WO2022152222A1 - 靶向pd-1的单域抗体及其衍生物和用途 - Google Patents

靶向pd-1的单域抗体及其衍生物和用途 Download PDF

Info

Publication number
WO2022152222A1
WO2022152222A1 PCT/CN2022/071865 CN2022071865W WO2022152222A1 WO 2022152222 A1 WO2022152222 A1 WO 2022152222A1 CN 2022071865 W CN2022071865 W CN 2022071865W WO 2022152222 A1 WO2022152222 A1 WO 2022152222A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
targeting
domain antibody
vhh
domain
Prior art date
Application number
PCT/CN2022/071865
Other languages
English (en)
French (fr)
Inventor
李玉俊
Original Assignee
立凌生物制药(苏州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 立凌生物制药(苏州)有限公司 filed Critical 立凌生物制药(苏州)有限公司
Publication of WO2022152222A1 publication Critical patent/WO2022152222A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • the present invention relates to the field of biotechnology. More specifically, the present invention relates to single domain antibodies targeting PD-1 and derivatives and uses thereof.
  • PD-1 Programmed cell death protein 1, PD-1
  • PD-L1 tumor immunity-associated PD-1 and PD-L1 are important targets of tumor immunity.
  • PD-1 and PD-L1 are a pair of immunosuppressive molecules, which are an important part of the immune system to prevent autoimmune overstimulation. The activation of their pathways can inhibit tumor immune responses, induce tumor-specific T cell apoptosis, and have a role in tumor development. close relationship.
  • the use of monoclonal antibodies to block PD-1 and PD-L1 pathways in the treatment of tumors has shown good clinical efficacy and safety.
  • a variety of antibody drugs have been approved for marketing, and the indications include melanoma, non-small cell Lung cancer, advanced renal cell carcinoma and other malignant tumors, and many ongoing clinical trials try to develop more new indications.
  • PD-1 In addition to being expressed on the surface of activated T cells, NKT cells, B cells and activated monocytes, PD-1 is also highly expressed on the surface of exhausted T cells. Its ligand PD-L1 is not only expressed in immune cells such as B cells, T cells, and dendritic cells, but also expressed in peripheral microvascular endothelial cells, heart and lung organs. In contrast, PD-L2 is only expressed on the surface of macrophages and dendritic cells. Notably, the expression of PD-L1 has been widely found in human tumors, including human lung cancer, melanoma, ovarian cancer, and colon cancer.
  • blocking the PD-L1/PD-1 pathway can significantly increase T cell proliferation, cytokine secretion, and T cell killing of tumor cells.
  • blocking the PD-L1/PD-1 pathway significantly blocked tumor growth.
  • Blockade of PD-1/PD-L1 signaling pathway using PD-1 antibody significantly enhanced tumor-specific T cell expansion and tumor infiltration. The above results demonstrate the important application value of PD-1/PD-L1 blocking antibodies in tumor therapy.
  • bispecific antibodies based on the PD-1/PD-L1 pathway generally take the form of diabodies.
  • the bispecific antibody prepared by this method generally has a relatively large molecular weight, which makes it difficult for the antibody to infiltrate the tumor tissue, which may affect the clinical effect.
  • Double-chain-based bispecific antibodies often suffer from mismatches in production, causing great problems for downstream production and purification.
  • the current PD-1/PD-L1 monoclonal antibody has problems such as large dosage and low overall response rate.
  • Single domain antibodies are a special class of antibodies that contain only one antibody heavy chain. Similar to traditional diabodies, it can selectively bind to specific antigens. Single-domain antibodies were first discovered in camelids, and later in cartilaginous fish such as nurse sharks. Single domain antibody single heavy chain antibody variable domain (VHH) is a single functional domain that can completely bind antigen, only 12-15kDa. VHH has a simple structure and has the advantages of high specificity, high affinity, low immunogenicity, good permeability, and the ability to contact relatively hidden targets that cannot be contacted by conventional antibodies during tumor treatment. In addition, because single-domain antibodies have only one chain, there are no mismatching problems associated with diabody fusions.
  • the purpose of the present invention is to provide a specific single domain antibody targeting PD-1.
  • the present invention also aims to provide a specific humanized single domain antibody targeting PD-1.
  • the present invention also aims to provide the use of the single domain antibody or the humanized single domain antibody in the treatment of tumors or the preparation of medicines for the treatment of tumors.
  • the present invention provides a VHH chain of a single-domain antibody targeting PD-1, the VHH chain comprising CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3 and SEQ ID CDR3 shown in NO:4.
  • the PD-1 is human PD-1.
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid and can retain the derivative sequence that binds to PD-1 with high affinity and blocks the binding of PD-L1 to PD-1.
  • the VHH chain further comprises framework regions FR1, FR2, FR3 and FR4, and the FR1, FR2, FR3 and FR4 are shown in positions 1-25 of the amino acid sequence shown in SEQ ID NO: 1, 36-49 bits, 67-98 bits, and 110-120 bits are shown.
  • amino acid sequence of the VHH chain of the single-domain antibody targeting PD-1 is shown in SEQ ID NO: 1.
  • the present invention provides a heavy chain variable region of an antibody targeting PD-1, the heavy chain variable region comprising CDR1 shown in SEQ ID NO:2, and shown in SEQ ID NO:3 CDR2 and CDR3 shown in SEQ ID NO:4.
  • amino acid sequence of the heavy chain variable region of the antibody targeting PD-1 is shown in SEQ ID NO:1.
  • the present invention provides a single-domain antibody targeting PD-1, which has the VHH chain described in the first aspect.
  • the present invention provides a humanized VHH chain of a single-domain antibody targeting PD-1, based on the VHH chain described in the first aspect, the framework regions FR1, FR2, FR3 and FR4 are humanized source.
  • the framework regions FR1, FR2, FR3 and FR4 of the VHH chain of the humanized single-domain antibody targeting PD-1 are as shown in SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO: 18, or SEQ ID NO: 20, or SEQ ID NO: 22, or positions 1-25, 36-49, 67-98 and 110-120 of the amino acid sequence shown in SEQ ID NO: 24 bit shown.
  • the VHH chain amino acid sequence of the humanized single-domain antibody targeting PD-1 is as SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO: 18, or SEQ ID NO:20, or SEQ ID NO:22, or SEQ ID NO:24.
  • the present invention provides an antibody targeting PD-1, the antibody comprising one or more VHH chains of the single-domain antibody targeting PD-1 according to the first aspect or the fourth aspect The VHH chain of a humanized single-domain antibody targeting PD-1.
  • the antibodies targeting PD-1 include monomeric, bivalent, and/or multivalent antibodies.
  • the present invention provides a bispecific antibody, the bispecific antibody includes a first antibody and a second antibody, the first antibody includes the PD-1 targeting single domain of the first aspect
  • the VHH chain of the antibody, or the variable region of the heavy chain of the antibody targeting PD-1 described in the second aspect, or the single domain antibody targeting PD-1 described in the third aspect, or the human described in the fourth aspect The VHH chain of a single-domain antibody targeting PD-1, or the antibody targeting PD-1 according to the fifth aspect.
  • the second antibody may bind the same or a different antigen as the first antibody, or bind a different epitope of the same antigen as the first antibody.
  • the second antibody is a single domain antibody, a single chain antibody or a diabody.
  • the bispecific antibody includes 2-4 single-domain antibodies targeting PD-1; preferably, including 2 single-domain antibodies targeting PD-1; more preferably, The two PD-1-targeting single-domain antibodies form a PD-1-targeting single-domain antibody dimer.
  • the present invention provides a fusion protein comprising the VHH chain of the single-domain antibody targeting PD-1 according to the first aspect and the antibody targeting PD-1 according to the second aspect
  • the variable region of the heavy chain, the single-domain antibody targeting PD-1 according to the third aspect, the VHH chain of the humanized single-domain antibody targeting PD-1 according to the fourth aspect, or the fifth aspect The antibody targeting PD-1, an optional linker sequence and an Fc fragment of an immunoglobulin.
  • the immunoglobulin is IgGl, IgG2, IgG3, IgG4; preferably IgG4.
  • the present invention provides a nucleic acid molecule encoding the VHH chain of the single-domain antibody targeting PD-1 according to the first aspect, and the antibody targeting PD-1 according to the second aspect
  • the variable region of the heavy chain, the single-domain antibody targeting PD-1 described in the third aspect, the VHH chain of the humanized single-domain antibody targeting PD-1 described in the fourth aspect, the VHH chain of the single-domain antibody targeting PD-1 described in the fifth aspect The antibody targeting PD-1, the bispecific antibody described in the sixth aspect, or the fusion protein described in the seventh aspect.
  • the present invention provides an expression vector comprising the nucleic acid molecule of the eighth aspect.
  • the present invention provides a host cell comprising the expression vector of the ninth aspect, or the nucleic acid molecule of the eighth aspect integrated into its genome.
  • the present invention provides a method for preparing the VHH chain of the PD-1-targeting single domain antibody described in the first aspect, and the heavy chain variable region of the PD-1-targeting antibody described in the second aspect , the single domain antibody targeting PD-1 described in the third aspect, the VHH chain of the humanized single domain antibody targeting PD-1 described in the fourth aspect, the targeting PD-1 described in the fifth aspect
  • the host cell as described in the tenth aspect is cultivated, thereby obtaining the VHH chain of the single-domain antibody targeting PD-1 and the variable heavy chain of the antibody targeting PD-1.
  • the VHH chain of the single-domain antibody targeting PD-1, the variable region of the heavy chain of the antibody targeting PD-1, the variable region of the heavy chain of the antibody targeting PD-1, and the PD-1 targeting antibody are isolated or recovered from the culture.
  • the present invention provides an immunoconjugate comprising:
  • Conjugation moieties selected from detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the coupling moiety is a drug or a toxin.
  • the immunoconjugate is an antibody-drug conjugate (Antibody-Drug-Conjugate, ADC).
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pre- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (eg, bivalent) VHH chain of the PD-1-targeting single domain antibody of claim 1 , the target of claim 2 VHH of the heavy chain variable region of the antibody to PD-1, the single-domain antibody targeting PD-1 according to claim 3, and the humanized single-domain antibody targeting PD-1 according to claim 4 chain, the PD-1 targeting antibody of claim 5, the bispecific antibody of claim 6, or the fusion protein of claim 7.
  • a multivalent (eg, bivalent) VHH chain of the PD-1-targeting single domain antibody of claim 1 the target of claim 2 VHH of the heavy chain variable region of the antibody to PD-1
  • the single-domain antibody targeting PD-1 according to claim 3 the humanized single-domain antibody targeting PD-1 according to claim 4 chain
  • the PD-1 targeting antibody of claim 5 the bispecific antibody of claim 6, or the fusion protein of claim 7.
  • the polyvalent means that the amino acid sequence of the immunoconjugate comprises a plurality of repeating moieties.
  • the present invention provides a pharmaceutical composition comprising a therapeutically or diagnostically effective amount of the VHH chain of the single-domain antibody targeting PD-1 according to the first aspect, the The heavy chain variable region of the antibody targeting PD-1, the single domain antibody targeting PD-1 described in the third aspect, the humanized single domain targeting PD-1 described in the fourth aspect
  • the pharmaceutical composition is used to treat tumors selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, Breast, colorectal, prostate, cervical, lymphoma, adrenal tumors, or bladder tumors.
  • the present invention provides the VHH chain of the PD-1 targeting single domain antibody of the first aspect, the heavy chain variable region of the PD-1 targeting antibody of the second aspect, and the third The single-domain antibody targeting PD-1 according to the aspect, the VHH chain of the humanized single-domain antibody targeting PD-1 according to the fourth aspect, and the antibody targeting PD-1 according to the fifth aspect , Use of the bispecific antibody described in the sixth aspect, the fusion protein described in the seventh aspect or the immunoconjugate described in the twelfth aspect, for the preparation of the following reagents:
  • the tumor is selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer , lymphoma, adrenal tumor, or bladder tumor.
  • the present invention provides a kit comprising:
  • the VHH chain of the PD-1-targeting single-domain antibody described in the first aspect, the heavy chain variable region of the PD-1-targeting antibody described in the second aspect, and the PD-targeting antibody described in the third aspect The single-domain antibody of -1, the VHH chain of the humanized single-domain antibody targeting PD-1 according to the fourth aspect, the antibody targeting PD-1 according to the fifth aspect, and the sixth aspect.
  • the present invention provides a method for preparing an antibody targeting an immune checkpoint, the method comprising the following steps:
  • the immune cells are primary immune cells.
  • the method further comprises the step of first activating immune cells.
  • the immune checkpoints include but are not limited to: PD-1, CTLA-4, TIM3, LAG3, KIR, GITR, VISTA, 4-1BB, CD28, OX40, ICOS and the like.
  • the immune cells are lymphocytes or phagocytes; preferably, the immune cells are T lymphocytes, B lymphocytes, K lymphocytes, NK lymphocytes, plasma cells, granulocytes, mast cells , antigen presenting cells or cells of the mononuclear phagocyte system (eg macrophages); more preferably T lymphocytes.
  • the mononuclear phagocyte system eg macrophages
  • the antibody is a polyclonal antibody, a monoclonal antibody, a single domain antibody; preferably a single domain antibody.
  • the animals include but are not limited to mice, rats, camels, alpacas, camels, rabbits; preferably mice, alpacas.
  • the present invention provides a method for detecting PD-1 protein in a sample, the method comprising the steps of:
  • the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the VHH chain of the single-domain antibody targeting PD-1 according to the first aspect , the heavy chain variable region of the antibody targeting PD-1 described in the second aspect, the single domain antibody targeting PD-1 described in the third aspect, the humanized targeting PD described in the fourth aspect
  • the VHH chain of the single domain antibody of -1, the antibody targeting PD-1 according to the fifth aspect, the bispecific antibody according to the sixth aspect, the fusion protein according to the seventh aspect, and the twelfth aspect The immunoconjugate or the pharmaceutical composition of the thirteenth aspect.
  • the subject includes a mammal; preferably a human.
  • Figure 1 shows the results of affinity detection of VHH-Fc fusion protein LL-VHH01-Fc binding to PD-1 antigen
  • Figure 2 shows the biological activity detection results of VHH-Fc fusion protein LL-VHH01-Fc blocking the binding of PD-L1 to PD-1 protein;
  • Figure 3 shows that all six humanized antibodies can bind PD-1 antigen well
  • Figure 4 shows that six humanized antibodies can well block PD-1 and PD-L1 signaling pathways at the cellular level
  • Figure 5 shows that 3 representative humanized antibodies can control tumor growth well in animals.
  • the inventors unexpectedly discovered a class of single-domain antibodies targeting PD-1.
  • the single-domain antibody of the present invention has better binding activity to PD-1 molecule, can block the interaction between PD-1 and PD-L1, and has good anti-tumor activity.
  • the inventors further developed a series of humanized single-domain antibodies, and found that the affinity and biological effect of these humanized single-domain antibodies were similar to those of the parent antibody Or better yet, with very good chemical and serum stability. The present invention has been completed on this basis.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • single domain antibody Single domain antibody
  • single domain antibody single domain antibody
  • VHH single-domain antibody
  • the VHH chain of the single-domain antibody targeting PD-1 of the present invention includes CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3 and CDR2 shown in SEQ ID NO:4 CDR3.
  • the amino acid sequence of the VHH chain of the single-domain antibody targeting PD-1 is shown in SEQ ID NO: 1.
  • the framework regions FR1, FR2, FR3 and FR4 are also included, and their amino acid sequences are as shown in SEQ ID NO: 1 at positions 1-25, 36-49, 67-98 and 110-120 bits are shown.
  • the inventors On the basis of the VHH chain of the single-domain antibody targeting PD-1 of the present invention, the inventors also humanized the VHH chain, thereby obtaining a humanized single-domain antibody targeting PD-1.
  • VHH chain In a specific embodiment, the amino acid sequence of the humanized VHH chain of the present invention is as SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO: 18, or SEQ ID NO: 20, or SEQ ID, respectively NO:22, or SEQ ID NO:24.
  • framework regions FR1, FR2, FR3 and FR4 whose amino acid sequences are respectively as SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO: 18, or Positions 1-25, 36-49, 67-98 and 110-120 of the amino acid sequence shown in SEQ ID NO:20, or SEQ ID NO:22, or SEQ ID NO:24.
  • the present invention also provides a PD-1 targeting antibody, which comprises one or more of the targeting The VHH chain of a single-domain antibody to PD-1 or the VHH chain of a humanized single-domain antibody targeting PD-1.
  • the present invention also provides a bispecific antibody, the bispecific antibody includes a first antibody and a second antibody, and the first antibody can be the VHH chain of the single domain antibody targeting PD-1 of the present invention, or humanized VHH chains.
  • the bispecific antibody includes a first antibody and a second antibody
  • the first antibody can be the VHH chain of the single domain antibody targeting PD-1 of the present invention, or humanized VHH chains.
  • Those skilled in the art can select the second antibody in the bispecific antibody according to actual needs.
  • the second antibody may bind the same or a different antigen as the first antibody; if the second antibody binds the same antigen as the first antibody, it preferably binds to a different epitope.
  • the second antibody can be a single-domain antibody, or a single-chain antibody or a diabody.
  • the VHH chain or humanized VHH chain of the PD-1-targeting single domain antibody of the present invention into a fusion protein, for example, a fusion protein further comprising an Fc fragment of an immunoglobulin.
  • the fusion protein thus obtained not only possesses the biological activity of the single domain antibody VHH chain itself, but also possesses other properties conferred by the Fc fragment of the immunoglobulin, such as prolonged plasma half-life, reduced immunogenicity, improved stability, and the like.
  • the fusion protein comprises the VHH chain or humanized VHH chain of the PD-1 targeting single domain antibody of the invention, an optional linker sequence, and an Fc fragment of an immunoglobulin.
  • the immunoglobulin is IgGl, IgG2, IgG3, IgG4; preferably IgG4.
  • the present invention includes not only intact antibodies, but also fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having PD-1 protein-binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein.
  • the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency The encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention.
  • the present invention also includes fragments of the single domain antibodies of the present invention.
  • the fragments have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to the following table.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxins; 3. cytokines, such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. 6. Liposomes; 7. Nanomagnetic particles; 8. Drug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. Therapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • immune checkpoint or immune checkpoint molecule
  • immune checkpoint refers to a series of molecules that are expressed on immune cells and can regulate the degree of immune activation. They play an important role in preventing autoimmunity (abnormal immune function, attacking normal cells). Therefore, one of the important functions of "immune checkpoint molecules" is to keep the activation of the immune system within the normal range to prevent the overactivation of the immune system.
  • immune checkpoint molecules The abnormal expression and function of immune checkpoint molecules is one of the important reasons for the occurrence of many diseases. For example, if the immune checkpoint molecules are overexpressed or function too strong, the immune function is suppressed, the immunity of the body is low, and people are prone to diseases such as tumors. On the contrary, if the immunosuppressive function of immune checkpoint molecules is too poor, the immune function of the body will also be abnormal.
  • Tumor cells express some substances to activate immune checkpoints. Once activated, antigens cannot be presented to T cells, blocking the process of presenting antigens in the tumor immune loop, thereby inhibiting the immune function of T cells. It allows tumor cells to escape immune surveillance and survive.
  • immune checkpoints include CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), PD-1, TIM3, LAG3, KIR, GITR, VISTA, 4-1BB, etc.
  • CTLA-4 cytotoxic T lymphocyte-associated antigen-4
  • PD-1 PD-1
  • TIM3 LAG3, KIR
  • GITR GITR
  • VISTA 4-1BB
  • immune cells and immune effector cells have the same meaning and are the same as conventionally understood by those skilled in the art. It refers to cells involved in or associated with an immune response, including lymphocytes and phagocytes.
  • the immune cells refer to lymphocytes that can recognize antigens to generate specific immune responses.
  • the lymphocytes are mainly T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes.
  • cells involved in the immune response are plasma cells, granulocytes, mast cells, antigen-presenting cells, and cells of the mononuclear phagocyte system (e.g., macrophages).
  • the present invention also provides an immunoconjugate comprising a VHH chain, a humanized VHH chain, etc. of the PD-1-targeting single domain antibody of the present invention and a coupling moiety.
  • the coupling moiety can be a detectable label, drug, toxin, cytokine, radionuclide or enzyme, etc., to achieve diagnostic, detection or treatment purposes, and the like.
  • the immunoconjugate is an antibody-drug conjugate (Antibody-Drug-Conjugate, ADC).
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-1 protein, thereby blocking the interaction between PD-1 and PD-L1. Therefore, the pharmaceutical composition of the present invention can be used to treat tumors.
  • the tumor is selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer , lymphoma, adrenal tumor, or bladder tumor.
  • the pharmaceutical compositions of the present invention may also be used in combination with other therapeutic agents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned single domain antibody (or its conjugate) of the present invention and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention also relates to a method for detecting PD-1 protein.
  • the steps of the method are roughly as follows: contact the sample to be tested with the VHH chain of the single-domain antibody targeting PD-1 of the present invention, or the humanized VHH chain, antibody, fusion protein or immunoconjugate, etc.; An antigen-antibody complex is formed, and if the complex is formed, it indicates the presence of PD-1 protein in the sample.
  • the present invention also provides a kit containing the VHH chain of the single-domain antibody targeting PD-1, or the humanized VHH chain, antibody, fusion protein or immunoconjugate of the present invention.
  • the kit further includes a container, instructions for use, buffers and the like.
  • the inventors found that the PD-1 single domain antibody obtained by directly immunizing animals with PD-1 protein has poor activity. However, when the inventors used T cells for immunization, a PD-1 single domain antibody with good activity was obtained.
  • the present invention also provides a special immunization method capable of preparing antibodies targeting immune checkpoints.
  • the method includes immunizing an animal with immune cells expressing an immune checkpoint.
  • the immune cells expressing the immune checkpoint may be activated prior to immunizing the animal with the immune cells expressing the immune checkpoint.
  • Methods or technical means for activating immune cells are well known to those skilled in the art.
  • the used immune cells are preferably primary immune cells.
  • the immunized animals include, but are not limited to, mice, camels, alpacas, rats, and rabbits; preferably mice, alpacas.
  • the antibodies obtained by the method of the present invention can be polyclonal antibodies, monoclonal antibodies, and single domain antibodies.
  • the single-domain antibody targeting PD-1 of the present invention has good binding affinity to PD-1, so that it can effectively block the binding of PD-1 and PD-L1;
  • the humanized single-domain antibody of the present invention can retain a good binding affinity to PD-1, and exhibits similar or even better biological effects than the parent antibody at the cellular level in vitro;
  • the humanized single domain antibody of the present invention has good chemical stability and serum stability
  • the PD-1-targeting single-domain antibody and humanized single-domain antibody of the present invention have wide biological application value and clinical application value, thereby laying a new foundation for the development of PD-1-targeting therapeutic or diagnostic drugs. Material basis.
  • cryopreserved human peripheral blood mononuclear cells purchased from Microton Biotech
  • concentration was adjusted to 2*10 6 /ml.
  • microbeads (Thermo Fisher Scientific) loaded with CD3 antibody and CD28 antibody were added according to the ratio recommended in the instructions to activate T cells in PBMCs.
  • T cells use a magnetic stand to demagnetize the beads; then centrifuge the cells at 1600 rpm for 5 minutes to collect the cells; use the freezing medium to freeze the cells.
  • the vector (pComb3XSS) and the target fragment amplified by PCR were digested with SfiI respectively, digested overnight at 50°C, and then the target fragment was recovered.
  • the VHH-linked vector was introduced into competent cells by electroporation to construct a heavy chain single-domain antibody phage display library. Plate after electroporation. To detect the insertion rate of the library, 48 clones were randomly selected on the plate for colony PCR. The results showed that the insertion rate reached 100%. By serial dilution plating, the size of the library volume was calculated to be 1.0 ⁇ 10 9 .
  • the phage library titer was measured to be 5.76 ⁇ 10 13 cfu/mL by the method of calculating the number of clones by serially diluting the phage library and then plating it.
  • the constructed alpaca immune library was subjected to affinity screening to obtain a specific phage library.
  • amino acid sequence of LL-VHH01 is SEQ ID NO:1
  • sequence of CDR1 is SEQ ID NO:2
  • sequence of CDR2 is SEQ ID NO:3
  • sequence of CDR3 is SEQ ID NO:4.
  • the amino acid sequence of the constant region of human immunoglobulin gamma4 (IgG4) was obtained from the database Uniprot (P01861).
  • the PD-1 single domain antibody VHH amino acid sequence and hIgG4-Fc amino acid sequence were linked to obtain a PD-1 VHH-Fc fusion protein, named: LL-VHH01-Fc.
  • the gene sequence of anti-human PD-1 single domain antibody VHH-Fc fusion protein LL-VHH01-Fc was obtained by gene synthesis.
  • the PD-1 VHH-Fc fusion protein gene was then cloned into the expression vector pCDNA4 (Invitrogen, Cat V86220).
  • the PD-1 single domain antibody VHH-Fc fusion protein LL-VHH01-Fc was expressed by transient transfection of HEK293 suspension cells. After the expression is completed, the supernatant is collected and purified using a Protein A affinity chromatography column to finally obtain the purified PD-1 single domain antibody Fc fusion protein LL-VHH01-Fc.
  • the gene sequence is: SEQ ID NO:10
  • amino acid sequence is: SEQ ID NO:11
  • the light chain amino acid sequence is SEQ ID NO: 13
  • the Pembrolizumab antibody analog was expressed and purified with reference to the method of antibody expression and purification in the patent.
  • the anti-PD-1 antibody analog of MSD Company was obtained and renamed as LL-Pos.
  • Embodiment 5 ELISA method detects the binding ability of PD-1 single domain antibody VHH-Fc fusion protein to PD-1 protein
  • human PD-1 protein purchased from Beijing Baipsis, product number PD1-H5221
  • PBS purchased from Hyclone Company
  • 100 ⁇ l/well was added to a 96-well ELISA plate for antigen coating , and incubated in a constant temperature incubator at 37°C for 60 min.
  • the plate was washed three times, and 200 ⁇ l/well of PBS containing 2% BSA was added for blocking, and incubated in a 37° C. constant temperature incubator for 60 min.
  • sample LL-VHH01-Fc and the control antibody LL-Pos were diluted to 100 ⁇ g/ml with diluent (PBS containing 2% BSA), and were sequentially diluted to 0.56 ng/ml in a 3-fold gradient on the sample dilution plate. Then, 100 ⁇ l/well of the sample was added to a 96-well microtiter plate and incubated in a constant temperature incubator at 37°C for 60 min. After the incubation, the plate was washed, and the secondary antibody, goat anti-human IgG (Fc specific)-HRP antibody (purchased from Sigma, Cat. No.
  • A-0170 was diluted 6000 times with 2% BSA, and added to the ELISA plate at 100 ⁇ l/well. Incubate in a 37°C constant temperature incubator for 30 min. After the incubation, wash the plate three times and add 100 ⁇ l/well of chromogenic solution, which is 100 ⁇ g/ml of TMB (3,3',5,5'-tetramethylbenzidine), and place it in a 37°C constant temperature incubator. Incubate in the dark for 15 min.
  • stop solution (2M/L hydrochloric acid solution) was added according to 100 ⁇ l/well and the absorbance value at 450nm/620nm was detected using a microplate reader (Thermo Fisher Scientific, Varioskan LUX), and the data was analyzed using Graphpad Prism.
  • Example 6 Detection of biological activity of VHH-Fc fusion protein using luciferase reporter gene method
  • CHO/PD-L1 cells purchased from Promega
  • F-12 nutrient mix complete medium containing 10% FBS and adjust the cell density to 5 ⁇ 10 5 cells using complete medium according to the cell count results. cells/ml.
  • the cell suspension was transferred to the sample addition tank and added to a 96-well plate at 100 ⁇ l/well using a multi-channel pipette, and placed in a 5% CO 2 incubator at 37° C. for 16-22 h.
  • Jurkat/PD-1 cells purchased from Promega were cultured and the cell density was adjusted to 2 ⁇ 10 6 cells/ml using assay medium (RPMI 1640 Medium+2% FBS) according to cell count results.
  • Assay medium RPMI 1640 Medium+2% FBS
  • the above Jurkat/PD-1 cell suspension was transferred to the sample addition tank and added to the cell culture plate at 40 ⁇ l/well, and cultured in a 5% CO 2 incubator at 37° C. for 4-6 h.
  • LL-VHH01-Fc can effectively block the binding of PD-1 and PD-L1, and activate the downstream pathway to generate signals.
  • the EC 50 of the dose-response curve is 0.35 ⁇ g/ml.
  • the positive control LL-Pos can also effectively block the binding of PD-1 and PD-L1, and activate the downstream pathway to generate signals, with an EC 50 of 1.09 ⁇ g/ml in the dose-response curve.
  • Fig. 1 and Fig. 2 show that the PD-1 single-domain antibody fusion protein of the present invention is equal to or better than the marketed one in terms of the affinity for the antigen PD-1 and the blocking effect on the binding of PD-1 and PD-L1 Therefore, it may have better clinical effect.
  • single-domain antibodies have no light chain and are very suitable for the development of diabodies or multispecific antibodies as important constituent antibodies, and are expected to develop antibody drugs with better efficacy.
  • LL-VHH01 is an alpaca-derived antibody. In order to improve the druggability of this single-domain antibody, LL-VHH01 was humanized. The basic steps:
  • IGHV3-23*04 was selected as the LL-VHH01 humanized parent vector
  • Example 3 six humanized antibodies fused to the human IgG4 Fc tag were expressed and purified, named respectively: huVHH3-1-Fc, huVHH3-2-Fc, huVHH3-3-Fc, huVHH3-4-Fc , huVHH3-5-Fc, huVHH3-6-Fc.
  • the sequences of the six humanized anti-PD-1 single domain antibodies fused to hIgG4Fc are as follows:
  • Example 5 the affinity of the humanized antibody to the antigen PD-1 was detected.
  • the test results are shown in Figure 3.
  • the results show that huVHH3-1-Fc, huVHH3-2-Fc, huVHH3-3-Fc, huVHH3-4-Fc, huVHH3-5-Fc, huVHH3-6-Fc bind to the EC of PD-1 protein 50 were 29.25ng/ml, 40.50ng/ml, 46.96ng/ml, 13.31ng/ml, 47.61ng/ml and 45.97ng/ml, respectively.
  • the experimental results showed that the affinity of the six humanized antibodies was similar to or better than that of the parent antibody after humanization.
  • Example 10 Detection of cellular activity of humanized antibodies blocking PD-1/PD-L1 signaling pathway in vitro
  • Example 6 Referring to the experimental steps of Example 6, the cellular activity of the humanized antibody to block the PD-1/PD-L1 signaling pathway in vitro was detected.
  • the test results are shown in Figure 4.
  • the experimental results showed that the six humanized antibodies showed similar or better biological effects than the parent antibodies at the cellular level in vitro after humanization.
  • the Octet RED 96e Molecular Interaction Analyzer of Fortebio Company was used.
  • the candidate antibody molecules (at a concentration of 5 ⁇ g/mL) were first captured using a Protein A probe (Fortebio).
  • the probes were then immersed in 30nM or 120nM human PD-1 antigen solution, respectively, so that the candidate antibody molecules were combined with the antigen for 180s.
  • the probes were then immersed in the buffer, the dissociation time was 1400 s, and the binding and dissociation signals were measured.
  • Affinity KD values were determined by fitting binding dissociation curves using the Octet data analysis software.
  • mice Fifty 6-8-week-old female hPD-1C57 (PD-1 gene humanized) humanized mice (Bioseda) were taken, and MC38WT cells were inoculated into the mice at a concentration of 1 ⁇ 10 6 /0.1 mL. Right subcutaneously, when the tumor grows to about 100mm 3 , the mice are randomly divided into groups according to the tumor volume, 5 mice in each group, 8 groups in total, respectively:
  • Group 1KLH IgG4 (0.3mg/kg) negative control group
  • group 2LL-Pos (0.3mg/kg) positive control group
  • group 3huVHH3-1-Fc (0.3mg/kg) treatment group
  • group 4huVHH3-3-Fc (0.3mg/kg) mg/kg) treatment group
  • group 5huVHH3-5-Fc (0.3 mg/kg) treatment group.
  • the route of administration in all groups was intraperitoneal injection, the dosage was 0.3 mg/kg, and the administration concentration was 0.03 mg/ml.
  • the drug was administered twice a week, 5 times in a row, and the experiment was terminated 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • T the mean tumor volume of the treatment group or the positive control group on the ith day of administration
  • T0 the mean tumor volume of the treatment group or the positive control group on the 0th day of administration
  • Vi the negative control group on the ith day of administration
  • V0 the mean tumor volume of the negative control group on the 0th day of administration
  • the average tumor volume in the negative control group was 1375 ⁇ 115 mm 3 and the average tumor volume in the positive control group was 494 ⁇ 267 mm 3 .
  • the inhibition rate was 69.10%; the mean tumor volumes in the treatment groups of group 3, group 4 and group 5 were 823 ⁇ 85mm 3 , 483 ⁇ 197mm 3 and 320 ⁇ 229mm 3 , respectively, compared with the negative control, the relative tumor inhibition rates were 43.30%, respectively , 70.00% and 82.75%, indicating that the above humanized anti-PD-1 single domain antibody can inhibit the growth of hPD-1 humanized mouse MC38-WT cells subcutaneously transplanted tumor in vivo, and is similar to or significantly better than the positive control antibody.
  • the PD-1 humanized single domain antibody in the present invention has good chemical stability and serum stability, and can be further developed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种靶向人程序性死亡因子PD-1的单域抗体及其人源化变体。所述VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。本发明的靶向PD-1的单域抗体能够阻断PD-1与PD-L1的相互作用，从而具有广泛的生物应用价值和临床应用价值，并为靶向PD-1的治疗或诊断药物的开发奠定了新的物质基础。

Description

靶向PD-1的单域抗体及其衍生物和用途 技术领域
本发明涉及生物技术领域。更具体地说，本发明涉及靶向PD-1的单域抗体及其衍生物和用途。
背景技术
PD-1(程序性死亡受体1，Programmed cell death protein 1，PD-1)及其配体PD-L1是肿瘤免疫的重要靶点。PD-1、PD-L1是一对免疫抑制分子，是免疫系统防止自身免疫过激的重要组成部分，其通路的激活具有抑制肿瘤免疫应答、诱导肿瘤特异性T细胞凋亡的作用,与肿瘤发展关系密切。利用单克隆抗体阻断PD-1、PD-L1通路治疗肿瘤，目前在临床上显示出良好的疗效和安全性，已有多种抗体类药物获批上市，适应症包括黑色素瘤、非小细胞肺癌、晚期肾细胞癌等多种恶性肿瘤，同时很多正在进行的临床试验尝试开发更多新的适应症。
PD-1除了在激活的T细胞、NKT细胞、B细胞和活化的单核细胞表面表达外，在耗竭的T细胞表面也会高表达。而其配体PD-L1除了在B细胞、T细胞、树突状细胞等免疫细胞表达外，还在外周微血管内皮细胞、心脏和肺脏等器官表达。而PD-L2只在巨噬细胞和树突状细胞表面表达。值得注意的是，PD-L1的表达已经在人类肿瘤中被广泛发现，包括人肺癌、黑色素瘤、卵巢癌和结肠癌等。在体外和体内试验中，阻断PD-L1/PD-1通路可以显著增加T细胞的增殖，细胞因子分泌和T细胞对肿瘤细胞的杀伤作用。在动物体内，阻断PD-L1/PD-1通路显著阻断肿瘤生长。使用PD-1抗体阻断PD-1/PD-L1信号通路，显著增强肿瘤特异T细胞的扩增和肿瘤浸润。以上结果证明了PD-1/PD-L1阻断抗体在肿瘤治疗中的重要应用价值。
目前，FDA已经批准三种PD-1抗体用于多种肿瘤的治疗。这些批准的抗体药物在肿瘤病人身上都取得了很好的治疗效果。但是，总体上临床反应率还比较低，一般在20％左右。提高PD-1/PD-L1阻断抗体的疗效的一种策略是采用双特异性抗体或者多靶点融合蛋白，同时阻断包括PD-1/PD-L1通路在内的其他免疫相关或者不相关通路，希望达到协同的更好的治疗效果。基于PD-1/PD-L1通路的双特异性抗体一般采用双链抗体的形式。这种方法制备的双特异性抗体一般分子量较大，使抗体不易浸润到肿瘤组织，很可能影响临床效果。基于双链的双特异性抗体在生产中常存在错配问题，给下游生产纯化造成很大问题。并且目前的PD-1/PD-L1单抗存在用药量大、总体反应率低等问题。
单域抗体(single domain antibody，sdAb)是一类特殊的，只包含一条抗体重链的抗体。和传统双链抗体类似，它可以选择性地与特定抗原结合。单域抗体最早在骆驼科动物中被发现，之后在护士鲨等软骨鱼纲动物中也被发现。单域抗体单个重链抗体可变区(VHH)是能完整结合抗原的单个功能域，只有12-15kDa。VHH结构简单，在与抗原结合时具有高特异性、高亲和力、免疫原性低、渗透性好以及在进行肿瘤治疗时具有接触到不能被常规抗体接触的较为隐蔽靶点的能力等优点。此外，因为单域抗体只有一条链，所以不会产生双链抗体融合 时的错配问题。
发明内容
本发明的目的在于提供一种靶向PD-1的特异性单域抗体。
本发明的目的还在于提供一种靶向PD-1的特异性人源化单域抗体。
本发明的目的还在于提供所述单域抗体或人源化单域抗体在治疗肿瘤或制备治疗肿瘤的药物中的用途。
在第一方面，本发明提供一种靶向PD-1的单域抗体的VHH链，所述VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
在优选的实施方式中，所述PD-1为人PD-1。
在优选的实施方式中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与PD-1高亲和力结合、阻断PD-L1与PD-1结合的衍生序列。
在优选的实施方式中，所述VHH链还包括框架区FR1、FR2、FR3和FR4，所述的FR1、FR2、FR3和FR4如SEQ ID NO:1所示的氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在优选的实施方式中，所述靶向PD-1的单域抗体的VHH链的氨基酸序列如SEQ ID NO:1所示。
在第二方面，本发明提供一种靶向PD-1的抗体的重链可变区，所述的重链可变区包括SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
在优选的实施方式中，所述靶向PD-1的抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示。
在第三方面，本发明提供一种靶向PD-1的单域抗体，其具有第一方面所述的VHH链。
在第四方面，本发明提供一种人源化的靶向PD-1的单域抗体的VHH链，以第一方面所述的VHH链为基础对框架区FR1、FR2、FR3和FR4进行人源化。
在优选的实施方式中，所述人源化的靶向PD-1的单域抗体的VHH链的框架区FR1、FR2、FR3和FR4分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在优选的实施方式中，所述人源化的靶向PD-1的单域抗体的VHH链氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示。
在第五方面，本发明提供一种靶向PD-1的抗体，所述抗体包括一个或多个第一方面所述的靶向PD-1的单域抗体的VHH链或第四方面所述的人源化的靶向PD-1的单域抗体的VHH链。
在优选的实施方式中，所述的靶向PD-1的抗体包括单体、二价抗体、和/或多价抗体。
在第六方面，本发明提供一种双特异性抗体，所述双特异性抗体包括第一抗体和第二抗体，所述第一抗体包括第一方面所述的靶向PD-1的单域抗体的VHH链、或第二方面所述的靶向PD-1的抗体的重链可变区、或第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、或第五方面所述的靶向PD-1的抗体。
在优选的实施方式中，所述第二抗体可以结合与第一抗体相同或不同的抗原，或结合与第一抗体相同抗原的不同表位。
在优选的实施方式中，所述第二抗体是单域抗体、单链抗体或双链抗体。
在优选的实施方式中，所述的双特异性抗体包括2-4个靶向PD-1的单域抗体；较佳地，包括2个靶向PD-1的单域抗体；更佳地，所述2个靶向PD-1的单域抗体形成靶向PD-1的单域抗体二聚体。
在第七方面，本发明提供一种融合蛋白，所述融合蛋白包括第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、或第五方面所述的靶向PD-1的抗体，任选的接头序列以及免疫球蛋白的Fc片段。
在优选的实施方式中，所述免疫球蛋白是是IgG1、IgG2、IgG3、IgG4；优选IgG4。
在第八方面，本发明提供一种核酸分子，所述核酸分子编码第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体或第七方面所述的融合蛋白。
在第九方面，本发明提供一种表达载体，所述表达载体包含第八方面所述的核酸分子。
在第十方面，本发明提供一种宿主细胞，所述宿主细胞包含第九方面所述的表达载体，或者其基因组上整合有第八方面所述的核酸分子。
在第十一方面，本发明提供一种制备第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、或第七方面所述的融合蛋白的方法，所述方法包括以下步骤：
1)在适合的条件下，培养如第十方面所述的宿主细胞，从而获得含有所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体、双特异性抗体或融合蛋白的培养物；以及
2)任选地，从所述培养物中分离或回收所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的单域抗体、双特异性抗体或融合蛋白。
在第十二方面，本发明提供一种免疫偶联物，所述免疫偶联物含有：
1)如第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的 抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体或第七方面所述的融合蛋白；和
2)选自以下的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在优选的实施方式中，所述偶联部分为药物或毒素。
在优选的实施方式中，所述免疫偶联物是抗体-药物偶联物(Antibody-Drug-Conjugate，ADC)。
在优选的实施方式中，所述偶联部分为可检测标记物。
在优选的实施方式中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在优选的实施方式中，所述免疫偶联物含有：多价(如二价)的如权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体或权利要求7所述的融合蛋白。
在优选的实施方式中，所述多价是指，在所述免疫偶联物的氨基酸序列中包含多个重复的部分。
在第十三方面，本发明提供一种药物组合物，所述药物组合物包含治疗或诊断有效量的第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物，和任选的药学上可接受的赋形剂。
在优选的实施方式中，所述的药物组合物用于治疗肿瘤，所述肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
在第十四方面，本发明提供第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物的用途，用于制备以下试剂：
1)检测PD-1的试剂；
2)阻断PD-1与PD-L1结合的试剂；
3)治疗肿瘤的药物。
在优选的实施方式中，所述肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。
在第十五方面，本发明提供一种试剂盒，所述试剂盒中包括：
1)第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白、第十二方面所述的免疫偶联物或第十三方面所述的药物组合物；
2)容器；和
3)任选的使用说明书。
在第十六方面，本发明提供一种制备靶向免疫检查点的抗体的方法，所述方法包括以下步骤：
a)利用表达所述免疫检查点的免疫细胞免疫动物；和
b)从步骤1)获得的经免疫动物中获得靶向免疫检查点的抗体。
在优选的实施方式中，所述免疫细胞是原代免疫细胞。
在优选的实施方式中，所述方法还包括先活化免疫细胞的步骤。
在优选的实施方式中，所述免疫检查点包括但不限于：PD-1、CTLA-4、TIM3、LAG3、KIR、GITR、VISTA、4-1BB、CD28、OX40、ICOS等。
在优选的实施方式中，所述免疫细胞是淋巴细胞或吞噬细胞；优选地，所述免疫细胞是T淋巴细胞、B淋巴细胞、K淋巴细胞、NK淋巴细胞、浆细胞、粒细胞、肥大细胞、抗原呈递细胞或单核吞噬细胞系统的细胞(例如巨噬细胞)；更优选T淋巴细胞。
在优选的实施方式中，所述抗体是多克隆抗体、单克隆抗体、单域抗体；优选单域抗体。
在优选的实施方式中，所述动物包括但不限于小鼠、大鼠、骆驼、羊驼、骆驼、家兔；优选小鼠、羊驼。
在第十七方面，本发明提供一种检测样品中PD-1蛋白的方法，所述方法包括步骤：
1)将待测样品与第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白或第十二方面所述的免疫偶联物接触；
2)检测是否形成抗原-抗体复合物，如果形成复合物就表示样品中存在PD-1蛋白。
在第十八方面，本发明提供一种治疗疾病的方法，所述方法包括，给有此需要的对象施用治疗有效量的第一方面所述的靶向PD-1的单域抗体的VHH链、第二方面所述的靶向PD-1的抗体的重链可变区、第三方面所述的靶向PD-1的单域抗体、第四方面所述的人源化的靶向PD-1的单域抗体的VHH链、第五方面所述的靶向PD-1的抗体、第六方面所述的双特异性抗体、第七方面所述的融合蛋白、第十二方面所述的免疫偶联物或第十三方面所述的 药物组合物。
在优选的实施方式中，所述的对象包括哺乳动物；优选人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了VHH-Fc融合蛋白LL-VHH01-Fc结合PD-1抗原的亲和力检测结果；
图2显示了VHH-Fc融合蛋白LL-VHH01-Fc阻断PD-L1与PD-1蛋白结合的生物活性检测结果；
图3显示了6种人源化抗体均能很好地结合PD-1抗原；
图4显示了6种人源化抗体在细胞水平可以很好地阻断PD-1和PD-L1信号通路；和
图5显示了3种代表性人源化抗体可以在动物体内很好地控制肿瘤生长。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现一类靶向PD-1的单域抗体。本发明的单域抗体与PD-1分子有较好的结合活性，同时能够阻断PD-1与PD-L1的相互作用，具有良好的抗肿瘤活性。在该靶向PD-1的单域抗体的基础上，本发明人进一步开发了一系列人源化单域抗体，结果发现这些人源化单域抗体的亲和力、生物学效果与母本抗体类似或者更好，并且具有很好的化学稳定性和血清稳定性。在此基础上完成了本发明。
术语定义
本文所用的术语具有与本领域技术人员常规理解的相同或相似的含义。为清晰起见，对其中的一些术语定义如下。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
单域抗体
在本文中，“单域抗体”、“单结构域抗体”、“纳米抗体”等具有相同或相似的含 义，均是指缺失抗体轻链、而只有重链可变区的一类抗体分子。单域抗体是最小抗原结合单元，即具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，从而构建仅由一个重链可变区组成的单域抗体(VHH)。
在具体的实施方式中，本发明的靶向PD-1的单域抗体VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。在优选的实施方式中，所述靶向PD-1的单域抗体的VHH链的氨基酸序列如SEQ ID NO:1所示。在所述VHH链中，还包括框架区FR1、FR2、FR3和FR4，它们的氨基酸序列如SEQ ID NO:1所示的氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在本发明的靶向PD-1的单域抗体VHH链的基础上，本发明人还对该VHH链进行了人源化，从而得到了人源化的靶向PD-1的单域抗体的VHH链。在具体的实施方式中，本发明的人源化VHH链的氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示。在所述人源化的VHH链中，还包括框架区FR1、FR2、FR3和FR4，它们的氨基酸序列分别如SEQ ID NO:14、或SEQ ID NO:16、或SEQ ID NO:18、或SEQ ID NO:20、或SEQ ID NO:22、或SEQ ID NO:24所示氨基酸序列的1-25位、36-49位、67-98位和110-120位所示。
在本发明的靶向PD-1的单域抗体VHH链或人源化的VHH链的基础上，本发明还提供了靶向PD-1的抗体，其包括一个或多个所述的靶向PD-1的单域抗体的VHH链或人源化的靶向PD-1的单域抗体的VHH链。本发明还提供了一种双特异性抗体，所述双特异性抗体包括第一抗体和第二抗体，所述第一抗体可以是本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链。本领域技术人员可以按照实际需要选择所述双特异性抗体中的第二抗体。例如，所述第二抗体可以结合与第一抗体相同或不同的抗原；如果第二抗体结合与第一抗体相同的抗原，则优选结合在不同的表位上。在具体的实施方式中，所述第二抗体可以是单域抗体，也可以是单链抗体或双链抗体。
本领域技术人员还可以将本发明的靶向PD-1的单域抗体VHH链或人源化的VHH链制成融合蛋白，例如制备成进一步包含免疫球蛋白的Fc片段的融合蛋白。如此得到的融合蛋白不仅具备单域抗体VHH链本身的生物学活性，还能够具备免疫球蛋白的Fc片段所赋予的其它特性，例如血浆半衰期延长、免疫原性降低、稳定性提高等等。在具体的实施方式中，所述融合蛋白包括本发明的靶向PD-1的单域抗体的VHH链或人源化的VHH链，任选的接头序列以及免疫球蛋白的Fc片段。在具体的实施方式中，所述免疫球蛋白是是IgG1、IgG2、IgG3、IgG4；优选IgG4。
本发明不仅包括完整的抗体，还包括所述抗体的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽， 或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域技术人员公知的范围。
本发明抗体指具有PD-1蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择 的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒素；3.细胞因子，如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；9.治疗剂(例如，顺铂)或任何形式的纳米颗粒等。
免疫检查点
在本文中，免疫检查点(checkpoint)或免疫检查点分子具有相同的含义，并且与本领域技术人员常规理解的相同。其是指在免疫细胞上表达、能调节免疫激活程度的一系列分子，它们对防止自身免疫(免疫功能发生异常，对正常细胞发动攻击)作用的发生起重要作用。因此，“免疫检查点分子”的重要功能之一就是使免疫系统的活化保持在正常的范围之内，以防止免疫系统的过度活化。
免疫检查点分子的表达和功能异常是很多疾病发生的重要原因之一，比如免疫检查点分子过度表达或功能过强，免疫功能受到抑制，机体的免疫力就低下，人就容易得肿瘤等疾病；反之，免疫检查点分子的这种免疫抑制功能如果太差，机体的免疫功能也会异常。肿瘤细胞会表达一些物质，来激活免疫检查点，后者一旦被激活，则抗原不能被提呈至T细胞，阻断了肿瘤免疫环中的提呈抗原过程，从而抑制T细胞的免疫功能，使得肿瘤细胞逃脱免疫监视，进而存活。
常用的免疫检查点有CTLA-4(细胞毒T淋巴细胞相关抗原-4，cytotoxic T lymphocyte-associated antigen-4)、PD-1、TIM3、LAG3、KIR、GITR、VISTA、4-1BB等。
免疫细胞
在本文中，免疫细胞和免疫效应细胞具有相同的含义，并且与本领域技术人员常规理解的相同。其是指参与免疫应答或与免疫应答相关的细胞，包括淋巴细胞和吞噬细胞。在具体的实施方式中，所述免疫细胞是指能识别抗原、从而产生特异性免疫应答的淋巴细胞。所述淋巴细胞主要是T淋巴细胞、B淋巴细胞、K淋巴细胞和NK淋巴细胞。除淋巴细胞外，参与免疫应答的细胞还有浆细胞、粒细胞、肥大细胞、抗原呈递细胞及单核吞噬细胞系统的 细胞(例如巨噬细胞)。
免疫偶联物
本发明还提供一种免疫偶联物，其含有本发明的靶向PD-1的单域抗体的VHH链、人源化的VHH链等以及偶联部分。在具体的实施方式中，所述偶联部分可以是可检测标记物、药物、毒素、细胞因子、放射性核素或酶等，从而实现诊断、检测或治疗等等目的。
在优选的实施方式中，所述免疫偶联物是抗体-药物偶联物(Antibody-Drug-Conjugate，ADC)。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-1蛋白，从而阻断PD-1和PD-L1的相互作用。因此，本发明的药物组合物可用于治疗肿瘤。在优选的实施方式中，所述肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、或膀胱肿瘤。此外，本发明的药物组合物还可与其他治疗剂联用。
本发明的药物组合物含有安全有效量(如0.001-99wt％、较佳地0.01-90wt％、更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。
活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测方法
本发明还涉及检测PD-1蛋白的方法。该方法步骤大致如下：将待测样品与本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链、抗体、融合蛋白或免疫偶联物等接触；随后检测是否形成抗原-抗体复合物，如果形成复合物就表示样品中存在PD-1蛋白。
试剂盒
本发明还提供了一种含有本发明的靶向PD-1的单域抗体的VHH链、或人源化的VHH链、抗体、融合蛋白或免疫偶联物等的试剂盒。在具体的实施方式中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
制备靶向免疫检查点的抗体的方法
在研究过程中，本发明人发现直接利用PD-1蛋白免疫动物得到的PD-1单域抗体的活性不佳。然而，当本发明人利用T细胞进行免疫时，获得了活性很好的PD-1单域抗体。
基于上述发现，本发明还提供了一种特殊的免疫方法，该方法能够制备靶向免疫检查点的抗体。所述方法包括利用表达免疫检查点的免疫细胞免疫动物。在优选的实施方式中，可以在利用表达免疫检查点的免疫细胞免疫动物之前先活化所述免疫细胞。活化免疫细胞的方法或技术手段是本领域技术人员熟知的。
本发明的制备靶向免疫检查点的抗体的方法中，利用的免疫细胞优选原代免疫细胞。在本发明的方法中，所免疫的动物包括但不限于小鼠、骆驼、羊驼、大鼠、家兔；优选小鼠、羊驼。本发明的方法获得的抗体可以是多克隆抗体、单克隆抗体、单域抗体。
本发明的优点：
1.本发明的靶向PD-1的单域抗体与PD-1具有良好的结合亲和力，从而能够有效阻断PD-1和PD-L1结合；
2.本发明的人源化单域抗体能够保留与PD-1的良好结合亲和力，并且在体外细胞水平表现出与母本抗体类似、甚至更好的生物学效果；
3.本发明的人源化单域抗体具有很好的化学稳定性和血清稳定性；和
4.本发明的靶向PD-1的单域抗体和人源化单域抗体具有广泛的生物应用价值和临床应用价值，从而为靶向PD-1的治疗或诊断药物的开发奠定了新的物质基础。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：噬菌体展示免疫库的构建
1.1动物免疫
将冻存的人外周血单个核细胞(购自妙通生物)复苏后，将浓度调整到2*10 6/ml。然后按照说明书推荐比例加入负载CD3抗体和CD28抗体的微型磁珠(Thermo Fisher Scientific)以 激活PBMCs中的T细胞。激活T细胞以后，使用磁力架去磁珠；之后按照1600rpm，5分钟条件离心收取细胞；使用冻存培养基冻存细胞。冻存培养基为：RPMI-1640:FBS:DMSO＝4:5:1。暂时将冻存的细胞储存于液氮中。
选取一只成年健康的羊驼(Alpaca)。首先将冻存的T细胞复苏，然后使用1*PBS洗涤细胞一次。使用缓冲液重悬细胞后对羊驼进行皮下免疫。之后采用相同的方法对羊驼进行五次免疫。
1.2噬菌体文库构建
免疫结束后，采集羊驼四免和五免之后的外周血各50mL，将两者混合后分离外周血单个核细胞(PBMCs)。进一步使用RNAiso Plus试剂(Takara，货号：9109)提取总RNA。使用PrimeScript TM II 1st Strand cDNA Synthesis Kit(Takara，货号：6210A)试剂盒，按照试剂盒提供的说明书，对提取的总RNA进行反转录。使用巢式PCR扩增羊驼重链可变区的DNA片段。共进行两轮巢式PCR。
将载体(pComb3XSS)与PCR扩增的目的片段分别使用SfiI进行酶切，50℃过夜酶切，然后回收目的片段。将酶切后的PCR产物和载体进行连接，连接摩尔比例为载体:PCR产物＝1:3。使用电转化的方法将连接有VHH的载体导入到感受态细胞中，构建重链单域抗体噬菌体展示文库。电转后进行铺板。为检测文库的插入率，随机选取板上48个克隆做菌落PCR。结果显示插入率达到100％。通过梯度稀释铺板，计算库容的大小为1.0×10 9。将细菌文库接入2×300mL 2YT+A+G(Amp:100ug/ml、Glu:1％)培养基中，至其初始OD 600＝0.1-0.2，37℃、230rpm培养至OD 600＝0.8以上。根据OD 600值加入辅助噬菌体M13KO7，(辅助噬菌体:细菌＝20:1)，制备噬菌体文库。通过梯度稀释噬菌体文库然后铺板，计算克隆数的方法测到噬菌体文库滴度为5.76×10 13cfu/mL。
实施例2：抗人PD-1单域抗体筛选
通过固相筛选的方法，对所构建的羊驼免疫库进行亲和力筛选，获得特异性噬菌体文库。
2.1亲和淘选
2.1.1淘选
1)使用pH值为9.6的碳酸盐缓冲液稀释靶抗原，终浓度为5μg/mL。然后按100μL/孔加入到酶标孔中，4℃包被过夜；
2)弃包被液，使用PBS洗涤3次。然后每孔中加入300μL 3％BSA-PBS封闭液，37℃条件封闭1小时；
3)之后弃掉封闭液，使用PBS洗涤3次。加入100μL噬菌体文库，37℃孵育1小时；
4)吸出未结合的噬菌体，用PBST洗涤平板6次，PBS洗涤平板2次；
5)加入100μL的Gly-HCl洗脱液到每个孔中，37℃孵育8min；
6)将洗脱液转移至1.5mL无菌离心管中，迅速用15μL Tris-HCl中和缓冲液中和；
7)取10μL中和后的溶液进行梯度稀释，测定噬菌体滴度，计算淘选回收率。将其余 洗脱物混合后进行扩增和纯化，用于下一轮亲和淘选。改变淘选条件，每一轮淘选条件如表1。
表1.亲和淘选条件
Figure PCTCN2022071865-appb-000001
2.1.2文库扩增
淘选后需要将文库进行扩增，主要步骤如下：
1)将淘选洗脱物与处于对数生长前期的E.coli TG1培养物5mL混匀，37℃条件静置30min，然后220r/min振荡培养30min；
2)1000g离心15min，去上清，用500μL 2×YT重悬涂布于200mm 2×YT-GA平板；
3)用10ml 2×YT液体培养基刮菌，取500μl悬液加入50ml 2×YT液体培养基中，37℃振摇30min；按cell:phage＝1:20的比例加入M13K07辅助噬菌体，37℃静置30min，220r/min振摇培养30min；
4)将培养物分装于离心管中，25℃，5000r/min，10min，细胞沉淀以50mL 2×YT-AK液体培养基重悬，30℃，230r/min振荡培养过夜；
5)将过夜培养物4℃，10000r/min离心20min，将上清转移至新离心管，加入1/5体积的PEG-NaCl，混匀后置于4℃2h以上；
6)离心，4℃，10000r/min，20min，去除上清，将沉淀重悬于1mL PBS中，加入1/5体积的PEG/NaCl，混匀后置于4℃1h以上；
7)4℃，12000r/min，2min，去除上清，将沉淀悬浮于200μL PBS中，即为扩增产物，测定滴度，用于下一轮淘选或者分析。
2.1.3噬菌体救援
1)从第二轮淘选洗脱物滴度的平板上用灭菌牙签随机挑取96个单克隆，接种于1mL2×YT-A中，37℃，220r/min振荡培养8h；
2)取200μL上述培养物，按cell:phage＝1:20的比例加入M13K07噬菌体，37℃，静置15min；
3)220r/min振荡培养45min；
4)补加800μL体积的2×YT-AK，30℃，剧烈振荡培养过夜；
5)第二天12000rpm离心2min；
6)取上清，用于单克隆ELISA鉴定。
2.1.4阳性噬菌体克隆鉴定
1)将PD-1抗原用pH值为9.6的碳酸盐缓冲液稀释至终浓度为2μg/mL，按100μL/ 孔加入酶标孔中，4℃包被过夜；
2)弃包被液，PBST洗涤3次；
3)每孔加入300μL 5％脱脂牛奶，37℃封闭1h；
4)PBST洗涤3次，每孔加入50μL噬菌体培养菌液上清和50μL 5％脱脂牛奶，37℃，孵育1h；
5)PBST洗涤5次，之后加入辣根过氧化物酶标记的抗M13抗体(用5％脱脂牛奶按1:10000稀释)，100μL/孔，37℃作用1h；
6)PBST洗板6次。加入TMB显色液显色，100μL/孔，37℃，7min；
7)加入终止液终止反应，50μL/孔，于450nm下测光密度。
2.2阳性克隆的序列确定
结果检测到三个阳性克隆，将三个阳性细菌克隆送苏州泓迅生物技术有限公司进行测序。其中一个克隆序列测序成功。测序结果分析得到抗PD-1抗体VHH区，将该VHH命名为LL-VHH01。使用Kabat命名法对LL-VHH01进行CDR区分析，得到对应的CDR区域序列。LL-VHH01的全长和CDR区的氨基酸序列见表2，核酸序列见表3。
表2.VHH的氨基酸序列和对应的CDR氨基酸序列
Figure PCTCN2022071865-appb-000002
LL-VHH01的氨基酸序列为SEQ ID NO:1，CDR1序列为SEQ ID NO:2，CDR2序列为SEQ ID NO:3、CDR3的序列为SEQ ID NO:4。
表3.LL-VHH01的核酸序列和对应的CDR核酸序列
Figure PCTCN2022071865-appb-000003
实施例3：制备PD-1单域抗体VHH-Fc融合蛋白
通过数据库Uniprot获得人免疫球蛋白gamma4(IgG4)的恒定区氨基酸序列(P01861)。
Figure PCTCN2022071865-appb-000004
将PD-1单域抗体VHH氨基酸序列和hIgG4-Fc氨基酸序列连接，得到PD-1VHH-Fc融合蛋白，命名为：LL-VHH01-Fc。通过基因合成方式得到抗人PD-1单域抗体VHH-Fc融合蛋白LL-VHH01-Fc的基因序列。
然后将PD-1VHH-Fc融合蛋白基因克隆至表达载体pCDNA4(Invitrogen，Cat V86220)中。使用瞬时转染HEK293悬浮细胞的方法对PD-1单域抗体VHH-Fc融合蛋白LL-VHH01-Fc进行表达。表达完成后，收取上清液，使用Protein A亲和层析柱纯化，最终获得纯化的PD-1单域抗体的Fc融合蛋白LL-VHH01-Fc。
LL-VHH01-Fc
基因序列为：SEQ ID NO:10
Figure PCTCN2022071865-appb-000005
氨基酸序列为：SEQ ID NO:11
Figure PCTCN2022071865-appb-000006
实施例4:制备PD-1阳性对照抗体
通过专利WO2008156712A1，获得Merck Sharp&Dohme(MSD)公司的抗PD-1抗体Pembrolizumab氨基酸序列。其中重链氨基酸序列为SEQ ID NO:12
Figure PCTCN2022071865-appb-000007
轻链氨基酸序列为SEQ ID NO:13
Figure PCTCN2022071865-appb-000008
同时参考专利中抗体的表达纯化的方法对Pembrolizumab抗体类似物进行表达纯化。得到MSD公司的抗PD-1抗体类似物，重新命名为LL-Pos。
实施例5:ELISA法检测PD-1单域抗体VHH-Fc融合蛋白对PD-1蛋白的结合能力
首先将人PD-1蛋白(购自北京百普赛斯，货号PD1-H5221)使用PBS(购自Hyclone公司)稀释至1.5μg/mL，按照100μl/孔加入96孔酶标板进行抗原包被，置于37℃恒温培养箱中孵育60min。孵育结束后洗板三次并按照200μl/孔加入含有2％BSA的PBS进行封闭，置于37℃恒温培养箱内孵育60min。同时，将样品LL-VHH01-Fc及对照抗体LL-Pos用稀释液(含有2％BSA的PBS)稀释至100μg/ml，并在样品稀释板上以3倍梯度依次稀释至0.56ng/ml。然后，按照100μl/孔将样品加入到96孔酶标板中并置于37℃恒温培养箱内孵育60min。孵育结束后洗板并将二抗山羊抗-人IgG(Fc特异性)-HRP抗体(购自Sigma公司，货号A-0170)用2％BSA稀释6000倍，按照100μl/孔加入酶标板并置于37℃恒温培养箱内孵育30min。孵育结束后洗板三次并按照100μl/孔加入显色液，显色液为100μg/ml的TMB(3,3',5,5'-四甲基联苯胺)，置于37℃恒温培养箱内避光孵育15min。最后，按照100μl/孔加入终止液(2M/L盐酸溶液)并使用酶标仪(Thermo Fisher Scientific，Varioskan LUX)检测450nm/620nm测吸收值，使用Graphpad Prism分析数据。
结果如图1所示，LL-Pos结合PD-1蛋白的EC 50为11.84ng/ml。而LL-VHH01-Fc的EC 50为29.69ng/ml。在该实验条件下，LL-VHH01-Fc结合PD-1蛋白的量效反应曲线的上平台值明显高于LL-Pos。综合来看，本发明VHH的Fc融合蛋白对PD-1抗原的亲和力与对照抗体LL-Pos基本相当。
实施例6：使用荧光素酶报告基因法检测VHH-Fc融合蛋白的生物学活性
培养CHO/PD-L1细胞(购自Promega公司)后消化并用含有10％FBS的F-12营养混合物完全培养基重悬细胞，根据细胞计数结果使用完全培养基调整细胞密度至5×10 5个细胞 /ml。随后将细胞悬液转移至加样槽中并使用多道移液器以100μl/孔加入到96孔板中，放置于37℃的5％CO 2培养箱中培养16～22h。同时，培养Jurkat/PD-1细胞(购自Promega公司)细胞并根据细胞计数结果使用分析培养基(RPMI 1640 Medium+2％FBS)调整细胞密度至2×10 6个细胞/ml。将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出并使用多道移液器每孔取出100μl培养液，随后按照40μl/孔加入梯度稀释(起始浓度为1650nM)的待测样品。然后，将上述Jurkat/PD-1细胞悬液转移至加样槽中并按照40μl/孔加入到细胞培养板中，置于37℃的5％CO 2培养箱培养4～6h。培养结束后取出细胞培养板，置于室温放置5～10min，然后每孔加入40μl One-Glo试剂(购自Promega公司，货号E6130)，置于混匀器上混匀5～10min，然后使用多功能酶标仪读取化学发光信号值，使用Graphpad Prism分析数据。
结果如图2所示，LL-VHH01-Fc可有效阻断PD-1和PD-L1结合，并激活下游通路产生信号，其量效曲线的EC 50为0.35μg/ml。阳性对照LL-Pos也可有效阻断PD-1和PD-L1结合，并激活下游通路产生信号，其量效曲线的EC 50为1.09μg/ml。结果表明LL-VHH01-Fc阻断PD-1和PD-L1结合的能力类似于阳性对照。
图1和图2的结果表明本发明的PD-1单域抗体融合蛋白在对抗原PD-1的亲和力和对PD-1和PD-L1结合的阻断作用方面都等同于或者优于已经上市的对照抗体，因此可能在临床上会有更好的效果。另外，单域抗体没有轻链，非常适合作为重要的组成抗体进行双抗或者多特异性抗体的开发，有望开发出疗效更好的抗体药物。
实施例7：单域抗体的人源化
LL-VHH01为羊驼来源抗体，为了提高该单域抗体的成药性，对LL-VHH01进行人源化。基本步骤：
1.使用IMGT数据库进行抗体序列比对。根据数据库序列比对结果，选取IGHV3-23*04作为LL-VHH01人源化母本载体；
2.将LL-VHH01单域抗体的CDR区移植到IGHV3-23*04；
3.对移植后的人源化抗体进行回复突变以确保人源化抗体的亲和力。
得到六个候选人源化抗体：
>huVHH3-1
Figure PCTCN2022071865-appb-000009
核酸序列：
Figure PCTCN2022071865-appb-000010
>huVHH3-2
Figure PCTCN2022071865-appb-000011
核酸序列：
Figure PCTCN2022071865-appb-000012
>huVHH3-3
Figure PCTCN2022071865-appb-000013
核酸序列：
Figure PCTCN2022071865-appb-000014
>huVHH3-4
Figure PCTCN2022071865-appb-000015
核酸序列：
Figure PCTCN2022071865-appb-000016
>huVHH3-5
Figure PCTCN2022071865-appb-000017
核酸序列：
Figure PCTCN2022071865-appb-000018
>huVHH3-6
Figure PCTCN2022071865-appb-000019
核酸序列：
Figure PCTCN2022071865-appb-000020
实施例8.制备人IgG4Fc标签的六种人源化抗体
参考实施例3的实验流程，表达、纯化融合人IgG4Fc标签的六种人源化抗体，分别命名为：huVHH3-1-Fc、huVHH3-2-Fc、huVHH3-3-Fc、huVHH3-4-Fc、huVHH3-5-Fc、huVHH3-6-Fc。六种人源化抗PD-1单域抗体融合hIgG4Fc的序列如下：
>huVHH3-1-Fc
Figure PCTCN2022071865-appb-000021
核酸序列：
Figure PCTCN2022071865-appb-000022
>huVHH3-2-Fc
Figure PCTCN2022071865-appb-000023
Figure PCTCN2022071865-appb-000024
核酸序列：
Figure PCTCN2022071865-appb-000025
>huVHH3-3-Fc
Figure PCTCN2022071865-appb-000026
核酸序列：
Figure PCTCN2022071865-appb-000027
>huVHH3-4-Fc
Figure PCTCN2022071865-appb-000028
核酸序列：
Figure PCTCN2022071865-appb-000029
>huVHH3-5-Fc
Figure PCTCN2022071865-appb-000030
核酸序列：
Figure PCTCN2022071865-appb-000031
Figure PCTCN2022071865-appb-000032
>huVHH3-6-Fc
Figure PCTCN2022071865-appb-000033
核酸序列：
Figure PCTCN2022071865-appb-000034
实施例9.检测人源化抗体对抗原PD-1的亲和力
参考实施例5的实验步骤，检测人源化抗体对抗原PD-1的亲和力。试验结果见图3，结果显示huVHH3-1-Fc、huVHH3-2-Fc、huVHH3-3-Fc、huVHH3-4-Fc、huVHH3-5-Fc、huVHH3-6-Fc结合PD-1蛋白的EC 50分别为29.25ng/ml、40.50ng/ml、46.96ng/ml、13.31ng/ml、47.61ng/ml和45.97ng/ml。实验结果说明，六种人源化抗体在人源化以后亲和力与母本抗体类似或者更好。
实施例10.检测人源化抗体在体外阻断PD-1/PD-L1信号通路的细胞活性
参考实施例6的实验步骤，检测人源化抗体在体外阻断PD-1/PD-L1信号通路的细胞活性。试验结果见图4。实验结果说明，六种人源化抗体在人源化以后在体外细胞水平表现出与母本抗体类似或者更好的生物学效果。
实施例11.六种人源化抗体的亲和力测定
本次实验使用Fortebio公司的Octet RED 96e型分子相互作用分析仪。使用Protein A探针(Fortebio)先分别捕获候选抗体分子(浓度为5μg/mL)。再将探针分别浸入30nM或120nM的人PD-1抗原溶液中，使候选抗体分子与抗原结合时间为180s。然后将探针浸入缓冲液中，解离时间为1400s，测量结合与解离信号。使用软件Octet data analysis software拟合结合解离曲线来确定亲和力KD值。
实验结果见表4。由表4的结果可以看出六种PD-1单域重组抗体都可以以较高的亲和力结合抗原并且对抗原结合的亲和力差别不大。
表4.六种人源化PD-1重组抗体与抗原的结合情况
Figure PCTCN2022071865-appb-000035
实施例12.人源化PD-1单域抗体的体内药效学评估
取50只6-8周龄雌性hPD-1C57(PD-1基因人源化)人源化小鼠(百奥塞图)，将MC38WT细胞以1×10 6个/0.1mL浓度接种小鼠的右侧皮下，待肿瘤生长到约100mm 3时将小鼠按肿瘤体积随机分组，每组5只，共8组，分别为：
组1KLH IgG4(0.3mg/kg)阴性对照组、组2LL-Pos(0.3mg/kg)阳性对照组、组3huVHH3-1-Fc(0.3mg/kg)治疗组、组4huVHH3-3-Fc(0.3mg/kg)治疗组、组5huVHH3-5-Fc(0.3mg/kg)治疗组。
所有组的给药途径均为腹腔注射，给药剂量为0.3mg/kg，给药浓度为0.03mg/ml。每周给药2次，连续给药5次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时将小鼠安乐死，计算相对肿瘤抑制率TGI％＝(1-(Ti-T0)/(Vi-V0))×100％。(Ti：治疗组或阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组或阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值)。
如图5所示，在小鼠接种肿瘤细胞后第28天，阴性对照组平均肿瘤体积为1375±115mm 3，阳性对照组平均肿瘤体积为494±267mm 3，与阴性对照组相比，相对肿瘤抑制率为69.10％；组3、组4、组5治疗组平均肿瘤体积分别为823±85mm 3、483±197mm 3和320±229mm 3，与阴性对照相比，相对肿瘤抑制率分别为43.30％、70.00％和82.75％，表明上述人源化抗PD-1单域抗体能够体内抑制hPD-1人源化小鼠MC38-WT细胞皮下移植瘤的生长，且类似或者明显优于阳性对照抗体。
实施例13.代表性人源化抗体的化学稳定性和血清稳定性检测
将候选分子置换到pH 5.5的缓冲液中(20mM醋酸-醋酸钠，220mM海藻糖，0.02％聚山梨酯80)，控制样品浓度在10mg/ml左右，分装一定样品体积(200μl/管)放置40℃恒温箱，考察0周、2周和4周稳定性，根据时间点送样检测SEC-HPLC纯度和结合ELISA活性。检测结果显示3个候选分子具有良好的化学稳定性，单体纯度和结合ELISA活性未见明显下降，详见表5。
表5.三种代表性人源化抗体的化学稳定性检测
Figure PCTCN2022071865-appb-000036
将候选分子添加到人血清中，控制样品浓度在1mg/ml左右，分装一定样品体积(200μl/管)放置37℃恒温箱，考察0周、2周和4周稳定性，根据时间点送样检测结合ELISA活性。检测结果显示3个候选分子具有良好的血清稳定性，结合ELISA活性未见明显下降，详见表6。
表6.三种代表性人源化抗体的血清稳定性检测
Figure PCTCN2022071865-appb-000037
以上结果说明，本发明中PD-1人源化单域抗体具有很好的化学稳定性和血清稳定性，可以进行后续开发。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种靶向PD-1的单域抗体的VHH链，所述VHH链包括SEQ ID NO:2所示的CDR1、SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
  2. 一种靶向PD-1的抗体的重链可变区，所述的重链可变区包括SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:4所示的CDR3。
  3. 一种靶向PD-1的单域抗体，其具有权利要求1所述的VHH链。
  4. 一种人源化的靶向PD-1的单域抗体的VHH链，其特征在于，以权利要求1所述的VHH链为基础对框架区FR1、FR2、FR3和FR4进行人源化。
  5. 一种靶向PD-1的抗体，所述抗体包括一个或多个如权利要求1所述的靶向PD-1的单域抗体的VHH链或如权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链。
  6. 一种双特异性抗体，所述双特异性抗体包括第一抗体和第二抗体，所述第一抗体包括权利要求1所述的靶向PD-1的单域抗体的VHH链、或权利要求2所述的靶向PD-1的抗体的重链可变区、或权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、或权利要求5所述的靶向PD-1的抗体。
  7. 一种融合蛋白，所述融合蛋白包括权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、或权利要求5所述的靶向PD-1的抗体，任选的接头序列以及免疫球蛋白的Fc片段。
  8. 一种核酸分子，所述核酸分子编码权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体或权利要求7所述的融合蛋白。
  9. 一种表达载体，所述表达载体包含权利要求8所述的核酸分子。
  10. 一种宿主细胞，所述宿主细胞包含权利要求9所述的表达载体，或者其基因组上整合有权利要求8所述的核酸分子。
  11. 一种制备权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体、或权利要求7所述的融合蛋白的方法，所述方法包括以下步骤：
    1)在适合的条件下，培养如权利要求10所述的宿主细胞，从而获得含有所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体、双特异性抗体或融合蛋白的培养物；以及
    2)任选地，从所述培养物中分离或回收所述的靶向PD-1的单域抗体的VHH链、靶向PD-1的抗体的重链可变区、靶向PD-1的单域抗体、人源化的靶向PD-1的单域抗体的VHH 链、靶向PD-1的单域抗体、双特异性抗体或融合蛋白。
  12. 一种免疫偶联物，所述免疫偶联物含有：
    1)如权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体或权利要求7所述的融合蛋白；和
    2)选自以下的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  13. 一种药物组合物，所述药物组合物包含治疗或诊断有效量的权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体、权利要求7所述的融合蛋白或权利要求12所述的免疫偶联物，和任选的药学上可接受的赋形剂。
  14. 权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体、权利要求7所述的融合蛋白或权利要求12所述的免疫偶联物的用途，用于制备以下试剂：
    1)检测PD-1的试剂；
    2)阻断PD-1与PD-L1结合的试剂；
    3)治疗肿瘤的药物。
  15. 一种试剂盒，所述试剂盒中包括：
    1)权利要求1所述的靶向PD-1的单域抗体的VHH链、权利要求2所述的靶向PD-1的抗体的重链可变区、权利要求3所述的靶向PD-1的单域抗体、权利要求4所述的人源化的靶向PD-1的单域抗体的VHH链、权利要求5所述的靶向PD-1的抗体、权利要求6所述的双特异性抗体、权利要求7所述的融合蛋白、权利要求12所述的免疫偶联物或权利要求13所述的药物组合物；
    2)容器；和
    3)任选的使用说明书。
  16. 一种制备靶向免疫检查点的抗体的方法，所述方法包括以下步骤：
    a)利用表达所述免疫检查点的免疫细胞免疫动物；和
    b)从步骤1)获得的经免疫动物中获得靶向免疫检查点的抗体。
PCT/CN2022/071865 2021-01-14 2022-01-13 靶向pd-1的单域抗体及其衍生物和用途 WO2022152222A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110048196.1 2021-01-14
CN202110048196 2021-01-14
CN202110343322.6A CN114763384B (zh) 2021-01-14 2021-03-30 靶向pd-1的单域抗体及其衍生物和用途
CN202110343322.6 2021-03-30

Publications (1)

Publication Number Publication Date
WO2022152222A1 true WO2022152222A1 (zh) 2022-07-21

Family

ID=82364535

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/071865 WO2022152222A1 (zh) 2021-01-14 2022-01-13 靶向pd-1的单域抗体及其衍生物和用途

Country Status (2)

Country Link
CN (1) CN114763384B (zh)
WO (1) WO2022152222A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115558029B (zh) * 2021-10-22 2024-01-30 立凌生物制药(苏州)有限公司 靶向pd-1的双特异性抗体、及其制备和应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017020802A1 (zh) * 2015-07-31 2017-02-09 苏州康宁杰瑞生物科技有限公司 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
CN110003336A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 Pd-1单域抗体、核苷酸序列及试剂盒
WO2019137541A1 (en) * 2018-01-15 2019-07-18 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against pd-1
WO2019158113A1 (zh) * 2018-02-14 2019-08-22 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
WO2020077257A1 (en) * 2018-10-11 2020-04-16 Inhibrx, Inc. Pd-1 single domain antibodies and therapeutic compositions thereof
WO2020127367A1 (en) * 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017020802A1 (zh) * 2015-07-31 2017-02-09 苏州康宁杰瑞生物科技有限公司 针对程序性死亡配体(pd-l1)的单域抗体及其衍生蛋白
CN108285485A (zh) * 2018-01-08 2018-07-17 乌鲁木齐恒康致远生物技术有限公司 一种抗pd-1的单域抗体及其应用
WO2019137541A1 (en) * 2018-01-15 2019-07-18 Nanjing Legend Biotech Co., Ltd. Single-domain antibodies and variants thereof against pd-1
WO2019158113A1 (zh) * 2018-02-14 2019-08-22 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
WO2020077257A1 (en) * 2018-10-11 2020-04-16 Inhibrx, Inc. Pd-1 single domain antibodies and therapeutic compositions thereof
WO2020127367A1 (en) * 2018-12-21 2020-06-25 Ose Immunotherapeutics Humanized anti-human-pd-1 antibody
CN110003336A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 Pd-1单域抗体、核苷酸序列及试剂盒

Also Published As

Publication number Publication date
CN114763384A (zh) 2022-07-19
CN114763384B (zh) 2023-03-17

Similar Documents

Publication Publication Date Title
CN109096396B (zh) 一种抗pd-l1人源化纳米抗体及其应用
US11292841B2 (en) Anti-PD-1 nano-antibody and application thereof
AU2017305366B2 (en) Anti-PD-L1 nanobody and use thereof
US11466085B2 (en) Anti-PD-L1 nanobody, coding sequence and use thereof
US11912770B2 (en) Blocking type PD-L1 single-domain camel antibody and application thereof
US20220002418A1 (en) Anti-pd-l1/vegf bifunctional antibody and use thereof
WO2018233575A1 (zh) 阻断型cd47纳米抗体及其用途
WO2019024911A1 (zh) B7h3抗体-药物偶联物及其医药用途
WO2021213478A1 (zh) 抗人b7-h3的单克隆抗体及其应用
CN114195900B (zh) 一种抗4-1bb/pd-l1双特异性抗体及其用途
WO2021219127A1 (zh) 一种靶向her2和pd-1的双特异性抗体及其应用
WO2022152222A1 (zh) 靶向pd-1的单域抗体及其衍生物和用途
WO2020108636A1 (zh) 全人抗gitr抗体及其制备方法
WO2023125842A1 (zh) 一种新型upar单域抗体的开发
CN115315445B (zh) 一种靶向人cd47的单域抗体及其用途
CA3224617A1 (en) Anti-trop2 single-domain antibody and use thereof
WO2021083248A1 (zh) 抗tspan8单克隆抗体及其用途
CN113461825A (zh) 一种抗pd-l2纳米抗体及其应用
WO2023116781A1 (zh) 一种新型pd1单域抗体的开发
WO2023125973A1 (zh) 一种新型pdl1单域抗体的开发
CN115558029B (zh) 靶向pd-1的双特异性抗体、及其制备和应用
WO2022142272A1 (zh) Cldn18.2抗体及其应用
WO2023103962A1 (zh) Tnfr2结合分子及其用途
CN114805581A (zh) 靶向il13ra2的抗体、嵌合抗原受体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22739098

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22739098

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 22739098

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 22739098

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 22/05/2024)