WO2023274183A1 - Cd16抗体及其应用 - Google Patents

Cd16抗体及其应用 Download PDF

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WO2023274183A1
WO2023274183A1 PCT/CN2022/101713 CN2022101713W WO2023274183A1 WO 2023274183 A1 WO2023274183 A1 WO 2023274183A1 CN 2022101713 W CN2022101713 W CN 2022101713W WO 2023274183 A1 WO2023274183 A1 WO 2023274183A1
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antibody
seq
antigen
binding fragment
binding
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French (fr)
Inventor
王琼
付雅媛
杨翠青
曹卓晓
唐任宏
任晋生
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Jiangsu Simcere Pharmaceutical Co Ltd
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Jiangsu Simcere Pharmaceutical Co Ltd
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Priority to EP22831997.6A priority Critical patent/EP4365199A4/en
Priority to JP2023580479A priority patent/JP2024524378A/ja
Priority to US18/574,812 priority patent/US20240343811A1/en
Priority to CN202280046034.7A priority patent/CN117580865A/zh
Publication of WO2023274183A1 publication Critical patent/WO2023274183A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/283—Immunoglobulins [IG], 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 Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)

Definitions

  • the present invention relates to the field of biomedicine, in particular to CD16 antibody and its application.
  • NK cells Natural killer cells
  • NK cells are a component of the innate immune system, accounting for approximately 5-15% of circulating lymphocytes. Unlike B cells and T cells, NK cells do not express somatically rearranged antigen receptors, but instead express a series of activating and inhibitory receptors, the integration of activating and inhibitory signals from the interaction of ligands and different receptors And the balance determines the state of NK cell activation.
  • Activated NK cells kill target cells by similar means as cytotoxic T cells, namely by cytolytic granules containing perforin and granzymes and by the death receptor pathway.
  • Activated NK cells also secrete inflammatory cytokines such as IFN- ⁇ and chemokines that promote the recruitment of other inflammatory cells to target tissues.
  • NK cells do not require antigen priming and recognize targets by activating receptors in the absence of MHC recognition.
  • MHC I Class I HLA
  • the human IgG Fc receptor CD16 (Fc ⁇ RIII) consists of two subtypes (CD16a/Fc ⁇ RIIIa and CD16b/Fc ⁇ RIIIb), encoded by two highly homologous genes.
  • CD16b (Fc ⁇ RIIIb), mainly expressed on neutrophils, is a GPI-anchored glycoprotein that lacks the intracellular signal transduction domain.
  • CD16b There are gene polymorphisms in CD16b, which can produce three allotypes, namely NA1, NA2 and SH.
  • CD16a is a low-affinity receptor for human IgG Fc, a single transmembrane protein that is involved in antibody-dependent cellular cytotoxicity (ADCC) and triggers specific lysis of target cells by natural killer (NK) cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCC is one of the dominant mechanisms of cytotoxicity by Fc ⁇ R-expressing effector cells to clear tumor cells.
  • a single nucleotide substitution (SNP) from G to T at position 559 of the CD16a gene in the cDNA of the CD16a gene resulted in two distinct Fc ⁇ RIIIa allotypes: at position 158 of the amino acid sequence, one encoded as valine Acid (V), the other encoded as phenylalanine (F).
  • V valine Acid
  • F phenylalanine
  • the presence of valine (V/V or V/F) enhances the binding affinity of NK cells for IgG1 or IgG3 antibodies compared to homozygous phenylalanine genotypes (F/F), resulting in higher NK Levels of cell-mediated ADCC.
  • NK cell-mediated ADCC is one of the mechanisms of anticancer effects of commonly used antibodies such as rituximab, trastuzumab, and cetuximab.
  • NK cells The ADCC function of NK cells has received high attention in antibody immunotherapy.
  • Bispecific antibodies that simultaneously recruit the ADCC receptor CD16a and recognize target antigens have been developed. Different formats of these bispecific antibodies are currently undergoing preclinical and clinical research, such as GTB-3550 targeting CD33 and CD16, AFM24 targeting EGFR and CD16, and AFM26 targeting BCMA and CD16 are currently in clinical research, these
  • the CD16 antibody part used in the antibody is from natural human phage display library.
  • Nanobody (Nb) is a genetically engineered antibody containing only a single domain. In 1993, Belgian scientist Hamers-Casterman C found a natural heavy chain antibody in camel blood that only contained heavy chains but no light chains.
  • nanobody technology is used to develop a CD16-targeted therapy that specifically recognizes CD16a and has comparable high binding affinity to the two allotypes of CD16a, relatively low binding activity to CD16b, and is not affected by human immunoglobulin in serum Sexual antibodies have broad prospects.
  • the invention provides an antibody or antigen-binding fragment specifically binding to CD16, a multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid fragment, a vector, a host cell, a pharmaceutical composition, a kit, and a preparation method and its application in treating diseases and detecting CD16.
  • the present invention discloses an antibody or antigen-binding fragment that specifically binds CD16, said antibody or antigen-binding fragment comprising CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 respectively comprising a group selected from SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the VHH domain shown in any one of 21-34 or SEQ ID NO: 90-101.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the Kabat, Chothia or IMGT numbering system; optionally, the HCDR1, HCDR2 and HCDR3 are selected from Table 9; optionally, the HCDR1 is selected from SEQ ID NO: 49, 52, 54, 57, 60, 62, 65, 66, 67, 68, 70, 72, 78, 79, 80, 81 or 88;
  • the HCDR2 is selected from SEQ ID NO : 50, 53, 55, 58, 61, 63, 69, 71, 73, 75, 76, 77, 82 or 85;
  • the HCDR3 is selected from SEQ ID NO: 51, 56, 59, 64, 74, 83, 84, 86 or 87;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 52, 53, 51 or SEQ ID NO: 54, 55, 56 according to the Kabat, Chothia or IMGT numbering system ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 57, 58, 59, SEQ ID NO: 60, 61, 59 or SEQ ID NO: 62, 63, 64 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 65, 53, 51 or SEQ ID NO: 66, 55, 56 according to the Kabat, Chothia or IMGT numbering system ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 67, 53, 51 or SEQ ID NO: 68, 55, 56 according to the Kabat, Chothia or IMGT numbering system ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 69, 51, SEQ ID NO: 70, 71, 51 or SEQ ID NO: 72, 73, 74 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 75, 51, SEQ ID NO: 52, 76, 51 or SEQ ID NO: 54, 77, 56 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 78, 53, 51 or SEQ ID NO: 79, 55, 56 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 80, 53, 51 or SEQ ID NO: 81, 55, 56 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 69, 51, SEQ ID NO: 65, 71, 51 or SEQ ID NO: 66, 73, 56 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 57, 82, 83, SEQ ID NO: 60, 61, 83 or SEQ ID NO: 62, 63, 84 according to the Kabat, Chothia or IMGT numbering system ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 57, 85, 86, SEQ ID NO: 60, 61, 86 or SEQ ID NO: 62, 63, 87 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 57, 82, 86, SEQ ID NO: 60, 61, 86 or SEQ ID NO: 62, 63, 87 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 88, 82, 83, SEQ ID NO: 60, 61, 83 or SEQ ID NO: 62, 63, 84 ;
  • said HCDR1, HCDR2 and HCDR3 are selected from SEQ ID NO: 49, 50, 51, SEQ ID NO: 80, 53, 51 or SEQ ID NO: 81, 55, 56 ;
  • the CDR1, CDR2 and/or CDR3 comprise an amino acid sequence in which 1, 2 or 3 mutations occur on the HCDR1, HCDR2 and/or HCDR3; the mutations may be selected from insertion , deletion and/or substitution, and the substitution is preferably a conservative amino acid substitution.
  • said CDR1, CDR2 and/or CDR3 comprise at least 80, 85%, 90%, 91%, 92%, 93%, 94% compared with said HCDR1, HCDR2 and/or HCDR3 %, 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • said antibody or antigen-binding fragment comprises a single domain antibody comprising said CDR1, CDR2 and CDR3.
  • the single domain antibody comprises a sequence selected from any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101;
  • the single domain antibody comprises at most 20, 19, 18, 17, 16 sequences compared with any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101. 1, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated sequence,
  • the mutation can be selected from insertion, deletion and/or substitution, and the substitution is preferably a conservative amino acid substitution;
  • the single domain antibody comprises at least 80, 85%, 90%, 91%, 92% of the sequence shown in any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101. , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences.
  • the antibody comprises the FR region in the VHH domain shown in any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101;
  • the antibody comprises at most 15, 14, 13, 12 FR regions in the VHH domain shown in any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101.
  • the antibody comprises at least 80%, 85%, 90%, 91% of the FR region in the VHH domain shown in any one of SEQ ID NO: 21-34 or SEQ ID NO: 90-101. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences.
  • the antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.
  • the antibody or antigen-binding fragment comprises or does not comprise an antibody heavy chain constant region; alternatively, the antibody heavy chain constant region may be selected from human, alpaca, mouse, rat, Rabbit or sheep; alternatively, the heavy chain constant region of the antibody may be selected from IgG, IgM, IgA, IgE or IgD, and the IgG may be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain The constant region can be selected from an Fc region, a CH3 region or a complete heavy chain constant region.
  • the heavy chain constant region is a human Fc region, preferably comprising any one of SEQ ID NO: 35-48 or SEQ ID NO: 102-113.
  • the sequence shown in item; preferably, the antibody or antigen-binding fragment is a heavy chain antibody.
  • the antibody or antigen-binding fragment is also coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulators, and the tracer selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulators
  • the tracer selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the antibody or antigen-binding fragment specifically binds to human CD16, monkey CD16 and/or mouse CD16, preferably, the KD of the antibody or antigen-binding fragment to human CD16 and/or monkey CD16 is less than 1E-6M, 1E-7M, 2E-7M, 3E-7M, 4E-7M, 5E-7M, 6E-7M, 8E-7M, 9E-7M, 1E-8M, 2E-8M, 3E-8M, 4E- 8M, 5E-8M, 6E-8M, 8E-8M, 9E-8M, or 1E-9M.
  • the antibody or antigen-binding fragment binds to CD16A, does not bind to CD16B or binds weakly to CD16B, and the CD16B is selected from CD16B(NA1), CD16B(NA2) or CD16B(SH).
  • the antibody or antigen-binding fragment is also linked with other functional molecules, preferably, the other functional molecules can be selected from one or more of the following: signal peptides, protein tags, cell factors, angiogenesis inhibitors, or immune checkpoint inhibitors.
  • the cytokine can be IL2, IL-6, IL-12, IL-15, IL-21, IFN or TNF ⁇ ;
  • the angiogenesis inhibitor can be endostatin;
  • the The immune checkpoint inhibitor can be SIRP ⁇ .
  • the present invention also discloses a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment, and an antigen-binding molecule that binds to an antigen other than CD16, or binds to the aforementioned Different CD16 epitopes of antibodies or antigen-binding fragments; optionally, other antigens other than CD16 can be selected from: CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, Phosphatidylserine (Phosphatidylserine, PS), C-Met, BCMA, HSA, GPRC5D, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FR ⁇ or Gucy2C;
  • said other antigen-binding molecule is an antibody or an antigen-binding fragment
  • said multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific;
  • the multispecific antigen binding molecule may be bivalent, tetravalent or hexavalent.
  • the present invention also discloses an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule.
  • the present invention also discloses a vector, which comprises the aforementioned nucleic acid fragment.
  • the present invention also discloses a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the present invention also discloses a method for preparing the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody, antigen-binding fragment or multispecific antigen-binding molecule expressed by the cell. Specific antigen-binding molecules.
  • the present invention also discloses a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, or product prepared according to the aforementioned method;
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional antineoplastic agent.
  • the present invention also discloses a method for treating tumors or cancers, inflammatory diseases or allergic diseases, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen Binding molecules, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods; preferably, the tumor or cancer is selected from non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, minimal residual disease, metastases.
  • the present invention also discloses that the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods are used in the preparation and treatment of tumors or cancers, inflammatory diseases or Use in allergy medicine; preferably, the tumor or cancer is selected from non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, minimal residual disease, metastases.
  • the present invention also discloses a kit comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, product or pharmaceutical composition prepared according to the aforementioned method .
  • the present invention also discloses a method for detecting the expression of CD16 in a biological sample, the method comprising making the biological A chemical sample is contacted with the antibody or antigen-binding fragment; preferably, the method further comprises detecting the formation of the complex, indicating the presence or expression level of CD16 in the sample.
  • the present invention also discloses the use of the aforementioned antibodies or antigen-binding fragments in the preparation of CD16 detection reagents.
  • CD16 refers to receptor type III for the Fc portion of immunoglobulin G (Fc ⁇ RIII), a group of differentiated molecules found on the surface of natural killer cells, neutrophils, monocytes, and macrophages.
  • CD16 has been identified as Fc receptors Fc ⁇ RIIIa (CD16a) and Fc ⁇ RIIIb (CD16b), involved in signal transduction.
  • Human Fc ⁇ RIIIA (CD16a) is a low-affinity receptor that can bind to IgG Fc expressed on human CD56 low-expressing natural killer (NK) cells, monocyte subsets, dendritic cells and rare T cells.
  • CD16b Human Fc ⁇ RIIIB (CD16b), encoded by different genes and mainly expressed in neutrophils, is a GPI-anchored glycoprotein that lacks the intracellular signal transduction domain.
  • CD16a is a type I membrane glycoprotein with a single transmembrane (TM) domain and a short cytoplasmic tail, whose expression on the cell surface depends on binding to the signaling molecules CD247 (TCR ⁇ ) and/or Fc- ⁇ RI- ⁇ , once they interact, they induce a series of signal transductions, leading to cytokine release and cell killing activity.
  • KD equilibrium dissociation constant
  • high affinity generally means having a KD of about 10 ⁇ 6 M or lower, 10 ⁇ 7 M or lower, about 10 ⁇ 8 M or lower, about 10 ⁇ 9 M or lower.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • antibody includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.
  • Antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that can interact with antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgM, IgD, IgG, IgA, and IgE immunoglobulins
  • their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos) and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptors Ig new antigen receptors, IgNAR
  • antibody herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, large sheep Camels, protozoans, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • heavy chain antibody herein refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • nanobody in this paper refers to the natural heavy chain antibody lacking the light chain in camels, etc., and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • VHH domain and “nanobody” and “single domain antibody” (sdAb) herein have the same meaning and are used interchangeably, referring to the variable region of a cloned heavy chain antibody, constructed A single domain antibody consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • multispecific herein refers to the ability of an antibody or antigen-binding fragment to bind, for example, different antigens or at least two different epitopes on the same antigen.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody can bind.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and thus can only bind the same epitope (rather than bind different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and thus can bind at least two different epitopes.
  • complementarity determining region refers to the antigen binding site of an antibody.
  • a single “specificity” may refer to one, two, three or more than three identical CDRs in a single antibody (the actual number of CDRs/binding sites in a single antibody molecule is referred to as " price").
  • a single native IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody comprises at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and has the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also proteins, e.g. antibodies, scaffolds which specifically bind three or more than three different epitopes, i.e. having three or more Antibodies with more than three paratopes/binding sites.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • exemplary, "antigen-binding fragment” or “antibody fragment” herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody and single domain Antibody.
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as rat, mouse, rabbit or alpaca) and derived from a different organism (such as human ) of the immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125(1-2):191-202; above incorporated by reference Incorporated into this article. See also patent US5807715A.
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity or enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • dasheavy chain variable region is used interchangeably with “VH” and “HCVR”
  • light chain variable region can be used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable domains hypervariable regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered within hypervariable regions under one set of criteria (such as IMGT or KABAT) but under a different set of criteria (such as KABAT or IMGT) outside the hypervariable region. One or more of these positions may also be found in extended hypervariable regions.
  • the invention includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR may be abbreviated as HCDR and the light chain variable region may be abbreviated as LCDR.
  • the variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site.
  • CDR CDR
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results).
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, e.g., Chothia, C., & Lesk, A.M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. Journal of molecular biology, 196(4), 901-917.).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (The international ImMunoGeneTics information system (IMGT)) initiated by Lefranc et al., see Lefranc, M.P., Pommié, C., Ruiz, M.,Giudicelli,V.,Foulquier,E.,Truong,L.,...& Lefranc,G.(2003).IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Developmental & Comparative Immunology, 27(1), 55-77.
  • IMGT international ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the “heavy chain constant region” may be selected from a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • typical "heavy chain constant region fragments" can be selected from Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include cleavage variants of the full-length heavy chain.
  • the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index).
  • the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region includes IgG CH2 and IgG CH3 domains, optionally, on this basis, it may also include a complete or partial hinge region, but does not include a CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • a "CH3 domain” comprises the stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service , National Institutes of Health, Bethesda, MD, 1991 described.
  • Fc variant herein refers to changes in the structure or function of Fc caused by one or more amino acid substitutions, insertions or deletion mutations at appropriate positions on the Fc.
  • Interaction between Fc variants refers to the space-filling effect, electrostatic guidance, hydrogen bond interaction, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • a preferred mutation design is a "Knob-into-Hole” style mutation design.
  • Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative ones are Cater et al. (Ridgway, J.B., Presta, L.G., & Carter, P. (1996). 'Knobs-into-holes' engineering of antibody CH3domains for heavy chain heterodimerization. Protein Engineering, Design and Selection, 9( 7), 617-621.) The "Knob-into-Hole" form proposed; Amgen's technicians use electrostatic guidance (Electrostatic Steering) to form a heterodimer form containing Fc (US 20100286374A1); Jonathan H.Davis et al.
  • SEED bodies fusion proteins based on strand-exchange engineered domain (SEED) CH3heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.
  • the Knob/Hole structure on the Fc variant fragments of the present invention means that the two Fc fragments are mutated respectively, and can be combined in a "Knob-into-Hole" form after the mutations. It is preferred to use the "knob-into-hole" model of Cater et al. to carry out site mutation modification on the Fc region, so that the obtained first Fc variant and the second Fc variant can be in the form of "knob-into-hole” Combine together to form heterodimers.
  • the selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • the Fc region of human antibody IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected for knob mutation and the other for hole mutation.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0) to determine the difference between two amino acid sequences or nucleotide sequences. The percent identity between.
  • nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg to identify other family member sequences or related sequences.
  • search sequences For example, Altschul, SF, Gish, W., Miller, W., Myers, EW, & Lipman, DJ (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), 403-410.
  • the NBLAST and XBLAST programs (version 2.0) of ® perform such searches.
  • Gapped BLAST and PSI-BLAST a new generation of protein database search programs. Nucleic acids research, 25(17), 3389 Use Gapped BLAST as described in -3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as an antibody The heavy chain variable region and/or the light chain variable region, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler, CR, H., Celik, L., Hebich, B., Roth, AS, Roth, RP,... & Sahin, U. (2017). Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nature medicine, 23 (7), 815-817. or EP2101823B1).
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, Agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, which are known to those skilled in the art (see For example, Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions, such as cancers and tumors, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject herein refers to an organism receiving treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, a primate (eg, monkey) or a non-primate mammal, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • Figure 1 shows human CD16a(158F)-Fc, human CD16a(158V)-Fc, CD16a(158F)-his, human CD16a(158V)-his, human CD16b(NA1)-his, human CD16b(NA2)-his, human CD16b(SH)-his and cynomolgus monkey CD16-his protein samples SDS-PAGE reducing and non-reducing gum detection results.
  • Lanes 1 and 2 are the protein bands of human CD16a(158F)-Fc under reducing and non-reducing conditions respectively
  • lanes 3 and 4 are protein bands of human CD16a(158V)-Fc under reducing and non-reducing conditions respectively
  • lane 5 and 6 are the protein bands of human CD16a(158F)-his under reducing and non-reducing conditions, respectively
  • lanes 7 and 8 are the protein bands of human CD16a(158V)-his under reducing and non-reducing conditions, respectively
  • lanes 9 and 10 Respectively, the protein bands of human CD16b(NA1)-his under reducing and non-reducing conditions
  • lanes 11 and 12 are the protein bands of human CD16b(NA2)-his under reducing and non-reducing conditions, respectively
  • lanes 13 and 14 are respectively
  • lanes 15 and 16 are the protein bands of cynomolgus monkey CD16-his under reducing and non-reducing conditions, respectively
  • Figure 2A is the FACS detection result of FlpinCHO cells transfected with human CD16a (158F) protein
  • Figure 2B is the FACS detection result of FlpinCHO cells transfected with human CD16a (158V) protein
  • Figure 2C is the FACS detection result of FlpinCHO cells transfected with human CD16b (NA1) protein
  • Figure 2D is the FACS detection result of FlpinCHO cells transfected with human CD16b (NA2) protein
  • Fig. 2E is the FACS detection result of FlpinCHO cells transfected with cynomolgus monkey CD16 protein
  • Figure 3A is the binding of CD16 VHH-Fc antibody to FlpinCHO-human CD16a (158F) cells;
  • Figure 3B is the binding of CD16 VHH-Fc antibody to FlpinCHO-human CD16a (158V) cells;
  • Figure 4A is the combination of biotin-labeled CD16 VHH-Fc antibody and FlpinCHO-human CD16a (158V) cells;
  • Fig. 4B is the combination of biotin-labeled CD16 VHH-Fc antibody and FlpinCHO-human CD16a (158V) cells in the presence of 10mg/mL human immunoglobulin;
  • Figure 4C is the combination of biotin-labeled CD16 VHH-Fc antibody and FlpinCHO-human CD16a (158F) cells;
  • Figure 4D is the binding of biotin-labeled CD16 VHH-Fc antibody to FlpinCHO-human CD16a (158F) cells in the presence of 10 mg/mL human immunoglobulin;
  • Figure 5A is the binding reaction of FACS detection CD16 VHH-Fc antibody and FlpinCHO-human CD16b (NA1) cells;
  • Figure 5B is the FACS detection of the binding reaction of CD16 VHH-Fc antibody to FlpinCHO-human CD16b (NA2) cells.
  • Figure 6A is the binding of CD16 VHH-Fc antibody to human NK cells
  • Figure 6B is the binding of biotin-labeled CD16 VHH-Fc antibody to human NK cells
  • Figure 6C is the binding of biotin-labeled CD16 VHH-Fc antibody to human NK cells in the presence of 10 mg/mL human immunoglobulin;
  • Fig. 7 is that FACS detects the binding reaction of CD16 VHH-Fc antibody and FlpinCHO-monkey CD16 cell;
  • Figure 8 shows the activation of Jurkat-NFAT cells detected by the luciferase reporter system.
  • Figure 9A is the binding of humanized CD16 VHH-Fc antibody to FlpinCHO-human CD16a (158F) cells;
  • Figure 9B is the binding of humanized CD16 VHH-Fc antibody to FlpinCHO-human CD16a (158V) cells;
  • Figure 10A is the binding reaction between humanized CD16 VHH-Fc antibody and FlpinCHO-human CD16b (NA1) cells;
  • Figure 10B is the binding reaction of humanized CD16 VHH-Fc antibody to FlpinCHO-human CD16b (NA2) cells.
  • Figure 11A is the binding of biotin-labeled CD16 VHH-Fc antibody to NK cells
  • Figure 11B is the binding of biotin-labeled CD16 VHH-Fc antibody to NK cells in the presence of 10 mg/mL human immunoglobulin;
  • Figure 12 is the binding of humanized CD16 VHH-Fc antibody to human neutrophils
  • Figure 13 is the binding reaction of humanized CD16 VHH-Fc antibody and FlpinCHO-monkey CD16 cells
  • Figure 14 is the activation of Jurkat-NFAT cells detected by the luciferase reporter system detected by the humanized CD16 VHH-Fc antibody.
  • the extracellular domain (ECD, extra-cellular domain) of the above protein was sequentially connected with human IgG1 Fc (N297A) or His tag, (G 3 S) 2 linker and AVI tag to obtain immunity and
  • the antigen sequence for antibody screening and identification (the amino acid sequence is shown in Table 2 as SEQ ID NO: 7-14), and General Biosystems (Anhui) Co., Ltd. was commissioned to synthesize the nucleotide sequence corresponding to the amino acid sequence and clone it into The pTT5 vector containing the signal peptide, and the plasmid was prepared according to established standard molecular biology methods.
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PI Polysciences, Cat. No.: 24765-1
  • FreeStyle TM 293 Thermofisher scientific, Cat. No.: 12338018
  • the culture supernatant was loaded onto the protein A chromatography column (Protein A filler AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borglon, and the article numbers were AA0273 and B-1620 respectively) , washed with PBS phosphate buffer (pH 7.4), then washed with 20mM PB, 1M NaCl (pH 7.2), and finally eluted with pH 3.4 citric acid buffer, collected from the protein A chromatography column
  • the eluted Fc-labeled antibody was neutralized with 1/10 volume of 1M Tris at pH 8.0, dialyzed with PBS overnight at 4°C, and the dialyzed protein was sterile filtered through a 0.22-micron filter membrane and dispensed in Store at -80°C. That is, the purified human CD16-Fc protein was obtained, and the target bands of samples detected by SDS-PAGE reducing gel and non-reducing gel were shown in FIG.
  • the culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, article number: GE17-3712-06), and the UV absorbance (A280nm )The change.
  • the dialyzed protein was sterile-filtered with a 0.22-micron filter membrane and stored at -80°C to obtain the purified human CD16-his protein.
  • the target bands of the samples detected by SDS-PAGE reducing gel and non-reducing gel are shown in Figure 1 .
  • biotin-labeled protein a part of the His-tagged and AVI-tagged CD16 protein purified in the previous step was taken out for biotin-labeling in vitro.
  • biotin labeling kit BirA biotin-protein ligase kit, Avidity, product number: BirA500.
  • the uppercase bold font is the extracellular region
  • the uppercase underline is the Fc tag
  • GGGSGGGS is the linker
  • HHHHHHHHHH is the His tag
  • the lowercase bold font is the AVI tag.
  • the mouse antibody 3G8 that recognizes human CD16 was obtained by immunizing mice with PMN (Fleit H.B.et al., Proc Natl Acad Sci U S A.1982 May), which can recognize human CD16a (158F and 158V) and CD16b (NA1, NA2 and SH), and has cross-binding activity with cynomolgus monkey CD16.
  • the VL and VH of 3G8 and P2C47 and the human IgG1 Fc were connected in order from N-terminal to C-terminal, wherein VL and VH were connected through three GGGGS linkers to form scFv-hFc.
  • the corresponding amino acid sequence information is as follows 3.
  • NC is the negative control antibody scFv-hFc (L234A, L235A, D265A), which is an antibody against chicken egg lysozyme, and is commissioned to Taizhou Baiying Biotechnology Co., Ltd. for custom production.
  • the full-length amino acid sequence encoding human CD16a158F (Uniprot ID: P08637, SEQ ID NO: 1), the full-length amino acid sequence of human CD16a158V (NCBI ID: AAH17865.1, SEQ ID NO: 2), the full-length human CD16b (NA1) Amino acid sequence (NCBI ID: AAA35881.1, SEQ ID NO: 3), human CD16b (NA2) full-length amino acid sequence (Uniprot ID: O75015, SEQ ID NO: 4) and cynomolgus monkey CD16 full-length amino acid sequence (NCBI ID : NP_001270121.1, the nucleotide sequence of SEQ ID NO: 6) was cloned into the pcDNA5-FRT vector (Thermofisher scientific, article number: V601020) and the plasmid was prepared (completed by Sangon Bioengineering (Shanghai) Co., Ltd.), the preparation was completed The plasmids were co
  • the transfected FlipinCHO cells were placed in a 37°C, 5% (v/v) CO 2 incubator, and contained 800 ⁇ g/ml hygromycin and Selective culture in F12 medium with 10% (w/w) fetal bovine serum, after about 2 weeks, some cells were taken to detect the expression of cell surface antigens by flow cytometry, and the cell lines that recovered growth continued to be expanded and cultured. Nitrogen freezer.
  • the IgG subtype control is a mouse IgG1 control (purchased from Taizhou Baiying Biotechnology Co., Ltd., article number: B118301).
  • the 3G8 antibody can interact with Flpin CHO-human CD16a (158F), Flpin CHO-human CD16a (158V), Flpin CHO-human CD16b (NA1), Flpin CHO-human CD16b (NA2) and Flpin CHO-monkey CD16 cell line combination
  • Table 4 lists the average fluorescence intensity of cells stained with the transfected cell line 3G8 antibody and fluorescent secondary antibody, indicating that these cell lines have been able to stably express the corresponding CD16 antigen, which can be used for subsequent antibody screening and identification.
  • Human CD16a(158F)-hFc and human CD16a(158V)-hFc proteins were used for immunization.
  • One alpaca (Llama) was selected for immunization, and each alpaca was immunized four times with an interval of 3 weeks.
  • peripheral blood was collected and serum was separated, and the enzyme-linked immunosorbent assay ( ELISA) was used to detect the antibody titer and specificity against human CD16 in the serum, and the results are shown in Table 5.
  • ELISA enzyme-linked immunosorbent assay
  • Table 5 shows that the sera of alpacas immunized with human CD16 have varying degrees of binding to the immunogen after immunization, showing antigen-antibody reactions, and the highest dilution is about 5.9 million.
  • the blank control is 1% (w/w) BSA, and the batch refers to the alpaca serum on the seventh day after the third (TB3) and fourth (TB4) immunization, and the data in the table are OD450nm values.
  • RNAiso Plus reagent (Takara, catalog number: #9108/9109) to extract total RNA, and use PrimeScript TM II 1st Strand The cDNA Synthesis Kit (Takara, Cat. No.: 6210A) reverse-transcribed the extracted RNA into cDNA.
  • Nested PCR was used to amplify the variable region nucleic acid fragment encoding the Nanobody and recover the target Nanobody nucleic acid fragment, and electrotransform the Nanobody nucleic acid fragment together with the linearized yeast display vector pYDC2 (from Sichuan Apak Biotechnology Co., Ltd.) into In vivo recombination was carried out in yeast competent cells EYB100, and a nanobody yeast display library targeting CD16 was constructed and tested. By serial dilution plating, the calculated stock size was 3.26 ⁇ 10 8 . To test the insertion rate of the library, 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
  • the yeast display library was coated with magnetic beads with biotin-labeled human CD16b(NA1)-his, CD16b(NA2)-his and CD16b(SH)-his proteins prepared in Example 1.1, and then reverse-screened with biological
  • the fluorescently labeled human CD16a(158F)-his and CD16a(158V)-his proteins were coated with magnetic beads for forward screening, and after the enrichment of yeast clones that specifically bind to human CD16a, the fluorescein-labeled Reverse screening of human CD16b(NA1)-his, CD16b(NA2)-his and CD16b(SH)-his proteins and forward screening of human CD16a(158F)-his and CD16a(158V)-his proteins to obtain specific binding to CD16a of yeast clones.
  • CD16a(158F) and CD16a(158V) binding-positive yeasts were plated and amplified and single clones were selected, and then 96 single colonies were selected for expansion and culture respectively.
  • Biotin-labeled CD16a(158F)-his protein was mixed with excess SA-iFluor 647 (purchased from ThermoFisher scientific, catalog number: S21374), biotin-labeled CD16b(NA1)-his, CD16b(NA2)-his and CD16b( SH)-his protein was incubated with excess SA-Alexa Fluor 488 (purchased from ThermoFisher scientific, catalog number: S11223) for 1 hour for fluorescent labeling.
  • yeast cells After washing, the expressed yeast cells were divided into two parts, and a part of the yeast cells were incubated with human immunoglobulin (Shanghai Institute of Biological Products) and the aforementioned fluorescein-labeled CD16a and CD16b antigens at 4°C for 1 hour. Another part of yeast cells was incubated with Alexa Fluor 488-labeled anti-HA antibody (purchased from ThermoFisher scientific, catalog number: A-21287) for 1 hour at 4°C. After the yeast cells were washed, they were analyzed by flow cytometry and the fluorescence intensity (MFI) was calculated respectively.
  • human immunoglobulin Sethai Institute of Biological Products
  • Alexa Fluor 488-labeled anti-HA antibody purchased from ThermoFisher scientific, catalog number: A-21287
  • the MFI values of the fluorescently labeled CD16a and CD16b were divided by the MFI of the fluorescently labeled anti-HA to obtain the normalized MFI values of CD16a and CD16b.
  • the normalized MFI values of some clones are shown in Table 6.
  • Clones positive for CD16a were selected for sequencing.
  • the sequencing results were analyzed using MOE software, and an evolutionary tree was constructed based on the amino acid sequence of the VHH-encoded protein. After eliminating sequences that were close to each other on the evolutionary tree according to the sequence similarity, 14 clones were obtained by screening.
  • the nanobody sequences are shown in Table 7.
  • Table 8 shows the amino acid sequences of VHH and Fc segment fusions.
  • the CDRs of the sequence were analyzed by KABAT, Chothia or IMGT software respectively, and the CDR sequence information is shown in Table 9 below. Subsequently, the production identification of the VHH Nanobody Fc fusion protein was carried out according to the method in Example 1.2.
  • the results are shown in Table 10 and Figures 3A-3B.
  • the CD16 VHH-hFc antibody has good binding activity to both FlpinCHO-CD16a(158F) and FlpinCHO-CD16a(158V) cells, and the EC50 for binding to the two cells is equivalent.
  • the EC50 of most antibodies is less than 1nM.
  • the antibody to be tested was labeled in advance according to the instructions of the biotinylation kit (EZ-Link TM NHS-PEG4 Biotinylation Kit, purchased from Thermofisher Scientific, product number: 21455). After collecting the cells, the cells were directly incubated with the antibody to be tested in serial dilution , or the cells were first incubated with 10 mg/mL human immunoglobulin (Shanghai Institute of Biological Products) and then incubated with the antibody to be tested in a gradient dilution, and detected according to the method of Example 3.1, wherein the biotin-labeled antibody was incubated with fluorescein APC-labeled Streptavidin (High Concentration) (purchased from Biolegend, catalog number: 405243) was used for detection.
  • fluorescein APC-labeled Streptavidin High Concentration
  • Table 11-1 FACS detection of the effect of human immunoglobulin on the binding of VHH-Fc antibody to FlpinCHO-CD16a (158V) cells
  • the binding of the antibody to be tested to FlpinCHO-human CD16b cells was detected according to the method in Example 3.1. As shown in Figures 5A-5B and Table 12, the 3G8 antibody is a positive antibody binding to CD16b, and the binding activity of all the tested antibodies to FlpinCHO-human CD16b (NA1) and FlpinCHO-human CD16b (NA2) is weaker than that of the 3G8 antibody.
  • Example 3.2 According to the method of Example 3.2, the results are shown in Figure 6B and Table 14.
  • the binding activity of the control antibody 3G8 to NK cells was completely lost, and VHH-05, VHH-06 and VHH-11
  • the EC50 of antibody-cell binding activity decreased by 2-3 times, and the EC50 of the control antibody P2C47 decreased by about 4 times.
  • the binding activity of VHH-05, VHH-06, VHH-11 and VHH-12 to NK cells was stronger than that of P2C47 antibody.
  • the Jurkat-NFAT luciferase reporter cell line stably expressing human CD16a (158V) (purchased from BPS Bioscience, Cat. No. 60541) was used to detect the signal pathway in the luciferase reporter cell line activated by the CD16 antibody, and the luciferase signal was detected according to Bright -Glo TM Luciferase Assay System kit (Promega, catalog number: E2620) operating instructions.
  • the experimental method is as follows: 2 ⁇ g/mL anti-human Fc tag antibody (Jackson Immune, catalog number: 109-006-098) was coated overnight at 4°C to capture the antibody to be tested with the human Fc tag, and the next day the antibody to be detected was The antibody was diluted with RIPM 1640 medium containing 5% FBS, the initial antibody concentration was 4nM, and the antibody was diluted 1:6, and then added to a 96-well flat-bottomed cell culture plate at 50 ⁇ l/well.
  • the collected For the luciferase reporter cell line in the logarithmic growth phase adjust the cell concentration to 8 ⁇ 10 5 /mL with medium containing 2% FBS (RPMI 1640, purchased from Gibco, catalog number 12633012), add the cells to the culture plate, 50 ⁇ l /hole.
  • RPMI 1640 purchased from Gibco, catalog number 12633012
  • VHH-hFc form antibody against human CD16 was captured using a Protein A chip (GE Helthcare; 29-127-558).
  • Sample and running buffer was HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD16 antigenic protein was injected to record the binding and dissociation process of the antibody and the antigenic protein, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration. Binding was measured by injecting different concentrations of recombinant human CD16-ECD his protein in solution for 240 seconds, with a flow rate of 30 ⁇ L/min, starting from 200 nM (see detailed results for actual concentrations tested), diluted 1:1, for a total of 5 concentration. The dissociation phase is monitored for up to 600 seconds and is triggered by switching from sample solution to running buffer.
  • binding rate (ka), dissociation rate (kd) and binding affinity (KD) of the antibody to be tested and the human CD16b-ECD his protein were detected according to the method of Example 7.1, as shown in Table 18 and Table 19.
  • Example 7.1 According to the method of Example 7.1, the binding rate (ka), dissociation rate (kd) and binding affinity (KD) of the antibody to be tested and monkey CD16-ECD his protein are shown in Table 20.
  • IGHV3-30*02 and IGHV3-7*01 were selected as alpaca antibody VHH-06 and IGHV3-7*01 respectively.
  • the CDRs of alpaca antibodies were grafted into the FRs of their human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the FR region sequence of the humanized antibody were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity, and a humanized anti-CD16 single domain antibody was obtained.
  • the numbering of amino acid residues and CDR regions of the antibodies in this example are determined and annotated by the IMGT numbering system (see http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi for details).
  • the humanized antibody VHHs designed by the above methods are shown in Table 21 and Table 22, respectively.
  • M39V means that the 39th M of Graft is mutated into V, and so on.
  • the numbering of backmutated amino acids is the IMGT numbering.
  • S40G means that the 40th position of Graft is mutated into G, and so on.
  • the numbering of backmutated amino acids is the IMGT numbering.
  • the humanized VHH sequences are shown in Table 23 and Table 24.
  • the humanized antibody sequences were prepared according to the method in Example 1.2 to prepare VHH-Fc antibodies.
  • the humanized VHH-Fc sequences are shown in Table 25 and Table 26.
  • the binding of the humanized antibody to FlpinCHO-human CD16a cells was detected according to the method in Example 3.1. As shown in Figures 9A-9B and Table 27, it has good binding activity to both FlpinCHO-CD16a(158F) and FlpinCHO-CD16a(158V) cells, and the EC50 of binding to the two cells is equivalent, both less than 1nM.
  • the binding of the antibody to be tested to FlpinCHO-human CD16b cells was detected according to the method in Example 3.1. It has been detected in Example 3 that the binding activity of P2C47 to FlpinCHO-human CD16b (NA1) and FlpinCHO-human CD16b (NA1) is significantly weaker than that of CD16a and CD16b positive antibody 3G8. As shown in Figures 10A-10B and Table 28, the binding of the humanized VHH antibody to FlpinCHO-human CD16b (NA1) and FlpinCHO-human CD16b (NA2) is equivalent to that of the control antibody P2C47, so the binding activity of the humanized antibody can be inferred Also weaker than 3G8 antibody.
  • NA indicates weak antibody binding activity and poor curve fitting.
  • the detection was carried out according to the method of Example 3.2, and the results are shown in Figures 11A-11B and Table 29.
  • the binding activity of the control antibody 3G8 to NK cells was completely lost, and VHH-05, VHH-06 and VHH
  • the EC50 of the binding activity of the -11 antibody to cells decreased by 3-4 times, and the EC50 of the control antibody P2C47 decreased by about 4-5 times.
  • the binding activity of VHH06-03, VHH06-04, VHH12-05 and VHH12-06 to NK cells was stronger than that of P2C47 antibody.
  • the humanized CD16 antibody activates the signaling pathway in the luciferase reporter cell line, and the results are shown in Table 31 and Figure 13.
  • all humanized antibodies can activate Jurkat cells The downstream signaling pathway, and the activation activity is stronger than that of P2C47 antibody.
  • the affinity between the humanized CD16 antibody and human CD16a(158F)-ECD-his protein was detected according to the method in Example 7.1.
  • the binding rate (ka), dissociation rate (kd) and binding affinity (KD) of the antibody to be tested and human CD16a(158F)-ECD his protein are shown in Table 32.

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Abstract

涉及CD16抗体及其应用。具体而言，公开一种特异性结合CD16的抗体或抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于治疗疾病的用途，例如治疗肿瘤中的用途。对于CD16抗体治疗药物和检测试剂的开发具有重要意义。

Description

CD16抗体及其应用
本申请要求2021年6月29日向中国国家知识产权局提交的，专利申请号为202110732732.X，发明名称为《CD16抗体及其应用》的中国专利申请的优先权。上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及生物医药领域，具体而言，涉及CD16抗体及其应用。
背景技术
自然杀伤细胞(Natural killer cell,NK cell)是先天性免疫系统的组成部分，占循环淋巴细胞的大约5-15％。与B细胞和T细胞不同，NK细胞不表达体细胞重排的抗原受体，而是表达一系列激活型和抑制型受体，来自配体和不同受体相互作用的激活和抑制信号的整合以及平衡决定了NK细胞激活的状态。激活的NK细胞通过与细胞毒性T细胞相似的手段杀伤靶细胞，即通过含有穿孔素和颗粒酶的细胞溶解颗粒以及通过死亡受体途径。激活的NK细胞还分泌炎性细胞因子例如IFN-γ和促进其他炎性细胞向靶组织招募的趋化因子。与T细胞不同，NK细胞不需要抗原启动，并通过在没有MHC识别的情况下激活受体来识别目标。
肿瘤细胞表面的I类HLA(MHC I)分子通过结合NK细胞表面的抑制性受体来抑制NK细胞的激活。然而，许多肿瘤细胞通过下调MHC I分子的表达来逃避细胞毒性CD8 +T细胞的监测；因此，在肿瘤微环境中，当T细胞无法识别肿瘤细胞时，NK细胞由于缺乏MHC I诱导的抑制信号，可以通过“丢失自我”机制来杀死肿瘤细胞。
人IgG Fc受体CD16(FcγRIII)由两种亚型(CD16a/FcγRIIIa和CD16b/FcγRIIIb)组成，由两个高度同源的基因编码。CD16b(FcγRIIIb)主要在中性粒细胞上表达，是一种GPI锚定糖蛋白，缺失胞内信号转导结构域。CD16b存在基因多态性，可以产生三种同种异型体，分别为NA1、NA2和SH。CD16a是人IgG Fc的低亲和力受体，单次跨膜蛋白，参与抗体依赖性细胞毒性(ADCC)并触发自然杀伤(NK)细胞对靶细胞的特异裂解。ADCC是由表达FcγR的效应细胞清除肿瘤细胞的细胞毒性主导机制之一。CD16a基因的cDNA在核苷酸序列位置的559处从G到T的单核苷酸取代(SNP)产生两种不同的FcγRIIIa同种异型：在氨基酸序列的第158位，一种编码为缬氨酸(V)，另一种编码为苯丙氨酸(F)。与纯合苯丙氨酸基因型(F/F)相比，缬氨酸(V/V或V/F)的存在可增强NK细胞对IgG1或IgG3抗体的结合亲和力，从而导致更高的NK细胞介导ADCC的水平。在基于抗体的免疫治疗中，NK细胞介导的ADCC是利妥昔单抗、曲妥珠单抗和西妥昔单抗等常用抗体抗癌作用的机制之一。针对几项临床研究的分析，证明了在非霍奇金淋巴瘤、HER-2/neu阳性转移性乳腺癌、转移性结直肠癌或头颈癌中，具有V/V多态性的患者与具有F/F的患者相比，具有改善的无进展生存期。
NK细胞的ADCC功能在抗体免疫疗法中受到高度关注。同时具有募集ADCC受体CD16a和识别靶抗原的双特异性抗体已经在开发中。目前这些双特异性抗体的不同形式正在进行临床前和临床研究，例如靶向CD33和CD16的GTB-3550、靶向EGFR和CD16的AFM24以及靶向BCMA和CD16的AFM26目前均处于临床研究，这些抗体所使用的CD16抗体部分均为天然人噬菌体展示文库来源。纳米抗体(Nanobody，Nb)是一种只含有单个结构域的基因 工程抗体。1993年比利时科学家Hamers-Casterman C在骆驼血液中发现一种只含重链不含轻链的天然重链抗体,重链抗体和普通的抗体相比虽然缺失了轻链,但是依然保留结合抗原的能力。克隆骆驼体内重链抗体的可变区后,得到的仅由一个重链可变区组成的单域抗体(single domain antibody，sdAb)，称为纳米抗体或VHH抗体(variable heavy chain domain of a heavy chain antibody)。纳米抗体不仅分子量只是普通抗体的1/10，而且化学性质也更加灵活，稳定性好，可溶性高，表达容易，肿瘤组织穿透性高。
综上，应用纳米抗体技术研发特异性识别CD16a且对CD16a两种同种异型具有相当的高结合亲和力、对CD16b的结合活性相对低、不受血清中人免疫球蛋白影响的靶向CD16的治疗性抗体具有广阔的前景。
发明内容
本发明提供一种特异性结合CD16的抗体或抗原结合片段、多特异性抗原结合分子、嵌合抗原受体、免疫效应细胞、核酸片段、载体、宿主细胞、药物组合物、试剂盒、制备方法及其在治疗疾病以及检测CD16中的应用。
在第一方面，本发明公开一种特异性结合CD16的抗体或抗原结合片段，所述抗体或抗原结合片段包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别包含选自SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域的HCDR1、HCDR2和HCDR3。
在一些具体的实施方式中，根据Kabat、Chothia或IMGT编号系统确定所述HCDR1、HCDR2和HCDR3；可选地，所述HCDR1、HCDR2和HCDR3选自表9；可选地，所述HCDR1选自SEQ ID NO：49、52、54、57、60、62、65、66、67、68、70、72、78、79、80、81或88；可选地，所述HCDR2选自SEQ ID NO：50、53、55、58、61、63、69、71、73、75、76、77、82或85；可选地，所述HCDR3选自SEQ ID NO：51、56、59、64、74、83、84、86或87；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：52、53、51或SEQ ID NO：54、55、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、58、59，SEQ ID NO：60、61、59或SEQ ID NO：62、63、64；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:49、50、51，SEQ ID NO：65、53、51或SEQ ID NO：66、55、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:49、50、51，SEQ ID NO：67、53、51或SEQ ID NO：68、55、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、69、51，SEQ ID NO：70、71、51或SEQ ID NO：72、73、74；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、75、51，SEQ ID NO：52、76、51或SEQ ID NO：54、77、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：78、53、51或SEQ ID NO：79、55、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：80、53、51或SEQ ID NO：81、55、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、69、51，SEQ ID NO：65、71、51或SEQ ID NO：66、73、56；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、82、83，SEQ ID NO：60、61、83或SEQ ID NO：62、63、84；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、85、86，SEQ ID NO：60、61、86或SEQ ID NO：62、63、87；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、82、86，SEQ ID NO：60、61、86或SEQ ID NO：62、63、87；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：88、82、83，SEQ ID NO：60、61、83或SEQ ID NO：62、63、84；
优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：80、53、51或SEQ ID NO：81、55、56；
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生1个、2个或3个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些具体的实施方式中，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
在一些具体的实施方式中，所述单域抗体包含选自SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列；
可选地，所述单域抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
可选地，所述单域抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列相比具有至少80、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中，所述抗体包含SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区；
可选地，所述抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换， 所述替换优选为保守氨基酸的替换；
可选地，所述抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
在一些具体的实施方式中所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些具体的实施方式中，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区，优选包含如SEQ ID NO：35～48或SEQ ID NO：102～113任一项所示的序列；优选地，所述抗体或抗原结合片段为重链抗体。
在一些具体的实施方式中，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在一些具体的实施方式中，所述抗体或抗原结合片段特异性结合人CD16、猴CD16和/或鼠CD16，优选地，所述抗体或抗原结合片段与人CD16和/或猴CD16的KD小于1E-6M、1E-7M、2E-7M、3E-7M、4E-7M、5E-7M、6E-7M、8E-7M、9E-7M、1E-8M、2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、8E-8M、9E-8M或1E-9M。
在一些具体的实施方式中，所述抗体或抗原结合片段与CD16A结合，不与CD16B结合或与CD16B弱结合，所述CD16B选自CD16B(NA1)、CD16B(NA2)或CD16B(SH)。
在一些具体的实施方式中，所述抗体或抗原结合片段还连接有其他功能性分子，优选地，所述其他功能性分子可选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂或免疫检查点抑制剂。
在一些具体的实施方式中，所述细胞因子可为IL2、IL-6、IL-12、IL-15、IL-21、IFN或TNFα；所述血管生成抑制剂可为内皮抑制素；所述免疫检查点抑制剂可为SIRPα。
在第二方面，本发明还公开一种多特异性抗原结合分子，所述多特异性抗原结合分子包含前述的抗体或抗原结合片段，以及结合CD16以外其他抗原的抗原结合分子，或结合与前述抗体或抗原结合片段不同的CD16表位；可选地，所述CD16以外的其他抗原可选自：CD137、CD258、PD-1、PD-L1、4-1BB、CD40、CD64、EGFR、VEGF、HER2、HER1、HER3、IGF-1R、磷脂酰丝氨酸(Phosphatidylserine，PS)、C-Met、BCMA、HSA、GPRC5D、MSLN、血脑屏障受体、GPC3，PSMA，CD33，GD2，ROR1，ROR2，FRα或Gucy2C；
优选地，所述其他抗原结合分子为抗体或抗原结合片段；
优选地，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性；
优选地，所述多特异性抗原结合分子可为二价、四价或六价。
在第三方面，本发明还公开一种分离的核酸片段，所述核酸片段编码前述抗体或抗原结 合片段或多特异性抗原结合分子。
在第四方面，本发明还公开一种载体(vector)，所述载体包含前述的核酸片段。
在第五方面，本发明还公开一种宿主细胞，所述宿主细胞包含前述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在第六方面，本发明还公开一种制备前述抗体或抗原结合片段或多特异性抗原结合分子的方法，所述方法包括培养前述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
在第七方面，本发明还公开一种药物组合物，所述药物组合物包含前述的抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体或根据前述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
在第八方面，本发明还公开一种治疗肿瘤或癌症、炎性疾病或过敏症疾病的方法，所述方法包括向受试者施用有效量的前述的抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或药物组合物；优选地，肿瘤或癌症选自非霍奇金淋巴瘤、慢性淋巴细胞白血病、霍奇金氏病、微小残留病、转移瘤。
在第九方面，本发明还公开前述抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或药物组合物在制备治疗肿瘤或癌症、炎性疾病或过敏症药物中的用途；优选地，肿瘤或癌症选自非霍奇金淋巴瘤、慢性淋巴细胞白血病、霍奇金氏病、微小残留病、转移瘤。
在第十方面，本发明还公开一种试剂盒，所述试剂盒包含前述的抗体或抗原结合片段、多特异性抗原结合分子、核酸片段、载体、根据前述方法制备获得的产品或药物组合物。
在第十一方面，本发明还公开一种检测生物学样品中CD16表达的方法，所述方法包括在前述的抗体或抗原结合片段与CD16之间能够形成复合物的条件下，使所述生物学样品与所述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中CD16的存在或表达水平。
在第十二方面，本发明还公开前述抗体或抗原结合片段在制备CD16检测试剂中的用途。
术语定义和说明
除非本文另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“CD16”是指免疫球蛋白G Fc部分的受体Ⅲ型(FcγRⅢ)，是一组在自然杀伤细胞、中性粒细胞、单核细胞和巨噬细胞表面发现的分化分子。CD16已被鉴定为Fc受体FcγRⅢa(CD16a)和FcγRⅢb(CD16b)，参与信号转导。人FcγRIIIA(CD16a)是在人CD56低表达自然杀伤(NK)细胞、单核细胞亚群、树突状细胞和稀有T细胞表达的可以与IgG Fc结合的低亲和力受体。人FcγRIIIB(CD16b)由不同的基因编码，主要在中性粒细胞表达，是一种GPI锚定糖蛋白，缺失胞内信号转导结构域。CD16a是I型膜糖蛋白，具有单个跨膜(TM)结构域和短胞质尾，其在细胞表面的表达取决于与信号转导分子CD247(TCRζ)和/或Fc-εRI-γ的结合，它们一旦发生相互作用则诱导一系列信号转导，导致细胞因子释放和细胞杀伤活性。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10 -6M或更低、10 -7M或更低、约10 -8M或更低、约10 -9M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer,I.P.,Beste,G.& Skerra,A.(2003).Proteins,53,121–129.；Roque,A.C.A.,Lowe,C.R.& Taipa,M.A.Antibodies and genetically engineered related molecules:production and purification.Biotechnol.Prog.20,639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
术语“抗体”包括完整抗体及其任何抗原结合片段(即“抗原结合部分”)或单链。“抗体”是指包含通过二硫键互相连接在一起的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合部分。每条重链由重链可变区(在此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在此缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步再分为高变区，称为互补决定区(CDR)，CDR散布在被称为构架区(FR)的更加保守的区域中。每个VH和VL，均由三个CDR和四个FR组成，它们从氨基端向羧基端以如下顺序排列：FR1，CDR1，FR2， CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有可与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，该宿主组织或因子包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一成分(C1q)。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文术语“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
本文术语“纳米抗体”是指骆驼等体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“VHH结构域”和“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“单域抗体”、“VHH结构域”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.363,446-8(1993).Naturally occurring antibodies devoid of light chains.；Muyldermans的综述文章(J Biotechnol.2001Jun；74(4):277-302.Single domain camel antibodies:current status)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO 94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文术语“多特异性”是指抗体或抗原结合片段结合例如不同抗原或同一抗原上的至少 两种不同表位的能力。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体可以结合的不同表位的数目。例如，常规的单特异性IgG型抗体具有两个相同的抗原结合位点(互补位)，因此仅可以结合相同的表位(而不是结合不同的表位)。相比之下，多特异性抗体具有至少两种不同类型的互补位/结合位点，因此可以结合至少两种不同的表位。如本文所述，“互补决定区”是指抗体的抗原结合位点。此外，单个“特异性”可以指单个抗体中的一个、两个、三个或多于三个相同的互补决定区(一个单个抗体分子中的互补决定区/结合位点的实际数量称为“价”)。例如，单个天然IgG抗体是单特异性和二价的，因为它具有两个相同的互补位。相应地，多特异性抗体包含至少两种(不同的)互补决定区/结合位点。因此，术语“多特异性抗体”是指具有多于一个互补位并具有结合两种或多于两种不同表位的能力的抗体。术语“多特异性抗体”特别地包括如上文所定义的双特异性抗体，但是通常还包括蛋白质，例如特别结合三种或多于三种不同的表位的抗体、支架，即具有三种或多于三种互补位/结合位点的抗体。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。示例性地，本文“抗原结合片段”或“抗体片段”包括但不限于Fab、F(ab’)2、Fab’、Fab’-SH、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
本文术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠、小鼠、兔或羊驼)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-1207.Transfectomas Provide Novel Chimeric Antibodies；Gillies等人，J Immunol Methods.1989 Dec 20；125(1-2):191-202；以上通过援引加入并入本文。亦可参见专利US5807715A。
本文术语“人源化抗体”是指经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力或增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。单个VH或VL域可足以赋予抗原结合特异性。
本文术语“互补决定区”与“CDR”可互换使用，通常指在轻链和重链可变结构域中均发现的高变区(HVR)。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。重链可变区CDR可缩写为HCDR，轻链可变区可缩写为LCDR。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成。
对于CDR的进一步描述，参考Kabat,E.A.,Wu,T.T.,& Bilofsky,H.(1977).Unusual distributions of amino acids in complementarity determining(hypervariable)segments of heavy and light chains of immunoglobulins and their possible roles in specificity of antibody-combining sites.Journal of Biological Chemistry,252(19),6609-6616.；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia,C.,& Lesk,A.M.(1987).Canonical structures for the hypervariable regions of immunoglobulins.Journal of molecular biology,196(4),901-917.；Al-Lazikani,B.,Lesk,A.M.,& Chothia,C.(1997).Standard conformations for the canonical structures of immunoglobulins.Journal of molecular biology,273(4),927-948.；MacCallum,R.M.,Martin,A.C.,& Thornton,J.M.(1996).Antibody-antigen interactions:contact analysis and binding site topography.Journal of molecular biology,262(5),732-745.；Abhinandan,K.R.,& Martin,A.C.(2008).Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains.Molecular immunology,45(14),3832-3839.；Lefranc,M.P.,Pommié,C.,Ruiz,M.,Giudicelli,V.,Foulquier,E.,Truong,L.,...& Lefranc,G.(2003).IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Developmental & Comparative Immunology,27(1),55-77.；以及Honegger,A.,&
Figure PCTCN2022101713-appb-000001
un,A.(2001).Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool.Journal of molecular biology,309(3),657-670.。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#resul ts)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia,C.,& Lesk,A.M.(1987).Canonical structures for the hypervariable regions of immunoglobulins.Journal of molecular biology,196(4),901-917.)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc,M.P.,Pommié,C.,Ruiz,M.,Giudicelli,V.,Foulquier,E.,Truong,L.,...& Lefranc,G.(2003).IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Developmental & Comparative Immunology,27(1),55-77.。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”可选自CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文中的术语“Fc区”用于定义抗体重链中含有恒定区的至少一部分的C端区域。该术语包括天然序列Fc区和变体Fc区。示例性地，人IgG重链Fc区可自Cys226或Pro230延伸至重链的羧基末端。然而，由宿主细胞生成的抗体可经历翻译后切割，自重链的C端切除一个或多个，特别是一个或两个氨基酸。因此，通过编码全长重链的特定核酸分子的表达由宿主细胞生成的抗体可包括全长重链，或者它可包括全长重链的切割变体。当重链的最终两个C端氨基酸是甘氨酸(G446)和赖氨酸(K447，编号方式依照Kabat EU索引)时可能就是这种情况。因此，Fc区的C端赖氨酸(Lys447)，或C端甘氨酸(Gly446)和赖氨酸(Lys447)可以存在或不存在。典型地，IgG Fc区包含IgG CH2和IgG CH3域，可选地，在此基础上还可包含完整或部分铰链区，但不包含CH1域。人IgG Fc区的“CH2域”通常自约位置231处的氨基酸残基延伸至约位置340处的氨基酸残基。在一个实施方案中，碳水化合物链附着于CH2域。本文中的CH2域可以是天然序列CH2域或变体CH2域。“CH3域”包含Fc区中在CH2域C端的那段残基(即自IgG的约位置341处的氨基酸残基至约位置447处的氨基酸残基)。本文中的CH3区可以是天然序列CH3域或变体CH3域(例如具有在其一条链中引入的“隆起”“节”，(knob)和在其另一条链中相应引入的“空腔”“穴”，(hole)的CH3域；参见美国专 利No.5,821,333，通过援引明确收入本文)。如本文中描述的，此类变体CH3域可用于促进两条不相同抗体重链的异二聚化。
除非本文中另有规定，Fc区或恒定区中的氨基酸残基的编号依照EU编号系统，也称作EU索引，如Kabat et al.,Sequences of Proteins of Immunological Interest,5 th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991中描述的。
本文术语“Fc变体”是指，通过在Fc上合适的位点处存在一个或者多个氨基酸替换、插入或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指的是经突变设计的Fc变体之间，可以形成空间填充效应、静电导引、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体蛋白。优选的突变设计为“Knob-into-Hole”形式的突变设计。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Ridgway,J.B.,Presta,L.G.,& Carter,P.(1996).‘Knobs-into-holes’engineering of antibody CH3domains for heavy chain heterodimerization.Protein Engineering,Design and Selection,9(7),617-621.)提出的“Knob-into-Hole”形式；Amgen公司技术人员利用静电导引(Electrostatic Steering)形成含Fc的异源二聚体形式(US 20100286374A1)；Jonathan H.Davis等人(Davis,J.H.,Aperlo,C.,Li,Y.,Kurosawa,E.,Lan,Y.,Lo,K.M.,& Huston,J.S.(2010).SEEDbodies:fusion proteins based on strand-exchange engineered domain(SEED)CH3heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies.Protein Engineering,Design & Selection,23(4),195-202.)提出的通过IgG/IgA链交换形成的异源二聚体形式(SEEDbodies)；Genmab公司DuoBody(Gramer,M.J.,van den Bremer,E.T.,van Kampen,M.D.,Kundu,A.,Kopfmann,P.,Etter,E.,...& Parren,P.W.(2013,November).Production of stable bispecific IgG1by controlled Fab-arm exchange:scalability from bench to large-scale manufacturing by application of standard approaches.In MAbs(Vol.5,No.6,pp.962-973).Taylor & Francis.)平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(Moore,G.L.,Bautista,C.,Pong,E.,Nguyen,D.H.T.,Jacinto,J.,Eivazi,A.,...& Lazar,G.A.(2011,November).A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens.In MAbs(Vol.3,No.6,pp.546-557).Taylor & Francis.)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob/Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-into-Hole”形式进行结合。优选用Cater等人的“knob-into-hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“knob-into-hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、 疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法，确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.,& Lipman,D.J.(1990).Basic local alignment search tool.Journal of molecular biology,215(3),403-410.的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul,S.F.,Madden,T.L.,
Figure PCTCN2022101713-appb-000002
A.A.,Zhang,J.,Zhang,Z.,Miller,W.,& Lipman,D.J.(1997).Gapped BLAST and PSI-BLAST:anew generation of protein database search programs.Nucleic acids research,25(17),3389-3402.中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其至少包含(1)细胞外抗原结合结构域，例如抗体的重链可变区和/或轻链可变区，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5’至3’。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler,C.R.,
Figure PCTCN2022101713-appb-000003
H.,Celik,L.,Hebich,B.,Roth,A.S.,Roth,R.P.,...& Sahin,U.(2017).Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.Nature medicine,23(7),815-817.或EP2101823B1)。
本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“药学上可接受的载体”包括任何和所有溶剂、分散介质、包衣材料、表面活性剂、抗氧化剂、防腐剂(例如，抗细菌剂、抗真菌剂)、等渗剂、吸收延迟剂、盐、防腐剂、药物稳定剂、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料等及其组合，其为本领域技术人员所知(参见例如，Remington's Pharmaceutical Sciences,第18版.MackPrinting  Company,1990,第1289-1329页)。除了与活性成分不相容的情况之外，考虑任一常规载体在治疗或药物组合物中的应用。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本文所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
附图说明
图1为人CD16a(158F)-Fc、人CD16a(158V)-Fc、CD16a(158F)-his、人CD16a(158V)-his、人CD16b(NA1)-his、人CD16b(NA2)-his、人CD16b(SH)-his和食蟹猴CD16-his蛋白样品SDS-PAGE还原胶和非还原胶检测结果。泳道1和2分别为还原和非还原条件下人CD16a(158F)-Fc的蛋白条带、泳道3和4分别为还原和非还原条件下人CD16a(158V)-Fc的蛋白条带，泳道5和6分别为还原和非还原条件下人CD16a(158F)-his的蛋白条带，泳道7和8分别为还原和非还原条件下人CD16a(158V)-his的蛋白条带，泳道9和10分别为还原和非还原条件下人CD16b(NA1)-his的蛋白条带，泳道11和12分别为还原和非还原条件下人CD16b(NA2)-his的蛋白条带，泳道13和14分别为还原和非还原条件下人CD16b(SH)-his的蛋白条带，泳道15和16分别为还原和非还原条件下食蟹猴CD16-his的蛋白条带，泳道M为蛋白maker条带；
图2A为人CD16a(158F)蛋白转染的FlpinCHO细胞FACS检测结果；
图2B为人CD16a(158V)蛋白转染的FlpinCHO细胞FACS检测结果；
图2C为人CD16b(NA1)蛋白转染的FlpinCHO细胞FACS检测结果；
图2D为人CD16b(NA2)蛋白转染的FlpinCHO细胞FACS检测结果；
图2E为食蟹猴CD16蛋白转染的FlpinCHO细胞FACS检测结果；
图3A为CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158F)细胞的结合；
图3B为CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158V)细胞的结合；
图4A为生物素标记的CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158V)细胞的结合；
图4B为10mg/mL人免疫球蛋白存在下，生物素标记的CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158V)细胞的结合；
图4C为生物素标记的CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158F)细胞的结合；
图4D为10mg/mL人免疫球蛋白存在下，生物素标记的CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158F)细胞的结合；
图5A为FACS检测CD16 VHH-Fc抗体与FlpinCHO-人CD16b(NA1)细胞的结合反应；
图5B为FACS检测CD16 VHH-Fc抗体与FlpinCHO-人CD16b(NA2)细胞的结合反应。
图6A为CD16 VHH-Fc抗体与人NK细胞的结合；
图6B为生物素标记的CD16 VHH-Fc抗体与人NK细胞的结合；
图6C为10mg/mL人免疫球蛋白存在下，生物素标记的CD16 VHH-Fc抗体与人NK细胞的结合；
图7为FACS检测CD16 VHH-Fc抗体与FlpinCHO-猴CD16细胞的结合反应；
图8为荧光素酶报告系统检测抗体对Jurkat-NFAT细胞的激活。
图9A为人源化CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158F)细胞的结合；
图9B为人源化CD16 VHH-Fc抗体与FlpinCHO-人CD16a(158V)细胞的结合；
图10A为人源化CD16 VHH-Fc抗体与FlpinCHO-人CD16b(NA1)细胞的结合反应；
图10B为人源化CD16 VHH-Fc抗体与FlpinCHO-人CD16b(NA2)细胞的结合反应。
图11A为生物素标记的CD16 VHH-Fc抗体与NK细胞的结合；
图11B为10mg/mL人免疫球蛋白存在下，生物素标记的CD16 VHH-Fc抗体与NK细胞的结合；
图12为人源化CD16 VHH-Fc抗体与人中性粒细胞的结合；
图13为人源化CD16 VHH-Fc抗体与FlpinCHO-猴CD16细胞的结合反应；
图14为荧光素酶报告系统检测人源化CD16 VHH-Fc抗体对Jurkat-NFAT细胞的激活。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该 理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 CD16相关抗原和抗体的制备以及稳转细胞株的构建
1.1人和食蟹猴CD16抗原的制备
编码人CD16a(158F)全长氨基酸序列(Uniprot ID：P08637，SEQ ID NO：1)、人CD16a(158V)全长氨基酸序列(NCBI ID：AAH17865.1，SEQ ID NO：2)、人CD16b(NA1)全长氨基酸序列(NCBI ID：AAA35881.1，SEQ ID NO：3)、人CD16b(NA2)全长氨基酸序列(Uniprot ID：O75015，SEQ ID NO：4)、人CD16b(SH)(SEQ ID NO：5)全长氨基酸序列来自专利WO-2016177846，以及食蟹猴CD16全长氨基酸序列(NCBI ID：NP_001270121.1，SEQ ID NO：6)如表1所示。按照从N端到C端的顺序，将上述蛋白的胞外区(ECD，extra-cellular domain)依次与人IgG1 Fc(N297A)或His标签、(G 3S) 2linker以及AVI标签相连获得免疫和抗体筛选鉴定用抗原序列(氨基酸序列如表2 SEQ ID NO：7-14所示)，并委托通用生物系统(安徽)有限公司将氨基酸序列所对应的核苷酸序列进行基因合成并分别克隆到含信号肽的pTT5载体，并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293E细胞(购自苏州益研生物科技有限公司)进行瞬时转染(PEI，Polysciences，货号：24765-1)，并使用FreeStyle TM 293(Thermofisher scientific，货号：12338018)在37℃下进行扩大培养。6天后收集细胞培养液，离心去除细胞成分，获得含抗原蛋白胞外区的培养上清液用于下步纯化。
对于Fc标签的蛋白将培养上清液上样到蛋白A层析柱(蛋白A填料AT Protein A Diamond和层析柱BXK16/26均购自博格隆，货号分别为：AA0273和B-1620)，使用PBS磷酸盐缓冲液(pH7.4)清洗后再用20mM PB,1M NaCl(pH 7.2)进行清洗，最后使用pH3.4的柠檬酸缓冲液进行洗脱，收集从蛋白A层析柱上洗脱下来的带Fc标签的抗体，用1/10体积的pH8.0的1M Tris中和，用PBS在4℃条件透析过夜，透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存。即获得纯化的人CD16-Fc蛋白，SDS-PAGE还原胶和非还原胶检测样品目的条带如图1所示。
对于His标签的抗原将培养上清液上样到镍离子亲和层析柱HisTrap TM Excel(GE Healthcare，货号：GE17-3712-06)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用20mM PB，0.5M NaCl(pH7.4)清洗镍离子亲和层析柱直到紫外吸收值回到基线，然后用buffer A：20mM PB，0.5M NaCl(pH7.4)和buffer B：20mM PB，0.5M NaCl，500mM咪唑进行梯度洗脱(2％，4％，8％，16％，50％，100％)，收集从镍离子亲和层析柱上洗脱下来的带His标签的人CD16蛋白，用PBS磷酸盐缓冲液(pH7.4)在4℃冰箱透析过夜。透析后的蛋白经0.22微米滤膜无菌过滤后分装于-80℃保存，即获得纯化的人CD16-his蛋白，SDS-PAGE还原胶和非还原胶检测样品目的条带如图1所示。
对于生物素标记蛋白的制备，将上步纯化获得的带有His标签和AVI标签的CD16蛋白取出一部分进行体外生物素标记。按照生物素标记试剂盒(BirA biotin-protein ligase kit，Avidity，货号：BirA500)的操作说明书进行。
表1 CD16蛋白全长氨基酸序列
Figure PCTCN2022101713-appb-000004
表2 CD16胞外区带标签抗原氨基酸序列
Figure PCTCN2022101713-appb-000005
Figure PCTCN2022101713-appb-000006
注：大写加粗字体为胞外区，大写下划线为Fc标签，GGGSGGGS为linker，HHHHHHHHHH为His标签，小写加粗字体为AVI标签。
1.2 CD16检测抗体的制备
识别人CD16的小鼠抗体3G8是用PMN免疫小鼠获得的(Fleit H.B.et al.,Proc Natl Acad Sci U S A.1982May)，能够能识别人CD16a(158F和158V)和CD16b(NA1，NA2和SH)，且与食蟹猴CD16具有交叉结合活性。将3G8和P2C47的VL和VH以及人IgG1 Fc按照从N端到C端的顺序连接，其中VL和VH之间通过3个GGGGS连接子连接，形成scFv-hFc的形式，对应的氨基酸序列信息如下表3所示。将其对应的核苷酸序列分别克隆到pTT5载体(由通用生物系统(安徽)有限公司完成)并按照实施例1.1的方法在HEK293E细胞(购自苏州益研生物科技有限公司)中进行表达并纯化。NC为阴性对照抗体scFv-hFc(L234A，L235A，D265A)，是针对鸡卵溶菌酶的抗体，委托泰州市百英生物科技有限公司定制生产。
表3 抗人CD16的抗体序列信息
Figure PCTCN2022101713-appb-000007
注：加粗字体为人hIgG1 Fc序列
1.3 CD16稳转Flipin CHO细胞株的构建
分别将编码人CD16a158F全长氨基酸序列(Uniprot ID：P08637，SEQ ID NO：1)、人CD16a158V全长氨基酸序列(NCBI ID：AAH17865.1，SEQ ID NO：2)、人CD16b(NA1)全长氨基酸序列(NCBI ID：AAA35881.1，SEQ ID NO：3)、人CD16b(NA2)全长氨基酸序列(Uniprot ID：O75015，SEQ ID NO：4)以及食蟹猴CD16全长氨基酸序列(NCBI ID：NP_001270121.1，SEQ ID NO：6)的核苷酸序列克隆到pcDNA5-FRT载体(Thermofisher scientific,货号：V601020)并制备质粒(由生工生物工程(上海)股份有限公司完成)，制备完成的质粒分别和POG44酶载体(Thermofisher scientific,货号：V600520)一起共转染FlpinCHO细胞系(Thermofisher scientific,货号：R75807)，转染方法按照转染试剂(
Figure PCTCN2022101713-appb-000008
3000 Transfection Kit，Invitrogen，货号：L3000-015)的操作说明进行，将转染后的FlipinCHO细胞置于37℃，5％(v/v)CO 2培养箱中，并在含800μg/ml hygromycin以及10％(w/w)胎牛血清的F12培养基中选择性培养，大约2周后取部分细胞采用流式细胞术检测细胞表面抗原表达情况，并对恢复生长的细胞系继续扩大培养并液氮冻存。
如表4和图2所示，IgG亚型对照为鼠IgG1对照(购自泰州市百英生物科技有限公司，货号：B118301)。如图2所示，3G8抗体能与Flpin CHO-人CD16a(158F)、Flpin CHO-人CD16a(158V)、Flpin CHO-人CD16b(NA1)、Flpin CHO-人CD16b(NA2)和Flpin CHO–猴CD16细胞株结合，表4列出了转染细胞株3G8抗体和荧光二抗染色的细胞平均荧光强度，说明这些细胞株已经能稳定表达对应的CD16抗原，可以用于后续的抗体筛选鉴定。
表4 CD16全长蛋白的Flpin CHO稳转细胞系FACS检测结果
Figure PCTCN2022101713-appb-000009
实施例2 针对人CD16的单域抗体的筛选
2.1羊驼的免疫和血清效价检测
人CD16a(158F)-hFc和人CD16a(158V)-hFc蛋白用于免疫。选取一只羊驼(Llama)进行免疫，每只羊驼免疫四次，每次间隔3周，第三次免疫后和第四次免疫后采集外周血并分离血清，用酶联免疫吸附实验(ELISA)检测血清中针对人CD16的抗体效价和特异性，结果如表5所示。表5说明，经人CD16免疫的羊驼，免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在五百九十万左右。其中空白对照为1％(w/w)BSA，其中批次指第三次(TB3)和第四次(TB4)免疫后第七天的羊驼血清，表中的数据为OD450nm值。
表5 ELISA检测人CD16蛋白免疫后羊驼血清抗体效价
Figure PCTCN2022101713-appb-000010
Figure PCTCN2022101713-appb-000011
2.2文库的构建
采集三次免疫后和四次免疫后的羊驼外周血共100mL；使用淋巴细胞分离液分离PBMC，并使用RNAiso Plus试剂(Takara，货号：#9108/9109)提取总RNA，使用PrimeScript TM II 1st Strand cDNA Synthesis Kit(Takara，货号：6210A)将提取的RNA反转录成cDNA。用巢式PCR扩增编码纳米抗体的可变区核酸片段并回收目标纳米抗体核酸片段，将纳米抗体核酸片段与线性化酵母展示用载体pYDC2(来自四川阿帕克生物科技有限公司)一起电转化至酵母感受态细胞EYB100中进行体内重组，构建针对CD16的纳米抗体酵母展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为3.26×10 8。为检测文库的插入率，随机选取48个克隆做菌落PCR。结果显示插入率达到100％。
2.3针对CD16的纳米抗体VHH的淘选
将酵母展示文库用实施例1.1中制备的生物素标记的人CD16b(NA1)-his、CD16b(NA2)-his和CD16b(SH)-his蛋白包被磁珠后进行反向筛选，然后用生物素标记的人CD16a(158F)-his和CD16a(158V)-his蛋白包被磁珠进行正向筛选，富集特异性结合人CD16a的酵母克隆后，利用流式细胞术继续进行荧光素标记的人CD16b(NA1)-his、CD16b(NA2)-his和CD16b(SH)-his蛋白反向筛选和人CD16a(158F)-his和CD16a(158V)-his蛋白正向筛选，获得特异性结合CD16a的酵母克隆。
2.4流式细胞术方法筛选特异性单个阳性克隆
淘选后，将获得的CD16a(158F)和CD16a(158V)结合阳性的酵母铺板扩增并挑选单个克隆，随后挑选96个单菌落分别扩增培养。分别将生物素标记的CD16a(158F)-his蛋白与过量SA-iFluor 647(购自ThermoFisher scientific，货号：S21374)、生物素标记的CD16b(NA1)-his、CD16b(NA2)-his和CD16b(SH)-his蛋白与过量SA-Alexa Fluor 488(购自ThermoFisher scientific，货号：S11223)孵育1小时进行荧光标记备用。将表达后的酵母细胞洗涤后分成两份，一部分酵母细胞与人免疫球蛋白(上海生物制品研究所)以及前述荧光素标记的CD16a和CD16b抗原一起在4℃孵育1小时。另一部分酵母细胞与Alexa Fluor 488标记的抗HA抗体(购自ThermoFisher scientific，货号：A-21287)4℃孵育1小时。洗涤酵母细胞后，使用流式细胞仪进行分析，分别计算荧光强度(MFI)。将荧光标记的CD16a和CD16b的MFI分别除以荧光标记的抗HA的MFI，获得均一化后的CD16a和CD16b的MFI 值，部分克隆的均一化MFI值如表6所示。
表6 单克隆酵母细胞均一化后的CD16a和CD16b荧光强度值
Figure PCTCN2022101713-appb-000012
挑选CD16a阳性的克隆进行测序。对测序结果使用MOE软件进行分析，根据VHH编码蛋白氨基酸序列构建进化树，根据序列相似性剔除在进化树上距离较近的序列后，筛选获得14个克隆，纳米抗体序列如表7所示，表8示出VHH和Fc区段融合的氨基酸序列。其序列的CDRs分别用KABAT、Chothia或IMGT软件分析，CDR序列信息如下表9所示。随后按照实施例1.2的方法进行VHH纳米抗体Fc融合蛋白的生产鉴定。
表7 VHH抗体氨基酸序列
Figure PCTCN2022101713-appb-000013
Figure PCTCN2022101713-appb-000014
表8 VHH-Fc抗体氨基酸序列
Figure PCTCN2022101713-appb-000015
Figure PCTCN2022101713-appb-000016
Figure PCTCN2022101713-appb-000017
Figure PCTCN2022101713-appb-000018
注：加粗字体为人hIgG1Fc序列。
表9 CDR序列信息
Figure PCTCN2022101713-appb-000019
Figure PCTCN2022101713-appb-000020
Figure PCTCN2022101713-appb-000021
实施例3 CD16抗体的结合活性检测
3.1流式细胞实验(FACS)检测人CD16抗体与CD16a不同突变体表达细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至对数生长期，吸除培养基，用PBS缓冲液洗涤2次，然后用胰酶消化细胞，终止消化后用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞沉淀用[PBS+2％(w/w)BSA]封闭液重悬至2×10 6个细胞/毫升，按50μl/孔加入到96孔FACS反应板中，待测抗体的起始浓度100nM，用PBS按照1:5的比例进行梯度稀释，加入不同浓度的待测抗体50μl/孔，冰上孵育1小时。用PBS缓冲液离心洗涤3次，加入50μl/孔iFluor 647标记的抗人IgG的二抗(购自Jackson immune，货号：109-605-088)，冰上孵育1小时。用PBS缓冲液离心洗涤5次，用FACS(FACS Canto II，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50值。
结果如表10以及图3A-3B所示，CD16 VHH-hFc抗体，与FlpinCHO-CD16a(158F)和FlpinCHO-CD16a(158V)细胞均有很好的结合活性，与两种细胞结合的EC50相当，大部分抗体的EC50均小于1nM。
表10 FACS检测VHH-Fc抗体与FlpinCHO-CD16a细胞的结合反应
Figure PCTCN2022101713-appb-000022
Figure PCTCN2022101713-appb-000023
3.2流式细胞实验(FACS)检测人免疫球蛋白对CD16抗体与细胞结合的影响
待测抗体提前按照生物素标记试剂盒(EZ-Link TM NHS-PEG4 Biotinylation Kit，购自Thermofisher Scientific,货号：21455)说明书进行标记，收集细胞后，将细胞直接与梯度稀释的待测抗体进行孵育，或者细胞先与10mg/mL人免疫球蛋白(上海生物制品研究所)孵育后再与梯度稀释的待测抗体孵育，并按照实施例3.1的方法进行检测，其中生物素标记的抗体用荧光素APC标记的Streptavidin(High Concentration)(购自Biolegend，货号：405243)进行检测。在人免疫球蛋白存在下，对照抗体3G8与FlpinCHO-人CD16a(158V)的结合活性完全丢失，VHH抗体与细胞的结合活性EC50下降程度在2-4倍之间，对照抗体P2C47的EC50下降程度在4-5倍左右，结果如图4A-4B和表11-1所示。在人免疫球蛋白存在下，对照抗体3G8与FlpinCHO-人CD16a(158F)的结合活性完全丢失，大部分VHH抗体与细胞的结合活性EC50下降程度在2-4倍之间，对照抗体P2C47的EC50下降程度在7-8倍左右，结果如图4C-4D和表11-2所示。
表11-1 FACS检测人免疫球蛋白对VHH-Fc抗体与FlpinCHO-CD16a(158V)细胞的结合影响
Figure PCTCN2022101713-appb-000024
Figure PCTCN2022101713-appb-000025
表12-2 FACS检测人免疫球蛋白对VHH-Fc抗体与FlpinCHO-CD16a细胞的结合影响
Figure PCTCN2022101713-appb-000026
3.3流式细胞实验(FACS)检测人CD16抗体与CD16b不同突变体表达细胞的结合
按照实施例3.1的方法检测待测抗体与FlpinCHO-人CD16b细胞的结合。如图5A-5B和表12所示，3G8抗体是结合CD16b的阳性抗体，所有待测抗体与FlpinCHO-人CD16b(NA1)和FlpinCHO-人CD16b(NA2)的结合活性都弱于3G8抗体。
表13 FACS检测VHH-Fc抗体与FlpinCHO-CD16b细胞的结合反应
Figure PCTCN2022101713-appb-000027
Figure PCTCN2022101713-appb-000028
实施例4 CD16抗体与人原代NK细胞的结合活性检测
4.1流式细胞实验(FACS)检测人CD16抗体与静息NK细胞的结合
按照实施例3.1的方法检测待测抗体与新鲜制备的人NK细胞(妙顺(上海)生物科技有限公司，货号：PB56-N-Custom)的结合。如图6A和表13所示，所有检测抗体均与NK细胞有很好的结合活性，EC50小于1nM。
表14 FACS检测VHH-hFc抗体与NK细胞的结合反应
Figure PCTCN2022101713-appb-000029
4.2流式细胞实验(FACS)检测人免疫球蛋白存在时对人CD16抗体与静息NK细胞结合的影响
按照实施例3.2的方法进行检测，结果如图6B和表14所示，在人免疫球蛋白存在下， 对照抗体3G8与NK细胞的结合活性完全丢失，VHH-05、VHH-06和VHH-11抗体与细胞的结合活性EC50下降程度在2-3倍之间，对照抗体P2C47的EC50下降程度在4倍左右。并且在人免疫球蛋白存在时，VHH-05、VHH-06、VHH-11和VHH-12与NK细胞的结合活性均强于P2C47抗体。
表15 人免疫球蛋白存在下，FACS检测VHH-hFc抗体与NK细胞的结合反应
Figure PCTCN2022101713-appb-000030
实施例5 CD16抗体种属交叉活性的检测
5.1 FACS检测CD16抗体与食蟹猴CD16表达细胞的结合
将Flipin CHO-猴CD16细胞按照实施例3.1的方法进行FACS检测与数据分析。分析结果如表15以及图7所示，VHH-11、VHH-12、VHH-13和VHH-14抗体均与过表达猴CD16的Flipin CHO细胞均有结合活性，EC50显示结合活性最高达到0.18nM。
表16 FACS检测VHH-hFc抗体与FlpinCHO-猴CD16细胞的结合反应
Figure PCTCN2022101713-appb-000031
实施例6 人CD16抗体的功能鉴定
6.1荧光素酶报告基因(report assay)检测人CD16抗体的功能
利用稳定表达人CD16a(158V)的Jurkat-NFAT荧光素酶报告细胞系(购自BPS Bioscience，货号60541)检测CD16抗体激活荧光素酶报告细胞系内的信号通路，荧光素酶的信号检测按照Bright-Glo TM Luciferase Assay System试剂盒(Promega，货号：E2620)操作说明进行。实验方法如下：将2μg/mL抗人Fc标签的抗体(Jackson Immune，货号：109-006-098)于4℃包被过夜用于捕获带人Fc标签的待测抗体，次日将待检测的抗体用含5％FBS的RIPM 1640培养基稀释，抗体起始浓度4nM，1:6梯度稀释，然后以50μl/孔加到96孔平底细胞培 养板，37℃预孵育15分钟后，将收集的处于对数生长期的荧光素酶报告细胞系，用含2％FBS的培养基(RPMI 1640，采购于Gibco，货号12633012)调节细胞浓度至8×10 5/mL，将细胞加入培养板，50μl/孔。在37℃的培养箱中孵育6小时后400g离心5分钟，取上清20uL至黑色不透明96孔检测板(Greiner，货号：655090)中，加入50uL检测试剂，通过PerkiElmer Ensight-HH3400酶标仪读取响应值，再通过软件(GraphPad Prism8)分析，进行数据拟合，计算EC50值。结果如表16及图8所示，在report assay中，所有抗体能激活Jurkat细胞的下游信号通路，且激活活性与P2C47抗体相当或更强。
表17 荧光素酶报告基因检测CD16抗体的激活活性
Figure PCTCN2022101713-appb-000032
实施例7 CD16抗体亲和力测定
7.1 CD16抗体与人CD16a-ECD-his蛋白亲和力测定
使用Protein A芯片(GE Helthcare；29-127-558)捕获抗人CD16的VHH-hFc形式抗体。样品和运行缓冲液是HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(GE Healthcare；BR-1006-69)。流经池设置为25℃。样品块设置为16℃。两者都用运行缓冲液预处理。在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的CD16抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5(GE Helthcare；BR-1003-54)完成芯片再生。通过注射溶液中不同浓度的重组人CD16-ECD his蛋白持续240秒来测量结合，其中流速为30μL/分钟，从200nM起始(测试的实际浓度见详细结果)，以1:1稀释，总共5个浓度。监测解离相长达600秒，并通过从样品溶液切换到运行缓冲液触发。通过用10mM甘氨酸溶液(pH 1.5)以30μL/分钟的流速洗涤30秒，再生表面。通过减去从山羊抗人Fc表面获得的响应来校正本体折射率(Bulk refractive index)差异。也减去空白注射(＝双重参照)。为了计算表观KD和其他动力学参数，使用Langmuir 1:1模型。待测抗体与人CD16-ECD his蛋白的结合速率(ka)、解离速率(kd)及结合亲和力(KD)如表17所示。
表18 Biacore检测CD16抗体与人CD16a蛋白的结合
Figure PCTCN2022101713-appb-000033
7.2 CD16抗体与人CD16b-ECD-his蛋白亲和力测定
按照实施例7.1的方法检测待测抗体与人CD16b-ECD his蛋白的结合速率(ka)、解离速率(kd)及结合亲和力(KD)如表18和表19所示。
表19 Biacore检测CD16抗体与人CD16b蛋白的结合
Figure PCTCN2022101713-appb-000034
表20 Biacore检测CD16抗体与人CD16b蛋白的结合
Figure PCTCN2022101713-appb-000035
7.3 CD16抗体与猴CD16-ECD-his蛋白亲和力测定
按照实施例7.1的方法检测待测抗体与猴CD16-ECD his蛋白的结合速率(ka)、解离速率(kd)及结合亲和力(KD)如表20所示。
表21 Biacore检测CD16抗体与猴CD16蛋白的结合
Figure PCTCN2022101713-appb-000036
实施例8 抗CD16单域抗体的人源化设计以及制备
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选IGHV3-30*02和IGHV3-7*01分别作为羊驼抗体VHH-06和VHH-12选择的人源化重链模板。首先，将羊驼抗体的CDR分别移植到其人源模板的FR中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据抗体的三维结构模拟，将人源化抗体的FR区序列中关键氨基酸进行回复突变为羊驼抗体对应的氨基酸，以保证原有的亲和力，得到人源化抗CD16单域抗体。本实施例抗体氨基酸残基的编号和CDR区均由IMGT编号系统确定并注释(详见http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)。通过上述方法设计得到的人源化抗体VHH分别见表21和表22。
表21 VHH06的人源化VH的设计
VH FR Template FR mutations
VH3 IGHV3-30*02 M39V,H40R,V42Y,G49Q,L50R,Y66N,L87V,V101D,Y103I
VH4 IGHV3-30*02 M39V,H40R,V42Y,G49Q,L50R,Y66N,A68P,L87V,V101D,Y103I
VH5 IGHV3-30*02 M39V,H40R,V42Y,G49Q,L50R,Y66N,S83A,L87V,V101D,Y103I
VH6 IGHV3-30*02 M39V,H40R,V42Y,G49Q,L50R,Y66N,I78V,L87V,V101D,Y103I
VH7 IGHV3-30*02 M39V,H40R,V42Y,G49Q,L50R,Y66N,L87V,V101D,Y103I,M123Q
注：M39V表示将Graft第39位M突变成V，其它依此类推。回复突变氨基酸的编号为IMGT编号。
表22 VHH12的人源化VH的设计
VH FR Template FR mutations
VH2 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A
VH3 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N
VH4 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N,V68A,S70F
VH5 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N,D81V
VH6 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N,L87V
VH7 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N,M123Q
VH8 IGHV3-30*01 S40G,V42Y,G49Q,L50R,W52L,N55A,Y66N,V68A,S70F,D81V
注：S40G表示将Graft第40位S突变成G，其它依此类推。回复突变氨基酸的编号为IMGT编号。
人源化VHH序列如表23和表24所示，将人源化抗体序列按照实施例1.2的方法制备VHH-Fc抗体，人源化VHH-Fc序列如表25和表26所示。
表23 VHH06人源化抗体序列
Figure PCTCN2022101713-appb-000037
Figure PCTCN2022101713-appb-000038
表24 VHH12人源化抗体序列
Figure PCTCN2022101713-appb-000039
表25 VHH06-Fc人源化抗体序列
Figure PCTCN2022101713-appb-000040
Figure PCTCN2022101713-appb-000041
注：加粗字体为人hIgG1 Fc序列
表26 VHH12-Fc人源化抗体序列
Figure PCTCN2022101713-appb-000042
Figure PCTCN2022101713-appb-000043
注：加粗字体为人hIgG1 Fc序列
实施例9 CD16抗体的结合活性检测
9.1流式细胞实验(FACS)检测人CD16抗体与CD16a不同突变体表达细胞的结合
按照实施例3.1的方法检测人源化抗体与FlpinCHO-人CD16a细胞的结合。如图9A-9B和表27所示，与FlpinCHO-CD16a(158F)和FlpinCHO-CD16a(158V)细胞均有很好的结合活性，与两种细胞结合的EC50相当，均小于1nM。
表27 FACS检测VHH-Fc抗体与FlpinCHO-CD16a细胞的结合反应
Figure PCTCN2022101713-appb-000044
Figure PCTCN2022101713-appb-000045
9.2流式细胞实验(FACS)检测人CD16抗体与CD16b不同突变体表达细胞的结合
按照实施例3.1的方法检测待测抗体与FlpinCHO-人CD16b细胞的结合。在实施例3中已经检测过P2C47与FlpinCHO-人CD16b(NA1)和FlpinCHO-人CD16b(NA1)的结合活性明显弱于CD16a和CD16b阳性抗体3G8。如图10A-10B和表28所示，人源化VHH抗体与FlpinCHO-人CD16b(NA1)和FlpinCHO-人CD16b(NA2)的结合与对照抗体P2C47相当，因此可推测人源化抗体的结合活性也弱于3G8抗体。
表28 FACS检测VHH-Fc抗体与FlpinCHO-CD16b细胞的结合反应
Figure PCTCN2022101713-appb-000046
注：NA表示抗体结合活性弱，曲线拟合差。
实施例10 CD16抗体与人原代NK细胞以及粒细胞的结合活性检测
10.1流式细胞实验(FACS)检测人免疫球蛋白存在时对人CD16抗体与静息NK细胞结合的影响
按照实施例3.2的方法进行检测，结果如图11A-11B和表29所示，在人免疫球蛋白存在下，对照抗体3G8与NK细胞的结合活性完全丢失，VHH-05、VHH-06和VHH-11抗体与细 胞的结合活性EC50下降程度在3-4倍之间，对照抗体P2C47的EC50下降程度在4-5倍左右。并且在人免疫球蛋白存在时，VHH06-03、VHH06-04、VHH12-05和VHH12-06与NK细胞的结合活性均强于P2C47抗体。
表29 人免疫球蛋白存在下，FACS检测VHH-hFc抗体与NK细胞的结合反应
Figure PCTCN2022101713-appb-000047
实施例11 CD16抗体种属交叉活性的检测
11.1 FACS检测CD16抗体与食蟹猴CD16表达细胞的结合
将Flipin CHO-猴CD16细胞按照实施例3.1的方法进行FACS检测与数据分析。分析结果如表30以及图12所示，VHH-12的人源化抗体均与过表达猴CD16的Flipin CHO细胞均有结合活性，EC50均小于1nM。
表30 FACS检测VHH-hFc抗体与FlpinCHO-猴CD16细胞的结合反应
Figure PCTCN2022101713-appb-000048
实施例12 人CD16抗体的功能鉴定
12.1荧光素酶报告基因(report assay)检测人CD16抗体的功能
按照实施例6.1的方法检测人源化CD16抗体激活荧光素酶报告细胞系内的信号通路，结果如表31及图13所示，在report assay中，所有人源化抗体均能激活Jurkat细胞的下游信号通路，且激活活性强于P2C47抗体。
表31 荧光素酶报告基因检测CD16抗体的激活活性
Figure PCTCN2022101713-appb-000049
Figure PCTCN2022101713-appb-000050
实施例13 CD16抗体亲和力测定
按照实施例7.1的方法检测人源化CD16抗体与人CD16a(158F)-ECD-his蛋白亲和力。待测抗体与人CD16a(158F)-ECD his蛋白的结合速率(ka)、解离速率(kd)及结合亲和力(KD)如表32所示。
表32 Biacore检测CD16抗体与人CD16a蛋白的结合
Figure PCTCN2022101713-appb-000051

Claims (25)

  1. 一种特异性结合人CD16的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含CDRs组合，所述CDRs组合包含：CDR1、CDR2和CDR3；所述CDR1、CDR2和CDR3具有选自SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH序列的HCDR1、HCDR2和HCDR3。
  2. 根据权利要求1所述的抗体或抗原结合片段，其特征在于，根据Kabat、Chothia或IMGT编号系统确定所述HCDR1、HCDR2和HCDR3；可选地，所述HCDR1、HCDR2和HCDR3选自表9；
    可选地，所述HCDR1选自SEQ ID NO：49、52、54、57、60、62、65、66、67、68、70、72、78、79、80、81或88；
    可选地，所述HCDR2选自SEQ ID NO：50、53、55、58、61、63、69、71、73、75、76、77、82或85；
    可选地，所述HCDR3选自SEQ ID NO：51、56、59、64、74、83、84、86或87；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：52、53、51或SEQ ID NO：54、55、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、58、59，SEQ ID NO：60、61、59或SEQ ID NO：62、63、64；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:49、50、51，SEQ ID NO：65、53、51或SEQ ID NO：66、55、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO:49、50、51，SEQ ID NO：67、53、51或SEQ ID NO：68、55、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、69、51，SEQ ID NO：70、71、51或SEQ ID NO：72、73、74；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、75、51，SEQ ID NO：52、76、51或SEQ ID NO：54、77、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：78、53、51或SEQ ID NO：79、55、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：80、53、51或SEQ ID NO：81、55、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、69、51，SEQ ID NO：65、71、51或SEQ ID NO：66、73、56；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、82、83，SEQ ID NO：60、61、83或SEQ ID NO：62、63、84；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、85、86，SEQ ID NO：60、61、86或SEQ ID NO：62、63、87；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：57、82、86，SEQ ID NO：60、61、86或SEQ ID NO：62、63、87；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：88、82、83，SEQ ID NO：60、61、83或SEQ ID NO：62、63、84；
    优选地，根据Kabat、Chothia或IMGT编号系统，所述HCDR1、HCDR2和HCDR3选自SEQ ID NO：49、50、51，SEQ ID NO：80、53、51或SEQ ID NO：81、55、56；
  3. 根据权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述CDR1、CDR2和/或CDR3包含在所述HCDR1、HCDR2和/或HCDR3上发生1个、2个或3个突变的氨基酸序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  4. 根据权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述CDR1、CDR2和/或CDR3包含与所述HCDR1、HCDR2和/或HCDR3相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  5. 根据权利要求1～4任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含单域抗体，所述单域抗体包含所述CDR1、CDR2和CDR3。
  6. 根据权利要求5所述的抗体或抗原结合片段，其特征在于，所述单域抗体包含选自SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列；
    可选地，所述单域抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列相比具有至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述单域抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示的序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  7. 根据权利要求1～6任一项所述的抗体或抗原结合片段，其特征在于，所述抗体包含SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区；
    可选地，所述抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区相比具有至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列，所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    可选地，所述单域抗体包含与SEQ ID NO：21～34或SEQ ID NO：90～101任一项所示VHH结构域中的FR区序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列。
  8. 根据权利要求1～7任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  9. 根据权利要求1～8任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含或不包含抗体重链恒定区；可选地，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或山羊；可选地所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1、IgG2、IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区，优选包含如SEQ ID NO：35～48或SEQ ID NO：102～113任一项所示的序列；优选地，所述抗体或抗原结合片段为重链抗体。
  10. 根据权利要求1～9任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、细胞毒性剂、化疗药物或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  11. 根据权利要求1～10任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段特异性结合人CD16或猴CD16，优选地所述抗体或抗原结合片段与人的CD16或猴CD16的KD小于1E-6M、1E-7M、2E-7M、3E-7M、4E-7M、5E-7M、6E-7M、8E-7M、9E-7M、1E-8M、2E-8M、3E-8M、4E-8M、5E-8M、6E-8M、8E-8M、9E-8M或1E-9M。
  12. 根据权利要求1～11任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段与CD16A结合，不与CD16B结合或与CD16B弱结合，所述CD16B选自CD16B(NA1)、CD16B(NA2)或CD16B(HS)。
  13. 根据权利要求1～12任一项所述抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段还连接有其他功能性分子，优选地，所述其他功能性分子可选自以下的一种或多种：信号肽、蛋白标签、细胞因子、血管生成抑制剂或免疫检查点抑制剂。
  14. 根据权利要求13所述的抗体或抗原结合片段，其特征在于所述细胞因子可为IL-2、IL-6、IL-12、IL-15、IL-21、IFNγ或TNFα；所述血管生成抑制剂可为内皮抑制素；所述免疫检查点抑制剂可为SIRPα。
  15. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含权利要求1～14的抗体或抗原结合片段、以及结合CD16以外其它抗原的抗原结合分子，或结合与权利要求1～14所述的抗体或抗原结合片段不同的CD16表位；可选地，所述CD16以外其它抗原可选自：CD137、CD258、PD-1、PD-L1、4-1BB、CD40、CD64、EGFR、VEGF、HER2、HER1、HER3、IGF-1R、磷脂酰丝氨酸(Phosphatidylserine，PS)、C-Met、BCMA、HSA、GPRC5D、MSLN、血脑屏障受体、GPC3，PSMA，CD33，GD2，ROR1，ROR2，FRα或Gucy2C；
    优选地，所述其他抗原结合分子为抗体或抗原结合片段；
    优选地，所述多特异性抗原结合分子可为双特异性、三特异性或四特异性；
    优选地，所述多特异性结合分子可为二价、四价或六价。
  16. 一种分离的核酸片段，其特征在于，所述核酸片段编码权利要求1～14任一项所述抗体或抗原结合片段或权利要求15所述多特异性抗原结合分子。
  17. 一种载体(vector)，其特征自在于，所述载体包含权利要求16所述的核酸片段。
  18. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求17所述的载体；优选地，所述宿主细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
  19. 一种制备权利要求1～14任一项所述抗体或抗原结合片段、权利要求15所述多特异性抗原结合分子的方法，其特征在于，所述方法包括培养权利要求18所述细胞，以及分离所述细胞表达的抗体、抗原结合片段或多特异性抗原结合分子。
  20. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求16所述的核酸片 段、权利要求17所述载体或根据权利要求19所述方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的载体(carrier)、稀释剂或助剂；可选地，所述药物组合物还包含额外的抗肿瘤剂。
  21. 一种治疗肿瘤或癌症、炎性疾病或过敏症的方法，其特征在于，所述方法包括向受试者施用有效量的权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求16所述的核酸片段、权利要求17所述的载体、根据权利要求19所述方法制备获得的产品或权利要求20所述的药物组合物；优选地，肿瘤或癌症选自非霍奇金淋巴瘤、慢性淋巴细胞白血病、霍奇金氏病、微小残留病、转移瘤。
  22. 权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求16所述的核酸片段、权利要求17所述的载体、根据权利要求19所述方法制备获得的产品或权利要求20所述的药物组合物在制备治疗肿瘤或癌症、炎性疾病或过敏症药物中的用途；优选地，肿瘤或癌症选自非霍奇金淋巴瘤、慢性淋巴细胞白血病、霍奇金氏病、微小残留病、转移瘤。
  23. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1～14任一项所述的抗体或抗原结合片段、权利要求15所述的多特异性抗原结合分子、权利要求16所述的核酸片段、权利要求17所述的载体、根据权利要求19所述方法制备获得的产品或权利要求20所述的药物组合物。
  24. 一种检测生物学样品中CD16表达的方法，其特征在于，所述方法包括在权利要求1～14任一项所述的抗体或抗原结合片段与CD16之间能够形成复合物的条件下，使所述生物学样品与权利要求1～14任一项所述的抗体或抗原结合片段接触；优选地，所述方法还包括检测所述复合物的形成，指示样品中CD16的存在或表达水平。
  25. 权利要求1～14任一项所述的抗体或抗原结合片段在制备CD16检测试剂中的用途。
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STADLER, C. R.BAHR-MAHMUD, H.CELIK, L.HEBICH, B.ROTH, A. S.ROTH, R. P.SAHIN, U.: "Elimination of large tumours in mice by mRNA-encoded bispecific antibodies", NATURE MEDICINE, vol. 23, no. 7, 2017, pages 815 - 817
VAN FAASSEN HENK, JO DONG-HYEON, RYAN SHANNON, LOWDEN MICHAEL J., RAPHAEL SHALINI, MACKENZIE C. ROGER, LEE SEUNG-HWAN, HUSSACK GRE: "Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC", MOLECULAR PHARMACEUTICS, AMERICAN CHEMICAL SOCIETY, US, vol. 18, no. 6, 7 June 2021 (2021-06-07), US , pages 2375 - 2384, XP093018471, ISSN: 1543-8384, DOI: 10.1021/acs.molpharmaceut.1c00208 *

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WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024183635A1 (en) * 2023-03-03 2024-09-12 Beigene, Ltd. Muc1 and cd16a antibodies and methods of use
WO2024183636A1 (en) * 2023-03-03 2024-09-12 Beigene Switzerland Gmbh Cd16a antibodies and methods of use
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions
WO2025076127A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles
WO2025076113A1 (en) 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025108079A1 (zh) * 2023-11-21 2025-05-30 南京融捷康生物科技有限公司 一种抗CD16a的单域抗体及其用途
WO2025179294A2 (en) 2024-02-22 2025-08-28 Capstan Therapeutics, Inc. Immune engineering amplification
WO2025217454A2 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Ionizable cationic lipids and lipid nanoparticles
WO2025217452A1 (en) 2024-04-11 2025-10-16 Capstan Therapeutics, Inc. Constrained ionizable cationic lipids and lipid nanoparticles

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US20240343811A1 (en) 2024-10-17

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