WO2021143741A1 - 靶向多肽-药物缀合物及其用途 - Google Patents

靶向多肽-药物缀合物及其用途 Download PDF

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WO2021143741A1
WO2021143741A1 PCT/CN2021/071595 CN2021071595W WO2021143741A1 WO 2021143741 A1 WO2021143741 A1 WO 2021143741A1 CN 2021071595 W CN2021071595 W CN 2021071595W WO 2021143741 A1 WO2021143741 A1 WO 2021143741A1
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antibody
conjugate
group
seq
mab
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PCT/CN2021/071595
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English (en)
French (fr)
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王英召
刘长茹
张旭
刘艳玲
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北京海步医药科技有限公司
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Priority to CN202180008220.7A priority Critical patent/CN115052632A/zh
Publication of WO2021143741A1 publication Critical patent/WO2021143741A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, in particular to a conjugate of a targeted polypeptide and a small molecule therapeutic drug, and its use for preparing drugs.
  • Camptothecin is an alkaloid extracted by American chemists from the bark and fruit of Chinese Davidia family plant Camptotheca acuminata in 1966. It has excellent anti-tumor activity and its chemical structure is
  • Camptothecin derivatives have improved anti-tumor activity through reasonable modification, and can be used as a potential drug candidate molecule.
  • small molecule drugs usually have problems such as low targeting, being cleared by the body's circulatory system before reaching the tumor site, killing normal cells, or having serious side effects.
  • Antibody-drug conjugate composed of antibodies and medicinal chemicals through linkers, not only has the anti-tumor efficacy of highly cytotoxic small molecule drugs, but also combines the high selectivity, stability and beneficial drugs of antibodies Generation dynamics characteristics.
  • ADC Antibody-drug conjugate
  • DAR drug-to-antibody ratio
  • This application provides a targeted polypeptide-drug conjugate, and a preparation method and composition thereof.
  • the conjugates, methods, and compositions disclosed in this application can be used to treat a variety of diseases and conditions, and can include those for which selective targeting moieties exist, such as tumors.
  • the application also provides the use of the conjugate together with other tumor therapies or drugs in the preparation of drugs for the treatment of tumors.
  • the conjugate of the present application has at least one of the following properties: (1) can bind tumor antigens with high affinity, (2) excellent in vitro killing target cell activity, inhibit tumor cell growth, (3) excellent in vivo inhibition of tumor cell growth The activity and (4) good uniformity.
  • the application provides a conjugate having the structure shown in Formula I:
  • R 1 is methyl substituted by halogen
  • R 2 is selected from hydrogen, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl
  • R 3 is selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 alkyl
  • Mab is the targeting polypeptide
  • L is the linker.
  • the conjugate has the structure shown in Formula II:
  • R 1 is methyl substituted by halogen
  • R 2 is selected from hydrogen, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl
  • R 3 is selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 alkyl.
  • R 1 in the conjugate is -CF 2 H.
  • the conjugate has the structure shown in Formula III:
  • the Mab includes an antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab) 2 , Fv and ScFv fragments.
  • the Mab specifically binds to a tumor-specific antigen.
  • the tumor-specific antigen is selected from the group consisting of CLDN18.2, Nectin 4, Her2 and Trop2.
  • the Mab includes the antibody heavy chain HCDR3, and the HCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 19, 25, 31, and 39.
  • the Mab includes the antibody heavy chain HCDR2, and the HCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 18, 24, 30, and 38.
  • the Mab includes the antibody heavy chain HCDR1, and the HCDR1 includes the amino acid sequence shown in any one of SEQ ID NOs: 17, 23, 29, and 37.
  • the Mab includes the antibody heavy chain variable region VH, and the VH includes the amino acid sequence shown in any one of SEQ ID NOs: 1, 5, 32, and 40.
  • the Mab includes an antibody heavy chain, and the antibody heavy chain includes the amino acid sequence shown in any one of SEQ ID NOs: 9, 11, 13, and 15.
  • the Mab comprises the antibody light chain LCDR3, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 22, 28, 35, and 43.
  • the Mab includes the antibody light chain LCDR2, and the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 21, 27, 34, and 42.
  • the Mab comprises the antibody light chain LCDR1, and the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 20, 26, 33, and 41.
  • the Mab includes the antibody light chain variable region VL, and the VL includes the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 36, and 44.
  • the Mab includes an antibody light chain, and the antibody light chain includes the amino acid sequence shown in any one of SEQ ID NOs: 10, 12, 14, and 16.
  • the present application provides a method for producing the conjugate, which includes:
  • R 1 is methyl substituted by halogen
  • R 2 is selected from hydrogen, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl
  • R 3 is selected from hydrogen, amino, C 1 -C 6 Alkyl and substituted C 1 -C 6 alkyl
  • Mab is a targeting polypeptide
  • L is a linker
  • the compound represented by formula IV is conjugated with the Mab to obtain the conjugate.
  • the present application provides a pharmaceutical composition comprising the conjugate described in the present application.
  • this application provides the use of the conjugate or the pharmaceutical composition described in this application in the preparation of a drug for the treatment of tumors.
  • the application provides the use of the conjugate together with other tumor therapies or drugs in the preparation of drugs for the treatment of tumors.
  • the other tumor therapy or drug is selected from the group consisting of chemotherapy, radiotherapy, miRNA and oligonucleotide.
  • the tumor includes a solid tumor and/or a non-solid tumor.
  • the tumor is a solid tumor.
  • the tumor is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, prostate cancer, and gastric cancer.
  • Figure 1 shows the binding activity of the conjugate described in this application with Her2 on Ba/F2 cells
  • Figure 2 shows the binding activity of the conjugate described in this application with Trop2 on 293T cells
  • Figure 3 shows that the conjugate of the present application inhibits the activity of SK-BR-3 cells in vitro
  • Figure 4 shows that the conjugate of the present application inhibits the activity of NCI-N87 cells in vitro
  • Figure 5 shows that the conjugate of the present application inhibits the activity of MDA-MB-468 cells in vitro
  • Figure 6 shows that the conjugate of the present application inhibits the activity of BXPC-3 cells in vitro
  • Figure 7 shows that the conjugate of the present application inhibits the activity of COLO-205 cells in vitro
  • Figure 8 shows that the conjugates described in this application inhibit the activity of HCC1954 cells in vitro.
  • Figure 9 shows that HB010 inhibits breast cancer growth
  • Figure 10 shows the weight change of breast cancer mice after administration of the conjugate of the present application
  • Figure 11 shows that the conjugate HB060 of the present application inhibits the growth of breast cancer
  • Figure 12 shows the weight change of breast cancer mice after administration of the conjugate of the present application
  • Figure 13 shows that the conjugate HB030 described in this application inhibits the growth of breast cancer
  • Figure 14 shows the weight change of breast cancer mice after administration of the conjugate of the present application
  • Figure 15 shows that the conjugate HB030 described in this application inhibits the growth of breast cancer
  • Figure 16 shows the weight change of breast cancer mice after administration of the conjugate of the present application
  • Figure 17 shows that the conjugate HB010 of the present application inhibits the growth of gastric cancer
  • Figure 18 shows the weight change of mice with gastric cancer after administration of the conjugate of the present application
  • Figure 19 shows that the conjugate HB050 of the present application inhibits the growth of gastric cancer
  • Figure 20 shows the weight change of mice with gastric cancer after administration of the conjugate of the present application
  • Figure 21 shows that the conjugate HB050 described in this application inhibits the growth of gastric cancer
  • Figure 22 shows the weight change of mice with gastric cancer after administration of the conjugate of the present application
  • Figure 23 shows that the conjugate HB030 described in this application inhibits the growth of pancreatic cancer
  • Figure 24 shows the weight change of pancreatic cancer mice after administration of the conjugate of the present application
  • Figure 25 shows that the conjugate HB050 of the present application inhibits the growth of pancreatic cancer
  • Figure 26 shows the weight change of pancreatic cancer mice after administration of the conjugate of the present application
  • Figure 27 shows that the conjugate HB060 described in this application inhibits the growth of prostate cancer
  • Figure 28 shows the weight change of prostate cancer mice after administration of the conjugate of the present application
  • Figure 29 shows that the conjugate HB060 described in this application inhibits the growth of prostate cancer
  • Figure 30 shows the weight change of prostate cancer mice after administration of the conjugate of the present application.
  • targeting polypeptide generally refers to a polypeptide that specifically binds or selectively binds to a target molecule, cell, particle, tissue, or aggregate.
  • the targeting polypeptide may be an antibody, antibody fragment, bispecific antibody, antigen-binding fragment, or other antibody-based molecules or compounds.
  • targeting moieties may also be known in the art, such as aptamers, avimers, receptor binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins.
  • humanized antibody generally refers to a recombinant protein in which the CDRs of an antibody from a species (such as a murine antibody) are transferred from the heavy and light chains of that species to the human heavy chain And in the variable region of the light chain.
  • the constant region of the antibody molecule is derived from the constant region of a human antibody.
  • specific residues in the variable region of a humanized antibody can be modified, especially residues that contact or are close to the CDR sequence, for example, from primitive murines, rodents, and non-primates. Or substitute the corresponding residues of other antibodies.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies in the group are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • single chain antibody refers generally to an antibody molecule comprising a heavy chain variable region through (V H) and a linker antibody light chain variable region (V L) of.
  • Such scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -VH- linker -VL-COOH.
  • antibody generally includes whole antibodies and any antigen-binding fragments or single chains thereof. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibody can also encompass antibodies, digested fragments, designated parts and variants thereof, including antibody mimics or antibody parts that mimic the structure and/or function of antibodies or designated fragments or parts thereof, including single-chain antibodies and single domains.
  • Functional fragments include antigen-binding fragments against preselected targets.
  • antigen-binding fragment refers to a part of a complete antibody and refers to the epitope variable region of the complete antibody.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv and single-chain Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antigen-binding fragments.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may be composed of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv generally refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of a VH domain.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • linker generally refers to a bond, molecule, or group of molecules that binds two separate entities (such as the targeting polypeptide and the camptothecin small molecule in this application) to each other.
  • the linker can provide the best separation between the two entities, or it can also provide an unstable connection that separates the two entities from each other.
  • Unstable linkages include photo-cleavable groups, acid-labile moieties, base-labile moieties, and enzyme-cleavable groups.
  • the linker may refer to any reagent or molecule that connects the targeting polypeptide and the pharmaceutically active moiety (eg, camptothecin small molecule).
  • the linker can form a covalent bond or a non-covalent bond with two physical parts.
  • the ideal linker can be any agent or molecule that remains stable, does not affect the functionality of the targeted polypeptide or pharmacologically active part, or does not produce additional undesired functions.
  • the linker can be cleavable or non-cleavable.
  • Common linkers can include, but are not limited to, valine-citrulline (vc), valine-alanine (va), maleimide Maleimidomethyl cyclohexane-1-carboxylate (Maleimidomethyl cyclohexane-1-carboxylate, MCC), maleimidocaproyl (mc), N-succinimidyl-4-(2-pyridine N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate Ester (N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate, sulfo-SPDB) or 4-(4-acetylphenoxy)butanoic acid-N , AcBut-N).
  • vc valine-citrulline
  • chimeric antibody generally refers to an antibody in which a part of the heavy chain and/or light chain is the same or the same as the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass. Source, and the rest of the chain is the same or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. Fragments of the antibodies are also included, which exhibit the required biological activity (ie, the ability to specifically bind to tumor antigens).
  • Fully human antibody generally refers to an antibody that is expressed by transferring a human antibody-encoding gene to a genetically engineered antibody gene-deficient animal. All parts 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 the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • the term "specific binding” generally refers to binding to a specific target without cross-reactivity to other targets.
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • the antibody when an antibody binds to an epitope through its antigen-binding domain more easily than it will bind to a random, unrelated epitope, the antibody is said to "specifically bind" the antigen.
  • the selective reactivity usually refers to preferential binding to a specific target.
  • tumor-specific antigen generally refers to a protein that is mainly or only expressed on tumor cells.
  • Main expression refers to relatively higher expression on tumor cells than on normal somatic cells, while only expression refers to the expression on tumor cells of a protein that is not detected on normal somatic cells by standard means known in the art .
  • conjugate refers to a polypeptide (e.g., peptide, nucleic acid, protein, enzyme, sugar, polysaccharide, lipid, Glycoprotein and Lipoprotein) and the chemical part.
  • the chemical moiety that can be included in the conjugate can be a therapeutic agent or a cytotoxic agent, non-limiting examples of which can be: mitosis inhibitors, anti-tumor antibiotic immunomodulators, vectors for gene therapy, alkylating agents, Antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, antihormones, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors Agents and radiosensitizers.
  • Her2 generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, ErbB2 or Neu (Slamon, et al., Science 235 (1987) ) 177-182; Swiss-ProtP04626).
  • the term “Her2” can also cover homologs, variants and isoforms of Her2, including splicing isoforms.
  • Her2 also includes proteins with one or more sequences of Her2 homologs, variants and isoforms, as well as fragments of the sequence, as long as the variant protein (including isoforms), homologous protein And/or fragments can be recognized by one or more Her-specific antibodies (such as Pertuzumab or Trastuzumab).
  • Her2 is associated with tumor transformation in human breast cancer cells.
  • the overexpression of Her2 protein has been detected in patients with breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, peritoneal cancer, or colon cancer.
  • the heavy chain variable region of an exemplary Pertuzumab may include the amino acid sequence shown in SEQ ID NO.
  • an exemplary Pertuzumab may include the amino acid sequence shown in SEQ ID NO. 4;
  • An exemplary heavy chain variable region of trastuzumab may include the amino acid sequence shown in SEQ ID NO. 1, and an exemplary light chain variable region of trastuzumab may include the amino acid sequence shown in SEQ ID NO.2.
  • Trop2 and "TROP2” generally refer to a single-way transmembrane type I cell membrane protein, also known as tumor-associated calcium signal transducer 2 (TACSTD2), GA733-1, EGP-1 or MIS1.
  • Trop2 can also cover homologs, variants and isoforms of Trop2, including splicing isoforms.
  • the term “Trop” also includes proteins with one or more sequences of Trop 2 homologs, variants and isoforms, as well as fragments of the sequence, as long as the variant protein (including isoforms), homologous
  • the protein and/or fragment can be recognized by one or more Trop-specific antibodies (such as hRS7 or hTINA1).
  • Trop2 can be human Trop2, and the DNA sequence and amino acid sequence of human Trop2 can be obtained on public databases, for example, see NCBI accession numbers NM_002353 and NP_002344.
  • the exemplary heavy chain variable region of hRS7 may include the amino acid sequence shown in SEQ ID NO. 5, and the exemplary light chain variable region of hRS7 may include the amino acid sequence shown in SEQ ID NO. 6; the exemplary heavy chain of hTINA1 may The variable region may include the amino acid sequence shown in SEQ ID NO. 7, and the exemplary light chain variable region of hTINA1 may include the amino acid sequence shown in SEQ ID NO. 8.
  • the application provides a conjugate having the structure of Formula I:
  • the conjugate of the present application has the structure of Formula I, wherein Mab is a targeting polypeptide.
  • the Mab may be an antibody (including fully human antibodies, humanized antibodies, non-human antibodies, or chimeric antibodies), or antigen-binding fragments thereof (including enzymatically or recombinantly produced fragments), or incorporated There are binding proteins derived from sequences of antibodies or antigen-binding fragments thereof.
  • the antibody or its antigen binding protein may be multivalent and multispecific or multivalent and monospecific.
  • the Mab may be a monoclonal antibody.
  • the targeting polypeptide may be a multivalent and/or multispecific monoclonal antibody.
  • the targeting polypeptide can be a murine, chimeric, humanized or fully human monoclonal antibody, and the antibody can be a complete, fragment (Fab', Fab, F(ab) 2 , F(ab') 2 , Fv or scFv) or sub-fragment (single-chain construct) form, or IgG1, IgG2, IgG3, IgG4, IgA isotype, or submolecules thereof.
  • the Mab may be a chimeric antibody.
  • a chimeric antibody is a recombinant protein in which the variable region of a human antibody is replaced with a variable region of, for example, a murine antibody, and includes the complementarity determining region (CDR) of a murine antibody. Chimeric antibodies exhibit reduced immunogenicity and increased stability when administered to a subject. Methods of constructing chimeric antibodies are well known in the art.
  • Chimeric antibodies can be humanized by transferring the mouse CDRs of the variable regions of the mouse immunoglobulin heavy chain and light chain to the corresponding domains of the human antibody.
  • the mouse framework region (FR) of the chimeric antibody was also replaced with a human FR sequence.
  • the Mab may be a humanized antibody.
  • one or more human FR residues can be replaced with mouse equivalent residues.
  • Humanized antibodies can be used for therapeutic treatment of subjects. The technology for producing humanized antibodies is well known in the art.
  • the Mab may be a fully human antibody.
  • Methods of producing fully human antibodies using combinatorial methods or using transgenic animals transformed with human immunoglobulin loci are known in the art.
  • the fully human antibodies are expected to exhibit less side effects than chimeric antibodies or humanized antibodies, and act as substantially endogenous human antibodies in vivo.
  • the Mab described in this application may be an antibody fragment.
  • Antibody fragments can be obtained by conventional methods, such as pepsin or papain digestion of the full-length antibody.
  • antibody fragments can be produced by digesting the antibody with pepsin to provide a 5S fragment denoted as F(ab')2.
  • the fragment can be further cleaved with a thiol reducing agent, and optionally a blocking group of a mercapto group due to disulfide bond cleavage, to produce a 3.5S Fab' monovalent fragment.
  • a thiol reducing agent optionally a blocking group of a mercapto group due to disulfide bond cleavage
  • the Mab may be a single chain antibody.
  • These single-chain antibodies can be prepared by constructing a structural gene comprising a DNA sequence encoding a VH domain and a VL domain connected by an oligonucleotide linker sequence. The structural gene is inserted into an expression vector, and then the expression vector is introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain in which the linker peptide bridges the two variable domains. Methods for generating scFv are well known in the art.
  • the Mab may be modified, for example, by deleting, adding or substituting parts of the antibody.
  • the modification can change certain properties of the antibody, such as making the number of small molecule moieties connected to the targeting moiety reach the desired number, reducing ADCC after enhancing, or enhancing or reducing CDC.
  • the sequence of the antibody such as the Fc portion of the antibody, can be altered to optimize the physiological characteristics of the conjugate, such as serum half-life. Methods of substituting amino acid sequences in proteins are widely known in the art, for example by site-directed mutagenesis.
  • the changes may include the addition or removal of one or more glycosylation sites in the Fc sequence.
  • specific amino acid substitutions can be made in the Fc sequence.
  • the Mab can recognize or bind markers or tumor-associated antigens, and can mainly or only recognize expression on cells that are diseased relative to normal tissues, and can be internalized by cells.
  • the target or antigen that the Mab can recognize or bind may include: carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, Her2, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g.
  • ILGF insulin-like growth factor
  • IGF-1R Ia, ganglioside, HCG, L243 bound HLA-DR antigen, CD66 antigen (CD66a-d or a combination thereof), MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage cell migration inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5, placental growth factor (P1GF), PSA (prostate specific antigen), PSMA, PAM4 antigen, NCA-95, NCA- 90, A3, A33, Ep
  • the Mab can specifically bind to tumor-specific antigens.
  • the targeting polypeptide may be an antibody or an antigen-binding fragment thereof that reacts with an antigen or epitope expressed on tumor cells.
  • Available tumor-specific antigens include the tumor-specific antigens that can be selected from CLDN18.2, Nectin 4, CD22, CD30, CD33, Her2, Mesothelin, PSMA and Trop2.
  • the tumor-specific antigen can be selected from the group consisting of CLDN18.2, Nectin 4, Her2 and Trop2.
  • the Mab can be internalized, then re-expressed, processed, and presented on the cell surface so that the cell can continuously take up the conjugate and increase the conjugate circulation.
  • Targeting polypeptides that can be used in this application include but are not limited to the following polypeptides or antibodies: LL1 antibody (anti-CD74), LL2 and RFB4 antibodies (anti-CD22), RS7 antibody (anti-epiglycoprotein-1 (EGP-1) ), PAM-4 and KC4 antibodies (both anti-mucin), MN-14 antibody (anti-carcinoembryonic antigen (CEA, also known as CD66e), Mu-9 antibody (anti-colon-specific antigen-p ), Immu31 antibody (anti-alpha-fetoprotein), TAG-72 antibody (such as CC49), Tn antibody, J591 or HuJ591 (anti-PSMA (prostate specific membrane antigen)), AB-PG1-XG1-026 (anti-PSMA Dimer), D2/B (anti-PSMA
  • the Mab may include the antibody HCDR3, and the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 19, 25, 31, and 39.
  • the Mab may include the antibody HCDR2, and the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 18, 24, 30, and 38.
  • the Mab may include the antibody HCDR1, and the HCDR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 17, 23, 29, and 37.
  • the Mab in the conjugate of the present application may include HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 each include any set of amino acid sequences selected from the following groups:
  • HCDR1 SEQ ID NO: 17, HCDR2: SEQ ID NO: 18 and HCDR3: SEQ ID NO: 19;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30 and HCDR3: SEQ ID NO: 31;
  • HCDR1 SEQ ID NO: 37
  • HCDR2 SEQ ID NO: 38
  • HCDR3 SEQ ID NO: 39.
  • the Mab in the conjugate of the present application may include the antibody heavy chain variable region VH, and the VH may include the amino acid sequence shown in any one of SEQ ID NOs: 1, 5, 32, and 40.
  • the Mab may include the antibody LCDR3, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 22, 28, 35, and 43.
  • the Mab may include the antibody LCDR2, and the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 21, 27, 34, and 42.
  • the Mab may include the antibody LCDR1, and the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 20, 26, 33, and 41.
  • the Mab in the conjugate of the present application may include LCDR1, LCDR2, and LCDR3, and the LCDR1, LCDR2, and LCDR3 each include any set of amino acid sequences selected from the following groups:
  • LCDR1 SEQ ID NO: 20
  • LCDR2 SEQ ID NO: 21
  • LCDR3 SEQ ID NO: 22;
  • LCDR1 SEQ ID NO: 26
  • LCDR2 SEQ ID NO: 27
  • LCDR3 SEQ ID NO: 28;
  • LCDR1 SEQ ID NO: 33
  • LCDR2 SEQ ID NO: 34
  • LCDR3 SEQ ID NO: 35;
  • LCDR1 SEQ ID NO: 41
  • LCDR2 SEQ ID NO: 42
  • LCDR3 SEQ ID NO: 43.
  • the Mab in the conjugate of the present application may include the antibody light chain variable region VL, and the VL may include the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 36, and 44.
  • the Mab in the conjugate of the present application may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each include any group of amino acids selected from the following groups sequence:
  • HCDR1 SEQ ID NO: 17, HCDR2: SEQ ID NO: 18, HCDR3: SEQ ID NO: 19, LCDR1: SEQ ID NO: 20, LCDR2: SEQ ID NO: 21 and LCDR3: SEQ ID NO: 22 ;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 24
  • HCDR3 SEQ ID NO: 25
  • LCDR1 SEQ ID NO: 26
  • LCDR2 SEQ ID NO: 27
  • LCDR3 SEQ ID NO: 28 ;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31, LCDR1: SEQ ID NO: 33, LCDR2: SEQ ID NO: 34 and LCDR3: SEQ ID NO: 35 ;and
  • HCDR1 SEQ ID NO: 37
  • HCDR2 SEQ ID NO: 38
  • HCDR3 SEQ ID NO: 39
  • LCDR1 SEQ ID NO: 41
  • LCDR2 SEQ ID NO: 42
  • LCDR3 SEQ ID NO: 43 .
  • the Mab in the conjugate of the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 1, and the VL may include the amino acid sequence shown in SEQ ID NO: 2. .
  • the Mab in the conjugate of the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 5, and the VL may include the amino acid sequence shown in SEQ ID NO: 6 .
  • the Mab in the conjugate of the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 32, and the VL may include the amino acid sequence shown in SEQ ID NO: 36 .
  • the Mab in the conjugate described in the present application may include VH and VL, and the VH may include the amino acid sequence shown in SEQ ID NO: 40, and the VL may include the amino acid sequence shown in SEQ ID NO: 44 .
  • the Mab in the conjugate described in the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 9, and the antibody light chain may include SEQ ID NO: The amino acid sequence shown in 10.
  • the Mab in the conjugate of the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 11, and the antibody light chain may include SEQ ID The amino acid sequence shown as NO: 12.
  • the Mab in the conjugate of the present application may include an antibody heavy chain and an antibody light chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 13, and the antibody light chain may include SEQ ID The amino acid sequence shown as NO:14.
  • the Mab in the conjugate of the present application may include an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 15
  • the antibody light chain may include SEQ ID The amino acid sequence shown as NO:16.
  • R 1 can be a methyl substituted by halogen (for example, a methyl substituted by chlorine, a methyl substituted by fluorine, a methyl substituted by bromine, or a methyl substituted by iodine.
  • R 2 can be selected from hydrogen, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl;
  • R 3 can be selected from hydrogen, amino, C 1 -C 6 alkyl and substituted ⁇ C 1 -C 6 alkyl group.
  • the present application may include a -NH 2 amino group with a nitrogen atom attached to two hydrogen atoms may also be included in the group after a -NH 2 or two hydrogen atoms are substituted with an alkyl group, e.g., -N (CH 3 ) 2 , NHCH 3 , -N(CH 2 CH 3 ) 2 , -N(CH 2 CH 2 CH 3 ) 2 , -NHCH 2 CH 3 or -NHCH 2 CH 2 CH 3 and so on.
  • an alkyl group e.g., -N (CH 3 ) 2 , NHCH 3 , -N(CH 2 CH 3 ) 2 , -N(CH 2 CH 2 CH 3 ) 2 , -NHCH 2 CH 3 or -NHCH 2 CH 2 CH 3 and so on.
  • the C 1 -C 6 alkyl group may be a straight-chain alkyl group or a branched-chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, tert-butyl and the like.
  • substituted C 1 -C 6 alkyl may refer to the group at least one hydrogen atom C 1- C 6 alkyl substituted with halogen, amino, hydroxy, alkoxy, or aryl group group.
  • R 3 can be selected from hydrogen, amino, C 1 -C 4 alkyl or substituted C 1 -C 4 alkyl; in other cases, R 3 can be selected from hydrogen, amino, or Wherein, R 4 and R 5 can be selected from hydrogen, C1-C6 alkyl, substituted C1-C6 alkyl or aryl, respectively.
  • R 3 binding site may be any one of an unsubstituted benzene ring three sites, including the ortho or meta position R1.
  • the binding site of R 3 can be the C 9 position of a small molecule.
  • R 3 can be hydrogen.
  • R 2 may be selected from hydrogen, C 1 -C 6 alkyl, and substituted C 1 -C 6 alkyl.
  • the C 1 -C 6 alkyl group may be a straight-chain alkyl group or a branched-chain alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, tert-butyl and the like.
  • the substituted C 1 -C 6 alkyl group may refer to a group in which at least one hydrogen atom in the C1-C6 alkyl group is substituted by a halogen, an amino group, a hydroxyl group, an alkoxy group, or an aryl group.
  • R 2 can be selected from hydrogen, C 1 -C 4 alkyl, or substituted C 1 -C 4 alkyl; for example, R 2 can be selected from Et, wherein, R 4 and R 5 can be selected from hydrogen, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl or aryl, respectively.
  • R 2 can be -CH 2 CH 3 .
  • R 1 may be a methyl substituted with halogen.
  • R 1 may be methyl substituted with fluorine, chlorine, bromine or iodine.
  • R1 can be methyl substituted with 1, 2, or 3 halogens.
  • R 1 may be selected from -CF 2 H, -CCl 2 H, -CBr 2 H, -CFH 2 , -CBrH 2 , -CClH 3 , -CF 3 , -CCl 3 or -CBr 3 .
  • R 1 may be -CF 2 H.
  • the small molecule in the conjugate described in this application may have the following structure:
  • the targeting polypeptide and the small molecule part can be connected via a linker L.
  • the linker L may include a defined polyethylene glycol (PEG) moiety, L-amino acid, and another spacer.
  • the determined PEG portion is a PEG containing a determined number of monomer units, where the determined PEG may be a low molecular weight PEG, and in some cases, it may include 1-30 monomer units, For example, it contains 1-12 monomer units.
  • the defined PEG may have a reactive group (for example, a carboxylic acid or a hydroxyl group) at one end.
  • the L-amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid , Histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine and proline.
  • the number of L-amino acids may be 0, 1, 2, 3, 4 or more.
  • the number of the L-amino acid may be one, and the one L-amino acid may be selected from any one of the following: glycine, alanine, valine, leucine, isoleucine, benzene Alanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine Acid, cysteine and proline.
  • the additional spacer may be selected from ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, or substituted or unsubstituted ethylenediamine. If it includes a hydroxyl group, it is attached to the hydroxyl or amino group of the drug in the form of carbonate or carbamate, respectively. In some cases, the additional spacer is a substituted ethylenediamine derived from an L-amino acid in which the carboxylic acid group is replaced by a hydroxymethyl moiety.
  • the additional spacer may be derived from any one of the following L-amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, Aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine and proline.
  • the additional spacer may be a foldable moiety injected with 4-aminobenzyl alcohol or substituted 4-aminobenzyl alcohol substituted with C1-C10 alkyl at the benzylic position, substituted 4-amino Benzyl alcohol is linked to an L-amino acid or a polypeptide comprising multiple L-amino acid moieties via its amino group; wherein the N-terminus is connected to a cross-linker terminating in the targeting polypeptide moiety.
  • the additional spacer may be derived from substituted 4-aminobenzyl alcohol, the substituted 4-aminobenzyl alcohol being hydrogen or an alkane selected from a C 1 -C 10 alkyl group. Substitution.
  • the linker L may have the following structure:
  • the present application provides a method for producing the conjugate, which may include: obtaining a compound represented by formula IV:
  • R 1 can be a methyl substituted by halogen
  • R 2 can be selected from hydrogen, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl
  • R 3 can be selected from hydrogen, amino, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl
  • Mab may be a targeting polypeptide
  • L may be a linker
  • the compound represented by formula IV is conjugated with the Mab to obtain the conjugate Compound.
  • This application provides a method for preparing compound IV, which may include: 1) preparing a small molecule represented by formula I, 2) synthesizing a linker, and 3) obtaining a compound represented by formula IV.
  • the preparation of the small molecule represented by formula I may include reacting 10-hydroxycamptothecin compounds with a sulfonylation reagent to obtain sulfonic acid ester intermediates.
  • the structural formula of 10-hydroxycamptothecin compounds can be
  • R 1 can be selected from hydrogen, amino, C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl, and the binding site of R 1 can be one of the three unsubstituted positions on the benzene ring Any one;
  • R 2 can be selected from hydrogen, C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl;
  • R 3 is selected from hydrogen, acyl, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl;
  • R 6 can be selected from methanesulfonyl, p-toluenesulfonyl or trifluoromethanesulfonyl.
  • Sulfonylation reagents can sulfonylate the C10 hydroxyl group of 10-hydroxycamptothecin compounds to obtain leaving groups -OTf, -OTs or -OMs, which are convenient for substitution with halogen-substituted methyl groups in the subsequent steps .
  • Sulfonylation reagents can include sulfonyl chloride reagents, sulfonic acid reagents, sulfonic anhydride reagents, and sulfonamide reagents.
  • sulfonyl groups they can include methanesulfonyl chloride, p-toluenesulfonyl chloride, trifluoromethanesulfonyl chloride, and methanesulfonic acid. , At least one of p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride, and N-phenylbis(trifluoromethanesulfonimide).
  • the reaction temperature of the 10-hydroxycamptothecin compound and the sulfonylation reagent can be 60-100°C, and the reaction time is 2-8h.
  • the sulfonylation reaction is carried out under the above reaction conditions, the reaction is more complete, and the sulfonic acid ester intermediate can be obtained in a high yield.
  • the molar ratio of 10-hydroxycamptothecin compound to sulfonylation reagent can be 1:1-1.5. Excessive sulfonylation reagent can ensure the full reaction of 10-hydroxycamptothecin compounds and improve the utilization rate of raw materials.
  • the preparation of the small molecule represented by Formula I may also include reacting the sulfonic acid ester intermediate with a halogen-substituted methyl reagent to obtain a 10-halomethyl camptothecin compound.
  • the preparation method described in this application may further include synthesizing the linker L.
  • a certain polyethylene glycol (PEG) part that is, a PEG containing a certain number of monomer units
  • the certain PEG is a low molecular weight PEG.
  • 1-30 monomer units are included, for example, 1-12 monomer units are included.
  • the linker can enhance the solubility of small molecules.
  • one end of the defined polyethylene glycol portion of the L may carry different reactive groups, for example, a carboxyl group or a hydroxyl group.
  • the polyethylene glycol moiety can be connected to the amino group of the amino alcohol, and the hydroxyl group of the amino alcohol is connected to the hydroxyl group in the form of carbonate on the small molecule.
  • one end of the different reactive group of the polyethylene glycol moiety can be connected to the N-terminus of the L-amino acid or polypeptide, the C-terminus is connected to the amino group of the amino alcohol, and the hydroxyl group of the amino alcohol is respectively connected to the small molecule.
  • the targeting polypeptide-coupling group is designed as a thiol or thiol reactive group.
  • the preparation method described in the present application may further include obtaining the compound represented by formula IV, and conjugate the targeting moiety and the small molecule of formula I through the linker L.
  • the present application provides a pharmaceutical composition comprising the conjugate.
  • the conjugate or drug can be formulated according to known methods for preparing pharmaceutically usable compositions, whereby the immunoconjugate is mixed with pharmaceutically suitable excipients in the mixture.
  • pharmaceutically suitable excipients in the mixture.
  • Sterile phosphate buffered saline is an example of a pharmaceutically suitable excipient.
  • Other suitable excipients are well known to those skilled in the art.
  • the application provides the use of the conjugate or pharmaceutical composition described in the application in the preparation of drugs for preventing or treating tumors.
  • the application also provides a method of preventing or treating tumors, the method comprising administering the conjugate or pharmaceutical composition described in the application to a subject in need.
  • the pharmacologically treatable tumors that can be prepared using the conjugates or pharmaceutical compositions described in this application may include solid tumors and/or non-solid tumors.
  • the solid tumors may be selected from the following group: prostate cancer, breast cancer, Pancreatic cancer, colon cancer and stomach cancer.
  • the conjugates described in this application can be used to inhibit the growth, progression and/or metastasis of tumors, especially those cancers listed above.
  • the application provides the use of the conjugate together with other tumor therapies or drugs in the preparation of drugs for preventing or treating tumors.
  • This application also provides a method for preventing or treating tumors, the method comprising administering the conjugate or pharmaceutical composition described in this application, and other tumor therapies or drugs to a subject in need.
  • the tumor may include a solid tumor and/or a non-solid tumor, for example, the solid tumor may be selected from the following group: prostate cancer, breast cancer, pancreatic cancer, colon cancer, and gastric cancer.
  • the conjugate can be administered single or repeatedly, and can also be used in combination with other tumor therapies or drugs.
  • the other tumor therapies or drugs may include, for example, surgery, external Radiation, radioimmunotherapy, chemotherapy, antisense therapy, interfering RNA therapy, gene therapy, etc. Each combination will be suitable for tumor type, stage, patient condition and previous therapy, as well as other factors considered by the attending physician.
  • the other tumor therapy or drug is selected from the group consisting of chemotherapy, radiotherapy, miRNA and oligonucleotide.
  • the chemotherapy that can be used in combination with the conjugate includes chemotherapeutic drugs, such as vinca alkaloids, allium ring antibiotics, epipodophyllotoxins, taxols, antimetabolites, alkylating agents, antibiotics, Cox-2 Inhibitors, antimitotic agents, antiangiogenic agents, and pro-apoptotic agents, such as doxorubicin, methotrexate, paclitaxel, other camptothecins, and other forms of these and other types of anticancer agents.
  • chemotherapeutic drugs such as vinca alkaloids, allium ring antibiotics, epipodophyllotoxins, taxols, antimetabolites, alkylating agents, antibiotics, Cox-2 Inhibitors, antimitotic agents, antiangiogenic agents, and pro-apoptotic agents, such as doxorubicin, methotrexate, paclitaxel, other camptothecins, and other forms of these and other types of anticancer agents.
  • chemotherapeutic drugs may include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones and the like.
  • the chemotherapy may be part of the conjugate described in this application, or alternatively may be used in combination before, at the same time or after the conjugate.
  • the radiotherapy that can be used in combination with the conjugates described in this application can include any mechanism for inducing local DNA damage in tumor cells, such as gamma-radiation, X-rays, UV-radiation, microwaves, electron emission, and the like. Radiotherapy may also include the use of combination therapy that directs the delivery of radioisotopes to tumor cells, and may be used in combination with or as part of the conjugate. Optionally, the radiotherapy can be administered as a single dose or as multiple consecutive doses.
  • Oligonucleotides that can be used in conjunction with the conjugates described in this application can include any suitable short-chain nucleotides (including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA), for example, antisense oligonucleotides Nucleotides, small interfering RNAs, ribozymes, deoxyribozymes, antigenes, CpG oligonucleotides, transcription factor decoys and nucleic acid aptamers.
  • nucleotides including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA
  • antisense oligonucleotides Nucleotides, small interfering RNAs, ribozymes, deoxyribozymes, antigenes, CpG oligonucleotides, transcription factor decoys and nucleic acid aptamers.
  • Suitable routes of administration of the conjugate include, but are not limited to, oral, parenteral, rectal, transmucosal, enteral administration, intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, Intranasal or intraocular injection.
  • the route of administration is parenteral.
  • compound V was prepared from compound IV (camptothecin derivative, please refer to CN108690036A for the preparation method).
  • Method 1 or Method 2 can be used to obtain lightly reduced antibodies antiHer2 antibody trastuzumab, pertuzumab, antiTrop2 antibody hRS7, hTINA1.
  • the amino acid sequence of the heavy chain variable region of trastuzumab is shown in SEQ ID NO: 1 and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO: 2; the heavy chain of the Pertuzumab antibody is variable
  • the amino acid sequence of the region is shown in SEQ ID NO: 3, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 4; the amino acid sequence of the hRS7 antibody heavy chain variable region is shown in SEQ ID NO: 5, the antibody light chain
  • the variable region amino acid sequence is shown in SEQ ID NO: 6; the hTINA1 antibody heavy chain variable region amino acid sequence is shown in SEQ ID NO: 7, and the antibody light chain variable region amino acid sequence is shown in SEQ ID NO: 8.
  • Method 2 The antibody was reduced with Tris (2-carboxyethyl) phosphine (TCEP, CAS#51805-45-9) in a phosphate buffer in the range of pH 5-7.
  • Tris (2-carboxyethyl) phosphine TCEP, CAS#51805-45-9
  • the reduced antibody was reacted with a 10-15-fold molar excess of Compound VIII, and incubated at room temperature for 20 minutes.
  • the conjugate was purified by molecular exclusion chromatography through hydrophobic strains and finally by ultrafiltration-dialysis.
  • the absorbance of camptothecin of the product was measured by the absorbance of UV spectrophotometer at 366nm, and it was correlated with the standard value.
  • the protein concentration was judged from the absorbance at 280nm, and the absorption of camptothecin was removed. In order to determine the substitution ratio of small molecule drugs/antibodies.
  • the purified conjugate was stored as a lyophilized preparation in a glass vial, capped under vacuum, and stored in a refrigerator at -20°C.
  • HB010 antibody is trastuzumab
  • HB020 antibody is Pertuzumab
  • HB030 antibody is hRS7
  • HB040 antibody is hTINA1
  • HB050 anti-CLDN18. 2 antibody
  • HB060 anti-Nectin 4 antibody
  • HB011 antibody is trastuzumab
  • HB021 antibody is Pertuzumab
  • HB031 antibody is hRS7
  • HB041 antibody is hTINA1
  • HB051 anti-CLDN18.2 antibody
  • HB061 anti-Nectin 4 antibody
  • the difluoromethyl compound IV is replaced with a monofluoromethyl compound, a chlorinated compound, and a brominated compound to prepare a conjugate.
  • the prepared conjugate and drug antibody ratios are as follows:
  • the binding activity of the conjugate obtained in Example 1 to the antigen was tested.
  • the binding activity of the sample to be tested with the antigen is shown in Figure 1-2 and Table 2 below.
  • the results show that the binding EC 50 of the conjugates HB010 and Her2 described in the present application is not significantly different from the control molecule HB011, and the binding EC 50 of HB030 and TROP2 is slightly higher than that of the control molecule HB031.
  • the in vitro anti-tumor activity of the conjugate obtained in Example 1 was tested.
  • the luminescence detection method is used to collect the cells in the logarithmic growth phase and use a platelet counter to count the cells. Use the trypan blue exclusion method to detect the cell viability to ensure that the cell viability is above 90%.
  • the selected commercially available cell strains, culture media, and cell inoculation numbers are shown in Table 3 below.
  • HB010 and HB030 have obvious inhibitory effects on a variety of tumor cells.
  • HB010 has no significant difference in the anti-tumor effect of breast cancer and gastric cancer cell lines from the control molecule HB011.
  • HB030 has obvious effects on breast cancer, pancreatic cancer, colon cancer and gastric cancer.
  • the anti-tumor effect of the cell line is better than the control molecule HB031.
  • Example 1 The conjugate obtained in Example 1 was intravenously administered to mice, blood samples were collected before administration and within a certain time after the injection, the serum was separated, the concentration of the drug in the serum was determined by the Elisa method, and the pharmacokinetic parameters were calculated by software.
  • mice with breast cancer, pancreatic cancer, colon cancer or gastric cancer xenografts were treated with the conjugate prepared in Example 1. Observe the therapeutic effect of the conjugate. The results show that the conjugate described in this application can effectively treat tumors in vitro.
  • Example 6 The conjugate of the present application inhibits tumor growth in mice with breast cancer
  • Tumor volume (mm 3 ) 1/2 ⁇ long diameter (mm) ⁇ [short diameter (mm)] 2
  • Percentage of tumor volume change (weekly tumor volume-tumor volume at the first dose)/tumor volume at the first dose ⁇ 100%
  • the body weight of nude mice in each group increased positively with time, and there was no significant difference in body weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume of the HB010 (0.5mg/kg) group became smaller, smaller than that of the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB010 (0.05mg/kg)
  • the tumor volume of the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB011 (0.5mg/kg) group.
  • the results are shown in Figures 11-12.
  • the weight of nude mice in each group increased positively with time, and there was no significant difference in weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume in the HB060 (0.5mg/kg) group became smaller and smaller than the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB060 (0.05mg/kg)
  • the tumor volume in the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB061 (0.5mg/kg) group.
  • HB060 is better than HB061 in inhibiting breast cancer growth at the same dose.
  • the average percentage of tumor volume in the HB060 (0.5mg/kg) group is compared with the other groups *P ⁇ 0.05, #P ⁇ 0.001; in Figure 12, the average weight of the HB060 (0.5mg/kg) group is There was no statistically significant difference between the other groups.
  • the effect of HB030 in inhibiting breast cancer growth at the same dose is similar to that of HB031 and even slightly better than HB031.
  • the average percentage of tumor volume in the HB030 (0.5mg/kg) group was compared with the other groups *P ⁇ 0.05, #P ⁇ 0.001; in Figure 14, the average body weight of the HB030 (0.5mg/kg) group was compared with that of the other groups. There was no statistically significant difference between the other groups.
  • HB030 (0.2mg/kg) is significantly better than other groups in suppressing tumors, and its anti-tumor activity is at least 2.5 times that of HB031 (0.5mg/kg). And there was no toxic reaction in each group.
  • Example 7 The conjugate of the present application inhibits tumor growth in mice with gastric cancer
  • the results are shown in Figures 17-18.
  • the body weight of nude mice in each group increased positively over time, and there was no significant difference in body weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume of the HB010 (0.5mg/kg) group became smaller, smaller than that of the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB010 (0.05mg/kg)
  • the tumor volume of the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB011 (0.5mg/kg) group.
  • HB010 has a better inhibitory effect on NCI-N87 gastric cancer than irinotecan and HB011, and has a certain dose-effect relationship, and no toxicity is seen.
  • the results are shown in Figures 19-20.
  • the body weight of nude mice in each group increased positively with time, and there was no significant difference in body weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume of the HB050 (0.5mg/kg) group became smaller and smaller than that of the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB050 (0.05mg/kg)
  • the tumor volume of the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB051 (0.5mg/kg) group.
  • HB050 has a better inhibitory effect on NCI-N87 gastric cancer than irinotecan and HB051. It has a certain dose-effect relationship, and no toxicity is seen.
  • Example 8 The conjugate of the present application inhibits tumor growth in mice with pancreatic cancer
  • the results are shown in Figures 23-24.
  • the weight of nude mice in each group increased positively over time, and there was no significant difference in weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume of the HB030 (0.5mg/kg) group became smaller, smaller than that of the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB030 (0.05mg/kg)
  • the tumor volume of the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB031 (0.5mg/kg) group.
  • the results are shown in Figures 25-26.
  • the body weight of nude mice in each group increased positively with time, and there was no significant difference in body weight between the groups at each time point, and no serious toxic reaction was seen in each group.
  • the tumor volume of the HB050 (0.5mg/kg) group became smaller and smaller than that of the other groups, and its average percentage change was statistically significant (P ⁇ 0.05 or 0.001) compared with the other groups (P ⁇ 0.05 or 0.001); HB050 (0.05mg/kg)
  • the tumor volume of the) group was smaller than that of the saline group, but larger than that of the irinotecan (5mg/kg) group or the HB051 (0.5mg/kg) group.
  • HB050 has a better inhibitory effect on BXPC-3 pancreatic cancer than irinotecan and HB051, and has a certain dose-effect relationship, and no toxicity is seen.
  • Example 9 The conjugate of the present application inhibits tumor growth in mice with prostate cancer

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Abstract

提供了一种靶向多肽-药物缀合物，所述缀合物具有式I所示的结构。以及所述缀合物的制备方法和包含其的组合物。所述缀合物和组合物可用于治疗多种疾病和病症，如肿瘤。还提供了所述靶向多肽-药物缀合物，或与其他肿瘤疗法或药物共同，在制备治疗肿瘤的药物中的用途。

Description

靶向多肽-药物缀合物及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向多肽和小分子治疗药物的缀合物，及其用于制备药物的用途。
背景技术
喜树碱(camptothecin，CPT)是1966年美国化学家从中国珙桐科植物喜树的皮和果实中提取的一种生物碱，具有优异的抗肿瘤活性，其化学结构式为
Figure PCTCN2021071595-appb-000001
其能够抑制拓扑异构酶I，发挥抗肿瘤效果。喜树碱衍生物通过合理的修饰，抗肿瘤活性得到提高，可作为一种有潜力的候选药物分子。但是小分子药物通常存在靶向性低、未到达肿瘤部位便被体内循环系统清除、杀伤正常细胞、或毒副作用严重等问题。
随着更有效地靶向并杀灭肿瘤细胞的药物开发，肿瘤治疗取得显著的进步。抗体-药物缀合物(ADC)，由抗体和药物化学物通过连接子组成，不仅具有高度细胞毒性的小分子药物的抗肿瘤效力，也结合了抗体的高选择性、稳定性和有利的药代动力学特征。然而，由于肿瘤细胞表面上抗原数量有限(每个细胞约5,000-10 6个抗原)且ADC的平均药物与抗体比(DAR)仅限于3.5-4，即ADC转入肿瘤细胞的药物量很低，合并细胞毒性药物的ADC可能因此造成临床失败。因此，亟需开发更有效的抗体-药物缀合物。
发明内容
本申请提供了一种靶向多肽-药物缀合物，及其制备方法和组合物。本申请公开的缀合物、方法和组合物可用于治疗多种疾病和病症，可包括用于存在可选择性的靶向部分的疾病和病症，如肿瘤。本申请还提供了所述缀合物与其他肿瘤疗法或药物共同在制备治疗肿瘤的药物中的用途。本申请的缀合物具有至少一种以下性质：(1)能够以高亲和力结合肿瘤抗原，(2)优异的体外杀伤靶细胞活性，抑制肿瘤细胞生长，(3)优异的体内抑制肿瘤细胞生长的活性 和(4)良好的均一性。
一方面，本申请提供了一种缀合物，其具有式I所示的结构：
Figure PCTCN2021071595-appb-000002
其中，R 1为经卤素取代的甲基；R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基；Mab为靶向多肽；L为连接子。
在某些实施方式中，所述的缀合物具有式II所示的结构：
Figure PCTCN2021071595-appb-000003
其中，R 1为经卤素取代的甲基；R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基。
在某些实施方式中，所述缀合物中的R 1为-CF 2H。
在某些实施方式中，所述缀合物具有式III所示的结构：
Figure PCTCN2021071595-appb-000004
在某些实施方式中，所述Mab包括抗体或其抗原结合片段。
在某些实施方式中，所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述抗原结合片段选自下组：Fab、Fab’、F(ab) 2、Fv和ScFv片段。
在某些实施方式中，所述Mab特异性结合肿瘤特异性抗原。
在某些实施方式中，所述肿瘤特异性抗原选自下组：CLDN18.2、Nectin 4、Her2和Trop2。
在某些实施方式中，所述Mab包含抗体重链HCDR3，且所述HCDR3包含SEQ ID NO: 19、25、31和39中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体重链HCDR2，且所述HCDR2包含SEQ ID NO:18、24、30和38中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体重链HCDR1，且所述HCDR1包含SEQ ID NO:17、23、29和37中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体重链可变区VH，且所述VH包含SEQ ID NO:1、5、32和40中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体重链，且所述抗体重链包含SEQ ID NO:9、11、13和15中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体轻链LCDR3，且所述LCDR3包含SEQ ID NO:22、28、35和43中任一项所示的氨基酸序列。
在某些实施方式中，述Mab包含抗体轻链LCDR2，且所述LCDR2包含SEQ ID NO:21、27、34和42中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体轻链LCDR1，且所述LCDR1包含SEQ ID NO:20、26、33和41中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体轻链可变区VL，且所述VL包含SEQ ID NO:2、6、36和44中任一项所示的氨基酸序列。
在某些实施方式中，所述Mab包含抗体轻链，且所述抗体轻链包含SEQ ID NO:10、12、14和16中任一项所示的氨基酸序列。
另一方面，本申请提供了一种产生所述的缀合物的方法，其包括：
获得式IV所示的化合物：
Figure PCTCN2021071595-appb-000005
其中，R 1为经卤素取代的甲基；R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基；Mab为靶向多肽；L为连接子；使式IV所示的化合物与所述Mab缀合，以得到所述的缀合物。
另一方面，本申请提供了药物组合物，其包含本申请所述的缀合物。
另一方面，本申请提供了本申请所述的缀合物或所述的药物组合物在制备治疗肿瘤的药 物中的用途。
另一方面，本申请提供了所述的缀合物与其他肿瘤疗法或药物共同在制备治疗肿瘤的药物中的用途。
在某些实施方式中，所述其他肿瘤疗法或药物选自下组：化疗、放疗、miRNA和寡核苷酸。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。例如，所述肿瘤为实体瘤。
在某些实施方式中，所述肿瘤选自以下组：乳腺癌、胰腺癌、结肠癌、前列腺癌和胃癌。
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1显示的是本申请所述缀合物与Ba/F2细胞上的Her2的结合活性；
图2显示的是本申请所述缀合物与293T细胞上的Trop2的结合活性；
图3显示的是本申请所述缀合物体外抑制SK-BR-3细胞的活性；
图4显示的是本申请所述缀合物体外抑制NCI-N87细胞的活性；
图5显示的是本申请所述缀合物体外抑制MDA-MB-468细胞的活性；
图6显示的是本申请所述缀合物体外抑制BXPC-3细胞的活性；
图7显示的是本申请所述缀合物体外抑制COLO-205细胞的活性；
图8显示的是本申请所述缀合物体外抑制HCC1954细胞的活性。
图9显示的是HB010抑制乳腺癌生长；
图10显示的是施用本申请的缀合物后乳腺癌小鼠的体重变化；
图11显示的是本申请所述缀合物HB060抑制乳腺癌生长；
图12显示的是施用本申请的缀合物后乳腺癌小鼠的体重变化；
图13显示的是本申请所述缀合物HB030抑制乳腺癌生长；
图14显示的是施用本申请的缀合物后乳腺癌小鼠的体重变化；
图15显示的是本申请所述缀合物HB030抑制乳腺癌生长；
图16显示的是施用本申请的缀合物后乳腺癌小鼠的体重变化；
图17显示的是本申请所述缀合物HB010抑制胃癌生长；
图18显示的是施用本申请的缀合物后胃癌小鼠的体重变化；
图19显示的是本申请所述缀合物HB050抑制胃癌生长；
图20显示的是施用本申请的缀合物后胃癌小鼠的体重变化；
图21显示的是本申请所述缀合物HB050抑制胃癌生长；
图22显示的是施用本申请的缀合物后胃癌小鼠的体重变化；
图23显示的是本申请所述缀合物HB030抑制胰腺癌生长；
图24显示的是施用本申请的缀合物后胰腺癌小鼠的体重变化；
图25显示的是本申请所述缀合物HB050抑制胰腺癌生长；
图26显示的是施用本申请的缀合物后胰腺癌小鼠的体重变化；
图27显示的是本申请所述缀合物HB060抑制前列腺癌生长；
图28显示的是本申请的施用缀合物后前列腺癌小鼠的体重变化；
图29显示的是本申请所述缀合物HB060抑制前列腺癌生长；
图30显示的是施用本申请的缀合物后前列腺癌小鼠的体重变化。
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“靶向多肽”通常是指与靶分子、细胞、颗粒、组织或聚集物特异性结合或选择性结合的多肽。例如，靶向多肽可以是抗体、抗体片段、双特异性抗体、抗原结合片段或其他基于抗体的分子或化合物。其他的靶向部分的示例还可以是本领域已知的，例如适体、avimer、受体结合配体、核酸、生物素-亲和素结合对、结合肽或蛋白。
在本申请中，术语“人源化抗体”通常是指这样的重组蛋白，其中将来自于一个物种的抗体(如鼠抗体)的CDR从该物种的重链和轻链上转移到人重链和轻链的可变区中。该抗体分子的恒定区源于人抗体的恒定区。在某些实施方式中，可修饰人源化抗体可变区的特定 残基，尤其是接触或接近CDR序列的残基，例如用来自原始的鼠科动物、啮齿类动物、非灵长类动物或其他抗体的相应残基代替。
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。
在本申请中，术语“单链抗体”或“scFv”通常是指包含通过连接体连接的抗体重链可变区(V H)和抗体轻链可变区(V L)的分子。此类scFv分子可具有一般结构：NH 2-V L-连接体-V H-COOH或NH 2-VH-连接体-VL-COOH。
本文所用的术语“抗体”通常包括全抗体和任何抗原结合片段或其单链。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。术语“抗体”还可涵盖抗体、其消化片段、指定部分及变体，包括抗体模拟物或包含模拟抗体或其指定片段或部分的结构和/或功能的抗体部分，包括单链抗体和单域抗体及其片段。功能片段包括针对预选靶标的抗原结合片段。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。抗原结合片段的实例包括但不限于Fab、Fab'、F(ab')2、Fv和单链Fv片段、线性抗体、单链抗体以及由抗原结合片段形成的多特异性抗体。
在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性 肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“连接子”通常是指将两个分离的实体(如本申请中的靶向多肽和喜树碱小分子)彼此结合的键、分子或分子组。连接子可以为两个实体提供最佳间隔，或者还可提供使两个实体彼此分开的不稳定连接。不稳定连接包括光可切割基团、酸不稳定部分、碱不稳定部分和酶可切割基团。某些实施方案中，连接子可以指将靶向多肽和药学活性部分(如，喜树碱小分子)连接的任何试剂或分子。连接子可以与两个实体部分形成共价键或非共价键。理想的连接子可以是保持稳定的、不影响所述靶向多肽或药学活性部分的功能性的、或不会产生另外的不需要的功能的任何试剂或分子。连接子可以是可降解的(cleavable)或不可降解的(non-cleavable)。常见的连接子可包括但不限于，缬氨酸-瓜氨酸连接子(valine-citrulline，vc)、缬氨酸-丙氨酸连接子(valine-alanine，va)、马来酰亚胺基甲基环己烷-1-羧酸酯(Maleimidomethyl cyclohexane-1-carboxylate，MCC)、马来酰亚胺基己酰基(maleimidocaproyl，mc)、N-琥珀酰亚胺基-4-(2-吡啶基二硫代)丁酸酯(N-succinimidyl-4-(2-pyridyldithio)butanoate，SPDB)、N-琥珀酰亚胺基-4-(2-吡啶基二硫代)-2-磺基丁酸酯(N-succinimidyl-4-(2-pyridyldithio)-2-sulfo butanoate，sulfo-SPDB)或4-(4-乙酰基苯氧基)丁酸(4-(4-acetylphenoxy)butanoic acid-N，AcBut-N)。
在本申请中，术语“嵌合抗体”通常是指一种抗体，其中重链和/或轻链的一部分与来源于特定物种或属于特定抗体种类或亚类的抗体中的相应序列相同或同源，而所述链的其余部分与来源于另一物种或属于另一抗体种类或亚类的抗体中的相应序列相同或同源。也包括所述抗体的片段，它们显示出需要的生物活性(即，特异性结合肿瘤抗原的能力)。
在本申请中，术语“全人源抗体”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”通常是指结合特定靶而对其他靶没有交叉反应性。例如，当抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。而选择反应性通常是指优先结合特定靶。
在本申请中，术语“肿瘤特异性抗原”通常是指主要或只能在肿瘤细胞上表达的蛋白。主要表达是指在肿瘤细胞上比在正常体细胞上相对更高表达，而只能表达是指通过本领域已知的标准手段在正常体细胞上没有检测到的蛋白而在肿瘤细胞上的表达。
在本申请中，术语“缀合物”是指共价或非共价(例如，通过接头)连接成一个更大分子的多肽(例如肽、核酸、蛋白质、酶、糖、多糖、脂质、糖蛋白和脂蛋白)和化学部份。可包括在缀合物中的化学部分可以是为治疗剂或细胞毒性剂，其非限制性实例可以为：有丝分裂抑制剂、抗肿瘤抗生素免疫调节剂、用于基因治疗的载体、烷化剂、抗血管生成剂、抗代谢物、含硼剂、化学保护剂、激素、抗激素剂、皮质类固醇、光活性治疗剂、寡核苷酸、放射性核素剂、拓扑异构酶抑制剂、激酶抑制剂和放射增敏剂。
在本申请中，术语“Her2”通常是指一种属于表皮生长因子受体家族的I型跨膜蛋白，也称为c-erbB2，ErbB2或Neu(Slamon，等，Science(科学)235(1987)177-182；Swiss-ProtP04626)。在本申请中，术语“Her2”还可涵盖Her2的同源物、变体和同工型，包括剪接同工型。术语“Her2”还包括具有Her2同源物、变体和同工型中的一个或多个序列的蛋白，以及该序列的片段，只要是该变体蛋白(包括同工型)、同源蛋白和/或片段能够被一种或多种Her特异性抗体(如帕妥珠单抗(Pertuzumab)或曲妥珠单抗(Trastuzumab))识别。Her2与人乳腺癌细胞中的肿瘤转化相关，如已在乳腺癌、胃癌、胰腺癌、卵巢癌、腹膜癌或结肠肠癌的患者中检测到Her2蛋白的过量表达。示例性的帕妥珠单抗的重链可变区可包含SEQIDNO.3所示的氨基酸序列，示例性的帕妥珠单抗的轻链可变区可包含SEQIDNO.4所示的氨基酸序列；示例性的曲妥珠单抗的重链可变区可包含SEQIDNO.1所示的氨基酸序列，示例性的曲妥珠单抗的轻链可变区可包含SEQIDNO.2所示的氨基酸序列。
在本申请中，术语“Trop2”、“TROP2”通常是指单程跨膜I型细胞膜蛋白，也称为肿瘤相关钙信号转导子2(TACSTD2)、GA733-1、EGP-1或MIS1。在本申请中，术语“Trop2”还可涵盖Trop 2的同源物、变体和同工型，包括剪接同工型。术语“Trop”还包括具有Trop 2同源物、变体和同工型中的一个或多个序列的蛋白，以及该序列的片段，只要是该变体蛋白(包括同工型)、同源蛋白和/或片段能够被一种或多种Trop特异性抗体(如hRS7或hTINA1)识别。Trop2可以是人Trop2，人Trop2的DNA序列和氨基酸序列可以在公共数据库上获得，例如可参见NCBI登录号NM_002353和NP_002344。示例性的hRS7的重链可变区可包含SEQIDNO.5所示的氨基酸序列，示例性的hRS7的轻链可变区可包含SEQIDNO.6所示的氨基酸序列；示例性的hTINA1的重链可变区可包含SEQIDNO.7所示的氨基酸序列，示例性的hTINA1的轻链可变区可包含SEQIDNO.8所示的氨基酸序列。
发明详述
一方面，本申请提供一种缀合物，其具有式I的结构：
Figure PCTCN2021071595-appb-000006
靶向多肽
本申请的缀合物具有式I的结构，其中，Mab为靶向多肽。
在某些情形中，Mab可以是抗体(包括全人源抗体、人源化抗体、非人抗体或嵌合抗体)、或其抗原结合片段(包括酶促或重组产生的片段)、或掺入有来自抗体或其抗原结合片段的序列的结合蛋白。抗体或其抗原结合蛋白可以是多价的和多特异性的或多价的和单特异性的。
在某些情形中，所述Mab可以是单克隆抗体。在更具体的情形中，靶向多肽可以是多价和/或多特异性的单克隆抗体。靶向多肽可以是鼠、嵌合、人源化或全人源的单克隆抗体，所述抗体可以是完整的、片段(Fab’、Fab、F(ab) 2、F(ab’) 2、Fv或scFv)或亚片段(单链构建体)形式，或为IgG1、IgG2、IgG3、IgG4、IgA同种型，或其亚分子。本领域技术人员将认识到，本申请所述的缀合物、方法、组合物和用途可利用本领域已知的多种抗体的任意一种抗体。使用的抗体可以从多种已知来源商购获得。
在某些情形中，所述Mab可以是嵌合抗体。嵌合抗体是其中人抗体的可变区被例如鼠抗体的可变区取代，而包括鼠抗体的互补决定区(CDR)的重组蛋白。嵌合抗体在被施用至受治疗者时表现出降低的免疫原性和增加的稳定性。构建嵌合抗体的方法为本领域所众所周知的。
嵌合抗体可通过将小鼠免疫球蛋白重链和轻链可变区的小鼠CDR转移至人抗体的相应结构域进行人源化。所述嵌合抗体的小鼠骨架区(FR)也被替换为人FR序列。在某些情形中，所述Mab可以是人源化抗体。为了保留人源化抗体的稳定性和抗原特异性，可将一个或多个人FR残基替换为小鼠的对等残基。人源化抗体可用于受治疗者的治疗性治疗。生产人化抗体的技术为本领域众所周知的。
在某些情形中，所述Mab可以是全人源抗体。使用组合方法或使用人免疫球蛋白基因座转化的转基因动物生产全人源抗体的方法为本领域已知的。所述全人源抗体预期表现出比嵌 合抗体或人源化抗体更小的副作用，并在体内充当基本上内源的人抗体。
本申请所述的Mab可以是抗体片段。可通过常规方法，例如将全长抗体通过胃蛋白酶或木瓜蛋白酶消化获得抗体片段。例如，抗体片段可通过用胃蛋白酶酶切抗体生产，以提供表示为F(ab') 2的5S片段。该片段可进一步使用硫醇还原剂切割，并且任选地使用因二硫键切割产生的琉基的封闭基团，以生产3.5S的Fab'单价片段。例如，利用胃蛋白酶酶切产生两个单价Fab片段和一个Fc片段。也可使用其他切割抗体的方法，例如分离重链以形成单价轻链-重链片段，进一步切割片段，或者其他酶促技术、化学技术或基因技术，只要所述片段结合被完整抗体所识别的抗原即可。在某些情形中，所述Mab可以是单链抗体。这些单链抗体可可通过构建包含由寡核苷酸接头序列连接的编码VH结构域和VL结构域的DNA序列的结构基因来制备。将结构基因插入表达载体，随后将表达载体引入宿主细胞诸如大肠杆菌。重组的宿主细胞合成单条多肽链，其中接头肽桥接两个可变结构域。用于产生scFv的方法是本领域公知的。
在某些情形中，所述Mab可以被修饰，例如，被通过删除、增加或取代抗体的部分而修饰过。所述修饰可以改变抗体的某种性质，如使靶向部分连接的小分子部分数目达到所希望的数目、增强后降低ADCC、增强或降低CDC。在某些实施方案中，抗体的序列，诸如抗体的Fc部分，可被改变以优化缀合物的生理学特征，诸如血清半衰期。取代蛋白质中的氨基酸序列的方法是本领域广泛已知的，例如通过定点突变。在某些实施方案中，变化可包括在Fc序列中添加或去除一个或多个糖基化位点。在另一些实施方案中，可在Fc序列中进行特定的氨基酸取代。
在某些情形中，所述Mab能够识别或结合标记物或与肿瘤相关的抗原，并且主要或者只能识别在相对正常组织为病变的细胞上表达，并且能够被细胞内化。
在某些情形中，所述Mab可识别或结合的靶点或抗原可以包括：碳酸酐酶IX、B7、CCCL19、CCCL21、CSAp、Her2、BrE3、CD1、CDla、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20(如，C2B8、hA20、lF5MAb)、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD67、CD70、CD74、CD79a、CD80、CD83、CD95、CD126、CD133、CD138、CD147、CD154、CEACAM5、CEACAM-6、甲胎蛋白(AFP)、VEGF、ED-B纤连蛋白(如L19)、EGP-1、EGP-2(如，17-1A)、EGF受体(ErbB1)、ErbB2、ErbB3、因子H、FHL-1、Flt-3、叶酸受体、Ga733、GROB-1、缺氧诱导因子(HIF)、HM1.24、胰岛素样生长因子(ILGF)、IFN-γ、IFN-α、IFNβ、IL-2R、IL-4R、IL-6R、IL-13R、IL-15R、IL- 17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-25、IP-10、IGF-1R、Ia、、神经节糖苷、HCG、L243结合的HLA-DR抗原、CD66抗原(CD66a-d或其组合)、MAGE、mCRP、MCP-1、MIP-1A、MIP-1B、巨隧细胞移动抑制因子(MIF)、MUC1、MUC2、MUC3、MUC4、MUC5、胎盘生长因子(P1GF)、PSA(前列腺特异性抗原)、PSMA、PAM4抗原、NCA-95、NCA-90、A3、A33、Ep-CAM、KS-1、Le(y)、间皮素、S100、腱生蛋白、TAC、Tn抗原、Thomas-Friedenreich抗原、肿瘤坏死抗原、肿瘤血管生成抗原、TNF-α、TRAIL受体(R1和R2)、VEGFR、RANTES、T101、以及癌干细胞抗原、补体因子C3、C3a、C3b、C5a、C5、和致癌基因产物。
在某些情形中，所述Mab可以特异性结合肿瘤特异性抗原。在某些情形中，所述靶向多肽可以是与肿瘤细胞上表达的抗原或抗原表位反应的抗体或其抗原结合片段。可利用的肿瘤特异性抗原包括所述肿瘤特异性抗原可以选自CLDN18.2、Nectin 4、CD22、CD30、CD33、Her2、Mesothelin、PSMA与Trop2。例如，所述肿瘤特异性抗原可选自下组：CLDN18.2、Nectin 4、Her2和Trop2。
在某些情形中，所述Mab能内化，然后重新表达、加工并且呈递到细胞表面上，使得细胞能够连续摄入所述缀合物，并且增加所述缀合物循环。可用于本申请的靶向多肽包括但不限于以下多肽或抗体：LL1抗体(抗-CD74)、LL2和RFB4抗体(抗-CD22)、RS7抗体(抗-上皮糖蛋白-1(EGP-1))、PAM-4和KC4抗体(均为抗-粘蛋白)、MN-14抗体(抗-癌胚抗原(CEA，也称为CD66e)、Mu-9抗体(抗-结肠-特异性抗原-p)、Immu31抗体(抗甲胎蛋白)、TAG-72抗体(如CC49)、Tn抗体、J591或HuJ591(抗-PSMA(前列腺特异性膜抗原))、AB-PG1-XG1-026(抗-PSMA二聚体)、D2/B(抗-PSMA)、G250(抗-碳酸酐酶IXMab)和hL243(抗-HLA-DR)。
例如，所述Mab可包含抗体HCDR3，所述HCDR3可包含SEQ ID NO:19、25、31和39中任一项所示的氨基酸序列。
例如，所述Mab可包含抗体HCDR2，所述HCDR2可包含SEQ ID NO:18、24、30和38中任一项所示的氨基酸序列。
例如，所述Mab可包含抗体HCDR1，所述HCDR1可包含SEQ ID NO:17、23、29和37中任一项所示的氨基酸序列。
例如，本申请的缀合物中的Mab可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3分别包含选自以下组的任意一组氨基酸序列：
(1)HCDR1:SEQ ID NO:17，HCDR2:SEQ ID NO:18和HCDR3:SEQ ID NO:19；
(2)HCDR1:SEQ ID NO:23，HCDR2:SEQ ID NO:24和HCDR3:SEQ ID NO:25；
(3)HCDR1:SEQ ID NO:29，HCDR2:SEQ ID NO:30和HCDR3:SEQ ID NO:31；和
(4)HCDR1:SEQ ID NO:37，HCDR2:SEQ ID NO:38和HCDR3:SEQ ID NO:39。
本申请缀合物中的Mab可包含抗体重链可变区VH，所述VH可包含SEQ ID NO:1、5、32和40中任一项所示的氨基酸序列。
例如，所述Mab可包含抗体LCDR3，所述LCDR3可包含SEQ ID NO:22、28、35、43中任一项所示的氨基酸序列。
例如，所述Mab可包含抗体LCDR2，所述LCDR2可包含SEQ ID NO:21、27、34和42中任一项所示的氨基酸序列。
例如，所述Mab可包含抗体LCDR1，所述LCDR1可包含SEQ ID NO:20、26、33和41中任一项所示的氨基酸序列。
例如，本申请的缀合物中的Mab可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3分别包含选自以下组的任意一组氨基酸序列：
(1)LCDR1:SEQ ID NO:20，LCDR2:SEQ ID NO:21和LCDR3:SEQ ID NO:22；
(2)LCDR1:SEQ ID NO:26，LCDR2:SEQ ID NO:27和LCDR3:SEQ ID NO:28；
(3)LCDR1:SEQ ID NO:33，LCDR2:SEQ ID NO:34和LCDR3:SEQ ID NO:35；和
(4)LCDR1:SEQ ID NO:41，LCDR2:SEQ ID NO:42和LCDR3:SEQ ID NO:43。
本申请缀合物中的Mab可包含抗体轻链可变区VL，所述VL可包含SEQ ID NO:2、6、36和44中任一项所示的氨基酸序列。
例如，本申请的缀合物中的Mab可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别包含选自以下组的任意一组氨基酸序列：
(1)HCDR1:SEQ ID NO:17，HCDR2:SEQ ID NO:18，HCDR3:SEQ ID NO:19，LCDR1:SEQ ID NO:20，LCDR2:SEQ ID NO:21和LCDR3:SEQ ID NO:22；
(2)HCDR1:SEQ ID NO:23，HCDR2:SEQ ID NO:24，HCDR3:SEQ ID NO:25，LCDR1:SEQ ID NO:26，LCDR2:SEQ ID NO:27和LCDR3:SEQ ID NO:28；
(3)HCDR1:SEQ ID NO:29，HCDR2:SEQ ID NO:30，HCDR3:SEQ ID NO:31，LCDR1:SEQ ID NO:33，LCDR2:SEQ ID NO:34和LCDR3:SEQ ID NO:35；和
(4)HCDR1:SEQ ID NO:37，HCDR2:SEQ ID NO:38，HCDR3:SEQ ID NO:39，LCDR1:SEQ ID NO:41，LCDR2:SEQ ID NO:42和LCDR3:SEQ ID NO:43。
例如，本申请所述缀合物中的Mab可包含VH和VL，且所述VH可包含SEQ ID NO:1所示的氨基酸序列，所述VL可包含SEQ ID NO:2所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含VH和VL，且所述VH可包含SEQ ID NO:5所示的氨基酸序列，所述VL可包含SEQ ID NO:6所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含VH和VL，且所述VH可包含SEQ ID NO:32所示的氨基酸序列，所述VL可包含SEQ ID NO:36所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含VH和VL，且所述VH可包含SEQ ID NO:40所示的氨基酸序列，所述VL可包含SEQ ID NO:44所示的氨基酸序列。
本申请所述缀合物中的Mab可包含抗体重链和抗体轻链，且所述抗体重链可包含SEQ ID NO:9所示的氨基酸序列，所述抗体轻链可包含SEQ ID NO:10所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含抗体重链和抗体轻链，且所述抗体重链可包含SEQ ID NO:11所示的氨基酸序列，所述抗体轻链可包含SEQ ID NO:12所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含抗体重链和抗体轻链，且所述抗体重链可包含SEQ ID NO:13所示的氨基酸序列，所述抗体轻链可包含SEQ ID NO:14所示的氨基酸序列。例如，本申请所述缀合物中的Mab可包含抗体重链和抗体轻链，且所述抗体重链可包含SEQ ID NO:15所示的氨基酸序列，所述抗体轻链可包含SEQ ID NO:16所示的氨基酸序列。
喜树碱衍生物
本申请缀合物具有式I的结构，其中，R 1可以为经卤素取代的甲基(例如，经氯取代的甲基，经氟取代的甲基，经溴取代的甲基，经碘取代的甲基)；R 2可以选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3可以选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基。本申请所述的氨基可以包括氮原子与两个氢原子相连的-NH 2，也可包括-NH 2中的一个或两个氢原子被烷基取代后的基团，例如，-N(CH 3) 2、NHCH 3、-N(CH 2CH 3) 2、-N(CH 2CH 2CH 3) 2、-NHCH 2CH 3或-NHCH 2CH 2CH 3等。C 1-C 6烷基可以是直链烷基也可以是支链烷基，包括但不限于甲基、乙基、正丙基、异丙基、叔丁基等。
在本申请，术语“经取代的C 1-C 6烷基”可以是指C 1-C 6烷基中的至少一个氢原子被卤素、氨基、羟基、烷氧基或芳基取代后的基团。
在某些情形中，R 3可以选自氢、氨基、C 1-C 4烷基或经取代的C 1-C 4烷基；在另一些情形中，R 3可选自氢、氨基或
Figure PCTCN2021071595-appb-000007
其中，R 4和R 5可分别选自氢、C1-C6烷基，经取代的C1-C6烷基或芳基。R 3的结合位点可以为苯环上未取代的三个位点中的任意一个，包括R1 的邻位或间位。例如，R 3的结合位点可以为小分子的C 9位置。例如，R 3可以是氢。
在某些情形中，R 2可以选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基。同样的，C 1-C 6烷基可以是直链烷基也可以是支链烷基，包括但不限于甲基、乙基、正丙基、异丙基、叔丁基等。经取代的C 1-C 6烷基可以是指C1-C6烷基中的至少一个氢原子被卤素、氨基、羟基、烷氧基或芳基取代后的基团。在某些实施方式中，R 2可选自氢、C 1-C 4烷基或经取代的C 1-C 4烷基；例如，R 2可选自Et、
Figure PCTCN2021071595-appb-000008
其中，R 4和R 5可分别选自氢、C 1-C 6烷基、经取代的C 1-C 6烷基或芳基。例如，R 2可以是-CH 2CH 3。
在某些情形中，R 1可以为经卤素取代的甲基。R 1可以为经氟、氯、溴或碘取代的甲基。在某些情形中，R1可以是经1个、2个或3个卤素取代的甲基。例如，R 1可选自-CF 2H、-CCl 2H、-CBr 2H、-CFH 2、-CBrH 2、-CClH 3、-CF 3、-CCl 3或-CBr 3。在某些实施方式中，R 1可以是-CF 2H。
例如，本申请所述缀合物中的小分子可以是如下的结构：
Figure PCTCN2021071595-appb-000009
连接子
靶向多肽和小分子部分之间可通过连接子L连接。在某些实施方式中，所述连接子L可包含确定的聚乙二醇(PEG)部分，L-氨基酸，以及另外的间隔物。
在某些情形中，所述确定的PEG部分即包含确定数目的单体单元的PEG，其中确定的PEG可以是低分子量的PEG，在某些情况下，可包含1-30个单体单元，例如，包含1-12个单体单元。所述确定的PEG在一端可带有反应性基团(例如，羧酸或羟基)。
在某些情形中，所述L-氨基酸可选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、组胺酸、天冬酰胺、谷氨酸、赖氨酸、谷胺酰胺、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸和脯氨酸。在某些实施方式中，所述L-氨基酸的数量可以是0个、1个、2个、3个、4个或更多个。例如，所述L-氨基酸的数量可以是1个，且所述1个L-氨基酸可选自以下任意一种：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、组胺酸、天冬酰胺、谷氨酸、赖氨酸、谷胺酰胺、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸和脯氨酸。
在某些情形中，所述另外的间隔物可以选自乙醇胺、4-羟基苄醇、4-氨基苄醇或取代的或 未取代的乙二胺。如果其包括羟基，则其分别连接于以碳酸酯或氨基甲酸形式的药物的羟基或氨基。在某些情况下，所述另外的间隔物是衍生自L-氨基酸的取代的乙二胺，其中的羧酸基团被羟甲基部分代替。所述另外的间隔物可衍生自以下L-氨基酸的任意一种：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、组胺酸、天冬酰胺、谷氨酸、赖氨酸、谷胺酰胺、甲硫氨酸、精氨酸、丝氨酸、苏氨酸、半胱氨酸和脯氨酸。在另一些情形中，所述另外的间隔物可以是可折叠部分注入4-氨基苄醇或在苄型位置用C1-C 10烷基取代的取代的4-氨基苄醇，取代的4-氨基苄醇经由其氨基连接于L-氨基酸或包含多个L-氨基酸部分的多肽；其中N末端与终止于靶向多肽部分的交联接头连接。例如，在所述连接子L中，所述另外的间隔物可以衍生自取代的4-氨基苄醇，所述取代的4-氨基苄醇被氢或选自C 1-C 10烷基的烷基取代。
在某些实施方式中，所述连接子L可以具有如下结构：
Figure PCTCN2021071595-appb-000010
方法
另一方面，本申请提供了一种产生所述的缀合物的方法，其可包括：获得式IV所示的化合物：
Figure PCTCN2021071595-appb-000011
其中，R 1可以为经卤素取代的甲基；R 2可以选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3可以选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基；Mab可以为靶向多肽；L可以为连接子；使式IV所示的化合物与所述Mab缀合，以得到所述的缀合物。
本申请提供了化合物IV的制备方法，其可以包括，1)制备式I所示的小分子，2)合成连接子，以及3)获得式IV所示的化合物。
制备式I所示的小分子可包括采用10-羟基喜树碱类化合物与磺酰化试剂反应得到磺酸酯类中间体。在某些情形中，10-羟基喜树碱类化合物的结构式可以为
Figure PCTCN2021071595-appb-000012
磺酸酯类中间体的结构式可以为
Figure PCTCN2021071595-appb-000013
其中，R 1可选自氢、氨基、C 1-C 6烷基或经取代的C 1-C 6烷基，R 1的结合位点可以为苯环上未取代的三个位点中的任一个；R 2可选自氢、C 1-C 6烷基或经取代的C 1-C 6烷基；R 3选自氢、酰基、C 1-C 6烷基、经取代的C 1-C 6烷基；R 6可选自甲磺酰基、对甲苯磺酰基或三氟甲磺酰基。
磺酰化试剂可以对10-羟基喜树碱类化合物的C10位羟基进行磺酰化，得到离去基团-OTf，-OTs或-OMs，便于后续步骤中用经卤素取代的甲基进行取代。磺酰化试剂可包括磺酰氯试剂、磺酸试剂、磺酸酐试剂以及磺酰胺试剂，根据具体的磺酰基来说，可包括甲磺酰氯、对甲苯磺酰氯、三氟甲磺酰氯、甲磺酸、对甲苯磺酸、三氟甲磺酸、甲磺酸酐、对甲苯磺酸酐、三氟甲磺酸酐和N-苯基双(三氟甲磺酰亚胺)中的至少一种。
10-羟基喜树碱类化合物与磺酰化试剂反应的温度可以为60-100℃，反应时间为2-8h。在上述反应条件下进行磺酰化反应，反应更为充分，能够高收率地得到磺酸酯类中间体。例如，10-羟基喜树碱类化合物与磺酰化试剂的摩尔比可以为1:1-1.5。过量的磺酰化试剂可以保证10-羟基喜树碱类化合物的充分反应，提高原料利用率。
制备式I所示的小分子还可包括将磺酸酯类中间体与经卤素取代的甲基试剂反应，得到10-卤代甲基喜树碱类化合物。
本申请所述制备方法还可包括合成连接子L。在本申请中，在小分子与靶向多肽之间放置确定的聚乙二醇(PEG)部分(即，包含确定数目的单体单元的PEG)，其中确定的PEG是低分子量的PEG，在某些情况下，包含1-30个单体单元，例如，包含1-12个单体单元。该连接子可增强小分子的溶解度。
在某些实施方式中，所述L的确定的聚乙二醇部分的一端可带有不同反应性基团，例如，羧基或羟基。所述聚乙二醇部分可与氨基醇的氨基连接，氨基醇的羟基与小分子上碳酸酯形式的羟基连接。在某些情形中，所述聚乙二醇部分的不同反应性基团的一端可与L-氨基酸或多肽N末端连接，C末端连接于氨基醇的氨基，氨基醇的羟基分别连接于小分子上所连接的 碳酸酯或氨基甲酸酯。在某些情形中，可经由乙炔-叠氮化物环加成反应与小分子反应以提供可用于与靶向多肽共轭的最终的缀合物。在某些实施方式中，靶向多肽-偶联基团设计为硫醇或硫醇反应性基团。
本申请所述制备方法还可包括获得式IV所示的化合物，通过所述连接子L将所述靶向部分和式I中的小分子共轭。
药物组合物和用途
另一方面，本申请提供了一种药物组合物，其包含所述的缀合物。所述缀合物或药物可根据制备药学上可用的组合物的已知方法配制，由此所述免疫缀合物在混合物中与药学上合适的赋形剂混合。无菌磷酸缓冲盐水是药学上合适的赋形剂的一个实例。其他合适的赋形剂是本领域技术人员众所周知的。
另一方面，本申请提供了本申请所述缀合物或药物组合物在制备预防或治疗肿瘤的药物中的用途。本申请还提供了预防或治疗肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述缀合物或药物组合物。可使用本申请所述的缀合物或药物组合物制备的药物可治疗的肿瘤可以包括实体瘤和/或非实体瘤，例如，所述实体瘤可以选自以下组：前列腺癌、乳腺癌、胰腺癌、结肠癌和胃癌。本申请所述缀合物可用于抑制肿瘤的生长、进展和/或转移，尤其是以上列出的那些癌症。
另一方面，本申请提供了所述的缀合物与其他肿瘤疗法或药物共同在制备预防或治疗肿瘤的药物中的用途。本申请还提供了预防或治疗肿瘤的方法，所述方法包括向有需要的受试者施用本申请所述缀合物或药物组合物，以及其他肿瘤疗法或药物。所述肿瘤可以包括实体瘤和/或非实体瘤，例如，所述实体瘤可以选自以下组：前列腺癌、乳腺癌、胰腺癌、结肠癌和胃癌。取决于疾病状态和缀合物的耐受性，所述缀合物可单次给予或重复给予，还可以与其他肿瘤疗法或药物联用，所述其他肿瘤疗法或药物可包括例如手术、外部辐射、放射免疫疗法、化疗、反义疗法、干扰RNA疗法、基因疗法等。每种组合将适合于肿瘤类型、阶段、患者情况和此前疗法，以及主治医生考虑的其他因素。在某些实施方式中，所述其他肿瘤疗法或药物选自下组：化疗、放疗、miRNA和寡核苷酸。
可与所述的缀合物联用的化疗包括化疗药物，如长春花生物碱类、蔥环类抗生素、表鬼臼毒素类、紫杉醇类、抗代谢物、烷化剂、抗生素、Cox-2抑制剂、抗有丝分裂剂、抗血管生成剂和促细胞调亡剂，如阿霉素、氨甲喋呤、紫杉醇、其他喜树碱类、以及其他形式的这些和其它类别的抗癌剂等。其他化疗药物可包括氮芥类、烷基磺酸酯类、亚硝基脲类、三氮烯类、叶酸类似物、嘧啶类似物、嘌呤类似物、铂配位复合物、激素等。所述化疗可以是本申 请所述的缀合物的一部分，或可选地可在所述缀合物之前、同时或之后联合使用。
可与本申请所述的缀合物联用的放疗可包括用于诱导肿瘤细胞内局部DNA损伤的任何机制，诸如γ-辐射、X-射线、UV-辐射、微波、电子发射等。放疗还可包括使用将放射性同位素定向递送至肿瘤细胞的组合疗法，并且可以与所述缀合物组合使用或作为其一部分使用。任选地，所述放疗可以作为单一剂量或作为多个连续剂量施用。
可与本申请所述的缀合物联用的寡核苷酸可包括任意合适的短链核苷酸(包括脱氧核糖核酸DNA或核糖核酸RNA内的核苷酸)，例如，反义寡核苷酸、小干扰RNA、核酶、脱氧核酶、反基因、CpG寡核苷酸、转录因子诱饵和核酸适配体。
缀合物的合适的施用途径包括但不限于口服、胃肠外、直肠、经粘膜、肠施用、肌内、皮下、髓内、鞘内、直接心室内、静脉内、玻璃体内、腹膜内、鼻内或眼内注射。例如，施用途径是胃肠外。人们可能以局部方式而不是系统方式施用化合物，例如，经由直接注射化合物到实体瘤中。
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融合蛋白、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缀合物的制备
1.1 Linker合成
在室温将商购获得的1.5gFmoc-Lys-(MMT)-OH(CAS#167393-62-6)、0.3g4-氨基苄醇(CAS#623-04-1)溶于15ml二氯甲烷，在混合物中加入EEDQ(CAS#16357-59-8)，并搅拌4h。萃取后通过快速色谱得到1.67g中间产物(化合物I)，为白色泡沫。化合物I表征如下：ESI-MSm/e 745.8(M+H)。
Figure PCTCN2021071595-appb-000014
将1.5g该中间产物溶解在二乙胺(10mL，CAS#109-89-7)中，并搅拌2h。去除溶剂后，用己烷洗涤残留物，得到无色沉淀的0.97g中间产物(化合物II)。化合物II表征如下：ESI-MS m/e 549.2(M+Na)。
Figure PCTCN2021071595-appb-000015
利用EEDQ(CAS#16357-59-8)将0.57g化合物II与商购获得的0.63g PEG-N3(O-(2-叠氮基乙基)-O’-(N-二乙醇酰基-2-氨乙基)庚乙二醇)在二氯甲烷中偶联。除去溶剂快速色谱法得到0.99g的linker(化合物III)，为淡黄色油状物，产率85％。化合物III表征如下：ESI-MS：m/e 1082.7(M+Na),m/e 1061.3(M+H)。
Figure PCTCN2021071595-appb-000016
1.2 Linker-小分子制备
利用三光气(CAS#1122-58-3)和DMAP(CAS#32315-10-9)由化合物IV(喜树碱衍生物，制备方法请参见CN108690036A)制备得到化合物V。
Figure PCTCN2021071595-appb-000017
在氩气保护下将0.5g化合物V和0.95g化合物III在10mL二氯甲烷中反应10min。由快速色谱法纯化混合物，获得0.98g淡黄色油状物(化合物VI，产率68％)。化合物VI表征如下：ESI-MS:m/e 1513.7(M+H)。
Figure PCTCN2021071595-appb-000018
在CuBr(0.01g，CAS#7787-70-4)、DIEA(0.01mL，CAS#7087-68-5)和三苯基膦(0.02g，CAS#603-35-0)存在下，将化合物VI(0.88g)与4-(N-马来酰亚胺基甲基)-N-(炔丙基)-环己烷-1-酰胺(0.25g)在二氯甲烷中反应18h。然后用EDTA(CAS#139-33-3)洗涤。快速色谱法得到黄色泡沫的化合物VII(0.95g，产率85％)。化合物VII表征如下：ESI-MS m/e 1788.1(M+H)。
Figure PCTCN2021071595-appb-000019
将二氯乙酸(0.6mL，CAS#79-43-6)和苯甲醚(0.06mL，CAS#100-66-3)溶于二氯甲烷(6mL)，将化合物VII0.5g脱保护，用乙醚沉淀，得到化合物VIII淡黄色粉末0.4g，产率为94％。化合物VIII表征如下：ESI-MS m/e 1517.2(M+2H)，m/e 758.8([M+2H]/2)。
Figure PCTCN2021071595-appb-000020
1.3抗体还原
可利用方法1或方法2得到轻度还原的抗体antiHer2抗体曲妥珠单抗、帕妥珠单抗、antiTrop2抗体hRS7、hTINA1。其中，曲妥珠单抗重链可变区氨基酸序列如SEQ ID NO:1所示，抗体轻链可变区氨基酸序列如SEQ ID NO:2所示；帕妥珠单抗抗体重链可变区氨基酸序列如SEQ ID NO:3所示，抗体轻链可变区氨基酸序列如SEQ ID NO:4所示；hRS7抗体重链可变区氨基酸序列如SEQ ID NO:5所示，抗体轻链可变区氨基酸序列如SEQ ID NO:6所示；hTINA1抗体重链可变区氨基酸序列如SEQ ID NO:7所示，抗体轻链可变区氨基酸序列如SEQ ID NO:8所示。
方法1：在含有5.4mMEDTA(CAS#139-33-3)的40mMPBS(Na 2HPO 4、NaH 2PO 4和NaCl，pH＝7.4)中，将每种抗体用50-70倍摩尔过量的二硫苏糖醇(DTT，CAS#3483-12-3)还原45min。由分子排阻色谱、透析过滤纯化还原产物。然后将缓冲液置换到pH6.5的适当缓冲液中。最后，由Ellman’s检验(DTNB，CAS69-78-3)确定硫醇含量，测定412nm处的吸光度。
方法2：将抗体用Tris(2-羧乙基)膦(TCEP，CAS#51805-45-9)在pH5-7范围内的磷酸盐缓冲液中还原。
M Ab→Reduced M Ab
1.4共轭
利用7-15％v/v的DMSO作为共溶剂，将还原的抗体与10-15倍摩尔过量的化合物VIII反应，并在室温孵育20min。将共轭物利用分子排阻色谱，通过疏水株，最后通过超滤-透析纯化。利用UV分光光度仪在366nm处的吸光度测定产物的喜树碱吸收，将其与标准值关联，蛋白浓度从280nm处的吸光度判断，去除喜树碱吸收。从而确定小分子药物/抗体的取代比。
将纯化的共轭物作为冻干制剂储存在玻璃小瓶中，在真空下加帽，储存在-20℃冰箱中。
Figure PCTCN2021071595-appb-000021
得到的缀合物命名为HB010(抗体为曲妥珠单抗)、HB020(抗体为帕妥珠单抗)、HB030(抗体为hRS7)、HB040(抗体为hTINA1)、HB050(抗体为抗CLDN18.2抗体)和HB060(抗体为抗Nectin 4抗体)。
同时，将化合物IV替换为SN-38(CAS#86639-52-3)，按照同样的方法制备对照缀合物HB011(抗体为曲妥珠单抗)、HB021(抗体为帕妥珠单抗)、HB031(抗体为hRS7)、HB041(抗体为hTINA1)、HB051(抗体为抗CLDN18.2抗体)和HB061(抗体为抗Nectin 4抗体)。
按照上述方法将二氟甲基化合物IV替换为一氟甲基化合物、氯代化合物、溴代化合物，制备得到缀合物。
制备的缀合物及药物抗体比率如下：
表1示例性的缀合物及其DAR值
Figure PCTCN2021071595-appb-000022
Figure PCTCN2021071595-appb-000023
实施例2细胞结合活性
检测实施例1得到的缀合物与抗原的结合活性。收获处于对数生长期的细胞，并用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上。1000r/min速度下，离心5min后，弃上清；使用PBS洗涤细胞一次；使用FACS Buffer重悬细胞，并计数；使用FACS缓冲液配制密度为5×10 6cells/mL的细胞悬液；向96孔板中分别加入80μL的细胞悬液；设置空白对照组(不加抗体、二抗)、二抗对照组(不加抗体，加二抗)和实验组(加抗体和二抗)。
向实验组中加入20μL不同梯度浓度的受试物，终浓度起始浓度为：20μg/ml，3.16倍稀释，8个浓度。其他对照组中加入等体积的FACS缓冲液；混匀后，放于4℃避光孵育40min；使用FACS缓冲液洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用100μL FACS缓冲液重悬细胞；然后向实验组、二抗对照组中加入5μL的PE标记的二抗(PE抗人IgG Fc抗体)，空白对照组加入等体积的FACS缓冲液；混匀后，放于4℃避光孵育40min；接着，使用FACS缓冲液洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用250μL FACS缓冲液重悬细胞；流式细胞仪检测YEL-HLog(Excitation Laser：488nm Blue Laser)。最后用FlowJo软件分析FACS数据。
待测样品与抗原的结合活性如图1-2和下表2所示。结果显示，本申请所述的缀合物HB010和Her2的结合EC 50值与对照分子HB011无明显差别，HB030和TROP2的结合EC 50稍高于对照分子HB031。
结果表明，连接不同Her2抗体或Trop2抗体的缀合物也能与抗原有效结合。将二氟甲基取代替换为其他卤素(如氯、溴)取代后，所得到的缀合物也具有抗原结合活性。
表2缀合物与抗原的结合活性
Figure PCTCN2021071595-appb-000024
实施例3体外抗肿瘤活性
检测实施例1得到的缀合物的体外抗肿瘤活性。采用冷光检测法，收集处于对数生长期的细胞并采用血小板计数器进行细胞计数，用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上。选用的可商购获得的细胞株、培养基及细胞接种数如下表3所示。
表3癌细胞系的培养条件
细胞株 细胞类型 细胞数量/孔 培养基
MDA-MB-468 乳腺癌 3000 RPMI1640+10％FBS
HCC1954 乳腺癌 3000 RPMI1640+10％FBS
SK-BR-3 乳腺癌 5000 RPMI1640+10％FBS
BXPC-3 胰腺癌 5000 DMEM+10％FBS
COLO205 结肠癌 3000 RPMI1640+10％FBS
NCI-N87 胃癌 5000 RPMI1640+10％FBS
分别添加90μl细胞悬液至96孔板中，将96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下培养过夜。配制浓度梯度稀释的待测药物溶液，在接种有细胞的96孔板中每孔加入10μl待测药物，每个浓度设置三个复孔。将已加药的96孔板中的细胞置于37℃、5％CO 2、95％湿度条件下继续培养72h，之后加入测定发光细胞活力的CTG试剂(Promega，Cat#G7572)进行分析。融化CTG试剂并平衡细胞板至室温30min，每孔加入等体积的CTG溶液，在定轨摇床上震动5min使细胞裂解，将细胞放置于室温20min冷光信号，用SpectraMax(MD， 2104-0010A)多标记微孔板检测仪读取冷光值。使用GraphPad Prism5.0软件分析数据，利用非线性S曲线回归来拟合数据得出剂量-效应曲线，并由此计算IC50值，计算结果如图3-8和下表4所示。
结果显示，HB010和HB030对于多种肿瘤细胞均具有明显的抑制作用，HB010对乳腺癌和胃癌细胞株的抗肿瘤效果与对照分子HB011无明显差异，HB030对乳腺癌、胰腺癌、结肠癌和胃癌细胞株的抗肿瘤效果比对照分子HB031好。
结果表明，连接不同Her2抗体或Trop2抗体的缀合物也能有效抑制肿瘤细胞生长。将二氟甲基取代替换为其他卤素(如氯、溴)取代后，所得到的缀合物对癌细胞株也具有抑制作用。
表4缀合物体外抑制癌细胞生长的活性
Figure PCTCN2021071595-appb-000025
实施例4药代动力学实验
将实施例1得到的缀合物静脉注射给予小鼠，于给药前和注射结束后一定时间内采集血样，分离血清，Elisa法测定血清中药物的浓度，并用软件计算药代动力学参数。
结果显示了所述缀合物在小鼠体内的半衰期。
实施例5体内抗肿瘤活性
对具有乳腺癌、胰腺癌、结肠癌或胃癌异种移植物的小鼠用实施例1制备的缀合物治疗。观察缀合物的治疗效果。结果显示，本申请所述缀合物能在体外有效治疗肿瘤。
实施例6本申请缀合物抑制乳腺癌小鼠肿瘤生长
6.1 SK-BR-3乳腺癌
(1)50只5～6周龄雌性BALB/c裸鼠，SPF级条件下适应5日后，腋下接种4×10 6个 SK-BR-3乳腺癌细胞。喂食灭菌饲料、10ppm次氯酸钠灭菌自来水。待肿瘤体积约130mm 3时，随机分为5组，每组10只：生理盐水组、伊立替康(5mg/kg)组、HB011(0.5mg/kg)组、HB010(0.5mg/kg)组、HB010(0.05mg/kg)组。按相应剂量，每周每只鼠尾静脉注射给药2次，连续给药4周，停药后持续观察10日。每周用电子数显卡尺测量肿瘤长径和短径1次，用电子秤称量裸鼠体重1次。
肿瘤体积(mm 3)＝1/2×长径(mm)×[短径(mm)] 2
肿瘤体积变化百分比(％)＝(每周肿瘤体积-第1次给药肿瘤体积)/第1次给药肿瘤体积×100％
结果如图9-10所示：各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB010(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB010(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB011(0.5mg/kg)组的大。且同剂量下HB010的抑制乳腺癌生长的效果略优于HB011。图9中，HB010(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图10中，HB010(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB010对SK-BR-3乳腺癌的抑制作用优于伊立替康和HB011，具有一定量效关系，未见显现出毒性反应。
(2)或者，接种6×10 6个SK-BR-3乳腺癌细胞至雌性裸鼠，停药后持续观察10日，检测HB060和HB061对肿瘤的抑制效果。
结果如图11-12所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB060(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB060(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB061(0.5mg/kg)组的大。且同剂量下HB060的抑制乳腺癌生长的效果优于HB061。图11中，HB060(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图12中，HB060(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
6.2 MDA-MB-468乳腺癌
(1)按照6.1的方法接种2×10 6个MDA-MB-468乳腺癌细胞至雌性裸鼠，停药后持续观察17日，按照6.1的方式给药并检测小鼠肿瘤体积变化和平均体重。
结果如图13-14所示：各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB030(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB030(0.05mg/kg)组的肿瘤体积比生理盐水组的小，与伊立替康(5mg/kg)组相近，比HB031(0.5mg/kg)组的大。且同剂量下HB030的抑制乳腺癌生长的效果与HB031相近甚至略优于HB031。图13中，HB030(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图14中，HB030(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
(2)按照6.1的方法接种6×10 6个MDA-MB-468乳腺癌细胞至雌性裸鼠，每5天尾静脉注射给药1次，停药后持续观察10日。结果如图13-14所示，HB030(0.2mg/kg)的抑制肿瘤效果显著优于其它各组，其抗肿瘤活性至少是HB031(0.5mg/kg)的2.5倍。且未见各组显示出毒性反应。图15中，HB030(0.2mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.001；图16中，HB030(0.2mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
图13-16的结果说明HB030对MDA-MB-468乳腺癌的抑制作用优于伊立替康、抗Trop2抗体和HB031，具有一定量效关系，未见显现出毒性反应。
实施例7本申请缀合物抑制胃癌小鼠肿瘤生长
7.1 NCI-N87胃癌
(1)按照实施例6的方法接种4×10 6个NCI-N87胃癌细胞至雌性裸鼠，停药后持续观察10日，检测HB010和HB011对肿瘤的抑制效果。
结果如图17-18所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB010(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB010(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB011(0.5mg/kg)组的大。且同剂量下HB010的抑制胃癌生长的效果略优于HB011。图17中，HB010(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图18中，HB010(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB010对NCI-N87胃癌的抑制作用优于伊立替康和HB011，具有一定量效关系，未见显现出毒性反应。
(2)按照实施例6的方法接种6×10 6个NCI-N87胃癌细胞至雌性裸鼠，停药后持续观 察17日，检测HB050和HB051对肿瘤的抑制效果。
结果如图19-20所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB050(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB050(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB051(0.5mg/kg)组的大。且同剂量下HB050的抑制胃癌生长的效果优于HB051。图19中，HB050(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图20中，HB050(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB050对NCI-N87胃癌的抑制作用优于伊立替康和HB051，具有一定量效关系，未见显现出毒性反应。
(3)按照实施例6的方法接种6×10 6个NCI-N87胃癌细胞至雄性裸鼠，每5天尾静脉注射给药1次，停药后持续观察10日，结果如图21-22所示，HB050(0.2mg/kg)的抑制肿瘤效果显著优于其它各组，其抗肿瘤活性至少是HB051(0.5mg/kg)的2.5倍。未见各组显示出毒性反应。图21中，HB050(0.2mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.001；图22中，HB050(0.2mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
实施例8本申请缀合物抑制胰腺癌小鼠肿瘤生长
8.1 BXPC-3胰腺癌
(1)按照实施例6的方法接种4×10 6个BXPC-3胰腺癌细胞至雌性裸鼠，停药后持续观察17日，检测HB030和HB031对肿瘤的抑制效果。
结果如图23-24所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB030(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB030(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB031(0.5mg/kg)组的大。且同剂量下HB030的抑制胰腺癌生长的效果优于HB031。图23中，HB030(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图24中，HB030(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB030对BXPC-3胰腺癌的抑制作用优于伊立替康和HB031，具有一定量效关系，未见显现出毒性反应。
(1)按照实施例6的方法接种6×10 6个BXPC-3胰腺癌细胞至雌性裸鼠，停药后持续 观察17日，检测HB050和HB051对肿瘤的抑制效果。
结果如图25-26所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB050(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB050(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB051(0.5mg/kg)组的大。且同剂量下HB050的抑制胰腺癌生长的效果优于HB051。图25中，HB050(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图26中，HB050(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB050对BXPC-3胰腺癌的抑制作用优于伊立替康和HB051，具有一定量效关系，未见显现出毒性反应。
实施例9本申请缀合物抑制前列腺癌小鼠肿瘤生长
9.1 PC3前列腺癌
(1)按照实施例6的方法接种6×10 6个PC3前列腺癌细胞至雄性裸鼠，停药后持续观察10日，检测HB050和HB051对肿瘤的抑制效果。
结果如图27-28所示，各组裸鼠体重随时间呈正向增长，各时间点各组间体重无显著差异，未见各组显现出严重毒性反应。HB050(0.5mg/kg)组的肿瘤体积变小，小于其它各组，其平均变化百分比与其它各组的相比统计学上有显著差异(P<0.05或0.001)；HB050(0.05mg/kg)组的肿瘤体积比生理盐水组的小，但比伊立替康(5mg/kg)组的或HB051(0.5mg/kg)组的大。且同剂量下HB050的抑制前列腺癌生长的效果优于HB051。图27中，HB050(0.5mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.05，#P＜0.001；图28中，HB050(0.5mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
结果说明HB050对PC3前列腺癌的抑制作用优于伊立替康和HB051，具有一定量效关系，未见显现出毒性反应。
(2)按照实施例6的方法接种6×10 6个PC3前列腺癌细胞至雄性裸鼠，每5天尾静脉注射给药1次，停药后持续观察10日，结果如图29-30显示，HB060(0.2mg/kg)的抑制肿瘤效果显著优于其它各组，其抗肿瘤活性至少是HB061(0.5mg/kg)的2.5倍。未见各组显示出毒性反应。图29中，HB060(0.2mg/kg)组的肿瘤体积平均百分比与其他各组的相比*P＜0.001；图30中，HB060(0.2mg/kg)组的平均体重与其他各组的相比统计学上无显著差异。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本 申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (27)

  1. 缀合物，其具有式I所示的结构：
    Figure PCTCN2021071595-appb-100001
    其中，R 1为经卤素取代的甲基；
    R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；
    R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基；
    Mab为靶向多肽；
    L为连接子。
  2. 根据权利要求1所述的缀合物，其具有式II所示的结构：
    Figure PCTCN2021071595-appb-100002
    其中，R 1为经卤素取代的甲基；
    R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；
    R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基。
  3. 根据权利要求1-2中任一项所述的缀合物，其中R 1为-CF 2H。
  4. 根据权利要求1-3中任一项所述的缀合物，其具有式III所示的结构：
    Figure PCTCN2021071595-appb-100003
  5. 根据权利要求1-4中任一项所述的缀合物，其中所述Mab包括抗体或其抗原结合片段。
  6. 根据权利要求5所述的缀合物，其中所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
  7. 根据权利要求1-6中任一项所述的缀合物，其中所述抗原结合片段选自下组：Fab、Fab’、 F(ab) 2、Fv和ScFv片段。
  8. 根据权利要求1-7中任一项所述的缀合物，其中所述Mab特异性结合肿瘤特异性抗原。
  9. 根据权利要求8所述的缀合物，其中所述肿瘤特异性抗原选自下组：Her2、Trop2、CLDN18.2和Nectin 4。
  10. 根据权利要求1-9中任一项所述的缀合物，其中所述Mab包含抗体重链HCDR3，且所述HCDR3包含SEQ ID NO:19、25、31和39中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的缀合物，其中所述Mab包含抗体重链HCDR2，且所述HCDR2包含SEQ ID NO:18、24、30和38中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的缀合物，其中所述Mab包含抗体重链HCDR1，且所述HCDR1包含SEQ ID NO:17、23、29和37中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的缀合物，其中所述Mab包含抗体重链可变区VH，且所述VH包含SEQ ID NO:1、5、32和40中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的缀合物，其中所述Mab包含抗体重链，且所述抗体重链包含SEQ ID NO:9、11、13和15中任一项所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的缀合物，其中所述Mab包含抗体轻链LCDR3，且所述LCDR3包含SEQ ID NO:22、28、35和43中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的缀合物，其中所述Mab包含抗体轻链LCDR2，且所述LCDR2包含SEQ ID NO:21、27、34和42中任一项所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的缀合物，其中所述Mab包含抗体轻链LCDR1，且所述LCDR1包含SEQ ID NO:20、26、33和41中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的缀合物，其中所述Mab包含抗体轻链可变区VL，且所述VL包含SEQ ID NO:2、6、36和44中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的缀合物，其中所述Mab包含抗体轻链，且所述抗体轻链包含SEQ ID NO:10、12、14和16中任一项所示的氨基酸序列。
  20. 产生权利要求1-19中任一项所述的缀合物的方法，其包括：
    获得式IV所示的化合物：
    Figure PCTCN2021071595-appb-100004
    其中，R 1为经卤素取代的甲基；R 2选自氢、C 1-C 6烷基和经取代的C 1-C 6烷基；R 3选自氢、氨基、C 1-C 6烷基和经取代的C 1-C 6烷基；Mab为靶向多肽；L为连接子；
    使式IV所示的化合物与所述Mab缀合，以得到权利要求1-19中任一项所述的缀合物。
  21. 药物组合物，其包含权利要求1-19中任一项所述的缀合物。
  22. 权利要求1-19中任一项所述的缀合物或权利要求21所述的药物组合物在制备预防或治疗肿瘤的药物中的用途。
  23. 权利要求1-19中任一项所述的缀合物与其他肿瘤疗法或药物共同在制备预防或治疗肿瘤的药物中的用途。
  24. 根据权利要求23所述的用途，其中所述其他肿瘤疗法或药物选自下组：化疗、放疗、miRNA和寡核苷酸。
  25. 根据权利要求23-24中任一项所述的用途，其中所述肿瘤为实体瘤。
  26. 根据权利要求23-25中任一项所述的用途，其中所述肿瘤选自以下组：乳腺癌、胰腺癌、结肠癌、胃癌和前列腺癌。
  27. 预防或治疗肿瘤的方法，包括施用权利要求1-19中任一项所述的缀合物和/或权利要求21所述的药物组合物。
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