WO2023174312A1 - 抗人pd-l1和tlr7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 - Google Patents

抗人pd-l1和tlr7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 Download PDF

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WO2023174312A1
WO2023174312A1 PCT/CN2023/081459 CN2023081459W WO2023174312A1 WO 2023174312 A1 WO2023174312 A1 WO 2023174312A1 CN 2023081459 W CN2023081459 W CN 2023081459W WO 2023174312 A1 WO2023174312 A1 WO 2023174312A1
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seq
antibody
nanobody
cdr2
cdr1
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PCT/CN2023/081459
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English (en)
French (fr)
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宫丽崑
尉骁璐
黄蔚
李佳
龙益如
唐峰
胡超
李方林
刘志
秦秋平
孙建华
徐俊玖
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中国科学院上海药物研究所
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Publication of WO2023174312A1 publication Critical patent/WO2023174312A1/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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention relates to the field of biomedicine, and more specifically to an anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drug and its application in anti-tumor.
  • PD-1/PD-L1 immune checkpoint blockade therapy is effective for many cancer types, but it suffers from low response rate and drug-resistant relapse. Therefore, determining appropriate treatment strategies to improve efficacy is a key issue in the field of tumor immunotherapy. Important tasks.
  • TLRs Toll-like receptors
  • APCs antigen-presenting cells
  • NKs natural killer cells
  • TLR agonists can effectively activate APCs, increase phagocytosis and antigen-presentation functions, thereby promoting T Cell activation plays a tumor suppressive role.
  • the application of TLR agonists in tumor immunity is expected to turn cold tumors into hot tumors and solve the problem of low response rate to single immune checkpoint inhibitors.
  • Nanobodies namely heavy chain single domain antibodies VHH, are natural heavy chains of camelids (camels, llamas, alpacas and their close relatives) that lack light chains.
  • the variable region of an antibody is the smallest naturally occurring unit known to bind an antigen.
  • nanobodies Compared with monoclonal antibodies, nanobodies have superior tumor targeting and stability, and can achieve targeted delivery of drugs to achieve precise treatment.
  • the purpose of the present invention is to provide a novel anti-human PD-L1 Nanobody drug.
  • the purpose of the present invention is to provide a new and effective anti-human PD-L1 nanobody conjugated drug.
  • the purpose of the present invention is to provide an anti-human PD-L1 Nanobody and a TLR7 agonist for combined anti-tumor treatment and to provide a PD-L1 and TLR7 dual-targeting Nanobody conjugate drug.
  • Another object of the present invention is to provide the application of anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drugs in tumor prevention and treatment, especially in tumors with low response rate to PD-L1 antibodies.
  • an antibody-drug conjugate or a pharmaceutically acceptable salt thereof is provided.
  • the structure of the antibody-drug conjugate is shown in Formula I:
  • Ab is PD-L1 antibody
  • U are each independently a TLR agonist
  • J is a chemical bond or linker
  • n 0 or a positive integer
  • the PD-L1 antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies (such as trispecific antibodies).
  • the PD-L1 antibody includes: monoclonal antibody, single chain antibody (scFv), and Nanobody.
  • the PD-L1 antibody includes monovalent, bivalent or multivalent antibodies.
  • the PD-L1 antibody includes a multimeric antibody.
  • the PD-L1 antibody specifically binds to PD-L1.
  • the PD-L1 antibodies include PD-L1 monovalent Nanobodies, bivalent Nanobodies and/or multivalent Nanobodies.
  • the PD-L1 antibody includes blocking type (can block the binding of PD-L1 and PD-1), non-blocking type (can not block the binding of PD-L1 and PD-1 ), or a combination thereof.
  • the PD-L1 antibody is a blocking antibody.
  • the PD-L1 antibody blocks the binding of PD-1 to PD-L1.
  • the PD-L1 is human PD-L1 or non-human mammalian PD-L1 (such as mouse PD-L1).
  • the PD-L1 antibody is a human or non-human mammalian antibody.
  • the non-human mammal is selected from the group consisting of camels, alpacas, mice, and cynomolgus monkeys.
  • the PD-L1 antibody is a PD-L1 Nanobody or a derivative antibody thereof, preferably a targeting human PD-L1 Nanobody or a derivative antibody thereof.
  • the derivative antibody is a modification of the PD-L1 Nanobody, including but not limited to connecting the PD-L1 Nanobody to an Fc fragment, human serum albumin, polyethylene glycol PEG, or the like.
  • valent antibodies and/or polyvalent antibodies are examples of the PD-L1 Nanobody, including but not limited to connecting the PD-L1 Nanobody to an Fc fragment, human serum albumin, polyethylene glycol PEG, or the like.
  • the Nanobodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.
  • the PD-L1 Nanobody is a Nanobody that specifically binds to human PD-L1, and the complementarity determining region CDR of the VHH chain in the Nanobody is selected from the following group:
  • any one of the above amino acid sequences also includes at least one optionally added, deleted, modified and/or substituted (such as 1-3, preferably 1-2, more preferably 1) amino acid and retain the ability to bind to PD-L1.
  • the PD-L1 Nanobody is a Nanobody that specifically binds to human PD-L1, and the complementarity determining region CDR of the VHH chain in the Nanobody is selected from the following group:
  • CDR1 represented by SEQ ID NO: 106, CDR2 represented by SEQ ID NO: 7, CDR3 represented by SEQ ID NO: 8;
  • the Nanobody that specifically binds to human PD-L1 includes a Nanobody that has undergone reverse mutation of human PD-L1, and the reverse mutation is selected from the group consisting of VHHs shown in SEQ ID NO.1.
  • the back mutation is selected from the group consisting of SEQ ID NO.1
  • the VHH shown has an amino acid back mutation selected from the following group: C103K, C103M, C103W, G99R
  • amino acid sequence of the VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the following group:
  • the Nanobody sequence contains at least 80%, preferably at least 90%, of SEQ ID NO: 1, 5, 9, 13, 17, 20, 24, 28, 32, 36 or 39. , more preferably at least 95%, even more preferably at least 99% sequence similarity between amino acid sequences.
  • the specific binding human PD-L1 Nanobody is a humanized specifically binding human PD-L1 Nanobody, and the amino acid sequence of the VHH chain it contains is selected from the following group:
  • the Nanobody sequence includes SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71 have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99 % sequence similarity between amino acid sequences.
  • the Nanobody that specifically binds to human PD-L1 is an affinity-matured Nanobody that specifically binds to human PD-L1, and the amino acid sequence of the VHH chain it contains is selected from the following group:
  • the Nanobody sequence includes SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97 having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% Amino acid sequence.
  • the "affinity maturation” refers to the affinity of the anti-human PD-L1 Nanobody modified by affinity maturation for PD-L1 relative to the affinity of the anti-human PD-L1 Nanobody before modification for PD-L1.
  • the affinity 12 times, at least 20 times, or at least 25 times.
  • the complementarity determining region CDR of the VHH chain of the Nanobody is composed of CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4 .
  • VHH chain sequence of the Nanobody is shown in SEQ ID NO.: 1.
  • the complementarity determining region CDR of the VHH chain of the Nanobody is composed of CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8 .
  • VHH chain sequence of the Nanobody is shown in SEQ ID NO.: 5.
  • the TLR agonist is a macromolecule (protein or nucleic acid) or small molecule agonist.
  • the TLR agonists include, but are not limited to, TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists and TLR9 agonists. agent.
  • n is the average number of conjugated drugs in the antibody-drug conjugate, preferably n is 1 to 9, preferably 2.5 to 6.5, and more preferably 3.5 to 5.5.
  • the TLR agonist is a TLR7 agonist.
  • the TLR agonist does not have TLR8 agonistic activity.
  • the TLR7 agonist is a host endogenous agonist or an exogenous agonist.
  • the TLR7 agonist is a small molecule agonist.
  • the TLR7 agonist includes: SZU-101:
  • the TLR7 agonist is a derivative compound of SZU-101, including but not limited to the substitution, modification or deletion of one or more groups based on SZU-101.
  • the TLR7 agonist is a multivalent compound of SZU-101.
  • the TLR7 agonist (such as SZU-101) is connected to the terminal amino group or the side chain amino group of the heavy chain constant region or heavy chain variable domain (VHH) of the PD-L1 antibody.
  • the TLR7 agonist (such as SZU-101) is connected to the sulfhydryl group of the PD-L1 antibody.
  • the SZU-101 is connected to the amino group of the PD-L1 antibody and forms the structure shown in S1:
  • the SZU-101 is connected to the sulfhydryl group of the PD-L1 antibody and forms the structure shown in S2:
  • the TLR7 agonist is site-specifically and/or randomly connected to the PD-L1 antibody (ie, in Formula I, the U is site-specifically and/or randomly connected to Z).
  • the U fixed point is connected to Z.
  • the U is connected to an amino acid position of the PD-L1 antibody Z selected from the following group: G, K, L, A, C or a combination thereof.
  • the chemical bond is polyethylene glycol PEG.
  • the chemical bond is a derivative compound of PEG, including but not limited to substitution, modification or deletion of one or more groups based on SZU-101.
  • the degree of polymerization of the PEG chemical bond is a positive integer greater than or equal to 1.
  • the antibody-drug conjugate increases the PD-L1 level of cells within the tumor.
  • the antibody-drug conjugate activates immune cells.
  • the activation is in vitro activation.
  • the in vitro activation includes culturing the immune cells for a period of time (such as 6-48 hours) in the presence of the antibody-drug conjugate, thereby obtaining immune-activated immunity. cell.
  • the immune cells are selected from, but are not limited to: CD8+ T cells, natural killer cells (NK), dendritic cells, lymphocytes, monocytes/macrophages, granulocytes, or combinations thereof.
  • NK natural killer cells
  • dendritic cells lymphocytes, monocytes/macrophages, granulocytes, or combinations thereof.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is used to prepare a composition or preparation, and the composition or preparation is used for:
  • the remodeling of the tumor immune microenvironment is to coordinate the innate immune and adaptive immune anti-tumor immune responses within the tumor.
  • the reshaping of the tumor immune microenvironment is to increase the infiltration of anti-tumor immune cells and reduce the proportion of immunosuppressive cells.
  • the anti-tumor immune cells include, but are not limited to, CD8+ T cells and NK cells that secrete granzymes and IFN- ⁇ , activated dendritic cells, and CD4+ T cells that secrete IFN- ⁇ . , M1 macrophages.
  • the immunosuppressive cells include but are not limited to M2 macrophages, Treg cells, and TGF- ⁇ -secreting leukocytes.
  • the PD-L1 level includes cell surface PD-L1 level and intracellular PD-L1 level.
  • the tumor with low PD-L1 expression is a solid tumor or a hematological tumor.
  • a PD-L1 Nanobody is provided, the PD-L1 Nanobody specifically binds to human PD-L1, and the complementarity determining region CDR of the VHH chain in the Nanobody is selected from the following: One or more of the group:
  • any one of the above amino acid sequences also includes at least one optionally added, deleted, modified and/or substituted (such as 1-3, preferably 1-2, more preferably 1) amino acid and retain the ability to bind to PD-L1.
  • amino acid sequence of the VHH chain that specifically binds to the human PD-L1 Nanobody is selected from the following group:
  • the Nanobody sequence contains at least 80%, preferably at least 90%, SEQ ID NO: 1, 5, 9, 13, 17, 20, 24, 28, 32, 36 or 39. , more preferably at least 95%, even more preferably at least 99% sequence similarity between amino acid sequences.
  • the specific binding human PD-L1 Nanobody is a humanized specifically binding human PD-L1 Nanobody, and the amino acid sequence of the VHH chain it contains is selected from the following group:
  • the Nanobody sequence includes SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71 have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99 % sequence similarity between amino acid sequences.
  • the specific binding human PD-L1 Nanobody is an affinity matured specific binding human PD-L1 Nanobody, and the amino acid sequence of the VHH chain it contains is selected from the following group:
  • the Nanobody sequence includes SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96 or 97 having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% Amino acid sequence.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition further includes:
  • the other biologically active drugs promote the anti-tumor functions of CD8+ T cells and NK cells.
  • the pharmaceutical composition includes single drugs, compound drugs, or synergistic drugs.
  • the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spray and mist Inhaled.
  • the pharmaceutical composition is administered by combining the pharmaceutical composition with immune cells (such as dendritic cells, natural killer cells, lymphocytes, monocytes/macrophages, granulocytes, etc. ) after co-culture, the immune cells are isolated for in vivo reinfusion.
  • immune cells such as dendritic cells, natural killer cells, lymphocytes, monocytes/macrophages, granulocytes, etc.
  • the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.
  • the pharmaceutical composition is used for anti-tumor treatment.
  • the pharmaceutical composition is used to treat tumors with low PD-L1 expression.
  • low expression of PD-L1 means that the amount of PD-L1 expressed by the tumor E1 is lower than the amount of PD-L1 expressed by the normal tumor E0, preferably E1/E0 ⁇ 1/2, More preferably ⁇ 1/3, more preferably ⁇ 1/4.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma cancer, bone cancer, adrenal gland tumors, or bladder tumors.
  • an immunoconjugate is provided, and the immunoconjugate contains:
  • the other coupling moieties are selected from the following group: small molecule compounds, PEG, fluorescein, radioisotopes, contrast agents, fatty acid chains, protein fragments, or combinations thereof.
  • the components (a) and (b) are operably connected.
  • the coupling moiety includes chemical markers and biological markers.
  • the chemical label is selected from isotopes, immunotoxins and/or chemical drugs.
  • the biomarker is selected from biotin, avidin or enzyme markers.
  • the small molecule compound is selected from drugs or toxins for treating tumors or autoimmune diseases.
  • the radioactive isotopes include:
  • Diagnostic isotope the said diagnostic isotope is selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • Therapeutic isotope the therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.
  • the radioactive isotopes include but are not limited to iodine-131, indium-111 and lutetium-177.
  • the contrast agent is used for MRI or CT.
  • the protein fragments include but are not limited to antibody Fc, biotin, avidin, HRP, antibodies, enzymes, cytokines and other biologically active proteins or polypeptides.
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or is capable of producing Enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, and nanomagnetic particles that can detect products , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticle.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • a fusion protein comprising:
  • the polypeptide molecules or fragments with therapeutic functions include but are not limited to: targeting PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB, CD47, TIM3, Polypeptide molecules or fragments of CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, and HER2.
  • polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cells factor.
  • the polypeptide molecule or fragment with therapeutic function includes a single-chain antibody (scFv), a diabody, a monoclonal antibody, or a chimeric antibody.
  • scFv single-chain antibody
  • diabody diabody
  • monoclonal antibody monoclonal antibody
  • chimeric antibody a single-chain antibody
  • the fusion protein also contains a tag sequence to assist expression and/or purification.
  • the tag sequence is selected from the following group: 6His tag, GGGS sequence, and FLAG tag.
  • the fusion protein includes bispecific antibodies and chimeric antibodies.
  • a multispecific antibody comprising:
  • the multispecific antibody further includes a second antigen-binding region targeting a target selected from the following group: PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB , CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2, or combinations thereof.
  • a target selected from the following group: PD-1, IL-4R, IL-4R ⁇ , TNF- ⁇ , VEGF, 4-1BB , CD47, TIM3, CTLA4, IL-17A, CD19, CD22, CD28, CD38, CD40, CD47, B7-H3, TSLP, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, HER2, or combinations thereof.
  • the second antigen-binding region is a Nanobody.
  • the multispecific antibody includes one or more second antigen-binding regions.
  • the multispecific antibody further includes the Fc segment of the antibody.
  • the seventh aspect of the present invention there is provided a method for preparing the antibody-drug conjugate according to the first aspect of the present invention, the method comprising the steps:
  • reaction time is 3h-10h.
  • the molar ratio of the antibody to the drug molecule is 1-2:3-20; preferably 1:6-10.
  • SZU-101, EDCI and NHS are dissolved in DMSO and stirred at room temperature for three hours to react to prepare SZU-101-NHS active ester.
  • the PD-L1 nanobody and SZU-101-NHS active ester are 1:10.
  • the molar ratio dose was stirred and reacted at 4°C for 4 hours to prepare the nanobody-conjugated drug.
  • a pharmaceutical kit in an eighth aspect of the present invention, includes:
  • a method for preventing or treating tumors is provided, which is to administer the Nanobody conjugated drug as described in the first aspect of the present invention, or the Nanobody conjugated drug as described in the second aspect of the present invention to a subject in need.
  • the tumor is a tumor expressing PD-L1.
  • the tumor is selected from the following group: tumors with high expression of PD-L1, tumors with medium expression of PD-L1, and tumors with low expression of PD-L1.
  • the tumor is a tumor with medium expression of PD-L1 or a tumor with low expression of PD-L1.
  • the tumor is a tumor with low expression of PD-L1.
  • high expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0)>1, more preferably ⁇ 1.5, preferably ⁇ 2.0.
  • “moderate expression of PD-L1” means that the ratio of the amount E1 of PD-L1 expressed by the tumor to the amount E0 of PD-L1 expressed by the normal tumor (E1/E0) is 0.5-1.1, more Optimally, it is 0.7-1.0, and more preferably, it is 0.8-0.9.
  • low expression of PD-L1 means that the ratio of the amount of PD-L1 expressed by the tumor E1 to the amount of PD-L1 expressed by the normal tumor E0 (E1/E0) ⁇ 1/2, more Best land ⁇ 1/3, better land ⁇ 1/4.
  • the tumors include but are not limited to: breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumor, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma cancer, bone cancer, adrenal gland tumors, or bladder tumors.
  • an antibody-drug conjugate according to the first aspect of the present invention such as the PD-L1 Nanobody according to the second aspect of the present invention, such as the third aspect of the present invention
  • the pharmaceutical composition, the immunoconjugate as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, or the multispecific antibody as described in the sixth aspect of the present invention Use for preparing reagents, test plates or kits; or for preparing drugs for preventing and/or treating PD-L1 related diseases.
  • the PD-L1-related disease is a tumor/cancer expressing PD-L1.
  • Figure 1 shows the human PD-L1 binding activity of candidate anti-human PD-L1 Nanobodies, where blank is the negative control.
  • Figure 2 shows the blocking activity assay of candidate anti-human PD-L1 Nanobodies on human PD-1/PD-L1 binding, in which blank and blank+ligand are the control groups.
  • Figure 3 shows the IC 50 curve of candidate anti-human PD-L1 Nanobodies blocking human PD-1/PD-L1 binding, in which KN035 is the positive control group.
  • Figure 4 shows the mass spectrometry identification of the candidate anti-human PD-L1 Nanobody conjugated drug before and after the degree of conjugation (4A-D) and the mass spectrometry identification of the positive control group KN035 (4E).
  • Figure 5 shows the tumor inhibitory effects of the candidate anti-human PD-L1 Nanobodies h_Nb1 and h_Nb2, the positive control antibody KN035, and the Nanobody conjugated drugs h_Nb1-SZU-101 and h_Nb2-SZU-101.
  • 5A represents different groups of tumors. Growth curve, 5B is the end-point tumor weight of each group; 5C is the tumor inhibition rate of each group, 5D-5I is the tumor growth curve of each mouse in different groups, 5J-5K is the nanobody conjugated drug h_Nb1- Tumor growth curves of SZU-101 and h_Nb2-SZU-101 tumor regression mice in re-tumor bearing experiments.
  • the inventor screened and identified an anti-human PD-L1 nanobody, and developed an anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drug.
  • the present invention uses phage display technology and flow cytometry screening to obtain anti-human PD-L1 Nanobodies and their derivative molecules, and uses antibody coupling technology to prepare and obtain new dual-targeting Nanobody conjugated drugs and their derivatives. Derived molecules, it was found that anti-human PD-L1 nanobodies and dual-targeting nanobody conjugates have excellent anti-tumor activity.
  • the present invention found that dual-targeted nanobody-conjugated drugs can reshape the tumor immune microenvironment and promote the expression of PD-L1 by antigen-presenting cells, including macrophages, which is beneficial to the treatment of "cold" tumors with low expression of PD-L1 molecules.
  • the PD-L1 and TLR7 dual-targeting nanobody conjugate drug developed by the present invention shows outstanding anti-tumor effect and novel mechanism of action, and has clinical development and application value. On this basis, the present invention was completed.
  • TLR receptor refers to Toll-like receptors, which are an important type of innate immune pattern recognition receptors in the immune system of organisms and can specifically recognize relatively conserved antigen molecules during the evolution of pathogenic microorganisms ( Or called pathogen-related molecular patterns), to achieve effective detection of pathogenic microorganism invasion and induction of innate immune responses.
  • TLR1-TLR10 TLR3-TLR10
  • TLR3, TLR7, TLR8, and TLR9 are located on the endosomes and lysosomal membranes of cells, and the others are located on the cytoplasmic membrane.
  • TLR7 is preferably used as one of the drug molecular targets.
  • the natural ligand of TLR7 molecules is single-stranded linear RNA.
  • TLR receptor agonist refers to a macromolecule (protein or nucleic acid) or small molecule agonist that can specifically bind to and activate TLR receptors, promote the transduction of downstream signals of TLR receptors, and achieve intrinsic waived Activation of immune cells.
  • TLR7 agonists are preferred to construct Nanobody-conjugated drugs.
  • available TLR7 agonists also include imiquimod, R848, and the like.
  • Nanobody of the invention As used herein, the terms “Nanobody of the invention”, “Nanobody targeting PD-L1 of the invention” and “anti-PD-L1 Nanobody of the invention” are used interchangeably and all refer to specific recognition and binding to Nanobodies against PD-L1 (including human or mouse PD-L1). Particularly preferred are Nanobodies (h_Nb1 or h_Nb2) whose amino acid sequence of the VHH chain is shown in SEQ ID NO: 1 or SEQ ID NO: 5.
  • Nanobody conjugated drug of the present invention As used herein, the terms "Nanobody conjugated drug of the present invention”, “dual-targeting Nanobody conjugated drug of the present invention”, “PD-L1 and TLR7 dual-targeting Nanobody conjugated drug of the present invention” are interchangeable. Used interchangeably, they all refer to new drug molecules formed by nanobodies and their derivative proteins coupled to TLR7 agonists that specifically recognize and bind to PD-L1 (including human or mouse PD-L1). Nanobodies in Nanobody conjugated drugs are particularly preferably Nanobodies whose amino acid sequence of the VHH chain is shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • single domain antibody sdAb, or VHH
  • nanobody a single domain antibody
  • VHH single domain antibody
  • VHH single domain antibody
  • Nanobody/single domain antibody is a new type of small molecule antibody fragment, which is obtained by cloning the heavy chain variable region (VHH) of camelid natural heavy chain antibody.
  • Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15kDa, which is one-tenth that of a complete antibody. It has good tissue penetration, high specificity and good water solubility. Because of its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, almost perfectly overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and harsh storage conditions, and has gradually become an emerging new generation of antibody therapy. power, showing broad application prospects in immunodiagnosis and therapy.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effects. response function, such as involvement in antibody-dependent cellular cytotoxicity.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules formed by combining with the antibodies of the present invention or fragments thereof of conjugates.
  • Nanobody drug conjugates are a special form of antibody-drug conjugates, which are nanobodies or derivative proteins conjugated to drugs, toxins, cytokines, radionuclides, enzymes and other diagnostics. Or the form of drug molecules formed by therapeutic molecules, which can be used for tumor treatment, drug delivery and in vivo imaging, etc., and has broad clinical application value.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the invention protein of the invention
  • polypeptide of the invention are used interchangeably, and all refer to polypeptides that specifically bind to PD-L1, such as proteins with heavy chain variable regions or Peptides. They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to the heavy chain of the antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), and the amino acid sequences of the four FRs. Relatively conservative and not directly involved in binding reactions. These CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least part of them is involved in binding the antigen. Accordingly, the present invention includes those molecules having antibody heavy chain variable regions with CDRs as long as the CDRs are more than 90% (preferably more than 95%, optimally more than 98%) homologous to the CDRs identified herein sex.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of the antibodies of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) in one or more amino acids A polypeptide with substituent groups in the residues, or (iii) a polypeptide formed by the fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to A polypeptide formed from this polypeptide sequence (such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein source sequence, or a fusion protein formed with
  • the antibody of the present invention refers to a polypeptide that has PD-L1 binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above-described CDR regions that have the same function as the antibodies of the invention. These variant forms include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the DNA encoding the antibody of the invention under high or low stringency conditions.
  • the invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also encompasses fragments of the Nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally at most 3 Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the invention may be in DNA form or RNA form.
  • Forms of DNA include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • Polynucleotides encoding mature polypeptides of the present invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include polynucleotides encoding such polypeptides, or may also include polynucleotides that also include additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone, or may be combined or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing a detectable product Enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. Therapeutic agents (for example, , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • ADCs Antibody-drug conjugates
  • the invention also provides antibody-drug conjugates (antibody-drug conjugates, ADC) based on the antibodies of the invention.
  • the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled.
  • the effector molecule is preferably a drug with therapeutic activity or a drug with immune-promoting function.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc.
  • the coupling agent utilizing carboxyl groups may be any one or more of aconitic anhydride coupling agents (such as aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is an acyl hydrazone).
  • antibodies are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizer (e.g. such as ethylene glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorescent groups
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizer e.g. such as ethylene glycol polymers
  • therapeutic agents e.g. such as ethylene glycol polymers
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent can be linked to the antibody directly or indirectly through a linker.
  • Antibodies can be conjugated with drugs to form antibody drug conjugates (ADCs).
  • ADCs contain a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers.
  • Degradable linkers are typically susceptible to degradation in the intracellular environment, such as at the target site, allowing the drug to be released from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases, or sugar linkers, such as those that can be degraded by glucuronides. Enzymatic degradation of glucuronide-containing linkers.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.
  • the linker Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g.
  • Linkers may include, for example, maleimides linked to the antibody via thiosuccinimide.
  • the drug may generally be any cytotoxic, cytostatic or immunosuppressive drug.
  • drugs are drugs that activate or promote immune responses, such as activating innate immune responses to assist activation of adaptive immunity.
  • the drug is a TLR receptor agonist.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has reactive groups before being attached to the antibody.
  • Useful drug classes include, for example, TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonists, and TLR9 agonists, such as SZU-101, TLR9 agonists, Quimod, R848, CpG, etc.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive part of the linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug, forming an ADC.
  • linker typically, functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety.
  • the drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) alcohols reaction); and activated esters, such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines) alcohols reaction
  • activated esters such as N-hydroxysuccinimide ester (suitable for reaction with amines and alcohols).
  • the present invention also provides a method for preparing an ADC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • methods of the invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the methods of the present invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is PD-L1 antibody
  • U are each independently a TLR agonist
  • J is a chemical bond or linker
  • n 0 or a positive integer
  • the invention provides uses of the antibodies of the invention, for example, for the preparation of diagnostic preparations or for the preparation of medicaments for the prevention and/or treatment of PD-L1 related diseases.
  • the PD-L1 related diseases include inflammatory diseases, autoimmune diseases, etc., including but not limited to breast cancer, liver cancer, gastric cancer, colorectal cancer, leukemia, lung cancer, kidney tumors, small intestine cancer, prostate cancer, colorectal cancer, prostate cancer, Cervical cancer, lymphoma, bone cancer, adrenal gland tumors, or bladder tumors.
  • cancers such as pancreatic cancer or prostate cancer that do not respond to treatment with one or more checkpoint inhibitors (such as antibodies that bind PD-L1, CTLA-4, or CD47, etc.) are termed cold Tumor.
  • Such tumors are thought to have higher levels of tumor-infiltrating lymphocytes (TILs) and/or a higher tumor mutational load compared to tumors that do not respond to checkpoint inhibitor treatment.
  • TILs tumor-infiltrating lymphocytes
  • the invention provides a preferred antibody-drug conjugate, which has excellent anti-tumor activity, and also shows extremely significant anti-tumor activity and response rate in cold tumors and tumor models with low expression of PD-L1.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier or excipient, and optionally of other biologically active substances.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable amount.
  • Acceptable carrier or excipient include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. Additionally, the polypeptides of the invention may be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the anti-human PD-L1 nanobody of the present invention can be highly specific against the human PD-L1 protein with the correct spatial structure, has strong affinity, and is easy to produce.
  • the present invention provides a combined treatment plan for PD-L1 nanobodies and TLR7 agonists, which exhibits significant anti-tumor activity in vivo, indicating that PD-L1 antibody therapy and TLR7 immune agonists are rational in combination and can synergistically fight against Tumor.
  • the present invention has developed a dual-targeting nanobody conjugate drug targeting human PD-L1 and TLR7, which can promote the up-regulation of PD-L1 expression in cells in tumors and coordinate intratumoral innate immunity and adaptive anti-tumor immune responses, This enables it to exhibit excellent tumor growth inhibition in a variety of tumors for which PD-L1 antibody treatment is less effective, such as "cold" tumors and tumors with low PD-L1 expression.
  • the PD-L1 and TLR7 dual-targeting nanobody conjugate drug provided by the present invention can target the tumor immune microenvironment, reshape the tumor immune microenvironment, increase the infiltration of anti-tumor immune cells, and reduce the infiltration of immunosuppressive cells. .
  • auxiliary phage to the Nanobody library with 10 times the library capacity to amplify the Nanobody display phage.
  • the 96-well microplate was coated with 5 ⁇ g/mL NeutrAvidin solution (100 ⁇ L per well) at 4°C overnight. The next day, block with 2% skimmed milk powder at room temperature for 2 hours, and wash 5 times with 20mM HEPES (pH 7.5) and 150mM NaCl solution.
  • the eluted phages were infected into the TG1 competent cells in the logarithmic phase of growth, serially diluted, spread on plates and cultured overnight.
  • the 96-well microplate was coated with 5 ⁇ g/mL NeutrAvidin solution at 4°C, 700 rpm, overnight.
  • the candidate Nanobody sequences were homologously recombined into the pFUSE-mIgG2b-Fc and pFUSE-hIgG1-Fc vectors, and then mammalian cells HEK293F were used to express the candidate Nanobodies. .
  • the 11 preferred anti-human PD-L1 Nanobodies obtained in the present invention are h_Nb1, h_Nb2, h_Nb4, h_Nb5, h_Nb6, h_Nb9, h_Nb12, h_Nb13, h_Nb19, h_Nb26, and h_Nb30.
  • VHH sequences of h_Nb1, h_Nb2, h_Nb4, h_Nb5, h_Nb6, h_Nb9, h_Nb12, h_Nb13, h_Nb19, h_Nb26, and h_Nb30 are as follows: SEQ ID NO.: 1, 5, 9, 13, 17, 20, 24, 28, 32 , 36 and 39, the CDR part is shown in Table 1.
  • TLR7 small molecule agonist used to conjugate antibodies is as follows:
  • SZU-101-Mal can be prepared in a similar manner.
  • the activated ester is dissolved in DMSO, and the antibody and small molecule are dosed and reacted at a molar ratio of 1:10.
  • a certain amount of small molecule activated ester is added to the candidate antibody, and the reaction is stirred at 4°C for 4 hours.
  • PBS was added to the mixture, and small molecules were removed by filtration with a 10kD biological filter to obtain novel dual-targeting nanobody coupling compounds h_Nb1-SZU-101 and h_Nb2-SZU-101.
  • the antibody is first denatured to open the disulfide bond, and then the sample is identified using the XevoG2XSQTOF mass spectrometer.
  • the coupling degree is mainly calculated based on the increased molecular weight of the new coupled compound compared to the uncoupled antibody.
  • Example 7 In vitro activity assay of PD-L1 and TLR7 dual-targeting Nanobody drug conjugates
  • Example 8 Evaluation of in vivo anti-tumor activity of anti-human PD-L1 and TLR7 dual-targeting Nanobody conjugate drugs
  • anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drugs h_Nb1-SZU-101 and h_Nb2-SZU-101 were prepared.
  • the candidate nanobodies h_Nb1 and h_Nb2 and the positive control antibody KN035 all have high purity.
  • Mass spectrometry identified the coupling degree of the nanobody conjugate h_Nb1-SZU-101 as approximately 3, and the conjugation degree of h_Nb2-SZU-101 as determined by mass spectrometry.
  • the joint degree is about 5.
  • mice with tumor regression were re-carried with tumors 28 days after the last dose of the drug efficacy test.
  • Six BALB/c-hPD1/hPDL1 mice that had not been exposed to tumors were re-selected as the control group and were loaded with the mice with tumor regression at the same time.
  • tumor ie: CT26/hPD-L1 tumor
  • mice with tumor regression no longer grew tumors within 45 days of tumor bearing ( Figures 5J-5K), indicating that the conjugated compound induced effective anti-tumor immune memory.
  • the CDR Grafting method is used to replace camel antibody FR with human antibody FR to achieve the purpose of reducing immunogenicity.
  • homology modeling is performed on the candidate antibodies to identify the key amino acid residue sites.
  • the candidate Nanobody sequence is used as a template to search for homologous structures in the structural database, and the optimal structural sequence is selected for sequence replacement and finally the human source is obtained.
  • the sequence of the antibody must be optimized, and key sites in the framework region that may affect the CDR function must be considered.
  • the 11 preferred anti-human PD-L1 Nanobodies obtained were humanized respectively.
  • the corresponding sequences after the humanized antibodies are as follows:
  • h_Nb1 The humanized sequences of h_Nb1 are h_Nb1_1, h_Nb1_2, h_Nb1_3, h_Nb1_4, and h_Nb1_5;
  • h_Nb2 The humanized sequences of h_Nb2 are h_Nb2_1, h_Nb2_2, h_Nb2_3, h_Nb2_4, and h_Nb2_5;
  • h_Nb4 The humanized sequences of h_Nb4 are h_Nb4_1 and h_Nb4_2 respectively;
  • h_Nb5 The humanized sequences of h_Nb5 are h_Nb5_1, h_Nb5_2, and h_Nb5_3 respectively;
  • h_Nb6_1 and h_Nb6_2 are humanized sequences of h_Nb6_1 and h_Nb6_2 respectively;
  • h_Nb9 The humanized sequences of h_Nb9 are h_Nb9_1 and h_Nb9_2 respectively;
  • h_Nb12 The humanized sequences of h_Nb12 are h_Nb12_1 and h_Nb12_2 respectively;
  • h_Nb13_1 and h_Nb13_2 The humanized sequences of h_Nb13 are h_Nb13_1 and h_Nb13_2 respectively;
  • h_Nb19 The humanized sequences of h_Nb19 are h_Nb19_1 and h_Nb19_2 respectively;
  • h_Nb26 The humanized sequences of h_Nb26 are h_Nb26_1 and h_Nb26_2 respectively;
  • h_Nb30 The humanized sequences of h_Nb30 are h_Nb30_1 and h_Nb30_2 respectively.
  • the humanized antibody sequences are shown in Table 2.
  • the CDR region and FR region of the antibody were annotated by the CCG method; through the antibody homology pattern Build, select appropriate FR region and CDR region templates, construct and select the optimal nanobody three-dimensional protein structure; from the PDB protein number
  • the primary antibody corresponding to the sequence shown in SEQ ID NO.72-86 is h_Nb1 VHH (SEQ ID NO.1); the primary antibody corresponding to the sequence shown in SEQ ID NO.87-97 is h_Nb2 VHH (SEQ ID NO. .5).
  • the present invention has developed anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drugs, which can coordinate the innate immunity and adaptive immune response, and target Transform and reshape the tumor immune microenvironment and increase the PD-L1 expression level in tumor tissues, so that it also shows extremely significant anti-tumor activity and response rate in "cold" tumors and tumor models with low PD-L1 expression, showing Its clinical application value.
  • the present invention provides an anti-human PD-L1 and TLR7 dual-targeting nanobody conjugate drug, which can be used for subsequent development of tumor immunotherapy drugs, and is especially suitable for tumors with low immunogenicity and/or low expression of PD-L1.

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Abstract

提供一种抗人PD-L1的纳米抗体及其应用，以及抗人PD-L1和TLR7双靶向纳米抗体偶联药物及其制备方法和应用。具体地，提供抗人PD-L1的纳米抗体及其衍生蛋白用于抗肿瘤治疗的用途和方案，同时提供新型抗人PD-L1和TLR7双靶向纳米抗体药物偶联物及其衍生分子的设计、制备和鉴定方案及其在抗肿瘤治疗中的作用。所述抗人PD-L1和TLR7双靶向纳米抗体药物偶联物可发挥显著的抗肿瘤药效。

Description

抗人PD-L1和TLR7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 技术领域
本发明涉及生物医药领域，更具体地涉及一种抗人PD-L1和TLR7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用。
背景技术
PD-1/PD-L1免疫检查点阻断疗法(ICB)对许多癌症类型有效，但其存在响应率低以及耐药复发的问题，因此，确定合适的治疗策略提高疗效是肿瘤免疫治疗领域的重要任务。
Toll样受体(TLR)是先天免疫重要靶点，主要表达在抗原呈递细胞(APC)以及自然杀伤细胞(NK)上，TLR激动剂可有效激活APC，增加吞噬功能和抗原呈递功能进而促进T细胞激活，发挥肿瘤抑制作用。此外，TLR激动剂应用到肿瘤免疫中，有望将冷肿瘤变热，解决单免疫检查点抑制剂响应率低等问题。
抗体小型化是抗体基因工程领域的研究方向之一，纳米抗体，即重链单域抗体VHH，是骆驼科动物(骆驼、大羊驼、羊驼及其近亲物种)缺失轻链的天然重链抗体的可变区，是目前已知的天然存在的结合抗原的最小单位。与单克隆抗体相比，纳米抗体具有更优越的肿瘤靶向性和稳定性，可实现药物的靶向递送从而实现精准治疗。
综上，本领域急需开发一种新型、有效的抗人PD-L1纳米抗体和TLR7偶联药物。
发明内容
本发明的目的在于提供一种新型的抗人PD-L1的纳米抗体药物。
本发明的目的在于提供一种新型、有效的抗人PD-L1纳米抗体偶联药物。
本发明的目的在于提供一种抗人PD-L1纳米抗体和TLR7激动剂联合抗肿瘤治疗用途以及提供一种PD-L1和TLR7双靶向纳米抗体偶联药物。
本发明的另一目的在于提供抗人PD-L1和TLR7双靶向纳米抗体偶联药物在肿瘤预防和治疗中的应用，特别是PD-L1抗体低响应率的肿瘤中的应用。
在本发明的第一方面，提供了一种抗体-药物偶联物或其药学上可接受的盐，所述的抗体-药物偶联物结构如式Ⅰ所示：
Ab-(J-U)n     (Ⅰ)
式中，
Ab为PD-L1抗体；
U各自独立地为TLR激动剂；
J为化学键或连接子；
n为0或正整数；
“-”为化学键或接头或连接子。
在另一优选例中，所述的PD-L1抗体包括单特异性抗体、双特异性抗体、多特异性抗体(如三特异性抗体)。
在另一优选例中，所述的PD-L1抗体包括：单克隆抗体、单链抗体(scFv)、纳米抗体。
在另一优选例中，所述的PD-L1抗体包括单价、二价或多价抗体。
在另一优选例中，所述的PD-L1抗体包括多聚体形式的抗体。
在另一优选例中，所述的PD-L1抗体特异性结合PD-L1。
在另一优选例中，所述的PD-L1抗体包括PD-L1单价纳米抗体、二价纳米抗体和/或多价纳米抗体。
在另一优选例中，所述的PD-L1抗体包括阻断型(可阻断PD-L1和PD-1的结合)、非阻断型(不阻断PD-L1和PD-1的结合)、或其组合。
在另一优选例中，所述的PD-L1抗体为阻断型抗体。
在另一优选例中，所述的PD-L1抗体阻断PD-1与PD-L1的结合。
在另一优选例中，所述的PD-L1为人PD-L1或非人哺乳动物的PD-L1(如小鼠PD-L1)。
在另一优选例中，所述的PD-L1抗体为人或非人哺乳动物抗体。
在另一优选例中，所述非人哺乳动物选自下组：骆驼、羊驼、小鼠、食蟹猴。
在另一优选例中，所述的PD-L1抗体为PD-L1纳米抗体或其衍生抗体，优选为靶向人PD-L1纳米抗体或其衍生抗体。
在另一优选例中，所述的衍生抗体为针对PD-L1纳米抗体的修饰改造，包括但不限于将PD-L1纳米抗体连接Fc片段、人血清白蛋白、聚乙二醇PEG、形成二价抗体和/或多价抗体。
在另一优选例中，所述的纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在另一优选例中，所述的PD-L1纳米抗体为特异性结合人PD-L1的纳米抗体，且所述纳米抗体中的VHH链的互补决定区CDR选自下组：
(1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
(2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
(4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
(5)SEQ ID NO：2所示的CDR1、SEQ ID NO：18所示的CDR2，SEQ ID NO：19所示的CDR3；
(6)SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2，SEQ ID NO：23所示的CDR3；
(7)SEQ ID NO：25所示的CDR1、SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3；
(8)SEQ ID NO：29所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：31所示的CDR3；
(9)SEQ ID NO：33所示的CDR1、SEQ ID NO：34所示的CDR2，SEQ ID NO：35所示的CDR3；
(10)SEQ ID NO：37所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：38所示的CDR3；
(11)SEQ ID NO：40所示的CDR1、SEQ ID NO：41所示的CDR2，SEQ ID NO：42所示的CDR3；
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍生序列。
在另一优选例中，所述的PD-L1纳米抗体为特异性结合人PD-L1的纳米抗体，且所述纳米抗体中的VHH链的互补决定区CDR选自下组：
(1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
(2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(3)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：98所示的CDR3；
(4)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：99所示的CDR3；
(5)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：100所示的CDR3；
(6)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：101所示的CDR3；
(7)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：102所示的CDR3；
(8)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：103所示的CDR3；
(9)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：104所示的CDR3；
(10)SEQ ID NO：105所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(11)SEQ ID NO：106所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(12)SEQ ID NO：107所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(13)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：108所示的CDR3；
(14)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：109所示的CDR3；
(15)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：110所示的CDR3；
(16)SEQ ID NO：111所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(17)SEQ ID NO：112所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(18)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：113所示的CDR3；
(19)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：114所示的CDR3；或
(20)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：115所示的CDR3。
在另一优选例中，所述特异性结合人PD-L1的纳米抗体包括经过回复突变的人PD-L1的纳米抗体，所述回复突变选自在SEQ ID NO.1所示的VHH上具有选自下组的氨基酸回复突变：A61N、A61Q、A61K、D59R、D59M、D59F、D62W、D62Y、C103K、C103M、C103W、G99R、G99Q、G99W、G99Y；或在SEQ ID NO.5所示的VHH上具有选自下组的氨基酸回复突变：G30R、G30F、G30W、G101R、G101W、G101Y、F29R、F29W、P100R、P100Q、P100E；较佳地，所述回复突变选自在SEQ ID NO.1所示的VHH上具有选自下组的氨基酸回复突变：C103K、C103M、C103W、G99R、G99Q、G99W、G99Y；或在SEQ ID NO.5所示的VHH上具有选自下组的氨基酸回复突变：G30R、G30F、G30W、G101R、G101W、G101Y、F29R、F29W、P100R、P100Q、P100E。
在另一优选例中，所述特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:1、5、9、13、17、20、24、28、32、36、39所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:1、5、9、13、17、20、24、28、32、36或39具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的特异性结合人PD-L1纳米抗体为人源化的特异性结合人PD-L1纳米抗体，其包含的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70或71具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的特异性结合人PD-L1纳米抗体为亲和力成熟的特异性结合人PD-L1的纳米抗体，其包含的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96或97所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96或97具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述“亲和力成熟”是指，亲和力成熟化改造的抗人PD-L1纳米抗体对PD-L1的亲和力相对于改造前的抗人PD-L1纳米抗体对PD-L1的亲和力，提高了至少1倍、至少2倍、至少3倍、至少4倍、至少5倍、至少6倍、至少7倍、至少8倍、至少9倍、至少10倍、至少11倍、至少12倍、至少20倍、或至少25倍。
在另一优选例中，所述纳米抗体的VHH链的互补决定区CDR由SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3组成。
在另一优选例中，所述纳米抗体的VHH链序列如SEQ ID NO.:1所示。
在另一优选例中，所述纳米抗体的VHH链的互补决定区CDR由SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3组成。
在另一优选例中，所述纳米抗体的VHH链序列如SEQ ID NO.:5所示。
在另一优选例中，所述TLR激动剂为大分子(蛋白质或核酸)或小分子激动剂。
在另一优选例中，所述TLR激动剂包括但不限于TLR1激动剂、TLR2激动剂、TLR3激动剂、TLR4激动剂、TLR5激动剂、TLR6激动剂、TLR7激动剂、TLR8激动剂和TLR9激动剂。
在另一优选例中，n为所述抗体-药物偶联物中的药物平均偶联数量，较佳地n为1～9，优选为2.5～6.5，更优选为3.5～5.5。
在另一优选例中，所述的TLR激动剂为TLR7激动剂。
在另一优选例中，所述的TLR激动剂不具有TLR8激动活性。
在另一优选例中，所述的TLR7激动剂为宿主内源性激动剂或外源性激动剂。
在另一优选例中，所述的TLR7激动剂为小分子激动剂。
在另一优选例中，所述的TLR7激动剂包括：SZU-101：
在另一优选例中，所述TLR7激动剂为SZU-101的衍生化合物，包括但不限于在SZU-101基础进行的一个或多个基团的替换、修饰或删除。
在另一优选例中，所述TLR7激动剂为SZU-101的多价化合物。
在另一优选例中，所述TLR7激动剂(如SZU-101)连接于PD-L1抗体的重链恒定区或重链可变结构域(VHH)的末端氨基或侧链氨基。
在另一优选例中，所述TLR7激动剂(如SZU-101)连接于PD-L1抗体的巯基。
在另一优选例中，所述SZU-101连接于PD-L1抗体的氨基，并形成S1所示结构：
或者
所述的SZU-101连接于PD-L1抗体的巯基，并形成S2所示结构：
在另一优选例中，所述的TLR7激动剂定点和/或随机地连接于所述的PD-L1抗体(即式I中，所述的U定点和/或随机地连接于Z)。
在另一优选例中，所述的U定点连接于Z。
在另一优选例中，所述的U定点连接于PD-L1抗体Z的选自下组的氨基酸位点：G、K、L、A、C或其组合。
在另一优选例中，所述化学键为聚乙二醇PEG。
在另一优选例中，所述化学键为PEG的衍生化合物，包括但不限于在SZU-101的基础上进行的一个或多个基团的替换、修饰或删除。
在另一优选例中，所述PEG化学键的聚合度为大于等于1的正整数。
在另一优选例中，所述抗体-药物偶联物提高肿瘤内细胞的PD-L1水平。
在另一优选例中，所述抗体-药物偶联物激活免疫细胞。
在另一优选例中，所述的激活为体外激活。
在另一优选例中，所述的体外激活包括：在所述的抗体-药物偶联物存在下，培养所述的免疫细胞一段时间(如6-48小时)，从而获得经免疫激活的免疫细胞。
在另一优选例中，所述的免疫细胞选自但不限于：CD8+T细胞、自然杀伤细胞NK、树突状细胞、淋巴细胞、单核/巨噬细胞、粒细胞、或其组合。
在另一优选例中，所述的抗体-药物偶联物或其药学上可接受的盐用于制备一种组合物或制剂，所述组合物或制剂用于：
(a)促进树突状细胞的成熟；
(b)增加肿瘤浸润细胞毒性细胞(CD8+T细胞和NK细胞)的功能；
(c)促进肿瘤浸润细胞毒性细胞颗粒酶B和IFN-γ的表达；
(d)促使肿瘤相关巨噬细胞重极化；
(e)减少TGF-β+巨噬细胞的浸润；
(f)促进IFN-γ+CD4+T细胞的浸润；
(g)促进瘤内巨噬细胞表达PD-L1；
(h)靶向并重塑肿瘤免疫微环境；
(i)提高肿瘤细胞的PD-L1水平；和/或
(j)用于治疗中表达或低表达PD-L1的肿瘤。
在另一优选例中，所述的重塑肿瘤免疫微环境为协调肿瘤内先天免疫和适应性免疫抗肿瘤免疫应答。
在另一优选例中，所述的重塑肿瘤免疫微环境为提高抗肿瘤免疫细胞的浸润，和降低免疫抑制细胞的比例。
在另一优选例中，所述的抗肿瘤免疫细胞包括但不限于分泌颗粒酶及IFN-γ的CD8+T细胞和NK细胞、活化的树突状细胞、分泌IFN-γ的CD4+T细胞、M1巨噬细胞。
在另一优选例中，所述的免疫抑制细胞包括但不限于M2巨噬细胞、Treg细胞、分泌TGF-β的白细胞。
在另一优选例中，所述的PD-L1水平包括细胞表面PD-L1水平和细胞内PD-L1水平。
在另一优选例中，所述的低表达PD-L1的肿瘤为实体瘤或血液瘤。
在本发明的第二方面，提供了一种PD-L1纳米抗体，所述PD-L1纳米抗体特异性结合人PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
(1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
(2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
(3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
(4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
(5)SEQ ID NO：2所示的CDR1、SEQ ID NO：18所示的CDR2，SEQ ID NO：19所示的CDR3；
(6)SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2，SEQ ID NO：23所示的CDR3；
(7)SEQ ID NO：25所示的CDR1、SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3；
(8)SEQ ID NO：29所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：31所示的CDR3；
(9)SEQ ID NO：33所示的CDR1、SEQ ID NO：34所示的CDR2，SEQ ID NO：35所示的CDR3；
(10)SEQ ID NO：37所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：38所示的CDR3；
(11)SEQ ID NO：40所示的CDR1、SEQ ID NO：41所示的CDR2，SEQ ID NO：42所示的CDR3；
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍生序列。
在另一优选例中，所述特异性结合人PD-L1纳米抗体的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:1、5、9、13、17、20、24、28、32、36、39所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:1、5、9、13、17、20、24、28、32、36或39具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少 99％的序列相似性的氨基酸序列。
在另一优选例中，所述的特异性结合人PD-L1纳米抗体为人源化的特异性结合人PD-L1纳米抗体，其包含的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70或71具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的特异性结合人PD-L1纳米抗体为亲和力成熟的特异性结合人PD-L1纳米抗体，其包含的VHH链的氨基酸序列选自下组：
(a)具有SEQ ID NO:72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96或97所示的氨基酸序列；
(b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
在另一优选例中，所述的纳米抗体序列包含与SEQ ID NO:72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96或97具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在本发明的第三方面，提供了一种药物组合物，所述药物组合物包含：
(a)如本发明第一方面所述的抗体-药物偶联物或其药学上可接受的盐、或如本发明第二方面所述的PD-L1纳米抗体；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物还包含：
(c)其他生物活性的药物，如治疗肿瘤的药物。
在另一优选例中，所述其他生物活性的药物促进CD8+T细胞和NK细胞的抗肿瘤功能。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物的施用方式选自下组：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述药物组合物的施用方式为，将所述药物组合物和免疫细胞(如树突状细胞、自然杀伤细胞、淋巴细胞、单核/巨噬细胞、粒细胞等)共培养后，分离免疫细胞进行体内回输。
在另一优选例中，所述的药物组合物的剂型选自下组：液态、固体、或凝胶态。
在另一优选例中，所述的药物组合物用于抗肿瘤治疗。
在另一优选例中，所述的药物组合物用于治疗PD-L1低表达的肿瘤。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1低于正常肿瘤表达PD-L1的量E0，较佳地E1/E0≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、前列腺癌、结直肠癌、前列腺癌、宫颈癌、淋巴癌、骨癌、肾上腺肿瘤、或膀胱肿瘤。
在本发明的第四方面，提供了一种免疫偶联物，所述的免疫偶联物含有：
(a)如本发明第一方面所述的抗体-药物偶联物；和
(b)其他偶联部分。
在另一优选例中，所述其他的偶联部分选自下组：小分子化合物、PEG、荧光素、放射性同位素、造影剂、脂肪酸链、蛋白片段、或其组合。
在另一优选例中，所述的组分(a)和(b)可操作性连接。
在另一优选例中，所述的偶联部分包括化学标记和生物标记。
在另一优选例中，所述化学标记选自同位素、免疫毒素和/或化学药物。
在另一优选例中，所述生物标记选自生物素、亲和素或酶标记。
在另一优选例中，所述小分子化合物选自治疗肿瘤或自身免疫性疾病药物或毒素。
在另一优选例中，所述的放射性同位素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
在另一优选例中，所述的放射性同位素包括但不限于碘131、铟111和镥177。
在另一优选例中，所述的造影剂用于MRI或CT。
在另一优选例中，所述的蛋白片段包括但不限于抗体Fc、生物素、亲和素、HRP、抗体、酶、细胞因子及其他生物活性蛋白或多肽。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂，或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在本发明的第五方面，提供了一种融合蛋白，所述的融合蛋白包含：
(a)如本发明第二方面所述的PD-L1纳米抗体；和
(b)任选的具有治疗功能的多肽分子和蛋白片段。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括但不限于：靶向PD-1、IL-4R、IL-4Rα、TNF-α、VEGF、4-1BB、CD47、TIM3、CTLA4、IL-17A、CD19、CD22、CD28、CD38、CD40、CD47、B7-H3、TSLP、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、HER2的多肽分子或片段。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括但不限于：胰岛素、IL-2、干扰素、降钙素、GHRH肽、肠肽类似物、白蛋白、抗体片段、细胞因子。
在另一优选例中，所述具有治疗功能的多肽分子或片段包括单链抗体(scFv)、双链抗体、单克隆抗体、或嵌合抗体。
在另一优选例中，所述融合蛋白还包含协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列选自下组：6His标签、GGGS序列、FLAG标签。
在另一优选例中，所述的融合蛋白包括双特异性抗体、嵌合抗体。
在本发明的第六方面，提供了一种多特异性抗体，所述的多特异性抗体包含：
(a)如本发明第二方面所述的PD-L1纳米抗体；和
(b)任选的靶向第二抗原的抗体分子。
在另一优选例中，多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：PD-1、IL-4R、IL-4Rα、TNF-α、VEGF、4-1BB、CD47、TIM3、CTLA4、IL-17A、CD19、CD22、CD28、CD38、CD40、CD47、B7-H3、TSLP、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、HER2或其组合。
在另一优选例中，所述的第二抗原结合区为纳米抗体。
在另一优选例中，所述多特异性抗体包括一个或多个第二抗原结合区。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
在本发明的第七方面，提供了一种制备本发明第一方面所述的抗体-药物偶联物的方法，所述方法包括步骤：
配置反应体系，所述反应体系中包括抗体和游离的药物分子，然后进行偶联反应，从而制得所述抗体-药物偶联物，其中，所述药物分子包括TLR激动剂、接头。
在另一优选例中，反应时间为3h-10h。
在另一优选例中，所述抗体与药物分子的摩尔比为1-2：3-20；优选为1：6-10。
在另一优选例中，将SZU-101、EDCI和NHS溶于DMSO中室温搅拌三小时反应制备SZU-101-NHS活性酯，PD-L1纳米抗体和SZU-101-NHS活性酯按照1:10的摩尔比剂量4℃搅拌反应4小时，制备获得纳米抗体偶联药物。
在本发明的第八方面，提供了一种药盒，所述的药盒包括：
(1)第一容器，以及位于所述第一容器内的如本发明的第二方面所述的PD-L1纳米抗体，以及药学上可用的载体；
(2)第二容器，以及位于所述第二容器内的TLR7激动剂，以及药学上可用的载体；
以及(3)任选的使用说明书。
在本发明的第九方面，提供了一种预防或治疗肿瘤的方法，向有需要的受试者施用如本发明第一方面所述的纳米抗体偶联药物、如本发明的第二方面所述的PD-L1纳米抗体或如本发明的第八方面所述的药盒。
在另一优选例中，所述的肿瘤为表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤选自下组：高表达PD-L1的肿瘤、中表达PD-L1的肿瘤、低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为中表达PD-L1的肿瘤或低表达PD-L1的肿瘤。
在另一优选例中，所述的肿瘤为低表达PD-L1的肿瘤。
在另一优选例中，“高表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)＞1，更佳地≥1.5，更佳地≥2.0。
在另一优选例中，“中表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)为0.5-1.1，更佳地为0.7-1.0，更佳地为0.8-0.9。
在另一优选例中，“低表达PD-L1”指，所述肿瘤表达的PD-L1的量E1与正常肿瘤表达PD-L1的量E0之比(E1/E0)≤1/2，更佳地≤1/3，更佳地≤1/4。
在另一优选例中，所述的肿瘤包括但不限于：乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、前列腺癌、结直肠癌、前列腺癌、宫颈癌、淋巴癌、骨癌、肾上腺肿瘤、或膀胱肿瘤。
在本发明的第十方面，提供了一种本发明第一方面所述的抗体-药物偶联物，如本发明的第二方面所述的PD-L1纳米抗体，如本发明的第三方面所述的药物组合物，如本发明的第四方面所述的免疫偶联物，如本发明的第五方面所述的融合蛋白，或如本发明的第六方面所述的多特异性抗体的用途，用于制备试剂、检测板或试剂盒；或用于制备预防和/或治疗PD-L1相关疾病的药物。
在另一优选例中，所述PD-L1相关疾病为表达PD-L1的肿瘤/癌症。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中 具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了候选抗人PD-L1纳米抗体的human PD-L1结合活性，其中blank为阴性对照。
图2显示了候选抗人PD-L1纳米抗体对human PD-1/PD-L1结合的阻断活性测定，其中blank和blank+ligand为对照组。
图3显示了候选抗人PD-L1的纳米抗体阻断human PD-1/PD-L1结合的IC50曲线，其中KN035为阳性对照组。
图4显示了候选抗人PD-L1的纳米抗体偶联药物偶联度前后的质谱鉴定(4A-D)以及阳性对照组KN035的质谱鉴定图(4E)。
图5显示了候选抗人PD-L1的纳米抗体h_Nb1、h_Nb2与阳性对照抗体KN035以及纳米抗体偶联药物h_Nb1-SZU-101和h_Nb2-SZU-101的肿瘤抑制作用，5A为不同组别的肿瘤生长曲线，5B为各组的终点肿瘤重量；5C为各组别的肿瘤抑制率，5D-5I为不同组别的每只小鼠的肿瘤生长曲线，5J-5K为纳米抗体偶联药物h_Nb1-SZU-101和h_Nb2-SZU-101肿瘤消退小鼠再荷瘤实验的肿瘤生长曲线。
具体实施方式
发明人通过广泛而深入的研究，筛选并鉴定了一种抗人PD-L1纳米抗体，并开发了一种抗人PD-L1和TLR7双靶向纳米抗体偶联药物。具体地，本发明利用噬菌体展示技术和流式细胞术筛选获得了抗人PD-L1的纳米抗体及其衍生分子，并利用抗体偶联技术制备获取了新型双靶向纳米抗体偶联药物及其衍生分子，发现抗人PD-L1的纳米抗体和双靶向纳米抗体偶联药物均具有优异的抗肿瘤活性。此外，本发明发现双靶向纳米抗体偶联药物可重塑肿瘤免疫微环境，促进抗原递呈细胞包括巨噬细胞表达PD-L1，有利于治疗低表达PD-L1分子的“冷”肿瘤。本发明开发的PD-L1和TLR7双靶向纳米抗体偶联药物表现出突出的抗肿瘤效果和新颖的作用机制，具备临床开发和应用价值。在此基础上完成了本发明。
TLR受体及TLR受体激动剂
如本文所用，术语“TLR受体”是指Toll样受体，是生物体免疫系统中一类重要的固有免疫模式识别受体，可特异性的识别病原微生物进化过程中相对保守的抗原分子(或称为病原相关分子模式)，实现病原微生物入侵的有效检测和固有免疫应答诱导。在人体中已发现十种TLR受体，即TLR1-TLR10,其中TLR3、TLR7、TLR8、TLR9定位于细胞的内体、溶酶体膜上，其余定位于细胞质膜上。在本发明实施方式中，优选TLR7作为药物分子靶标之一。TLR7分子的天然配体为单链线性RNA。
如本文所用，术语“TLR受体激动剂”是指可以特异性结合并激活TLR受体的大分子(蛋白质或核酸)或小分子激动剂，促进TLR受体的下游信号的转导，实现固有免 疫细胞的活化。在本发明实施方式中，优选TLR7激动剂构建纳米抗体偶联药物。除本发明实施方式中应用的SZU-101外，可用的TLR7激动剂还包括咪喹莫特和R848等。
如本文所用，术语“本发明纳米抗体”、“本发明的靶向PD-L1的纳米抗体”、“本发明的抗PD-L1纳米抗体”可互换使用，均指特异性识别和结合于PD-L1(包括人或鼠PD-L1)的纳米抗体。特别优选的是VHH链的氨基酸序列如SEQ ID NO:1或SEQ ID NO:5所示的纳米抗体(h_Nb1或h_Nb2)。
如本文所用，术语“本发明纳米抗体偶联药物”、“本发明的双靶向纳米抗体偶联药物”、“本发明的PD-L1和TLR7双靶向纳米抗体偶联药物”，可互换使用，均指特异性识别和结合于PD-L1(包括人或鼠PD-L1)的纳米抗体及其衍生蛋白偶联TLR7激动剂形成的新型药物分子。纳米抗体偶联药物中的纳米抗体特别优选的是VHH链的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示的纳米抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体(single domain antibody，sdAb，或VHH)”、“纳米抗体”(nanobody)具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。
纳米抗体/单域抗体(Nanobody)作为一种新型的小分子抗体片段，由驼类天然的重链抗体重链可变区(VHH)克隆获得。Nanobody(Nb)具有优良的生物学特性，分子量12-15kDa，是完整抗体的十分之一，具有很好的组织穿透性，特异性高，水溶性好。因其特殊的结构性质，兼具了传统抗体与小分子药物的优势，几乎完美克服了传统抗体的开发周期长，稳定性较低，保存条件苛刻等缺陷，逐渐成为新一代抗体治疗中的新兴力量，在免疫诊断和治疗中显示出广阔的应用前景。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效 应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。
如本文所用，术语“纳米抗体偶联药物”与“纳米抗体药物偶联物”可互换使用。如本领域技术人员所知，纳米抗体偶联物是一种特殊的抗体药物偶联药物形式，其是将纳米抗体或者衍生蛋白偶联药物、毒素、细胞因子、放射性核素、酶和其他诊断或治疗分子形成的药物分子形式，可用于肿瘤治疗、药物递送和体内成像等，具有广阔的临床应用价值。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region，CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸 残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含纳米抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明纳米抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A

本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为 原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.疗剂(例如，顺铂)或任何形式的纳米颗粒等。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体-药物偶联物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物或具有促进免疫功能的药物。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例 如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
应理解，药物通常可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在本发明中，药物为激活或促进免疫反应的药物，例如激活固有免疫反应从而辅助适应性免疫的激活。在具体的实施方式中，药物为TLR受体激动剂。
在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，TLR1激动剂、TLR2激动剂、TLR3激动剂、TLR4激动剂、TLR5激动剂、TLR6激动剂、TLR7激动剂、TLR8激动剂和TLR9激动剂，例如SZU-101、咪喹莫特、R848、CpG等。在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类 反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Ab-(J-U)n        (Ⅰ)
式中，
Ab为PD-L1抗体；
U各自独立地为TLR激动剂；
J为化学键或连接子；
n为0或正整数；
“-”为化学键或接头或连接子。
应用
本发明提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗PD-L1相关疾病的药物。所述PD-L1相关疾病包括炎症疾病、自身免疫疾病等，包括但不限于乳腺癌、肝癌、胃癌、大肠癌、白血病、肺癌、肾脏肿瘤、小肠癌、前列腺癌、结直肠癌、前列腺癌、宫颈癌、淋巴癌、骨癌、肾上腺肿瘤、或膀胱肿瘤。
应理解，对一种或多种检查点抑制剂(例如结合PD-L1、CTLA-4或CD47等的抗体)的治疗无反应的癌症，例如胰腺癌或前列腺癌，这种肿瘤被称为冷肿瘤。对用一种或多种检查点抑制剂，例如结合PD-L1、CTLA-4或CD47等的抗体的治疗有反应的癌症，此类肿瘤也称为温或热肿瘤。与不响应检查点抑制剂治疗的肿瘤相比，此类肿瘤被认为具有更高的肿瘤浸润淋巴细胞(TIL)水平和/或更高的肿瘤突变负荷。
本发明提供的一种优选的抗体-药物偶联物，其具有优异的抗肿瘤活性，且在冷肿瘤和低表达PD-L1的肿瘤模型中同样表现出极其显著的抗肿瘤活性和响应率。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体或赋形剂，以及任选 的其他生物活性物质。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括
(a)本发明抗人PD-L1纳米抗体可高特异性针对人的具有正确空间结构的PD-L1蛋白，且亲和力强，生产简便。
(b)本发明提供了PD-L1纳米抗体和TLR7激动剂的联合治疗方案，表现出了显著的体内抗肿瘤活性，表明PD-L1抗体疗法和TLR7免疫激动剂具有联合合理性，可协同抗肿瘤。
(c)本发明开发了靶向人PD-L1和TLR7双靶向纳米抗体偶联药物，其可以促进肿瘤中细胞上调表达PD-L1，并协调瘤内先天免疫和适应性抗肿瘤免疫应答，使得其在“冷”肿瘤和低PD-L1表达的肿瘤等PD-L1抗体治疗效果较差的多种肿瘤中表现出极佳的抑制肿瘤生长作用。
(d)本发明提供的PD-L1和TLR7双靶向纳米抗体偶联药物可靶向肿瘤免疫微环境，并重塑肿瘤免疫微环境，提高抗肿瘤免疫细胞的浸润，减少免疫抑制细胞的浸润。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：抗人PD-L1的纳米抗体筛选
1.1 human PD-L1蛋白表达
构建human PD-L1(ECD)-pFUSE-hIgG1-Fc载体并进行质粒扩增。利用哺乳动物细胞HEK293F表达human PD-L1蛋白并利用Protein A亲和柱纯化蛋白。
1.2免疫动物和文库构建
将1.1中获得的抗原蛋白免疫骆驼，5次免疫结束后，抽取外周血，分离外周血单个核细胞，提取RNA并反转撸获得cDNA；利用巢氏PCR扩增获得VHH基因片段，并连接至噬菌体质粒载体上；将连接产物电转化至大肠杆菌电转感受态TG1中，完成抗human PD-L1蛋白的纳米抗体文库的构建。
1.3文库淘选
向10倍库容的纳米抗体库中加入辅助噬菌体扩增得到纳米抗体展示噬菌体。96孔酶标板用5μg/mL NeutrAvidin溶液(每孔100μL)包被，4℃,包被过夜。第二天，用2％脱脂奶粉室温封闭2h，用20mM HEPES(pH7.5),150mM NaCl溶液洗5次。设置对照组和实验组，加入100μL 200nM PD-L1-biotin蛋白稀释液，室温振荡(700rpm)并孵育15min，用20mM HEPES(pH7.5),150mM NaCl溶液洗5次。之后加入100μL噬菌体稀释液(1×1013cfu/mL)，室温振荡(700rpm)并孵育2h，用20mM HEPES(pH7.5),150mM NaCl溶液洗涤5次除去不结合的噬菌体。后加入100μL0.25mg/mL的胰酶室温振荡(700rpm)消化30min将特异性结合的噬菌体解离下，加入抑制剂终止消化。将洗脱的噬菌体感染TG1细胞，以便第二轮panning使用。重复上述操作2-3轮，直至阳性克隆被富集。
1.4 ELISA鉴定阳性克隆
几轮淘选过后，将洗脱下来的噬菌体感染处于生长对数期的TG1感受态，梯度稀释并涂布在平板上培养过夜。分别挑取96个克隆接种到每孔100μL培养基的96孔圆底板中静置过夜作为母板，之后吸取10μL过夜培养的菌液到每孔1mL培养基的96孔深底板中，诱导纳米抗体表达并粗纯。96孔酶标板用5μg/mL NeutrAvidin溶液包被，4℃,700rpm,过夜。第二天，加入2％脱脂奶粉室温封闭板子，用含牛血清白蛋白BSA的溶液洗3次。加入100μL 3μg/mL PD-L1蛋白室温孵育30分钟后洗涤，加入粗提的纳米抗体室温孵育1h后洗涤，加入小鼠抗HA一抗室温孵育1小时后洗涤，加入山羊抗小鼠碱性磷酸酶标记二抗室温孵育1h后洗涤，加入碱性磷酸酶显色液反应10min，在酶标仪405nm处检测吸收值，初步判定吸收值是对照组吸收值的3倍以上的即为阳性孔，将阳性克隆转移到摇菌管中培养以便提取质粒并进行测序，最终获得11株候选阳性克隆。
实施例2：抗人PD-L1纳米抗体的表达和鉴定
制备线性化pFUSE-mIgG2b-Fc和pFUSE-hIgG1-Fc载体后，将候选纳米抗体序列同源重组至pFUSE-mIgG2b-Fc和pFUSE-hIgG1-Fc载体中，随后利用哺乳动物细胞HEK293F表达候选纳米抗体。
本发明中获得的11株优选的抗人PD-L1纳米抗体，分别为h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、h_Nb12、h_Nb13、h_Nb19、h_Nb26、h_Nb30。
其中，h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、h_Nb12、h_Nb13、h_Nb19、h_Nb26、h_Nb30的VHH序列分别如SEQ ID NO.:1、5、9、13、17、20、24、28、32、36、39所示，CDR部分见表1。
表1.抗人PD-L1抗体的VHH及CDR序列


实施例3：抗人PD-L1的纳米抗体的初步体外活性评价
3.1候选抗人PD-L1纳米抗体的human PD-L1结合活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×106细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×105细胞/样品；样品中分别加入不同的抗人PD-L1的候选纳米抗体(浓度为10μg/ml)；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-human IgG Fc(FITC)作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到候选纳米抗体与human PD-L1的结合活性，如图1所示。
实验结果提示，候选纳米抗体h_Nb1、h_Nb2、h_Nb4、h_Nb5、h_Nb6、h_Nb9、h_Nb12、h_Nb13、h_Nb26共9株纳米抗体均与human PD-L1保持良好的结合活性。
3.2候选抗人PD-L1纳米抗体的human PD-1/PD-L1阻断活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×106细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×105细胞/样品；样品中分别加入human PD-1-his蛋白(浓度为1μg/mL)和抗人PD-L1的候选纳米抗体(浓度为10μg/ml)；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-his-APC作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到候选纳米抗体的PD-1/PD-L1阻断活性，如图2所示。
实验结果提示，候选纳米抗体h_Nb1、h_Nb2可有效阻断PD-1/PD-L1的结合活性，具有明显的阻断效果。
实施例4：抗人PD-L1的纳米抗体的体外活性评价
4.1候选抗人PD-L1的纳米抗体阻断human PD-1/PD-L1结合的IC50活性测定
消化处理稳转细胞株HEK293T/hPD-L1制备细胞样品；样品中分别加入不同浓度的纳米抗体h_Nb1或h_Nb2或阳性对照抗体KN035(序列来自IMGT数据库)，以及human PD-1-his蛋白(浓度为1μg/mL)；孵育离心结束后，以anti-his-APC作为二抗，孵育染色；后用流式细胞仪上机检测，得到候选纳米抗体阻断human PD-1/PD-L1结合的IC50值，如图3所示。
实验结果显示，候选纳米抗体h_Nb1、h_Nb2与阳性对照抗体KN035的IC50活性相当，甚至h_Nb2的体外活性较优于KN035。
实施例5：TLR7激动剂SZU-101的制备
TLR7小分子激动剂用于偶联抗体的分子结构如下：
SZU-101及上述用于偶联的SZU-101衍生物的合成可采用以下方法或类似方法。
将化合物20-1溶解于无水DMSO中，于10℃冷却加入等当量的丁二酸酐，混合物自然室温搅拌24小时。混合反应物倒与20倍体积水中，析出大量白色固体化合物SZU-101。
将SZU-101(1eq),NHS(1.2eq)和EDC(1.3eq)溶于无水DMF，室温下搅拌4h，结束 反应，将反应液倒入二氯甲烷中，抽滤干燥，得化合物23，即SZU-101-NHS，为白色固体。
SZU-101-Mal可用类似方法制备。
实施例6：PD-L1和TLR7双靶向纳米抗体偶联药物的制备
将SZU-101、EDCI和NHS溶于DMSO中，室温下搅拌3h，LC-MS监测反应进程，反应完全后，加入十倍量的双蒸水，抽滤，真空干燥即可得到SZU-101-NHS活性酯。
之后将活化酯用DMSO溶解，抗体和小分子按照1：10的摩尔比剂量反应，投入一定量的小分子活化酯至候选抗体中，4℃搅拌反应4小时。反应结束后，在混合物中加入PBS混合，用10kD的生物滤膜过滤除去小分子，得新型双靶向纳米抗体偶联化合物h_Nb1-SZU-101和h_Nb2-SZU-101。反应制得的偶联化合物，先将抗体变性打开二硫键，之后用XevoG2XSQTOF质谱仪鉴定样品，主要根据新型偶联化合物比未偶联抗体增加的分子量计算偶联度。
实施例7：PD-L1和TLR7双靶向纳米抗体偶联药物的体外活性测定
7.1 PD-L1和TLR7双靶向纳米抗体偶联药物的PD-L1结合活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×106细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×105细胞/样品；样品中分别加入不同的浓度的偶联化合物h_Nb1-SZU-101或h_Nb2-SZU-101以及其对应的裸抗；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-human IgG Fc(FITC)作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到偶联前后纳米抗体的EC50值。
7.2 PD-L1和TLR7双靶向纳米抗体偶联药物的PD-1/PD-L1阻断活性测定
消化处理稳转细胞株HEK293T/hPD-L1，离心去上清；用PBS清洗1遍并调节细胞密度为2.5×106细胞/mL；每个细胞样品加入100μL细胞悬液，即包含2.5×105细胞/样品；样品中分别加入human PD-1-his蛋白(浓度为1μg/mL)和不同的浓度的h_Nb1-SZU-101或h_Nb2-SZU-101以及其对应的裸抗；4℃冰箱孵育20min；离心，去上清，用PBS洗一遍；用稀释后抗体anti-his-APC作为二抗，重悬上述细胞；4℃冰箱孵育20min；离心，去上清，用PBS洗两遍，转移至流式管中；用流式细胞仪上机检测，得到偶联前后纳米抗体的IC50值。
实施例8：抗人PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性评价
如实施例6所述方法，制备获取抗人PD-L1和TLR7双靶向纳米抗体偶联药物h_Nb1-SZU-101和h_Nb2-SZU-101。
如图4所示，候选纳米抗体h_Nb1、h_Nb2与阳性对照抗体KN035均具有较高纯度，质谱鉴定纳米抗体偶联物h_Nb1-SZU-101的偶联度约为3，h_Nb2-SZU-101的偶联度约为5。
为进一步评价抗人PD-L1和TLR7双靶向纳米抗体偶联药物的体内抗肿瘤活性，我们采用PD-1/PD-L1靶点双人源化BALB/c小鼠建立的CT26/hPD-L1肿瘤模型；当平均肿瘤体积达到80-100mm3时，小鼠根据肿瘤体积随机分6组，每组6只；分组当天定义为D0天，并于D0、D3、D7、D10、D13、D16给药，共给药6次，其中给药剂量为10mg/kg、给药方式为腹腔给药。每周2-3次测量肿瘤大小，绘制肿瘤生长曲线，并于Day17解剖肿瘤未消失的瘤子，计算抑瘤率，如图5A-5I所示。
实验结果显示，候选纳米抗体h_Nb1(SEQ ID NO.1)、h_Nb2(SEQ ID NO.5)与阳性对照抗体KN035的抑瘤率分别为81.17％，85.14％，65.83％，h_Nb1和h_Nb2的抑制活性优于对照抗体KN035。令人意外地，纳米抗体偶联药物h_Nb1-SZU-101和h_Nb2-SZU-101的抑瘤活性显著优于h_Nb1和h_Nb2，抑瘤率分别为100.00％，93.26％，极强力地遏制了肿瘤体内进展；此外，h_Nb1-SZU-101会使100％的荷瘤小鼠出现肿瘤消退(图5H)，h_Nb2-SZU-101会使66.7％的荷瘤小鼠出现肿瘤消退(图5I)。肿瘤消退小鼠于于药效试验最后一次给药28天后进行再荷瘤实验，重新选取6只未荷过瘤的BALB/c-hPD1/hPDL1小鼠作为对照组，与肿瘤消退小鼠同时荷瘤(即：CT26/hPD-L1肿瘤)，观察小鼠肿瘤生长曲线。结果显示，肿瘤消退小鼠再荷瘤45天内不再生长肿瘤(图5J-5K)，表明偶联化合物诱导了有效的抗肿瘤免疫记忆。
实施例9：抗人PD-L1的纳米抗体的人源化
利用CDR Grafting方法，将人抗体FR替换驼抗体FR，以达到降低免疫原性的目的。首先对候选抗体进行同源模建，鉴别得到关键氨基酸残基位点，而后以候选纳米抗体序列为模板在结构数据库中进行同源结构的搜索，选择最优结构序列进行序列替换最终得到人源化抗体序列,同时需考虑保留框架区潜在影响CDR作用的关键位点。
本发明中分别对获得的11株优选的抗人PD-L1纳米抗体进行人源化，其中，人源化抗体后的对应序列如下所示：
h_Nb1人源化后序列分别为h_Nb1_1、h_Nb1_2、h_Nb1_3、h_Nb1_4、h_Nb1_5；
h_Nb2人源化后序列分别为h_Nb2_1、h_Nb2_2、h_Nb2_3、h_Nb2_4、h_Nb2_5；
h_Nb4人源化后序列分别为h_Nb4_1、h_Nb4_2；
h_Nb5人源化后序列分别为h_Nb5_1、h_Nb5_2、h_Nb5_3；
h_Nb6人源化后序列分别为h_Nb6_1、h_Nb6_2；
h_Nb9人源化后序列分别为h_Nb9_1、h_Nb9_2；
h_Nb12人源化后序列分别为h_Nb12_1、h_Nb12_2；
h_Nb13人源化后序列分别为h_Nb13_1、h_Nb13_2；
h_Nb19人源化后序列分别为h_Nb19_1、h_Nb19_2；
h_Nb26人源化后序列分别为h_Nb26_1、h_Nb26_2；
h_Nb30人源化后序列分别为h_Nb30_1、h_Nb30_2。
人源化后抗体序列见表2。
表2.抗人PD-L1抗体人源化后的VHH序列

实施例10：纳米抗体CDR亲和力成熟优化
将根据以上实施例优选的特异性结合人PD-L1纳米抗体的全长氨基酸序列(SEQ ID No.1和SEQ ID No.5)，CCG法注释抗体CDR区和FR区；通过抗体同源模建，选择合适的FR区和CDR区模板，构建并选择最优的纳米抗体三维蛋白结构；从PDB蛋白数 据库中获取人PD-L1蛋白晶体结构，通过蛋白复合物同源模建和蛋白-蛋白分子对接方法，获取优选的PD-L1纳米抗体-PD-L1蛋白复合物结构候选库；依据优选抗体的PD-1/PD-L1体外竞争结合特征，优选合适的对接角度，确定最优的对接构象；对选取的纳米抗体-PD-L1蛋白复合物结构分析相互作用关键位点，关注范德华力、氢键、离子键、疏水作用等；重点关注纳米抗体与PD-L1上作用位点的亲和力能量值较低的作用位点对，作为亲和力成熟点突变对象；选取符合以上条件的纳米抗体-PD-L1相互作用残基对，将纳米抗体的对应氨基酸位点进行点突变，分别测试突变为其他19种天然氨基酸，分析突变后位点与PD-L1上对应位点的相互作用力改变，选取使得亲和力显著提高的突变方式；将纳米抗体CDR区上与PD-L1蛋白存在弱相互作用的位点均进行以上点突变筛选，选取优良的突变组合；计算机水平筛选的候选亲和力成熟方案产生的序列，经由基因合成、克隆、转染和蛋白表达纯化获取候选抗体，通过ELISA方法，比较候选抗体和原抗体(SEQ ID NO.1和SEQ ID NO.5)与PD-L1的亲和力水平和阻断PD-1/PD-L1活性；最终确定优选抗体的CDR区亲和力成熟方案，亲和力成熟后抗体的序列为SEQ ID No.72–SEQ ID NO.97。其中，SEQ ID NO.72-86所示的序列对应的原抗体为h_Nb1 VHH(SEQ ID NO.1)；SEQ ID NO.87-97所示的序列对应的原抗体为h_Nb2 VHH(SEQ ID NO.5)。
表3.抗人PD-L1抗体亲和力成熟后的VHH序列


讨论
科学合理且安全有效的联合治疗方案同样可指导新型药物分子的设计，本发明开发了抗人PD-L1和TLR7双靶向纳米抗体偶联药物，可以协调统筹先天免疫和适应性免疫应答，靶向并重塑肿瘤免疫微环境，提高肿瘤组织的PD-L1表达水平，使得在“冷”肿瘤和低表达PD-L1的肿瘤模型中同样表现出极其显著的抗肿瘤活性和响应率，显示了其临床应用价值。
本发明提供了一种抗人PD-L1和TLR7双靶向纳米抗体偶联药物，可后续进行肿瘤免疫治疗药物开发，尤其适用于低免疫原性和/或低表达PD-L1的肿瘤。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的抗体-药物偶联物结构如式Ⅰ所示：
    Ab-(J-U)n    (Ⅰ)
    式中，
    Ab为PD-L1抗体；
    U各自独立地为TLR激动剂；
    J为化学键或连接子；
    n为0或正整数；
    “-”为化学键或接头或连接子。
  2. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的PD-L1抗体为PD-L1纳米抗体或其衍生抗体，优选为靶向人PD-L1纳米抗体或其衍生抗体；且所述纳米抗体中的VHH链的互补决定区CDR选自下组：
    (1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
    (2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
    (3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
    (4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
    (5)SEQ ID NO：2所示的CDR1、SEQ ID NO：18所示的CDR2，SEQ ID NO：19所示的CDR3；
    (6)SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2，SEQ ID NO：23所示的CDR3；
    (7)SEQ ID NO：25所示的CDR1、SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3；
    (8)SEQ ID NO：29所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：31所示的CDR3；
    (9)SEQ ID NO：33所示的CDR1、SEQ ID NO：34所示的CDR2，SEQ ID NO：35所示的CDR3；
    (10)SEQ ID NO：37所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：38所示的CDR3；和
    (11)SEQ ID NO：40所示的CDR1、SEQ ID NO：41所示的CDR2，SEQ ID NO：42所示的CDR3；
    上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍 生序列。
  3. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的TLR激动剂为TLR7激动剂。
  4. 如权利要求3所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的TLR7激动剂包括：SZU-101：
  5. 如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其特征在于，所述的抗体-药物偶联物或其药学上可接受的盐用于制备一种组合物或制剂，所述组合物或制剂用于：
    (a)促进树突状细胞的成熟；
    (b)增加肿瘤浸润细胞毒性细胞(CD8+T细胞和NK细胞)的功能；
    (c)促进肿瘤浸润细胞毒性细胞颗粒酶B和IFN-γ的表达；
    (d)促使肿瘤相关巨噬细胞重极化；
    (e)减少TGF-β+巨噬细胞的浸润；
    (f)促进IFN-γ+CD4+T细胞的浸润；
    (g)促进瘤内巨噬细胞表达PD-L1；
    (h)靶向并重塑肿瘤免疫微环境；
    (i)提高肿瘤细胞的PD-L1水平；和/或
    (j)用于治疗中表达或低表达PD-L1的肿瘤。
  6. 一种PD-L1纳米抗体，其特征在于，所述PD-L1纳米抗体特异性结合人PD-L1，且所述纳米抗体中的VHH链的互补决定区CDR选自下组中的一种或多种：
    (1)SEQ ID NO：2所示的CDR1、SEQ ID NO：3所示的CDR2、SEQ ID NO：4所示的CDR3；
    (2)SEQ ID NO：6所示的CDR1、SEQ ID NO：7所示的CDR2、SEQ ID NO：8所示的CDR3；
    (3)SEQ ID NO：10所示的CDR1、SEQ ID NO：11所示的CDR2，SEQ ID NO：12所示的CDR3；
    (4)SEQ ID NO：14所示的CDR1、SEQ ID NO：15所示的CDR2，SEQ ID NO：16所示的CDR3；
    (5)SEQ ID NO：2所示的CDR1、SEQ ID NO：18所示的CDR2，SEQ ID NO：19所示的CDR3；
    (6)SEQ ID NO：21所示的CDR1、SEQ ID NO：22所示的CDR2，SEQ ID NO：23所示 的CDR3；
    (7)SEQ ID NO：25所示的CDR1、SEQ ID NO：26所示的CDR2，SEQ ID NO：27所示的CDR3；
    (8)SEQ ID NO：29所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：31所示的CDR3；
    (9)SEQ ID NO：33所示的CDR1、SEQ ID NO：34所示的CDR2，SEQ ID NO：35所示的CDR3；
    (10)SEQ ID NO：37所示的CDR1、SEQ ID NO：30所示的CDR2，SEQ ID NO：38所示的CDR3；和
    (11)SEQ ID NO：40所示的CDR1、SEQ ID NO：41所示的CDR2，SEQ ID NO：42所示的CDR3；
    上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-L1结合能力的衍生序列。
  7. 如权利要求6所述的PD-L1纳米抗体，其特征在于，所述的特异性结合人PD-L1纳米抗体为人源化的特异性结合人PD-L1纳米抗体，其包含的VHH链的氨基酸序列选自下组：
    (a)具有SEQ ID NO:43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71所示的氨基酸序列；
    (b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
  8. 如权利要求6所述的PD-L1纳米抗体，其特征在于，所述的特异性结合人PD-L1的纳米抗体，其包含的VHH链的氨基酸序列选自下组：
    (a)具有SEQ ID NO:72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96或97所示的氨基酸序列；
    (b)对(a)中的氨基酸序列进行一个或多个氨基酸添加、一个或多个氨基酸的取代或1-3个氨基酸缺失所形成的衍生抗体或活性片段，所述衍生抗体或活性片段保留与PD-L1特异性结合能力。
  9. 一种药物组合物，其特征在于，所述药物组合物包含：
    (a)如权利要求1所述的抗体-药物偶联物或其药学上可接受的盐、或如权利要求6所述的PD-L1纳米抗体；和
    (b)药学上可接受的载体。
  10. 如权利要求9所述的药物组合物，其特征在于，所述的药物组合物用于治疗PD-L1低表达的肿瘤。
  11. 一种免疫偶联物，其特征在于，所述的免疫偶联物含有：
    (a)如权利要求1所述的抗体-药物偶联物；和
    (b)其他偶联部分。
  12. 一种融合蛋白，其特征在于，所述的融合蛋白包含：
    (a)如权利要求6所述的PD-L1纳米抗体；和
    (b)任选的具有治疗功能的多肽分子和蛋白片段。
  13. 一种多特异性抗体，所述的多特异性抗体包含：
    (a)如权利要求6所述的PD-L1纳米抗体；和
    (b)任选的靶向第二抗原的抗体分子。
  14. 一种药盒，其特征在于，所述的药盒包括：
    (1)第一容器，以及位于所述第一容器内的如权利要求6所述的PD-L1纳米抗体，以及药学上可用的载体；
    (2)第二容器，以及位于所述第二容器内的TLR7激动剂，以及药学上可用的载体；
    以及(3)任选的使用说明书。
  15. 如权利要求1所述的抗体-药物偶联物，如权利要求6所述的PD-L1纳米抗体，如权利要求9所述的药物组合物，如权利要求11所述的免疫偶联物，如权利要求12所述的融合蛋白，或如权利要求13所述的多特异性抗体的用途，其特征在于，用于制备试剂、检测板或试剂盒；或用于制备预防和/或治疗PD-L1相关疾病的药物。
PCT/CN2023/081459 2022-03-15 2023-03-14 抗人pd-l1和tlr7双靶向纳米抗体偶联药物及其在抗肿瘤中的应用 WO2023174312A1 (zh)

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