Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • 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
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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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • 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
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • 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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell
• • 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
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • 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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  • 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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  • 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.

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