WO2022015110A1 - Fl118 약물이 산 민감성 링커에 연결된 접합체 및 이를 이용한 면역접합체 - Google Patents

Fl118 약물이 산 민감성 링커에 연결된 접합체 및 이를 이용한 면역접합체 Download PDF

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WO2022015110A1
WO2022015110A1 PCT/KR2021/009204 KR2021009204W WO2022015110A1 WO 2022015110 A1 WO2022015110 A1 WO 2022015110A1 KR 2021009204 W KR2021009204 W KR 2021009204W WO 2022015110 A1 WO2022015110 A1 WO 2022015110A1
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drug
formula
immunoconjugate
acid
cancer
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French (fr)
Korean (ko)
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정두영
이진수
조현용
최신혜
이병성
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Pinotbio Inc
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Pinotbio Inc
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Priority to US18/016,331 priority Critical patent/US20230270867A1/en
Priority to CA3189462A priority patent/CA3189462A1/en
Priority to JP2023503070A priority patent/JP7660186B2/ja
Priority to CN202180062303.4A priority patent/CN116075321A/zh
Priority to EP21841752.5A priority patent/EP4183418A4/en
Priority to AU2021308834A priority patent/AU2021308834A1/en
Publication of WO2022015110A1 publication Critical patent/WO2022015110A1/ko
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/54—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 organic compound
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/6855—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 the tumour determinant being from breast cancer cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages

Definitions

  • the present invention relates to a conjugate in which a FL118 drug is linked to an acid-sensitive linker and an immunoconjugate using the same.
  • the antibody-drug conjugate is a new drug platform that selectively delivers a payload with strong anticancer efficacy only to cancer tissues by using the high tissue selectivity of an antibody.
  • ADC selectively delivers a strong payload that kills cancer cells even at a low concentration of pM to cancer tissues, and minimizes systemic drug exposure, thereby securing anticancer efficacy and safety at the same time.
  • ADC development started in the 1970s, and many efforts are underway, such as discovery of new antigen/antibody, development of payload for ADC, development of linker for antibody-payload conjugation, improvement of antibody for uniform quality, and pioneering of CMC route. As a result, most of the current ADCs are being developed using the 2nd or 3rd generation ADC platform.
  • ADCs that can be used effectively for the treatment of various cancers, especially solid cancers, centering on companies such as Genetech, Seattle Genetics, and Legochem Bio in Korea, but the therapeutic window and / Or there are still very important unmet requirements, such as the generation of immunity.
  • Immunomedics and Daiichi-Sankyo developed the third-generation ADC using a payload that secured a sufficient therapeutic window as a single-administered anticancer agent in the human body, and Trodelvy and Enhertu received FDA approval as an ADC for solid cancer. was obtained.
  • Valine-Citrulllin (Val-Cit) linker developed by Seattle Genetics in the United States is used.
  • Representative ADCs using this include Adcetris, Polivy, Padcev, and the like (FIG. 13).
  • Val-Cit linker is highly toxic as Vedotin and other drugs. Although a very stable linker system such as Val-Cit was introduced to solve the problem of high toxicity, there is still a limitation in securing a therapeutic window due to the toxicity of the strong payload.
  • a new ADC anticancer agent has been developed that introduces a target anticancer agent with high potential for therapeutic development as an ADC payload instead of the powerful Toxin, which was mainly used in second-generation anticancer drugs. development is in progress (FIG. 14).
  • FIG. 14 illustrates ADCs (Enhertu (top), Trodelvy (bottom)) using a topoisomerase I inhibitor Camptothecin-based compound as a payload as a target anticancer agent.
  • the anticancer efficacy is limited due to the use of payloads, which have relatively limited efficacy compared to the payloads used by the existing 2nd generation ADCs, or they are inconvenient to use at large doses, and resistance develops in patients with solid cancer
  • payloads which have relatively limited efficacy compared to the payloads used by the existing 2nd generation ADCs, or they are inconvenient to use at large doses, and resistance develops in patients with solid cancer
  • the present invention is a new payload-linker that has a mechanism of action that is differentiated from existing anticancer drugs/payloads, has sufficient anticancer efficacy and the potential to overcome resistance, and has sufficient safety even when used alone to secure a wide therapeutic window We would like to provide a conjugate and a new ADC development platform using the same.
  • the immunoconjugate of the present invention is a hydrophobic small molecule drug, and due to the combined use of the cell membrane-permeable FL118 drug and the acid-sensitive linker, the internalization process by the antigen-antibody complex is inefficient. It can solve the problem of not going to the cancer tissue and the problem of the antibody that does not penetrate well into the deep part of the cancerous tissue, and it can maximize the by-stander effect that enters not only the targeted cells but also the surrounding cells.
  • the immunoconjugate of the present invention can overcome the problem of a narrow therapeutic window that appears in the development of resistance to ADC and the relatively weak efficacy of the payload used in the third generation ADC, among the problems identified in the development process of the 1st - 3rd generation ADC. . That is, the immunoconjugate of the present invention can show an optimized therapeutic window by balancing strong anticancer efficacy and in vivo safety compared to the existing ADC payload due to the combined use of the FL118 drug with various resistance overcoming mechanisms and an acid-sensitive linker.
  • Figure 2 is a schematic diagram showing the mechanism of efficient drug release and resistance overcoming in the immunoconjugate of the present invention.
  • FIG. 4 is a schematic diagram of an example of a FL118 drug-carrier-drug conjugate comprising an acid-sensitive linker.
  • 5 is a DAR 4 ADC production confirmation result (5a) and a DAR 8 ADC production confirmation result (5b) using SEC, HIC, and LC-MS.
  • Trastuzumab-CL2A-FL118 (DAR 8) of Example 5 in the JIMT-1 Xenograft model.
  • Trastuzumab-FL118 ADC exhibits excellent efficacy in a Kadcyla-resistant JIMT-1 (Her2 over-expression) In Vivo Xenograft model.
  • Example 9 is an evaluation result in the MDA-MB-468 cell line of Cetumximab-CL2A-FL118 (DAR 8) of Example 8.
  • Example 10 is an evaluation result in the MDA-MB-468 Xenograft model of Cextuximab-CL2A-FL118 (DAR 8) of Example 8. Cetuximab-FL118 ADC exhibits excellent efficacy in the MDA-MB-468 (EGFR over-expression) In Vivo Xenograft model.
  • Example 11 is an evaluation of Sacituzumab-CL2A-FL118 (DAR 8) of Example 9 in the MDA-MB-468 cell line.
  • Sacituzumab-CL2A-FL118 DAR 8 of Example 9. Sacituzumab-FL118 ADC showed superior efficacy compared to Trodelvy, an approved TROP2 target ADC in the MDA-MB-468 (TROP2 over-expression) In Vivo Xenograft model.
  • FIG 13 shows ADCs developed using the Val-Cit linker: Adcetris (top), Polivy (middle), Padcev (bottom).
  • FIG. 14 is a schematic diagram of ADC using as a payload a compound of the Camptothecin series, a topoisomerase I inhibitor, as a target anticancer agent: Enhertu (top), Trodelvy (bottom)
  • a first aspect of the present invention is an immunoconjugate comprising [FL118 drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof] or a pharmaceutically acceptable salt thereof.
  • one or more FL118 drugs of Formula 1 are linked to an antibody or antigen-binding site-containing fragment thereof through an acid-sensitive linker, and after being targeted to cancer cells by an antigen-binding site that targets the antigen of cancer cells, an acidic atmosphere around the cancer (pH ⁇ 7), the acid-sensitive linker is decomposed to liberate at least a part of the FL118 drug of Formula 1, the free FL118 drug of Formula 1 penetrates the cell membrane and moves into the cell, and the FL118 drug of Formula 1 is an efflux pump By inhibiting the action of the intracellular free FL118 drug of Formula 1 is enriched (enrichment), and optionally (optionally), the immunoconjugate to which the FL118 drug of Formula 1 is linked is internalized into the cell to form lysosomes ) in which the
  • 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.
  • a third aspect of the present invention is FL118 of Formula 1 Provided is a drug-linker conjugate or a pharmaceutically acceptable salt thereof, wherein a drug is linked to an acid-sensitive linker of the following formula (2).
  • 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 antigen-binding site-containing fragment thereof, wherein at least one FL118 drug of Formula 1 is linked to the antibody or antigen-binding site-containing fragment through an acid-sensitive linker of Formula 2; or Provided are pharmaceutically acceptable salts thereof.
  • a fifth aspect of the present invention is the drug-linker conjugate of the first aspect or a pharmaceutically acceptable salt thereof, wherein one or more FL118 drugs of Formula 1 are linked to a carrier through an acid-sensitive linker of Formula 2 It provides a method for producing a carrier-drug conjugate (Carrier-Drug Conjugate) characterized in that.
  • the present invention relates to an immunoconjugate comprising [the FL118 drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof],
  • one or more FL118 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 is degraded in an acidic atmosphere (pH ⁇ 7) around the cancer to liberate at least a part of the FL118 drug of Formula 1, free formula
  • the FL118 drug of 1 penetrates the cell membrane and moves into the cell
  • the FL118 drug of Formula 1 inhibits the action of an efflux pump, and enriches the intracellular free FL118 drug of Formula 1,
  • the immunoconjugate to which the FL118 drug of Formula 1 is linked is internalized into a cell to release the FL118 drug of Formula 1 from lysosomes (FIG. 2).
  • the immunoconjugate of the present invention uses an acid-sensitive linker to efficiently release a drug not only from within the cancer cell but also from the surrounding cancer tissue after antigen binding, thereby overcoming the resistance mechanism related to ADC processing.
  • the FL118 drug of Formula 1 released from the immunoconjugate of the present invention is not released out of the cell by ABCG2, an efflux pump, and can block resistance by various anti-apoptic proteins.
  • the FL118 drug of Formula 1 directly targets Topoisomerase I, a well-established anticancer target, but simultaneously inhibits the Bcl family such as Survivin, a resistance protein involved in the resistance mechanism, and suppresses the action of the efflux pump. It is a triple-targeted anticancer drug.
  • the acid-sensitive linker is degraded in an acidic atmosphere (pH ⁇ 7) around the cancer, so that at least a part of the FL118 drug of Formula 1 is liberated, Since the free drug FL118 of Formula 1 is a hydrophobic low molecular weight, it can penetrate the cell membrane and move into the cell while penetrating deep into the cancer tissue.
  • the immunoconjugate of the present invention can rapidly release the drug from the tumor microenvironment around the cancer by using an acid-sensitive linker that is degraded in an acidic atmosphere (pH ⁇ 7) around the cancer, and the free FL118 drug is an antibody with a low molecular weight
  • an acid-sensitive linker that is degraded in an acidic atmosphere (pH ⁇ 7) around the cancer
  • the free FL118 drug is an antibody with a low molecular weight
  • the cells to which the free FL118 drug of Formula 1 migrates into cells may be targeted cancer cells and/or surrounding cells thereof.
  • the FL118 drug of Formula 1 is (a) a hydrophobic small molecule capable of penetrating a cell membrane, and (b) does not leak out of the cell through an efflux pump.
  • FL118 drug released from the drug rapidly accumulates in cancer tissues and maintains a high concentration for a long time. It penetrates the cell membrane and exerts cytotoxicity inside the cell to cause apoptosis, and then is released and then continuously penetrates the cell membrane to the surrounding cells and moves into the cell. may work.
  • the drug-linker conjugate of the present invention is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)
  • Another feature is that the organic mechanism of action of the FL118 drug-acid sensitive linker is utilized through its combined use.
  • the FL118 drug of Formula 1 and the acid-sensitive linker are linked by a carbonate or ester bond so that they are decomposed in an acidic atmosphere (pH ⁇ 7) and the free FL118 drug of Formula 1 is released upon decomposition of the acid-sensitive linker (FIG. 4).
  • the [acid-sensitive linker]-[antibody or antigen-binding fragment-containing fragment thereof] is linked with a thiol group contained in the antibody or antigen-binding fragment thereof, maleimide group of the acid-sensitive linker Alternatively, it may be bonded to a maleic hydrazide group through the “click” reaction of Scheme 1.
  • the present invention provides a drug-linker conjugate in which the FL118 drug is linked with various acid-sensitive linkers, and using this, the FL118 drug is linked to various carriers through various acid-sensitive linkers - a drug conjugate (Carrier- Drug Conjugate) is provided.
  • the present invention relates to one or more FL118 drugs of Formula 1 through the acid-sensitive linker of Formula 2 to a carrier using the above-described drug-linker conjugate of the present invention or a pharmaceutically acceptable salt thereof. It also provides a method for producing a carrier-drug conjugate (Carrier-Drug Conjugate).
  • FIG. 4 The structure of the carrier-drug conjugate linked to the carrier using the drug-linker conjugate according to the present invention is illustrated in FIG. 4 .
  • the immunoconjugate of the present invention is targeted to a cancer cell by an antigen-targeting antigen-binding site of the cancer cell, and then begins outside the cell, mainly in an acidic atmosphere (pH ⁇ 7) around the cancer and/or some acid-sensitive linkers are degraded in the blood
  • an acidic atmosphere pH ⁇ 7
  • the competitive advantage of the free drug FL118 of Formula 1 is as follows.
  • FL118 drug of Formula 1 is a strong Topoisomerase I inhibitor and has the same Camptothecin parent nucleus structure as SN-38, Topotecan, Exatecan, etc., which are the existing commercially available Top 1 inhibitors (FIG. 1), but even when administered alone, SN-38 in various cancer cells and animal models It is an anticancer drug that has secured a wide therapeutic window with differentiated anticancer efficacy and excellent safety.
  • FL118 drug has a Top1 inhibitory effect equivalent 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, that is, it has a low IC 50 level and cytotoxicity (Cytotoxicity). ) (FIG. 3), and the evaluation results of 140 cell lines originating from various carcinomas also show very strong anticancer efficacy with an IC 50 of ⁇ 100 nM against the majority of cancer cells.
  • FL118 drug can overcome the various mechanisms of resistance action of SN-38/Exatecan.
  • Camptothecin-based anticancer drugs such as SN-38 show resistance in the way that the drug is released out of the cell due to overexpression of ABCG2 Transporter, and FL118 drug is not affected by ABCG2 Transporter, so it is possible to overcome this resistance.
  • Camptothecin-based anticancer drugs show excellent anticancer response in the early stage when used in patients, but strong resistance to these drugs is shown through Epigenetic Silencing of Top1 gene and Top2 dependence of cancer cells.
  • FL118 drug showed strong efficacy in the Xenograft model of cancer cell lines in which Top1 is not expressed through epigenetic silencing or knock-out.
  • FL118 drug can block the expression of resistance by strongly inhibiting the expression of Anti-apoptic Protein (Survivin, cIAP2, XIAP, etc.), which is another major cause of resistance to anticancer drugs, at low concentrations.
  • Anti-apoptic Protein Survivin, cIAP2, XIAP, etc.
  • FL118 drug has an optimal PK/safety profile for targeted drug delivery (eg, carrier-drug conjugate) applications.
  • targeted drug delivery eg, carrier-drug conjugate
  • FL118 drug When FL118 drug is administered systemically, it is rapidly metabolized/excreted from the blood and shows only a low concentration, but it accumulates rapidly in cancer tissues immediately after administration and maintains a high concentration for a long time.
  • the ADC application ensures maximum selectivity between tumor tissue and normal tissue.
  • FL118 drug has excellent safety through the GLP-toxicity test in rats and beagle dogs, and is suitable for ADC payload with a therapeutic window.
  • FL118 drug showed superior efficacy compared to SN-38 in various cancer cell line Xenograft models.
  • FL118 drug When administered in vivo in the same amount as SN-38, FL118 drug showed strong tumor regression efficacy compared to SN-38 in colorectal cancer, head and neck cancer, and pancreatic cancer.
  • FL118 drug had strong anticancer efficacy even when FL118 was administered after induction of SN-38 resistance in tumor xenograft. The possibility of overcoming various mechanisms of resistance of FL118 drug to SN-38/Exatecan was confirmed in vivo.
  • the FL118 drug has a competitive advantage as a payload in the following respects:
  • Biomarkers (DDX5, K-ras, p53) and companion diagnostic techniques that can predict anticancer response in patients have already been established. It showed strong efficacy in p53/K-ras mutant cancer cells with poor prognosis.
  • the FL118 drug of Formula 1 (i) simultaneously inhibits the Bcl family such as Survivin, which is a resistance protein, and/or (ii) through an additional mechanism of action that inhibits the action of the efflux pump, the efficacy of the main drug target modulation can be maximized.
  • the FL118 drug linked to the carrier through an acid-sensitive linker targets one drug target with a single drug, deviating from the existing drug development method in which therapeutic efficacy is limited, multiple drug targets that need to be controlled together It was selected based on a well-designed polypharmacology-based approach that obtains excellent therapeutic efficacy by simultaneously targeting, and can treat intractable diseases in which existing therapeutics have not shown a clear therapeutic effect.
  • the FL118 drug which is linked through an acid-sensitive linker, utilizes two or more well-selected drug targets to show synergy in terms of drug efficacy and ensure sufficient safety at the same time.
  • the immunoconjugate according to the present invention uses the FL118 drug that inhibits the action of the efflux pump by linking it through an acid-sensitive linker, so that the competitive advantage of the aforementioned FL118 drug compared to Camptothecin-based anticancer drugs with a similar structure used in Enhertu and Trodelvy By reflecting this, it is possible to overcome resistance that is differentiated from the currently developed third-generation ADCs (Enhertu, Trodelvy) and secure a wider therapeutic window.
  • the immunoconjugate according to the present invention through the FL118 drug, in addition to the inhibition of the proven tumor target Topoisomerase I, the cancer resistance-inducing proteins DDX5, UBE2T, USP2a through the additional inhibition of strong resistance overcoming efficacy can be shown. Therefore, the immunoconjugate according to the present invention can be used as an ADC for overcoming resistance in fields where conventional SN-38-based ADCs such as Trop-2/CEACAM-5 are successfully developed.
  • Trop-2-SN-38 ADC is currently being successfully developed in a number of carcinomas such as TNBC, Bladder, and Gastric cancer. apoptic protein increase, etc.) remains, and an immunoconjugate comprising [the FL118 drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment] according to the present invention is utilized This can overcome the above problem.
  • the immunoconjugate according to the present invention utilizes related biomarkers such as DDX5, K-ras, and p53 to identify the optimal response patient group among the antigen-expressing patient groups such as Trop-2 and CEACAM-5 to secure the optimal therapeutic response.
  • related biomarkers such as DDX5, K-ras, and p53
  • the present invention selects the FL118 drug as a payload for various carrier-drug conjugates:
  • Exatecan, SN-38, etc. are actually developed as payloads for ADCs among drugs of the same class;
  • Topoisomerase I inhibitors (Irinotecan, Topotecan, etc.), which are anticancer mechanisms whose efficacy/safety has been verified in clinical practice. Anti-cancer efficacy has been verified;
  • Cancer cells stay in the cancer tissue for a long time, but have a PK profile that is quickly removed when discharged into the blood. Stable even when separated from
  • Linkers commonly used in ADC include acid-labile hydrazones, protease-labile peptides, and disulfides sensitive to reducing agents. Also, so-called “non-cleavable” thioether linkers that are not cleaved are used in ADCs.
  • the Stable Linker system is used, which aims to minimize the separation of the drug from the blood before reaching the cancer tissue (characteristic of the second-generation ADC) did
  • the principle that most second-generation ADCs work in cancer cells is that, in the first step, the antibody part constituting 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 by antigen-antibody reaction is endocytosed.
  • the antigen-antibody portion is decomposed in the lysosome, the enzyme (cathepsin B) drug is released, and in the last step, the cancer cell is killed by the drug released from inside the cancer cell. .
  • ADCs against cancer-specific antigens such as Slow Internalizing antigens such as CEACAM-5 and/or NY-ESO-1/HLA Complex and/or for the development of ADCs targeting new antigens beyond established drug targets such as Trop-2, Her2, and Folate Receptor.
  • cancer-specific antigens such as Slow Internalizing antigens such as CEACAM-5 and/or NY-ESO-1/HLA Complex
  • ADCs targeting new antigens beyond established drug targets such as Trop-2, Her2, and Folate Receptor.
  • antibody-drug binding maintains stability in the blood and/or the surrounding environment of normal tissues, but it is necessary to rapidly release the drug in the environment around cancer cells, such as the tumor microenvironment. do.
  • the drug can be rapidly released from the tumor microenvironment around the cancer, it can also be advantageously used for targeting various antigens (typically CEACAM-5, various Cancer Specific Antigen-HLA Complex, etc.) that have limitations in the ADC uptake rate. have.
  • various antigens typically CEACAM-5, various Cancer Specific Antigen-HLA Complex, etc.
  • the immunoconjugate using the FL118 drug is faster and more efficient after reaching the cancer tissue, rather than the stability that minimizes the blood separation of the FL118 drug.
  • the release profile that delivers the drug is more stringent on the linker.
  • the acid-sensitive linker used in the present invention is decomposed in an acidic atmosphere (pH ⁇ 7) around the cancer, and since the free FL118 drug of Formula 1 is a hydrophobic low molecule, it penetrates deep into the cancer tissue and moves into the cell through the cell membrane. It was selected based on the fact that it is possible.
  • the antibody to which the FL118 drug of Formula 1 is linked through an acid-sensitive linker binds the antibody to the antigen overexpressed on the surface of cancer cells in the same way as in the first step in which the second-generation ADC operates in cancer cells, but some are the same as the second-generation ADC It undergoes an intracellular processing step, and a significant part of it can release the drug due to the low pH around the cancer cells.
  • the drug released from the cancer tissue then moves to the inside of the cancer cell by diffusion, does not go through endosomes and lysosomes, and acts directly on the cancer cell without an enzyme (cathepsin B) reaction to induce apoptosis.
  • the antigen selectivity of cancer cells is the same as compared to second-generation ADCs, which can release drugs only by enzymatic reaction, but it is possible to maximize drug release and delivery efficiency into cancer cells by using a pH-sensitive linker. is the main characteristic of
  • the [FL118 drug of Formula 1]-[acid-sensitive linker] of the present invention is such that, upon decomposition of the acid-sensitive linker, the free FL118 drug of Formula 1 is released. It is preferable
  • Blood has a constant pH between 7.3 and 7.4. Therefore, the FL118 drug is not cleaved from the acid-sensitive linker in the blood, and even if cleaved, the release rate of the FL118 drug from the ADC at the neutral pH of the serum is much reduced than in the tumor tissue in an acidic atmosphere.
  • mouse plasma has high levels of esterase activity. activity) and thus easily cleavage of ester and carbonate functional groups, although it may be a problem in the mouse model experiment of Example 12, the FL118 drug-containing ADCs showed weight loss due to normal cell attack upon separate release of FL118 from blood. It was confirmed that there was no side effect (Example 12).
  • CL2A a linker used in Trodelvy, an existing FDA-approved ADC, has (i) storage stability after manufacture, (ii) stability in blood upon administration (little free payload exposure during plasma), and (iii) payload stability in cancer tissue. It is a linker that satisfies all properties such as fast release.
  • the acid-sensitive linker used in the present invention can utilize the CL2A linker that has already been successfully applied to the ADC development of Camptothecin-based compounds through the successful development of Trodelvy and IMMU-130 and the like, and the FL118 drug can be applied to cancer tissues. It can be designed as shown in Formula 2 below to selectively and efficiently deliver. That is, in the present invention, the acid-sensitive linker may be derived from a compound of Formula 2 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;
  • 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 when out of the above range, if there is no significant difference in effect according to the change in linker length, all of them are naturally included within the equivalent scope of the present invention.
  • the acid-sensitive linker of Formula 2 is a customized linker that can be optimized according to the characteristics of the target, payload, and carrier.
  • the FL118 drug of Formula 1 has various linkers and sites for easy attachment for the preparation of carrier-drug conjugates.
  • an alcohol group site of the drug of Formula 1 may be used as an attachment site with a linker.
  • [FL118 drug of Formula 1]-[acid-sensitive linker] may be one in which the alcohol group of the FL118 drug of Formula 1 and the alcohol group of the acid-sensitive linker of Formula 2 are connected.
  • the stability of the obtained ADC was evaluated, and it was confirmed that it can be stored stably at low temperature when stored in the form of a freeze-dried powder or solution that can be re-constitutional.
  • the [FL118 drug of Formula 1]-[acid-sensitive linker] conjugate may be any one selected from the group consisting of compounds represented by Formulas 3 to 5 below.
  • n is each independently an integer from 5 to 9.
  • the acid-sensitive linker of the present invention developed and applied a multivalent linker system that can overcome the problem of low DAR, which is considered a disadvantage of site-specific conjugation, and applied 2-3 payloads to one attachment site.
  • site-specific conjugation it is possible to secure a method for manufacturing high DAR ADC of DAR 4-12.
  • a Bridgeable Linker system as exemplified in Chemical Formulas 4 and 5 may be used as a linker.
  • This bridgeable linker system can increase the efficiency of CMC and simplify the process so that the ADC of DAR 4 can be easily manufactured while maintaining the fast release characteristic of the payload in cancer tissue, which is the greatest advantage of the CL2A linker system.
  • a site-specific antibody-drug complex of DAR 4 can be prepared without performing separate antibody engineering. When disulfide (-SS-) present in the antibody is reduced, two thiols (-SH) are formed, and the thus generated thiols form a 2:1 conjugate with a new bridgeable linker-FL118 compound, By reacting all disulfide of dogs, the antibody-drug complex of DAR 4 can be easily prepared as a single product.
  • 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 description of the ADC and the description of the immunoconjugate may be used interchangeably.
  • drug-linker conjugate refers to a material for the production of an immunoconjugate or carrier-drug conjugate, wherein the antibody, antigen-binding site-containing fragment or carrier is not linked, and the purpose Any antibody, antigen-binding site-containing fragment, or carrier may be used as an immunoconjugate or carrier-drug conjugate as required.
  • an antibody or antigen-binding site-containing fragment thereof which is a component thereof, binds to a target antigen and then releases the drug so that the drug can act on the target cell and/or surrounding cells, so that the target drug As a result, excellent drug efficacy and reduced side effects can be expected.
  • Factors that significantly influence the effectiveness of immunoconjugates are, in particular, (1) drug potency, (2) drug linker stability, and (3) efficient on-target drug release. etc. Since several factors have a complex effect on the effect, it is very difficult to predict the effect of an immunoconjugate, a combination of them, based only on known facts about each factor.
  • Immunoconjugates designed to release a drug after internalization have a problem in that a sufficient concentration of active drug cannot be delivered into the cell when the internalization process is inefficient, and even if a hydrophobic drug is adopted as a cytotoxic drug, It has a disadvantage that it is difficult to expect a -stander cell-killing phenomenon.
  • the immunoconjugate of the present invention is
  • FL118 drug may exhibit cytotoxicity to cancer cells as a topoisomerase I inhibitor, but an example of preparing an immunoconjugate using the drug has not been reported at all to date.
  • the FL118 drug of Formula 1 is (a) a hydrophobic small molecule that can permeate cell membranes and (b) does not leak out of the cells through an efflux pump, so it accumulates rapidly in cancer tissues and can maintain a high concentration for a long time. It is released after killing cells by exerting high concentration and cytotoxicity from the inside, and can continuously move through the cell membrane to the surrounding cells and move into the cell to act.
  • the immunoconjugate of the present invention is targeted to a cancer cell by an antigen-targeting antigen-binding site of the cancer cell, and then some acid-sensitive linkers are degraded mainly in an acidic atmosphere (pH ⁇ 7) around the cancer and/or in blood, Since the mechanism of action of the FL118 drug of 1 is mainly initiated outside the cell, exhibiting the above-described excellent anticancer efficacy and stability is a surprising result that could not be predicted until the results were confirmed by experiments.
  • FL118 drug of Formula 1 is (4S)-4-ethyl-4-hydroxy-8,9-methylenedioxy-1H-pyrano[3',4':6,7]indolizino[1,2- b] Also named quinoline-3,14(4H,12H)-dione (PubChem CID 72403).
  • the immunoconjugate of the present invention has technical characteristics in using the FL118 drug and an acid-sensitive linker. Therefore, the immunoconjugate can be designed by binding any antibody or antigen-binding site-containing fragment thereof according to a desired purpose. All of these are included within the scope of the present invention.
  • an immunoconjugate was prepared by binding trastuzumab, cetuximab and sacituzumab to the FL118-linker conjugate of the present invention, and their excellent anticancer effects were confirmed.
  • the immunoconjugate may have an average drug-to-antibody ratio (DAR) of 2 to 12, preferably 4 to 12 DAR.
  • DAR drug-to-antibody ratio
  • the ADC platform according to the present invention may be designed under the following basic design concept.
  • the FL118 drug-CL2A linker conjugate can be synthesized in two main steps.
  • step 1 the intermediate N 3 -PEG 8 -Lys(MMT)-PABOH is synthesized, and in step 2, it is combined with FL118 to synthesize the final material, FL118-CL2A.
  • step 1 Fmoc-Lys(MMT)-OH and 4-Aminobenzyl alcohol were synthesized by amide coupling using EEDQ, and after removing the protecting group Fmoc, N 3 -PEG 8 -COOH and EEDQ were used. After the reaction proceeds, separation and purification are performed to synthesize an intermediate, N 3 -PEG 8 -Lys(MMT)-PABOH.
  • N 3 -PEG 8 -Lys(MMT)-PABOH and FL118 synthesized previously were synthesized using Triphosgene and DMAP to synthesize N 3 -PEG 8 -Lys(MMT)-PABC-FL118, and binding to the antibody was performed.
  • MCC-alkyne a necessary part, is synthesized by performing a click reaction with the azide of N 3 -PEG 8 -Lys(MMT)-PABOH using CuBr, PPh 3 , and DIPEA. After that, MMT, the amine protecting group of Lysine, is removed to synthesize the final material, FL118 drug-CL2A linker conjugate.
  • FL118 drug - In order to connect an antibody (e.g., Her2 target monoclonal antibody Trastuzumab, EGFR target monoclonal antibody Cetumximab, Trop-2 target antibody sacituzumab) to the CL2A linker conjugate, it is produced by reducing the disulfide bond of the antibody. Conjugation is carried out through the bond between the cysteine residue and the maleimide functional group of the FL118 drug-CL2A linker conjugate.
  • an antibody e.g., Her2 target monoclonal antibody Trastuzumab, EGFR target monoclonal antibody Cetumximab, Trop-2 target antibody sacituzumab
  • reaction buffer 10 or 12 times (DAR 4, 8 ADC, respectively) of FL118-CL2A compared to the antibody is reacted with the reduced antibody.
  • the remaining FL118-CL2A is removed and the buffer is replaced with 18mM MES (pH 6.5).
  • the prepared ADC is sterile filtered using a 0.2 ⁇ m PVDF membrane filter, 0.07mM Polysorbate 80 (PS80) and 20mM Trehalose dehydrate are added, freeze-dried and stored.
  • the target target can be extended not only to cancer cells, but also to infectious disease organisms and/or cells associated with autoimmune diseases.
  • the cell targeted by the antibody or antigen-binding site-containing fragment thereof may be a cancer cell, an infectious disease organism and/or a cell associated with an autoimmune disease.
  • Non-limiting examples of target antigens include antigens selectively distributed on the surface of cancer, such as Her2, FolR, PSMA, and cancer cell overexpression antigens, which are also distributed in a small number in normal tissues, such as Trop2.
  • Cancer cell specific 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,
  • the target antigen may be an antigen distributed 10 times or more in cancer cells compared to normal cells.
  • the present invention utilizes a next-generation ADC platform focusing on the FL118 drug of Formula 1, a new payload that can overcome the shortcomings of existing ADCs, and targets Trop-2, an antigen selectively overexpressed on the surface of solid cancer. It is possible to provide the Best-in-class ADC and the first-in-class ADC targeting EGFR, which is a well-known cancer-related antigen but has not yet been developed due to issues such as safety/efficacy.
  • the immunoconjugate comprising [the FL118 drug of Formula 1]-[acid-sensitive linker]-[antibody or antigen-binding site-containing fragment thereof] is a cancer cell
  • an organic mechanism of action according to the combination use of the above-mentioned [FL118 drug of Formula 1]-[acid-sensitive linker] can be exerted.
  • the free FL118 drug showed cytotoxicity with a low IC 50 value that was 5 to 20 times more potent than SN-38 in various cancer cell lines, and the evaluation results of 140 cell lines originating from various carcinomas It is because they share a very strong anticancer efficacy with an IC 50 of ⁇ 100 nM against the majority of cancer cells in .
  • FL118 drug showed superior efficacy compared to SN-38 in various cancer cell line xenograft models, and excellent safety was secured through GLP-toxicity tests in rats and beagle dogs.
  • the FL118 drug utilizes an inhibitor of Topoisomerase I, an anticancer mechanism whose efficacy/safety has been verified in clinical practice, and Topoisomerase I inhibitors (Irinotecan, Topotecan, etc.) are used in clinical trials for various intractable solid cancers such as colorectal cancer, lung cancer, breast cancer, and ovarian cancer. It has been proven to have excellent anticancer efficacy in
  • the anticancer efficacy of the antibody-FL118 conjugate was verified using antibodies targeting Her2, EGFR, and TROP2, respectively.
  • the term "antibody” refers to a protein molecule serving as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule having immunological reactivity with a specific antigen, polyclonal antibody, monoclonal antibody, Includes 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 having a binding function to FcRn, scaps, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds.
  • the whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond.
  • the whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including 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 having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1 domain), and has one antigen-binding site.
  • Fab' differs from Fab in that it has a hinge region including one or more cysteine residues at the C terminus of the heavy chain CH1 domain.
  • the F(ab') 2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'.
  • Fv variable fragment refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region.
  • dsFv double disulfide Fv
  • scFv single chain Fv
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the whole antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab') 2 fragments), preferably It can be produced through genetic recombination technology.
  • the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry, for example, a salt of an inorganic ion including sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc.
  • inorganic acids such as perhydrochloric acid, phosphoric acid, sulfuric acid, and others 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.
  • salts of organic acids and salts of amino acids such as lysine, arginine, and guanidine.
  • salts of organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyl trimethyl ammonium, and benzethonium, which can be used in pharmaceutical reactions, purification and separation processes.
  • organic ions such as tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium, tetrabutyl ammonium, benzyl trimethyl ammonium, and benzethonium.
  • the types of salts in the present invention are not limited by these listed salts.
  • the [FL118 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 is well By using a carrier with the characteristics of being able to penetrate and easily CMC, it can be well utilized for various applications.
  • the carrier may be an antibody, a repeater body, and/or an aptamer.
  • Aptamers are DNA/RNA-based biopolymers and, like antibodies, have an antigen binding affinity of several nM levels, so they can be used as cancer tissue selective carriers for drugs.
  • Repebody is a new class of immune protein discovered in fish that can recognize antigens like antibodies and nanobodies (single chain antibodies derived from mammals such as llama), and has an antigen binding affinity of several nM to the antigen. Therefore, it can be used as a cancer tissue selective carrier for drugs. In particular, since it has no immunogenicity, it can be used as a drug carrier, and at the same time, it can increase the penetration of cancer cells with a low molecular weight, and it is easy to continuously produce a repeat body of uniform quality, so it is suitable for the manufacture of a drug complex with increased penetration into cancer tissues. .
  • the ADC of the present invention can also serve as a basic therapeutic antibody, which has been playing an important role in the treatment of diseases such as cancer and autoimmunity for the past 10 years.
  • Antibodies have the ability to differentiate between antigens and cells in our body, so they have an excellent selection function that selectively acts on antigens.
  • Antibody therapeutics are the ones that utilize these antigen-selective binding properties of antibodies to treat diseases.
  • Antibodies have a Y-shape, and antigen binding sites are present on each arm, and complementarity determining regions (CDRs) allow the antibody to selectively recognize a target antigen.
  • CDRs complementarity determining regions
  • Antibody therapeutics can remove antigens while selectively responding to antigens.
  • various mechanisms are used, and the characteristics of the representative mechanisms used in antibody therapeutics are as follows.
  • the first mechanism is the blocking function (Blocking).
  • the antibody selectively binds to the target receptor of the cell and blocks the physiological function of the target cell.
  • Antibodies bind to ligands or receptors expressed on the cell surface and block the target signal transduction pathway, causing loss of cell activity, proliferation inhibition, and apoptosis due to reduced signal transduction.
  • the antibody When the antibody binds to the receptor by targeting the receptor on the cell surface, it changes the structure of the receptor or prevents the binding of factors that should bind to the receptor, thereby inhibiting intracellular signal transduction and, through this, cell growth. and differentiation can be controlled. In addition, when the antibody binds to the receptor, the receptor enters the cell by the intracellular influx mechanism, and the number of receptors on the cell surface decreases, thereby regulating the physiological mechanism of the cell.
  • Trastuzumab (Herceptin®), which targets HER2-positive breast cancer, which is one of the Human epidermal growth factor receptors (HER/EGFR/ERBB), is a representative antibody treatment that shows excellent effects through this mechanism.
  • the second mechanism is an antibody-dependent cell-mediated cytotoxicity mechanism (Antibody-Dependent Cellmediated Cytotoxicity, ADCC).
  • Cancer cells have specific receptors or overexpressed receptors that are different from normal cells, and the immune system recognizes them and produces antibodies and binds to the cell surface.
  • NK cells are activated after recognizing the Fc receptor (Fc ⁇ RIII), apoptosis is induced.
  • the third mechanism is complement-dependent cytotoxicity (Complement-Dependent Cytotoxicity, CDC). It binds to the surface of cancer cells and activates the complement protein (C1q), triggering a classical pathway to attack the cell membrane, creating pores and causing target cell lysis. In addition, there are mechanisms such as opsonization, neutralization, and inflammatory response.
  • C1q complement protein
  • Therapeutic antibodies are generally made in an optimized form by re-engineering the antibody from various aspects for a specific therapeutic purpose. And the antibody engineering technology applied to the development of such therapeutic monoclonal antibodies has been extended and applied to the development of optimized ADCs. For example, most of the current therapeutic antibodies or ADCs in clinical development are IgG 1, and only a few are using IgG 2 and IgG 4. Antibody isotype selection is equally important for therapeutic antibodies and ADCs. The reason is that the antibody isotype affects the functions of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Also, IgG 4 becomes functionally monovalent by Fab arm exchange in vivo. In addition, the efficacy of the ADC by isotype and the in vivo efficacy of the ADC can be improved by applying Fc-domain engineering of the antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • 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.
  • the immunoconjugate of the present invention specifically binds to the antigen of cancer cells and exhibits cytotoxicity by releasing drugs inside and outside the cancer cells, and thus can be usefully used for the treatment or prevention of cancer.
  • the anticancer activity of the immunoconjugate of the present invention is as described above.
  • the cancer is one or more cancer-associated survival genes selected from the group consisting of topoisomerase I inhibition, and/or survivin, Mcl-1, XIAP and cIAP2. genes) that can be treated by suppression of genes, and may be solid cancers or blood cancers.
  • pseudomyxoma intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, labial cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell cancer, ovarian cancer Epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla Barter cancer, bladder cancer, peritoneal cancer, Parathyroid cancer, adrenal cancer, nasal sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, juvenile brain cancer, juvenile lymphom
  • a method for treating or preventing cancer comprising administering a therapeutically effective amount of the immunoconjugate to a subject in need thereof.
  • the subject may be a mammal including a human.
  • the term "therapeutically effective amount” refers to the amount of the immunoconjugate effective for the treatment or prevention of cancer. Specifically, “therapeutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and effective dose level includes the subject type and severity, age, sex, type of disease, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field may be determined according to factors.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. and may be administered single or multiple.
  • the active ingredient of the pharmaceutical composition of the present invention is excellent in safety, it can be used even more than 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 manufacture of a drug may be mixed with an acceptable adjuvant, diluent, carrier, etc., and may have a synergistic action of the active ingredients by being prepared as a complex formulation together with other active agents.
  • Step 1 (9H-fluoren-9-yl)methyl(S)-(1-((4-(hydroxy methyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl) Synthesis of amino)-1-oxohexan-2-yl)carbamate
  • Step 2 Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide
  • Step 3 (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido) Synthesis of -N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide
  • Step 4 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn-1-yl)cyclohexane-1 -Synthesis of carboxamide
  • Succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (1.01 g, 3.02 mmol) was dissolved in 20 mL of dichloromethane, propargylamine (0.213 mL, 3.32 mmol) and N,N-diisopropyl Ethylamine (0.528 mL, 3.02 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with 100 mL dichloromethane, washed with 100 mL 1N HCl and then washed with 100 mL brine. The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo.
  • Step 5 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15 , 18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamidobenzyl ((S)-7-ethyl-8,11-dioxo-7,8,11 ,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl ) synthesis of carbonate
  • Step 6 4-((S)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1- carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8 -dioxo-6,12,15,18,21,24,27,30,33-nona oxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8, 11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1 ,2-b] quinolin-7-yl) synthesis of carbonate
  • reaction mixture quickly turned into a light yellow suspension, and stirred for 28 hours.
  • the reaction mixture was then concentrated in vacuo.
  • the crude product was taken up in DMSO and purified by basic preparative HPLC. The product fractions were combined and lyophilized to give 62 mg, 80% of the title compound as a white solid.
  • Step 7 4-((S)-2-(4-aminobutyl)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1- yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21, 24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetra Synthesis of hydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl) carbonate
  • Step 1 Synthesis of tert-butyl ((4-(prop-2-yn-1-ylcarbamoyl)cyclohexyl)methyl)carbamate
  • Step 3 4-((3,4-Dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-N-(prop-2-yn- Synthesis of 1-yl)cyclohexane-1-carboxamide
  • Step 4 (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido) Synthesis of -N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide
  • SC_ACID_M1200 1082 [M+Na] + , 1058 [MH] -
  • Step 5 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15 , 18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8, 11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-7- 1) Synthesis of carbonate
  • Step 6 4-((S)-35-(4-((4-((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) )methyl)cyclohexane-1-carboxamido)-methyl)-1H-1,2,3-triazol-1-yl)-2-(4-(((4-methoxyphenyl)diphenylmethyl) amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S )-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6 Synthesis of ,7]indolizino[1,2-b]quino
  • Step 1 (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl) Synthesis of amino)-1-oxohexan-2-yl)carbamate
  • Step 2 Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide
  • Step 3 (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azadotriacontanamido) Synthesis of -N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide
  • SC_ACID_M1200 1082 [M+Na] + , 1058 [MH] -
  • Step 4 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15 , 18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8, 11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-7- 1) Synthesis of carbonate
  • Step 5 4-((S)-35-amino-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15, 18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11 ,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl ) synthesis of carbonate
  • Step 6 Synthesis of 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoic acid
  • Step 7 4-((S)-39-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)-2 -(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nona oxa-3,9,36-triazanonacontanamido)benzyl ((S)-7-ethyl-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1, 3] Synthesis of dioxolo [4,5-g] pyrano [3 ', 4': 6, 7] indolizino [1,2-b] quinolin-7-yl) carbonate
  • reaction mixture was directly purified by acidic preparative MPLC, the product fractions were combined and lyophilized to give the product as a white solid (120 mg). Analysis confirmed the product as a 4/1 mixture of the title compound and MMT-deprotected compound.
  • Step 1 (9H-fluoren-9-yl)methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl) Synthesis of amino)-1-oxohexan-2-yl)carbamate
  • Step 2 (9H-fluoren-9-yl)methyl ((S)-1-((4-(((((((S)-7-ethyl-8,11-dioxo-7,8,11) ,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-7-yl Synthesis of )oxy)carbonyl)oxy)methyl)phenyl)amino)-6-(((4-methoxyphenyl)diphenylmethyl)amino)-1-oxohexan-2-yl)carbamate
  • Step 3 4-((S)-2-amino-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamido)benzyl ((S)-7-ethyl-8,11-di Oxo-7,8,11,13-tetrahydro-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2- b] quinolin-7-yl) synthesis of carbonate
  • Step 4 Synthesis of DBCO-PEG-Acid
  • Step 5 Synthesis of Dibenzocyclooctyne-PEG-Lys(MMT)-PABC-FL118
  • Step 6 Synthesis of Dibenzocyclooctyne-FEG-Lys-PABC-FL118 (FL118-Linker 4 Conjugate)
  • step 5 The compound obtained in step 5 was dissolved in dichloromethane (0.5 mL) and dried over Na 2 SO 4 overnight. The solution was then transferred to a reaction tube and evaporated to dryness. Dichloromethane (4 mL) in anisole (0.052 mL, 0.472 mmol) and dichloroacetic acid (0.039 mL, 0.472 mmol) was added and the yellow reaction mixture was stirred at room temperature for 30 min. tert -Butylmethylether (15 mL) was added to form a suspension, but the product did not sufficiently precipitate and the solvent was evaporated at room temperature under reduced pressure.
  • Anti-HER2 antibody in original buffer (20 mM histidine, 150 mM NaCl, pH 6.0) was reduced with 5 mM TCEP, and the reaction vial was placed in an incubator shaker at 22 °C at 60 rpm rotation speed for 6 hours. . After reduction, TCEP was removed through a spin desalting column (40 K, 2 mL).
  • DMSO and FL118-Linker 1 compound solution (10 mg/mL stock in DMSO, 12 eq. for antibody) was added to the reduced antibody solution to a final DMSO concentration of 10%. The reaction mixture was mixed properly and the reaction vial was left at 4° C. for 2 hours.
  • the reaction mixture was placed in exchange buffer with 18 mM MES (pH 6.5) through a spin desalting column (40 K, 5 mL).
  • the product was then sterile filtered through a 0.2 ⁇ m PVDF disposable filter.
  • the resulting immunoconjugates were characterized, 0.07 mM PS80 and 20 mM trehalose dehydrate were added, and the resulting immunoconjugates were freeze-dried.
  • the DAR of the immunoconjugate of Example 5 was determined using HIC and MS, and it was confirmed that the average MS-DAR value was 7.14 and the HIC-DAR value was 8.00 (Table 1 and FIG. 5B ).
  • Anti-HER2 antibody in original buffer (20 mM histidine, 150 mM NaCl, pH 6.0) was reduced with 5 mM TCEP and the reaction vial was placed in an incubator shaker at 22 °C at 60 rpm rotation speed for 3 hours. After reduction, TCEP was removed through a spin desalting column (40 K, 2 mL).
  • DMSO and FL118-linker compound solutions (10 mg/mL stock in DMSO, 10 eq. for antibody) were added to the reduced antibody solution to a final DMSO concentration of 10%.
  • the reaction mixture was mixed properly and the reaction vial was left at 4° C. for 2 hours.
  • the reaction mixture was placed in exchange buffer with 18 mM MES (pH 6.5) through a spin desalting column (40 K, 5 mL).
  • the product was then sterile filtered through a 0.2 ⁇ m PVDF disposable filter.
  • the resulting immunoconjugates were characterized, 0.07 mM PS80 and 20 mM trehalose dehydrate were added, and the resulting immunoconjugates were freeze-dried.
  • the DAR of the immunoconjugate of Example 6 was determined using HIC and MS, and the average MS-DAR value was found to be 4.02 (Table 2, and FIG. 5A ).
  • the reaction was purified through an SEC column under phosphate buffer conditions, and it was confirmed that the immunoconjugates of Example 7 were prepared using SDS PAGE, SEC, and HIC.
  • Anti-EGFR antibody (cetuximab) in original buffer (20 mM histidine, 150 mM NaCl, pH 6.0) was reduced with 5 mM TCEP and the reaction vial was placed in an incubator shaker at 22 °C at 60 rpm rotation speed for 3 hours. After reduction, TCEP was removed through a spin desalting column (40 K, 2 mL x 2).
  • DMSO and FL118-linker compound solutions (10 mg/mL stock of DMSO, 12 eq. for antibody) were added to the reduced antibody solution to a final DMSO concentration of 10%.
  • the reaction mixture was mixed properly and the reaction vial was left at 4° C. for 2 hours.
  • the reaction mixture was placed in exchange buffer with 18 mM MES (pH 6.5) through a spin desalting column (40 K, 10 mL).
  • the product was then sterile filtered through a 0.2 ⁇ m PVDF disposable filter.
  • the resulting immunoconjugates were characterized, 0.07 mM PS80 and 20 mM trehalose dehydrate were added, and the resulting immunoconjugates were freeze-dried.
  • the DAR of the immunoconjugate of Example 8 was determined using HIC and MS, and the average MS-DAR value was found to be 8.00 (Table 3).
  • Anti- Trop-2 antibody-linker 1-FL118 conjugate (DAR 8) was prepared (Table 4).
  • Example 10 In vitro ( in vitro ) cytotoxicity and in vivo ( in vivo ) preparation of animal models and materials used to evaluate drug efficacy
  • Example 5 (molecular weight 159 kDa, purity 97%), Example 6 (molecular weight 154 kDa, purity 98%), Example 8 (molecular weight 160 kDa, purity 99%) and Example 9 (molecular weight 160 kDa, purity 99.6%) ), each prepared as a freeze-dried powder stored at -80 °C was used.
  • Trastuzumab (molecular weight 148 kDa, purity 97%) supplied from BCD as a solution stored at -80 ° C was dissolved in a buffer (20 mM Histidine, 150 mM NaCl, pH 6.0) (concentration 10.09 mg/mL).
  • cetuximab (molecular weight 148 kDa, purity 100%) was dissolved in a buffer (20 mM Histidine, 150 mM NaCl, pH 6.0) (concentration 10.47 mg/mL)
  • sacituzumab ( Molecular weight 148 kDa, purity 100%) was used in a state of being dissolved in a buffer.
  • DS8201 (molecular weight 156 kDa, purity 99%) supplied from BCD in a solution state stored at -80°C was dissolved in a buffer (25 mM Histidine, pH 5.5) (concentration 2.87 mg/mL) and used as a control.
  • DS8201 is an immunoconjugate of trastuzumab and deruxtecan, the immunoconjugates of Examples 5 and 6 and the anti-HER2 targeting antibody are the same as trastuzumab, and the cytotoxic drug (payload) deruxtecan is topo As an isomerase I inhibitor, there are similarities in structure and function to FL118 of Example.
  • DS8201 is different from the immunoconjugates of Examples 5 and 6 in the structure and properties of the linker by using a cleavable tetrapeptide-based linker. DS8201 is internalized and then cleaved to release the drug, and the released drug is known to exhibit cytotoxicity to surrounding cancer cells regardless of target expression due to high cell membrane permeability (Cancer Sci 2016; 107: 1039-1046) ). In addition, an excellent effect was recently reported in a phase 2 clinical trial in patients with HER2-positive metastatic breast cancer (N Engl J Med 2020; 382: 610-621), and HER2-positive in 2017 and 2020 from the US FDA, respectively. It has been designated as a breakthrough treatment for relapsed/metastatic breast cancer and HER2-positive relapsed/metastatic gastric cancer.
  • Example 11 In vitro ( in vitro ) cytotoxicity assessment
  • cytotoxicity analysis was performed on the HER2-overexpressing JIMT-1 breast cancer cell line, the EGFR-overexpressing MDA-MB-468 breast cancer cell line or the MDA-MB-468 cell line. It was carried out in the same process as
  • Immunoconjugate-mediated cytotoxicity to JIMT-1 cells or MDA-MB-468 cells was determined by cell viability measurement. On day 0, 6 x 10 3 per well of JIMT-1 in a 96-well plate (Greinier, # 655090) with 50 ⁇ L DMEM (Gibco, # 11995-065) containing 10% FBS (HyClone, SV30087.03) was dispensed. Dog cells (for 3 days culture) or 3 x 10 3 cells per well (for 6 days culture) were plated.
  • Cell viability was detected on day 4 or day 7 using CellTiter-Glo (Promega, # G7573) according to the manufacturer's instructions. After the plate was equilibrated to room temperature for approximately 15 minutes, 50 ⁇ L/well of CellTiter-Glo solution was added. The plate was gently shaken for about 1 minute and then incubated for 10 minutes at room temperature. Luminescence was then read using Envision (PerkinElmer).
  • the cytotoxic effect obtained with each immunoconjugate was calculated by comparing it with the luminescence value obtained from the control well (cell only) by the following formula.
  • Cytotoxicity % 100 x (V cell only -V sample ) / V cell only
  • Example 6 Even in the case of the immunoconjugate of Example 6, when it was added to JIMT-1 cells and cultured for 3 or 6 days, both showed a cytotoxic effect and induced cell death. As in Example 5, the immunoconjugate of Example 6 also exhibited a significantly superior cytotoxic effect than DS8201 (positive control) ( FIG. 7 ).
  • Example 12 In vivo ( in vivo ) efficacy evaluation
  • JIMT-1 breast cancer cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin at 5% CO 2 , 37 ° C., and treated with trypsin-EDTA twice a week in a routine manner. subcultured. Cells growing in exponential growth phase were harvested and counted for tumor inoculation. MDA-MB-468 breast cancer cells were cultured with RPMI containing 10% FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin under the conditions of 5% CO 2 , 37 ° C, and routinely treated with trypsin-EDTA twice a week. It was subcultured in this way. Cells growing in exponential growth phase were harvested and counted for tumor inoculation.
  • JIMT-1 tumor cells (1 x 10 7 ) or MDA-MB-468 cells (1 x 10 7 ) in 0.2 mL of DPBS with Matrigel were inoculated subcutaneously into the right anterior flank of each mouse. Animals were randomly grouped (Tables 5-7) when the mean tumor volume reached about 215 mm 3 , and then treatment for drug efficacy studies was initiated.
  • JIMT-1 process Dosage (mg/kg) route of administration number of horses (n) Group 1 (negative control) saline - I.V. 3 Group 2 (positive control) Trastuzumab 10 I.V. 3 group 3
  • animals were evaluated for normal behaviors such as mobility, food and water consumption (observation only), weight changes (weighed twice a week), eye/hair matting, tumor growth, and effects of treatment. was checked daily. Mortality and observed clinical signs were recorded based on the number of animals in each subset.
  • the main endpoint was when it could be determined whether tumor growth could be delayed or if mice could be treated.
  • TGI Tumor growth inhibition
  • TGI (%) [1 - (T i / T 0 ) / (V i / V 0 )] ⁇ 100
  • T i is the mean tumor volume of the treated group on a given day
  • T 0 is the treatment day treatment group mean tumor volume
  • V i is the average tumor in the vehicle group the mean tumor volume
  • V 0 is treated first day of the day vehicle control, such as T i of volume).
  • Animals with a weight loss of 15% or greater or a tumor volume greater than or equal to 3000 mm 3 were euthanized as humane endpoints.
  • Results are expressed as mean and standard error (mean ⁇ SEM). Data were analyzed using two-way RM ANOVA Dunnett's multiple comparison test using Graphpad Prism 6.0 software and p ⁇ 0.05 was considered statistically significant.
  • MDA-MB-468 process Dosage (mg/kg) route of administration number of horses (n) Group 1 (negative control) saline - I.V. 3 Group 2 (positive control) cetuximab 10 I.V. 3 group 3 Immunoconjugate of Example 8 One I.V. 3 group 4 Immunoconjugate of Example 8 5 I.V. 3 group 5 Immunoconjugate of Example 8 10 I.V. 3
  • mouse plasma has a high level of esterase activity and can easily cleave acid-sensitive bonds such as ester or carbonate functional groups. It was predicted that side effects such as reduction would occur, but surprisingly, no such side effects were observed. From the above results, it was found that the immunoconjugate of the present invention did not show toxicity to normal cells even when FL118 was separated and released from the blood.
  • Tumor size (mm 3 ) (day 0) Tumor size (mm 3 ) (day 21) TGI (%) (day 21) significance a group 1 217.07 ⁇ 2.79 1336.32 ⁇ 361.41 NA NA group 2 216.90 ⁇ 5.63 671.06 ⁇ 92.85 59.41 NS group 3 216.88 ⁇ 6.84 370.14 ⁇ 71.86 86.31 ** group 4 216.71 ⁇ 10.87 102.20 ⁇ 18.12 110.23 *** group 5 216.32 ⁇ 12.67 73.87 ⁇ 12.01 112.73 ***
  • Tumor size (mm 3 ) (day 0) Tumor size (mm 3 ) (day 21) TGI (%) (day 21) significance a group 1 214.67 ⁇ 37.46 390.24 ⁇ 45.45 NA NA group 2 215.45 ⁇ 17.45 363.07 ⁇ 44.38 15.92 NS group 3 212.05 ⁇ 28.35 361.79 ⁇ 28.44 14.71 NS group 4 212.77 ⁇ 16.17 24.59 ⁇ 12.8 207.18 *** group 5 212.28 ⁇ 42.78 7.56 ⁇ 7.56 216.60 ****
  • Tumor size (mm 3 ) (day 0) Tumor size (mm 3 ) (day 21) TGI (%) (day 21) significance a group 1 214.67 ⁇ 37.46 390.24 ⁇ 45.45 NA NA group 2 215.45 ⁇ 17.45 363.07 ⁇ 44.38 15.92 NS group 3 212.05 ⁇ 28.35 361.79 ⁇ 28.44 14.71 NS group 4 212.77 ⁇ 16.17 24.59 ⁇ 12.8 207.18 *** group 5 212.28 ⁇ 42.78 7.56 ⁇ 7.56 216.60 ****
  • Tumor size (mm 3 ) (day 0) Tumor size (mm 3 ) (day 21) TGI (%) (day 21) significance a group 1 200.81 ⁇ 27.71 613.76 ⁇ 125.11 NA NA group 2 200.20 ⁇ 30.90 433.08 ⁇ 126.82 43.60 not significant group 3 200.86 ⁇ 22.49 129.35 ⁇ 14.70 117.32 **** group 4 200.66 ⁇ 28.55 234.34 ⁇ 64.31 91.85 **** group 5 200.11 ⁇ 26.13 77.45 ⁇ 1.46 129.70 **** group 6 200.69 ⁇ 36.42 74.61 ⁇ 11.55 130.53 ****
  • results of tumor growth curves of JIMT-1 or MDA-MB-468 tumor model mice are shown in FIGS. 8, 10 and 12, respectively.
  • the mean tumor size of group 1, the saline control group was 1336.32 ⁇ 361.41 mm 3 and 390.24 ⁇ 45.45 mm 3 at 21 days after treatment, respectively.
  • Example 5 had a mean tumor of 370.14 ⁇ 71.86, 102.20 ⁇ 18.12 and 73.87 ⁇ 12.01 mm 3 at doses of 1 mg/kg, 5 mg/kg and 10 mg/kg (groups 3, 4 and 5), respectively.
  • the immunoconjugate of Example 8 was administered at doses of 5 mg/kg and 10 mg/kg (groups 4 and 5), respectively , with an average tumor volume of 24.59 ⁇ 12.8 and 7.56 ⁇ 7.56 mm 3
  • the immunoconjugate of Example 9 was administered At the doses of 5 mg/kg and 7 mg/kg (groups 5 and 6), it was confirmed that the average tumor volume of 77.45 ⁇ 1.46 and 74.61 ⁇ 11.55 mm 3 , respectively, exhibited a remarkably excellent antitumor effect (the immunoconjugate of Example 5).
  • the FL118-linker conjugate of the present invention can form an immunoconjugate by binding to an antibody or antigen-binding fragment thereof against a desired antigen.
  • the immunoconjugates of Examples 5 and 6 prepared in an embodiment of the present invention contain the same antibody (trastuzumab) as DS8201, a known immunoconjugate, and contain a drug (payload) similar in structure and function in vitro. It was confirmed that it showed overwhelmingly superior cytotoxicity to cancer cells compared to DS8201.
  • the immunoconjugate of Example 8 containing cetuximab prepared as an example of the present invention was also confirmed to exhibit strong cytotoxicity in vitro. It was also confirmed that the immunoconjugate of Example 9 containing sacituzumab prepared according to an embodiment of the present invention exhibits strong cytotoxicity in vitro.
  • trastuzumab , cetuximab and sasituzumab also showed some anticancer effects, but it was confirmed that the immunoconjugate of the present invention showed significantly excellent anticancer activity at all administered doses and did not exhibit side effects such as weight loss.
  • the immunoconjugate of the present invention can be very usefully used for the prevention or treatment of cancer, and the FL118-linker conjugate of the present invention can be very usefully used for the purpose of preparing the immunoconjugate.

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PCT/KR2021/009204 2020-07-16 2021-07-16 Fl118 약물이 산 민감성 링커에 연결된 접합체 및 이를 이용한 면역접합체 Ceased WO2022015110A1 (ko)

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JP2023503070A JP7660186B2 (ja) 2020-07-16 2021-07-16 Ｆｌ１１８薬物が酸感受性リンカーに結合された複合体及びそれを用いた免疫複合体
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WO2024014837A1 (ko) * 2022-07-11 2024-01-18 주식회사 피노바이오 Ddx5 단백질에 결합하는 캄토테신 유도체 및 이의 프로드럭
WO2024049220A1 (ko) 2022-08-30 2024-03-07 주식회사 피노바이오 캄토테신계 약물이, 항원 결합 친화도가 낮은 항체에 링커를 통해 연결된 항체-약물 접합체
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US20230270867A1 (en) 2023-08-31
CA3189462A1 (en) 2022-01-20
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AU2021308834A1 (en) 2023-02-16
KR20220009928A (ko) 2022-01-25
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EP4183418A4 (en) 2025-02-12
EP4183418A1 (en) 2023-05-24

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