WO2023109514A1 - 膜整合型融合蛋白、含有其的细胞及用途 - Google Patents

膜整合型融合蛋白、含有其的细胞及用途 Download PDF

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WO2023109514A1
WO2023109514A1 PCT/CN2022/135389 CN2022135389W WO2023109514A1 WO 2023109514 A1 WO2023109514 A1 WO 2023109514A1 CN 2022135389 W CN2022135389 W CN 2022135389W WO 2023109514 A1 WO2023109514 A1 WO 2023109514A1
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cells
fusion protein
cell
sequence
nucleic acid
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French (fr)
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王海鹰
刘芳
胡红明
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上海恒润达生生物科技股份有限公司
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    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Definitions

  • the invention relates to the field of immune cell culture and stimulation, in particular to membrane-integrated fusion proteins, cells containing the fusion proteins and applications thereof.
  • NK cells Natural killer cells
  • T cells Unlike T cells, NK cells have natural killer cytotoxicity, and can initiate the elimination of foreign components such as tumor cells and bacteria by recognizing non-self without activation or antigen presentation.
  • NK Natural killer cells
  • NK cells account for a low proportion of peripheral blood mononuclear cells (1-32.6%), and researchers have developed a variety of methods to increase the number of NK cells in vitro expansion and anti-tumor function.
  • RetroNectin is a fragment of recombinant human fibronectin (FN), including three functional regions: cell binding domain, heparin binding domain II and CS1 site. It can not only enhance the infection efficiency of the virus, but also stimulate lymphocytes to multiply by thousands of times. But so far no one has reported the effect of RetroNectin or its fragments on NK proliferation.
  • FN human fibronectin
  • the invention discloses a membrane-integrated fusion protein and a method for integrating cytokines on the cell surface by using the fusion protein to obtain feeder layer cells that can stimulate large-scale expansion of immune cells in vitro.
  • the invention also discloses a method for using the cells to expand immune cells in vitro and an application in the field of cell therapy.
  • the first aspect of the present invention provides a membrane-integrated fusion protein, comprising an integration domain and a functional polypeptide fused therewith, and the integration domain comprises a heparin-binding domain II and a CS1-binding domain of FN.
  • the first aspect of the present invention provides a fusion protein, comprising an integration domain and a functional polypeptide fused therewith, and the integration domain comprises the heparin binding domain II and CS1 binding domain of FN.
  • the integration domain is located in the N-terminal direction of the functional polypeptide.
  • the integration domain integrates the fusion protein on the cell membrane.
  • the integration domain further comprises the cell binding domain of FN.
  • the fusion protein further comprises a signal peptide.
  • the signal peptide is located at the N-terminal of the fusion protein.
  • the fusion protein further comprises a transmembrane region.
  • the fusion protein comprises a signal peptide, an integration domain and a functional polypeptide from the N-terminus to the C-terminus.
  • the fusion protein comprises a signal peptide, an integration domain, a functional polypeptide and a transmembrane region from the N-terminus to the C-terminus.
  • linker between the integration domain and the functional polypeptide, preferably having the sequence shown in SEQ ID NO:3.
  • the signal peptide is a CD8 signal peptide.
  • the signal peptide is a CD8 signal peptide.
  • the integration domain has the sequence set forth in SEQ ID NO: 2 or a variant having at least 90% sequence identity thereto.
  • the functional polypeptide is a cytokine.
  • cytokines include one or more selected from the group consisting of interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF), and tumor growth factor (TGF)
  • TNF tumor necrosis factor
  • IFN interferon
  • CSF colony stimulating factor
  • TGF tumor growth factor
  • the interleukins include, but are not limited to, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL- 18 and IL-21;
  • the tumor necrosis factor includes but not limited to TNF- ⁇ and TNF- ⁇ ;
  • the interferon includes but not limited to IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ;
  • the colony-stimulating factor includes but Not limited to M-CSF, G-CSF and GM-CSF; said tumor growth factors include but not limited to TGF- ⁇ , TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3.
  • the cytokine is IL-21.
  • IL-21 Preferably having the sequence shown in SEQ ID NO: 4 or a variant having at least 90% sequence identity thereto.
  • transmembrane regions include, but are not limited to, CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R ⁇ , IL-2R ⁇ , IL-4R ⁇ , IL-7R ⁇ , IL-10R, IL-12R, IL-15R, IL-21R, The transmembrane region of either CD27 and CD40.
  • the transmembrane region is a CD8 transmembrane region.
  • the transmembrane region is connected to its adjacent structure (eg, cytokine) by a hinge region or linker.
  • a hinge region or linker is a CD8 hinge region, preferably having the sequence set forth in SEQ ID NO: 5 or a variant having at least 90% sequence identity thereto.
  • the invention also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:
  • the fragment is a primer.
  • the coding sequence is DNA or RNA.
  • the invention also provides a nucleic acid construct comprising a nucleic acid molecule according to any embodiment of the invention.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector, or an integrating vector.
  • the present invention also provides a host cell, the host cell:
  • the host cell also expresses or secretes a costimulatory molecule or a protein comprising a costimulatory molecule.
  • the co-stimulatory molecule comprises one or more selected from the group consisting of: CD28, CD134/OX40, CD86, CD137/4-1BB, 4-1BBL.
  • the cells express or secrete CD86 and 41BBL.
  • the host cells are feeder cells for immune cells.
  • Feeder cells for NK cells are preferred.
  • the host cell is K562.
  • the present invention also provides a method for culturing or stimulating immune cells, comprising the step of: incubating immune cells or a sample containing immune cells in the presence of the fusion protein and/or host cells described in any embodiment herein, the fusion protein
  • the functional polypeptides in are cytokines.
  • the ratio of host cells to immune cells is 0.5:1-10:1.
  • the incubation is for at least 1 week or at least 10 days.
  • the sample is a peripheral blood product or PBMC.
  • the immune cells are NK cells.
  • the method further comprises the step of sorting NK cells, the sorting marker is CD3-CD56+.
  • the NK cells are CD3-CD56+ NK cells.
  • the present invention also provides an immune cell culture, which contains the fusion protein described in any embodiment herein and/or host cells and a culture medium suitable for immune cells, and the functional polypeptide in the fusion protein is a cytokine.
  • the immune cells are NK cells.
  • the medium is SCGM supplemented with FBS and IL-2.
  • the present invention also provides a pharmaceutical composition, comprising the immune cells obtained by the method of cultivating or stimulating immune cells of the present invention and pharmaceutically acceptable auxiliary materials.
  • the immune cells are NK cells.
  • the present invention also provides a kit for culturing or stimulating immune cells, comprising the fusion protein and/or host cells described in any embodiment herein, and the functional polypeptide in the fusion protein is a cytokine.
  • the kit further includes reagents required for culturing immune cells, including but not limited to culture medium, cytokines, activating antibodies, and the like.
  • the kit further includes reagents for obtaining immune cells in the sample, including but not limited to culture medium, buffer, immune cell screening antibodies and the like.
  • the sample is a peripheral blood product or PBMC.
  • the immune cells are NK cells.
  • the present invention also provides the application of reagents in the preparation of immune cells, the reagents containing:
  • nucleic acid molecule nucleic acid construct or host cell of any embodiment herein.
  • the immune cells are NK cells.
  • Another aspect of the present invention provides the application of a reagent in the preparation of anti-tumor drugs for prevention and/or treatment, the reagent comprising: (1) the fusion protein described in any embodiment herein, or (2) the fusion protein described in any embodiment herein
  • the reagent comprising: (1) the fusion protein described in any embodiment herein, or (2) the fusion protein described in any embodiment herein
  • the tumors include solid tumors and hematological tumors.
  • FN21 membrane-integrated cytokines
  • heparin-binding domain II and CS-1 domain of FN1 were combined with cytokines to form integrated cytokines.
  • Figure 1 Schematic diagram of IL21 sequence combination in different feeder cells
  • Figure 2 Flow cytometric detection of the positive rate of co-stimulatory factors on the surface of feeder cells. a. Flow cytometry diagram of positive rate of CD86 on the surface of feeder layer cells; b. Flow cytometry diagram of positive rate of 41BBL on the surface of feeder layer cells.
  • Figure 3 Flow cytometric detection of IL-21 positive rate on the surface of FN21 cells.
  • Figure 4 Flow cytometric detection of IL-21 positive rate on the surface of FN-mbIL21 cells.
  • Figure 5 Flow cytometric detection of IL-21 positive rate on the surface of GFP-mbIL21 cells.
  • Figure 6 Flow cytometric detection of IL-21 positive rate on the surface of CS1-IL21 cells.
  • Figure 7 Flow cytometric detection of IL-21 positive rate on the surface of HBD-IL21 cells.
  • Figure 8 Comparison of the effects of FN21 and other feeder cells and different culture methods on NK amplification folds
  • Figure 10 NK cell ratio. a, the ratio of CD3-CD56+NK stimulated by FN21, FN-mbIL-21 and GFP-mbIL-21; b, the ratio of CD16+CD56+NK stimulated by FN21, FN-mbIL-21 and GFP-mbIL-21.
  • Figure 11 NK cell ratio.
  • a The ratio of CD56+NKp30+NK stimulated by FN21, FN-mbIL-21 and GFP-mbIL-21;
  • b the ratio of CD56+NKp44+NK stimulated by FN21, FN-mbIL-21 and GFP-mbIL-21;
  • c FN21, FN-mbIL-21 and GFP-mbIL-21 stimulated to produce CD56+NKp46+NK ratio;
  • d FN21, FN-mbIL-21 and GFP-mbIL-21 stimulated to produce CD56+NKG2D+NK ratio;
  • e FN21, FN-mbIL-21 and GFP-mbIL-21 stimulated to produce CD56+NKG2A+NK ratio;
  • f FN21, FN-mbIL-21 and GFP-mbIL-21 stimulated to produce CD56+KIR+NK ratio;
  • g FN21, FN- mbIL-21 and GFP-mb
  • Figure 12 The proportion of NK stimulated by FN21, FN-mbIL21 and GFP-mbIL21 meeting the target cell K562 to produce CD107a.
  • Figure 13 The proportion of NK stimulated by FN21, FN-mbIL21 and GFP-mbIL21 meeting target cell K562 to produce cytokine IFN- ⁇ .
  • Figure 14 NK stimulated by FN21, FN-mbIL21 and GFP-mbIL21 met the target cell K562-luc-GFP cytotoxicity test.
  • the invention discloses a feeding layer cell capable of stimulating large-scale expansion of immune cells in vitro, and a fusion protein containing FN fragments and cytokines is integrated on the membrane.
  • the feeder cells in this paper can expand NK cells more than 200,000 times in vitro in 24 days, providing a very efficient tool for NK cells as a general-purpose cell therapy product.
  • the NK cells prepared by the method of the invention have higher tumor cell killing ability and cytokine secretion.
  • a fusion protein is integrated on the membrane of the feeder cells of the present invention, and the fusion protein includes an integration domain and cytokines fused therewith.
  • membrane “integration” is distinguished from membrane “association”.
  • Membrane association generally refers to the process by which molecules are bound between cell membrane phospholipid bimolecules through covalent attachment to transmembrane proteins, transmembrane domains, or membrane proteins.
  • Membrane integration is fixed to the cell surface through integration domain and integrin for ligand receptor integration. The inventors found that in order to achieve effective membrane integration, the integration domain needs the receptor of the FN heparin binding domain II to cooperate with the VLA-4 integration receptor of the CS-1 functional domain to exert the maximum integration advantage.
  • the present invention also provides a membrane-integrated fusion protein, comprising an integration domain and a functional polypeptide fused therewith, and the integration domain comprises the heparin-binding domain II and CS1-binding domain of FN.
  • the functional polypeptide can be any polypeptide molecule that needs to be immobilized on the membrane, such as cytokines, tumor-related proteins, and viral proteins.
  • the inventors used the above-mentioned membrane-integrated fusion protein to integrate the cytokines on the membrane of the feeder cells.
  • the inventors found that integrated membrane surface expressed cytokines stimulate immune cells more strongly than membrane bound cytokines and free cytokines.
  • cytokines are integrated on the cell membrane surface by coupling with integration domains.
  • the integration domain comprises the heparin binding domain II and/or the CS1 binding domain of FN (fibronectin), optionally further comprising the cell binding domain of FN.
  • the integration domain is located in the N-terminal direction of the cytokine.
  • the integration domain has the sequence set forth in SEQ ID NO: 2 or a variant having at least 90% sequence identity thereto. There is a spacer (linker or hinge) between the integration domain and the cytokine.
  • the length of the spacer is 10-70aa, preferably 12-66aa, such as 12aa, 54aa, 55aa or 66aa or a range between any two numbers above.
  • the spacer may comprise a linker, such as a polypeptide sequence consisting of G and/or S, such as a polypeptide repeated n times (eg, 3 times) of GS, GSG, GSSG, SGS, GGGGS.
  • exemplary linkers have the sequence set forth in SEQ ID NO: 3, or a sequence having at least 90% sequence identity thereto.
  • cytokines described herein can be used conventionally in the art to stimulate or activate immune cells, including but not limited to: interleukins, tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF), and tumor growth factors (TGF).
  • TNF tumor necrosis factor
  • IFN interferon
  • CSF colony stimulating factor
  • TGF tumor growth factors
  • the interleukins include but are not limited to IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18 and IL-21;
  • the tumor Necrosis factors include but not limited to TNF- ⁇ and TNF- ⁇ ;
  • the interferons include but not limited to IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ;
  • the colony-stimulating factors include but not limited to M-CSF, G-CSF and GM-CSF; said tumor growth factors include but are not limited to TGF- ⁇ , TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3.
  • the cytokine is IL-21, preferably having the sequence set forth in SEQ ID NO: 4 or a variant having at least 90% sequence identity thereto.
  • the fusion protein may also include a signal peptide.
  • the signal peptide is located at the N-terminus of the fusion protein.
  • the signal peptide can be any polypeptide known in the art for directing the movement of proteins.
  • the signal peptide is a CD8 signal peptide, preferably having the sequence set forth in SEQ ID NO: 1 or a variant having at least 90% sequence identity thereto.
  • the fusion protein herein is an integrative fusion protein
  • the fusion protein may still contain a transmembrane region.
  • Transmembrane regions suitable for fusion proteins herein include, but are not limited to: CD28, CD8, CD134 (OX40), CD137 (4-1BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R ⁇ , IL-2R ⁇ , IL-4R ⁇ , IL-7R ⁇ , IL-10R, IL-12R, IL-15R, IL-21R, CD27 and The transmembrane region of any of CD40.
  • the transmembrane region is a CD8 transmembrane region.
  • the transmembrane region is connected to its adjacent structures (such as cytokines) by hinge regions or linkers.
  • Hinge regions include, but are not limited to: CD4 extracellular hinge region, CD8 extracellular hinge region, CD28 extracellular hinge region, IgG1 Fc hinge region and IgG4 Fc hinge region.
  • An exemplary hinge region is the CD8 hinge region having the sequence set forth in SEQ ID NO: 5, or a variant having at least 90% sequence identity thereto.
  • the fusion protein may also contain an intracellular signaling region if extracellular signaling is desired.
  • the intracellular signal region can be the intracellular region of transmembrane proteins that can respond to extracellular signals to cause intracellular effects in any signaling pathway, including but not limited to: CD8, CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS , DAP10, CD3 ⁇ or the intracellular signaling region of Fc310, preferably the intracellular signaling region of CD8.
  • the fusion protein comprises signal peptide, integration domain and cytokine from N-terminus to C-terminus; or comprises signal peptide, integration domain, cytokine and transmembrane region from N-terminus to C-terminus; or from N-terminal to C-terminal contains signal peptide, integration domain, cytokines, transmembrane region and intracellular signal region.
  • the fusion protein sequentially comprises from N-terminus to C-terminus: CD8 signal peptide, heparin binding domain II and CS1 binding domain of FN, linker and IL-21; or CD8 signal peptide, heparin binding domain of FN Domain II and CS1 binding domain, linker, IL-21, CD8 hinge region and CD8 transmembrane region.
  • the fusion protein has a sequence consisting of any one of the following sequences in series: (1) SEQ ID NO: 1, 2, 3, 4, (2) SEQ ID NO: 1, 7, 3, 4, (3) SEQ ID NO: 1, 8, 3, 4, (4) SEQ ID NO: 1, 2, 3, 4, 5, 6.
  • polypeptide herein such as signal peptide, heparin binding domain II of FN, CS1 binding domain, cytokines, hinge region, transmembrane region and intracellular signal region, etc.
  • a linker sequence known in the art is ligated, such as the linker sequence containing G and S described above.
  • the polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide.
  • Polypeptides of the invention can be the product of chemical synthesis, or produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of said polypeptides or domains.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the polypeptide.
  • Fragments, derivatives or analogs of polypeptides or domains may be (i) polypeptides or domains having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substitutions
  • the amino acid residues of which may or may not be encoded by the genetic code, or (ii) a polypeptide or domain having a substitution group in one or more amino acid residues, or (iii) a mature polypeptide or domain with another Polypeptides formed by the fusion of a compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide or domain sequence (such as a leader sequence or secretory sequence or with To purify the polypeptide sequence or protein sequence, or the fusion protein formed with the antigen IgG fragment).
  • a compound such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol
  • mutant refers to a peptide or polypeptide that has an amino acid sequence altered by insertion, deletion or substitution of one or more amino acids, but retains at least one biological activity, compared to a reference sequence.
  • the mutant described in any embodiment herein comprises at least 70%, preferably at least 80%, preferably at least 85%, preferably at least Amino acid sequences that have 90%, preferably at least 95%, preferably at least 97% sequence identity and retain the biological activity of the reference sequence (eg as chimeric antigen receptors, surface cytokine expressed proteins, signal transducing receptors). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • Mutants also include amino acid sequences having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of a reference sequence while retaining the biological activity of the reference sequence.
  • the plurality of mutations usually refers to within 1-10, such as 1-8, 1-5 or 1-3.
  • Substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains, which families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains, chain amino acids (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acid), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), with Amino acids with ⁇ -branched side chains (eg threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).
  • amino acids with basic side chains e.g., lysine,
  • Truncated variants of polypeptides or domains can also be used in the present invention as long as the variants retain substantially the same biological function or activity as the polypeptide or domain, for example FN heparin binding domain II that retains the membrane integration function of the integration domain and CS1 binding domain truncated variants.
  • the polypeptides described herein may be modified polypeptides.
  • Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • Nucleic acid molecules encoding such polypeptides or domains are also within the scope of this document.
  • a nucleic acid molecule can be in the form of DNA or RNA.
  • the nucleic acid molecules described herein include sequences altered by codon optimization, as long as the amino acid sequence encoded by the nucleic acid molecule remains unchanged. Codon-optimized sequences may exhibit better expressivity for a particular species. Methods for codon-optimizing the sequence of nucleic acid molecules are well known in the art.
  • the coding region sequence encoding the mature polypeptide may be of degenerate variants. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes the amino acid sequence shown in SEQ ID NO: 1-8 or a domain fragment thereof, but differs in base sequence.
  • a nucleic acid molecule encoding a mature polypeptide includes: the coding sequence encoding the mature polypeptide alone; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.
  • the term "nucleic acid molecule encoding a polypeptide" may include a nucleic acid molecule encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • a nucleic acid molecule of the invention may be a coding sequence for a polypeptide or domain, or an expression cassette for a polypeptide or domain.
  • the coding sequence refers to the part of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (such as signal peptide, integration domain, hinge region, transmembrane region, intracellular signal region or cytokine protein).
  • the boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotes) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA.
  • a coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • the expression cassette refers to the complete elements required to express the gene of interest, including the promoter, gene coding sequence and PolyA tailing signal sequence.
  • the coding sequence or expression cassette is integrated into the genome of the cell.
  • the cell described herein has stably integrated into its genome an expression cassette comprising an encoding polypeptide described herein.
  • the present invention also relates to variants of the above-mentioned nucleic acid molecules, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, 85%, 90%, 95% identity between the two sequences.
  • the invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • nucleic acid fragments are at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR) to identify and/or isolate polynucleotides encoding desired polypeptides or domains.
  • “Hybridization” as used herein mainly refers to the pairing of nucleic acid sequences under stringent conditions. Exemplary stringent conditions are 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS solution, hybridization at 65° C. and membrane washing.
  • the nucleic acid molecule is a nucleic acid construct comprising the coding sequences for the fusion proteins described herein, and one or more regulatory sequences operably linked to these sequences.
  • the regulatory sequence may be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from an extracellular sequence that encodes either homologous or heterologous to the host cell. Or intracellular polypeptide gene acquisition.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. A terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used herein.
  • suitable vectors will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites and one or more selectable markers.
  • the retroviral vector typically contains an origin of replication, a 3'LTR, a 5'LTR, a coding sequence for a fusion protein described herein, and optionally a selectable marker.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequence, elongated growth factor-1 (EF-1), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rouss sarcoma virus promoter, and human gene promoters such as But not limited to actin promoter, myosin promoter, heme promoter, creatine kinase promoter and the like.
  • CMV immediate early cytomegalovirus
  • EF-1 elongated growth factor-1
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV40 human immunodeficiency virus
  • LTR human immunodeficiency virus
  • MoMuLV promoter avian leukemia virus
  • Selectable markers include either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells infected with the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.
  • the nucleic acid molecules described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
  • the nucleic acid molecules described herein can also be directly synthesized.
  • nucleic acid molecules can be introduced into host cells by conventional methods, including microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation wait.
  • host cells contain the nucleic acid molecules described herein.
  • Host cells include not only feeder cells that are ultimately used as co-cultured immune cells, but also various cells used in the process of producing feeder cells, such as E. coli cells, for providing the coding sequence of the protein of the present invention or providing the present invention. the carrier described.
  • this document provides a feeder cell for NK cells stably expressing the fusion protein described herein, such as K562 cells, 721.221, PC3 and other HLA-deleted cell lines.
  • K562 etc. are irradiated, for example, 100Gy gamma rays, so as to lose the ability to divide.
  • the functional polypeptide in the fusion protein contained in the host cell is the cytokine IL-21, which is used to provide the host cell with the ability to stimulate NK cells.
  • the host cell fusion protein can contain other corresponding cytokines, which is within the knowledge of those skilled in the art.
  • the host cell also expresses (eg, membrane expresses) or secretes a co-stimulatory molecule or a protein comprising a co-stimulatory molecule.
  • costimulatory molecules include one or more selected from the following: CD28, CD134/OX40, CD86, CD137/4-1BB, 4-1BBL.
  • the host cell also expresses or secretes CD86 and 41BBL.
  • nucleic acid molecules described herein can be prepared by conventional methods in the art.
  • lentiviruses that include the expression cassettes described herein and are capable of integrating the expression cassettes described herein into the genome of the host cell.
  • the lentiviruses described herein can be prepared by methods well known in the art. For example, firstly, a lentiviral vector containing the expression cassette described herein is prepared, then the virus is packaged in a suitable host cell, and the desired lentivirus is obtained by isolation and purification.
  • Reagents for lentiviral packaging are well known in the art, for example, conventional lentiviral vector systems include pCCL and plasmids expressing RRE-SIV, REV, and VSVG.
  • a NK cell culture comprising a host cell as described herein and a suitable medium.
  • the medium can be a medium conventionally used in the art for culturing NK cells, such as SCGM.
  • SCGM a medium conventionally used in the art for culturing NK cells
  • the medium is SCGM supplemented with FBS (eg, at least 2%) and IL-2 (eg, at least 100 IU/mL).
  • the present invention also includes a method for culturing or stimulating NK cells, comprising the step of: incubating NK cells or a sample containing NK cells in the presence of the fusion protein and/or host cells described herein.
  • a method for culturing or stimulating NK cells comprising the step of: incubating NK cells or a sample containing NK cells in the presence of the fusion protein and/or host cells described herein.
  • the ratio of host cells to NK cells is 0.5:1-10:1, preferably 1:1-5:1; the culture medium is a serum-containing medium (such as the aforementioned SCGM).
  • the incubation lasts for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 10 days, 15 days, 21 days, 24 days.
  • Such samples include, but are not limited to, blood or bodily fluids derived from the immune system, such as peripheral blood, umbilical cord blood, or PBMCs.
  • Methods for extracting NK cells or PMBCs from samples are well known in the art.
  • the method also includes the step of sorting NK cells, and the sorting marker is CD3-CD56+. Sorting NK cells can use conventional techniques in the field such as flow cytometry.
  • This article also provides a pharmaceutical composition, which contains NK cells obtained by the method for culturing or stimulating NK cells of the present invention and pharmaceutically acceptable auxiliary materials.
  • pharmaceutically acceptable excipients refer to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: pH regulators, surfactants, Adjuvant, ionic strength enhancer. More specifically, the suitable pharmaceutically acceptable adjuvant can be the adjuvant commonly used in NK cell administration in this field.
  • a therapeutically effective amount of NK cells is included in the pharmaceutical composition.
  • a therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of a disease or condition in a subject.
  • the therapeutically effective dose can be determined according to factors such as the patient's age, sex, disease and its severity, and other physical conditions of the patient.
  • a subject or a patient generally refers to a mammal, especially a human.
  • diseases suitable for treatment with the NK cells and pharmaceutical compositions described herein include solid tumors and blood tumors, such as adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer , gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, as well as leukemias and lymphomas, such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myeloid leukemia leukemia and chronic myeloid leukemia.
  • B-cell lymphoma such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, acute myeloid leukemia leukemia and chronic myeloid leukemia.
  • the present invention also provides a kit comprising the fusion protein, nucleic acid molecule and/or host cell described herein for culturing or stimulating NK cells.
  • the kit may also include: various reagents suitable for transfecting the nucleic acid molecule into host cells (such as K562), medium suitable for culturing host cells, various reagents suitable for incubating NK cells (including but not limited to medium, cytokines, activating antibodies, etc.), reagents suitable for obtaining NK cells in samples (including but not limited to medium, buffer, NK cell screening antibodies, etc.). And optional instructions to guide those skilled in the art to transfect host cells, extract and incubate NK cells.
  • host cells such as K562
  • medium suitable for culturing host cells such as medium suitable for culturing host cells
  • various reagents suitable for incubating NK cells including but not limited to medium, cytokines, activating antibodies, etc.
  • reagents suitable for obtaining NK cells in samples including but not limited to medium, buffer, NK cell screening antibodies, etc.
  • the present invention uses lentiviral infection co-stimulatory factors CD86 (NCBI CCDS ID: CCDS 3009.1), 41BBL (NCBI CCDS ID: CCDS 12169.1) on the human chronic myeloid leukemia cell K562 with the deletion of MHC class I molecules, and at the same time Construct the FN heparin-binding domain II_CS-1_IL21 fusion protein expression vector, package it into lentivirus, infect K562, make it express and integrate into the cell surface, and form a new integrated feeder cell, named FN21.
  • FN21 was irradiated with 100Gy ⁇ -rays to make it lose its ability to divide, and it was used as a feeder cell bank for future use.
  • NK can be stimulated to expand more than 200,000 times, and the purity of NK is as high as 99%, and the phenotype of NK cells stimulated by FN21 is consistent with that of the membrane-bound cytokine (mbIL21) group.
  • NK cells stimulated by FN21 can kill more than 95% of tumor cells in 4 hours when the effect-to-target ratio is 10:1; when the effect-to-target ratio is 1:1, the ratio of CD107a in cell degranulation is as high as 50%, and the secretion of cytokine IFN ⁇ is as high as 20% %above.
  • CD86 and 41BBL lentiviral vectors PCR was performed using human cDNA as a template to obtain CD86 (NCBI CCDS ID: CCDS 3009.1) with a fragment length of 990 nt, which was purified and cloned into pCCL lentiviral main plasmid; the same method was used to clone the fragment length
  • the 765nt 41BBL (NCBI CCDS ID: CCDS 12169.1) was inserted into the pCCL lentiviral expression vector.
  • FN21 plasmid cDNA sequence of CD8 leader peptide (SEQ ID NO: 1), and FN1 heparin binding region II-CS1 functional region (FN HBDII-CS1, 1812-2107aa) (SEQ ID NO: 2) was carried out Codon optimization, the formed DNA sequence is linked together by chemical synthesis using the gene linker sequence GS (SEQ ID NO: 3) and IL-21 (SEQ ID NO: 4) with the secretion signal peptide removed, and cloned into pCCL lentiviral master in the plasmid.
  • FN-mbIL21 plasmid construction cDNA of CD8 leader peptide (SEQ ID NO:1), and FN1 heparin binding region II-CS1 functional region (FN HBDII-CS1, 1812-2107aa) (SEQ ID NO:2) The sequence is codon-optimized, and the formed DNA sequence utilizes the gene linker sequence GS (SEQ ID NO: 3) and the IL-21 (SEQ ID NO: 4) and CD8 hinge region (SEQ ID NO: 5) with the secretion signal peptide removed, The CD8 transmembrane region (SEQ ID NO:6) was linked together by chemical synthesis and cloned into the pCCL lentiviral master plasmid.
  • Recombinant GFP-mbIL21 plasmid construction the CD8 leader peptide (SEQ ID NO: 1), and the GFP gene sequence were used to remove the secretory signal peptide IL-21 (SEQ ID NO :4), CD8 hinge region (SEQ ID NO: 5), and CD8 transmembrane region (SEQ ID NO: 6) were linked together by chemical synthesis and cloned into pCCL lentiviral main plasmid.
  • Recombinant CS1-IL21 plasmid construction CD8 leader peptide (SEQ ID NO: 1), and FN1 gene CS1 functional region (2083-2202aa) (SEQ ID NO: 7) using the gene linker sequence GS (SEQ ID NO: 3 ) and IL-21 (SEQ ID NO:4) with the secretion signal peptide removed were linked together by chemical synthesis, and cloned into the pCCL lentiviral main plasmid.
  • Recombinant HBD-IL21 plasmid construction the cDNA sequence of CD8 leader peptide (SEQ ID NO: 1), FN1 heparin-binding region II functional region (1812-2082aa) (SEQ ID NO: 8) was used to connect the sequence GS (SEQ ID NO: 3) was linked with IL-21 (SEQ ID NO: 4) with the secretion signal peptide removed by chemical synthesis, and cloned into the pCCL lentiviral main plasmid.
  • PEI cationic polymer
  • Cell line construction strategy first construct K562-CD86-41BBL, and then overexpress FN21, FN-mbIL-21, GFP-mbIL21 on the basis of this cell, and the feeder cells were named K562-CD86-41BBL, respectively, according to the different combinations of IL21.
  • FN21, FN-mbIL-21, GFP-mbIL21, CS1-IL21, HBD-IL21 The schematic diagram of the combined sequence of IL21 expressed in the cells is shown in FIG. 1 .
  • Infection process Adjust the cell density of K562 (Cell Bank of Chinese Academy of Sciences) to 5x105/mL, add 1ml K562 cell suspension and 1ml virus stock solution to a 24-well plate, add 1 ⁇ L polybrene to each well, centrifuge at 32°C, 2500rpm for 1.5h . The supernatant was discarded, and 1 ml of complete cell culture medium (RPMI1640+10% FBS) was added to each well. Place the culture plate in a 37°C, 5% CO2 incubator. 48h after infection, 5E5 cells were taken for the following flow cytometric detection.
  • K562 Cell Bank of Chinese Academy of Sciences
  • Feeder layer cell K562-CD86-41BBL characterization Flow cytometry results are shown in Figure 2, a and b: CD86 and 41BBL positive cells accounted for 96.2% and 93%;
  • Mononuclear cell separation is performed after checking the information of the collected umbilical cord blood or blood apheresis from healthy people.
  • each centrifuge tube was filled with sodium chloride injection to 50ml after the end, and the first washing was performed: after mixing up and down, centrifuge at 700g at room temperature for 5min.
  • the red blood cell lysis operation is required: gently discard the supernatant, and according to the counting result of the previous step, use 90-110 ⁇ l for every 1 ⁇ 108 total cells
  • the erythrocyte lysate was resuspended and mixed evenly, and placed at room temperature for 10 minutes for lysis.
  • sodium chloride injection was added to make up to 50ml, mixed at 450g at room temperature, and centrifuged for 6 minutes.
  • NK cells were isolated using the Miltenyi NK isolation kit (NK isolation kit, human, 130-092-657), and the flow pattern of the specific operation was as follows:
  • PBMC or CBMC cells in a 37° water bath for 2-3 minutes, transfer them to a centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, add an appropriate amount of buffer to resuspend, take 20 ⁇ L of cell suspension, add 20 ⁇ L of 0.1% Trypan blue was used for counting, and the living cell density and cell viability were recorded. Count the total number of cells according to the volume of the cell suspension. Add 40 ⁇ L Buffer and 10 ⁇ L NK Cell Biotin-Antibody Cocktail per 107 cells, mix well and incubate in the refrigerator (2-8°C) for 5 minutes.
  • NK cell suspension After mixing the received NK cell suspension, take 20 ⁇ L of the cell suspension, add 20 ⁇ L of 0.1% trypan blue for counting, and record the viable cell density and cell viability. Count the total number of cells according to the volume of the cell suspension.
  • Feeder layer cells K562-CD86-41BBL, K562-CD86-41BBL+20ng/mL sIL-21, K562-CD86-41BBL+Retronectin coating, FN21, FN-mbIL21 and GFP-mbIL21 stimulated two rounds of NK cell expansion for 14 days
  • the comparison of the amplification factor is shown in Figure 8,
  • the average expansion factor of the cells on D14 is only 598.6, adding rhIL-21, or adding the cells to the culture dish coated with Retronectin, the NK cell expansion factor doubles, but the statistics No significant difference on .
  • FN21, FN-mbIL21 and GFP-mbIL21 were used to stimulate NK amplification for 3 rounds for a total of 24 days.
  • the amplification curve is shown in Figure 9.
  • the average amplification factor of FN21 can reach 239994.95, which is better than that of FN-mbIL21 (125572) and GFP -mbIL21 (131121) nearly 2-fold.
  • Embodiment 4 NK cell purity detection
  • NKCD3-CD56+NK NKCD3-CD56+NK
  • CD56+CD16+NK cell detection specific operation process is as follows:
  • NK purity (CD3-CD56+) As shown in Figure 10, the ratio of CD3-CD56+NK produced by the three types of feeder layer cells is above 99%; the ratio of CD56+CD16+NK is about 80%
  • Embodiment 5 NK cell phenotype detection
  • NK cell CD56+ cells
  • NK-activated receptor NCRs NKp30/NKp44/NKp46
  • SONY SA3800 flow cytometer tumor recognition site NKG2D
  • inhibitory receptor NKG2A inhibitory receptor NKG2A
  • KIRs CD158a/h/g
  • mediates ADCC receptor CD16 see purity detection method
  • NK cells stimulated by feeder layer cells FN21 was detected by flow cytometry for NKp30/NKp44/NKp46/NKG2D/NKG2A/KIRs(CD158a/h/g)/CD69, and the results of the proportion of positive cells are shown in Figure 11
  • Embodiment 6 NK cell function detection
  • CD107a lysosome-associated membrane protein 1
  • CD107a working solution Take an appropriate amount of complete cell culture medium (RPMI1640+10% FBS) and add PE-anti-human CD107a (eBioscience, 12-1079-42) at a ratio of 1:100, and add GolgiStop Inhibitor at a ratio of 1:1000 (BD Bioscience, 51-2092KZ)
  • NK cells When NK cells encounter target cells, the secretion of cytokines will also increase, and the secretion of IFN- ⁇ can inhibit the proliferation of tumor cells.
  • BFA protein transport inhibitor Brefeldin A
  • GolgiPlug protein transport inhibitor Brefeldin A
  • GolgiPlug resuspension working solution Take an appropriate amount of complete cell culture medium (RPMI1640+10% FBS) and add GolgiPlug Inhibitor (BD Bioscience, 51-2301KZ) at a ratio of 1:1000
  • Intracellular factor staining Prepare IFN- ⁇ (Human IFN-r(gamma)PE, BD, 554701) staining solution with 1x BD Perm/Wash TM Buffer at a ratio of 1:100,
  • NK cell killing function detection (based on Luciferase killing detection)
  • Luciferase reporter gene system is a reporter system that uses luciferin as a substrate to detect firefly luciferase activity. Luciferase can catalyze the oxidation of luciferin to oxyluciferin, and during the oxidation of luciferin, bioluminescence will be emitted. The bioluminescence released during luciferin oxidation can then be measured by a fluorometer, also known as a luminometer or a liquid scintillation meter. Luciferin and luciferase, a bioluminescent system, can detect cell death and gene expression extremely sensitively and efficiently.
  • Luciferase-GFP gene Stable expression of Luciferase-GFP gene in target cells (GFP and Luciferase share the promoter, which can be used as a reference for Luciferase expression).
  • luciferin luciferin
  • chemiluminescence The magnitude of the overall fluorescence value.
  • Luciferase reporter gene system can be used to quantify the killing ability of NK cells.
  • luciferin substrate Bright-Glo luciferase detection system, Promega, E2620
  • a microplate reader Perkin ELmer, VICTOR NIVO 3F was used for fluorescence reading detection.
  • Killing efficiency (1-experimental group sample reading average value/background group sample reading average value)*100%
  • serial number illustrate SEQ ID NO: 1 CD8 SP amino acid sequence SEQ ID NO: 2 FN1 heparin-binding domain II-CS1 SEQ ID NO: 3 Linker SEQ ID NO: 4 IL-21 that removes the secretory signal peptide SEQ ID NO: 5 CD8 hinge region SEQ ID NO: 6 CD8 transmembrane region SEQ ID NO: 7 V region(type III connecting segment, IIICS) SEQ ID NO: 8 FN1 heparin-binding domain II

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Abstract

本发明涉及膜整合型融合蛋白、含有其的细胞及用途。具体提供膜整合型融合蛋白，包含整合域以及与其融合的功能多肽，所述整合域包含FN的肝素结合域Ⅱ和/或CS1结合域。还提供含融合蛋白的细胞，该细胞可以实现免疫细胞体外大量扩增。

Description

膜整合型融合蛋白、含有其的细胞及用途 技术领域
本发明涉及免疫细胞培养和刺激领域，具体涉及含膜整合型融合蛋白、含有该融合蛋白的细胞及其用途。
背景技术
近年来随着将免疫细胞转化为治疗手段的技术的发展，免疫细胞治疗已经显著地改变了当前的癌症治疗模式。自然杀伤细胞(NK细胞)是先天免疫系统的重要组成部分，对人体免疫能力起着关键的调节作用，并表现出强大的肿瘤细胞溶解功能。与T细胞不同，NK细胞具有天然杀伤细胞毒性，无需激活或抗原递呈，即可通过识别非自我方式启动清除肿瘤细胞、细菌等异己成分。
自然杀伤细胞(NK)在癌症过继免疫治疗中是一种很有前途的癌症治疗方法，引起了广泛的关注，其抗肿瘤功能也被大量临床试验证实。但是NK细胞大规模GMP级体外扩增方法的缺乏仍是限制其临床应用的主要因素。NK细胞在外周血单个核细胞中占比较低(1-32.6％)，研究人员已经开发出多种方法来提高NK细胞的体外扩增数量和抗肿瘤功能。这些方法包括使用细胞因子和抗体来刺激NK细胞的扩增，但是扩增倍数非常有限，也包括使用饲养层细胞进行NK细胞的扩增，扩增倍数最高报道如【Denman CJ,Senyukov VV,Somanchi SS,Phatarpekar PV,Kopp LM,et al.(2012)Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells.PLoS ONE 7(1):e30264.doi:10.1371/journal.pone.0030264】文献所述Clone 9.mbIL21滋养层细胞三周时间可激活NK细胞平均扩增47967倍(中位数为31,747)。
RetroNectin是重组人纤维连接蛋白(FN)的片断，包括细胞结合域，肝素结合域Ⅱ和CS1位点三个功能区域。不仅可以增强病毒的感染效率，还可以刺激淋巴细胞，使其获得上千倍的增殖。但至今没有人报道RetroNectin或其片段对NK增殖的报道。
发明内容
本发明公开了一种膜整合型融合蛋白，以及利用该融合蛋白在细胞表面整合细胞因子的方法，获得了可以刺激免疫细胞体外大量扩增的饲养层细胞。本发明还公开了利用该细胞进行免疫细胞体外扩增的方法和在细胞治疗领域的应用。
本发明第一方面提供一种膜整合的融合蛋白，包含整合域以及与其融合的功能多肽，所述整合域包含FN的肝素结合域Ⅱ和CS1结合域。
本发明第一方面提供一种融合蛋白，包含整合域以及与其融合的功能多肽，所述整合域包含FN的肝素结合域Ⅱ和CS1结合域。优选地，整合域位于功能多肽的N端方向。整合域将融合蛋白整合在细胞膜上。
在一个或多个实施方案中，整合域还包含FN的细胞结合域。
在一个或多个实施方案中，所述融合蛋白还包含信号肽。优选地，所述信号肽位于融合蛋白的N端。
在一个或多个实施方案中，所述融合蛋白还包含跨膜区。
在一个或多个实施方案中，所述融合蛋白从N端至C端包含信号肽、整合域和功能多肽。
在一个或多个实施方案中，所述融合蛋白从N端至C端包含信号肽、整合域、功能多肽和跨膜区。
在一个或多个实施方案中，整合域和功能多肽之间具有接头，优选具有SEQ ID NO:3所示的序列。
在一个或多个实施方案中，信号肽是CD8信号肽。优选具有SEQ ID NO:1所示的序列或与其具有至少90％序列相同性的变体。
在一个或多个实施方案中，整合域具有SEQ ID NO:2所示的序列或与其具有至少90％序列相同性的变体。
在一个或多个实施方案中，功能多肽是细胞因子。
在一个或多个实施方案中，细胞因子包括选自以下的一种或多种：白介素、肿瘤坏死因子(TNF)、干扰素(IFN)、集落刺激因子(CSF)和肿瘤生长因子(TGF)
在一个或多个实施方案中，所述白介素包括但不限于IL-2、IL-4、IL-6、IL-7、IL-8、IL-10、IL-12、IL-15、IL-18和IL-21；所述肿瘤坏死因子包括但不限于TNF-α和TNF-β；所述干扰素包括但不限于IFN-α、IFN-β和IFN-γ；所述集落刺激因子包括但不限于M-CSF、G-CSF和GM-CSF；所述肿瘤生长因子包括但不限于TGF-α、TGF-β1、TGF-β2和TGF-β3。
在一个或多个实施方案中，细胞因子是IL-21。优选具有SEQ ID NO:4所示的序列或与其具有至少90％序列相同性的变体。
在一个或多个实施方案中，跨膜区包括但不限于CD28、CD8、CD134(OX40)、CD137(4-1BB)、LCK、ICOS、DAP10、siglec-9、siglec-10、siglec-15、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、IL-2Rβ、IL-2Rγ、IL-4Rα、IL-7Rα、IL-10R、IL-12R、IL-15R、IL-21R、CD27和CD40中任一种的跨膜区。
在一个或多个实施方案中，跨膜区是CD8跨膜区。优选具有SEQ ID NO:6所示的序列或与其具有至少90％序列相同性的变体。
在一个或多个实施方案中，跨膜区与其相邻结构(例如细胞因子)通过铰链区或接头连接。示例性铰链区是CD8铰链区，优选具有SEQ ID NO:5所示的序列或与其具有至少90％序列相同性的变体。
本发明还提供核酸分子，包含选自以下的序列：
(1)本文任一实施方案所述融合蛋白的编码序列，
(2)(1)的互补序列，和
(3)(1)或(2)中任一序列的5-50bp的片段，
在一个或多个实施方案中，所述片段是引物。
在一个或多个实施方案中，所述编码序列是DNA或RNA。
本发明还提供核酸构建物，其包含本发明任一实施方案所述的核酸分子。
在一个或多个实施方案中，核酸构建物是载体，例如克隆载体、表达载体或整合载体。
本发明还提供一种宿主细胞，所述宿主细胞：
(1)包含、表达或分泌本文任一实施方案中所述的融合蛋白，和/或
(2)包含本文所述的核酸分子和/或核酸构建物。
在一个或多个实施方案中，所述宿主细胞还表达或分泌共刺激分子或分泌包含共刺激分子的蛋白。在一个或多个实施方案中，共刺激分子包括选自以下的一种或多种：CD28、CD134/OX40、CD86、CD137/4-1BB、4-1BBL。优选地，所述细胞表达或分泌CD86和41BBL。
在一个或多个实施方案中，所述宿主细胞是用于免疫细胞的饲养层细胞。优选NK细胞的饲养层细胞。
在一个或多个实施方案中，所述宿主细胞是K562。
本发明还提供一种培养或刺激免疫细胞的方法，包括步骤：在本文任一实施方案所述融合蛋白和/或宿主细胞存在的条件下孵育免疫细胞或含有免疫细胞的样品，所述融合蛋白中的功能多肽是细胞因子。
在一个或多个实施方案中，宿主细胞和免疫细胞的比例为0.5:1-10:1。
在一个或多个实施方案中，所述孵育持续至少1周或至少10天。
一个或多个实施方案中，所述样品是外周血制品或PBMC。
在一个或多个实施方案中，所述免疫细胞是NK细胞。
在一个或多个实施方案中，所述方法还包括分选NK细胞的步骤，分选标记为CD3-CD56+。
在一个或多个实施方案中，所述NK细胞是CD3-CD56+NK细胞。
本发明还提供一种免疫细胞培养物，该培养物含有本文任一实施方案所述融合蛋白和/或宿主细胞以及适用于免疫细胞的培养基，所述融合蛋白中的功能多肽是细胞因子。
在一个或多个实施方案中，所述免疫细胞是NK细胞。
在一个或多个实施方案中，所述培养基是补充有FBS和IL-2的SCGM。
本发明还提供一种药物组合物，包含本发明所述培养或刺激免疫细胞的方 法获得的免疫细胞和药学上可接受的辅料。
在一个或多个实施方案中，所述免疫细胞是NK细胞。
本发明还提供一种培养或刺激免疫细胞试剂盒，包含本文任一实施方案所述融合蛋白和/或宿主细胞，所述融合蛋白中的功能多肽是细胞因子。
在一个或多个实施方案中，所述试剂盒还包含培养免疫细胞所需的试剂，包括但不限于培养基、细胞因子、激活抗体等。
在一个或多个实施方案中，所述试剂盒还包含获取样品中的免疫细胞的试剂，包括但不限于培养基、缓冲液、免疫细胞筛选抗体等。
在一个或多个实施方案中，所述样品是外周血制品或PBMC。
在一个或多个实施方案中，所述免疫细胞是NK细胞。
本发明还提供试剂在制备免疫细胞中的应用，所述试剂含有：
(1)本文任一实施方案所述的融合蛋白，或
(2)本文任一实施方案所述的核酸分子、核酸构建物或宿主细胞。
在一个或多个实施方案中，所述免疫细胞是NK细胞。
本发明另一方面提供试剂在制备预防和/或治疗抗肿瘤药物中的应用，所述试剂含有：(1)本文任一实施方案所述的融合蛋白，或(2)本文任一实施方案所述的核酸分子、核酸构建物或宿主细胞。
在一个或多个实施方案中，所述肿瘤包括实体瘤和血液肿瘤。
本发明优点：
1)实现免疫细胞特别是NK细胞的体外大量扩增满足临床需求；
2)膜整合型细胞因子(FN21)相比分泌型和膜结合型细胞因子(FN-mbIL21、GFP-mbIL21)，对NK细胞形成更强的刺激；
3)首次将FN1肝素结合域Ⅱ、CS-1功能域与细胞因子组合，形成整合型细胞因子。
附图说明
图1：不同饲养层细胞中IL21序列组合示意图
图2：饲养层细胞表面共刺激因子阳性率流式检测图。a.饲养层细胞表面CD86阳性率流式检测图；b.饲养层细胞表面41BBL阳性率流式检测图。
图3：FN21细胞表面IL-21阳性率流式检测图。
图4：FN-mbIL21细胞表面IL-21阳性率流式检测图。
图5：GFP-mbIL21细胞表面IL-21阳性率流式检测图。
图6：CS1-IL21细胞表面IL-21阳性率流式检测图。
图7：HBD-IL21细胞表面IL-21阳性率流式检测图。
图8：FN21与其它饲养层细胞、不同培养方式对NK扩增倍数影响的比较
图9：FN21、FN-mbIL-21与GFP-mbIL-21刺激CB-NK扩增曲线
图10：NK细胞比例。a，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD3-CD56+NK比例；b，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD16+CD56+NK比例。
图11：NK细胞比例。a：FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+NKp30+NK比例；b，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+NKp44+NK比例；c，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+NKp46+NK比例；d，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+NKG2D+NK比例；e，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+NKG2A+NK比例；f，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+KIR+NK比例；g，FN21、FN-mbIL-21与GFP-mbIL-21刺激产生CD56+CD69+NK比例。
图12：FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562产生CD107a的比例。
图13：FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562产生细胞因子IFN-γ的比例。
图14：FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562-luc-GFP细胞毒性测试。
具体实施方式
本发明公开了一种可以刺激免疫细胞体外大量扩增的饲养层细胞，其膜上整合有含FN片段与细胞因子的融合蛋白。本文的饲养层细胞可使NK细胞24天体外扩增20万倍以上，为NK细胞作为通用型细胞治疗产品提供了非常高效的工具。本发明方法制备的NK细胞具有更高的肿瘤细胞杀伤能力和细胞因子分泌量。本发明饲养层细胞的膜上整合有融合蛋白，该融合蛋白包含与整合域以及与其融合的细胞因子。
本文中，膜“整合”与膜“结合”不同。膜结合通常指分子通过与跨膜蛋白、跨膜区或膜蛋白的共价连接而结合在细胞膜磷脂双分子之间的过程。而膜整合是通过整合结构域与整合素进行配体受体整合而固定到细胞表面。发明人发现，想要实现有效的膜整合，整合域需要FN肝素结合区域Ⅱ的受体与CS-1功能区域的VLA-4整合受体协同发挥最大整合优势。
因此，本发明还提供一种膜整合的融合蛋白，包含整合域以及与其融合的功能多肽，所述整合域包含FN的肝素结合域Ⅱ和CS1结合域。所述功能多肽可以是任何需要固定在膜上的多肽分子，例如细胞因子、肿瘤相关蛋白、病毒蛋白。
根据上述发现，为了使饲养层细胞可以激活待培养的细胞(例如NK细胞)而无需添加额外的细胞因子，发明人利用上述膜整合型融合蛋白将细胞因子整合在饲养层细胞的膜上。发明人发现，整合型膜表面表达细胞因子对免疫细胞的刺激比膜结合型细胞因子和游离细胞因子更强。
本文中，细胞因子通过与整合域偶联而整合在细胞膜表面。整合域包含FN(纤维连接蛋白)的肝素结合域Ⅱ和/或CS1结合域，任选还包含FN的细胞结合域。优选地，整合域位于细胞因子的N端方向。在一个或多个实施方案中，整合域具有SEQ ID NO:2所示的序列或与其具有至少90％序列相同性的变体。整合域和细胞因子之间具有间隔区(接头或铰链)。通常，间隔区的长度为10-70aa，优选12-66aa，例如为12aa、54aa、55aa或66aa或由上述任两个数字之间的范围。间隔区可以包含接头，例如由G和/或S组成的多肽序列，例如GS、GSG、GSSG、SGS、GGGGS的重复n次(如3次)的多肽。示例 性接头具有SEQ ID NO:3所示序列，或与其具有至少90％序列相同性的序列。
本文所述细胞因子可以本领域常规用于刺激或活化免疫细胞的细胞因子，包括但不限于：白介素、肿瘤坏死因子(TNF)、干扰素(IFN)、集落刺激因子(CSF)和肿瘤生长因子(TGF)。所述白介素包括但不限于IL-2、IL-4、IL-6、IL-7、IL-8、IL-10、IL-12、IL-15、IL-18和IL-21；所述肿瘤坏死因子包括但不限于TNF-α和TNF-β；所述干扰素包括但不限于IFN-α、IFN-β和IFN-γ；所述集落刺激因子包括但不限于M-CSF、G-CSF和GM-CSF；所述肿瘤生长因子包括但不限于TGF-α、TGF-β1、TGF-β2和TGF-β3。在示例性实施方案中，细胞因子是IL-21，优选具有SEQ ID NO:4所示的序列或与其具有至少90％序列相同性的变体。
所述融合蛋白还可包含信号肽。通常，所述信号肽位于融合蛋白的N端。信号肽可以是本领域已知的任何用于引导蛋白移动的多肽。在示例性实施方案中，信号肽是CD8信号肽，优选具有SEQ ID NO:1所示的序列或与其具有至少90％序列相同性的变体。
虽然本文的融合蛋白是整合型融合蛋白，但该融合蛋白仍可以含有跨膜区。适用于本文融合蛋白的跨膜区包括但不限于：CD28、CD8、CD134(OX40)、CD137(4-1BB)、LCK、ICOS、DAP10、siglec-9、siglec-10、siglec-15、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、IL-2Rβ、IL-2Rγ、IL-4Rα、IL-7Rα、IL-10R、IL-12R、IL-15R、IL-21R、CD27和CD40中任一种的跨膜区。在示例性实施方案中，跨膜区是CD8跨膜区。优选具有SEQ ID NO:6所示的序列或与其具有至少90％序列相同性的变体。
跨膜区与其相邻结构(例如细胞因子)通过铰链区或接头连接。铰链区包括但不限于：CD4胞外铰链区、CD8胞外铰链区、CD28胞外铰链区、IgG1Fc铰链区和IgG4Fc铰链区。示例性铰链区是具有SEQ ID NO:5所示的序列的CD8铰链区，或与其具有至少90％序列相同性的变体。
如果需要向胞内传递胞外信号，所述融合蛋白还可包含胞内信号区。胞内信号区可以是任何信号通路中能够响应胞外信号引起胞内效应的跨膜蛋白的胞内区，包括但不限于：CD8、CD28、CD134/OX40、CD137/4-1BB、LCK、ICOS、DAP10、CD3ζ或Fc310的胞内信号区，优选为CD8胞内信号区。
在优选实施方案中，所述融合蛋白：从N端至C端包含信号肽、整合域和细胞因子；或从N端至C端包含信号肽、整合域、细胞因子和跨膜区；或从N端至C端包含信号肽、整合域、细胞因子、跨膜区和胞内信号区。
在示例性实施方案中，所述融合蛋白从N端至C端依次包含：CD8信号肽、FN的肝素结合域Ⅱ和CS1结合域、接头和IL-21；或CD8信号肽、FN的肝素结合域Ⅱ和CS1结合域、接头、IL-21、CD8铰链区和CD8跨膜区。
在示例性实施方案中，所述融合蛋白具有由以下任一组所示序列顺序串联组成的序列：(1)SEQ ID NO:1、2、3、4，(2)SEQ ID NO:1、7、3、4，(3)SEQ ID NO:1、8、3、4，(4)SEQ ID NO:1、2、3、4、5、6。
形成本文多肽的上述各部分，如信号肽、FN的肝素结合域Ⅱ、CS1结合域、细胞因子、铰链区、跨膜区和胞内信号区等，相互之间可直接连接，或者可通过本领域周知的接头序列连接，例如前文所述的含G和S的接头序列。
本发明的多肽可以是重组多肽、合成多肽。本发明的多肽可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述多肽或结构域的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述多肽相同的生物学功能或活性的多肽。
多肽或结构域的片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽或结构域，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽或结构域，或(iii)成熟多肽或结构域与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽或结构域序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
术语“变体”或“突变体”是指与参照序列相比，通过一个或多个氨基酸 的插入、缺失或取代使氨基酸序列发生变化但保留至少一种生物活性的肽或多肽。本文任一实施方案所述的突变体包括与参照序列(如本文所述的SEQ ID NO:1-8所编码的氨基酸序列)具有至少70％，优选至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少97％的序列相同性并保留参照序列的生物学活性(如作为嵌合抗原受体、表面细胞因子表达蛋白、信号转换受体)的氨基酸序列。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。突变体还包括在参照序列的氨基酸序列中具有一个或多个突变(插入、缺失或取代)、同时仍保留参照序列生物学活性的氨基酸序列。所述多个突变通常指1-10个以内，例如1-8个、1-5个或1-3个。取代优选是保守性取代。例如，在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质或多肽的功能。“性能相近或相似的氨基酸”包括例如，具有相似侧链的氨基酸残基的家族，这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在本发明多肽中用来自同一侧链类的另一氨基酸残基替换一个或几个位点，将不会在实质上影响其活性。
多肽或结构域的截短变体也可以用于本发明，只要该变体基本上保持与多肽或结构域相同的生物学功能或活性，例如保持整合域的膜整合功能的FN肝素结合域Ⅱ和CS1结合域的截短变体。
本文所述多肽可以是经过修饰的多肽。修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
编码所述多肽或结构域的核酸分子也在本文的范围内。核酸分子可以是DNA形式或RNA形式。本文所述核酸分子包括经密码子优化而发生变化的序列，只要核酸分子所编码的氨基酸序列不变即可。经密码子优化的序列可对具体物种表现出更适合的表达性。本领域周知对核酸分子序列进行密码子优化的方法。编码成熟多肽的编码区序列可以是简并的变异体。如本文所用，“简并的变异体”是指编码SEQ ID NO:1-8所示氨基酸序列或其结构域片段，但碱基序列有差别的核酸序列。
编码成熟多肽的核酸分子包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。术语“编码多肽的核酸分子”可以是包括编码此多肽的核酸分子，也可以是还包括附加编码和/或非编码序列的核酸分子。
本发明的核酸分子可以是多肽或结构域的编码序列，或者是多肽或结构域的表达框。本文中，编码序列指核酸序列中直接确定其蛋白产物(例如信号肽、整合域、铰链区、跨膜区、胞内信号区或细胞因子蛋白)的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。本文中，表达框指表达感兴趣基因所需的完整元件，包括启动子、基因编码序列和PolyA加尾信号序列。在某些实施方案中，所述编码序列或表达框整合到细胞的基因组中。因此，在这些实施方案中，本文所述的细胞的基因组中稳定整合了包含编码本文所述多肽的表达框。
本发明还涉及上述核酸分子的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％、85％、90％、95％相同性的多核苷酸。本发明特别 涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明还涉及上述序列的互补序列或与上述序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码所需多肽或结构域的多聚核苷酸。本文所述“杂交”主要指在严谨条件下的核酸序列配对。示例性严谨条件为在0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
在某些实施方案中，所述核酸分子是核酸构建物，其含有本文所述融合蛋白的编码序列，以及与这些序列操作性连接的一个或多个调控序列。调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本文。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。例如，当使用逆转录病毒载体时，逆转录病毒载体通常含有复制起始位点、3’LTR、5’LTR、本文所述融合蛋白的编码序列以及任选的可选择的标记。
合适的启动子包括但不限于即时早期巨细胞病毒(CMV)启动子序列、延伸生长因子-1伸(EF-1因)、类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子等。
可选择的标记包括可选择的标记基因或报道基因中的任一个或两者，以便于从被病毒载体感染的细胞群中鉴定和选择表达细胞。有用的可选择标记基因包括例如抗生素抗性基因，如neo。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色荧光蛋白的基因。
本文所述的核酸分子通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。或者，也可直接合成本文所述的核酸分子。
可采用常规的方法将本文的核酸分子(尤其是载体)导入宿主细胞中，这些方法包括显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等。
本文所述，宿主细胞含有本文所述的核酸分子。宿主细胞既包括最终用作共培养免疫细胞的饲养层细胞，也包括生产饲养层细胞过程中使用到的各种细胞，如大肠杆菌细胞，以用于提供本发明蛋白的编码序列或提供本文所述的载体。在某些实施方案中，本文提供一种稳定表达本文所述融合蛋白的用于NK细胞的饲养层细胞，例如K562细胞、721.221、PC3等缺失HLA的细胞株。为了用作饲养层细胞，K562等经辐照，例如100Gyγ射线辐照，以使其失去分裂能力。
在示例性实施方案中，宿主细胞(饲养层细胞)所含的融合蛋白中的功能多肽是细胞因子IL-21，用于为宿主细胞提供刺激NK细胞的能力。但是，针对不同的免疫细胞，可以使宿主细胞的融合蛋白含有相应的其他细胞因子，这在本领域技术人员的知识范围内。
在优选实施方案中，所述宿主细胞还表达(例如膜表达)或分泌共刺激分子或分泌包含共刺激分子的蛋白。本文中，共刺激分子包括选自以下的一种或多种：CD28、CD134/OX40、CD86、CD137/4-1BB、4-1BBL。优选地，宿主细胞还表达或分泌CD86和41BBL。
本文也包括本文所述的核酸分子。如前文所述，可采用本领域常规的方法制备得到本文所述的核酸分子。在某些实施方案中，本文还包括慢病毒，其包 括本文所述的表达框，并能将本文所述的表达框整合到宿主细胞的基因组中。可采用本领域周知的方法制备本文所述的慢病毒。例如，首先制备得到含有本文所述表达框的慢病毒载体，然后在合适的宿主细胞中进行病毒包装，并分离纯化得到所需的慢病毒。用于慢病毒包装的试剂为本领域所周知，如常规的慢病毒载体系统包括pCCL和表达RRE-SIV、REV、VSVG的质粒。
本文还包括一种NK细胞培养物，该培养物含有本文所述的宿主细胞以及合适的培养基。培养基可以是本领域常规用于培养NK细胞的培养基，例如SCGM。示例性地，所述培养基是补充有FBS(例如至少2％)和IL-2(例如至少100IU/mL)的SCGM。
本发明还包括一种培养或刺激NK细胞的方法，包括步骤：在本文所述融合蛋白和/或宿主细胞存在的条件下孵育NK细胞或含有NK细胞的样品。所述孵育可以使用本领域常规用于培养NK细胞的条件。示例性地，宿主细胞和NK细胞的比例为0.5:1-10:1，优选1:1-5:1；培养基是含血清培养基(例如上述SCGM)。在一个或多个实施方案中，所述孵育持续至少1周、至少2周、至少3周，或至少10天、15天、21天、24天。所述样品包括但不限于血液或源自免疫系统的体液，例如外周血、脐带血或PBMC。从样品中提取NK细胞或PMBC的方法本领域周知。所述方法还包括分选NK细胞的步骤，分选标记为CD3-CD56+。分选NK细胞可使用流逝细胞术等本领域常规技术。
本文还提供一种药物组合物，该药物组合物中含有包含本发明所述培养或刺激NK细胞的方法获得的NK细胞和药学上可接受的辅料。本文中，药学上可接受的辅料是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。更具体而言，合适的药学上可接受的辅料可以是本领域常用于NK细胞给药的辅料。
通常，药物组合物中含有治疗有效量的NK细胞。治疗有效量是指可在受试者中实现治疗、预防、减轻和/或缓解疾病或病症的剂量。可根据患者年龄、性别、所患病症及其严重程度、患者的其它身体状况等因素确定治疗有效量。本文中，受试者或患者通常指哺乳动物，尤其指人。
本文中，适合使用本文所述的NK细胞以及药物组合物治疗的疾病包括实 体瘤和血液肿瘤，如腺癌、肺癌、结肠癌、大肠癌、乳腺癌、卵巢癌、宫颈癌、胃癌、胆管癌、胆囊癌、食管癌、胰腺癌和前列腺癌等实体瘤，以及白血病和淋巴瘤，如B细胞淋巴瘤、套细胞淋巴瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、多毛细胞白血病、急性髓性白血病和慢性髓系白血病等。
在某些实施方案中，本文还提供了一种试剂盒，所述试剂盒含有本文所述的融合蛋白、核酸分子和/或宿主细胞，用于培养或刺激NK细胞。
试剂盒还可包含：适用于将所述核酸分子转染入宿主细胞(例如K562)中的各种试剂，适用于培养宿主细胞的培养基，适用于孵育NK细胞的各种试剂(包括但不限于培养基、细胞因子、激活抗体等)，适用于获取样品中的NK细胞的试剂(包括但不限于培养基、缓冲液、NK细胞筛选抗体等)。以及任选的指导本领域技术人员转染宿主细胞、提取和孵育NK细胞的说明书。
在具体实施方案中，本发明在MHCⅠ类分子缺失的人慢性髓系白血病细胞K562上使用慢病毒感染共刺激因子CD86(NCBI CCDS ID:CCDS 3009.1)、41BBL(NCBI CCDS ID:CCDS 12169.1)，同时构FN肝素结合域Ⅱ_CS-1_IL21融合蛋白表达载体，包装成慢病毒，感染K562，使其表达并整合到细胞表面，形成全新的整合型饲养层细胞，命名为FN21。FN21经过100Gyγ射线辐照，使其失去分裂能力，作为饲养层细胞建库备用。
然后从脐带血或者健康人血液，分离出其中的单个核细胞，通过流式检测CD56+细胞比例，利用NK分选试剂盒将其中NK分离出来，在SCGM+10％FBS，添加200IU/mL IL-2条件下，与辐照后的FN21(IR-FN21)按照1:2比例混合均匀进行扩增。经过三轮共24天培养可刺激NK扩增20万倍以上，NK纯度高达99％以上，且FN21刺激产生的NK细胞表型与膜结合型细胞因子(mbIL21)组相比，表型一致。FN21刺激产生的NK细胞在效靶比10:1时4h杀伤肿瘤细胞可达95％以上；在效靶比1：1时细胞脱颗粒能力CD107a比例高达50％以上，细胞因子IFNγ分泌量高达20％以上。
实施例
实施例1：FN21饲养层细胞的制备
1.CD86、41BBL、FN21的慢病毒表达载体构建
1)CD86、41BBL慢病毒载体构建：以human cDNA为模板进行PCR，获得片段长度为990nt的CD86(NCBI CCDS ID:CCDS 3009.1)，纯化后克隆至pCCL慢病毒主质粒中；同样方法克隆片段长度为765nt的41BBL(NCBI CCDS ID:CCDS 12169.1)至pCCL慢病毒表达载体中。
2)重组FN21质粒构建：将CD8前导肽(SEQ ID NO:1)、与FN1肝素结合区域Ⅱ-CS1功能区域(FN HBDⅡ-CS1，1812-2107aa)(SEQ ID NO:2)的cDNA序列进行密码子优化，形成的DNA序列利用基因连接序列GS(SEQ ID NO:3)与去掉分泌信号肽的IL-21(SEQ ID NO:4)、通过化学合成连接到一起，克隆到pCCL慢病毒主质粒中。
3)重组FN-mbIL21质粒构建：将CD8前导肽(SEQ ID NO:1)、与FN1肝素结合区域Ⅱ-CS1功能区域(FN HBDⅡ-CS1，1812-2107aa)(SEQ ID NO:2)的cDNA序列进行密码子优化，形成的DNA序列利用基因连接序列GS(SEQ ID NO:3)与去掉分泌信号肽的IL-21(SEQ ID NO:4)、CD8铰链区(SEQ ID NO:5)，CD8跨膜区(SEQ ID NO:6)通过化学合成连接到一起，克隆到pCCL慢病毒主质粒中。
4)重组GFP-mbIL21质粒构建：将CD8前导肽(SEQ ID NO:1)、与GFP基因序列利用基因连接序列GS(SEQ ID NO:3)与去掉分泌信号肽的IL-21(SEQ ID NO:4)、CD8铰链区(SEQ ID NO:5)，CD8跨膜区(SEQ ID NO:6)通过化学合成连接到一起，克隆到pCCL慢病毒主质粒中。
5)重组CS1-IL21质粒构建：将CD8前导肽(SEQ ID NO:1)、与FN1基因CS1功能区域(2083-2202aa)(SEQ ID NO:7)利用基因连接序列GS(SEQ ID NO:3)与去掉分泌信号肽的IL-21(SEQ ID NO:4)通过化学合成连接到一起，克隆到pCCL慢病毒主质粒中。
6)重组HBD-IL21质粒构建：将CD8前导肽(SEQ ID NO:1)、FN1肝素结合区域Ⅱ功能区域(1812-2082aa)(SEQ ID NO:8)的cDNA序列利用基因连接序列GS(SEQ ID NO:3)与去掉分泌信号肽的IL-21(SEQ ID NO:4)通过化学合成连接到一起，克隆到pCCL慢病毒主质粒中。
2.CD86、41BBL、FN21、FN-mbIL21、GFP-mbIL21、CS1-IL21、HBD-IL21的慢病毒包装
用阳离子聚合物PEI包装慢病毒，流程如下：用OPTI-MEM分别稀释PEI和慢病毒包装质粒(慢病毒主质粒、RRE-SIV、REV、VSVG)；然后将PEI/OPTI-MEM加入质粒/OPTI-MEM混合物中，涡旋震荡混匀，在室温下静置10分钟；将质粒-PEI复合物加入预先铺板的293T细胞(中国科学院细胞库),混匀后放置于37℃，5％CO2培养箱中培养。转染后16h换液，48h后收集病毒上清，0.45um滤器过滤，留存原液用于后续感染实验。
3.CD86、41BBL、FN21、FN-mbIL21、GFP-mbIL21、CS1-IL21、HBD-IL21的慢病毒感染
细胞株构建策略：先构建K562-CD86-41BBL，在此细胞基础上再过表达FN21，FN-mbIL-21，GFP-mbIL21，饲养层细胞根据IL21不同组合形式分别命名为K562-CD86-41BBL、FN21，FN-mbIL-21，GFP-mbIL21、CS1-IL21、HBD-IL21。其中细胞中表达IL21组合序列示意图如图1所示。
感染流程：将K562(中国科学院细胞库)细胞密度调整为5x105/mL，在24孔板中分别加入1ml K562细胞悬液和1ml病毒原液，每孔加1μL polybrene，32℃，2500rpm，离心1.5h。弃去上清液，每孔加入1ml细胞完全培养基(RPMI1640+10％FBS)。将培养板置于37℃，5％CO2培养箱中培养。感染后48h，取5E5细胞进行以下流式检测。
4.K562细胞上CD86、41BBL、FN21、FN-mbIL21、GFP-mbIL21、CS1-IL21、HBD-IL21的蛋白的流式检测
1)取总活细胞数为5E5的细胞悬液，转移至对应标记的流式管中；
2)每管中各加入约3-5ml PBS缓冲液对细胞进行洗涤，450±50g离心5min；
3)配制抗体工作液，按照1:100比例，依次加入PE/Cy7 anti-human CD86 Antibody(BioLegend，374210),APC anti-human 41BBL Antibody
(BioLegend,311506),PE anti-human IL-21 Antibody(Biolegend,513004)或者PerCP/Cyanine5.5 anti-human IL-21 Antibody(Biolegend,513011)
4)吸弃上清，每个样品加100μL抗体工作液，混匀放置2-8℃冰箱，避光染色30min。
5)染色结束后，加入4ml的PBS缓冲液进行洗涤，450±50g离心5min。
6)吸弃上清，加入200μL的PBS缓冲液进行重悬然后进行上机检测
7)饲养层细胞K562-CD86-41BBL表征流式检测结果如图2，a、b所示：CD86、41BBL阳性细胞占比分布为96.2％、93％；
8)饲养层细胞FN21表征流式检测结果如图3所示，FN21阳性细胞占比分为86.1％；
9)饲养层细胞FN-mbIL21表征流式检测结果如图4所示，FN-mbIL21阳性细胞占比分为93.4％；
10)饲养层细胞GFP-mbIL21表征流式检测结果如图5所示，GFP-mbIL21阳性细胞占比分为93.3％。
11)饲养层细胞CS1-IL21、HBD-IL21流式未检测到膜整合的IL-21信号，检测结果分别如图6、图7所示，几乎检测不到IL21阳性细胞。因此，整合域需要FN肝素结合区域Ⅱ的受体与CS-1功能区域的VLA-4整合受体协同发挥最大整合优势。
实施例2：NK细胞分离
单个核细胞分离与冻存
将采集的脐带血或者健康人单采血核对信息后进行单个核细胞分离。
血液颠倒混匀后，用浸泡70％乙醇的棉球擦拭转移导管，然后用剪刀剪开，将脐带血或者单采血平均倒入50ml离心管中，室温1500g离心10min(降速为4)。离心过程中，将样本密度分离液分出15ml/管于新的50ml离心管中。离心结束后用移液管吸取上层血浆，转移至新的50ml离心管中，分离得到的血浆于56℃进行灭活30min；然后1500g离心10min后取上清并于-20℃冰箱储存备用。
离心下来血细胞用氯化钠注射液稀释，血细胞：氯化钠注射液(V/V)＝1:1，用移液管吹打混匀；用25ml规格的移液管向每支装有样本密度分离液离心管中慢慢加入上述稀释的血细胞，注意不要冲破界面(方法：将离心管倾斜45°，在样本密度分离液液面上0.5cm处，缓慢注入稀释的血细胞，不要打乱液面界面。将离心机升降速均调为0，室温850g离心20min。离心结束后，取出离心管，可见管内液体有分层，用10ml移液管吸弃上层，再缓慢吸取白细胞层， 合并至新的50ml离心管中，结束后每支离心管补氯化钠注射液至50ml，进行第一次洗涤：上下颠倒混匀后，室温700g，离心5min。
观察沉淀，弃上清，指弹混匀细胞，用10ml移液管每管加10ml氯化钠注射液重悬细胞，再补至50ml，进行第二次洗涤：混匀后，室温450g，离心6min。离心结束弃上清，加入10ml氯化钠注射液重悬，将细胞合并至一支50ml离心管中，补氯化钠注射液至50ml，混匀后，取10-20μl计数，其余细胞进行第三次洗涤：室温300g，离心8min。
离心结束后，观察细胞沉淀有无洗涤干净，若红细胞污染严重，则需进行红细胞裂解操作：轻柔弃干净上清，根据上一步骤的计数结果，每1×10 8个总细胞用90～110μl红细胞裂解液重悬混匀，室温放置裂解10分钟，裂解完成后加入氯化钠注射液补至50ml，混匀室温450g，离心6min。离心前取10-20μl计数；将取出的细胞悬液与0.1％台盼蓝按照1:1混合，然后取出计数板，往加样槽中加入20ul混悬液，待细胞沉降数秒后打开软件开始计数并记录结果。
根据计数结果进行细胞灌装：吸弃上清(注意将上清完全吸弃)，用自制冻存液将细胞沉淀重悬，调整细胞密度为1×10 8个活细胞/ml后分装，每支1mL。放置程序降温仪冻存结束转移至液氮罐长期保存。
单个核细胞中NK细胞分离
NK细胞分离是使用美天旎NK分选试剂盒(NK isolation kit,human,130-092-657)，具体操作流式如下：
将冻存的PBMC或者CBMC细胞放37°水浴锅速溶2-3min后，转入离心管中，300g离心5min，弃上清，加入适量缓冲液重悬，取20μL细胞悬液，加入20μL 0.1％台盼蓝进行计数，记录活细胞密度与细胞活率。根据细胞悬液体积，计数出细胞总数。按照每107cells加入40μL Buffer，10μL NK Cell Biotin-Antibody Cocktail，混匀后(2-8℃)冰箱孵育5min。之后，每107cells直接加入30μL buffer，加入20μL of NK Cell MicroBeads Cocktail，混匀后(2-8℃)冰箱孵育10min。10min中后，使用buffer补足体积至500μL。放置LS柱子至磁力架上，15mL离心管放置于LS柱子下方，使用3mL Buffer冲洗一次。等冲洗Buffer完全滴完，LS柱子下方换新离心管，加入细胞磁珠混合 悬液至LS柱子中，收集未标记的细胞，即NK细胞。
接收的NK细胞悬液混匀后，取20μL细胞悬液，加入20μL 0.1％台盼蓝进行计数，记录活细胞密度与细胞活率。根据细胞悬液体积，计数出细胞总数。
实施例3：NK细胞扩增
根据NK细胞的计数结果以及流式检测分选后NK细胞CD56+细胞比例，算出起始细胞中NK细胞的数量。按照NK：FC＝1:2(或者其它比例)加入对应量的NK细胞与100Gy辐照后饲养层细胞(FC,Feeder cell)，混匀后用完全培养基(SCGM+10％FBS+200IU/mL IL-2、或者SCGM+10％FBS+200IU/mL IL-2+20ng/mL rhIL-21)将总细胞密度调为2.5×106/mL左右，放于合适的培养容器中进行培养。培养的第三天、第五天进行换液，第七天或者第八天计数并计算第一轮扩增倍数。第七天或者第八天以同样方式开始第二轮培养，第15或16天按照除NK：FC＝1:1外同样方式进行第三轮培养，培养周期为21-24天。
饲养层细胞K562-CD86-41BBL、K562-CD86-41BBL+20ng/mL sIL-21、K562-CD86-41BBL+Retronectin coating、FN21、FN-mbIL21与GFP-mbIL21刺激NK细胞扩增两轮共14天扩增倍数比较如图8所示，
饲养层细胞若只表达CD86+41BBL，D14天细胞平均扩增倍数仅为598.6，添加rhIL-21，或者将细胞加入Retronectin包被的培养皿中，NK细胞扩增倍数上升1倍，但统计学上无明显差异。
若饲养层细胞FN21表面表达融合蛋白FN1 heparin binding domain-CS1-IL-21，NK扩增倍数D14可达3450倍，且与FN-mbIL21(2764)无显著差异(n＝5)，说明整合型FN21对NK的扩增效果显著。
接着用FN21、FN-mbIL21与GFP-mbIL21刺激NK扩增3轮，共24天，扩增曲线如图9所示，FN21平均扩增倍数可达239994.95，优于FN-mbIL21(125572)与GFP-mbIL21(131121)将近2倍。
实施例4：NK细胞纯度检测
培养至D14的细胞，用Sony SA3800流式细胞仪进行NK细胞比例的检 测，NKCD3-CD56+NK，CD56+CD16+NK细胞检测具体操作流程如下:
1)取总活细胞数为5E5的细胞悬液，转移至对应标记的流式管中；
2)每管中各加入约3-5ml PBS缓冲液对细胞进行洗涤，450±50g离心5min；
3)配制抗体工作液，按照1:100比例，依次加入PE/Cy7 anti-human CD56(NCAM)Antibody(BioLegend，318318),PerCP/Cyanine5.5 anti-human CD3 Antibody(BioLegend,300328),Brilliant Violet 421 TM anti-human CD16 Antibody(Biolegend,302038),
4)吸弃上清，每个样品加100μL抗体工作液，混匀放置2-8℃冰箱，避光染色30min。
5)染色结束后，加入4ml的PBS缓冲液进行洗涤，450±50g离心5min。
6)吸弃上清，加入200μL的PBS缓冲液进行重悬然后进行上机检测
7)NK纯度(CD3-CD56+)如图10所示，三种饲养层细胞产生CD3-CD56+NK比例均在99％以上；其中CD56+CD16+NK比例为80％左右
实施例5：NK细胞表型检测
培养至D14细胞，取总活细胞数为5E5的细胞悬液用SONY SA3800流式细胞仪进行NK细胞(CD56+细胞)表面标志物比例的检测，包括NK激活型受体NCRs(NKp30/NKp44/NKp46)、肿瘤识别位点NKG2D、抑制性受体NKG2A、KIRs(CD158a/h/g)，介导ADCC受体CD16(见纯度检测方法)，具体操作流程如下:
1)取总活细胞数为5E5的细胞悬液，转移至对应标记的流式管中；
2)每管中各加入约3-5ml PBS缓冲液对细胞进行洗涤，450±50g离心5min；
3)配制抗体工作液，按照1:100比例，在PBS缓冲液中依次加入所用抗体，如下表所示：
Figure PCTCN2022135389-appb-000001
Figure PCTCN2022135389-appb-000002
4)对饲养层细胞FN21刺激产生的NK细胞表型利用流式细胞术检测NKp30/NKp44/NKp46/NKG2D/NKG2A/KIRs(CD158a/h/g)/CD69，阳性细胞占比结果如图11所示：饲养层细胞FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK表型基本一致，NCRs、NKG2D均高表达，低表达抑制性受体NKG2A和KIRs.
实施例6：NK细胞功能检测
1.NK细胞脱颗粒功能CD107a检测
相关研究表明，在无靶细胞情况下，NK细胞膜表面溶酶体相关膜蛋白1(CD107a)表达量很低，几乎检测不到，但是当NK细胞杀伤靶细胞时，CD107a是会随着细胞毒性颗粒转运到细胞膜表面，可通过流式细胞术检测到细胞表面CD107a表达量的增加，因此，刺激后NK细胞表面CD107a表达量高低可反映NK细胞毒杀伤活性水平，具体检测流程如下所述：
1)实验设置本底组(只有NK细胞)和实验组(NK细胞+靶细胞)，本试验中NK细胞和靶细胞使用量均为4×10 5个活细胞/孔。NK细胞分别来自4个供者，靶细胞为K562。
2)配制CD107a工作液：取适量细胞完全培养基(RPMI1640+10％FBS)按照1:100比例加入PE-anti-human CD107a(eBioscience，12-1079-42)，按照1:1000比例加入GolgiStop Inhibitor(BD Bioscience，51-2092KZ)
3)将本底组(4×10 5个NK细胞)和实验组(4×10 5个NK细胞+4×10 5个靶细胞)离心后用100μL CD107a工作液重悬，加入到V底96孔板中
4)将各组细胞置于37℃，5％CO2培养箱中共孵育4小时后，将实验组和本底组细胞分别转移至对应的流式管中，450±50g离心5min。加入PE/Cy7  anti-human CD56(NCAM)Antibody(BioLegend，318318)标记，流式检测CD56+NK细胞表面CD107a表达量比例。
5)结果如图12所示：饲养层细胞FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562时，产生CD107a的比例分别为64.2％、63.4％、56.9％
2.NK细胞因子IFN-γ分泌量检测
NK细胞遇到靶细胞时，细胞因子分泌量也会增加，IFN-γ分泌可以抑制肿瘤细胞的增殖。NK与靶细胞孵育时，加入蛋白转运抑制剂布雷菲德菌素A(BFA)(GolgiPlug)，可使IFN-γ在高尔基体的累积，提高流式检测细胞因子分泌细胞的能力。细胞内因子流式细胞术检测流程如下所述：
1)实验设置本底组(只有NK细胞)和实验组(NK细胞+靶细胞)，本试验中NK细胞和靶细胞使用量均为4×105个活细胞/孔。NK细胞分别来自4个供者，靶细胞为K562。
2)配制GolgiPlug重悬工作液：取适量细胞完全培养基(RPMI1640+10％FBS)按照1:1000比例加入GolgiPlug Inhibitor(BD Bioscience,51-2301KZ)
3)将本底组(4×10 5个NK细胞)和实验组(4×10 5个NK细胞+4×10 5个靶细胞)离心后用100μLGolgiPlug工作液重悬，加入到V底96孔板中
4)将各组细胞置于37℃，5％CO2培养箱中共孵育4小时后，将实验组和本底组细胞分别转移至对应的流式管中，450±50g离心5min。加入PE/Cy7 anti-human CD56(NCAM)Antibody(BioLegend，318318)标记30min
5)细胞固定：每管加入固定液BD Fixation/Permeabilization solution(BD,51-2090KZ)200μL，混匀，4℃破膜20min。
6)细胞通透：将10x BD Perm/Wash TM Buffer(BD,51-2091KZ)用ddH2O配制1x BD Perm/Wash TM Buffer，每管加入约1mL左右1xBD Perm/Wash TM Buffer；室温通透10min；450±50g离心5min，吸弃上清。
7)细胞内因子染色：用1x BD Perm/Wash TM Buffer按照1:100比例配制IFN-γ(Human IFN-r(gamma)PE,BD,554701)染色工作液，
8)每管流式管中加入抗体工作液100μL/管，混匀后放置2-8℃避光孵育， 染色30min。
9)染色结束后，加入1ml的1x BD Perm/Wash TM Buffer进行洗涤，450±50g离心5min；
10)吸弃上清，加入200μL PBS缓冲液重悬细胞，流式上机检测细胞内IFN-γ聚集量。
6)检测结果如图13所示：饲养层细胞FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562时，产生细胞因子的比例分别为32.2％、26.2％、16.4％。
3.NK细胞杀伤功能检测(基于Luciferase杀伤检测)
荧光素酶报告基因Luciferase报告基因系统是以荧光素(luciferin)为底物来检测萤火虫荧光素酶(fire fly luciferase)活性的一种报告系统。荧光素酶可以催化luciferin氧化成oxyluciferin，在luciferin氧化的过程中，会发出生物荧光(bioluminescence)。然后可以通过荧光测定仪也称化学发光仪(luminometer)或液闪测定仪测定luciferin氧化过程中释放的生物荧光。荧光素和荧光素酶这一生物发光体系，可以极其灵敏、高效地检测细胞死活与基因表达。在靶细胞中稳定表达Luciferase-GFP基因(GFP与Luciferase共用启动子，可作为Luciferase表达参照物)，当所有细胞是活细胞状态时，加入荧光素(luciferin)后，可用化学发光仪检测检测到整体荧光值的大小。当一部分细胞被NK细胞杀伤后，Luciferase不再表达，荧光值会随着活细胞比例下降而下降，所以可以用Luciferase报告基因系统来定量NK细胞的杀伤能力。具体操作步骤如下：
1)实验设置对照组(3×10 4个靶细胞)和实验组(不同效靶比E:T＝10:1，5:1，2.5:1,1.25:1,0.625:1，0.31:1,0.16:1)本试验中效应细胞为NK细胞分别来自4个供者，靶细胞为K562-Luciferase-GFP。
2)取出效靶细胞，先对细胞进行镜检，观察细胞状态，另用移液管轻轻混匀从中取20μL细胞加20μL 0.1％台盼蓝1:1混匀，进行计数，记录下计数的结果
3)靶细胞K562-Luci-GFP的取用：根据实验设计，靶细胞每孔3×10 4个，每孔50μL，取相应体积细胞悬液，450±50g离心5min，吸弃上清；用适量新鲜完全培养基将靶细胞重悬，密度调整为6×10 5cells/mL。
4)效应细胞的取用：根据实验设计，按照两倍梯度稀释法，效应细胞第一排10:1孔需6×10 5个活细胞，共设2个复孔，共1.2×10 6个活细胞，取相应体积细胞悬液，450±50g离心5min，吸弃上清；按照每孔100μL，共2个孔，用200μL新鲜完全培养基将靶细胞重悬，密度调整为6×10 6cells/mL。
5)取一块不透光酶标板，实验组中除第一排10:1孔外，其余均加入50μL新鲜培养基，本底组加入50μL新鲜培养基。
6)效应细胞的梯度稀释：取重悬后的效应细胞悬液100μL，用排枪加入第一排10:1两个孔中，从第一排10:1孔取50μL加入第二排5:1孔中，直至第七个孔，多余50μL弃掉。
7)效应细胞与靶细胞的混合：所有孔每孔依次加入50μL密度为6×10 5cells/mL的靶细胞，第一排补50μL完全培养基，即效应细胞与靶细胞总体积为100μL/孔，加入后，轻轻混匀，37℃共孵育4h。
8)培养结束后，将细胞从CO2培养箱取出，向标记好的每孔中依次加入100μL荧光素底物(Bright-Glo萤光素酶检测系统，Promega,E2620)，反应3-5min，在酶标仪(PerkinELmer,VICTOR NIVO 3F)进行荧光读数检测。
9)数据分析方法：杀伤效率＝(1-实验组样品读数平均值/本底组样品读数平均值)*100％
10)杀伤曲线图详见图14，饲养层细胞FN21、FN-mbIL21与GFP-mbIL21刺激产生的NK遇到靶细胞K562-luc-GFP时，杀伤效果相当。
序列编号 说明
SEQ ID NO：1 CD8 SP氨基酸序列
SEQ ID NO：2 FN1肝素结合区域Ⅱ-CS1
SEQ ID NO：3 Linker
SEQ ID NO：4 去掉分泌信号肽的IL-21
SEQ ID NO：5 CD8铰链区
SEQ ID NO：6 CD8跨膜区
SEQ ID NO：7 V region(type III connecting segment,IIICS)
SEQ ID NO：8 FN1肝素结合区域Ⅱ

Claims (10)

  1. 一种膜整合型融合蛋白，包含整合域以及与其融合的功能多肽，所述整合域包含FN的肝素结合域Ⅱ和/或CS1结合域，
    优选地：
    整合域位于功能多肽的N端方向，
    整合域还包含FN的细胞结合域，
    所述融合蛋白还包含信号肽、跨膜区或胞内信号区中的一种或多种，
    更优选地：
    所述融合蛋白从N端至C端包含：(1)信号肽、整合域和细胞因子，或(2)信号肽、整合域、功能多肽和跨膜区；
    所述功能多肽是细胞因子；优选地，细胞因子包括选自以下的一种或多种：白介素、肿瘤坏死因子(TNF)、干扰素(IFN)、集落刺激因子(CSF)和肿瘤生长因子(TGF)；
    所述白介素包括但不限于IL-2、IL-4、IL-6、IL-7、IL-8、IL-10、IL-12、IL-15、IL-18和IL-21；所述肿瘤坏死因子包括但不限于TNF-α和TNF-β；所述干扰素包括但不限于IFN-α、IFN-β和IFN-γ；所述集落刺激因子包括但不限于M-CSF、G-CSF和GM-CSF；所述肿瘤生长因子包括但不限于TGF-α、TGF-β1、TGF-β2和TGF-β3；
    所述跨膜区包括选自CD28、CD8、CD134(OX40)、CD137(4-1BB)、LCK、ICOS、DAP10、siglec-9、siglec-10、siglec-15、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、IL-2Rβ、IL-2Rγ、IL-4Rα、IL-7Rα、IL-10R、IL-12R、IL-15R、IL-21R、CD27和CD40中任一种的跨膜区。
  2. 如权利要求1所述的融合蛋白，其特征在于，
    整合域和功能多肽之间具有接头，所述接头优选具有SEQ ID NO:3所示的序列，
    信号肽是具有SEQ ID NO:1所示的序列或与其具有至少90％序列相同性 的变体，
    整合域具有SEQ ID NO:2、7或8所示的序列或与其具有至少90％序列相同性的变体，
    功能多肽具有SEQ ID NO:4所示的序列或与其具有至少90％序列相同性的变体，
    跨膜区具有SEQ ID NO:6所示的序列或与其具有至少90％序列相同性的变体，
    跨膜区与功能多肽通过铰链区连接，所述铰链区具有SEQ ID NO:5所示的序列或与其具有至少90％序列相同性的变体。
  3. 一种核酸分子，包含选自以下的序列：
    (1)权利要求1或2所述的融合蛋白的编码序列，
    (2)(1)的互补序列，和
    (3)(1)或(2)中任一序列的5-50bp的片段，
    优选地，所述片段是引物，
    优选地，所述编码序列是DNA或RNA。
  4. 一种核酸构建物，其包含权利要求3所述的核酸分子，
    优选地，所述核酸构建物是载体，例如克隆载体、表达载体或整合载体。
  5. 一种宿主细胞，所述宿主细胞：
    (1)包含、表达或分泌权利要求1或2所述的融合蛋白，和/或
    (2)包含权利要求3所述的核酸分子和/或权利要求4所述的核酸构建物，
    优选地，所述宿主细胞还分泌共刺激分子或分泌包含共刺激分子的蛋白，
    优选地，所述宿主细胞是用于免疫细胞的饲养层细胞，更优选K562。
  6. 一种培养或刺激NK细胞的方法，包括步骤：在权利要求1或2所述的融合蛋白和/或权利要求5所述的宿主细胞存在的条件下孵育NK细胞或含有NK细胞的样品，
    优选地，所述方法还包括分选NK细胞的步骤，分选标记为CD3-CD56+，
    优选地，所述孵育持续至少1周或至少10天。
  7. 一种NK细胞培养物，该培养物含有权利要求1或2所述的融合蛋白和/或权利要求5所述的宿主细胞以及适用于NK细胞的培养基。
  8. 一种药物组合物，包含由权利要求6所述方法获得的NK细胞和药学上可接受的辅料。
  9. 一种培养或刺激NK细胞的试剂盒，包含权利要求1或2所述的融合蛋白和/或权利要求5所述的宿主细胞。
  10. 试剂在制备预防和/或治疗抗肿瘤药物中的应用，所述试剂含有：(1)权利要求1或2所述的融合蛋白，或(2)权利要求3所述的核酸分子、权利要求4所述的核酸构建物和/或权利要求5所述的宿主细胞。
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