) as a parent nucleus by combining it with any antibody, fragment containing an antigen-binding site, or carrier to form an immunoconjugate or carrier.
  • Formula 1 e.g., exatecan or Dxd
  • the present invention is characterized by providing a carrier-drug conjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker].
  • an immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof];
  • the carrier-drug conjugate containing [camptothecin-based drug of Formula 1]-[acid-sensitive linker] may have an acid-sensitive linker in an acidic environment (pH ⁇ 7) surrounding the cancer.
  • camptothecin-based drug of Formula 1 Since at least some of the camptothecin-based drug of Formula 1 is released by decomposition, in order to exert an appropriate bystander effect while penetrating deep into the tumor tissue or to control the degree of the effect, the camptothecin-based drug of Formula 1
  • the relative hydrophilic/hydrophobic nature has a significant impact on the solubility, absorption, distribution, metabolism, and excretion (ADME) of a drug. In particular, it is important how easily the drug crosses the cell membrane and its interaction with the drug target, the DDX5 protein and/or E3 ligase.
  • the hydrophobicity of various camptothecin-based drugs can be expressed as a partition coefficient ( p ).
  • Hydrophobicity has a large p value, and hydrophilicity (polar) has a small p value.
  • the log P value is used as a measure to evaluate hydrophobicity.
  • Clog P means calculating the log P value of a given compound through appropriate software. The smaller the cLogP value, the higher the polarity.
  • polar surface area (PSA) or topological polar surface area (TPSA) of a molecule is defined as the sum of the surfaces for all polar atoms or molecules, mainly oxygen and nitrogen, including attached hydrogen atoms.
  • tPSA is a commonly used medicinal chemistry measurement to optimize the cell-penetrating ability of drugs. Molecules with a polar surface area greater than 140 ⁇ 2 tend not to penetrate cell membranes. A tPSA of less than 90 ⁇ 2 is generally required for the molecule to cross the blood-brain barrier and act on receptors in the central nervous system.
  • the camptothecin-based drug of Formula 1 used as a payload in the present invention is a hydrophobic small molecule that can penetrate the cell membrane even if R 3 and R 4 of Formula 1 are modified, so when delivered to tumor tissue, it penetrates deep into the tumor tissue and produces high It is a molecular glue degrader that can be accumulated in high concentration and binds to DDX5 inside the cell after the camptothecin-based drug of Formula 1 or its prodrug, ADC, penetrates the cell membrane. During cell death, it is released into the extracellular fluid and can subsequently penetrate the cell membrane of surrounding cells and move into the cell, thereby exerting an apoptotic mechanism.
  • the camptothecin-based drug of Formula 1 released from the immunoconjugate or carrier-drug conjugate of the present invention, converts solid cancers with high cell density into scattered cancers with low cell density and/or activates cold tumors with low immune activity. This can transform it into a highly hot tumor. In addition, it has a high killing effect on surrounding cells, which is advantageous in the treatment of heterogeneous tumors.
  • Drug efficacy means that it remains in the body without being decomposed for the expected period of time to exert an effect on the target indication.
  • the in vivo efficacy of anticancer drugs can be affected by absorption, distribution, metabolism, and excretion (ADME) characteristics.
  • ADME absorption, distribution, metabolism, and excretion
  • a drug's ADME profile can affect its ability to reach and act on cancer cells.
  • the ADME characteristics of anticancer drugs can have a significant impact on in vivo efficacy.
  • the ability of a drug to penetrate tumor tissue may also be affected by factors such as the tumor's microenvironment, including blood flow and cell density.
  • ADCs Antibody-drug conjugates
  • Abs antibodies
  • Antibodies are large proteins produced naturally by the immune system, usually in response to foreign substances (antigens). Due to their size and complex structure, they have a long circulating half-life (several weeks to months) and may protect them from degradation and elimination. Antibodies are distributed throughout the body, including tissues, and can interact with target antigens with high specificity and affinity. Antibodies are primarily eliminated by catabolism in the reticuloendothelial system (RES), which includes the liver and spleen, as well as the kidneys and other organs.
  • RES reticuloendothelial system
  • ADCs consist of an antibody (usually a monoclonal antibody) conjugated to a cytotoxic drug molecule.
  • the antibody component provides specificity and targeting to the tumor or diseased tissue, while the drug component provides cytotoxic activity to kill target cells.
  • ADCs have shorter half-lives than antibodies, typically ranging from a few days to a week. This is because ADC is internalized into target cells, resulting in lysosomal degradation of ADC and release of the drug payload (Figure 3).
  • ADC is primarily eliminated by RES, the drug payload can also undergo metabolism and excretion via the liver and kidneys.
  • Antibodies are usually administered subcutaneously or intravenously and are absorbed into the bloodstream. They can be distributed throughout the body, including tissues, but are generally restricted to the extracellular space due to their large size. ADCs are also administered by injection and absorbed into the bloodstream, but the drug payload released from the ADC penetrates into cells and tissues after targeting and, in some cases, receptor-mediated endocytosis to tumors or diseased tissues due to the antibody component. can do. Furthermore, the metabolism and excretion of ADC varies depending on the specific structure and the specific drug, linker, or antibody used.
  • the acid-sensitive linker is stable in the neutral environment of blood, pH 7.3 to 7.5, but is internalized around tumor cells (pH 6.5 to 7.2) or within cells, such as endosomes (pH 5.0 to 6.5) and lysosomes. It refers to a linker that is hydrolyzed in a slightly acidic environment (pH 4.5 ⁇ 5.0) to release the drug. Therefore, the acid-sensitive linker in the present invention has a hydrophilic molecular structure to create a hydrolytic environment.
  • the acid-sensitive linker may comprise, for example, a polyethylene glycol (PEG) spacer.
  • Trastuzumab-CL2A-exadecane (Preparation Example 3), an ADC that connects a camptothecin-based drug of Formula 1-1 or Formula 1-2 to Trastuzumab, a type of HER2 antibody, through a CL2A linker, a type of acid-sensitive linker of Formula 3;
  • Trastuzumab-CL2A-dxd (Preparation Example 4) (DAR 7-8) was easily synthesized without aggregation problems.
  • 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). indicated.
  • the present invention provides a variety of camptothecin-based drugs of Formula 1 that bind to DDX5 protein and E3 ligase as molecular glue degraders as various payload candidates, thereby enabling selection of one or more appropriate drugs and their dosage-administration method.
  • the desired efficacy e.g., anticancer, combination therapy
  • side effects aggregation problems of camptothecin-based drugs
  • camptothecin-based drug of Formula 1 it may be important to exceed a specific drug concentration (high) within tumor tissue depending on the drug modality (small molecule, ADC), and in some cases, the specific drug concentration (low) within tumor tissue. ) or longer may be important.
  • the main routes of drug elimination are (1) hepatic metabolism, (2) biliary excretion, and (3) urinary excretion.
  • Carrier-drug conjugates such as antibody-drug conjugates (ADCs)
  • ADCs antibody-drug conjugates
  • drug nanoformulations such as artificial nanocarriers, liposomes, and polymer nanoparticles
  • limitations such as being phagocytosed by the reticuloendothelial system, one of the body's immune systems, and quickly eliminated from the blood circulation before reaching the disease site.
  • the reticuloendothelial system is also called the macrophage system and the mononuclear phagocyte system (MPS). These are cells that absorb specific substances from various parts of the human body. These cells form part of the body's defense mechanism.
  • Reticuloendothelial cells are created from progenitor cells in the bone marrow. Progenitor cells develop into monocytes, phagocytes that are released into the bloodstream. Some monocytes remain in the circulation, but most enter body tissues and become much larger phagocytes called macrophages. The majority of macrophages are immobile cells that remain within tissues, filtering out and destroying foreign substances. However, some break off and wander around in the circulatory system or the spaces between cells.
  • Macrophages within tissues have different shapes and names depending on where they are located. Reticular cells are found in lymph nodes, spleen, and bone marrow, while histiocytes are found in abundance in subcutaneous tissue. Microglia appear in nervous tissue, alveolar macrophages appear in the alveoli of the lung, and Kupffer cells appear in the liver. Through phagocytosis, macrophages form the first line of defense against harmful particles entering the body.
  • Nanoparticles aggregated in the body vary depending on the size of the organ they are ingested in and their distribution within the human body. Nanoparticles larger than 50 nm are phagocytosed by the Kupffer cells of the liver, an organ with a developed reticuloendothelial system, and rapidly accumulate in the liver.
  • camptothecin drug linked to the acid-sensitive linker in the liver is converted to free hydrophobic camptothecin drug. It is released as a tothecin-based drug and can cause liver toxicity problems by exerting cytotoxicity after penetrating the cell membrane of normal cells.
  • the present invention is to prevent a carrier-drug conjugate from being aggregated by a hydrophobic camptothecin-based drug linked through an acid-sensitive linker and accumulated in the liver by macrophage phagocytosis, by (1) a camptothecin-based payload linked to an acid-sensitive linker; , instead of SN-38, where aggregation is induced by ⁇ - ⁇ stacking of the aromatic rings formed by the A- and B-rings, successive carbons of hexagonal or heptagonal rings extend from the A- and B-rings.
  • A- and B-rings e.g., exatecan or Dxd
  • the present invention is designed so that the carrier-drug conjugate to which the camptothecin drug of Formula 1 is linked is not aggregated through the camptothecin drug and accumulated in the liver by macrophage phagocytosis, thereby producing an ADC (ADC) with SN-38 as a payload.
  • ADC ADC
  • it can solve the aggregation problem of hRS7-CL2A-SN-38) and the capacity limitation problem due to liver toxicity caused by this.
  • the present invention improves the water dispersibility of the camptothecin drug moiety of Formula 1 oriented through an acid-sensitive linker in the carrier-drug conjugate, thereby reducing phagocytosis by the reticuloendothelial system in the liver and thereby reducing the residence time in the bloodstream.
  • the drug concentration in the blood it can be maintained at a high concentration for a long time.
  • the hydrophobicity of the linker and the drug increases, problems such as increased cohesion of the ADC and a corresponding decrease in the therapeutic coefficient of the drug occur.
  • the desired hepatic clearance profile and blood circulation profile can be provided to the carrier-drug conjugate through hydrophilicity control through various combinations of the camptothecin-based drug of Formula 1 and the acid-sensitive linker of Formula 3.
  • FL118 of Formula 2 can act as a molecular glue degrader that degrades DDX5 by directly attaching DDX5 to the ubiquitination regulator.
  • DDX5 which acts as a molecular glue, directly binds to the tumor protein DDX5, a multifunctional master regulator, through the proteasome degradation pathway without reducing DDX5 mRNA, and has the function of dephosphorylating and decomposing it.
  • DDX5 silencing indicates that DDX5 is a master regulator that regulates the expression of several oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras.
  • FL118 indirectly controls DDX5 downstream targets to promote cancer initiation, development, metastasis, and treatment resistance with high efficacy, as demonstrated in studies using human colorectal cancer/pancreatic adenocarcinoma cells and tumor models. , recurrence and treatment resistance).
  • DDX5 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 showed that FL118 showed high efficacy in eliminating human PDAC and CRC tumors with high DDX5 expression, whereas FL118 was less effective in PDAC and CRC tumors with low DDX5 expression.
  • DDX5 protein is a direct target of the FL118 drug and may serve as a biomarker to predict PDAC and CRC tumor sensitivity to FL118.
  • the FL118 drug has a Top1 inhibitory effect equal to or higher than that of SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low IC 50 value and cytotoxicity.
  • the evaluation results of 140 cell lines originating from various carcinomas showed very strong anticancer efficacy with an IC 50 of ⁇ 100nM against the majority of cancer cells.
  • the FL118 drug has secured excellent safety through GLP-toxicity tests in rats and beagle dogs, and has shown superior efficacy compared to SN-38 in various cancer cell line Xenograft models.
  • camptothecin-based anticancer drugs show excellent anticancer responses initially when used in patients, but strong resistance to these drugs appears through Epigenetic Silencing of the Top1 gene and Top2 Dependence of cancer cells.
  • the FL118 drug showed strong efficacy even in the Xenograft model of cancer cell lines in which Top1 is not expressed through Epigenetic Silencing or Knock-out.
  • the FL118 drug directly targets type 1 topoisomerase, a well-established anticancer target, and targets the Bcl family, including Survivin, a resistance protein involved in resistance mechanisms. It is a triple target anticancer drug that simultaneously inhibits and inhibits the action of the efflux pump.
  • camptothecin-type anticancer drugs such as SN-38 show resistance in the way that the drug is released out of the cell due to overexpression of the ABCG2 Transporter.
  • camptothecin-type anticancer drugs such as SN-38 show resistance in the way that the drug is released out of the cell due to overexpression of the ABCG2 Transporter.
  • the FL118 drug can block the development of resistance by strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another main cause of anticancer drug resistance, at low concentrations.
  • the FL118 drug is not expelled out of the cell by ABCG2, an efflux pump, and can block resistance caused by various anti-apoptotic proteins.
  • the FL118 drug can overcome the various resistance mechanisms of SN-38/Exatecan.
  • the FL118 drug When administered in vivo in the same amount as SN-38, the FL118 drug showed stronger tumor regression efficacy than SN-38 in colon cancer, head and neck cancer, and pancreatic cancer.
  • the FL118 drug possessed strong anticancer efficacy even when FL118 was administered after inducing SN-38 resistance in tumor xenograft.
  • the FL118 drug has an optimal PK/safety profile for targeted drug delivery (e.g., Carrier-drug Conjugate) application.
  • targeted drug delivery e.g., Carrier-drug Conjugate
  • the FL118 drug is administered systemically alone, it is rapidly metabolized and excreted from the blood and only shows a low concentration, but it accumulates quickly in cancer tissue immediately after administration and maintains a high concentration for a long time. For example, when applying ADC, maximum selectivity between tumor tissue and normal tissue is ensured.
  • camptothecin-based drug of Formula 1 can be designed in various ways according to the present invention and used as a payload so that it can exhibit various advantages as an anticancer drug, as exemplified by the drug FL118.
  • Ubiquitin-proteasome system is an important pathway for the degradation of intracellular proteins that regulates a wide range of cellular processes.
  • Ubiquitin is a small protein that is covalently attached to lysine residues of substrate proteins by a series of enzymatic reactions involving ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process is called ubiquitination and is an important mechanism that regulates protein degradation, signal transduction, and trafficking.
  • Ubiquitin ligase is responsible for substrate specificity in the ubiquitination pathway.
  • camptothecin-based drug of Formula 1 is designed to bind to DDX5 protein and E3 ligase.
  • RNA helicases The DEAD-box family of RNA helicases is involved in several metabolic pathways, ranging from transcription and translation to cell proliferation, innate immunity, and stress response. Given their diverse roles, their deregulation or mutations have been linked to a variety of pathological conditions, including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation, indicating a tumor suppressive role, whereas in other situations, overexpression of the same enzyme favors cancer progression, acting as a typical oncogene.
  • DDX5 also known as p68
  • DDX5 is a multifunctional master regulator that acts through the following mechanisms: (1) direct interaction with various transcription factors (e.g. c-Myc) at oncogenic gene promoters; (2) a biological process that co-activates the transcription of many oncogenes through interaction, (2) a biological process that regulates miRNA and pre-RNA splicing (e.g. U1, U2, U3, ... snRNP) process, and (3) ribosome biogenesis (e.g., 32S rRNA, pre-ribosome).
  • transcription factors e.g. c-Myc
  • miRNA and pre-RNA splicing e.g. U1, U2, U3, ... snRNP
  • ribosome biogenesis e.g., 32S rRNA, pre-ribosome
  • camptothecin-based drug of Formula 1 binds to DDX5 protein without reducing DDX5 mRNA and functionally degrades it through dephosphorylation and proteasome degradation pathways, which means that the camptothecin-based drug of Formula 1 binds to DDX5 protein and ubiquitin-related proteins. This suggests that it can attach to both ubiquitin-involved protein stability/degradation regulators, acting as a “molecular adhesion degrader.”
  • DDX5 downstream protein targets are all known to be involved in cancer initiation, development, metastasis, recurrence, and treatment resistance. Therefore, if the DDX5 downstream target is indirectly blocked through decomposition of DDX5 protein by the camptothecin drug of Formula 1 according to the present invention, the camptothecin drug of Formula 1 may exhibit high antitumor efficacy.
  • DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and transcription cofactor. Therefore, when cancer occurs due to deregulation of the transcription factor due to the physiological state of DDX5, the cancer disease can be treated by selectively degrading DDX5 (p68), a transcription cofactor. Likewise, cancer disease can be prevented by selectively degrading DDX5 (p68), a transcription cofactor.
  • the DDX5 protein is the drug target of the camptothecin-based drug of Formula 1 according to the present invention, it can avoid drug resistance, resistance to targeted therapy, and/or resistance during treatment.
  • the transcriptional induction of anti-apoptotic genes can be turned off by the camptothecin-based drug of Formula 1.
  • the DDX5 protein, a transcription cofactor is degraded by the camptothecin-based drug of Formula 1, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy or radiotherapy.
  • One aspect of the present invention relates to an immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof],
  • the camptothecin-based drug of Formula 1 is a payload designed to bind to DDX5 protein and/or E3 ligase
  • camptothecin-based drugs of Formula 1 are linked to the antibody or antigen-binding site-containing fragment thereof through an acid-sensitive linker,
  • the acid-sensitive linker After being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ⁇ 7) surrounding the cancer, and at least part of the camptothecin-based drug of Formula 1 is liberated.
  • the camptothecin drug of formula 1 penetrates the cell membrane and moves into the cell.
  • the immunoconjugate to which the camptothecin drug of Formula 1 is linked is characterized in that it is internalized into cells and the camptothecin drug of Formula 1 is liberated from lysosomes.
  • the immunoconjugate of the present invention can efficiently release the camptothecin-based drug of Formula 1 not only inside cancer cells but also around cancer tissues after antigen binding, thereby overcoming the resistance mechanism related to ADC processing ( Figure 3 ).
  • the immunoconjugate of the present invention is targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, and then the acid-sensitive linker is decomposed in an acidic environment (pH ⁇ 7) surrounding the cancer, thereby liberating at least some of the camptothecin-based drug of Formula 1.
  • the free camptothecin-based drug of Formula 1 is a hydrophobic small molecule, but compared to SN-38, it has improved water dispersibility to prevent aggregation in body fluids such as blood and interstitial fluid, so it can penetrate deep into the tumor tissue and penetrate the cell membrane to move into cells. .
  • the immunoconjugate of the present invention can rapidly release the drug in the tumor microenvironment surrounding the cancer by using an acid-sensitive linker that decomposes in the acidic environment (pH ⁇ 7) surrounding the cancer, and the free camptothecin-based drug of Formula 1 Unlike antibodies, it has a low molecular weight and has a high ability to penetrate cancer tissue, so it can solve the problem of existing ADCs, which have problems with antibodies that do not penetrate deep into cancer tissue.
  • the cells into which the free camptothecin-based drug of Formula 1 moves intracellularly may be targeted cancer cells and/or their surrounding cells.
  • the camptothecin-based drug of Formula 1 is a hydrophobic small molecule that can penetrate cell membranes
  • the camptothecin-based drug of Formula 1 released from the immunoconjugate of the present invention through decomposition of the extracellular linker around the cancer rapidly accumulates in tumor tissue. It can maintain a high concentration for a long time, penetrates the cell membrane, exerts cytotoxicity inside the cell, kills the cell, and is then released and can subsequently penetrate the cell membrane and move into the cell to act on surrounding cells.
  • camptothecin drug-acid sensitive linker of Chemical Formula 1 is utilized in terms of anticancer efficacy and toxicity due to aggregation through use of this combination.
  • camptothecin-based drug of Formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the drug is decomposed in an acidic environment (pH ⁇ 7) and the free camptothecin-based drug of Formula 1 is released when the acid-sensitive linker is decomposed.
  • the linkage of [acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof] is performed by linking the thiol group contained in the antibody or antigen-binding fragment thereof to the maleimide group of the acid-sensitive linker.
  • it may be bound to a maleic hydrazide group through the “click” reaction of Scheme 1.
  • the present invention provides a drug-linker conjugate in which the camptothecin-based drug of Formula 1 is linked to various acid-sensitive linkers, using this to transport the camptothecin-based drug of Formula 1 through various acid-sensitive linkers to various carriers.
  • a carrier-drug conjugate linked to (Carrier-Drug Conjugate) is provided.
  • the present invention relates to one or more camptothecin drugs of Formula 1 through an acid-sensitive linker of Formula 3 to a carrier using the drug-linker conjugate of the present invention or a pharmaceutically acceptable salt thereof.
  • a method for manufacturing a carrier-drug conjugate is also provided.
  • Inhibitors of type 1 topoisomerase I are anticancer mechanisms with clinically proven efficacy/safety, and are excellent anticancer agents for various intractable solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer in clinical trials. Efficacy has been verified. Camptothecin-based drugs such as Exatecan and SN-38 have been developed as payloads for ADCs.
  • Exatecan of Formula 1-1 is a camptothecin derivative and is an anti-tumor small molecule compound that inhibits type 1 topoisomerase.
  • Exatecan is a substance that has been confirmed to have cellular cytotoxicity that is 5 to 10 times more powerful than SN-38.
  • Exatecan is different from irinotecan and does not require activation by enzymes.
  • it has stronger type 1 topoisomerase inhibitory activity than SN-38, the main medicinal product of irinotecan, and topotecan, which is used in clinical trials, and has stronger cytotoxic activity against various cancer cells in vitro. there is.
  • it was effective against cancer cells that were resistant to SN-38 and other drugs due to the expression of P-glycoprotein.
  • it showed a strong anti-tumor effect in a human tumor subcutaneous transplant model in mice, and clinical trials were conducted.
  • Dxd (Exatecan derivative for ADC) of Formula 1-2 is a potent DNA topoisomerase I inhibitor with an IC 50 of 0.31 ⁇ M, used as a conjugate drug for HER2 targeting ADC (DS-8201a).
  • the Stable Linker system In the case of ADCs using super toxins such as MMAE, Calicheamicin, and PBD, the Stable Linker system is used, which aims to minimize the separation of drugs from the blood before reaching cancer tissues (a feature of the 2nd generation ADC). did.
  • the principle behind how most second-generation ADCs work in cancer cells is that, in the first step, the antibody portion that makes up the ADC binds to the antigen overexpressed in the cancer cell, and in the second step, the ADC bound to the antigen on the surface of the cancer cell through an antigen-antibody reaction is endo-activated. It is transported inside the cancer cell through lysosomes. In the third step, the antigen-antibody portion is decomposed in the lysosome, the enzyme (cathepsin B) and the drug are released, and in the final step, the cancer cell is killed by the drug released inside the cancer cell. .
  • ADC In order to develop a new ADC that can be selectively used for various solid cancers, it is essential to utilize a linker system that exceeds the drug delivery efficiency of existing ADC.
  • ADC for slow internalizing antigens such as CEACAM-5 and/or cancer-specific antigens such as NY-ESO-1/HLA Complex. should be developed, but it is difficult to deliver a sufficient amount of drug with the limited drug delivery efficiency of the existing Val-Cit or MAC-glucuronide Linker system.
  • the antibody-drug bond maintains stability in the blood and/or the surrounding environment of normal tissues, but requires the characteristic of rapidly releasing the drug in the surrounding environment of cancer cells, such as the tumor microenvironment. do.
  • the drug can be released quickly even in the tumor microenvironment surrounding the cancer, it can be advantageously used to target various antigens (typically CEACAM-5, various Cancer Specific antigen-HLA Complexes, etc.) that have limitations in ADC uptake speed. there is.
  • various antigens typically CEACAM-5, various Cancer Specific antigen-HLA Complexes, etc.
  • the camptothecin-based drug of Formula 1 is a drug with improved water dispersibility to prevent aggregation in body fluids such as blood and interstitial fluid compared to SN-38, so it is a hydrophobic small molecule that can penetrate deep into tumor tissue and move into cells through cell membranes.
  • the present invention selects an acid-sensitive linker that decomposes in an acidic environment (pH ⁇ 7) around the cancer as a linker with a release profile that delivers the drug quickly and efficiently once it reaches the cancer tissue.
  • the antibody to which the camptothecin-based drug of Formula 1 is linked through an acid-sensitive linker binds to the antigen overexpressed on the surface of cancer cells in the same way as the first step in which the second-generation ADC operates in cancer cells, but some of the antibodies bind to the antigen overexpressed on the surface of the second-generation ADC.
  • a significant portion of the drug can be released due to the low pH around the cancer cells.
  • the drug released from the cancer tissue moves into the cancer cells by diffusion, bypassing endosomes and lysosomes, and acts directly on the cancer cells without enzymatic (cathepsin B) reaction, inducing apoptosis.
  • the antigen selectivity of cancer cells is the same, but the immunoconjugate of the present invention can maximize drug release and delivery efficiency into cancer cells by using a pH-sensitive linker. This is the main feature of .
  • the linker is stable in the bloodstream, preventing the drug from separating from the antibody, maintaining it in a prodrug state until it reaches the target, and minimizing damage to normal tissues.
  • the present invention creates a hydrolysis environment. By using an acid-sensitive linker with a hydrophilic molecular structure, the problem of ADC aggregation when combined with a hydrophobic drug can be alleviated.
  • the [camptothecin-based drug of Formula 1]-[acid-sensitive linker] of the present invention is a camptothecin-based drug of Formula 1 so that the free camptothecin-based drug of Formula 1 is released when the acid-sensitive linker is decomposed.
  • the acid-sensitive linker is preferably connected by a carbonate or ester bond.
  • carbamate bonds provide superior drug linker stability compared to ester and carbonate bonds with respect to hydrolysis.
  • carbamate bonds provide superior drug linker stability compared to ester and carbonate bonds with respect to hydrolysis.
  • carbamate bonds in order to design the camptothecin-based drug of Formula 1 to be separable from the drug linker both outside and inside the cells surrounding cancer cells in the acidic environment (pH) surrounding cancer cells, instead of carbamate bonds, It is characterized by the use of unstable ester or carbonate bonds.
  • the pH of blood is kept constant at 7.3 to 7.4. Therefore, the camptothecin-based drug of Formula 1 is not cleaved from the acid-sensitive linker in the blood, and even if cleaved, the release rate of the camptothecin-based drug of Formula 1 from the ADC at the neutral pH of serum is much reduced than in tumor tissue in an acidic environment. .
  • the present invention connects an acid-sensitive linker to the alpha hydroxyl group located at carbon 20 of the E-ring in the camptothecin-based drug of Formula 1. It is a characteristic.
  • the tetrapeptide linker showed a limit to the extent to which ADC aggregation could occur when combined with hydrophobic drugs.
  • CL2A the linker used in Trodelvy, an existing FDA-approved ADC, has (i) storage stability after manufacturing, (ii) stability in the blood upon administration (little exposure to free payload during plasma), and (iii) stability of payload in cancer tissue. It is a linker that satisfies all characteristics such as rapid release.
  • the acid-sensitive linker used in the present invention can utilize the CL2A linker, and can be designed as shown in Formula 3 below to selectively and efficiently deliver the camptothecin-based drug of Formula 1 to cancer tissue. That is, in the present invention, the acid-sensitive linker may be derived from a compound of formula 3 below:
  • X 1 and X 2 are each independently -H or -halogen
  • Y is -NH-, -NR A -, or nothing (null);
  • Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
  • W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
  • R A to R C are each independently C 1 -C 4 alkyl
  • n is an integer from 5 to 9.
  • X 1 and X 2 are each independently -H or -halogen
  • Y is -NR A -, or nothing (null);
  • Z is -C 1 -C 4 alkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, or -(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
  • W is -R B - or -R B -MR C -;
  • R A to R C are each independently C 1 -C 4 alkyl
  • n may be an integer from 5 to 9.
  • n may be an integer of 5 to 9
  • n may be an integer of 6 to 8
  • n may be 7, but is not limited thereto. Even in cases outside the above range, if there is no significant difference in effect due to change in linker length, all are naturally included within the equivalent scope of the present invention.
  • the acid-sensitive linker of Formula 3 is a customized linker that can be optimized according to the characteristics of the targeting target, payload, and carrier.
  • the camptothecin drug of Formula 1 has various linkers and sites that are easy to attach to for the production of carrier-drug conjugates.
  • the alcohol moiety of the camptothecin-based drug of Formula 1 can be used as an attachment site to the linker.
  • [camptothecin-based drug of Formula 1]-[acid-sensitive linker] may be the alcohol moiety of the camptothecin-based drug of Formula 1 and the alcohol moiety of the acid-sensitive linker of Formula 3.
  • the [camptothecin drug of Formula 1]-[acid sensitive linker] conjugate may be a compound represented by Formula 4 or 5 below.
  • n is each independently an integer from 5 to 9.
  • the acid-sensitive linker of the present invention develops and applies a multivalent linker system that can overcome the problem of low DAR, which is considered a disadvantage of site-specific conjugation, to deliver 2-3 payloads to one attachment site.
  • a method it is possible to secure a method to manufacture a high DAR ADC of DAR 4 - 12 even during site-specific conjugation.
  • the Bridgeable Linker system as exemplified in Formulas 4 and 5 can be used as a linker.
  • This Bridgeable Linker system can increase the efficiency of CMC and simplify the process so that DAR 4 ADC can be easily manufactured while maintaining the rapid release characteristics of the payload from cancer tissue, which is the greatest advantage of the CL2A linker system.
  • a site-specific antibody-drug complex of DAR 4 can be manufactured without performing separate antibody engineering. When the disulfide (-S-S-) present in the antibody is reduced, two thiols (-SH) are formed, and the thiols thus produced form a 2:1 conjugate with a new bridgeable linker - the camptothecin drug of Formula 1.
  • the antibody-drug complex of DAR 4 can be easily manufactured as a single product by reacting all four disulfides inside a general antibody.
  • UPS Ubiquitin proteasome system
  • ubiquitin acts as a marker to indicate which proteins need to be decomposed, and proteasome
  • the moth recognizes the ubiquitin tag and acts as a shredder that destroys the corresponding protein.
  • E3 ligase is an enzyme that initiates the protein degradation system in the body and is responsible for substrate specificity in the ubiquitination pathway.
  • Molecular glue degrader or molecular glue is a compound that functions as an adhesive to attach a target protein to a specific enzyme (E3 ligase) in our body.
  • E3 ligase a specific enzyme
  • One of the advantages of molecular glue is that it acts as a catalyst, decomposing a target protein and then separating again to degrade another target protein.
  • molecular adhesives for tumor proteins overcome drug resistance, which is a problem with targeted anticancer drugs, and have high therapeutic effects even at low administration doses.
  • the camptothecin-based drug of Formula 1 used as a payload according to the present invention is a molecular glue degrader that binds to DDX5 protein and E3 ligase, that is, tumor protein DDX5 or its phosphorylated DDX5 protein (p-DDX5). ) It is a molecular adhesive that activates decomposition ( Figures 1, 4 to 8).
  • a molecular glue degrader is not only a warhead that binds to a target protein, but can also act as an E3 ligase ligand (binder).
  • the camptothecin-based drug of Formula 1 is a molecular adhesive that activates the decomposition of the tumor protein DDX5, and can be used as a ligand targeting the DDX5 protein or a ligand that binds to the DDX5 protein.
  • molecular glue degraders are 'proximity-driven' and their ability to induce degradation depends on the formation of a temporary 'target protein-molecular glue-E3 ligase' triple complex. It is ‘event-driven’. After degradation occurs, the separated molecular glue forms an additional triple complex with the target protein, allowing multiple degradation processes to proceed until the target protein disappears.
  • molecular glue a low-molecular-weight compound that acts as an adhesive that binds tumor proteins and E3 ligase together, is a suitable modality for cancer treatment because it is resistant to resistance.
  • the camptothecin-based drug of Formula 1 is a drug that can accurately bind to the DDX5 tumor protein, and solves the resistance problem of targeted treatments through a molecular glue approach that selectively decomposes the protein. It can be avoided.
  • the camptothecin-based drug of Formula 1 according to the present invention has a binding strength to the target protein, DDX5 tumor protein, depending on its physicochemical properties, and is preferably an irreversible drug that binds so strongly that it cannot return to its previous state. .
  • the camptothecin-based drug of Formula 1 has a high affinity for DDX5, so it does not fall off easily and decomposes DDX5 through a molecular glue function, thereby irreversibly inhibiting signaling in cancer cells related to DDX5. Not only does it inhibit it, but it also controls the cell membrane permeability of the drug according to its physicochemical properties as desired, thereby exerting a bystander effect or controlling the degree of the effect, resulting in a high killing effect on surrounding cells, which is advantageous in the treatment of heterogeneous tumors. In addition, it can suppress long-term cancer progression and increase the treatment response rate by reducing the risk of resistance development.
  • the camptothecin-based drug of Formula 1 according to the present invention can bind to DDX5, which acts as an oncoprotein within cells, and induce cell death through DDX5 protein degradation (FIG. 11).
  • the camptothecin-based drug of Formula 1 decomposes DDX5 protein through its mechanism of action as a molecular glue degrader that binds to DDX5, which is an electron cofactor and an oncoprotein. Can induce cell death.
  • DDX5 protein degradation can downregulate the transcription of anti-apoptotic genes ( Figures 4 to 8). Therefore, unlike other targeted therapies, drug resistance caused by abnormally expressed cell proliferation and/or cell death-related transcription factors is less likely to occur, and/or uncontrolled activation of transcription factors during chemotherapy and/or radiotherapy. Acquired drug resistance induced through can be suppressed.
  • the anti-cancer mechanism of the camptothecin-based drug of Formula 1 according to the present invention can bypass the molecular mechanism of cancer treatment resistance, and thus can be free from the problem of drug resistance. Since the treatment effect is lower when resistance develops, the camptothecin-based drug of Formula 1 according to the present invention may be preferred as a standard treatment or first-line treatment after cancer diagnosis.
  • the camptothecin-based drug of Formula 1 used as a payload according to the present invention will be a targeted anticancer agent that acts on the DDX5 protein, which plays an important role in the growth, survival (cell survivor), proliferation, metastasis, and/or metabolism of cancer cells. You can.
  • immunoconjugate refers to a complex in which a cytotoxic drug-linker conjugate is linked to an antibody or antigen-binding fragment thereof.
  • ADC antibody-drug conjugate
  • the antibody or antigen-binding site-containing fragment thereof binds to the target antigen and then releases the drug, allowing the drug to act on target cells and/or surrounding cells, thereby producing the target drug. Excellent efficacy and reduced side effects can be expected.
  • Factors that significantly affect the effectiveness of immunoconjugates are, in particular, (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. etc. Because many factors have a complex effect on the effect, it is very difficult to predict the effect of an immunoconjugate that is a combination of these factors based only on what is known about each factor.
  • Immunoconjugates designed to release drugs after internalization have the problem of not being able to deliver a sufficient concentration of the active drug into the cell if the internalization process is inefficient, and even if a hydrophobic drug is used as a cytotoxic drug, the -It has the disadvantage that it is difficult to expect a stander cell-killing phenomenon.
  • Formula 1 is a hydrophobic drug that can penetrate the cell membrane. It is characterized by the use of camptothecin-based drugs ( Figure 4).
  • the camptothecin-based drug of Formula 1 is a topoisomerase I inhibitor and can exhibit cytotoxicity against cancer cells, but there are currently no examples of using it as a payload to manufacture an immunoconjugate through an acid-sensitive linker. It has not been reported at all so far.
  • camptothecin drug of Formula 1 is a hydrophobic small molecule that can penetrate the cell membrane, it accumulates rapidly in tumor tissue and can maintain a high concentration for a long time. It spreads inside the cell and exerts cytotoxicity to kill the cell and is then released and continuously released. As a result, it can penetrate the cell membrane of surrounding cells and move into the cell to act.
  • the immunoconjugate of the present invention has a technical feature of combining the camptothecin-based drug of Formula 1 with an acid-sensitive linker, so the immunoconjugate can be prepared by combining any antibody or fragment containing an antigen-binding site thereof according to the desired purpose. It can be designed, and it is all included within the scope of the present invention.
  • an immunoconjugate can be prepared by combining trastuzumab, cetuximab, and sacituzumab with the camptothecin drug-acid sensitive linker conjugate of Formula 1 of the present invention.
  • the immunoconjugate may have an average drug-to-antibody ratio (DAR) of 2 to 12, preferably DAR of 4 to 12.
  • DAR drug-to-antibody ratio
  • the target target may be expanded to include not only cancer cells, but also senescent cells, infectious disease organisms, and/or cells related to autoimmune diseases.
  • the cells targeted by the antibody or antigen-binding site-containing fragment thereof may be cancer cells, senescent cells, infectious disease organisms, and/or cells associated with autoimmune diseases.
  • target antigens include antigens selectively distributed on the surface of cancer, such as Her2, FolR, and PSMA, and antigens overexpressed in cancer cells, such as Trop2, which are distributed in small numbers in normal tissues.
  • Cancer cell target antigens include, for example, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1.
  • the target antigen may be an antigen that is more than 10 times more distributed in cancer cells than in normal cells.
  • Antibody or fragment containing antigen-binding site thereof [Antibody or fragment containing antigen-binding site thereof]
  • the immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof] is an antibody or antigen-binding site-containing fragment thereof.
  • the organic mechanism of action can be exerted by using the combination of [camptothecin-based drug of Formula 1] and [acid-sensitive linker] described above, regardless of the type.
  • the free exatecan or Dxd drug shares very strong anticancer efficacy with a low IC 50 value, 5 to 20 times more potent than SN-38 in various cancer cell lines.
  • Exatecan or Dxd drugs utilize inhibitors of Topoisomerase I, an anticancer mechanism whose efficacy/safety has been clinically proven, and Topoisomerase I inhibitors (Irinotecan, Topotecan, etc.) have been clinically used to treat colon cancer, lung cancer, breast cancer, and ovarian cancer. This is because its excellent anticancer efficacy has been proven in various intractable solid cancers, such as:
  • antibody refers to a protein molecule that acts as a ligand that specifically recognizes an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, including polyclonal antibodies, monoclonal antibodies, Contains all whole antibodies.
  • the term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies and tetrabodies.
  • the term further includes single chain antibodies possessing a binding function to FcRn, scaps, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds.
  • a full antibody is structured with two full-length light chains and two full-length heavy chains, with each light chain linked to the heavy chain by a disulfide bond.
  • the total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4.
  • the term “antigen binding site-containing fragment” 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 included in the Fab fragment.
  • the Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen binding site.
  • Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C terminus of the heavy chain CH1 domain.
  • F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond.
  • Fv variable fragment refers to the minimum antibody fragment containing only the heavy chain variable region and the light chain variable region.
  • dsFv double disulfide Fv
  • scFv single chain Fv
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragments can be obtained by digestion with pepsin), and are preferred. It can be produced through genetic recombination technology.
  • proteolytic enzymes for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragments can be obtained by digestion with pepsin
  • salts refer to salts commonly used in the pharmaceutical industry, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc.
  • inorganic acids such as perhydrochloric acid, phosphoric acid, and sulfuric acid, as well as salts such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, and acetylsalicylic acid.
  • organic acids and amino acid salts such as lysine, arginine, and guanidine.
  • salts of organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyltrimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes.
  • organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyltrimethyl ammonium, and benzethonium
  • the types of salts meant in the present invention are not limited to these salts listed.
  • prodrugs include, but are not limited to, prodrugs that are biologically active or become more active after being placed in a predetermined physiological environment.
  • a physiologically active substance or a therapeutically active organic compound is chemically modified and the parent compound is liberated or released under enzymatic or other conditions in vivo.
  • it is a useful drug, it has unsuitable properties in terms of side effects, stability, solubility, absorption, and duration of action, and chemical modifications are added to make it possible for clinical use.
  • Carrier-drug conjugate is also a type of prodrug.
  • [Camptothecin-based 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, and unlike antibodies, it can be used to penetrate deep into cancer tissue. By using a carrier that can penetrate well and has the characteristics of easy CMC, it can be utilized for a variety of applications.
  • camptothecin drug of Formula 1 is delivered to a carrier through the linker of Formula 3. It is characterized by linking one or more tothecin drugs.
  • the carrier may be an antibody, peptide, lipibody, and/or aptamer.
  • antibody-drug conjugate utilizes the high tissue selectivity of antibodies that specifically bind to specific antigens expressed on the surface of cancer cells to selectively deliver a payload with strong anticancer efficacy only to cancer tissues. It is a new drug platform. ADC selectively delivers a powerful payload that kills cancer cells even at concentrations as low as pM, and minimizes systemic exposure to the drug, ensuring anticancer efficacy and safety at the same time.
  • ADC a prodrug of a camptothecin-based drug of Formula 1 that targets the DDX5 protein according to the present invention, can be a new treatment method that can solve anti-HER2 treatment resistance.
  • the prodrug of the present invention may be an immunoconjugate containing an antibody or an antigen-binding site-containing fragment thereof, designed to release the camptothecin-based drug of Formula 1 in vivo, or a pharmaceutically acceptable salt thereof.
  • the immunoconjugate of the present invention binds specifically to the antigen of cancer cells and exhibits cytotoxicity by releasing the drug inside and outside the cancer cells, so it can be usefully used in the treatment or prevention of cancer.
  • Aptamer-Drug Conjugate is an aptamer introduced instead of an antibody in ADC.
  • Aptamers are single-stranded nucleic acids with a three-dimensional structure. It is discovered through the 'SELEX' (Systematic Evolution of Ligands by Exponential enrichment) process. Selex is a technology that obtains functional nucleic acids that bind to target protein molecules in a compound library.
  • Aptamers can bind to targets very strongly and selectively, so they are also called chemical antibodies. Aptamers are about 20 kDa in size and are known to have excellent cell penetration and low immunogenicity compared to antibodies.
  • aptamers can be chemically synthesized, precise design of the conjugation location and number of drug conjugates is possible when manufacturing aptamer-drug conjugates.
  • the production cost is lower than that of ADC.
  • Aptamers are generally made of natural nucleic acids, so they are degraded by nucleic acid degrading enzymes in the body, making them less stable in vivo.
  • nucleic acid degrading enzymes in the body, making them less stable in vivo.
  • the limitations on the stability of modified aptamers can be overcome.
  • PDC Peptide-Drug Conjugate
  • ADC ADC in which peptides are introduced instead of antibodies.
  • Peptides are composed of amino acids and have a size ranging from 500 to 5000 Da (Dalton). This is a very small size compared to antibodies of 150 kDa (kilodaltons) or more. Therefore, peptide-based PDC has superior cell penetration ability compared to ADC, and the possibility of immunogenicity is very low. Additionally, peptides can be chemically synthesized. For this reason, PDC not only has a very low production cost, but also allows precise control of the conjugation position and ratio of peptide and drug.
  • peptides are easily degraded by proteolytic enzymes and therefore have a short biological half-life.
  • strategies using modified peptides such as cyclic peptides and introduction of non-natural amino acids are being proposed.
  • Repebody is a type of artificial antibody that does not have an antibody skeleton but has the function of recognizing antigens like an antibody. Lipibodies specific to target proteins can be discovered through phage display technology.
  • Phage display is a technology that expresses desired proteins on the surface of bacteriophage.
  • Lipibody is about 30kDa in size, which is 20% of an antibody drug. Therefore, it is known to have relatively low immunogenicity and improved cell penetration compared to antibodies. In addition, it is expected that structural stability can be improved by controlling the thermal and pH stability of Lipibody. Compared to antibodies, production costs are also assessed to be relatively low. Because of these advantages of Lipibodies, interest in the development of Lipibody-drug conjugates (Repebody-DC) as a strategy to replace antibodies with Lipibodies is also increasing.
  • Repebody-DC Lipibody-drug conjugates
  • the ADC of the present invention can also serve as a basic therapeutic antibody, which has played an important role in the treatment of diseases such as cancer and autoimmunity over the past 10 years.
  • Antibodies have the ability to distinguish between antigens and cells in our body, so they have an excellent screening function that acts selectively on antigens.
  • Antibody therapeutics utilize the antigen-selective binding characteristics of antibodies to treat diseases.
  • Antibodies are Y-shaped, with an antigen binding site on each arm, and complementarity determining regions (CDRs) allow the antibody to selectively recognize the target antigen. Additionally, there are five major types of antibodies (IgM, IgD, IgG, IgA, and IgE / Immunoglobulin (Ig)), and IgG is mainly used in antibody treatments.
  • Antibody treatments can selectively react with antigens and remove them.
  • various mechanisms are used, and the characteristics of the representative mechanisms used in antibody therapeutics are as follows.
  • the first mechanism is blocking.
  • Antibodies selectively bind to target receptors on cells 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, and reduced signal transduction causes loss of cell activity, inhibition of proliferation, and apoptosis.
  • an antibody When an antibody targets a receptor on the cell surface and binds to the receptor, it changes the structure of the receptor or prevents factors that must bind to the receptor from binding, thereby inhibiting intracellular signal transduction and thereby promoting cell growth. and differentiation can be controlled. Additionally, when an antibody binds to a receptor, the receptor enters the cell through an intracellular influx mechanism, which reduces the number of receptors on the cell surface, thereby controlling the physiological mechanism of the cell.
  • a representative antibody treatment that shows excellent effectiveness through this mechanism is Trastuzumab (Herceptin®), which targets HER2-positive breast cancer, one of the human epidermal growth factor receptors (HER/EGFR/ERBB).
  • ADCC Antibody-Dependent Cellmediated Cytotoxicity
  • 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 lysis of target cells. In addition, there are mechanisms such as opsonization, neutralization, and inflammatory response.
  • C1q complement protein
  • Therapeutic antibodies are generally made into an optimized form by re-engineering the antibody from various perspectives for a specific therapeutic purpose. And the antibody engineering technology applied to the development of these therapeutic monoclonal antibodies is being expanded and applied to the development of optimized ADCs.
  • most therapeutic antibodies or ADCs currently in clinical development are IgG 1 , with only a small number using IgG 2 and IgG 4 .
  • Selection of the antibody isotype is equally important for therapeutic antibodies and ADCs. The reason is that the isotype of an antibody affects the functions of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Additionally, IgG 4 is functionally monovalent through in vivo Fab arm exchange. Additionally, the in vivo efficacy of ADC can be improved by applying the efficacy of ADC by isotype and Fc-domain engineering of antibodies.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocyto
  • composition for preventing or treating cancer [ Pharmaceutical composition for preventing or treating cancer ]
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-described immunoconjugate according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.
  • a method for treating or preventing cancer comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof.
  • the subject may be a mammal, including humans.
  • the immunoconjugate of the present invention binds specifically to the antigen of cancer cells and exhibits cytotoxicity by releasing the drug inside and outside the cancer cells, so it can be usefully used in the treatment or prevention of cancer.
  • the anticancer activity of the immunoconjugate of the present invention is as described above.
  • the cancer includes all cancers treatable by inhibition of topoisomerase I, and may be solid cancer or hematological cancer.
  • solid cancer or hematological cancer.
  • pseudomyxoma intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, oral cavity cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovary.
  • Epithelial cancer ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, Parathyroid cancer, adrenal cancer, sinonasal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenum Cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, ure
  • the term “therapeutically effective amount” refers to the amount of the immunoconjugate effective for treating or preventing cancer. Specifically, “therapeutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, type of disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. And it can be administered single or multiple times.
  • the immunoconjugate of the present invention exhibits a dose-dependent effect, the administered dose is determined by the patient's condition, age, gender, and It can be easily determined by a person skilled in the art depending on various factors such as complications. Since the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it can be used at a dose exceeding the determined dosage.
  • the present invention provides a use of the immunoconjugate for use in the manufacture of a medicament for use in the treatment or prevention of cancer.
  • the immunoconjugate for the preparation of a drug can be mixed with acceptable auxiliaries, diluents, carriers, etc., and can be prepared as a complex preparation with other active agents to have a synergistic effect of the active ingredients.
  • the camptothecin-based drug of Formula 1 designed to bind to the DDX5 protein according to the present invention, not only binds to the DDX5 protein within cells and induces cell death through DDX5 protein degradation, but is preferably used as a payload.
  • it can solve the problem of aggregation of its carrier-drug conjugate.
  • the carrier-drug conjugate according to the present invention is phagocytosed by macrophages in the route of administration, distribution, and elimination in the body after in vivo injection when connected to the camptothecin drug of formula 1 through the acid-sensitive linker of formula 3. It is precisely designed to prevent phagocytosis, and is not absorbed into the liver, spleen, bone marrow, or lymph nodes due to the phagocytosis of macrophages in reticuloendothelial organs, and can provide the desired blood circulation profile. Therefore, the immunoconjugate of the present invention can be administered at a dose of 11 mg/kg or more.
  • the immunoconjugate of the present invention is a hydrophobic small molecule drug that is permeable to cell membranes due to the combined use of the camptothecin-based drug of Formula 1 and an acid-sensitive linker, so the internalization process by the antigen-antibody complex is inefficient, so it is maintained at a sufficient concentration. It can solve the problem of drugs not entering the cells and the problems of antibodies not penetrating deep into cancerous tissue, and can maximize the by-stander effect of entering not only targeted cells but also surrounding cells.
  • the immunoconjugate of the present invention can overcome the problems of resistance to ADCs and a narrow therapeutic window resulting from the relatively weak efficacy of the payload used in 3rd generation ADCs. . That is, the immunoconjugate of the present invention can exhibit an optimized therapeutic window by balancing strong anticancer efficacy and in vivo safety compared to existing ADC payloads due to the combined use of the camptothecin-based drug of Formula 1 and an acid-sensitive linker.
  • Figure 1 shows the structural formulas of various camptothecin anticancer drugs (SN-38, Exatecan, Dxd, FL118).
  • FIG. 1 shows the structural formula of camptothecin (CPT) and its binding to type 1 topoisomerase-1.
  • Figure 3 is a schematic diagram showing an efficient drug release mechanism from an immunoconjugate using an acid-sensitive linker according to the present invention.
  • Figures 4 to 7 show the degradation of DDX5 and p-DDX5 proteins of various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line. This is a Western blot result showing the presence/degree of inhibition of various anti-apoptotic proteins.
  • Figures 5 and 7 show the results of western blot experiments conducted on the FaDu cell line and the A549 cell line ( Figures 4 and 6), graphically quantifying the concentration.
  • Figure 8 shows the Western blot results of Tra-CL2A-FL118/Tra-CL2A-Exatecan.
  • Figures 9 and 10 show various camptothecin-based drugs and ADCs using them as payload, namely Tra-CL2A-FL118/Tra-CL2A-Exatecan, in MDA-MB-453 (HER2++) cell line and SK-BR-3 cell line, respectively. This is the result of in vitro cell viability comparative evaluation.
  • Figure 11 is a detailed classification diagram of cell death related to programmed cell death.
  • Triethylamine (0.098 mL, 0.705 mmol) was added to a suspension of exatecan 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.
  • reaction mixture was diluted with DMSO and purified by basic preparative MPLC (XSelect40-80), lyophilized, and obtained as an off-white solid (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%) was obtained.
  • reaction mixture was stirred at room temperature overnight. Additional amount of copper(I) bromide (7.3 mg, 0.051 mmol) was added. After stirring for 3 hours, the product increased in sample-2 on LCMS. The reaction mixture was stirred for an additional 5 hours and concentrated under reduced pressure. The residue was purified by basic preparative MPLC (XSelect50-100) and lyophilized to obtain the product (133 mg, 75%) as an off-white solid.
  • AN_ACID m/z 763.0 [M+2H] 2+ /2
  • reaction mixture was diluted with DMSO and purified by basic preparative MPLC (XSelect30-70), concentrated from a mixture of acetonitrile and water (1:1, 10 mL), lyophilized, and then 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 was obtained as an off-white solid. Yield: 89 mg, 68%.
  • DMSO and compound solutions of Preparation 1 and Preparation 2 were added to the reduced antibody solution at a final DMSO concentration of 10% at 12 eq. relative to antibody.
  • the reaction mixture was mixed appropriately and the reaction vial was left at 25° C. for 1 hour.
  • reaction mixture was passed through a spin desalting column (PD-10) to remove unreacted compounds and only the ADC was separated.
  • the product was then sterile filtered through a 0.2 ⁇ m PVDF disposable filter.
  • the resulting immunoconjugate was characterized and 0.07 mM PS80 and 20 mM trehalose dehydrate were added.
  • the DAR of the immunoconjugate of Preparation Example 3 was determined using HIC and MS, and the average MS-DAR value was confirmed to be 7.14 and the HIC-DAR value was found to be 8.00.
  • Example 1 Confirmation of inhibitory activity of DDX5, p-DDX5, survivin, Mcl-1, XIAP and cIAP2 expression of cancer-related survival genes through Western blot
  • Tra-CL2A-FL118 and Tra-CL2A-SN-38 were prepared in the same manner as Preparation Example 3, except that the drug FL118 and SN-38 were used instead of the drug Exatecan, respectively.
  • FaDu cell lines were seeded per well in a 6-well plate and incubated at constant temperature (37°C, 5% CO 2 ). After 24 hours, the drug (FL118, Exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN) was administered at concentrations of 0 nM, 10 nM, and 100 nM. -38 and Tra-CL2A-Exatecan) were treated in each well. It was incubated at constant temperature (37°C, 5% CO 2 ) for 24 hours. The well was treated with 100ul of RIPA buffer dissolved in protease inhibitor cocktail.
  • the plate was placed on ice and incubated for 2 hours on an orbital shaker.
  • RIPA buffer containing lysed cells was transferred to an ep tube and centrifuged (16000 rcf, 20 min, 4°C). Afterwards, only the supernatant was transferred to a new EP tube. Protein concentration was confirmed through protein assay.
  • Protein sample and 4x SDS-PAGE Loading Buffer were mixed in a 3:1 ratio, boiled at 95°C for 10 minutes, and cooled. Samples were loaded into gel wells so that the amount of protein was the same. Electrophoresis was performed on the gel at 60V.
  • the transferred membrane was immersed in blocking buffer and incubated at constant temperature (RT, 1h). Next, it was incubated (4°C, overnight) in the primary antibody solution. Rinsed with TBST buffer for 3 minutes (repeated 3 times). Incubation was performed (RT, 1h) in a secondary antibody solution conjugated with HRP. Rinsed with TBST buffer for 3 minutes (repeated 3 times).
  • the signal was confirmed using the ChemiDocTM MP Imaging System. HRP bound to the secondary antibody oxidized the Luminol of ECL, and the light emitted was detected and displayed as an image. Since the thickness of the band is proportional to the amount of protein, the amount of protein can be compared through the thickness of the band.
  • the drugs FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 and We confirmed how the protein expression level changes by treatment (Tra-CL2A-Exatecan).
  • GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells. It is a gene that is always expressed in cells and its expression level does not change easily, and it is an indicator that shows whether the same amount of protein was loaded on the gel in the samples.
  • camptothecin-based compounds degrade DDX5 or phosphorylated DDX5 (p-DDX5), which are known to be factors directly involved in the expression of anti-apoptotic proteins.
  • p-DDX5 phosphorylated DDX5
  • anti-apoptotic proteins such as Survivin, Mcl-1, cIAP2, and It was confirmed that it is a compound with a dual mechanism of action.
  • Trastuzumab (molecular weight 148 kDa, purity 97%), supplied from BCD as a solution stored at -80°C, was dissolved in a buffer solution (20mM Histidine, 150mM NaCl, pH 6.0) (concentration 10.09 mg/mL). It was used as a control.
  • Her2-high cell lines (MDA-MB-453) and HER2 positive breast cancer-derived cell lines (SK-BR-3) were seeded per well in a 96-well plate and incubated at constant temperature (37°C, 5% CO 2 ). did. After 24 hours, 100ul of the drug at 9 concentrations (serial dilution of 1/5 from 1000nM) was treated with the cells. At this time, trastuzumab, Tra-CL2A-FL118, and Tra-CL2A-Exatecan were treated. Cell viability was observed through constant temperature incubation (37°C, 5% CO 2 ) for 3 or 6 days.
  • 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). showed.
  • ADCs using various camptothecin-based compounds of formula 1, such as FL118 to exatecan, as payloads and connected with an acid-sensitive linker such as CL2A showed excellent cytotoxicity and operated with high efficiency.

Abstract

The present invention relates to a conjugate of a camptothecin-based drug (e.g., exatecan and dxd) of chemical formula 1, designed to bind to a DDX5 protein, linked to an acid-sensitive linker, and a carrier-drug conjugate using same. The camptothecin-based drug of chemical formula 1 designed to bind to a DDX5 protein, according to the present invention, binds to a DDX5 protein in cells and not only can induce cell death through breakdown of the DDX5 protein, but also, preferably, can solve problems of aggregation of the acid-sensitive linker and the carrier-drug conjugate thereof, when used as a payload. In addition, the carrier-drug conjugate according to the present invention is precisely designed not to be phagocytized by phagocytes in the pathways of administration, distribution and elimination in the body after in vivo injection when linked to the camptothecin-based drug of chemical formula 1 via the acid-sensitive linker, and does not undergo absorption by the liver, spleen, bone marrow, lymph nodes, etc. caused by the phagocytosis of macrophages of the reticuloendothelial organs, and can provide a desired blood circulation profile.

Description

DDX5 단백질에 결합하는 캄토테신계 약물이 산 민감성 링커에 연결된 접합체 및 이를 이용한 면역접합체A conjugate in which a camptothecin-based drug that binds to the DDX5 protein is linked to an acid-sensitive linker and an immunoconjugate using the same.
본 발명은 DDX5 단백질에 결합하도록 설계된 화학식 1의 캄토테신계 약물(예, Exatecane 및 dxd)이 산 민감성(acid-sensitive) 링커에 연결된 접합체 및 이를 이용한 운반체-약물 접합체(Carrier-Drug Conjugate)에 관한 것이다.The present invention relates to a conjugate in which a camptothecin drug of formula 1 (e.g., Exatecane and dxd) designed to bind to DDX5 protein is linked to an acid-sensitive linker, and a carrier-drug conjugate using the same. will be.
본 발명에 따라 DDX5 단백질에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 세포 내에서 DDX5 단백질에 결합하여 DDX5 단백질 분해를 통해 세포사멸(cell death)을 유도할 수 있을 뿐만 아니라, 바람직하게는 페이로드(payload)로 사용시 산 민감성 링커와 함께 이의 운반체-약물 접합체가 응집되는 문제를 해결할 수 있다.The camptothecin-based drug of Formula 1, designed to bind to the DDX5 protein according to the present invention, not only binds to the DDX5 protein within cells and induces cell death through DDX5 protein degradation, but is preferably used as a payload. When used as a payload, it can solve the problem of aggregation of its carrier-drug conjugate with an acid-sensitive linker.
암 조절 유전자(Cancer Genes)는 원래는 세포의 정상적인 기능을 조절하기 위하여 존재하지만, 변이가 일어나거나 비 정상적으로 조절되면 암이 발생할 수 있는 유전자를 의미한다. 정상적인 세포 기능의 하나로 세포사멸 기능이 있으며, 이와 관련하여 사멸 촉진인자와 사멸 억제인자가 있다.Cancer genes are genes that originally exist to control the normal functions of cells, but can cause cancer if mutated or regulated abnormally. One of the normal cell functions is apoptosis, and related to this, there are death promoting factors and death suppressing factors.
예컨대, 암 조절 유전자(Cancer Genes)에는 (i) 돌연변이가 발생했을 때 세포 분열을 활발하게 촉진하는 종양유전자(Oncogenes) 및 (ii) 돌연변이가 일어나면 세포 분열을 억제하지 못하는 종양 억제 유전자(Tumor suppressor genes)가 있다. 종양유전자(Oncogenes)의 일례로, Src, EGFR, HER2, RAS, APC, BCL-2 등이 있고, 종양 억제 유전자(Tumor suppressor genes)의 일례로 p53, BRCA, Rb, PTEN, BAX 등이 있다.For example, cancer genes include (i) oncogenes that actively promote cell division when mutated, and (ii) tumor suppressor genes that do not suppress cell division when mutated. ). Examples of oncogenes include Src, EGFR, HER2, RAS, APC, and BCL-2, and examples of tumor suppressor genes include p53, BRCA, Rb, PTEN, and BAX.
암은 성장과 분열의 세포주기가 조절되지 않는 질병 그룹이다. 도 11에 도시된 바와 같이, 프로그램된 세포 사멸(예, 아폽토시스) 메커니즘이 손상되면 암이 발생할 수 있다. Cancer is a group of diseases in which the cell cycle of growth and division is dysregulated. As shown in Figure 11, cancer can occur when programmed cell death (e.g., apoptosis) mechanisms are impaired.
암은 단백질 생성물이 세포 주기 조절에 관여하는 유전자의 돌연변이 발생으로 인해 발생한다. 많은 암은 특정 유전자의 과발현 또는 돌연변이 단백질 산물의 비정상적인 활동과 관련이 있다(Oncogenes). 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).
바이러스 종양유전자들(viral oncogenes)에 의해 코딩되는 단백질들은 중요한 조절 기능을 가진 세포 단백질과 유사하다. 이러한 세포 상동체를 원종양유전자(proto-oncogenes) 또는 정상세포 종양유전자(normal cellular oncogenes)라고 한다. Proto-oncogenes의 돌연변이는 세포 증식을 적극적으로 촉진한다. 돌연변이 c-H-ras 단백질에는 GTP를 가수분해하는 능력을 손상시키는 돌연변이가 있다. 이것은 돌연변이 단백질을 활성 신호 모드로 유지하고 세포 분열을 자극한다. c-ras의 돌연변이 버전은 많은 유형의 종양에서 발견되었다.Proteins encoded by viral oncogenes are similar to cellular proteins that have important regulatory functions. These cellular homologs are called proto-oncogenes or normal cellular oncogenes. Mutations in proto-oncogenes actively promote cell proliferation. The mutant cH-ras protein contains mutations that impair its ability to hydrolyze GTP. This keeps the mutant protein in an active signaling mode and stimulates cell division. Mutant versions of c- ras have been found in many types of tumors.
단일 돌연변이는 일반적으로 암을 유발하지 않는다. 일반적으로 세포 성장을 조절하는 여러 유전자들이 암 상태가 발생하기 전에 돌연변이된다.Single mutations usually do not cause cancer. Typically, several genes that regulate cell growth become mutated before the cancerous condition develops.
유방 상피세포에 HER2가 비정상적으로 많이 발현하는 HER2 양성 유방암은 유방암 환자 전체의 ¼ 정도이고, 여성호르몬 관련 유방암에 비해 화학 항암제 치료 시 재발율이 높고 예후가 나쁘다. HER2 표적치료제인 허셉틴(Herceptin, 성분명: Trastuzumab)은 세포 표면의 HER2의 수를 줄이고, 면역세포가 HER2 양성 유방암 세포를 죽이도록 도와 줌으로써, 유방암을 치료한다.HER2-positive breast cancer, in which HER2 is expressed abnormally in breast epithelial cells, accounts for about ¼ of all breast cancer patients, and has a higher recurrence rate and worse prognosis when treated with chemotherapy than female hormone-related breast cancer. Herceptin (generic name: Trastuzumab), a HER2-targeted treatment, treats breast cancer by reducing the number of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.
HER2 양성 유방암은 ErbB 수용체의 멤버인 HER2 (ErbB2) 암유전자의 증폭(amplification) 또는 과발현(overexpression)을 보유한 유방암이다. 트라스투주맙(허셉틴)은 HER2 양성유방암의 치료효과를 증대시켰으나, HER2 양성 유방암은 다른 유방암에 비해 공격적인 양상을 나타내며 환자의 과반수 이상이 기존 표적치료에 저항성을 갖거나 치료 중 내성이 생긴다. HER2-positive breast cancer is breast cancer that has amplification or overexpression of the HER2 (ErbB2) oncogene, a member of the ErbB receptor. Trastuzumab (Herceptin) has increased the treatment effect of HER2-positive breast cancer, but HER2-positive breast cancer shows an aggressive pattern compared to other breast cancers, and more than half of patients are resistant to existing targeted treatments or develop resistance during treatment.
세포막에 부착된 ErbB 수용체의 리간드들로는 NRG1, HB-EGF가 있으며, 활성화된 ErbB 리간드들은 이들의 수용체(EGFR, HER3)에 결합하여, 수용체들 간의 결합(EGFR-HER2, HER2-HER3)에 의한 인산화(phosphorylation, P)를 촉매하여, 세포 내 성장증식 신호를 유도한다. 이러한 과도한 ErbB 수용체의 활성화에 의해 항HER2 치료제에 내성을 나타내게 된다. Ligands of the ErbB receptor attached to the cell membrane include NRG1 and HB-EGF, and activated ErbB ligands bind to their receptors (EGFR, HER3) and undergo phosphorylation by binding between receptors (EGFR-HER2, HER2-HER3). It catalyzes phosphorylation (P) and induces growth and proliferation signals within cells. This excessive activation of the ErbB receptor results in resistance to anti-HER2 treatment.
STAT(signal transducer 및 activator of transcription의 약자) 전사 인자 패밀리에는 STAT1, STAT2, STAT3, STAT4, STAT5 및 STAT6이 포함된다. STAT 단백질은 성장 인자, 사이토카인, 인터페론 또는 발암 유전자로 자극 시 JAK 키나아제에 의해 활성화되어 인터페론 신호 전달을 조절할 수 있다. STAT의 DNA 결합 도메인은 특정 DNA 표적 서열에 대한 결합을 매개하는 면역글로빈 유사 구조이다. 활성화 동안 JAK 키나아제에 의한 STAT3의 인산화는 이합체화를 활성화한다. 그런 다음 이합체는 핵 구획으로 이동하고 표적 프로모터의 서열에 결합하여 표적 유전자의 발현을 유도한다. STAT3 표적 유전자에는 서바이빈(survivin), bcl-xl, mcl-1, 사이클린 D1, MMP2, MMP9, VEGF, Myc, Sox2 등이 포함된다. STAT 단백질은 세포 성장, 세포 사멸, 분화 및 면역을 포함한 많은 생물학적 과정을 조절한다. STAT 계열 내에서 STAT3 및 STAT5는 암 진행에 관여했다.The STAT (short for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6. STAT proteins can be activated by JAK kinases upon stimulation with growth factors, cytokines, interferons, or oncogenes to regulate interferon signaling. The DNA binding domain of STAT is an immunoglobin-like structure that mediates binding to specific DNA target sequences. During activation, phosphorylation of STAT3 by JAK kinases activates dimerization. The dimer then moves to the nuclear compartment and binds to the sequence of the target promoter, leading to expression of the target gene. STAT3 target genes include survivin, bcl-xl, mcl-1, cyclin D1, MMP2, MMP9, VEGF, Myc, and Sox2. STAT proteins regulate many biological processes, including cell growth, apoptosis, differentiation, and immunity. Within the STAT family, STAT3 and STAT5 have been implicated in cancer progression.
조절되지 않은 STAT3 및 활성화된 STAT5는 발암유전자로 간주되어 혈관신생을 증가시키고 암세포의 생존을 향상시킨다. cBioPortal 데이터베이스의 많은 샘플을 사용한 연구에서 STAT3는 정상 조직에 비해 폐암, 난소암, 위암, 조혈암 및 뇌암에서 더 자주 과발현되는 것으로 나타났으며, STAT3의 과발현은 낮은 전체 생존율과 관련이 있었다. JAK 키나아제에 의한 STAT3의 인산화는 STAT3 활성화의 첫 번째 단계이다. 교모세포종 환자 90명을 대상으로 한 또 다른 연구에서는 높은 p-STAT3 수치가 무진행 생존율 및 전체 생존율 저하와 유의한 관련이 있음을 발견했다. 다변량 생존 분석은 높은 p-STAT3 수준이 불량한 무진행 생존 및 전체 생존에 대한 중요한 예후 지표 역할을 할 수 있음을 보여주었다.Dysregulated STAT3 and activated STAT5 are considered oncogenes, increasing angiogenesis and improving cancer cell survival. In a study using a large number of samples from the cBioPortal database, STAT3 was found to be more frequently overexpressed in lung, ovarian, gastric, hematopoietic, and brain cancers compared to normal tissues, and overexpression of STAT3 was associated with poor overall survival. Phosphorylation of STAT3 by JAK kinases is the first step in STAT3 activation. Another study of 90 patients with glioblastoma found that high p-STAT3 levels were significantly associated with poorer progression-free and overall survival. Multivariate survival analysis showed that high p-STAT3 levels could serve as an important prognostic indicator for poor progression-free survival and overall survival.
제1형 토포이소머라아제(Topoisomerase I)의 저해제(Irinotecan, Topotecan 등)는 임상에서 효능/안전성이 검증된 항암기전이며, 임상에서 대장암, 폐암, 유방암, 난소암 등 다양한 난치성 고형암에서 뛰어난 항암 효능이 검증되었다. Inhibitors of type 1 topoisomerase I (Irinotecan, Topotecan, etc.) are anticancer mechanisms whose efficacy/safety has been proven clinically, and are excellent anticancer agents for various intractable solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer in clinical trials. Efficacy has been proven.
이와 관련하여, 제1형 토포이소머라제(topoisomerase-1)를 저해하여 항종양 작용을 발현하는 저분자 화합물인 캄토테신 유도체가 알려져 있다.In this regard, camptothecin derivatives, which are low-molecular-weight compounds that exhibit anti-tumor activity by inhibiting type 1 topoisomerase (topoisomerase-1), are known.
캄토테신(camptothecin)은 DNA의 복제 및 재조합 등에 관여하는 이성질화 효소인 제1형 토포이소머라제(topoisomerase-1)의 선택적인 억제제이다. 이미 1966년 미국의 Wall 등에 의해 중국 원산지의 희수 나무(Camptotheca acuminata)에서 단리된 천연 항종양 알카로이드이다. in vitro에서 강력한 세포독성(cytotoxicity)을 나타낸 것으로 판명된 이래 미국 암연구센터(NCI) 등에서 임상시험을 통한 개발에 착수되었으나 극히 난용성이라는 한계로 인해 그와 관련된 골수억제 및 출혈성 방광염 등의 다양한 부작용을 보임에 따라 개발이 중단되었다. 그러나 1990년 이후, 캄토테신이 가지는 독특한 작용기전, 즉 DNA 제2형 토포이소머라제(topoisomerase-2) 저해기전과는 달리 DNA 제1형 토포이소머라제를 선택적으로 억제하여 항종양효과를 나타냄이 확인되었다.Camptothecin is a selective inhibitor of type 1 topoisomerase-1, an isomerase involved in DNA replication and recombination. It is a natural anti-tumor alkaloid isolated from Camptotheca acuminata, native to China, by Wall et al. in the United States in 1966. Since it was found to exhibit strong cytotoxicity in vitro, development through clinical trials was initiated at the National Cancer Research Center (NCI), etc., but due to the limitation of extremely poor solubility, various side effects such as bone marrow suppression and hemorrhagic cystitis were associated with it. As a result, development was halted. However, since 1990, camptothecin has a unique mechanism of action, that is, unlike the DNA type 2 topoisomerase (topoisomerase-2) inhibition mechanism, it selectively inhibits DNA type 1 topoisomerase, showing an antitumor effect. This has been confirmed.
DNA 토포이소머라제는 기라아제(gyrase) 효소군의 일원이다. 이들은 핵내의 효소들로서, 복제나 전사를 위해 세포가 유전 물질에 접근이 요구될 때 일시적으로 DNA의 절단하거나 이중 나선을 푸는 역할을 한다. 이들은 또한 염색체 농축 및 재조합, DNA 수리 등 다양한 세포내 활동에 참여한다. 토포이소머라제의 유전암호는 종들 간에 상당히 보존적이다.DNA topoisomerase is a member of the gyrase enzyme family. These are enzymes in the nucleus that temporarily cut DNA or unwind the double helix when the cell needs access to genetic material for replication or transcription. They also participate in a variety of intracellular activities, including chromosome condensation and recombination, and DNA repair. The genetic code of topoisomerase is highly conserved among species.
캄토테신의 약물 표적인 제1형 토포이소머라제는 다양한 악성 종양에서 그 수준이 증가하는 것이 관찰되었다. 이 약물은 유리효소를 억제하지는 못하나 토포이소머라제-DNA 복합체의 공유결합을 안정화시켜 절단된 DNA 조각들이 다시 연결되는 것을 방해한다. 따라서, 이러한 토포이소머라제-대상 약물에 대한 세포의 민감성은 핵내 존재하는 효소의 수준과 관련이 있다. 이 약물은 DNA의 재결합을 방해함으로써 전사가 진행되는 것을 불가능하게 한다. 제1형 토포이소머라제의 양이 더 많을수록, 더 많은 절단가능한 복합체를 형성하며 이는 약제 감수성이 높음을 의미한다. 이것은 중요한 임상적 관련성을 가지는데, 제1형 토포이소머라제 억제제는 제2형 토포이소머라제의 발현을 증가시키는데 사용되어 이는 제2형 토포이소머라제 억제제에 더욱 감수성을 가지게 한다. 이러한 결과는 제1형 토포이소머라제와 제2형 토포이소머라제 간의 대립 관계에 의해 지지된다. 이러한 제1형 토포이소머라제는 제2형 토포이소머라제와 달리 정상 조직에서는 증식과 밀접한 관련이 없으며, 임파종으로 포함한 대장암, 난소암, 식도암 등의 세포분열이 왕성한 “S”기의 고형암에 주변의 정상 조직에 비해 다량 존재하며, 실제적으로 세포주기상“S”기에 종양세포 내에 유전자의 복제 및 전사를 차단함으로써 세포사를 유발할 수 있다고 알려져 있다. Type 1 topoisomerase, the drug target of camptothecin, has been observed to have increased levels in various malignant tumors. This drug does not inhibit free enzymes, but stabilizes the covalent bond of the topoisomerase-DNA complex, preventing the reconnection of cut DNA fragments. Therefore, the sensitivity of cells to these topoisomerase-targeting drugs is related to the level of the enzyme present in the nucleus. This drug prevents transcription from proceeding by interfering with DNA recombination. The higher the amount of type 1 topoisomerase, the more cleavable complexes are formed, which means higher drug sensitivity. This has important clinical relevance, as type 1 topoisomerase inhibitors are used to increase the expression of type 2 topoisomerases, making them more susceptible to type 2 topoisomerase inhibitors. This result is supported by the antagonistic relationship between type 1 and type 2 topoisomerases. Unlike type 2 topoisomerase, type 1 topoisomerase is not closely related to proliferation in normal tissues, and is an “S” stage solid cancer with active cell division, such as colon cancer, ovarian cancer, and esophageal cancer, including lymphoma. It is present in large quantities compared to surrounding normal tissues, and is known to actually cause cell death by blocking the replication and transcription of genes in tumor cells during the “S” phase of the cell cycle.
지금까지 확인된 모든 캄토테신 유도체들은 세포독성에 필수적인 5 개의 고리를 갖는 모 구조를 함유한다(도 1). 분자구조상 E-고리와 A-, B-고리 부위가 중요부위로 확인되었다. 캄토테신은 세포독성에 필수적인 E-고리에 펜타사이클릭 락톤구조를 포함한다. 이중 E-고리의 20번 탄소에 위치한 락톤기와 알파수산화기는 제1형 토포이소머라제-DNA 부산물의 안정을 위해 중요하며, A- 및 B-고리의 변형이 수용성과 활성도를 증가시킬 수 있다는 사실이 증명되었다. 첫 번째 고리상의 변경은, 예를 들어 상기 언급한 약물의 경우에 물에 대한 용해도를 증가시키고 보다 큰 허용성을 허용하는 것으로 입증되었다.All camptothecin derivatives identified to date contain a parent structure with five rings essential for cytotoxicity (Figure 1). In terms of molecular structure, the E-ring and A- and B-ring regions were identified as important regions. Camptothecin contains a pentacyclic lactone structure in the E-ring, which is essential for cytotoxicity. Among them, the lactone group and alpha hydroxyl group located at carbon 20 of the E-ring are important for the stability of type 1 topoisomerase-DNA by-products, and the fact that modification of the A- and B-rings can increase water solubility and activity. This has been proven. Modifications on the first ring have been proven to increase the solubility in water and allow greater tolerability, for example in the case of the drugs mentioned above.
CKD-602 역시 수용성 및 항암효과의 증가를 위해 7번 탄소의 B-고리 부위의 치환을 시도하였다. Lee 등은 CKD-602가 광범위한 암세포주에서 캄토테신(camptothecin)과 토포테칸(topotecan)에 비해 우수한 항암효과가 있다고 하였다. 또한 L1210 백혈병 누드마우스모델에서 최대내약용량(maximum tolerated dose, MTD)이 25mg/kg로 비교적 안전한 약물임을 확인하였다. 일반적으로 알려진 캄토테신계 약물의 부작용은 크게 혈액학적 부작용과 비혈액학적 부작용으로 분류할 수 있다. 혈액학적 부작용으로는 발열을 동반한 호중구감소증, 패혈증, 출혈 등이 있으며, 비혈액학적 부작용으로는 구역, 구토, 탈모 등 피부계 부작용 및 위장관, 신장, 신경계에 대한 독성을 들 수 있다. Kim 등은 CKD-602의 동물 연구에서 이러한 캄토테신계 약물의 부작용 중 임상적용이 가능한 용량보다 10배 이상의 고용량의 투여시에도 위액분비 증가를 제외한 이상약물반응은 발견되지 않았다. 또한 최근 국내 임상 연구들에서도 심각한 전신적인 독성 보다는 호중구감소증 및 백혈구감소증의 가역적이며 조절가능한 수준의 부작용만이 보고되는 등 비교적 안정성이 입증되고 있다. 하지만 현재까지는 표준 화학요법에 실패하거나 표준 화학요법을 시행할 수 없는 환자, 즉 표준치료 후에 재발 또는 악화되어 더 이상의 항암화학요법, 수술로 효과를 보기 어려울 것으로 판단되는 저항성(refractory) 또는 재발성(recurrent) 난소암 및 대장암의 치료, 1차화학요법에 실패한 저항성 또는 재발성 제한병기(limited disease) 소세포성 폐암의 치료, 진행병기(extensive disease) 소세포성 폐암의 치료에 주된 적응증을 두고 제한적으로 사용되고 있다.CKD-602 also attempted to substitute the B-ring portion at carbon 7 to increase water solubility and anticancer effect. Lee et al. reported that CKD-602 has superior anticancer effects compared to camptothecin and topotecan in a wide range of cancer cell lines. In addition, it was confirmed to be a relatively safe drug with a maximum tolerated dose (MTD) of 25 mg/kg in the L1210 leukemia nude mouse model. The generally known side effects of camptothecin-based drugs can be broadly classified into hematological side effects and non-hematological side effects. Hematological side effects include neutropenia with fever, sepsis, and bleeding, while non-hematological side effects include skin side effects such as nausea, vomiting, and hair loss, and toxicity to the gastrointestinal tract, kidneys, and nervous system. In an animal study of CKD-602, Kim et al. found no adverse drug reactions other than increased gastric secretion, even when administered at doses 10 times higher than the clinically applicable dose, among the side effects of these camptothecin drugs. In addition, recent domestic clinical studies have proven its relative safety, with only reversible and controllable side effects such as neutropenia and leukopenia reported rather than serious systemic toxicity. However, to date, patients who have failed standard chemotherapy or cannot undergo standard chemotherapy, that is, patients who have relapsed or worsened after standard treatment and are unlikely to benefit from further chemotherapy or surgery, are resistant (refractory) or relapsed (refractory). Recurrent) Treatment of ovarian cancer and colon cancer, treatment of resistant or recurrent limited disease small cell lung cancer that has failed primary chemotherapy, and treatment of advanced stage small cell lung cancer (extensive disease). It is being used.
항체-약물 접합체(ADC)는 항체의 높은 조직 선택성을 이용하여 강한 항암 효능을 가진 페이로드(payload)를 암 조직에만 선택적으로 전달하는 신약 플랫폼이다. ADC는 pM 수준의 낮은 농도에서도 암세포를 사멸하는 강력한 Payload를 암 조직에만 선택적으로 전달하고, 전신으로의 약물 노출은 최소화하여 항암 효능과 안전성을 동시에 확보할 수 있다.Antibody-drug conjugate (ADC) is a new drug platform that utilizes the high tissue selectivity of antibodies to selectively deliver a payload with strong anticancer efficacy only to cancerous tissues. ADC selectively delivers a powerful payload that kills cancer cells even at concentrations as low as pM, and minimizes systemic exposure to the drug, ensuring anticancer efficacy and safety at the same time.
ADC는 약물, 단클론 항체, 그리고 항체와 약물을 연결하는 링커 (linker)를 포함한 세 가지 구성 요소로 구성되어 있으며, ADC 테크놀로지는 암세포의 표면에 발현된 특정 항원에 특이적으로 결합하는 항체를 사용하여 약물을 종양세포에 전달하는 방법이다. 대부분 ADC의 세포 내 도입은 clathrin-coated pit 기능으로 진행된다. 세포 내로 이동된 ADC는 clathrin에서 떨어지고 세포 내 다른 vesicles와 융합한 다음 endosome-lysosome 경로로 진행된다. 이어서 endosomes의 산성 환경에 있는 proteases가 링커를 절단하고, 활성화(active)된 “free” 약물은 lysosomal membrane을 통과하여 cytoplasm로 이동한 후 약물의 molecular 타겟에 결합함으로써 종양세포의 세포주기는 정지되고 apoptosis로 인해 암세포가 죽게 된다. 이중 일정 양의 약물은 세포에서 수동적 확산되거나(passive diffusion), 능동적으로 수송되거나(active transport), 또는 죽은 세포를 통해 세포 밖으로 유출된다. 이때, 유출된 약물이 세포막 투과성을 지니면 주변 세포에도 들어가 소위 by-stander cell-killing 현상이 일어날 수도 있다.ADC consists of three components, including a drug, a monoclonal antibody, and a linker that connects the antibody and the drug. ADC technology uses antibodies that specifically bind to specific antigens expressed on the surface of cancer cells. This is a method of delivering drugs to tumor cells. In most cases, intracellular transduction of ADC proceeds through the clathrin-coated pit function. ADC moved into the cell detaches from clathrin, fuses with other vesicles within the cell, and then proceeds to the endosome-lysosome pathway. Then, proteases in the acidic environment of endosomes cleave the linker, and the activated “free” drug passes through the lysosomal membrane, moves to the cytoplasm, and binds to the drug’s molecular target, thereby arresting the cell cycle of tumor cells and causing apoptosis. This causes cancer cells to die. Among these, a certain 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 may enter surrounding cells and cause so-called by-stander cell-killing.
링커는 혈류에 안정(stable)하여 약물이 항체로부터 분리되는 것을 막아 타겟에 도달할 때까지 prodrug 상태로 유지되어 정상적인 조직에 입히는 피해를 최소화해야 한다. 또한, ADC는 항체가 약물과 결합되기 전의 항체와 같은 친화력을 유지해야 한다. 즉, 항체에 결합된 약물로 인해 항체-항원 결합에 지장이 없어야 한다. The linker must be stable in the bloodstream, preventing the drug from separating from the antibody, maintaining it in a prodrug state until it reaches the target, and minimizing damage to normal tissues. Additionally, the ADC must maintain the same affinity as the antibody before it is combined with the drug. In other words, the drug bound to the antibody should not interfere with antibody-antigen binding.
표적 세포에 결합한 후에는, receptor-mediated endocytosis라는 프로세스에 의해 ADC는 세포 내로 내재화(internalize)된다. 이때 충분한 농도의 활성 약물이 세포 내로 들어가야 하지만, 항원-항체 complex에 의한 internalization 과정은 일반적으로 비효율적이고 세포 표면에 있는 항원의 수도 일반적으로 <1 × 105 receptors/cell로 제한되어 있어서, 매우 강력한 약물을 사용하여 낮은 농도의 약물에서도 충분히 종양 세포를 죽일 수 있어야 한다. 따라서 항체에 결합되어 ADC로 사용될 약물은 보편적으로 사용되는 항암제보다 100-1000배 이상 세포독성이 있는 약물을 사용한다.After binding to the target cell, ADC is internalized into the cell by a process called receptor-mediated endocytosis. At this time, a sufficient concentration of active drug must enter the cell, but the internalization process by the antigen-antibody complex is generally inefficient and the number of antigens on the cell surface is generally limited to <1 × 10 5 receptors/cell, making it a very powerful drug. It must be possible to sufficiently kill tumor cells even at low concentrations of the drug. Therefore, drugs bound to antibodies and used as ADCs are drugs that are 100 to 1,000 times more cytotoxic than commonly used anticancer drugs.
강력한 세포독성약물을 특정 암세포에만 전달하기 위해서는 표적화할 항원을 결정하는 것이 ADC 개발의 첫 번째 주요 단계이다. 항체를 사용함으로써 표적에 대한 높은 특이성과 긴 반감기로 장기적인 전신 순환을 가능하게 하는데 이로 인해 세포독성약물을 종양세포에만 선택적으로 축적이 가능하게 하고 정상조직의 노출을 최소화해 손상을 줄여 부작용을 줄이고 치료효과를 증가시킬 수 있다. 이를 위해 종양세포를 특정할 수 있는 표적 항원을 찾아야 하는데 다음과 같은 조건이 필요하다. 첫 번째로 표적 항원은 종양 세포 표면에서 균일하게 과발현되어야 하며 정상 세포에서는 상대적으로 발현이 적거나 없어야 한다. 대표적인 예로 Human epidermal growth factor receptor 2 (HER2)가 있으며 HER2 양성 유방암에서는 정상 세포보다 100배 이상 더 많이 발현이 되는 것으로 알려져 있다. 그래서 항체를 만들기 전에 다양한 프로파일링을 통해 표적 항원의 종양 발현을 분석하여 특정 항원의 과발현을 확인하면 이 항원을 인식하는 단일 클론 항체를 생성한다. 두 번째는 항원에 대한 결합력인데 수용체를 매개로 내재화가 일어나는 항체의 특징으로 인해 항원의 에피토프 (epitope)에 결합하는 힘이 강할수록 더 많은 내재화가 일어날 수 있어 치료효과를 증가시킬 수 있기 때문이다. 추가적으로 낮은 면역원성이 있다. In order to deliver powerful cytotoxic drugs only to specific cancer cells, determining the antigen to be targeted is the first major step in ADC development. By using antibodies, high specificity for the target and long half-life enable long-term systemic circulation. This allows cytotoxic drugs to selectively accumulate only in tumor cells and minimizes exposure to normal tissues, thereby reducing damage, reducing side effects and providing treatment. The effect can be increased. To achieve this, a target antigen that can identify tumor cells must be found, and the following conditions are required. First, the target antigen must be uniformly overexpressed on the surface of tumor cells and have relatively low or no expression on normal cells. A representative example is Human epidermal growth factor receptor 2 (HER2), and it is known to be expressed more than 100 times more in HER2-positive breast cancer than in normal cells. Therefore, before making an antibody, the tumor expression of the target antigen is analyzed through various profiling, and if overexpression of a specific antigen is confirmed, a monoclonal antibody that recognizes this antigen is generated. The second is the binding ability to the antigen. Due to the characteristic of antibodies that internalization occurs through receptors, the stronger the binding force to the epitope of the antigen, the more internalization can occur, which can increase the therapeutic effect. Additionally, there is low immunogenicity.
암세포의 항원을 인지한 항체는 약물과 함께 세포 내재화가 일어나야 한다. 암세포에서 세포 내재화를 높이기 위해 이중특이성 항체(bispecific antibody)가 개발되고 있다. Antibodies that recognize antigens on cancer cells must be internalized into cells together with the drug. Bispecific antibodies are being developed to increase cell internalization in cancer cells.
SN-38은 여러 세포주에서 나노몰 범위의 IC50 값을 갖는 강력한 토포이소머라제-I 억제제이다. 결장직장암 치료에 사용되는 전구약물인 이리노테칸의 활성 형태이며 폐암, 유방암 및 뇌암에서도 활성을 나타낸다. Trop-2-SN-38 ADC의 경우 현재 TNBC, 방광암, 위암 등 다수의 암종에서 성공적으로 개발되고 있으나, 여전히 Drug efflux transporter 과발현, epigenetic silencing of Top1, anti-apoptotic protein 증가 등으로 인한 내성 문제는 남아 있다.SN-38 is a potent topoisomerase-I inhibitor with IC 50 values in the nanomolar range in several cell lines. It is the active form of irinotecan, a prodrug used to treat colorectal cancer, and is also active in lung, breast, and brain cancer. In the case of Trop-2-SN-38 ADC, it is currently being successfully developed in a number of cancer types such as TNBC, bladder cancer, and stomach cancer, but resistance problems due to drug efflux transporter overexpression, epigenetic silencing of Top1, and increased anti-apoptotic protein still remain. there is.
US 9629926 B에 따르면, hRS7-CL2A-SN-38의 체내분포는 모 hRS7 IgG와 유사한 종양에 의해 흡수(tumor uptake)되나, SN-38의 소수성으로 인해 2배 더 높은 간 흡수로 빠르게 제거된다. 상기 ADC는 간을 통해 제거됨에 따라, 간 및 위장 독성은 용량 제한적인 것으로 예상되었다. According to US 9629926 B, the biodistribution of hRS7-CL2A-SN-38 is uptaken by tumors similar to the parent hRS7 IgG, but is quickly eliminated with a two-fold higher liver uptake due to the hydrophobicity of SN-38. As the ADC is eliminated through the liver, hepatic and gastrointestinal toxicity was expected to be dose limiting.
한편, 다이이치산쿄(Daiichi Sankyo)는 엔허투(Enhertu®)의 개발에 SN-38보다 암세포에서 10배 정도 활성이 높은 세포독성약물인 DXD (exatecan계)를 사용하였다. DXD는 용해성이 좋고 비교적 안전하며 주변세포살상효과가 높아서 비균질종양의 치료에 이점이 있다. 그러나, 오프타겟 효과를 줄일 수 있는 반감기는 짧다. DXD는 항-HER2 항체의 시스테인 잔기에 maleimide 링커로 생접합되었는데 균질 DAR 값이 8에 달한다. 높은 DAR 값에도 불구하고 DXD가 혈장에서 21일 동안 단 2.1%만 방출되었을 정도로 안정성이 높다(Ogitani et al., 2016). 엔허투는 2019년 US FDA에서 허가를 받았는데 대상 환자는 과거 2번 이상 HER2 표적치료를 받은 전력이 있는 절제 불가능한 전이성 Her2 양성 유방암 성인 환자이다.Meanwhile, Daiichi Sankyo used DXD (exatecan), a cytotoxic drug that is 10 times more active in cancer cells than SN-38, in the development of Enhertu ® . DXD has good solubility, is relatively safe, and has a high killing effect on surrounding cells, making it advantageous in the treatment of heterogeneous tumors. However, the half-life to reduce off-target effects is short. DXD was bioconjugated to the cysteine residue of the anti-HER2 antibody with a maleimide linker, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD is highly stable, with only 2.1% released in plasma over 21 days (Ogitani et al., 2016). Enhertu was approved by the US FDA in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have received HER2-targeted therapy at least twice in the past.
Trodelvy, Enhertu 등 캄토테신 유도체를 Payload로 사용하는 항체-약물 접합체 (ADC)들은 암세포에만 선택적으로 강력한 항암 효능을 보이는 캄토테신 화합물을 전달하여 심한 전신 부작용 없이도 우수한 항암 효능을 가져오는데 성공하고 있으나, 아직 여러 부족한 부분을 보이고 있으며, 가장 대표적으로는 ABCG2 Drug Efflux Pump의 과발현에 의한 저항성 발생을 들 수 있다. 많은 캄토테신 유도체들은 ABCG2에 의하여 세포 밖으로 빠르게 배출되며, 이로 인하여 ABCG2를 발현하지 않거나 정상 수준으로 낮게 발현하고 있는 암 세포에 비하여, ABCG2를 과발현하는 암세포에서는 치료 효능이 심각하게 낮아지는 문제를 보인다. 이러한 ABCG2의 과발현은 Irinotecan, Topotecan 등 small molecule 캄토테신 화합물뿐아니라, 캄토테신 화합물을 payload로 사용하는 Trodelvy, Enhertu 등의 ADC에서도 치료 효능을 떨어뜨리는 주된 원인이 되고는 한다.Antibody-drug conjugates (ADCs) that use camptothecin derivatives as payload, such as Trodelvy and Enhertu, have succeeded in delivering excellent anticancer efficacy without severe systemic side effects by selectively delivering camptothecin compounds that show strong anticancer efficacy only to cancer cells. It shows several shortcomings, the most representative of which is resistance caused by overexpression of the ABCG2 Drug Efflux Pump. Many camptothecin derivatives are quickly exported out of cells by ABCG2, and as a result, cancer cells that overexpress ABCG2 show a problem of seriously reduced therapeutic efficacy compared to cancer cells that do not express ABCG2 or express ABCG2 at a low normal level. Overexpression of ABCG2 is the main cause of reduced therapeutic efficacy not only in small molecule camptothecin compounds such as Irinotecan and Topotecan, but also in ADCs such as Trodelvy and Enhertu that use camptothecin compounds as payload.
한편, 캐사일라의 DM1과 대조적으로 엔허투의 DXd는 막 투과성이 높았고 그 결과 세포 내부에서 방출된 페이로드가 표적 세포 근처에 인접한 HER2를 발현하지 않는 세포로 전달된다는 연구 결과 또한 보고됐다. 이는 엔허투가 페이로드의 특성으로 인해 잠재적인 방관자살상효과(bystander effect)를 가지고 허셉틴 혹은 캐사일라 불응성 환자에게도 임상적 이점을 제공할 수 있다.Meanwhile, in contrast to Kadsyla's DM1, Enhertu's DXd had high membrane permeability, and as a result, research results showed that the payload released inside the cell was delivered to cells that do not express HER2 adjacent to the target cell. Due to the nature of Enhertu's payload, this could provide clinical benefit to Herceptin or Kaesyla refractory patients as well, with a potential bystander effect.
본 발명자들은 탁월한 제1형 토포이소머라제 저해 능력과 함께 종양단백질(oncoprotein) DDX5(p68)를 분해하는 dual MoA를 갖는 캄토테신계 약물인 FL118 화합물을 합성하여, 이의 물리화학적 특성에 대한 다양한 연구를 계속 수행하고 있었다. 그러나, FL118 화합물은 물리화학적 특성의 제한으로 인해 제형 개발의 한계가 있었다. The present inventors synthesized compound FL118, a camptothecin-based drug with excellent type 1 topoisomerase inhibition ability and dual MoA that decomposes the oncoprotein DDX5 (p68), and conducted various studies on its physicochemical properties. was continuing to be performed. However, the FL118 compound had limitations in formulation development due to limitations in physicochemical properties.
이에 본 발명자들은 SN38 약물과 차별화되는 FL118 화합물의 구조를 바탕으로 FL118 화합물의 장점인 제1형 토포이소머라제 저해 및 DDX5 분해 측면에서 이중 작용기전(dual MoA)를 발휘하는 구조의 캄토테신 유도체를 설계하는 도중, 엑사테칸 또는 Dxd과 같은 캄토테신 유도체도 DDX5 단백질을 분해하는 작용기전(MoA)를 발휘한다는 것을 발견하였다(도 4 및 도 5). Accordingly, based on the structure of the FL118 compound, which is differentiated from the SN38 drug, the present inventors developed a camptothecin derivative with a structure that exhibits 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, it was discovered that camptothecin derivatives such as exatecan or Dxd also exert a mechanism of action (MoA) to degrade DDX5 protein (Figures 4 and 5).
따라서, 본 발명은 제1형 토포이소머라제 저해 능력과 함께 종양단백질(oncoprotein) DDX5를 분해하는 이중 작용기전(dual MoA)을 가지는 화학식 1의 캄토테신계 약물을 종양 조직과 같은 표적 부위에서만 주로 방출하도록 산 민감성 링커를 통해 연결시킨 이의 프로드럭인 항체-약물 접합체(ADC)를 체액 내 응집문제 없이 새롭게 설계 및 제공하고자 한다.Therefore, the present invention provides a camptothecin-based drug of Formula 1, which has a type 1 topoisomerase inhibitory ability and a dual MoA to decompose the oncoprotein DDX5, mainly only at target sites such as tumor tissue. We aim to design and provide a new antibody-drug conjugate (ADC), which is a prodrug linked through an acid-sensitive linker for release, without aggregation problems in body fluids.
또한, 본 발명은 SN-38를 페이로드로 하는 ADC(예, hRS7-CL2A-SN-38)의 응집문제 및 간 독성으로 인한 용량 제한 문제를 해결하기 위해 및/또는 SN-38의 상대적으로 약한(충분하지 않은) 효력을 개선하기 위해서, 산 민감성 링커의 일 말단을 캄토테신계 유도체의 E-고리의 20번 탄소에 위치한 알파수산화기에 연결 시 SN-38 보다 분자구조상 수소결합 강도가 강화된 작용기 및 배향을 가질 뿐만아니라 표적 세포를 사멸시키는 약물 후보로, DDX5 단백질에 결합하도록 설계된 화학식 1의 캄토테신계 약물(예, 엑사테칸 또는 Dxd)를 페이로드로 사용하고자 하며, 이로인해 화학식 1의 캄토테신계 약물을 산 민감성(acid-sensitive) 링커에 연결시킴으로써 접합체의 수분산성을 향상시키고자 한다. In addition, the present invention is intended to solve the aggregation problem of ADC (e.g., hRS7-CL2A-SN-38) using SN-38 as a payload and the capacity limitation problem due to liver toxicity and/or the relatively weak SN-38. In order to improve the efficacy (which is not sufficient), when one end of the acid-sensitive linker is connected to the alpha hydroxyl group located at carbon 20 of the E-ring of the camptothecin derivative, a functional group with stronger hydrogen bonding strength in the molecular structure than SN-38 is created. As a drug candidate that not only has and orientation but also kills target cells, the camptothecin-based drug of Formula 1 (e.g., exatecan or Dxd) designed to bind to the DDX5 protein is intended to be used as a payload, resulting in the drug of Formula 1 We aim to improve the water dispersibility of the conjugate by linking a camptothecin-based drug to an acid-sensitive linker.
또한, 본 발명은 (i) 원하는 간청소율(hepatic clearance) 프로파일 및 혈액 순환 프로파일을 제공하도록 및/또는 (ii) 종양조직 심부까지 침투하면서 주변세포살상효과(bystander effect)가 발휘 또는 그 효과의 정도가 제어되도록, 화학식 1의 캄토테신계 약물(예, 엑사테칸 또는 Dxd)이 산 민감성(acid-sensitive) 링커에 연결된 다양한 페이로드-산 민감성 링커 조합의 접합체를 설계하고자 한다. In addition, the present invention (i) provides a desired hepatic clearance profile and blood circulation profile and/or (ii) penetrates deep into the tumor tissue to exert a bystander effect or the extent of the effect. In order to control , we intend to design conjugates of various payload-acid sensitive linker combinations in which a camptothecin drug of Formula 1 (e.g., exatecan or Dxd) is connected to an acid-sensitive linker.
나아가, 본 발명은 엑사테칸을 모핵으로 하는 화학식 1의 캄토테신 유도체가 방관자 효과(bystander effect)와 함께, 세포 내에서 DDX5 단백질에 결합하여 DDX5 단백질 분해를 통해 세포사멸(cell death)을 유도하게 함으로써, 내인성 또는 후천성 약물 내성 이전에 암세포 및/또는 주변세포를 사멸시키는 새로운 모달리티로서 항암기전을 제공하고자 한다.Furthermore, the present invention provides that the camptothecin derivative of Formula 1, which has exatecan as its parent nucleus, has a bystander effect and binds to DDX5 protein within cells to induce cell death through DDX5 protein degradation. By doing so, we aim to provide an anti-cancer mechanism as a new modality that kills cancer cells and/or surrounding cells before endogenous or acquired drug resistance.
본 발명의 제1양태는 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하여, 화학식 1의 캄토테신계 약물을 생체내 표적부위에서 방출하도록 설계된 면역접합체 또는 이의 약학적으로 허용가능한 염으로서, The first aspect of the present invention is a camptothecin-based drug of Formula 1, including [camptothecin-based drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof]. An immunoconjugate or a pharmaceutically acceptable salt thereof designed to be released at a target site in vivo,
화학식 1의 캄토테신계 약물은 DDX5 단백질 및/또는 E3 리가아제에 결합하도록 설계된 페이로드(payload)이고,The camptothecin-based drug of Formula 1 is a payload designed to bind to DDX5 protein and/or E3 ligase,
항체 또는 이의 항원결합부위 함유 단편에 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물이 하나 이상 연결되어 있고,At least one camptothecin-based drug of Formula 1 is linked to the antibody or its antigen-binding site-containing fragment through an acid-sensitive linker,
암세포의 항원을 표적화하는 항원결합부위에 의해 암세포로 표적화된 후, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되고, 유리형 화학식 1의 캄토테신계 약물은 세포막을 관통해 세포내로 이동하고, After being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ≤ 7) surrounding the cancer, and at least part of the camptothecin-based drug of Formula 1 is released, and the free form of Formula 1 Camptothecin drugs penetrate the cell membrane and move into the cell.
선택적(optionally)으로, 화학식 1의 캄토테신계 약물이 연결되어 있는 면역접합체는 세포안으로 내재화(internalization)되어 리소좀(lysosomes)에서 화학식 1의 캄토테신계 약물이 유리되는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염을 제공한다.Optionally, the immunoconjugate to which the camptothecin drug of Formula 1 is linked is internalized into cells and the camptothecin drug of Formula 1 is released from lysosomes. An immunoconjugate or pharmaceutical thereof Provides an acceptable salt.
[화학식 1][Formula 1]
Figure PCTKR2023006170-appb-img-000001
Figure PCTKR2023006170-appb-img-000001
X1 및 X3는 각각 독립적으로 탄소, 산소, 질소, 또는 황이고, X1 및 X3는 동일 또는 상이할 수 있으며,X 1 and X 3 are each independently carbon, oxygen, nitrogen, or sulfur, and X 1 and X 3 may be the same or different,
X2는 탄소, 산소, 질소, 황, 단일결합 또는 이중결합이고,X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond,
X1, (X2)n 및 X3는 5각, 6각 또는 7각 고리를 형성할 수 있으며(n=0~2의 값),X 1 , (X 2 )n and
Y1, Y2 및 Y3는 각각 독립적으로 수소이거나, 또는 산소, 질소, 인 또는 황을 포함하는 작용기일 수 있음.Y 1 , Y 2 and Y 3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.
본 발명의 제2양태는 제1양태의 면역접합체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공한다.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.
예컨대, HER2 양성 암, 유방암, 폐암, 대장암을 치료하기 위해 투여하는 것일 수 있다.For example, it may be administered to treat HER2-positive cancer, breast cancer, lung cancer, and colon cancer.
바람직하게는, 암 예방 또는 치료용 약학 조성물은 암 진단 후 1차 치료제로 사용하거나 또는 암 조직에서 DDX5을 (과)발현하는 개체에 투여할 수 있다.Preferably, the pharmaceutical composition for preventing or treating cancer can be used as a primary treatment after cancer diagnosis or administered to an individual that (over)expresses DDX5 in cancer tissue.
본 발명의 제3양태는 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 화학식 3의 산 민감성 링커에 연결된, 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염을 제공한다.The third aspect of the present invention is a camptothecin system of Formula 1-1 or Formula 1-2. Provided is a drug-linker conjugate, or a pharmaceutically acceptable salt thereof, wherein the drug is linked to an acid-sensitive linker of formula (3).
[화학식 1-1][Formula 1-1]
Figure PCTKR2023006170-appb-img-000002
Figure PCTKR2023006170-appb-img-000002
[화학식 1-2][Formula 1-2]
Figure PCTKR2023006170-appb-img-000003
Figure PCTKR2023006170-appb-img-000003
[화학식 3][Formula 3]
Figure PCTKR2023006170-appb-img-000004
Figure PCTKR2023006170-appb-img-000004
여기서, X1 및 X2는 각각 독립적으로 -H 또는 -할로젠이고;Here, X 1 and X 2 are each independently -H or -halogen;
Y는 -NH-, -NRA-, 또는 아무 것도 아니며 (null); Y is -NH-, -NR A -, or nothing (null);
Z는 -C1-C4알킬-, -C3-C6시클로알킬-, -(C1-C2알킬)-(C3-C6시클로알킬)-, -(C3-C6시클로알킬)-(C1-C2알킬)-, 또는 -(C1-C2알킬)-(C3-C6시클로알킬)-(C1-C2알킬)-이고; Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
W는 -RB-, -M- -RB-M-, -M-RB- 또는 -RB-M-RC-이며; W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
RA 내지 RC는 각각 독립적으로 C1-C4알킬이고, R A to R C are each independently C 1 -C 4 alkyl,
M은
Figure PCTKR2023006170-appb-img-000005
이며; 및M is
Figure PCTKR2023006170-appb-img-000005
and; and
n은 5 내지 9의 정수임.n is an integer from 5 to 9.
본 발명의 제4양태는 (a) 제3양태의 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염; 및 (b) 항체 또는 이의 항원결합부위 함유 단편을 포함하되, 항체 또는 이의 항원결합부위 함유 단편에 화학식 3의 산 민감성 링커를 통해 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 하나 이상 연결되어 있는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염을 제공한다.A fourth aspect of the present invention provides (a) the drug-linker conjugate of the third aspect or a pharmaceutically acceptable salt thereof; and (b) an antibody or an antigen-binding site-containing fragment thereof, wherein the antibody or an antigen-binding site-containing fragment contains at least one camptothecin-based drug of Formula 1-1 or Formula 1-2 through an acid-sensitive linker of Formula 3. An immunoconjugate characterized by being linked or a pharmaceutically acceptable salt thereof is provided.
본 발명의 제5양태는 제3양태의 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염을 사용하여, 운반체(Carrier)에 화학식 3의 산 민감성 링커를 통해 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물을 하나 이상 연결시키는 것이 특징인 운반체-약물 접합체(Carrier-Drug Conjugate)의 제조방법을 제공한다.The fifth aspect of the present invention uses the drug-linker conjugate of the third aspect or a pharmaceutically acceptable salt thereof to form a drug of Formula 1-1 or Formula 1-2 through an acid-sensitive linker of Formula 3 in a carrier. A method for manufacturing a carrier-drug conjugate characterized by linking one or more camptothecin drugs is provided.
이하, 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 명세서에서, 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물은 각각 엑사테칸(exatecan) 또는 Dxd로 혼용 사용한다.In this specification, camptothecin-based drugs of Formula 1-1 or Formula 1-2 are used interchangeably as exatecan or Dxd, respectively.
본 명세서에서, 암 및 종양은 서로 혼용하여 사용될 수 있다.In this specification, cancer and tumor may be used interchangeably.
본 발명에서, 용어 "약물-링커 접합체"는 면역접합체 또는 운반체-약물 접합체(Carrier-Drug Conjugate)의 제조를 위한 물질로서 항체, 이의 항원결합부위 함유 단편 또는 운반체가 연결되지 않은 것을 의미할 뿐만아니라, 목적하는 바에 따라 임의의 항체, 이의 항원결합부위 함유 단편, 또는 운반체와 결합하여 면역접합체 또는 운반체-약물 접합체(Carrier-Drug Conjugate)로도 사용될 수 있다. In the present invention, the term "drug-linker conjugate" refers to a material for the production of an immunoconjugate or carrier-drug conjugate, which not only refers to an antibody, an antigen-binding site-containing fragment thereof, or a carrier that is not linked. Depending on the purpose, it can be used as an immunoconjugate or carrier-drug conjugate by combining with any antibody, antigen-binding site-containing fragment thereof, or carrier.
본 명세서에서, 화학식 1의 캄토테신계 약물은In this specification, the camptothecin-based drug of Formula 1 is
(1) 화학식 1로 표시되는 캄토테신계 골격을 모핵으로 포함한 화합물 라이브러리에서 제1형 토포이소머라제를 저해하는 작용기전(MoA) 및/또는 종양단백질(oncoprotein) DDX5를 분해하는 작용기전(MoA)을 갖도록 설계된 활성형 캄토테신 유도체를 선택하고/하거나,(1) A mechanism of action (MoA) to inhibit type 1 topoisomerase and/or to decompose the oncoprotein DDX5 (MoA) in a compound library containing the camptothecin-based skeleton represented by Formula 1 as a parent nucleus. ) and/or select an active camptothecin derivative designed to have
(2) 화학식 1로 표시되는 캄토테신계 골격을 모핵으로 포함한 화합물이 제1형 토포이소머라제 저해 및/또는 종양단백질(oncoprotein) DDX5를 분해시키는지 인비트로(in vitro) 실험 및/또는 인비보(in vivo) 실험을 통해 확인하는 단계를 통해 제공될 수 있다.(2) In vitro and/or in vitro experiments to determine whether a compound containing the camptothecin-based skeleton represented by Formula 1 as its mother nucleus inhibits type 1 topoisomerase and/or decomposes the oncoprotein DDX5. This can be provided through confirmation through in vivo experiments.
약의 특성을 알아야 약을 제대로 사용할 수 있다. 약의 특성을 이해하는데 약물의 약력학, 약동학적 파라미터는 도움을 준다. Knowing the properties of a drug can help you use it properly. Pharmacodynamic and pharmacokinetic parameters of a drug are helpful in understanding the properties of a drug.
약력학은 약이 수용체에 결합한 후 일어나는 세포나 몸에서 일어나는 변화(약효, effect) (cell viability, clinical effect)(therapeutic action, toxic effect, adverse effect )의 크기와 양상을 약물농도와의 관계로 설명한다. Pharmacodynamics explains the size and pattern of changes (cell viability, clinical effect) (therapeutic action, toxic effect, adverse effect) that occur in cells or the body after a drug binds to a receptor in terms of their relationship with drug concentration. .
PK(Pharmaco-kinetics)는 약물 또는 약물의 모달리티에 따라 ADME을 통해 신체의 다른 구획(Compartment)을 통해 이동할 때 약물 농도는 어떻게 변하는지를 보여준다.Pharmaco-kinetics (PK) shows how drug concentration changes as it moves through different compartments of the body through ADME, depending on the drug or its modality.
약동학적 측면에서 신약 개발시 실패이유는, 독성 약물(Toxic drugs)이 축적될 수 있으며, 유용한 약물(Useful drugs)은 치료를 확립하기에는 복용량(doses)이 너무 적기 때문에 이점이 없을 수 있고, 약물이 빠르게 대사될 수 있기 때문이다.In terms of pharmacokinetics, the reasons for failure when developing new drugs are that toxic drugs may accumulate, useful drugs may have no benefit because the doses are too small to establish treatment, and the drugs may not be effective. This is because it can be metabolized quickly.
따라서, 약을 제대로 사용하기 위해 적절한 약물을 선택 및 적절한 용량(dose)-용법(dosage)을 적용하여야 한다. Therefore, in order to use the drug properly, the appropriate drug must be selected and the appropriate dose-administration must be applied.
용량(Dose)은 한 번에 투여되는 약물의 양을 나타낸다. 약물의 용량은 일반적으로 환자의 나이, 체중 및 건강 상태와 같은 요인에 따라 의료 제공자가 처방한다. 반면, 용법(Dosage)은 약물 투여 빈도와 기간을 나타낸다. 일정 기간 동안 주어진 총 약물 양을 측정한 것으로 일반적으로 일일 또는 주간 양으로 표시된다. 용량(Dose)과 용법(Dosage)은 약물의 안전하고 효과적인 사용에 중요한 고려 사항이다.Dose refers to the amount of drug administered at one time. The dosage of the drug is usually prescribed by a health care provider based on factors such as the patient's age, weight, and health status. On the other hand, dosage refers to the frequency and duration of drug administration. A measure of the total amount of drug given over a period of time, usually expressed as daily or weekly amounts. Dose and dosage are important considerations in the safe and effective use of drugs.
한편, 항암제의 생체 내 효능(in vivo efficacy) 및 생체 내 부작용(in vivo side effects)은 항암제의 흡수, 분포, 대사 및 배설(ADME) 특성과 밀접한 관련이 있다. 약물의 ADME는 약물이 신체(body)를 통해 이동하고 조직 및 기관과 상호 작용하는 방식을 설명하는 약동학을 결정한다.Meanwhile, the in vivo efficacy and in vivo side effects of anticancer drugs are closely related to the absorption, distribution, metabolism and excretion (ADME) characteristics of anticancer drugs. A drug's ADME determines its pharmacokinetics, which describes how the drug moves through the body and interacts with tissues and organs.
약물의 흡수(Absorption)는 효능과 부작용에 영향을 미칠 수 있다. 잘 흡수되지 않는 약물은 표적 조직(target tissue)에서 치료 농도에 도달하지 못하여 효능이 감소될 수 있다. 반면에 흡수가 잘 되는 약물은 전신노출을 증가시켜 비표적 부작용을 일으킬 수 있다.Absorption of a drug can affect its efficacy and side effects. Drugs that are poorly absorbed may not reach therapeutic concentrations in target tissues, resulting in reduced efficacy. On the other hand, drugs that are well absorbed may increase systemic exposure and cause off-target side effects.
약물의 분포(Distribution)도 효능과 부작용에 영향을 미칠 수 있다. 표적 조직에 잘 분포되지 않는 약물은 효과가 없을 수 있고, 비표적 조직(non-target tissues)에 광범위하게 분포하는 약물은 해당 조직에서 독성을 유발할 수 있다. 또한 혈장 단백질과 결합력이 높은 약물은 표적 조직으로의 분포가 감소하여 효능이 감소할 수 있다.Distribution of a drug can also affect efficacy and side effects. Drugs that are poorly distributed in target tissues may be ineffective, and drugs that are widely distributed in non-target tissues may cause toxicity in those tissues. Additionally, drugs with high binding affinity to plasma proteins may have reduced distribution to target tissues, resulting in reduced efficacy.
신체 내 약물 분포는 크기, 전하 및 친유성을 포함한 물리화학적 특성에 의해 영향을 받을 수 있다. 종양 조직에 침투하는 약물의 능력은 혈류 및 세포 밀도를 포함한 종양의 미세 환경과 같은 요인에 의해 영향을 받을 수도 있다. 일부 항암제는 특정 조직에 축적되어 독성 효과를 유발할 수 있다. Drug distribution in the body can be influenced by its physicochemical properties, including size, charge, and lipophilicity. The ability of a drug to penetrate tumor tissue may also be affected by factors such as the tumor's microenvironment, including blood flow and cell density. Some anticancer drugs can accumulate in certain tissues and cause toxic effects.
따라서, 항암제의 ADME 특성을 분석하여 생체 내에서 효능을 최적화하고 부작용을 최소화하는 것이 필수적이다. 즉, ADME 프로파일을 평가하고 최적화하면 이러한 약물의 치료 지수를 개선하여 암 환자에게 더 나은 결과를 가져올 수 있다.Therefore, it is essential to analyze the ADME properties of anticancer drugs to optimize their efficacy and minimize side effects in vivo. In other words, evaluating and optimizing the ADME profile can improve the therapeutic index of these drugs, resulting in better outcomes for cancer patients.
놀랍게도, 본 발명자들은 후술하는 바와 같이 SN38 약물과 차별화되는 화학식 2의 FL118 약물의 구조를 바탕으로 FL118 약물의 장점인 제1형 토포이소머라제 저해 및 DDX5 분해 측면에서 이중 작용기전(dual MoA)를 발휘하는 구조의 캄토테신 유도체를 설계하는 도중(도 1), 엑사테칸 또는 Dxd도 DDX5 단백질을 분해하는 작용기전(MoA)를 발휘한다는 것을 발견하였다(도 4 및 도 5). Surprisingly, the present inventors, as described later, based on the structure of the FL118 drug of Chemical Formula 2, which is differentiated from the SN38 drug, developed a dual mechanism of action (dual MoA) in terms of type 1 topoisomerase inhibition and DDX5 degradation, which are the advantages of the FL118 drug. While designing a camptothecin derivative with a structure that exerts a structure (Figure 1), it was discovered that exatecan or Dxd also exerts a mechanism of action (MoA) to degrade the DDX5 protein (Figures 4 and 5).
[화학식 2][Formula 2]
Figure PCTKR2023006170-appb-img-000006
Figure PCTKR2023006170-appb-img-000006
놀랍게도, 엑사테칸(Exatecan) 약물은 FL118 약물과 같이, 또 다른 항암제 내성의 주원인인 항세포사멸 단백질(anti-apoptotic protein)(Survivin, cIAP2, XIAP 등)의 발현을 낮은 농도에서 강하게 억제할 뿐만 아니라, 이로인해 약물 내성 기전을 차단할 수 있다는 것을 발견하였다(도 4 및 도 5). Surprisingly, the Exatecan drug, like the FL118 drug, not only strongly inhibits the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another main cause of anticancer drug resistance, at low concentrations. Rather, it was discovered that this could block the drug resistance mechanism (Figures 4 and 5).
본 발명은 이러한 발견들에 기초한 것이다.The present invention is based on these findings.
따라서, 본 발명에서 [산민감성 링커]에 연결하여 페이로드로 사용하는 화학식 1의 캄토테신계 약물은 일반식 1에서 A고리상의 R1 및 R2 (Group A)가 화학식 2의 FL118 화합물과 유사하면서, 화학식 1-1의 화합물(엑사데칸) 및 화학식 1-2의 화합물(dxd)과 동일하게 설계함으로써(도 4), 세포내 종양단백질(oncoprotein) DDX5를 분해하는 항암기전을 발휘시키는 것이 특징이다(실시예 1).Therefore, in the present invention, the camptothecin-based drug of Formula 1 used as a payload by connecting to an [acid-sensitive linker] has R 1 and R 2 (Group A) on ring A in General Formula 1 similar to the FL118 compound of Formula 2. Meanwhile, by designing it in the same way as the compound of Formula 1-1 (exadecane) and the compound of Formula 1-2 (dxd) (Figure 4), it is characterized by exerting an anti-cancer mechanism that decomposes the intracellular oncoprotein DDX5. (Example 1).
[일반식 1][General Formula 1]
Figure PCTKR2023006170-appb-img-000007
Figure PCTKR2023006170-appb-img-000007
제1형 토포이소머라제 저해 능력과 함께 종양단백질(oncoprotein) DDX5를 분해하는 이중 작용기전(dual MoA)을 가지는 활성형 캄토테신 유도체의 합성 설계 개념(synthetic design concept)은, 도 1에 도시된 바와 같이 SAR(Structure Activity Relationship)을 기준으로 제1형 토포이소머라제 저해 영역인 Group C의 구조를 유지하고, DDX5 분해를 위한 결합 부위인 Group A 역시 FL118과 유사하면서, 화학식 1-1의 화합물(엑사데칸) 및 화학식 1-2의 화합물(dxd)과 동일한 구조를 유지하되, 일반식 1 중 Group B의 구조(R3 및 R4)를 화학식 1과 같이 조절함으로써 약물의 응집 문제를 해결하도록 약물의 물리화학적 특성을 개선할 뿐만 아니라, 약물의 세포독성(cytotoxicity) 및/또는 약물의 종양조직 침투 및/또는 세포막 투과도를 조절하여 방관자 살상효과(bystander effect)를 정밀하게 제어할 수 있는 구조로 디자인할 수 있다.The synthetic design concept of an active camptothecin derivative with a dual MoA that decomposes the oncoprotein DDX5 along with the ability to inhibit type 1 topoisomerase is shown in Figure 1. As shown, based on SAR (Structure Activity Relationship), the structure of Group C, which is the type 1 topoisomerase inhibition region, is maintained, and Group A, which is the binding site for DDX5 decomposition, is also similar to FL118, and the compound of Formula 1-1 (exadecane) and the compound (dxd) of Formula 1-2, but maintain the same structure, and adjust the structure of Group B (R 3 and R 4 ) in Formula 1 as in Formula 1 to solve the drug aggregation problem. It is a structure that not only improves the physicochemical properties of the drug, but also precisely controls the bystander effect by controlling the cytotoxicity of the drug and/or the tumor tissue penetration and/or cell membrane permeability of the drug. You can design it.
일반식 1 또는 화학식 2에서 Group C 부위는 제1형 토포이소머라제에 결합하고 Group A 부위는 DNA에 결합하여 토포이소머라제-DNA 복합체의 공유결합을 안정화시켜 절단된 DNA 조각들이 다시 연결되는 것을 방해하고/하거나, 일반식 1 또는 화학식 2에서 Group A 부위는 DDX5에 결합하고 Group C 부위는 E3 리가아제에 결합하여 DDX5 분해를 유도하는 것일 수 있다(본 명세서에 통합되는 PCT/KR2023/005380 참조).In General Formula 1 or Formula 2, the Group C site binds to type 1 topoisomerase and the Group A site binds to DNA to stabilize the covalent bond of the topoisomerase-DNA complex, allowing the cut DNA fragments to be reconnected. and/or, in General Formula 1 or Formula 2, the Group A site binds to DDX5 and the Group C site binds to E3 ligase to induce DDX5 degradation (PCT/KR2023/005380, incorporated herein by reference) reference).
본 발명은 일반식 1 중 R3 및/또는 R4 변형을 통해 세포막 투과도를 원하는 대로 조절하여 종양조직에서 적절한 방관자 효과(bystander effect)가 발휘되도록 화학식 1의 캄토테신계 약물을 설계하는 것이 바람직하다.In the present invention, it is desirable to design the camptothecin-based drug of Formula 1 to exert an appropriate bystander effect in tumor tissue by controlling the cell membrane permeability as desired through modification of R 3 and/or R 4 in Formula 1. .
따라서, 다양한 부작용 뿐만아니라 치료계수감소와 같은 문제점까지 고려하여, 적절한 항암 효능을 발휘하는 ADC의 페이로드의 선택 범위를, 분자접착제(molecular glue degrader)로 DDX5 단백질 및 E3 리가아제에 결합하도록 설계된, 화학식 1의 활성형 캄토테신 유도체들을 포함하는 다양한 후보군으로 확장시킬 수 있게 하는 것도 본 발명의 주요 특징이다.Therefore, considering not only various side effects but also problems such as reduced therapeutic coefficient, the selection range of ADC payload that exhibits appropriate anticancer efficacy is designed to bind to the DDX5 protein and E3 ligase as a molecular glue degrader, Another key feature of the present invention is that it can be expanded to a variety of candidates including active camptothecin derivatives of Formula 1.
[화학식 1][Formula 1]
Figure PCTKR2023006170-appb-img-000008
Figure PCTKR2023006170-appb-img-000008
X1 및 X3는 각각 독립적으로 탄소, 산소, 질소, 또는 황이고, X1 및 X3는 동일 또는 상이할 수 있으며,X 1 and X 3 are each independently carbon, oxygen, nitrogen, or sulfur, and X 1 and X 3 may be the same or different,
X2는 탄소, 산소, 질소, 황, 단일결합 또는 이중결합이고,X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond,
X1, (X2)n 및 X3는 5각, 6각 또는 7각 고리를 형성할 수 있으며(n=0~2의 값),X 1 , (X 2 )n and
Y1, Y2 및 Y3는 각각 독립적으로 수소이거나, 또는 산소, 질소, 인 또는 황을 포함하는 작용기일 수 있음.Y 1 , Y 2 and Y 3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.
여기서, 산소, 질소, 인 또는 황을 포함하는 작용기(functional group)의 비제한적인 예들은 -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR1)(OR2)(R1, R2=CsHtNuOwSxPyXz, X = -F, -Cl, -Br 또는 -I, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -SO3H, -OSO3H, -NO2, -N3, -NR3OH(R=CnH2n+1, 0≤n≤16), 　-NR3 +X-(R= CnHm, 0≤n≤16, 0≤m≤34, X = OH, Cl 또는 Br), NR4 +X-(R= CnHm, 0≤n≤16, 0≤m≤34, X = OH, Cl 또는 Br), 　-COSH, -COOCO-, -CORCO- (R = ClHm, 0≤l≤3, 0≤m≤2l+1), -COOR, -CN, -N3, -N2, -NROH(R = CsHtNuOwSxPyXz, X = -F, -Cl, -Br 또는 -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), -NR1NR2R3(R1, R2, R3　= CsHtNuOwSxPyXz, X = -F, -Cl, -Br 또는 -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), -CONHNR1R2(R1, R2　= CsHtNuOwSxPyXz, X = -F, -Cl, -Br 또는 -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), -NR1R2R3X’(R1, R2, R3　= CsHtNuOwSxPyXz, X = -F, -Cl, -Br 또는 -I, X’= F-,Cl-,Br-,또는 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 또는 I), -COOCO-, -CONH-, -CN, -SCOCH3, -SCN, -NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, 에폭시기, -하이드라존, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C≡C- 및 탄소 수 2개 이상의 탄화수소로 구성된 군으로부터 선택되는 작용기를 포함하는 것일 수 있다. Here, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur include -CHO, -COOH, -NH 2 , -SH, -CONH 2 , -PO 3 H, -PO 4 H 2 , -OPO 4 H, -PO 2 (OR 1 )(OR 2 ) (R 1 , R 2 =C s H t N u O w S x P y -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), -SO 3 H, -OSO 3 H, -NO 2 , -N 3 , -NR 3 OH (R=C n H 2n+1 , 0≤n≤16 ) , -NR 3 + = C n H m , 0≤n≤16, 0≤m≤34, X = OH, Cl or Br), NR 4 + X - (R= C n H m , 0≤n≤16, 0≤m≤ 34 , -N 3 , -N 2 , -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 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 ≤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-, -NO 2 , -COX (X = F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH 3 , -SCN, - NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, epoxy group, -hydrazone, -ONO 2 , -PO(OH) 2 , -C=NNH 2 , -HC= It may contain a functional group selected from the group consisting of CH-, -C=C-, -C≡C-, and hydrocarbons having 2 or more carbon atoms.
본 발명에 따라 DDX5 단백질 및/또는 E3 리가아제에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 분자접착제(molecular glue degrader) 기전(MoA)을 통해 DDX5 단백질을 발현하는 표적 세포를 사멸시킬 수 있다. The camptothecin-based drug of Formula 1, which is designed to bind to DDX5 protein and/or E3 ligase according to the present invention, can kill target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).
표적 세포는 암세포 또는 노화세포일 수 있다. 노화세포에는 장기 고유의 특징적인 기능을 수행하지 않는 세포도 포함한다.Target cells may be cancer cells or senescent cells. Senescent cells also include cells that do not perform organ-specific functions.
바람직하게는, 본 발명에 따라 DDX5 단백질 및/또는 E3 리가아제에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 제1형 토포이소머라제 저해 능력과 함께 종양단백질(oncoprotein) DDX5를 분해하는 다중 작용기전(MoA)를 갖는 것일 수 있다.Preferably, the camptothecin-based drug of Formula 1 designed to bind to DDX5 protein and/or E3 ligase according to the present invention has the ability to inhibit type 1 topoisomerase and has multiple actions to degrade the oncoprotein DDX5. It may have a mechanism (MoA).
본 발명에 따라 화학식 1, 예컨대 화학식 1-1 및 1-2로 표시되는 캄토테신 유도체는 도 1에 도시된 캄토테신과 같이 세포독성에 필수적인 E-고리에 락톤을 갖는 펜타사이클릭 구조를 가지며, 제1형 토포이소머라제-DNA 부산물의 안정을 위해 중요한 E-고리의 20번 탄소에 위치한 락톤기와 알파수산화기는 유지하도록 설계된 것으로, 일반식 1 중 Exatecan계 약물의 구조적 특징, 즉 (1) DDX5 단백질에 결합하는, FL118 약물(-OCH2O- (methylenedioxo) 5각형 링)과 배향 구조가 유사한 R1 및 R2 의 구조적 특징(CH3-C=C-F)을 유지하면서, (2) R3 및 R4을 통해, A- 및 B-고리가 형성하는 방향족 고리들의 π-π적층(stacking)에 의해 응집이 유도되는 SN-38 대비, 예컨대 A- 및 B-고리로부터 확장된 6각 또는 7각 고리의 연속적인 탄소-탄소 단일 결합의 다양한 배향들이 동적 평형을 이루어 A- 및 B-고리가 형성하는 방향족 고리들의 적층(stacking)이 약화 또는 억제가능하다.According to the present invention, camptothecin derivatives represented by Formula 1, such as Formulas 1-1 and 1-2, have a pentacyclic structure having a lactone in the E-ring essential for cytotoxicity, like camptothecin shown in Figure 1, Type 1 topoisomerase-DNA is designed to maintain the lactone group and alpha hydroxyl group located at carbon 20 of the E-ring, which are important for the stability of DNA by-products, and is designed to maintain the structural characteristics of Exatecan drugs in General Formula 1, namely (1) DDX5 While maintaining the structural features (CH 3 -C=CF) of R 1 and R 2 , which are similar in orientation to the protein-binding FL118 drug (-OCH 2 O- (methylenedioxo) pentagonal ring), (2) R 3 and through R 4 , compared to SN-38, where aggregation is induced by π-π stacking of aromatic rings formed by the A- and B-rings, such as hexagonal or 7-membered rings extended from the A- and B-rings. The various orientations of the successive carbon-carbon single bonds of each ring are in dynamic equilibrium so that the stacking of aromatic rings formed by the A- and B-rings can be weakened or suppressed.
이에 더하여, 화학식 1-1로 표시되는 화합물은, A- 및 B-고리로부터 확장된 6각 고리에 있는 탄소-탄소 단일 결합에 대하여 분자 결합의 회전이 가능하여 자유도가 큰 -NH2가 물(H2O)에 노출되어 (+) 전하를 띠거나 물과 수소결합하여 수분산성(water dispersibility)을 증가시킬 수 있다.In addition, the compound represented by Formula 1-1 is capable of rotating the molecular bond with respect to the carbon-carbon single bond in the hexagonal ring extended from the A- and B-rings, so that -NH 2 with a large degree of freedom is water ( When exposed to H 2 O), water dispersibility can be increased by taking on a (+) charge or by hydrogen bonding with water.
이에 더하여, 화학식 1-2 로 표시되는 화합물은, A- 및 B-고리로부터 확장된 6각 고리에 있는 탄소-탄소 단일 결합에 대하여 분자 결합의 회전이 가능하여 자유도가 큰 -NH2의 Lactic acid 형태의 작용기 CH2(OH)CONH-가 물(H2O)에 노출되어 프로펠러처럼 회전하면서 물과 수소결합하여 수분산성을 증가시킬 수 있다.In addition, the compound represented by Formula 1-2 is a Lactic acid of -NH 2 with a large degree of freedom as the molecular bond can be rotated about the carbon-carbon single bond in the hexagonal ring extending from the A- and B-rings. The functional group in the form of CH 2 (OH)CONH- is exposed to water (H 2 O) and rotates like a propeller, forming a hydrogen bond with water to increase water dispersibility.
한편, Enhertu에 사용되는 DXd payload는 원래는 ABCG2에 영향을 거의 받지 않는 Exatecan 화합물로부터 만들어졌지만, Exatecan을 DXd로 전환시키기 위하여 사용된 glycolic acid (alpha-hydroxy acetic acid) 관능기의 존재로 인하여 ABCG2에 의하여 강하게 영향을 받게 되었다. 하지만, glycolic acid 관능기는 Enhertu의 우수한 안전성/효력 프로파일에 매우 중요한 역할을 하는 것으로서, 이를 제거할 경우 ADC 제조 시의 난점과 동시에 동물 모델, 임상 시험에서 ADC의 성능이 나빠지는 (안전성 문제 대두 또는 효력의 감소) 문제를 가지고 온다. Meanwhile, the DXd payload used in Enhertu was originally made from the compound Exatecan, which is almost unaffected by ABCG2, but due to the presence of the glycolic acid (alpha-hydroxy acetic acid) functional group used to convert Exatecan to DXd, it is converted to DXd by ABCG2. was strongly influenced. However, the glycolic acid functional group plays a very important role in Enhertu's excellent safety/efficacy profile, and if it is removed, it will cause difficulties in ADC manufacturing and at the same time deteriorate the performance of ADC in animal models and clinical trials (resulting in safety issues or efficacy). decrease) brings problems.
도 4 내지 도 7에 도시된 바와 같이, ABCG2를 발현하지 않는 Her2-low/mid 암 세포인 FaDu와 ABCG2를 과발현하고 있는 암세포인 A549에서 각종 캄토테신계 약물들을 다양한 농도로 처리하여, 세포내 DDX5 단백질의 분해정도 및 이로 인한 survivin, Mcl-1, XIAP 및 cIAP2의 암-관련 생존 유전자 (cancer-associated survival genes)의 발현 억제 활성을 확인할 수 있고, 간접적으로 세포막 투과도를 비교 확인할 수 있었다.As shown in Figures 4 to 7, FaDu, a Her2-low/mid cancer cell that does not express ABCG2, and A549, a cancer cell that overexpresses ABCG2, were treated with various camptothecin-based drugs at various concentrations to increase intracellular DDX5. The degree of protein degradation and the resulting inhibitory activity on the expression of cancer-related survival genes of survivin, Mcl-1,
또한, 본 발명은 화학식 1로 표시되는 캄토테신계 골격을 모핵으로 포함하는 화합물(예, 엑사테칸 또는 Dxd)을 임의의 항체, 이의 항원결합부위 함유 단편, 또는 운반체와 결합하여 면역접합체 또는 운반체-약물 접합체(Carrier-Drug Conjugate)의 페이로드로 사용 시, (1) 세포내 DDX5 단백질을 분해하는 작용기전(MoA)을 통한 우수한 세포 사멸 효과; (2) 종양조직 심부까지 침투하면서 최적화된 주변세포살상효과(bystander effect); 및 (3) 높은 DAR ADC 생성에 적합한 우수한 물리화학적 특성과 같은 특징을 최적화 또는 극대화하기 위해, [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하는 면역접합체; 또는 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]를 포함하는 운반체-약물 접합체(Carrier-Drug Conjugate)를 제공하는 것이 특징이다.In addition, the present invention relates to a compound comprising the camptothecin-based skeleton represented by Formula 1 (e.g., exatecan or Dxd) as a parent nucleus by combining it with any antibody, fragment containing an antigen-binding site, or carrier to form an immunoconjugate or carrier. -When used as a payload of a carrier-drug conjugate, (1) excellent cell death effect through the mechanism of action (MoA) that decomposes intracellular DDX5 protein; (2) Optimized bystander effect while penetrating deep into the tumor tissue; and (3) to optimize or maximize features such as excellent physicochemical properties suitable for high DAR ADC production, [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen thereof an immunoconjugate comprising a binding site-containing fragment]; Alternatively, the present invention is characterized by providing a carrier-drug conjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker].
본 발명에 따라 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하는 면역접합체; 또는 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]를 포함하는 운반체-약물 접합체(Carrier-Drug Conjugate)는, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되므로, 종양조직 심부까지 침투하면서 적절한 주변세포살상효과(bystander effect)가 발휘 또는 그 효과의 정도가 제어하기 위해, 화학식 1의 캄토테신계 약물의 상대적인 친수성/소수성 성질은 약물의 용해도, 흡수, 분포, 대사, 배설 (ADME)에 중요한 영향을 미친다. 특히, 약물이 세포막을 얼마나 쉽게 통과하는지, 그리고 약물 표적인 DDX5 단백질 및/또는 E3 리가아제와의 상호 작용에서 중요하다.According to the present invention, an immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof]; Alternatively, the carrier-drug conjugate containing [camptothecin-based drug of Formula 1]-[acid-sensitive linker] may have an acid-sensitive linker in an acidic environment (pH ≤ 7) surrounding the cancer. Since at least some of the camptothecin-based drug of Formula 1 is released by decomposition, in order to exert an appropriate bystander effect while penetrating deep into the tumor tissue or to control the degree of the effect, the camptothecin-based drug of Formula 1 The relative hydrophilic/hydrophobic nature has a significant impact on the solubility, absorption, distribution, metabolism, and excretion (ADME) of a drug. In particular, it is important how easily the drug crosses the cell membrane and its interaction with the drug target, the DDX5 protein and/or E3 ligase.
도 2에 예시된 바와 같이, 각종 캄토테신계 약물의 소수성은 분배계수(partition coefficient, p)로 나타낼 수 있다. As illustrated in Figure 2, the hydrophobicity of various camptothecin-based drugs can be expressed as a partition coefficient ( p ).
소수성은 p값이 크고, 친수성(극성)인 경우 p값이 작다. 실제로 그 log P 값은 소수성을 평가하는 척도로 사용한다. Clog P는 적절한 소프트웨어를 통하여 주어진 화합물의 log P 값을 구한 것을 의미한다. cLogP 값이 작아질수록 극성이 높다.Hydrophobicity has a large p value, and hydrophilicity (polar) has a small p value. In fact, the log P value is used as a measure to evaluate hydrophobicity. Clog P means calculating the log P value of a given compound through appropriate software. The smaller the cLogP value, the higher the polarity.
분자의 극성 표면적(PSA) 또는 위상 극성 표면적(TPSA)은 부착된 수소 원자를 포함하여 주로 산소와 질소인 모든 극성 원자 또는 분자에 대한 표면 합으로 정의된다. tPSA는 약물의 세포 투과 능력을 최적화하기 위해 일반적으로 사용되는 의약 화학 측정법이다. 140 Å2보다 큰 극성 표면적을 가진 분자는 세포막을 투과하지 못하는 경향이 있다. 분자가 혈액-뇌 장벽을 통과하여 중추 신경계의 수용체에 작용하려면 일반적으로 90Å2 미만의 tPSA가 필요하다.The polar surface area (PSA) or topological polar surface area (TPSA) of a molecule is defined as the sum of the surfaces for all polar atoms or molecules, mainly oxygen and nitrogen, including attached hydrogen atoms. tPSA is a commonly used medicinal chemistry measurement to optimize the cell-penetrating ability of drugs. Molecules with a polar surface area greater than 140 Å 2 tend not to penetrate cell membranes. A tPSA of less than 90 Å 2 is generally required for the molecule to cross the blood-brain barrier and act on receptors in the central nervous system.
본 발명에서 페이로드로 사용되는 화학식 1의 캄토테신계 약물은 일반식 1의 R3 및 R4를 변형하더라도 세포막을 투과할 수 있는 소수성 저분자이므로, 종양조직으로 전달되면 종양조직 심부까지 침투하면서 높은 농도로 축적 가능하고, 화학식 1의 캄토테신계 약물 또는 이의 프로드럭인 ADC이 세포막을 관통한 후 화학식 1의 캄토테신계 약물이 세포 내부에서 DDX5에 결합하는 분자 접착 분해제(molecular glue degrader)로서 세포 사멸시키면서 세포 외액으로 방출되어 연속적으로 주변 세포에도 세포막을 관통해 세포내로 이동하여 세포사멸 기전을 발휘할 수 있다. 따라서, 본 발명의 면역접합체 또는 운반체-약물 접합체에서 유리된 화학식 1의 캄토테신계 약물은 세포 밀도가 높아 뭉친 고형암을 세포 밀도가 낮은 흩어진 암로 전환시키고/시키거나 면역활성이 낮은 cold tumor를 면역활성이 높은 hot tumor로 전환시킬 수 있다. 또한, 주변세포살상효과가 높아서 비균질종양의 치료에 이점이 있다.The camptothecin-based drug of Formula 1 used as a payload in the present invention is a hydrophobic small molecule that can penetrate the cell membrane even if R 3 and R 4 of Formula 1 are modified, so when delivered to tumor tissue, it penetrates deep into the tumor tissue and produces high It is a molecular glue degrader that can be accumulated in high concentration and binds to DDX5 inside the cell after the camptothecin-based drug of Formula 1 or its prodrug, ADC, penetrates the cell membrane. During cell death, it is released into the extracellular fluid and can subsequently penetrate the cell membrane of surrounding cells and move into the cell, thereby exerting an apoptotic mechanism. Therefore, the camptothecin-based drug of Formula 1, released from the immunoconjugate or carrier-drug conjugate of the present invention, converts solid cancers with high cell density into scattered cancers with low cell density and/or activates cold tumors with low immune activity. This can transform it into a highly hot tumor. In addition, it has a high killing effect on surrounding cells, which is advantageous in the treatment of heterogeneous tumors.
약물 효능이란 대상 적응증에 효과를 나타내는 기대 시간 동안 분해되지 않고 체내에 남아 있는 것을 의미한다.Drug efficacy means that it remains in the body without being decomposed for the expected period of time to exert an effect on the target indication.
항암제의 생체 내 효능(in vivo efficacy)은 흡수, 분포, 대사 및 배설(Absorption, Distribution, Metabolism, and Excretion, ADME) 특성에 의해 영향을 받을 수 있다. 약물의 ADME 프로파일은 암세포에 도달하여 작용하는 능력에 영향을 줄 수 있다. 즉, 항암제의 ADME 특성은 생체 내 효능(in vivo efficacy)에 상당한 영향을 미칠 수 있다. The in vivo efficacy of anticancer drugs can be affected by absorption, distribution, metabolism, and excretion (ADME) characteristics. A drug's ADME profile can affect its ability to reach and act on cancer cells. In other words, the ADME characteristics of anticancer drugs can have a significant impact on in vivo efficacy.
종양 조직에 침투하는 약물의 능력은 혈류 및 세포 밀도를 포함한 종양의 미세 환경과 같은 요인에 의해 영향을 받을 수도 있다.The ability of a drug to penetrate tumor tissue may also be affected by factors such as the tumor's microenvironment, including blood flow and cell density.
따라서, 항암제의 ADME 특성을 이해하고 약물/약물 모달리티 선택 및 용량/용법을 최적화하면 이러한 항암제의 치료 지수를 개선하여 암 환자에게 더 나은 결과를 가져올 수 있다.Therefore, understanding the ADME properties of anticancer drugs and optimizing drug/drug modality selection and dosage/regimen can improve the therapeutic index of these anticancer drugs, resulting in better outcomes for cancer patients.
항체-약물 접합체(ADC)는, 이의 페이로드인 약물 그 자체 뿐만아니라 링커와의 다양한 조합에 따라, 항체(Ab)와는 서로 다른 구조와 작용 메커니즘으로 인해 서로 다른 수명(life time)과 ADME 프로파일을 갖는다.Antibody-drug conjugates (ADCs) have different life times and ADME profiles due to their different structures and mechanisms of action from antibodies (Abs), depending on not only the drug itself, which is its payload, but also various combinations with the linker. have
항체는 통상 외부 물질(항원)에 반응하여 면역 체계에 의해 자연적으로 생성되는 큰 단백질이다. 크기와 복잡한 구조로 인해 순환 반감기가 길며(몇 주에서 몇 달) 분해 및 제거로부터 보호할 수 있다. 항체는 조직을 포함하여 신체 전체에 분포하며 높은 특이성과 친화력으로 표적 항원과 상호 작용할 수 있다. 항체들은 주로 간과 비장을 포함하는 세망내피계(RES)와 신장 및 기타 기관의 이화작용에 의해 제거된다.Antibodies are large proteins produced naturally by the immune system, usually in response to foreign substances (antigens). Due to their size and complex structure, they have a long circulating half-life (several weeks to months) and may protect them from degradation and elimination. Antibodies are distributed throughout the body, including tissues, and can interact with target antigens with high specificity and affinity. Antibodies are primarily eliminated by catabolism in the reticuloendothelial system (RES), which includes the liver and spleen, as well as the kidneys and other organs.
ADC는 세포독성 약물 분자에 접합된 항체(일반적으로 단클론 항체)로 구성된다. 항체 성분은 종양 또는 질병 조직에 대한 특이성과 표적화를 제공하는 반면, 약물 성분은 표적 세포를 죽이는 세포독성 활성을 제공한다. ADC는 항체보다 짧은 반감기를 가지며 일반적으로 며칠에서 일주일에 이른다. 이는 ADC가 표적 세포로 내재화되어 ADC의 리소좀 분해 및 약물 페이로드의 방출을 초래하기 때문이다(도 3). ADC는 주로 RES에 의해 제거되지만, 약물 페이로드는 간과 신장을 통해 대사 및 배설(Metabolism and Excretion)을 겪을 수도 있다.ADCs consist of an antibody (usually a monoclonal antibody) conjugated to a cytotoxic drug molecule. The antibody component provides specificity and targeting to the tumor or diseased tissue, while the drug component provides cytotoxic activity to kill target cells. ADCs have shorter half-lives than antibodies, typically ranging from a few days to a week. This is because ADC is internalized into target cells, resulting in lysosomal degradation of ADC and release of the drug payload (Figure 3). Although ADC is primarily eliminated by RES, the drug payload can also undergo metabolism and excretion via the liver and kidneys.
항체는 일반적으로 피하 또는 정맥 주사로 투여되며 혈류로 흡수된다. 그들은 조직을 포함하여 몸 전체에 분포할 수 있지만 일반적으로 크기 때문에 세포외 공간으로 제한된다. ADC도 주사로 투여되어 혈류로 흡수되지만, 항체 성분으로 인해 종양 또는 질병 조직에 표적화 및 경우에 따라서는 수용체 매개 내재화(Receptor-mediated endocytosis)된 후 ADC에서 방출된 약물 페이로드는 세포와 조직으로 침투할 수 있다. 나아가, ADC의 대사 및 배설은 특정 구조 (specific structure) 및 사용된 특정 약물, 링커 또는 항체(specific drug or antibody)에 따라 다르다.Antibodies are usually administered subcutaneously or intravenously and are absorbed into the bloodstream. They can be distributed throughout the body, including tissues, but are generally restricted to the extracellular space due to their large size. ADCs are also administered by injection and absorbed into the bloodstream, but the drug payload released from the ADC penetrates into cells and tissues after targeting and, in some cases, receptor-mediated endocytosis to tumors or diseased tissues due to the antibody component. can do. Furthermore, the metabolism and excretion of ADC varies depending on the specific structure and the specific drug, linker, or antibody used.
본 발명에서, 산 민감성(acid-sensitive) 링커는 혈액의 중성 환경인 pH 7.3~7.5에서는 안정적이지만 종양세포 주변 (pH 6.5~7.2)이나 세포내 내재화가 일어나 엔도좀 (pH 5.0~6.5)과 리소좀(pH 4.5~5.0) 같은 약산성 환경에서 가수분해되어 약물을 방출하는 링커를 의미한다. 따라서, 본 발명에서 산 민감성 링커는 가수분해 환경을 조성하도록 친수성 분자 구조를 가진다. 이를 위해, 산 민감성 링커는 예컨대 폴리에틸렌글리콜 (PEG) 스페이서를 포함할 수 있다.In the present invention, the acid-sensitive linker is stable in the neutral environment of blood, pH 7.3 to 7.5, but is internalized around tumor cells (pH 6.5 to 7.2) or within cells, such as endosomes (pH 5.0 to 6.5) and lysosomes. It refers to a linker that is hydrolyzed in a slightly acidic environment (pH 4.5~5.0) to release the drug. Therefore, the acid-sensitive linker in the present invention has a hydrophilic molecular structure to create a hydrolytic environment. For this purpose, the acid-sensitive linker may comprise, for example, a polyethylene glycol (PEG) spacer.
화학식 3의 산 민감성 링커의 일종인 CL2A 링커를 통해 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물을 HER2 항체의 일종인 Trastuzumab에 연결시킨 ADC인 Trastuzumab-CL2A-exadecane(제조예 3) 및 Trastuzumab-CL2A-dxd(제조예 4) (DAR 7-8)은 응집 문제 없이 쉽게 합성되었다. Trastuzumab-CL2A-exadecane (Preparation Example 3), an ADC that connects a camptothecin-based drug of Formula 1-1 or Formula 1-2 to Trastuzumab, a type of HER2 antibody, through a CL2A linker, a type of acid-sensitive linker of Formula 3; Trastuzumab-CL2A-dxd (Preparation Example 4) (DAR 7-8) was easily synthesized without aggregation problems.
도 8에 도시된 바와 같이, ABCG2를 발현하지 않는 Her2-low/mid 암 세포인 FaDu와 ABCG2를 과발현하고 있는 암세포인 A549에서 Trastuzumab-CL2A-exadecane(제조예 3)은 세포내 DDX5 단백질의 분해정도 및 이로 인한 survivin, Mcl-1, XIAP 및 cIAP2의 암-관련 생존 유전자 (cancer-associated survival genes)의 발현 억제 활성이 농도 의존적으로 나타났다.As shown in Figure 8, Trastuzumab-CL2A-exadecane (Preparation Example 3) in FaDu, a Her2-low/mid cancer cell that does not express ABCG2, and A549, a cancer cell that overexpresses ABCG2, decomposes intracellular DDX5 protein. And the resulting inhibitory activity of survivin, Mcl-1, XIAP and cIAP2 expression of cancer-related survival genes was shown in a concentration-dependent manner.
도 9 및 도 10에 나타난 바와 같이, Trastuzumab-CL2A-exadecane(제조예 3)은 Her2-high 세포주(MDA-MB-453) 및 HER2 positive breast cancer 유래 세포주(SK-BR-3)에서 세포 독성을 나타냈다. As shown in Figures 9 and 10, 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). indicated.
따라서, 본 발명은 분자접착제(molecular glue degrader)로 DDX5 단백질 및 E3 리가아제에 결합하는 다양한 화학식 1의 캄토테신계 약물들을 다양한 페이로드 후보로 제공함으로써, 하나 이상의 적절한 약물을 선택 및 이의 용량-용법을 적절히 적용하여, 원하는 효능(예, 항암, 병용요법) 및 부작용(캄토테신계 약물의 응집 문제)을 정밀하게 제어할 수 있다.Therefore, the present invention provides a variety of camptothecin-based drugs of Formula 1 that bind to DDX5 protein and E3 ligase as molecular glue degraders as various payload candidates, thereby enabling selection of one or more appropriate drugs and their dosage-administration method. By appropriately applying, the desired efficacy (e.g., anticancer, combination therapy) and side effects (aggregation problems of camptothecin-based drugs) can be precisely controlled.
예컨대, 화학식 1의 캄토테신계 약물은 약물 모달리티(small molecule, ADC)에 따라 종양조직 내에서 특정 약물 농도(高)를 넘는 것이 중요할 수 있고, 경우에 따라서 종양조직 내에서 특정 약물 농도(低) 이상으로 유지된 시간이 중요할 수 있다.For example, for the camptothecin-based drug of Formula 1, it may be important to exceed a specific drug concentration (high) within tumor tissue depending on the drug modality (small molecule, ADC), and in some cases, the specific drug concentration (low) within tumor tissue. ) or longer may be important.
[화학식 1의 캄토테신계 약물 및 화학식 3의 산민감성 링커의 다양한 조합을 통한 운반체-약물 접합체의 분포(Distribution) 및 제거(Excretion) 제어][ Distribution and Excretion Control of Carrier-Drug Conjugates through Various Combinations of Camptothecin Drug of Formula 1 and Acid-Sensitive Linker of Formula 3 ]
약물이 체내에 들어가면 제거과정이 시작된다. 약물제거 주요경로는 (1) 간장대사, (2) 담즙배설, (3) 요중배설이다. Once the drug enters the body, the elimination process begins. The main routes of drug elimination are (1) hepatic metabolism, (2) biliary excretion, and (3) urinary excretion.
항체-약물 접합체(ADC)와 같은 운반체-약물 접합체(Carrier-Drug Conjugate)는 약물을 질병 부위에 선택적으로 전달하거나 더 많은 양의 약물을 질병 부위에 전달할 수 있는 가능성을 갖고 있다. 하지만, 인공 나노 캐리어, 리포솜 및 고분자 나노입자 등 대부분의 약물 나노 제형은 질병 부위에 도달하기 전에 체내 면역 체계 중 하나인 망상내피계에 의해 탐식되어 혈액 순환계에서 빠르게 제거되는 등 한계가 있다.Carrier-drug conjugates, such as antibody-drug conjugates (ADCs), have the potential to selectively deliver drugs to disease sites or deliver larger amounts of drugs to disease sites. However, most drug nanoformulations, such as artificial nanocarriers, liposomes, and polymer nanoparticles, have limitations such as being phagocytosed by the reticuloendothelial system, one of the body's immune systems, and quickly eliminated from the blood circulation before reaching the disease site.
망상내피계는 대식세포계, 단핵식세포계(mononuclear phagocyte system, MPS)라고도 한다. 인체의 여러 부분에서 특정 물질들을 흡수하는 세포들이다. 이 세포들은 인체방어 메커니즘의 일부를 이룬다. The reticuloendothelial system is also called the macrophage system and the mononuclear phagocyte system (MPS). These are cells that absorb specific substances from various parts of the human body. These cells form part of the body's defense mechanism.
망상내피세포는 골수에 있는 전구세포로부터 만들어진다. 전구세포는 혈류로 방출되는 식세포인 단핵구로 발달되는데, 일부 단핵구는 순환계에 남지만 대부분은 체조직으로 들어가서 대식세포(macrophage)라고 하는 훨씬 더 큰 식세포가 된다. 대식세포 대다수는 움직이지 않는 세포로 조직 안에 남아서, 이물질을 걸러 파괴시킨다. 그러나 일부는 떨어져 나와 순환계나 세포사이 공간 안에서 떠돌아다닌다. Reticuloendothelial cells are created from progenitor cells in the bone marrow. Progenitor cells develop into monocytes, phagocytes that are released into the bloodstream. Some monocytes remain in the circulation, but most enter body tissues and become much larger phagocytes called macrophages. The majority of macrophages are immobile cells that remain within tissues, filtering out and destroying foreign substances. However, some break off and wander around in the circulatory system or the spaces between cells.
조직 내의 대식세포는 그 세포가 위치한 곳에 따라 모양과 이름이 다르다. 망상세포는 림프 절동과 비장, 골수에 있는 반면, 조직구는 피하조직에서 많이 발견된다. 신경소교세포(microglia)는 신경조직에, 폐포대식세포(alveolar macrophage)는 폐의 폐포에, 쿠퍼세포는 간에서 나타난다. 식세포작용을 통해 대식세포는 인체 내부에 들어온 해로운 입자들에 대한 첫 방어선을 형성한다. Macrophages within tissues have different shapes and names depending on where they are located. Reticular cells are found in lymph nodes, spleen, and bone marrow, while histiocytes are found in abundance in subcutaneous tissue. Microglia appear in nervous tissue, alveolar macrophages appear in the alveoli of the lung, and Kupffer cells appear in the liver. Through phagocytosis, macrophages form the first line of defense against harmful particles entering the body.
체내에서 응집된 나노입자는 크기에 따라 섭취되는 장기와 그에 따른 인체 내 분포가 달라진다. 50 nm 이상의 나노입자는 망상내피계가 발달한 장기인 간의 쿠퍼셀에 탐식되어 빠르게 간에 축적된다.Nanoparticles aggregated in the body vary depending on the size of the organ they are ingested in and their distribution within the human body. Nanoparticles larger than 50 nm are phagocytosed by the Kupffer cells of the liver, an organ with a developed reticuloendothelial system, and rapidly accumulate in the liver.
운반체-약물 접합체(Carrier-Drug Conjugate)가 링커를 통해 연결된 소수성 캄토테신계 약물에 의해 응집되어 대식세포 탐식에 의해 간에 축적되면, 간에서 산 민감성 링커에 연결된 캄토테신계 약물 일부가 유리형 소수성 캄토테신계 약물로 방출되어 정상 세포의 세포막 투과 후 세포독성을 발휘하는 간 독성 문제를 일으킬 수 있다.When the carrier-drug conjugate is aggregated by the hydrophobic camptothecin drug linked through the linker and accumulated in the liver by macrophage phagocytosis, some of the camptothecin drug linked to the acid-sensitive linker in the liver is converted to free hydrophobic camptothecin drug. It is released as a tothecin-based drug and can cause liver toxicity problems by exerting cytotoxicity after penetrating the cell membrane of normal cells.
본 발명은 운반체-약물 접합체가 산 민감성 링커를 통해 연결된 소수성 캄토테신계 약물에 의해 응집되어 대식세포 탐식에 의해 간에 축적되지 않도록 하기 위해, (1) 산 민감성 링커에 연결되는 캄토테신계 페이로드로, A- 및 B-고리가 형성하는 방향족 고리들의 π-π적층(stacking)에 의해 응집이 유도되는 SN-38 대신, A- 및 B-고리로부터 확장된 6각 또는 7각 고리의 연속적인 탄소-탄소 단일 결합의 다양한 배향들이 동적 평형을 이루어 A- 및 B-고리가 형성하는 방향족 고리들의 적층(stacking)이 억제가능한, 화학식 1로 표시되는 캄토테신계 골격을 모핵으로 포함하는 화합물(예, 엑사테칸 또는 Dxd)를 사용하고, (2) 바람직하게는 링커의 일 말단을 화학식 1의 캄토테신계 약물의 E-고리의 20번 탄소에 위치한 알파수산화기에 연결함으로써, A- 및 B-고리로부터 확장된 6각 또는 7각 고리에 있는 탄소-탄소 단일 결합에 대하여 분자 결합의 회전이 가능하여 자유도가 큰 -NH2 또는 -OH가 수 환경(aqueous environment), 즉 물(H2O)에 노출되어 (+) 전하를 띠거나 물과 수소결합되도록 설계하여, 운반체-약물 접합체에 산 민감성 링커를 통해 배향된 화학식 1의 캄토테신계 약물 부위의 수 분산성을 향상시키는 것이 특징이다. The present invention is to prevent a carrier-drug conjugate from being aggregated by a hydrophobic camptothecin-based drug linked through an acid-sensitive linker and accumulated in the liver by macrophage phagocytosis, by (1) a camptothecin-based payload linked to an acid-sensitive linker; , instead of SN-38, where aggregation is induced by π-π stacking of the aromatic rings formed by the A- and B-rings, successive carbons of hexagonal or heptagonal rings extend from the A- and B-rings. - A compound containing the camptothecin-based skeleton represented by Formula 1 as a parent nucleus, in which various orientations of carbon single bonds are in dynamic equilibrium to suppress the stacking of aromatic rings formed by A- and B-rings (e.g., exatecan or Dxd), and (2) preferably by connecting one end of the linker to the alpha hydroxyl group located at carbon 20 of the E-ring of the camptothecin drug of Formula 1, thereby forming the A- and B-rings. -NH 2 or -OH, which has a large degree of freedom due to the rotation of the molecular bond with respect to the carbon-carbon single bond in the hexagonal or heptagonal ring extended from It is designed to be exposed and carry a (+) charge or hydrogen bond with water, and is characterized by improving the water dispersibility of the camptothecin drug moiety of Formula 1, which is oriented through an acid-sensitive linker in the carrier-drug conjugate.
즉, 본 발명은 화학식 1의 캄토테신계 약물이 연결된 운반체-약물 접합체가, 캄토테신계 약물을 통해 응집되어 대식세포 탐식에 의해 간에 축적되지 않도록 설계함으로써, SN-38를 페이로드로 하는 ADC(예, hRS7-CL2A-SN-38)의 응집문제 및 이로 인해 야기되는 간 독성으로 인한 용량 제한 문제를 해결할 수 있다.That is, the present invention is designed so that the carrier-drug conjugate to which the camptothecin drug of Formula 1 is linked is not aggregated through the camptothecin drug and accumulated in the liver by macrophage phagocytosis, thereby producing an ADC (ADC) with SN-38 as a payload. For example, it can solve the aggregation problem of hRS7-CL2A-SN-38) and the capacity limitation problem due to liver toxicity caused by this.
또한, 본 발명은 운반체-약물 접합체에 산 민감성 링커를 통해 배향된 화학식 1의 캄토테신계 약물 부위의 수 분산성을 향상시킴으로써, 간 내 망상내피계에 의한 탐식을 줄이고 이에 따라 혈류에서의 체류시간을 증대시켜 장시간 혈중 약물농도를 고농도로 유지할 수 있다. 또한, 링커와 약물의 소수성이 증가되면, ADC의 응집력 증가와 이에 따른 약물의 치료계수감소와 같은 문제점을 야기된다. 따라서, 화학식 1의 캄토테신계 약물 및 화학식 3의 산민감성 링커의 다양한 조합으로 인한 친수성 조절을 통해, 운반체-약물 접합체에 원하는 간청소율(hepatic clearance) 프로파일 및 혈액 순환 프로파일을 제공할 수 있다.In addition, the present invention improves the water dispersibility of the camptothecin drug moiety of Formula 1 oriented through an acid-sensitive linker in the carrier-drug conjugate, thereby reducing phagocytosis by the reticuloendothelial system in the liver and thereby reducing the residence time in the bloodstream. By increasing the drug concentration in the blood, it can be maintained at a high concentration for a long time. In addition, if the hydrophobicity of the linker and the drug increases, problems such as increased cohesion of the ADC and a corresponding decrease in the therapeutic coefficient of the drug occur. Accordingly, the desired hepatic clearance profile and blood circulation profile can be provided to the carrier-drug conjugate through hydrophilicity control through various combinations of the camptothecin-based drug of Formula 1 and the acid-sensitive linker of Formula 3.
[DDX5에 결합하는 분자 접착 분해제(molecular glue degrader)로서 FL118 약물의 항암 기전][ Anticancer mechanism of FL118 drug as a molecular glue degrader that binds to DDX5 ]
화학식 2의 FL118은 DDX5와 유비퀴틴화 조절자를 직접 접착하여 DDX5를 분해하는 분자 접착 분해제(molecular glue degrader) 역할을 할 수 있다.FL118 of Formula 2 can act as a molecular glue degrader that degrades DDX5 by directly attaching DDX5 to the ubiquitination regulator.
분자 접착제(molecular glue) 역할을 하는 FL118은, DDX5 mRNA를 감소시키지 않고 프로테아좀 분해 경로를 통해 다기능 마스터 조절인자(multifunctional master regulator)인 종양단백질 DDX5에 직접 결합하고 이를 탈인산화 및 분해하는 기능을 가지고 있으며, DDX5의 사일런싱은, DDX5가 서바이빈, Mcl-1, XIAP, cIAP2, c-Myc 및 돌연변이 Kras를 비롯한 여러 발암성 단백질의 발현을 조절하는 마스터 조절인자임을 나타낸다.FL118, which acts as a molecular glue, directly binds to the tumor protein DDX5, a multifunctional master regulator, through the proteasome degradation pathway without reducing DDX5 mRNA, and has the function of dephosphorylating and decomposing it. DDX5 silencing indicates that DDX5 is a master regulator that regulates the expression of several oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc, and mutant Kras.
또한, 대부분의 암에 공통된 내성발생 기전인 항세포사멸 단백질(anti-apoptotic protein)의 과발현을 원천적으로 차단하여 내성 회피가 가능하고; 환자에서의 항암 반응을 예측할 수 있는 바이오마커 (DDX5, K-ras, p53), 동반진단 기법 등이 이미 확립되어 있으며, 맞춤형 바이오마커 확보로 동반 진단을 통한 개인 맞춤형 치료 가능하고, 특히 예후가 좋지 않은 p53/K-ras 돌연변이 암세포들에서 강력한 효능을 보인다.In addition, it is possible to avoid resistance by fundamentally blocking the overexpression of anti-apoptotic protein, which is a common resistance development mechanism in most cancers; Biomarkers (DDX5, K-ras, p53) and companion diagnostic techniques that can predict anticancer response in patients have already been established, and by securing customized biomarkers, personalized treatment is possible through companion diagnosis, especially in patients with poor prognosis. It shows strong efficacy against p53/K-ras mutant cancer cells.
FL118은 DDX5 다운스트림 표적들을 간접적으로 제어하여 인간 결장 직장암/췌관 선암종 세포 및 종양 모델을 사용한 연구에서 입증된 바와 같이 높은 효능으로 암 개시, 발달, 전이, 재발 및 치료 내성(cancer initiation, development, metastasis, recurrence and treatment resistance)을 억제한다.FL118 indirectly controls DDX5 downstream targets to promote cancer initiation, development, metastasis, and treatment resistance with high efficacy, as demonstrated in studies using human colorectal cancer/pancreatic adenocarcinoma cells and tumor models. , recurrence and treatment resistance).
PDAC 세포에서 DDX5의 유전적 조작은 종양 성장에 영향을 미친다. DDX5 KO가 있는 PDAC 세포는 FL118 처리에 내성이 있다. 인간 종양 동물 모델 연구에서 FL118이 DDX5 발현이 높은 인간 PDAC 및 CRC 종양을 제거하는 데 높은 효능을 나타내는 반면, FL118은 DDX5 발현이 낮은 PDAC 및 CRC 종양에서 덜 효과적인 것으로 나타났다.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 showed that FL118 showed high efficacy in eliminating human PDAC and CRC tumors with high DDX5 expression, whereas FL118 was less effective in PDAC and CRC tumors with low DDX5 expression.
DDX5 단백질은 FL118 약물의 직접 표적이며 FL118에 대한 PDAC 및 CRC 종양 감수성(tumour sensitivity)을 예측하기 위한 바이오마커 역할을 할 수 있다. DDX5 protein is a direct target of the FL118 drug and may serve as a biomarker to predict PDAC and CRC tumor sensitivity to FL118.
한편, FL118 약물은 암세포에서 SN-38과 동등 이상 수준의 Top1 저해 효능을 가지고 있으며, 다양한 암 세포주에서 SN-38에 비하여 5 - 20배 강력한, 즉 낮은 수준의 IC50 수치로 세포독성(Cytotoxicity)를 보이며, 다양한 암종에서 기원한 140개의 세포주에 대한 평가 결과에서도 대다수의 암세포에 대하여 < 100nM의 IC50를 보이는 매우 강력한 항암 효능을 보였다. FL118 약물은 쥐와 비글견에서의 GLP-독성 시험을 통하여 우수한 안전성이 확보되었으며, 다양한 암 세포주 Xenograft 모델에서 SN-38 대비 탁월한 효능을 보였다. 한편, Camptothecin계 항암제의 경우 환자에 사용 시 초기에는 우수한 항암 반응을 보이나 Top1 유전자의 Epigenetic Silencing과 암세포의 Top2 Dependence를 통하여 이들 약물에 대한 강력한 내성이 나타난다. 반면, FL118 약물은 Top1이 Epigenetic Silencing 또는 Knock-out을 통하여 발현되지 않는 암 세포주의 Xenograft 모델에서도 강력한 효능을 보였다.Meanwhile, the FL118 drug has a Top1 inhibitory effect equal to or higher than that of SN-38 in cancer cells, and is 5 to 20 times more potent than SN-38 in various cancer cell lines, i.e., has a low IC 50 value and cytotoxicity. , and the evaluation results of 140 cell lines originating from various carcinomas showed very strong anticancer efficacy with an IC 50 of <100nM against the majority of cancer cells. The FL118 drug has secured excellent safety through GLP-toxicity tests in rats and beagle dogs, and has shown superior efficacy compared to SN-38 in various cancer cell line Xenograft models. Meanwhile, camptothecin-based anticancer drugs show excellent anticancer responses initially when used in patients, but strong resistance to these drugs appears through Epigenetic Silencing of the Top1 gene and Top2 Dependence of cancer cells. On the other hand, the FL118 drug showed strong efficacy even in the Xenograft model of cancer cell lines in which Top1 is not expressed through Epigenetic Silencing or Knock-out.
또한, FL118 약물은 잘 확립된 항암 타깃인 제1형 토포이소머라제를 직접 표적으로 하면서, 내성 기전에 관여하는 내성 단백질인 Survivin 등 Bcl family를 동시에 억제하고, 유출펌프(efflux pump)의 작용을 억제하는 삼중 표적 항암제이다.In addition, the FL118 drug directly targets type 1 topoisomerase, a well-established anticancer target, and targets the Bcl family, including Survivin, a resistance protein involved in resistance mechanisms. It is a triple target anticancer drug that simultaneously inhibits and inhibits the action of the efflux pump.
구체적으로, SN-38 등 Camptothecin 계열 항암제들은 ABCG2 Transporter의 과발현에 의해서 약물이 세포 밖으로 배출되는 방식의 내성을 보이게 되는데, FL118 약물은 ABCG2 Transporter에 영향을 받지 않기 때문에 이에 의한 내성 극복이 가능하다. FL118 약물은 또 다른 항암제 내성의 주원인인 항세포사멸 단백질(anti-apoptotic protein)(Survivin, cIAP2, XIAP 등)의 발현을 낮은 농도에서 강하게 억제함으로써 내성의 발현을 차단할 수 있다. Specifically, camptothecin-type anticancer drugs such as SN-38 show resistance in the way that the drug is released out of the cell due to overexpression of the ABCG2 Transporter. However, since the FL118 drug is not affected by the ABCG2 Transporter, it is possible to overcome this resistance. The FL118 drug can block the development of resistance by strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another main cause of anticancer drug resistance, at low concentrations.
따라서, FL118 약물은 유출펌프(efflux pump)인 ABCG2에 의해 세포 밖으로 배출되지 않으며, 다양한 항세포사멸 단백질(anti-apoptotic protein)에 의한 내성을 차단할 수 있다. 그 결과, FL118 약물은 SN-38/Exatecan의 다양한 내성 작용 기전을 극복할 수 있다.Therefore, the FL118 drug is not expelled out of the cell by ABCG2, an efflux pump, and can block resistance caused by various anti-apoptotic proteins. As a result, the FL118 drug can overcome the various resistance mechanisms of SN-38/Exatecan.
FL118 약물은 SN-38과 동일한 양으로 in vivo 투여 시 대장암/두경부암/췌장암 등에서 SN-38 대비 강력한 tumor regression 효능을 보였다.When administered in vivo in the same amount as SN-38, the FL118 drug showed stronger tumor regression efficacy than SN-38 in colon cancer, head and neck cancer, and pancreatic cancer.
FL118 약물은 tumor xenograft에서 SN-38 내성 유도 후 FL118 투여 시에도 강력한 항암 효능을 보유하였다. The FL118 drug possessed strong anticancer efficacy even when FL118 was administered after inducing SN-38 resistance in tumor xenograft.
더욱이, FL118 약물은 targeted drug delivery (예, Carrier-drug Conjugate) 적용에 최적인 PK/safety profile을 가지고 있다. FL118 약물을 단독으로 전신투여 시 혈액에서 빠르게 대사/배출되어 낮은 농도만을 보이나 암 조직에는 투여 직후부터 빠르게 축적되어 장시간 높은 농도를 유지한다. 예컨대, ADC 적용시 종양 조직과 정상 조직 사이의 최대 선택성을 보장한다. Moreover, the FL118 drug has an optimal PK/safety profile for targeted drug delivery (e.g., Carrier-drug Conjugate) application. When the FL118 drug is administered systemically alone, it is rapidly metabolized and excreted from the blood and only shows a low concentration, but it accumulates quickly in cancer tissue immediately after administration and maintains a high concentration for a long time. For example, when applying ADC, maximum selectivity between tumor tissue and normal tissue is ensured.
이를 기반으로 하여, FL118 약물에서 예시되는 항암제로서의 다양한 잇점들을 발휘할 수 있도록, 본 발명에 따라 화학식 1의 캄토테신계 약물을 다양하게 설계하여, 페이로드로 사용할 수 있다. Based on this, the camptothecin-based drug of Formula 1 can be designed in various ways according to the present invention and used as a payload so that it can exhibit various advantages as an anticancer drug, as exemplified by the drug FL118.
[DDX5에 결합하는 분자 접착 분해제(molecular glue degrader)로서 항암기전][ Anticancer mechanism as a molecular glue degrader that binds to DDX5 ]
유비퀴틴-프로테아좀 시스템(UPS)은 광범위한 세포 과정을 조절하는 세포내 단백질의 분해를 위한 중요한 경로이다. 유비퀴틴은 유비퀴틴 활성화 효소(E1s), 유비퀴틴 접합 효소(E2s) 및 유비퀴틴 리가아제(E3s)를 포함하는 일련의 효소 반응에 의해 기질 단백질의 라이신 잔기에 공유 결합되는 작은 단백질이다. 이 과정을 유비퀴틴화(ubiquitination)라고 하며 단백질 분해, 신호 전달 및 트래피킹을 조절하는 중요한 메커니즘이다.The ubiquitin-proteasome system (UPS) is an important pathway for the degradation of intracellular proteins that regulates a wide range of cellular processes. Ubiquitin is a small protein that is covalently attached to lysine residues of substrate proteins by a series of enzymatic reactions involving ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process is called ubiquitination and is an important mechanism that regulates protein degradation, signal transduction, and trafficking.
유비퀴틴 리가아제는 유비퀴틴화 경로에서 기질 특이성을 담당한다.Ubiquitin ligase is responsible for substrate specificity in the ubiquitination pathway.
이와 관련하여, 본 발명에 따른 화학식 1의 캄토테신계 약물은 DDX5 단백질 및 E3 리가아제에 결합하도록 설계된 것이다.In this regard, the camptothecin-based drug of Formula 1 according to the present invention is designed to bind to DDX5 protein and E3 ligase.
정상 세포가 암성 세포(cancerous cell)로 변형되는 것은, 단백질-단백질 상호 작용의 복잡한 네트워크를 포함하는 상이한 대사 경로가 조절되지 않음 (deregulation)으로써 발생한다. 세포 효소 및 DDX5는 정상적인 세포 대사 유지에 중요한 역할을 하지만 이들이 조절되지 않으면(deregulation), 종양 변형을 가속화할 수 있다. DDX5는 모두 수백 가지의 다른 세포 단백질과 상호 작용하며, 관련된 특정 경로에 따라 두 단백질 모두 종양 억제 유전자 또는 종양유전자로 작용할 수 있다. The transformation of normal cells into cancerous cells occurs through deregulation of different metabolic pathways involving a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cellular metabolism, but when deregulated, they can accelerate tumor transformation. Together, DDX5 interacts with hundreds of other cellular proteins, and depending on the specific pathway involved, both proteins can act as tumor suppressor genes or oncogenes.
DEAD-box 계열의 RNA 헬리카제는, 전사 및 번역뿐만아니라, 세포 증식, 선천적 면역 및 스트레스 반응에 이르기까지, 여러 대사 경로에 관여한다. 그들의 다양한 역할을 감안할 때 그들이 조절되지 않거나(deregulation) 또는 그들의 돌연변이가 암을 포함한 다양한 병리학적 상태(pathological conditions)와 연결되어 있다. 그러나 어떤 경우에는 주어진 DEAD-box helicase의 기능 상실이 종양 변형(tumor transformation)을 촉진하여 종양 억제 역할을 나타내는 반면, 다른 상황에서는 동일한 효소의 과발현이 암 진행을 선호하여 전형적인 발암 유전자로 작용한다.The DEAD-box family of RNA helicases is involved in several metabolic pathways, ranging from transcription and translation to cell proliferation, innate immunity, and stress response. Given their diverse roles, their deregulation or mutations have been linked to a variety of pathological conditions, including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation, indicating a tumor suppressive role, whereas in other situations, overexpression of the same enzyme favors cancer progression, acting as a typical oncogene.
DDX5(p68이라고도 함)는 하기와 같은 기전에 작용하는 다기능 마스터 조절자(multifunctional master regulator)이다: (1) 발암 유전자 프로모터(oncogenic gene promoters)에서 다양한 전사 인자들(예: c-Myc)와의 직접적인 상호작용을 통해 많은 종양 유전자들의 전사를 함께 활성화(co-activation)시키는 생물학적 과정, (2) miRNA 및 pre-RNA 스플라이싱(예: U1, U2, U3, ... snRNP)을 조절하는 생물학적 과정, 및 (3) 리보솜 생합성(예: 32S rRNA, pre-ribosome). DDX5 (also known as p68) is a multifunctional master regulator that acts through the following mechanisms: (1) direct interaction with various transcription factors (e.g. c-Myc) at oncogenic gene promoters; (2) a biological process that co-activates the transcription of many oncogenes through interaction, (2) a biological process that regulates miRNA and pre-RNA splicing (e.g. U1, U2, U3, ... snRNP) process, and (3) ribosome biogenesis (e.g., 32S rRNA, pre-ribosome).
본 발명에 따른 화학식 1의 캄토테신계 약물은 DDX5 mRNA 감소 없이 DDX5 단백질에 결합하여 기능적으로 탈인산화 및 프로테아좀 분해 경로를 통해 분해시키는데, 이는 화학식 1의 캄토테신계 약물이 DDX5 및 유비퀴틴 관련 단백질 안정성/분해 조절제(ubiquitin-involved protein stability/degradation regulators) 모두에 붙을 수 있다는 것을 시사하며, "분자 접착 분해제" 역할을 한다.The camptothecin-based drug of Formula 1 according to the present invention binds to DDX5 protein without reducing DDX5 mRNA and functionally degrades it through dephosphorylation and proteasome degradation pathways, which means that the camptothecin-based drug of Formula 1 binds to DDX5 protein and ubiquitin-related proteins. This suggests that it can attach to both ubiquitin-involved protein stability/degradation regulators, acting as a “molecular adhesion degrader.”
DDX5 다운스트림 단백질 표적들(DDX5 downstream protein targets)은 모두 암 개시, 발달, 전이, 재발 및 치료 내성에 관여하는 것으로 알려져 있다. 따라서, 본 발명에 따른 화학식 1의 캄토테신계 약물에 의한 DDX5 단백질의 분해를 통해 DDX5 다운스트림 표적을 간접적으로 차단하면, 화학식 1의 캄토테신계 약물은 높은 항종양 효능이 나타날 수 있다.DDX5 downstream protein targets are all known to be involved in cancer initiation, development, metastasis, recurrence, and treatment resistance. Therefore, if the DDX5 downstream target is indirectly blocked through decomposition of DDX5 protein by the camptothecin drug of Formula 1 according to the present invention, the camptothecin drug of Formula 1 may exhibit high antitumor efficacy.
DDX5(p68)는 잘 알려진 다기능 DEAD-box RNA helicase이자 전사 보조인자(transcription cofactor)이다. 따라서, DDX5의 생리학적 상태에 의해 전사 인자가 조절되지 아니하여(deregulation) 암이 발병한 경우, 전사 보조인자(transcription cofactor)인 DDX5(p68)를 선택적으로 분해시킴으로써, 암 질환을 치료할 수 있다. 마찬가지로, 전사 보조인자(transcription cofactor)인 DDX5(p68)를 선택적으로 분해시킴으로써, 암 질환을 예방할 수 있다.DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and transcription cofactor. Therefore, when cancer occurs due to deregulation of the transcription factor due to the physiological state of DDX5, the cancer disease can be treated by selectively degrading DDX5 (p68), a transcription cofactor. Likewise, cancer disease can be prevented by selectively degrading DDX5 (p68), a transcription cofactor.
본 발명에 따른 화학식 1의 캄토테신계 약물은 DDX5 단백질이 약물 표적이므로, 약물 내성, 표적치료에 저항성 및/또는 치료 중 내성을 피할 수 있게 할 수 있다. 또한, 화학식 1의 캄토테신계 약물에 의해, 항세포사멸 유전자(anti-apoptotic genes)의 전사 유도를 차단(off)시킬 수 있다. 나아가, 화학식 1의 캄토테신계 약물에 의해, 전사 보조인자(transcription cofactor)인 DDX5 단백질이 분해되어, 화학 요법이나 방사선 요법에 대한 암 세포의 민감도를 유지 또는 향상시킬 수 있다.Since the DDX5 protein is the drug target of the camptothecin-based drug of Formula 1 according to the present invention, it can avoid drug resistance, resistance to targeted therapy, and/or resistance during treatment. In addition, the transcriptional induction of anti-apoptotic genes can be turned off by the camptothecin-based drug of Formula 1. Furthermore, the DDX5 protein, a transcription cofactor, is degraded by the camptothecin-based drug of Formula 1, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy or radiotherapy.
[본 발명의 면역접합체의 작용기전(MoA)][ Mechanism of action (MoA) of the immunoconjugate of the present invention ]
본 발명의 일 양태는 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하는 면역접합체에 관한 것으로,One aspect of the present invention relates to an immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof],
(i) 화학식 1의 캄토테신계 약물은 DDX5 단백질 및/또는 E3 리가아제에 결합하도록 설계된 페이로드(payload)이고,(i) The camptothecin-based drug of Formula 1 is a payload designed to bind to DDX5 protein and/or E3 ligase,
(ii) 항체 또는 이의 항원결합부위 함유 단편에 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물이 하나 이상 연결되어 있고,(ii) one or more camptothecin-based drugs of Formula 1 are linked to the antibody or antigen-binding site-containing fragment thereof through an acid-sensitive linker,
(iii) 암세포의 항원을 표적화하는 항원결합부위에 의해 암세포로 표적화된 후, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되고, 유리형 화학식 1의 캄토테신계 약물은 세포막을 관통해 세포내로 이동하고, (iii) After being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ≤ 7) surrounding the cancer, and at least part of the camptothecin-based drug of Formula 1 is liberated. The camptothecin drug of formula 1 penetrates the cell membrane and moves into the cell.
(iv) 선택적(optionally)으로, 화학식 1의 캄토테신계 약물이 연결되어 있는 면역접합체는 세포안으로 내재화(internalization)되어 리소좀(lysosomes)에서 화학식 1의 캄토테신계 약물이 유리되는 것이 특징이다.(iv) Optionally, the immunoconjugate to which the camptothecin drug of Formula 1 is linked is characterized in that it is internalized into cells and the camptothecin drug of Formula 1 is liberated from lysosomes.
본 발명의 면역접합체는 산 민감성 링커를 사용함으로써 항원결합 이후 암세포 내부에서뿐만 아니라 암 조직 주변에서도 효율적으로 화학식 1의 캄토테신계 약물을 방출하여, ADC 프로세싱과 관련한 내성 기전을 극복할 수 있다(도 3). By using an acid-sensitive linker, the immunoconjugate of the present invention can efficiently release the camptothecin-based drug of Formula 1 not only inside cancer cells but also around cancer tissues after antigen binding, thereby overcoming the resistance mechanism related to ADC processing (Figure 3 ).
ADC가 효율적으로 작동하기 위해서는 단순히 in vitro에서 세포 대상으로 강력한 항원 선택적 세포독성을 최대화하는 것만으로는 부족하며, in vivo 내지는 실제 환자에서 암 조직에 잘 침투하여 효율적으로 약물을 전달할 수 있어야 한다.In order for an ADC to operate efficiently, it is not enough to simply maximize powerful antigen-selective cytotoxicity against cells in vitro; it must also be able to penetrate cancer tissue well and efficiently deliver the drug in vivo or in actual patients.
본 발명의 면역접합체는 암세포의 항원을 표적화하는 항원결합부위에 의해 암세포로 표적화된 후, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되고, 유리형 화학식 1의 캄토테신계 약물은 소수성 저분자이나 SN-38 대비 혈액, 간질액 등 체액에서 응집되지 않도록 수분산성이 향상된 약물이므로 종양조직 심부까지 침투하면서 세포막을 관통해 세포내로 이동할 수 있다.The immunoconjugate of the present invention is targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, and then the acid-sensitive linker is decomposed in an acidic environment (pH ≤ 7) surrounding the cancer, thereby liberating at least some of the camptothecin-based drug of Formula 1. The free camptothecin-based drug of Formula 1 is a hydrophobic small molecule, but compared to SN-38, it has improved water dispersibility to prevent aggregation in body fluids such as blood and interstitial fluid, so it can penetrate deep into the tumor tissue and penetrate the cell membrane to move into cells. .
따라서, 본 발명의 면역접합체는 암 주변 산성 환경(pH ≤ 7)에서 분해되는 산 민감성 링커를 사용함으로써 암 주변의 종양 미세 환경에서 빠르게 약물을 방출할 수 있고, 유리형 화학식 1의 캄토테신계 약물은 낮은 분자량으로 항체와 달리 암 조직 침투력이 높으므로, 암 조직의 심부까지 침투가 잘 되지 않는 항체의 문제점을 보유한 기존의 ADC 문제점을 해결할 수 있다. Therefore, the immunoconjugate of the present invention can rapidly release the drug in the tumor microenvironment surrounding the cancer by using an acid-sensitive linker that decomposes in the acidic environment (pH ≤ 7) surrounding the cancer, and the free camptothecin-based drug of Formula 1 Unlike antibodies, it has a low molecular weight and has a high ability to penetrate cancer tissue, so it can solve the problem of existing ADCs, which have problems with antibodies that do not penetrate deep into cancer tissue.
따라서, 유리형 화학식 1의 캄토테신계 약물이 세포내로 이동하는 세포는 표적화된 암세포 및/또는 이의 주변 세포일 수 있다.Accordingly, the cells into which the free camptothecin-based drug of Formula 1 moves intracellularly may be targeted cancer cells and/or their surrounding cells.
또한, 화학식 1의 캄토테신계 약물은 세포막을 투과할 수 있는 소수성 저분자이므로, 암 주변 세포 밖 링커 분해를 통해 본 발명의 면역접합체에서 유리된 화학식 1의 캄토테신계 약물은 종양조직에 빠르게 축적되어 장시간 높은 농도를 유지 가능하고, 세포막을 관통해 세포 내부에서 세포독성을 발휘하여 세포 사멸시킨 후 방출되어 연속적으로 주변 세포에도 세포막을 관통해 세포내로 이동하여 작용할 수 있다. In addition, since the camptothecin-based drug of Formula 1 is a hydrophobic small molecule that can penetrate cell membranes, the camptothecin-based drug of Formula 1 released from the immunoconjugate of the present invention through decomposition of the extracellular linker around the cancer rapidly accumulates in tumor tissue. It can maintain a high concentration for a long time, penetrates the cell membrane, exerts cytotoxicity inside the cell, kills the cell, and is then released and can subsequently penetrate the cell membrane and move into the cell to act on surrounding cells.
요컨대, 본 발명의 운반체-약물 접합체에 적용가능한, 본 발명의 [화학식 1의 캄토테신계 약물]-[산 민감성 링커] 접합체는 In short, the [camptothecin-based drug of Formula 1]-[acid sensitive linker] conjugate of the present invention, which is applicable to the carrier-drug conjugate of the present invention, is
(i) 산성 환경(pH ≤ 7)인 암 주변의 종양 미세 환경에서, 세포막을 관통할 수 있고 세포 내부에서 제역할을 할 수 있는 소수성 저분자 약물을 유리시킨 후, 다량의 유리형 소수성 저분자 약물을 세포 안으로 도입 및/또는 조직 심부까지 침투시키기 위해, 화학식 3의 CL2A 링커와 같은 산 민감성(acid-sensitive) 링커를 사용하는 점; 및(i) In the tumor microenvironment surrounding the cancer, which is an acidic environment (pH ≤ 7), a hydrophobic small molecule drug that can penetrate the cell membrane and play a role inside the cell is liberated, and then a large amount of the free hydrophobic small molecule drug is released. The use of an acid-sensitive linker, such as the CL2A linker of Formula 3, for introduction into cells and/or penetration deep into tissues; and
(ii) 산성 환경(pH ≤ 7)인 암 주변의 종양 미세 환경에서 산 민감성 링커가 분해되어 유리된 다량의 유리형 약물이 종양조직 심부까지 침투가능하고 세포막을 관통해 세포내로 이동하고 세포내 고 풍부화(enrichment)되기 위해, 세포막을 관통할 수 있는 소수성 저분자 약물이지만, 혈액, 간질액 등 체액에서 응집되지 않도록 SN-38 대비 수분산성이 향상된 화학식 1의 캄토테신계 약물을 사용한 점을 특징으로 하며,(ii) In the tumor microenvironment surrounding the cancer, which is an acidic environment (pH ≤ 7), a large amount of free drug released by decomposition of the acid-sensitive linker can penetrate deep into the tumor tissue, penetrate the cell membrane, move into the cell, and remain in the cell. For enrichment, it is a hydrophobic low-molecular-weight drug that can penetrate cell membranes, but it is characterized by using a camptothecin-based drug of Formula 1 that has improved water dispersibility compared to SN-38 to prevent aggregation in body fluids such as blood and interstitial fluid. ,
이의 조합 사용을 통해 항암 효능 및 응집에 의한 독성 측면에서 화학식 1의 캄토테신계 약물-산 민감성 링커의 유기적인 작용기전을 활용하는 점이 또다른 특징이다.Another feature is that the organic mechanism of action of the camptothecin drug-acid sensitive linker of Chemical Formula 1 is utilized in terms of anticancer efficacy and toxicity due to aggregation through use of this combination.
산성 환경(pH ≤ 7)에서 분해되고 산 민감성 링커 분해시 유리형 화학식 1의 캄토테신계 약물이 방출되도록, 화학식 1의 캄토테신계 약물과 산 민감성 링커는 탄산염 또는 에스테르 결합으로 연결된 것이 바람직하다. It is preferable that the camptothecin-based drug of Formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the drug is decomposed in an acidic environment (pH ≤ 7) and the free camptothecin-based drug of Formula 1 is released when the acid-sensitive linker is decomposed.
본 발명에서, [산 민감성 링커]-[항체 또는 이의 항원결합부위 함유 단편]의 연결은 상기 항체 또는 이의 항원 결합 단편에 포함된 티올기가 산 민감성(acid-sensitive) 링커의 말레이미드기 (maleimide) 또는 말레익 하이드라자이드기 (maleic hydrazide)에 반응식 1 의“click” 반응을 통해 결합된 것일 수 있다.In the present invention, the linkage of [acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof] is performed by linking the thiol group contained in the antibody or antigen-binding fragment thereof to the maleimide group of the acid-sensitive linker. Alternatively, it may be bound to a maleic hydrazide group through the “click” reaction of Scheme 1.
[반응식 1][Scheme 1]
Figure PCTKR2023006170-appb-img-000009
Figure PCTKR2023006170-appb-img-000009
또한, 본 발명은 화학식 1의 캄토테신계 약물이 다양한 산 민감성 링커와 연결되어 있는 약물-링커 접합체를 제공하며, 이를 이용하여 다양한 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물이 다양한 운반체(Carrier)에 연결된 운반체-약물 접합체(Carrier-Drug Conjugate)를 제공한다.In addition, the present invention provides a drug-linker conjugate in which the camptothecin-based drug of Formula 1 is linked to various acid-sensitive linkers, using this to transport the camptothecin-based drug of Formula 1 through various acid-sensitive linkers to various carriers. ) Provides a carrier-drug conjugate linked to (Carrier-Drug Conjugate).
나아가, 본 발명은 전술한 본 발명의 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염을 사용하여, 운반체(Carrier)에 화학식 3의 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물을 하나 이상 연결시키는 것이 특징인 운반체-약물 접합체(Carrier-Drug Conjugate)의 제조방법도 제공한다.Furthermore, the present invention relates to one or more camptothecin drugs of Formula 1 through an acid-sensitive linker of Formula 3 to a carrier using the drug-linker conjugate of the present invention or a pharmaceutically acceptable salt thereof. A method for manufacturing a carrier-drug conjugate is also provided.
[다양한 운반체-약물 접합체 (Carrier-Drug Conjugate)용 Payload][ Payload for various carrier-drug conjugates ]
제1형 토포이소머라아제(Topoisomerase I)의 저해제(Irinotecan, Topotecan 등)는 임상에서 효능/안전성이 검증된 항암기전으로, 임상에서 대장암, 폐암, 유방암, 난소암 등 다양한 난치성 고형암에서 뛰어난 항암 효능이 검증되었다. 캄토테신계 약물로 Exatecan, SN-38 등이 ADC 용 Payload로 개발되어 있다.Inhibitors of type 1 topoisomerase I (Irinotecan, Topotecan, etc.) are anticancer mechanisms with clinically proven efficacy/safety, and are excellent anticancer agents for various intractable solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer in clinical trials. Efficacy has been verified. Camptothecin-based drugs such as Exatecan and SN-38 have been developed as payloads for ADCs.
화학식 1-1의 엑사테칸(Exatecan)은 캄토테신 유도체로서, 제1형 토포이소머라아제를 저해하는 항종양성 저분자 화합물이다. 엑사테칸은 SN-38 보다 5 - 10배 강력한 세포독성(Cellular Cytotoxicity)이 확인된 물질이다.Exatecan of Formula 1-1 is a camptothecin derivative and is an anti-tumor small molecule compound that inhibits type 1 topoisomerase. Exatecan is a substance that has been confirmed to have cellular cytotoxicity that is 5 to 10 times more powerful than SN-38.
엑사테칸은 이리노테칸과는 상이하고, 효소에 의한 활성화가 불필요하다. 또, 이리노테칸의 약효 본체인 SN-38이나, 동 임상에서 사용되고 있는 토포테칸보다 제1형 토포이소머라아제 저해 활성이 강하고, in vitro 에서 여러 가지의 암 세포에 대해, 보다 강한 세포독성 활성을 가지고 있다. 특히 P-glycoprotein의 발현에 의해 SN-38 등에 내성을 나타내는 암 세포에 대해서도 효과를 나타냈다. 또, 마우스의 인간 종양 피하 이식 모델에서도 강한 항종양 효과를 나타내어, 임상 시험이 실시되었다. Exatecan is different from irinotecan and does not require activation by enzymes. In addition, it has stronger type 1 topoisomerase inhibitory activity than SN-38, the main medicinal product of irinotecan, and topotecan, which is used in clinical trials, and has stronger cytotoxic activity against various cancer cells in vitro. there is. In particular, it was effective against cancer cells that were resistant to SN-38 and other drugs due to the expression of P-glycoprotein. In addition, it showed a strong anti-tumor effect in a human tumor subcutaneous transplant model in mice, and clinical trials were conducted.
화학식 1-2의 Dxd(ADC용 Exatecan 유도체)는, HER2 표적 ADC(DS-8201a)의 접합 약물로 사용되는 IC50이 0.31μM인, 강력한 DNA 토포이소머라제 I 억제제이다.Dxd (Exatecan derivative for ADC) of Formula 1-2 is a potent DNA topoisomerase I inhibitor with an IC 50 of 0.31 μM, used as a conjugate drug for HER2 targeting ADC (DS-8201a).
이에 제한되는 것은 아니나, 상기 화학식 1의 화합물에 C1-3 알킬, 히드록시, 할로젠, 아민기 등의 간단한 작용기를 부가하거나 혹은 이미 존재하는 히드록시, 에틸, 옥소 작용기를 다른 작용기로 치환하거나 제거하더라도, 본 발명의 면역접합체 또는 운반체-약물 접합체와 동등한 효능을 나타내는 한 본 발명의 균등범위에 포함됨은 자명하다.It is not limited thereto, but simple functional groups such as C 1-3 alkyl, hydroxy, halogen, and amine groups can be added to the compound of Formula 1, or existing hydroxy, ethyl, and oxo functional groups can be substituted with other functional groups. Even if removed, it is obvious that it is included in the scope of equivalents of the present invention as long as it exhibits equivalent efficacy to the immunoconjugate or carrier-drug conjugate of the present invention.
[효율적 약물 전달이 가능한 링커 기술][ Linker technology enabling efficient drug delivery ]
기존 MMAE, Calicheamicin, PBD 등 슈퍼톡신(super toxin)을 활용한 ADC의 경우 약물이 암 조직에 도달하기 전에 혈중에서 분리되는 것을 최소화하는 것을 목표로 하는 Stable Linker 시스템을 활용(2세대 ADC의 특징)하였다.In the case of ADCs using super toxins such as MMAE, Calicheamicin, and PBD, the Stable Linker system is used, which aims to minimize the separation of drugs from the blood before reaching cancer tissues (a feature of the 2nd generation ADC). did.
대부분의 2세대 ADC가 암세포에서 작동하는 원리는 첫 단계로 ADC를 구성하는 항체부분이 암세포에서 과발현된 항원에 결합하고, 두번째 단계로 항원-항체 반응에 의해 암세포 표면에서 항원에 결합된 ADC가 엔도좀, 리소좀을 거치며 암세포 내부로 이송되며, 세번째 단계로 리소좀에서 항원-항체 부분이 분해되고, 효소 (cathepsin B) 약물이 방출되며, 마지막 단계로 암세포 내부에서 방출된 약물에 의해 암세포가 사멸하게 된다.The principle behind how most second-generation ADCs work in cancer cells is that, in the first step, the antibody portion that makes up the ADC binds to the antigen overexpressed in the cancer cell, and in the second step, the ADC bound to the antigen on the surface of the cancer cell through an antigen-antibody reaction is endo-activated. It is transported inside the cancer cell through lysosomes. In the third step, the antigen-antibody portion is decomposed in the lysosome, the enzyme (cathepsin B) and the drug are released, and in the final step, the cancer cell is killed by the drug released inside the cancer cell. .
다양한 고형암에 선택적으로 사용할 수 있는 새로운 ADC의 개발을 위해서는 기존 ADC의 약물전달 효율을 능가하는 링커 시스템의 활용이 필수적이다. 이미 정립된 약물 표적인 Trop-2, Her2, Folate Receptor 등을 넘어서 새로운 항원 대상의 ADC 개발을 위해서는 CEACAM-5 등 Slow Internalizing 항원 및/또는 NY-ESO-1/HLA Complex 등 암특이 항원에 대한 ADC의 개발이 이루어져야 하나 기존 Val-Cit 또는 MAC-glucuronide Linker 시스템의 제한된 약물전달 효율로는 충분한 양의 약물을 전달하기가 어렵다.In order to develop a new ADC that can be selectively used for various solid cancers, it is essential to utilize a linker system that exceeds the drug delivery efficiency of existing ADC. In order to develop ADC targeting new antigens beyond already established drug targets such as Trop-2, Her2, and Folate Receptor, ADC for slow internalizing antigens such as CEACAM-5 and/or cancer-specific antigens such as NY-ESO-1/HLA Complex. should be developed, but it is difficult to deliver a sufficient amount of drug with the limited drug delivery efficiency of the existing Val-Cit or MAC-glucuronide Linker system.
특히 CEACAM-5 등의 항원에 대해서 효율적인 약물전달을 위해서는 혈중 및/또는 정상 조직의 주변환경에서는 항체-약물 결합이 안정성을 유지하지만, 종양 미세환경 등 암세포 주변 환경에서는 빠르게 약물을 방출하는 특성이 필요하다.In particular, for efficient drug delivery to antigens such as CEACAM-5, the antibody-drug bond maintains stability in the blood and/or the surrounding environment of normal tissues, but requires the characteristic of rapidly releasing the drug in the surrounding environment of cancer cells, such as the tumor microenvironment. do.
암 주변의 종양 미세 환경에서도 빠르게 약물을 방출할 수 있는 경우에는 ADC uptake 속도에 제한이 있는 여러 항원들 (대표적으로는 CEACAM-5, 각종 Cancer Specific antigen-HLA Complex 등)의 타겟팅에도 유리하게 사용할 수 있다.If the drug can be released quickly even in the tumor microenvironment surrounding the cancer, it can be advantageously used to target various antigens (typically CEACAM-5, various Cancer Specific antigen-HLA Complexes, etc.) that have limitations in ADC uptake speed. there is.
따라서, 화학식 1의 캄토테신계 약물은 SN-38 대비 혈액, 간질액 등 체액에서 응집되지 않도록 수분산성이 향상된 약물이므로 종양조직 심부까지 침투하면서 세포막을 관통해 세포내로 이동할 수 있는 소수성 저분자라는 점을 십분 활용하도록, 본 발명은 암 조직에 다다른 후에는 빠르고 효율적으로 약물을 전달하는 방출 프로파일(Release Profile)의 링커로 암 주변 산성 환경(pH ≤ 7)에서 분해되는 산 민감성 링커를 선정한 것이다. Therefore, the camptothecin-based drug of Formula 1 is a drug with improved water dispersibility to prevent aggregation in body fluids such as blood and interstitial fluid compared to SN-38, so it is a hydrophobic small molecule that can penetrate deep into tumor tissue and move into cells through cell membranes. To take full advantage, the present invention selects an acid-sensitive linker that decomposes in an acidic environment (pH ≤ 7) around the cancer as a linker with a release profile that delivers the drug quickly and efficiently once it reaches the cancer tissue.
본 발명에 따라 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물이 연결된 항체는 2세대 ADC가 암세포에서 작동하는 첫 단계와 동일하게 암세포 표면에 과발현된 항원에 항체가 결합하지만, 일부는 2세대 ADC와 동일하게 세포 내 처리 단계를 거치고 이외의 상당 부분은 암세포 주위의 낮은 pH에 의해 약물을 방출할 수 있다. 이후 암 조직에서 방출된 약물은 확산에 의해 암세포 내부로 이동하여 엔도좀, 리소좀을 거치지 않을 뿐만 아니라 효소 (cathepsin B) 반응 없이 바로 암세포에 작동하여 세포사멸을 유도한다. 이로인해 효소 반응에 의해서만 약물 방출이 가능한 2세대 ADC들과 비교하면 암세포의 항원선택성은 동일하지만, pH 민감성 링커를 사용하여 약물 방출 및 암세포 내로의 전달 효율을 극대화시킬 수 있다는 것이 본 발명의 면역 접합체의 주요한 특징이다.According to the present invention, the antibody to which the camptothecin-based drug of Formula 1 is linked through an acid-sensitive linker binds to the antigen overexpressed on the surface of cancer cells in the same way as the first step in which the second-generation ADC operates in cancer cells, but some of the antibodies bind to the antigen overexpressed on the surface of the second-generation ADC. Apart from going through the same intracellular processing steps, a significant portion of the drug can be released due to the low pH around the cancer cells. Afterwards, the drug released from the cancer tissue moves into the cancer cells by diffusion, bypassing endosomes and lysosomes, and acts directly on the cancer cells without enzymatic (cathepsin B) reaction, inducing apoptosis. As a result, compared to second-generation ADCs that can release drugs only through enzymatic reactions, the antigen selectivity of cancer cells is the same, but the immunoconjugate of the present invention can maximize drug release and delivery efficiency into cancer cells by using a pH-sensitive linker. This is the main feature of .
또한, 링커는 혈류에 안정(stable)하여 약물이 항체로부터 분리되는 것을 막아 타겟에 도달할 때까지 prodrug 상태로 유지되어 정상적인 조직에 입히는 피해를 최소화해야 함에도 불구하고, 본 발명은 가수분해 환경을 조성하도록 친수성 분자 구조를 가진 산 민감성 링커를 사용하여, 소수성 약물과 결합하여 ADC 응집이 일어나는 문제를 완화시킬 수 있다.In addition, the linker is stable in the bloodstream, preventing the drug from separating from the antibody, maintaining it in a prodrug state until it reaches the target, and minimizing damage to normal tissues. However, the present invention creates a hydrolysis environment. By using an acid-sensitive linker with a hydrophilic molecular structure, the problem of ADC aggregation when combined with a hydrophobic drug can be alleviated.
링커의 종류에 따라 상이한 대사체(catabolite)가 형성된다. 이와 관련하여, 본 발명의 [화학식 1의 캄토테신계 약물]-[산 민감성 링커]는, 산 민감성 링커 분해시 유리형 화학식 1의 캄토테신계 약물이 방출되도록, 화학식 1의 캄토테신계 약물과 산 민감성 링커는 탄산염 또는 에스테르 결합으로 연결된 것이 바람직하다.Different metabolites are formed depending on the type of linker. In this regard, the [camptothecin-based drug of Formula 1]-[acid-sensitive linker] of the present invention is a camptothecin-based drug of Formula 1 so that the free camptothecin-based drug of Formula 1 is released when the acid-sensitive linker is decomposed. The acid-sensitive linker is preferably connected by a carbonate or ester bond.
일반적으로 가수분해와 관련하여 에스테르 및 탄산염 결합에 비해 카르바메이트 결합(Carbamate bonds)이 우수한 약물 링커 안정성을 제공한다. 그러나, 본 발명은 화학식 1의 캄토테신계 약물이 암세포 주변 산성 환경(pH)에서 암세포 주변 세포 밖 및 세포 내에서 모두 약물 링커로부터 분리가능하게 설계하기 위해, 카르바메이트 결합(Carbamate bonds) 대신, 불안정한 에스테르 또는 탄산염 결합(carbonate bonds)를 사용하는 것이 특징이다. In general, carbamate bonds provide superior drug linker stability compared to ester and carbonate bonds with respect to hydrolysis. However, in the present invention, in order to design the camptothecin-based drug of Formula 1 to be separable from the drug linker both outside and inside the cells surrounding cancer cells in the acidic environment (pH) surrounding cancer cells, instead of carbamate bonds, It is characterized by the use of unstable ester or carbonate bonds.
혈액은 pH가 7.3~7.4로 일정하게 유지된다. 따라서, 혈액 중에서 산 민감성 링커로부터 화학식 1의 캄토테신계 약물은 절단되지 않으며, 절단되더라도 혈청의 중성 pH에서 화학식 1의 캄토테신계 약물의 ADC로부터 방출 속도는 산성 환경의 종양 조직에서 보다 훨씬 감소된다.The pH of blood is kept constant at 7.3 to 7.4. Therefore, the camptothecin-based drug of Formula 1 is not cleaved from the acid-sensitive linker in the blood, and even if cleaved, the release rate of the camptothecin-based drug of Formula 1 from the ADC at the neutral pH of serum is much reduced than in tumor tissue in an acidic environment. .
또한, 소수성 약물에 의한 혈장 내 ADC 응집을 감소하기 위해, 본 발명은 화학식 1의 캄토테신계 약물에서 E-고리의 20번 탄소에 위치한 알파수산화기에 산 민감성(acid-sensitive) 링커를 연결하는 것이 특징이다.In addition, in order to reduce ADC aggregation in plasma caused by hydrophobic drugs, the present invention connects an acid-sensitive linker to the alpha hydroxyl group located at carbon 20 of the E-ring in the camptothecin-based drug of Formula 1. It is a characteristic.
이경우, 캄토테신계 약물에서 A- 및 B-고리로부터 확장된 6각 또는 7각 고리에 있는 예컨대 탄소-탄소 단일 결합에 대하여 분자 결합의 회전이 가능하여 자유도가 큰 -NH2 또는 -OH가 수 환경(aqueous environment), 즉 물(H2O)에 노출되어 (+) 전하를 띠거나 물과 수소결합됨으로써, 산 민감성 링커를 통해 배향된 엑사테칸 또는 Dxd 약물 부위의 수 분산성을 향상시킬 수 있다. In this case, in camptothecin-based drugs, rotation of the molecular bond with respect to, for example, a carbon-carbon single bond in a hexagonal or heptagonal ring extended from the A- and B-rings is possible, so that -NH 2 or -OH, which has a large degree of freedom, can be formed. By being exposed to the aqueous environment, that is, water (H 2 O), it can acquire a (+) charge or be hydrogen bonded with water, thereby improving the water dispersibility of the drug moiety of exatecan or Dxd oriented through an acid-sensitive linker. You can.
Tetrapeptide 링커는 소수성 약물과 결합하여 ADC 응집이 일어날 수 있는 한계가 나타났다.The tetrapeptide linker showed a limit to the extent to which ADC aggregation could occur when combined with hydrophobic drugs.
기존 FDA 승인된 ADC인 Trodelvy에 사용된 링커인 CL2A는 (i) 제조 후 보관 안정성, (ii) 투여 시 혈중에서의 안정성 (Plasma 중에 Free Payload 노출이 거의 없음), (iii) 암 조직에서 Payload의 빠른 방출 등의 특성을 모두 만족하는 링커이다.CL2A, the linker used in Trodelvy, an existing FDA-approved ADC, has (i) storage stability after manufacturing, (ii) stability in the blood upon administration (little exposure to free payload during plasma), and (iii) stability of payload in cancer tissue. It is a linker that satisfies all characteristics such as rapid release.
최초 CL2 유도체에 삽입된 Phe-Lys 펩타이드는 카텝신 B를 통해 절단을 가능하게 한다. 합성 과정을 간소화시키기 위한 일환으로, CL2A에서 페닐알라닌이 제거되었고, 이에 따라 카텝신 B 절단 부위가 제거되었다. 이러한 변화는 접합체 결합, 안정성, 또는 효능에 대해 영향을 주지 않았다. 이는 CL2에서의 카텝신 B 절단 부위가 아니라, SN-38의 락톤 고리에 대한 pH 민감성 탄산 벤질 결합(pH-sensitive benzyl carbonate bond)의 절단에 의해 주로 접합체로부터 방출되었음을 시사한다.The Phe-Lys peptide inserted into the original CL2 derivative enables cleavage through cathepsin B. In an effort to simplify the synthesis process, phenylalanine was removed from CL2A, thereby eliminating the cathepsin B cleavage site. These changes had no effect on conjugate binding, stability, or efficacy. This suggests that it was released from the conjugate primarily by cleavage of the pH-sensitive benzyl carbonate bond to the lactone ring of SN-38, rather than through the cathepsin B cleavage site in CL2.
본 발명에서 사용되는 산 민감성 링커는, CL2A 링커를 활용할 수 있으며, 화학식 1의 캄토테신계 약물을 암 조직에 선택적, 효율적으로 전달하도록 하기 화학식 3과 같이 설계될 수 있다. 즉, 본 발명에서 산 민감성 링커는 하기 화학식 3의 화합물로부터 유래되는 것일 수 있다: The acid-sensitive linker used in the present invention can utilize the CL2A linker, and can be designed as shown in Formula 3 below to selectively and efficiently deliver the camptothecin-based drug of Formula 1 to cancer tissue. That is, in the present invention, the acid-sensitive linker may be derived from a compound of formula 3 below:
[화학식 3][Formula 3]
Figure PCTKR2023006170-appb-img-000010
Figure PCTKR2023006170-appb-img-000010
여기서, X1 및 X2는 각각 독립적으로 -H 또는 -할로젠이고;Here, X 1 and X 2 are each independently -H or -halogen;
Y는 -NH-, -NRA-, 또는 아무 것도 아니며 (null); Y is -NH-, -NR A -, or nothing (null);
Z는 -C1-C4알킬-, -C3-C6시클로알킬-, -(C1-C2알킬)-(C3-C6시클로알킬)-, -(C3-C6시클로알킬)-(C1-C2알킬)-, 또는 -(C1-C2알킬)-(C3-C6시클로알킬)-(C1-C2알킬)-이고; Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
W는 -RB-, -M- -RB-M-, -M-RB- 또는 -RB-M-RC-이며; W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
RA 내지 RC는 각각 독립적으로 C1-C4알킬이고, R A to R C are each independently C 1 -C 4 alkyl,
M은
Figure PCTKR2023006170-appb-img-000011
이며; 및M is
Figure PCTKR2023006170-appb-img-000011
and; and
n은 5 내지 9의 정수임.n is an integer from 5 to 9.
바람직하게는 상기 화학식 3에서, Preferably in Formula 3,
X1 및 X2는 각각 독립적으로 -H 또는 -할로젠이고;X 1 and X 2 are each independently -H or -halogen;
Y는 -NRA-, 또는 아무 것도 아니며 (null); Y is -NR A -, or nothing (null);
Z는 -C1-C4알킬-, -(C1-C2알킬)-(C3-C6시클로알킬)-, 또는 -(C3-C6시클로알킬)-(C1-C2알킬)-이고; Z is -C 1 -C 4 alkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, or -(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
W는 -RB- 또는 -RB-M-RC-이며; W is -R B - or -R B -MR C -;
RA 내지 RC는 각각 독립적으로 C1-C4알킬이고; R A to R C are each independently C 1 -C 4 alkyl;
M은
Figure PCTKR2023006170-appb-img-000012
이며; 및M is
Figure PCTKR2023006170-appb-img-000012
and; and
n은 5 내지 9의 정수일 수 있다.n may be an integer from 5 to 9.
본 발명에서, 링커의 길이, 즉 상기 화학식 3에서 n은 5 내지 9의 정수일 수 있고, 구체적으로 n은 6 내지 8의 정수일 수 있으며, 더욱 구체적으로 n은 7일 수 있으나, 이에 제한되지 않는다. 위 범위를 벗어나는 경우라도, 링커 길이 변화에 따른 별 다른 효과 차이가 없는 경우에는 당연히 모두 본 발명의 균등 범위 내에 포함된다.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, and more specifically n may be 7, but is not limited thereto. Even in cases outside the above range, if there is no significant difference in effect due to change in linker length, all are naturally included within the equivalent scope of the present invention.
약물의 수분산도는 약물과 운반체 사이에 폴리에틸렌글리콜 (PEG) 스페이서를 배치하여 향상될 수 있으므로, 화학식 3의 링커는 한정된 수(n=5 내지 9)의 PEG 모노머를 포함하는 저분자량 PEG 모이어티를 포함한다.Since the aqueous dispersibility of the drug can be improved by placing a polyethylene glycol (PEG) spacer between the drug and the carrier, the linker of Formula 3 contains a low molecular weight PEG moiety containing a limited number (n = 5 to 9) of PEG monomers. Includes.
화학식 3의 산 민감성 링커는 Targeting 대상, Payload, Carrier의 특성에 따라 최적화 가능한 맞춤형 링커이다.The acid-sensitive linker of Formula 3 is a customized linker that can be optimized according to the characteristics of the targeting target, payload, and carrier.
화학식 1의 캄토테신계 약물은 운반체-약물 접합체 제작을 위하여 다양한 링커와 부착이 용이한 Site를 가지고 있다. 예컨대, 화학식 1의 캄토테신계 약물의 알코올기 부위를 링커와의 부착 부위(site)로 사용할 수 있다.The camptothecin drug of Formula 1 has various linkers and sites that are easy to attach to for the production of carrier-drug conjugates. For example, the alcohol moiety of the camptothecin-based drug of Formula 1 can be used as an attachment site to the linker.
따라서, 본 발명에서 [화학식 1의 캄토테신계 약물]-[산 민감성 링커]는 화학식 1의 캄토테신계 약물의 알코올기 부위와 화학식 3의 산 민감성 링커의 알코올기 부위가 연결된 것일 수 있다.Therefore, in the present invention, [camptothecin-based drug of Formula 1]-[acid-sensitive linker] may be the alcohol moiety of the camptothecin-based drug of Formula 1 and the alcohol moiety of the acid-sensitive linker of Formula 3.
본 발명에서 [화학식 1의 캄토테신계 약물]-[산 민감성 링커] 접합체는 하기 화학식 4 또는 5로 표시되는 화합물일 수 있다.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.
[화학식 4][Formula 4]
Figure PCTKR2023006170-appb-img-000013
Figure PCTKR2023006170-appb-img-000013
[화학식 5][Formula 5]
Figure PCTKR2023006170-appb-img-000014
Figure PCTKR2023006170-appb-img-000014
(여기서, n은 각각 독립적으로 5 내지 9의 정수임).(where n is each independently an integer from 5 to 9).
약물전달 효율 개선을 위해 본 발명의 산 민감성 링커는, site-specific conjugation의 단점으로 생각되는 낮은 DAR의 문제를 극복할 수 있는 multivalent linker 시스템을 개발, 적용하여 1개의 attachment site에 2-3개의 payload를 부착하는 방식으로 Site-specific conjugation 시에도 DAR 4 - 12의 high DAR ADC를 제조할 수 있는 방법을 확보할 수 있다.To improve drug delivery efficiency, the acid-sensitive linker of the present invention develops and applies a multivalent linker system that can overcome the problem of low DAR, which is considered a disadvantage of site-specific conjugation, to deliver 2-3 payloads to one attachment site. By attaching a method, it is possible to secure a method to manufacture a high DAR ADC of DAR 4 - 12 even during site-specific conjugation.
이를 위해, 본 발명에서는 화학식 4 및 5에 예시된 바와 같은 Bridgeable Linker 시스템을 링커로 사용할 수 있다.For this purpose, in the present invention, the Bridgeable Linker system as exemplified in Formulas 4 and 5 can be used as a linker.
이러한 Bridgeable Linker 시스템은, CL2A 링커 시스템의 최대 장점인, 암 조직에서 Payload의 빠른 방출 특성을 그대로 유지하면서 DAR 4의 ADC를 용이하게 제조할 수 있도록 CMC의 효율을 높이고 공정을 단순화시킬 수 있다. 별도의 항체 engineering을 수행하지 않고 DAR 4의 site-specific 항체-약물 복합체를 제조할 수 있다. 항체 내에 존재하는 disulfide(-S-S-)를 환원시키면 두 개의 Thiol(-SH)이 형성되고, 이렇게 생성된 Thiol은 새로운 bridgeable linker-화학식 1의 캄토테신계 약물 과 2:1로 conjugate를 형성하므로, 일반 항체 내부의 4개의 disulfide를 모두 반응시키는 방법으로 DAR 4의 항체-약물 복합체를 단일 product로 용이하게 제조할 수 있다.This Bridgeable Linker system can increase the efficiency of CMC and simplify the process so that DAR 4 ADC can be easily manufactured while maintaining the rapid release characteristics of the payload from cancer tissue, which is the greatest advantage of the CL2A linker system. A site-specific antibody-drug complex of DAR 4 can be manufactured without performing separate antibody engineering. When the disulfide (-S-S-) present in the antibody is reduced, two thiols (-SH) are formed, and the thiols thus produced form a 2:1 conjugate with a new bridgeable linker - the camptothecin drug of Formula 1. The antibody-drug complex of DAR 4 can be easily manufactured as a single product by reacting all four disulfides inside a general antibody.
[분자 접착 분해제(molecular glue degrader)라는 약물 모달리티의 잇점] [ Advantages of the drug modality called molecular glue degrader ]
몸속 세포 내 단백질들은 제 기능을 수행한 후 수시간에서 수일 내 자연 분해된다. 체내의 모든 세포에는 단백질을 분해시키는 유비퀴틴 프로테아좀 시스템(Ubiquitin proteasome system, UPS)이라는 정화작용이 존재하는데, 이 과정에서 유비퀴틴은 분해되어야 하는 단백질을 알려주는 표식(marker) 역할을 하며, 프로테아좀은 유비퀴틴 표식을 인지하고 해당 단백질을 파괴하는 분쇄기 역할을 한다. 즉, 제 역할을 다한 단백질 옆에 유비퀴틴(Ubiquitin)이라는 물질 여러 개가 표식처럼 붙고, 프로테아좀(Proteasome)이라는 물질이 이 표식을 가진 단백질만 골라서 분쇄기처럼 분해해버린다. E3 리가아제는 체내 단백질 분해 시스템을 일으키는 효소로서, 유비퀴틴화 경로에서 기질 특이성을 담당한다.Proteins within the cells of the body are naturally decomposed within a few hours to a few days after performing their function. All cells in the body have a purification system called the Ubiquitin proteasome system (UPS) that decomposes proteins. In this process, ubiquitin acts as a marker to indicate which proteins need to be decomposed, and proteasome The moth recognizes the ubiquitin tag and acts as a shredder that destroys the corresponding protein. In other words, several substances called ubiquitin are attached like a mark next to a protein that has completed its function, and a substance called proteasome selects only the proteins with this mark and breaks them down like a shredder. E3 ligase is an enzyme that initiates the protein degradation system in the body and is responsible for substrate specificity in the ubiquitination pathway.
분자 접착 분해제(molecular glue degrader) 또는 분자접착제(molecular glue)는 표적 단백질과 우리 몸의 특정 효소(E3 리가아제)를 서로 붙이는 접착제 기능을 하는 화합물이다. 분자접착제(molecular glue)의 장점 중 하나는 촉매 역할로, 표적 단백질을 분해한 뒤 다시 분리돼 또 다른 표적 단백질을 분해할 수 있다는 것이다. Molecular glue degrader or molecular glue is a compound that functions as an adhesive to attach a target protein to a specific enzyme (E3 ligase) in our body. One of the advantages of molecular glue is that it acts as a catalyst, decomposing a target protein and then separating again to degrade another target protein.
분자접착제(molecular glue)를 통해 종양단백질에 E3 리가아제 효소가 붙으면 종양단백질이 분해되고, 표적인 종양단백질이 없어질 때까지 연속적으로 다른 종양단백질들이 분해되기 때문에 암세포 증식을 막을 수 있다. 따라서, 종양단백질에 대한 분자접착제는 표적 항암제의 문제점인 약물 내성을 극복하고, 적은 투여 용량으로도 치료 효과가 높다.When the E3 ligase enzyme attaches to a tumor protein through molecular glue, the tumor protein is decomposed, and other tumor proteins are successively degraded until the target tumor protein disappears, thereby preventing cancer cell proliferation. Therefore, molecular adhesives for tumor proteins overcome drug resistance, which is a problem with targeted anticancer drugs, and have high therapeutic effects even at low administration doses.
본 발명에 따라 페이로드로 사용되는 화학식 1의 캄토테신계 약물은 DDX5 단백질 및 E3 리가아제에 결합하는 분자접착 분해제(molecular glue degrader), 즉 종양단백질 DDX5 또는 이의 인산화된 DDX5 단백질(p-DDX5) 분해를 활성화하는 분자접착제이다(도 1, 도 4 내지 도 8).The camptothecin-based drug of Formula 1 used as a payload according to the present invention is a molecular glue degrader that binds to DDX5 protein and E3 ligase, that is, tumor protein DDX5 or its phosphorylated DDX5 protein (p-DDX5). ) It is a molecular adhesive that activates decomposition (Figures 1, 4 to 8).
분자접착제(molecular glue degrader)는 표적 단백질에 결합하는 리간드(warhead)일 뿐만아니라, E3 리가아제 리간드(binder) 역할을 수행할 수 있다. A molecular glue degrader is not only a warhead that binds to a target protein, but can also act as an E3 ligase ligand (binder).
따라서, 본 발명에 따른 화학식 1의 캄토테신계 약물은 종양단백질 DDX5 분해를 활성화하는 분자접착제이므로, DDX5 단백질을 표적화하는 리간드 또는 DDX5 단백질에 결합하는 리간드로서 사용될 수 있다. Therefore, the camptothecin-based drug of Formula 1 according to the present invention is a molecular adhesive that activates the decomposition of the tumor protein DDX5, and can be used as a ligand targeting the DDX5 protein or a ligand that binds to the DDX5 protein.
분자접착제(molecular glue degrader)은 키나제 저해제와 달리 '위치 주도형 (proximity-driven)'이고 분해 유도 능력은 일시적인 '표적단백질- 분자접착제(molecular glue)-E3 리가아제' 삼중 복합체 형성에 달려 있는 '발생 주도형(event-driven)'이다. 분해 발생 이후에 분리된 분자접착제(molecular glue)이 표적 단백질과 추가적인 삼중 복합체를 형성하여 표적 단백질이 없어질 때까지 여러 번의 분해 과정을 진행할 수 있다.Unlike kinase inhibitors, molecular glue degraders are 'proximity-driven' and their ability to induce degradation depends on the formation of a temporary 'target protein-molecular glue-E3 ligase' triple complex. It is ‘event-driven’. After degradation occurs, the separated molecular glue forms an additional triple complex with the target protein, allowing multiple degradation processes to proceed until the target protein disappears.
대부분 약물 타겟이 되는 단백질은 약물에 적응해 진화하는 내성이 발생한다. 그러나, 종양단백질과 E3 리가아제를 서로 붙이는 접착제 기능을 하는 저분자 화합물인 분자접착제(molecular glue)은 내성에 강하기 때문에 암 치료제에 적합한 모달리티이다.Most proteins that are drug targets develop resistance that evolves by adapting to the drug. However, molecular glue, a low-molecular-weight compound that acts as an adhesive that binds tumor proteins and E3 ligase together, is a suitable modality for cancer treatment because it is resistant to resistance.
유전자 변이가 일어날 때마다 약물 타겟 단백질의 모양은 조금씩 바뀐다. 한 가지 약물로 모든 변이체의 활성을 막으면 좋겠지만 선택성의 문제 때문에 가능하지 않기에, 한 가지 약물이 모든 변이의 활성을 막기 위해, 약물 타겟 단백질을 분해시켜 아예 제거해 버리는 것이다. Each time a genetic mutation occurs, the shape of the drug target protein changes slightly. It would be nice to block the activity of all variants with one drug, but this is not possible due to selectivity issues. In order to block the activity of all variants with one drug, the drug target protein is decomposed and completely removed.
따라서, 본 발명에 따른 화학식 1의 캄토테신계 약물은 DDX5 종양단백질에 정확하게 결합할 수 있는 약물로, 바로 선택적으로 단백질을 분해하는 분자접착제(molecular glue) 접근 방법을 통해, 표적치료제들의 내성 문제를 피할 수 있다. Therefore, the camptothecin-based drug of Formula 1 according to the present invention is a drug that can accurately bind to the DDX5 tumor protein, and solves the resistance problem of targeted treatments through a molecular glue approach that selectively decomposes the protein. It can be avoided.
본 발명에 따른 화학식 1의 캄토테신계 약물은 이의 물리화학적 특성에 따라 표적 단백질인 DDX5 종양단백질에 결합강도가 결정되며, 너무 강하게 결합해 다시 이전의 상태로 돌아오지 못하는 비가역적 약물인 것이 바람직하다.The camptothecin-based drug of Formula 1 according to the present invention has a binding strength to the target protein, DDX5 tumor protein, depending on its physicochemical properties, and is preferably an irreversible drug that binds so strongly that it cannot return to its previous state. .
본 발명에 따른 화학식 1의 캄토테신계 약물은 기존 SN38 약물과 달리 DDX5와의 친화도가 높아 쉽게 떨어지지 않고 분자접착제(molecular glue) 기능을 통해 DDX5을 분해시켜 DDX5과 관련된 암세포의 신호전달을 비가역적으로 억제할 뿐만 아니라, 이의 물리화학적 특성에 따라 약물의 세포막 투과도를 원하는대로 조절하여, 방관자 효과(bystander effect)를 발휘 또는 그 효과의 정도를 제어시킴으로써 주변세포살상효과가 높아서 비균질종양의 치료에 이점이 있을 뿐만 아니라, 장기간 암 진행을 억제하고, 내성 발현의 위험을 줄여 치료 반응률을 높일 수 있다. Unlike existing SN38 drugs, the camptothecin-based drug of Formula 1 according to the present invention has a high affinity for DDX5, so it does not fall off easily and decomposes DDX5 through a molecular glue function, thereby irreversibly inhibiting signaling in cancer cells related to DDX5. Not only does it inhibit it, but it also controls the cell membrane permeability of the drug according to its physicochemical properties as desired, thereby exerting a bystander effect or controlling the degree of the effect, resulting in a high killing effect on surrounding cells, which is advantageous in the treatment of heterogeneous tumors. In addition, it can suppress long-term cancer progression and increase the treatment response rate by reducing the risk of resistance development.
본 발명에 따른 화학식 1의 캄토테신계 약물은 세포 내에서 종양단백질(oncoprotein)로 작동하는 DDX5에 결합하여 DDX5 단백질 분해를 통해 세포사멸(cell death)을 유도할 수 있다(도 11).The camptothecin-based drug of Formula 1 according to the present invention can bind to DDX5, which acts as an oncoprotein within cells, and induce cell death through DDX5 protein degradation (FIG. 11).
암 치료에서 본질적인 약물 내성은 세포 증식과 세포 사멸의 결정적인 조절자인 비정상적으로 발현된 전사 인자에 의해 야기될 수 있다. 따라서, 본 발명에 따른 화학식 1의 캄토테신계 약물은, 전자인자 보조인자이면서 종양단백질(oncoprotein)인 DDX5에 결합하는 분자 접착분해제(molecular glue degrader)로서의 작용기전을 통해 DDX5 단백질 분해 및 이로인한 세포사멸(cell death)을 유도할 수 있다. 이때, DDX5 단백질 분해는 항세포사멸 유전자(anti-apoptotic genes)의 전사를 하향조절할 수 있다(도 4 내지 도 8). 따라서, 다른 표적치료제와 달리 비정상적으로 발현되는, 세포 증식 및/또는 세포 사멸 관련 전사 인자에 의해 야기되는 약물 내성이 잘 일어나지 않고/않거나, 화학요법 및/또는 방사선요법 동안 조절되지 않은 전사 인자의 활성화를 통해 유도되는 후천적 약물 내성을 억제할 수 있다. In cancer treatment, intrinsic drug resistance may be caused by abnormally expressed transcription factors, which are critical regulators of cell proliferation and apoptosis. Therefore, the camptothecin-based drug of Formula 1 according to the present invention decomposes DDX5 protein through its mechanism of action as a molecular glue degrader that binds to DDX5, which is an electron cofactor and an oncoprotein. Can induce cell death. At this time, DDX5 protein degradation can downregulate the transcription of anti-apoptotic genes (Figures 4 to 8). Therefore, unlike other targeted therapies, drug resistance caused by abnormally expressed cell proliferation and/or cell death-related transcription factors is less likely to occur, and/or uncontrolled activation of transcription factors during chemotherapy and/or radiotherapy. Acquired drug resistance induced through can be suppressed.
즉, 본 발명에 따른 화학식 1의 캄토테신계 약물의 항암기전은 암 치료 저항성의 분자 메커니즘을 우회할 수 있으므로, 약물 내성 문제에서 자유로울 수 있다. 내성이 발생하면 이전보다 치료 효과가 떨어지기 때문에, 본 발명에 따른 화학식 1의 캄토테신계 약물은, 암 진단 후 표준 치료제 또는 1차 치료제로 선호될 수 있다. In other words, the anti-cancer mechanism of the camptothecin-based drug of Formula 1 according to the present invention can bypass the molecular mechanism of cancer treatment resistance, and thus can be free from the problem of drug resistance. Since the treatment effect is lower when resistance develops, the camptothecin-based drug of Formula 1 according to the present invention may be preferred as a standard treatment or first-line treatment after cancer diagnosis.
요컨대, 본 발명에 따라 페이로드로 사용되는 화학식 1의 캄토테신계 약물은 암세포의 성장, 생존(cell survivor), 증식, 전이 및/또는 대사에 중요한 역할을 하는 DDX5 단백질에 작용하는 표적 항암제가 될 수 있다.In short, the camptothecin-based drug of Formula 1 used as a payload according to the present invention will be a targeted anticancer agent that acts on the DDX5 protein, which plays an important role in the growth, survival (cell survivor), proliferation, metastasis, and/or metabolism of cancer cells. You can.
[면역접합체][Immunoconjugate]
본 발명에서, 용어 "면역접합체"는 항체 또는 이의 항원 결합 단편에 세포 독성 약물-링커 접합체가 연결된 복합체를 의미한다.In the present invention, the term “immunoconjugate” refers to a complex in which a cytotoxic drug-linker conjugate is linked to an antibody or antigen-binding fragment thereof.
항체-약물 접합체(ADC)은 면역접합체의 일례이므로, 본 발명에서 ADC에 대한 설명 및 면역접합체에 대한 설명은 서로 혼용하여 사용될 수 있다.Since antibody-drug conjugate (ADC) is an example of an immunoconjugate, the description of ADC and the description of immunoconjugate may be used interchangeably in the present invention.
상기 면역접합체는 생체 내 투여될 경우 그 일 구성인 항체 또는 이의 항원결합부위 함유 단편이 표적하는 항원에 결합한 후 약물을 방출함으로써 표적 세포 및/또는 주변세포들에 약물이 작용할 수 있도록 하여, 표적 약물로서 우수한 약효와 감소된 부작용을 기대할 수 있다.When the immunoconjugate is administered in vivo, the antibody or antigen-binding site-containing fragment thereof binds to the target antigen and then releases the drug, allowing the drug to act on target cells and/or surrounding cells, thereby producing the target drug. Excellent efficacy and reduced side effects can be expected.
면역접합체의 효과에 중요한 영향을 미치는 요소는 특히, (1) 약물 효능 (drug potency), (2) 약물 링커 안정성 (drug linker stability), (3) 효율적인 표적 약물 방출 (efficient on-target drug release) 등이 있다. 효과에 여러 요소가 복합적으로 영향을 미치기 때문에 각각의 요소에 대해 알려진 사실만을 바탕으로 이들의 조합인 면역접합체의 효과를 예측하는 것은 대단히 곤란하다.Factors that significantly affect the effectiveness of immunoconjugates are, in particular, (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. etc. Because many factors have a complex effect on the effect, it is very difficult to predict the effect of an immunoconjugate that is a combination of these factors based only on what is known about each factor.
내재화 (internalization)된 후 약물을 방출하도록 설계된 면역접합체는 내재화 과정이 비효율적인 경우 충분한 농도의 활성 약물이 세포 내부로 전달되지 못하는 문제점이 있고, 소수성 약물을 세포 독성 약물로 채택하더라도 주변 세포에 대한 by-stander cell-killing 현상을 기대하기 어렵다는 단점을 가진다.Immunoconjugates designed to release drugs after internalization have the problem of not being able to deliver a sufficient concentration of the active drug into the cell if the internalization process is inefficient, and even if a hydrophobic drug is used as a cytotoxic drug, the -It has the disadvantage that it is difficult to expect a stander cell-killing phenomenon.
이러한 문제점을 해결하기 위해, 본 발명의 면역접합체는 In order to solve this problem, the immunoconjugate of the present invention
(i) 암 주변의 종양 미세 환경(pH ≤ 7)에서, 세포막을 관통할 수 있고 세포 내부에서 제역할을 할 수 있는 소수성 약물을 유리시킨 후, 다량의 유리형 소수성 약물을 세포 안으로 도입시키기 위해, 산 민감성(acid-sensitive) 링커를 사용하고; (i) In the tumor microenvironment (pH ≤ 7) around the cancer, to liberate the hydrophobic drug that can penetrate the cell membrane and play a role inside the cell, and then to introduce a large amount of the free hydrophobic drug into the cell. , using an acid-sensitive linker;
(ii) 암세포 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 약물을 유리하고 다량의 유리형 약물이 세포막을 관통해 세포내에 농축시키기 위해, 세포막을 관통할 수 있는 소수성 약물인 화학식 1의 캄토테신계 약물을 사용한 것이 특징이다(도 4).(ii) In the acidic environment (pH ≤ 7) surrounding cancer cells, the acid-sensitive linker is decomposed to release the drug, and a large amount of the free drug penetrates the cell membrane to concentrate within the cell. Formula 1 is a hydrophobic drug that can penetrate the cell membrane. It is characterized by the use of camptothecin-based drugs (Figure 4).
화학식 1의 캄토테신계 약물은 토포아이소머라제 I 억제제로서 암 세포에 대한 세포 독성을 나타낼 수 있으나, 이를 페이로드로 이용하여 산 민감성(acid-sensitive) 링커를 통해 면역접합체를 제조한 예는 현재까지 전혀 보고된 바 없다.The camptothecin-based drug of Formula 1 is a topoisomerase I inhibitor and can exhibit cytotoxicity against cancer cells, but there are currently no examples of using it as a payload to manufacture an immunoconjugate through an acid-sensitive linker. It has not been reported at all so far.
화학식 1의 캄토테신계 약물은 세포막을 투과할 수 있는 소수성 저분자이므로, 종양조직에 빠르게 축적되어 장시간 높은 농도를 유지 가능하고, 세포 내부로 확산 및 세포독성을 발휘하여 세포를 사멸시킨 후 방출되어 연속적으로 주변 세포에도 세포막을 관통해 세포내로 이동하여 작용할 수 있다. Since the camptothecin drug of Formula 1 is a hydrophobic small molecule that can penetrate the cell membrane, it accumulates rapidly in tumor tissue and can maintain a high concentration for a long time. It spreads inside the cell and exerts cytotoxicity to kill the cell and is then released and continuously released. As a result, it can penetrate the cell membrane of surrounding cells and move into the cell to act.
본 발명의 면역접합체는 전술한 바와 같이 화학식 1의 캄토테신계 약물과 산 민감성 링커를 조합하는 것에 기술적 특징이 있으므로, 원하는 목적에 따라 임의의 항체 또는 이의 항원결합부위 함유 단편을 결합하여 면역접합체를 설계할 수 있으며, 이는 모두 본 발명의 범주 내에 포함된다. 예컨대, 우수한 항암 효과를 위해, 본 발명의 화학식 1의 캄토테신계 약물-산 민감성 링커 접합체에 트라스투주맙, 세툭시맙 및 사시투주맙 (sacituzumab)을 결합시켜 면역접합체를 제조할 수 있다. As described above, the immunoconjugate of the present invention has a technical feature of combining the camptothecin-based drug of Formula 1 with an acid-sensitive linker, so the immunoconjugate can be prepared by combining any antibody or fragment containing an antigen-binding site thereof according to the desired purpose. It can be designed, and it is all included within the scope of the present invention. For example, for excellent anticancer effect, an immunoconjugate can be prepared by combining trastuzumab, cetuximab, and sacituzumab with the camptothecin drug-acid sensitive linker conjugate of Formula 1 of the present invention.
본 발명에서, 상기 면역접합체는 평균 약물/항체 비 (Drug-to-antibody ratio, DAR)가 2 내지 12일 수 있고, 바람직하게는 DAR 4 내지 12일 수 있다.In the present invention, the immunoconjugate may have an average drug-to-antibody ratio (DAR) of 2 to 12, preferably DAR of 4 to 12.
[표적 항원_약물 표적][Target antigen_drug target]
본 발명의 항체-약물 접합체(ADC) 또는 면역접합체(Immunoconjugate) 설계시 표적화하는 대상은 암세포뿐만 아니라, 노화세포, 감염성 질환 유기체 및/또는 자가면역 질환과 관련된 세포까지 확장될 수 있다.When designing the antibody-drug conjugate (ADC) or immunoconjugate of the present invention, the target target may be expanded to include not only cancer cells, but also senescent cells, infectious disease organisms, and/or cells related to autoimmune diseases.
따라서, 항체 또는 이의 항원결합부위 함유 단편이 표적화하는 세포는, 암 세포, 노화세포, 감염성 질환 유기체 및/또는 자가면역 질환과 관련된 세포일 수 있다.Accordingly, the cells targeted by the antibody or antigen-binding site-containing fragment thereof may be cancer cells, senescent cells, infectious disease organisms, and/or cells associated with autoimmune diseases.
표적 항원의 비제한적인 예로, Her2, FolR, PSMA 등 암 표면에 선택적으로 분포하는 항원 및 Trop2 등 정상 조직에도 소수 분포하는 암세포 과발현 항원이 있다.Non-limiting examples of target antigens include antigens selectively distributed on the surface of cancer, such as Her2, FolR, and PSMA, and antigens overexpressed in cancer cells, such as Trop2, which are distributed in small numbers in normal tissues.
암 세포 표적 항원은 예컨대, 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, 아로마타제 (aromatase), ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), 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, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (플렉틴), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2 (D6/JAB61), CCL1 (1-309), CCLI1 (에오탁신), 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/에오탁신-2), CCL25 (TECK), CCL26(에오탁신-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 또는 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, CD69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH1 (E-카드헤린), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wap1/Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB, 키티나제 (Chitinase), CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN6, CLDN7 (클라우딘-7), CLDN18, CLEC5A, CLEC6A, CLEC11A, CLEC14A, CLN3, CLU (클러스테린), 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-카테닌), CTSB (카텝신 B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO), CXCLI1 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9/CKR-L2), CXCR4, CXCR6 (TYMSTR/STRL33/Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2 (HER-2), ERBB3, ERBB4, EREG, ERK8, 에스 트로겐 수용체, Earl, ESR2, F3 (TF), FADD, FAP, 파르네실트란스퍼라제, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF (VEGFD), FIL1(EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (피브로넥틴), FLT1, FLT-3, FOLR1, FOS, FOSL1(FRA-1), FR-알파, FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1, GGT1, GM-CSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, 히스타민 및 히스타민 수용체, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFN감마, 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(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (당단백질 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 인테그린), ITGAV, ITGB3, ITGB4 (β4 인테그린), 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 (케라틴 19), KRT2A, KRTHB6 (헤어 (hair)-특이적 타입 II 케라틴), L1CAM, LAG3, LAMA5, LAMP1, LEP (렙틴), Lewis Y 항원 ("LeY"), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, LY75, LYPD3, MACMARCKS, MAG 또는 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 (메탈로티오넥틴-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, 뉴로칸 (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, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI, P2RX7, PAP, PART1, PATE, PAWR, P-카드헤린, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-아스파라기나제, PF4 (CXCL4), PGF, PGR, 포스파칸 (phosphacan), PIAS2, PI3 키나제, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-베타, PPBP (CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2 (COX-2), PTN, PVRIG, RAC2 (P21Rac2), RANK, RANK 리간드, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB 1D2 (리포필린 B), SCGB2A1 (맘마글로빈 2), SCGB2A2 (맘마글 로빈 1), SCYE1 (내피 단핵구-활성화 사이토카인), SDF2, SERPENA1, SERPINA3, SERPINB5 (마스핀), 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 (트롬보스폰딘-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, 조직 인자 (tissue factor), TLR1, TLR2, TLR3, 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 리간드), TNFSF5 (CD40 리간드), TNFSF6 (FasL), TNFSF7 (CD27 리간드), TNFSF8 (CD30 리간드), TNFSF9 (4-1BB 리간드), TOLLIP, Toll-유사 수용체, TOP2A (토포이소머라제 Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, 티로시나제 (Tyrosinase), uPAR, VEGF, VEGFB, VEGFC, 베르시칸 (versican), VHL C5, VLA-4, WT1, Wnt-1, XCL1 (림포탁틴), XCL2 (SCM-Ib), XCRI (GPR5/CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (덱틴-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (덱틴-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), 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), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPA, SIRPB1 (CD172B), TREM1 (CD354), TREM2, KLRF1 (NKp80), 17-1A, SLAM7, MSLN, CTAG1B/NY-ESO-1, MAGEA3/A6, ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (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), CALU (O35887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1A (Q8R180), 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 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), 또는 CH10 (Q64433)일 수 있으나, 이에 제한되지 않는다.Cancer cell target antigens include, 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), 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, BMP2, BMP3B (GDFIO ), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRIN-1, CASP1, CASP4 , CAV1, CCBP2 (D6/JAB61), CCL1 (1-309), CCLI1 (eotaxin), 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/Eotaxin-2), CCL25 (TECK), CCL26 (Eotaxin-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, CD69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86 , CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH1 (E-cadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8 , CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (P21WAP1/CIP1), CDKN1B (P27KIP1), CDKN1C, CDKN2A (P16INK4A), CDKN2B, CDKN2B KN3, 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 , CLEC11A, 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), CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9/CKR-L2), CXCR4, CXCR6 (TYMSTR/STRL33/Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3, EpCAM, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2 ( HER-2), ERBB3, ERBB4, EREG, ERK8, estrogen receptor, Earl, ESR2, F3 (TF), FADD, FAP, farnesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1 ( aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5 , FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF (VEGFD), FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1 , FLT-3, FOLR1, FOS, FOSL1(FRA-1), FR-alpha, FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1, GGT1, GM-CSF, GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG , ICOSL, ID2,IFN-a,IFNA1,IFNA2,IFNA4,IFNA5,EFNA6,BFNA7,IFNB1,IFNgamma,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 (CD129), IL-10, IL10RA (CD210), IL10RB (CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, 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 integrins), 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 (hair-specific type II 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 (metallothionectin-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, 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, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2 , NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI, P2RX7, PAP, PART 1, PATE , PAWR, P-cadherin, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphatase phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2 (COX-2), PTN, 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 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), 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, TLR1, TLR2, TLR3, 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 (Trace), TNFSF12 (APO3L), TNFSF13 (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 (lymphotactin), XCL2 (SCM-Ib), ), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 ( CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), 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), NCR2 (CD336, NKp) 44) , Pilrb, SIGLEC1 (CD169, SN), SIGLEC5, SIGLEC6, SIGLEC7, SIGLEC8, SIGLEC9, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC14, SIGLEC15 (CD33L3), SIGLEC 16, Sirpa, Sirpb1 (CD172B), Trem1 (CD354), Trem2, KLRF1 (NKp80), 17-1A, SLAM7, MSLN, CTAG1B/NY-ESO-1, MAGEA3/A6, ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (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), CALU (O35887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1A (Q8R180), 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 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433).
표적 항원은 정상세포 대비 암세포에 10배 이상 많이 분포하는 항원일 수 있다.The target antigen may be an antigen that is more than 10 times more distributed in cancer cells than in normal cells.
[항체 또는 이의 항원결합부위 함유 단편][Antibody or fragment containing antigen-binding site thereof]
본 발명에 따라 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하는 면역접합체는, 항체 또는 이의 항원결합부위 함유 단편이 암세포의 항원을 표적화하기만 하면, 그 종류와 상관없이 전술한 [화학식 1의 캄토테신계 약물]-[산 민감성 링커]의 조합사용에 따른 유기적인 작용기전을 발휘할 수 있다. 특히, 유리형 엑사테칸 또는 Dxd 약물이 다양한 암 세포주에서 SN-38에 비하여 5 - 20배 강력한, 즉 낮은 수준의 IC50 수치로 매우 강력한 항암 효능을 공유하기 때문이다. 또한, 엑사테칸 또는 Dxd 약물이 임상에서 효능/안전성이 검증된 항암기전인 Topoisomerase I의 저해제를 활용한 것이며, Topoisomerase I 저해제(Irinotecan, Topotecan 등)는 임상에서 대장암, 폐암, 유방암, 난소암 등 다양한 난치성 고형암에서 뛰어난 항암 효능이 검증되어 있기 때문이다. According to the present invention, the immunoconjugate comprising [camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof] is an antibody or antigen-binding site-containing fragment thereof. As long as the antigen of these cancer cells is targeted, the organic mechanism of action can be exerted by using the combination of [camptothecin-based drug of Formula 1] and [acid-sensitive linker] described above, regardless of the type. In particular, the free exatecan or Dxd drug shares very strong anticancer efficacy with a low IC 50 value, 5 to 20 times more potent than SN-38 in various cancer cell lines. In addition, Exatecan or Dxd drugs utilize inhibitors of Topoisomerase I, an anticancer mechanism whose efficacy/safety has been clinically proven, and Topoisomerase I inhibitors (Irinotecan, Topotecan, etc.) have been clinically used to treat colon cancer, lung cancer, breast cancer, and ovarian cancer. This is because its excellent anticancer efficacy has been proven in various intractable solid cancers, such as:
본 명세서에서, 용어 "항체"는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하는, 항원을 특이적으로 인식하는 리간드 역할을 하는 단백질 분자를 의미하며, 다클론 항체, 단일클론 항체, 전체 (whole) 항체를 모두 포함한다. 또한, 상기 용어는 키메라성 항체 및 이가 (bivalent) 또는 이중특이성 분자, 디아바디, 트리아바디 및 테트라바디를 포함한다. 상기 용어는 추가로 FcRn에 대한 결합 기능을 보유한 단쇄 항체, 스캡, 항체 불변영역의 유도체 및 단백질 스캐폴드에 기초한 인공 항체를 포함한다. 전체 항체는 2 개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 상기 전체 항체는 IgA, IgD, IgE, IgM 및 IgG를 포함하며, IgG는 아형 (subtype)으로, IgG1, IgG2, IgG3 및 IgG4를 포함한다. As used herein, the term “antibody” refers to a protein molecule that acts as a ligand that specifically recognizes an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, including polyclonal antibodies, monoclonal antibodies, Contains all whole antibodies. The term also includes chimeric antibodies and bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. The term further includes single chain antibodies possessing a binding function to FcRn, scaps, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds. A full antibody is structured with two full-length light chains and two full-length heavy chains, with each light chain linked to the heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4.
본 명세서에서 용어 "항원결합부위 함유 단편"은 항체의 항원-결합 활성을 보유하는 항체의 임의의 단편일 수 있다. 예시적인 항체 단편은 단일 쇄 항체, Fd, Fab, Fab', F(ab')2, dsFv 또는 scFv를 포함하나 이에 제한되지 않는다. 상기 Fd는 Fab 단편에 포함되어 있는 중쇄 부분을 의미한다. 상기 Fab는 경쇄 및 중쇄의 가변 영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역 (CH1 도메인)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C 말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역 (hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv (variable fragment)는 중쇄 가변부위 및 경쇄 가변부위만을 가지고 있는 최소의 항체조각을 의미한다. 이중디설파이드 Fv (dsFv)는 디설파이드 결합으로 중쇄 가변부위와 경쇄 가변부위가 연결되어 있고, 단쇄 Fv (scFv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 경쇄의 가변 영역이 공유 결합으로 연결되어 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고 (예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 바람직하게는 유전자 재조합 기술을 통하여 제작할 수 있다.As used herein, the term “antigen binding site-containing fragment” 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 included in the Fab fragment. The Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv (variable fragment) refers to the minimum antibody fragment containing only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and light chain variable region are connected by a disulfide bond, and in single chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently connected through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragments can be obtained by digestion with pepsin), and are preferred. It can be produced through genetic recombination technology.
[약학적으로 허용가능한 염][ Pharmaceutically acceptable salt ]
본 명세서에서, 약학적으로 허용가능한 염은 제약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 나트륨, 칼륨, 칼슘, 마그네슘, 리튬, 구리, 망간, 아연, 철 등을 비롯한 무기이온의 염과 염산, 인산, 황산과 같은 무기산의 염이 있으며, 그 외에 아스코르브산, 시트르산, 타르타르산, 락트산, 말레산, 말론산, 푸마르산, 글리콜산, 숙신산, 프로피온산, 아세트산, 오로테이트산, 아세틸살리실산과 같은 유기산의 염 등과 라이신, 아르기닌, 구아니딘 등의 아미노산 염이 있다. 또한 약학적인 반응, 정제 및 분리과정에서 사용될 수 있는 테트라메틸 암모늄, 테트라에틸 암모늄, 테트라프로파일 암모늄, 테트라부틸 암모늄, 벤질 트리메틸 암모늄, 벤제토늄 등의 유기이온의 염이 있다. 다만, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다.In this specification, pharmaceutically acceptable salts refer to salts commonly used in the pharmaceutical industry, for example, salts of inorganic ions including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc. There are salts of inorganic acids such as perhydrochloric acid, phosphoric acid, and sulfuric acid, as well as salts such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotate acid, and acetylsalicylic acid. There are salts of organic acids and amino acid salts such as lysine, arginine, and guanidine. There are also salts of organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyltrimethyl ammonium, and benzethonium that can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts meant in the present invention are not limited to these salts listed.
[다양한 운반체-약물 접합체(Carrier-Drug Conjugate)][ Various Carrier-Drug Conjugate ]
본 명세서에서, 프로드럭은 미리 결정된 생리학적 환경에 배치된 후 생물학적으로 활성화되거나 더 활성화되는 프로드럭(pro-drug)을 포함하나 이에 제한되지 않는다.As used herein, prodrugs include, but are not limited to, prodrugs that are biologically active or become more active after being placed in a predetermined physiological environment.
예를 들면, 생리 활성 물질(physiologically active substance) 또는 치료적 활성 유기 화합물(therapeutically active organic compound)을 화학적으로 수식하고, 생체 내에서 효소적 또는 그 외의 조건하에서 모 화합물(parent compound)을 유리 혹은 방출하도록 설계된 화합물을 의미한다. 프로드럭은 투여후에 생체내에서 목적으로 하는 화합물로 변화된다. 유용한 약물임에도 불구하고 부작용, 안정성, 용해성, 흡수성, 작용시간 등에서 적합치 않는 성질을 가지고 있는 것에 화학적 수식을 가해서 임상사용 가능하게 한다.For example, a physiologically active substance or a therapeutically active organic compound is chemically modified and the parent compound is liberated or released under enzymatic or other conditions in vivo. refers to a compound designed to A prodrug is converted into the target compound in vivo after administration. Although it is a useful drug, it has unsuitable properties in terms of side effects, stability, solubility, absorption, and duration of action, and chemical modifications are added to make it possible for clinical use.
운반체-약물 접합체(Carrier-Drug Conjugate)도 일종의 프로드럭이다.Carrier-drug conjugate is also a type of prodrug.
본 발명에 따른 [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커] 접합체는 항체 이외에 다양한 종류의 약물 운반체(Carrier)에 적용이 가능하며, 항체와는 다르게 암 조직의 심부까지 잘 침투가 가능하고 CMC가 용이한 특성을 가지고 있는 운반체를 사용하여, 다양한 용도로의 응용에 잘 활용될 수 있다.[Camptothecin-based 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, and unlike antibodies, it can be used to penetrate deep into cancer tissue. By using a carrier that can penetrate well and has the characteristics of easy CMC, it can be utilized for a variety of applications.
따라서, 본 발명의 운반체-약물 접합체(Carrier-Drug Conjugate)의 제조방법은Therefore, the method for producing the carrier-drug conjugate of the present invention is
본 발명에 따른 [화학식 1의 캄토테신계 약물]-[화학식 2의 산 민감성 링커] 접합체 또는 이의 약학적으로 허용가능한 염을 사용하여, 운반체(Carrier)에 화학식 3의 링커를 통해 화학식 1의 캄토테신계 약물을 하나 이상 연결시키는 것이 특징이다.Using the [camptothecin-based drug of Formula 1]-[acid sensitive linker of Formula 2] conjugate or a pharmaceutically acceptable salt thereof according to the present invention, the camptothecin drug of Formula 1 is delivered to a carrier through the linker of Formula 3. It is characterized by linking one or more tothecin drugs.
이때 운반체(Carrier)는 항체, 펩타이드, 리피바디, 및/또는 압타머일 수 있다.At this time, the carrier may be an antibody, peptide, lipibody, and/or aptamer.
Figure PCTKR2023006170-appb-img-000015
Figure PCTKR2023006170-appb-img-000015
예컨대, 항체-약물 접합체(ADC)는 암세포의 표면에 발현된 특정 항원에 특이적으로 결합하는 항체의 높은 조직 선택성을 이용하여 강한 항암 효능을 가진 페이로드(payload)를 암 조직에만 선택적으로 전달하는 신약 플랫폼이다. ADC는 pM 수준의 낮은 농도에서도 암세포를 사멸하는 강력한 Payload를 암 조직에만 선택적으로 전달하고, 전신으로의 약물 노출은 최소화하여 항암 효능과 안전성을 동시에 확보할 수 있다. For example, antibody-drug conjugate (ADC) utilizes the high tissue selectivity of antibodies that specifically bind to specific antigens expressed on the surface of cancer cells to selectively deliver a payload with strong anticancer efficacy only to cancer tissues. It is a new drug platform. ADC selectively delivers a powerful payload that kills cancer cells even at concentrations as low as pM, and minimizes systemic exposure to the drug, ensuring anticancer efficacy and safety at the same time.
HER2 양성 암의 경우, 본 발명에 따라 DDX5 단백질을 약물 표적으로 하는 화학식 1의 캄토테신계 약물의 프로드럭인 ADC은 항HER2 치료 내성을 해결할 수 있는 새로운 치료법이 될 수 있다.In the case of HER2-positive cancer, ADC, a prodrug of a camptothecin-based drug of Formula 1 that targets the DDX5 protein according to the present invention, can be a new treatment method that can solve anti-HER2 treatment resistance.
본 발명의 프로드럭은 화학식 1의 캄토테신계 약물을 생체내에서 방출하도록 설계된, 항체 또는 이의 항원결합부위 함유 단편을 포함하는 면역접합체 또는 이의 약학적으로 허용가능한 염일 수 있다. The prodrug of the present invention may be an immunoconjugate containing an antibody or an antigen-binding site-containing fragment thereof, designed to release the camptothecin-based drug of Formula 1 in vivo, or a pharmaceutically acceptable salt thereof.
본 발명의 면역접합체는 암 세포의 항원에 특이적으로 결합하고 암 세포 내외에서 약물을 방출하여 세포 독성을 나타내므로, 암의 치료 또는 예방에 유용하게 사용될 수 있다. The immunoconjugate of the present invention binds specifically to the antigen of cancer cells and exhibits cytotoxicity by releasing the drug inside and outside the cancer cells, so it can be usefully used in the treatment or prevention of cancer.
압타머-약물 접합체(Aptamer-Drug Conjugate, ApDC)는 ADC의 항체 대신 압타머를 도입한 것이다. 압타머는 3차원 입체구조를 갖는 단일 가닥 핵산이다. 'SELEX'(Systematic Evolution of Ligands by Exponential enrichment) 과정을 통해 발굴된다. 셀렉스는 화합물 라이브러리에 표적하는 단백질 분자를 넣어 이와결합하는 기능성 핵산을 얻는 기술이다.Aptamer-Drug Conjugate (ApDC) is an aptamer introduced instead of an antibody in ADC. Aptamers are single-stranded nucleic acids with a three-dimensional structure. It is discovered through the 'SELEX' (Systematic Evolution of Ligands by Exponential enrichment) process. Selex is a technology that obtains functional nucleic acids that bind to target protein molecules in a compound library.
압타머는 표적에 매우 강력하고 선택적으로 결합할 수 있어 화학적 항체(chemical antibody)라고도 불린다. 압타머는 20kDa 정도의 크기이며, 항체에 비해 우수한 세포 침투성과 낮은 면역원성을 가진 것으로 알려져 있다.Aptamers can bind to targets very strongly and selectively, so they are also called chemical antibodies. Aptamers are about 20 kDa in size and are known to have excellent cell penetration and low immunogenicity compared to antibodies.
압타머는 화학적 합성이 가능하므로 압타머·약물 접합체의 제조 시 결합 약물의 접합 위치 및 개수에 대한 정밀한 설계가 가능하다. ADC에 비해 생산비용이 낮다.Since aptamers can be chemically synthesized, precise design of the conjugation location and number of drug conjugates is possible when manufacturing aptamer-drug conjugates. The production cost is lower than that of ADC.
압타머는 일반적으로 천연 핵산으로 이루어져 있어서 체내 핵산분해효소로 분해돼 생체 내 안정성이 떨어진다. 하지만 압타머의 화학적 변형이 용이하다는 점을 이용해 변형된 압타머의 안정성에 대한 한계를 극복할 수 있다.Aptamers are generally made of natural nucleic acids, so they are degraded by nucleic acid degrading enzymes in the body, making them less stable in vivo. However, by taking advantage of the ease of chemical modification of aptamers, the limitations on the stability of modified aptamers can be overcome.
펩타이드-약물 접합체(Peptide-Drug Conjugate, PDC)는 ADC에서 항체 대신 펩타이드를 도입한 형태다. 펩타이드는 아미노산으로 구성되며, 500~5000Da(달톤) 범위의 크기를 가진다. 이는 150kDa(킬로달톤)이상인 항체와 비교해 매우 작은 크기다. 따라서 펩타이드 기반인 PDC는 ADC에 비해 우수한 세포 침투 능력을 가지며, 면역원성이 발생할 가능성이 매우 낮다. 또한 펩타이드는 화학적 합성이 가능하다. 이 때문에 PDC는 생산비용이 매우 낮을 뿐 아니라 펩타이드와 약물의 접합 위치와 비율을 정밀하게 조절할 수 있다.Peptide-Drug Conjugate (PDC) is a form of ADC in which peptides are introduced instead of antibodies. Peptides are composed of amino acids and have a size ranging from 500 to 5000 Da (Dalton). This is a very small size compared to antibodies of 150 kDa (kilodaltons) or more. Therefore, peptide-based PDC has superior cell penetration ability compared to ADC, and the possibility of immunogenicity is very low. Additionally, peptides can be chemically synthesized. For this reason, PDC not only has a very low production cost, but also allows precise control of the conjugation position and ratio of peptide and drug.
일반적으로 펩타이드는 단백질분해효소에 쉽게 분해되므로 짧은 생물학적 반감기를 갖는다. 이러한 펩타이드 기반 약물접합체의 한계를 극복하기 위해 고리형 펩타이드, 비천연아미노산 도입 등 변형된 펩타이드를 활용하는 전략이 제시되고 있다.In general, peptides are easily degraded by proteolytic enzymes and therefore have a short biological half-life. To overcome these limitations of peptide-based drug conjugates, strategies using modified peptides such as cyclic peptides and introduction of non-natural amino acids are being proposed.
리피바디(Repebody)는 항체 골격을 갖고 있지는 않지만 항체와 같이 항원을 인식하는 기능을 갖는 일종의 인공항체다. 표적 단백질에 특이적인 리피바디는 파지디스플레이(phage display) 기술을 통해 발굴할 수 있다.Repebody is a type of artificial antibody that does not have an antibody skeleton but has the function of recognizing antigens like an antibody. Lipibodies specific to target proteins can be discovered through phage display technology.
파지디스플레이는 박테리오파지의 표면에 원하는 단백질을 발현시키는 기술이다. 리피바디는 항체의약 20% 수준인 30kDa 정도의 크기다. 따라서 항체에 비해 상대적으로 낮은 면역원성과 향상된 세포침투성을 갖는다고 알려져 있다. 또한, 리피바디의 열적·pH 안정성을 조절할 수 있어 구조적 안정성을 높일 수 있을 것으로 기대된다. 항체에 비해 생산 비용 또한 비교적 낮은 것으로 평가된다. 이러한 리피바디의 장점 때문에 항체를 리피바디로 대체하는 전략으로서 리피바디-약물 접합체(Repebody-DC)개발에 대한 관심도 높아지고 있다.Phage display is a technology that expresses desired proteins on the surface of bacteriophage. Lipibody is about 30kDa in size, which is 20% of an antibody drug. Therefore, it is known to have relatively low immunogenicity and improved cell penetration compared to antibodies. In addition, it is expected that structural stability can be improved by controlling the thermal and pH stability of Lipibody. Compared to antibodies, production costs are also assessed to be relatively low. Because of these advantages of Lipibodies, interest in the development of Lipibody-drug conjugates (Repebody-DC) as a strategy to replace antibodies with Lipibodies is also increasing.
[ADC 경우 항체 치료제로서의 역할][ In the case of ADC, its role as an antibody treatment ]
본 발명의 ADC는 지난 10년동안 암, 자가면역 등의 질병 치료에서 중요한 역할을 하고 있는, 기본적인 항체 치료제(Therapeutic antibody)로서 역할도 수행할 수 있다.The ADC of the present invention can also serve as a basic therapeutic antibody, which has played an important role in the treatment of diseases such as cancer and autoimmunity over the past 10 years.
항체는 항원과 우리 몸의 체내 세포를 구별하는 능력을 갖고 있기 때문에 항원에 대해 선택적으로 작용하는 우수한 선별 기능을 갖고 있다. 항체의 이러한 항원 선택적 결합 특성을 질병 치료제에 활용한 것이 바로 항체치료제이다. 항체는 Y자 모양을 하고 있는데 양 팔에 각각 항원 결합 부위 (Antigen binding site)가 존재하며 상보적 결정부위 (Complementarity determining regions, CDR)에 의해 항체는 표적 항원을 선택적으로 인지하게 된다. 또한 항체는 크게 5종류(IgM, IgD, IgG, IgA 및 IgE / Immunoglobulin(Ig))가 있으며 주로 IgG가 항체 치료제에 사용되고 있다.Antibodies have the ability to distinguish between antigens and cells in our body, so they have an excellent screening function that acts selectively on antigens. Antibody therapeutics utilize the antigen-selective binding characteristics of antibodies to treat diseases. Antibodies are Y-shaped, with an antigen binding site on each arm, and complementarity determining regions (CDRs) allow the antibody to selectively recognize the target antigen. Additionally, there are five major types of antibodies (IgM, IgD, IgG, IgA, and IgE / Immunoglobulin (Ig)), and IgG is mainly used in antibody treatments.
항체 치료제는 항원에 대해 선택적으로 반응을 하면서 항원을 제거할 수 있다. 이를 위해 다양한 기전을 활용하게 되는데 항체 치료제에서 이용하는 대표적인 기전의 특징들은 다음과 같다. Antibody treatments can selectively react with antigens and remove them. For this purpose, various mechanisms are used, and the characteristics of the representative mechanisms used in antibody therapeutics are as follows.
첫 번째 기전은 차단 기능이다 (Blocking). 항체는 세포의 표적 수용체에 선택적으로 결합하게 되고 표적세포의 생리학적 기능을 차단한다. 세포 표면에서 발현되는 리간드 또는 수용체에 항체가 결합하여 표적 신호 전달 경로를 차단하고, 감소된 신호 전달로 인해 세포활성상실, 증식억제 및 세포사멸 등을 일으킨다. The first mechanism is blocking. Antibodies selectively bind to target receptors on cells 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, and reduced signal transduction causes loss of cell activity, inhibition of proliferation, and apoptosis.
세포 표면의 수용체 (Receptor)를 표적으로 하여 항체가 수용체에 결합을 하게 되면 수용체의 구조를 변화시키거나 수용체에 결합해야 하는 인자가 결합을 하지 못하게 하여 세포 내 신호 전달을 억제하고 이를 통해 세포의 성장 및 분화를 통제할 수 있게 된다. 또한 항체가 수용체에 결합하게 되면 수용체가 세포 내 유입 기전에 의해 세포 내로 들어가게 되어 세포 표면의 수용체 수가 감소하게 되면서 세포의 생리 기전을 조절할 수 있다. 이 기전으로 우수한 효과를 나타내는 대표적인 항체 치료제는 Human epidermal growth factor Receptor(HER/EGFR/ERBB) 중 하나인 HER2 양성 유방암을 표적으로 하는 Trastuzumab(Herceptin®)이 있다.When an antibody targets a receptor on the cell surface and binds to the receptor, it changes the structure of the receptor or prevents factors that must bind to the receptor from binding, thereby inhibiting intracellular signal transduction and thereby promoting cell growth. and differentiation can be controlled. Additionally, when an antibody binds to a receptor, the receptor enters the cell through an intracellular influx mechanism, which reduces the number of receptors on the cell surface, thereby controlling the physiological mechanism of the cell. A representative antibody treatment that shows excellent effectiveness through this mechanism is Trastuzumab (Herceptin®), which targets HER2-positive breast cancer, one of the human epidermal growth factor receptors (HER/EGFR/ERBB).
두 번째 기전은 항체-의존적 세포-매개 세포 독성 기전이다 (Antibody-Dependent Cellmediated Cytotoxicity, ADCC). 암세포의 경우 일반 세포와 다른 특이한 수용체 또는 과발현 수용체를 가지고 있으며 면역계가 이를 인식하여 항체를 생산하고 세포 표면에 결합하게 된다. NK cell이 Fc receptor(FcγRIII)를 인식 후 활성화되면 세포사멸 (Apoptosis)을 유도한다. The second mechanism is Antibody-Dependent Cellmediated Cytotoxicity (ADCC). Cancer cells have unique receptors or overexpressed receptors that are different from normal cells, and the immune system recognizes them, produces antibodies, and binds to the cell surface. When NK cells recognize Fc receptor (FcγRIII) and become activated, they induce apoptosis.
세 번째 기전은 보체-의존적 세포독성이다 (Complement-Dependent Cytotoxicity, CDC). 암세포 표면에 결합하여 보체 단백질 (C1q)을 활성화시켜 classical pathway를 일으켜 세포막을 공격해 구멍(pore)을 만들어 표적세포의 용해를 일으킨다. 이외에도 옵소닌 작용 (Opsonization), 중화 작용 (Neutralization)과 염증반응 등의 기전이 있다.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 lysis of target cells. In addition, there are mechanisms such as opsonization, neutralization, and inflammatory response.
치료용 항체는 일반적으로 특정 치료 목적을 위해 항체를 여러 관점에서 다시 engineering하여 최적화된 형태로 만들어진다. 그리고 이러한 치료용 단클론 항체 개발에 적용되었던 항체공학 기술이 최적화된 ADC 개발에도 확장하여 적용되고 있다. 예를 들어 현재 치료용 항체나 임상 개발에 있는 ADC는 대부분 IgG1이고 소수만 IgG2와 IgG4를 사용하고 있다. 항체의 isotype 선택은 치료 용 항체와 ADC에 있어 동일하게 중요하다. 그 이유는 항체의 isotype이 antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) 및 complement-dependent cytotoxicity (CDC)의 기능에 영향을 미치기 때문이다. 또한 IgG4은 생체내 Fab arm 교환으로 functionally monovalent된다. 또한 isotype에 의한 ADC의 효능과 항체의 Fc-domain engineering을 응용하여 ADC의 in vivo 효능이 향상될 수 있다.Therapeutic antibodies are generally made into an optimized form by re-engineering the antibody from various perspectives for a specific therapeutic purpose. And the antibody engineering technology applied to the development of these therapeutic monoclonal antibodies is being expanded and applied to the development of optimized ADCs. For example, most therapeutic antibodies or ADCs currently in clinical development are IgG 1 , with only a small number using IgG 2 and IgG 4 . Selection of the antibody isotype is equally important for therapeutic antibodies and ADCs. The reason is that the isotype of an antibody affects the functions of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Additionally, IgG 4 is functionally monovalent through in vivo Fab arm exchange. Additionally, the in vivo efficacy of ADC can be improved by applying the efficacy of ADC by isotype and Fc-domain engineering of antibodies.
[암의 예방 또는 치료용 약학적 조성물][ Pharmaceutical composition for preventing or treating cancer ]
본 발명은 전술한 본 발명에 따른 면역접합체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-described immunoconjugate according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.
또한 본 발명의 일 구체예에 따르면, 상기 면역접합체의 치료학적으로 유효한 양을, 이를 필요로 하는 대상 (subject)에게 투여하는 단계를 포함하는, 암의 치료 또는 예방하는 방법을 제공한다. 상기 대상 (subject)은 인간을 포함하는 포유류일 수 있다.According to one embodiment of the present invention, a method for treating or preventing cancer is provided, comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof. The subject may be a mammal, including humans.
본 발명의 면역접합체는 암 세포의 항원에 특이적으로 결합하고 암 세포 내외에서 약물을 방출하여 세포 독성을 나타내므로, 암의 치료 또는 예방에 유용하게 사용될 수 있다. 본 발명의 면역접합체가 갖는 항암 활성은 전술한 바와 같다.The immunoconjugate of the present invention binds specifically to the antigen of cancer cells and exhibits cytotoxicity by releasing the drug inside and outside the cancer cells, so it can be usefully used in the treatment or prevention of cancer. The anticancer activity of the immunoconjugate of the present invention is as described above.
본 발명에서, 상기 암은 토포아이소머라제 I의 억제로 치료 가능한 모든　암을 포함하며, 고형암　또는 혈액암일 수 있다. 예컨대, 가성점액종, 간내 담도암, 간모세포종, 간암, 갑상선암, 결장암, 고환암, 골수이형성증후군, 교모세포종, 구강암, 구순암, 균상식육종, 급성골수성백혈병, 급성림프구성백혈병, 기저세포암, 난소상피암, 난소생식세포암, 남성유방암, 뇌암, 뇌하수체선종, 다발성골수종, 담낭암, 담도암, 대장암, 만성골수성백혈병, 만성림프구백혈병, 망막모세포종, 맥락막흑색종, 바터팽대부암, 방광암, 복막암, 부갑상선암, 부신암, 비부비동암, 비소세포폐암, 설암, 성상세포종, 소세포폐암, 소아뇌암, 소아림프종, 소아백혈병, 소장암, 수막종, 식도암, 신경교종, 신우암, 신장암, 심장암, 십이지장암, 악성 연부조직 암, 악성골암, 악성림프종, 악성중피종, 악성흑색종, 안암, 외음부암, 요관암, 요도암, 원발부위불명암, 위림프종, 위암, 위유암종, 위장관간질암, 윌름스암, 유방암, 육종, 음경암, 인두암, 임신융모질환, 자궁경부암, 자궁내막암, 자궁육종, 전립선암, 전이성골암, 전이성뇌암, 종격동암, 직장암, 직장유암종, 질암, 척수암, 청신경초종, 췌장암, 침샘암, 카포시 육종, 파제트병, 편도암, 편평상피세포암, 폐선암, 폐암, 폐편평상피세포암, 피부암, 항문암, 횡문근육종, 후두암, 흉막암, 혈액암, 및 흉선암으로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있으나, 이에 제한되지 않는다. 또한, 상기　암은 원발성　암뿐 아니라 전이성　암도 포함한다.In the present invention, the cancer includes all cancers treatable by inhibition of topoisomerase I, and may be solid cancer or hematological cancer. For example, pseudomyxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, oral cavity cancer, mycosis fungoides, acute myeloid leukemia, acute lymphocytic leukemia, basal cell carcinoma, ovary. Epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, Parathyroid cancer, adrenal cancer, sinonasal cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenum Cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid, gastrointestinal stromal cancer, Wilms cancer. , breast cancer, sarcoma, penile cancer, oropharyngeal 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 cancer, acoustic neuroma, Pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleura cancer, blood cancer, and thymic cancer. It may be one or more types selected from the group consisting of, but is not limited thereto. Additionally, the cancer includes not only primary cancer but also metastatic cancer.
본 발명에서 사용되는 "치료학적으로 유효한 양"이라는 용어는 암의 치료 또는 예방에 유효한 상기 면역접합체의 양을 나타낸다. 구체적으로, "치료학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 시판되는 치료제와는 순차적으로 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 본 발명의 면역접합체는 용량 의존적인 효과를 나타내므로 투여 용량은 환자의 상태, 연령, 성별 및 합병증 등의 다양한 요인에 따라 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 약학적 조성물의 유효성분은 안전성이 우수하므로, 결정된 투여 용량 이상으로도 사용될 수 있다.As used herein, the term “therapeutically effective amount” refers to the amount of the immunoconjugate effective for treating or preventing cancer. Specifically, “therapeutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, type of disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. And it can be administered single or multiple times. Considering 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. Since the immunoconjugate of the present invention exhibits a dose-dependent effect, the administered dose is determined by the patient's condition, age, gender, and It can be easily determined by a person skilled in the art depending on various factors such as complications. Since the active ingredient of the pharmaceutical composition of the present invention has excellent safety, it can be used at a dose exceeding the determined dosage.
또한 본 발명의 일 구체예에 따르면, 본 발명은 암의 치료 또는 예방에 사용하기 위한 약제 (medicament)의 제조에 사용하기 위한, 상기 면역접합체의 용도 (use)를 제공한다. 약제의 제조를 위한 상기 면역접합체는 허용되는 보조제, 희석제, 담체 등을 혼합할 수 있으며, 기타 활성제제와 함께 복합 제제로 제조되어 활성 성분들의 상승 작용을 가질 수 있다.In addition, according to one embodiment of the present invention, the present invention provides a use of the immunoconjugate for use in the manufacture of a medicament for use in the treatment or prevention of cancer. The immunoconjugate for the preparation of a drug can be mixed with acceptable auxiliaries, diluents, carriers, etc., and can be prepared as a complex preparation with other active agents to have a synergistic effect of the active ingredients.
본 발명의 용도, 조성물, 치료 방법에서 언급된 사항은 서로 모순되지 않는 한 동일하게 적용된다.Matters mentioned in the uses, compositions, and treatment methods of the present invention apply equally unless they contradict each other.
본 발명에 따라 DDX5 단백질에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 세포 내에서 DDX5 단백질에 결합하여 DDX5 단백질 분해를 통해 세포사멸(cell death)을 유도할 수 있을 뿐만 아니라, 바람직하게는 페이로드로 사용시 산 민감성 링커와 함께 이의 운반체-약물 접합체가 응집되는 문제를 해결할 수 있다.The camptothecin-based drug of Formula 1, designed to bind to the DDX5 protein according to the present invention, not only binds to the DDX5 protein within cells and induces cell death through DDX5 protein degradation, but is preferably used as a payload. When used as an acid-sensitive linker, it can solve the problem of aggregation of its carrier-drug conjugate.
본 발명에 따른 운반체-약물 접합체(Carrier-Drug Conjugate)는 화학식 3의 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물 연결시 생체 내 주입 후 체내 투여, 분포 및 제거 경로에서 대식세포에 의해 탐식(phagocytosis)되지 않도록 정밀하게 설계한 것으로, 세망내피계 기관의 대식세포 식세포작용으로 인해 간, 비장, 골수, 림프절 등에 흡수되지 아니하며, 원하는 혈액 순환 프로파일을 제공할 수 있다. 따라서, 본 발명의 면역접합체는 11mg/kg 이상의 용량으로 투여가능하다.The carrier-drug conjugate according to the present invention is phagocytosed by macrophages in the route of administration, distribution, and elimination in the body after in vivo injection when connected to the camptothecin drug of formula 1 through the acid-sensitive linker of formula 3. It is precisely designed to prevent phagocytosis, and is not absorbed into the liver, spleen, bone marrow, or lymph nodes due to the phagocytosis of macrophages in reticuloendothelial organs, and can provide the desired blood circulation profile. Therefore, the immunoconjugate of the present invention can be administered at a dose of 11 mg/kg or more.
또한 본 발명의 면역접합체는 소수성 저분자 약물로 세포막 투과성인 화학식 1의 캄토테신계 약물 및 산 민감성 링커의 조합사용으로 인해, 항원-항체 복합체(complex)에 의한 내재화(internalization) 과정이 비효율적이라 충분한 농도의 약물이 세포 내로 들어가지 않는 문제점 및 암 조직의 심부까지 침투가 잘 되지 않는 항체의 문제점을 해결할 수 있고 표적화된 세포뿐만아니라 주변 세포에도 들어가는 by-stander 효과를 극대화할 수 있다.In addition, the immunoconjugate of the present invention is a hydrophobic small molecule drug that is permeable to cell membranes due to the combined use of the camptothecin-based drug of Formula 1 and an acid-sensitive linker, so the internalization process by the antigen-antibody complex is inefficient, so it is maintained at a sufficient concentration. It can solve the problem of drugs not entering the cells and the problems of antibodies not penetrating deep into cancerous tissue, and can maximize the by-stander effect of entering not only targeted cells but also surrounding cells.
본 발명의 면역접합체는 1-3세대 ADC의 개발 과정에서 확인된 문제들 중 ADC에 대한 내성발생과 3세대 ADC에서 사용되는 payload의 상대적으로 약한 효능에서 나타나는 좁은 therapeutic window의 문제를 극복할 수 있다. 즉, 본 발명의 면역접합체는 화학식 1의 캄토테신계 약물과 산 민감성 링커의 조합 사용으로 인해 기존 ADC payload에 비하여 강력한 항암 효능과 in vivo 안전성의 균형을 이루어 최적화된 therapeutic window를 보일 수 있다. Among the problems identified during the development of 1st-3rd generation ADCs, the immunoconjugate of the present invention can overcome the problems of resistance to ADCs and a narrow therapeutic window resulting from the relatively weak efficacy of the payload used in 3rd generation ADCs. . That is, the immunoconjugate of the present invention can exhibit an optimized therapeutic window by balancing strong anticancer efficacy and in vivo safety compared to existing ADC payloads due to the combined use of the camptothecin-based drug of Formula 1 and an acid-sensitive linker.
도 1은 다양한 Camptothecin계 항암제(SN-38, Exatecan, Dxd, FL118)의 구조식이다.Figure 1 shows the structural formulas of various camptothecin anticancer drugs (SN-38, Exatecan, Dxd, FL118).
도 2는 캄토테신(camptothecin, CPT)의 구조식 및 이의 제1형 토포이소머라제(topoisomerase-1)와의 결합을 도시한 것이다.Figure 2 shows the structural formula of camptothecin (CPT) and its binding to type 1 topoisomerase-1.
도 3은 본 발명에 따라 산민감성 링커를 사용한 면역접합체에서 효율적인 약물 방출 기전을 도시한 모식도이다.Figure 3 is a schematic diagram showing an efficient drug release mechanism from an immunoconjugate using an acid-sensitive linker according to the present invention.
도 4 내지 도 7은 FaDu 세포주 및 A549 세포주에서 다양한 캄토테신계 약물(FL118 약물, SN-38 약물, Exatecan 약물, PBX-7011, PBX-7014, PBX-7016)의 DDX5 및 p-DDX5 단백질의 분해 여부/정도, 이외 다양한 anti-apoptotic protein들의 저해 정도를 보여주는 웨스턴 블럿 결과이다. 도 5 및 도 7은 FaDu 세포주 및 A549 세포주에서 실시한 western blot 실험결과(도 4 및 도 6)에서 농도를 그래프로 수치화한 결과이다.Figures 4 to 7 show the degradation of DDX5 and p-DDX5 proteins of various camptothecin drugs (FL118 drug, SN-38 drug, Exatecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line. This is a Western blot result showing the presence/degree of inhibition of various anti-apoptotic proteins. Figures 5 and 7 show the results of western blot experiments conducted on the FaDu cell line and the A549 cell line (Figures 4 and 6), graphically quantifying the concentration.
도 8은 Tra-CL2A-FL118/Tra-CL2A-Exatecan의 Western blot 결과이다.Figure 8 shows the Western blot results of Tra-CL2A-FL118/Tra-CL2A-Exatecan.
도 9 및 도 10은 각각 MDA-MB-453(HER2++) 세포주, SK-BR-3 세포주에서 다양한 캄토테신계 약물 및 이를 payload로 하는 ADC, 즉 Tra-CL2A-FL118/Tra-CL2A-Exatecan에 대해 in vitro cell viability 비교 평가 결과이다. Figures 9 and 10 show various camptothecin-based drugs and ADCs using them as payload, namely Tra-CL2A-FL118/Tra-CL2A-Exatecan, in MDA-MB-453 (HER2++) cell line and SK-BR-3 cell line, respectively. This is the result of in vitro cell viability comparative evaluation.
도 11은 프로그램된 세포사멸(programmed cell death)와 관련한 세포 사멸의 상세 분류도이다.Figure 11 is a detailed classification diagram of cell death related to programmed cell death.
이하, 본 발명을 실시예를 통하여 보다 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것일 뿐 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.
제조예 1: CL2A-Exatecan의 합성Preparation Example 1: Synthesis of CL2A-Exatecan
Figure PCTKR2023006170-appb-img-000016
Figure PCTKR2023006170-appb-img-000016
1-1: 화합물 2의 합성1-1: Synthesis of Compound 2
Figure PCTKR2023006170-appb-img-000017
Figure PCTKR2023006170-appb-img-000017
아르곤 분위기 하에 실온에서 N,N-디메틸포름아미드(건조)(3mL) 중 엑사테칸 메실레이트 이수화물(100mg, 0.176mmol) 현탁액에 트리에틸아민(0.098mL, 0.705mmol)을 첨가하였다. 이어서, MMTrCl(109 mg, 0.352 mmol)을 첨가하였다. 반응 혼합물을 DMSO로 희석하고 염기성 분취용 MPLC(XSelect40-80)로 정제하여, 동결 건조 후, 회백색 고체로 (1S,9S)-9-에틸-5-플루오로-9-히드록시-1-(((4-메톡시페닐)디페닐메틸)아미노)-4-메틸-1,2,3,9,12,15-헥사히드로-10H,13H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-10,13-디온(103 mg, 83%)을 수득하였다.Triethylamine (0.098 mL, 0.705 mmol) was added to a suspension of exatecan 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 DMSO and purified by basic preparative MPLC (XSelect40-80), lyophilized, and obtained as an off-white solid (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%) was obtained.
SC_BASE: m/z 708.4 [M+H]+ SC_BASE: m/z 708.4 [M+H] +
1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 10.9 Hz, 1H), 7.58 - 7.49 (m, 4H), 7.45 - 7.38 (m, 2H), 7.37 - 7.27 (m, 5H), 7.27 - 7.20 (m, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.49 (s, 1H), 5.42 (s, 2H), 5.17 (d, J = 19.2 Hz, 1H), 4.91 (d, J = 19.2 Hz, 1H), 4.00 - 3.90 (m, 1H), 3.74 (s, 3H), 3.63 (d, J = 7.4 Hz, 1H), 3.23 - 3.11 (m, 1H), 2.80 - 2.68 (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 = 7.3 Hz, 3H). 1 H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 10.9 Hz, 1H), 7.58 - 7.49 (m, 4H), 7.45 - 7.38 (m, 2H), 7.37 - 7.27 (m, 5H) , 7.27 - 7.20 (m, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.49 (s, 1H), 5.42 (s, 2H), 5.17 (d, J = 19.2 Hz, 1H), 4.91 ( d, J = 19.2 Hz, 1H), 4.00 - 3.90 (m, 1H), 3.74 (s, 3H), 3.63 (d, J = 7.4 Hz, 1H), 3.23 - 3.11 (m, 1H), 2.80 - 2.68 (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 = 7.3 Hz, 3H) ).
1-2: 화합물 4의 합성1-2: Synthesis of Compound 4
Figure PCTKR2023006170-appb-img-000018
Figure PCTKR2023006170-appb-img-000018
질소 분위기 하 건조된 플라스크에 (1S,9S)-9-에틸-5-플루오로-9-히드록시-1-(((4-메톡시페닐)디페닐메틸)아미노)-4-메틸-1,2,3,9,12,15-헥사히드로-10H,13H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-10,13-디온 (85 mg, 0.120 mmol) 및 DMAP(55mg, 0.456mmol)을 첨가하고 디클로로메탄(6mL)에 용해시켰다. 5분 동안 교반한 후, 디클로로메탄(0.750m) 중 트리포스겐(14.2mg, 0.048mmol)의 용액을 한번에 첨가하고 반응 혼합물을 실온에서 15분 동안 교반하였다. 10분 후 LCMS 분석(MeOH 중의 샘플)에 따르면, 메틸 카보네이트로 양호하게 전환되었다. (S)-2-(32-아지도-5-옥소-3,9,12,15,18,21,24,27,30-노나옥사-6-아자도트리아콘탄아미도)-N-(4-(하이드록시메틸) 페닐)-6-(((4-메톡시페닐)디페닐메틸)아미노)헥산아미드 용액(140mg, 0.132mmol)을 디클로로메탄(1.5mL)에 첨가하고, 반응 혼합물을 실온에서 30분 동안 교반하였다. 반응 혼합물을 감압 하에 농축하고, DMSO에 용해시키고, 염기성 분취용 MPLC(XSelect50-100)로 정제하여 동결건조 후 생성물(153 mg, 71%)을 회백색 고체로서 수득하였다.(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((4-methoxyphenyl)diphenylmethyl)amino)-4-methyl-1 in a flask dried under a nitrogen atmosphere. ,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 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 m) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. After 10 minutes there was good conversion to methyl carbonate according to LCMS analysis (sample in MeOH). (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azadotriacontanamido)-N- (4-(hydroxymethyl) phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide solution (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 DMSO, and purified by basic preparative MPLC (XSelect50-100) to give the product (153 mg, 71%) as an off-white solid after lyophilization.
SC_BASE_M1800: m/z 1793.5 [M+H]+ SC_BASE_M1800: m/z 1793.5 [M+H] +
1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.69 (d, J = 10.6 Hz, 1H), 7.59 - 7.49 (m, 6H), 7.44 (d, J = 8.4 Hz, 6H), 7.38 - 7.28 (m, 8H), 7.24 - 7.09 (m, 6H), 6.89 - 6.74 (m, 4H), 5.65 (d, J = 17.3 Hz, 1H), 5.36 (d, J = 17.2 Hz, 1H), 5.11 (d, J = 12.0 Hz, 1H), 4.97 (d, J = 12.1 Hz, 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 = 7.4 Hz, 3H). 1H NMR (400 MHz, CDCl 3 ) δ 8.60 (s, 1H), 7.69 (d, J = 10.6 Hz, 1H), 7.59 - 7.49 (m, 6H), 7.44 (d, J = 8.4 Hz, 6H) , 7.38 - 7.28 (m, 8H), 7.24 - 7.09 (m, 6H), 6.89 - 6.74 (m, 4H), 5.65 (d, J = 17.3 Hz, 1H), 5.36 (d, J = 17.2 Hz, 1H) ), 5.11 (d, J = 12.0 Hz, 1H), 4.97 (d, J = 12.1 Hz, 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 = 7.4 Hz, 3H).
1-3. 화합물 5의 합성1-3. Synthesis of compound 5
Figure PCTKR2023006170-appb-img-000019
Figure PCTKR2023006170-appb-img-000019
디클로로메탄(9 mL) 중 4-((S)-35-아지도-2-(4-(((4-메톡시페닐)디페닐메틸)아미노)부틸)-4,8-디옥소-6,12,15,18,21,24,27,30,33-노나옥사-3,9-디아자펜타트리아콘탄아미도)벤질 ((1S,9S)-9-에틸-5-플루오로-1-(((4-메톡시페닐)디페닐메틸)아미노)-4-메틸- 10,13-디옥소-2,3,9,10,13,15-헥사히드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-9-일)카보네이트 용액(0.153 g, 0.085 mmol)에, 4-((2,5-디옥소-2,5-디히드로-1H-피롤-1-일)메틸)-N-(프로프-2-인-1-일)시클로헥산-1-카르복사미드(0.070g, 0.256mmol), 브롬화구리(I)(7.3mg, 0.051mmol) 및 DIPEA(0.045mL, 0.256mmol)를 첨가하였다. 반응 혼합물을 실온에서 밤새 교반하였다. 추가량의 브롬화구리(I)(7.3mg, 0.051mmol)를 첨가하였다. 3시간 동안 교반한 후, LCMS 상의 샘플-2에서 생성물이 증가하였다. 반응 혼합물을 추가로 5시간 동안 교반하고 감압 하에 농축하였다. 잔류물을 염기성 분취용 MPLC(XSelect50-100)로 정제하여 동결건조 후 회백색 고체로서 생성물(133 mg, 75%)을 얻었다.4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 in dichloromethane (9 mL) ,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontanamido)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-benzo[ de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate solution (0.153 g, 0.085 mmol), 4-((2,5- Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1-carboxamide (0.070g, 0.256mmol) , copper(I) bromide (7.3 mg, 0.051 mmol) and DIPEA (0.045 mL, 0.256 mmol) were added. The reaction mixture was stirred at room temperature overnight. Additional amount of copper(I) bromide (7.3 mg, 0.051 mmol) was added. After stirring for 3 hours, the product increased in sample-2 on LCMS. The reaction mixture was stirred for an additional 5 hours and concentrated under reduced pressure. The residue was purified by basic preparative MPLC (XSelect50-100) and lyophilized to obtain the product (133 mg, 75%) as an off-white solid.
SC_BASE_M1800: m/z 1795.5 [M-MMT+H]+, 1523.5 [M-2xMMT+H]+ SC_BASE_M1800: m/z 1795.5 [M-MMT+H] + , 1523.5 [M-2xMMT+H] +
1H NMR (400 MHz, DMSO-d6) δ 10.16 (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 = 8.5 Hz, 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 = 5.6 Hz, 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 = 7.3 Hz, 4H), 0.87 - 0.80 (m, 1H). 1H NMR (400 MHz, DMSO-d6) δ 10.16 (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 = 8.5 Hz, 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 = 5.6 Hz, 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 = 7.3 Hz, 4H), 0.87 - 0.80 (m, 1H).
1-4. CL2A-Exatecan 화합물의 합성1-4. Synthesis of CL2A-Exatecan compound
Figure PCTKR2023006170-appb-img-000020
Figure PCTKR2023006170-appb-img-000020
디클로로메탄(무수)(1.5mL) 중 4-((S)-35-(4-((4-((2,5-디옥소-2,5-디히드로-1H-피롤-1-일)메틸)시클로헥산-1-카르복사미도)메틸)-1H-1,2,3-트리아졸-1-일)-2-(4-(((4-메톡시페닐)디페닐메틸)아미노)부틸)-4,8-디옥소-6,12,15,18,21,24,27,30,33-노나옥사-3,9-디아자펜타트리아콘탄아미도)벤질 ((1S,9S)-9-에틸-5-플루오로-1-(((4-메톡시페닐)디페닐메틸)아미노)-4-메틸-10,13-디옥소-2,3,9,10,13,15-헥사히드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b] 퀴놀린-9-일)카보네이트 용액(100mg, 0.048mmol)에 아니솔(0.528mL, 4.83mmol)을 실온에서 불활성 분위기 하에 첨가한 다음, 디클로로아세트산(0.160 mL, 1.934mmol)을 적가하였다. 이어서, MTBE(~3mL)를 첨가했다. 반응 혼합물이 미세한 현탁액으로 변하였다. 헵탄(~3mL)을 첨가하였다. 피펫으로 용매를 최대한 제거하였다. 잔류물을 MTBE/헵탄(1:1, ~4 mL) 혼합물로 여러 번 세척했다. 습윤 잔류물을 감압 하에 밤새 건조시켜 옅은 녹색 고체를 얻었다(85 mg, 89%).4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) in dichloromethane (anhydrous) (1.5 mL) 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-diazapentatriacontanamido)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-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] quinolin-9-yl)carbonate solution (100mg, 0.048mmol) Anisole (0.528 mL, 4.83 mmol) was added under an inert atmosphere at room temperature, and then dichloroacetic acid (0.160 mL, 1.934 mmol) was added dropwise. MTBE (~3 mL) was then added. The reaction mixture turned into a fine suspension. Heptane (~3 mL) was added. The solvent was removed as much as possible with 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 overnight to give a pale green solid (85 mg, 89%).
AN_ACID: m/z 763.0 [M+2H]2+/2AN_ACID: m/z 763.0 [M+2H] 2+ /2
1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.26 - 8.14 (m, 2H), 8.08 (t, J = 5.7 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H), 7.81 (s, 1H), 7.78 - 7.50 (m, 5H), 7.31 (d, J = 8.3 Hz, 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 = 5.6 Hz, 2H), 4.09 - 3.94 (m, 4H), 3.78 (t, J = 5.3 Hz, 2H), 3.55 - 3.41 (m, 33H), 3.25 - 3.19 (m, 4H), 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). 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.26 - 8.14 (m, 2H), 8.08 (t, J = 5.7 Hz, 1H), 7.89 (d, J = 10.8 Hz, 1H) ), 7.81 (s, 1H), 7.78 - 7.50 (m, 5H), 7.31 (d, J = 8.3 Hz, 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 = 5.6 Hz, 2H), 4.09 - 3.94 (m, 4H), 3.78 (t, J = 5.3 Hz, 2H), 3.55 - 3.41 (m, 33H), 3.25 - 3.19 (m, 4H), 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).
제조예 2: CL2A-Dxd의 합성Preparation Example 2: Synthesis of CL2A-Dxd
Figure PCTKR2023006170-appb-img-000021
Figure PCTKR2023006170-appb-img-000021
2-1: 화합물 1의 합성2-1: Synthesis of Compound 1
Figure PCTKR2023006170-appb-img-000022
Figure PCTKR2023006170-appb-img-000022
2-히드록시아세트산(133mg, 1.749mmol) 및 트리에틸아민(0.729ml, 5.25mmol)을 디클로로메탄(4ml)에 용해시켰다. 반응 혼합물을 0℃로 냉각시켰다. 그 다음, 디클로로메탄(4.00ml) 중 MMTrCl(702mg, 2.273mmol)의 용액을 아르곤 분위기 하에 첨가하였다. 반응 혼합물을 천천히 실온에 도달하게 하고 2일 동안 교반하였다. 반응 혼합물을 감압 하에 농축시켰다. 잔류물을 플래시 크로마토그래피(실리카, 디클로로메탄 중 0% 내지 10% 메탄올 + 1% TEA)로 정제하여 잔류 트리에틸아민으로 오염된 백색 고체로 생성물을 얻었다. 수율: 500 mg, 30%(잔여 트리에틸아민에 대해 보정됨). 생성물을 추가 정제 없이 사용하였다.2-Hydroxyacetic acid (133 mg, 1.749 mmol) and triethylamine (0.729 ml, 5.25 mmol) were dissolved in dichloromethane (4 ml). The reaction mixture was cooled to 0°C. Then, a solution of MMTrCl (702 mg, 2.273 mmol) in dichloromethane (4.00 ml) was added under argon atmosphere. The reaction mixture was slowly allowed to reach room temperature and stirred for 2 days. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 0% to 10% methanol in dichloromethane + 1% TEA) to give the product as a white solid contaminated with residual triethylamine. Yield: 500 mg, 30% (corrected for residual triethylamine). The product was used without further purification.
SC_BASE: m/z 347.2 [M+H]+ SC_BASE: m/z 347.2 [M+H] +
1H NMR (400 MHz, CDCl3) δ 7.59 - 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). 1 H NMR (400 MHz, CDCl 3 ) δ 7.59 - 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).
2-2: 화합물 2의 합성2-2: Synthesis of Compound 2
Figure PCTKR2023006170-appb-img-000023
Figure PCTKR2023006170-appb-img-000023
N,N-디메틸포름아미드(건조)(1.2mL) 중 2-((4-메톡시페닐)디페닐메톡시)아세트산(126mg, 0.254mmol)의 용액에 NHS(29.2mg, 0.254mmol) 및 EDCI·HCl(48.7 mg, 0.254 mmol)을 첨가하였다. 반응 혼합물을 실온에서 1시간 동안 교반하였다. 이어서, 이를 N,N-디메틸포름아미드(건조)(1.2mL) 중 엑사테칸 메실레이트(90mg, 0.169mmol) 및 트리에틸아민(0.026mL, 0.186mmol)의 현탁액에 첨가하고 실온에서 밤새 교반하였다. 반응 혼합물을 DMSO로 희석하고 염기성 분취용 MPLC(XSelect30-70)로 정제하여, 아세토니트릴과 물의 혼합물(1:1, 10mL)로부터 농축 및 동결건조 후 N-((1S,9S)-9-에틸-5-플루오로-9-히드록시-4-메틸-10,13-디옥소-2,3,9,10,13,15-헥사하이드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-1-일)- 2-((4-메톡시페닐)디페닐메톡시)아세트아미드를 회백색 고체로 얻었다. 수율: 89 mg, 68%.NHS (29.2 mg, 0.254 mmol) and EDCI in a solution of 2-((4-methoxyphenyl)diphenylmethoxy)acetic acid (126 mg, 0.254 mmol) in N,N-dimethylformamide (dry) (1.2 mL). ·HCl (48.7 mg, 0.254 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. This was then added to a suspension of exatecan 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 DMSO and purified by basic preparative MPLC (XSelect30-70), concentrated from a mixture of acetonitrile and water (1:1, 10 mL), lyophilized, and then 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 was obtained as an off-white solid. Yield: 89 mg, 68%.
SC_BASE: m/z 766.4 [M+H]+ SC_BASE: m/z 766.4 [M+H] +
1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 10.6 Hz, 1H), 7.61 (s, 1H), 7.26 - 7.09 (m, 13H), 6.90 (d, J = 9.1 Hz, 1H), 6.75 - 6.69 (m, 2H), 5.76 (d, J = 16.4 Hz, 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 = 7.4 Hz, 3H). 1H NMR (400 MHz, CDCl 3 ) δ 7.74 (d, J = 10.6 Hz, 1H), 7.61 (s, 1H), 7.26 - 7.09 (m, 13H), 6.90 (d, J = 9.1 Hz, 1H) , 6.75 - 6.69 (m, 2H), 5.76 (d, J = 16.4 Hz, 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 = 7.4 Hz, 3H).
2-3: 화합물 4의 합성2-3: Synthesis of Compound 4
Figure PCTKR2023006170-appb-img-000024
Figure PCTKR2023006170-appb-img-000024
질소 분위기 하에 건조된 플라스크에 N-((1S,9S)-9-에틸-5-플루오로-9-히드록시-4-메틸-10,13-디옥소-2,3,9,10,13,15-헥사히드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-1-일)-2-((4-메톡시페닐)디페닐메톡시)아세트아미드 (80 mg, 0.104 mmol) 및 DMAP(48.5 mg, 0.397 mmol)을 디클로로메탄(6 mL)에 용해시켰다. 5분 동안 교반한 후, 디클로로메탄(0.750mL) 중 트리포스겐(12.4mg, 0.042mmol)의 용액을 한번에 첨가하고 반응 혼합물을 실온에서 15분 동안 교반하였다. 디클로로메탄 (1.5 mL) 중 (S)-2-(32-아지도-5-옥소-3,9,12,15,18,21,24,27,30-노나옥사-6-아자도트리아콘탄아미도)-N-(4-(하이드록시메틸))페닐)-6-(((4-메톡시페닐)디페닐메틸)아미노)헥산아미드 (122 mg, 0.115 mmol)의 용액을 첨가하고 반응 혼합물을 실온에서 30분 동안 교반하였다. 반응 혼합물을 감압하에 농축하고, DMSO에 용해시키고, 염기성 분취용 MPLC(XSelect50-100)로 정제하여 동결건조 후 무색 고체를 얻었다. 수율: 0.129g, 66%.N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 was placed in a flask dried under a nitrogen atmosphere. ,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) in dichloromethane (0.750 mL) was added in one portion and the reaction mixture was stirred at room temperature for 15 minutes. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxa-6-azadotriacone in dichloromethane (1.5 mL) Add a solution of tanamido)-N-(4-(hydroxymethyl))phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (122 mg, 0.115 mmol) The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, dissolved in DMSO, and purified by basic preparative MPLC (XSelect50-100) to obtain a colorless solid after lyophilization. Yield: 0.129 g, 66%.
SC_BASE_M1900: m/z 1580.0 [M-MMT+H]+ SC_BASE_M1900: m/z 1580.0 [M-MMT+H] +
1H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.10 - 8.01 (m, 1H), 7.80 (d, J = 10.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 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 = 12.2 Hz, 2H), 4.47 - 4.40 (m, 1H), 4.00 (s, 2H), 3.96 (d, J = 4.2 Hz, 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 = 7.4 Hz, 3H). 1H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.32 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.10 - 8.01 (m, 1H) ), 7.80 (d, J = 10.9 Hz, 1H), 7.58 (d, J = 8.5 Hz, 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 = 12.2 Hz, 2H), 4.47 - 4.40 (m , 1H), 4.00 (s, 2H), 3.96 (d, J = 4.2 Hz, 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 = 7.4 Hz, 3H).
2-4: 화합물 5의 합성2-4: Synthesis of Compound 5
Figure PCTKR2023006170-appb-img-000025
Figure PCTKR2023006170-appb-img-000025
디클로로메탄(7 mL) 중 4-((S)-35-아지도-2-(4-(((4-메톡시페닐)디페닐메틸)아미노)부틸)-4,8-디옥소-6,12,15,18,21,24,27,30,33-노나옥사-3,9-디아자펜타트리아콘탄아미도)벤질 ((1S,9R)-9-에틸-5-플루오로-1-(2-((4-메톡시페닐)디페닐메톡시)아세트아미도)-4- 메틸-10,13-디옥소-2,3,9,10,13,15-헥사히드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b] 퀴놀린-9-일)카보네이트(0.129g, 0.070 mmol)의 용액에, 4-((2,5-디옥소-2,5-디히드로-1H-피롤-1-일)메틸)-N-(프로프-2-인-1-일)시클로헥산-1-카르복사미드 (57mg, 0.209mmol), 브롬화구리(I)(6mg, 0.042mmol) 및 DIPEA(0.036mL, 0.209mmol)를 첨가하였다. 반응 혼합물을 실온에서 밤새 교반하였다. 추가량의 브롬화구리(I)(6 mg, 0.042 mmol)를 첨가하였다. 추가로 3시간 동안 교반한 후, 반응 혼합물을 감압 하에 농축시켰다. 잔류물을 염기성 분취용 MPLC(XSelect50-100)로 정제하였다. 생성물 분획을 동결건조하여 회백색 고체를 얻었다. 수율: 106mg, 71%4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6 in dichloromethane (7 mL) ,12,15,18,21,24,27,30,33-nonoxa-3,9-diazapentatriacontanamido)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-1H In a solution of 12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b] quinolin-9-yl)carbonate (0.129g, 0.070 mmol), 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. The reaction mixture was stirred at room temperature overnight. Additional amount of copper(I) bromide (6 mg, 0.042 mmol) was added. After stirring for an additional 3 hours, the reaction mixture was concentrated under reduced pressure. The residue was purified by basic preparative MPLC (XSelect50-100). The product fraction was lyophilized to obtain an off-white solid. Yield: 106 mg, 71%
SC_BASE: m/z 1854.2 [M-MMT+H]+, 1582.0 [M-2xMMT+H]+ SC_BASE: m/z 1854.2 [M-MMT+H] + , 1582.0 [M-2xMMT+H] +
1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.34 (d, J = 8.6 Hz, 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 = 9.7 Hz, 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 = 7.0 Hz, 4H).1H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.34 (d, J = 8.6 Hz, 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 = 9.7 Hz, 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 = 7.0 Hz, 4H).
2-5: 화합물 CL2A-Dxd의 합성2-5: Synthesis of compound CL2A-Dxd
Figure PCTKR2023006170-appb-img-000026
Figure PCTKR2023006170-appb-img-000026
디클로로메탄(무수)(1.14ml) 중 4-((S)-35-(4-((4-((2,5-디옥소-2,5-디히드로-1H-피롤-1-일)메틸)시클로헥산-1-카르복사미도)메틸)-1H-1,2,3-트리아졸-1-일)-2-(4-(((4-메톡시페닐)디페닐메틸)아미노)부틸)-4,8-디옥소-6,12,15,18,21,24,27,30,33-노나옥사-3,9-디아자펜타트리아콘탄아미도)벤질((1S,9R)-9-에틸-5-플루오로-1-(2-((4-메톡시페닐)디페닐메톡시)아세트아미도)-4-메틸-10,13-디옥소-2,3,9,10,13,15-헥사히드로-1H,12H-벤조[de]피라노[3',4':6,7]인돌리지노[1,2-b]퀴놀린-9-일)카보네이트(80mg, 0.038mmol) 의 용액에, 아니솔(0.411ml, 3.76mmol)을 실온에서 아르곤 분위기하에서 첨가한 후, 디클로로아세트산(0.047 ml, 0.564 mmol)을 적가하였다. 1시간 동안 교반한 후, MTBE(~3 mL)를 첨가하였다. 반응 혼합물은 미세한 현탁액으로 변하였다. 헵탄(~3mL)을 첨가하여 침전을 일으켰다. 피펫으로 용매를 최대한 제거하였다. 잔류물을 MTBE/헵탄(1:1, ~4 mL) 혼합물로 여러 번 세척했다. 습윤 잔류물을 감압 하에 건조시키고, 바이알로 옮기고, 진공 하에 밤새 건조시켜 회백색 고체를 수득하였다. 수율: 57 mg, 96%. 순도(LC-UV: 78%). 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) in dichloromethane (anhydrous) (1.14 ml) 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-diazapentatriacontanamido)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-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (80mg , 0.038 mmol), anisole (0.411 ml, 3.76 mmol) was added at room temperature under an argon atmosphere, and then dichloroacetic acid (0.047 ml, 0.564 mmol) was added dropwise. After stirring for 1 hour, MTBE (~3 mL) was added. The reaction mixture turned into a fine suspension. Heptane (~3 mL) was added to cause precipitation. The solvent was removed as much as possible with 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 give an off-white solid. Yield: 57 mg, 96%. Purity (LC-UV: 78%).
AN_ACID: 792.0 [M+2H]2+/2AN_ACID: 792.0 [M+2H] 2+ /2
1H NMR analysis contains the expected signals. 1 H NMR analysis contains the expected signals.
1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.43 (d, J = 9.0 Hz, 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 = 5.7 Hz, 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 = 7.1 Hz, 7H), 2.40 (s, 3H), 2.27 - 1.98 (m, 7H), 1.82 - 1.44 (m, 11H), 1.28 (d, J = 12.2 Hz, 6H), 0.95 - 0.81 (m, 6H). 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.43 (d, J = 9.0 Hz, 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 = 5.7 Hz, 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 = 7.1 Hz, 7H), 2.40 (s, 3H), 2.27 - 1.98 (m, 7H), 1.82 - 1.44 (m, 11H), 1.28 (d, J = 12.2 Hz, 6H), 0.95 - 0.81 (m, 6H).
제조예 3: 항-HER2 항체-CL2A 링커-Exatecan/Dxd 면역접합체(DAR 8)의 제조Preparation Example 3: Preparation of anti-HER2 antibody-CL2A linker-Exatecan/Dxd immunoconjugate (DAR 8)
반응 버퍼 (20 mM 히스티딘, 150 mM NaCl, pH 6.0)중 2mg/ml의 항-HER2 항체 (트라스투주맙)를 825uM TCEP으로 2hrs 동안 25℃에서 환원시키고, 항체의 disulfide로부터 반응에 필요한 thiol 자리를 만들었다. 환원 후, 스핀 탈염 컬럼 (PD-10)을 통해 TCEP를 제거하였다.2mg/ml anti-HER2 antibody (trastuzumab) in reaction buffer (20mM histidine, 150mM NaCl, pH 6.0) was reduced with 825uM TCEP for 2hrs at 25°C, and the thiol site required for the reaction was converted from the disulfide of the antibody. made. After reduction, TCEP was removed through a spin desalting column (PD-10).
DMSO 및 제조예 1 및 제조예 2의 화합물 용액 (DMSO 중 5mM 스톡)을 최종 DMSO 농도가 10 %인 환원된 항체 용액에 항체 대비 12 eq.로 첨가했다. 반응 혼합물을 적절히 혼합하고 반응 바이알을 25 ℃에서 1 시간 동안 방치하였다.DMSO and compound solutions of Preparation 1 and Preparation 2 (5mM stock in DMSO) were added to the reduced antibody solution at a final DMSO concentration of 10% at 12 eq. relative to antibody. The reaction mixture was mixed appropriately and the reaction vial was left at 25° C. for 1 hour.
접합 후, 반응 혼합물을 스핀 탈염 컬럼 (PD-10)을 통해 미반응 화합물을 제거하고 ADC만을 분리했다. 이어서 생성물을 0.2 μm PVDF 일회용 필터를 통해 멸균 여과시켰다. 결과물인 면역접합체를 특성화하였으며, 0.07 mM PS80 및 20 mM 트레할로스 탈수물을 첨가했다. After conjugation, the reaction mixture was passed through a spin desalting column (PD-10) to remove unreacted compounds and only the ADC was separated. The product was then sterile filtered through a 0.2 μm PVDF disposable filter. The resulting immunoconjugate was characterized and 0.07 mM PS80 and 20 mM trehalose dehydrate were added.
HIC 및 MS를 이용하여 제조예 3의 면역접합체의 DAR을 결정하였으며, 평균 MS-DAR 값은 7.14, HIC-DAR 값은 8.00으로 확인되었다.The DAR of the immunoconjugate of Preparation Example 3 was determined using HIC and MS, and the average MS-DAR value was confirmed to be 7.14 and the HIC-DAR value was found to be 8.00.
실시예 1. Western blot을 통한 DDX5, p-DDX5, survivin, Mcl-1, XIAP 및 cIAP2의 암-관련 생존 유전자 (cancer-associated survival genes)의 발현 억제 활성 확인Example 1. Confirmation of inhibitory activity of DDX5, p-DDX5, survivin, Mcl-1, XIAP and cIAP2 expression of cancer-related survival genes through Western blot
FL118, Exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 및 Tra-CL2A-Exatecan(제조예 3)에 대해, 하기와 같이 anti-apoptotic protein들의 발현 저해 분석을 수행하였다.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), as follows: Analysis of inhibition of expression of anti-apoptotic proteins was performed as follows.
Tra-CL2A-FL118 및 Tra-CL2A-SN-38는 각각 Exatecan 약물 대신 FL118 약물 및 SN-38을 사용한 것을 제외하고 제조예 3와 동일한 방법을 제조하였다.Tra-CL2A-FL118 and Tra-CL2A-SN-38 were prepared in the same manner as Preparation Example 3, except that the drug FL118 and SN-38 were used instead of the drug Exatecan, respectively.
(1) protein extraction(1) protein extraction
6well plate에 well당 200000개의 FaDu 세포주를 파종(seeding)하고 항온배양(37℃, 5% CO2)하였다. 24시간 후, 0 nM, 10 nM, 100 nM의 농도로 약물(FL118, Exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 및 Tra-CL2A-Exatecan)을 well에 각각 처리하였다. 24시간동안 항온배양(37℃, 5% CO2) 시켰다. well에 protease inhibitor cocktail을 녹인 RIPA buffer 100ul를 처리하였다. plate를 얼음 위에 박고 orbital shaker에서 2시간동안 항온배양하였다. lysis된 cell이 포함된 RIPA buffer를 ep tube에 옮기고 원심분리 (16000rcf, 20min, 4℃)시켰다. 이후 상층액만 새로운 ep tube에 옮겼다. protein assay를 통해 단백질의 농도를 확인하였다.200,000 FaDu cell lines were seeded per well in a 6-well plate and incubated at constant temperature (37°C, 5% CO 2 ). After 24 hours, the drug (FL118, Exatecan, SN-38, Dxd, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN) was administered at concentrations of 0 nM, 10 nM, and 100 nM. -38 and Tra-CL2A-Exatecan) were treated in each well. It was incubated at constant temperature (37°C, 5% CO 2 ) for 24 hours. The well was treated with 100ul of RIPA buffer dissolved in protease inhibitor cocktail. The plate was placed on ice and incubated for 2 hours on an orbital shaker. RIPA buffer containing lysed cells was transferred to an ep tube and centrifuged (16000 rcf, 20 min, 4°C). Afterwards, only the supernatant was transferred to a new EP tube. Protein concentration was confirmed through protein assay.
(2) 전기영동을 통한 단백질 분리(2) Protein separation through electrophoresis
protein sample과 4x SDS-PAGE Loading Buffer를 3:1비율로 섞고 95℃에서 10분간 끓인 후 식혔다. 단백질의 양이 동일하게끔 샘플들을 gel의 well에 로딩하였다. 60V로 gel에서 전기영동하였다.Protein sample and 4x SDS-PAGE Loading Buffer were mixed in a 3:1 ratio, boiled at 95°C for 10 minutes, and cooled. Samples were loaded into gel wells so that the amount of protein was the same. Electrophoresis was performed on the gel at 60V.
(3) 단백질을 gel에서 membrane으로 transfer(3) Transfer of protein from gel to membrane
Trans-Blot® Turbo™ Transfer System의 cassettes에 활성화된 filter paper 7장, PVDF membrane, gel, filter paper 7장을 차례로 놓고 뚜껑을 닫고, 기계에 꽂은 후 protocol을 작동시켰다.7 sheets of activated filter paper, PVDF membrane, gel, and 7 sheets of filter paper were placed in order in the cassettes of the Trans-Blot® Turbo™ Transfer System, closed the lid, inserted into the machine, and started the protocol.
(4) Antibody incubation(4) Antibody incubation
transfer된 membrane을 blocking buffer에 담그고 항온배양(RT,1h) 시켰다. 그다음, 1차 antibody solution에서 항온배양(4℃, overnight)하였다. TBST buffer로 3min동안 헹구었다(3회 반복). HRP가 conjugation된 2차 antibody solution에서 항온배양(RT,1h)시켰다. TBST buffer로 3min동안 헹구었다(3회 반복).The transferred membrane was immersed in blocking buffer and incubated at constant temperature (RT, 1h). Next, it was incubated (4℃, overnight) in the primary antibody solution. Rinsed with TBST buffer for 3 minutes (repeated 3 times). Incubation was performed (RT, 1h) in a secondary antibody solution conjugated with HRP. Rinsed with TBST buffer for 3 minutes (repeated 3 times).
(5) Imaging과 결과 분석(5) Imaging and result analysis
membrane을 ECL substrate에 3~5min정도 담군 후 ChemiDoc™ MP Imaging System을 이용하여 signal을 확인하였다. 2차 항체에 결합된 HRP가 ECL의 Luminol을 산화시켜 방출되는 빛을 detection하여 이미지로 보여주었다. band의 굵기는 단백질의 양과 비례하므로 band의 굵기를 통해 단백질의 양을 비교할 수 있다. After soaking the membrane in the ECL substrate for 3 to 5 minutes, the signal was confirmed using the ChemiDoc™ MP Imaging System. HRP bound to the secondary antibody oxidized the Luminol of ECL, and the light emitted was detected and displayed as an image. Since the thickness of the band is proportional to the amount of protein, the amount of protein can be compared through the thickness of the band.
도 4 및 도 5에 도시된 바와 같이, 약물(FL118 약물, SN-38 약물, exatecan 약물, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 및 Tra-CL2A-Exatecan) 처리에 의해 단백질의 발현량이 어떻게 변하는지 확인하였다. GAPDH는 세포에서 필수적인 대사과정인 glycolysis에 관여하는 효소이며, 세포내에서 항상 발현되면서 그 발현량이 잘 변하지 않는 유전자로 샘플들이 동일양의 단백질이 gel 로딩되었는지 알 수 있는 지표이다.As shown in Figures 4 and 5, the drugs (FL118 drug, SN-38 drug, exatecan drug, PBX-7011, PBX-7014, PBX-7016, Tra-CL2A-FL118, Tra-CL2A-SN-38 and We confirmed how the protein expression level changes by treatment (Tra-CL2A-Exatecan). GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells. It is a gene that is always expressed in cells and its expression level does not change easily, and it is an indicator that shows whether the same amount of protein was loaded on the gel in the samples.
동일한 방법으로 ABCG2를 발현하지 않는 FaDu 세포주 대신 ABCG2를 과발현하고 있는 A549를 파종(seeding)하고, protein 4ug loading하여, Western blot을 수행하였다(도 6 및 도 7, 도 8).In the same way, A549, which overexpresses ABCG2, was seeded instead of the FaDu cell line, which does not express ABCG2, and 4ug of protein was loaded, and Western blot was performed (Figures 6, 7, and 8).
다양한 캄토테신계 화합물들이 항세포사멸단백질의 발현에 직접 관여하는 인자로 알려진 DDX5 내지 인산화된 DDX5(p-DDX5)를 분해하는 것을 확인하였다. 뿐만 아니라, 항세포사멸단백질들인 Survivin, Mcl-1, cIAP2, XIAP 등의 발현도 일부 억제하는 것을 확인함으로써, 다양한 캄토테신계 화합물들이 토포아이소머라아제 I을 저해할 뿐만 아니라 항세포사멸단백질 억제제로서 작용하는 이중기전을 갖는 화합물임을 확인하였다.It was confirmed that various camptothecin-based compounds degrade DDX5 or phosphorylated DDX5 (p-DDX5), which are known to be factors directly involved in the expression of anti-apoptotic proteins. In addition, it was confirmed that the expression of anti-apoptotic proteins such as Survivin, Mcl-1, cIAP2, and It was confirmed that it is a compound with a dual mechanism of action.
실시예 2. Tra-CL2A-FL118 및 Tra-CL2A-Exatecan의 in vitro cell viability test Example 2. In vitro cell viability test of Tra-CL2A-FL118 and Tra-CL2A-Exatecan
-80 ℃에서 저장된 용액 상태로 BCD에서 공급받은 트라스투주맙 (분자량 148 kDa, 순도 97 %)은 완충액 (20 mM Histidine, 150 mM NaCl, pH 6.0)에 용해된 상태 (농도 10.09 mg/mL)로 대조군으로 사용하였다.Trastuzumab (molecular weight 148 kDa, purity 97%), supplied from BCD as a solution stored at -80°C, was dissolved in a buffer solution (20mM Histidine, 150mM NaCl, pH 6.0) (concentration 10.09 mg/mL). It was used as a control.
96well plate에 well당 3000개의 Her2-high 세포주(MDA-MB-453) 및 HER2 positive breast cancer 유래 세포주(SK-BR-3)를 각각 파종(seeding)하고 항온배양(37℃, 5% CO2)하였다. 24시간 후, 9개 농도(1000nM부터 1/5씩 serial dilution)의 약물 100ul를 셀에 처리하였다. 이 때, 트라스투주맙, Tra-CL2A-FL118 및 Tra-CL2A-Exatecan을 처리하였다. 3일 또는 6일동안 항온배양(37℃, 5% CO2) 을 통해 cell viability를 관찰하였다. well에 CellTiter-Glo reagent(CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)를 사용) 100ul씩 추가한 후 파이펫팅해 주었다. 10분동안 항온배양 (RT)후 luminescence를 측정하였다. 약물농도가 0일 때의 발광 값을 100%로 볼 때, 50%의 발광 값을 나타내는 농도가 IC50값이다.3,000 Her2-high cell lines (MDA-MB-453) and HER2 positive breast cancer-derived cell lines (SK-BR-3) were seeded per well in a 96-well plate and incubated at constant temperature (37°C, 5% CO 2 ). did. After 24 hours, 100ul of the drug at 9 concentrations (serial dilution of 1/5 from 1000nM) was treated with the cells. At this time, trastuzumab, Tra-CL2A-FL118, and Tra-CL2A-Exatecan were treated. Cell viability was observed through constant temperature incubation (37°C, 5% CO 2 ) for 3 or 6 days. 100ul of CellTiter-Glo reagent (using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7571)) was added to each well and pipetted. After incubation at temperature (RT) for 10 minutes, luminescence was measured. When considering the luminescence value when the drug concentration is 0 as 100%, the concentration that represents a luminescence value of 50% is the IC 50 value.
도 9 및 도 10에 나타난 바와 같이, Trastuzumab-CL2A-exadecane(제조예 3)은 Her2-high 세포주(MDA-MB-453) 및 HER2 positive breast cancer 유래 세포주(SK-BR-3)에서 세포 독성을 나타냈다.As shown in Figures 9 and 10, 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). showed.
이로부터, FL118 내지 엑사테칸과 같은 다양한 화학식 1의 캄토테신계 화합물을 페이로드로 하고 CL2A와 같은 산민감성 링커로 연결한 ADC가 우수한 세포 독성을 보이며 높은 효율로 작동하는 것을 확인하였다.From this, it was confirmed that ADCs using various camptothecin-based compounds of formula 1, such as FL118 to exatecan, as payloads and connected with an acid-sensitive linker such as CL2A showed excellent cytotoxicity and operated with high efficiency.

Claims (28)

  1. [화학식 1의 캄토테신계 약물]-[산 민감성(acid-sensitive) 링커]-[항체 또는 이의 항원결합부위 함유 단편]을 포함하여, 화학식 1의 캄토테신계 약물을 생체내 표적부위에서 방출하도록 설계된 면역접합체 또는 이의 약학적으로 허용가능한 염으로서, [Camptothecin-based drug of Formula 1] - [acid-sensitive linker] - [antibody or antigen-binding site-containing fragment thereof] to release the camptothecin-based drug of Formula 1 at the target site in vivo. A designed immunoconjugate or a pharmaceutically acceptable salt thereof,
    화학식 1의 캄토테신계 약물은 DDX5 단백질 및/또는 E3 리가아제에 결합하도록 설계된 페이로드(payload)이고,The camptothecin-based drug of Formula 1 is a payload designed to bind to DDX5 protein and/or E3 ligase,
    항체 또는 이의 항원결합부위 함유 단편에 산 민감성 링커를 통해 화학식 1의 캄토테신계 약물이 하나 이상 연결되어 있고,At least one camptothecin-based drug of Formula 1 is linked to the antibody or its antigen-binding site-containing fragment through an acid-sensitive linker,
    암세포의 항원을 표적화하는 항원결합부위에 의해 암세포로 표적화된 후, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되고, 유리형 화학식 1의 캄토테신계 약물은 세포막을 관통해 세포내로 이동하고, After being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ≤ 7) surrounding the cancer, and at least part of the camptothecin-based drug of Formula 1 is released, and the free form of Formula 1 Camptothecin drugs penetrate the cell membrane and move into the cell.
    선택적(optionally)으로, 화학식 1의 캄토테신계 약물이 연결되어 있는 면역접합체는 세포안으로 내재화(internalization)되어 리소좀(lysosomes)에서 화학식 1의 캄토테신계 약물이 유리되는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염:Optionally, the immunoconjugate to which the camptothecin drug of Formula 1 is linked is internalized into cells and the camptothecin drug of Formula 1 is released from lysosomes. An immunoconjugate or pharmaceutical thereof Acceptable salts:
    [화학식 1][Formula 1]
    Figure PCTKR2023006170-appb-img-000027
    Figure PCTKR2023006170-appb-img-000027
    X1 및 X3는 각각 독립적으로 탄소, 산소, 질소, 또는 황이고, X1 및 X3는 동일 또는 상이할 수 있으며,X 1 and X 3 are each independently carbon, oxygen, nitrogen, or sulfur, and X 1 and X 3 may be the same or different,
    X2는 탄소, 산소, 질소, 황, 단일결합 또는 이중결합이고,X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond,
    X1, (X2)n 및 X3는 5각, 6각 또는 7각 고리를 형성할 수 있으며(n=0~2의 값),X 1 , (X 2 )n and
    Y1, Y2 및 Y3는 각각 독립적으로 수소이거나, 또는 산소, 질소, 인 또는 황을 포함하는 작용기일 수 있음.Y 1 , Y 2 and Y 3 may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.
  2. 제1항에 있어서, 세포 내에서 DDX5이 종양단백질(oncoprotein)로 작동하는 환자군에 투여하기 위한 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, which is intended for administration to a group of patients in which DDX5 acts as an oncoprotein in cells.
  3. 제1항에 있어서, 화학식 1의 캄토테신계 약물은 X1, (X2)n, X3, Y1, Y2 및/또는 Y3 변형을 통해 세포막 투과도를 원하는 대로 조절하여, 종양조직에서 방관자 효과(bystander effect)가 발휘 또는 그 효과의 정도가 제어되도록 설계된 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 1 , wherein the camptothecin-based drug of Formula 1 adjusts the cell membrane permeability as desired through modification of X 1 , (X 2 )n, An immunoconjugate or a pharmaceutically acceptable salt thereof characterized by being designed to exert a bystander effect or to control the degree of the effect.
  4. 제1항에 있어서, 화학식 1의 캄토테신계 약물은 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물인 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염:The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the camptothecin-based drug of Formula 1 is a camptothecin-based drug of Formula 1-1 or Formula 1-2:
    [화학식 1-1][Formula 1-1]
    Figure PCTKR2023006170-appb-img-000028
    Figure PCTKR2023006170-appb-img-000028
    [화학식 1-2][Formula 1-2]
    Figure PCTKR2023006170-appb-img-000029
    Figure PCTKR2023006170-appb-img-000029
  5. 제1항에 있어서, 산 민감성 링커는 혈액의 중성 환경인 pH 7.3~7.5에서는 안정적이지만 종양세포 주변 (pH 6.5~7.2)이나 세포내 내재화 후 엔도좀 (pH 5.0~6.5) 또는 리소좀(pH 4.5~5.0)에서 가수분해되어 화학식 1의 활성형 캄토테신계 약물을 방출하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.According to claim 1, the acid-sensitive linker is stable in the neutral environment of blood, pH 7.3 to 7.5, but is stored around tumor cells (pH 6.5 to 7.2) or after internalization within the cell, in endosomes (pH 5.0 to 6.5) or lysosomes (pH 4.5 to 4.5). 5.0) An immunoconjugate or a pharmaceutically acceptable salt thereof, characterized in that it is hydrolyzed to release the active camptothecin-based drug of Formula 1.
  6. 제1항에 있어서, 암세포의 항원을 표적화하는 항원결합부위에 의해 암세포로 표적화된 후, 암 주변 산성 환경(pH ≤ 7)에서 산 민감성 링커가 분해되어 화학식 1의 캄토테신계 약물이 적어도 일부 유리되고, 유리형 화학식 1의 캄토테신계 약물은 종양조직 심부까지 침투하면서 세포막을 관통해 세포내로 이동하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 1, wherein after targeting to cancer cells by an antigen-binding site that targets the antigen of cancer cells, the acid-sensitive linker is decomposed in an acidic environment (pH ≤ 7) surrounding the cancer, thereby liberating at least some of the camptothecin-based drug of Formula 1. The free camptothecin-based drug of Formula 1 is an immunoconjugate or a pharmaceutically acceptable salt thereof, which is characterized by penetrating deep into the tumor tissue and moving into the cell through the cell membrane.
  7. 제1항에 있어서, 산 민감성 링커 분해시 유리형 화학식 1의 캄토테신계 약물이 방출되도록, 화학식 1의 캄토테신계 약물과 산 민감성 링커는 탄산염 또는 에스테르 결합으로 연결된 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The immunoconjugate or pharmaceutical thereof according to claim 1, wherein the camptothecin-based drug of Formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that the free camptothecin-based drug of Formula 1 is released when the acid-sensitive linker is decomposed. Salts that are generally acceptable.
  8. 제1항에 있어서, 유리형 화학식 1의 캄토테신계 약물이 세포내로 이동하는 세포는 표적화된 암세포 및/또는 이의 주변 세포인 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the cells into which the free camptothecin-based drug of Formula 1 moves intracellularly are targeted cancer cells and/or their surrounding cells.
  9. 제1항에 있어서, 항체 또는 이의 항원결합부위는 세포 표면의 수용체 (Receptor)에 결합하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염. The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody or antigen-binding site thereof binds to a receptor on the cell surface.
  10. 제1항에 있어서, 항체 또는 이의 항원결합부위는 암 표면에 선택적으로 분포하는 항원 또는 정상 조직에도 소수 분포하는 암세포 과발현 항원을 표적항원으로 하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody or antigen-binding site thereof uses an antigen selectively distributed on the surface of cancer or an antigen overexpressed in cancer cells distributed in small numbers in normal tissues as a target antigen.
  11. 제1항에 있어서, 항체 또는 이의 항원결합부위는 Human epidermal growth factor Receptor(HER/EGFR/ERBB) 중 하나인 HER2, FolR, PSMA 또는 Trop-2를 표적화하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 1, wherein the antibody or its antigen-binding site is an immunoconjugate or a pharmaceutically derived immunoconjugate characterized by targeting HER2, FolR, PSMA or Trop-2, which is one of the Human epidermal growth factor receptors (HER/EGFR/ERBB). Acceptable salts.
  12. 제1항에 있어서, 산 민감성 링커는 하기 화학식 3의 화합물로부터 유래되는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염:The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein the acid-sensitive linker is derived from a compound of formula 3:
    [화학식 3][Formula 3]
    Figure PCTKR2023006170-appb-img-000030
    Figure PCTKR2023006170-appb-img-000030
    여기서, X1 및 X2는 각각 독립적으로 -H 또는 -할로젠이고;Here, X 1 and X 2 are each independently -H or -halogen;
    Y는 -NH-, -NRA-, 또는 아무 것도 아니며 (null); Y is -NH-, -NR A -, or nothing (null);
    Z는 -C1-C4알킬-, -C3-C6시클로알킬-, -(C1-C2알킬)-(C3-C6시클로알킬)-, -(C3-C6시클로알킬)-(C1-C2알킬)-, 또는 -(C1-C2알킬)-(C3-C6시클로알킬)-(C1-C2알킬)-이고; Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
    W는 -RB-, -M- -RB-M-, -M-RB- 또는 -RB-M-RC-이며; W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
    RA 내지 RC는 각각 독립적으로 C1-C4알킬이고, R A to R C are each independently C 1 -C 4 alkyl,
    M은
    Figure PCTKR2023006170-appb-img-000031
    이며; 및    M is
    Figure PCTKR2023006170-appb-img-000031
    and; and
    n은 5 내지 9의 정수임.n is an integer from 5 to 9.
  13. 제12항에 있어서, 화학식 1의 캄토테신계 약물의 20번 탄소에 위치한 알코올기 부위와 화학식 3의 산 민감성 링커의 알코올기 부위가 연결된 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The immunoconjugate of claim 12, or a pharmaceutically acceptable salt thereof, wherein the alcohol moiety located at carbon 20 of the camptothecin drug of Formula 1 is linked to the alcohol moiety of the acid-sensitive linker of Formula 3.
  14. 제1항에 있어서, [화학식 1의 캄토테신계 약물]-[산 민감성 링커]는 하기 화학식 4 또는 화학식 5로 표시되는 화합물로부터 유래된 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염:The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein [camptothecin-based drug of Formula 1]-[acid sensitive linker] is derived from a compound represented by Formula 4 or Formula 5 below:
    [화학식 4][Formula 4]
    Figure PCTKR2023006170-appb-img-000032
    Figure PCTKR2023006170-appb-img-000032
    [화학식 5][Formula 5]
    Figure PCTKR2023006170-appb-img-000033
    Figure PCTKR2023006170-appb-img-000033
    여기서, n은 각각 독립적으로 5 내지 9의 정수임.Here, n is each independently an integer from 5 to 9.
  15. 제1항에 있어서, [산 민감성 링커]-[항체 또는 이의 항원결합부위 함유 단편]의 연결은 상기 항체 또는 이의 항원 결합 단편에 포함된 티올기가 산 민감성 링커의 말레이미드기 (maleimide) 또는 말레익 하이드라자이드기 (maleic hydrazide)에 결합된 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 1, wherein the linkage of [acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof] is formed by linking the thiol group contained in the antibody or antigen-binding fragment thereof with the maleimide group or maleimide group of the acid-sensitive linker. An immunoconjugate or a pharmaceutically acceptable salt thereof characterized by being bound to a hydrazide group (maleic hydrazide).
  16. 제1항에 있어서, DDX5 단백질 및 E3 리가아제에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 분자접착제(molecular glue degrader) 기전(MoA)을 통해 DDX5 단백질을 발현하는 표적 세포를 사멸시키는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 1, wherein the camptothecin-based drug of Formula 1, which is designed to bind to DDX5 protein and E3 ligase, kills target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA). Immunoconjugate or pharmaceutically acceptable salt thereof.
  17. 제1항에 있어서, DDX5 단백질 및 E3 리가아제에 결합하도록 설계된 화학식 1의 캄토테신계 약물은 제1형 토포이소머라제 저해 능력과 함께 종양단백질(oncoprotein) DDX5를 분해하는 작용기전(MoA)를 갖는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.According to claim 1, the camptothecin-based drug of Formula 1, which is designed to bind to the DDX5 protein and E3 ligase, has the ability to inhibit type 1 topoisomerase and has a mechanism of action (MoA) to degrade the oncoprotein DDX5. An immunoconjugate or a pharmaceutically acceptable salt thereof.
  18. 제17항에 있어서, (1) 일반식 1에서, 선택적으로(optionally) Group C 부위는 제1형 토포이소머라제에 결합하고, Group A 부위는 DNA에 결합하여 토포이소머라제-DNA 복합체의 공유결합을 안정화시켜 절단된 DNA 조각들이 다시 연결되는 것을 방해하고/하거나, (2) 일반식 1에서 Group A 부위는 DDX5 단백질에 결합하고, 선택적으로(optionally) Group C 부위는 E3 리가아제에 결합하여 DDX5 단백질 분해를 유도하는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 17, wherein (1) in General Formula 1, the Group C site optionally binds to type 1 topoisomerase, and the Group A site binds to DNA to form a topoisomerase-DNA complex. Stabilizes the covalent bond and prevents the cut DNA fragments from being reconnected, or (2) in General Formula 1, the Group A site binds to the DDX5 protein, and optionally, the Group C site binds to the E3 ligase. An immunoconjugate or a pharmaceutically acceptable salt thereof characterized by inducing DDX5 protein degradation.
    [일반식 1][General Formula 1]
    Figure PCTKR2023006170-appb-img-000034
    Figure PCTKR2023006170-appb-img-000034
  19. 제1항 내지 제18항 중 어느 한 항의 면역접합체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, comprising the immunoconjugate of any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof as an active ingredient.
  20. 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 화학식 3의 산 민감성 링커에 연결된, 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염:Camptothecin system of Formula 1-1 or Formula 1-2 A drug-linker conjugate, or a pharmaceutically acceptable salt thereof, wherein the drug is linked to an acid-sensitive linker of Formula 3:
    [화학식 1-1][Formula 1-1]
    Figure PCTKR2023006170-appb-img-000035
    Figure PCTKR2023006170-appb-img-000035
    [화학식 1-2][Formula 1-2]
    Figure PCTKR2023006170-appb-img-000036
    Figure PCTKR2023006170-appb-img-000036
    [화학식 3][Formula 3]
    Figure PCTKR2023006170-appb-img-000037
    Figure PCTKR2023006170-appb-img-000037
    여기서, X1 및 X2는 각각 독립적으로 -H 또는 -할로젠이고;Here, X 1 and X 2 are each independently -H or -halogen;
    Y는 -NH-, -NRA-, 또는 아무 것도 아니며 (null); Y is -NH-, -NR A -, or nothing (null);
    Z는 -C1-C4알킬-, -C3-C6시클로알킬-, -(C1-C2알킬)-(C3-C6시클로알킬)-, -(C3-C6시클로알킬)-(C1-C2알킬)-, 또는 -(C1-C2알킬)-(C3-C6시클로알킬)-(C1-C2알킬)-이고; Z is -C 1 -C 4 alkyl-, -C 3 -C 6 cycloalkyl-, -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-, -(C 3 -C 6 cyclo alkyl)-(C 1 -C 2 alkyl)-, or -(C 1 -C 2 alkyl)-(C 3 -C 6 cycloalkyl)-(C 1 -C 2 alkyl)-;
    W는 -RB-, -M- -RB-M-, -M-RB- 또는 -RB-M-RC-이며; W is -R B -, -M- -R B -M-, -MR B - or -R B -MR C -;
    RA 내지 RC는 각각 독립적으로 C1-C4알킬이고, R A to R C are each independently C 1 -C 4 alkyl,
    M은
    Figure PCTKR2023006170-appb-img-000038
    이며; 및    M is
    Figure PCTKR2023006170-appb-img-000038
    and; and
    n은 5 내지 9의 정수임.n is an integer from 5 to 9.
  21. 제20항에 있어서, 산 민감성 링커 분해시 유리형 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 방출되도록, 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물과 산 민감성 링커는 탄산염 또는 에스테르 결합으로 연결된 것이 특징인 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염.The method of claim 20, wherein the camptothecin drug of Formula 1-1 or Formula 1-2 and the acid-sensitive linker are used so that the free camptothecin drug of Formula 1-1 or Formula 1-2 is released when the acid-sensitive linker is decomposed. A drug-linker conjugate characterized by being linked by a carbonate or ester bond, or a pharmaceutically acceptable salt thereof.
  22. 제20항에 있어서, 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물의 20번 탄소에 위치한 알코올기 부위와 화학식 3의 산 민감성 링커의 알코올기 부위가 연결된 것이 특징인 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염.The drug-linker conjugate according to claim 20, wherein the alcohol moiety located at carbon 20 of the camptothecin drug of Formula 1-1 or Formula 1-2 is connected to the alcohol moiety of the acid-sensitive linker of Formula 3, or A pharmaceutically acceptable salt thereof.
  23. 제20항에 있어서, 상기 약물-링커 접합체는 하기 화학식 4 또는 화학식 5로 표시되는 화합물인 것이 특징인 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염:The drug-linker conjugate or pharmaceutically acceptable salt thereof according to claim 20, wherein the drug-linker conjugate is a compound represented by the following Formula 4 or Formula 5:
    [화학식 4][Formula 4]
    Figure PCTKR2023006170-appb-img-000039
    Figure PCTKR2023006170-appb-img-000039
    [화학식 5][Formula 5]
    Figure PCTKR2023006170-appb-img-000040
    Figure PCTKR2023006170-appb-img-000040
    여기서, n은 각각 독립적으로 5 내지 9의 정수임.Here, n is each independently an integer from 5 to 9.
  24. 제20항 내지 제23항 중 어느 한 항에 있어서, 산성 환경(pH ≤ 7)에서 화학식 3의 산 민감성 링커가 분해되어 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 유리되는 것이 특징인 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염.The method according to any one of claims 20 to 23, wherein the acid-sensitive linker of Formula 3 is decomposed in an acidic environment (pH ≤ 7) to release the camptothecin-based drug of Formula 1-1 or Formula 1-2. Phosphorus drug-linker conjugate or a pharmaceutically acceptable salt thereof.
  25. (a) 제20항 내지 제23항 중 어느 한 항의 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염; 및 (a) the drug-linker conjugate of any one of claims 20 to 23 or a pharmaceutically acceptable salt thereof; and
    (b) 항체 또는 이의 항원결합부위 함유 단편(b) Antibody or antigen-binding site-containing fragment thereof
    을 포함하되, Including,
    항체 또는 이의 항원결합부위 함유 단편에 화학식 3의 산 민감성 링커를 통해 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물이 하나 이상 연결되어 있는 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.An immunoconjugate or a pharmaceutically acceptable salt thereof, characterized in that one or more camptothecin drugs of Formula 1-1 or Formula 1-2 are linked to an antibody or fragment containing an antigen-binding site thereof through an acid-sensitive linker of Formula 3 .
  26. 제25항에 있어서, 항체는 트라스투주맙(trastzumab), 세툭시맙(cetuximab), 또는 사시투주맙 (sacituzumab)인 것이 특징인 면역접합체 또는 이의 약학적으로 허용가능한 염.The immunoconjugate or pharmaceutically acceptable salt thereof according to claim 25, wherein the antibody is trastzumab, cetuximab, or sacituzumab.
  27. 제20항 내지 제23항 중 어느 한 항의 약물-링커 접합체 또는 이의 약학적으로 허용가능한 염을 사용하여, 운반체(Carrier)에 화학식 3의 산 민감성 링커를 통해 화학식 1-1 또는 화학식 1-2의 캄토테신계 약물을 하나 이상 연결시키는 것이 특징인 운반체-약물 접합체(Carrier-Drug Conjugate)의 제조방법.Using the drug-linker conjugate of any one of claims 20 to 23 or a pharmaceutically acceptable salt thereof, the drug of Formula 1-1 or Formula 1-2 is attached to a carrier through an acid-sensitive linker of Formula 3. A method for producing a carrier-drug conjugate characterized by linking one or more camptothecin drugs.
  28. 제27항에 있어서, 운반체는 항체 또는 이의 항원결합부위 함유 단편, 리피바디, 압타머인, 및/또는 펩타이드인 것이 특징인 운반체-약물 접합체의 제조방법.The method of claim 27, wherein the carrier is an antibody or an antigen-binding site-containing fragment thereof, a lipibody, an aptamerine, and/or a peptide.
PCT/KR2023/006170 2022-05-04 2023-05-04 Conjugate of ddx5 protein-binding camptothecin-based drug linked to acid-sensitive linker and immunoconjugate using same WO2023214849A1 (en)

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CN111001012A (en) * 2018-10-19 2020-04-14 四川百利药业有限责任公司 Hydrophilic carbonate type antibody coupling drug
KR20210006362A (en) * 2018-04-06 2021-01-18 시애틀 지네틱스, 인크. Camptothecin peptide conjugates
WO2021147993A1 (en) * 2020-01-22 2021-07-29 江苏恒瑞医药股份有限公司 Anti-trop-2 antidody-exatecan analog conjugate and medical use thereof
WO2022001864A1 (en) * 2020-06-28 2022-01-06 昆山新蕴达生物科技有限公司 Antibody-drug conjugate and preparation method therefor and use thereof
KR20220009916A (en) * 2020-07-16 2022-01-25 주식회사 피노바이오 Conjugate of FL118 drug linked to acid-sensitive linker and immunoconjugates using the same

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KR20210006362A (en) * 2018-04-06 2021-01-18 시애틀 지네틱스, 인크. Camptothecin peptide conjugates
CN111001012A (en) * 2018-10-19 2020-04-14 四川百利药业有限责任公司 Hydrophilic carbonate type antibody coupling drug
WO2021147993A1 (en) * 2020-01-22 2021-07-29 江苏恒瑞医药股份有限公司 Anti-trop-2 antidody-exatecan analog conjugate and medical use thereof
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