WO2018028157A1 - 一种vc-car分子及在清除hiv-1感染细胞中的应用 - Google Patents

一种vc-car分子及在清除hiv-1感染细胞中的应用 Download PDF

Info

Publication number
WO2018028157A1
WO2018028157A1 PCT/CN2017/072266 CN2017072266W WO2018028157A1 WO 2018028157 A1 WO2018028157 A1 WO 2018028157A1 CN 2017072266 W CN2017072266 W CN 2017072266W WO 2018028157 A1 WO2018028157 A1 WO 2018028157A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
car
car molecule
hiv
cell
Prior art date
Application number
PCT/CN2017/072266
Other languages
English (en)
French (fr)
Inventor
张辉
刘炳峰
邹帆
Original Assignee
中山大学
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 中山大学 filed Critical 中山大学
Priority to US16/322,470 priority Critical patent/US10633432B2/en
Publication of WO2018028157A1 publication Critical patent/WO2018028157A1/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
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • 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
    • 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
    • C12N5/0636—T lymphocytes
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env

Definitions

  • the invention relates to the technical field of tumor immunotherapy, in particular to a VC-CAR molecule and application thereof in eliminating HIV-1 infected cells.
  • HIV-1 infection human immunodeficiency virus type 1 (HIV-1) infection
  • cART combined antiretroviral therapy
  • the current research hotspot is to activate latently infected viruses through specific latent-reversing agents (LRAs), and then to treat or induce the body's immune system to kill infected cells.
  • LRAs latent-reversing agents
  • This intervention strategy is called "shock and kill.”
  • HIV-1 can rapidly mutate to evade immune recognition.
  • Studies have shown that among those infected with cART, even if they successfully activate their latent infection, CD8+ T lymphocytes in the body cannot completely eliminate the infected cells due to the lack of an effective immune response to HIV-1. Therefore, in the "shock and kill” strategy, in order to better clear the latent infection repository, it is necessary to reconstruct a strong immune monitoring function in the infected person.
  • CAR chimeric antigen receptor
  • This strategy can also be applied to antiviral therapy, including the treatment of viral infections such as HIV-1, HBV (hepatitis B virus) and HCV (hepatitis C virus).
  • viral infections such as HIV-1, HBV (hepatitis B virus) and HCV (hepatitis C virus).
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • the present invention provides a novel CAR molecule, called a VC-CAR molecule, which is a scFv sequence derived from HIV-1 broad-spectrum neutralizing antibody VRC01 and a conventional CAR molecule.
  • the intracellular sequences are joined together as extracellular and intracellular structures, respectively.
  • Another object of the invention is to provide a CD8 + T cell engineered with a VC-CAR molecule.
  • HIV-1 broad-spectrum neutralizing antibody VRC01-derived scFv sequence shown in SEQ ID NO: 1 in the extracellular antigen binding domain of a CAR molecule.
  • a VC-CAR molecule which is composed of the scFv sequence derived from the HIV-1 broad-spectrum neutralizing antibody VRC01 represented by SEQ ID NO: 1 and the intracellular sequence of the CAR molecule, and the HIV-1 broad-spectrum neutralizing antibody VRC01
  • the source scFv sequence is at the N-terminus and the CAR molecule intracellular sequence is at the C-terminus.
  • a VC-CAR molecule having a nucleotide sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.
  • the structure of the VC-CAR molecule shown in SEQ ID NO: 2 is shown as VR C01-28BBZ-1 in Figure 1; the structure of the VC-CAR molecule shown in SEQ ID NO: 3 is shown in Figure 1 as VR C01- 28BBZ-2; the structure of the VC-CAR molecule shown by SEQ ID NO: 4 is shown as VR C01-28BBZ-3 in FIG.
  • VC-CAR molecule having a nucleotide sequence as set forth in SEQ ID NO:4.
  • An engineered CD8 + T cell specifically prepared using the VC-CAR molecule transduced CD8 + T cells designed in accordance with the present invention.
  • the cleavage effect of the CD8 + cell-mediated expression of the gp120 cell line by VC-CAR was more pronounced.
  • the invention also claims the use of CD8 + T cells engineered with VC-CAR molecules for the clearance of HIV-1 infected cells.
  • a modified CD8 + T cell was prepared by the following methods: (1) collecting peripheral blood mononuclear cells and isolating CD8 + T cells enriched therein, and activating CD8 + using anti-CD3, anti-CD28 and IL-2 T cells; (2) 48 hours after cell activation, the VC-CAR molecular recombinant virus concentrate was added at a ratio of 1 ml / 1 ⁇ 10 6 cells for infection, and the polybrene solution was added, and the culture was continued after centrifugation; 8-12 After a few hours, a second round of viral infection was performed.
  • the concentration of the anti-CD3 is 1 ⁇ g/mL
  • the concentration of anti-CD28 is 1 ⁇ g/mL
  • the concentration of IL-2 is 10 ng/mL.
  • the concentration of the polybrene solution is 8 ⁇ g/mL.
  • a method for amplifying VC-CAR molecularly engineered CD8 + T cells comprises the following steps: (1) CD8 + T cells are infected by VC-CAR molecule recombinant virus for 12 hours after transformation, centrifuged, washed and cultured The virus in the base, resuspended the cells in fresh medium, and added IL-2 and IL-7 to maintain the cell state; (2) on the 3rd and 5th day after the viral infection transformation, according to the cell state and proliferation, The cells were supplemented with complete RMPI1640 medium, the cell concentration was maintained at 2 ⁇ 10 6 /ml, and IL-2 and IL-7 were supplemented, and the cells were further cultured and passaged in time to further expand the cells.
  • the concentration of IL-2 is 10 ng/mL and the concentration of IL-7 is 10 ng/mL.
  • the present invention has the following beneficial effects:
  • the newly engineered VC-CAR-T cells of the present invention are co-cultured with cell lines expressing HIV-1 envelope protein or HIV-1 infected CD4 + T cells, and VC-CAR-T cells can be specifically activated and abundantly Cytokines associated with cytotoxicity (including IFN- ⁇ , and Granzyme B) are secreted, which in turn strongly mediates lysis of target cells.
  • the newly engineered VC-CAR-T cells in the present invention have a more pronounced cleavage effect on the gp120 cell line mediated by VC-CAR-T cells than the reported CD4-CAR-T cells.
  • the IL-2+IL-7 cytokine combination used in the present invention can further increase the CD8 + T cell expansion factor of HIV-1 infected patients compared to the conventional amplification method of applying IL-2 alone.
  • Figure 1 is a schematic view showing the structure of a constructed VC-CAR molecule.
  • Figure 2 is a schematic diagram showing the structure of the pCPPT-IRES-mStrewbeery lentiviral vector.
  • Figure 3 shows the expression of VC-CAR in CD8 + T cells.
  • Figure 4 is a comparison of the killing effect of VC-CAR and CD4-CAR engineered CD8 + T cells mixed with target cell lines, respectively.
  • Figure 5 shows the secretion of IFN- ⁇ and Granzyme-B after mixed culture of VC-CAR-T cells with target cell lines expressing HIV-1 envelope protein.
  • Figure 6 shows the killing activity of VC-CAR-T cells by detecting the release of LDH after mixed culture of VC-CAR-T cells with target cell lines expressing HIV-1 envelope protein.
  • Figure 7 shows the ratio of VC-CAR-T cells mixed with wild-type HIV-1 NL4-3 infected CD4 + T lymphocytes by flow cytometry to detect Gag + CD4 + T lymphocytes. The killing activity of VC-CAR-T cells was verified.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • a VC-CAR molecule having VRC01 as an extracellular antigen binding domain the nucleotide sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.
  • the structure of the VC-CAR molecule shown in SEQ ID NO: 2 is shown as VR C01-28BBZ-1 in Figure 1; the structure of the VC-CAR molecule shown in SEQ ID NO: 3 is shown in Figure 1 as VR C01- 28BBZ-2; the structure of the VC-CAR molecule shown by SEQ ID NO: 4 is shown as VR C01-28BBZ-3 in FIG.
  • HIV-1 broad-spectrum neutralizing antibody VRC01-derived scFv sequence is shown in SEQ ID NO: 1.
  • the structure design of the VC-CAR molecule of the present invention is described in detail below with the VC-CAR molecule shown in SEQ ID NO: 4.
  • the sequence N' end of the VC-CAR molecule is the scFv sequence derived from the HIV-1 broad-spectrum neutralizing antibody VRC01, which specifically binds to the CD4 binding domain of the viral envelope protein; the C-end of the VC-CAR molecular sequence is a three-generation CAR structure.
  • CD8 nucleotides 460-660, GenBank NM_006139.3
  • CD137 nucleotides 640-765, GenBank NM_001561.5
  • CD3 ⁇ nucleotides 160-492, Genbank NM_198053.2
  • VC-CAR molecular recombination pCPPT-IRES-mStrewbeery lentiviral vector
  • VC-CAR was synthesized according to the sequence shown in SEQ ID NO: 4, and the synthesized VC-CAR was digested with NheI and NotI, and inserted into pCPPT-IRES-mStrewbeery.
  • the viral vector see Fig. 2
  • specific steps of digestion and ligation are referred to conventional methods in the art
  • pCPPT-IRES-mStrewbeery lentiviral vector after recombinant VC-CAR molecule is obtained.
  • HEK293T cells A growing state of HEK293T cells was plated evenly in a 10 cm culture dish treated with polylysine (cell density of about 6.5 x 10 4 /cm 2 ), requiring a single uniform distribution of cells. After about 24 hours of culture, the cell confluence should be close to 80%, at which time each dish was exchanged with 12 ml of complete medium while adding 3.75 ul of chloroquine (100 mM, 4000 x). After changing the solution, prepare the calcium phosphate-DNA mixture according to Table 1. After mixing for several times, let stand for 1 minute, then quickly add the cells to the cell culture medium at 3 ml/dish, shake while stirring, and add quickly by drop.
  • Table 1 shows the formula of calcium phosphate-DNA mixture system
  • each dish was changed with 12 ml of fresh medium, and 90 ⁇ l of sodium butyrate (1 M, 100 ⁇ ) was added.
  • 90 ⁇ l of sodium butyrate (1 M, 100 ⁇ ) was added.
  • all 34 ml HEK293T cell supernatants were collected, filtered through a 0.45 ⁇ m filter, and placed in a 50 ml centrifuge tube.
  • the mixture was added to the PEG-NaCl-PBS mixture in the same manner as in Table 2, and the mixture was inverted and mixed. Leave at °C for 1.5 hours, mix once every 20 to 30 minutes or mix by inversion and place at 4 °C overnight.
  • the mixture was centrifuged at 7000 g for 10 minutes at 4 ° C. A white precipitate was observed on the tube wall. All the supernatant was carefully removed, and an appropriate volume (300 ul to 3 ml) of fresh medium was added, and the precipitate was dissolved by shaking gently to obtain VC-
  • the recombinant virus concentrate of CAR is immediately used or stored at -80 ° C.
  • CD8 + T cells Peripheral blood mononuclear cells were isolated from peripheral blood samples, and CD8 + T lymphocytes were isolated and enriched by biotinylated CD8 antibody, counted, and centrifuged. Resuspend in RPMI1640 complete medium and spread evenly to the cell culture plate at a cell concentration of 2 x 10 6 /ml. Cells were stimulated with anti-CD3 (final concentration 1 ⁇ g/ml), anti-CD28 (final concentration 1 ⁇ g/ml) antibody and IL2 (final concentration 10 ng/ml). After 48 hours, cells were harvested and used for The infection of a pseudovirus.
  • Appropriate amount of the target cell suspension in good growth state was placed in a centrifuge tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded, and the pseudovirus concentrate was added at a ratio of 1 ml/1 ⁇ 10 6 cells, while adding Polybrene at a final concentration of 8 ⁇ g/ml, and gently pipetting and mixing. The cell suspension was transferred to a Petri dish, centrifuged at 350 x g for 90 minutes at 37 ° C, and after centrifugation, the culture was carried out by returning to the incubator. After about 8-12 hours, the second round of infection was carried out. The procedure was the same as above.
  • the solution was centrifuged, the virus in the medium was washed away with PBS, and the cells were resuspended in fresh medium and added to a final concentration of 10 ng. /ml of IL-2 and IL-7 maintain cell status.
  • the cells were completely RMPI1640-cultured according to the state of the cells and the proliferation, and the cell concentration was maintained at 2 ⁇ 10 6 /ml. And supplemented with IL-2 and IL-7, the final concentration was 10ng/ml.
  • the cells were expanded according to the procedure on the third day.
  • VC-CAR-T cells The expression of the fluorescent protein mStrewbeery was detected by flow cytometry to determine the infection efficiency of VC-CAR, and it was usually necessary to ensure that the infection rate reached 50% or more (Fig. 3). On day 5 post-infection, the cells were used for subsequent experimental testing or continued culture and cryopreservation. Hereinafter, for the sake of convenience, the following experiments are referred to as VC-CAR-T cells.
  • CD4-CAR CAR-T cells
  • CD4-CAR-T cells Since the CD4 molecule is the natural receptor for the HIV-1 envelope glycoprotein gp120, CD4-CAR-T cells are capable of cleaving target cells expressing gp120.
  • CD4-CAR-T cells we constructed CD4-CAR by replacing the scFv sequence of VC-CAR with the extracellular domain of CD4 molecule according to previous reports.
  • CD8 + T lymphocytes were transduced under the same conditions, and then mixed with a target cell line expressing HIV-1 envelope protein (Jurkat-gp160 NL4-3 ), and a cell killing experiment was carried out in a U-bottom 96-well plate.
  • the target cell number was 10 4 /well and the RMPI 1640 complete medium volume was 200 ⁇ l/well.
  • the release activity of the modified CD8 + T lymphocytes of each experimental group was determined by detecting the release of lactate dehydrogenase within 24 hours after a given target ratio (8:1 to 2:1).
  • the results showed that VC-CAR-T cells have stronger target cell lysis ability than CD4-CAR-T cells.
  • the likely result of this result is that the HIV-1 broad-spectrum neutralizing antibody-derived scFv has a stronger affinity for HIV-1 gp120 than the native CD4 molecule (see Figure 4).
  • VC-CAR-T cells were mixed with Jurkat-gp160 NL4-3 for 24 hours, and within a given range of effective target ratios (4:1-2:1), granzyme B ELISA experiments also showed that with the target Increased cells (Jurkat-gp160 NL4-3 ), cytokine secretion by VC-CAR-T cells increased in a dose-dependent manner, whereas control target cells (Jurkat-GFP) did not stimulate VC-CAR-T The cells secrete IL-2 and granzyme B. The results indicate that VC-CAR-T cells have the ability to efficiently secrete antiviral cytokines under specific antigen stimulation (Fig. 5).
  • Detection of killing activity of VC-CAR-T cells mixed with target cell lines expressing HIV-1 envelope protein To further detect the function of VC-CAR, we expressed VC-CAR-T cells with two strains of HIV.
  • the target cell number was 10 4 /well and the RMPI 1640 complete medium volume was 200 ⁇ l/well.
  • We tested the cytotoxicity of VC-CAR-T cells against HIV-1 envelope protein-expressing cells by the release of LDH within a given range of efficacy targets (8:1 - 0.5:1).
  • VC-CAR-T cells significantly killed two target cells expressing HIV-1 gp120 in a dose-dependent manner (Jurkat-gp160 NL4-3 and in a dose-dependent range from 8:1 to 0.5:1).
  • Jurkat-gp160 BaL while the control target cells (Jurkat-GFP) had no significant killing effect, indicating that the effect of VC-CAR-T cell killing target cells is HIV-1 gp120 specific.
  • Jurkat-GFP the control effector cells showed no significant killing effect, further illustrating the specificity of VC-CAR-T cell action (Fig. 6).
  • Detection of killing activity of VC-CAR-T cells mixed with wild-type HIV-1 NL4-3 infected CD4 + T lymphocytes To further demonstrate VC-CAR in clearing wild-type HIV-1 infected cells -T cell availability, using wild-type HIV-1 NL4-3 to infect CD4 + T lymphocytes isolated from healthy human blood samples.
  • the volume of RMPI1640 complete medium at the time of infection was 1 ml/well (24-well plate) containing 2 x 10 6 cells corresponding to 200 ng (p24) of wild-type virus. Change the fluid 3 hours after infection.
  • the cells were mixed with VC-CAR engineered homologous CD8 + T lymphocytes at a ratio of 1:2 and 1:4, and cell killing experiments were performed in 24-well plates.
  • the target cell number was 10 6 /well, and the RMPI 1640 complete medium volume was 500 ⁇ l/well.
  • the ratio of Gag + CD4 + T lymphocytes was measured by flow cytometry to verify the killing effect of VC-CAR-T cells.
  • the results showed that VC-CAR-T cells had a clearance rate of 78% for HIV-1 infected cells, showing a significant killing effect, while the value of control effector cells was less than 30%. The difference was statistically significant (Fig. 7 ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • AIDS & HIV (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)

Abstract

本发明属于肿瘤免疫治疗技术领域，具体公开了一种VC-CAR分子及在清除HIV-1感染细胞中的应用。所述VC-CAR分子是将HIV-1广谱中和抗体VRC01来源的scFv序列和传统的CAR分子的胞内段序列连接在一起，分别作为胞外和胞内结构。利用VC-CAR分子改造后的T细胞可以被特异性激活并大量分泌细胞毒性相关的细胞因子，进而强有力地介导靶细胞的裂解，能更好的用于抗感染过继免疫治疗。

Description

一种VC-CAR分子及在清除HIV-1感染细胞中的应用 技术领域
本发明涉及肿瘤免疫治疗技术领域，具体地，涉及一种VC-CAR分子及在清除HIV-1感染细胞中的应用。
背景技术
人类免疫缺陷病毒1型(Human Immunodificiency Viruse 1,HIV-1)感染后，联合抗逆转录病毒疗法(combined antiretroviral therapy,cART)可以有效地抑制病毒复制。然而，由于病毒整合在被感染细胞中并形成一个稳定的潜伏感染储存库，被感染者一旦停止cART治疗，病毒血症即在短时间内再爆发，这构成了治愈HIV-1感染的主要障碍。
当前的研究热点是通过特异性的潜伏感染逆转药物(latency-reversing agents,LRAs)激活潜伏感染的病毒，进而药物治疗或诱导机体免疫系统杀灭被感染细胞。这种干预策略被称为“shock and kill”。然而，HIV-1可以迅速的发生突变以逃避免疫识别。研究显示在经过cART治疗的感染者当中，即使成功激活了其潜伏感染，体内的CD8+T淋巴细胞由于缺乏对HIV-1有效的免疫应答，所以不能完全清除被感染的细胞。因此，在“shock and kill”策略中，为了更好的清除潜伏感染储存库，需要在被感染者体内重建强有力的免疫监控机能。
近年来，由于具有高亲和力、TCR(T cell receptor)非依赖和MHC(major histocompatibility complex)非限制等特点，嵌合抗原受体(chimeric antigen receptor,CAR)的免疫细胞疗法成为了杀伤肿瘤细胞的全新途径。CAR是由抗体靶向区域与T细胞激活胞内信号区融合而成，从而赋予细胞特异性抗原识别能力。通过在患者自体免疫细胞中表达识别肿瘤天然抗原的CAR分子并对进行过继免疫回输，可以特异性的靶向杀伤患者体内的肿瘤细胞。CAR-T细胞疗法已在白血病和淋巴瘤的临床治疗中证明了有效性，并且获得了令人鼓舞的成功。该策略也可应用于抗病毒治疗，包括HIV-1、HBV(hepatitis B virus)和HCV(hepatitis C virus)等病毒感染的治疗。但是，传统构建的CAR分子还不能完全满足各种疾病的免疫治疗。
发明内容
本发明为了克服现有技术的上述不足，提供一种新的CAR分子，称作VC-CAR分子，VC-CAR分子是将HIV-1广谱中和抗体VRC01来源的scFv序列和传统的CAR分子的胞内段序列连接在一起，分别作为胞外和胞内结构。
本发明的另一个目的是提供一种经VC-CAR分子改造的CD8+T细胞。
本发明的再一个目的是提供一种经VC-CAR分子改造的CD8+T细胞在清除HIV-1感染细胞的应用。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
SEQ ID NO：1所示的HIV-1广谱中和抗体VRC01来源的scFv序列在作为CAR分子的胞外抗原结合域中的应用。
一种VC-CAR分子，由SEQ ID NO：1所示的HIV-1广谱中和抗体VRC01来源的scFv序列和CAR分子的胞内段序列连接而成，HIV-1广谱中和抗体VRC01来源的scFv序列在N端，CAR分子胞内段序列在C端。
优选地，一种VC-CAR分子，其核苷酸序列如SEQ ID NO：2或SEQ ID NO：3或SEQ ID NO：4所示。SEQ ID NO：2所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-1所示；SEQ ID NO：3所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-2所示；SEQ ID NO：4所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-3所示。
更优选地，一种VC-CAR分子，其核苷酸序列如SEQ ID NO：4所示。
一种改造的CD8+T细胞，具体为使用本发明设计的VC-CAR分子转导CD8+T细胞制备得到。VC-CAR改造的CD8+细胞介导表达gp120细胞系的裂解效果更加明显。
本发明还要求保护以VC-CAR分子改造的CD8+T细胞在清除HIV-1感染细胞的应用。
一种改造的CD8+T细胞，由以下方法制备得到：(1)收集外周血单个核细胞并分离富集其中的CD8+T细胞，利用anti-CD3、anti-CD28和IL-2激活CD8+T细胞；(2)细胞激活48小时后，以1ml/1×106细胞的比例加入VC-CAR分子重组的病毒浓缩液进行感染，同时加入聚凝胺溶液，离心后继续培养；8～12小时后，进行第二轮病毒感染。
优选地，所述anti-CD3的浓度为1μg/mL，anti-CD28的浓度为1μg/mL，IL-2的浓度为10ng/mL。
优选地，所述聚凝胺溶液的浓度为8μg/mL。
一种扩增VC-CAR分子改造的CD8+T细胞的方法，包括如下步骤：(1)CD8+T细胞被VC-CAR分子重组的病毒感染改造后的12小时，离心换液，洗去培养基中的病毒，以新鲜培养基重悬细胞，并加入IL-2和IL-7保持细胞状态；(2)病毒感染改造后的第3天和第5天，根据细胞状态和增殖情况，对细胞添加完全RMPI1640培养基，细胞浓度维持在2×106/ml，并补充IL-2和IL-7，继续培养并及时传代，进一步扩增细胞。
优选地，所述IL-2的浓度为10ng/mL，IL-7的浓度为10ng/mL。
与现有技术相比，本发明具有如下有益效果：
本发明中全新改造的VC-CAR-T细胞与表达HIV-1包膜蛋白的细胞系或HIV-1感染的CD4+T细胞进行共培养，VC-CAR-T细胞可以被特异性激活并大量分泌细胞毒性相关的细胞因子(包括IFN-γ、和Granzyme B)，进而强有力地介导靶细胞的裂解。
本发明中全新改造的VC-CAR-T细胞相比已报道的CD4-CAR-T细胞，VC-CAR-T细胞介导表达gp120细胞系的裂解效果更加明显。
本发明使用的IL-2+IL-7细胞因子组合，相比单独施加IL-2的传统扩增方法，可以进一步提高HIV-1感染患者CD8+T细胞扩增倍数。
附图说明
图1为构建的VC-CAR分子的结构示意图。
图2为pCPPT-IRES-mStrewbeery慢病毒载体结构示意图。
图3为VC-CAR在CD8+T细胞中的表达情况。
图4为VC-CAR和CD4-CAR改造的CD8+T细胞分别与靶细胞系混合后杀伤效果的比较。
图5为VC-CAR-T细胞与表达HIV-1包膜蛋白的靶细胞系混合培养后，IFN-γ和Granzyme-B的分泌情况。
图6为VC-CAR-T细胞与表达HIV-1包膜蛋白的靶细胞系混合培养后，通过检测LDH的释放，检测VC-CAR-T细胞的杀伤活性。
图7为VC-CAR-T细胞与野生型HIV-1NL4-3感染的CD4+T淋巴细胞进行混合培养，通过流式检测Gag+CD4+T淋巴细胞的比例。验证VC-CAR-T细胞的杀伤活性。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
一种以VRC01为胞外抗原结合域的VC-CAR分子，其核苷酸序列如SEQ ID NO：2或SEQ ID NO：3或SEQ ID NO：4所示。SEQ ID NO：2所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-1所示；SEQ ID NO：3所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-2所示；SEQ ID NO：4所示的VC-CAR分子的结构如图1中的VR C01-28BBZ-3所示。
HIV-1广谱中和抗体VRC01来源的scFv序列如SEQ ID NO：1所示。
下面以SEQ ID NO：4所示的VC-CAR分子详细介绍一下本发明的VC-CAR分子的结构设计。VC-CAR分子的序列N’端是HIV-1广谱中和抗体VRC01来源的scFv序列，可特异性结合病毒包膜蛋白的CD4结合域；VC-CAR分子序列C’端是以三代CAR结构为基础，包括CD28(nucleotides 460-660,GenBank NM_006139.3)，CD137(nucleotides 640-765,GenBank NM_001561.5)和CD3ζ(nucleotides 160-492,Genbank NM_198053.2)胞内结构域串联组成，通过CD28分子的跨膜结构域将scFv和胞内信号分子相连。
实施例2
一、VC-CAR分子重组pCPPT-IRES-mStrewbeery慢病毒载体：按照SEQ ID NO：4所示序列合成VC-CAR，合成的VC-CAR通过NheI和NotI双酶切，插入pCPPT-IRES-mStrewbeery慢病毒载体中(见图2)，酶切、连接的具体步骤参考本领域的常规方法；得到VC-CAR分子重组后的pCPPT-IRES-mStrewbeery慢病毒载体。
将一个生长状态的HEK293T细胞平均铺于用多聚赖氨酸处理的10cm培养皿中(细胞密度约为6.5×104/cm2)，要求细胞呈单个均匀分布。培养约24小时后，细胞汇合度应接近80％，此时各皿均以12ml完全培养基换液，同时加入3.75ul氯喹(100mM，4000×)。换液后按表1配制磷酸钙-DNA混合液，颠倒数次混匀后静置1分钟，然后以3ml/皿迅速加入各皿细胞培养液中，边加边摇匀，逐滴 快速加入。
表1为磷酸钙-DNA混合液体系配方
Figure PCTCN2017072266-appb-000001
转染后12小时，细胞汇合度应接近100％，此时各皿均以12ml新鲜培养基换液，同时加入90μl丁酸钠(1M，100×)。转染后约48小时，收集全部34ml HEK293T细胞上清液，以0.45μm的过滤器过滤后装入50ml离心管，按下表2比例加入PEG-NaCl-PBS混合液，颠倒混匀后于4℃放置1.5小时，期间每20～30分钟混匀一次或颠倒混匀后于4℃放置过夜。
表2
Figure PCTCN2017072266-appb-000002
将混合液于4℃，7000g离心10分钟，管壁上可见白色沉淀，小心去除全部上清液，加入适量体积(300ul～3ml)的新鲜培养基，轻轻摇晃使沉淀溶解，即得到VC-CAR分子重组的病毒浓缩液，立即使用或分装后于-80℃保存。
二、VC-CAR在CD8+T细胞中的表达情况：从外周血样本中分离外周血单个核细胞，然后通过偶联生物素的CD8抗体分离富集CD8+T淋巴细胞，计数，离心。用RPMI1640完全培养基重悬，然后按2×106/ml的细胞浓度，均匀铺至细胞培养板中。利用anti-CD3(终浓度为1μg/ml)、anti-CD28(终浓度为1μg/ml)抗体和IL2(终浓度为10ng/ml)对细胞进行刺激，作用48小时后，收集细胞，用于假病毒的感染。
取适量生长状态良好的目标细胞悬液，放入离心管中300g离心5分钟。弃去上清液，以1ml/1×106细胞的比例加入假病毒浓缩液，同时加入终浓度8μg/ml 的Polybrene，轻轻吹打混匀。将细胞悬液移入培养皿，37℃、350×g离心90分钟，离心结束后，放回培养箱中继续培养。约8～12小时后，进行第二轮感染，步骤同上，约12小时后，离心换液，用PBS洗去培养基中的病毒，以新鲜培养基重悬细胞，并加入终浓度均为10ng/ml的IL-2和IL-7保持细胞状态。继续培养并及时传代，进一步扩增细胞。感染后的第3天，根据细胞状态和增殖情况，对细胞添加完全RMPI1640培养基，细胞浓度维持在2×106/ml。并补充IL-2和IL-7，终浓度均为10ng/ml。感染后的第5天，按照第3天的流程继续扩增细胞。并利用流式检测荧光蛋白mStrewbeery的表达，确定VC-CAR的感染效率，通常需确保感染率达到50％以上(图3)。感染后的第5天，细胞用于后续实验检测或继续培养以及冻存。此后，为了方便起见，以下实验均称其为VC-CAR-T细胞。
实施例3
一、VC-CAR和CD4-CAR改造的CD8+T细胞分别与靶细胞系混合后杀伤效果的比较：早前有研究利用CD4分子的胞外结构为基础，构建CAR-T细胞(CD4-CAR-T细胞)。由于CD4分子是HIV-1包膜糖蛋白gp120的天然受体，CD4-CAR-T细胞能够裂解表达gp120的靶细胞。为了对比VC-CAR-T细胞和CD4-CAR-T细胞效应，我们根据前人报道，将VC-CAR的scFv序列替换为CD4分子的胞外结构域构建了CD4-CAR。以同样的条件转导CD8+T淋巴细胞，然后与表达HIV-1包膜蛋白的靶细胞系(Jurkat-gp160NL4-3)混合，在U形底的96孔板中进行细胞杀伤实验。靶细胞数量为104/孔，RMPI1640完全培养基体积为200μl/孔。在给定的效靶比范围内(8：1至2：1)，24小时后，通过检测乳酸脱氢酶的释放，以确定每个实验组经过改造CD8+T淋巴细胞的杀伤活性。结果显示：VC-CAR-T细胞比CD4-CAR-T细胞具有更强的靶细胞裂解能力。该结果可能的原因为HIV-1广谱中和抗体来源的scFv对HIV-1gp120亲和力强于天然的CD4分子(见图4)。
二、VC-CAR-T细胞与表达HIV-1包膜蛋白的靶细胞系混合培养后特异性细胞因子的分泌情况：为了进一步检测VC-CAR的功能，我们将VC-CAR-T细胞与表达HIV-1包膜蛋白的细胞系Jurkat-gp160NL4-3在预包被IFN-γ抗体的96孔PVDF板中进行混合培养，效应细胞数量为104/孔，在给定的效靶比范围内(4：1和2：1)，24小时后，利用ELIspot实验检测VC-CAR-T细胞IFN-γ的分泌。 结果显示：与两株靶细胞混合培养的VC-CAR-T细胞IFN-γ的分泌显著提高，而与HIV-1包膜蛋白阴性的对照细胞系Jurkat-GFP混合培养的VC-CAR-T细胞则不会分泌IFN-γ。另一方面，与表达HIV-1包膜蛋白的靶细胞系混合培养的对照效应细胞没有明显的IFN-γ分泌，从而进一步证明IFN-γ的分泌是VC-CAR-T细胞特异性的(图5)。
将VC-CAR-T细胞与Jurkat-gp160NL4-3分别混合培养24小时，在给定的效靶比范围内(4：1-2：1)，granzyme B的ELISA实验也显示，随着靶细胞增加(Jurkat-gp160NL4-3)，VC-CAR-T细胞的细胞因子分泌量会以剂量依赖的方式随之增加，而对照靶细胞(Jurkat-GFP)则不会刺激VC-CAR-T细胞分泌IL-2和granzyme B。结果说明，在特异性抗原刺激下，VC-CAR-T细胞具有高效分泌抗病毒细胞因子的能力(图5)。
三、VC-CAR-T细胞与表达HIV-1包膜蛋白的靶细胞系混合培养后杀伤活性的检测：为了进一步检测VC-CAR的功能，我们将VC-CAR-T细胞与两株表达HIV-1包膜蛋白的细胞系Jurkat-gp160NL4-3和Jurkat-gp160BaL分别进行混合培养，在U形底的96孔板中进行细胞杀伤实验。靶细胞数量为104/孔，RMPI1640完全培养基体积为200μl/孔。在给定的效靶比范围内(8：1-0.5：1)，24小时后，通过LDH的释放，我们检测VC-CAR-T细胞对HIV-1包膜蛋白表达细胞的细胞毒性。结果显示：在从8：1到0.5：1的效靶比区间范围内，VC-CAR-T细胞以剂量依赖的方式显著杀伤两株表达HIV-1gp120的靶细胞(Jurkat-gp160NL4-3和Jurkat-gp160BaL)，而对照靶细胞(Jurkat-GFP)没有显著的杀伤效果，说明VC-CAR-T细胞杀伤靶细胞作用是HIV-1gp120特异性的。无论Jurkat-gp160NL4-3和Jurkat-gp160BaL还是对照靶细胞Jurkat-GFP，对照效应细胞均没有显示明显杀伤作用，从而进一步说明了VC-CAR-T细胞作用的特异性(图6)。
四、VC-CAR-T细胞与野生型HIV-1NL4-3感染的CD4+T淋巴细胞进行混合培养后杀伤活性的检测：为了进一步证明在清除野生型HIV-1感染的细胞方面VC-CAR-T细胞的有效性，利用野生型HIV-1NL4-3，对健康人血液样本中分离的CD4+T淋巴细胞进行感染。进行感染时RMPI1640完全培养基的体积为1ml/孔(24孔板)，包含2×106细胞，对应200ng(p24)的野生型病毒。感染后3小时换液。感染后的第8天，该细胞与VC-CAR改造的同源CD8+T淋巴细胞以1：2和1：4的比例混合，在24孔板中进行细胞杀伤实验。靶细胞数量为106/孔， RMPI1640完全培养基体积为500μl/孔。48小时后，通过流式细胞术检测Gag+CD4+T淋巴细胞的比例，验证VC-CAR-T细胞的杀伤作用。结果显示：VC-CAR-T细胞对HIV-1感染的细胞的清除率高达78％，表现了显著的杀伤效果，同时对照效应细胞的数值不足30％，两者差异具有统计学意义(图7)。

Claims (10)

  1. SEQ ID NO：1所示的HIV-1广谱中和抗体VRC01来源的scFv序列在作为CAR分子的胞外抗原结合域中的应用。
  2. 一种VC-CAR分子，其特征在于，由SEQ ID NO：1所示的HIV-1广谱中和抗体VRC01来源的scFv序列和CAR分子胞内段序列连接而成，HIV-1广谱中和抗体VRC01来源的scFv序列在N端，CAR分子胞内段序列在C端。
  3. 根据权利要求2所述的VC-CAR分子，其特征在于，所述VC-CAR分子的核苷酸序列如SEQ ID NO：2或SEQ ID NO：3或SEQ ID NO：4所示。
  4. 根据权利要求2所述的VC-CAR分子，其特征在于，所述VC-CAR分子的核苷酸序列如SEQ ID NO：4所示。
  5. 一种改造的CD8+T细胞，其特征在于，使用权利要求2至4任一项所述的VC-CAR分子转导至CD8+T细胞中制备得到。
  6. 权利要求5所述的改造的CD8+T细胞在制备清除HIV-1感染细胞的制剂中的应用。
  7. 根据权利要求5所述的改造的CD8+T细胞，其特征在于，由以下方法制备得到：(1)收集外周血单个核细胞并分离富集其中的CD8+T细胞，利用anti-CD3、anti-CD28和IL-2激活CD8+T细胞；(2)细胞激活48小时后，以1ml/1×106细胞的比例加入VC-CAR分子重组的病毒浓缩液进行感染，同时加入聚凝胺溶液，离心后继续培养；8～12小时后，进行第二轮病毒感染。
  8. 根据权利要求7所述的改造的CD8+T细胞，其特征在于，所述anti-CD3的浓度为1μg/mL，anti-CD28的浓度为1μg/mL，IL-2的浓度为10ng/mL。
  9. 一种扩增权利要求5或7或8所述的改造的CD8+T细胞的方法，其特征在于，包括如下步骤：(1)CD8+T细胞被VC-CAR分子重组的病毒感染改造后的12小时，离心换液，洗去培养基中的病毒，以新鲜培养基重悬细胞，并加入IL-2和IL-7保持细胞状态；(2)病毒感染改造后的第3天和第5天，根据细胞状态和增殖情况，对细胞添加完全RMPI1640培养基，细胞浓度维持在2×106/ml，并补充IL-2和IL-7，继续培养并及时传代，进一步扩增细胞。
  10. 根据权利要求9所述的方法，其特征在于，所述IL-2的浓度为10ng/mL，IL-7的浓度为10ng/mL。
PCT/CN2017/072266 2016-08-10 2017-01-23 一种vc-car分子及在清除hiv-1感染细胞中的应用 WO2018028157A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/322,470 US10633432B2 (en) 2016-08-10 2017-01-23 VC-CAR molecule and use thereof in removing HIV-1 infected cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610652995.9 2016-08-10
CN201610652995.9A CN106279432B (zh) 2016-08-10 2016-08-10 一种vc-car分子及在清除hiv-1感染细胞中的应用

Publications (1)

Publication Number Publication Date
WO2018028157A1 true WO2018028157A1 (zh) 2018-02-15

Family

ID=57668046

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/072266 WO2018028157A1 (zh) 2016-08-10 2017-01-23 一种vc-car分子及在清除hiv-1感染细胞中的应用

Country Status (3)

Country Link
US (1) US10633432B2 (zh)
CN (1) CN106279432B (zh)
WO (1) WO2018028157A1 (zh)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106279432B (zh) * 2016-08-10 2019-09-20 深圳市再生之城生物医药技术有限公司 一种vc-car分子及在清除hiv-1感染细胞中的应用
CN107098969B (zh) * 2017-06-28 2018-10-12 武汉波睿达生物科技有限公司 一种治疗hiv感染的嵌合抗原受体的重组基因构建及其应用
CN107619820A (zh) * 2017-09-07 2018-01-23 河南大学淮河医院 Hiv病毒潜伏库双靶向性嵌合抗原受体修饰的t细胞及其制备方法和应用
CN108070033A (zh) * 2017-12-12 2018-05-25 武汉波睿达生物科技有限公司 一种3bnc-car分子的构建及其在杀灭hiv-1感染细胞中的应用
CA3129100A1 (en) * 2019-02-08 2020-08-13 Biontech Cell & Gene Therapies Gmbh Treatment involving car-engineered t cells and cytokines
CN112522322A (zh) * 2020-12-24 2021-03-19 深圳市人民医院 一种nrp1配体的修饰技术

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103483453A (zh) * 2012-06-12 2014-01-01 上海吴孟超医学科技基金会 结合egfr家族蛋白的嵌合抗原受体、其组合物及用途
CN103502438A (zh) * 2011-03-23 2014-01-08 弗雷德哈钦森癌症研究中心 用于细胞免疫治疗的方法和组合物
WO2015084859A1 (en) * 2013-12-02 2015-06-11 Aaron Diamond Aids Research Center Improved hiv-1-neutralizing antibody potency and breadth via cell receptor anchoring using bispecific antibodies with native architecture
CN105518143A (zh) * 2013-03-15 2016-04-20 北京凯得尔森生物技术有限公司 融合蛋白分子和它的使用方法
CN106279432A (zh) * 2016-08-10 2017-01-04 中山大学 一种vc‑car分子及在清除hiv‑1感染细胞中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2013204922B2 (en) * 2012-12-20 2015-05-14 Celgene Corporation Chimeric antigen receptors
CN105377897A (zh) * 2013-05-03 2016-03-02 俄亥俄州创新基金会 Cs1-特异性嵌合抗原受体工程化的免疫效应细胞
CN104788573B (zh) * 2015-05-08 2018-10-16 中国医学科学院血液病医院（血液学研究所） 嵌合抗原受体hCD19scFv-CD8α-CD28-CD3ζ及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103502438A (zh) * 2011-03-23 2014-01-08 弗雷德哈钦森癌症研究中心 用于细胞免疫治疗的方法和组合物
CN103483453A (zh) * 2012-06-12 2014-01-01 上海吴孟超医学科技基金会 结合egfr家族蛋白的嵌合抗原受体、其组合物及用途
CN105518143A (zh) * 2013-03-15 2016-04-20 北京凯得尔森生物技术有限公司 融合蛋白分子和它的使用方法
WO2015084859A1 (en) * 2013-12-02 2015-06-11 Aaron Diamond Aids Research Center Improved hiv-1-neutralizing antibody potency and breadth via cell receptor anchoring using bispecific antibodies with native architecture
CN106279432A (zh) * 2016-08-10 2017-01-04 中山大学 一种vc‑car分子及在清除hiv‑1感染细胞中的应用

Also Published As

Publication number Publication date
CN106279432B (zh) 2019-09-20
US10633432B2 (en) 2020-04-28
CN106279432A (zh) 2017-01-04
US20190367587A1 (en) 2019-12-05

Similar Documents

Publication Publication Date Title
WO2018028157A1 (zh) 一种vc-car分子及在清除hiv-1感染细胞中的应用
EP3027755B1 (en) Engineering antiviral t cell immunity through stem cells and chimeric antigen receptors
JP7162895B2 (ja) Ｈｉｖ予備免疫化および免疫療法
JP6890664B2 (ja) Ｈｉｖ感染を治療するキメラ抗原受容体の組換え遺伝子構築およびその応用
WO2018223600A1 (zh) Octs-car双靶向嵌合抗原受体、编码基因、重组表达载体及其构建和应用
US11957715B2 (en) Method for NK cell transduction
US11993781B2 (en) Stable pseudotyped lentiviral particles and uses thereof
JP2020515234A (ja) 事前の免疫化ステップのないｈｉｖ免疫療法
WO2020216229A1 (zh) 同种异体car-t细胞、其制备及应用
JP2017505621A (ja) Ｔ細胞受容体を発現する細胞を生産する方法および組成物
JP2024073653A (ja) 遺伝子改変リンパ球の製造方法
WO2017144621A1 (en) T cell receptors from the hiv-specific repertoire, means for their production and therapeutic uses thereof
Li et al. Preclinical development and clinical-scale manufacturing of HIV Gag-specific, lentivirusmodified CD4 T cells for HIV functional cure
US20180371057A1 (en) Compositions and methods for improving immunotherapy
CN110343175B (zh) 抑制HIV-1病毒感染的scFv序列及其应用
WO2022177966A1 (en) Compositions and methods for the treatment of hiv
Strack Transcriptional Targeting of Dendritic Cells as a New Therapeutic Vaccine against HIV-1
Powell Genetically Modified Immune Cells Secreting Broadly Neutralizing Antibodies against HIV: Restoration of Systemic Immunity
Mahesh The role and impact of Nef and Nef inhibitor in HIV-induced impairment of CD4 T-cells
Van Tulleken Characterisation of Lentiviral Vpr Function and Mechanism
WO2024078995A1 (en) Transduction of gammadelta t cells with pseudotyped retroviral vectors
Abduljawad Optimizing a Universal Mosaic T cell vaccine for HIV-1

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: 17838294

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17838294

Country of ref document: EP

Kind code of ref document: A1