WO2023186120A1 - 抗血清白蛋白纳米抗体及其衍生物 - Google Patents

抗血清白蛋白纳米抗体及其衍生物 Download PDF

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WO2023186120A1
WO2023186120A1 PCT/CN2023/085559 CN2023085559W WO2023186120A1 WO 2023186120 A1 WO2023186120 A1 WO 2023186120A1 CN 2023085559 W CN2023085559 W CN 2023085559W WO 2023186120 A1 WO2023186120 A1 WO 2023186120A1
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amino acid
seq
antigen
acid residues
nanobody
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PCT/CN2023/085559
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English (en)
French (fr)
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吴凡
缪小牛
罗羿
陈连娣
许英达
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普米斯生物技术(珠海)有限公司
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
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    • C12N2510/00—Genetically modified cells
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to Nanobodies or antigen-binding fragments thereof that specifically bind to serum albumin, derivatives containing the Nanobodies or antigen-binding fragments thereof, nucleic acids encoding the antibodies or antigen-binding fragments thereof, and host cells containing the same, and related uses. Furthermore, the invention relates to the therapeutic use of these antibodies or antigen-binding fragments thereof, or derivatives thereof.
  • PK pharmacokinetics
  • PEG polyethylene glycol
  • fusion of transferrin etc.
  • these chemical methods have some drawbacks.
  • PEG may shield or block the antigen-binding site of the antibody, reducing the number of antibodies or treatments.
  • the drug efficacy of the protein may even lead to the loss of antibody activity.
  • Human serum albumin is the most abundant protein in human plasma, accounting for about half of serum proteins. It is soluble in water and exists in monomeric form. The half-life of serum albumin in the body is approximately 21 days. It consists of 585 amino acids and has a molecular weight of approximately 66.5KD. Human serum albumin is stable in the body, safe, non-toxic, and has low immunogenicity, making it an ideal drug carrier. For antibody drugs, human serum albumin can be modified to increase the half-life of the drug to improve patient compliance.
  • HSA and IgG are internalized into vascular endothelial cells through pinocytosis.
  • the pH of the endosome is about 6.0, which promotes the binding of membrane-bound FcRn to HSA. It is then recycled to the top or bottom of the endothelial cells and reaches a blood pH of 7.0 through exocytosis. Under 4 conditions, membrane-bound FcRn rapidly dissociates from HSA for recycling.
  • the inventor of the present application has screened and obtained a series of anti-serum albumin Nanobodies through extensive research.
  • the Nanobodies have high binding activity to serum albumin and have the ability to bind to human, monkey and/or mouse serum albumin. Cross-reactivity.
  • the binding of the Nanobody of the present invention to serum albumin is pH-independent, and it can effectively participate in FcRn-mediated serum albumin circulation, thereby extending the in vivo half-life of the Nanobody. Therefore, it can be passed
  • the Nanobody of the present application is coupled (eg, fused) to a drug molecule (eg, a polypeptide drug), thereby extending the half-life of the drug molecule in the body.
  • a drug molecule eg, a polypeptide drug
  • the nanobody also has the characteristics of small molecular weight and good stability. Compared with traditional common normal antibodies in drug development and other aspects, it has good tissue infiltration, flexible administration methods, high degree of humanization, and easy recombinant protein. Construction and many other advantages.
  • the present application provides a Nanobody or an antigen-binding fragment thereof capable of specifically binding to serum albumin, the Nanobody or an antigen-binding fragment thereof comprising:
  • CDR1 having a structure selected from the group consisting of : X 1 ), GFTFSIYS (SEQ ID NO: 31), GSIFTFYR (SEQ ID NO: 33);
  • CDR2 having a structure selected from: IX 6 SSGGX 7 X 8 (Formula II, SEQ ID NO:73), ITVDGST (SEQ ID NO:46), ISSGGSP (SEQ ID NO:40), ITTDTST ( SEQ ID NO:42);
  • CDR3 having a structure selected from the group consisting of: AAAX 9 LECRTX 10 , AAALLECRVRSWPSDN (SEQ ID NO: 49), AAAVLECRAAEYVNS (SEQ ID NO: 54), DIRNVGGDY (SEQ ID NO: 57), SPITSIFKA (SEQ ID NO: 48), NVRNVERDY (SEQ ID NO: 50);
  • X 1 is selected from (i) amino acid residues G, R and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 2 is selected from (i) amino acid residues F, A, S, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 3 is selected from (i) amino acid residues T, Q and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 4 is selected from (i) amino acid residues D, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 5 is selected from (i) amino acid residue A and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 6 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 7 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 8 is selected from (i) amino acid residue T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 9 is selected from (i) amino acid residues T, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 10 is selected from (i) amino acid residues T, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 11 is selected from (i) amino acid residues V, I, L and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 12 is selected from (i) amino acid residues R, V, T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 13 is selected from (i) amino acid residues G, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 14 is selected from (i) amino acid residues D, A and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 15 is selected from (i) amino acid residues L, F and (ii) amino acid residues that are conservatively substituted relative to (i).
  • the Nanobody or antigen-binding fragment thereof comprises CDR1 as set forth in any one of SEQ ID NOs: 30-38; CDR2 as set forth in any one of SEQ ID NOs: 39-46; and , such as the CDR3 shown in any one of SEQ ID NOs: 47-59.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR2 which has the structure shown as IX6SSGGX7X8 (Formula II, SEQ ID NO:73) ;
  • CDR3 having a structure selected from the group consisting of: AAAX 9 LECRTX 10 , AAALLECRVRSWPSDN (SEQ ID NO: 49), AAAVLECRAAEYVNS (SEQ ID NO: 54);
  • X 1 is selected from (i) amino acid residues G, R and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 2 is selected from (i) amino acid residues F, A, S, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 3 is selected from (i) amino acid residues T, Q and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 4 is selected from (i) amino acid residues D, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 5 is selected from (i) amino acid residue A and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 6 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 7 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 8 is selected from (i) amino acid residue T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 9 is selected from (i) amino acid residues T, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 10 is selected from (i) amino acid residues T, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 11 is selected from (i) amino acid residues V, I, L and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 12 is selected from (i) amino acid residues R, V, T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 13 is selected from (i) amino acid residues G, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 14 is selected from (i) amino acid residues D, A and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 15 is selected from (i) amino acid residues L, F and (ii) amino acid residues that are conservatively substituted relative to (i).
  • the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in any one of SEQ ID NOs: 30, 32, 34-36, 38; such as SEQ ID NOs: 39, 41, 43 CDR2 shown in any one of -45; and, CDR3 shown in any one of SEQ ID NOs: 47, 49, 51-56, 58-59.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR2 which has the structure shown as IX6SSGGX7X8 (Formula II, SEQ ID NO:73) ;
  • CDR3 having a structure selected from the group consisting of: AAAX 9 LECRTX 10 , AAALLECRVRSWPSDN (SEQ ID NO: 49), AAAVLECRAAEYVNS (SEQ ID NO: 54);
  • X 1 is selected from (i) amino acid residues G, R and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 2 is selected from (i) amino acid residues F, E, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 3 is selected from (i) amino acid residue T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 4 is selected from (i) amino acid residue D and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 5 is selected from (i) amino acid residue A and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 6 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 7 is selected from (i) amino acid residues A, S and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 8 is selected from (i) amino acid residue T and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 9 is selected from (i) amino acid residues T, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 10 is selected from (i) amino acid residue V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 11 is selected from (i) amino acid residues V, I and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 12 is selected from (i) amino acid residues R, V and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 13 is selected from (i) amino acid residues G, E and (ii) amino acid residues that are conservatively substituted relative to (i);
  • X 14 is selected from (i) amino acid residue D and (ii) an amino acid residue that is a conservative substitution relative to (i);
  • X 15 is selected from (i) amino acid residues L, F and (ii) amino acid residues that are conservatively substituted relative to (i).
  • the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in any one of SEQ ID NOs: 30, 32, 34-36; such as SEQ ID NOs: 39, 41, 43-45 CDR2 shown in any one; and, CDR3 shown in any one of SEQ ID NOs: 47, 49, 51, 52, 54-56, 58.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 which has: a sequence as shown in SEQ ID NO:32, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:32 Sequence with substitution, deletion or addition of one or three amino acids);
  • CDR2 which has: a sequence as shown in SEQ ID NO:41, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2) compared with the sequence shown in SEQ ID NO:41 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:49, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:49 substitution, deletion or addition of one or three amino acids).
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO:32, CDR2 as shown in SEQ ID NO:41, CDR3 as shown in SEQ ID NO:49 .
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 which has: a sequence as shown in SEQ ID NO: 32 or 34, or one or several amino acid substitutions, deletions or additions compared to the sequence as shown in SEQ ID NO: 32 or 34 (e.g. Sequences with 1, 2 or 3 amino acid substitutions, deletions or additions);
  • CDR2 which has: a sequence as shown in SEQ ID NO:43, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2) compared to the sequence shown in SEQ ID NO:43 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:52, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:52 substitution, deletion or addition of one or three amino acids).
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • ( a ) CDR1 which has a structure shown as X 1 Amino acid residue F or E and (ii) are conservatively substituted amino acid residues relative to (i); X 3 is amino acid residue T; X 4 is amino acid residue D; X 5 is amino acid residue A;
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 as set forth in SEQ ID NO:32 or 34; CDR2 as set forth in SEQ ID NO:43; and, as set forth in SEQ ID Ns:52 CDR3 shown.
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 which has: a sequence as shown in SEQ ID NO:37, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:37 Sequence with substitution, deletion or addition of one or three amino acids);
  • CDR2 which has: a sequence as shown in SEQ ID NO:46, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:46 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:57, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2) compared with the sequence shown in SEQ ID NO:57 substitution, deletion or addition of one or three amino acids).
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO:37, CDR2 as shown in SEQ ID NO:46, CDR3 as shown in SEQ ID NO:57 .
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 which has: a sequence as shown in SEQ ID NO:31, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:31 Sequence with substitution, deletion or addition of one or three amino acids);
  • CDR2 which has: a sequence as shown in SEQ ID NO:40, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2) compared to the sequence shown in SEQ ID NO:40 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:48, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:48 substitution, deletion or addition of one or three amino acids).
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO:31, CDR2 as shown in SEQ ID NO:40, CDR3 as shown in SEQ ID NO:48 .
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 which has: the sequence shown in SEQ ID NO:33, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2) compared with the sequence shown in SEQ ID NO:33 Sequence with substitution, deletion or addition of one or three amino acids);
  • CDR2 which has: a sequence as shown in SEQ ID NO:42, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:42 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:50, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:50 substitution, deletion or addition of one or three amino acids).
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO:33, CDR2 as shown in SEQ ID NO:42, CDR3 as shown in SEQ ID NO:50 .
  • the Nanobody or antigen-binding fragment thereof comprises:
  • CDR1 as shown in SEQ ID NO:30; CDR2 as shown in SEQ ID NO:39; and CDR3 as shown in SEQ ID NO:47;
  • CDR1 as shown in SEQ ID NO:32; CDR2 as shown in SEQ ID NO:41; and, as CDR3 shown in SEQ ID NO:52;
  • CDR1 as shown in SEQ ID NO:32; CDR2 as shown in SEQ ID NO:41; and, CDR3 as shown in SEQ ID NO:58; or
  • CDR1 as shown in SEQ ID NO:38; CDR2 as shown in SEQ ID NO:41; and CDR3 as shown in SEQ ID NO:59.
  • the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from:
  • the substitutions are conservative substitutions.
  • the Nanobody or antigen-binding fragment thereof is humanized.
  • the Nanobody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene ), the heavy chain framework region optionally includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from Back mutation of human residues to camel residues.
  • a human immunoglobulin e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene
  • the heavy chain framework region optionally includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from Back mutation of human residues to camel residues.
  • the Nanobody or antigen-binding fragment thereof comprises an amino acid sequence selected from:
  • the substitutions are conservative substitutions.
  • the serum albumin is selected from human serum albumin (HSA), murine serum albumin (MSA), and/or cynomolgus serum albumin (CSA).
  • HSA human serum albumin
  • MSA murine serum albumin
  • CSA cynomolgus serum albumin
  • the binding of the Nanobody or antigen-binding fragment thereof to the serum albumin is non- pH dependent.
  • the Nanobody or antigen-binding fragment thereof is capable of specifically binding to the serum albumin in the range of pH 5-8 (e.g., pH 5.5-7.4).
  • the present application also provides a polypeptide construct that specifically binds serum albumin, comprising a Nanobody or an antigen-binding fragment thereof as described above, and an immunoglobulin Fc domain.
  • the Fc domain also called Fc region, refers to the part of the heavy chain constant region that contains CH2 and CH3.
  • the Fc domain includes hinge, CH2, and CH3.
  • the hinge mediates dimerization between the two Fc-containing polypeptides.
  • the Fc domain can be of any antibody heavy chain constant region isotype.
  • the Fc domain is IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain comprised by the polypeptide construct of the invention is a native Fc region, which contains an amino acid sequence consistent with the amino acid sequence of an Fc region found in nature.
  • the Fc domain may be a native sequence human IgG1 Fc region, a native sequence human IgG2 Fc region, a native sequence human IgG3 Fc region, or a native sequence human IgG4 Fc region. Native Fc regions can have effector functions.
  • effector functions include binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; response to cell surface receptors (e.g., Downregulation of B cell receptors); and B cell activation, etc.
  • Functional changes can be produced by replacing at least one amino acid residue in the native Fc region with a different residue or by chemical modification, e.g., altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function. (e.g. lower or boost).
  • the Fc domain comprised by the polypeptide construct of the invention may also be a variant Fc region, which may comprise one or more (e.g., 1-10, e.g., 1-10) compared to the native Fc region. 5) Amino acid mutation or chemical modification to change one or more of the following properties of the antibody of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. .
  • the Fc domain comprised by the polypeptide construct of the invention possesses ADCC activity. In certain embodiments, the Fc domain comprised by the polypeptide construct of the invention does not possess ADCC activity.
  • the immunoglobulin Fc domain is optionally linked to the N-terminus and/or C-terminus (eg, C-terminus) of the Nanobody or antigen-binding fragment thereof via a peptide linker.
  • the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain).
  • the immunoglobulin Fc domain comprises, or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, the sequence set forth in SEQ ID NO: 68. , a sequence that has at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or several amino acid substitutions compared thereto , missing or added (for example, 1, 2, Sequences with 3, 4 or 5 amino acid substitutions, deletions or additions).
  • the serum albumin is selected from human serum albumin (HSA), murine serum albumin (MSA), and/or cynomolgus serum albumin (CSA).
  • HSA human serum albumin
  • MSA murine serum albumin
  • CSA cynomolgus serum albumin
  • binding of the polypeptide construct to the serum albumin is pH independent.
  • the polypeptide construct is capable of specifically binding to the serum albumin in the range of pH 5-8 (e.g., pH 5.5-7.4).
  • the present application also provides a fusion protein comprising a Nanobody or an antigen-binding fragment thereof as described above or a polypeptide construct as described above, and an additional peptide domain.
  • the additional peptide domains are selected from polypeptide domains with therapeutic effects.
  • the additional polypeptide domain is optionally linked to the N-terminus and/or C-terminus of the Nanobody or antigen-binding fragment thereof or the polypeptide construct via a peptide linker.
  • the present application also provides an isolated nucleic acid molecule encoding a Nanobody or an antigen-binding fragment thereof as described above, a polypeptide construct as described above, or a fusion protein as described above.
  • the application also provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the present application also provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., small mouse cells, human cells, etc.).
  • the Nanobody or polypeptide construct or fusion protein of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology.
  • the DNA molecule encoding the Nanobody or polypeptide construct or fusion protein of the present invention is obtained through chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody or polypeptide construct or fusion protein of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact Nanobody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985) ). Alternatively, these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )). Book Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the present application also provides a method for preparing a Nanobody or an antigen-binding fragment thereof as described above, a polypeptide construct as described above, or a fusion protein as described above, which includes, under conditions allowing protein expression
  • the host cell as described above is cultured, and the Nanobody or antigen-binding fragment thereof or the polypeptide construct is recovered from the cultured host cell culture.
  • the present application also provides bispecific or multispecific antibodies comprising Nanobodies or antigen-binding fragments thereof as described above or polypeptide constructs as described above.
  • the bispecific or multispecific antibody specifically binds serum albumin and additionally specifically binds one or more other targets.
  • the bispecific or multispecific antibody further comprises at least one second antibody, or antigen-binding fragment thereof, having a second binding specificity for a second target.
  • the bispecific or multispecific antibody comprises:
  • Nanobodies or antigen-binding fragments thereof as described above or polypeptide constructs as described above for example, Nanobodies or antigen-binding fragments thereof containing the sequence shown in any one of SEQ ID NOs: 10, 4, 78 fragment or polypeptide construct) as the first antigen-specific binding domain;
  • a second antigen-specific binding domain (such as Fab) comprising a light chain as shown in SEQ ID NO:79 and a heavy chain as shown in SEQ ID NO:80;
  • a third antigen-specific binding domain (e.g., Nanobody VHH) as shown in SEQ ID NO:81.
  • the bispecific or multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises (e.g., from N-terminus to C-terminus): the light chain or heavy chain (such as VH and CH1) of the second antigen-specific binding domain, the third antigen-specific binding domain, the first antigen-specific binding domain; and, the third antigen-specific binding domain;
  • the two polypeptide chains comprise (e.g.
  • the heavy chain such as VH and CH1 or light chain of the second antigen-specific binding domain, the third antigen-specific binding domain; wherein , the first polypeptide chain and the second polypeptide chain pair to form the complete second antigen-specific binding domain.
  • the various domains in the first polypeptide chain and/or the second polypeptide chain are connected by a linker (e.g., a peptide linker comprising one or more glycines and/or one or more serines) .
  • the bispecific or multispecific antibody comprises:
  • the first peptide is present in a first peptide chain
  • the second peptide is present in a second peptide chain
  • the first peptide chain and the second peptide are Chains form complexes.
  • the present application also provides a conjugate comprising a Nanobody or an antigen-binding fragment thereof or a polypeptide construct as described above, and a conjugate constructed with the Nanobody or an antigen-binding fragment thereof or a polypeptide construct as described above.
  • Therapeutic agents for body junctions comprising a Nanobody or an antigen-binding fragment thereof or a polypeptide construct as described above, and a conjugate constructed with the Nanobody or an antigen-binding fragment thereof or a polypeptide construct as described above.
  • the therapeutic agent is selected from the group consisting of antineoplastic agents, such as cytotoxic agents, hormonal agents, biological response modifiers, additional antibodies, or antigen-binding fragments thereof.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a Nanobody or an antigen-binding fragment thereof, a polypeptide construct, a fusion protein, an isolated nucleic acid molecule, a vector, a host cell, a bispecific or a polypeptide as described above. Specific antibodies or conjugates.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • the pharmaceutical composition comprises a Nanobody or an antigen-binding fragment thereof, a polypeptide construct as described above, or a nucleic acid molecule, vector, or encoding the Nanobody or an antigen-binding fragment thereof or a polypeptide construct. host cell.
  • the pharmaceutical composition comprises a fusion protein as described above, or a nucleic acid molecule, vector or host cell encoding the fusion protein.
  • the pharmaceutical compositions comprise a bispecific or multispecific antibody as described above, or a nucleic acid molecule, vector or host cell encoding the bispecific or multispecific antibody.
  • the pharmaceutical composition comprises a conjugate as described above.
  • the application also provides Nanobodies or antigen-binding fragments thereof, polypeptide constructs, fusion proteins, isolated nucleic acid molecules, vectors, host cells, bispecific or multispecific antibodies or conjugates as described above.
  • the drug can directly or indirectly participate in FcRn-mediated serum albumin recycling in the subject.
  • the drug exhibits an extended in vivo half-life relative to a corresponding drug lacking the Nanobody or antigen-binding fragment thereof.
  • the drug is a proteinaceous drug.
  • the subject is a mammal, such as a human, a mouse, or a cynomolgus monkey.
  • the present application also provides a method for extending the in vivo half-life of a drug, which includes: connecting the Nanobody or antigen-binding fragment thereof or the polypeptide construct as described above to the drug.
  • the increase in in vivo half-life is relative to the in vivo half-life of the drug in the absence of the Nanobody or antigen-binding fragment thereof.
  • the drug is selected from macromolecular drugs (eg, peptide drugs).
  • the subject is a mammal, such as a human, a mouse, or a cynomolgus monkey.
  • Nanobodies has the meaning commonly understood by those skilled in the art and refers to an antibody consisting of a single monomeric variable antibody domain (eg, a single heavy chain variable region) Fragments, usually derived from the variable region of a heavy chain antibody such as a camelid antibody or a shark antibody.
  • a single monomeric variable antibody domain eg, a single heavy chain variable region
  • Fragments usually derived from the variable region of a heavy chain antibody such as a camelid antibody or a shark antibody.
  • Nanobodies are composed of 4 framework regions and 3 complementarity determining regions, with the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus so that they contain only part of FR1 and/or FR4, or lack one or both of those backbone regions, as long as they substantially retain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAb), and the two are used interchangeably.
  • the term "antigen-binding fragment" of a Nanobody refers to a polypeptide comprising a fragment of a Nanobody that retains the ability to specifically bind to the same antigen to which the Nanobody binds, and/or competes with the Nanobody. Specific binding of antigen, which is also called the "antigen-binding moiety". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology Or an antigen-binding fragment of an antibody of the invention is generated by enzymatic or chemical cleavage of a Nanobody of the invention.
  • the "antigen-binding fragment" of the Nanobody can be at the N-terminal or C-terminus compared to the full-length Nanobody.
  • the ends are truncated so that they contain only part of FRl and/or FR4, or lack one or both of those backbone regions, as long as they substantially retain antigen binding and specificity.
  • Antigen-binding fragments of a Nanobody can be obtained from a given Nanobody (eg, a Nanobody provided by the invention) using conventional techniques known to those skilled in the art (eg, recombinant DNA technology or enzymatic or chemical fragmentation methods) and Antigen-binding fragments of Nanobodies are screened for specificity in the same manner as for intact Nanobodies.
  • Nanobody includes not only intact Nanobodies, but also antigen-binding fragments of Nanobodies, unless the context clearly indicates otherwise.
  • CDR complementarity determining region
  • Nanobodies contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the term "Fc domain” or "Fc region” means a portion of the heavy chain constant region that includes CH2 and CH3.
  • the Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; effects on cell surface receptors Down-regulation of receptors (e.g., B-cell receptors); and B-cell activation, etc.
  • the Fc region includes hinge, CH2, and CH3. When the Fc region includes a hinge, the hinge mediates dimerization between the two Fc-containing polypeptides.
  • the Fc region can be of any antibody heavy chain constant region isotype, such as IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain may include either a native Fc region or a variant Fc region.
  • a native Fc region includes an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature, e.g., a native sequence.
  • a human Fc region includes a native sequence. Human IgG1 Fc region (non-A and A allotype); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region, and naturally occurring variants thereof.
  • a variant Fc region includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification.
  • a variant Fc region may possess altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to the native Fc region. .
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology to that of a human antibody.
  • all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Immunoglobulins receptor antibodies.
  • the CDR regions of the humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and/or constant region) are derived from a human source.
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody may be a camelid antibody with desired properties (eg, antigen specificity, affinity, reactivity, etc.).
  • the CDR regions of the immunized animal can be inserted into human framework sequences using methods known in the art.
  • humanized antibodies may refer to humanized VHHs, ie VHHs in which one or more backbone regions have been substantially replaced by human backbone regions. In some cases, certain framework regions (FRs) of human immunoglobulins are replaced by corresponding non-human residues. Additionally, a humanized VHH may contain residues that are not found in either the original VHH or the human backbone sequence, but are included to further improve and optimize the performance of the VHH or VHH-containing polypeptide.
  • the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence.
  • Determination of percent identity between two sequences can also be accomplished using mathematical algorithms.
  • One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877.
  • Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can determine the interaction The equilibrium dissociation constant (K D ) of the action is expressed.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • K D , kon and kdis values can be measured by any valid method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to,
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine Acid, proline, Phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan acid, histidine) amino acids.
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% (w/v) NaCl
  • dextrose solutions eg 5% glucose
  • surfactant containing solutions eg 0.01% polysorbate 20
  • pH buffer solutions eg phosphate buffer solution
  • Ringer's solution any combination thereof.
  • the term "in vivo half-life” has a meaning well known to those skilled in the art and may generally be defined as a 50% reduction in the serum concentration of a molecule in the body (e.g. due to degradation of the ligand by natural mechanisms and/or ligand the time required for removal or chelation). Methods for determining half-life in vivo are well known to those skilled in the art and can be determined, for example, by pharmacokinetic analysis.
  • the present invention provides Nanobodies with high binding activity to serum albumin, which have cross-reactivity with human, monkey and/or mouse serum albumin.
  • the binding of the Nanobody of the present invention to serum albumin is pH-independent, and it can effectively participate in FcRn-mediated serum albumin circulation, thereby extending the in vivo half-life of the Nanobody. Therefore, the half-life of the drug molecule in the body can be extended by coupling (eg, fusion) the Nanobody of the present application to a drug molecule (eg, a polypeptide drug).
  • the nanobody also has the characteristics of small molecular weight and good stability. Compared with traditional common normal antibodies in drug development and other aspects, it has good tissue infiltration, flexible administration methods, high degree of humanization, and easy recombinant protein. Construction and many other advantages.
  • Figure 1 shows the concentration changes of Bi-340-352, Bi-340-353, Bi-340-354 and Bi-340-356 in mouse serum over time.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out as described in FMAusubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer.
  • restriction enzymes was in accordance with the conditions recommended by the product manufacturer.
  • HSA human serum albumin
  • Freund's adjuvant in equal volumes, and immunize 2 alpacas once a week for a total of 4 times to stimulate B cells to express the antigen.
  • Specific Nanobodies After four rounds of immunization, 50 ml of alpaca peripheral blood was extracted, and lymphocytes were separated using lymphocyte separation solution. Total RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen). The total cDNA of alpaca lymphocytes was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).
  • the first round PCR product was subjected to agarose gel electrophoresis, and the 750 bp fragment was recovered by cutting the gel for the second round of VHH sequence amplification.
  • the primers for the second round of PCR amplification are shown in Table 2:
  • the target fragment was recovered using a PCR purification kit (purchased from QIAGEN).
  • the linearized yeast display vector (artificially synthesized) and the third round of PCR products were mixed and electrotransformed into Saccharomyces cerevisiae (purchased from ATCC).
  • Anti-HSA nanobody libraries from two animals were constructed and the library volumes were measured.
  • the library sizes were respectively are 2.1 ⁇ 10 7 and 3.2 ⁇ 10 7 .
  • HSA-H5220 Take an appropriate volume of double-distilled water to dissolve the HSA protein (purchased from AcroBiosystems, product number: HSA-H5220). According to the product instructions of the biotin labeling kit (purchased from Thermo), dissolve the biotin and mix it with the protein solution, and incubate at 4°C. 2 hours. Use a desalting column (purchased from Thermo) to remove excess biotin. The desalting column pretreatment and sample collection operations are performed according to the product instructions.
  • MACS enriches yeasts that specifically bind to HSA
  • SD-CAA amplification medium 1L SD-CAA amplification medium contains 6.7g YNB, 5g tyrosine, 13.62g Na 2 HPO 4 ⁇ 12H 2 O, 7.44g NaH 2 PO 4 and 2% glucose), incubate overnight at 30°C, 225 rpm. Take an appropriate amount of yeast cells, centrifuge at 3000 rpm ⁇ 5 min (the parameters for the following centrifugation operations are the same as here), remove the culture medium, resuspend the yeast cells in SD-CAA induction medium, and induce overnight. Determine the library concentration after induction, take an appropriate amount of yeast cells, and centrifuge to remove the culture medium. Resuspend the yeast cells in 50 ml of PBS and centrifuge to remove the supernatant. Resuspend yeast cells in 10 ml PBS.
  • MACS-enriched yeast cells were inoculated into SD-CAA expansion medium. Incubate in shake flask at 30°C, 225rpm overnight. Resuspend yeast cells in SD-CAA induction medium and induce overnight. Add anti-c-Myc mouse antibody (purchased from Thermo, Cat. No.: MA1-980) and 100 nM biotin-labeled HSA antigen, and incubate for 10 minutes. Add PBS to wash the yeast 3 times, then add goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen, Catalog No.: A32723) and streptavidin APC conjugate fluorescent antibody (purchased from Invitrogen, Catalog No. : SA1005), incubate for 15 minutes. Add PBS to resuspend the cells, and use the BD FACSAria II instrument to sort the yeast to obtain yeast with higher binding ability to HSA antigen.
  • anti-c-Myc mouse antibody purchased from Ther
  • the yeast liquid with high binding ability to HSA antigen obtained through MACS and FACS enrichment was cultured overnight in SD-CAA amplification medium at 30°C and 225 rpm.
  • yeast plasmid extraction kit purchased from Tiangen
  • Top10 competent cells purchased from Tiangen
  • Single clones were selected and sequenced to obtain the VHH amino acid sequence, and the CDR region sequence was determined according to the IMGT numbering system.
  • the sequence information of the obtained monoclonal Nanobodies is shown in Table 4.
  • VHH gene sequence Connect the VHH gene sequence to the Fc segment of human IgG1 (LALA mutation) (the amino acid sequence is shown in SEQ ID NO: 68), and use homologous recombinase (purchased from Vazyme, Cat. No.: c115-02) to construct a combination with EcoR I/Not In the linearized pCDNA3.1 vector digested by I double enzyme, the process follows the product instructions.
  • the homologous recombination products were transformed into Top10 competent cells, spread on ampicillin-resistant plates, cultured at 37°C overnight, single clones were picked for sequencing, and plasmids were extracted.
  • sequence of SEQ ID NO:68 is as follows:
  • the extracted plasmid obtained in the above steps was transferred into Expi-CHO cells.
  • the transfection method was in accordance with the product instructions. After 5 days of cell culture, the supernatant was collected using protein A magnetic beads (purchased from Thermo).
  • the target protein was purified by sorting method from GenScript. Resuspend the magnetic beads in an appropriate volume (1-4 times the volume of the magnetic beads) of binding buffer (PBS+0.1% Tween 20, pH 7.4), add it to the sample to be purified, and incubate at room temperature for 1 hour, shaking gently during the period.
  • the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer.
  • Add elution buffer (0.1M sodium citrate, pH 3.2) at 3-5 times the volume of magnetic beads and shake at room temperature for 5-10 minutes. Place it back on the magnetic stand. Collect the elution buffer and transfer it to the neutralizer. Buffer (1M Tris, pH 8.54) was mixed in a collection tube to obtain the target protein.
  • This example further detects the cross-reactivity of candidate molecules with serum albumin of different species, including mouse serum albumin (Mouse Serum Albumin, MSA, purchased from AcroBiosystems, product number: MSA-M52H8), cynomolgus monkey serum albumin (Cynomolgus Serum Albumin, CSA, purchased from AcroBiosystems, product number: CSA-C52H4).
  • MSA mouse Serum Albumin
  • CSA cynomolgus monkey serum albumin
  • Affinity data are shown in Table 6.
  • HSA-59 In order to reduce the immunogenicity of monoclonal antibodies in the human body, three antibodies, HSA-59, HSA-M-27 and HSA-PH-8, were humanized.
  • the humanization method adopts the VHH humanized universal framework transplantation method, and is also based on the literature (Vincke, C., et al., General strategy for humanizing camel single domain antibodies and identifying universal humanized nanobody scaffolds. J Biol Chem 284 (5): 3273-3284) The reported method is to mutate some amino acids of the antibody framework 2 (framework2).
  • the sequence information of humanized antibodies is shown in Table 7.
  • the protein construction, expression and purification methods were the same as in Example 2, and the purity of the protein was obtained by HPLC detection.
  • the purity detection results are shown in Table 9.
  • the HPLC method is as follows, mobile phase: 150mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m).
  • This example tests the binding affinity of the purified anti-HSA humanized sample to HSA.
  • the experimental method is the same as in Example 3.
  • the experimental results are shown in Table 10.
  • the results show that the anti-HSA humanized sample has good binding activity to HSA protein. .
  • This embodiment simultaneously detects the binding affinity of the purified anti-HSA humanized sample to MSA/CSA.
  • the experimental method is the same as in Example 3.
  • the experimental results are shown in Table 11.
  • HSA The recycling mechanism of HSA is similar to that of IgG. It is internalized into vascular endothelial cells through pinocytosis. The pH of the endosome is about 6.0, which promotes the binding of membrane-bound FcRn to HSA. The endosomal complex is then recycled into the blood. Under pH 7.4 conditions, membrane-bound FcRn rapidly dissociates from HSA, and HSA is recycled.
  • This example detects the affinity between the purified anti-HSA humanized sample and HSA under different pH conditions.
  • the experimental method is the same as in Example 3.
  • the experimental results are shown in Table 12.
  • the results show that the anti-HSA humanized sample and HSA protein are in It has good binding activity under different pH conditions.
  • Multispecific antibodies Bi-340-352, Bi-340-353 and Bi-340-354 are each independently composed of peptide chain #1 and peptide chain #2, and contain three antigen-binding domains, respectively.
  • the structure and sequence of the multispecific antibody are shown in Tables 13-1 and 13-2, in which the domains in peptide chain #1 and peptide chain #2 are connected through a linker containing GGGGS.
  • the gene sequences encoding peptide chain #1 and peptide chain #2 of Bi-340-352, Bi-340-353, Bi-340-354 and Bi-340-356 respectively were synthesized by Jinweizhi Company.
  • the coding gene sequences were constructed into the pCDNA3.1 vector linearized by EcoR I/Not I double enzyme digestion using homologous recombinase (purchased from Vazyme), and the construction process was in accordance with the product instructions.
  • the homologous recombination products of peptide chain #1 and peptide chain #2 of the multispecific antibodies were co-transformed into Top10 competent cells, coated on ampicillin-resistant plates, cultured at 37°C overnight, and single clones were picked for sequencing. .
  • the plasmid was transferred into Expi-CHO cells using the ExpiCHO TM expression system kit (purchased from Thermo).
  • the transfection method was in accordance with the product instructions.
  • the supernatant was collected and purified using a KappaSelect (GE) affinity chromatography column.
  • the specific method is as follows: Use a syringe to filter the sample through a 0.2um sterile syringe filter PES. Equilibrate the chromatography column with 5 times the column volume of equilibrium buffer (20mM PB+0.15M NaCl, pH 7.4) until the conductivity and pH of the effluent remain unchanged. Load the sample at a flow rate of 0.5ml/minute.
  • HPLC HPLC to detect protein purity.
  • the HPLC method is as follows, mobile phase: 150mM Na2HPO4 ⁇ 12H2O, pH7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m).
  • the injection dose for mice is 10mg/Kg.
  • Blood collection time points venous blood collection at 3 minutes, 30 minutes, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, and 168 hours after administration.
  • Whole blood samples were placed at 2-8°C for 30 minutes, centrifuged at 12,000 rpm for 5 minutes to collect serum.
  • the resulting serum was centrifuged at 2-8°C, 12,000 rpm for 5 minutes, and stored at -80°C.
  • the molecular weight of free antibodies in the serum was detected by ELISA. The results are shown in Figure 1.
  • mice The results show that the half-life of molecules with anti-HSA nanobodies in mice is approximately 23.7 hours (Bi-340-352), 24.2 hours (Bi-340-353) and 10.9 hours (Bi- 340-354); while the molecule with non-HSA-related Nanobodies (Bi-340-356) has a half-life of approximately 1.4 hours. Knot The results show that anti-HSA nanobodies can significantly increase the in vivo half-life of the molecule.

Abstract

本发明涉及特异性结合血清白蛋白的纳米抗体或其抗原结合片段，含有所述纳米抗体或其抗原结合片段的衍生物，编码所述抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段，或所述衍生物的治疗用途。

Description

抗血清白蛋白纳米抗体及其衍生物 技术领域
本发明涉及特异性结合血清白蛋白的纳米抗体或其抗原结合片段，含有所述纳米抗体或其抗原结合片段的衍生物，编码所述抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段，或所述衍生物的治疗用途。
背景技术
随着技术的发展，抗体药物早已成为独立于化药，细胞治疗等之外的重要疾病疗法。在抗体药物研发中，药代动力学(pharmacokinetics,PK)是描述机体对抗体作用的重要领域，包括吸收、分布及清除等，其中半衰期就是PK的一个重要参数。通常，较长的体内半衰期可以提高抗体的药效，但也可能增加毒性，而对某些靶点的紧密结合可以导致抗体被快速内吞至胞内降解，缩短体内的半衰期。
延长大分子药物体内的半衰期可以在提高药物本身作用时间的基础上减少给药频率，降低病人的医疗负担。目前提高药物长效化的设计包括聚乙二醇(PEG)修饰、融合转铁蛋白等，但这些化学方法存在一些弊端，比如PEG可能会屏蔽或阻挡抗体的抗原结合位点，降低抗体或治疗蛋白的药效，甚至可能导致抗体活性丧失。
人血清白蛋白(HSA)是人体血浆中含量最丰富的蛋白质，约占血清蛋白的一半，可溶于水并以单体形式存在。血清白蛋白在体内的半衰期大约为21天，其由585个氨基酸组成，分子量约66.5KD。人血清白蛋白在体内稳定、安全无毒、免疫原性低，是理想的药物载体。对于抗体药物，可以通过人血清白蛋白修饰从而提高药物半衰期，以提高患者顺应性。
HSA的回收机制和IgG类似，都是通过FcRn介导完成的。HSA和IgG通过胞饮作用内化到血管内皮细胞，内体的pH为6.0左右，促进膜结合的FcRn与HSA结合，之后经过再循环至内皮细胞顶端或底部，通过胞吐作用在血液pH7.4条件下，膜结合的FcRn与HSA快速解离进行再循环。
发明内容
本申请的发明人经过大量的研究，筛选获得了系列抗血清白蛋白的纳米抗体，所述纳米抗体与血清白蛋白具有高结合活性，并且具备与人、猴和/或小鼠血清白蛋白的交叉反应活性。特别地，本发明的纳米抗体与血清白蛋白的结合是非pH依赖性的，其能有效参与FcRn介导的血清白蛋白循环，从而延长所述纳米抗体的体内半衰期。因此，可通过 将本申请的纳米抗体与药物分子(例如，多肽药物)进行偶联(例如，融合)，从而延长所述药物分子在体内的半衰期。
此外，所述纳米抗体还具有分子量小，稳定性好等特点，在药物研发等方面相比于传统常见的正常抗体有着组织浸润性好、给药方式灵活、人源化程度高、容易重组蛋白改造等诸多优势。
基于此，本申请还提供了含有所述纳米抗体或其抗原结合片段的组合物，编码所述纳米抗体或其抗原结合片段的核酸及包含其的宿主细胞，以及相关用途。
纳米抗体及其抗原结合片段
因此，在一方面，本申请提供了能够特异性结合血清白蛋白的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有选自以下的结构：X1X2X3LX4YYX5(式I，SEQ ID NO:72)，SGFTLDYYA(SEQ ID NO:30)，GSIWGIYH(SEQ ID NO:37)，GFTFSIYS(SEQ ID NO:31)，GSIFTFYR(SEQ ID NO:33)；
(b)CDR2，其具有选自以下的结构：IX6SSGGX7X8(式II，SEQ ID NO:73)，ITVDGST(SEQ ID NO:46)，ISSGGSP(SEQ ID NO:40)，ITTDTST(SEQ ID NO:42)；
(c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)，DIRNVGGDY(SEQ ID NO:57)，SPITSIFKA(SEQ ID NO:48)，NVRNVERDY(SEQ ID NO:50)；
其中，
X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
X2选自(i)氨基酸残基F，A，S，E和(ii)相对于(i)是保守置换的氨基酸残基；
X3选自(i)氨基酸残基T，Q和(ii)相对于(i)是保守置换的氨基酸残基；
X4选自(i)氨基酸残基D，E和(ii)相对于(i)是保守置换的氨基酸残基；
X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
X10选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
X11选自(i)氨基酸残基V，I，L和(ii)相对于(i)是保守置换的氨基酸残基；
X12选自(i)氨基酸残基R，V，T和(ii)相对于(i)是保守置换的氨基酸残基；
X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
X14选自(i)氨基酸残基D，A和(ii)相对于(i)是保守置换的氨基酸残基；
X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含如SEQ ID NOs:30-38任一项所示的CDR1；如SEQ ID NOs:39-46任一项所示的CDR2；以及，如SEQ ID NOs:47-59任一项所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)或SGFTLDYYA(SEQ ID NO:30)所示的结构；
(b)CDR2，其具有如IX6SSGGX7X8(式II，SEQ ID NO:73)所示的结构；
(c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)；
其中，
X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
X2选自(i)氨基酸残基F，A，S，E和(ii)相对于(i)是保守置换的氨基酸残基；
X3选自(i)氨基酸残基T，Q和(ii)相对于(i)是保守置换的氨基酸残基；
X4选自(i)氨基酸残基D，E和(ii)相对于(i)是保守置换的氨基酸残基；
X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
X10选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
X11选自(i)氨基酸残基V，I，L和(ii)相对于(i)是保守置换的氨基酸残基；
X12选自(i)氨基酸残基R，V，T和(ii)相对于(i)是保守置换的氨基酸残基；
X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
X14选自(i)氨基酸残基D，A和(ii)相对于(i)是保守置换的氨基酸残基；
X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基。
在某些实施方案中，X1选自氨基酸残基G或R；X2选自氨基酸残基F，A，S或E；X3选自氨基酸残基T或Q；X4选自氨基酸残基D或E；X5为氨基酸残基A；X6选自氨基酸残基A或S；X7选自氨基酸残基A或S；X8为氨基酸残基T；X9选自氨基酸残基T或 V；X10选自氨基酸残基T或V；X11选自氨基酸残基V，I或L；X12选自氨基酸残基R，V或T；X13选自氨基酸残基G或E；X14选自氨基酸残基D或A；X15选自氨基酸残基L或F。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NOs:30、32、34-36、38任一项所示的CDR1；如SEQ ID NOs:39、41、43-45任一项所示的CDR2；以及，如SEQ ID NOs:47、49、51-56、58-59任一项所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)或SGFTLDYYA(SEQ ID NO:30)所示的结构；
(b)CDR2，其具有如IX6SSGGX7X8(式II，SEQ ID NO:73)所示的结构；
(c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)；
其中，
X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
X2选自(i)氨基酸残基F，E，S和(ii)相对于(i)是保守置换的氨基酸残基；
X3选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
X4选自(i)氨基酸残基D和(ii)相对于(i)是保守置换的氨基酸残基；
X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
X10选自(i)氨基酸残基V和(ii)相对于(i)是保守置换的氨基酸残基；
X11选自(i)氨基酸残基V，I和(ii)相对于(i)是保守置换的氨基酸残基；
X12选自(i)氨基酸残基R，V和(ii)相对于(i)是保守置换的氨基酸残基；
X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
X14选自(i)氨基酸残基D和(ii)相对于(i)是保守置换的氨基酸残基；
X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基。
在某些实施方案中，X1选自氨基酸残基G或R；X2选自氨基酸残基F，S或E；X3为氨基酸残基T；X4为氨基酸残基D；X5为氨基酸残基A；X6选自氨基酸残基A或S；X7选自氨基酸残基A或S；X8为氨基酸残基T；X9选自氨基酸残基T或V；X10为氨基酸残基V；X11选自氨基酸残基V或I；X12选自氨基酸残基R或V；X13选自氨基酸残基G 或E；X14为氨基酸残基D；X15选自氨基酸残基L或F。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NOs:30、32、34-36任一项所示的CDR1；如SEQ ID NOs:39、41、43-45任一项所示的CDR2；以及，如SEQ ID NOs:47、49、51、52、54-56、58任一项所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有：如SEQ ID NO:32所示的序列，或与SEQ ID NO:32所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:41所示的序列，或与SEQ ID NO:41所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:49所示的序列，或与SEQ ID NO:49所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:32所示的CDR1、如SEQ ID NO:41所示的CDR2、如SEQ ID NO:49所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有：如SEQ ID NO:32或34所示的序列，或与SEQ ID NO:32或34所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:43所示的序列，或与SEQ ID NO:43所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:52所示的序列，或与SEQ ID NO:52所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)所示的结构，其中，X1为氨基酸残基G；X2选自(i)氨基酸残基F或E和(ii)相对于(i)是保守置换的氨基酸残基；X3为氨基酸残基T；X4为氨基酸残基D；X5为氨基酸残基A；
(b)CDR2，其具有如IASSGGST(SEQ ID NO:43)所示的结构；
(c)CDR3，其具有如AAAVLECRTVVRGYDY(SEQ ID NO:52)所示的结构。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:32或34所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID Ns:52所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有：如SEQ ID NO:37所示的序列，或与SEQ ID NO:37所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:46所示的序列，或与SEQ ID NO:46所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:57所示的序列，或与SEQ ID NO:57所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:37所示的CDR1、如SEQ ID NO:46所示的CDR2、如SEQ ID NO:57所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有：如SEQ ID NO:31所示的序列，或与SEQ ID NO:31所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:40所示的序列，或与SEQ ID NO:40所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:48所示的序列，或与SEQ ID NO:48所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:31所示的CDR1、如SEQ ID NO:40所示的CDR2、如SEQ ID NO:48所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(a)CDR1，其具有：如SEQ ID NO:33所示的序列，或与SEQ ID NO:33所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:42所示的序列，或与SEQ ID NO:42所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:50所示的序列，或与SEQ ID NO:50所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:33所示的CDR1、如SEQ ID NO:42所示的CDR2、如SEQ ID NO:50所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含：
(1)如SEQ ID NO:30所示的CDR1；如SEQ ID NO:39所示的CDR2；以及，如SEQ ID NO:47所示的CDR3；
(2)如SEQ ID NO:31所示的CDR1；如SEQ ID NO:40所示的CDR2；以及，如SEQ ID NO:48所示的CDR3；
(3)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:49所示的CDR3；
(4)如SEQ ID NO:33所示的CDR1；如SEQ ID NO:42所示的CDR2；以及，如SEQ ID NO:50所示的CDR3；
(5)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:51所示的CDR3；
(6)如SEQ ID NO:32或34所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:52所示的CDR3；
(7)如SEQ ID NO:35所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:52所示的CDR3；
(8)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:53所示的CDR3；
(9)如SEQ ID NO:36所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:54所示的CDR3；
(10)如SEQ ID NO:35所示的CDR1；如SEQ ID NO:44所示的CDR2；以及，如SEQ ID NO:55所示的CDR3；
(11)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:45所示的CDR2；以及，如SEQ ID NO:56所示的CDR3；
(12)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如 SEQ ID NO:52所示的CDR3；
(13)如SEQ ID NO:37所示的CDR1；如SEQ ID NO:46所示的CDR2；以及，如SEQ ID NO:57所示的CDR3；
(14)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:58所示的CDR3；或
(15)如SEQ ID NO:38所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:59所示的CDR3。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
(i)如SEQ ID NOs:1-3、7-9、14-24、28-29任一项所示的序列；
(ii)与SEQ ID NOs:1-3、7-9、14-24、28-29任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NOs:1-3、7-9、14-24、28-29任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述纳米抗体或其抗原结合片段是人源化的。
在某些实施方案中，所述纳米抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，所述重链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至驼源残基的回复突变。
在某些实施方案中，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
(i)如SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列；
(ii)与SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述血清白蛋白选自人血清白蛋白(HSA)，鼠血清白蛋白(MSA)和/或食蟹猴血清白蛋白(CSA)。
在某些实施方案中，所述纳米抗体或其抗原结合片段与所述血清白蛋白的结合是非 pH依赖性的。
在某些实施方案中，所述纳米抗体或其抗原结合片段能与所述血清白蛋白在pH 5-8(例如pH 5.5-7.4)的范围内特异性结合。
多肽构建体
在另一方面，本申请还提供了特异性结合血清白蛋白的多肽构建体，其包含如上所述的纳米抗体或其抗原结合片段，以及免疫球蛋白Fc结构域。
在本文中，所述Fc结构域也称为Fc区，是指包含CH2和CH3的重链恒定区的一部分。在一些实施方案中，Fc结构域包含铰链、CH2和CH3。当Fc结构域包含铰链时，铰链调节两个含Fc的多肽之间的二聚作用。Fc结构域可为任何抗体重链恒定区同型。在一些实施方案中，Fc结构域是IgG1、IgG2、IgG3或IgG4。
在某些实施方案中，本发明多肽构建体所包含的Fc结构域是天然Fc区，其包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列。例如，所述Fc结构域可以是天然序列人类IgG1Fc区、天然序列人类IgG2Fc区、天然序列人类IgG3Fc区或天然序列人类IgG4Fc区。天然Fc区可具有效应子功能。示例性“效应子功能”包括与Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。可以通过将天然Fc区中的至少一个氨基酸残基替换为不同残基或化学修饰，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低或增强)。
因此，在某些实施方案中，本发明多肽构建体所包含的Fc结构域也可以是变异Fc区，其与天然Fc区相比可以包含一个或多个(例如1-10个，例如1-5个)氨基酸突变或化学修饰以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。
在某些实施方案中，本发明多肽构建体所包含的Fc结构域具备ADCC活性。在某些实施方案中，本发明多肽构建体所包含的Fc结构域不具备ADCC活性。
在某些实施方案中，所述免疫球蛋白Fc结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段的N端和/或C端(例如C端)。
在某些实施方案中，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域)。
在某些实施方案中，所述免疫球蛋白Fc结构域包含SEQ ID NO：68所示的序列，或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、 3个、4个或5个氨基酸置换、缺失或添加)的序列。
在某些实施方案中，所述血清白蛋白选自人血清白蛋白(HSA)，鼠血清白蛋白(MSA)和/或食蟹猴血清白蛋白(CSA)。
在某些实施方案中，所述多肽构建体与所述血清白蛋白的结合是非pH依赖性的。
在某些实施方案中，所述多肽构建体能与所述血清白蛋白在pH 5-8(例如pH 5.5-7.4)的范围内特异性结合。
融合蛋白
在另一方面，本申请还提供了融合蛋白，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，以及，另外的肽结构域。
在某些实施方案中，所述另外的肽结构域选自具有治疗效应的多肽结构域。
在某些实施方案中，所述另外的多肽结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段或所述多肽构建体的N端和/或C端。
分离的核酸分子
在另一方面，本申请还提供了分离的核酸分子，其编码如上所述的纳米抗体或其抗原结合片段，如上所述的多肽构建体，或如上所述的融合蛋白。
载体
在另一方面，本申请还提供了载体，其包含如上所述的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
宿主细胞
在另一方面，本申请还提供了宿主细胞，其包含如上所述的核酸分子或如上所述的载体。此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
制备方法
本发明的纳米抗体或多肽构建体或融合蛋白可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明纳米抗体或多肽构建体或融合蛋白的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体或多肽构建体或融合蛋白。
本发明的抗原结合片段可以通过水解完整的纳米抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。本 领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
在另一方面，本申请还提供了制备如上所述的纳米抗体或其抗原结合片段，如上所述的多肽构建体，或如上所述的融合蛋白的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述纳米抗体或其抗原结合片段或所述多肽构建体。
双特异性或多特异性抗体
在另一方面，本申请还提供了双特异性或多特异性抗体，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体。
在某些实施方案中，所述双特异性或多特异性抗体特异性结合血清白蛋白，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述双特异性或多特异性抗体还包含至少一种具有针对第二靶标的第二结合特异性的第二抗体或其抗原结合片段。
在某些实施方案中，所述双特异性或多特异性抗体包含：
(i)如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体(例如，含有如SEQ ID NOs:10、4、78任一项所示的序列的纳米抗体或其抗原结合片段或多肽构建体)作为第一抗原特异性结合结构域；
(ii)包含如SEQ ID NO:79所示的轻链和如SEQ ID NO:80所示的重链的第二抗原特异性结合结构域(如Fab)；
以及，
(iii)如SEQ ID NO:81所示的第三抗原特异性结合结构域(例如，纳米抗体VHH)。
在某些实施方案中，所述双特异性或多特异性抗体包含第一多肽链和第二多肽链，其中，所述第一多肽链包含(例如从N端至C端)：所述第二抗原特异性结合结构域的轻链或重链(如VH和CH1)，所述第三抗原特异性结合结构域，所述第一抗原特异性结合结构域；并且，所述第二多肽链包含(例如从N端至C端)：所述第二抗原特异性结合结构域的重链(如VH和CH1)或轻链，所述第三抗原特异性结合结构域；其中，所述第一多肽链与第二多肽链配对形成完整的所述第二抗原特异性结合结构域。在某些实施方案中，所述第一多肽链和/或第二多肽链中的各个结构域通过接头(例如包含一个或多个甘氨酸和/或一个或多个丝氨酸的肽接头)连接。
在某些实施方案中，所述双特异性或多特异性抗体包含:
(1)序列如SEQ ID NO:69所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽；
(2)序列如SEQ ID NO:70所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽；
或者,
(3)序列如SEQ ID NO:71所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽。
在某些实施方案中，所述第一肽存在于第一肽链，所述第二肽存在于第二肽链；在某些实施方案中，所述第一肽链与所述第二肽链形成复合物。
缀合物
在另一方面，本申请还提供了缀合物，其包含如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的治疗剂。
在某些实施方案中，所述治疗剂选自抗肿瘤药物，例如细胞毒剂、激素类药物、生物反应调节剂、另外的抗体或其抗原结合片段。
药物组合物
在另一方面，本申请还提供了药物组合物，其包含如上所述的纳米抗体或其抗原结合片段、多肽构建体、融合蛋白、分离的核酸分子、载体、宿主细胞、双特异性或多特异性抗体或缀合物。
在某些实施方案中，所述药物组合物还包含药学上可接受的载体和/或赋形剂。
在某些示例性实施方案中，所述药学上可接受的载体和/或赋形剂包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
在某些实施方案中，所述药物组合物包含如上所述的纳米抗体或其抗原结合片段、多肽构建体、或编码所述纳米抗体或其抗原结合片段或多肽构建体的核酸分子、载体或宿主细胞。
在某些实施方案中，所述药物组合物包含如上所述的融合蛋白、或编码所述融合蛋白的核酸分子、载体或宿主细胞。
在某些实施方案中，所述药物组合物包含如上所述的双特异性或多特异性抗体、或编码所述双特异性或多特异性抗体的核酸分子、载体或宿主细胞。
在某些实施方案中，所述药物组合物包含如上所述的缀合物。
用途
在另一方面，本申请还提供了如上所述的纳米抗体或其抗原结合片段、多肽构建体、融合蛋白、分离的核酸分子、载体、宿主细胞、双特异性或多特异性抗体或缀合物、或药物组合物用于制备药物的用途。
在某些实施方案中，所述药物在受试者体内能直接或间接参与FcRn介导的血清白蛋白循环。
在某些实施方案中，所述药物显示相对于缺少所述纳米抗体或其抗原结合片段的相应的药物延长的体内半衰期。
在某些实施方案中，所述药物是蛋白质类药物。
在某些实施方案中，所述受试者为哺乳动物，例如人，小鼠或食蟹猴。
延长药物的体内半衰期的方法
在另一方面，本申请还提供了一种延长药物的体内半衰期的方法，其包括：将如上所述的纳米抗体或其抗原结合片段或如上所述的多肽构建体与所述药物进行连接。
在某些实施方案中，所述体内半衰期的延长是相对于缺少所述纳米抗体或其抗原结合片段时所述药物的体内半衰期。
在某些实施方案中，所述药物选自大分子药物(例如多肽类药物)。
在某些实施方案中，所述受试者为哺乳动物，例如人，小鼠或食蟹猴。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文中所使用的，术语“纳米抗体(nanobody)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，通常来源于重链抗体(例如骆驼科动物抗体或鲨鱼抗体)的可变区。典型地，纳米抗体由4个构架区和3个互补性决定区组成，具有FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。纳米抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。纳米抗体也称为单域抗体(single-domain antibody,sdAb)，两者可互换使用。
如本文中所使用的，术语纳米抗体的“抗原结合片段”是指包含纳米抗体的片段的多肽，其保持特异性结合纳米抗体所结合的相同抗原的能力，和/或与纳米抗体竞争对 抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过本发明纳米抗体的酶促或化学断裂产生本发明抗体的抗原结合片段。在一些实施方案中，所述纳米抗体的“抗原结合片段”与全长纳米抗体相比可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的纳米抗体(例如本发明提供的纳米抗体)获得纳米抗体的抗原结合片段，并且以与用于完整纳米抗体的方式相同的方式就特异性筛选纳米抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“纳米抗体”时，其不仅包括完整纳米抗体，而且包括纳米抗体的抗原结合片段。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在纳米抗体中含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia & Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的纳米抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。在本文中，纳米抗体的CDR优选地通过IMGT编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“Fc结构域”或“Fc区”意指，包含CH2和CH3的重链恒定区的一部分。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。由Fc区介导的“效应子功能”包括Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。在一些实施方案中，Fc区包含铰链、CH2和CH3。当Fc区包含铰链时，铰链调节两个含Fc的多肽之间的二聚作用。Fc区可为任何抗体重链恒定区同型，例如IgG1、IgG2、IgG3或IgG4。
Fc结构域既可以包括天然Fc区，也可以包括变异Fc区。天然Fc区包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列，例如天然序列人类Fc区包括天然序列 人类IgG1Fc区(非A和A同种异型)；天然序列人类IgG2Fc区；天然序列人类IgG3Fc区；及天然序列人类IgG4Fc区，以及其天然存在的变异体。变异Fc区包含因至少一个氨基酸修饰而与天然序列Fc区的氨基酸序列不同的氨基酸序列。在一些实施方案中，变异Fc区可具备相比于天然Fc区改变的效应子功能(例如Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。在某些实施方案中，人源化抗体的CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。在本申请中，供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的驼源抗体。为制备人源化抗体，可以使用本领域已知的方法将免疫动物的CDR区插入人源框架序列。在纳米抗体的语境下，人源化抗体可指人源化VHH，即其中一或多个骨架区已基本上由人类骨架区替换的VHH。在一些情形中，人类免疫球蛋白的某些骨架区(FR)由相应非人类残基替换。此外，人源化VHH可包含在初始VHH或人类骨架序列中均未发现、但被包括在内以进一步改进和优化VHH或含VHH的多肽的性能的残基。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990,Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人,1990,J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互 作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、 苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％(w/v)NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“体内半衰期”具有本领域技术人员公知的含义，通常可以定义为分子在体内的血清浓度减少50％(例如由于通过天然机制导致配体的降解和/或配体的清除或螯合)所需要的时间。测定体内半衰期的方法是本领域技术人员所熟知的，例如可通过药物代谢动力学分析进行测定。
发明的有益效果
本发明提供了与血清白蛋白具有高结合活性的纳米抗体，其具备与人、猴和/或小鼠血清白蛋白的交叉反应活性。特别地，本发明的纳米抗体与血清白蛋白的结合是非pH依赖性的，其能有效参与FcRn介导的血清白蛋白循环，从而延长所述纳米抗体的体内半衰期。因此，可通过将本申请的纳米抗体与药物分子(例如，多肽药物)进行偶联(例如，融合)，从而延长所述药物分子在体内的半衰期。
此外，所述纳米抗体还具有分子量小，稳定性好等特点，在药物研发等方面相比于传统常见的正常抗体有着组织浸润性好、给药方式灵活、人源化程度高、容易重组蛋白改造等诸多优势。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了Bi-340-352、Bi-340-353、Bi-340-354及Bi-340-356在小鼠血清中的浓度随时间的变化。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley & Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗HSA纳米抗体筛选
1.1纳米抗体文库的构建
动物免疫
将l mg人血清白蛋白(Human serum albumin，HSA)抗原(购自AcroBiosystems)与弗氏佐剂等体积混合，免疫2只羊驼，每周一次，共免疫4次，剌激B细胞表达抗原特异性的纳米抗体。4次免疫结束后，提取50ml羊驼外周血，采用淋巴细胞分离液分离得到淋巴细胞。采用RNA提取试剂Trizol(购自Invitrogen)提取总RNA。使用cDNA合成试剂盒(购自Invitrogen)反转录获得羊驼淋巴细胞总cDNA。
纳米抗体基因扩增
进行第一轮PCR，从上一步骤获得的cDNA中扩增出IgG2、IgG3序列，扩增用引物如表1所示：
表1.第一轮PCR引物
将第一轮PCR产物进行琼脂糖凝胶电泳，切胶回收750bp处的片段用于第二轮VHH序列扩增。第二轮PCR扩增引物如表2所示：
表2.第二轮PCR引物
以第二轮PCR产物为模板，进行第三轮PCR，为VHH基因加上与展示载体同源的同源臂序列。第三轮PCR扩增引物如表3所示：
表3.第三轮PCR引物
利用PCR纯化试剂盒(购自QIAGEN)回收目的片段。
文库构建
将线性化的酵母展示载体(人工合成)和第三轮的PCR产物混合后电转化入酿酒酵母(购自ATCC)中，构建来自两个动物的抗HSA纳米抗体文库并测定库容，库容大小分别为2.1×107和3.2×107。
1.2 HSA纳米抗体的筛选
HSA蛋白的生物素化标记
取适量体积的双蒸水溶解HSA蛋白(购自AcroBiosystems，货号：HSA-H5220)，按照生物素标记试剂盒(购自Thermo)产品说明书，将生物素溶解后与蛋白溶液混合，于4℃孵育2小时。用脱盐柱(购自Thermo)去除多余的生物素，脱盐柱预处理及样品收集操作均参考产品说明书步骤进行。
MACS富集能与HSA特异性结合的酵母
将上述步骤1.1中构建的VHH文库接种于SD-CAA扩增培养基(1L SD-CAA扩增培养基中含6.7g YNB、5g酪氨基酸、13.62g Na2HPO4·12H2O、7.44g NaH2PO4和2％葡萄糖)中，30℃，225rpm培养过夜。取适量酵母细胞，3000rpm×5min离心(以下离心操作的参数与此处相同)去除培养基，用SD-CAA诱导培养基重悬酵母细胞，诱导过夜。测定诱导后的文库浓度，取适量酵母细胞，离心去除培养基。用50ml PBS重悬酵母细胞，离心去除上清。用10ml PBS重悬酵母细胞。
加入如上述步骤获得的生物素标记的HSA蛋白(终浓度100mM)，室温孵育30min，离心收集酵母细胞，并用50ml PBS洗涤酵母3遍。用5ml清洗液重悬酵母细胞，并加入200μl SA磁珠(购自美天旎)，颠倒孵育10min。用PBS洗涤酵母和磁珠混合物3遍，将混合物加入LS纯化柱(购自美天旎)中。将LS纯化柱放在磁力架上，用PBS洗涤去除非特异性结合的酵母细胞。将纯化柱从磁力架上取出，加入PBS洗脱酵母。洗脱下来的酵母离心后转入SD-CAA扩增培养基中进行扩增。
流式细胞分选获得高亲和力酵母细胞
将经过MACS富集的酵母细胞接种于SD-CAA扩增培养基中。30℃，225rpm摇瓶培养过夜。用SD-CAA诱导培养基重悬酵母细胞，诱导过夜。加入抗c-Myc鼠源抗体(购自Thermo，货号：MA1-980)和100nM生物素标记的HSA抗原，孵育10min。加入PBS清洗酵母3遍，加入羊抗小鼠IgG(H+L)Alexa Fluor Plus 488荧光抗体(购自Invitrogen，货号：A32723)和链霉亲和素APC结合物荧光抗体(购自Invitrogen，货号：SA1005)，孵育15min。加入PBS重悬细胞，使用BD FACSAriaII仪器进行分选获得可与HSA抗原有较高结合能力的酵母。
HSA纳米抗体候选分子抗体基因的调取
通过MACS和FACS富集得到的能与HSA抗原有较高结合能力的酵母菌液，在SD-CAA扩增培养基中30℃，225rpm培养过夜，按照酵母质粒抽提试剂盒(购自天根)操 作抽提酵母质粒。质粒通过电转化入Top10感受态细胞(购自天根)，涂布氨苄抗性平板，于37℃培养过夜。挑取单克隆测序获得VHH氨基酸序列，并根据IMGT编号系统确定CDR区序列。所获得的单克隆纳米抗体的序列信息如表4所示。


实施例2.重链抗体的构建及表达纯化
2.1.抗体基因构建入pCDNA3.1表达载体
将VHH基因序列和人IgG1(LALA突变)Fc段(氨基酸序列如SEQ ID NO:68所示)相连，利用同源重组酶(购自Vazyme，货号：c115-02)构建在和EcoR I/Not I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序，并抽提质粒。
SEQ ID NO:68序列如下：
2.2.细胞转染及蛋白纯化
采用ExpiCHOTM表达系统试剂盒(购自Thermo)，将上述步骤获得的抽提质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积(1-4倍磁珠体积)的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M柠檬酸钠,pH3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀，获得目的蛋白。
实施例3.HSA抗体亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于高通量法的抗体-抗原亲和力和表位结合的测定.MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器上。再将传感器放入100nM的HSA抗原(购自AcroBiosystems，货号：HSA-H5220)中作用5min，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。亲和力数据如表5所示。
表5.候选分子与HSA的亲和力

本实施例进一步检测了候选分子与不同种属血清白蛋白的交叉反应，包括鼠血清白蛋白(Mouse Serum Albumin，MSA，购自AcroBiosystems，货号：MSA-M52H8)，食蟹猴血清白蛋白(Cynomolgus Serum Albumin，CSA，购自AcroBiosystems，货号：CSA-C52H4)。亲和力数据如表6所示。
表6.候选分子与MSA/CSA的亲和力

实施例4.抗HSA抗体人源化构建
为了减少单克隆抗体在人体内产生免疫原性，将HSA-59,HSA-M-27和HSA-PH-8三个抗体进行人源化。人源化方法采用VHH人源化通用框架移植法，同时根据文献(Vincke,C.,等人，人源化骆驼单域抗体和鉴定通用人源化纳米抗体支架的一般策略.J Biol Chem 284(5):3273-3284)报道的方法对抗体框架2(framework2)的部分氨基酸进行突变完成。人源化抗体的序列信息如表7所示。

本研究利用IMGT(http://www.imgt.org)对HSA-59,HSA-M-27和HSA-PH-8及人源化序列进行了人源化水平评估，结果如表8所示，所有人源化后的样品人源化水平均有所提高，符合后期药物开发要求。
表8.HSA人源化序列和人的同源性
蛋白构建及表达纯化方法同实施例2，利用HPLC检测获得蛋白的纯度，纯度检测结果如表9所示。HPLC方法如下，流动相：150mM Na2HPO4·12H2O,pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20min，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm,3μm)。
表9.Anti-HSA人源化抗体的纯度检测结果
实施例5.抗HSA人源化样品亲和力测定
5.1.人源化抗体的亲和力测定
本实施例检测纯化获得的抗HSA人源化样品与HSA结合亲和力，实验方法同实施例3，实验结果如表10所示，结果表明抗HSA人源化样品与HSA蛋白有很好的结合活性。
表10.人源化分子与HSA的亲和力
本实施例同时检测了纯化获得的抗HSA人源化样品与MSA/CSA结合亲和力，实验方法同实施例3，实验结果如表11所示。
表11.人源化分子与MSA/CSA的亲和力
5.2.人源化抗体在不同pH下的亲和力测定
HSA的回收机制与IgG类似，通过胞饮作用内化到血管内皮细胞，内体的pH为6.0左右，促进膜结合的FcRn与HSA结合。之后内体的复合物再循环到血液中，在pH7.4的条件下，膜结合的FcRn与HSA快速解离，HSA进行再循环。
本实施例检测纯化获得的抗HSA人源化样品与HSA在不同pH条件下的亲和力，实验方法同实施例3，实验结果如表12所示，结果表明抗HSA人源化样品与HSA蛋白在不同pH条件下均有很好的结合活性。
表12.人源化分子与HSA在不同pH条件下的亲和力
实施例6.抗HSA纳米抗体延长半衰期效果检测
6.1.分子构建和蛋白制备
为了证实抗HSA的纳米抗体的半衰期延长作用，在本实施例中，构建了多特异性抗体。多特异性抗体Bi-340-352、Bi-340-353及Bi-340-354各自独立地由肽链#1和肽链#2组成，共包含三个抗原结合结构域，分别为靶向第一抗原的Fab，靶向第二抗原的纳米抗体(VHH1)，以及靶向第三抗原的纳米抗体(VHH2)，所述VHH2为Hu-HSA-M-27-1、Hu-HSA-59-1、Hu-HSA-PH-8-5或作为对照的非HSA相关VHH。多特异性抗体的结构及序列如表13-1和13-2所示，其中，肽链#1和肽链#2中的各结构域通过包含GGGGS的接头连接。
表13-1多特异性抗体结构

表13-2多特异性抗体相关序列


分别编码Bi-340-352、Bi-340-353、Bi-340-354及Bi-340-356的肽链#1和肽链#2的基因序列由金维智公司合成。将所述编码基因序列分别利用同源重组酶(购自Vazyme)构建至EcoR I/Not I双酶切线性化的pCDNA3.1载体中，构建流程按照商品说明书。所述多特异性抗体各自的肽链#1同源重组产物和肽链#2同源重组产物共转化入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序。
采用ExpiCHOTM表达系统试剂盒(购自Thermo)，将质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用KappaSelect(GE)亲和层析柱进行纯化。具体方法如下：样品用注射器通过0.2um无菌针头式过滤器PES进行样品过滤。用5倍柱体积的平衡缓冲液(20mM PB+0.15M NaCl，pH 7.4)平衡层析柱，至流出液电导和pH不变。流速0.5ml/分钟上样。上样完毕后继续用平衡缓冲液冲洗层析柱，冲洗至穿透完全，UV值不再下降。用洗脱缓冲液(0.1M glycine-HCl，pH 3.0)洗脱，收集流出液。洗脱后，应立刻用碱性缓冲液(如1M Tris/HCl,pH 8.0)将收集到的抗体溶液中和到抗体稳定的pH。
利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na2HPO4·12H2O，pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20min，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm，3μm)。
6.2.蛋白半衰期检测
小鼠注射剂量为10mg/Kg。取血时间点：给药后3分钟、30分钟、2小时、6小时、24小时、48小时、96小时、168小时静脉采血。全血样品2-8℃放置30分钟，12000rpm离心5min收集血清，所得血清再于2-8℃，12000rpm离心5min，-80℃保存，ELISA检测血清中游离抗体分子量。结果如图1所示，结果表明带有抗HSA纳米抗体的分子在小鼠体能的半衰期约为23.7小时(Bi-340-352)、24.2小时(Bi-340-353)及10.9小时(Bi-340-354)；而带有非HSA相关的纳米抗体的分子(Bi-340-356)半衰期约为1.4小时。结 果显示，抗HSA纳米抗体可以显著提高分子的体内半衰期。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (25)

  1. 能够特异性结合血清白蛋白的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有选自以下的结构：X1X2X3LX4YYX5(式I，SEQ ID NO:72)，SGFTLDYYA(SEQ ID NO:30)，GSIWGIYH(SEQ ID NO:37)，GFTFSIYS(SEQ ID NO:31)，GSIFTFYR(SEQ ID NO:33)；
    (b)CDR2，其具有选自以下的结构：IX6SSGGX7X8(式II，SEQ ID NO:73)，ITVDGST(SEQ ID NO:46)，ISSGGSP(SEQ ID NO:40)，ITTDTST(SEQ ID NO:42)；
    (c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)，DIRNVGGDY(SEQ ID NO:57)，SPITSIFKA(SEQ ID NO:48)，NVRNVERDY(SEQ ID NO:50)；
    其中，
    X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
    X2选自(i)氨基酸残基F，A，S，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X3选自(i)氨基酸残基T，Q和(ii)相对于(i)是保守置换的氨基酸残基；
    X4选自(i)氨基酸残基D，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
    X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
    X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X10选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X11选自(i)氨基酸残基V，I，L和(ii)相对于(i)是保守置换的氨基酸残基；
    X12选自(i)氨基酸残基R，V，T和(ii)相对于(i)是保守置换的氨基酸残基；
    X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X14选自(i)氨基酸残基D，A和(ii)相对于(i)是保守置换的氨基酸残基；
    X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基；
    优选地，所述纳米抗体或其抗原结合片段包含如SEQ ID NOs:30-38任一项所示的CDR1；如SEQ ID NOs:39-46任一项所示的CDR2；以及，如SEQ ID NOs:47-59任一项 所示的CDR3。
  2. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)或SGFTLDYYA(SEQ ID NO:30)所示的结构；
    (b)CDR2，其具有如IX6SSGGX7X8(式II，SEQ ID NO:73)所示的结构；
    (c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)；
    其中，
    X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
    X2选自(i)氨基酸残基F，A，S，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X3选自(i)氨基酸残基T，Q和(ii)相对于(i)是保守置换的氨基酸残基；
    X4选自(i)氨基酸残基D，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
    X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
    X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X10选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X11选自(i)氨基酸残基V，I，L和(ii)相对于(i)是保守置换的氨基酸残基；
    X12选自(i)氨基酸残基R，V，T和(ii)相对于(i)是保守置换的氨基酸残基；
    X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X14选自(i)氨基酸残基D，A和(ii)相对于(i)是保守置换的氨基酸残基；
    X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基；
    优选地，X1选自氨基酸残基G或R；X2选自氨基酸残基F，A，S或E；X3选自氨基酸残基T或Q；X4选自氨基酸残基D或E；X5为氨基酸残基A；X6选自氨基酸残基A或S；X7选自氨基酸残基A或S；X8为氨基酸残基T；X9选自氨基酸残基T或V；X10选自氨基酸残基T或V；X11选自氨基酸残基V，I或L；X12选自氨基酸残基R，V或T；X13选自氨基酸残基G或E；X14选自氨基酸残基D或A；X15选自氨基酸残基L或F；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NOs:30、32、34-36、38任一项所示的CDR1；如SEQ ID NOs:39、41、43-45任一项所示的CDR2；以及，如 SEQ ID NOs:47、49、51-56、58-59任一项所示的CDR3。
  3. 权利要求1或2的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)或SGFTLDYYA(SEQ ID NO:30)所示的结构；
    (b)CDR2，其具有如IX6SSGGX7X8(式II，SEQ ID NO:73)所示的结构；
    (c)CDR3，其具有选自以下的结构：AAAX9LECRTX10X11X12X13YX14Y(式III，SEQ ID NO:74)，AAATX15ECRGRSSSYDY(式IV，SEQ ID NO:75)，AAALLECRVRSWPSDN(SEQ ID NO:49)，AAAVLECRAAEYVNS(SEQ ID NO:54)；
    其中，
    X1选自(i)氨基酸残基G，R和(ii)相对于(i)是保守置换的氨基酸残基；
    X2选自(i)氨基酸残基F，E，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X3选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
    X4选自(i)氨基酸残基D和(ii)相对于(i)是保守置换的氨基酸残基；
    X5选自(i)氨基酸残基A和(ii)相对于(i)是保守置换的氨基酸残基；
    X6选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X7选自(i)氨基酸残基A，S和(ii)相对于(i)是保守置换的氨基酸残基；
    X8选自(i)氨基酸残基T和(ii)相对于(i)是保守置换的氨基酸残基；
    X9选自(i)氨基酸残基T，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X10选自(i)氨基酸残基V和(ii)相对于(i)是保守置换的氨基酸残基；
    X11选自(i)氨基酸残基V，I和(ii)相对于(i)是保守置换的氨基酸残基；
    X12选自(i)氨基酸残基R，V和(ii)相对于(i)是保守置换的氨基酸残基；
    X13选自(i)氨基酸残基G，E和(ii)相对于(i)是保守置换的氨基酸残基；
    X14选自(i)氨基酸残基D和(ii)相对于(i)是保守置换的氨基酸残基；
    X15选自(i)氨基酸残基L，F和(ii)相对于(i)是保守置换的氨基酸残基；
    优选地，X1选自氨基酸残基G或R；X2选自氨基酸残基F，S或E；X3为氨基酸残基T；X4为氨基酸残基D；X5为氨基酸残基A；X6选自氨基酸残基A或S；X7选自氨基酸残基A或S；X8为氨基酸残基T；X9选自氨基酸残基T或V；X10为氨基酸残基V；X11选自氨基酸残基V或I；X12选自氨基酸残基R或V；X13选自氨基酸残基G或E；X14为氨基酸残基D；X15选自氨基酸残基L或F；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NOs:30、32、34-36任一 项所示的CDR1；如SEQ ID NOs:39、41、43-45任一项所示的CDR2；以及，如SEQ ID NOs:47、49、51、52、54-56、58任一项所示的CDR3。
  4. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有：如SEQ ID NO:32所示的序列，或与SEQ ID NO:32所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:41所示的序列，或与SEQ ID NO:41所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:49所示的序列，或与SEQ ID NO:49所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:32所示的CDR1、如SEQ ID NO:41所示的CDR2、如SEQ ID NO:49所示的CDR3。
  5. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有：如SEQ ID NO:32或34所示的序列，或与SEQ ID NO:32或34所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:43所示的序列，或与SEQ ID NO:43所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:52所示的序列，或与SEQ ID NO:52所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有如X1X2X3LX4YYX5(式I，SEQ ID NO:72)所示的结构，其中，X1为氨基酸残基G；X2选自(i)氨基酸残基F或E和(ii)相对于(i)是保守置换的氨基酸残基；X3为氨基酸残基T；X4为氨基酸残基D；X5为氨基酸残基A；
    (b)CDR2，其具有如IASSGGST(SEQ ID NO:43)所示的结构；
    (c)CDR3，其具有如AAAVLECRTVVRGYDY(SEQ ID NO:52)所示的结构；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:32或34所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID Ns:52所示的CDR3。
  6. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有：如SEQ ID NO:37所示的序列，或与SEQ ID NO:37所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:46所示的序列，或与SEQ ID NO:46所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:57所示的序列，或与SEQ ID NO:57所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:37所示的CDR1、如SEQ ID NO:46所示的CDR2、如SEQ ID NO:57所示的CDR3。
  7. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有：如SEQ ID NO:31所示的序列，或与SEQ ID NO:31所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:40所示的序列，或与SEQ ID NO:40所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:48所示的序列，或与SEQ ID NO:48所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:31所示的CDR1、如SEQ ID NO:40所示的CDR2、如SEQ ID NO:48所示的CDR3。
  8. 权利要求1的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含：
    (a)CDR1，其具有：如SEQ ID NO:33所示的序列，或与SEQ ID NO:33所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:42所示的序列，或与SEQ ID NO:42所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:50所示的序列，或与SEQ ID NO:50所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述纳米抗体或其抗原结合片段包含：如SEQ ID NO:33所示的CDR1、如SEQ ID NO:42所示的CDR2、如SEQ ID NO:50所示的CDR3。
  9. 权利要求1-8任一项的纳米抗体或其抗原结合片段，其包含：
    (1)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:49所示的CDR3；
    (2)如SEQ ID NO:32或34所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:52所示的CDR3；
    (3)如SEQ ID NO:37所示的CDR1；如SEQ ID NO:46所示的CDR2；以及，如SEQ ID NO:57所示的CDR3；
    (4)如SEQ ID NO:30所示的CDR1；如SEQ ID NO:39所示的CDR2；以及，如SEQ ID NO:47所示的CDR3；
    (5)如SEQ ID NO:31所示的CDR1；如SEQ ID NO:40所示的CDR2；以及，如SEQ ID NO:48所示的CDR3；
    (6)如SEQ ID NO:33所示的CDR1；如SEQ ID NO:42所示的CDR2；以及，如SEQ ID NO:50所示的CDR3；
    (7)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:51所示的CDR3；
    (8)如SEQ ID NO:35所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:52所示的CDR3；
    (9)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:43所示的CDR2；以及，如SEQ ID NO:53所示的CDR3；
    (10)如SEQ ID NO:36所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:54所示的CDR3；
    (11)如SEQ ID NO:35所示的CDR1；如SEQ ID NO:44所示的CDR2；以及，如SEQ ID NO:55所示的CDR3；
    (12)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:45所示的CDR2；以及，如SEQ ID NO:56所示的CDR3；
    (13)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:52所示的CDR3；
    (14)如SEQ ID NO:32所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:58所示的CDR3；或
    (15)如SEQ ID NO:38所示的CDR1；如SEQ ID NO:41所示的CDR2；以及，如SEQ ID NO:59所示的CDR3。
  10. 权利要求1-9任一项的纳米抗体或其抗原结合片段，其包含选自下列的氨基酸序列：
    (i)如SEQ ID NOs:3、9、24、1-2、7-8、14-23、28-29任一项所示的序列；
    (ii)与SEQ ID NOs:3、9、24、1-2、7-8、14-23、28-29任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NOs:3、9、24、1-2、7-8、14-23、28-29任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换。
  11. 权利要求1-9任一项的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段是人源化的；
    优选地，所述纳米抗体或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，所述重链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至驼源残基的回复突变。
  12. 权利要求11的纳米抗体或其抗原结合片段，所述纳米抗体或其抗原结合片段包含选自下列的氨基酸序列：
    (i)如SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列；
    (ii)与SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NOs:4-6、10-13、25-27、78任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换。
  13. 权利要求1-12任一项的纳米抗体或其抗原结合片段，其中，所述血清白蛋白选自人血清白蛋白(HSA)，鼠血清白蛋白(MSA)和/或食蟹猴血清白蛋白(CSA)；
    优选地，所述纳米抗体或其抗原结合片段与所述血清白蛋白的结合是非pH依赖性的；
    优选地，所述纳米抗体或其抗原结合片段能与所述血清白蛋白在pH 5-8(例如pH 5.5-7.4)的范围内特异性结合。
  14. 特异性结合血清白蛋白的多肽构建体，其包含权利要求1-13任一项所述的纳米抗体或其抗原结合片段，以及免疫球蛋白Fc结构域；
    优选地，所述免疫球蛋白Fc结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段的N端和/或C端(例如C端)；
    优选地，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域)；
    优选地，所述免疫球蛋白Fc结构域包含SEQ ID NO：68所示的序列，或与其相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个或5个氨基酸置换、缺失或添加)的序列。
  15. 权利要求14的多肽构建体，其中，所述血清白蛋白选自人血清白蛋白(HSA)，鼠血清白蛋白(MSA)和/或食蟹猴血清白蛋白(CSA)；
    优选地，所述多肽构建体与所述血清白蛋白的结合是非pH依赖性的；
    优选地，所述多肽构建体能与所述血清白蛋白在pH 5-8(例如pH 5.5-7.4)的范围内 特异性结合。
  16. 融合蛋白，其包含权利要求1-13任一项所述的纳米抗体或其抗原结合片段或权利要求13或14所述的多肽构建体，以及，另外的肽结构域；
    优选地，所述另外的肽结构域选自具有治疗效应的多肽结构域；
    优选地，所述另外的多肽结构域任选地通过肽接头连接至所述纳米抗体或其抗原结合片段或所述多肽构建体的N端和/或C端。
  17. 分离的核酸分子，其编码权利要求1-13任一项所述的纳米抗体或其抗原结合片段，权利要求14或15所述的多肽构建体，或权利要求16的融合蛋白。
  18. 载体，其包含权利要求17所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  19. 宿主细胞，其包含权利要求17所述的核酸分子或权利要求18所述的载体。
  20. 制备权利要求1-13任一项所述的纳米抗体或其抗原结合片段，权利要求14或15所述的多肽构建体，或权利要求16的融合蛋白的方法，其包括，在允许蛋白表达的条件下，培养权利要求19所述的宿主细胞，和从培养的宿主细胞培养物中回收所述纳米抗体或其抗原结合片段或所述多肽构建体。
  21. 双特异性或多特异性抗体，其包含权利要求1-13任一项所述的纳米抗体或其抗原结合片段或权利要求14或15所述的多肽构建体；
    优选地，所述双特异性或多特异性抗体特异性结合血清白蛋白，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性抗体还包含至少一种具有针对第二靶标的第二结合特异性的第二抗体；
    优选地，所述双特异性或多特异性抗体包含：
    (i)如权利要求1-13任一项所述的纳米抗体或其抗原结合片段或如权利要求14或15所述的多肽构建体(例如，含有如SEQ ID NOs:10、4、78任一项所示的序列的纳米抗体或其抗原结合片段或多肽构建体)作为第一抗原特异性结合结构域；
    (ii)包含如SEQ ID NO:79所示的轻链和如SEQ ID NO:80所示的重链的第二抗原 特异性结合结构域(如Fab)；
    以及，
    (iii)如SEQ ID NO:81所示的第三抗原特异性结合结构域(例如，纳米抗体VHH)；
    优选地，所述双特异性或多特异性抗体包含第一多肽链和第二多肽链，其中，所述第一多肽链包含(例如从N端至C端)：所述第二抗原特异性结合结构域的轻链或重链(如VH和CH1)，所述第三抗原特异性结合结构域，所述第一抗原特异性结合结构域；并且，所述第二多肽链包含(例如从N端至C端)：所述第二抗原特异性结合结构域的重链(如VH和CH1)或轻链，所述第三抗原特异性结合结构域；其中，所述第一多肽链与第二多肽链配对形成完整的所述第二抗原特异性结合结构域；优选地，所述第一多肽链和/或第二多肽链中的各个结构域通过接头(例如包含一个或多个甘氨酸和/或一个或多个丝氨酸的肽接头)连接；
    优选地，所述双特异性或多特异性抗体包含:
    (1)序列如SEQ ID NO:69所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽；
    (2)序列如SEQ ID NO:70所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽；
    或者,
    (3)序列如SEQ ID NO:71所示的第一肽，和，序列如SEQ ID NO:76所示的第二肽；
    优选地，所述第一肽存在于第一肽链，所述第二肽存在于第二肽链；优选地，所述第一肽链与所述第二肽链形成复合物。
  22. 缀合物，其包含权利要求1-13任一项所述的纳米抗体或其抗原结合片段或权利要求14或15所述的多肽构建体，以及与所述纳米抗体或其抗原结合片段或多肽构建体连接的治疗剂；
    优选地，所述治疗剂选自抗肿瘤药物，例如细胞毒剂、激素类药物、生物反应调节剂、另外的抗体或其抗原结合片段。
  23. 药物组合物，其包含权利要求1-13任一项的纳米抗体或其抗原结合片段、权利要求14或15所述的多肽构建体、权利要求16的融合蛋白、权利要求17的分离的核酸分子、权利要求18的载体、权利要求19所述的宿主细胞、权利要求21所述的双特异性或多特异性抗体或权利要求22所述的缀合物；
    优选地，所述药物组合物还包含药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物包含权利要求1-13任一项的纳米抗体或其抗原结合片段、权利要求14或15所述的多肽构建体、或编码所述纳米抗体或其抗原结合片段或多肽构建体的核酸分子、载体或宿主细胞；
    优选地，所述药物组合物包含权利要求16所述的融合蛋白、或编码所述融合蛋白的核酸分子、载体或宿主细胞；
    优选地，所述药物组合物包含权利要求21所述的双特异性或多特异性抗体、或编码所述双特异性或多特异性抗体的核酸分子、载体或宿主细胞；
    优选地，所述药物组合物包含权利要求22所述的缀合物。
  24. 权利要求1-13任一项的纳米抗体或其抗原结合片段、权利要求14或15所述的多肽构建体、权利要求16的融合蛋白、权利要求17的分离的核酸分子、权利要求18的载体、权利要求19所述的宿主细胞、权利要求21所述的双特异性或多特异性抗体或权利要求22所述的缀合物、或权利要求23所述的药物组合物用于制备药物的用途；
    优选地，所述药物在受试者体内能直接或间接参与FcRn介导的血清白蛋白循环；
    优选地，所述药物显示相对于缺少所述纳米抗体或其抗原结合片段的相应的药物延长的体内半衰期；
    优选地，所述药物是蛋白质类药物；
    优选地，所述受试者为哺乳动物，例如人，小鼠或食蟹猴。
  25. 一种延长药物的体内半衰期的方法，其包括：将权利要求1-13任一项所述的纳米抗体或其抗原结合片段或权利要求14或15所述的多肽构建体与所述药物进行连接；
    优选地，所述体内半衰期的延长是相对于缺少所述纳米抗体或其抗原结合片段时所述药物的体内半衰期；
    优选地，所述药物选自大分子药物(例如多肽类药物)；
    优选地，所述受试者为哺乳动物，例如人，小鼠或食蟹猴。
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