WO2023098691A1 - 抗体药物偶联物及其用途 - Google Patents

抗体药物偶联物及其用途 Download PDF

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WO2023098691A1
WO2023098691A1 PCT/CN2022/135216 CN2022135216W WO2023098691A1 WO 2023098691 A1 WO2023098691 A1 WO 2023098691A1 CN 2022135216 W CN2022135216 W CN 2022135216W WO 2023098691 A1 WO2023098691 A1 WO 2023098691A1
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ch2ch2
dxd
ch2ch2o
ch2ch2ch2
antibody
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PCT/CN2022/135216
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English (en)
French (fr)
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瞿爱东
梁红远
沈文妍
祝婧烨
邱建华
周旭
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上海生物制品研究所有限责任公司
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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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present application relates to the field of biomedicine, in particular to a linker and an antibody drug conjugate (antibody-drug conjugate, ADC) comprising it.
  • ADC antibody-drug conjugate
  • ADC antibody drug conjugates
  • ADC is generally composed of three parts: antibody, linker, and drug, in which the antibody is connected to the drug with a linker.
  • the mechanism of action of the antibody-drug complex is to first recognize the specific or overexpressed antigen on the tumor cell through the high recognition ability of the antibody to the corresponding antigen and bind to it.
  • the linked Cytotoxic drug molecules are also simultaneously brought into tumor cells.
  • the antibody is decomposed or the linker is broken to release the cytotoxic drug molecules, and then kill the target tumor cells to achieve selective poisoning.
  • Linkers used in ADCs need to meet several requirements: when circulating in human plasma, linkers need to be stable to prevent early release of the drug; are cleaved to release the drug, whereas for non-cleavable linkers, the drug moiety is released in an active form comprising the drug, linker, and amino acid residues derived from the protease-degrading ligand.
  • cleavable antibody-drug conjugates Since the main mechanism of action of cleavable antibody-drug conjugates is to use antibodies to target specific cell surface antigens, after binding, they enter the endoplasmic reticulum system by endocytosis, and are cleaved by proteases in endosomes or lysosomes. After that, small molecule drugs are released to achieve the purpose of targeting and killing tumor cells. For solid tumors, due to the heterogeneity of tumors, some tumors do not express or low-express target antigens, so small molecules with membrane-penetrating activity can kill nearby tumor cells after being released, which is called bystander. org effect.
  • This protease belongs to the cysteine protease hydrolase family. Homologous with papain (trypsin), the optimum pH is acidic. Since this enzyme participates in the dissolution of the extracellular matrix, thereby affecting the invasion and metastasis of tumors, it is found to be highly expressed in various tumor cells, such as gastric cancer, lung cancer, colon cancer, breast cancer, prostate cancer, and kidney cancer. Therefore, when ADC drugs enter tumor cells, they can be rapidly cleaved by a large amount of cathepsin B in the cells, and small molecules are quickly released to kill cells.
  • Non-optimal properties may lead to reduced ADC potency, insufficient immunological specificity of the conjugate, and increased toxicity due to non-specific release of drug from the conjugate. Therefore, there is a need for new linker technologies and conjugates that can be used in targeted therapies.
  • the application provides a linker, which includes a self-degrading spacer, that is, an amino acid unit, and the amino acid unit includes a substrate component GFG preferred by Cathepsin B, and a VA sequence is added at the N-terminal of the GFG fragment to enhance the cleavage activity with Cathepsin B Regional combination, while considering that the length of the polypeptide affects the activity of the enzyme on the substrate, one S and two S can be added between VA and GF to form three polypeptide sequences, namely VAGFG, VASGFG, and VASSGFG. Through the optimization of the polypeptide sequence, ADC can be achieved.
  • the purpose of drugs is to release small molecule drugs more efficiently in tumor cells.
  • the present application also provides linker drugs, conjugates and uses thereof comprising the aforementioned linkers.
  • the present application provides a linker, the linker includes an amino acid unit (RL), the RL includes: -Val-Ala-Q-Gly-Phe-Gly-, wherein Q is a bond, a single amino acid or two peptide.
  • RL amino acid unit
  • RL can be cleaved by cathepsin B (Cathepsin B).
  • RL comprises -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser- Ser-Gly-Phe-Gly-(VASSGFG).
  • the linker is selected from the following structures: -Z-A-S*-RL- and -Z-A-S*-RL-Y-; wherein, the Z is an extender unit, and the A is a bond or a linker unit ; The S* is a bond or a distribution agent; Y is a spacer subunit.
  • the -(succinimide-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure indicates the attachment position with the ligand unit, and the wavy line at the N atom at position 1 indicates the attachment position with A.
  • the A is optionally substituted by a basic unit (BU), and the basic unit is -(CH 2 ) x NH 2 , -(CH 2 ) x NHR a or -(CH 2 ) x NR a 2 ; wherein x is any integer of 1-4; and each R a is independently selected from C 1- C 6 alkyl and C 1- C 6 haloalkyl, or two R a groups are attached to them
  • the nitrogens combine to form a 4- to 6-membered heterocycloalkyl ring, or an azetidinyl, pyrrolidinyl or piperidinyl group.
  • linker can comprise the following structure:
  • a is any integer from 1 to 8.
  • the Lb represents -CR 2 (-R 3 )- or a single bond, wherein R 2 and R 3 each independently represent H, -NH 2 , C 1 to C 6 alkylene, C 1 to C 6 alkylene -amino, C 1 -C 6 alkylene-carboxyl, or C 1 -C 6 alkylene-hydroxyl, and R 2 and R 3 are not -NH 2 at the same time;
  • n 1 represents an integer from 0 to 6;
  • said linker is -Z-A-S*-RL- or -Z-A-S*-RL-Y-.
  • said S* comprises a PEG unit.
  • the S* has the formula:
  • the left side of S* is connected to A, and the right side is connected to RL, and b is any integer from 2 to 20.
  • the linker is selected from the following structures:
  • the -(succinimidyl-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure represents the attachment position with the ligand unit, and the wavy line on the N atom at position 1 represents the attachment position with A;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG).
  • the linker drug is selected from the following structures:
  • the present application provides a linker medicine, which comprises the linker described in the present application.
  • the linker drug is selected from the following structures: Z'-A-S*-RL-D and Z'-A-S*-RL-Y-D; wherein, the Z' is the front of the extender unit body, the A is a bond or a linker unit; the S* is a bond or a distributing agent; Y is a spacer unit; D is a drug unit.
  • the Drug Unit includes a cytotoxic agent.
  • the drug unit includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicin ( calicheamicins), camptothecins, cemadotins, colchicines, colchicines, colcimids, combretastatins, cryptophycins, pumice sponges Discodermolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, eleutherobins ), epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate , netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (pyrrolo[2,1-c][1,4]benzodi-azepines; PBDs), puromycins, rhizoxins,
  • the pharmaceutical unit comprises a DNA topoisomerase I inhibitor.
  • the drug unit comprises camptothecin or a derivative thereof.
  • the drug unit has the following structure:
  • the nitrogen atom of the amino group at the 1-position is the group at the attachment position.
  • linker drug unit has the following structure:
  • the (maleimide-N-yl)- has the following structure: wherein the nitrogen atom is the group at the attachment site;
  • the (pyrrolidin-2,5-dione-N-yl)- has the following structure: wherein the nitrogen atom is the group at the attachment site;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG);
  • the Dxd has the following structure: Wherein the nitrogen atom of the amino group at position 1 is the group at the attachment position;
  • Said M represents a halogen atom.
  • linker drug unit is selected from the following structures:
  • the present application provides a conjugate comprising the linker described in the present application.
  • the conjugate comprises a ligand.
  • the ligand targets a cell surface receptor or a tumor-associated antigen.
  • the cell surface receptors or tumor-associated antigens include: HER2, HER3, claudin18.2, folate receptor alpha (FR ⁇ ), BCMA, PSMA, TROP-2, CD19, CD20, CD22, CD30, CD79b, EGFR, c-Met, or CEACAM5.
  • the ligand comprises an antibody or antigen-binding fragment thereof.
  • the antibodies include monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies, intact antibodies, antibody fragments, human antibodies, humanized antibodies, or chimeric antibodies.
  • said antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the antibody is a monoclonal antibody.
  • the antibody is a human antibody, a humanized antibody or a chimeric antibody.
  • the conjugate includes a ligand-drug conjugate and has the following structure: C-(LD) m , wherein the C represents a ligand unit, L represents a linker, and D represents Drug unit, m represents any number from 1 to 10.
  • the ligand drug conjugate is an antibody drug conjugate (ADC), and has the following structure: Ab-(LD) m , wherein the Ab represents an antibody or an antigen-binding fragment thereof, L represents a linker, D represents a drug unit, and m represents any number from 1 to 10.
  • ADC antibody drug conjugate
  • the Drug Unit includes a cytotoxic agent.
  • the drug unit includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicin ( calicheamicins), camptothecins, cemadotins, colchicines, colchicines, colcimids, combretastatins, cryptophycins, pumice sponges Discodermolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, eleutherobins ), epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate , netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (pyrrolo[2,1-c][1,4]benzodi-azepines; PBDs), puromycins, rhizoxins,
  • the pharmaceutical unit comprises a DNA topoisomerase I inhibitor.
  • the drug unit comprises camptothecin or a derivative thereof.
  • the drug unit has the following structure:
  • the nitrogen atom of the amino group at the 1-position is the group at the attachment position.
  • antibody drug conjugate is selected from the following structures:
  • the -(succinimidyl-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure represents the attachment position with the ligand unit, and the wavy line on the N atom at position 1 represents the attachment position with A;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG);
  • the Dxd has the following structure: Wherein the nitrogen atom of the amino group at position 1 is the group at the attachment position;
  • the Ab represents an antibody or an antigen-binding fragment thereof, and m represents any number from 1 to 10.
  • antibody drug conjugate is selected from the following structures:
  • the Ab represents an antibody or an antigen-binding fragment thereof, and m is any number from 1 to 10.
  • the antibodies or antigen-binding fragments thereof include: anti-HER2 antibodies, anti-HER3 antibodies, anti-claudin18.2 antibodies, anti-FR ⁇ antibodies or antigen-binding fragments thereof.
  • the present application provides a pharmaceutical composition, which comprises the conjugate described in the present application and optionally a pharmaceutically acceptable carrier.
  • the present application provides the use of the conjugate described in the present application or the pharmaceutical composition described in the present application in the preparation of a medicament for treating a disease or a disease.
  • the disease or condition comprises a tumor
  • the disease or condition includes solid tumors and hematological tumors.
  • the solid tumor includes: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney, tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer carcinoma, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer leukemia, kidney, tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer carcinoma, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small cell lung
  • said hematological tumor comprises: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell Lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B cell Lymphoma
  • Hodgkin's lymphoma or a combination thereof.
  • the present application provides a method for treating a disease or condition of a subject in need thereof, comprising administering to the subject an effective amount of the conjugate described in the present application.
  • the disease or condition comprises a tumor
  • the disease or condition includes solid tumors and hematological tumors.
  • the solid tumor includes: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, leukemia, kidney, tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer carcinoma, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer peritoneal metastasis of gastric cancer
  • liver cancer leukemia, kidney, tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer carcinoma, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small cell lung
  • said hematological tumor comprises: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B cell Lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse large B cell Lymphoma
  • Hodgkin's lymphoma or a combination thereof.
  • Figure 1A shows the HIC-HPLC chromatogram of HER3-ADC04 described in Example 1 of the present application.
  • Figure 1B shows the HIC-HPLC chromatogram of HER3-ADC07 described in Example 1 of the present application.
  • Figure 1C shows the HIC-HPLC chromatogram of HER3-ADC08 described in Example 1 of the present application.
  • Figure 1D shows the HIC-HPLC chromatogram of HER3-ADC09 described in Example 1 of the present application.
  • Figure 1E shows the HIC-HPLC chromatogram of Claudin18.2-ADC08 described in Example 1 of the present application.
  • Figure 1F shows the HIC-HPLC chromatogram of Claudin18.2-ADC09 described in Example 1 of the present application.
  • Figure 1G shows the HIC-HPLC chromatogram of FR ⁇ -ADC08 described in Example 1 of the present application.
  • Figure 1H shows the HIC-HPLC chromatogram of FR ⁇ -ADC09 described in Example 1 of the present application.
  • Figure 2A shows the in vitro killing effect of HER3-ADC04 described in the present application on human colon cancer cells SW620;
  • Figure 2B shows the killing effect of different linker-Dxd drugs described in the present application on human colon cancer cells SW620 in vitro;
  • Figure 3 shows the killing effect of anti-HER3 ADC containing different linkers described in this application on human colon cancer cells SW620 in vitro
  • Figure 4 shows the in vitro killing effect of anti-HER3 ADC containing different linkers on non-target cells CHO-K1;
  • Figure 5 shows the in vitro killing effect of the anti-HER3 ADC described in this application on SW620 target cells after plasma incubation
  • Figure 6 shows the therapeutic effect of the anti-HER3 ADC described in this application on the SW620 transplanted tumor model
  • Figure 7 shows the changes in body weight of mice in the SW620 transplanted tumor model described in the present application during anti-HER3 ADC treatment
  • Figure 8 shows the in vitro killing effect of anti-Claudin18.2 ADC described in the present application on human gastric cancer cell NUGC4;
  • Figure 9 shows the in vitro killing effect of the anti-Claudin18.2 ADC described in the present application on the human gastric cancer cell NUGC4 overexpressing the recombinant Claudin18.2 molecule;
  • Figure 10 shows the in vitro killing effect of the anti-Claudin18.2 ADC described in the present application on HEK293 cells overexpressing recombinant Claudin18.2 molecules;
  • Figure 11 shows the in vitro killing effect of anti-Claudin18.2 ADC described in the present application on HEK293 cells overexpressing Claudin18.2 molecules after plasma incubation;
  • Figure 12 shows the growth inhibitory effect of the anti-claudin18.2 ADC described in the present application on the NUGC4 gastric cancer tumor transplantation model
  • Figure 13 shows the in vitro killing effect of the anti-FR ⁇ ADC described in the present application on human ovarian cancer cell OVCAR3;
  • Figure 14 shows the in vitro killing effect of anti-FR ⁇ ADC described in this application on anti-FR ⁇ ADC after plasma incubation
  • Figure 15 shows the growth inhibitory effect of the anti-FR ⁇ ADC described in the present application on the subcutaneously transplanted tumor model of OVCAR3 mice.
  • the linker L can include a cleavable moiety between the antibody or antigen-binding fragment and the Drug unit. Exemplary cleavable linkers are described and exemplified herein.
  • an “antibody” is generally used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibody), and antibody fragments, as long as they show the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861).
  • Antibodies can be murine, human, humanized, chimeric, or derived from other species.
  • an “antibody” may typically comprise a protein of at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • each light chain comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL comprises three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • variable domains of native heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), mostly in a ⁇ -sheet configuration, connected by three CDRs, Loop links are formed and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are in close proximity by the FR regions and, together with the CDRs from the other chain, form the antigen-binding site of the antibody.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • an antigen-binding fragment or “functional fragment of an antibody” (also referred to herein as “targeting moiety” or “antigen-binding portion”) generally refers to a portion of an antibody molecule that contains the Amino acids that specifically bind to antigens.
  • the portion of an antigen that is specifically recognized and bound by an antibody is called an "epitope" as described above.
  • An antigen-binding fragment can typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it need not comprise both.
  • antigen-binding fragments of antibodies include: (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab') 2 fragments, having Bivalent fragment of two Fab fragments connected by disulfide bridge; (3) Fd fragment with two VH and CH1 domains; (4) Fv fragment with VL and VH domains of antibody single arm, (5) dAb Fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which is hereby incorporated by reference in its entirety), which has a VH domain (6) Isolated Complementarity Determining Regions (CDRs); (7) Single-chain Fv (scFv), for example derived from a scFv-library.
  • CDRs Isolated Complementarity Determining Regions
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows it to be produced as a single protein in which the VL and VH regions pair to form a monovalent molecule chain (termed single-chain Fv (scFv)) (see, e.g., Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc.
  • scFv single-chain Fv
  • VHH relates to variable antigens from heavy chain antibodies of the family Camelidae (camel, dromedary, llama, alpaca, etc.) Binding domain (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92 , 389-407). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments can be assessed in the same manner as intact antibodies.
  • variable generally refers to the fact that certain parts of the sequence of the variable domains of antibodies vary strongly, which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of variable domains are called the frameworks (FRs).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • the CDRs of antibodies can be defined by various methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Besse Star, MD (1991)), Chothia definition rules based on the location of structural ring regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on the IMGT ontology (IMGT-ONTOLOGY) and KABAT definition rules for IMGT Scientific chart rules.
  • Kabat definition rules based on sequence variability see, Kabat et al., Protein Sequences in Immunology, 5th edition, National Institutes of Health, Besse Star, MD (1991)
  • Chothia definition rules based on the location of structural ring regions see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on the IMGT ontology (IM
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for minor natural mutations that may be present.
  • Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants.
  • monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • monoclonal denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody”), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parental (e.g., mouse-derived) antibody. Less likely to trigger an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen.
  • a humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigen specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region.
  • donor antibody such as mouse, rat
  • rabbit or non-human primate residue substitution in the CDR region such as mouse, rat
  • FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.
  • Fully human antibody or “human antibody” generally refers to the antibody that is expressed by transferring the gene encoding the human antibody into a genetically engineered animal lacking the antibody gene. All portions of the antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • binding when referring to the interaction of a binding molecule (such as an antibody) with its binding partner (such as an antigen) generally means that the interaction depends on a specific structure on the binding partner (such as an antigen). determinant or epitope).
  • an antibody will preferentially bind or recognize a binding partner even when the binding partner is present in a mixture of other molecules or organisms. Binding can be mediated by covalent or non-covalent interactions or a combination of both.
  • specifically binds generally means immunospecifically binding to an antigenic determinant or epitope and not immunospecifically binding to other antigenic determinants or epitopes.
  • a binding molecule that immunospecifically binds an antigen may bind other peptides or polypeptides with lower affinity, as determined by, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIACORE, or other assays known in the art. Determination. A binding molecule or fragment thereof that immunospecifically binds an antigen may cross-react with a related antigen bearing the same epitope. In certain instances, a binding molecule or fragment thereof that immunospecifically binds an antigen does not cross-react with other antigens.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • BIACORE enzyme-linked immunosorbent assay
  • linker generally refer to any chemical compound capable of covalently joining a compound (typically a drug unit) to another moiety such as an antibody or antigen-binding fragment. part. Linkers may be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage under conditions that render the compound or antibody active.
  • the term "drug unit” or “D” generally refers to any compound with the desired biological activity and reactive functional groups, which can be used to incorporate drugs into the conjugates of the present application.
  • the drug unit represents a cytotoxic drug for cancer treatment; a protein or polypeptide with desired biological activity, such as a toxin such as abrin, ricin A, pseudomonas exotoxin, and diphtheria toxin; other suitable proteins, including tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, and biological response modifiers, such as: Lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleuk
  • cytotoxic agent generally refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu); chemotherapy agents or drugs (eg, methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, phentermine Ammonium mustard, daunomycin or other intercalating agents; growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, vegetable or animal origin , including fragment
  • pharmaceutically acceptable form generally refers to the form of the disclosed compound, including but not limited to its pharmaceutically acceptable salt, ester, hydrate, solvate, polymorph, isomorph conformers, prodrugs and isotopically labeled derivatives.
  • pharmaceutically acceptable form includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs and isotopically labeled derivatives thereof.
  • pharmaceutically acceptable form includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives thereof.
  • linkers, linker drugs and conjugates of the present application may include any pharmaceutically acceptable form thereof, such as pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, pro Drugs or isotope-labeled derivatives.
  • pharmaceutically acceptable forms are also within the scope of the present application.
  • the term “pharmaceutically acceptable salt” generally refers to a pharmaceutically acceptable organic or inorganic salt of a compound (eg, drug, linker drug or conjugate).
  • the compounds may contain at least one amino group, and thus may form acid addition salts with amino groups.
  • Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid Phosphate, Isonicotinate, Lactate, Salicylate, Citrate, Tartrate, Oleate, Tannate, Pantothenate, Bitartrate, Ascorbate, Succinate, Maleic Acid Salt, gentisate, fumarate, gluconate, glucuronate, sucrose salt, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzene Sulfonate, p-toluenesulfonate, and pamoate (ie, 1,1'-methylenebis-(2-hydroxy-3-naphthoate)).
  • a pharmaceutically acceptable salt may involve the incorporation of another molecule such as acetate, succinate or other counterion.
  • a counterion can be any organic or inorganic moiety that will stabilize the charge on the parent compound.
  • a pharmaceutically acceptable salt can have more than one charged atom in its structure. Instances in which multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.
  • derivative generally refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions.
  • prodrug generally refers to a biologically less or inactive compound that is converted in vivo to a more biologically active compound via a chemical or biological process (i.e., chemical reaction or enzymatic biotransformation) .
  • isotopically labeled compound or “isotopically labeled derivative” generally refers to a compound wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature
  • isotopes that may be incorporated into the presently disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • the term "pharmaceutically acceptable carrier” generally refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations will generally contain salts, buffers, preservatives, compatible carriers, adjuvants and, optionally, other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also generally contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for human administration.
  • pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol and ethanol.
  • Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in these carriers.
  • alkyl or “alkyl group” generally refers to a fully saturated straight, branched or cyclic hydrocarbon chain.
  • an alkyl group may contain 1-8 carbon atoms (“C 1 -C 8 alkyl”).
  • an alkyl group may contain 1-6 carbon atoms (“C 1 -C 6 alkyl”).
  • alkyl groups contain 1-3 carbon atoms.
  • the alkyl group contains 2-3 carbon atoms, and in still other embodiments, the alkyl group contains 1-2 carbon atoms.
  • alkylalkoxy means an alkyl group substituted with an alkoxy group.
  • alkoxy refers to an alkyl group as previously defined attached to the main carbon chain via an oxygen (“alkoxy”) atom.
  • alkylhydroxyl means an alkyl group substituted with a hydroxyl group.
  • hydroxy or hydroxyl refers to -OH.
  • a carbocyclic group contains 3-10 carbon atoms ("3-10 membered carbocycle”). In certain embodiments, carbocyclic groups contain 3-8 carbon atoms ("3-8 membered carbocycles"). In certain embodiments, a carbocyclic group contains 3-6 carbon atoms ("3-6 membered carbocycle”). In certain embodiments, a carbocyclic group contains 3-5 carbon atoms ("3-5 membered carbocycle”).
  • haloalkyl refers to an alkyl group substituted with one or more halogen atoms.
  • halogen refers to any halogen group, such as -F, -Cl, -Br or -I.
  • heterocycle herein, the term “heterocycle (heterocyclic or heterocyclic)", “heterocyclyl” means a monocyclic heterocycle, a bicyclic heterocycle or a tricyclic heterocycle containing at least one heteroatom in the ring.
  • PEG unit is generally an organic moiety consisting of repeating ethylene-oxyl subunits (PEG or PEG subunits) and may be polydisperse, monodisperse, or discrete (i.e., with discrete number of ethylene-oxyl subunits).
  • Polydisperse PEGs are a heterogeneous mixture of sizes and molecular weights, while monodisperse PEGs are usually purified from heterogeneous mixtures and thus have a single chain length and molecular weight.
  • the PEG units of the present application may comprise one or more polyethylene glycol chains, each polyethylene glycol chain consisting of one or more ethyleneoxy subunits covalently attached to each other.
  • the polyethylene glycol chains may be linked together, for example, in a linear, branched or star configuration.
  • substituted whether preceded by the term “optionally” or not, generally means that one or more hydrogens of the designated moiety are replaced by a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be replaced by more than one When a substituent is substituted, the substituent at each position may be the same or different.
  • Combinations of substituents envisioned in accordance with the present disclosure are preferably those combinations of substituents that result in the formation of stable or chemically feasible compounds.
  • substituted or “substituted” or “absent” includes the following implicit provisos: the substitution or absence is based on the permissible valences of the substituted atoms and substituents, and the substitution or absence Stable compounds are produced, eg, that do not undergo transformations spontaneously, such as by rearrangement, cyclization, elimination, and the like.
  • a heteroatom such as nitrogen may have a hydrogen substituent and/or any permissible substituent of an organic compound described herein that satisfies the valence of the heteroatom.
  • prevention and/or treatment includes not only preventing and/or treating a disease, but also generally preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing down one or more symptoms associated with a disease onset, reduction and/or alleviation of one or more symptoms associated with the disease, reduction of the severity and/or duration of the disease and/or any symptoms associated therewith and/or prevention of the disease and/or any symptoms associated therewith prevent, reduce or reverse any physiological impairment caused by the disease, and generally any pharmacological effect that is beneficial to the patient being treated.
  • the compositions of the present application need not achieve a complete cure or eradicate any symptom or manifestation of a disease to form a viable therapeutic agent.
  • drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic agent.
  • a treatment administered prophylactically need not be fully effective in preventing the onset of the disorder to constitute a viable prophylactic. Simply reducing the effects of disease in a subject (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing disease occurrence or The possibility of deterioration is enough.
  • administration generally refers to the delivery of proteins, including immunoglobulins, to a human or animal in need thereof by any route known in the art.
  • Pharmaceutically acceptable carriers and formulations or compositions are also well known in the art. Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral or mucosal.
  • the term “contacting” generally means that two or more substances of different types are brought into contact together in any order, in any manner, and for any length of time.
  • the term “contacting” generally refers to the means by which an antibody or antigen-binding fragment thereof, drug, linker drug, conjugate, and/or pharmaceutical composition of the present application is delivered to or placed in direct proximity to a target cell methods, the delivery may be in vitro or in vivo and may involve the use of recombinant vector systems.
  • Contact can occur in vivo, ex vivo or in vitro.
  • the term “effective amount” or “effective dose” generally refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is typically one that, when used alone or in combination with another therapeutic agent, promotes regression of disease (this is achieved by a reduction in the severity of symptoms of the disease, frequency of asymptomatic periods of the disease) any amount of drug that is evidenced by an increase in the degree and duration of the disease, or by the prevention of impairment or disability due to the presence of a disease.
  • a “prophylactically effective amount” or “prophylactically effective dose” of a drug generally refers to the amount of the drug that, alone or in combination with another therapeutic agent, inhibits the development or recurrence of the disease when administered to a subject at risk of disease development or disease recurrence .
  • the ability of a therapeutic or prophylactic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems Efficacy in humans is predicted, or by assaying the activity of the agent in an in vitro assay.
  • tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • the term "subject” generally refers to a human or non-human animal (including mammals), such as a human, a non-human primate (ape, or , gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits , guinea pig).
  • Human subjects include fetal, neonatal, infant, adolescent and adult subjects.
  • Subjects can include animal disease models.
  • the term “about” generally means approximately, in the region of, roughly, or around.
  • a cut-off or a specific value is used to indicate that the stated value may vary by as much as 10% from the recited value.
  • the term “about” may be used to encompass a variation of ⁇ 10% or less, ⁇ 5% or less, ⁇ 1% or less, ⁇ 0.5% or less, or ⁇ 0.5% or less from the specified value. 0.1% or less variation.
  • the application provides a novel linker (conjugating linker) structure, which includes amino acid units (RL), the RL includes: -Val-Ala-Q-Gly-Phe-Gly-, where Q is a bond, a single amino acid or dipeptides.
  • RL amino acid units
  • RL can be cleaved by cathepsin B (Cathepsin B).
  • RL comprises -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser- Ser-Gly-Phe-Gly-(VASSGFG).
  • the linker is selected from the following structures: -Z-A-S*-RL- and -Z-A-S*-RL-Y-; wherein, the Z is an extender unit, and the A is a bond or a linker unit ; The S* is a bond or a distribution agent; Y is a spacer subunit.
  • the -(succinimide-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure indicates the attachment position with the ligand unit, and the wavy line at the N atom at position 1 indicates the attachment position with A.
  • the A is optionally substituted by a basic unit (BU), and the basic unit is -(CH 2 ) x NH 2 , -(CH 2 ) x NHR a or -(CH 2 ) x NR a 2 ; wherein x is any integer of 1-4; and each R a is independently selected from C 1- C 6 alkyl and C 1- C 6 haloalkyl, or two R a groups are attached to them
  • the nitrogens combine to form a 4- to 6-membered heterocycloalkyl ring, or an azetidinyl, pyrrolidinyl or piperidinyl group.
  • said Z may be -(succinimide-3-yl-N)-
  • said ZA Can contain the following structures:
  • a is any integer from 1 to 8.
  • the a may be any integer from 2 to 5.
  • Z-A- could have the following structure:
  • Aryl represents a divalent aromatic hydrocarbon group
  • cyc.Het represents a divalent cyclic heterocyclic group
  • the Lb represents -CR 2 (-R 3 )- or a single bond, wherein R 2 and R 3 each independently represent H, -NH 2 , C 1 to C 6 alkylene, C 1 to C 6 alkylene - amino, C 1 -C 6 alkylene - carboxyl, or C 1 -C 6 alkylene - hydroxyl, and R 2 and R 3 are not - NH 2 at the same time;
  • n 1 represents an integer from 0 to 6;
  • the spacer can be a functional group that will facilitate the attachment of the RL to the Drug unit, or it can provide an additional structural component to further facilitate the release of the Drug unit from the rest of the conjugate (e.g., self-digesting p-aminobenzyl (PAB ) component).
  • PAB self-digesting p-aminobenzyl
  • Y can contain the following structures:
  • said S* is a distributing agent
  • said linker is -Z-A-S*-RL- or -Z-A-S*-RL-Y-.
  • the partitioning agent moiety can be used, for example, to mask the hydrophobicity of the Drug Unit or other Linked Unit components.
  • Exemplary dispensing agents may include polyethylene glycol (PEG) units, cyclodextrin units, polyamides, hydrophilic peptides, polysaccharides, and dendrimers.
  • PEG polyethylene glycol
  • cyclodextrin units polyamides
  • hydrophilic peptides polysaccharides
  • dendrimers may be organized as "in line” components or It exists as a side chain or branched component.
  • said S* comprises a PEG unit.
  • the S* has the formula:
  • b is any integer from 2 to 20.
  • b can be 2, 4, 8 or 12.
  • subscript b is 2.
  • subscript b is 4.
  • subscript b is 8.
  • subscript b is 12.
  • the linker can be selected from the following structures:
  • the -(succinimidyl-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure represents the attachment position with the ligand unit, and the wavy line on the N atom at position 1 represents the attachment position with A;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG).
  • the linker can have the following structure:
  • the present application provides a linker medicine, which comprises the linker described in the present application.
  • the linker drug is selected from the following structures: Z'-A-S*-RL-D and Z'-A-S*-RL-Y-D; wherein, the Z' is the precursor of the extender unit,
  • the A is a bond or a linker unit;
  • the S* is a bond or a distributing agent;
  • Y is a spacer unit;
  • D is a drug unit.
  • the Drug Unit includes a cytotoxic agent.
  • the drug unit includes: amanitins, anthracyclines, auristatins, baccatins, calicheamicin ( calicheamicins), camptothecins, cemadotins, colchicines, colchicines, colcimids, combretastatins, cryptophycins, pumice sponges Discodermolides, duocarmycins, docetaxel, doxorubicin, duocarmycins, echinomycins, eleutherobins ), epothilones, estramustines, lexitropsins, maytansines, maytansinoids, methotrexate , netropsins, pyrrolo[2,1-c][1,4]benzodiazepines (pyrrolo[2,1-c][1,4]benzodi-azepines; PBDs), puromycins, rhizoxins,
  • the pharmaceutical unit comprises a DNA topoisomerase I inhibitor.
  • the drug unit comprises camptothecin or a derivative thereof.
  • the drug unit has the following structure:
  • the nitrogen atom of the amino group at the 1-position is the group at the attachment position.
  • linker drug unit has the following structure:
  • the (maleimide-N-yl)- has the following structure: wherein the nitrogen atom is the group at the attachment site;
  • the (pyrrolidin-2,5-dione-N-yl)- has the following structure: wherein the nitrogen atom is the group at the attachment site;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG);
  • the Dxd has the following structure: Wherein the nitrogen atom of the amino group at position 1 is the group at the attachment position;
  • Said M represents a halogen atom.
  • linker drug unit is selected from the following structures:
  • a Ligand unit is present.
  • the Ligand unit (C) is a targeting agent that specifically binds to the target moiety.
  • the Ligand unit specifically and selectively binds to a cellular component (cell-binding agent) or other target molecule of interest.
  • the role of the Ligand unit is to target and present the Drug unit (such as Dxd) to interact with the Ligand unit due to the presence of its target component or molecule and to allow free drug to be subsequently inside the target cell (i.e., intracellular) or nearby (ie, extracellular) release of specific target cell populations.
  • Ligand unit C includes, but is not limited to, proteins, polypeptides and peptides.
  • Suitable Ligand units include, for example, antibodies, such as full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules such as but not limited to transferrin, or any Other cell-binding molecules or substances.
  • Ligand unit (C) is an antibody or non-antibody protein targeting agent.
  • the Ligand unit is bonded to an L (Linker unit) comprising a releasable peptide linker.
  • L Linker unit
  • other linking components may also be present in the conjugates described herein to provide additional space between the Drug unit and the Ligand unit (e.g., the Stretcher unit and optionally the Linker unit A) or to provide Composition properties to increase solubility (eg, partitioning agent S*).
  • the Ligand unit is bonded to Z of the Linker unit via a heteroatom of the Ligand unit.
  • Heteroatoms that may be present on the Ligand unit for this bonding include sulfur (in one embodiment, from the sulfhydryl group of the targeting ligand), oxygen (in one embodiment, from the carboxyl group of the targeting ligand), or hydroxyl groups) and nitrogen, which is optionally substituted (in one embodiment, from a primary or secondary amine function of the targeting ligand, or in another embodiment, from an optionally substituted amide nitrogen).
  • These heteroatoms can be present on the targeting ligand in its native state (eg, in a naturally occurring antibody), or can be introduced into the targeting ligand via chemical modification or bioengineering.
  • the Ligand unit has a thiol functional group such that the Ligand unit is bonded to the Linker unit via the sulfur atom of the thiol functional group.
  • the Ligand unit has one or more lysine residues capable of an activated ester with the Extender unit precursor of the camptothecin-Linker compound intermediate (such esters include, but are not limited to N-hydroxysuccinimide, pentafluorophenyl and p-nitrophenyl ester) react and thus provide an amide bond consisting of the nitrogen atom of the Ligand unit and the C ⁇ O group of the Extender unit of the Linker unit.
  • esters include, but are not limited to N-hydroxysuccinimide, pentafluorophenyl and p-nitrophenyl ester
  • the Ligand unit is capable of forming a covalent bond between the Extender unit (Z) and the Ligand unit corresponding to the targeting ligand by interacting with a reactive functional group on the Extender unit precursor (Z').
  • the functional group of Z' having the ability to interact with the targeting ligand will depend on the nature of the Ligand unit.
  • the reactive group is a maleimide, which is present on the Stretcher unit (ie, the maleimide moiety of the Stretcher unit precursor) prior to attachment to form the Ligand unit.
  • Covalent attachment of the Ligand unit to the Extender unit is achieved through the interaction of the sulfhydryl functionality of the Ligand unit with the maleimide functionality of Z' to form a sulfur-substituted succinimide.
  • the thiol functional group may be present on the Ligand Unit in its native state (eg, in a naturally occurring residue) or may be introduced into the Ligand Unit via chemical modification or by bioengineering.
  • the Ligand unit is an antibody and the sulfhydryl groups are generated by reduction of interchain disulfides of the antibody. Accordingly, in some embodiments, the Linker unit is conjugated to a cysteine residue from a reduced interchain disulfide.
  • the Ligand unit is an antibody, and the sulfhydryl functional group is chemically introduced into the antibody, eg, through the introduction of cysteine residues.
  • the Linker unit (with or without an attached Drug unit) is conjugated to the Ligand unit through an introduced cysteine residue of the Ligand unit.
  • the ligand targets a cell surface receptor or a tumor-associated antigen.
  • the cell surface receptors or tumor-associated antigens may include HER2, HER3, claudin18.2, folate receptor alpha (FR ⁇ ), BCMA, PSMA, TROP-2, CD19, CD20, CD22, CD30, CD79b, EGFR, c-Met, or CEACAM5.
  • ADC Antibody Drug Conjugate
  • an antibody-drug conjugate which has the following structure: Ab-(LD) m , wherein the Ab represents an antibody or an antigen-binding fragment thereof, L represents a linker, and D represents a drug unit, m represents any number from 1 to 10.
  • the antibodies include monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies, intact antibodies, antibody fragments, human antibodies, humanized antibodies, or chimeric antibodies.
  • said antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the antibody is a monoclonal antibody.
  • the antibody is a human antibody, a humanized antibody or a chimeric antibody.
  • the Drug Unit includes a cytotoxic agent.
  • the drug unit can be a microtubule-interfering drug, such as auristatin, such as monomethyl auristatin E (monomethyl auristatin E; MMAE), monomethyl auristatin F (monomethyl auristatin F; MMAF), and auristatin Auristatin F (auristatin F; AF).
  • auristatin such as monomethyl auristatin E (monomethyl auristatin E; MMAE), monomethyl auristatin F (monomethyl auristatin F; MMAF), and auristatin Auristatin F (auristatin F; AF).
  • the drug unit can be a microtubule disrupting drug, such as a maytansinoid, such as DM1, DM3, and DM4.
  • the drug unit can be a DNA damaging agent such as calicheamicins, duocarmycins, SN-38, and pyrrolo[2,1-c][1,4]benzene Pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs).
  • a DNA damaging agent such as calicheamicins, duocarmycins, SN-38, and pyrrolo[2,1-c][1,4]benzene Pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs).
  • the drug unit can be amanitins, anthracyclines, baccatins, camptothecins, cemadotins, colchicines ), colcimids, combretastatins, cryptophycins, discodermolides, docetaxel, doxorubicin, echinomycin ( echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate , netropsins, puromycins, rhizoxins, taxanes, tubulysins, or vinca alkaloids.
  • doxorubicin echinomycin ( echinomycins, eleutherobins, epothilones, estramustines, lexitropsins, maytansines, methotrexate , netropsins, puromycins, rhizox
  • the drug unit is a topoisomerase-I inhibitor, such as a camptothecin derivative DXd.
  • antibody drug conjugate is selected from the following structures:
  • the -(succinimidyl-3-yl-N)- has the following structure: Wherein the wavy line at position 3 of the structure represents the attachment position with the ligand unit, and the wavy line on the N atom at position 1 represents the attachment position with A;
  • the -RL- represents the following structure: -Val-Ala-Gly-Phe-Gly-(VAGFG), -Val-Ala-Ser-Gly-Phe-Gly-(VASGFG), or -Val-Ala-Ser-Ser -Gly-Phe-Gly-(VASSGFG);
  • the Dxd has the following structure: Wherein the nitrogen atom of the amino group at position 1 is the group at the attachment position;
  • the Ab represents an antibody or an antigen-binding fragment thereof, and m represents any number from 1 to 10.
  • antibody drug conjugate is selected from the following structures:
  • the Ab represents an antibody or an antigen-binding fragment thereof, and m is any number from 1 to 10.
  • the antibodies or antigen-binding fragments thereof include: anti-HER2 antibodies, anti-HER3 antibodies, anti-claudin18.2 antibodies, anti-FR ⁇ antibodies or antigen-binding fragments thereof.
  • Antibody drug conjugate (ADC) compounds of the present disclosure include ADC compounds having anticancer activity.
  • ADC compounds include antibodies or antigen-binding fragments (including antigen-binding fragments thereof) conjugated (i.e., covalently linked via a linker) to a Drug Unit (such as a cytotoxic agent), e.g., wherein the cytotoxic agent is not bound to the antibody or antigen Cytotoxic or cytostatic when bound to the fragment.
  • the ADC compounds of the present application can selectively deliver effective doses of cytotoxic or cytostatic agents to cancer cells or tumor tissues. It has been found that the disclosed ADCs are directed against genes expressing respective target antigens (e.g. HER2, HER3, claudin18.2, folate receptor alpha (FR ⁇ ), CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1 , KIT, MET, MUC16, SLC39A6, SLC44A4 or STEAP1) cells have potent cytotoxic and/or cytostatic activity. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC is dependent on expression of the target antigen in the cell.
  • target antigens e.g. HER2, HER3, claudin18.2, folate receptor alpha (FR ⁇ ), CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR
  • the disclosed ADCs are particularly effective at killing cancer cells expressing target antigens while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and/or cytostatic effects on cancer cells that do not express the target antigen.
  • Examples of types of cancer to which the anti-HER3 antibody-drug conjugate of the present invention can be applied include lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, pleomorphic glucocorticoid Gymoma, ovarian cancer, pancreatic cancer, breast cancer, metastatic breast cancer, luminal breast cancer, melanoma, liver cancer, bladder cancer, gastric (stomach) cancer , gastrointestinal stromal tumor, cervical cancer, head and neck cancer, esophageal cancer, epidermoid cancer, peritoneal cancer, adult glioblastoma multiforme, hepatic cancer, hepatocellular carcinoma Carcinoma, colon cancer, rectal cancer, colon and rectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, hepatic cancer , anal cancer, penile cancer.
  • the treatment using the anti-HER3 antibody-drug conjugate of the present invention can target the cancer cells expressing the HER3 protein recognizable by the antibody of the antibody-drug conjugate in the cancer cells of the treatment subject.
  • a cancer expressing a HER3 protein refers to a cancer containing cells having a HER3 protein on its surface or a cancer secreting a HER3 protein into blood.
  • Examples of types of cancer to which the anti-claudin18.2 antibody-drug conjugate of the present invention can be applied include gastrointestinal tract cancer, pancreatic cancer, esophageal cancer, non-small cell lung cancer, liver cancer, ovarian cancer, lung cancer, gallbladder cancer, or head and neck cancer .
  • Examples of types of cancer to which the anti-FR ⁇ antibody-drug conjugate of the present invention can be applied include ovarian cancer, breast cancer, lung cancer, colorectal cancer, or kidney cancer.
  • cancer types to which the anti-HER2 antibody-drug conjugates of the present invention are applicable include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., Uterine Serous Endometrial Carcinoma), Salivary Duct Carcinoma, Cervical Cancer, Endometrial Cancer, and Ovarian Cancer (English et al. (2013) Mol Diagn Ther. [Molecular Diagnosis and Therapy] 17:85-99).
  • lung cancer e.g., lung adenocarcinoma
  • uterine cancer e.g., Uterine Serous Endometrial Carcinoma
  • Salivary Duct Carcinoma e.g., Cervical Cancer, Endometrial Cancer, and Ovarian Cancer
  • antibodies may target cell surface receptors or tumor-associated antigens.
  • antibodies can include full-length antibodies or antibody fragments that bind, reactively associate, or complex with a receptor, antigen, or other target cell-associated portion.
  • An antibody can be any protein, protein-like molecule, or polypeptide that binds, complexes, or reacts with a portion of a cell population for which therapeutic modification is sought.
  • the antibody can be a chimeric antibody or a functionally active fragment thereof, a humanized antibody or a functionally active fragment thereof, a human antibody or a functionally active fragment thereof.
  • an antibody can also be a functionally active fragment, derivative or analog of an antibody, wherein the antibody can immunospecifically bind to a target antigen (e.g., cancer antigen, viral antigen, microbial antigen, or other antigen bound to cells or matrix).
  • a target antigen e.g., cancer antigen, viral antigen, microbial antigen, or other antigen bound to cells or matrix.
  • functional activity means that the fragment, derivative or analog can recognize the same antigen and recognize antibodies derived from the fragment, derivative or analog of the antigen.
  • Other useful antibodies include fragments of antibodies such as, but not limited to: F(ab')2 fragments, Fab fragments, Fab', Fv fragments and heavy and light chain dimers of antibodies, or Fv fragments or single chain antibodies ( scFv).
  • the antibody can be a fusion protein of an antibody, or a functionally active fragment thereof.
  • Antibodies of the present application may include, but are not limited to, antibodies against the following antigens (exemplary cancers are indicated in parentheses): CA125 (ovarian cancer), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (cancer ), Lewis Y (cancer), Lewis X (cancer), alpha-fetoprotein (cancer), CA 242 (colorectal cancer), placental alkaline phosphatase (cancer), prostate cancer-specific membrane antigen (prostate cancer), Prostatic Acid Phosphatase (Prostate Cancer), Epidermal Growth Factor (Cancer), MAGE-1 (Cancer), MAGE-2 (Cancer), MAGE-3 (Cancer), MAGE-4 (Cancer), Anti-transferrin Receptor (cancer), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal cancer), gp100 (melanoma), MART1 (melanoma), prostate-specific antibody (PSA) (prostate cancer
  • Antibodies of the present application may also include analogs and derivatives that are modified or unmodified (ie, by covalent attachment of any molecule), so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity.
  • analogs and derivatives of antibodies including further modifications such as: glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by conventional protecting/blocking groups, proteases Cleavage, linking to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be accomplished using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, and the like. Additionally, an analog or derivative may include one or more unnatural amino acids. In some embodiments, antibodies may have modifications (eg, substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors.
  • the antibody or antigen-binding fragment thereof comprises: an anti-HER2 antibody, an anti-claudin18.2 antibody, an anti-FR ⁇ antibody, an anti-HER3 antibody, or an antigen-binding fragment thereof.
  • the antibody is an anti-HER3 antibody
  • the anti-HER3 antibody comprises VH
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • said anti-HER3 antibody comprises VH
  • said VH comprises the amino acid sequence shown in SEQ ID NO: 15.
  • the anti-HER3 antibody comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region may comprise a constant region derived from human IgG.
  • the antibody heavy chain constant region may include a constant region derived from human IgG1, IgG2, IgG3 or IgG4.
  • said anti-HER3 antibody comprises an antibody heavy chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO:17.
  • said anti-HER3 antibody comprises VL
  • said VL comprises LCDR1, LCDR2 and LCDR3
  • said LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4
  • said LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 5
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6.
  • said anti-HER3 antibody comprises VH and VL
  • said VH comprises HCDR1, HCDR2 and HCDR3
  • said VL comprises LCDR1, LCDR2 and LCDR3
  • said HCDR1 comprises SEQ ID NO: 1
  • the amino acid sequence shown, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:2
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:4
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.
  • the anti-HER3 antibody comprises a VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the anti-HER3 antibody comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:15, and the VL comprises the amino acid sequence shown in SEQ ID NO:16.
  • the anti-HER3 antibody comprises an antibody light chain constant region.
  • the antibody light chain constant region may comprise a human Ig ⁇ constant region or a human Ig ⁇ constant region.
  • said anti-HER3 antibody comprises an antibody light chain
  • said antibody light chain comprises the amino acid sequence described in SEQ ID NO: 18.
  • said anti-HER3 antibody comprises an antibody heavy chain and an antibody light chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO: 17
  • said antibody light chain comprises SEQ ID NO: The amino acid sequence described in 18.
  • the antibody is an anti-Claudin18.2 antibody
  • the anti-Claudin18.2 antibody comprises VH
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises SEQ ID NO:19
  • the amino acid sequence of the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:21.
  • said anti-Claudin18.2 antibody comprises VH, wherein said VH comprises the amino acid sequence shown in SEQ ID NO:32.
  • the anti-Claudin18.2 antibody comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region may comprise a constant region derived from human IgG.
  • the antibody heavy chain constant region may include a constant region derived from human IgG1, IgG2, IgG3 or IgG4.
  • said anti-Claudin18.2 antibody comprises an antibody heavy chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO:34.
  • said anti-Claudin18.2 antibody comprises VL
  • said VL comprises LCDR1, LCDR2 and LCDR3
  • said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:22
  • said LCDR2 comprises SEQ ID The amino acid sequence shown in NO:23
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • said anti-Claudin18.2 antibody comprises VH and VL, wherein said VH comprises HCDR1, HCDR2 and HCDR3, said VL comprises LCDR1, LCDR2 and LCDR3; wherein said HCDR1 comprises SEQ ID NO: The amino acid sequence shown in 19, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:20, the HCDR3 includes the amino acid sequence shown in SEQ ID NO:21, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO:22 Sequence, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:23, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the anti-Claudin18.2 antibody comprises a VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:33.
  • the anti-Claudin18.2 antibody comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:33 .
  • the anti-Claudin18.2 antibody comprises an antibody light chain constant region.
  • the antibody light chain constant region may comprise a human Ig ⁇ constant region or a human Ig ⁇ constant region.
  • said anti-Claudin18.2 antibody comprises an antibody light chain
  • said antibody light chain comprises the amino acid sequence described in SEQ ID NO:35.
  • said anti-Claudin18.2 antibody comprises an antibody heavy chain and an antibody light chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO: 34
  • said antibody light chain comprises SEQ ID The amino acid sequence described in NO:35.
  • the antibody is an anti-FR ⁇ antibody
  • the anti-FRA antibody comprises VH
  • the VH comprises HCDR1, HCDR2 and HCDR3
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:37
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:38.
  • the anti-FR ⁇ antibody comprises VH
  • the VH comprises the amino acid sequence shown in SEQ ID NO:49.
  • the anti-FR ⁇ antibody comprises an antibody heavy chain constant region.
  • the antibody heavy chain constant region may comprise a constant region derived from human IgG.
  • the antibody heavy chain constant region may include a constant region derived from human IgG1, IgG2, IgG3 or IgG4.
  • said anti-FR ⁇ antibody comprises an antibody heavy chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO:51.
  • said anti-FR ⁇ antibody comprises VL
  • said VL comprises LCDR1, LCDR2 and LCDR3
  • said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:39
  • said LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 40
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:41.
  • said anti-FR ⁇ antibody comprises VH and VL
  • said VH comprises HCDR1, HCDR2 and HCDR3
  • said VL comprises LCDR1, LCDR2 and LCDR3
  • said HCDR1 comprises SEQ ID NO:36
  • the amino acid sequence shown the HCDR2 includes the amino acid sequence shown in SEQ ID NO:37
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:38
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:39
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:40
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:41.
  • the anti-FR ⁇ antibody comprises a VL
  • the VL comprises the amino acid sequence shown in SEQ ID NO:50.
  • the anti-FR ⁇ antibody comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:49, and the VL comprises the amino acid sequence shown in SEQ ID NO:50.
  • the anti-FR ⁇ antibody comprises an antibody light chain constant region.
  • the antibody light chain constant region may comprise a human Ig ⁇ constant region or a human Ig ⁇ constant region.
  • said anti-FR ⁇ antibody comprises an antibody light chain
  • said antibody light chain comprises the amino acid sequence described in SEQ ID NO:52.
  • said anti-FR ⁇ antibody comprises an antibody heavy chain and an antibody light chain
  • said antibody heavy chain comprises the amino acid sequence described in SEQ ID NO:51
  • said antibody light chain comprises SEQ ID NO: The amino acid sequence described in 52.
  • the present application provides a pharmaceutical composition, which comprises the conjugate described in the present application and optionally a pharmaceutically acceptable carrier.
  • carrier generally refers to a diluent, adjuvant, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil.
  • the carrier can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea.
  • adjuvants, stabilizers, thickeners, lubricants and colorants may be used.
  • the compound or composition and the pharmaceutically acceptable carrier are sterile when administered to a patient.
  • the conjugates of the present application may be in any form that allows the compound (drug unit) to be administered to a patient for the treatment of a disorder associated with the expression of the antigen to which the Ligand unit binds.
  • the conjugate can be in liquid or solid form.
  • An exemplary route of administration is parenteral. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the composition is administered parenterally.
  • the conjugate is administered intravenously. Administration may be by any convenient route, for example by infusion or bolus injection.
  • compositions may be nontoxic in the amounts used.
  • optimal dosages of one or more active ingredients in pharmaceutical compositions will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (eg, human), the particular form of the compound, the mode of administration, and the composition employed.
  • the composition may, for example, be in liquid form. Liquids are available for delivery by injection. In the composition for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may also be included. Water is an exemplary carrier when the composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk , Glycerin, Propylene, Ethylene Glycol, Water, Ethanol.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk , Glycerin, Propylene, Ethylene Glycol, Water, Ethanol.
  • the compositions of the present invention can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can be formulated such that the compound is bioavailable after administration of the composition to a patient.
  • Compositions may be in the form of one or more dosage units.
  • the composition may comprise from about 0.01 to about 100 mg of conjugate per kg body weight of the subject.
  • the dosage may be even lower, such as 1.0 ⁇ g/kg to 5.0 mg/kg, 4.0 mg/kg, 3.0 mg/kg, 2.0 mg/kg or 1.0 mg/kg, or 1.0 ⁇ g/kg to 500.0 ⁇ g/kg subject body weight.
  • the dosage of the conjugate administered to a patient is generally about 0.01 mg/kg to about 100 mg/kg of the subject's body weight, or 1.0 ⁇ g/kg to 5.0 mg/kg of the subject's body weight. In some embodiments, the dose administered to a patient is between about 0.01 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dose administered to a patient is between about 0.1 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dose administered to a patient is between about 0.1 mg/kg to about 20 mg/kg of the subject's body weight. In some embodiments, the dose administered is between about 0.1 mg/kg to about 5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's body weight.
  • the present application provides the use of the conjugate described in the present application or the pharmaceutical composition described in the present application in the preparation of a medicament for treating a disease or a disease.
  • the present application provides a method for treating a disease or condition of a subject in need thereof, comprising administering to the subject an effective amount of the conjugate described in the present application.
  • the disease or condition comprises a tumor
  • hematopoietic cancers such as, for example, lymphomas (Hodgkin's and non-Hodgkin's) as well as leukemias and solid tumors.
  • lymphomas Hodgkin's and non-Hodgkin's
  • leukemias and solid tumors examples include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophagus cancer, stomach cancer, oral cancer, nose cancer, throat cancer, squamous cell Carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver
  • the tumor comprises a tumor with abnormal expression of HER3.
  • the tumor comprises a tumor that highly expresses HER3.
  • the tumor comprises a tumor with abnormal expression of claudin18.2.
  • the tumor comprises a tumor that highly expresses claudin18.2.
  • the tumor comprises a tumor with abnormal expression of FRa.
  • the tumor comprises a tumor that highly expresses FRa.
  • linker-drug solution (04-Dxd, 07-Dxd, 08-Dxd, 09-Dxd, wherein the amino acid unit in 04-Dxd is replaced by GGFG)
  • Linker-drug solution (04-Dxd, 07-Dxd, 08-Dxd, 09-Dxd, wherein the amino acid unit in 04-Dxd is replaced by GGFG)
  • GGFG linker-drug solution
  • HIC-HPLC Use the HIC-HPLC method to detect, carry out sample loading and elution under the condition of pH 7.0 mobile phase, and detect the absorption peak area at the specified wavelength.
  • the specific test method is shown in Table 3, and the DAR of the ADC product is between 7-8.
  • Human colon cancer tumor cells SW620 in the logarithmic growth phase were digested with trypsin, resuspended with the corresponding medium, adjusted to a density of 2 ⁇ 104/ml, and plated on a 96-well microplate at 100 ⁇ l/well, so that each The number of cells in the well was 2 ⁇ 10 3 , and 200 ⁇ l of medium was added to each well of the rest.
  • the linker-small molecule drugs (04-Dxd, 07-Dxd, 08-Dxd, 09-Dxd), HER3ADC Drugs (HER3-ADC04, HER3-ADC07, HER3-ADC08, HER3-ADC09), the monoclonal antibody control was diluted, and the initial concentration was 400nM, and the 2-fold gradient dilution was started, and there were 9 concentration gradients in total; the diluted sample was added, 100 ⁇ l /hole.
  • Figure 2A shows that the HER3 monoclonal antibody conjugated with Dxd has an obvious killing effect on SW620 target cells compared with naked antibody (RS-HT-002).
  • Figure 2B shows that different linkers-Dxd have killing effects on human colon cancer cell SW620, and the killing effects of 07-Dxd, 08-Dxd, and 09-Dxd are significantly better than 04-Dxd.
  • Figure 3 shows that anti-HER3 ADCs containing different linkers have killing effects on human colon cancer cells SW620, and the killing effects of HER3-ADC07, HER3-ADC08, and HER3-ADC09 are significantly better than HER3-ADC04.
  • Figure 4 shows that anti-HER3 ADCs containing different linkers have no obvious killing effect on non-target cells CHO-K1.
  • this study used human plasma incubation in vitro to simulate the in vivo environment for detection. Specifically, HER3 ADC was added to 10% human plasma so that the final antibody concentration was 1 mg/ml, incubated at 37° C. for 3 days and 7 days, and then the cell killing assay was performed. Digest the SW620 target cells in the logarithmic growth phase and plate them at a certain density, culture at 37°C, 5% CO 2 for 48 hours, then add ADC incubated with gradiently diluted plasma, and use the unincubated ADC with the same plasma dilution as a control for the experiment .
  • Figure 5 shows that after HER3-ADC08 and HER3-ADC09 were incubated in plasma for 3 days or 7 days, no significant change was found in the killing activity of the cells.
  • the small molecule control group was given the same molar amount of 10 mg/kg ADC drugs, and the frequency of administration was once a week, of which 6, 10 mg/kg The 2 mg/kg group was given four times of administration, and the 2mg/kg group was given eight times of administration by intraperitoneal injection.
  • the tumor was measured twice a week, and the tumor volume and mouse body weight data were recorded before each administration.
  • Figure 6 shows that HER3-ADC07, HER3-ADC08, and HER3-ADC09 can significantly inhibit the growth of SW620 transplanted tumors.
  • the mouse tumors in the 6 and 10mg/kg administration groups all disappeared completely after four administrations, and individual mouse tumors began to grow three weeks after the administration was stopped.
  • the 2mg/kg administration group can inhibit tumor growth, and HER3-ADC08 has a consistent effect on tumors than HER3-ADC07 and HER3-ADC09.
  • Figure 7 shows that during the treatment, there was no significant difference in the body weight change of the mice in the ADC treatment group compared with the PBS control group.
  • HEK293-hCLDN18.2, NUGC4, and NUGC4-hCLDN18.2 in the logarithmic growth phase were digested with trypsin, resuspended with the corresponding medium, adjusted to a density of 2 ⁇ 10 4 /ml, and carried out at 100 ⁇ l/well for 96
  • the wells were plated in a microwell plate so that the number of cells per well was 2 ⁇ 10 3 , and 200 ⁇ l of medium was added to each well of the rest.
  • the supernatant in the cell culture plate was aspirated, and the linker-small molecule drug (08-Dxd, 09-Dxd), ADC drug (Claudin18.2-ADC08, Claudin18.2-ADC09), the monoclonal antibody control (hu782) were diluted, and the initial concentration was 400nM, and a 2-fold gradient dilution was started, with a total of 9 concentration gradients; the diluted sample was added, 100 ⁇ l/well.
  • the linker-small molecule drug (08-Dxd, 09-Dxd)
  • ADC drug Claudin18.2-ADC08, Claudin18.2-ADC09
  • the monoclonal antibody control hu782
  • this study used human plasma incubation in vitro to simulate the in vivo environment for detection. Specifically, Claudin18.2-ADC08 was added to 10% human plasma so that the final antibody concentration was 1 mg/ml, incubated at 37°C for 3 days and 7 days, and then the cell killing assay was performed. HEK293-hCLDN18.2 target cells in the logarithmic growth phase were digested and plated according to a certain density, cultured at 37°C and 5% CO 2 for 48 hours, and then diluted plasma was added to incubate Claudin18.2-ADC08. Claudin18.2-ADC08 was incubated as a control for experiments.
  • the small molecule control group was administered in the same molar amount of 10 mg/kg ADC drug, and the dosage of Claudin18.2-ADC08 was administered in three dose groups of 1 mg/kg, 3 mg/kg, and 10 mg/kg, and the administration frequency was once a week, for a total of The drug was given four times, and the measurement was continued after the drug was stopped, twice a week, the intraperitoneal injection was administered, and the tumor volume and mouse body weight data were recorded before each administration.
  • Figure 12 shows that Claudin18.2-ADC08 has anti-tumor growth activity in the subcutaneously transplanted tumor model of NUGC4 mice, and has a certain dose effect.
  • Ovary cancer OVCAR3 cells in the logarithmic growth phase were digested with trypsin, resuspended in the corresponding medium, adjusted to a density of 2 ⁇ 10 4 /ml, and plated on a 96-well microplate at 100 ⁇ l/well, so that each well The number of cells is 2 ⁇ 10 3 , and 200 ⁇ l medium is added to each well of the rest.
  • the supernatant in the cell culture plate was aspirated, and the linker-small molecule drug (08-Dxd, 09-Dxd), ADC drug (FR ⁇ -ADC08, FR ⁇ - ADC09), the monoclonal antibody control (M9346A) was diluted, and the initial concentration was 400nM, and a 2-fold gradient dilution was started, with a total of 9 concentration gradients; the diluted sample was added, 100 ⁇ l/well.
  • Figure 13 shows that FR ⁇ antibody conjugates FR ⁇ -ADC08 and FR ⁇ -ADC09 have significant killing activity on OVCAR3 cells.
  • this study used human plasma incubation in vitro to simulate the in vivo environment for detection. Specifically, FR ⁇ -ADC08 was added to 50% human plasma so that the final antibody concentration was 1 mg/ml, incubated at 37°C for 3 days and 7 days, and then the cell killing assay was performed. Digest the OVCAR3 target cells in the logarithmic growth phase and plate them at a certain density. After culturing at 37°C and 5% CO for 48 hours, add serially diluted plasma to incubate FR ⁇ -ADC08, and use the same plasma dilution of unincubated FR ⁇ -ADC08 as a control conduct experiment.
  • Figure 14 shows that after FR ⁇ -ADC08 was incubated in plasma for 3 days or 7 days, no significant change was found in the cell killing activity.
  • OVCAR3 cells in the logarithmic growth phase were digested and washed twice with serum-free 1640 medium, diluted to 3 ⁇ 10 7 /ml and mixed with an equal volume of Matrigel, and subcutaneously inoculated with 0.2ml of 3 ⁇ 10 6 cells in the right axilla of the mouse cells/only.
  • the blank model group was PBS control.
  • the small molecule control group was given the same molar amount of 5 mg/kg ADC drug, and the dose of FR ⁇ -ADC08 was given according to the two dose groups of 2 mg/kg and 5 mg/kg.
  • the frequency of administration was once a week, and the measurement was performed twice a week. After injection, the tumor volume and mouse body weight were recorded before each administration.
  • Figure 15 shows that FR ⁇ -ADC08 has an inhibitory effect on tumor growth in the OVCAR3 mouse subcutaneously transplanted tumor model.

Abstract

一种连接子，其包括氨基酸单元（RL），所述RL包括：-Val-Ala-Q-Gly-Phe-Gly-，其中Q为键，单个氨基酸或二肽，通过多肽序列优化，达到ADC药物在肿瘤细胞内更有效释放小分子药物的目的。此外还涉及了包含前述连接子的连接子药物、偶联物及它们的用途。

Description

抗体药物偶联物及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种连接子及包含其的抗体药物偶联物(antibody-drug conjugate，ADC)。
背景技术
在癌症治疗方法中，许多细胞毒性药物分子因为无法选择性地毒杀癌细胞而无法用于癌症治疗。因此，已发展出抗体药物偶联物(ADC)作为新型的癌症治疗药物。ADC一般是由抗体、连接子、和药物三个部分所组成，其中抗体以连接子和药物连接。抗体药物复合物的作用机制是先通过抗体对于相应抗原的高度辨识能力来识别肿瘤细胞上专一或过度表现的抗原而与之结合，形成的结合物以内吞的方式进入肿瘤细胞时，连接的细胞毒性药物分子也同时被带入肿瘤细胞。在肿瘤细胞中，抗体被分解或是连接子断裂使得细胞毒性药物分子释放，进而杀死目标肿瘤细胞，达到选择性的毒杀作用。
ADC中使用的连接子需要满足几个要求：当在人血浆中循环时，连接子需要稳定以防止药物的早期释放；在内化到肿瘤细胞中时，可切割的连接子在一定条件下可被切割以释放药物，而对于不可切割的连接子，药物部分以包含药物、连接子、和衍生自蛋白酶降解配体的氨基酸残基的活性形式释放。
由于可裂解的抗体偶联药物的主要作用机制是利用抗体靶向特定细胞表面抗原，结合后由内吞作用进入细胞内质网系统，通过内体(Endosome)或溶酶体中的蛋白酶类切割后释放小分子药物，达到靶向杀伤肿瘤细胞的目的。对实体瘤而言，由于肿瘤的异质性，有部分肿瘤不表达或低表达靶向抗原，因而有穿透膜活性的小分子被释放后，能连带杀伤旁边的肿瘤细胞，被称为旁观者效应。
在Endosome或溶酶体内一系列蛋白酶，其中组织蛋白酶B为重要成分之一。该蛋白酶属于半胱氨酸蛋白酶水解酶家族。与papain(胰乳蛋白酶)同源，最适PH值为酸性。由于该酶参与溶解细胞外基质，进而影响肿瘤的浸润转移，被发现在多种肿瘤细胞，如胃癌、肺癌、肠癌、乳腺癌、前列腺癌、肾癌中有较高表达。因而ADC药物进入肿瘤细胞中可被胞内大量的组织蛋白酶B快速裂解，快速释放小分子杀伤细胞。
非最佳特性可能导致ADC效能的降低、偶联物免疫学特异性的不足以及由于药物从偶联物的非特异性释放所致的毒性的增加。因此，存在对可用于靶向疗法的新连接子技术和偶联物的需要。
发明内容
本申请提供了一种连接子，其包含自降解间隔子即氨基酸单元，所述氨基酸单元包含Cathepsin B偏好的底物成分GFG，以及在GFG片段N端增加VA序列，增强与Cathepsin B的裂解活性区域结合，同时考虑多肽长度影响酶对底物的活性，可在VA与GF间分别加了一个S及二个S，形成三个多肽序列即VAGFG、VASGFG、VASSGFG，通过多肽序列优化，达到ADC药物在肿瘤细胞内更有效释放小分子药物的目的。
本申请还提供了包含前述连接子的连接子药物、偶联物及它们的用途。
一方面，本申请提供一种连接子，所述连接子包括氨基酸单元(RL)，所述RL包括：-Val-Ala-Q-Gly-Phe-Gly-，其中Q为键、单个氨基酸或二肽。
在某些实施方式中，其中RL可被酶裂解。
在某些实施方式中，其中RL可被组织蛋白酶B(Cathepsin B)裂解。
在某些实施方式中，其中所述Q为键。
在某些实施方式中，其中所述Q为-Ser-或-Ser-Ser-。
在某些实施方式中，其中RL包括-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
在某些实施方式中，所述连接子选自以下结构：-Z-A-S*-RL-和-Z-A-S*-RL-Y-；其中，所述Z为延伸子单元，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元。
在某些实施方式中，其中所述Z包含以下结构：-(琥珀酰亚胺-3-基-N)-，-CH 2-C(＝O)-NH-或-C(＝O)-；
其中，所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000001
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置。
在某些实施方式中，其中所述A选自以下结构：
-C 1-C 10亚烷基-、-C 1-C 10亚杂烷基-、-C 3-C 8碳环基-、-O-(C 1-C 8亚烷基)-、-亚芳基-、-C 1-C 10亚烷基-亚芳基-、-亚芳基-C 1-C 10亚烷基-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-、-C 3-C 8杂环基-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-、-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚杂烷基-C(＝O)-、-C 3-C 8碳环基-C(＝O)-、-O-(C 1-C 8亚烷基)-C(＝O)-、-亚芳基-C(＝O)-、-C 1-C 10亚烷基-亚芳基-C(＝O)-、-亚芳基-C 1- C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-C(＝O)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-C(＝O)-、-C 3-C 8杂环基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-C(＝O)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-NH-、-C 1-C 10亚杂烷基-NH-、-C 3-C 8碳环基-NH-、-O-(C 1-C 8亚烷基)-NH-、-亚芳基-NH-、-C 1-C 10亚烷基-亚芳基-NH-、-亚芳基-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-NH-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-NH-、-C 3-C 8杂环基-NH-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-NH-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-S-、-C 1-C 10亚杂烷基-S-、-C 3-C 8碳环基-S-、-O-(C 1-C 8亚烷基)-S-、-亚芳基-S-、-C 1-C 10亚烷基-亚芳基-S-、-亚芳基-C 1-C 10亚烷基-S-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-S-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-S-、-C 3-C 8杂环基-S-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-S-或-(C 3-C 8杂环基)-C 1-C 10亚烷基-S-；
其中，所述A任选地被碱性单元(BU)所取代，所述碱性单元为-(CH 2) xNH 2、-(CH 2) xNHR a或-(CH 2) xNR a 2；其中x为1-4的任意整数；并且每个R a独立地选自C 1-C 6烷基和C 1-C 6卤代烷基，或者两个R a基团与它们所附连的氮组合形成4-至6-元杂环烷基环、或者氮杂环丁烷基、吡咯烷基或哌啶基基团。
在某些实施方式中，其中所述A选自以下结构：为-(C 1-C 10)亚烷基-C(＝O)-，其中A的所述亚烷基部分任选地被所述碱性单元(BU)所取代。
在某些实施方式中，其中所述连接子可以包含以下结构：
Figure PCTCN2022135216-appb-000002
其中a为1至8中的任意整数。
在某些实施方式中，其中所述a为2至5中的任意整数。
在某些实施方式中，其中所述Y包含-NH-(CH 2)n 1-La-Lb-Lc-所示的结构，其中所述La表示-C(＝O)-NH-、-NR 1-(CH 2)n 2-、-O-或单键，R 1表示H、C 1-C 6烷基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基；
所述Lb表示-CR 2(-R 3)-或单键，其中R 2及R 3各自独立地表示H、-NH 2、C 1～C 6亚烷基、C 1～C 6亚烷基-氨基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基，且R 2和R 3不同时为-NH 2；
n 1表示0～6的整数；
Lc表示-CH 2-或-C(＝O)-。
在某些实施方式中，其中所述Y包含NH-(CH 2)n 1-O-(CH 2)n 2-C(＝O)-，其中n 1或n 2各自独立地为0至5的任意整数。
在某些实施方式中，其中所述Y包含-NH-CH 2-C(＝O)-、-NH-CH 2CH 2-C(＝O)-、-NH-(CH 2) 3-C(＝O)-、-NH-(CH 2) 4-C(＝O)-、-NH-(CH 2) 5-C(＝O)-、-NH-CH 2-O-CH 2-C(＝O)-、或-NH-(CH 2) 2-O-CH 2-C(＝O)-。
在某些实施方式中，其中所述S*为键，且所述连接子为-Z-A-RL-或-Z-A-RL-Y-。
在某些实施方式中，其中所述S*为分配剂，且所述连接子为-Z-A-S*-RL-或-Z-A-S*-RL-Y-。
在某些实施方式中，其中所述S*包含PEG单元。
在某些实施方式中，所述S*具有下式：
-NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)-；
-NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；或
-NH-(CH 2CH 2O) b-CH 2CH 2NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；
其中所述S*左侧与A连接，右侧与RL连接，并且b为2至20的任意整数。
在某些实施方式中，其中所述b为2、4、8或12。
在某些实施方式中，所述连接子选自以下结构：
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2- C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-C(＝O)-CH 2CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
其中，
所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000003
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
在某些实施方式中，所述连接子药物选自以下结构：
Figure PCTCN2022135216-appb-000004
另一方面，本申请提供一种连接子药物，其包含本申请所述的连接子。
在某些实施方式中，所述连接子药物选自以下结构：Z’-A-S*-RL-D和Z’-A-S*-RL-Y- D；其中，所述Z’为延伸子单元前体，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元；D为药物单元。
在某些实施方式中，其中所述延伸子单元前体Z’选自(马来酰亚胺-N-基)-、(吡咯烷-2,5-二酮-N-基)-、M-CH 2-C(＝O)-NH-和HS-(CH 2) 1-5-C(＝O)-，其中M为卤素。
在某些实施方式中，其中所述药物单元包括细胞毒性剂。
在某些实施方式中，其中所述药物单元包括：瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱、(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂
Figure PCTCN2022135216-appb-000005
(pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、长春花生物碱(vincaalkaloids)、或其衍生物。
在某些实施方式中，其中所述药物单元包括DNA拓扑异构酶I抑制剂。
在某些实施方式中，其中所述药物单元包括喜树碱或其衍生物。
在某些实施方式中，其中所述药物单元具有以下结构：
Figure PCTCN2022135216-appb-000006
其中1位的氨基的氮原子为附接位置的基团。
在某些实施方式中，其中所述连接子药物单元具有以下结构：
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)- Dxd；
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；和
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
其中，
所述(马来酰亚胺-N-基)-具有以下结构：
Figure PCTCN2022135216-appb-000007
其中氮原子是附接位置的基团；
所述(吡咯烷-2,5-二酮-N-基)-具有以下结构：
Figure PCTCN2022135216-appb-000008
其中氮原子是附接位置的基团；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
所述Dxd具有以下结构：
Figure PCTCN2022135216-appb-000009
其中1位的氨基的氮原子为附接位置的基团；
所述M表示卤原子。
在某些实施方式中，其中所述连接子药物单元选自以下结构：
Figure PCTCN2022135216-appb-000010
Figure PCTCN2022135216-appb-000011
另一方面，本申请提供一种偶联物，其包含本申请所述的连接子。
在某些实施方式中，所述的偶联物包含配体。
在某些实施方式中，其中所述配体靶向细胞表面受体或肿瘤相关抗原。
在某些实施方式中，其中所述细胞表面受体或肿瘤相关抗原包括：HER2、HER3、claudin18.2、叶酸受体α(FRα)、BCMA、PSMA、TROP-2、CD19、CD20、CD22、CD30、CD79b、EGFR、c-Met或CEACAM5。
在某些实施方式中，其中所述配体包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体包括单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体、完整抗体、抗体片段、人抗体、人源化抗体或嵌合抗体。
在某些实施方式中，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，其中所述抗体为单克隆抗体。
在某些实施方式中，其中所述抗体为人抗体、人源化抗体或嵌合抗体。
在某些实施方式中，所述的偶联物包括配体药物偶联物，并具有以下结构：C-(L-D) m，其中，所述C表示配体单元，L表示连接子，D表示药物单元，m表示1至10中的任意数。
在某些实施方式中，所述配体药物偶联物为抗体药物偶联物(ADC)，并具有以下结构：Ab-(L-D) m，其中，所述Ab表示抗体或其抗原结合片段，L表示连接子，D表示药物单 元，m表示1至10中的任意数。
在某些实施方式中，其中所述药物单元包括细胞毒性剂。
在某些实施方式中，其中所述药物单元包括：瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱、(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂
Figure PCTCN2022135216-appb-000012
(pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、长春花生物碱(vincaalkaloids)、或其衍生物。
在某些实施方式中，其中所述药物单元包括DNA拓扑异构酶I抑制剂。
在某些实施方式中，其中所述药物单元包括喜树碱或其衍生物。
在某些实施方式中，其中所述药物单元具有以下结构：
Figure PCTCN2022135216-appb-000013
其中1位的氨基的氮原子为附接位置的基团。
在某些实施方式中，其中所述抗体药物偶联物选自以下结构：
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；和
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
其中，
所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000014
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
所述Dxd具有以下结构：
Figure PCTCN2022135216-appb-000015
其中1位的氨基的氮原子为附接位置的基团；
所述Ab表示抗体或其抗原结合片段，m表示1至10的任意数。
在某些实施方式中，其中所述抗体药物偶联物选自以下结构：
Figure PCTCN2022135216-appb-000016
Figure PCTCN2022135216-appb-000017
其中，所述Ab表示抗体或其抗原结合片段，m为1至10的任意数。
在某些实施方式中，所述抗体或其抗原结合片段包括：抗HER2抗体、抗HER3抗体、抗claudin18.2抗体、抗FRα抗体或其抗原结合片段。
另一方面，本申请提供一种药物组合物，其包含本申请所述的偶联物以及任选地药学上可接受的载体。
另一方面，本申请提供本申请所述的偶联物或本申请所述的药物组合物在制备药物中的用途，所述药物用于治疗疾病或病症。
在某些实施方式中，其中所述疾病或病症包括肿瘤。
在某些实施方式中，其中所述疾病或病症包括实体瘤和血液瘤。
在某些实施方式中，其中所述实体瘤包括：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏、肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在某些实施方式中，其中所述血液瘤包括：急性髓细胞白血病(AML)、多发性骨髓瘤 (MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、霍奇金淋巴瘤、或其组合。
另一方面，本申请提供一种治疗有需要的受试者的疾病或病症的方法，其包括给予所述受试者有效量的本申请所述的偶联物。
在某些实施方式中，其中所述疾病或病症包括肿瘤。
在某些实施方式中，其中所述疾病或病症包括实体瘤和血液瘤。
在某些实施方式中，其中所述实体瘤包括：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏、肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在某些实施方式中，其中所述血液瘤包括：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)、霍奇金淋巴瘤、或其组合。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A显示的是本申请实施例1所述HER3-ADC04的HIC-HPLC色谱图。
图1B显示的是本申请实施例1所述HER3-ADC07的HIC-HPLC色谱图。
图1C显示的是本申请实施例1所述HER3-ADC08的HIC-HPLC色谱图。
图1D显示的是本申请实施例1所述HER3-ADC09的HIC-HPLC色谱图。
图1E显示的是本申请实施例1所述Claudin18.2-ADC08的HIC-HPLC色谱图。
图1F显示的是本申请实施例1所述Claudin18.2-ADC09的HIC-HPLC色谱图。
图1G显示的是本申请实施例1所述FRα-ADC08的HIC-HPLC色谱图。
图1H显示的是本申请实施例1所述FRα-ADC09的HIC-HPLC色谱图。
图2A显示的是本申请所述HER3-ADC04对人结肠癌细胞SW620体外杀伤作用；
图2B显示的是本申请所述不同连接子-Dxd药物对人结肠癌细胞SW620体外杀伤作用；
图3显示的是本申请所述含不同连接子的anti-HER3 ADC对人结肠癌细胞SW620体外杀伤作用
图4显示的是本申请所述含不同连接子的anti-HER3 ADC对非靶细胞CHO-K1体外杀伤作用；
图5显示的是本申请所述anti-HER3 ADC在血浆孵育后对SW620靶细胞的体外杀伤作用；
图6显示的是本申请所述anti-HER3 ADC对SW620移植肿瘤模型的治疗作用；
图7显示的是本申请所述SW620移植肿瘤模型在anti-HER3 ADC治疗过程中小鼠体重变化；
图8显示的是本申请所述anti-Claudin18.2 ADC对人胃癌细胞NUGC4的体外杀伤作用；
图9显示的是本申请所述anti-Claudin18.2 ADC对重组过表达Claudin18.2分子的人胃癌细胞NUGC4的体外杀伤作用；
图10显示的是本申请所述anti-Claudin18.2 ADC对重组过表达Claudin18.2分子的HEK293细胞的体外杀伤作用；
图11显示的是本申请所述anti-Claudin18.2 ADC在血浆孵育后对过表达Claudin18.2分子的HEK293细胞的体外杀伤作用；
图12显示的是本申请所述anti-claudin18.2 ADC对NUGC4胃癌肿瘤移植模型的生长抑制作用；
图13显示的是本申请所述anti-FRαADC对人卵巢癌细胞OVCAR3的体外杀伤作用；
图14显示的是本申请所述anti-FRαADC在血浆孵育后对anti-FRαADC的体外杀伤作用；
图15显示的是本申请所述anti-FRαADC对OVCAR3小鼠皮下移植肿瘤模型的生长抑制作用。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“抗体药物偶联物(抗体-药物偶联物)”、“抗体药物缀合物(抗体-药物缀合物)”和“ADC”可互换使用，通常是指连接至一种或多种抗体或抗原结合片段的一种或多种治疗性化合物(例如Dxd)且其由以下通式定义：Ab-(L-D) m，其中Ab＝抗体或抗原结合片段，L＝连接子，D＝药物单元，且m＝每个抗体或抗原结合片段的药物部分的数目，可以为1至10中的任意数。在一些实施例中，连接子L可包括介于抗体或抗原结合片段与药物单元之间的可裂解部分。示例性可裂解接头描述且例示于本文中。
在本申请中，术语“抗体”通常是在最广泛的意义上加以使用，并且具体地涵盖单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体(例如，双特异性抗体)、和抗体片段，只要它们显示所期望的生物活性(Milleretal(2003)Jour.ofImmunology170:4854-4861)。抗体可以是鼠、人、人源化、嵌合抗体，或源于其它物种。非限制性的，“抗体”典型地可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。天然重链和轻链的可变结构域各自包含四个FR区(HFR1，HFR2，HFR3，HFR4，LFR1，LFR2，LFR3，LFR4)，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。
在本申请中，术语“抗原结合片段”或“抗体的功能片段”(在本文中也被称作“靶向部分”或“抗原结合部分”)通常是指抗体分子的一部分，其包含负责抗体与抗原之间的特异性结合的氨基酸。抗原中由抗体特异性地识别和结合的部分是称作如上文所述的“表位”。抗原结合片段可典型地包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，其并非必须包含两者。抗体的抗原结合片段的实例包括：(1)Fab片段，具有VL、VH、恒定轻链(CL)和CH1结构域的单价片段；(2)F(ab’) 2片段，具有由铰链区的二硫桥连接的两个Fab片段的二价片段；(3)具有两个VH和CH1结构域的Fd片段；(4)具有抗体单臂的VL和VH结构域的Fv 片段，(5)dAb片段(Ward等人,“Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,”Nature 341:544-546(1989)，其以引用的方式整体并入本申请)，其具有VH结构域；(6)分离的互补决定区(CDR)；(7)单链Fv(scFv)，例如源于scFV-文库。尽管Fv片段的两个结构域VL和VH是由独立基因编码，但其可通过合成连接子使用重组方法接合，合成连接子使得其被制备为其中VL和VH区配对以形成单价分子的单一蛋白链(称为单链Fv(scFv))(可参见例如Huston等人,“Protein Engineering of Antibody Binding Sites:Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))；和(8)VHH，“VHH”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域(参见Nguyen V.K.等人，2000，The EMBO Journal，19，921-930；Muyldermans S.，2001，J Biotechnol.，74，277-302以及综述Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。这些抗体片段使用所属领域的技术人员已知的常规技术获得，且能够以与完整抗体相同的方式评估所述片段的功能。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))、基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)和基于IMGT本体论(IMGT-ONTOLOGY)的概念和IMGT Scientific图表规则的KABAT定义规则。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”或“人抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“特异性结合”在提及结合分子(例如抗体)与其结合配偶体(例如抗原)的相互作用时，通常是指该相互作用取决于结合配偶体上特定结构(例如抗原决定簇或表位)的存在。换言之，即使在结合配偶体存在于其他分子或有机体的混合物中时，抗体仍会优先结合或识别结合配偶体。结合可通过共价或非共价相互作用或二者的组合介导。换言之，术语“特异性结合”通常是指免疫特异性结合抗原决定簇或表位且不免疫特异性结合其他抗原决定簇或表位。免疫特异性结合抗原的结合分子可以较低亲和力结合其他肽或多肽，如通过(例如)放射免疫分析(RIA)、酶联免疫吸附分析(ELISA)、BIACORE或本领域中已知的其他分析所测定。免疫特异性结合抗原的结合分子或其片段可与带有相同表位的相关抗原交叉反应。在某些情形中，免疫特异性结合抗原的结合分子或其片段不与其他抗原交叉反应。
在本申请中，术语“连接子”、“接头”、“linker”或“L”通常是指能够将化合物 (通常药物单元)共价接合至另一部分(诸如抗体或抗原结合片段)的任何化学部分。接头可能在使化合物或抗体保持活性的条件下易于发生或实质上抵抗酸诱导的裂解、肽酶诱导的裂解、基于光的裂解、酯酶诱导的裂解和/或二硫键裂解。
在本申请中，术语“药物单元”或“D”通常是指具有期望的生物学活性和反应性官能团的任何化合物，其可用于将药物并入本申请的偶联物。于一些实施例中，药物单元表示用于癌症治疗的细胞毒性药物；具有所需生物活性的蛋白质或多肽，例如毒素像是相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素、和白喉毒素；其它合适的蛋白质，包括肿瘤坏死因子、α-干扰素、β-干扰素、神经生长因子、血小板衍生的生长因子、组织纤溶酶原激活剂、和生物反应调节剂，例如：淋巴因子、白细胞介素-1(IL-1)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(G-CSF)、或其他生长因子。
在本申请中，术语“细胞毒性剂”通常是指抑制或防止细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂包括但不限于放射性同位素(例如，At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212以及Lu的放射性同位素)；化学治疗剂或药物(例如，氨甲蝶呤、阿霉素、长春花生物碱(长春新碱、长春花碱、依托泊苷)、多柔比星、美法仑、丝裂霉素C、苯丁酸氮芥、道诺霉素或其他插入剂；生长抑制剂；酶和其片段，诸如核苷酸分解酶；抗生素；毒素，诸如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体；以及下文所公开的各种抗肿瘤或抗癌剂。
在本申请中，术语“药学上可接受的形式”通常是指所公开的化合物的形式，包括但不限于其药学上可接受的盐、酯、水合物、溶剂化物、多晶型物、异构体、前药和同位素标记衍生物。在一个实施方式中，“药学上可接受的形式”包括但不限于其药学上可接受的盐、酯、前药和同位素标记衍生物。在一些实施方式中，“药学上可接受的形式”包括但不限于其药学上可接受的异构体和立体异构体、前药和同位素标记衍生物。
本申请的连接子、连接子药物及偶联物可包含其药学上可接受的任何形式，如药学上可接受的盐、酯、水合物、溶剂化物、多晶型物、异构体、前药或同位素标记衍生物。上述药学上可接受的形式也在本申请的范围内。
在本申请中，术语“药学上可接受的盐”通常是指化合物(例如，药物、连接子药物或偶联物)的药学上可接受的有机或无机盐。在一些方面，化合物可含至少一个氨基基团，并因此可与氨基基团形成酸加成盐。示例性的盐包括但不限于硫酸盐、三氟乙酸盐、柠檬酸盐、乙酸盐、草酸盐、氯化物、溴化物、碘化物、硝酸盐、硫酸氢盐、磷酸盐、酸式磷酸 盐、异烟酸酯、乳酸盐、水杨酸盐、柠檬酸盐、酒石酸盐、油酸盐、鞣酸盐、泛酸盐、酒石酸氢盐、抗坏血酸盐、琥珀酸盐、马来酸盐、龙胆酸盐、富马酸盐、葡萄糖酸盐、葡糖醛酸盐、蔗糖盐、甲酸盐、苯甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐和扑酸盐(即，1,1’-亚甲基双-(2-羟基-3-萘甲酸盐))。药学上可接受的盐可能涉及另一分子如乙酸根离子、琥珀酸根离子或其他抗衡离子的引入。抗衡离子可以是任何将稳定母体化合物上的电荷的有机或无机部分。此外，药学上可接受的盐可在其结构中具有不止一个带电荷的原子。其中多个带电荷的原子是药学上可接受的盐的一部分的情况可具有多个抗衡离子。因此，药学上可接受的盐可具有一个或多个带电荷的原子和/或一个或多个抗衡离子。
在本申请中，术语“衍生物”通常是指通过一种或多种化学反应由母化合物制成的化学化合物或分子。
在本申请中，术语“前药”通常是指生物活性较低或无活性的化合物，其经由化学或生物过程(即，化学反应或酶促生物转化)在体内转化为生物活性更高的化合物。
在本申请中，术语“同位素标记化合物”或“同位素标记衍生物”通常是指其中一个或多个原子被具有的原子质量或质量数不同于自然界通常发现的原子质量或质量数的原子所替代的目前公开的化合物，包括各自如本文所述的药物盐和前药。可引入到目前公开的化合物中的同位素的实例包括氢、碳、氮、氧、磷、氟和氯的同位素，如分别为 2H、 3H、 13C、 14C、 15N、 18O、 17O、 31P、 32P、 35S、 18F和 36Cl。
在本申请中，术语“药学上可接受的载体”通常是指不干扰活性成分生物学活性的有效性的一种或多种无毒物质。这类制剂通常可含有盐、缓冲剂、防腐剂、相容性载体、佐剂和任选的其它治疗剂。这类药学上可接受的制剂通常也可包含适合给予人的相容性固体或液体填料、稀释剂或包囊材料。非限制性的，药学上可接受的载体可包括液体，例如水、盐水、甘油和乙醇。这些载体中也可存在辅助物质，例如润湿剂或乳化剂、pH缓冲物质等。
在本申请中，术语“烷基”或“烷基基团”通常是指完全饱和的直链、分支链或环烃链。在某些实施例中，烷基可含有1-8个碳原子(“C 1-C 8烷基”)。在某些实施例中，烷基可含有1-6个碳原子(“C 1-C 6烷基”)。在某些实施例中，烷基含有1-3个碳原子。在又其他实施例中，烷基含有2-3个碳原子，且在又其他实施例中，烷基含有1-2个碳原子。
在本申请中，术语“烷基烷氧基”意谓经烷氧基取代的烷基。
在本申请中，术语“烷氧基”是指经由氧(“烷氧基”)原子连接至主碳链的如先前所定义的烷基。
在本申请中，术语“烷基羟基”意谓经羟基取代的烷基。
在本申请中，术语“羟基(hydroxy或hydroxyl)”是指-OH。
在本申请中，术语“碳环”或“碳环基”包括芳族基团(例如芳基)及非芳族基团(例如环烷基)两者。在某些实施例中，碳环基团含有3-10个碳原子(“3至10元碳环”)。在某些实施例中，碳环基团含有3-8个碳原子(“3至8元碳环”)。在某些实施例中，碳环基团含有3-6个碳原子(“3至6元碳环”)。在某些实施例中，碳环基团含有3-5个碳原子(“3至5元碳环”)。
在本申请中，术语“卤烷基”是指经一个或多个卤素原子取代的烷基。
在本申请中，术语“卤素”是指任何卤素基团，例如-F、-Cl、-Br或-I。
在本申请中，术语“杂环(heterocycle或heterocyclyl或heterocyclic)”、“杂环基”意谓在环中含有至少一个杂原子的单环杂环、双环杂环或三环杂环。术语“杂环(heterocycle或heterocyclyl或heterocyclic)”、“杂环基”涵盖杂芳基。“杂芳基”是指具有一个或多个闭合环且在这类环中的至少一个中具有一个或多个杂原子(氧、氮或硫)的环状部分，其中这类环中的至少一个为芳族环，且其中该环或这类环可独立地经稠合和/或桥连。实例包括不限于苯基、噻吩基、三唑基、吡啶基、嘧啶基、哒嗪基及吡嗪基。
在本申请中，术语“PEG单元”通常为由重复的乙烯-氧基亚单元(PEG或PEG亚单元)组成的有机部分并可以是多分散的、单分散的或离散的(即，具有离散数目的乙烯-氧基亚单元)。多分散PEG为大小和分子量的非均匀混合物，而单分散PEG通常是从非均匀混合物纯化的并因此具有单一的链长和分子量。本申请的PEG单元可以包含一个或多个聚乙二醇链，每个聚乙二醇链由一个或多个彼此共价附连的乙烯氧基亚单元组成。聚乙二醇链可例如以线形、支化或星形构型连接在一起。
在本申请中，术语“取代的”无论前面是否有术语“任选”通常表示指定部分的一个或多个氢经合适取代基置换。除非另外指示，否则“任选取代的”基团可在该基团的各可取代位置处具有合适取代基，且当任何给定结构中的超过一个位置可经超过一个选自指定群组的取代基取代时，在各位置处的取代基可相同或不同。依据本披露预想的取代基组合较佳为引起形成稳定或化学上可行的化合物的取代基组合。
本领域普通技术人员应理解，“取代”或“经取代”或“不存在”包括以下隐含限制条件：该取代或不存在根据经取代原子及取代基的准许价数，且取代或不存在产生稳定化合物，例如该稳定化合物不自发地经历诸如通过重排、环化、消除等进行的转型。出于本披露的目的，诸如氮的杂原子可具有氢取代基和/或满足杂原子价数的本文所描述的有机化合物的任何容许取代基。
在本申请中，术语“预防和/或治疗”不仅包括预防和/或治疗疾病，还通常包括预防疾病的发作，减缓或逆转疾病的进展，预防或减缓与疾病相关的一种或多种症状的发作，减少和/或减轻与疾病相关的一种或多种症状，降低疾病和/或与其相关的任何症状的严重程度和/或持续时间和/或预防疾病和/或与其相关的任何症状的严重程度的进一步增加，预防、减少或逆转由疾病引起的任何生理损伤，以及通常对正在治疗的患者有益的任何药理学作用。本申请的组合物形成可行的治疗剂不需要实现完全治愈或根除疾病的任何症状或表现。如在相关领域中所认识到的，用作治疗剂的药物可降低给定疾病状态的严重程度，但不需要消除疾病的每种表现才能被认为是有用治疗剂。类似地，预防性施用的治疗构成可行的预防剂不需要完全有效地预防病症的发作。简单地在受试者中减少疾病的影响(例如，通过减少其症状的数量或严重程度，或通过提高另一种治疗的有效性，或通过产生另一种有益效果)，或减少疾病发生或恶化的可能性就足够了。
在本申请中，术语“施用”通常是指通过本领域已知的任意途径，将蛋白(包括免疫球蛋白)递送给有此需要的人或动物。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的或粘膜的。
在本申请中，术语“接触”通常是指两种两个或更多个不同类型的物质以任何顺序、任何方式以及任何时长接触在一起。当应用于细胞时，术语“接触”通常指借以将本申请的抗体或其抗原结合片段、药物、连接子药物、偶联物和/或药物组合物递送到靶细胞或与靶细胞直接靠近放置的方法，该递送可以是体外的或体内的并且可以涉及使用重组载体系统。“接触”可以发生在体内(in vivo)、间接体内(ex vivo)或体外(in vitro)。
在本申请中，术语“有效量”或“有效剂量”通常是指足以实现或至少部分实现所需效果的量。药物或治疗剂的“治疗有效量”或“治疗有效剂量”通常是当单独使用或与另一种治疗剂组合使用时促进疾病消退(这通过疾病症状严重程度的降低、疾病无症状期的频度和持续时间的增加、或者由于罹患疾病而引起的损害或残疾的预防来证明)的任何药物量。药物的“预防有效量”或“预防有效剂量”通常是指当单独或与另一种治疗剂组合给有疾病发展或疾病复发的风险的受试者施用时抑制疾病的发展或复发的药物量。可以使用本领域技术人员已知的多种方法对治疗剂或预防剂促进疾病消退或抑制疾病发展或复发的能力进行评估，比如在处于临床试验期间的人类受试者中、在动物模型系统中预测对人类的功效、或者通过在体外测定中测定药剂的活性。
在本申请中，术语“肿瘤”通常是指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”,“癌”,“细胞增 殖性病症”,“增殖性病症”和“肿瘤”在本文中提到时并不互相排斥。在本申请中，肿瘤可以是实体瘤或非实体瘤。
在本申请中，术语“受试者”通常是指需要诊断、预后、改善、预防和/或治疗疾病的人或非人动物(包括哺乳动物)，诸如人、非人灵长类动物(猿、长臂猿、大猩猩、黑猩猩、猩猩、猕猴)、家畜(狗和猫)、农场动物(家禽如鸡和鸭、马、牛、山羊、绵羊、猪)和实验动物(小鼠、大鼠、兔、豚鼠)。人受试者包括胎儿、新生儿、婴儿、青少年和成人受试者。受试者可以包括动物疾病模型。
在本申请中，术语“包括”、“包含”、“具有”、“可以”、“含有”及其变体通常旨在是开放式过渡性短语、术语或词语，其不排除额外行为或结构的可能性。术语“由……组成”通常表示不能存在别的组分(或同样地，特征、整数、步骤、等)。除非上下文另有明确规定，单数形式如英文的“a”，“an”，“the”，中文的“一个”、“一种”和“所述/该”一般包括所指代事物的复数形式。
在本申请中，术语“约”通常意指大约(approximately)、在......的附近(in the region of)、粗略地(roughly)、或左右(around)。当术语“约”当用于指涉数值范围时，截值或特定数值用于指示所载明的数值可与该列举数值有多达10％的差异。因此，术语“约”可用于涵盖自特定值±10％或更少的变异、±5％或更少的变异、±1％或更少的变异、±0.5％或更少的变异、或±0.1％或更少的变异。
发明详述
连接子(linker)
本申请提供了一种新颖的连接子(conjugating linker)结构，其包括氨基酸单元(RL)，所述RL包括：-Val-Ala-Q-Gly-Phe-Gly-，其中Q为键，单个氨基酸或二肽。
在某些实施方式中，其中RL可被酶裂解。
在某些实施方式中，其中RL可被组织蛋白酶B(Cathepsin B)裂解。
在某些实施方式中，其中所述Q为键。
在某些实施方式中，其中所述Q为-Ser-或-Ser-Ser-。
在某些实施方式中，其中RL包括-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
在某些实施方式中，所述连接子选自以下结构：-Z-A-S*-RL-和-Z-A-S*-RL-Y-；其中，所述Z为延伸子单元，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元。
在某些实施方式中，其中所述Z包含以下结构：-(琥珀酰亚胺-3-基-N)-，-CH 2-C(＝O)-NH-或-C(＝O)-；
其中，所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000018
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置。
在某些实施方式中，其中所述A选自以下结构：
-C 1-C 10亚烷基-、-C 1-C 10亚杂烷基-、-C 3-C 8碳环基-、-O-(C 1-C 8亚烷基)-、-亚芳基-、-C 1-C 10亚烷基-亚芳基-、-亚芳基-C 1-C 10亚烷基-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-、-C 3-C 8杂环基-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-、-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚杂烷基-C(＝O)-、-C 3-C 8碳环基-C(＝O)-、-O-(C 1-C 8亚烷基)-C(＝O)-、-亚芳基-C(＝O)-、-C 1-C 10亚烷基-亚芳基-C(＝O)-、-亚芳基-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-C(＝O)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-C(＝O)-、-C 3-C 8杂环基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-C(＝O)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-NH-、-C 1-C 10亚杂烷基-NH-、-C 3-C 8碳环基-NH-、-O-(C 1-C 8亚烷基)-NH-、-亚芳基-NH-、-C 1-C 10亚烷基-亚芳基-NH-、-亚芳基-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-NH-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-NH-、-C 3-C 8杂环基-NH-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-NH-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-S-、-C 1-C 10亚杂烷基-S-、-C 3-C 8碳环基-S-、-O-(C 1-C 8亚烷基)-S-、-亚芳基-S-、-C 1-C 10亚烷基-亚芳基-S-、-亚芳基-C 1-C 10亚烷基-S-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-S-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-S-、-C 3-C 8杂环基-S-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-S-或-(C 3-C 8杂环基)-C 1-C 10亚烷基-S-；
其中，所述A任选地被碱性单元(BU)所取代，所述碱性单元为-(CH 2) xNH 2、-(CH 2) xNHR a或-(CH 2) xNR a 2；其中x为1-4的任意整数；并且每个R a独立地选自C 1-C 6烷基和C 1-C 6卤代烷基，或者两个R a基团与它们所附连的氮组合形成4-至6-元杂环烷基环、或者氮杂环丁烷基、吡咯烷基或哌啶基基团。
在某些实施方式中，其中所述A选自以下结构：为-(C 1-C 5)亚烷基-C(＝O)-，其中A的所述亚烷基部分任选地被所述碱性单元(BU)所取代。
例如，所述Z包含可以是-(琥珀酰亚胺-3-基-N)-，所述A可以是-(C 1-C 10)亚烷基-C(＝ O)-，所述Z-A可以包含以下结构：
Figure PCTCN2022135216-appb-000019
其中a为1至8中的任意整数。例如，其中所述a可以为2至5中的任意整数。
又例如，Z-A-可以具有以下结构：
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2-C(＝O)-
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2-C(＝O)-
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-
-CH 2C(＝O)NH-CH 2-C(＝O)-、
-CH 2C(＝O)NH-CH 2CH 2-C(＝O)-
-CH 2C(＝O)NH-CH 2CH 2CH 2-C(＝O)-
-CH 2C(＝O)NH-CH 2CH 2CH 2CH 2-C(＝O)-
-CH 2C(＝O)NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-
-C(＝O)-cyc.Hex(1,4)-CH 2-(N-ly-3-diminiccuS)-
-C(＝O)-Aryl-CH 2-(N-ly-3-diminiccuS)-
-C(＝O)-cyc.Het-CH 2-(N-ly-3-diminiccuS)-
-C(＝O)-CH 2CH 2-C(＝O)-
-C(＝O)-CH 2CH 2CH 2-C(＝O)-
-C(＝O)-CH 2CH 2CH 2CH 2-C(＝O)-
-C(＝O)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-
-C(＝O)-CH 2CH 2CH 2CH 2CH 2CH 2-C(＝O)-等，
(Aryl表示2价的芳香族烃基，cyc.Het表示2价的环状杂环基)。
在某些实施方式中，其中所述Y包含-NH-(CH 2)n 1-La-Lb-Lc-所示的结构，其中所述La表示-C(＝O)-NH-、-NR 1-(CH 2)n 2-、-O-或单键，R 1表示H、C 1-C 6烷基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基；
所述Lb表示-CR 2(-R 3)-或单键，其中R 2及R 3各自独立地表示H、-NH 2、C 1～C 6亚烷基、C 1～C 6亚烷基-氨基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基，且R 2和R 3不同时为- NH 2；
n 1表示0～6的整数；
Lc表示-CH 2-或-C(＝O)-。
在某些实施方式中，其中所述Y包含NH-(CH 2)n 1-O-(CH 2)n 2-C(＝O)-，其中n 1或n 2各自独立地为0至5的任意整数。间隔子可以是将促进RL与药物单元的附连的官能团，或者其可提供附加的结构组分以进一步促进药物单元从缀合物的其余部分的释放(例如，自消解对氨基苄基(PAB)组分)。
在某些实施方式中，其中所述Y包含-NH-CH 2-C(＝O)-、-NH-CH 2CH 2-C(＝O)-、-NH-(CH 2) 3-C(＝O)-、-NH-(CH 2) 4-C(＝O)-、-NH-(CH 2) 5-C(＝O)-、-NH-CH 2-O-CH 2-C(＝O)-、或-NH-(CH 2) 2-O-CH 2-C(＝O)-。
例如，Y可以包含以下结构：
-NH-CH 2-
-NH-CH(-Me)-
-NH-C(-Me) 2-
-NH-CH 2-CHMe-
-NH-CH(-CH 2OH)-
-NH-CH(-CH 2COOH)-
-NH-CH(-CH 2CH 2COOH)-
-NH-CH(-CH 2CH 2CH 2CH 2NH 2)-
-NH-CH 2CH 2-
-NH-CH 2-O-CH 2-
-NH-CH 2CH 2-O-
-NH-CH 2CH 2-O-CH 2-
-NH-CH 2CH 2C(-Me) 2-
-NH-CH 2CH 2NH-
-NH-CH 2CH 2NH-CH 2-
-NH-CH 2CH 2NMe-CH 2-
-NH-CH 2CH 2NH-CH 2CH 2-
-NH-CH 2CH 2NMe-CH 2CH 2-
-NH-CH 2CH 2N(-CH 2COOH)-CH 2-
-NH-CH 2CH 2N(-CH 2CH 2OH)-CH 2-
-NH-CH 2CH 2N(-CH 2CH 2OH)-CH 2CH 2-
-NH-CH 2CH 2CH 2C(＝O)-NHCH(-CH 2OH)-
-NH-CH 2CH 2CH 2C(＝O)-NHCH(-CH 2COOH)-
-NH-CH 2CH 2CH 2C(＝O)-NHCH(-CH 2CH 2CH 2CH 2NH 2)-
-NH-CH 2CH 2CH 2-
-NH-CH 2CH 2CH 2CH 2-
-NH-CH 2CH 2CH 2CH 2CH 2-
-NH-CH 2CH 2CH 2CH 2CH(NH 2)-等。
在某些实施方式中，其中所述S*为键，且所述连接子为-Z-A-RL-或-Z-A-RL-Y-。
在某些实施方式中，其中所述S*为分配剂，且所述连接子为-Z-A-S*-RL-或-Z-A-S*-RL-Y-。分配剂部分可用于例如掩盖药物单元或其他连接单元组分的疏水性。
示例性的分配剂可以包括聚乙二醇(PEG)单元、环糊精单元、聚酰胺、亲水性肽、多糖和树状聚合物。当连接子中包含聚乙二醇(PEG)单元、环糊精单元、聚酰胺、亲水性肽、多糖或树状聚合物时，这些基团可能以“串联(in line)”组分或者以侧链或支化组分存在。
在某些实施方式中，其中所述S*包含PEG单元。
在某些实施方式中，所述S*具有下式：
-NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)-；
-NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；或
-NH-(CH 2CH 2O) b-CH 2CH 2NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；
其中所述S*左侧与A连接，右侧与RL连接，并且b为2至20的任意整数。例如，所述b可以为2、4、8或12。在一些实施方式中，下标b为2。在一些实施方式中，下标b为4。在一些实施方式中，下标b为8。在一些实施方式中，下标b为12。
例如，所述连接子可以选自以下结构：
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
-C(＝O)-CH 2CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
其中，
所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000020
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
又例如，所述连接子可以具有以下结构：
Figure PCTCN2022135216-appb-000021
Figure PCTCN2022135216-appb-000022
连接子药物(linker-payload)
另一方面，本申请提供一种连接子药物，其包含本申请所述的连接子。
在某些实施方式中，所述连接子药物选自以下结构：Z’-A-S*-RL-D和Z’-A-S*-RL-Y-D；其中，所述Z’为延伸子单元前体，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元；D为药物单元。
在某些实施方式中，其中所述延伸子单元前体Z’选自(马来酰亚胺-N-基)-、(吡咯烷-2,5-二酮-N-基)-、M-CH 2-C(＝O)-NH-和HS-(CH 2) 1-5-C(＝O)-，其中M为卤素。
在某些实施方式中，其中所述药物单元包括细胞毒性剂。
在某些实施方式中，其中所述药物单元包括：瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱、(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂
Figure PCTCN2022135216-appb-000023
(pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、长春花生物碱(vincaalkaloids)、或其衍生物。
在某些实施方式中，其中所述药物单元包括DNA拓扑异构酶I抑制剂。
在某些实施方式中，其中所述药物单元包括喜树碱或其衍生物。
在某些实施方式中，其中所述药物单元具有以下结构：
Figure PCTCN2022135216-appb-000024
其中1位的氨基的氮原子为附接位置的基团。
在某些实施方式中，其中所述连接子药物单元具有以下结构：
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
(吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；和
HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
其中，
所述(马来酰亚胺-N-基)-具有以下结构：
Figure PCTCN2022135216-appb-000025
其中氮原子是附接位置的基团；
所述(吡咯烷-2,5-二酮-N-基)-具有以下结构：
Figure PCTCN2022135216-appb-000026
其中氮原子是附接位置的基团；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
所述Dxd具有以下结构：
Figure PCTCN2022135216-appb-000027
其中1位的氨基的氮原子为附接位置的基团；
所述M表示卤原子。
在某些实施方式中，其中所述连接子药物单元选自以下结构：
Figure PCTCN2022135216-appb-000028
Figure PCTCN2022135216-appb-000029
配体单元
在本申请的一些实施方式中，存在配体单元。配体单元(C)是与靶部分特异性结合的靶向剂。在一些实施方式中，配体单元特异性地并选择性地与细胞组分(细胞结合剂)或感兴趣的其他靶分子结合。配体单元的作用是将药物单元(如Dxd)靶向并呈递给因其靶组分或分子的存在而与该配体单元相互作用并允许游离药物随后在靶细胞内(即，细胞内)或附近(即，细胞外)释放的特定靶细胞群体。配体单元C包括但不限于蛋白质、多肽和肽。
合适的配体单元包括例如抗体，例如全长抗体及其抗原结合片段、干扰素、淋巴因子、激素、生长因子和集落刺激因子、维生素、营养转运分子(如但不限于转铁蛋白)或任何其他细胞结合分子或物质。在一些实施方式中，配体单元(C)为抗体或非抗体蛋白靶向剂。
在一些实施方式中，配体单元与包含可释放肽连接子的L(连接子单元)键合。如上所述，本文描述的偶联物中还可存在其他连接组分以用于在药物单元与配体单元之间提供额外的空间(例如，延伸子单元和任选地接头单元A)或提供组合物属性以增加溶解度(例如，分配剂S*)。在这些实施方式中的一些中，配体单元经由配体单元的杂原子与连接子单元的Z键合。可能存在于配体单元上用于该键合的杂原子包括硫(在一个实施方式中，来自靶向配体的巯基基团)、氧(在一个实施方式中，来自靶向配体的羧基或羟基基团)和氮，其任选被取代(在一个实施方式中，来自靶向配体的伯或仲胺官能团，或在另一个实施方式中，来自任选被取代的酰胺氮)。这些杂原子可以配体的天然状态(例如，在天然存在的抗体中)存在于靶向配体上，或者可经由化学修饰或生物工程引入到靶向配体中。
在一个实施方式中，配体单元具有巯基官能团，使得配体单元经由巯基官能团的硫原子与连接子单元键合。
在另一个实施方式中，配体单元具有一个或多个赖氨酸残基，其能够与喜树碱-连接子化合物中间体的延伸子单元前体的活化酯(这样的酯包括但不限于N-羟基琥珀酰亚胺、五氟苯基和对硝基苯基酯)反应并因此提供由配体单元的氮原子和连接子单元的延伸子单元的C＝O基团组成的酰胺键。
在一些方面，配体单元能够通过与延伸子单元前体(Z’)上的反应性官能团相互作用而在延伸子单元(Z)与对应于靶向配体的配体单元之间形成共价键。具有与靶向配体相互作用的能力的Z'的官能团将取决于配体单元的性质。在一些实施方式中，反应性基团为马来酰亚胺，其在附连以形成配体单元之前存在于延伸子单元上(即，延伸子单元前体的马来酰亚胺部分)。配体单元与延伸子单元的共价附连通过配体单元的巯基官能团与Z’的马来酰亚胺官能团相互作用形成硫取代的琥珀酰亚胺而实现。巯基官能团可以配体单元的天然状态(例如，在天然存在的残基中)存在于配体单元上，或者可经由化学修饰或通过生物工程引入到配体单元中。
在又一个实施方式中，配体单元为抗体，并且巯基基团通过抗体的链间二硫化物的还原生成。相应地，在一些实施方式中，连接子单元与来自经还原的链间二硫化物的半胱氨酸残基缀合。
在又一个实施方式中，配体单元为抗体，并且巯基官能团以化学方式引入到抗体中，例如，通过半胱氨酸残基的引入。相应地，在一些实施方式中，连接子单元(具有或不具有附连的药物单元)通过配体单元的引入的半胱氨酸残基与配体单元缀合。
在一些实施方式中，所述配体靶向细胞表面受体或肿瘤相关抗原。
在一些实施方式中，所述细胞表面受体或肿瘤相关抗原可以包括HER2、HER3、claudin18.2、叶酸受体α(FRα)、BCMA、PSMA、TROP-2、CD19、CD20、CD22、CD30、CD79b、EGFR、c-Met或CEACAM5。
抗体药物偶联物(ADC)
另一方面，本申请提供一种抗体药物偶联物(ADC)，具有以下结构：Ab-(L-D) m，其中，所述Ab表示抗体或其抗原结合片段，L表示连接子，D表示药物单元，m表示1至10中的任意数。
在某些实施方式中，所述抗体包括单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体、完整抗体、抗体片段、人抗体、人源化抗体或嵌合抗体。
在某些实施方式中，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
在某些实施方式中，其中所述抗体为单克隆抗体。
在某些实施方式中，其中所述抗体为人抗体、人源化抗体或嵌合抗体。
在某些实施方式中，其中所述药物单元包括细胞毒性剂。
例如，药物单元可以是微管干扰药物，例如澳瑞他汀，像是单甲基澳瑞他汀E(monomethyl auristatin E；MMAE)、单甲基澳瑞他汀F(monomethyl auristatin F；MMAF)、和澳瑞他汀F(auristatin F；AF)。又例如，药物单元可以是微管破坏药物，例如美登木素生物碱，像是DM1、DM3、和DM4。又例如，药物单元可以是DNA损伤剂，像是加利车霉素(calicheamicins)、多卡霉素(duocarmycins)、SN-38、和吡咯并[2,1-c][1,4]苯并二氮杂(pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)。又例如，药物单元可以是瓢菌素(amanitins)、蒽环类物(anthracyclines)、浆果赤霉素(baccatins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、或长春花生物碱(vinca alkaloids)。
在某些实施方式中，所述药物单元为拓扑异构酶-Ⅰ抑制剂，如喜树碱衍生物DXd。
在某些实施方式中，其中所述抗体药物偶联物选自以下结构：
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O- CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2-O-CH 2-C(＝O)- Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；和
Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
其中，
所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
Figure PCTCN2022135216-appb-000030
其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
所述Dxd具有以下结构：
Figure PCTCN2022135216-appb-000031
其中1位的氨基的氮原子为附接位置的基团；
所述Ab表示抗体或其抗原结合片段，m表示1至10的任意数。
在某些实施方式中，其中所述抗体药物偶联物选自以下结构：
Figure PCTCN2022135216-appb-000032
Figure PCTCN2022135216-appb-000033
其中，所述Ab表示抗体或其抗原结合片段，m为1至10的任意数。
在某些实施方式中，所述抗体或其抗原结合片段包括：抗HER2抗体、抗HER3抗体、抗claudin18.2抗体、抗FRα抗体或其抗原结合片段。
本披露的抗体药物缀合物(ADC)化合物包括具有抗癌活性的ADC化合物。具体地，ADC化合物包括结合(即通过接头共价连接)至药物单元(如细胞毒性剂)的抗体或抗原结合片段(包括其抗原结合片段)，例如其中细胞毒性剂在不结合至抗体或抗原结合片段时具有细胞毒性或细胞生长抑制作用。
本申请的ADC化合物可将有效剂量的细胞毒性剂或细胞生长抑制剂选择性递送至癌细胞或肿瘤组织。已发现，所披露的ADC针对表达各别靶标抗原(例如HER2、HER3、claudin18.2、叶酸受体α(FRα)、CD138、EPHA2、MSLN、FOLH1、CDH6、CEACAM5、CFC1B、ENPP3、FOLR1、HAVCR1、KIT、MET、MUC16、SLC39A6、SLC44A4或STEAP1)的细胞具有强效细胞毒性和/或细胞生长抑制活性。在一些实施例中，ADC的细胞毒性和/或细胞生长抑制活性视细胞中的靶标抗原表达而定。在一些实施例中，所披露的ADC特别有效地杀灭表达靶标抗原的癌细胞，同时将脱靶杀灭减至最少。在一些实施例中，所披露的ADC不对不表达靶标抗原的癌细胞展现细胞毒性和/或细胞生长抑制作用。
可应用本发明的抗HER3抗体-药物偶联物的癌的种类的实例包括肺癌、肾癌(kidney cancer)、尿路上皮癌、结直肠癌(colorectal cancer)、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、转移性乳腺癌(metastatic breast cancer)、管腔乳腺癌(luminal breast cancer)、黑素瘤、肝癌、膀胱癌、胃癌(gastric(stomach)cancer)、胃肠道间质瘤、宫颈癌、头颈癌、食道癌、表皮样癌(epidermoid)、腹膜癌、成体多形性成胶质细胞瘤(adult glioblastoma multiforme)、肝癌(hepatic cancer)、肝细胞癌、结肠癌、直肠癌、结肠和直肠癌(colon and rectal cancer)、子宫内膜癌、子宫癌、唾液腺癌、肾脏癌(renal cancer)、外阴癌、甲状腺癌、肝恶性肿瘤(hepatic  carcinoma)、肛门癌、阴茎癌。使用本发明的抗HER3抗体-药物偶联物的治疗可靶向在治疗对象的癌细胞中表达抗体-药物偶联物的抗体可识别的HER3蛋白质的癌细胞。在本说明书中，“表达HER3蛋白的癌”是指，包含在其表面上具有HER3蛋白的细胞的癌或向血液中分泌HER3蛋白的癌。
可应用本发明的抗claudin18.2抗体-药物偶联物的癌的种类的实例包括胃肠道癌、胰腺癌、食道癌、非小细胞肺癌、肝癌、卵巢癌、肺癌、胆囊癌或头颈癌。
可应用本发明的抗FRα抗体-药物偶联物的癌的种类的实例包括卵巢癌、乳腺癌、肺癌、结直肠癌或肾癌。
可应用本发明的抗HER2抗体-药物偶联物的癌的种类的实例包括但不限于乳癌、胃癌、膀胱癌、尿道上皮细胞癌、食道癌、肺癌(例如肺腺癌)、子宫癌(例如子宫浆液性子宫内膜癌)、唾腺管癌、子宫颈癌、子宫内膜癌及卵巢癌(English等人(2013)Mol Diagn Ther.[分子诊断与疗法]17:85-99)。
在本申请中，抗体可以靶向细胞表面受体或肿瘤相关抗原。此外，抗体可以包括全长抗体或与受体、抗原、或其他目标细胞相关的部分结合、反应性相关、或复合的抗体片段。抗体可以是任何蛋白质、类蛋白质分子、或多肽，其与寻求治疗性修饰的细胞群的一部分结合、复合、或反应。在一些实施方式中，所述抗体可为嵌合抗体或其功能活性片段、人源化抗体或其功能活性片段、人抗体或其功能活性片段。
在一些实施方式中，抗体也可为功能性活性片段、抗体的衍生物或类似物，其中该抗体可免疫特异性地结合至目标抗原(例如：癌症抗原、病毒抗原、微生物抗原、或其他可结合至细胞或基质的抗原)。在此，“功能性活性”代表所述片段、衍生物或类似物可辨认相同的抗原，辨认衍生自抗原的片段、衍生物或类似物的抗体。其他有用的抗体包括抗体的片段，例如但不限于：F(ab’)2片段、Fab片段、Fab’、Fv片段及抗体的重链及轻链二聚体、或Fv片段或单链抗体(scFv)。于其他实施例中，抗体可为抗体的融合蛋白、或其功能性活性片段。
本申请的抗体可包括但不限于对抗下述抗原的抗体(在括号中指出示例性癌症)：CA125(卵巢癌)、CA15-3(癌；carcinomas)、CA19-9(癌)、L6(癌)、Lewis Y(癌)、Lewis X(癌)、α-胎儿蛋白(癌)、CA 242(大肠直肠癌)、胎盘碱性磷酸酶(癌)、前列腺癌特异性膜抗原(前列腺癌)、前列腺酸性磷酸酶(前列腺癌)、表皮生长因子(癌)、MAGE-1(癌)、MAGE-2(癌)、MAGE-3(癌)、MAGE-4(癌)、抗输铁蛋白受体(癌)、p97(黑色素瘤)、MUC1-KLH(乳癌)、CEA(大肠直肠癌)、gp100(黑色素瘤)、MART1(黑色素瘤)、前列腺特异性抗体(PSA)(前列腺癌)、IL-2受体(T-细胞白血病和淋巴瘤)、CD20(非霍奇金氏淋巴瘤)、CD52(白血病)、CD33(白血病)、CD22(淋 巴瘤)、人绒毛膜促性腺激素(癌)、CD38(多发性骨髓瘤)、CD40(淋巴瘤)、黏蛋白(癌)、P21(癌)、MPG(黑色素瘤)、及Neu癌基因产物(癌)。
本申请的抗体也可包括经修饰或未经修饰(亦即，通过任何分子的共价连接)的类似物和衍生物，只要这样的共价连接允许抗体保留其抗原结合免疫特异性。例如但不限于：抗体的类似物和衍生物，包括经过进一步修饰，例如：糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过习知的保护/阻隔基衍生化、蛋白酶切割、连接至细胞抗体单元或其他蛋白质等。可利用习知技术实现任何大量的化学修饰，包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素存在下的代谢合成等。此外，类似物或衍生物可包括一种或多种非天然氨基酸。在一些实施方式中，抗体可在与Fc受体作用的氨基酸残基中具有修饰(例如：取代、删除、或增加)。
在某些实施方式中，所述抗体或其抗原结合片段包括：抗HER2抗体、抗claudin18.2抗体、抗FRα抗体、抗HER3抗体、或其抗原结合片段。
在一个实施例中，其中所述抗体为抗HER3抗体，其中所述抗HER3抗体包含VH，其中所述VH包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含VH，其中所述VH包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含抗体重链恒定区。例如，其中所述抗体重链恒定区可以包括源自人IgG的恒定区。又例如，其中所述抗体重链恒定区可以包括源自人IgG1，IgG2，IgG3或IgG4的恒定区。
在某些实施方式中，其中所述抗HER3抗体包含抗体重链，所述抗体重链包括SEQ ID NO:17所述的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含VL，其中所述VL包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含VH和VL，其中所述VH包含HCDR1，HCDR2和HCDR3，所述VL包含LCDR1，LCDR2和LCDR3；其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列， 所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含VL，其中所述VL包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含VH和VL，其中所述VH包含SEQ ID NO:15所示的氨基酸序列，所述VL包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含抗体轻链恒定区。例如，其中所述抗体轻链恒定区可以包括人Igκ恒定区或人Igλ恒定区。
在某些实施方式中，其中所述抗HER3抗体包含抗体轻链，所述抗体轻链包括SEQ ID NO:18所述的氨基酸序列。
在某些实施方式中，其中所述抗HER3抗体包含抗体重链和抗体轻链，所述抗体重链包括SEQ ID NO:17所述的氨基酸序列，且所述抗体轻链包括SEQ ID NO:18所述的氨基酸序列。
在一个实施例中，其中所述抗体为抗Claudin18.2抗体，其中所述抗Claudin18.2抗体包含VH，其中所述VH包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2包含SEQ ID NO:20所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:21所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含VH，其中所述VH包含SEQ ID NO:32所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含抗体重链恒定区。例如，其中所述抗体重链恒定区可以包括源自人IgG的恒定区。又例如，其中所述抗体重链恒定区可以包括源自人IgG1，IgG2，IgG3或IgG4的恒定区。
在某些实施方式中，其中所述抗Claudin18.2抗体包含抗体重链，所述抗体重链包括SEQ ID NO:34所述的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含VL，其中所述VL包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:22所示的氨基酸序列，所述LCDR2包含SEQ ID NO:23所示的氨基酸序列，所述LCDR3包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含VH和VL，其中所述VH包含HCDR1，HCDR2和HCDR3，所述VL包含LCDR1，LCDR2和LCDR3；其中所述HCDR1包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2包含SEQ ID NO:20所示的氨基酸序 列，所述HCDR3包含SEQ ID NO:21所示的氨基酸序列，所述LCDR1包含SEQ ID NO:22所示的氨基酸序列，所述LCDR2包含SEQ ID NO:23所示的氨基酸序列，所述LCDR3包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含VL，其中所述VL包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含VH和VL，其中所述VH包含SEQ ID NO:32所示的氨基酸序列，所述VL包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含抗体轻链恒定区。例如，其中所述抗体轻链恒定区可以包括人Igκ恒定区或人Igλ恒定区。
在某些实施方式中，其中所述抗Claudin18.2抗体包含抗体轻链，所述抗体轻链包括SEQ ID NO:35所述的氨基酸序列。
在某些实施方式中，其中所述抗Claudin18.2抗体包含抗体重链和抗体轻链，所述抗体重链包括SEQ ID NO:34所述的氨基酸序列，且所述抗体轻链包括SEQ ID NO:35所述的氨基酸序列。
在一个实施例中，其中所述抗体为抗FRα抗体，其中所述抗FRΑ抗体包含VH，其中所述VH包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:36所示的氨基酸序列，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含VH，其中所述VH包含SEQ ID NO:49所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含抗体重链恒定区。例如，其中所述抗体重链恒定区可以包括源自人IgG的恒定区。又例如，其中所述抗体重链恒定区可以包括源自人IgG1，IgG2，IgG3或IgG4的恒定区。
在某些实施方式中，其中所述抗FRα抗体包含抗体重链，所述抗体重链包括SEQ ID NO:51所述的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含VL，其中所述VL包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:39所示的氨基酸序列，所述LCDR2包含SEQ ID NO:40所示的氨基酸序列，所述LCDR3包含SEQ ID NO:41所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含VH和VL，其中所述VH包含HCDR1，HCDR2和HCDR3，所述VL包含LCDR1，LCDR2和LCDR3；其中所述HCDR1包含SEQ  ID NO:36所示的氨基酸序列，所述HCDR2包含SEQ ID NO:37所示的氨基酸序列，所述HCDR3包含SEQ ID NO:38所示的氨基酸序列，所述LCDR1包含SEQ ID NO:39所示的氨基酸序列，所述LCDR2包含SEQ ID NO:40所示的氨基酸序列，所述LCDR3包含SEQ ID NO:41所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含VL，其中所述VL包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含VH和VL，其中所述VH包含SEQ ID NO:49所示的氨基酸序列，所述VL包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含抗体轻链恒定区。例如，其中所述抗体轻链恒定区可以包括人Igκ恒定区或人Igλ恒定区。
在某些实施方式中，其中所述抗FRα抗体包含抗体轻链，所述抗体轻链包括SEQ ID NO:52所述的氨基酸序列。
在某些实施方式中，其中所述抗FRα抗体包含抗体重链和抗体轻链，所述抗体重链包括SEQ ID NO:51所述的氨基酸序列，且所述抗体轻链包括SEQ ID NO:52所述的氨基酸序列。
组合物
另一方面，本申请提供一种药物组合物，其包含本申请所述的偶联物以及任选地药学上可接受的载体。
术语“载体”一般是指与化合物一起施用的稀释剂、佐剂或赋形剂。这样的药物载体可以是液体，如水和油，包括石油、动物、植物或合成来源的那些，如花生油、大豆油、矿物油、芝麻油。载体可以是盐水、阿拉伯树胶、明胶、淀粉糊、滑石、角蛋白、胶体氧化硅、尿素。另外，可使用辅助剂、稳定剂、增稠剂、润滑剂和着色剂。在一个实施方式中，当向患者施用时，化合物或组合物和药学上可接受的载体是无菌的。
本申请的偶联物可呈允许将化合物(药物单元)施用给患者以治疗与配体单元结合的抗原的表达相关的病症的任何形式。例如，偶联物可呈液体或固体的形式。示例性的施用途径是肠胃外。肠胃外施用包括皮下注射、静脉内、肌肉内、胸骨内注射或输注技术。在一个方面，组合物肠胃外施用。在一个方面，缀合物静脉内施用。可通过任何方便的途径进行施用，例如通过输注或推注。
在药物组合物的制备中使用的材料在使用的量上可以是无毒的。对于本领域普通技术人员显而易见的是，药物组合物中一种或多种活性成分的最佳剂量将取决于多种因素。相关因素包括但不限于动物的类型(例如，人)、化合物的特定形式、施用的方式和所采用的组合物。
组合物可例如呈液体形式。液体可用于通过注射递送。在用于通过注射施用的组合物中，还可包含表面活性剂、防腐剂、湿润剂、分散剂、悬浮剂、缓冲剂、稳定剂和等渗剂中的一种或多种。当组合物静脉内施用时，水是示例性的载体。盐水溶液以及右旋糖和甘油水溶液也可用作液体载体，特别是对于可注射溶液。合适的药物载体还包括赋形剂如淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇。如果需要，本发明的组合物还可含少量的湿润剂或乳化剂或pH缓冲剂。
可配制药物组合物以使得在向患者施用组合物后化合物是可生物利用的。组合物可呈一个或多个剂量单位的形式。对于静脉内施用，组合物可包含约0.01至约100mg偶联物每kg受试者体重。取决于所使用的药物，剂量可甚至更低，例如1.0μg/kg至5.0mg/kg、4.0mg/kg、3.0mg/kg、2.0mg/kg或1.0mg/kg、或1.0μg/kg至500.0μg/kg受试者体重。
通常，施用给患者的偶联物的剂量通常为约0.01mg/kg至约100mg/kg受试者体重或者1.0μg/kg至5.0mg/kg受试者体重。在一些实施方式中，施用给患者的剂量在约0.01mg/kg至约15mg/kg受试者体重之间。在一些实施方式中，施用给患者的剂量在约0.1mg/kg至约15mg/kg受试者体重之间。在一些实施方式中，施用给患者的剂量在约0.1mg/kg至约20mg/kg受试者体重之间。在一些实施方式中，施用的剂量在约0.1mg/kg至约5mg/kg或约0.1mg/kg至约10mg/kg受试者体重之间。
用途
另一方面，本申请提供本申请所述的偶联物或本申请所述的药物组合物在制备药物中的用途，所述药物用于治疗疾病或病症。
另一方面，本申请提供一种治疗有需要的受试者的疾病或病症的方法，其包括给予所述受试者有效量的本申请所述的偶联物。
在某些实施方式中，其中所述疾病或病症包括肿瘤。
可用本申请ADC治疗的癌症包括但不限于造血系统癌症，如例如淋巴瘤(霍奇金淋巴瘤和非霍奇金淋巴瘤)以及白血病和实体瘤。造血系统癌症的实例包括滤泡性淋巴瘤、间变性大细胞淋巴瘤、套细胞淋巴瘤、急性粒细胞性白血病、慢性粒细胞性白血病、慢性淋巴细胞性白血病、弥漫性大B细胞淋巴瘤和多发性骨髓瘤。实体瘤的实例包括纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、成骨肉瘤、脊索瘤、血管肉瘤、内皮肉瘤、淋巴管肉瘤、淋巴管内皮肉瘤、滑膜瘤、间皮瘤、尤因氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、结直肠癌、肾癌、胰腺癌、骨癌、乳腺癌、卵巢癌、前列腺癌、食道癌、胃癌、口腔癌、鼻癌、咽喉癌、鳞状细胞 癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、肝癌、胆管癌、绒毛膜癌、精原细胞瘤、胚胎癌、威尔姆斯氏瘤、宫颈癌、子宫癌、睾丸癌、小细胞肺癌、膀胱癌、肺癌、上皮癌、胶质瘤、多形性胶质母细胞瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质细胞瘤、脑膜瘤、皮肤癌、黑色素瘤、神经母细胞瘤和视网膜母细胞瘤。
在某些实施方式中，其中所述肿瘤包括HER3表达异常的肿瘤。
在某些实施方式中，其中所述肿瘤包括高表达HER3的肿瘤。
在某些实施方式中，其中所述肿瘤包括claudin18.2表达异常的肿瘤。
在某些实施方式中，其中所述肿瘤包括高表达claudin18.2的肿瘤。
在某些实施方式中，其中所述肿瘤包括FRα表达异常的肿瘤。
在某些实施方式中，其中所述肿瘤包括高表达FRα的肿瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的连接子、连接子药物、偶联物、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1
1.1 ADC的构建方法
使用换液装置(脱盐柱，超滤管等)将抗HER3抗体、抗Claudin18.2抗体、抗叶酸受体FRα抗体置换至目标buffer(柠檬酸缓冲液或盐酸组氨酸缓冲液，pH6.0-6.5)中并测量抗体浓度。保持抗体浓度在5-20mg/mL，与还原剂0.04-0.4mM TCEP混合孵育，在10-37℃下，维持1-5小时，以使抗体链间二硫键完全打开。随后使用磷酸将还原后抗体溶液pH调至6.0,温度将至室温。用DMSO溶解连接子-小分子药物，计算其浓度。按照合适的药物分子/抗体分子比例(摩尔比>10)，将连接子-药物溶液(04-Dxd、07-Dxd、08-Dxd、09-Dxd，其中04-Dxd中氨基酸单元替换为GGFG)与完全还原后的抗体溶液混合，保持DMSO比例不超过10％(v/v)，维持0.5-2小时，反应结束。ADC粗产品溶液过滤后，使用纯化装置(脱盐柱，超滤管，切向流系统等)进行纯化，去除溶液中的有机小分子杂质和多余的DMSO，然后将ADC产品置换至保存缓冲液中。
表1抗体轻链及重链序列
Figure PCTCN2022135216-appb-000034
Figure PCTCN2022135216-appb-000035
表2连接子药物结构
Figure PCTCN2022135216-appb-000036
Figure PCTCN2022135216-appb-000037
1.2 ADC产物DAR检测
使用HIC-HPLC方法检测，在pH 7.0流动相条件下进行上样洗脱，检测指定波长下的吸收峰面积，具体测试方法表3，ADC产物DAR在7-8之间。
表3：HIC-HPLC检测方法
Figure PCTCN2022135216-appb-000038
1.3 HIC-HPLC DAR值检测结果
Figure PCTCN2022135216-appb-000039
实施例2
2.1 HER3 ADC体外活性评价
将处于对数生长期的人结肠癌肿瘤细胞SW620用胰酶消化后，用对应的培养基重悬，调整密度为2×104/ml，按照100μl/孔进行96孔微孔板铺板，使得每孔细胞个数为2×10 3个，其余每孔加200μl培养基。37℃、5％CO 2培养48h后，将细胞培养板中的上清吸出，用培养基将连接子-小分子药物(04-Dxd、07-Dxd、08-Dxd、09-Dxd)、HER3ADC药物(HER3-ADC04、HER3-ADC07、HER3-ADC08、HER3-ADC09)，单抗对照进行稀释，按照起始浓度为400nM开始2倍梯度稀释，共9个浓度梯度；加入稀释好的样品，100μl/孔。继续培养4、6天进行 显色，将细胞板取出，在室温下平衡15-20min，加入100μl的CellTiter-
Figure PCTCN2022135216-appb-000040
Luminescent Cell Viability分析液(购自promega)，200rpm振荡器震荡2min，避光18min后，用M5酶标仪进行读数。
Figure PCTCN2022135216-appb-000041
然后以浓度的对数为横坐标，杀伤率为纵坐标进行四参数拟合作图。
结果：图2A显示HER3单抗偶联Dxd后相比裸抗(RS-HT-002)对SW620靶细胞具有明显的杀伤作用。
图2B显示不同连接子-Dxd对人结肠癌细胞SW620均具有杀伤作用，07-Dxd、08-Dxd、09-Dxd杀伤作用明显优于04-Dxd。
图3显示含不同连接子的anti-HER3 ADC对人结肠癌细胞SW620均具有杀伤作用，HER3-ADC07、HER3-ADC08、HER3-ADC09杀伤作用明显优于HER3-ADC04。
图4显示含不同连接子的anti-HER3 ADC对非靶细胞CHO-K1均无明显杀伤作用。
2.2 HER3 ADC血浆稳定性评价
为了检测ADC药物在体内的稳定性，本研究在体外采用人血浆孵育模拟体内环境，进行检测。具体地，将HER3ADC加入10％人血浆使得抗体终浓度为1mg/ml，37℃孵育3天、7天，然后进行细胞杀伤检测。将对数生长期SW620靶细胞消化后按照一定的密度进行铺板，37℃、5％CO 2培养48h后加入梯度稀释的血浆孵育过的ADC，同时以同样血浆稀释的未孵育ADC作为对照进行实验。
结果：图5显示HER3-ADC08、HER3-ADC09在血浆中孵育3天或者7天后，细胞杀伤活性未发现明显变化。
2.3 HER3 ADC体内活性评价
选用雌性SPF级BALB/c裸小鼠，4～6周龄，适应性饲养3-4天后准备种瘤。将对数生长期人结肠癌SW620细胞，消化后用无血清1640培养基洗2次后，稀释到2.5×10 7/ml，小鼠右腋窝皮下接种0.2ml共5×10 6个细胞/只。接种1周后肿瘤长至150±50mm 3时进行随机区组设计分组，每组5只。空白模型组为PBS对照，ADC药物给药剂量为2、6、10mg/kg，小分子对照组按照10mg/kg ADC药物同等摩尔量给药，给药频率一周一次，其中6、10mg/kg剂量组共给药四次，2mg/kg组共给药八次，腹腔注射给药，每周测量肿瘤2次，每次给药前记录肿瘤体积和小鼠体重数据。
肿瘤体积计算：V(mm 3)＝(W 2×L)/2，W为肿瘤近中线短直径，L为肿瘤近中线长直径。当肿瘤体积大于2000mm 3，体重减轻大于20％或观察到对重要生理机能的干扰或肿瘤性溃疡 坏死时颈椎脱臼法处死小鼠。
结果：图6显示HER3-ADC07、HER3-ADC08、HER3-ADC09均能显著抑制SW620移植肿瘤的生长。其中6、10mg/kg给药组四次给药后小鼠肿瘤均发生完全消退，停止给药三周后有个别小鼠肿瘤开始生长。2mg/kg给药组均能抑制肿瘤生长，HER3-ADC08对肿瘤一致作用优于HER3-ADC07和HER3-ADC09。图7显示治疗过程中，ADC治疗组相比PBS对照组，小鼠体重变化未发生明显差异。
实施例3
3.1 Claudin18.2 ADC体外活性评价
将处于对数生长期的HEK293-hCLDN18.2、NUGC4、NUGC4-hCLDN18.2用胰酶消化后，用对应的培养基重悬，调整密度为2×10 4/ml，按照100μl/孔进行96孔微孔板铺板，使得每孔细胞个数为2×10 3个，其余每孔加200μl培养基。37℃、5％CO 2培养48h后，将细胞培养板中的上清吸出，用培养基将连接子-小分子药物(08-Dxd、09-Dxd)、ADC药物(Claudin18.2-ADC08、Claudin18.2-ADC09)，单抗对照(hu782)进行稀释，按照起始浓度为400nM开始2倍梯度稀释，共9个浓度梯度；加入稀释好的样品，100μl/孔。继续培养4、6天进行显色，将细胞板取出，在室温下平衡15-20min，加入100μl的CellTiter-
Figure PCTCN2022135216-appb-000042
Luminescent Cell Viability分析液(购自promega)，200rpm振荡器震荡2min，避光18min后，用M5酶标仪进行读数。
结果处理：
Figure PCTCN2022135216-appb-000043
然后以浓度的对数为横坐标，杀伤率为纵坐标进行四参数拟合作图。
结果：图8、图9、图10显示Claudin18.2-ADC08、Claudin18.2-ADC09抗体偶联药物对NUGC4、NUGC4-hCLDn18.2、HEK293-hCLDn18.2三个Claudin18.2阳性靶细胞具有明显的杀伤作用。
3.2 Claudin18.2 ADC血浆稳定性评价
为了检测ADC药物在体内的稳定性，本研究在体外采用人血浆孵育模拟体内环境，进行检测。具体地，将Claudin18.2-ADC08加入10％人血浆使得抗体终浓度为1mg/ml，37℃孵育3天、7天，然后进行细胞杀伤检测。将对数生长期HEK293-hCLDN18.2靶细胞消化后按照一定的密度进行铺板，37℃、5％CO 2培养48h后加入梯度稀释的血浆孵育Claudin18.2-ADC08，同时以同样血浆稀释的未孵育Claudin18.2-ADC08作为对照进行实验。
结果：图11显示Claudin18.2-ADC08在血浆中孵育3天或者7天后，细胞杀伤活性未发现明显变化。
3.3 Claudin18.2 ADC体内活性评价
选用雌性SPF级BALB/c裸小鼠，4～6周龄，适应性饲养3-4天后准备种瘤。将对数生长期NUGC4细胞，消化后用无血清1640培养基洗2次后，稀释到2.5×10 7/ml，小鼠右腋窝皮下接种0.2ml共5×10 6个细胞/只。接种1周后肿瘤长至150±50mm 3时进行随机区组设计分组，每组5只。空白模型组为PBS对照。小分子对照组按照10mg/kg ADC药物同等摩尔量给药，Claudin18.2-ADC08给药剂量按照1mg/kg、3mg/kg、10mg/kg三个剂量组给药，给药频率一周一次，共给药四次，停药后继续测量，每周测量2次，腹腔注射给药，每次给药前记录肿瘤体积和小鼠体重数据。
肿瘤体积计算：V(mm 3)＝(W 2×L)/2，W为肿瘤近中线短直径，L为肿瘤近中线长直径。当肿瘤体积大于2000mm 3，体重减轻大于20％或观察到对重要生理机能的干扰或肿瘤性溃疡坏死时颈椎脱臼法处死小鼠。
结果：图12显示在NUGC4小鼠皮下移植肿瘤模型中，Claudin18.2-ADC08具有抗肿瘤生长活性，具有一定的剂量效应。
实施例4
4.1叶酸受体FRαADC药物体外活性评价
将处于对数生长期的卵巢癌OVCAR3细胞用胰酶消化后，用对应的培养基重悬，调整密度为2×10 4/ml，按照100μl/孔进行96孔微孔板铺板，使得每孔细胞个数为2×10 3个，其余每孔加200μl培养基。37℃、5％CO 2培养48h后，将细胞培养板中的上清吸出，用培养基将连接子-小分子药物(08-Dxd、09-Dxd)、ADC药物(FRα-ADC08、FRα-ADC09)，单抗对照(M9346A)进行稀释，按照起始浓度为400nM开始2倍梯度稀释，共9个浓度梯度；加入稀释好的样品，100μl/孔。继续培养4、6天进行显色，将细胞板取出，在室温下平衡15-20min，加入100μl的CellTiter-
Figure PCTCN2022135216-appb-000044
Luminescent Cell Viability分析液(购自promega)，200rpm振荡器震荡2min，避光18min后，用M5酶标仪进行读数。
结果处理：
Figure PCTCN2022135216-appb-000045
然后以浓度的对数为横坐标，杀伤率为纵坐标进行四参数拟合作图。
结果：图13显示FRα抗体偶联物FRα-ADC08、FRα-ADC09均对OVCAR3细胞具有显著杀伤活性。
4.2叶酸受体FRαADC血浆稳定性评价
为了检测ADC药物在体内的稳定性，本研究在体外采用人血浆孵育模拟体内环境，进 行检测。具体地，将FRα-ADC08加入50％人血浆使得抗体终浓度为1mg/ml，37℃孵育3天、7天，然后进行细胞杀伤检测。将对数生长期OVCAR3靶细胞消化后按照一定的密度进行铺板，37℃、5％CO 2培养48h后加入梯度稀释的血浆孵育FRα-ADC08，同时以同样血浆稀释的未孵育FRα-ADC08作为对照进行实验。
结果：图14显示FRα-ADC08在血浆中孵育3天或者7天后，细胞杀伤活性未发现明显变化。
4.3叶酸受体FRαADC体内活性评价
选用雌性SPF级BALB/c裸小鼠，3～4周龄，适应性饲养3-4天后准备种瘤。将对数生长期OVCAR3细胞，消化后用无血清1640培养基洗2次后，稀释到3×10 7/ml与等体积基质胶混合，小鼠右腋窝皮下接种0.2ml共3×10 6个细胞/只。接种1周后肿瘤长至150±50mm 3时进行随机区组设计分组，每组5只。空白模型组为PBS对照。小分子对照组按照5mg/kg ADC药物同等摩尔量给药，FRα-ADC08给药剂量按照2mg/kg、5mg/kg两个剂量组给药，给药频率一周一次，每周测量2次，腹腔注射给药，每次给药前记录肿瘤体积和小鼠体重数据。
肿瘤体积计算：V(mm 3)＝(W 2×L)/2，W为肿瘤近中线短直径，L为肿瘤近中线长直径。当肿瘤体积大于2000mm 3，体重减轻大于20％或观察到对重要生理机能的干扰或肿瘤性溃疡坏死时颈椎脱臼法处死小鼠。
结果：图15显示在OVCAR3小鼠皮下移植肿瘤模型中，FRα-ADC08对肿瘤生长具有抑制作用。

Claims (57)

  1. 连接子，所述连接子包括氨基酸单元(RL)，所述RL包括：-Val-Ala-Q-Gly-Phe-Gly-，其中Q为键、单个氨基酸或二肽。
  2. 根据权利要求1所述的连接子，其中RL可被酶裂解。
  3. 根据权利要求1-2中任一项所述的连接子，其中RL可被组织蛋白酶B(Cathepsin B)裂解。
  4. 根据权利要求1-3中任一项所述的连接子，其中所述Q为键。
  5. 根据权利要求1-3中任一项所述的连接子，其中所述Q为-Ser-或-Ser-Ser-。
  6. 根据权利要求1-5中任一项所述的连接子，其中RL包括-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
  7. 根据权利要求1-6中任一项所述的连接子，所述连接子选自以下结构：-Z-A-S*-RL-和-Z-A-S*-RL-Y-；其中，所述Z为延伸子单元，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元。
  8. 根据权利要求7所述的连接子，其中所述Z包含以下结构：-(琥珀酰亚胺-3-基-N)-，-CH 2-C(＝O)-NH-或-C(＝O)-；
    其中，所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
    Figure PCTCN2022135216-appb-100001
    其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置。
  9. 根据权利要求7-8中任一项所述的连接子，其中所述A选自以下结构：
    -C 1-C 10亚烷基-、-C 1-C 10亚杂烷基-、-C 3-C 8碳环基-、-O-(C 1-C 8亚烷基)-、-亚芳基-、-C 1-C 10亚烷基-亚芳基-、-亚芳基-C 1-C 10亚烷基-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-、-C 3-C 8杂环基-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-、-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚杂烷基-C(＝O)-、-C 3-C 8碳环基-C(＝O)-、-O-(C 1-C 8亚烷基)-C(＝O)-、-亚芳基-C(＝O)-、-C 1-C 10亚烷基-亚芳基-C(＝O)-、-亚芳基-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-C(＝O)-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-C(＝O)-、-C 3-C 8杂环基-C(＝O)-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-C(＝O)-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-C(＝O)-、-C 1-C 10亚烷基-NH-、-C 1-C 10亚杂烷基-NH-、-C 3-C 8碳环基-NH-、-O-(C 1-C 8亚烷基)-NH-、-亚芳基-NH-、-C 1-C 10亚烷基-亚芳基-NH-、-亚芳基-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-NH-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-NH-、-C 3-C 8杂环 基-NH-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-NH-、-(C 3-C 8杂环基)-C 1-C 10亚烷基-NH-、-C 1-C 10亚烷基-S-、-C 1-C 10亚杂烷基-S-、-C 3-C 8碳环基-S-、-O-(C 1-C 8亚烷基)-S-、-亚芳基-S-、-C 1-C 10亚烷基-亚芳基-S-、-亚芳基-C 1-C 10亚烷基-S-、-C 1-C 10亚烷基-(C 3-C 8碳环基)-S-、-(C 3-C 8碳环基)-C 1-C 10亚烷基-S-、-C 3-C 8杂环基-S-、-C 1-C 10亚烷基-(C 3-C 8杂环基)-S-或-(C 3-C 8杂环基)-C 1-C 10亚烷基-S-；
    其中，所述A任选地被碱性单元(BU)所取代，所述碱性单元为-(CH 2) xNH 2、-(CH 2) xNHR a或-(CH 2) xNR a 2；其中x为1-4的任意整数；并且每个R a独立地选自C 1-C 6烷基和C 1-C 6卤代烷基，或者两个R a基团与它们所附连的氮组合形成4-至6-元杂环烷基环、或者氮杂环丁烷基、吡咯烷基或哌啶基基团。
  10. 根据权利要求7-9中任一项所述的连接子，其中所述A选自以下结构：为-(C 1-C 10)亚烷基-C(＝O)-，其中A的所述亚烷基部分任选地被所述碱性单元(BU)所取代。
  11. 根据权利要求7-10中任一项所述的连接子，其中所述连接子包含以下结构：
    Figure PCTCN2022135216-appb-100002
    其中a为1至8中的任意整数。
  12. 根据权利要求11所述的连接子，其中所述a为2至5中的任意整数。
  13. 根据权利要求7-12中任一项所述的连接子，其中所述Y包含-NH-(CH 2)n 1-La-Lb-Lc-所示的结构，其中所述La表示-C(＝O)-NH-、-NR 1-(CH 2)n 2-、-O-或单键，R 1表示H、C 1-C 6烷基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基；
    所述Lb表示-CR 2(-R 3)-或单键，其中R 2及R 3各自独立地表示H、-NH 2、C 1～C 6亚烷基、C 1～C 6亚烷基-氨基、C 1-C 6亚烷基-羧基、或C 1-C 6亚烷基-羟基，且R 2和R 3不同时为-NH 2；
    n 1表示0～6的整数；
    Lc表示-CH 2-或-C(＝O)-。
  14. 根据权利要求7-13中任一项所述的连接子，其中所述Y包含NH-(CH 2)n 1-O-(CH 2)n 2-C(＝O)-，其中n 1或n 2各自独立地为0至5的任意整数。
  15. 根据权利要求7-14中任一项所述的连接子，其中所述Y包含-NH-CH 2-C(＝O)-、-NH-CH 2CH 2-C(＝O)-、-NH-(CH 2) 3-C(＝O)-、-NH-(CH 2) 4-C(＝O)-、-NH-(CH 2) 5-C(＝O)-、-NH-CH 2-O-CH 2-C(＝O)-、或-NH-(CH 2) 2-O-CH 2-C(＝O)-。
  16. 根据权利要求7-15中任一项所述的连接子，其中所述S*为键，且所述连接子为-Z-A-RL-或-Z-A-RL-Y-。
  17. 根据权利要求7-15中任一项所述的连接子，其中所述S*为分配剂，且所述连接子为-Z-A-S*-RL-或-Z-A-S*-RL-Y-。
  18. 根据权利要求17所述的连接子，其中所述S*包含PEG单元。
  19. 根据权利要求18所述的连接子，所述S*具有下式：
    -NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)-；
    -NH-(CH 2CH 2O) b-CH 2CH 2C(＝O)NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；或
    -NH-(CH 2CH 2O) b-CH 2CH 2NH-(CH 2CH 2O)-CH 2CH 2C(＝O)-；
    其中所述S*左侧与A连接，右侧与RL连接，并且b为2至20的任意整数。
  20. 根据权利要求19所述的连接子，其中所述b为2、4、8或12。
  21. 根据权利要求1-20中任一项所述的连接子，所述连接子选自以下结构：
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    -(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-；
    -CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    -C(＝O)-CH 2CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-；
    其中，
    所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
    Figure PCTCN2022135216-appb-100003
    其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
    所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)。
  22. 根据权利要求1-21中任一项所述的连接子，所述连接子药物选自以下结构：
    Figure PCTCN2022135216-appb-100004
  23. 连接子药物，其包含权利要求1-22中任一项所述的连接子。
  24. 根据权利要求23项所述的连接子药物，所述连接子药物选自以下结构：
    Z-A-S*-RL-D和Z-A-S*-RL-Y-D；
    其中，所述Z’为延伸子单元前体，所述A为键或接头单元；所述S*为键或分配剂；Y为间隔子单元；D为药物单元。
  25. 根据权利要求23-24中任一项所述的连接子药物，其中所述延伸子单元前体Z’选自(马来 酰亚胺-N-基)-、(吡咯烷-2,5-二酮-N-基)-、M-CH 2-C(＝O)-NH-和HS-(CH 2) 1-5-C(＝O)-，其中M为卤素。
  26. 根据权利要求23-25中任一项所述的连接子药物，其中所述药物单元包括细胞毒性剂。
  27. 根据权利要求23-26中任一项所述的连接子药物，其中所述药物单元包括：瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱、(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂
    Figure PCTCN2022135216-appb-100005
    (pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、长春花生物碱(vincaalkaloids)、或其衍生物。
  28. 根据权利要求23-27中任一项所述的连接子药物，其中所述药物单元包括DNA拓扑异构酶I抑制剂。
  29. 根据权利要求23-28中任一项所述的连接子药物，其中所述药物单元包括喜树碱或其衍生物。
  30. 根据权利要求23-29中任一项所述的连接子药物，其中所述药物单元具有以下结构：
    Figure PCTCN2022135216-appb-100006
    其中1位的氨基的氮原子为附接位置的基团。
  31. 根据权利要求30所述的连接子药物，其中所述连接子药物单元具有以下结构：
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (马来酰亚胺-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    (吡咯烷-2,5-二酮-N-基)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    M-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd
    HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；和
    HS-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd；
    其中，
    所述(马来酰亚胺-N-基)-具有以下结构：
    Figure PCTCN2022135216-appb-100007
    其中氮原子是附接位置的基团；
    所述(吡咯烷-2,5-二酮-N-基)-具有以下结构：
    Figure PCTCN2022135216-appb-100008
    其中氮原子是附接位置的基团；
    所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
    所述-Dxd具有以下结构：
    Figure PCTCN2022135216-appb-100009
    其中1位的氨基的氮原子为附接位置的基团；
    所述M表示卤原子。
  32. 根据权利要求23-31中任一项所述的连接子药物，其中所述连接子药物单元选自以下结构：
    Figure PCTCN2022135216-appb-100010
  33. 偶联物，其包含权利要求1-22中任一项所述的连接子。
  34. 根据权利要求33所述的偶联物，其包含配体。
  35. 根据权利要求34所述的偶联物，其中所述配体靶向细胞表面受体或肿瘤相关抗原。
  36. 根据权利要求35所述的偶联物，其中所述细胞表面受体或肿瘤相关抗原包括：HER2、HER3、claudin18.2、叶酸受体α(FRα)、BCMA、PSMA、TROP-2、CD19、CD20、CD22、 CD30、CD79b、EGFR、c-Met、CEACAM5。
  37. 根据权利要求34-36中任一项所述的偶联物，其中所述配体包括抗体或其抗原结合片段。
  38. 根据权利要37所述的偶联物，所述抗体包括单克隆抗体、多克隆抗体、二聚体、多聚体、多特异性抗体、完整抗体、抗体片段、人抗体、人源化抗体或嵌合抗体。
  39. 根据权利要求37所述的偶联物，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
  40. 根据权利要求37-39中任一项所述的偶联物，其中所述抗体为单克隆抗体。
  41. 根据权利要求37-40中任一项所述的偶联物，其中所述抗体为人抗体、人源化抗体或嵌合抗体。
  42. 根据权利要求33-41中任一项所述的偶联物，其包括配体药物偶联物，并具有以下结构：C-(L-D) m，其中，所述C表示配体单元，L表示连接子，D表示药物单元，m表示1至10中的任意数。
  43. 根据权利要求33-42中任一项所述的偶联物，所述配体药物偶联物为抗体药物偶联物(ADC)，并具有以下结构：Ab-(L-D) m，其中，所述Ab表示抗体或其抗原结合片段，L表示连接子，D表示药物单元，m表示1至10中的任意数。
  44. 根据权利要求42-43中任一项所述的偶联物，其中所述药物单元包括细胞毒性剂。
  45. 根据权利要求42-44中任一项所述的偶联物，其中所述药物单元包括：瓢菌素(amanitins)、蒽环类物(anthracyclines)、澳瑞他汀(auristatins)、浆果赤霉素(baccatins)、加利车霉素(calicheamicins)、喜树碱(camptothecins)、西马多丁(cemadotins)、秋水仙碱、(colchicines)、秋水仙胺(colcimids)、考布他汀(combretastatins)、隐菲辛(cryptophycins)、圆皮海绵内酯(discodermolides)、多卡霉素(duocarmycins)、多烯紫杉醇(docetaxel)、阿霉素(doxorubicin)、多卡霉素(duocarmycins)、棘霉素(echinomycins)、艾榴塞洛素(eleutherobins)、埃博霉素(epothilones)、雌莫司汀(estramustines)、偏端霉素(lexitropsins)、美登素(maytansines)、美登木素生物碱(maytansinoids)、氨甲蝶呤(methotrexate)、纺锤菌素(netropsins)、吡咯并[2,1-c][1,4]苯并二氮杂
    Figure PCTCN2022135216-appb-100011
    (pyrrolo[2,1-c][1,4]benzodi-azepines；PBDs)、嘌呤霉素(puromycins)、根瘤菌素(rhizoxins)、SN-38、紫杉烷(taxanes)、微管蛋白裂解素(tubulysins)、长春花生物碱(vincaalkaloids)、或其衍生物。
  46. 根据权利要求42-45中任一项所述的偶联物，其中所述药物单元包括DNA拓扑异构酶I抑制剂。
  47. 根据权利要求42-46中任一项所述的偶联物，其中所述药物单元包括喜树碱或其衍生 物。
  48. 根据权利要求42-47中任一项所述的偶联物，其中所述药物单元具有以下结构：
    Figure PCTCN2022135216-appb-100012
    其中1位的氨基的氮原子为附接位置的基团。
  49. 根据权利要求43-48中任一项所述的偶联物，其中所述抗体药物偶联物选自以下结构：
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2CH 2CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-(琥珀酰亚胺-3-基-N)-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-VASSGFG-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-CH 2-C(＝O)-NH-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m
    Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2CH 2CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2-O-CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；和
    Ab-[-S-CH 2CH 2-C(＝O)-NH-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2O-CH 2CH 2-C(＝O)-RL-NH-CH 2CH 2CH 2-C(＝O)-Dxd] m；
    其中，
    所述-(琥珀酰亚胺-3-基-N)-具有以下结构：
    Figure PCTCN2022135216-appb-100013
    其中该结构的3位的波浪线表示与配体单元的附接位置，在1位的N原子上的波浪线表示与A的附连位置；
    所述-RL-表示以下结构：-Val-Ala-Gly-Phe-Gly-(VAGFG)、-Val-Ala-Ser-Gly-Phe-Gly-(VASGFG)、或-Val-Ala-Ser-Ser-Gly-Phe-Gly-(VASSGFG)；
    所述-Dxd具有以下结构：
    Figure PCTCN2022135216-appb-100014
    其中1位的氨基的氮原子为附接位置的基团；
    所述Ab表示抗体或其抗原结合片段，m表示1至10的任意数。
  50. 根据权利要求43-49中任一项所述的偶联物，其中所述抗体药物偶联物选自以下结构：
    Figure PCTCN2022135216-appb-100015
    其中，所述Ab表示抗体或其抗原结合片段，m为1至10的任意数。
  51. 根据权利要求37-50中任一项所述的偶联物，所述抗体或其抗原结合片段包括：抗HER2抗体、抗HER3抗体、抗claudin18.2抗体、抗FRα抗体或其抗原结合片段。
  52. 药物组合物，其包含前述权利要求33-51中任一项所述的偶联物以及任选地药学上可接 受的载体。
  53. 权利要求1-22中任一项的连接子、权利要求23-32中任一项的连接子药物、权利要求33-51中任一项的偶联物或权利要求52所述的药物组合物在制备药物中的用途，所述药物用于治疗疾病或病症。
  54. 根据权利要求53所述的用途，其中所述疾病或病症包括肿瘤。
  55. 根据权利要求54所述的用途，其中所述疾病或病症包括实体瘤和血液瘤。
  56. 治疗有需要的受试者的疾病或病症的方法，其包括给予所述受试者有效量的权利要求33-51中任一项的偶联物。
  57. 根据权利要求56所述的方法，其中所述疾病或病症包括肿瘤。
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