WO2020043076A1 - 一种不依赖细胞因子培养的自然杀伤细胞系silk-nk - Google Patents

一种不依赖细胞因子培养的自然杀伤细胞系silk-nk Download PDF

Info

Publication number
WO2020043076A1
WO2020043076A1 PCT/CN2019/102690 CN2019102690W WO2020043076A1 WO 2020043076 A1 WO2020043076 A1 WO 2020043076A1 CN 2019102690 W CN2019102690 W CN 2019102690W WO 2020043076 A1 WO2020043076 A1 WO 2020043076A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell line
natural killer
seq
silk
killer cell
Prior art date
Application number
PCT/CN2019/102690
Other languages
English (en)
French (fr)
Inventor
吴亚松
高斌
司远
Original Assignee
天津天锐生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 天津天锐生物科技有限公司 filed Critical 天津天锐生物科技有限公司
Publication of WO2020043076A1 publication Critical patent/WO2020043076A1/zh

Links

Images

Classifications

    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the field of adoptive immunotherapy of tumors, and in particular relates to a natural killer cell line SILK-NK which does not depend on cytokine culture and a preparation method thereof.
  • NK (Nature Killer) cells are an important class of cytotoxic killer cells in the immune system. They can release perforin and cytokines to induce cancer cell apoptosis. NK cells do not have MHC restriction or tumor-associated antigen dependency on target cells, and therefore have a broader spectrum of anti-tumor capabilities than T cells.
  • the non-MHC restriction of NK cells allows it to support allogeneic transplantation and to kill cancer cells that evade T cell killing by reducing MHC molecule expression. NK cells do not secrete a large amount of IL-6, so they do not cause a strong cytokine storm.
  • NK cells only account for about 10% of blood cells, which requires a tedious process of blood cell isolation and enrichment, and the low transfection efficiency of NK cells also increases the difficulty of genetic modification.
  • NK-92 is a NK cell line derived from patients with non-Hodgkin's lymphoma. It has the advantages of NK cells and overcomes the disadvantages of NK cells. It has both MHC non-restrictive and tumor-associated antigen-independent broad-spectrum antitumor capabilities, it is very easy to cultivate on a large scale, and it is easier to perform genetic manipulation. More importantly, the clinical phase I data of NK-92 infusion patients have shown high safety, and no serious adverse reactions occurred even at a dose of 1 ⁇ 10 10 / m 2 .
  • cytokines especially interleukin-2 (IL-2) or IL-15.
  • IL-2 stimulates Treg cell proliferation and induces T cell apoptosis, which has certain toxic and side effects in clinical applications.
  • IL-15 is an indispensable cytokine responsible for NK development, differentiation, and maintenance.
  • the NK-92 cell line expressing IL-15 showed stronger proliferation ability and cancer cell killing ability, but still could not get rid of its dependence on IL-2.
  • the purpose of the present invention is to provide a natural killer cell line SILK-NK independent of cytokine culture and a preparation method thereof, so as to change the biological characteristics of NK-92 cells, get rid of dependence on IL-2, and improve the proliferation and killing ability , Make NK-92 cells more suitable for clinical applications.
  • the present invention claims a method for preparing a natural killer cell line SILK-NK that does not rely on cytokine culture.
  • the method for preparing a natural killer cell line SILK-NK independent of cytokine culture may include the following steps: a protein complex containing an IL-15 protein and an IL-15 binding region of IL-15R ⁇ is bound by a membrane It was expressed on the surface of NK-92 cell line in this manner, and then cultured in a medium without IL-2. The resulting cloned cell line was the natural killer cell line SILK-NK.
  • the protein complex is composed of an IL-15 binding region, a transmembrane region, and an IL-15 protein of IL-15R ⁇ in sequence from the N-terminus to the C-terminus (as shown in FIG. 1).
  • the transmembrane region may be, but is not limited to, a CD8 ⁇ transmembrane region.
  • the transmembrane region is a CD8 ⁇ transmembrane region.
  • amino acid sequence of the IL-15 binding region of the IL-15R ⁇ may be SEQ ID No. 1, or the amino acid sequence shown in SEQ ID No. 1 has more than 99%, more than 95%, and 90% A sequence having the same function as above, more than 85%, more than 80%, or more than 75%, or a substitution and / or deletion and / or addition of one or several amino acid residues.
  • the amino acid sequence of the CD8 ⁇ transmembrane region may be SEQ ID No. 2, or the amino acid sequence shown in SEQ ID No.
  • the amino acid sequence of the IL-15 protein may be SEQ ID No. 3, or the amino acid sequence shown in SEQ ID No. 3 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more Or more than 75% homology, or a sequence that has the same function after substitution and / or deletion and / or addition of one or several amino acid residues.
  • amino acid sequence of the protein complex may be SEQ ID No. 4, or the amino acid sequence shown in SEQ ID No. 4 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more than 75% homology, or a sequence that has the same function after substitution and / or deletion and / or addition of one or several amino acid residues.
  • the method may include the steps of: inserting a coding gene of the protein complex into a lentiviral expression vector, and then using the obtained recombinant lentiviral vector to package a lentivirus (for example, by obtaining the obtained recombinant lentiviral vector)
  • the lentivirus was prepared by co-transfecting 293T cells with a packaging plasmid), the NK-92 cell line was infected with the lentivirus, cultured in an IL-2-free medium and obtained by limiting dilution method Said cloned cell line.
  • the IL-2-free medium is specifically GT-T551 containing a serum substitute (eg, product number: HPCFDCGL50, a product of Helios) at a final concentration of 2.5% (volume percentage).
  • a serum substitute eg, product number: HPCFDCGL50, a product of Helios
  • -H3 medium such as article number: WK593S, product of TAKARA company.
  • the nucleotide sequence of the IL-15 binding region encoding the IL-15R ⁇ may be positions 1 to 216 of SEQ ID No. 5, or the same as SEQ ID No. 5. Sequences at positions 1 to 216 of 5 having a homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more and encoding the IL-15 binding region of the IL-15R ⁇ .
  • the nucleotide sequence encoding the CD8 ⁇ transmembrane region may be positions 217-423 of SEQ ID No. 5, or positions 217-423 of SEQ ID No.
  • nucleotide sequence encoding the IL-15 protein may be positions 490-831 of SEQ ID No. 5 or positions 490-831 of SEQ ID No. 5 A sequence having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more homology and encoding the IL-15 protein.
  • the nucleotide sequence of the coding gene of the protein complex may be SEQ ID No. 5 or have SEQ ID No. 5 with 99% or more, 95% or more, 90% or more, 85% or more, 80 % Or more than 75% homology and a sequence encoding the protein complex.
  • the present invention claims the natural killer cell line SILK-NK, which is prepared by the method described above and does not rely on cytokine culture.
  • the STR phenotype of the natural killer cell line SILK-NK is
  • Amelogenin X, Y
  • mRNA reverse transcription PCR can detect the expression of IL-15 binding region of IL-15 and IL-15R ⁇ .
  • Its morphology is suspended clones or dispersed cells.
  • the natural killer cell line SILK-NK is specifically a cell line deposited in the General Microbial Center of the China Microbial Species Collection Management Committee and registered in the register number CGMCC No.15583.
  • the natural killer cell line SILK-NK may be genetically modified to express a chimeric antigen receptor A, and the modified natural killer cell line SILK-NK is referred to as cell line A; use The targeting of the chimeric antigen receptor A mediates specific killing of the cancer cell expressing the target antigen by the cell line A.
  • the natural killer cell line SILK-NK may be genetically modified to express a chimeric antigen receptor A, and the modified natural killer cell line SILK-NK is referred to as cell line A;
  • the cell line A is infused into a patient for cell therapy.
  • the present invention claims a method of treatment (tumor adoptive immunotherapy, cell therapy).
  • the treatment method claimed in the present invention is completed by using the natural killer cell line SILK-NK described above.
  • the method may include the following steps: genetically modifying the natural killer cell line SILK-NK so that it expresses a chimeric antigen receptor A, and the modified natural killer cell line SILK-NK is described as Cell line A; utilizing the targeting of the chimeric antigen receptor A to mediate the cell line A to specifically kill cancer cells expressing the target antigen.
  • the method may include the following steps: genetically modifying the natural killer cell line SILK-NK so that it expresses a chimeric antigen receptor A, and the modified natural killer cell line SILK-NK is described as Cell line A; cell therapy by injecting said cell line A into a patient.
  • the invention claims a product for adoptive tumor immunotherapy and / or cell therapy.
  • the claimed product for adoptive tumor immunotherapy and / or cell therapy contains cell line A; the cell line A is prepared according to a method including the following steps: for the natural killer cell line SILK- NK is genetically modified to express a chimeric antigen receptor A, and the natural killer cell line SILK-NK after modification is the cell line A.
  • FIG. 1 is a structural diagram of a protein complex of the present invention.
  • A IL-15 binding region of IL-15R ⁇
  • B transmembrane region
  • C IL-15.
  • FIG. 2 is a schematic structural diagram of a coding nucleic acid sequence of a protein complex of the present invention.
  • FIG. 3 is a map of a lentiviral vector loaded with the nucleic acid sequence of IL-15R ⁇ -TM-P2A-IL-15 of the present invention.
  • FIG. 4 is a morphological photograph of SILK-NK-D8 cells and NK-92 cells of the present invention.
  • A is NK-92 cells
  • B is SILK-NK-D8 cells.
  • FIG. 5 is a comparison of the doubling of 3-day increments of SILK-NK-D8 cells and NK-92 at different culture time points according to the present invention.
  • A is a 3-day multiplication curve of NK-92 plus 100 U / mL IL-2 culture;
  • B is a 3-day multiplication curve of SILK-NK-D8 without IL-2 culture.
  • FIG. 6 is an electrophoresis diagram of mRNA expression of the IL-15 binding region of IL-15 and IL-15R ⁇ identified by RT-PCR of the present invention.
  • A is NK-92 cells, and B is SILK-NK-D8 cells.
  • P1 is primer 1 + primer 2 and P2 is primer 3 + primer 4.
  • FIG. 7 is a killing experiment of NK-92 and SILK-NK-D8 cells of the present invention on K562 cells expressing GFP. Fluorescence showed K562 cells.
  • FIG. 8 is a killing experiment of NK-92 and SILK-NK-D8 cells of the present invention on K562 cells expressing firefly luciferase.
  • A is NK-92 cells
  • B is SILK-NK-D8 cells.
  • FIG. 9 is a result of STR analysis of SILK-NK-D8 cells of the present invention.
  • NK-92 cells were purchased from the Chinese Type Culture Collection Center of Wuhan University, and the culture medium was GT-T551-H3 medium (article number: WK593S, product of TAKARA company), which can be used to culture T lymphocytes and NK cells.
  • GT-T551-H3 medium article number: WK593S, product of TAKARA company
  • HPCFDCGL50 a product of Helios
  • 293T medium was supplemented with 10% (volume percentage) fetal bovine serum for DMEM.
  • pLTR-abg019 Nanjing Erbin Violet Biotechnology Co., Ltd., article number abg019.
  • pCMV-dR8.2 plasmid Addgene, Cat. No. 8455.
  • pCMV-VSV-G plasmid Addgene, Cat. No. 8454.
  • K562 cell line K562-GFP-Luc expressing GFP and luciferase Nanjing Erbinjing Biotechnology Co., Ltd., article number 562-GL.
  • Example 1 Preparation and identification of a natural killer cell line SILK-NK independent of cytokine culture
  • the DNA sequence of IL-15R ⁇ -TM-P2A-IL-15 was synthesized from Nanjing Kingsray Biotechnology Co., Ltd., with BamHI (5 ') And MluI (3'), the synthetic sequence and pLTR-abg019 plasmid were digested with BamH I and Mlu I, respectively, and then ligated and transformed into DH5 ⁇ competent bacteria.
  • the primers (SEQ ID No. 6) and the downstream primers (SEQ ID No. 7) in the sequence of IL-15 were used to identify positive clones by colony PCR and then sequenced to obtain the expression plasmid pLTR-IL15R ⁇ -IL15.
  • the structure of the recombinant plasmid pLTR-IL15R ⁇ -IL15 is described as: a recombinant plasmid obtained by replacing the small fragment between the restriction sites BamH1 and MluI of pLTR-abg019 with the DNA fragment shown in SEQ ID No.5.
  • SEQ ID No. 5 is the coding gene sequence of the IL-15R ⁇ -TM-P2A-IL-15 protein complex, in which position 1-216 is the coding gene of the IL-15 binding region of IL-15R ⁇ , positions 217-423 It is the encoding gene of CD8 ⁇ transmembrane region, and the 490-831th is encoding gene of IL-15 protein.
  • SEQ ID No. 5 encodes the protein shown in SEQ ID No. 4. Among them, positions 1 to 72 of SEQ ID No. 4 are the amino acid sequence of the IL-15 binding region of the IL-15R ⁇ (ie, SEQ ID No. 1); positions 73 to 141 of SEQ ID No. 4 are all The amino acid sequence of the CD8 ⁇ transmembrane region (ie, SEQ ID No. 2); positions 164-277 of SEQ ID No. 4 are the amino acid sequence of the IL-15 protein (ie, SEQ ID No. 3).
  • the recombinant vector pLTR-IL15R ⁇ -IL15, packaging plasmids pCMV-dR8.2, and pCMV-VSV-G constructed in step 1 were transfected into 293T cells with PEI (1 ⁇ g / ⁇ l) at a ratio (mass ratio) of 15: 10: 1.
  • Virus stock solution was added with 1/3 volume of 40% PEG, and mixed at 4 ° C overnight. Centrifuge the next day at 1800g for 45 minutes at 4 ° C. Discard the supernatant and resuspend the virus pellet with 1/10 volume of NK-92 medium (with a final concentration of 100U / ml IL-2). Concentrated virus suspension.
  • NK-92 cells Take 3 ⁇ 10 5 NK-92 cells with 1 mL of the 10-fold concentrated virus prepared in step two, resuspend and add 8 ⁇ g / ml polybrane, transfer to a well of a 24-well plate, centrifuge at 1500g at 32 ° C for 45 minutes, and transfer to carbon dioxide culture. Incubate in a box. Centrifuge the supernatant after 3 hours and replace with NK-92 medium with a final concentration of 100U / ml IL-2. Repeat the infection the next day and replace with NK with a final concentration of 100U / ml IL-2. -92 medium, and then pass it every 3 days.
  • IL-2 was not added to the medium during the passage after 2 weeks, and the culture was continued for 2 weeks.
  • the cells that survived were the target cells SILK-NK (Specific Interleukin Linked Killer).
  • the obtained cells were diluted serially in a 96-well plate, and five monoclonal cell lines were identified: A6, A9, D8, F6, F7, and clone D8 (hereinafter referred to as SILK-NK-D8) was used in the following examples.
  • SILK-NK-D8 was deposited on April 3, 2018 at the General Microbiology Center of the China Microbial Species Collection Management Committee, and its registration number is CGMCC No.15583. It is proposed to classify and name it as human natural killer cell line; Co., Ltd.) is SILK.
  • SILK-NK-D8 has a similar cell morphology as NK-92, and tends to form loose clones. As shown in Figure 5, SILK-NK-D8 can reach the medium without IL-2. The proliferation rate doubled in 2-3 days, the same as NK-92.
  • Oligo (dT) 18 was used for reverse transcription to obtain cDNA, and primers targeting the IL-15 binding region of IL-15R ⁇ (P1 is primer 1 + primer 2, P2 Primer 3 + Primer 4) PCR, as shown in Figure 6, 1% agarose gel electrophoresis showed that SILK-NK-D8 had a positive band of about 500bp in size and NK-92 did not.
  • Primer 1 5'-atgtccgtggaacacgcagac-3 '(SEQ ID No. 6);
  • Primer 2 5'-cttgaggtcg gagatgacgt tc-3 '(SEQ ID No. 7);
  • Primer 3 5'-cccagtctcaaatgcattagagacc-3 '(SEQ ID No. 8);
  • Primer 4 5'-ggtgtcatggatgctggcg-3 '(SEQ ID No. 9).
  • K562-GFP-Luc a K562 cell line expressing GFP and luciferase
  • SILK-NK-D8 and NK-92 were 10: 1, 5: 1, 1: 1 and 1 ⁇ 10 4 targets, respectively.
  • Cells were incubated in 100 ⁇ l of 96-well plates, and GFP fluorescence and luciferase were detected after 24 hours. The results are shown in Fig. 7 and Fig. 8.
  • SILK-NK-D8 and NK-92 have the same killing activity on K562 cells.
  • SILK-NK-D8 cells were tested for STR. The results are shown in Figure 9.
  • the STR phenotype is
  • mRNA reverse transcription PCR can detect the expression of IL-15 binding region of IL-15 and IL-15R ⁇ .
  • the present invention establishes a new type of killer cell called SILK-NK (Specific Interleukin Linked Killer, SILK-NK) by trying to express the complex of IL-15 and IL-15R ⁇ in the IL-15 binding region simultaneously on NK-92.
  • SILK-NK Specific Interleukin Linked Killer
  • This complex stimulates NK-92 in a membrane-bound form, cis-presented, allowing it to proliferate in a culture environment without the addition of cytokines, and on the other hand, trans-presented to stimulate the host's own T
  • cytokines and on the other hand, trans-presented to stimulate the host's own T
  • the proliferation and activation of cells and NK cells improve their own anti-tumor ability.
  • the present invention starts with cytokines that play a regulatory role in the differentiation and development of NK cells because 1.IL-15 is expressed in a variety of tissues in the human body and plays an important role in the differentiation and development of NK cells; 2.NK-92 It is an undifferentiated mature NK cell, which can express the IL-15 receptor on the cell surface; 3. The toxic and side effects of IL-15 are significantly less than that of IL-2; 4. The IL-15 binding region of IL-15R ⁇ can be used for IL-15 activity Activator.
  • the present invention infects NK-92 cells with a viral vector that expresses a membrane-bound IL-15 and IL-15R ⁇ complex of the IL-15 binding region, in order to obtain the expression of membrane-bound IL-15 and IL-15R ⁇ -IL-15 binding.
  • NK-92 cells of the complex through the trans and homeostatic activation of the IL-15 binding domain complex of IL-15 and IL-15R ⁇ , the culture of NK-92 cells is completely free of dependence on IL-2, which improves the NK-92 cells' proliferative ability, killing ability, and reduced toxic and side effects significantly increase their potential for clinical use.
  • NK-92 expressing IL-15 can increase the cytotoxicity of NK-92 cells, but its NK-92 cells still cannot get rid of its dependence on IL-2.
  • the complex that stably expresses membrane-bound IL-15 and IL-15R ⁇ -IL-15 binding region in NK-92 cells uses the super-agonist action of IL-15R ⁇ -15 binding region on IL-15.
  • the NK-92 cells can proliferate for a long time under IL-2 free culture conditions and have higher killing activity, which is conducive to large-scale culture and clinical application.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK。提供了一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK的制备方法，包括如下步骤：将含有IL-15蛋白和IL-15Rα的IL-15结合区的蛋白复合物以膜结合的方式表达于NK-92细胞系的表面，然后用无IL-2的培养基进行培养，得到的克隆化细胞系即为自然杀伤细胞系SILK-NK。该细胞系可在完全不添加IL-2的培养基中稳定传代扩增，并且能够保持NK-92的杀伤活性，因此成为具有临床应用价值的工程细胞系，可用于肿瘤的过继免疫治疗。

Description

一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK 技术领域
本发明涉及肿瘤的过继免疫治疗领域，具体涉及一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK及其制备方法。
背景技术
NK(Nature Killer)细胞是免疫系统中一类重要的细胞毒性杀伤细胞，它能够释放穿孔素和细胞因子诱导癌症细胞凋亡。NK细胞对靶细胞的识别不具有MHC限制性和肿瘤相关抗原依赖性，因此具有比T细胞更广谱的抗肿瘤能力。NK细胞的非MHC限制性使得它可以支持异体移植，并且能够杀伤那些通过降低MHC分子表达来躲避T细胞杀伤的癌细胞。NK细胞不会大量分泌IL-6，因此不会造成强烈的细胞因子风暴。但是NK细胞在血液细胞中只占10％左右，需要繁琐的血细胞分离与富集过程，而且NK细胞的转染效率较低，也增加了其基因修饰的难度。
NK-92是一株来源于非霍奇金淋巴瘤病人的NK细胞系，它具有NK细胞的优点并克服了NK细胞的缺点。它既拥有MHC非限制性和肿瘤相关抗原非依赖性的广谱抗肿瘤能力，又非常易于大规模培养，而且更容易进行基因操作。更重要的是，NK-92输注病人的临床一期数据已经显示了很高的安全性，即使在1×10 10/m 2的剂量条件下都未出现严重的不良反应。
NK细胞的增殖和激活需要细胞因子，尤其白细胞介素-2(IL-2)或IL-15。IL-2会刺激Treg细胞增殖，并诱发T细胞凋亡，在临床应用中存在一定的毒副作用。IL-15与IL-2的功能有很多相同的地方，且IL-15是负责NK发育、分化、维持而不可缺少的细胞因子。表达IL-15的NK-92细胞系表现出了更强的增殖能力和癌细胞杀伤能力，但仍然不能摆脱对IL-2的依赖。
发明公开
本发明的目的是提供一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK及其制备方法，以改变NK-92细胞的生物学特性，摆脱对IL-2的依赖，提高增殖和杀伤能力，使NK-92细胞更适于临床应用。
第一方面，本发明要求保护一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK的制备方法。
本发明所提供的不依赖细胞因子培养的自然杀伤细胞系SILK-NK的制备方法，可包括如下步骤：将含有IL-15蛋白和IL-15Rα的IL-15结合区的蛋白复合物以膜结合的方式表达于NK-92细胞系的表面，然后用无IL-2的培养基进行培养，得到的克隆化细胞系即为自然杀伤细胞系SILK-NK。
进一步地，所述蛋白复合物自N端到C端依次由IL-15Rα的IL-15结合区、跨膜区和IL-15蛋白组成(如图1所示)。
其中，所述跨膜区可为但不限于CD8α跨膜区。在本发明的具体实施例方式中，所述跨膜区为CD8α跨膜区。
更进一步地，所述IL-15Rα的IL-15结合区的氨基酸序列可为SEQ ID No.1，或者为与SEQ ID No.1所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。所述CD8α跨膜区的氨基酸序列可为SEQ ID No.2，或者为与SEQ ID No.2所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。所述IL-15蛋白的氨基酸序列可为SEQ ID No.3，或者为与SEQ ID No.3所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
更加具体的，所述蛋白复合物的氨基酸序列可为SEQ ID No.4，或者为与SEQ ID No.4所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
进一步地，所述方法可包括如下步骤：将所述蛋白复合体的编码基因插入到慢病毒表达载体，然后利用所得到的重组慢病毒载体包装出慢病毒(如通过将所得的重组慢病毒载体与包装质粒共转染293T细胞的方式制备得到所述慢病毒)，用所述慢病毒感染所述NK-92细胞系，在无IL-2的培养基中培养并通过有限稀释的方法得到所述克隆化细胞系。
在本发明的具体实施方式中，所述无IL-2的培养基具体为含有终浓度为2.5％(体积百分含量)的血清替代物(如货号：HPCFDCGL50，Helios公司产品)的GT-T551-H3培养基(如货号：WK593S，TAKARA公司产品)。
其中，所述蛋白复合体的编码基因中，编码所述IL-15Rα的IL-15结合区的核苷酸序列可为SEQ ID No.5的第1-216位，或者为与SEQ ID No.5的第1-216位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述IL-15Rα的IL-15结合区的序列。所述蛋白复合体的编码基因中，编码所述CD8α跨膜区的核苷酸序列可为SEQ ID No.5的第217-423位，或者为与SEQ ID No.5的第217-423位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述CD8α跨膜区的序列。所述蛋白复合体的编码基因中，编码所述IL-15蛋白的核苷酸序列可为SEQ ID No.5的第490-831位，或者为与SEQ ID No.5的第490-831位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述IL-15蛋白的序列。
具体地，所述蛋白复合体的编码基因的核苷酸序列可为SEQ ID No.5，或者为与SEQ ID No.5具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述蛋白复合体的序列。
第二方面，本发明要求保护由前文所述方法制备得到的不依赖细胞因子培养的自然杀伤细胞系SILK-NK。
所述自然杀伤细胞系SILK-NK的STR表型为
Amelogenin：X,Y
CSF1PO：11,12
D13S317：9,12
D16S539：11,12
D5S818：12,13
D7S820：10,11
THO1：6,9.3
TPOX：8
vWA：18
D21S11：31.2,32。
其mRNA反转录PCR可检测到IL-15及IL-15Rα的IL-15结合区的表达。
其形态为悬浮的克隆团或分散细胞。
进一步地，所述自然杀伤细胞系SILK-NK具体为保藏于中国微生物菌种保藏管理委员会普通微生物中心，登记入册编号为CGMCC No.15583的细胞系。
第三方面，前文所述的自然杀伤细胞系SILK-NK在如下任一中的应用也属于本发明的保护范围：
(A1)制备用于肿瘤过继免疫治疗的产品；
(A2)制备用于细胞治疗的相关产品；
(A3)肿瘤过继免疫治疗；
(A4)细胞治疗。
所述应用中，可对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；利用所述嵌合抗原受体A的靶向性介导所述细胞系A对表达靶抗原的癌细胞进行特异性杀伤。
所述应用中，可对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；通过将所述细胞系A给病人输注来进行细胞治疗。
第四方面，本发明要求保护一种治疗的方法(肿瘤过继免疫治疗、细胞治疗)。
本发明所要求保护的治疗方法是利用前文所述的自然杀伤细胞系SILK-NK完成的。
进一步地，所述方法可包括如下步骤：对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；利用所述嵌合抗原受体A的靶向性介导所述细胞系A对表达靶抗原的癌细胞进行特异性杀伤。
进一步地，所述方法可包括如下步骤：对所述自然杀伤细胞系SILK-NK进 行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；通过将所述细胞系A给病人输注来进行细胞治疗。
第五方面，本发明要求保护一种用于肿瘤过继免疫治疗和/或细胞治疗的产品。
本发明要求保护的用于肿瘤过继免疫治疗和/或细胞治疗的产品含有细胞系A；所述细胞系A是按照包括如下步骤的方法制备得到的：对前文所述的自然杀伤细胞系SILK-NK进行基因改造后使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK即为所述细胞系A。
保藏说明
建议分类命名：人自然杀伤细胞系
参椐的生物材料(株)：SILK
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2018年04月03日
保藏中心登记入册编号：CGMCC No.15583
附图说明
图1为本发明的蛋白复合体结构图。A：IL-15Rα的IL-15结合区，B：跨膜区，C：IL-15。
图2为本发明的蛋白复合体的编码核酸序列结构示意图。A：IL-15Rα的IL-15结合区，B：跨膜区，C：IL-15。
图3为本发明的装载了IL-15Rα-TM-P2A-IL-15核酸序列的慢病毒载体图谱。
图4为本发明的SILK-NK-D8细胞与NK-92细胞的形态照片。A是NK-92细胞，B是SILK-NK-D8细胞。
图5为本发明的SILK-NK-D8细胞与NK-92在不同培养时间点测定的3天增值倍数的比较。A是NK-92加100U/mL IL-2培养的3天增值倍数曲线图；B是SILK-NK-D8无IL-2培养的3天增值倍数曲线图。
图6为本发明的RT-PCR鉴定IL-15及IL-15Rα的IL-15结合区的mRNA表达的电泳图。A是NK-92细胞，B是SILK-NK-D8细胞。P1是引物1+引物2，P2是引物3+引物4。
图7为本发明的NK-92和SILK-NK-D8细胞对表达GFP的K562细胞的杀伤实验。荧光显示K562细胞。
图8为本发明的NK-92和SILK-NK-D8细胞对表达萤火虫荧光素酶的K562细胞的杀伤实验。A是NK-92细胞，B是SILK-NK-D8细胞。
图9为本发明的SILK-NK-D8细胞的STR分析结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
NK-92细胞购自武汉大学中国典型培养物保藏中心，培养基为可用于培养T淋巴细胞和NK细胞的GT-T551-H3培养基(货号：WK593S，TAKARA公司产品)，添加血清替代物(货号：HPCFDCGL50，Helios公司产品))至其终浓度为2.5％(体积百分含量)。
293T培养基为DMEM添加10％(体积百分含量)胎牛血清。
pLTR-abg019：南京尔宾堇生物技术有限公司，货号abg019。
pCMV-dR8.2质粒：Addgene，货号8455。
pCMV-VSV-G质粒：Addgene，货号8454。
表达GFP和荧光素酶的K562细胞系K562-GFP-Luc：南京尔宾堇生物技术有限公司，货号562-GL。
实施例1、不依赖细胞因子培养的自然杀伤细胞系SILK-NK的制备及鉴定
一、pLTR-IL15Rα-IL15慢病毒表达载体的构建
如图2和图3所示，IL-15Rα-TM-P2A-IL-15的DNA序列(SEQ ID No.5)合成自南京金斯瑞生物科技股份有限公司，两端加上了BamHI(5’)和MluI(3’)，合成的序列与pLTR-abg019质粒分别用BamH I和Mlu I双酶切后连接并转化DH5α感受态细菌，用IL-15Rα的IL-15结合区序列内的上游引物(SEQ ID No.6)和IL-15序列内的下游引物(SEQ ID No.7)做菌落PCR鉴定阳性克隆后测序鉴定得到目的表达质粒pLTR-IL15Rα-IL15。
重组质粒pLTR-IL15Rα-IL15的结构描述为：将pLTR-abg019质粒的酶切位点BamH I和Mlu I之间的小片段替换为SEQ ID No.5所示DNA片段后得到的重组质粒。
SEQ ID No.5为IL-15Rα-TM-P2A-IL-15蛋白复合体的编码基因序列，其中第1-216位为IL-15Rα的IL-15结合区的编码基因，第217-423位为CD8α跨膜区的编码基因，第490-831位为IL-15蛋白的编码基因。SEQ ID No.5编码SEQ ID No.4所示的蛋白质。其中，SEQ ID No.4的第1-72位为所述IL-15Rα的IL-15结合区的氨基酸序列(即SEQ ID No.1)；SEQ ID No.4的第73-141位为所述CD8α跨膜区的氨基酸序列(即SEQ ID No.2)；SEQ ID No.4的第164-277位为所述IL-15蛋白的氨基酸序列(即SEQ ID No.3)。
二、pLTR-IL15Rα-IL15质粒包装病毒
将步骤一构建的重组载体pLTR-IL15Rα-IL15、包装质粒pCMV-dR8.2和pCMV-VSV-G以15：10：1的比例(质量比)用PEI(1μg/μl)转染293T细胞。
具体实施为：第0天传代293T细胞8×10 6个细胞至T75培养瓶；第1天：1ml Opti-MEM中加入三种质粒pLTR-IL15Rα-IL15：pCMV-dR8.2： pCMV-VSV-G＝15μg：10μg：1μg，混匀后加入78μl PEI，震荡混匀后室温放置15分钟，之后加入到培养293T的T75培养瓶中，轻柔摇晃混匀。转染后12-16小时换新鲜培养基。转染后48小时即第3天收集培养基，0.45μM滤膜过滤去除细胞得到病毒原液。病毒原液添加1/3体积的40％PEG，混匀后4℃放置过夜。第二天离心，4℃，1800g，45分钟，弃上清后用病毒原液1/10体积的NK-92培养基(添加终浓度为100U/ml IL-2)重悬病毒沉淀，得到10倍浓缩的病毒悬液。
三、感染NK-92细胞
取3×10 5NK-92细胞用1mL步骤二制备得到的10倍浓缩病毒重悬并添加8μg/ml polybrane，转移到24孔板一个孔中，32℃，1500g离心45分钟后转移到二氧化碳培养箱培养，3小时后离心去上清，换添加终浓度为100U/ml IL-2的NK-92培养基；第二天再重复一次感染后换添加终浓度为100U/ml IL-2的NK-92培养基，之后保持3天传代一次。2周后传代时培养基不再添加IL-2，继续培养两周，活下来的细胞即为得到的目的细胞SILK-NK(Specific Interleukin Linked Killer)。得到的细胞在96孔板做梯度稀释，鉴定出5个单克隆细胞系：A6，A9，D8，F6，F7，后续实施例中是用的克隆D8(后续称为SILK-NK-D8)。
SILK-NK-D8于2018年04月03日保藏于中国微生物菌种保藏管理委员会普通微生物中心，登记入册编号为CGMCC No.15583，建议分类命名为人自然杀伤细胞系；参椐的生物材料(株)为SILK。
四、细胞形态与增殖
如图4，SILK-NK-D8具有和NK-92类似的细胞形态，倾向于形成松散的克隆团，如图5所示，SILK-NK-D8在不添加IL-2的培养基中可以达到2-3天倍增的增殖速率，与NK-92相同。
五、外源基因表达鉴定
SILK-NK-D8和NK-92分别提取总RNA，用Oligo(dT)18做反转录得到cDNA，用针对IL-15Rα的IL-15结合区的引物(P1是引物1+引物2，P2是引物3+引物4)做PCR，如图6所示，1％琼脂糖胶电泳显示SILK-NK-D8有约500bp大小的阳性条带而NK-92没有。
引物1：5’-atgtccgtggaacacgcagac-3’(SEQ ID No.6)；
引物2：5’-cttgaggtcg gagatgacgt tc-3’(SEQ ID No.7)；
引物3：5’-cccagtctcaaatgcattagagacc-3’(SEQ ID No.8)；
引物4：5’-ggtgtcatggatgctggcg-3’(SEQ ID No.9)。
六、细胞杀伤能力检测
用表达GFP和荧光素酶的K562细胞系K562-GFP-Luc做靶细胞，SILK-NK-D8和NK-92分别以10:1,5:1,1:1的比例与1×10 4靶细胞在96孔板100μl体系中孵育，24小时后检测GFP荧光和荧光素酶。结果如图7和图8所示，SILK-NK-D8与NK-92对K562细胞有相同的杀伤活性。
SILK-NK-D8细胞做STR检测，结果如图9所示，STR表型为
“Amelogenin：X,Y
CSF1PO：11,12
D13S317：9,12
D16S539：11,12
D5S818：12,13
D7S820：10,11
THO1：6,9.3
TPOX：8
vWA：18
D21S11：31.2,32”。
其mRNA反转录PCR可检测到IL-15及IL-15Rα的IL-15结合区的表达。
以上述依据本发明的理想实施例为启示，通过上述的说明内容，相关工作人员完全可以在不偏离本项发明技术思想的范围内，进行多样的变更以及修改。本项发明的技术性范围并不局限于上述具体实施方试中的内容，必须要根据权利要求和说明书范围来确定其技术性范围。
工业应用
本发明通过尝试将IL-15与IL-15Rα的IL-15结合区复合物同时表达于NK-92，建立一种称作SILK-NK的新型杀伤细胞(Specific Interleukin Linked Killer,SILK-NK)，利用IL-15Rα的IL-15结合区作为IL-15的超级激活剂，用IL-15与IL-15Rα的IL-15结合区组成的复合体来激活NK-92细胞并提高NK-92细胞的癌细胞杀伤能力。此复合体一方面以膜结合的形式顺式(cis-presented)刺激NK-92，使其在没有添加细胞因子的培养环境下得以增殖，另一方面反式(trans-presented)刺激宿主自体T细胞、NK细胞的增殖与激活，提高其自身抗肿瘤能力。
本发明从NK细胞分化发育过程中起调节作用的细胞因子入手，因为1.IL-15在人体内多种组织都有表达，对NK细胞的分化发育过程起着重要作用；2.NK-92是未分化成熟的NK细胞，细胞表面能够表达IL-15受体；3.IL-15的毒副作用显著小于IL-2；4.IL-15Rα的IL-15结合区能够作为IL-15活性的激活剂。所以，本发明采用表达膜结合的IL-15与IL-15Rα的IL-15结合区复合体的病毒载体感染NK-92细胞，以期获得表达膜结合IL-15与IL-15Rα的IL-15结合区复合体的NK-92细胞，通过IL-15与IL-15Rα的IL-15结合区复合体的反式和顺势激活作用，使NK-92细胞的培养完全摆脱对IL-2的依赖，提高NK-92细胞的增殖能力、杀伤能力以及降低毒副作用，显著提高其在临床上的使用潜力。
现有技术使NK-92表达IL-15可以提高NK-92的细胞杀伤毒性，但其NK-92 细胞仍不能摆脱对IL-2的依赖。本发明在NK-92细胞稳定表达膜结合的IL-15与IL-15Rα的IL-15结合区的复合体，利用IL-15Rα的IL-15结合区对IL-15的超级激动剂作用，可以使NK-92细胞在无IL-2的培养条件下长期增殖并拥有更高的杀伤活性，有利于大规模培养和临床应用。

Claims (24)

  1. 一种不依赖细胞因子培养的自然杀伤细胞系SILK-NK的制备方法，包括如下步骤：将含有IL-15蛋白和IL-15Rα的IL-15结合区的蛋白复合物以膜结合的方式表达于NK-92细胞系的表面，然后用无IL-2的培养基进行培养，得到的克隆化细胞系即为自然杀伤细胞系SILK-NK。
  2. 根据权利要求1所述的方法，其特征在于：所述蛋白复合物自N端到C端依次由IL-15Rα的IL-15结合区、跨膜区和IL-15蛋白组成。
  3. 根据权利要求1或2所述的方法，其特征在于：所述IL-15Rα的IL-15结合区的氨基酸序列为SEQ ID No.1，或者为与SEQ ID No.1所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
  4. 根据权利要求1-3中任一所述的方法，其特征在于：所述跨膜区为CD8α跨膜区。
  5. 根据权利要求4所述的方法，其特征在于：所述CD8α跨膜区的氨基酸序列为SEQ ID No.2，或者为与SEQ ID No.2所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
  6. 根据权利要求1-5中任一所述的方法，其特征在于：所述IL-15蛋白的氨基酸序列为SEQ ID No.3，或者为与SEQ ID No.3所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
  7. 根据权利要求1-6中任一所述的方法，其特征在于：所述蛋白复合物的氨基酸序列为SEQ ID No.4，或者为与SEQ ID No.4所示的氨基酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性，或者经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的序列。
  8. 根据权利要求1-7中任一所述的方法，其特征在于：所述方法包括如下步骤：将所述蛋白复合体的编码基因插入到慢病毒表达载体，然后利用所得到的重组慢病毒载体包装出慢病毒，用所述慢病毒感染所述NK-92细胞系，在无IL-2的培养基中培养并通过有限稀释的方法得到所述克隆化细胞系。
  9. 根据权利要求8所述的方法，其特征在于：所述无IL-2的培养基具体为含有终浓度为2.5％(体积百分含量)的血清替代物的GT-T551-H3培养基。
  10. 根据权利要求8或9所述的方法，其特征在于：所述蛋白复合体的编码基因中，编码所述IL-15Rα的IL-15结合区的核苷酸序列为SEQ ID No.5的第1-216位，或者为与SEQ ID No.5的第1-216位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述IL-15Rα的IL-15结 合区的序列。
  11. 根据权利要求1-10中任一所述的方法，其特征在于：所述蛋白复合体的编码基因中，编码所述CD8α跨膜区的核苷酸序列为SEQ ID No.5的第217-423位，或者为与SEQ ID No.5的第217-423位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述CD8α跨膜区的序列。
  12. 根据权利要求1-11中任一所述的方法，其特征在于：所述蛋白复合体的编码基因中，编码所述IL-15蛋白的核苷酸序列为SEQ ID No.5的第490-831位，或者为与SEQ ID No.5的第490-831位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述IL-15蛋白的序列。
  13. 根据权利要求1-12中任一所述的方法，其特征在于：所述蛋白复合体的编码基因的核苷酸序列为SEQ ID No.5，或者为与SEQ ID No.5具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上同源性且编码所述蛋白复合体的序列。
  14. 由权利要求1-13中任一所述方法制备得到的不依赖细胞因子培养的自然杀伤细胞系SILK-NK。
  15. 根据权利要求14所述的自然杀伤细胞系SILK-NK，其特征在于：所述自然杀伤细胞系SILK-NK的STR表型为
    Amelogenin：X,Y
    CSF1PO：11,12
    D13S317：9,12
    D16S539：11,12
    D5S818：12,13
    D7S820：10,11
    THO1：6,9.3
    TPOX：8
    vWA：18
    D21S11：31.2,32；
    其mRNA反转录PCR能检测到IL-15及IL-15Rα的IL-15结合区的表达。
  16. 根据权利要求14或15所述的自然杀伤细胞系SILK-NK，其特征在于：所述自然杀伤细胞系SILK-NK的形态为悬浮的克隆团或分散细胞。
  17. 根据权利要求14-16中任一所述的自然杀伤细胞系SILK-NK，其特征在于：所述自然杀伤细胞系SILK-NK为保藏于中国微生物菌种保藏管理委员会普通微生物中心，登记入册编号为CGMCC No.15583的细胞系。
  18. 权利要求14-17中任一所述的自然杀伤细胞系SILK-NK在如下任一中的应用：
    (A1)制备用于肿瘤过继免疫治疗的产品；
    (A2)制备用于细胞治疗的相关产品；
    (A3)肿瘤过继免疫治疗；
    (A4)细胞治疗。
  19. 根据权利要求18所述的应用，其特征在于：所述应用中，对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；利用所述嵌合抗原受体A的靶向性介导所述细胞系A对表达靶抗原的癌细胞进行特异性杀伤。
  20. 根据权利要求18所述的应用，其特征在于：所述应用中，对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；通过将所述细胞系A给病人输注来进行细胞治疗。
  21. 一种治疗的方法，是利用权利要求14-17中所述的自然杀伤细胞系SILK-NK完成的。
  22. 根据权利要求21所述的方法，其特征在于：所述方法包括如下步骤：对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；利用所述嵌合抗原受体A的靶向性介导所述细胞系A对表达靶抗原的癌细胞进行特异性杀伤。
  23. 根据权利要求21所述的方法，其特征在于：所述方法包括如下步骤：对所述自然杀伤细胞系SILK-NK进行基因改造使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK，记为细胞系A；通过将所述细胞系A给病人输注来进行细胞治疗。
  24. 一种用于肿瘤过继免疫治疗和/或细胞治疗的产品，含有细胞系A；所述细胞系A是按照包括如下步骤的方法制备得到的：对权利要求14-17中任一所述的自然杀伤细胞系SILK-NK进行基因改造后使其表达一个嵌合抗原受体A，改造后的所述自然杀伤细胞系SILK-NK即为所述细胞系A。
PCT/CN2019/102690 2018-08-27 2019-08-27 一种不依赖细胞因子培养的自然杀伤细胞系silk-nk WO2020043076A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810981706.9 2018-08-27
CN201810981706.9A CN110862967A (zh) 2018-08-27 2018-08-27 一种不依赖细胞因子培养的自然杀伤细胞系silk-nk

Publications (1)

Publication Number Publication Date
WO2020043076A1 true WO2020043076A1 (zh) 2020-03-05

Family

ID=69642895

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/102690 WO2020043076A1 (zh) 2018-08-27 2019-08-27 一种不依赖细胞因子培养的自然杀伤细胞系silk-nk

Country Status (2)

Country Link
CN (1) CN110862967A (zh)
WO (1) WO2020043076A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12104178B2 (en) 2017-03-03 2024-10-01 Obsidian Therapeutics, Inc. DHFR tunable protein regulation

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105408473A (zh) * 2013-05-14 2016-03-16 得克萨斯州大学系统董事会 工程化嵌合抗原受体(car)t细胞的人应用
CN106459924A (zh) * 2014-04-23 2017-02-22 得克萨斯州大学系统董事会 用于疗法中的嵌合抗原受体(car)及其制备方法
WO2017214333A1 (en) * 2016-06-08 2017-12-14 Intrexon Corporation Cd33 specific chimeric antigen receptors
WO2017222593A1 (en) * 2016-06-24 2017-12-28 Icell Gene Therapeutics Llc Chimeric antigen receptors (cars), compositions and methods thereof
WO2018058067A1 (en) * 2016-09-23 2018-03-29 The Regents Of The University Of California Autologous irradiated whole cell tumor vaccines lentivirally engineered to express cd80, il-15 and il-15 receptor alpha
CN108250302A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多功能蛋白质
CN108250301A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多靶点嵌合抗原受体
CN108264567A (zh) * 2016-12-30 2018-07-10 天津天锐生物科技有限公司 一种识别cd19阳性肿瘤的嵌合抗原受体及细胞

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1777294A1 (en) * 2005-10-20 2007-04-25 Institut National De La Sante Et De La Recherche Medicale (Inserm) IL-15Ralpha sushi domain as a selective and potent enhancer of IL-15 action through IL-15Rbeta/gamma, and hyperagonist (IL15Ralpha sushi -IL15) fusion proteins

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105408473A (zh) * 2013-05-14 2016-03-16 得克萨斯州大学系统董事会 工程化嵌合抗原受体(car)t细胞的人应用
CN106459924A (zh) * 2014-04-23 2017-02-22 得克萨斯州大学系统董事会 用于疗法中的嵌合抗原受体(car)及其制备方法
WO2017214333A1 (en) * 2016-06-08 2017-12-14 Intrexon Corporation Cd33 specific chimeric antigen receptors
WO2017222593A1 (en) * 2016-06-24 2017-12-28 Icell Gene Therapeutics Llc Chimeric antigen receptors (cars), compositions and methods thereof
WO2018058067A1 (en) * 2016-09-23 2018-03-29 The Regents Of The University Of California Autologous irradiated whole cell tumor vaccines lentivirally engineered to express cd80, il-15 and il-15 receptor alpha
CN108250302A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多功能蛋白质
CN108250301A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多靶点嵌合抗原受体
CN108264567A (zh) * 2016-12-30 2018-07-10 天津天锐生物科技有限公司 一种识别cd19阳性肿瘤的嵌合抗原受体及细胞

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12104178B2 (en) 2017-03-03 2024-10-01 Obsidian Therapeutics, Inc. DHFR tunable protein regulation

Also Published As

Publication number Publication date
CN110862967A (zh) 2020-03-06

Similar Documents

Publication Publication Date Title
CN105624107B (zh) 一种多种淋巴细胞亚群的扩增方法及其应用
CN111566221B (zh) 用于nk细胞转导的方法
CN110464841B (zh) 免疫增强的药物组合物及其应用
US9670459B2 (en) Production method for cell populations
CN112204133B (zh) Car nk细胞
WO2012027376A2 (en) Method for expansion of stem cells and the use of such cells
WO2009139413A1 (ja) サイトカイン誘導キラー細胞含有細胞集団の製造方法
CN107624119B (zh) 一种分子、表达其的细胞及其制备方法和用途
EP3170896B1 (en) Production method for pluripotent stem cells having antigen-specific t cell receptor gene
CN111139222B (zh) 一种重组间充质干细胞及其制备方法和用途
WO2005019450A1 (ja) 細胞傷害性リンパ球の製造方法
CN112210539A (zh) 一种第四代car-t细胞及其应用
WO2020043076A1 (zh) 一种不依赖细胞因子培养的自然杀伤细胞系silk-nk
CN114729320B (zh) 用于将细胞重新程序化为能够呈递抗原的树突细胞2型的组合物、方法及其用途
CN104892770A (zh) 一种对t细胞和造血干细胞具有高效感染和促增殖能力的慢病毒载体
JP5911810B2 (ja) 制御性ｔ細胞の製造方法
WO2020164166A1 (zh) 一种通用型car-t细胞及其制备方法和用途
CN114249807B (zh) 缺氧触发的人工转录因子、转录控制系统及其应用
CN116254230A (zh) 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途
CN117157391A (zh) 由用于导入T细胞受体基因的iPS细胞构成的细胞库
CN113122555A (zh) 嵌合抗原受体(CAR)修饰的γδT细胞的制备方法、产品、抗肿瘤药物及其应用
CN112592898A (zh) 一种重编程nk饲养细胞及其制备方法和应用
CN112608901B (zh) 一种人工抗原呈递细胞及其制备方法和应用
CN117003855B (zh) 一种t细胞受体及其应用
WO2024190905A1 (ja) Ｈｐｖ特異的細胞傷害性ｔ細胞のｔ細胞受容体又はその機能的断片

Legal Events

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

Ref document number: 19853522

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

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

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 205A DATED 08.06.2021)

122 Ep: pct application non-entry in european phase

Ref document number: 19853522

Country of ref document: EP

Kind code of ref document: A1