WO2023115896A1 - 一种双特异性的nk细胞激动剂及制备方法和应用 - Google Patents

一种双特异性的nk细胞激动剂及制备方法和应用 Download PDF

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WO2023115896A1
WO2023115896A1 PCT/CN2022/102876 CN2022102876W WO2023115896A1 WO 2023115896 A1 WO2023115896 A1 WO 2023115896A1 CN 2022102876 W CN2022102876 W CN 2022102876W WO 2023115896 A1 WO2023115896 A1 WO 2023115896A1
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fusion protein
cells
linker
1bbl
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French (fr)
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肖卫华
田志刚
李洋阳
谢思奇
陈敏华
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上海恩凯细胞技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—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
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to a bispecific NK cell agonist and its preparation method and application.
  • 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 Inhibitory signals in the NK cells can restore the normal activation state and exert anti-tumor or anti-virus functions
  • 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 a strong activation effect and enhancement of anti-tumor ability to NK cells, and at the same time, it will temporarily increase the expression of 4-1BB receptor.
  • the 4-1BB receptor is a member of the TNF receptor superfamily. 4-1BB is mainly expressed in the form of a trimer on activated NK cells and T cells. Its ligand is 4-1BBL, which is the only one found so far. When 4-1BBL is combined with 4-1BB, the naturally occurring ligand will activate the 4-1BB receptor, and this process will bring a certain activation effect to NK cells and enhance the ADCC effect.
  • NK cell agonists At present, there are few research reports on NK cell agonists, and the existing NK cell agonists have limited ability to activate NK cells and need to be further improved.
  • an object of the present disclosure is to provide a fusion protein that uses CD16A receptor and 4-1BB receptor as targets to obtain a new bispecific NK cell agonist drug for activating NK cells , enhance its anti-virus and anti-tumor ability, and has the potential to promote the proliferation of NK cells and enhance the function of T cells.
  • the first aspect of the present disclosure provides a fusion protein.
  • the fusion protein includes a CD16A single-chain antibody and a 4-1BBL extracellular region, wherein the CD16A single-chain antibody is connected to the 4-1BBL extracellular region, and the 4-1BBL extracellular region includes At least 1-3 4-1BBL extracellular domains.
  • the inventors found that by using the CD16A receptor and the 4-1BB receptor as targets, the single-chain CD16A antibody and the extracellular region of 4-1BBL were selected to design a novel bispecific NK cell agonist drug scfv-CD16A/ Trimer-4-1BBL. After the single-chain CD16A antibody recognizes the CD16A receptor, the NK cell agonist drug recognizes the 4-1BB receptor through the extracellular region of the 4-1BBL at the other end of the fusion protein, thereby increasing the proliferation of NK cells.
  • the single-chain anti-CD16A antibody is connected with 1-3, preferably 3, 4-1BBL extracellular segments to construct a bispecific NK cell agonist drug, which is used to activate NK cells and enhance their anti-virus and Anti-tumor ability, at the same time has the potential to promote NK cell proliferation and enhance the effect of T cell function.
  • the scfv-CD16A/Trimer-4-1BBL fusion protein provided by the present disclosure synergistically activates NK cells by binding to CD16A receptors and 4-1BB receptors, reverses the exhaustion state of NK cells, and enhances the anti-tumor ability of NK cells.
  • the scfv-CD16A/Trimer-4-1BBL fusion protein provided by the present disclosure can effectively expand NK cells, and improve the purity and amplification factor of NK cells.
  • the fusion protein also includes at least one of the following additional technical features:
  • the 4-1BBL extracellular region includes three 4-1BBL extracellular domains, and the three 4-1BBL extracellular domains are connected in sequence.
  • scfv-CD16A-Trimer-4-1BBL Three extracellular domains of 4-1BBL were linked with CD16A single-chain antibody to form scfv-CD16A-Trimer-4-1BBL.
  • scfv-CD16A-Trimer-4-1BBL has the activity of activating and expanding NK cells in vitro, and the expanded NK cells have better cytotoxicity.
  • the CD16A single chain antibody and the extracellular region of 4-1BBL are linked by a flexible linker.
  • the three 4-1BBL extracellular domains are connected by a flexible linker.
  • the fusion protein is expressed by a non-mammalian cell expression system.
  • the non-mammalian cell expression system includes at least one of a prokaryotic expression system and a lower eukaryotic expression system.
  • the non-mammalian cell expression system includes at least one of an Escherichia coli expression system and a yeast expression system.
  • the yeast expression system comprises a Pichia pastoris expression system.
  • the CD16A single-chain antibody includes a VH region and a VL region, the amino acid sequence of the VH region is shown in SEQ ID NO: 1, and the amino acid sequence of the VL region is shown in SEQ ID NO:2 shown.
  • the VH region and the VL region are connected by a first linker.
  • the first joint is a flexible joint.
  • the length of the first linker is 10-20 amino acids.
  • amino acid sequence of the first linker is shown in SEQ ID NO:3.
  • the amino acid sequence of the VH region of the CD16A single-chain antibody is shown in the following SEQ ID NO: 1:
  • amino acid sequence of the VL region of the CD16A single-chain antibody is shown in the following SEQ ID NO: 2:
  • amino acid sequence of the first linker is shown in the following SEQ ID NO:3:
  • one end of the VH region or VL region is connected to the 4-1BBL extracellular region through a second linker.
  • the second joint is a flexible joint.
  • the length of the second linker is 10-20 amino acids.
  • amino acid sequence of the second linker is shown in SEQ ID NO:4.
  • amino acid sequence of the second linker is shown in the following SEQ ID NO:4:
  • amino acid sequence of the 4-1BBL extracellular domain is shown in SEQ ID NO:5.
  • amino acid sequence of the 4-1BBL extracellular domain is shown in the following SEQ ID NO:5:
  • two of the three 4-1BBL extracellular domains are respectively connected by a third linker and a fourth linker, and the third linker and the fourth linker are the same or different.
  • both the third joint and the fourth joint are flexible joints.
  • the length of the third linker and the fourth linker is 15-25 amino acids.
  • amino acid sequence of the third linker is shown in SEQ ID NO:6.
  • amino acid sequence of the fourth linker is shown in SEQ ID NO:7.
  • amino acid sequence of the third linker is shown in the following SEQ ID NO:6:
  • amino acid sequence of the fourth linker is shown in the following SEQ ID NO:7:
  • amino acid sequence of the fusion protein is shown in SEQ ID NO:8.
  • amino acid sequence of the fusion protein is shown in the following SEQ ID NO: 8:
  • a second aspect of the present disclosure provides a nucleic acid.
  • the nucleic acid encodes the fusion protein described in the first aspect.
  • the nucleic acid is a codon-optimized nucleic acid for a yeast expression system.
  • nucleotide sequence of the nucleic acid is shown in SEQ ID NO:10.
  • nucleotide sequence encoding the fusion protein without codon optimization is shown in the following SEQ ID NO:9:
  • the third aspect of the present disclosure provides an expression vector.
  • the expression vector comprises the nucleic acid of the second aspect.
  • the fourth aspect of the present disclosure provides a recombinant cell.
  • the recombinant cell carries the nucleic acid described in the second aspect, the expression vector described in the third aspect, or expresses the fusion protein described in the first aspect.
  • the fifth aspect of the present disclosure provides the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of medicines.
  • the fusion protein described in the first aspect the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of medicines.
  • the tumor comprises at least one of the following: ovarian cancer, lung cancer, rectal cancer, breast cancer, leukemia.
  • the inflammatory disease comprises a bacterial or viral infection.
  • the bacterial infection includes at least one of Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus infection.
  • the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.
  • the sixth aspect of the present disclosure provides the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in culturing and expanding NK cells in vitro .
  • the NK cells are obtained from peripheral blood or induced 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, NK cells induced from human embryonic stem cells, human NK cells induced by iPSCs.
  • the seventh aspect of the present disclosure provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect or the recombinant cell described in the fourth aspect.
  • the pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases.
  • the tumor comprises at least one of the following: ovarian cancer, lung cancer, rectal cancer, breast cancer, leukemia.
  • the inflammatory disease comprises a bacterial or viral infection.
  • the bacterial infection includes at least one of Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus infection.
  • the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.
  • the eighth aspect of the present disclosure provides a kit.
  • the kit includes the fusion protein described in the first aspect.
  • the kit is used for detecting CD16A and/or 4-1BB.
  • a ninth aspect of the present disclosure provides a method of treating or preventing a tumor or an inflammatory disease.
  • the method comprises administering to a subject having or suspected of having a neoplastic or inflammatory disease at least one of the following:
  • the tumor comprises at least one of the following: ovarian cancer, lung cancer, rectal cancer, breast cancer, leukemia.
  • the inflammatory disease comprises a bacterial or viral infection.
  • the bacterial infection includes at least one of Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus infection.
  • the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.
  • the tenth aspect of the present disclosure provides the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, and the pharmaceutical composition described in the seventh aspect Use in the treatment or prevention of neoplastic or inflammatory diseases.
  • the tumor comprises at least one of the following: ovarian cancer, lung cancer, rectal cancer, breast cancer, leukemia.
  • the inflammatory disease comprises a bacterial or viral infection.
  • the bacterial infection includes at least one of Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus infection.
  • the viral infection includes at least one of HPV, HBV, influenza virus, and coronavirus infection.
  • Figure 1 shows a schematic diagram of protein expression vector and scfv-CD16A-Trimer-4-1BBL fusion protein
  • Figure 2 shows the screening results of scfv-CD16A-Trimer-4-1BBL fusion protein expression clones, wherein (A) is the result of dot blot (Dot Blot); (B) Western Blot (Western Blot) result (anti-His tag antibody) ;
  • Figure 3 shows the results of fermentation expression of scfv-CD16A-Trimer-4-1BBL fusion protein, wherein Figure (A) shows the monitoring of fermentation process parameters, and Figure (B) shows the detection results of the target protein accumulation time point;
  • Figure 4 shows the purification and physical and chemical identification of the scfv-CD16A-Trimer-4-1BBL fusion protein
  • Figure (A) is the SDS-PAGE test result of the purity of the scfv-CD16A-Trimer-4-1BBL fusion protein
  • Figure (B) is the scfv - CD16A-Trimer-4-1BBL fusion protein dynamic light scattering detection results
  • Figure (C) is the scfv-CD16A-Trimer-4-1BBL fusion protein HPLC detection results;
  • Figure 5 shows the detection results of scfv-CD16A-Trimer-4-1BBL fusion protein in vitro activation of NK cell activity
  • Figure 6 shows the evaluation results of scfv-CD16A-Trimer-4-1BBL fusion protein in vitro induction of NK cell expansion and expansion effect
  • Figure (A) is the scfv-CD16A-Trimer-4-1BBL fusion protein in vitro induction of NK cell expansion NK purity test results after growth
  • picture (B) is scfv-CD16A-Trimer-4-1BBL fusion protein in vitro induced NK cell expansion multiple test results
  • picture (C) is scfv-CD16A-Trimer-4-1BBL fusion protein in vitro
  • the picture (D) is the detection result of the cell subpopulation after the scfv-CD16A-Trimer-4-1BBL fusion protein induces NK cell expansion in vitro
  • picture (E) is the scfv-CD16A -Trimer-4-1BBL fusion protein induces NK cell expansion in vitro and detects
  • 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 disclosure provides a fusion protein comprising CD16A single-chain antibody and 4-1BBL extracellular region, wherein the CD16A single-chain antibody and 4-1BBL extracellular region are linked, and the 4 - The 1BBL extracellular region includes at least 1-3 4-1BBL extracellular domains.
  • the 4-1BBL extracellular region includes three 4-1BBL extracellular domains, and the three 4-1BBL extracellular domains are connected in sequence to form a scfv-CD16A-Trimer-4-1BBL fusion protein.
  • the fusion protein provided by this disclosure uses CD16A receptor and 4-1BB receptor as targets to obtain a new bispecific NK cell agonist drug, scfv-CD16A-Trimer-4-1BBL, which has Activating and expanding the activity of NK cells in vitro enhances their anti-virus and anti-tumor capabilities, and has the potential to promote the proliferation of NK cells and enhance the function of T cells, and the expanded NK cells have better cytotoxicity.
  • the term "antibody” is an immunoglobulin molecule capable of binding to a specific antigen. It includes two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable, with little change, called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively, the C region of the L chain is CL, and the C region of the H chain includes CH1, CH2 and CH3 regions.
  • peripheral blood mononuclear cells are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.
  • the first, second, third and fourth connecting peptides are flexible polypeptides composed of glycine and serine.
  • the pharmaceutical composition provided by the present disclosure further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient Agents, dispersants, flavoring or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., suitable for specific purposes dosage form.
  • a pharmaceutically acceptable carrier including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient Agents, dispersants, flavoring or suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., suitable for specific purposes dosage form.
  • any conventional adjuvant is incompatible with the fusion protein of the present disclosure, such as any adverse biological effects produced or interacted in a harmful manner with any other components of the pharmaceutically
  • fusion proteins of the disclosure can be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms.
  • liquid, semi-solid and solid dosage forms and the like including but not limited to liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the fusion protein can be administered by intravenous infusion or intramuscular or subcutaneous injection.
  • treatment or prevention do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prophylaxis that would be considered by one of ordinary skill in the art to be of potential benefit or therapeutic effect.
  • treatment or prevention provided by the present disclosure may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as cancer. Additionally, for purposes herein, "prevention” may encompass delaying the onset of a disease or a symptom or condition thereof.
  • Embodiments of the present disclosure are described in detail below.
  • the embodiments described below are exemplary only for explaining the present disclosure and should not be construed as limiting the present disclosure. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.
  • Example 1 Construction of fusion protein expression vector of bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL
  • scfv-CD16A includes V H and V L , and V H and V L are connected by linker1, wherein the amino acid sequence of V H is shown in SEQ ID NO: 1, and the nucleotide sequence of V L As shown in SEQ ID NO: 2, the nucleotide sequence of linker1 is shown in SEQ ID NO: 3), and the 4-1BBL extracellular segment 71-254 sequence was obtained from NCBI (as shown in SEQ ID NO: 5),
  • the scfv-CD16A is connected to three 4-1BBL extracellular segments by linker2 (as shown in SEQ ID NO:4), and the three 4-1BBL extracellular segment domains are sequentially connected by linker3 (as shown in SEQ ID NO:6 Shown) and linker4 (shown as SEQ ID NO:7) are connected.
  • the original scfv-CD16A-Trimer-4-1BBL fusion gene obtained without codon optimization is shown in SEQ ID NO:
  • Adjust and transform the original scfv-CD16A-Trimer-4-1BBL fusion gene sequence optimize the codon based on Pichia pastoris and add Xhol and NotI restriction sites at both ends, optimize and adjust the gene sequence such as SEQ ID Shown in NO: 10; the gene sequence is then subjected to whole gene synthesis to obtain the scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence.
  • Both the scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence obtained in the previous step and the Pichia pastoris expression plasmid PGAPz ⁇ -rDNA-NTS were double-digested with restriction endonucleases Xhol and NotI.
  • Enzyme digestion of scfv-CD16A-Trimer-4-1BBL fusion protein gene sequence product gene was recovered by PCR product recovery kit (Axygen Company), and the digested plasmid was recovered by DNA gel recovery kit (Axygen Company).
  • DNA gel recovery kit Axygen Company
  • the fusion protein gene obtained after recovery is ligated with the digested carrier by using T4 DNA ligase.
  • 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.
  • PGAP-z ⁇ -scfv-CD16A-Trimer-4-1BBL protein expression vector as shown in Figure 1
  • the bacterial liquid corresponding to the positive clone was extracted with a small amount of plasmid extraction kit, and sent to General Biology and Jinweizhi Sequencing.
  • Example 2 Expression and purification of the fusion protein of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL
  • Ethanol precipitation steps 1) Add twice the volume of absolute ethanol and 0.1 times the volume of 3M sodium acetate to the enzyme digestion reaction system, and precipitate at -20°C for more than 2 hours. 2) Centrifuge at 12000 g for 10 minutes, discard the supernatant. 3) Resuspend the pellet in 300 ⁇ L of 70% ethanol, centrifuge at 12,000 g for 10 minutes, and discard the supernatant. 4) Dry at 37°C, resuspend in deionized water and measure the concentration, adjust the concentration between 0.5-1.0 ⁇ g/ ⁇ L.
  • 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 1ml of YPD, 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
  • yeast strains and then spread yeast strains with relatively high expression levels (e1, c7, c11, d1, f13) on the YPD plate containing zeocin, and then select multiple clones from them after the clones grow out, and shake in the above-mentioned way Bacteria and the supernatant of the bacterial liquid were obtained, and the Anti-his tag antibody was used for Western Blot identification and analysis to screen out high-expression strains, and the remaining 1mL bacterial liquid of the corresponding strains was frozen and preserved; this high-expression strain (f13-3) was able to Pichia strain expressing scFv-CD16A-Trimer-4-1BBL 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 70ml/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 nickel 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 the chromatographic column with a gradient of PBS solutions containing 10mM, 20mM, 30mM, and 40mM imidazole, and wash 1-1.5 column volumes for each concentration. 6) Elution of the target protein, the eluent is a PBS solution containing 200 mM imidazole, and the collected eluent is concentrated for subsequent purification.
  • Fine purification use AKTA PURE 25, equilibrate Superdex200 molecular sieves with PBS buffer, after equilibration, load the neutral eluent, wash with PBS buffer and collect the eluted samples. Subsequently, the sample was concentrated and passed through Superdex75 molecular sieves in the same manner to obtain scfv-CD16A-Trimer-4-1BBL recombinant protein.
  • Recombinant protein identification The purified protein was identified by SDS-PAGE, HPLC, and dynamic light scattering.
  • Figure 2 shows the yeast expression strains that obtained the fusion protein after screening on YPDZ-resistant culture plates. Using Dot blot for preliminary screening, some clones with relatively high expression levels can be initially obtained, and then further confirmed and compared by Western Blot, yeast expression clones with high expression levels can be screened out for subsequent protein expression and purification.
  • Fig. 3 shows the monitoring of each parameter of the fermentation process. The whole fermentation process lasted 50 hours, and the fermentation process is stable and easy to repeat.
  • a figure shows that from the end of the basic glycerol culture stage, the glycerol feeding restriction culture induces protein expression ( 20h), the protein continued to accumulate throughout the process, and there was little difference in the amount of protein accumulation from 43 to 50h, and the fermentation end point was determined (B in Figure 3 shows the scfv-CD16A- The amount of Trimer-4-1BBL recombinant protein);
  • the protein purity can reach more than 95% ( Figures A and C in Figure 4), and the molecular weight of the scfv-CD16A-Trimer-4-1BBL protein is about 95kDa, in line with expectations;
  • the molecular diameter of scfv-CD16A-Trimer-4-1BBL is about 10 nm, and the intermolecular uniformity is good ( Figure 4, B).
  • Example 3 Functional verification of the fusion protein of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL inducing NK cell activation in vitro
  • Human PBMCs were isolated by Ficoll density gradient centrifugation. The isolated PBMCs were diluted to 1.0 ⁇ 10 6 cells/mL with RPMI 1640 containing 10% FBS. Add 10 mL of diluted cells to the T25 culture flask. After 12 hours of incubation, the scFv-CD16A-Trimer-4-1BBL fusion protein was added to the flasks at a final concentration of 20 nM. After 24 hours of culture, the phenotype changes of NK cells were detected by flow cytometry.
  • Example 4 The fusion protein of the bispecific NK cell agonist scfv-CD16A-Trimer-4-1BBL induces NK cell expansion in vitro and evaluation of the expansion effect
  • Blocking Add 90 ⁇ l of mouse serum to the surface molecularly labeled cells, mix well, add 20 ⁇ l of mouse serum to the intracellular molecularly labeled cells, mix well, and let stand at room temperature for 15-30 minutes.
  • 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, several batches of cells can be used to equalize the number of cells in tubes 1-8. Labeling after mixing, the amount of cells per tube: 0.8-1 ⁇ 10 6 , sample tube 1 is surface molecular markers, the amount of cells: 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).
  • Detection 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 ⁇ 104 cells were collected in each sample gate.
  • Target cell K562 suspension preparation
  • 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) to terminate the staining, and keep it on ice 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;
  • scfv-CD16A-Trimer-4-1BBL can effectively amplify NK cells, improve the purity and amplification factor of NK cells, and improve the NK cell purity and amplification factor during the entire culture process.
  • the highest proportion of NK cells can reach about 60%;
  • the subgroup analysis of the amplified cells shows that the PBMC cells amplified after scfv-CD16A-Trimer-4-1BBL treatment, the proportion of NK cells can reach 55%, and the proportion of NKT About 20%, the total CD56-positive cells are about 75%, and the remaining cell subsets account for a relatively low proportion in the expanded PBMC cells;

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Abstract

本公开涉及生物医药领域，具体涉及一种双特异性的NK细胞激动剂及制备方法和应用。本公开提供一种融合蛋白，其以CD16A受体和4-1BB受体作为靶点，获得一种新的双特异性的NK细胞激动剂药物，即scfv-CD16A-Trimer-4-1BBL，其具有在体外活化扩增NK细胞的活性，增强其抗病毒和抗肿瘤的能力，同时具有潜在的促进NK细胞增殖及增强T细胞功能的效应，具有体外培养扩增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

一种双特异性的NK细胞激动剂及制备方法和应用
优先权信息
本申请请求2021年年12月20日向中国国家知识产权局提交的、专利申请号为202111566165.1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
本公开涉及生物医药领域，具体地，涉及一种双特异性的NK细胞激动剂及制备方法和应用。
背景技术
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，同时会使4-1BB受体表达出现短暂的上调。4-1BB受体是TNF受体超家族成员之一，4-1BB主要在活化后的NK细胞，T细胞上以三聚体的形式表达，其配体为4-1BBL，也是目前发现的唯一的天然存在的配体，当4-1BBL与4-1BB结合后，会使4-1BB受体活化，该过程会给NK细胞带来一定的活化效应以及ADCC效应的增强。
目前，关于NK细胞激动剂的研究报道尚少，现有的NK细胞激动剂活化NK细胞能力有限，仍有待进一步改进。
发明内容
本公开旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本公开的一个目的在于提供一种融合蛋白，其以CD16A受体和4-1BB受体作为靶点，获得一种新的双特异性的NK细胞激动剂药物，用于活化NK细胞，增强其抗病毒和抗肿瘤的能力，同时具有潜在的促进NK细胞增殖及增强T细胞功能的效应。
为此本公开第一方面提供一种融合蛋白。根据本公开的实施方案，所述融合蛋白包括CD16A单链抗体和4-1BBL胞外区，其中，所述CD16A单链抗体和4-1BBL胞外区连接，所述4-1BBL胞外区包括至少1-3个4-1BBL胞外结构域。
发明人发现，以CD16A受体和4-1BB受体作为靶点，选择单链CD16A抗体和4-1BBL的胞外段区域，设计一种新型双特异性的NK细胞激动剂药物scfv-CD16A/Trimer-4-1BBL。该NK细胞激动剂药物在单链CD16A抗体识别CD16A受体后，通过融合蛋白另一端的4-1BBL的胞外段区域识别4-1BB受体，从而导致NK细胞的增殖增加。其中将单链的anti-CD16A抗体与1-3个，优选3个4-1BBL胞外段连接，构建出的双特异性的NK细胞激动剂药物，用于活化NK细胞，增强其抗病毒和抗肿瘤的能力，同时具有潜在的促进NK细胞增殖及增强T细胞功能的效应。本公开提供的scfv-CD16A/Trimer-4-1BBL融合蛋白通过与CD16A受体和4-1BB受体的结合来协同活化NK细胞，逆转NK细胞的耗竭状态，增强NK细胞的抗肿瘤能力。本公开提供的scfv-CD16A/Trimer-4-1BBL融合蛋白可有效扩增NK细胞，提高NK细胞纯度及扩增倍数。
根据本公开的实施方案，所述融合蛋白还包括以下附加技术特征中的至少之一：
根据本公开的实施方案，所述4-1BBL胞外区包括3个4-1BBL胞外结构域，3个所述4-1BBL胞外 结构域顺次连接。
3个4-1BBL胞外结构域与CD16A单链抗体相连形成scfv-CD16A-Trimer-4-1BBL。scfv-CD16A-Trimer-4-1BBL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。
根据本公开的实施方案，所述CD16A单链抗体和4-1BBL胞外区通过柔性接头相连。
根据本公开的实施方案，3个所述4-1BBL胞外结构域之间通过柔性接头相连。
根据本公开的实施方案，所述融合蛋白是由非哺乳细胞表达体系表达的。
根据本公开的实施方案，所述非哺乳细胞表达体系包括原核表达体系和低级真核表达体系中的至少之一。
根据本公开的实施方案，所述非哺乳细胞表达体系包括大肠杆菌表达体系、酵母表达体系中的至少之一。
根据本公开的实施方案，所述酵母表达体系包括毕赤酵母表达体系。
根据本公开的实施方案，所述CD16A单链抗体包括V H区和V L区，所述V H区的氨基酸序列如SEQ ID NO:1所示，所述V L区的氨基酸序列如SEQ ID NO:2所示。
根据本公开的实施方案，所述V H区和V L区之间通过第一接头连接。
根据本公开的实施方案，所述第一接头为柔性接头。
根据本公开的实施方案，所述第一接头的长度为10～20个氨基酸。
根据本公开的实施方案，所述第一接头的氨基酸序列如SEQ ID NO:3所示。
CD16A单链抗体的V H区的氨基酸序列如以下SEQ ID NO:1所示：
Figure PCTCN2022102876-appb-000001
CD16A单链抗体的V L区的氨基酸序列如以下SEQ ID NO:2所示：
Figure PCTCN2022102876-appb-000002
第一接头的氨基酸序列如以下SEQ ID NO:3所示：
Figure PCTCN2022102876-appb-000003
根据本公开的实施方案，所述V H区或V L区的一端与所述4-1BBL胞外区通过第二接头相连。
根据本公开的实施方案，所述第二接头为柔性接头。
根据本公开的实施方案，所述第二接头的长度为10～20个氨基酸。
根据本公开的实施方案，所述第二接头的氨基酸序列如SEQ ID NO:4所示。
第二接头的氨基酸序列如以下SEQ ID NO:4所示：
Figure PCTCN2022102876-appb-000004
根据本公开的实施方案，所述4-1BBL胞外结构域的氨基酸序列如SEQ ID NO:5所示。
4-1BBL胞外结构域的氨基酸序列如以下SEQ ID NO:5所示：
Figure PCTCN2022102876-appb-000005
根据本公开的实施方案，3个所述4-1BBL胞外结构域之间两两分别通过第三接头和第四接头连接，所述第三接头和第四接头之间相同或不同。
根据本公开的实施方案，所述第三接头和第四接头均为柔性接头。
根据本公开的实施方案，所述第三接头和第四接头的长度为15～25个氨基酸。
根据本公开的实施方案，所述第三接头的氨基酸序列如SEQ ID NO:6所示。
根据本公开的实施方案，所述第四接头的氨基酸序列如SEQ ID NO:7所示。
第三接头的氨基酸序列如以下SEQ ID NO:6所示：
Figure PCTCN2022102876-appb-000006
第四接头的氨基酸序列如以下SEQ ID NO:7所示：
Figure PCTCN2022102876-appb-000007
根据本公开的实施方案，所述融合蛋白的氨基酸序列如SEQ ID NO:8所示。
融合蛋白的氨基酸序列如以下SEQ ID NO:8所示：
Figure PCTCN2022102876-appb-000008
本公开第二方面提供一种核酸。根据本公开的实施方案，所述核酸编码第一方面所述的融合蛋白。根据本公开的实施方案，所述核酸为经过酵母表达体系密码子优化的核酸。
根据本公开的实施方案，所述核酸的核苷酸序列如SEQ ID NO:10所示。
未经密码子优化的编码上述融合蛋白的核苷酸序列如以下SEQ ID NO:9所示：
Figure PCTCN2022102876-appb-000009
Figure PCTCN2022102876-appb-000010
经过密码子优化的编码上述融合蛋白的核苷酸序列如以下SEQ ID NO:10所示：
Figure PCTCN2022102876-appb-000011
Figure PCTCN2022102876-appb-000012
本公开第三方面提供一种表达载体。根据本公开的实施方案，所述表达载体包含第二方面所述的核酸。
本公开第四方面提供一种重组细胞。根据本公开的实施方案，所述重组细胞携带第二方面所述的核酸、第三方面所述的表达载体或表达第一方面所述的融合蛋白。
本公开第五方面提供第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体或第四方面所述的重组细胞在制备药物中的用途，所述药物用于治疗或预防肿瘤或炎性疾病。
根据本公开的实施方案，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病。
根据本公开的实施方案，所述炎性疾病包括细菌或病毒感染。
根据本公开的实施方案，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一。
根据本公开的实施方案，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
本公开第六方面提供第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体或第四方面所述的重组细胞在体外培养扩增NK细胞中的用途。
根据本公开的实施方案，所述NK细胞来源于外周血或诱导干细胞获得。根据本公开的实施方案，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。
本公开第七方面提供一种药物组合物。根据本公开的实施方案，所述药物组合物包含第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体或第四方面所述的重组细胞。
根据本公开的实施方案，所述药物组合物用于治疗或预防肿瘤或炎性疾病。
根据本公开的实施方案，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病。
根据本公开的实施方案，所述炎性疾病包括细菌或病毒感染。
根据本公开的实施方案，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一。
根据本公开的实施方案，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
本公开第八方面提供一种试剂盒。根据本公开的实施方案，所述试剂盒包括第一方面所述的融合蛋白。
根据本公开的实施方案，所述试剂盒用于检测CD16A和/或4-1BB。
本公开第九方面提供一种治疗或预防肿瘤或炎性疾病的方法。根据本公开的实施方案，所述方法包括向患有或疑似患有肿瘤或炎性疾病的受试者施用以下中的至少之一：
第一方面所述的融合蛋白；
第二方面所述的核酸；
第三方面所述的表达载体；
第四方面所述的重组细胞；
第七方面所述的药物组合物。
根据本公开的实施方案，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病。
根据本公开的实施方案，所述炎性疾病包括细菌或病毒感染。
根据本公开的实施方案，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一。
根据本公开的实施方案，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
本公开第十方面提供第一方面所述的融合蛋白、第二方面所述的核酸、第三方面所述的表达载体、第四方面所述的重组细胞、第七方面所述的药物组合物在治疗或预防肿瘤或炎性疾病中的用途。
根据本公开的实施方案，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病。
根据本公开的实施方案，所述炎性疾病包括细菌或病毒感染。
根据本公开的实施方案，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一。
根据本公开的实施方案，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
本公开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，或通过本公开的实践了解到。
附图说明
本公开的上述和/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显示了蛋白表达载体及scfv-CD16A-Trimer-4-1BBL融合蛋白示意图；
图2显示了scfv-CD16A-Trimer-4-1BBL融合蛋白表达克隆筛选结果，其中图(A)为斑点杂交(Dot Blot)结果；(B)免疫印迹(Western Blot)结果(抗His tag抗体)；
图3显示了scfv-CD16A-Trimer-4-1BBL融合蛋白发酵表达结果，其中图(A)显示了发酵过程参数监测，图(B)显示了目的蛋白累积时间点检测结果；
图4显示了scfv-CD16A-Trimer-4-1BBL融合蛋白纯化与理化鉴定，其中图(A)为scfv-CD16A-Trimer-4-1BBL融合蛋白纯度SDS-PAGE检测结果，图(B)为scfv-CD16A-Trimer-4-1BBL融合蛋白动态光散射检测结果，图(C)为scfv-CD16A-Trimer-4-1BBL融合蛋白HPLC检测结果；
图5显示了scfv-CD16A-Trimer-4-1BBL融合蛋白体外活化NK细胞活性检测结果；
图6显示了scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK细胞扩增及扩增效果评价结果，其中图(A)为scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK细胞扩增后NK纯度检测结果，图(B)为scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK细胞扩增倍数检测结果，图(C)为scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK扩增后总细胞扩增倍数检测结果，图(D)为scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK细胞扩增后细胞亚群检测结果，图(E)为scfv-CD16A-Trimer-4-1BBL融合蛋白体外诱导NK细胞扩增后细胞杀伤活性检测结果。
公开详细描述
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
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。在本公开的描述中，“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。
根据本公开的一些具体实施方案，本公开提供一种融合蛋白，包括CD16A单链抗体和4-1BBL胞外区，其中，所述CD16A单链抗体和4-1BBL胞外区连接，所述4-1BBL胞外区包括至少1-3个4-1BBL胞外结构域。例如，所述4-1BBL胞外区包括3个4-1BBL胞外结构域，3个所述4-1BBL胞外结构域顺次连接，形成了scfv-CD16A-Trimer-4-1BBL融合蛋白。
本公开提供的融合蛋白，以CD16A受体和4-1BB受体作为靶点，获得一种新的双特异性的NK细胞激动剂药物，即scfv-CD16A-Trimer-4-1BBL，其具有在体外活化扩增NK细胞的活性，增强其抗病毒和抗肿瘤的能力，同时具有潜在的促进NK细胞增殖及增强T细胞功能的效应，且扩增后的NK细胞具 有较好的细胞毒性。
本文中，术语“抗体”是能够与特异性抗原结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链，重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中，肽链的氨基端(N端)氨基酸序列变化很大，称为可变区(V区)，羧基端(C端)相对稳定，变化很小，称为恒定区(C区)。L链和H链的V区分别称为VL和VH，L链的C区为CL，H链的C区包含CH1区、CH2区和CH3区。
在本公开中，外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)是外周血中具有单个核的细胞，包括淋巴细胞和单核细胞。
根据本公开的一些具体实施方案，所述第一、第二、第三、第四连接肽为由甘氨酸和丝氨酸组成的柔性多肽。
根据本公开的一些具体实施方案，本公开提供的药物组合物进一步包括药学上可接受的载体，包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂，等等，适合于特有的目标剂型。除了任何常规的辅料与本公开的融合蛋白不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本公开所考虑的范围。
例如，本公开的融合蛋白可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。所述融合蛋白可通过静脉输注射或肌肉内或皮下注射来施用。
本文使用的术语“治疗”和“预防”以及源自于此的词不必暗示100％或完全治疗或预防。相反，存在不同程度的治疗或预防，本领域普通技术人员认为所述治疗或预防具有潜在的益处或治疗效果。而且，本公开提供的治疗或预防可包括正在治疗或预防的疾病如癌症的一种或多种病患或症状的治疗或预防。另外，为了本文的目的，“预防”可涵盖延缓疾病或其症状或病患的发作。
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。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NK细胞激动剂scfv-CD16A-Trimer-4-1BBL的融合蛋白表达载体的构建
1、获取scfv-CD16A-Trimer-4-1BBL融合基因
获取scfv-CD16A的序列信息(scfv-CD16A包括V H和V L，且V H和V L通过linker1相连，其中V H的氨基酸序列如SEQ ID NO:1所示，V L的核苷酸序列如SEQ ID NO:2所示，linker1的核苷酸序列如SEQ ID NO:3所示)，从NCBI上获取4-1BBL胞外段71-254序列(如SEQ ID NO:5所示)，将scfv-CD16A与三个4-1BBL胞外段通过linker2相连(如SEQ ID NO:4所示)，且三个4-1BBL胞外段结构域之间依次通过linker3(如SEQ ID NO:6所示)和linker4(如SEQ ID NO:7所示)相连。获得的未经密码子优化的原始scfv-CD16A-Trimer-4-1BBL融合基因如SEQ ID NO:9所示。
对原始scfv-CD16A-Trimer-4-1BBL融合基因序列进行调整改造，进行基于毕赤酵母密码子优化及在两端添加Xhol和NotⅠ两个酶切位点，优化调整后的基因序列如SEQ ID NO:10所示；之后将该基因序列进行全基因合成，获得scfv-CD16A-Trimer-4-1BBL融合蛋白基因序列。
2、构建scfv-CD16A-Trimer-4-1BBL融合蛋白的毕赤酵母表达载体
将上步获得的scfv-CD16A-Trimer-4-1BBL融合蛋白基因序列，与毕赤酵母表达质粒PGAPzα-rDNA-NTS皆使用限制性内切酶Xhol和NotⅠ进行双酶切。酶切scfv-CD16A-Trimer-4-1BBL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T4DNA连接酶连接。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-scfv-CD16A-Trimer-4-1BBL(蛋白表达载体如图1所示)，将阳性克隆对应的菌液用质粒小量抽提试剂盒进行抽提，并送通用生物和金唯智测序。
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NK细胞激动剂scfv-CD16A-Trimer-4-1BBL的融合蛋白的表达与纯化
1、融合蛋白表达
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-scfv-CD16A-Trimer-4-1BBL。将上步制备的表达质粒PGAPzα-scfv-CD16A-Trimer-4-1BBL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)300μL 70％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2mL BMGY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His tag抗体进行Dot blot鉴定初筛选(结果如图2所示)，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(e1、c7、c11、d1、f13)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-his tag抗体进行Western Blot鉴定分析，筛选出高表达菌株，将对应菌株的剩余1mL菌液冻存保种；此高表达菌株(f13-3)为能表达scfv-CD16A-Trimer-4-1BBL复合体蛋白的毕赤酵母菌株。
2、融合蛋白发酵、纯化与鉴定
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 6.0，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：70ml/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。
蛋白捕获：使用亲和色谱柱镍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： 使用含有10mM,20mM,30mM,40mM的咪唑的PBS溶液梯度冲洗色谱柱，每个浓度冲洗1～1.5个柱体积。6)洗脱目的蛋白，洗脱液为含有浓度为200mM咪唑的PBS溶液，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。随后，将该样品浓缩后按相同方式过Superdex75分子筛，获得scfv-CD16A-Trimer-4-1BBL重组蛋白。
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。
图2显示了经过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，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。
图3显示了发酵过程各参数监测，整个发酵过程持续了50个小时，发酵过程稳定易重复，图3中A图显示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(图3中B图分别显示了发酵20h、28h、35h、43h、50h发酵上清液中scfv-CD16A-Trimer-4-1BBL重组蛋白的量)；
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4中A图和C图)，且scfv-CD16A-Trimer-4-1BBL蛋白分子量约95kDa左右，与预期相符；
经过动态光散射鉴定，scfv-CD16A-Trimer-4-1BBL分子直径约10nm左右，分子间均一性较好(图4中B图)。
实施例3、双特异性NK细胞激动剂scfv-CD16A-Trimer-4-1BBL的融合蛋白体外诱导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个/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scfv-CD16A-Trimer-4-1BBL融合蛋白。培养24小时后，流式细胞仪检测NK细胞表型变化。
结果如图5所示，相比于对照组PBS，添加scfv-CD16A-Trimer-4-1BBL融合蛋白的实验组中，NK细胞表面主要活化性分子如CD69、NKp30及杀伤性分子4-1BB、TRAIL、Granzyme B、趋化性分子CX3CR1表达显著上调，据此判断NK细胞被scfv-CD16A-Trimer-4-1BBL融合蛋白有效活化。
实施例4、双特异性NK细胞激动剂scfv-CD16A-Trimer-4-1BBL的融合蛋白体外诱导NK细胞扩增及扩增效果评价
1、融合蛋白扩增NK细胞的纯度及数量检测：
1)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。
2)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scfv-CD16A-Trimer-4-1BBL的KBM581培养基加入1％的IL2(1000IU/ml)和5％对应的PBMC自体血浆。
3)第3天，补充相应培养体积1％的IL2因子。
4)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scfv-CD16A-Trimer-4-1BBL的KBM581培养基并添加补液体积1％的IL2(1000IU/ml)因子。
5)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(CD3/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scfv-CD16A-Trimer-4-1BBL的基础培养基，并添加1％的IL2和2％的自体血浆。
6)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KBM581培养基并添加补液体积1％的IL2因子。
7)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。
8)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)。
9)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(CD3/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。
10)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)。
11)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(CD3/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。
12)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。
13)第17天，取样进行计数，对其进行染色标记(CD3/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天的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。
2、融合蛋白扩增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％CO 2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)。
表1、细胞表面分子检测样本管及实验操作表
Figure PCTCN2022102876-appb-000013
Figure PCTCN2022102876-appb-000014
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1×104个细胞。
3、融合蛋白扩增NK细胞的杀伤活性检测
靶细胞(K562)悬液制备
1)细胞计数：培养的K562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；
2)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；
3)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；
4)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；
5)计数：细胞重悬，计数，调整细胞密度为2×10 5/mL；
6)铺板：在96孔圆底板中每孔中加入100μlK562悬液，使每孔最终细胞数为20000个/孔；
NK细胞的准备与加入
7)配制成合适的NK细胞密度；
8)取出E-Plate 16，置于超净台中；
9)将100μl的NK细胞悬液以不同的CD56+：K562效靶比(1:1、2:1、4:1、8:1)加入培养板内，此外还设置三组对照：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)；
10)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。
11)孵育结束后，加入5μl PI，混匀，避光孵育5min，上机检测。
12)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％)
4、结果分析
如图6的A～C图所示，使用scfv-CD16A-Trimer-4-1BBL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60％左右；
如图6中D图所示，对扩增后的细胞进行亚群分析，可知经scfv-CD16A-Trimer-4-1BBL处理扩增后的PBMC细胞，其中NK细胞比例可达55％，NKT比例约在20％左右，总的CD56阳性细胞约在75％左右，其余细胞亚群在扩增后的PBMC细胞中占比较低；
如图6中E图所示，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，这些结果证明scfv-CD16A-Trimer-4-1BBL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公开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公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (25)

  1. 一种融合蛋白，包括CD16A单链抗体和4-1BBL胞外区，其中，所述CD16A单链抗体和4-1BBL胞外区连接，所述4-1BBL胞外区包括至少1-3个4-1BBL胞外结构域。
  2. 根据权利要求1所述的融合蛋白，其中，所述4-1BBL胞外区包括3个4-1BBL胞外结构域，3个所述4-1BBL胞外结构域顺次连接。
  3. 根据权利要求1或2所述的融合蛋白，其中，所述CD16A单链抗体和4-1BBL胞外区通过柔性接头相连；
    任选地，3个所述4-1BBL胞外结构域之间通过柔性接头相连。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其中，所述融合蛋白是由非哺乳细胞表达体系表达的；
    任选地，所述非哺乳细胞表达体系包括原核表达体系和低级真核表达体系中的至少之一；
    任选地，所述非哺乳细胞表达体系包括大肠杆菌表达体系、酵母表达体系中的至少之一；
    任选地，所述酵母表达体系包括毕赤酵母表达体系。
  5. 根据权利要求1-4中任一项所述的融合蛋白，其中，所述CD16A单链抗体包括V H区和V L区，所述V H区的氨基酸序列如SEQ ID NO:1所示，所述V L区的氨基酸序列如SEQ ID NO:2所示；
    任选地，所述V H区和V L区之间通过第一接头连接；
    任选地，所述第一接头为柔性接头；
    任选地，所述第一接头的长度为10～20个氨基酸；
    任选地，所述第一接头的氨基酸序列如SEQ ID NO:3所示。
  6. 根据权利要求5所述的融合蛋白，其中，所述V H区或V L区的一端与所述4-1BBL胞外区通过第二接头相连；
    任选地，所述第二接头为柔性接头；
    任选地，所述第二接头的长度为10～20个氨基酸；
    任选地，所述第二接头的氨基酸序列如SEQ ID NO:4所示。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中，所述4-1BBL胞外结构域的氨基酸序列如SEQ ID NO:5所示。
  8. 根据权利要求1-7中任一项所述的融合蛋白，其中，3个所述4-1BBL胞外结构域之间两两分别通过第三接头和第四接头连接，所述第三接头和第四接头之间相同或不同；
    任选地，所述第三接头和第四接头均为柔性接头；
    任选地，所述第三接头和第四接头的长度为15～25个氨基酸；
    任选地，所述第三接头的氨基酸序列如SEQ ID NO:6所示；
    任选地，所述第四接头的氨基酸序列如SEQ ID NO:7所示。
  9. 根据权利要求1-8中任一项所述的融合蛋白，其中，所述融合蛋白的氨基酸序列如SEQ ID NO:8所示。
  10. 一种核酸，所述核酸编码权利要求1-9中任一项所述的融合蛋白。
  11. 根据权利要求10所述的核酸，其中，所述核酸为经过酵母表达体系密码子优化的核酸；
    任选地，所述核酸的核苷酸序列如SEQ ID NO:10所示。
  12. 一种表达载体，包含权利要求10或11所述的核酸。
  13. 一种重组细胞，携带权利要求10或11所述的核酸、权利要求12所述的表达载体或表达权利要求1-9中任一项所述的融合蛋白。
  14. 权利要求1-9中任一项所述的融合蛋白、权利要求10或11所述的核酸、权利要求12所述的表达载体、权利要求13所述的重组细胞在制备药物中的用途，所述药物用于治疗或预防肿瘤或炎性疾病。
  15. 根据权利要求14所述的用途，其中，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病；
    任选地，所述炎性疾病包括细菌或病毒感染；
    任选地，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一；
    任选地，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
  16. 权利要求1-9中任一项所述的融合蛋白、权利要求10或11所述的核酸、权利要求12所述的表达载体、权利要求13所述的重组细胞在体外培养扩增NK细胞中的用途。
  17. 根据权利要求16所述的用途，其中，所述NK细胞来源于外周血或诱导干细胞获得；
    任选地，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。
  18. 一种药物组合物，包含权利要求1-9中任一项所述的融合蛋白、权利要求10或11所述的核酸、权利要求12所述的表达载体或权利要求13所述的重组细胞。
  19. 根据权利要求18所述的药物组合物，其中，所述药物组合物用于治疗或预防肿瘤或炎性疾病；
    任选地，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病；
    任选地，所述炎性疾病包括细菌或病毒感染；
    任选地，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一；
    任选地，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
  20. 一种试剂盒，包括：权利要求1-9中任一项所述的融合蛋白。
  21. 根据权利要求20所述的试剂盒，其中，所述试剂盒用于检测CD16A和/或4-1BB。
  22. 一种治疗或预防肿瘤或炎性疾病的方法，包括向患有或疑似患有肿瘤或炎性疾病的受试者施用以下中的至少之一：
    权利要求1-9中任一项所述的融合蛋白；
    权利要求10或11所述的核酸；
    权利要求12所述的表达载体；
    权利要求13所述的重组细胞；
    权利要求18或19所述的药物组合物。
  23. 根据权利要求22所述的方法，其中，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病；
    任选地，所述炎性疾病包括细菌或病毒感染；
    任选地，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一；
    任选地，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
  24. 权利要求1-9中任一项所述的融合蛋白、权利要求10或11所述的核酸、权利要求12所述的表达载体、权利要求13所述的重组细胞、权利要求18或19所述的药物组合物在治疗或预防肿瘤或炎性疾病中的用途。
  25. 根据权利要求24所述的用途，其中，所述肿瘤包括下列中的至少之一：卵巢癌、肺癌、直肠癌、乳腺癌、白血病；
    任选地，所述炎性疾病包括细菌或病毒感染；
    任选地，所述细菌感染包括肺炎克雷伯、李斯特菌、金黄色葡萄球菌感染中的至少之一；
    任选地，所述病毒感染包括HPV、HBV、流感病毒、冠状病毒感染中的至少之一。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110461357A (zh) * 2017-02-28 2019-11-15 阿菲姆德股份有限公司 抗cd16a抗体与细胞因子的组合
WO2020028572A2 (en) * 2018-08-01 2020-02-06 Unum Therapeutics Inc. ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF
CN112119097A (zh) * 2018-04-13 2020-12-22 艾菲默德有限责任公司 自然杀伤细胞接合抗体融合构建体
AU2019328575A1 (en) * 2018-08-30 2021-02-25 HCW Biologics, Inc. Single-chain and multi-chain chimeric polypeptides and uses thereof
CN114349869A (zh) * 2021-12-20 2022-04-15 上海恩凯细胞技术有限公司 一种双特异性的nk细胞激动剂及制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW202115124A (zh) * 2019-06-26 2021-04-16 瑞士商赫孚孟拉羅股份公司 結合至cea之新穎抗原結合分子

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110461357A (zh) * 2017-02-28 2019-11-15 阿菲姆德股份有限公司 抗cd16a抗体与细胞因子的组合
CN112119097A (zh) * 2018-04-13 2020-12-22 艾菲默德有限责任公司 自然杀伤细胞接合抗体融合构建体
WO2020028572A2 (en) * 2018-08-01 2020-02-06 Unum Therapeutics Inc. ANTIBODY-COUPLED T CELL RECEPTORS (ACTRs) IN COMBINATION WITH TRANS CO-STIMULATORY MOLECULES AND THERAPEUTIC USES THEREOF
AU2019328575A1 (en) * 2018-08-30 2021-02-25 HCW Biologics, Inc. Single-chain and multi-chain chimeric polypeptides and uses thereof
CN114349869A (zh) * 2021-12-20 2022-04-15 上海恩凯细胞技术有限公司 一种双特异性的nk细胞激动剂及制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TANG TIAN, RAO WEI: "Progress of Natural Killer Cells and Their Application in Immunotherapy: from the Bench to Clinical", CHINA CANCER, vol. 26, no. 1, 5 January 2017 (2017-01-05), pages 44 - 52, XP093075725, ISSN: 1004-0242, DOI: 10.11735/j.issn.1004-0242.2017.01.A007 *
ZHU HUANG, BLUM ROBERT H., BJORDAHL RYAN, GAIDAROVA SVETLANA, ROGERS PAUL, LEE TOM TONG, ABUJAROUR RAMZEY, BONELLO GREGORY B., WU : "Pluripotent stem cell–derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity", BLOOD, vol. 135, no. 6, 6 February 2020 (2020-02-06), US , pages 399 - 410, XP055967123, ISSN: 0006-4971, DOI: 10.1182/blood.2019000621 *

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