WO2023077943A1 - Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof - Google Patents

Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof Download PDF

Info

Publication number
WO2023077943A1
WO2023077943A1 PCT/CN2022/117012 CN2022117012W WO2023077943A1 WO 2023077943 A1 WO2023077943 A1 WO 2023077943A1 CN 2022117012 W CN2022117012 W CN 2022117012W WO 2023077943 A1 WO2023077943 A1 WO 2023077943A1
Authority
WO
WIPO (PCT)
Prior art keywords
chimeric antigen
hiv
antigen receptor
bispecific chimeric
envelope protein
Prior art date
Application number
PCT/CN2022/117012
Other languages
French (fr)
Chinese (zh)
Inventor
徐建青
张晓燕
应天雷
毛蕴玉
廖启彬
Original Assignee
复旦大学
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 复旦大学 filed Critical 复旦大学
Publication of WO2023077943A1 publication Critical patent/WO2023077943A1/en

Links

Images

Classifications

    • 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
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • C07K14/70514—CD4
    • 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
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of immunotherapy, in particular to a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and its application.
  • AIDS is an infectious disease caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus, HIV-1) infection that threatens the safety of human life, and it is also one of the most important public health challenges facing our country.
  • Highly active anti-retroviral therapy (HAART) is the first revolution in the history of HIV-1/AIDS treatment. The load is lower than the detection line, thereby limiting the AIDS disease process.
  • HAART still has many limitations that cannot be ignored, such as severe drug side effects and restrictions on life-long medication. Not only that, but its biggest limitation is that it cannot target or clear the latent HIV-1 virus in resting CD4+ T cells in patients. Therefore, once HAART treatment is interrupted, the viral load level in the blood of infected patients will rebound rapidly, which constitutes a major obstacle to the cure of HIV-1/AIDS.
  • Chimeric antigen receptor T cells is one of the latest technologies in the current adoptive cell therapy technology, which has achieved progress from basic immunological mechanism research to clinical immunotherapy application .
  • the CAR-T products CTL019 (Novartis) and KTEC19 (Kite) for the treatment of leukemia and lymphoma were approved by the US FDA in 2017, bringing new hope for the application of immune cell therapy technology. Therefore, genetically modified CAR-T cells may become a powerful weapon to clear the latent library of HIV-1 virus.
  • studies on the use of gene-modified T cell adoptive transfer (adoptive cell transfer) to treat patients with advanced AIDS have begun.
  • CD4 sequence as the extracellular recognition domain of CAR and prepared T cells expressing the CD4-CD3 ⁇ chimeric receptor (first-generation CAR), although it has the ability to target and kill HIV-1 in vitro The ability to infect or express HIV-1 Env cells, but was subsequently unsuccessful in phase I and II clinical trials. Scholler et al. followed up patients in 3 clinical trials from 1998 to 2005 and found that autologous CD4+ and CD8+ T lymphocytes expressing CD4-CD3 ⁇ CAR can survive stably for at least 11 years in HIV-1 infected patients, showing that CAR-T has a positive effect on HIV. -1 Strong potential in the field of infectious cell therapy.
  • the main reasons for the failure of the first generation of HIV-1 CAR-T cells can be attributed to the following aspects: (1) researchers at the time lacked understanding of the signaling requirements and co-stimulatory signals for effective T cell function, for example, by providing The co-stimulatory signal CD28, which is beneficial to cell proliferation, cytokine secretion and cell survival, improves the performance of CAR-T cells in vivo; (2) CAR molecular design itself has defects, and the extracellular recognition domain of CAR directly copies the extracellular recognition domain of CD4 protein. The structural domain may make CAR-T cells the target of HIV-1 attack; (3) The transduction efficiency of retroviral vectors is too low.
  • the technical problem to be solved by the present invention is: in order to solve the deficiencies in the prior art, enhance the activation ability of CAR-T cells and the killing ability of HIV-1 infected target cells, and further improve and optimize the bispecific CAR-T targeting HIV-1 Structural design of sexual CAR to enhance its control of HIV-1 infection and clearance of latent pool.
  • the technical solution provided by the present invention is to provide a bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein, said bispecific chimeric antigen receptor comprising: target Recognition unit to HIV-1 gp120 protein co-receptor binding site, connecting peptide, recognition unit targeting other binding sites of HIV-1 gp120, hinge region and transmembrane region, intracellular signaling domain, one or more costimulatory signal domain; the tandem sequence of the extracellular segment of the chimeric antigen receptor is the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site at the far membrane end, and the HIV-1 gp120 other at the near membrane end The recognition unit for the binding site.
  • the recognition unit targeting HIV-1 gp120 protein co-receptor binding site is single domain antibody m36.4, 17b or X5.
  • the connecting peptide is (G 4 S) n or G n .
  • the recognition unit targeting other binding sites of HIV-1 gp120 includes but not limited to CD4 binding site, mutant CD4 D1 domain, V1V2 glycan region, V3 glycan region, gp120-gp41 interface or on gp41 The outer region of the proximal end of the membrane.
  • the hinge region and transmembrane region are derived from the hinge region and transmembrane region of CD28, CD8 ⁇ or CD3 ⁇ .
  • the co-stimulatory signal domain is selected from one or more of CD28, 4-1BB, CD27, ICOS, CD160, CD69, TLR2, CD27, CD40L, CD30, OX40, and TIM1.
  • the intracellular signaling domain is a CD3 ⁇ intracellular signaling domain.
  • the biheterogeneous chimeric antigen receptor includes a recognition unit targeting HIV-1 gp120 protein co-receptor binding site, a connecting peptide, and other binding sites targeting HIV-1 gp120 from N-terminus to C-terminus Point recognition unit, hinge and transmembrane regions, one or more co-stimulatory signaling domains and intracellular signaling domains.
  • the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site is a single domain antibody m36.4, the amino acid sequence of which is shown in SEQ ID No.1; the HIV-1 gp120 other
  • the recognition unit of the binding site is the mutant CD4 D1 domain, and its amino acid sequence is shown in SEQ ID No.2.
  • the connecting peptide is 3 ⁇ G 4 S; the hinge region and transmembrane region are the hinge region and transmembrane region of CD28, and the amino acid sequence is shown in SEQ ID No.3; the co-stimulatory signal domain It is the co-stimulatory signal domain of CD28 and 4-1BB, and its amino acid sequence is shown in SEQ ID No.4 and SEQ ID No.5; the intracellular signaling domain is CD3 ⁇ intracellular signaling domain, and its amino acid sequence is shown in SEQ ID No.6 shown.
  • the chimeric antigen receptor further includes a signal peptide and a tag, the tag is located between the signal peptide and the extracellular recognition domain, the signal peptide is the signal peptide of CD8, and the tag is a Flag tag.
  • the amino acid sequence of the bispecific chimeric antigen receptor is shown in SEQ ID NO.7.
  • the nucleotide sequence of the gene encoding the bispecific chimeric antigen receptor is shown in SEQ ID NO.8.
  • a carrier can express the above-mentioned bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein, and the carrier includes the nucleotide sequence shown in SEQ ID NO.8.
  • the present invention also provides a genetically engineered T lymphocyte, which contains the above vector and can express a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein.
  • the preparation method of the above-mentioned genetically engineered T lymphocytes comprises the following steps: connecting the above-mentioned bispecific chimeric antigen receptor coding sequence targeting HIV-1 virus envelope protein to a carrier, packaging to obtain lentiviral particles, and using The lentivirus infects T lymphocytes to obtain bispecific chimeric antigen receptor-modified T lymphocytes.
  • the present invention overcomes the defects of existing designs, and utilizes recognition units targeting HIV-1 gp120 protein co-receptor binding sites and other binding sites of HIV-1 gp120 (including but not limited to CD4 binding sites) , V1V2 glycan region, V3 glycan region, gp120-gp41 interface or near-membrane external region on gp41), which greatly improves the broad-spectrum and performance of chimeric antigen receptors in recognizing HIV-1 virus envelope proteins Specificity, and proposed that the dominant tandem order of the two is that the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site is far away from the cell membrane, and the recognition unit targeting other HIV-1 gp120 binding sites is close to the cell membrane.
  • the present invention overcomes the defects in the early design of chimeric antigen receptors targeting HIV-1, and the proposed bispecific chimeric antigen receptors contain co-stimulatory signals in cells, which is conducive to improving HIV-1-specific CAR-T Cell survival, proliferation and killing ability, increase clinical effectiveness.
  • Figure 1 is a schematic diagram of the structure of the M31-CAR bispecific chimeric antigen receptor.
  • Figure 2 is a schematic diagram of the structure of the M13-CAR bispecific chimeric antigen receptor.
  • Figure 3 is a graph showing the expression of M31 CAR and M13 CAR on the membrane surface of primary T cells, where UTD is the control of T cells that were not transduced with lentivirus.
  • Figure 4 is a diagram of M31 CAR-T and M13 CAR-T specifically recognizing target cell MT4-gp145 and secreting cytokines IFN- ⁇ and IL-2, where UTD is the T cell control that was not transduced with lentivirus.
  • Figure 5 is a diagram of the killing efficiency of the gp145 overexpression cell line MT4-gp145 by M31 CAR-T and M13 CAR-T, where UTD is the T cell control that was not transduced with lentivirus.
  • Figure 6 is a graph showing the A549-gp145 curves of M31 CAR-T and M13 CAR-T killing gp145 overexpression cells.
  • Figure 7 is a graph showing the killing efficiency of HIV latent cells reactivated by M31 CAR-T and M13 CAR-T, where UTD is the control of T cells not transduced with lentivirus.
  • Figure 8 is a graph showing the susceptibility of M31 CAR-T to HIV-1 virus, in which the CD4 + T cell group is a positive control.
  • DMEM medium and RPMI1640 medium were purchased from Corning Company, and lymphocyte medium X-VIVO 15 was purchased from Lonza Company.
  • T cell growth medium consists of basal medium and cytokines
  • the basal medium is lymphocyte medium X-VIVO 15
  • the cytokines are IL-7 at a final concentration of 5ng/mL
  • IL-15 at 10ng/mL and 30ng/mL mL of IL-21.
  • cytokines IL-7 and IL-15 were purchased from R&D Company
  • IL-21 was purchased from Nearshore Protein Technology Co., Ltd.
  • Fetal bovine serum was purchased from BI Company.
  • Lenti-X lentivirus concentrated reagent was purchased from Takara Company.
  • Synthetic genes were purchased from Shanghai Jierui Bioengineering Co., Ltd.
  • the lentiviral expression plasmid pKL was provided by Kanglin Biotechnology (Hangzhou) Co., Ltd., and the packaging plasmid psPAX2 and envelope plasmid PMD2.G were purchased from Addgene.
  • Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • the endotoxin-free plasmid mini-prep kit and the endotoxin-free plasmid mid-prep kit were purchased from OMEGA and Macherey Nagel, respectively.
  • RTCA Real-time label-free cell function analyzer
  • the M31 CAR and M13 CAR genes (shown in SEQ ID No: 8 and SEQ ID No: 9, respectively) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and cloned into a blank lentiviral expression plasmid (pKL) to obtain pKL-M31- CAR and pKL-M13-CAR recombinant lentiviral expression plasmids, the bispecific chimeric antigen receptor structure is shown in Figures 1 and 2.
  • HEK293T cell treatment 24 hours before transfection, collect HEK293T cells in the logarithmic growth phase, inoculate them in a 10 cm cell culture dish (6-8 ⁇ 10 6 cells), and place the cells in 10 mL of complete DMEM medium Grow, culture at 37°C, 5% CO 2 for 18-24 hours, and transfection can be carried out when the cell density reaches 70-90%.
  • T cells expressing bispecific chimeric antigen receptors secrete high levels of the cytokines IFN- ⁇ and IL-2 only when co-cultured with MT4-gp145, indicating expression of bispecific Chimeric antigen receptor T cells can specifically recognize HIV-1 envelope protein and be activated. Comparing the cytokine levels of T cells expressing M31 CAR and M13 CAR, it can be found that M31 CAR can better mediate the activation of modified T cells, and its secreted cytokine level is almost twice that of M13 CAR.
  • Example 5 T cells expressing bispecific chimeric antigen receptors kill gp145 overexpressing cell lines
  • the cell killing efficiency was detected by flow cytometry and real-time label-free cell analysis (RTCA, Real Time Cellular Analysis) technology.
  • RTCA Real Time Cellular Analysis
  • MT4-gp145 was labeled with PKH26 dye (1:1000, 37° C. water bath for 10 minutes). Then 1 ⁇ 10 5 labeled MT4-gp145 cells were plated in a 96-well cell culture U-bottom plate, and untransduced lentiviral T cells (UTD) or expression The T cells of the bispecific chimeric antigen receptor (M31 CAR-T or M13 CAR-T) were added to the wells containing the target cells, and half of the cells in the wells were aspirated every 24 hours for flow cytometry detection.
  • PKH26 dye 1:1000, 37° C. water bath for 10 minutes.
  • UTD untransduced lentiviral T cells
  • T cells of the bispecific chimeric antigen receptor M31 CAR-T or M13 CAR-T
  • T cells expressing bispecific chimeric antigen receptors can effectively kill MT4-gp145 cells, and the killing efficiency is as high as 90% at 48 hours;
  • the killing efficiency of M31 CAR and T cells expressing M13 CAR can be found that the killing response of M31 CAR-T to target cells is faster, and the killing efficiency at 24 hours is 22.2% higher than that of M13 CAR-T.
  • A549 cells 5 ⁇ 10 4 cells, wells
  • M13 CAR-T bispecific chimeric antigen receptors
  • T cells expressing bispecific chimeric antigen receptors can rapidly decrease the cell growth curve and effectively kill tumor cells overexpressing gp145 protein.
  • expressing M31 - The killing ability of CAR T cells on target cells is significantly better than that of T cells expressing M13-CAR, with a killing rate as high as 92.5% (the killing efficiency of M13 CAR-T cells is 88.3%).
  • Example 6 T cells expressing bispecific chimeric antigen receptors kill reactivated HIV-1 latent cell lines ACH2
  • the HIV-1 latent cell line ACH2 was plated in a 6-well plate at a concentration of 1 ⁇ 10 6 cells/mL, activated by adding PMA at a concentration of 10 ng/mL to the culture medium, and the cells were collected after 48 hours of culture.
  • the reactivated ACH2 cell line was stained and marked with PKH26 dye (1:1000, 37°C water bath for 10 minutes).
  • T cells expressing bispecific chimeric antigen receptors can kill HIV-1 latent cell line ACH2, but the killing efficiency differs greatly between the two; 48 hours , the killing efficiency of T cells expressing M31-CAR reached 48.33%, which was 19% higher than that of M13 CAR-T.
  • Example 7 T cells expressing bispecific chimeric antigen receptors are not susceptible to HIV-1 virus

Landscapes

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

Abstract

Provided are a bispecific chimeric antigen receptor targeting an HIV-1 envelope protein. The chimeric antigen receptor comprises: (1) a recognition unit targeting an HIV-1gp120 protein co-receptor binding site; (2) a recognition unit targeting another HIV-1 gp120 binding site (including, but not limited to, a CD4 binding site, a V1V2 glycan region, a V3 glycan region, a gp120-gp41 interface, or a membrane proximal external region on gp41); (3) hinge and transmembrane regions; and (4) an intracellular signaling domain; optionally, the chimeric antigen receptor further comprises (5) one or more co-stimulatory signal domains; preferably, the extracellular recognition domains of the chimeric antigen receptor that are sequentially connected in series is the recognition unit targeting the HIV-1gp120 protein co-receptor binding site at the end away from a membrane, and the recognition unit targeting the another HIV-1 gp120 binding site at the end close to the membrane. Provided is a new construction method for a bispecific chimeric antigen receptor targeting an HIV-1 envelope protein, the immune cells modified by the bispecific chimeric antigen receptor have stronger activation and killing ability, and can specifically recognize and kill HIV-1 infected cells.

Description

一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体及其制备方法和应用A kind of bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein and its preparation method and application 技术领域technical field
本发明涉及免疫治疗技术领域，具体为一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体及其应用。The invention relates to the technical field of immunotherapy, in particular to a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and its application.
背景技术Background technique
艾滋病是由人类免疫缺陷病毒1型(Human Immunodeficiency Virus,HIV-1)感染引起的一种威胁人类生命安全的传染病，也是我国面临的最重大的公共卫生挑战之一。高效抗逆转录病毒疗法(highly active anti-retroviral therapy,HAART)是HIV-1/AIDS治疗史上的第一次革命，可以有效控制HIV-1感染者体内病毒复制水平，使感染者外周血中病毒载量低于检测线，从而限制AIDS疾病进程。然而，HAART仍然存在很多不可忽视的局限性，例如严重的药物毒副作用和终生服药的限制。不仅如此，其最大的局限性在于不能靶向或清除患者体内静息CD4+T细胞中潜伏的HIV-1病毒。因此，一旦中断HAART治疗，感染者血液中病毒载量水平将迅速反弹，这构成了HIV-1/AIDS治愈的主要障碍。AIDS is an infectious disease caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus, HIV-1) infection that threatens the safety of human life, and it is also one of the most important public health challenges facing our country. Highly active anti-retroviral therapy (HAART) is the first revolution in the history of HIV-1/AIDS treatment. The load is lower than the detection line, thereby limiting the AIDS disease process. However, HAART still has many limitations that cannot be ignored, such as severe drug side effects and restrictions on life-long medication. Not only that, but its biggest limitation is that it cannot target or clear the latent HIV-1 virus in resting CD4+ T cells in patients. Therefore, once HAART treatment is interrupted, the viral load level in the blood of infected patients will rebound rapidly, which constitutes a major obstacle to the cure of HIV-1/AIDS.
如何能够重新激活潜伏的HIV-1病毒使其被机体的免疫系统自发识别和清除，是治愈AIDS的关键。同时，研究发现，即使在接受HAART治疗的感染者体内，也出现了不可逆转的免疫损伤，特别是细胞毒性T细胞数量减少和功能耗竭，这表明需要通过联合增强机体HIV-1特异性免疫应答的方法来加强HIV-1储藏库的清除效果。How to reactivate the latent HIV-1 virus so that it can be automatically recognized and cleared by the body's immune system is the key to curing AIDS. At the same time, the study found that irreversible immune damage occurred even in infected patients who received HAART treatment, especially the decrease in the number and function exhaustion of cytotoxic T cells, which indicated the need to enhance the body's HIV-1-specific immune response through combination approach to enhance clearance of HIV-1 reservoirs.
嵌合抗原受体修饰的T细胞(Chimeric antigen receptor T cells,CAR-T cells)是当前过继性细胞治疗技术中最新的技术之一，实现了从基础免疫学机 制研究到临床免疫治疗应用的进步。其中，用于治疗白血病、淋巴瘤的CAR-T产品CTL019(诺华)和KTEC19(Kite)在2017年先后获得美国FDA批准上市，为免疫细胞治疗技术的应用带来新的曙光。因此，基因修饰的CAR-T细胞，有可能成为清除HIV-1病毒潜伏库的有利武器。事实上，自上世纪90年代开始，基因修饰T细胞过继转移(adoptive cell transfer)治疗晚期AIDS病人的研究就已开始。在1994年，Roberts等人选取CD4序列作为CAR的胞外识别域，制备了表达CD4-CD3ζ嵌合受体(第一代CAR)的T细胞，虽然具有在体外靶向和杀死HIV-1感染或HIV-1 Env表达的细胞的能力，但随后在I期和II期临床试验中没有成功。Scholler等对1998-2005年3项临床试验的患者随访发现，表达CD4-CD3ζCAR的自体CD4+和CD8+T淋巴细胞在HIV-1感染者体内可稳定存活至少11年，显示出CAR-T在HIV-1感染细胞治疗领域的强大潜力。第一代HIV-1 CAR-T细胞失败的主要原因可以归结为以下几个方面：(1)当时的研究者缺乏对有效T细胞功能的信号传导要求和共刺激信号的理解，例如，通过提供对细胞增殖、细胞因子分泌和细胞存活有利的共刺激信号CD28来改善CAR-T细胞在体内的表现；(2)CAR分子设计本身存在缺陷，CAR的胞外识别域直接照搬CD4蛋白的胞外结构域可能使CAR-T细胞成为HIV-1攻击的目标；(3)使用逆转录病毒载体转导效率过低，为了获得足够的可回输CAR-T细胞，进行过度的体外扩增导致回输后细胞过快耗竭和凋亡。Kim Anthony-Gonda等在一项HIV-1病毒靶向性的多特异性CAR-T细胞研究中报道了一种靶向HIV-1的双特异性CAR-T细胞的构建方法，CAR分子从N端至C端顺次串联了突变型CD4 D1结构域mD1.22、连接肽、单域抗体m36.4、CD8的铰链与穿膜区、4-1BB共刺激信号域和CD3ζ胞内信号传导结构域。但经研究比较发现，上述的串联顺序不仅影响CAR分子的膜表达水平，更是显著减弱 了CAR-T细胞的活化能力和对HIV-1感染靶细胞的杀伤能力，不利于HIV-1的感染控制。因此，仍然有必要进一步改进和优化靶向HIV-1的双特异性CAR的结构设计，增强其对HIV-1感染的控制以及潜伏库的清除能力Chimeric antigen receptor T cells (CAR-T cells) is one of the latest technologies in the current adoptive cell therapy technology, which has achieved progress from basic immunological mechanism research to clinical immunotherapy application . Among them, the CAR-T products CTL019 (Novartis) and KTEC19 (Kite) for the treatment of leukemia and lymphoma were approved by the US FDA in 2017, bringing new hope for the application of immune cell therapy technology. Therefore, genetically modified CAR-T cells may become a powerful weapon to clear the latent library of HIV-1 virus. In fact, since the 1990s, studies on the use of gene-modified T cell adoptive transfer (adoptive cell transfer) to treat patients with advanced AIDS have begun. In 1994, Roberts et al. selected the CD4 sequence as the extracellular recognition domain of CAR and prepared T cells expressing the CD4-CD3ζ chimeric receptor (first-generation CAR), although it has the ability to target and kill HIV-1 in vitro The ability to infect or express HIV-1 Env cells, but was subsequently unsuccessful in phase I and II clinical trials. Scholler et al. followed up patients in 3 clinical trials from 1998 to 2005 and found that autologous CD4+ and CD8+ T lymphocytes expressing CD4-CD3ζCAR can survive stably for at least 11 years in HIV-1 infected patients, showing that CAR-T has a positive effect on HIV. -1 Strong potential in the field of infectious cell therapy. The main reasons for the failure of the first generation of HIV-1 CAR-T cells can be attributed to the following aspects: (1) Researchers at the time lacked understanding of the signaling requirements and co-stimulatory signals for effective T cell function, for example, by providing The co-stimulatory signal CD28, which is beneficial to cell proliferation, cytokine secretion and cell survival, improves the performance of CAR-T cells in vivo; (2) CAR molecular design itself has defects, and the extracellular recognition domain of CAR directly copies the extracellular recognition domain of CD4 protein. The structural domain may make CAR-T cells the target of HIV-1 attack; (3) The transduction efficiency of retroviral vectors is too low. In order to obtain enough reinfused CAR-T cells, excessive in vitro expansion leads to Excessive cell exhaustion and apoptosis after infusion. Kim Anthony-Gonda et al. reported a method for the construction of bispecific CAR-T cells targeting HIV-1 in a study of HIV-1 virus-targeted multispecific CAR-T cells. The CAR molecules were derived from N The mutant CD4 D1 domain mD1.22, connecting peptide, single domain antibody m36.4, hinge and transmembrane region of CD8, 4-1BB co-stimulatory signal domain and CD3ζ intracellular signal transduction structure are connected in series from end to C end area. However, after research and comparison, it was found that the above-mentioned tandem sequence not only affects the membrane expression level of CAR molecules, but also significantly weakens the activation ability of CAR-T cells and the killing ability of HIV-1 infected target cells, which is not conducive to HIV-1 infection. control. Therefore, it is still necessary to further improve and optimize the structural design of HIV-1-targeting bispecific CARs to enhance their control of HIV-1 infection and the clearance of latent pools.
发明内容Contents of the invention
本发明所要解决的技术问题是：为解决现有技术中的不足，增强CAR-T细胞的活化能力和对HIV-1感染靶细胞的杀伤能力，进一步改进和优化靶向HIV-1的双特异性CAR的结构设计，增强其对HIV-1感染的控制以及潜伏库的清除能力。The technical problem to be solved by the present invention is: in order to solve the deficiencies in the prior art, enhance the activation ability of CAR-T cells and the killing ability of HIV-1 infected target cells, and further improve and optimize the bispecific CAR-T targeting HIV-1 Structural design of sexual CAR to enhance its control of HIV-1 infection and clearance of latent pool.
为解决上述技术问题，本发明所提供的技术方案是：提供一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，所述双特异性嵌合抗原受体包含：靶向HIV-1 gp120蛋白共受体结合位点的识别单元、连接肽、靶向HIV-1 gp120其它结合位点的识别单元、铰链区和跨膜区、胞内信号传导结构域、一个或多个共刺激信号域；所述嵌合抗原受体胞外段的串联顺序为远膜端的靶向HIV-1 gp120蛋白共受体结合位点的识别单元，近膜端的靶向HIV-1 gp120其它结合位点的识别单元。In order to solve the above technical problems, the technical solution provided by the present invention is to provide a bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein, said bispecific chimeric antigen receptor comprising: target Recognition unit to HIV-1 gp120 protein co-receptor binding site, connecting peptide, recognition unit targeting other binding sites of HIV-1 gp120, hinge region and transmembrane region, intracellular signaling domain, one or more costimulatory signal domain; the tandem sequence of the extracellular segment of the chimeric antigen receptor is the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site at the far membrane end, and the HIV-1 gp120 other at the near membrane end The recognition unit for the binding site.
所述靶向HIV-1 gp120蛋白共受体结合位点的识别单元为单域抗体m36.4、17b或X5。The recognition unit targeting HIV-1 gp120 protein co-receptor binding site is single domain antibody m36.4, 17b or X5.
所述连接肽为(G 4S) n或G n。 The connecting peptide is (G 4 S) n or G n .
所述靶向HIV-1 gp120其它结合位点的识别单元包括但不限于CD4结合位点、突变型CD4 D1结构域、V1V2聚糖区、V3聚糖区、gp120-gp41交界面或gp41上的近膜端外部区域。The recognition unit targeting other binding sites of HIV-1 gp120 includes but not limited to CD4 binding site, mutant CD4 D1 domain, V1V2 glycan region, V3 glycan region, gp120-gp41 interface or on gp41 The outer region of the proximal end of the membrane.
所述铰链区和跨膜区来源于CD28、CD8α或CD3ζ的铰链区和跨膜区。The hinge region and transmembrane region are derived from the hinge region and transmembrane region of CD28, CD8α or CD3ζ.
所述共刺激信号域选自CD28、4-1BB、CD27、ICOS、CD160、CD69、TLR2、CD27、CD40L、CD30、OX40、TIM1中的一种或多种。The co-stimulatory signal domain is selected from one or more of CD28, 4-1BB, CD27, ICOS, CD160, CD69, TLR2, CD27, CD40L, CD30, OX40, and TIM1.
所述胞内信号传导结构域为CD3ζ胞内信号传导域。The intracellular signaling domain is a CD3ζ intracellular signaling domain.
优选的，所述双异性嵌合抗原受体从N端到C端顺次包括靶向HIV-1 gp120蛋白共受体结合位点的识别单元、连接肽、靶向HIV-1 gp120其它结合位点的识别单元、铰链和跨膜区、一个或多个共刺激信号域和胞内信号传导结构域。Preferably, the biheterogeneous chimeric antigen receptor includes a recognition unit targeting HIV-1 gp120 protein co-receptor binding site, a connecting peptide, and other binding sites targeting HIV-1 gp120 from N-terminus to C-terminus Point recognition unit, hinge and transmembrane regions, one or more co-stimulatory signaling domains and intracellular signaling domains.
优选的，所述靶向HIV-1 gp120蛋白共受体结合位点的识别单元为单域抗体m36.4，其氨基酸序列如SEQ ID No.1所示；所述靶向HIV-1 gp120其它结合位点的识别单元为突变型CD4 D1结构域,其氨基酸序列如SEQ ID No.2所示。Preferably, the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site is a single domain antibody m36.4, the amino acid sequence of which is shown in SEQ ID No.1; the HIV-1 gp120 other The recognition unit of the binding site is the mutant CD4 D1 domain, and its amino acid sequence is shown in SEQ ID No.2.
优选的，所述的连接肽为3×G 4S；所述铰链区和跨膜区为CD28的铰链区和跨膜区，氨基酸序列如SEQ ID No.3所示；所述共刺激信号域为CD28和4-1BB的共刺激信号域，氨基酸序列如SEQ ID No.4和SEQ ID No.5所示；所述胞内信号传导结构域为CD3ζ胞内信号传导域，氨基酸序列如SEQ ID No.6所示。 Preferably, the connecting peptide is 3×G 4 S; the hinge region and transmembrane region are the hinge region and transmembrane region of CD28, and the amino acid sequence is shown in SEQ ID No.3; the co-stimulatory signal domain It is the co-stimulatory signal domain of CD28 and 4-1BB, and its amino acid sequence is shown in SEQ ID No.4 and SEQ ID No.5; the intracellular signaling domain is CD3ζ intracellular signaling domain, and its amino acid sequence is shown in SEQ ID No.6 shown.
优选的，所述嵌合抗原受体还包括信号肽和标签，标签位于信号肽和胞外识别域之间，信号肽为CD8的信号肽，标签为Flag标签。Preferably, the chimeric antigen receptor further includes a signal peptide and a tag, the tag is located between the signal peptide and the extracellular recognition domain, the signal peptide is the signal peptide of CD8, and the tag is a Flag tag.
所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.7所示。The amino acid sequence of the bispecific chimeric antigen receptor is shown in SEQ ID NO.7.
编码所述的双特异性嵌合抗原受体的基因核苷酸序列如SEQ ID NO.8所示。The nucleotide sequence of the gene encoding the bispecific chimeric antigen receptor is shown in SEQ ID NO.8.
一种载体，所述载体能够表达上述靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，所述载体包含SEQ ID NO.8所示核苷酸序列。此外本发明还提供了一种基因工程化T淋巴细胞，该T淋巴细胞包含上述载体，并且可以表达靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体。A carrier, the carrier can express the above-mentioned bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein, and the carrier includes the nucleotide sequence shown in SEQ ID NO.8. In addition, the present invention also provides a genetically engineered T lymphocyte, which contains the above vector and can express a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein.
上述基因工程化T淋巴细胞的制备方法，包括以下步骤：将上述的靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体编码序列连接到载体上，包装获得慢病毒颗粒，利用该慢病毒感染T淋巴细胞从而获得双特异性嵌合抗原受体修饰的T淋巴细胞。The preparation method of the above-mentioned genetically engineered T lymphocytes comprises the following steps: connecting the above-mentioned bispecific chimeric antigen receptor coding sequence targeting HIV-1 virus envelope protein to a carrier, packaging to obtain lentiviral particles, and using The lentivirus infects T lymphocytes to obtain bispecific chimeric antigen receptor-modified T lymphocytes.
上述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体在制备治疗或预防艾滋病药物方面的应用。The application of the above-mentioned bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein in the preparation of drugs for treating or preventing AIDS.
本发明的有益效果在于：The beneficial effects of the present invention are:
(1)本发明克服已有设计的缺陷，同时利用靶向HIV-1 gp120蛋白共受体结合位点的识别单元和靶向HIV-1 gp120其它结合位点(包括但不限于CD4结合位点、V1V2聚糖区、V3聚糖区、gp120-gp41交界面或gp41上的近膜端外部区域)的识别单元，大大提高嵌合抗原受体识别HIV-1病毒囊膜蛋白的广谱性和特异性，并提出了二者的优势串联顺序为靶向HIV-1 gp120蛋白共受体结合位点的识别单元远离细胞膜，靶向HIV-1 gp120其它结合位点的识别单元靠近细胞膜。(1) The present invention overcomes the defects of existing designs, and utilizes recognition units targeting HIV-1 gp120 protein co-receptor binding sites and other binding sites of HIV-1 gp120 (including but not limited to CD4 binding sites) , V1V2 glycan region, V3 glycan region, gp120-gp41 interface or near-membrane external region on gp41), which greatly improves the broad-spectrum and performance of chimeric antigen receptors in recognizing HIV-1 virus envelope proteins Specificity, and proposed that the dominant tandem order of the two is that the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site is far away from the cell membrane, and the recognition unit targeting other HIV-1 gp120 binding sites is close to the cell membrane.
(2)本发明克服了靶向HIV-1嵌合抗原受体早期设计的缺陷，提出的双特异性嵌合抗原受体胞内含有共刺激信号，有利于提高HIV-1特异性CAR-T细胞的存活、增殖和杀伤能力，增加临床有效性。(2) The present invention overcomes the defects in the early design of chimeric antigen receptors targeting HIV-1, and the proposed bispecific chimeric antigen receptors contain co-stimulatory signals in cells, which is conducive to improving HIV-1-specific CAR-T Cell survival, proliferation and killing ability, increase clinical effectiveness.
附图说明Description of drawings
以下，结合附图来详细说明本发明的实施方案，其中：Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
图1为M31-CAR双特异性嵌合抗原受体结构示意图。Figure 1 is a schematic diagram of the structure of the M31-CAR bispecific chimeric antigen receptor.
图2为M13-CAR双特异性嵌合抗原受体结构示意图。Figure 2 is a schematic diagram of the structure of the M13-CAR bispecific chimeric antigen receptor.
图3为M31 CAR和M13 CAR在原代T细胞膜表面的表达情况图，其中，UTD为未转导慢病毒的T细胞对照。Figure 3 is a graph showing the expression of M31 CAR and M13 CAR on the membrane surface of primary T cells, where UTD is the control of T cells that were not transduced with lentivirus.
图4为M31 CAR-T和M13 CAR-T特异性识别靶细胞MT4-gp145并分泌细胞因子IFN-γ和IL-2情况图，其中，UTD为未转导慢病毒的T细胞对照。Figure 4 is a diagram of M31 CAR-T and M13 CAR-T specifically recognizing target cell MT4-gp145 and secreting cytokines IFN-γ and IL-2, where UTD is the T cell control that was not transduced with lentivirus.
图5为M31 CAR-T和M13 CAR-T杀伤gp145过表达细胞系MT4-gp145效率图，其中，UTD为未转导慢病毒的T细胞对照。Figure 5 is a diagram of the killing efficiency of the gp145 overexpression cell line MT4-gp145 by M31 CAR-T and M13 CAR-T, where UTD is the T cell control that was not transduced with lentivirus.
图6为M31 CAR-T和M13 CAR-T杀伤gp145过表达细胞A549-gp145曲线图。Figure 6 is a graph showing the A549-gp145 curves of M31 CAR-T and M13 CAR-T killing gp145 overexpression cells.
图7为M31 CAR-T和M13 CAR-T杀伤重激活的HIV潜伏细胞效率图，其中，UTD为未转导慢病毒的T细胞对照。Figure 7 is a graph showing the killing efficiency of HIV latent cells reactivated by M31 CAR-T and M13 CAR-T, where UTD is the control of T cells not transduced with lentivirus.
图8为M31 CAR-T对HIV-1病毒的不易感性图，图中CD4 +T细胞组为阳性对照。 Figure 8 is a graph showing the susceptibility of M31 CAR-T to HIV-1 virus, in which the CD4 + T cell group is a positive control.
具体实施方式Detailed ways
以下实施例仅用于说明本发明，但不用来限制本发明的范围。The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention.
以下实施例的实验方法，如无特殊说明，均为本领域的常规实验方法。以下实施例中所使用的实验材料，若无特殊说明，均为自常规生化试剂销售公司购买所得，其中：The experimental methods in the following examples, unless otherwise specified, are routine experimental methods in the art. The experimental materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent sales companies, wherein:
DMEM培养基、RPMI1640培养基均购自Corning公司，淋巴细胞培养基X-VIVO 15购自Lonza公司。DMEM medium and RPMI1640 medium were purchased from Corning Company, and lymphocyte medium X-VIVO 15 was purchased from Lonza Company.
T细胞生长培养基由基础培养基和细胞因子组成，基础培养基为淋巴细胞培养基X-VIVO 15，细胞因子为终浓度5ng/mL的IL-7，10ng/mL的IL-15和30ng/mL的IL-21。其中，细胞因子IL-7和IL-15购自R&D公司，IL-21购自近岸蛋白质科技有限公司。T cell growth medium consists of basal medium and cytokines, the basal medium is lymphocyte medium X-VIVO 15, the cytokines are IL-7 at a final concentration of 5ng/mL, IL-15 at 10ng/mL and 30ng/mL mL of IL-21. Among them, cytokines IL-7 and IL-15 were purchased from R&D Company, and IL-21 was purchased from Nearshore Protein Technology Co., Ltd.
胎牛血清购自BI公司。Fetal bovine serum was purchased from BI Company.
TurboFect转染试剂盒购自Thermo Fisher Scientific公司。TurboFect transfection kit was purchased from Thermo Fisher Scientific.
Lenti-X慢病毒浓缩试剂购自Takara公司。Lenti-X lentivirus concentrated reagent was purchased from Takara Company.
合成基因购自上海捷瑞生物工程有限公司。Synthetic genes were purchased from Shanghai Jierui Bioengineering Co., Ltd.
慢病毒表达质粒pKL由康霖生物科技(杭州)有限公司提供，包装质粒psPAX2和包膜质粒PMD2.G购自Addgene公司。The lentiviral expression plasmid pKL was provided by Kanglin Biotechnology (Hangzhou) Co., Ltd., and the packaging plasmid psPAX2 and envelope plasmid PMD2.G were purchased from Addgene.
Stable 3化学感受态细胞购自上海唯地生物技术有限公司。Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
无内毒素质粒小提试剂盒和无内毒素质粒中提试剂盒分别购自OMEGA公司和Macherey Nagel公司。The endotoxin-free plasmid mini-prep kit and the endotoxin-free plasmid mid-prep kit were purchased from OMEGA and Macherey Nagel, respectively.
实时无标记细胞功能分析仪(RTCA)购自上海优者生物科技有限公司。Real-time label-free cell function analyzer (RTCA) was purchased from Shanghai Youzhe Biotechnology Co., Ltd.
实施例1慢病毒表达质粒的构建Example 1 Construction of lentiviral expression plasmid
由上海捷瑞生物工程有限公司合成M31 CAR和M13 CAR基因(分别如SEQ ID No:8和SEQ ID No:9所示)，并克隆至空白慢病毒表达质粒(pKL)分别获得pKL-M31-CAR和pKL-M13-CAR重组慢病毒表达质粒，双特异性嵌合抗原受体结构如图1和2所示。The M31 CAR and M13 CAR genes (shown in SEQ ID No: 8 and SEQ ID No: 9, respectively) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and cloned into a blank lentiviral expression plasmid (pKL) to obtain pKL-M31- CAR and pKL-M13-CAR recombinant lentiviral expression plasmids, the bispecific chimeric antigen receptor structure is shown in Figures 1 and 2.
实施例2慢病毒的包装、浓缩和滴度测定Packaging, concentration and titer determination of embodiment 2 lentivirus
1.1慢病毒的包装1.1 Packaging of lentivirus
HEK293T细胞处理：转染前24小时，收集处于对数生长期的HEK293T细胞，将其接种于10cm细胞培养皿中(6～8×10 6个细胞)，细胞在含有10mL的完全DMEM培养基中生长，置于37℃，5％CO 2条件下培养18～24小时，细胞密度达到70～90％以上即可进行转染。 HEK293T cell treatment: 24 hours before transfection, collect HEK293T cells in the logarithmic growth phase, inoculate them in a 10 cm cell culture dish (6-8×10 6 cells), and place the cells in 10 mL of complete DMEM medium Grow, culture at 37°C, 5% CO 2 for 18-24 hours, and transfection can be carried out when the cell density reaches 70-90%.
HEK293T细胞转染：在15mL离心管中加入1mL基础DMEM培养基，按照质量比为慢病毒表达质粒(pKL-M31-CAR或pKL-M13-CAR):包装质粒psPAX2:包膜质粒PMD2.G＝1:3:1配制转染混合液，质粒总量合计15μg/皿。以质粒量(μg)：转染试剂(μL)＝1：2的比例加入TurboFect转染试剂30μL，室温孵育15～20分钟后加至细胞培养皿中，置于37℃，5％CO 2细胞培养箱中培养48小时并收集病毒上清，1000×g，4℃离心10分钟，收集上清病毒。 HEK293T cell transfection: Add 1mL basal DMEM medium in a 15mL centrifuge tube, according to the mass ratio: lentiviral expression plasmid (pKL-M31-CAR or pKL-M13-CAR): packaging plasmid psPAX2: envelope plasmid PMD2.G= Prepare the transfection mixture at 1:3:1, and the total amount of plasmids is 15 μg/dish. Add 30 μL of TurboFect transfection reagent at a ratio of plasmid amount (μg): transfection reagent (μL) = 1:2, incubate at room temperature for 15-20 minutes, add to a cell culture dish, and place cells at 37°C, 5% CO 2 Cultivate in an incubator for 48 hours and collect the virus supernatant, centrifuge at 1000×g, 4° C. for 10 minutes, and collect the supernatant virus.
1.2慢病毒的浓缩1.2 Enrichment of lentivirus
将离心收集的病毒上清采用0.45μm滤器过滤，加入1/3病毒上清体积的Lenti-X慢病毒浓缩试剂，颠倒混匀数次，4℃孵育过夜，2000×g，4℃离心45分钟，离心管底部可见白色沉淀，即为病毒。小心弃除上清，以原病毒上清的1/50～1/100体积的空白RPMI1640培养基重悬白色沉淀，分装并于-80℃冻存备用。Filter the virus supernatant collected by centrifugation with a 0.45 μm filter, add 1/3 of the volume of the virus supernatant Lenti-X lentivirus concentration reagent, invert and mix several times, incubate overnight at 4°C, centrifuge at 2000×g for 45 minutes at 4°C , a white precipitate can be seen at the bottom of the centrifuge tube, which is the virus. Discard the supernatant carefully, resuspend the white precipitate with 1/50-1/100 volume of the original virus supernatant blank RPMI1640 medium, aliquot and freeze at -80°C for later use.
1.3慢病毒滴度测定将Jurkat T细胞按照1×10 5个/孔接种于96孔U底板上，将所收集的慢病毒浓缩液按10倍递增稀释。将100μL的病毒稀释液加入到相应孔中，加入促感染试剂硫酸鱼精蛋白并调整浓度至10μg/mL，1000×g，32℃离心感染90分钟，过夜培养后，更换培养液，继续培养48小时，流式细胞仪检测荧光阳性细胞比例，采用下面的公式计算病毒滴度：病毒滴度(TU/mL)＝1×10 5×荧光阳性细胞比例/100×1000×相应的稀释倍数。 1.3 Determination of lentivirus titer Jurkat T cells were inoculated on a 96-well U-bottom plate at 1×10 5 cells/well, and the collected lentivirus concentrate was diluted by 10 times. Add 100 μL of virus dilution to the corresponding wells, add the pro-infection reagent protamine sulfate and adjust the concentration to 10 μg/mL, centrifuge at 1000×g, 32°C for 90 minutes, after overnight incubation, replace the culture medium, and continue to cultivate for 48 After 1 hour, the proportion of fluorescence-positive cells was detected by flow cytometry, and the virus titer was calculated using the following formula: virus titer (TU/mL)=1×10 5 ×proportion of fluorescence-positive cells/100×1000×corresponding dilution factor.
实施例3 T细胞的感染和扩增Example 3 Infection and Expansion of T Cells
在48孔平底细胞培养板中(含有1×10 6个预先活化的外周血单个核细胞)，加入实施例2包装、浓缩的慢病毒载体(LV-M31-CAR和LV-M13-CAR)(MOI＝5～10)，添加促感染试剂硫酸鱼精蛋白10μg/mL，1000×g，32℃离心感染90分钟后，向孔板中加入1×10 6个αCD3/αCD28抗体预先包被的免疫磁珠，过夜培养。第二天，更换培养液为新鲜的T细胞生长培养基继续培养。每2～3天添加新鲜的T细胞生长培养基，并调整细胞密度至0.5～2×10 6个细胞。自感染后6～7天，移除活化T细胞的免疫磁珠，继续培养扩增双特异性嵌合受体(M31-CAR或M13-CAR)修饰的T细胞，待细胞静息(移除免疫磁珠6～7天)后方可利用流式细胞仪检测CAR分子在原代细胞表面的表达情况。结果如图3所示，M31 CAR分子在原代细胞膜表面的表达情况显著优于M13 CAR，其表达阳性率约为M13 CAR分子的5倍。 In a 48-well flat-bottomed cell culture plate (containing 1× 10 pre-activated peripheral blood mononuclear cells), add the packaged and concentrated lentiviral vectors (LV-M31-CAR and LV-M13-CAR) in Example 2 ( MOI=5～10), add the pro-infection reagent protamine sulfate 10 μg/mL, 1000×g, centrifuge at 32°C for 90 minutes, add 1×10 6 αCD3/αCD28 antibody pre-coated immune cells to the well plate Magnetic beads, overnight incubation. The next day, replace the culture medium with fresh T cell growth medium to continue culturing. Add fresh T cell growth medium every 2-3 days, and adjust the cell density to 0.5-2×10 6 cells. From 6 to 7 days after infection, remove the immunomagnetic beads of activated T cells, continue to culture and expand T cells modified with bispecific chimeric receptors (M31-CAR or M13-CAR), and wait for the cells to rest (remove Immunization of magnetic beads 6-7 days) before using flow cytometry to detect the expression of CAR molecules on the surface of primary cells. The results are shown in Figure 3. The expression of M31 CAR molecules on the surface of primary cell membranes is significantly better than that of M13 CAR molecules, and its positive expression rate is about 5 times that of M13 CAR molecules.
实施例4表达双特异性嵌合抗原受体的T细胞的活化与细胞因子分泌Example 4 Activation and cytokine secretion of T cells expressing bispecific chimeric antigen receptors
将1×10 5个野生型MT4细胞或过表达gp145蛋白的MT4细胞(MT4-gp145)铺于96孔细胞培养U底板中，将未转导慢病毒的T细胞(UTD)或表达双特异性嵌合抗原受体(M31 CAR-T或M13 CAR-T)的T细胞按效应细胞：靶细胞为1：1比例铺于有靶细胞的试验孔中，每孔200uL培养基。共培养24小时后，收取100uL上清，用ELISA方法检测其中细胞因子IFN-γ和IL-2的量。结果如图4所示，表达双特异性嵌合抗原受体的T细胞仅在与MT4-gp145共培养时，才会分泌高水平的细胞因子IFN-γ和IL-2，表明表达双特异性嵌合抗原受体的T细胞可特异性地识别HIV-1囊膜蛋白并被活化。对比表达M31 CAR与表达M13 CAR的T细胞的细胞因子水平可发现，M31 CAR可更好地介导被修饰T细胞的活化，其分泌细胞因子水平几乎是M13 CAR的2倍。 Spread 1×10 5 wild-type MT4 cells or MT4 cells overexpressing gp145 protein (MT4-gp145) in a 96-well cell culture U-bottom plate, and untransduced lentivirus T cells (UTD) or express bispecific Chimeric antigen receptor (M31 CAR-T or M13 CAR-T) T cells were plated in test wells with target cells at a 1:1 ratio of effector cells: target cells, with 200uL of medium per well. After co-cultivation for 24 hours, 100 uL of supernatant was collected, and the amount of cytokines IFN-γ and IL-2 was detected by ELISA method. The results are shown in Figure 4. T cells expressing bispecific chimeric antigen receptors secrete high levels of the cytokines IFN-γ and IL-2 only when co-cultured with MT4-gp145, indicating expression of bispecific Chimeric antigen receptor T cells can specifically recognize HIV-1 envelope protein and be activated. Comparing the cytokine levels of T cells expressing M31 CAR and M13 CAR, it can be found that M31 CAR can better mediate the activation of modified T cells, and its secreted cytokine level is almost twice that of M13 CAR.
实施例5表达双特异性嵌合抗原受体的T细胞杀伤gp145过表达细胞系Example 5 T cells expressing bispecific chimeric antigen receptors kill gp145 overexpressing cell lines
细胞杀伤效率由流式细胞术和实时无标记细胞分析(RTCA，Real Time Cellular Analysis)技术两种方法进行检测。The cell killing efficiency was detected by flow cytometry and real-time label-free cell analysis (RTCA, Real Time Cellular Analysis) technology.
流式细胞仪检测：首先，利用PKH26染料对MT4-gp145进行标记(1：1000，37℃水浴10分钟)。再将1×10 5个标记后的MT4-gp145细胞铺于96孔细胞培养U底板中，以效应细胞：靶细胞＝0.4：1的比例加入未转导慢病毒的T细胞(UTD)或表达双特异性嵌合抗原受体(M31 CAR-T或M13 CAR-T)的T细胞至含有靶细胞的孔中，每24小时吸取孔中一半的细胞进行流式细胞仪检测。结果如图5所示，表达双特异性嵌合抗原受体(M31 CAR-T或M13 CAR-T)的T细胞可有效杀伤MT4-gp145细胞，48小时时的杀伤效率高达90％；对比表达M31 CAR与表达M13 CAR的T细胞的杀伤效率可发现，M31 CAR-T对靶细胞的杀伤响应更快，在24小时时的杀伤效率比M13 CAR-T高22.2％。 Flow cytometry detection: First, MT4-gp145 was labeled with PKH26 dye (1:1000, 37° C. water bath for 10 minutes). Then 1×10 5 labeled MT4-gp145 cells were plated in a 96-well cell culture U-bottom plate, and untransduced lentiviral T cells (UTD) or expression The T cells of the bispecific chimeric antigen receptor (M31 CAR-T or M13 CAR-T) were added to the wells containing the target cells, and half of the cells in the wells were aspirated every 24 hours for flow cytometry detection. The results are shown in Figure 5, T cells expressing bispecific chimeric antigen receptors (M31 CAR-T or M13 CAR-T) can effectively kill MT4-gp145 cells, and the killing efficiency is as high as 90% at 48 hours; The killing efficiency of M31 CAR and T cells expressing M13 CAR can be found that the killing response of M31 CAR-T to target cells is faster, and the killing efficiency at 24 hours is 22.2% higher than that of M13 CAR-T.
RTCA技术：首先，在16孔E-Plate电极板上接种100μL过表达gp145蛋白的A549细胞(5×10 4个、孔)，使用RTCA动态监测细胞生长12～15小时。以效应细胞：靶细胞＝1：1的比例加入表达双特异性嵌合抗原受体(M31 CAR-T或M13 CAR-T)的T细胞至含有靶细胞的孔中，每隔15分钟记录一次测定结果，连续记录24小时。 RTCA technique: First, inoculate 100 μL of A549 cells (5×10 4 cells, wells) overexpressing gp145 protein on a 16-well E-Plate electrode plate, and use RTCA to dynamically monitor cell growth for 12 to 15 hours. Add T cells expressing bispecific chimeric antigen receptors (M31 CAR-T or M13 CAR-T) to wells containing target cells at a ratio of effector cells: target cells = 1:1, and record every 15 minutes The measurement results were recorded continuously for 24 hours.
结果如图6所示,表达双特异性嵌合抗原受体(M31-CAR或M13-CAR)的T细胞可使细胞生长曲线快速下降，有效杀伤过表达gp145蛋白的肿瘤细胞，其中，表达M31-CAR的T细胞对靶细胞的杀伤能力明显优于表达M13-CAR的T细胞，杀伤率高达92.5％(M13 CAR-T细胞的杀伤效率为88.3％)。The results are shown in Figure 6. T cells expressing bispecific chimeric antigen receptors (M31-CAR or M13-CAR) can rapidly decrease the cell growth curve and effectively kill tumor cells overexpressing gp145 protein. Among them, expressing M31 - The killing ability of CAR T cells on target cells is significantly better than that of T cells expressing M13-CAR, with a killing rate as high as 92.5% (the killing efficiency of M13 CAR-T cells is 88.3%).
实施例6表达双特异性嵌合抗原受体的T细胞杀伤重激活的HIV-1潜伏细胞系Example 6 T cells expressing bispecific chimeric antigen receptors kill reactivated HIV-1 latent cell lines ACH2ACH2
将HIV-1潜伏细胞系ACH2按1×10 6个细胞/mL浓度铺于6孔板中，在培养基中添加浓度为10ng/mL的PMA激活，培养48小时后收集细胞。利用PKH26染料对重激活的ACH2细胞系进行染色标记(1：1000，37℃水浴10分钟)。再将1×10 5个标记后的ACH2细胞铺于96孔细胞培养U底板中，以效应细胞：靶细胞＝0.4：1的比例加入未转导慢病毒的T细胞(UTD)或表达双特异性嵌合抗原受体(M31-CAR或M13-CAR)的T细胞至含有靶细胞的孔中，每24小时吸取孔中一半的细胞进行流式细胞仪检测。结果如图7所示，表达双特异性嵌合抗原受体(M31-CAR或M13-CAR)的T细胞均可杀伤HIV-1潜伏细胞系ACH2，但二者间杀伤效率相差巨大；48小时时，表达M31-CAR的T细胞的杀伤效率达48.33％，比M13 CAR-T高19％。 The HIV-1 latent cell line ACH2 was plated in a 6-well plate at a concentration of 1×10 6 cells/mL, activated by adding PMA at a concentration of 10 ng/mL to the culture medium, and the cells were collected after 48 hours of culture. The reactivated ACH2 cell line was stained and marked with PKH26 dye (1:1000, 37°C water bath for 10 minutes). Then, 1×10 5 labeled ACH2 cells were plated in a 96-well cell culture U-bottom plate, and T cells (UTD) that were not transduced with lentivirus were added at a ratio of effector cells: target cells = 0.4:1 or bispecific T cells of sexual chimeric antigen receptor (M31-CAR or M13-CAR) were added to wells containing target cells, and half of the cells in the wells were drawn every 24 hours for flow cytometry detection. The results are shown in Figure 7, T cells expressing bispecific chimeric antigen receptors (M31-CAR or M13-CAR) can kill HIV-1 latent cell line ACH2, but the killing efficiency differs greatly between the two; 48 hours , the killing efficiency of T cells expressing M31-CAR reached 48.33%, which was 19% higher than that of M13 CAR-T.
实施例7表达双特异性嵌合抗原受体的T细胞对HIV-1病毒不易感Example 7 T cells expressing bispecific chimeric antigen receptors are not susceptible to HIV-1 virus
将CD4 +T细胞与表达双特异性嵌合抗原受体的T细胞按1×10 6个细胞/mL浓度铺于细胞培养平底板中，按1：1比例加入抗CD3/CD28抗体包被的磁珠，活化48-72小时后收集细胞。将5×10 5个活化后的CD4 +T细胞或表达双特异性嵌合抗原受体的T细胞铺于48孔板中，按MOI＝0.2或2的比例加入HIV-1病毒(实验室适应株pNL4-3)，培养4天及8天后，收取孔中一半的细胞，经固定穿膜处理后，进行胞内p24蛋白染色，利用流式检测p24+细胞比例。结果如图8所示，无论感染复数为0.2还是2时，M31 CAR-T对HIV-1病毒均不易感，且M31 CAR-T对HIV-1病毒的不易感性优于M13 CAR-T。 Spread CD4 + T cells and T cells expressing bispecific chimeric antigen receptors on a cell culture flat bottom plate at a concentration of 1×10 6 cells/mL, and add anti-CD3/CD28 antibody-coated Magnetic beads, cells were collected after 48-72 hours of activation. Spread 5×10 5 activated CD4 + T cells or T cells expressing bispecific chimeric antigen receptors in a 48-well plate, and add HIV-1 virus at a ratio of MOI=0.2 or 2 (laboratory adaptation Strain pNL4-3), cultured for 4 days and 8 days, collected half of the cells in the wells, fixed and transmembrane treated, stained for intracellular p24 protein, and detected the proportion of p24+ cells by flow cytometry. The results are shown in Figure 8, no matter the multiplicity of infection is 0.2 or 2, M31 CAR-T is not susceptible to HIV-1 virus, and M31 CAR-T is less susceptible to HIV-1 virus than M13 CAR-T.
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible, which fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (18)

  1. 一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述双特异性嵌合抗原受体包含：靶向HIV-1 gp120蛋白共受体结合位点的识别单元、连接肽、靶向HIV-1gp120其它结合位点的识别单元、铰链区和跨膜区、胞内信号传导结构域、一个或多个共刺激信号域；所述嵌合抗原受体胞外段的串联顺序为远膜端的靶向HIV-1 gp120蛋白共受体结合位点的识别单元，近膜端的靶向HIV-1 gp120其它结合位点的识别单元。A bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein, characterized in that the bispecific chimeric antigen receptor comprises: targeting HIV-1 gp120 protein co-receptor binding site Recognition units, linking peptides, recognition units targeting HIV-1gp120 other binding sites, hinge regions and transmembrane regions, intracellular signaling domains, one or more co-stimulatory signal domains; the chimeric antigen receptor The tandem sequence of the extracellular segment is the recognition unit targeting the HIV-1 gp120 protein co-receptor binding site at the far membrane end, and the recognition unit targeting other HIV-1 gp120 binding sites at the near membrane end.
  2. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于所述靶向HIV-1 gp120蛋白共受体结合位点的识别单元为单域抗体m36.4、17b或X5。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, characterized in that the recognition unit targeting HIV-1 gp120 protein co-receptor binding site is a single Domain antibody m36.4, 17b or X5.
  3. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述连接肽为(G 4S) n或G n。 The bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, wherein the connecting peptide is (G 4 S) n or G n .
  4. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于所述靶向HIV-1 gp120其它结合位点的识别单元包括但不限于CD4结合位点、V1V2聚糖区、V3聚糖区、gp120-gp41交界面或gp41上的近膜端外部区域。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, characterized in that the recognition unit targeting HIV-1 gp120 other binding sites includes but is not limited to CD4 Binding site, V1V2 glycan region, V3 glycan region, gp120-gp41 interface, or membrane-proximal outer region on gp41.
  5. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述铰链区和跨膜区来源于CD28、CD8α或CD3ζ的铰链区和跨膜区。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, wherein the hinge region and the transmembrane region are derived from the hinge region and the CD28, CD8α or CD3ζ transmembrane region.
  6. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，共刺激信号域选自CD28、4-1BB、CD27、ICOS、CD160、CD69、TLR2、CD27、CD40L、CD30、OX40、TIM1中的一种或多种。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, wherein the co-stimulatory signal domain is selected from CD28, 4-1BB, CD27, ICOS, CD160, CD69 One or more of , TLR2, CD27, CD40L, CD30, OX40, TIM1.
  7. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于所述胞内信号传导结构域为CD3ζ胞内信号传导域。A bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein according to claim 1, characterized in that the intracellular signaling domain is a CD3ζ intracellular signaling domain.
  8. 根据权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于：所述双异性嵌合抗原受体从N端到C端顺次包括靶向HIV-1 gp120蛋白共受体结合位点的识别单元、连接肽、靶向HIV-1 gp120其它结合位点的识别单元、铰链和跨膜区、一个或多个共刺激信号域和胞内信号传导结构域。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, characterized in that: said biheterotropic chimeric antigen receptor includes target in sequence from N-terminal to C-terminal A recognition unit targeting the HIV-1 gp120 protein co-receptor binding site, a connecting peptide, a recognition unit targeting other HIV-1 gp120 binding sites, hinge and transmembrane regions, one or more co-stimulatory signaling domains and intracellular Signal transduction domain.
  9. 根据权利要求8所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述靶向HIV-1 gp120蛋白共受体结合位点的识别单元为单域抗体m36.4，其氨基酸序列如SEQ ID No.1所示；所述靶向HIV-1 gp120其它结合位点的识别单元为突变型CD4 D1结构域,其氨基酸序列如SEQ ID No.2所示。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 8, wherein the recognition unit targeting HIV-1 gp120 protein co-receptor binding site is The amino acid sequence of the single domain antibody m36.4 is shown in SEQ ID No.1; the recognition unit targeting other binding sites of HIV-1 gp120 is a mutant CD4 D1 domain, and its amino acid sequence is shown in SEQ ID No. 2.
  10. 根据权利要求8所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于所述的连接肽为3×G 4S；所述铰链区和跨膜区为CD28的铰链区和跨膜区，氨基酸序列如SEQ ID No.3所示；所述共刺激信号域为CD28和4-1BB的共刺激信号域，氨基酸序列如 SEQ ID No.4和SEQ ID No.5所示；所述胞内信号传导结构域为CD3ζ胞内信号传导域，氨基酸序列如SEQ ID No.6所示。 A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 8, characterized in that the connecting peptide is 3×G 4 S; the hinge region and the transmembrane region It is the hinge region and transmembrane region of CD28, the amino acid sequence is shown in SEQ ID No.3; the costimulatory signal domain is the costimulatory signal domain of CD28 and 4-1BB, and the amino acid sequence is shown in SEQ ID No.4 and SEQ ID Shown in No.5; the intracellular signaling domain is CD3ζ intracellular signaling domain, and the amino acid sequence is shown in SEQ ID No.6.
  11. 根据权利要求8所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述嵌合抗原受体还包括信号肽和标签，标签位于信号肽和胞外识别域之间，信号肽为CD8的信号肽，标签为Flag标签。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 8, wherein said chimeric antigen receptor also includes a signal peptide and a tag, and the tag is located between the signal peptide and the tag. Between the extracellular recognition domains, the signal peptide is the signal peptide of CD8, and the tag is the Flag tag.
  12. 根据权利要求8所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，所述双特异性嵌合抗原受体的氨基酸序列如SEQ ID NO.7所示。A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 8, characterized in that the amino acid sequence of said bispecific chimeric antigen receptor is as SEQ ID NO.7 shown.
  13. 根据权利要求8所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，其特征在于，编码所述的双特异性嵌合抗原受体的基因核苷酸序列如SEQ ID NO.8所示。A bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein according to claim 8, characterized in that the gene nucleotide sequence encoding the bispecific chimeric antigen receptor is as follows: Shown in SEQ ID NO.8.
  14. 一种载体，其特征在于，所述载体能够表达权利要求1所述靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体，所述载体包含SEQ ID NO.8所示核苷酸序列。A carrier, characterized in that, said carrier can express the bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein according to claim 1, said carrier comprising nucleosides shown in SEQ ID NO.8 acid sequence.
  15. 一种基因工程化T淋巴细胞，其特征在于，所述T淋巴细胞包含权利要求14所述载体，并且可以表达靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体。A genetically engineered T lymphocyte, characterized in that the T lymphocyte comprises the vector according to claim 14 and can express a bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein.
  16. 一种权利要求15所述基因工程化T淋巴细胞的制备方法，其特征在于，该方法包括以下步骤：将权利要求14所述的靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体编码序列连接到载体上，包装获得慢病毒颗粒，利用该慢病毒感染T淋巴细胞从而获得双特异性嵌合抗原受体修饰的T淋巴细胞。A method for preparing genetically engineered T lymphocytes according to claim 15, characterized in that the method comprises the steps of: applying the bispecific chimeric antigen targeting HIV-1 virus envelope protein according to claim 14 The receptor coding sequence is connected to the carrier, and lentiviral particles are packaged, and the lentivirus is used to infect T lymphocytes to obtain bispecific chimeric antigen receptor-modified T lymphocytes.
  17. 权利要求1所述一种靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体在制备治疗或预防艾滋病药物方面的应用。The application of the bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein in claim 1 in the preparation of AIDS treatment or prevention drugs.
  18. 权利要求8所述靶向HIV-1病毒囊膜蛋白的双特异性嵌合抗原受体的制备方法，其具体步骤如下所示：将分别编码靶向HIV-1 gp120蛋白共受体结合位点的识别单元、连接肽、靶向HIV-1 gp120其它结合位点的识别单元、铰链和跨膜区、共刺激信号域和胞内信号传导结构域的核苷酸序列按5’至3’方向顺次串联，利用基因合成克隆至空白慢病毒表达载体pKL上，从而获得表达双特异性嵌合抗原受体的慢病毒质粒。The preparation method of the bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein as claimed in claim 8, its specific steps are as follows: coding target HIV-1 gp120 protein co-receptor binding site respectively The nucleotide sequences of recognition units, connecting peptides, recognition units targeting other binding sites of HIV-1 gp120, hinge and transmembrane regions, co-stimulatory signal domains and intracellular signaling domains are arranged in the 5' to 3' direction Sequentially tandem, using gene synthesis and cloning to a blank lentiviral expression vector pKL, thereby obtaining a lentiviral plasmid expressing a bispecific chimeric antigen receptor.
PCT/CN2022/117012 2021-11-08 2022-09-05 Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof WO2023077943A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111312730.1 2021-11-08
CN202111312730.1A CN114163534B (en) 2021-11-08 2021-11-08 Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof

Publications (1)

Publication Number Publication Date
WO2023077943A1 true WO2023077943A1 (en) 2023-05-11

Family

ID=80478205

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/117012 WO2023077943A1 (en) 2021-11-08 2022-09-05 Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof

Country Status (2)

Country Link
CN (1) CN114163534B (en)
WO (1) WO2023077943A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114163534B (en) * 2021-11-08 2023-07-18 复旦大学 Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130108636A1 (en) * 2010-05-21 2013-05-02 Dimiter S. Dimitrov High-affinity fully functional soluble single-domain human cd4, antibodies, and related fusion proteins
CN111196858A (en) * 2020-02-05 2020-05-26 武汉科技大学 Bispecific chimeric antigen receptor for treating hematological tumor complicated with HIV infection, gene, construction method and application thereof
CN111954677A (en) * 2017-12-20 2020-11-17 莱蒂恩技术公司 Compositions and methods for treating HIV/AIDS with immunotherapy
CN112368052A (en) * 2018-07-03 2021-02-12 吉利德科学公司 Antibodies targeting HIV GP120 and methods of use
CN114163534A (en) * 2021-11-08 2022-03-11 复旦大学 Bispecific chimeric antigen receptor of targeted HIV-1 virus envelope protein and preparation method and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107619821B (en) * 2017-10-13 2020-02-07 北京舜雷科技有限公司 CIK modified by similar chimeric antigen receptor and preparation method and application thereof
CN111253493B (en) * 2020-03-05 2021-03-23 武汉科技大学 Chimeric antigen receptor targeting HIV virus envelope double-site, expression vector and application thereof
CN112552404B (en) * 2020-07-20 2022-02-08 北京鼎成肽源生物技术有限公司 Single-chain antibody targeting c-Met, chimeric antigen receptor, recombinant vector, CAR-T cell and application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130108636A1 (en) * 2010-05-21 2013-05-02 Dimiter S. Dimitrov High-affinity fully functional soluble single-domain human cd4, antibodies, and related fusion proteins
CN111954677A (en) * 2017-12-20 2020-11-17 莱蒂恩技术公司 Compositions and methods for treating HIV/AIDS with immunotherapy
CN112368052A (en) * 2018-07-03 2021-02-12 吉利德科学公司 Antibodies targeting HIV GP120 and methods of use
CN111196858A (en) * 2020-02-05 2020-05-26 武汉科技大学 Bispecific chimeric antigen receptor for treating hematological tumor complicated with HIV infection, gene, construction method and application thereof
CN114163534A (en) * 2021-11-08 2022-03-11 复旦大学 Bispecific chimeric antigen receptor of targeted HIV-1 virus envelope protein and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OLLERTON MATTHEW T., BERGER EDWARD A., CONNICK ELIZABETH, BURTON GREGORY F.: "HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 94, no. 10, 4 May 2020 (2020-05-04), US , XP093063257, ISSN: 0022-538X, DOI: 10.1128/JVI.00190-20 *

Also Published As

Publication number Publication date
CN114163534B (en) 2023-07-18
CN114163534A (en) 2022-03-11

Similar Documents

Publication Publication Date Title
JP6890664B2 (en) Recombinant gene construction of chimeric antigen receptor for treating HIV infection and its application
CN108384760B (en) Human T lymphocyte carrying CD20/CD19 bispecific chimeric antigen receptor and preparation method and application thereof
CN113913379B (en) T lymphocyte and application thereof
CN110606893B (en) Method for treating tumor by chimeric antigen receptor T cell targeting CD19 and CD20 double antigens
WO2018053885A1 (en) Preparation method for and use of enhanced slit2 car-t cell and car-nk cell
CN111560076A (en) Chimeric antigen receptor immune cell and preparation method and application thereof
WO2022007795A1 (en) Chimeric antigen receptor and use thereof
CN110684121A (en) Chimeric antigen receptor of targeted HER2 combined expression PD1-MICA fusion protein, expression vector and application thereof
CN108752482A (en) Carry Chimeric antigen receptor and its application of truncation or not truncated myeloid cell triggering property receptor signal structure
US20180185416A1 (en) Methods and compositions for treating hiv
Tal et al. An NCR1-based chimeric receptor endows T-cells with multiple anti-tumor specificities
WO2023077943A1 (en) Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof
CN111171160A (en) chimeric antigen receptor based on TGF- β modification and modified immune cell thereof
CN117567650B (en) CAR-T cell co-expressing intercellular adhesion molecule ICAM2 as well as preparation method and application thereof
WO2023039968A1 (en) Multifunctional anti-hiv-1 car-t cell, construction method therefor and application thereof
CN117003834A (en) Envelope glycoprotein for transducing NK cell pseudotyped lentiviral vector and application thereof
CN107974460B (en) Chimeric antigen receptor gene aiming at HIV-1, plasmid, T cell, kit and application with gene
CN115595310A (en) NK cell trophoblast, preparation method and application thereof
CN112521515B (en) CD19 and CD10 double-target chimeric antigen receptor and application thereof
CN117567651B (en) Chimeric antigen receptor for synergistically expressing vascular endothelial adhesion molecule VCAM1 and application thereof
CN114573712B (en) Chimeric antigen receptor, CAR-T cell and application thereof
CN114591443A (en) Chimeric receptor CSR based on scTv and application thereof
CN116041550A (en) NKG 2D-targeting chimeric antigen receptor and application thereof
CN114957483A (en) CD 155-targeted CAR (CAR-based protein) structural design and application thereof
CN117736335A (en) Double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof

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: 22888974

Country of ref document: EP

Kind code of ref document: A1