WO2023116010A1 - 双特异性抗体及其应用 - Google Patents

双特异性抗体及其应用 Download PDF

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WO2023116010A1
WO2023116010A1 PCT/CN2022/114434 CN2022114434W WO2023116010A1 WO 2023116010 A1 WO2023116010 A1 WO 2023116010A1 CN 2022114434 W CN2022114434 W CN 2022114434W WO 2023116010 A1 WO2023116010 A1 WO 2023116010A1
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cells
cancer
cd16a
recombinant
antibody
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French (fr)
Inventor
肖卫华
田志刚
李洋阳
谢思奇
陈敏华
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Shanghai NK Cell Tech Co Ltd
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Shanghai NK Cell Tech Co Ltd
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Priority to JP2024524501A priority Critical patent/JP7717275B2/ja
Priority to KR1020247008669A priority patent/KR20240046248A/ko
Priority to EP22909337.2A priority patent/EP4382541A4/en
Publication of WO2023116010A1 publication Critical patent/WO2023116010A1/zh
Priority to US18/640,012 priority patent/US20240254236A1/en
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    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A—HUMAN NECESSITIES
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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    • 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)
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    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention belongs to the field of biomedicine, specifically relates to bispecific antibodies and applications thereof, and more specifically relates to recombinant antibodies, immune cells, nucleic acids, expression vectors, recombinant cells, compositions, the use of the above substances in the preparation of medicines, Reagent test kit.
  • Bispecific antibodies are antibodies that can specifically bind two antigenic sites at the same time.
  • NK cells are the main effector cells of the innate immune system. Compared with T cells, NK cells can kill tumor cells and virus-infected cells without MHC restriction by using perforin, granzyme and related mechanisms without prior stimulation. NK cells express many activating receptors, which can effectively recognize the stress ligands produced by tumor cells or infected cells, thereby effectively killing tumor cells, but at the same time, tumor cells also secrete and produce many immunosuppressive molecules, forming immunosuppression The tumor microenvironment can inhibit the activity of NK cells and the anti-tumor ability in this environment; make NK cells in a state of exhaustion.
  • NK cells in the tumor microenvironment there are also many strategies to restore the depletion of NK cells in the tumor microenvironment, such as immune checkpoint blockade, activating antibodies, activating cytokines, etc., these methods can provide activation signals to NK cells or block the tumor microenvironment
  • the inhibitory signal in the NK cell restores the normal activation state and exerts the function of anti-tumor or anti-virus.
  • CD16a is an important class of activating receptors expressed on NK cells. When CD16a is activated by its ligand or antibody binding, it will induce various cascade activation reactions, release granzymes, perforin, inflammatory cytokines and This will bring about a powerful activation effect and enhancement of anti-tumor ability for NK cells.
  • IL-21R IL-21 receptor
  • IL-21 receptor is a cytokine receptor constitutively expressed on NK cells.
  • NK cells When it is recognized and activated by its corresponding ligand IL-21 molecule, for NK cells in mice, this process will promote These NK cells have more granules, up-regulate the expression of perforin, increase the secretion of IFN- ⁇ , and promote effector activity; for human NK cells, this process can enhance IFN- ⁇ secretion, increase proliferation and NK cytotoxic activity, and at the same time Accelerates NK cell maturation and expression of NK cell receptors. Therefore, the development of highly targeted bispecific antibodies is of great value for the prevention and treatment of tumors.
  • the inventors combined CD16a antibody and IL-21 molecule to design a recombinant bispecific antibody, which can specifically bind CD16a and IL-21 receptor (IL-21R), Furthermore, IL-21 molecules and CD16a antibodies are brought into the vicinity of NK cells in a targeted manner, and combined with CD16a and/or IL-21R on NK cells, thereby inducing various cascade activation reactions, activating NK cells, and restoring The depletion of NK cells in the tumor microenvironment simultaneously enhances the secretion of IFN- ⁇ , increases the proliferation and cytotoxic activity of NK, and accelerates the maturation of NK cells and the expression of NK cell receptors to kill tumor cells or cells infected by viruses, said The recombinant bispecific antibody has high CD16a and IL-21R binding activity, and has high anti-tumor and anti-virus capabilities.
  • the present invention proposes a recombinant antibody.
  • it includes: an IL-21 molecule; and a CD16a antibody
  • the CD16a antibody includes a CD16a single-chain antibody and an Fc region, and the N-terminal of the Fc region is connected to the C-terminal of the single-chain antibody, so
  • the CD16a single-chain antibody includes a heavy chain variable region and a light chain variable region; the C-terminal of the FC is connected to the N-terminal of the IL21 molecule.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R, has strong binding activity in vivo and in vitro, can effectively activate NK cells, has a long half-life in vivo, and has remarkable anti-tumor and anti-virus effects.
  • the invention proposes a nucleic acid.
  • the nucleic acid encodes the recombinant antibody described in the first aspect.
  • the recombinant antibody encoded by nucleic acid according to the embodiment of the present invention can effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activity, can effectively activate NK cells, has a long half-life in vivo, and is anti-tumor and anti-virus The effect is remarkable.
  • the present invention provides an expression vector.
  • the expression vector carries the nucleic acid described in the second aspect.
  • the expression vector may include optional control sequences operably linked to the nucleic acid molecule.
  • the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in the host.
  • the expression vectors proposed in the embodiments of the present invention can efficiently express the recombinant antibodies in suitable host cells, and then can effectively activate NK cells for the treatment or prevention of diseases caused by tumors or viral infections.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant antibody described in the first aspect.
  • the recombinant cells are obtained by transfecting or transforming the expression vector.
  • the recombinant cells can efficiently express the above-mentioned recombinant antibodies under suitable conditions, and the recombinant cells can be effectively used for the treatment or prevention of diseases caused by tumors or viral infections, and can effectively activate NK cells.
  • the invention proposes a composition.
  • it includes the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect or the recombinant cell described in the fourth aspect.
  • the recombinant antibody of the embodiment of the present invention can effectively bind to CD16a and IL-21R protein molecules, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a, so that NK cells can be effectively activated.
  • the composition containing the recombinant antibody also has significant effects on treating or preventing tumors or diseases caused by virus infection, and its anti-tumor and anti-virus capabilities are remarkable.
  • the present invention provides the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect or the fifth aspect Use of the composition described in the aspect in the preparation of medicaments.
  • the medicament is used for treating or preventing tumor or viral infection.
  • the recombinant antibodies of the embodiments of the present invention can effectively bind to CD16a and IL-21R proteins, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a.
  • the medicaments prepared from the corresponding series of substances also have significant effects on treating or preventing tumors or viral infections, and have remarkable antitumor and antiviral abilities.
  • the present invention provides the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, and the recombinant cell described in the fourth aspect.
  • Use in increasing NK cells can be used to promote the proliferation and expansion of the NK cells in vitro, and the expanded NK cells have better cytotoxicity.
  • the present invention proposes a medicine. According to an embodiment of the present invention, it includes: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, or the nucleic acid molecule described in the fifth aspect.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R proteins, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a, using the recombinant antibody and
  • the medicaments prepared from the corresponding series of substances also have significant effects on treating or preventing tumor or virus infection, and have remarkable antitumor and antiviral abilities.
  • the present invention provides a kit.
  • it includes: the recombinant antibody described in the first aspect.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R protein molecules, and then can specifically recognize NK cells that specifically highly express the IL-21R or CD16a. Therefore, the recombinant antibody can be used to prepare a kit for detecting CD16a and/or IL-21R, and the kit can be used for scientific research, such as qualitative or quantitative detection of CD16a and/or IL-21R protein molecules in biological samples, so as to obtain Biological samples that meet the requirements, etc.
  • the present invention proposes the use of the aforementioned recombinant antibody, nucleic acid molecule, expression vector, recombinant cell, composition or drug in the treatment or prevention of tumor or viral infection.
  • the recombinant antibody can specifically recognize the above-mentioned CD16a and IL-21R proteins, and then can specifically recognize the NK cells that specifically highly express the IL-21R or CD16a, so that the NK cells can be effectively activated and the IFN- ⁇ secretion, increase the proliferation and cytotoxic activity of NK, effectively kill tumor cells, accelerate the maturation of NK cells and the expression of NK cell receptors at the same time, can effectively restore the exhaustion of NK cells in the tumor microenvironment, therefore, according to the embodiment of the present invention Recombinant antibodies, compositions containing the recombinant antibodies, drugs, or a series of substances capable of obtaining the recombinant antibodies expressed under suitable conditions, such as the above-mentioned
  • the present invention provides a method for treating or preventing tumor or viral infection. According to some specific embodiments of the present invention, it includes administering to the subject at least one of the following: 1) the aforementioned recombinant antibody; 2) the aforementioned nucleic acid molecule; 3) the aforementioned expression vector; 4 ) the aforementioned recombinant cell; 5) the aforementioned composition; or 6) the aforementioned drug.
  • the recombinant antibody can specifically recognize the above-mentioned CD16a and IL-21R proteins, and then can specifically recognize the NK cells that specifically highly express the IL-21R or CD16a, so that the NK cells can be effectively activated and the IFN- ⁇ secretion, increase the proliferation and cytotoxic activity of NK, effectively kill tumor cells, accelerate the maturation of NK cells and the expression of NK cell receptors at the same time, can effectively restore the exhaustion of NK cells in the tumor microenvironment, therefore, according to the embodiment of the present invention
  • Recombinant antibodies, compositions containing the recombinant antibodies, drugs, or a series of substances capable of obtaining the recombinant antibodies expressed under suitable conditions, such as the above-mentioned nucleic acid molecules, expression vectors, recombinant cells, etc. can effectively activate NK cells, and then treat Or prevent tumor or viral infection.
  • Figure 1 is a schematic diagram of a bispecific antibody expression vector according to an embodiment of the present invention.
  • Fig. 2 is a schematic structural diagram of a recombinant bispecific antibody according to an embodiment of the present invention, wherein Linker1 indicates linker peptide 1, Linker2 indicates linker peptide 2, Anti-CD16A indicates CD16A single-chain antibody, and Hinge indicates the hinge region;
  • Fig. 3 is an analysis result diagram of the recombinant bispecific antibody anti-CD16A-FC ⁇ 4-IL21 expression clone screening according to an embodiment of the present invention, wherein:
  • Fig. A (3-A) is DB, Dot blot, dot blot detection result figure,
  • Figure B is WB: Western blot, Western blot (anti-human FC ⁇ 4 antibody) detection results;
  • Fig. 4 is an analysis result diagram of the fermentation expression of the recombinant bispecific antibody anti-CD16A-FC ⁇ 4-IL21 according to an embodiment of the present invention, wherein:
  • Figure A (4-A) is the result figure of each parameter monitoring of fermentation process
  • Figure B (4-B) is the detection result of the target protein accumulation time point
  • Figure 5 is a graph showing the purification and physical and chemical identification results of the recombinant bispecific antibody anti-CD16A-FC ⁇ 4-IL21 according to an embodiment of the present invention, wherein:
  • Figure A is the result of SDS-PAGE detection of anti-CD16A-FC ⁇ 4-IL21 protein purity
  • Figure B is a diagram of the dynamic light scattering detection results of anti-CD16A-FC ⁇ 4-IL21, in which 3 solid lines represent 3 repetitions of the same treatment,
  • Figure C is the result of HPLC detection of anti-CD16A-FC ⁇ 4-IL21
  • Figure 6 is a graph showing the results of the activity detection of NK cells activated in vitro by the recombinant bispecific antibody anti-CD16A-FC ⁇ 4-IL21 according to an embodiment of the present invention, wherein the abscissa indicates the NK cells in PBMCs activated by anti-CD16A-FC ⁇ 4-IL21 Molecules whose surface expression changes, the ordinate Positive rate of NK surface molecules (%) indicates the positive rate (expression) of NK cell surface molecules; and
  • Fig. 7 is a graph showing the detection results of NK cell expansion and expansion effect induced by bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21 in vitro according to an embodiment of the present invention, wherein:
  • Figure A (7-A) is the bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21 induced NK cell expansion in vitro, the abscissa Day indicates the number of days, and the ordinate NK Percentage (%) indicates the proportion of NK cells,
  • Figure B (7-B) is the results of NK cell expansion induced by bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21 in vitro, the abscissa Day indicates the number of days, and the ordinate Fold increase of NK cell number indicates the NK cell number in the expansion system the expansion of the cells,
  • Figure C (7-C) is the result of NK cell expansion induced by bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21 in vitro, the abscissa Day indicates the number of days, and the ordinate Fold increase of cell number indicates the total cells in the expansion system the amplification factor,
  • Figure D (7-D) is the results of cell subtype analysis obtained after the bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21 expanded PBMC-derived NK cells in vitro, the abscissa indicates the classification of cell subgroups, and the ordinate Percentage( %) represents the average cell ratio,
  • Figure E (7-E) is the result of cytotoxicity detection of NK cells obtained by the expansion of bispecific antibody molecule anti-CD16a-FC ⁇ 4-IL21.
  • the abscissa indicates the ratio of CD56 positive cells to K562 cells, and the ordinate indicates Specific killing ( %) represents the ratio of killing specific cells.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • the present invention provides a recombinant antibody, including: IL-21 molecule; and CD16a antibody, said CD16a antibody includes CD16a single-chain antibody and Fc region, the N-terminal of said Fc region is connected to said The C-terminals of the single-chain antibody are connected, and the CD16a single-chain antibody includes a heavy chain variable region and a light chain variable region; the C-terminal of the FC is connected with the N-terminal of the IL21 molecule.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R, has strong binding activity in vivo and in vitro, can effectively activate NK cells, has a long half-life in vivo, and has remarkable anti-tumor and anti-virus effects.
  • the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
  • it further includes a connecting peptide 1, the N-terminal of the connecting peptide 1 is connected to the C-terminal of the heavy chain variable region of the CD16a single-chain antibody, and the C-terminal of the connecting peptide 1 is connected to the C-terminal of the heavy chain variable region of the CD16a single-chain antibody.
  • the N-terminals of the light chain variable regions of the CD16a single-chain antibody are connected.
  • the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:1.
  • GGGGSGGGGSGGGGS SEQ ID NO: 1.
  • it further includes a connecting peptide 2, the N-terminal of the connecting peptide 2 is connected to the C-terminal of the IL-21 molecule, and the C-terminal of the connecting peptide 2 is connected to the N-terminal of the Fc region. end connected.
  • the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO:2.
  • GGGGSGGGGSGGGGS SEQ ID NO: 2.
  • At least a part of the Fc region is from at least one of murine antibodies, human antibodies, primate antibodies or mutants thereof.
  • At least a part of the Fc region is from human IgG or a mutant thereof.
  • At least a part of the Fc region is from Fc ⁇ 4 or a mutant thereof.
  • At least a part of the Fc region is from human Fc ⁇ 4 or a mutant thereof.
  • the Fc region has an amino acid sequence as shown in SEQ ID NO:3.
  • the recombinant antibody has the amino acid sequence shown in SEQ ID NO:4.
  • the present invention provides a nucleic acid encoding the aforementioned recombinant antibody.
  • the recombinant antibody encoded by nucleic acid according to the embodiment of the present invention can effectively bind to CD16a and IL-21R, has strong in vivo and in vitro binding activity, can effectively activate NK cells, has a long half-life in vivo, and is anti-tumor and anti-virus The effect is remarkable.
  • the nucleic acid has the nucleotide sequence shown in SEQ ID NO:5.
  • nucleic acid in the specification and claims of the present invention, those skilled in the art should understand that it actually includes any one or both of the complementary double strands.
  • nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the present invention proposes an expression vector carrying the aforementioned nucleic acid molecule.
  • the expression vector may include optional control sequences operably linked to the nucleic acid molecule.
  • the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in the host.
  • the expression vectors proposed in the embodiments of the present invention can efficiently express the recombinant antibodies in suitable host cells, and then can effectively activate NK cells for the treatment or prevention of diseases caused by tumors or viral infections.
  • the present invention provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or recombinant antibody.
  • the recombinant cells are obtained by transfecting or transforming the expression vector.
  • the recombinant cells can efficiently express the above-mentioned recombinant antibodies under suitable conditions, and the recombinant cells can be effectively used for the treatment or prevention of diseases caused by tumors or viral infections.
  • suitable conditions refer to the conditions suitable for the expression of the recombinant antibody described in the present application.
  • conditions suitable for recombinant antibody expression include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture Environment, suitable cell culture time.
  • suitable conditions are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibody according to the specific environment of the laboratory.
  • the present invention proposes a composition
  • a composition comprising the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell.
  • the recombinant antibody of the embodiment of the present invention can effectively bind to CD16a or IL-21R protein molecules, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a, so that NK cells can be effectively activated,
  • the composition containing the recombinant antibody also has significant effects on treating or preventing tumors or diseases caused by virus infection, and its anti-tumor and anti-virus capabilities are remarkable.
  • the composition is not particularly limited, as long as any one of the above-mentioned substances is used in combination with any other substance, it is within the scope of the composition described in this application, such as a food composition or a pharmaceutical composition.
  • the present invention proposes a medicament, comprising: the aforementioned recombinant antibody nucleic acid molecule, expression vector, recombinant cell or composition, and the medicament is used for treating or preventing tumor or viral infection.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R proteins, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a, using the recombinant antibody and
  • the medicaments prepared from the corresponding series of substances also have significant effects on treating or preventing tumor or virus infection, and have remarkable antitumor and antiviral abilities.
  • the tumor includes at least one of the following: rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer.
  • the viral infection includes at least one of the following: HPV, HBV, influenza virus and coronavirus.
  • the medicine provided according to some specific embodiments of the present invention includes a pharmaceutically acceptable carrier and an effective amount of the active ingredient of the recombinant antibody.
  • an effective amount or “effective dose” refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration, and the dosage form of the medicine of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent.
  • it can be prepared by a conventional method using physiological saline or an aqueous solution containing glucose and other auxiliary agents.
  • Said medicaments are preferably manufactured under sterile conditions.
  • the effective amount of the active ingredient in the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like.
  • the selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (for example, through clinical trials).
  • the factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, drug administration way etc. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.
  • the pharmaceutically acceptable carrier of the present invention includes (but not limited to): water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or its combination.
  • the choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.
  • the present invention proposes the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors and recombinant cells in culturing and expanding NK cells.
  • the recombinant antibodies, nucleic acid molecules, expression vectors and the recombinant cells according to some specific embodiments of the present invention can be used to promote the proliferation and expansion of the NK cells in vitro, and the expanded NK cells have better cytotoxicity.
  • the NK cells are obtained from peripheral blood or induced stem cells.
  • NK cells are mainly distributed in peripheral blood, and NK cells can also be induced from stem cells.
  • the NK cells include at least one of the following: NK cells derived from human peripheral blood mononuclear cells, NK cells induced from human umbilical cord blood stem cells, and NK cells induced from human embryonic stem cells , NK cells induced by human iPSCs.
  • NK cells Peripheral blood mononuclear cells include lymphocytes and monocytes.
  • the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors and the recombinant cells promote the proliferation of NK cells from the above sources in vitro , the expansion effect is better, and the expanded NK cells have better cytotoxicity.
  • the present invention proposes the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines for treating or preventing tumors or viral infections.
  • the recombinant antibodies of the embodiments of the present invention can effectively bind to CD16a and IL-21R proteins, and then can specifically recognize NK cells that highly express the IL-21R and/or CD16a.
  • the medicaments prepared from the corresponding series of substances also have significant effects on treating or preventing tumors or viral infections, and have remarkable antitumor and antiviral abilities.
  • the above use may further include at least one of the following additional technical features:
  • the tumor includes at least one of the following: rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer and ovarian cancer.
  • the viral infection includes at least one of the following: HPV, HBV, influenza virus and coronavirus.
  • the present invention proposes a kit comprising: the aforementioned recombinant antibody.
  • the recombinant antibody according to the embodiment of the present invention can effectively bind to CD16a and IL-21R protein molecules, and then can specifically recognize NK cells that specifically highly express the IL-21R or CD16a. Therefore, the recombinant antibody can be used to prepare a kit for detecting CD16a or IL-21R, and the kit can be used for scientific research, such as qualitative or quantitative detection of CD16a and/or IL-21R protein molecules in biological samples.
  • the kit is used for detecting CD16a and/or IL-21R.
  • the present invention proposes the use of the aforementioned recombinant antibody, nucleic acid molecule, expression vector, recombinant cell, composition or drug in treating or preventing tumor or viral infection.
  • the recombinant antibody can specifically recognize the above-mentioned CD16a and IL-21R proteins, and then can specifically recognize the NK cells that specifically highly express the IL-21R or CD16a, so that the NK cells can be effectively activated and the IFN- ⁇ secretion, increase the proliferation and cytotoxic activity of NK, effectively kill tumor cells, accelerate the maturation of NK cells and the expression of NK cell receptors at the same time, can effectively restore the exhaustion of NK cells in the tumor microenvironment, therefore, according to the embodiment of the present invention Recombinant antibodies, compositions containing the recombinant antibodies, drugs, or a series of substances capable of obtaining the recombinant antibodies expressed under suitable conditions, such as the above-mentioned nucleic acid
  • the tumor includes at least one of the following: rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer
  • the viral infection includes the following At least one of: HPV, HBV, influenza virus, and coronavirus.
  • the present invention provides a method of treating or preventing tumor or viral infection. According to some specific embodiments of the present invention, it includes administering to the subject at least one of the following: 1) the aforementioned recombinant antibody; 2) the aforementioned nucleic acid molecule; 3) the aforementioned expression vector; 4 ) the aforementioned recombinant cell; 5) the aforementioned composition; or 6) the aforementioned drug.
  • the recombinant antibody can specifically recognize the above-mentioned CD16a and IL-21R proteins, and then can specifically recognize the NK cells that specifically highly express the IL-21R or CD16a, so that the NK cells can be effectively activated and the IFN- ⁇ secretion, increase the proliferation and cytotoxic activity of NK, effectively kill tumor cells, accelerate the maturation of NK cells and the expression of NK cell receptors at the same time, can effectively restore the exhaustion of NK cells in the tumor microenvironment, therefore, according to the embodiment of the present invention
  • Recombinant antibodies, compositions containing the recombinant antibodies, drugs, or a series of substances capable of obtaining the recombinant antibodies expressed under suitable conditions, such as the above-mentioned nucleic acid molecules, expression vectors, recombinant cells, etc. can effectively activate NK cells, and then treat Or prevent tumor or viral infection.
  • the tumor includes at least one of the following: rectal cancer, lung cancer, breast cancer, liver cancer, gastric cancer, skin cancer, kidney cancer, pancreatic cancer, and ovarian cancer
  • the viral infection includes the following At least one of: HPV, HBV, influenza virus, and coronavirus.
  • the PCR program is: denaturation: 98 20s; 3 cycles : 98 10s ⁇ 54 20s ⁇ 72 40s (3 cycles); extension: 72 2min; add primers afterwards: denaturation: 98 20s; 3 cycles: 98 10s ⁇ 54 20s ⁇ 72 40s (30 cycles); extension: 7210min;
  • the amplified product was subjected to agarose gel electrophoresis, and then a DNA gel recovery kit was used to recover a gene with XhoI and NotI restriction sites at both ends, named Anti-CD16a-FC ⁇ 4-IL21;
  • the enzyme-digested anti-CD16a-FC ⁇ 4-IL21 fusion protein gene sequence product gene was recovered using a PCR product recovery kit (Axygen Company), and the enzyme-digested plasmid was recovered using a DNA gel recovery kit (Axygen Company). For the operation steps, refer to the kit instructions.
  • the recovered digested fusion protein gene was ligated with the digested vector using T4 DNA ligase, and the constructed plasmid structure is shown in Figure 1.
  • the enzyme-linked product was transformed into Escherichia coli TOP10 competent cells. The transformation procedure is as follows: Take 10 ⁇ L of the enzyme-linked product and add it to 100 ⁇ L of E. coli competent, mix well, let it stand on ice for 15-30 minutes, and bounce it every five minutes; after that, heat shock at 42°C for 90 seconds, and keep it on ice After 3 to 5 minutes, add 400ul of LB liquid medium without anti-antibody, revive and incubate on a shaker at 37°C for 45 minutes, and then coat Zeocin-resistant plates.
  • the obtained positive clone was named PGAP-z ⁇ -anti-CD16a-FC ⁇ 4-IL21, and the bacterial liquid corresponding to the positive clone was extracted with a plasmid mini-extraction kit, and sent to general biological sequencing. After sequencing, the sequencing results were consistent with the theoretically expected results, and the clone was constructed correctly.
  • Preparation of competent yeast production steps: 1) Select a well-frozen strain of Pichia pastoris X33, streak it on a YPD plate, and culture it in a yeast incubator at a constant temperature of 30°C. 2) After the clone grows to a diameter of about 1mm, pick the clone into liquid 4mL LYPD medium, and culture it on a yeast shaker at a constant temperature of 30°C. 3) Culture for 24-48 hours. Inoculate 1 mL of the bacterial solution into 50 mL of fresh YPD medium. 4) When the OD600 value reaches 1-1.5, transfer the bacterial solution to a 50mL centrifuge tube, centrifuge at 1500g for 5 minutes at 4°C, and discard the supernatant.
  • Electroporation step Take 100 ⁇ L of the above-mentioned competent cells into a sterile pre-cooled electroporation cup, add 10 ⁇ L of linearized plasmid (5-10 ⁇ g) therein, and mix well. Set the electrorotor parameters: 2000V, 200 ⁇ , 25 ⁇ F. Immediately after electroporation, add 1mL of 1M cold sorbitol to the electroporation cup, let it stand on ice for 5-10min, then transfer all the bacterial solution to a 50mL centrifuge tube containing 1mLYPD, recover at 30°C, 225rpm for 2h. After recovery, take 100-1000 ⁇ L of the above-mentioned bacterial solution to coat the YPDZ (plate containing Zeocin resistance) plate, and place the plate in a constant temperature yeast incubator at 30°C for cultivation.
  • YPDZ plate containing Zeocin resistance
  • Screening of positive expression clones After the above YPDZ plate grows yeast clones, pick several clones and put them in 2mL BMGY medium, and culture them on a yeast shaker at 28°C at a constant temperature until the bacteria liquid is milky white. 1mL of the bacterial liquid was frozen and preserved, and the remaining 1mL of the bacterial liquid was centrifuged at 12,000g for 5 minutes, and the supernatant was used for preliminary screening of Dot blot identification with anti-human FC ⁇ 4 antibody, and yeast strains with relatively high expression levels were selected, and then the relative expression levels were compared.
  • the taller yeast strains were spread on the YPD plate containing zeocin, and after the clones were grown, multiple clones were selected from them and shaken according to the above method to obtain the supernatant of the bacterial liquid, and the Anti-human FC ⁇ antibody was used for Western Blot identification analysis.
  • a high-expression strain (34-3) was screened out, and the remaining 1 mL of the corresponding strain was frozen for preservation; this high-expression strain was a Pichia strain that could express the anti-CD16a-FC ⁇ 4-IL21 complex protein.
  • Expression strain culture The fermentation of the recombinant strain was carried out according to the Invitrogen company's fermentation instruction manual. Inoculate the screened high-expression strain into a shaking tube containing 4 mL of YPD medium, and culture it on a shaking table at 30°C for 24-48 hours until the OD600 is 2-6, which is the first-grade fermentation seed liquid. Take 1mL of the seed liquid and transfer it into a Erlenmeyer flask containing 200mL of BMGY medium, and cultivate it on a shaker at 30°C for 12-24 hours, which is the secondary seed liquid for fermentation. All the 200mL secondary seed liquid was inoculated into the fermenter containing 6L BMGY medium.
  • BMGY medium is used in the growth stage of the bacteria. When the dissolved oxygen rises rapidly, it means that the basic glycerin in the medium is exhausted. Start adding glycerol at a rate of 90mL/h. Adjust the fermentation temperature to 25°C and continue until the end of fermentation .
  • Clarification of the culture medium After the fermentation, centrifuge at 10,000g for 30 minutes, collect the supernatant, and filter and clarify it with a 0.22 ⁇ m and 500kDa microporous membrane, and then adjust the pH to 7.4.
  • Protein capture Recombinant fusion proteins were captured using an affinity column Mabselect column. The specific steps are: 1) Washing the chromatographic column with deionized water for 5 column volumes. 2) Rinse the column with PBS for 3 column volumes. 3) Sample loading. 4) After loading the sample, rinse the chromatographic column for 3 column volumes with PBS. 5) Impurity washing: wash 5 column volumes with citric acid buffer at pH 5.5. 6) The target protein was eluted, and the eluent was citrate buffer at pH 3.0, and the collected eluate was concentrated and used for subsequent purification.
  • Fine purification use AKTA PURE 25, equilibrate Superdex200 molecular sieve with PBS buffer, after equilibration, load the neutral eluent, wash with PBS buffer and collect the eluted sample to obtain anti-CD16a-FC ⁇ 4-IL21 recombinant protein.
  • the purity of the protein can reach more than 95% as identified by SDS-PAGE and HPLC (Fig. 5A, C).
  • Recombinant protein identification The purified protein was identified by SDS-PAGE, HPLC, and dynamic light scattering. The specific experimental results are shown in Figure 4B. Among them, the molecular weight of anti-CD16a-FC ⁇ 4-IL21 protein is about 135kDa, which is in line with expectations; through dynamic light scattering identification, the molecular diameter of anti-CD16a-FC ⁇ 4-IL21 is about 10nm, and the molecular weight between Good uniformity.
  • Human PBMCs were isolated by Ficoll density gradient centrifugation. The isolated PBMCs were diluted to 1.0 ⁇ 10 6 /mL with RPMI 1640 containing 10% FBS. Add 10 mL of diluted cells to the T25 culture flask. After culturing for 12 hours, anti-CD16a-FC ⁇ 4-IL21 fusion protein was added to the culture flask at a final concentration of 20 nM. After 24 hours of culture, flow cytometry was used to detect the phenotype changes of NK cells, mainly to detect the expression of main activating molecules CD69, NKp44 and killing molecules 4-1BB, TRAIL, and Granzyme B on the surface of NK cells.
  • PBMC cells were isolated from the blood sample, they were counted (Mindray counter), and the percentages of each cell subgroup in the PBMC cells were recorded. Inoculate at a density of 1.5*10 ⁇ 6/mL (20 mL culture volume). The total number of cells is 30*10 ⁇ 6. This is Day 0. At the same time, 1% IL2 (1000IU/mL) and 5% corresponding PBMC autologous plasma were added to the KBM581 medium without anti-CD16a-FC ⁇ 4-IL21.
  • Labeled antibody Divide 0.09mL/tube of surface molecularly labeled cells into 9 flow tubes after blocking. If multiple batches of cells are detected at the same time, the cells in 1-8 tubes can be used for several batches of cells, etc. The amount of cells is mixed for labeling. The amount of cells in each tube is: 0.8-1 ⁇ 10 6 .
  • Sample tube 1 is for surface molecular markers, and the amount of cells is: 0.8-1 ⁇ 10 6 .
  • Sample tube 2 is for intracellular molecular markers, the amount of cells used is: 3-4 ⁇ 10 6 cells, and the corresponding fluorescently-labeled antibody is added, mixed well, and then placed at 4°C in the dark for 30 minutes (mixed every 15 minutes in the middle).
  • the specific experimental groups are shown in Table 2.
  • Table 2 Cell surface molecular detection sample tube and experimental operation table
  • the flow cytometer is calibrated and adjusted according to the instrument manual. Blank tube samples were used to adjust the forward and side scatter voltages. The single standard control sample is used to adjust the fluorescence compensation of each channel. After the cell gate was circled during detection, 1 ⁇ 10 4 cells were collected in each sample gate.
  • the specific experimental results are shown in Figure 7-D.
  • the subgroup analysis of the expanded cells shows that the proportion of NK cells in the PBMC cells cultured and expanded by anti-CD16a-FC ⁇ 4-IL21 can reach 61%.
  • the proportion is about 10%, the total CD56 positive cells are about 71%, and the proportion of other cell subgroups in the expanded PBMC cells is relatively low.
  • Cell counting Cells were resuspended in 1640 medium containing 0.5% FBS (the medium used for target cells), counted, and the cell density was adjusted to 1 ⁇ 106/mL;
  • CFSE staining Add CFSE (working concentration: 5 ⁇ M) to the cell suspension, pipette and mix immediately with a 1mL pipette gun, vortex to mix well, incubate in a 37°C incubator in the dark for 15 minutes, take it out every 5 minutes and vortex to mix once;
  • Termination of staining Add 5 times the volume of 4°C pre-cooled complete medium (the medium used for the target cells), terminate the staining, and ice-bath for 5 minutes. 140g, 4°C, 5min centrifugation;
  • Wash cells resuspend with 4°C pre-cooled complete medium (medium used for target cells), centrifuge cells at 140g, 4°C, 5min; repeat washing twice;
  • Counting Cells were resuspended, counted, and the cell density was adjusted to 2 ⁇ 105/mL;
  • Plating Add 100 ⁇ l of target cell suspension to each well of a 96-well round bottom plate, so that the final number of cells per well is 20,000/well;
  • NK cell suspension into the culture plate with different effector-target ratios (CD56+:K562) (1:1, 2:1, 4:1, 8:1), and set up three groups of controls: only CFSE-stained target cells (to detect the natural death rate of target cells); only effector cells (to prove that effector cells do not contain CFSE non-specific staining); target cells + Tween-20 (as a positive control for target cell apoptosis);
  • NK cells were added to each well according to the pre-designed sequence. Centrifuge at 120g at room temperature for 2min to fully contact the target cells. Place back into the 37°C incubator and incubate for 4 hours.
  • killing percentage [(experimental group target cell death rate (%)-target cell death rate (%))/(100%-target cell natural death rate (%)] ⁇ 100%).

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Abstract

一种双特异性抗体及其应用，该双特异性抗体包括：IL-21分子；以及CD16a抗体，所述CD16a抗体包括CD16a单链抗体以及FC，所述FC的N末端与所述单链抗体的C末端相连，所述CD16a单链抗体包括重链可变区、轻链可变区；所述FC的C末端与所述IL21分子的N端相连。所制备的双特异性抗体能同时靶向CD16和IL-21受体，活化NK细胞，且半衰期较长，较单靶抗体表现出更强的抗肿瘤、抗病毒的能力，同时具有促进NK细胞增殖，可用于体外培养扩增NK细胞的应用潜力，且扩增后的NK细胞具有较好的细胞毒性。

Description

双特异性抗体及其应用
优先权信息
本申请请求2021年12月20日向中国国家知识产权局提交的、专利申请号为202111564703.3的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于生物医药领域，具体地，涉及双特异性抗体及其应用，更具体地，涉及重组抗体、免疫细胞、核酸、表达载体、重组细胞、组合物、上述物质在制备药物中的用途、试剂盒。
背景技术
双特异性抗体是可同时特异性结合两个抗原位点的抗体。NK细胞是固有免疫系统的主要效应细胞，相比于T细胞，NK细胞能无需预先刺激，通过使用穿孔素和颗粒酶以及相关机制，可以无MHC限制性杀伤肿瘤细胞和病毒感染细胞。NK细胞表达众多的活化性受体，能有效识别肿瘤细胞或感染细胞产生的应激性配体，从而有效杀伤肿瘤细胞，但是同时肿瘤细胞也会分泌产生许多免疫抑制性的分子，形成免疫抑制性的肿瘤微环境，抑制处于该环境中NK细胞的活性及抗肿瘤能力的发挥；使NK细胞处于耗竭状态。目前，也有许多策略用于恢复肿瘤微环境中NK细胞的耗竭，例如免疫检查点阻断，活化性抗体，活化性细胞因子等等，这些方法可以给NK细胞提供活化信号或阻断肿瘤微环境中的抑制信号，使NK细胞恢复正常的活化状态，发挥抗肿瘤或抗病毒的功能。
CD16a是NK细胞上表达的一类重要的活化性受体，当CD16a被其配体或者抗体结合活化后，会诱导各种级联激活反应，释放颗粒酶，穿孔素、炎性细胞因子和趋化因子，这会为NK细胞带来强大的活化效应及抗肿瘤能力的增强。IL-21R(IL-21受体)是NK细胞上组成型表达的细胞因子受体，当其被其对应配体IL-21分子识别结合激活后，对于小鼠的NK细胞，该过程会促使这些NK细胞颗粒更多，上调穿孔素的表达，增加IFN-γ的分泌，会促进效应活性；对于人的NK细胞，该过程可以增强IFN-γ分泌，提高增殖和NK的细胞毒活性，同时加速NK细胞成熟和NK细胞受体的表达。因此，开发靶向性强的双特异性抗体对肿瘤的预防及治疗具有极大的价值。
发明内容
本本申请是基于发明人对以下事实和问题的发现和认识作出的：
本发明中，发明人对CD16a抗体和IL-21分子进行组合，设计了重组双特异性抗体，所述重组双特异性重组抗体可以特异性结合CD16a和IL-21受体(IL-21R)，进而靶向性地将IL-21分子和CD16a抗体带入到NK细胞附近，与NK细胞上的CD16a和/或IL-21R进行结合，从而诱导各种级联激活反应， 使NK细胞活化，恢复肿瘤微环境中NK细胞的耗竭，同时增强IFN-γ分泌，提高增殖和NK的细胞毒活性，同时加速NK细胞成熟和NK细胞受体的表达，以杀伤肿瘤细胞或被病毒感染细胞，所述重组双特异性抗体具有较高的CD16a和IL-21R结合活性，抗肿瘤、抗病毒能力较高。
因此，在本发明的第一方面，本发明提出了一种重组抗体。根据本发明的实施例，包括：IL-21分子；以及CD16a抗体，所述CD16a抗体包括CD16a单链抗体以及Fc区，所述Fc区的N末端与所述单链抗体的C末端相连，所述CD16a单链抗体包括重链可变区、轻链可变区；所述FC的C末端与所述IL21分子的N端相连。根据本发明实施例的重组抗体可以有效与CD16a、IL-21R进行结合，具有较强的体内、外结合活性，可有效活化NK细胞，具备较长的体内半衰期，抗肿瘤、抗病毒效果显著。
在本发明的第二方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码第一方面所述的重组抗体。根据本发明实施例的核酸编码的重组抗体可以有效与CD16a、IL-21R进行结合，具有较强的体内、外结合活性，可有效活化NK细胞，具备较长的体内半衰期，抗肿瘤、抗病毒效果显著。
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带第二方面所述的核酸。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导所述核酸分子在宿主中表达的一个或多个控制序列。本发明实施例所提出的表达载体可在适合的宿主细胞中高效表达所述重组抗体，进而可有效激活NK细胞，用于对肿瘤或病毒感染所引发疾病的治疗或预防。
在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带第二方面所述的核酸分子、第三方面所述的表达载体或第一方面所述的重组抗体。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明的实施例，所述重组细胞在合适条件下可高效表达上述重组抗体，所述重组细胞可有效用于对肿瘤或病毒感染所引发疾病的治疗或预防，并有效活化NK细胞。
在本发明的第五方面，本发明提出了一种组合物。根据本发明的实施例，包括第一方面所述的重组抗体、第二所述的核酸分子、第三方面所述的表达载体或第四方面所述的重组细胞。如前所述，本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白分子结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，使得NK细胞有效活化，包含所述重组抗体的组合物同样具有显著的治疗或预防肿瘤或病毒感染所引发疾病的的作用，其抗肿瘤和抗病毒能力显著。
在本发明的第六方面，本发明提出了第一方面所述的重组抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第五方面所述的组合物在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或预防肿瘤或病毒感染。如前所述，本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，利用所述重组抗体及其相应的一系列物质制备的药物同样具有显著的治疗或预防肿瘤或病毒感染的作用，其抗肿瘤和抗病毒能力显著。
在本发明的第七方面，本发明提出了第一方面所述的重组抗体、第二方面所述的核酸分子、第三方面所述的表达载体和第四方面所述的重组细胞在培养扩增NK细胞中的用途。根据本发明实施例的重组 抗体、核酸分子、表达载体以及所述重组细胞可用于体外促进所述NK细胞的增殖、扩增，且扩增后的NK细胞具有较好的细胞毒性。
在本发明的第八方面，本发明提出了一种药物。根据本发明的实施例，包括：第一方面所述的重组抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第五方面所述的组合物，所述药物用于治疗或预防肿瘤或病毒感染。如前所述，根据本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，利用所述重组抗体及其相应的一系列物质制备的药物同样具有显著的治疗或预防肿瘤或病毒感染的作用，其抗肿瘤和抗病毒能力显著。
在本发明的第九方面，本发明提出了一种试剂盒。根据本发明的实施例，包括：第一方面所述的重组抗体。根据本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白分子结合，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞。因此，所述重组抗体可用于制备检测CD16a和/或IL-21R的试剂盒，所述试剂盒可用科学研究，如定性或定量检测生物样本中的CD16a和/或IL-21R蛋白分子，从而获得符合要求的生物样本等。
在本发明的第十方面，本发明提出了前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物或药物在治疗或预防肿瘤或病毒感染中的用途。如前所述，所述重组抗体可以特异性识别上述CD16a和IL-21R蛋白，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞，使得NK细胞有效活化，增强IFN-γ分泌，提高增殖和NK的细胞毒活性，有效杀伤肿瘤细胞，同时加速NK细胞成熟和NK细胞受体的表达，可以有效恢复肿瘤微环境中NK细胞的耗竭，因此，根据本发明实施例的重组抗体、包含所述重组抗体的组合物、药物或在合适条件下表达能够获得所述重组抗体的一系列物质，如上述核酸分子、表达载体、重组细胞等均可以有效活化NK细胞，进而治疗或预防肿瘤或病毒感染。
在本发明的第十一方面，本发明提出了一种治疗或预防肿瘤或病毒感染的方法。根据本发明的一些具体实施例，包括向受试者施用以下中的至少之一：1)前面所述的重组抗体；2)前面所述的核酸分子；3)前面所述的表达载体；4)前面所述的重组细胞；5)前面所述的组合物；或6)前面所述的药物。如前所述，所述重组抗体可以特异性识别上述CD16a和IL-21R蛋白，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞，使得NK细胞有效活化，增强IFN-γ分泌，提高增殖和NK的细胞毒活性，有效杀伤肿瘤细胞，同时加速NK细胞成熟和NK细胞受体的表达，可以有效恢复肿瘤微环境中NK细胞的耗竭，因此，根据本发明实施例的重组抗体、包含所述重组抗体的组合物、药物或在合适条件下表达能够获得所述重组抗体的一系列物质，如上述核酸分子、表达载体、重组细胞等均可以有效活化NK细胞，进而治疗或预防肿瘤或病毒感染。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的双特异性抗体表达载体的示意图；
图2是根据本发明实施例的重组双特异性抗体的结构示意图，其中，Linker1表示连接肽1，Linker2表示连接肽2，Anti-CD16A表示CD16A单链抗体，Hinge表示铰链区；
图3是根据本发明实施例的重组双特异性抗体anti-CD16A-FCγ4-IL21表达克隆筛选的分析结果图，其中：
图A(3-A)为DB、Dot blot、点杂交检测结果图，
图B(3-B)为WB：Western blot，免疫印记(抗human FCγ4抗体)检测结果图；
图4是根据本发明实施例的重组双特异性抗体anti-CD16A-FCγ4-IL21发酵表达的分析结果图，其中：
图A(4-A)为发酵过程各个参数监测的结果图，
图B(4-B)为目的蛋白累积时间点检测结果图；
图5是根据本发明实施例的重组双特异性抗体anti-CD16A-FCγ4-IL21纯化与理化鉴定结果图，其中：
图A(5-A)为anti-CD16A-FCγ4-IL21蛋白纯度SDS-PAGE检测结果图，
图B(5-B)为anti-CD16A-FCγ4-IL21动态光散射检测结果图，其中，3条实线表示同样处理的3个重复，
图C(5-C)为anti-CD16A-FCγ4-IL21的HPLC检测结果图；
图6是根据本发明实施例的重组双特异性抗体anti-CD16A-FCγ4-IL21体外活化NK细胞的活性检测结果图，其中，横坐标表示经anti-CD16A-FCγ4-IL21活化后PBMC中NK细胞表面表达发生变化的分子，纵坐标Positive rate of NK surface molecules(％)表示NK细胞表面分子的阳性率(表达情况)；以及
图7是根据本发明实施例的双特异性抗体分子anti-CD16a-FCγ4-IL21体外诱导NK细胞扩增及扩增效果的检测结果图，其中：
图A(7-A)为双特异性抗体分子anti-CD16a-FCγ4-IL21体外诱导NK细胞扩增，横坐标Day表示天数，纵坐标NK Percentage(％)表示NK细胞的占比，
图B(7-B)为双特异性抗体分子anti-CD16a-FCγ4-IL21体外诱导NK细胞扩增的结果图，横坐标Day表示天数，纵坐标Fold increase of NK cell number表示扩增体系中NK细胞的扩增倍数，
图C(7-C)为双特异性抗体分子anti-CD16a-FCγ4-IL21体外诱导NK细胞扩增的结果图，横坐标Day表示天数，纵坐标Fold increase of cell number表示扩增体系中总细胞的扩增倍数，
图D(7-D)为双特异性抗体分子anti-CD16a-FCγ4-IL21体外扩增PBMC来源NK细胞后获得的细 胞亚类分析的结果图，横坐标表示细胞亚群分类，纵坐标Percentage(％)表示平均细胞比例，
图E(7-E)为双特异性抗体分子anti-CD16a-FCγ4-IL21扩增获得的NK细胞的细胞毒性检测结果图，横坐标表示CD56阳性细胞与K562细胞的比例，纵坐标Specific killing(％)表示杀伤特定细胞的比例。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
重组抗体
在本发明的一个方面，本发明提出了一种重组抗体，包括：IL-21分子；以及CD16a抗体，所述CD16a抗体包括CD16a单链抗体以及Fc区，所述Fc区的N末端与所述单链抗体的C末端相连，所述CD16a单链抗体包括重链可变区、轻链可变区；所述FC的C末端与所述IL21分子的N端相连。根据本发明实施例的重组抗体可以有效与CD16a、IL-21R进行结合，具有较强的体内、外结合活性，可有效活化NK细胞，具备较长的体内半衰期，抗肿瘤、抗病毒效果显著。
根据本发明的一些具体实施例，上述重组抗体还可以进一步包括如下附加技术特征至少之一：
根据本发明的一些具体实施例，进一步包括连接肽1，所述连接肽1的N端与所述CD16a单链抗体的重链可变区的C端相连，所述连接肽1的C端与所述CD16a单链抗体的轻链可变区的N端相连。
根据本发明的一些具体实施例，所述连接肽1具有如SEQ ID NO:1所示的氨基酸序列。
GGGGSGGGGSGGGGS(SEQ ID NO:1)。
根据本发明的一些具体实施例，进一步包括连接肽2，所述连接肽2的N端与所述IL-21分子的C端相连，所述连接肽2的C端与所述Fc区的N端相连。
根据本发明的一些具体实施例，所述连接肽2具有如SEQ ID NO:2所示的氨基酸序列。
GGGGSGGGGSGGGGS(SEQ ID NO:2)。
根据本发明的一些具体实施例，所述Fc区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
根据本发明的一些具体实施例，所述Fc区的至少一部分来自人源IgG或其突变体。
根据本发明的一些具体实施例，所述Fc区的至少一部分来自于Fcγ4或其突变体。
根据本发明的一些具体实施例，所述Fc区的至少一部分来自于人源Fcγ4或其突变体。
根据本发明的一些具体实施例，所述Fc区具有如SEQ ID NO:3所示的氨基酸序列。
Figure PCTCN2022114434-appb-000001
根据本发明的一些具体实施例，所述重组抗体具有SEQ ID NO：4所示的氨基酸序列。
Figure PCTCN2022114434-appb-000002
预防或治疗组合物
在本发明的一个方面，本发明提出了一种核酸，所述核酸编码前面所述的重组抗体。根据本发明实施例的核酸编码的重组抗体可以有效与CD16a、IL-21R进行结合，具有较强的体内、外结合活性，可有效活化NK细胞，具备较长的体内半衰期，抗肿瘤、抗病毒效果显著。
根据本发明的一些具体实施例，所述核酸具有SEQ ID NO：5所示的核苷酸序列。
Figure PCTCN2022114434-appb-000003
Figure PCTCN2022114434-appb-000004
需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本发明的另一方面，本发明提出了一种表达载体，所述表达载体携带前面所述的核酸分子。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导所述核酸分子在宿主中表达的一个或多个控制序列。本发明实施例所提出的表达载体可在适合的宿主细胞中高效表达所述重组抗体，进而可有效激活NK细胞，用于对肿瘤或病毒感染所引发疾病的治疗或预防。
在本发明的又一方面，本发明提出了一种重组细胞，所述重组细胞携带前面所述的核酸分子、表达载体或重组抗体。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明的一些具体实施例，所述重组细胞在合适条件下可高效表达上述重组抗体，所述重组细胞可有效用于对肿瘤或病毒感染所引发疾病的治疗或预防。
需要说明的是，本申请说明书中所述的“适合条件”，是指适合本申请所述重组抗体表达的条件。本 领域技术人员容易理解的是，适合重组抗体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述重组抗体表达的条件。
在本发明的再一方面，本发明提出了一种组合物，包括前面所述的重组抗体、核酸分子、表达载体或重组细胞。如前所述，本发明实施例的重组抗体可以有效与CD16a或IL-21R蛋白分子结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，使得NK细胞有效活化，包含所述重组抗体的组合物同样具有显著的治疗或预防肿瘤或病毒感染所引发疾病的的作用，其抗肿瘤和抗病毒能力显著。所述组合物不受特别限制，只要是上述任何一种物质与其它任何物质进行组合使用的均为本申请所述的组合物范围内，如食品组合物或药物组合物。
在本发明的一个方面，本发明提出了一种药物，包括：前面所述的重组抗体核酸分子、表达载体、重组细胞或组合物，所述药物用于治疗或预防肿瘤或病毒感染。如前所述，根据本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，利用所述重组抗体及其相应的一系列物质制备的药物同样具有显著的治疗或预防肿瘤或病毒感染的作用，其抗肿瘤和抗病毒能力显著。
根据本发明的一些具体实施例，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌胰腺癌和卵巢癌。
根据本发明的一些具体实施例，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
根据本发明的一些具体实施例提供的药物，包括药学上可接受的载体和有效量的所述重组抗体活性成分。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物宜在无菌条件下制造。
本发明所述的活性成分的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的 有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括(但不限于)：水、盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择应与给药方式相匹配，这些都是本领域的普通技术人员所熟知的。
用途
在本发明的一个方面，本发明提出了前面所述的重组抗体、核酸分子、表达载体和重组细胞在培养扩增NK细胞中的用途。根据本发明一些具体实施例的重组抗体、核酸分子、表达载体以及所述重组细胞可用于体外促进所述NK细胞的增殖、扩增，且扩增后的NK细胞具有较好的细胞毒性。
根据本发明的一些具体实施例，所述NK细胞来源于外周血或诱导干细胞获得。NK细胞主要分布于外周血中，另外，NK细胞也可以将干细胞进行诱导获得。
根据本发明的一些具体实施例，所述NK细胞包括下列中的至少之一：人外周血单个核细胞来源的NK细胞、人脐血干细胞诱导获得的NK细胞、人胚胎干细胞诱导获得的NK细胞、人iPSC诱导获得的NK细胞。NK细胞外周血单个核细胞包括淋巴细胞和单核细胞，根据本发明的一些具体实施例，前面所述的重组抗体、核酸分子、表达载体以及所述重组细胞体外促进上述来源的NK细胞的增殖、扩增的效果较好，且扩增后的NK细胞具有较好的细胞毒性。
在本发明的第七方面，本发明提出了前面所述的重组抗体、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途，所述药物用于治疗或预防肿瘤或病毒感染。如前所述，本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白结合，进而可特异性识别高表达所述IL-21R和/或CD16a的NK细胞，利用所述重组抗体及其相应的一系列物质制备的药物同样具有显著的治疗或预防肿瘤或病毒感染的作用，其抗肿瘤和抗病毒能力显著。
根据本发明的一些具体实施例，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的一些具体实施例，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌。
根据本发明的一些具体实施例，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
试剂盒
在本发明的最后一方面，本发明提出了一种试剂盒，包括：前面所述的重组抗体。根据本发明实施例的重组抗体可以有效与CD16a和IL-21R蛋白分子结合，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞。因此，所述重组抗体可用于制备检测CD16a或IL-21R的试剂盒，所述试剂盒可用于科学研究，如定性或定量检测生物样本中的CD16a和/或IL-21R蛋白分子。
根据本发明的一些具体实施例，所述试剂盒用于检测CD16a和/或IL-21R。
疾病治疗用途和方法
在本发明的一个方面，本发明提出了前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物或药物在治疗或预防肿瘤或病毒感染中的用途。如前所述，所述重组抗体可以特异性识别上述CD16a和IL-21R蛋白，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞，使得NK细胞有效活化，增强IFN-γ分泌，提高增殖和NK的细胞毒活性，有效杀伤肿瘤细胞，同时加速NK细胞成熟和NK细胞受体的表达，可以有效恢复肿瘤微环境中NK细胞的耗竭，因此，根据本发明实施例的重组抗体、包含所述重组抗体的组合物、药物或在合适条件下表达能够获得所述重组抗体的一系列物质，如上述核酸分子、表达载体、重组细胞等均可以有效活化NK细胞，进而治疗或预防肿瘤或病毒感染。
根据本发明的一些具体实施例，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
在本发明的另一方面，本发明提出了一种治疗或预防肿瘤或病毒感染的方法。根据本发明的一些具体实施例，包括向受试者施用以下中的至少之一：1)前面所述的重组抗体；2)前面所述的核酸分子；3)前面所述的表达载体；4)前面所述的重组细胞；5)前面所述的组合物；或6)前面所述的药物。如前所述，所述重组抗体可以特异性识别上述CD16a和IL-21R蛋白，进而可特异性识别特异性高表达所述IL-21R或CD16a的NK细胞，使得NK细胞有效活化，增强IFN-γ分泌，提高增殖和NK的细胞毒活性，有效杀伤肿瘤细胞，同时加速NK细胞成熟和NK细胞受体的表达，可以有效恢复肿瘤微环境中NK细胞的耗竭，因此，根据本发明实施例的重组抗体、包含所述重组抗体的组合物、药物或在合适条件下表达能够获得所述重组抗体的一系列物质，如上述核酸分子、表达载体、重组细胞等均可以有效活化NK细胞，进而治疗或预防肿瘤或病毒感染。
根据本发明的一些具体实施例，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
下面将对实施例作具体介绍。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1双特异性抗体分子anti-CD16a-FCγ4-IL21的设计及构建
本实施例的具体实验操作如下：
1.1 anti-CD16a-FCγ4-IL21融合基因的获取
(1)Anti-CD16a-FCγ4融合基因的获取
以Pgapza-CD16-FC4γ4质粒作为模板，使用引物1和引物2进行PCR获取anti-CD16a-FCγ4融合基因，其具有如SEQ ID NO:6所示的核苷酸序列，PCR程序为：变性:98℃ 20s；循环:98℃ 10s→54℃ 15s→72℃ 40s(30循环)延伸:72℃ 2min；
Figure PCTCN2022114434-appb-000005
(2)GS-IL21融合基因获取
以Ppicza-rdna-FC-Y407T-IL21质粒作为模板，使用引物3和引物4进行PCR获取GS-IL21融合基因，其具有如SEQ ID NO:7所示的核苷酸序列，PCR程序为：变性:98 20s；循环:98℃ 10s→54℃ 15s→72℃ 30s(30循环)延伸:72℃ 5min；
Figure PCTCN2022114434-appb-000006
Figure PCTCN2022114434-appb-000007
所述引物1-4的序列如表1所示：
表1：
编号 名称 序列
引物1 Fw-CD16-XhoI CTCTCGAGAAAAGACAAGTTCAATTGGTTC(SEQ ID NO:8)
引物2 RV-T407Y CGGTTAGTCTGCTGTAGAGGAAGAAG(SEQ ID NO:9)
引物3 FW-T407Y CTTCTTCCTCTACAGCAGACTAACCG(SEQ ID NO:10)
引物4 RV-NOTI TTCGCGGCCGCCTAGGAATCTTCACTTCCG(SEQ ID NO:11)
对上述两步骤扩增后的产物进行琼脂糖凝胶电泳，并根据DNA凝胶回收试剂盒的操作步骤，将两段目的基因对应的条带切胶并回收，获得Anti-CD16a-FCγ4及GS-IL21融合基因，即得第1个目的基因片段和第2个目的基因片段；
(3)Anti-CD16a-FCγ4-IL21融合基因获取
使用上述步骤获取的Anti-CD16a-FCγ4及GS-IL21融合基因作为模板，使用引物1和引物4，进行PCR获取Anti-CD16a-FCγ4-IL21融合基因，PCR程序为：变性:98 20s；3循环:98 10s→54 20s→72 40s(3循环)；延伸:72 2min；之后添加引物：变性:98 20s；3循环:98 10s→54 20s→72 40s(30循环)；延伸:7210min；对扩增产物进行琼脂糖凝胶电泳，而后采用DNA凝胶回收试剂盒回收得到一段基因，此基因两端带有XhoⅠ和NotⅠ酶切位点，命名为Anti-CD16a-FCγ4-IL21；
上述Pgapza-CD16-FC4γ4及Ppicza-rdna-FC-Y407T-IL21质粒均为发明人按照常规表达载体构建方法，自己构建的。
1.2 anti-CD16a-FCγ4-IL21融合蛋白的毕赤酵母表达载体构建
将步骤1.1获得的编码anti-CD16a-FCγ4-IL21融合蛋白的基因序列(SEQ ID NO:5)，与实验室保存的毕赤酵母表达质粒PGAPzα-rDNA-NTS皆使用限制性内切酶Xhol和NotⅠ进行双酶切。酶切anti-CD16a-FCγ4-IL21融合蛋白基因序列产物基因采用PCR产物回收试剂盒回收(Axygen公司)， 酶切质粒采用DNA凝胶回收试剂盒回收(Axygen公司)，操作步骤参见试剂盒说明。回收所得的酶切后融合蛋白基因与酶切后载体采用T4 DNA连接酶连接，构建后的质粒结构图如图1所示。酶连产物转化大肠杆菌TOP10感受态细胞。转化操作步骤为：取10μL酶连产物加入100μL大肠杆菌感受态中，混匀，冰上静置15-30分钟，每隔五分钟弹匀一次；之后42℃热激90秒，冰上静置3～5分钟，加入400ul无抗的LB液体培养基，37℃摇床复苏培养45分钟，之后涂布Zeocin抗性平板。待平板长出克隆菌落后，挑选单一菌落克隆至含LZ液体培养基(Zeocin抗性的LB培养基)的摇菌管中，37℃培养6-8h，而后以菌液为模板进行PCR鉴定，所得阳性克隆命名为PGAP-zα-anti-CD16a-FCγ4-IL21，将阳性克隆对应的菌液用质粒小量抽提试剂盒进行抽提，并送通用生物测序。经测序后，测序结果与理论预期结果一致，克隆构建正确。
实施例2双特异性抗体分子anti-CD16a-FCγ4-IL21的表达及纯化
2.1 anti-CD16a-FCγ4-IL21双特异性抗体分子的表达
使用50mL LZ液体培养基扩大培养实施例1中构建的阳性克隆，使用试剂盒(Axygen公司)中量提取质粒PGAP-zα-anti-CD16a-FCγ4-IL21。将上步制备的表达质粒PGAP-zα-anti-CD16a-FCγ4-IL21使用内切酶SpeⅠ进行线性化，并通过乙醇沉淀回收。乙醇沉淀步骤：1)向酶切反应体系中加入两倍体积的无水乙醇和0.1倍体积的3M醋酸钠，-20℃沉淀2小时以上。2)12000g离心10分钟，弃除上清。3)300μL70％乙醇重悬沉淀，12000g离心10分钟，弃除上清。4)37℃烘干，用去离子水重悬并测定浓度,调节浓度在0.5-1.0μg/μL之间。
制备酵母感受态：制作步骤：1)选取冻存良好的巴斯德毕赤酵母X33菌株，划线于YPD平板，酵母培养箱30℃恒温培养。2)待克隆生长至直径1mm左右，挑取克隆至液体4mLYPD培养基，酵母摇床30℃恒温培养。3)培养24-48小时。将该菌液1mL接种到50mL新鲜YPD培养基中。4)待OD600值达到1-1.5，将菌液转移至50mL离心管，4℃，1500g离心5分钟，弃上清。5)50mL冰水重悬，4℃，1500g离心5分钟，弃上清，重复一次。6)50mL1M冷山梨醇重悬，4℃，1500g离心5分钟，弃上清,重复一次。7)500μL1M冷山梨醇重悬。
电转化步骤：取上述感受态细胞100μL于无菌预冷电转杯中，向其中加入10μL线性化质粒(5-10μg)，混合均匀。设定电转仪参数：2000V，200Ω，25μF。电转，电转后立即向电转杯中加入1mL1M冷山梨醇，冰上静置5～10min,之后将全部菌液转移至装有1mLYPD的50mL离心管中，30℃，225rpm，复苏2h。复苏结束后，取100-1000μL上述菌液涂布YPDZ(含有Zeocin抗性的平板)平板，平板置于30℃恒温酵母培养箱培养。
阳性表达克隆筛选：待上述YPDZ平板长出酵母克隆后，挑取若干克隆至2mLBMGY培养基中，酵 母摇床28℃恒温培养至菌液乳白。将其中1mL菌液冻存保种，其余1mL菌液12000g离心5分钟后取上清使用抗human FCγ4抗体进行Dot blot鉴定初筛选，挑出表达量相对较高的酵母菌株，而后将表达量相对较高的酵母菌株涂布于含zeocin的YPD平板上，待长出克隆后再从中选取多个克隆按照上述方式摇菌并获取菌液上清，使用Anti-human FCγ抗体进行Western Blot鉴定分析，筛选出高表达菌株(34-3)，将对应菌株的剩余1mL菌液冻存保种；此高表达菌株为能表达anti-CD16a-FCγ4-IL21复合体蛋白的毕赤酵母菌株。
具体的实验结果如图3所示，经过YPDZ抗性培养板筛选，获得了融合蛋白的酵母表达菌株。使用Dot blot进行初步筛选，可以初步获取一些表达量相对较高的克隆(如图3B中的66；91；98；34号)，之后进一步通过Western Blot进一步确认比较，可以筛选出表达量较高的酵母表达克隆，用于接下来的蛋白表达纯化。
2.2 anti-CD16a-FCγ4-IL21双特异性抗体分子的发酵、纯化与鉴定
(1)发酵
表达菌株培养：重组菌株发酵按照Invitrogen公司发酵指导手册进行。将筛选到的高表达菌株接种到含有4mL的YPD培养基的摇菌管中，30℃摇床培养24-48小时，至OD600为2-6，即为发酵一级种子液。取1mL该种子液转接入含有200mL BMGY培养基的锥形瓶中，30℃摇床培养12-24小时，即为发酵二级种子液。将200mL二级种子液全部接种入含有6L BMGY培养基的发酵罐中。设定参数：培养温度30℃，pH 7.0，监控溶氧、转速等，开始发酵培养。菌体生长阶段采用BMGY培养基，溶氧急速上升时说明培养基中基础甘油消耗殆尽，开始进行甘油流加，甘油流加速度为：90mL/h，调整发酵温度至25℃，持续至发酵结束。
培养液澄清：发酵结束之后，10000g离心30分钟，收集上清，并用0.22μm和500kDa微孔滤膜过滤澄清，之后调节PH至7.4。
发酵过程各参数监测如图4所示，整个发酵过程持续了50个小时，发酵过程稳定易重复。由图4B可知，从基础甘油培养阶段结束时开始进行甘油流加限制性培养诱导蛋白表达(20h)，整个过程蛋白持续累积，43～50h蛋白累积量差异不大，判定发酵终点。
(2)纯化
蛋白捕获：使用亲和色谱柱Mabselect柱捕获重组融合蛋白。具体步骤为：1)去离子水冲洗色谱柱5个柱体积。2)PBS冲洗色谱柱3个柱体积。3)上样。4)上样结束后，PBS冲洗色谱柱3个柱体积。5)洗杂：使用PH5.5的柠檬酸缓冲液冲洗5个柱体积。6)洗脱目的蛋白，洗脱液为PH3.0的柠檬酸缓冲液，收集到的洗脱液浓缩后用于后续纯化。
精细纯化：使用AKTA PURE 25，用PBS缓冲液平衡Superdex200分子筛，平衡后，将中性洗脱液 上样，使用PBS缓冲液冲洗后收集洗脱样品获得anti-CD16a-FCγ4-IL21重组蛋白。
通过离心过滤和两步纯化，经SDS-PAGE，HPLC鉴定，蛋白纯度可达95％以上(图5A，C)。
(3)鉴定
重组蛋白鉴定：将纯化所得蛋白进行SDS-PAGE、HPLC、动态光散射鉴定。具体的实验结果如图4B所示，其中，anti-CD16a-FCγ4-IL21蛋白分子量约135kDa左右，与预期相符；经过动态光散射鉴定，anti-CD16a-FCγ4-IL21分子直径约10nm左右，分子间均一性较好。
实施例3双特异性抗体anti-CD16a-FCγ4-IL21体外诱导NK细胞活化功能验证
采用Ficoll密度梯度离心法分离人PBMC。将分离到的PBMC使用含10％FBS的RPMI 1640稀释到1.0×10 6/mL。向T25培养瓶中加入10mL稀释后的细胞。培养12小时后，以20nM的终浓度向培养瓶中加入anti-CD16a-FCγ4-IL21融合蛋白。培养24小时后，流式细胞仪检测NK细胞表型变化，主要是检测NK细胞表面主要活化性分子CD69、NKp44及杀伤性分子4-1BB、TRAIL、Granzyme B的表达情况。
具体的实验结果如图6所示，相比于对照组PBS(Control组)，添加anti-CD16a-FCγ4-IL21融合蛋白的实验组中，NK细胞表面的主要活化性分子CD69、NKp44及杀伤性分子4-1BB、TRAIL、Granzyme B，表达水平均显著上调，依此判断NK细胞被anti-CD16a-FCγ4-IL21融合蛋白有效活化。
实施例4双特异性抗体分子anti-CD16a-FCγ4-IL21体外诱导NK细胞扩增及扩增效果评价
4.1 anti-CD16a-FCγ4-IL21扩增NK细胞的纯度及数量检测
提前24h使用融合蛋白包被T75培养瓶，每瓶取7.5mL包被液，覆盖瓶身底部。4℃冰箱包被过夜。
从血样中分离出PBMC细胞后，计数(迈瑞计数仪)，记录PBMC细胞中各细胞亚群的百分比。按1.5*10^6/mL密度(20mL培养体积)进行接种。细胞总数为30*10^6。此为第0天。同时，不含anti-CD16a-FCγ4-IL21的KBM581培养基加入1％的IL2(1000IU/mL)和5％对应的PBMC自体血浆。
第3天，补充相应培养体积1％的IL2因子。
第5天，观察培养基颜色及T75瓶身底部细胞贴壁状态，按需补充5～10mL含有anti-CD16a-FCγ4-IL21的KBM581培养基，并添加补液体积1％的IL2(1000IU/mL)因子。
第6天，取样进行计数进行细胞传代扩大培养(1.3*10^6/mL)。对其进行染色标记(CD16a/CD45/CD56)，流式细胞仪检测其NK细胞亚群的百分比，CD56+细胞亚群百分比，并计算总细胞数量及根据流式纯度检测结果计算NK细胞数量。自第5天以后细胞传代扩大培养使用含有anti-CD16a-FCγ4-IL21的KBM581培养基，并添加1％的IL2和2％的自体血浆。
第7天，观察培养基颜色及瓶身细胞贴壁生长状况，按需进行补液(并添加补液体积1％的IL2因子)。
第8天，取样计数。并按需进行细胞传代扩大(T75或T175瓶子，1.4*10^6/mL)并添加培养基的1％IL2因子，2％的自体血浆。
第9天，观察培养基颜色及瓶身细胞贴壁生长状况，按需进行补液(并添加补液体积1％的IL2因子)。
第10天，取样进行计数，进行细胞传代扩大培养(1.5*10^6/mL)。并对其进行染色标记(CD16a/CD45/CD56)，流式细胞仪检测其NK细胞亚群的百分比，CD56+细胞亚群百分比，并计算总细胞数量及根据流式纯度检测结果计算NK细胞数量。
第12天，观察培养基颜色及瓶身细胞贴壁生长状况，按需进行补液(并添加补液体积1％的IL2因子)。
第13天，取样进行计数，进行细胞传代扩大培养(1.5*10^6/mL)。对其进行染色标记(CD16a/CD45/CD56)，流式细胞仪检测其NK细胞亚群的百分比，CD56+细胞亚群百分比，并计算总细胞数量及根据流式纯度检测结果计算NK细胞数量。
第15天，取样计数。并按需进行细胞传代扩大(T175瓶子或者培养袋，1.5*10^6/mL)并添加培养基的1％IL2因子。
第17天，取样进行计数，对其进行染色标记(CD16a/CD45/CD56)，流式细胞仪检测其NK细胞亚群的百分比，CD56+细胞亚群百分比，并计算总细胞数量及根据流式纯度检测结果计算NK细胞数量。通过第0，6，10，13，17的NK纯度及细胞数量绘制NK细胞比例及数量随时间变化的曲线图。
具体的实验结果如图7中的A～C图所示，使用anti-CD16a-FCγ4-IL21扩增PBMC来源的NK细胞，可有效扩增NK细胞，提高NK细胞纯度及扩增倍数，在整个培养过程中最高可使NK细胞比例达65％左右。
4.2双特异性抗体分子扩增PBMC来源NK细胞的亚群分析
(1)胞内分子标记细胞莫能霉素诱导：取培养的细胞4×10 6个(2mL)置于6孔板中，加莫能霉素(0.5μg/μL)10μL,37℃，5％CO2恒温培养箱内诱导4小时。
(2)单细胞悬液制备：表面分子标记细胞取培养的细胞9.0×10 6个置于15mL离心管中，400g离心8分钟收集细胞，10mL 1×PBS洗涤2次，最后用0.81mL 1×PBS重悬成单细胞悬液。胞内分子标记细胞莫能霉素诱导结束后，加5mL 1×PBS洗涤2次，最后用180μL 1×PBS重悬成单细胞悬液。
(3)封闭：表面分子标记细胞加入90μL小鼠血清，混匀，胞内分子标记细胞加入20μL小鼠血清，混匀，室温静置15～30分钟。
(4)标记抗体：将封闭后表面分子标记细胞按0.09mL/每管分装至9个流式管中，如果有多批细胞同时检测，则1-8管的细胞可以用几批细胞等量混合后进行标记，每管的细胞用量为：0.8-1×10 6个，样品管1为表面分子标记，细胞用量为：0.8-1×10 6个。样品管2为胞内分子标记，细胞用量为：3-4×10 6个并加入相应的荧光标记抗体，混匀后置4℃避光静置30分钟(中间每15分钟混匀一次)。具体的实验 分组如表2所示。
表2：细胞表面分子检测样本管及实验操作表
Figure PCTCN2022114434-appb-000008
(5)洗涤：表面分子标记细胞中每管加入1mL 1×PBS，4℃，400g离心8分钟收集细胞。用1mL 1×PBS重复洗涤2次。最后每管中加入200ul PBS重悬标记后的细胞，实验组加入DAPI(50μg/mL,40×)5μL，转管上机检测。
(6)检测：流式细胞仪按仪器说明书进行校对和调整。空白对照管样品用于调节前向散射和侧向散射的电压。单标对照样品用于调节各个通道荧光补偿。检测时圈定细胞门后，每个样品门内收集1×10 4个细胞。
具体的实验结果如图7-D所示，对扩增后的细胞进行亚群分析，可知经anti-CD16a-FCγ4-IL21培养扩增后的PBMC细胞，其中NK细胞比例可达61％，NKT比例约在10％左右，总的CD56阳性细胞约在71％左右，其余细胞亚群在扩增后的PBMC细胞中占比较低。
4.3双特异性抗体分子扩增NK细胞的杀伤活性检测
(1)靶细胞K562悬液制备
细胞计数：细胞用含0.5％FBS的1640培养基(靶细胞所用培养基)重悬，计数，调整细胞密度在1×106/mL；
CFSE染色：细胞悬液加入CFSE(工作浓度为5μM),立刻用1mL移液枪吹打混匀，涡旋充分混匀， 37℃培养箱避光孵育15min，每5min取出来涡旋混匀一次；
终止染色：加入5倍体积4℃预冷的完全培养基(靶细胞所用培养基)，终止染色，冰浴5分钟。140g，4℃，5min离心；
洗细胞：用4℃预冷的完全培养基(靶细胞所用培养基)，重悬，140g，4℃，5min离心细胞；重复洗两遍；
计数：细胞重悬，计数，调整细胞密度为2×105/mL；
铺板：在96孔圆底板中每孔中加入100μl靶细胞悬液，使每孔最终细胞数为20000个/孔；
(2)NK细胞的准备与加入
配制成合适的NK细胞密度；
取出E-Plate 16，置于超净台中；
将100μl的NK细胞悬液以不同的效靶比(CD56+:K562)(1:1、2:1、4:1、8:1)加入培养板内，此外还应设置三组对照：仅有CFSE染色的靶细胞(检测靶细胞的自然死亡率)；仅有效应细胞(证明效应细胞不含有CFSE非特异性染色)；靶细胞+Tween-20(作为靶细胞凋亡的阳性对照)；
共孵育，按照预先设计好的顺序在各孔加入NK细胞。离心，120g,室温，2min，使效靶细胞充分接触。放回37℃培养箱中孵育4小时。
孵育结束后，加入5μl PI，混匀，避光孵育5min，上机检测。
结果分析：杀伤百分比＝[(实验组靶细胞死亡率(％)-靶细胞的死亡率(％))/(100％-靶细胞自然死亡率(％))]×100％)。
具体的实验结果如图7中E图(7-E)所示，对扩增后的细胞的细胞毒性进行评价，扩增后的细胞可以有效杀伤K562细胞。
综上所述，本实施例的实验结果证明anti-CD16a-FCγ4-IL21具有在体外活化扩增NK细胞的活性，且扩增后的NK细胞具有较好的细胞毒性。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为 对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (26)

  1. 一种重组抗体，其特征在于，包括：
    IL-21分子；以及
    CD16a抗体，所述CD16a抗体包括CD16a单链抗体以及Fc区，所述Fc区的N末端与所述单链抗体的C末端相连，所述CD16a单链抗体包括重链可变区、轻链可变区；
    所述FC的C末端与所述IL21分子的N端相连。
  2. 根据权利要求1所述的重组抗体，其特征在于，进一步包括连接肽1，所述连接肽1的N端与所述CD16a单链抗体的重链可变区的C端相连，所述连接肽1的C端与所述CD16a单链抗体的轻链可变区的N端相连；
    任选地，所述连接肽1具有如SEQ ID NO:1所示的氨基酸序列。
  3. 根据权利要求1所述的重组抗体，其特征在于，进一步包括连接肽2，所述连接肽2的N端与所述IL-21分子的C端相连，所述连接肽2的C端与所述Fc区的N端相连；
    任选地，所述连接肽2具有如SEQ ID NO:2所示的氨基酸序列。
  4. 根据权利要求3所述的重组抗体，其特征在于，所述Fc区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。
  5. 根据权利要求3或4所述的重组抗体，其特征在于，所述Fc区的至少一部分来自人源IgG或其突变体。
  6. 根据权利要求3-5任一项所述的重组抗体，其特征在于，所述Fc区的至少一部分来自于人源Fcγ4或其突变体。
  7. 根据权利要求3-6任一项所述的重组抗体，其特征在于，所述Fc区具有如SEQ ID NO:3所示的氨基酸序列。
  8. 根据权利要求1-7任一项所述的重组抗体，其特征在于，具有SEQ ID NO：4所示的氨基酸序列。
  9. 一种核酸，其特征在于，所述核酸编码权利要求1-8任一项所述的重组抗体。
  10. 根据权利要求9所述的核酸，其特征在于，具有SEQ ID NO：5所示的核苷酸序列。
  11. 一种表达载体，其特征在于，所述表达载体携带权利要求9或10所述的核酸分子。
  12. 一种重组细胞，其特征在于，所述重组细胞携带权利要求9或10所述的核酸分子、权利要求11所述的表达载体或权利要求1-8任一项所述的重组抗体。
  13. 一种组合物，其特征在于，包括：权利要求1-8任一项所述的重组抗体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体或权利要求12所述的重组细胞。
  14. 权利要求1-8任一项所述的重组抗体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体、权利要求12所述的重组细胞或权利要求13所述的组合物在制备药物中的用途，所述药物用于治疗或预防肿瘤或病毒感染。
  15. 根据权利要求14所述的用途，其特征在于，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
  16. 权利要求1-8任一项所述的重组抗体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体和权利要求12所述的重组细胞在培养扩增NK细胞中的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述NK细胞来源于外周血或诱导干细胞获得。
  18. 根据权利要求16所述的用途，其特征在于，所述NK细胞包括下列中的至少之一：人外周血单个核细胞来源的NK细胞、人脐血干细胞诱导获得的NK细胞、人胚胎干细胞诱导获得的NK细胞和人iPSC诱导获得的NK细胞。
  19. 一种药物，其特征在于，包括：权利要求1-8任一项所述的重组抗体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体、权利要求12所述的重组细胞或权利要求13所述的组合物，所述药物用于治疗或预防肿瘤或病毒感染。
  20. 根据权利要求19所述的药物，其特征在于，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
  21. 一种试剂盒，其特征在于，包括：权利要求1-8任一项所述的重组抗体。
  22. 根据权利要求21所述的试剂盒，其特征在于，所述试剂盒用于检测CD16a和/或IL-21R。
  23. 权利要求1-8任一项所述的重组抗体、权利要求9或10所述的核酸分子、权利要求11所述的表达载体、权利要求12所述的重组细胞、权利要求13所述的组合物或权利要求19-20任一项所述的药物在治疗或预防肿瘤或病毒感染中的用途。
  24. 根据权利要求23所述的用途，其特征在于，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
  25. 一种治疗或预防肿瘤或病毒感染的方法，其特征在于，包括向受试者施用以下中的至少之一：
    1)权利要求1-8任一项所述的重组抗体；
    2)权利要求9或10所述的核酸分子；
    3)权利要求11所述的表达载体；
    4)权利要求12所述的重组细胞；
    5)权利要求13所述的组合物；或
    6)权利要求19-20任一项所述的药物。
  26. 根据权利要求25所述的方法，其特征在于，所述肿瘤包括下列中的至少之一：直肠癌、肺癌、乳腺癌、肝癌、胃癌、皮肤癌、肾癌、胰腺癌和卵巢癌，所述病毒感染包括下列中的至少之一：HPV、HBV、流感病毒和冠状病毒。
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