CN114163534B - Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof - Google Patents

Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof Download PDF

Info

Publication number
CN114163534B
CN114163534B CN202111312730.1A CN202111312730A CN114163534B CN 114163534 B CN114163534 B CN 114163534B CN 202111312730 A CN202111312730 A CN 202111312730A CN 114163534 B CN114163534 B CN 114163534B
Authority
CN
China
Prior art keywords
hiv
chimeric antigen
antigen receptor
bispecific chimeric
targeting
Prior art date
Legal status (The legal status 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 status listed.)
Active
Application number
CN202111312730.1A
Other languages
Chinese (zh)
Other versions
CN114163534A (en
Inventor
徐建青
张晓燕
应天雷
毛蕴玉
廖启彬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fudan University
Original Assignee
Fudan University
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 Fudan University filed Critical Fudan University
Priority to CN202111312730.1A priority Critical patent/CN114163534B/en
Publication of CN114163534A publication Critical patent/CN114163534A/en
Priority to PCT/CN2022/117012 priority patent/WO2023077943A1/en
Application granted granted Critical
Publication of CN114163534B publication Critical patent/CN114163534B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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

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

The present invention provides a bispecific chimeric antigen receptor targeting HIV-1 viral envelope proteins, comprising: (1) A recognition unit targeting the HIV-1gp120 protein co-receptor binding site; (2) A recognition unit targeting other binding sites of HIV-1gp120 (including but not limited to the CD4 binding site, the V1V2 glycan region, the V3 glycan region, the gp120-gp41 interface or the membrane proximal outer region on gp 41); (3) Hinge and transmembrane regions, and (4) intracellular signaling domains; optionally, the chimeric antigen receptor further comprises (5) one or more costimulatory signaling domains; preferably, the chimeric antigen receptor extracellular recognition domains are in tandem order, and the far membrane end is a recognition unit targeting the HIV-1gp120 protein co-receptor binding site, and the near membrane end is a recognition unit targeting other HIV gp120 binding sites. The invention provides a brand-new construction method of HIV-1 virus envelope protein bispecific chimeric antigen receptor, and the modified immune cells have stronger activation and killing capacity, can specifically identify and kill HIV-1 infected cells, and have good application prospects.

Description

Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof
Technical Field
The invention relates to the technical field of immunotherapy, in particular to a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and application thereof.
Background
AIDS is an infectious disease which threatens the safety of human life and is caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus, HIV-1) infection, and is also one of the most important public health challenges facing China. High-efficiency antiretroviral therapy (highly active anti-retroviral therapy, HAART) was the first revolution in the history of HIV-1/AIDS treatment, and was effective in controlling the level of viral replication in HIV-1 infected subjects, resulting in lower viral loads in peripheral blood of the infected subjects than the detection line, and limiting the progression of AIDS disease. However, HAART still has many non-negligible limitations, such as serious drug side effects and limitations of life-long dosing. Furthermore, its greatest limitation is the inability to target or clear latent HIV-1 virus in resting cd4+ T cells in a patient. Thus, once HAART treatment is discontinued, the level of viral load in the blood of the infected person will rebound rapidly, which constitutes a major obstacle to HIV-1/AIDS cure.
How to reactivate the latent HIV-1 virus and allow it to be spontaneously recognized and cleared by the immune system of the body is critical in curing AIDS. Meanwhile, studies have found that irreversible immune damage, particularly reduction in the number of cytotoxic T cells and functional exhaustion, occurs even in infected subjects receiving HAART treatment, suggesting a need to enhance the clearing effect of HIV-1 reservoirs by combining methods to enhance HIV-1 specific immune responses in the body.
Chimeric antigen receptor modified T cells (Chimeric antigen receptor T cells, CAR-T cells) are one of the latest technologies in current adoptive cell therapy technology, enabling advances from basic immunological mechanism research to clinical immunotherapy applications. Among them, CAR-T products CTL019 (nohua) and KTEC19 (kit) for treating leukemia and lymphoma are sequentially approved by the FDA in the united states in 2017 to be marketed, and bring new eosin for application of immune cell therapy technology. Thus, genetically modified CAR-T cells are likely to be a favorable weapon to clear the HIV-1 virus latency pool. In fact, since the 90 s of the last century, studies have been initiated to treat patients with advanced AIDS by adoptive transfer of genetically modified T cells (adoptive cell transfer). In 1994, roberts et al selected the CD4 sequence as the extracellular recognition domain of the CAR, prepared T cells expressing the CD4-CD3 ζ chimeric receptor (first generation CAR), while having the ability to target and kill HIV-1 infected or HIV-1Env expressing cells in vitro, were then unsuccessful in phase I and II clinical trials. Patient follow-up of 3 clinical trials in 1998-2005 by Scholler et al found that autologous CD4+ and CD8+ T lymphocytes expressing CD4-CD3 ζCAR could survive stably for at least 11 years in HIV-1 infected individuals, demonstrating the great potential of CAR-T in the field of HIV-1 infected cell therapy. The primary cause of first generation HIV-1CAR-T cell failure can be summarized in the following ways: (1) Researchers at that time lack understanding of signaling requirements and costimulatory signals for effective T cell function, e.g., improving CAR-T cell performance in vivo by providing costimulatory signal CD28 that is beneficial for cell proliferation, cytokine secretion, and cell survival; (2) CAR molecule design itself is deficient, and the extracellular recognition domain of CAR directly acts as the extracellular domain of CD4 protein, potentially making CAR-T cells the target of HIV-1 attack; (3) Transduction with retroviral vectors is inefficient, and excessive in vitro expansion results in excessive cell depletion and apoptosis after reinfusion in order to obtain adequate reinfusion CAR-T cells. Kim Anthony-Gonda et al reported in a multi-specific CAR-T cell study targeting HIV-1 virus a construction method of bispecific CAR-T cells targeting HIV-1, the CAR molecule was serially connected in sequence from N-terminus to C-terminus with mutant CD 4D 1 domain mD1.22, connecting peptide, single domain antibody m36.4, hinge and transmembrane region of CD8, 4-1BB costimulatory signaling domain and CD3 zeta intracellular signaling domain. However, the serial sequence not only affects the membrane expression level of the CAR molecule, but also obviously weakens the activation ability of the CAR-T cells and the killing ability of the HIV-1 infected target cells, which is not beneficial to the infection control of the HIV-1. Thus, there remains a need to further improve and optimize the structural design of HIV-1-targeting bispecific CARs, enhancing their control of HIV-1 infection and the clearance of latency libraries
Disclosure of Invention
The technical problems to be solved by the invention are as follows: to solve the deficiencies in the prior art, the activation capability of the CAR-T cells and the killing capability of the HIV-1 infected target cells are enhanced, the structural design of the dual-specificity CAR targeting HIV-1 is further improved and optimized, and the control of HIV-1 infection and the clearance capability of the latency library are enhanced.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: there is provided a bispecific chimeric antigen receptor targeting HIV-1 viral envelope proteins, comprising: a recognition unit targeting the binding site of the HIV-1gp120 protein co-receptor, a linker peptide, a recognition unit targeting other binding sites of HIV-1gp120, hinge and transmembrane regions, an intracellular signaling domain, one or more costimulatory signaling domains; the serial sequence of the extracellular segment of the chimeric antigen receptor is a far-membrane recognition unit targeting the HIV-1gp120 protein co-receptor binding site, and a near-membrane recognition unit targeting other HIV-1gp120 binding sites.
The recognition unit of the HIV-1gp120 protein co-receptor binding site is a single-domain antibody m36.4, 17b or X5.
The connecting peptide is ( G 4 S) n Or G n 。
The recognition units targeting other binding sites of HIV-1gp120 include, but are not limited to, the CD4 binding site, the mutant CD 4D 1 domain, the V1V2 glycan region, the V3 glycan region, the gp120-gp41 interface, or the membrane proximal outer region on gp 41.
The hinge and transmembrane regions are derived from the hinge and transmembrane regions of CD28, CD8 a or CD3 ζ.
The costimulatory signaling domain is selected from one or more of CD28, 4-1BB, CD27, ICOS, CD160, CD69, TLR2, CD27, CD40L, CD, OX40, TIM 1.
The intracellular signaling domain is a cd3ζ intracellular signaling domain.
Preferably, the bispecific chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a recognition unit targeting the HIV-1gp120 protein co-receptor binding site, a connecting peptide, a recognition unit targeting other binding sites of HIV-1gp120, a hinge and transmembrane region, one or more costimulatory signaling domains, and an intracellular signaling domain.
Preferably, the recognition unit of the HIV-1gp120 protein co-receptor binding site is a single-domain antibody m36.4, and the amino acid sequence of the recognition unit is shown as SEQ ID No. 1; the recognition unit of the other binding sites of the target HIV-1gp120 is a mutant CD 4D 1 structural domain, and the amino acid sequence of the recognition unit is shown as SEQ ID No. 2.
Preferably, the connecting peptide is 3 XG 4 S, S; the hinge region and the transmembrane region are the hinge region and the transmembrane region of CD28, and the amino acid sequence is shown as SEQ ID No. 3; the co-stimulusThe signal domain is the co-stimulatory signal domain of CD28 and 4-1BB, and the amino acid sequences are shown as SEQ ID No.4 and SEQ ID No. 5; the intracellular signaling domain is CD3 zeta intracellular signaling domain, and the amino acid sequence is shown as SEQ ID No. 6.
Preferably, the chimeric antigen receptor further comprises a signal peptide and a tag, the tag being located between the signal peptide and the extracellular recognition domain, the signal peptide being a signal peptide of CD8 and the tag being a Flag tag.
The amino acid sequence of the bispecific chimeric antigen receptor is shown as SEQ ID NO. 7.
The nucleotide sequence of the gene for encoding the bispecific chimeric antigen receptor is shown in SEQ ID NO. 8.
A vector capable of expressing the above bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein, comprising the nucleotide sequence set forth in SEQ ID No. 8. In addition, the invention also provides a genetically engineered T lymphocyte which comprises the vector and can express a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein.
The preparation method of the genetically engineered T lymphocyte comprises the following steps: the bispecific chimeric antigen receptor coding sequence of the target HIV-1 virus envelope protein is connected to a carrier, and is packaged to obtain lentiviral particles, and T lymphocytes are infected by using the lentivirus to obtain the bispecific chimeric antigen receptor modified T lymphocytes.
The application of the bispecific chimeric antigen receptor of the target HIV-1 virus envelope protein in preparing medicaments for treating or preventing AIDS.
The invention has the beneficial effects that:
(1) The invention overcomes the defects of the existing design, simultaneously utilizes the recognition unit of the target HIV-1gp120 protein co-receptor binding site and the recognition unit of the target HIV-1gp120 other binding site (including but not limited to CD4 binding site, V1V2 glycan region, V3 glycan region, gp120-gp41 interface or near membrane end external region on gp 41), greatly improves the broad spectrum and specificity of the chimeric antigen receptor for recognizing HIV-1 virus envelope protein, and proposes that the dominant tandem sequence of the recognition unit of the target HIV-1gp120 protein co-receptor binding site is far away from the cell membrane, and the recognition unit of the target HIV-1gp120 other binding site is close to the cell membrane.
(2) The invention overcomes the defect of early design of the target HIV-1 chimeric antigen receptor, and the proposed bispecific chimeric antigen receptor contains a co-stimulation signal in the cell, which is beneficial to improving the survival, proliferation and killing ability of HIV-1 specific CAR-T cells and increasing clinical effectiveness.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a schematic representation of the structure of a M31-CAR bispecific chimeric antigen receptor.
FIG. 2 is a schematic representation of the structure of a M13-CAR bispecific chimeric antigen receptor.
Fig. 3 is a graph of expression of M31CAR and M13CAR at the surface of primary T cell membranes, where UTD is a T cell control that does not transduce lentivirus.
FIG. 4 is a graph of M31 CAR-T and M13 CAR-T specific recognition of target cells MT4-gp145 and secretion of cytokines IFN-gamma and IL-2, where UTD is a T cell control that does not transduce lentivirus.
FIG. 5 is a graph of M31 CAR-T and M13 CAR-T killing gp145 over-expressing cell line MT4-gp145 efficiency, wherein UTD is a T cell control without transduced lentivirus.
FIG. 6 is a graph of M31 CAR-T and M13 CAR-T killer gp145 overexpressing cells A549-gp 145.
Figure 7 is a graph of M31 CAR-T and M13 CAR-T killing re-activated HIV latent cell efficiency, where UTD is a T cell control that does not transduce lentivirus.
FIG. 8 is a graph of M31 CAR-T non-susceptibility to HIV-1 virus, CD4 in the graph + T cell groups were positive controls.
Detailed Description
The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
The experimental methods of the following examples, unless otherwise specified, are all routine in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent sales companies, wherein:
DMEM medium, RPMI1640 medium, was purchased from Corning, and lymphocyte culture medium X-VIVO15 was purchased from Lonza.
The T cell growth medium consists of a basal medium and cytokines, wherein the basal medium is lymphocyte medium X-VIVO15, and the cytokines are IL-7 with a final concentration of 5ng/mL, IL-15 with a final concentration of 10ng/mL and IL-21 with a final concentration of 30 ng/mL. Among them, cytokines IL-7 and IL-15 were purchased from R & D, and IL-21 was purchased from offshore protein technologies Inc.
Fetal bovine serum was purchased from BI company.
The TurboFect transfection kit was purchased from Thermo Fisher Scientific company.
Lenti-X lentiviral concentrate reagent was purchased from Takara.
Synthetic genes were purchased from Shanghai Jieli bioengineering Co.
Lentiviral expression plasmid pKL was supplied by Kang Lin Biotechnology (Hangzhou) Inc., packaging plasmid psPAX2 and envelope plasmid PMD2.G were purchased from Addgene Inc.
Stable 3 chemically competent cells were purchased from Shanghai Biotechnology, inc.
The endotoxin-free plasmid miniprep kit and the endotoxin-free plasmid midprep kit were purchased from OMEGA and Macherey Nagel, respectively.
Real-time label-free cell function analyzers (RTCAs) were purchased from Shanghai major biotechnology limited.
EXAMPLE 1 construction of lentiviral expression plasmidsM31CAR and M13CAR genes (shown as 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, respectively, the bispecific chimeric antigen receptor structures being shown in FIGS. 1 and 2.
EXAMPLE 2 packaging, concentration and titre determination of lentiviruses
1.1 packaging of lentiviruses
HEK293T cell treatment: HEK293T cells in the logarithmic growth phase were collected 24 hours before transfection and inoculated into 10cm cell culture dishes (6 to 8X 10) 6 Individual cells) were grown in complete DMEM medium containing 10mL, placed at 37 ℃,5% co 2 Culturing for 18-24 hours under the condition that the cell density reaches over 70-90 percent, and then carrying out transfection.
HEK293T cell transfection: 1mL of basic DMEM medium is added into a 15mL centrifuge tube, and a transfection mixture is prepared according to the mass ratio of lentiviral expression plasmids (pKL-M31-CAR or pKL-M13-CAR) to packaging plasmids psPAX2 to envelope plasmids PMD2. G=1:3:1, and the total plasmid amount is 15 mug/dish. Plasmid amount (μg): transfection reagent (μl) =1: 2, 30 mu L of TurboFect transfection reagent is added in proportion, incubated for 15 to 20 minutes at room temperature, added into a cell culture dish and placed at 37 ℃ and 5 percent CO 2 The cells were cultured in a cell incubator for 48 hours and the virus supernatant was collected, centrifuged at 1000 Xg at 4℃for 10 minutes, and the supernatant virus was collected.
1.2 concentration of lentiviruses
Filtering the virus supernatant collected by centrifugation by a 0.45 mu m filter, adding a Lenti-X lentiviral concentration reagent with the volume of 1/3 of the virus supernatant, reversing and uniformly mixing for several times, incubating overnight at 4 ℃, centrifuging for 45 minutes at 2000 Xg at 4 ℃, and obtaining the virus by visible white precipitation at the bottom of a centrifuge tube. Carefully discarding the supernatant, re-suspending the white precipitate with 1/50-1/100 volume of blank RPMI1640 culture medium of proviral supernatant, sub-packaging and freezing at-80 ℃ for later use.
1.3 lentiviral titre assay Jurkat T cells were assayed as 1X 10 5 The lentiviral concentrate collected was diluted 10-fold in increments by inoculating the cells/well onto a 96-well U-bottom plate. 100 mu L of virus diluent is added into a corresponding hole, an infection-promoting reagent protamine sulfate is added, the concentration is adjusted to 10 mu g/mL, the concentration is 1000 Xg, the virus is centrifugally infected for 90 minutes at 32 ℃, after overnight culture, the culture solution is replaced, the culture is continued for 48 hours, the proportion of fluorescence positive cells is detected by a flow cytometer, and the virus titer is calculated by adopting the following formula: virus titer (TU/mL) =1×10 5 X fluorescence positive cell fraction/100 x 1000 x corresponding dilution.
EXAMPLE 3 infection and expansion of T cells
In 48 well flat bottom cell culture plates (containing 1X 10) 6 Pre-activated peripheral blood mononuclear cells), packed, concentrated lentiviral vectors (LV-M31-CAR and LV-M13-CAR) of example 2 (moi=5-10), 10 μg/mL of protamine sulfate, an pro-infective agent, 1000×g, centrifugation at 32 ℃ for 90 min, and 1×10 added to the well plate 6 The individual αCD3/αCD28 antibodies were pre-coated immunomagnetic beads and incubated overnight. The next day, the culture medium was replaced with fresh T cell growth medium for continued culture. Fresh T cell growth medium is added every 2 to 3 days, and the cell density is regulated to 0.5 to 2 multiplied by 10 6 Individual cells. From 6-7 days after infection, removing immunomagnetic beads of activated T cells, continuing to culture and expand T cells modified by bispecific chimeric receptor (M31-CAR or M13-CAR), and detecting the expression condition of CAR molecules on the surface of primary cells by using a flow cytometry after the cells are at rest (after the immunomagnetic beads are removed for 6-7 days). The results are shown in fig. 3, the expression of the M31CAR molecule on the surface of the primary cell membrane is significantly better than that of the M13CAR, and the expression positive rate is about 5 times that of the M13CAR molecule.
Example 4 activation of T cells expressing bispecific chimeric antigen receptor and cytokine secretion
Will be 1X 10 5 The wild type MT4 cells or MT4 cells overexpressing gp145 protein (MT 4-gp 145) were plated in 96-well cell culture U-plates, and non-lentiviral transduced T cells (UTD) or T cells expressing bispecific chimeric antigen receptor (M31 CAR-T or M13 CAR-T) were plated as effector cells: target cells were 1:1 were plated in test wells with target cells, 200uL of medium per well. After 24 hours of co-culture, 100uL of supernatant was collected and the amounts of cytokines IFN-. Gamma.and IL-2 therein were measured by ELISA. The results are shown in FIG. 4, where T cells expressing the bispecific chimeric antigen receptor secrete high levels of cytokines IFN-gamma and IL-2 only when co-cultured with MT4-gp145, indicating that T cells expressing the bispecific chimeric antigen receptor specifically recognize HIV-1 envelope proteins and are activated. Comparing the cytokine levels of M31 CAR-expressing and M13 CAR-expressing T cells, it can be seen that M31CAR can better mediate activation of modified T cells, which secrete cellsFactor levels were almost 2 times that of M13 CAR.
EXAMPLE 5T cell killing gp145 overexpressing cell lines expressing bispecific chimeric antigen receptorsCell killing efficiency was measured by both flow cytometry and real-time label-free cell analysis (RTCA, real Time Cellular Analysis) techniques.
Flow cytometer detection: first, MT4-gp145 was labeled with PKH26 dye (1:1000, 37℃water bath for 10 min). And 1×10 5 The labeled MT4-gp145 cells were plated in 96-well cell culture U-bottom plates to effect cells: target cell = 0.4:1 to the wells containing target cells, half of the cells in the wells were aspirated every 24 hours for flow cytometry detection. As shown in fig. 5, T cells expressing bispecific chimeric antigen receptor (M31 CAR-T or M13 CAR-T) can kill MT4-gp145 cells efficiently, with a killing efficiency of up to 90% at 48 hours; comparing the killing efficiency of M31 CAR-expressing versus M13 CAR-expressing T cells, it can be seen that M31 CAR-T has a faster killing response to target cells, with 22.2% higher killing efficiency at 24 hours than M13 CAR-T.
RTCA technology: first, 100. Mu.L of A549 cells overexpressing gp145 protein (5X 10) were inoculated onto 16-well E-Plate electrode plates 4 Well), cells were dynamically monitored for growth using RTCA for 12-15 hours. The following effector cells: target cell = 1:1 to the wells containing target cells, the T cells expressing bispecific chimeric antigen receptor (M31 CAR-T or M13 CAR-T) were added, the assay results were recorded once every 15 minutes, and 24 hours in succession.
As shown in fig. 6, the T cells expressing the bispecific chimeric antigen receptor (M31-CAR or M13-CAR) can quickly reduce the cell growth curve, and effectively kill the tumor cells over-expressing gp145 protein, wherein the killing capacity of the T cells expressing the M31-CAR to the target cells is obviously better than that of the T cells expressing the M13-CAR, and the killing rate is as high as 92.5% (the killing efficiency of the M13 CAR-T cells is 88.3%).
EXAMPLE 6T cell killing reactivation of bispecific chimeric antigen receptor expressionIs the HIV-1 latent cell line ACH2
ACH2 was prepared as a 1X 10 potential HIV-1 cell line 6 The cells were plated in 6-well plates at a concentration of 10ng/mL PMA and activated in the medium, and the cells were collected after culturing for 48 hours. The re-activated ACH2 cell lines were stained with PKH26 dye (1:1000, 37℃water bath for 10 min). And 1×10 5 The individually labeled ACH2 cells were plated in 96-well cell culture U-bottom plates to effect cells: target cell = 0.4:1 to the wells containing target cells, half of the cells in the wells were aspirated every 24 hours for flow cytometry detection. As shown in fig. 7, T cells expressing bispecific chimeric antigen receptor (M31-CAR or M13-CAR) can kill ACH2, which is an HIV-1 latent cell line, but the killing efficiency differs greatly; at 48 hours, the killing efficiency of T cells expressing M31-CAR reached 48.33%, 19% higher than that of M13 CAR-T.
EXAMPLE 7T cells expressing bispecific chimeric antigen receptor are not susceptible to HIV-1 Virus
CD4 + T cells and T cells expressing bispecific chimeric antigen receptor were mixed at 1X 10 6 The individual cell/mL concentration was plated on a cell culture flat bottom plate at a ratio of 1:1 proportion of anti-CD 3/CD28 antibody coated magnetic beads, and cells were collected after 48-72 hours of activation. Will be 5X 10 5 Activated CD4 + T cells or T cells expressing bispecific chimeric antigen receptor were plated in 48 well plates, HIV-1 virus (laboratory adapted strain pNL 4-3) was added at a ratio of moi=0.2 or 2, after 4 and 8 days of culture, half of the cells in the wells were harvested, after fixed membrane-penetrating treatment, intracellular p24 protein staining was performed, and the p24+ cell ratio was detected using flow. As shown in FIG. 8, M31 CAR-T was not susceptible to HIV-1 virus, and M31 CAR-T was not susceptible to HIV-1 virus better than M13 CAR-T, regardless of the multiplicity of infection of 0.2 or 2.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> university of double denier
<120> a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein, and preparation method and application thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 2
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Phe Asp Phe Ser Asp Tyr
20 25 30
Glu Met Ser Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Asp Ser Gly Asn Thr Ile Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Ile Tyr Gly Gly Asn Ser Gly Gly Glu Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 2
<211> 99
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Lys Lys Val Val Tyr Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys
1 5 10 15
Thr Ala Ser Gln Lys Lys Asn Ile Gln Phe His Trp Lys Asn Ser Asn
20 25 30
Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro
35 40 45
Ser Lys Leu Asn Asp Arg Val Asp Ser Arg Arg Ser Leu Trp Asp Gln
50 55 60
Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Pro Glu Asp Ser Asp
65 70 75 80
Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Val
85 90 95
Val Val Gly
<210> 3
<211> 66
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val
65
<210> 4
<211> 41
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 5
<211> 47
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
1 5 10 15
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
20 25 30
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40 45
<210> 6
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 7
<211> 529
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Tyr Lys Asp Asp Asp Asp Lys Gln Val Gln
20 25 30
Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
35 40 45
Leu Ser Cys Ala Ala Ser Ala Phe Asp Phe Ser Asp Tyr Glu Met Ser
50 55 60
Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile
65 70 75 80
Asn Asp Ser Gly Asn Thr Ile Tyr Asn Pro Ser Leu Lys Ser Arg Val
85 90 95
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
100 105 110
Thr Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ile Tyr Gly
115 120 125
Gly Asn Ser Gly Gly Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
130 135 140
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Lys Lys Val Val Tyr Gly Lys Lys Gly Asp Thr Val Glu Leu Thr
165 170 175
Cys Thr Ala Ser Gln Lys Lys Asn Ile Gln Phe His Trp Lys Asn Ser
180 185 190
Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly
195 200 205
Pro Ser Lys Leu Asn Asp Arg Val Asp Ser Arg Arg Ser Leu Trp Asp
210 215 220
Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Pro Glu Asp Ser
225 230 235 240
Asp Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu
245 250 255
Val Val Val Gly Ala Ser Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu
260 265 270
Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His
275 280 285
Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val
290 295 300
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
305 310 315 320
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
325 330 335
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
340 345 350
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
355 360 365
Ser Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
370 375 380
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
385 390 395 400
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
405 410 415
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
420 425 430
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
435 440 445
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
450 455 460
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
465 470 475 480
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
485 490 495
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
500 505 510
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
515 520 525
Arg
<210> 8
<211> 1587
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atggccctcc cagtgaccgc cctcctgctg ccactcgccc tgctgctgca cgccgccaga 60
cctgattaca aggatgacga cgataagcag gtgcagctgg tgcagtctgg gggaggcttg 120
gtacagcctg gagggtccct gagactctcc tgtgcagcct ctgctttcga tttctctgat 180
tatgaaatga gctgggtccg cgaggctcca gggaaggggc tggagtggat tggggaaatc 240
aatgatagtg gaaacaccat ttacaatccg tccctcaaga gtcgagtcac catctccaga 300
gacaattcca agaacacact gtatctgcaa atgaacaccc tgagagccga ggacacagcc 360
atatattact gtgcgatata tggtggtaac tccgggggag agtactgggg ccagggcacc 420
ctggtcaccg tctcctcagg tggaggcggt tcaggcggag gtggctctgg cggtggcgga 480
tcaaagaagg tggtgtacgg caagaagggc gacaccgtgg agctgacctg caccgccagc 540
cagaagaaga acatccagtt ccactggaag aacagcaacc agatcaagat cctgggcaac 600
cagggcagct tcctgaccaa gggacctagc aagctgaacg acagggttga cagccggcgg 660
agcctgtggg accagggaaa cttcccactg atcatcaaga acctgaagcc agaggacagc 720
gacacctaca tctgcgaggt ggaggaccag aaggaggagg tgcagctggt agtggtaggc 780
gctagcatcg aggtgatgta ccctccccct tacctggaca acgagaagag caacggcacc 840
atcatccacg tgaagggcaa gcacctgtgc cctagccccc tgttccccgg acctagcaag 900
cccttttggg tgctggtggt ggtgggcggc gtgctggcct gttactccct gctggtgacc 960
gtggccttca ttatcttctg ggtgaggagc aagaggagca ggctgctgca cagcgactac 1020
atgaacatga cacccaggag acctggcccc accagaaagc actaccagcc ctatgccccc 1080
cccagagact ttgccgccta cagaagcagg ttcagcgtgg tgaagagggg caggaagaag 1140
ctgctgtaca tcttcaagca gcccttcatg aggcccgtgc agaccaccca ggaggaggac 1200
ggctgcagct gcaggttccc cgaggaggag gaaggcggat gcgagctgag agtgaagttc 1260
tccagaagcg ctgacgcccc tgcctaccag cagggacaga accagctgta taacgagctg 1320
aacctgggca ggagagagga gtacgatgtc ctggacaaga ggagaggacg tgatcctgag 1380
atgggcggca agccccaaag gagaaagaac ccccaggagg gactgtacaa tgagctgcag 1440
aaggacaaga tggccgaggc ctactccgaa atcggcatga aaggcgagag gagaaggggc 1500
aaaggccacg atggcctgta ccagggcctg agcacagcca ccaaagacac atacgacgcc 1560
ctgcacatgc aggccctgcc ccctagg 1587
<210> 9
<211> 1566
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggccctcc cagtgaccgc cctcctgctg ccactcgccc tgctgctgca cgccgccaga 60
cctaagaagg tggtgtacgg caagaagggc gacaccgtgg agctgacctg caccgccagc 120
cagaagaaga acatccagtt ccactggaag aacagcaacc agatcaagat cctgggcaac 180
cagggcagct tcctgaccaa gggacctagc aagctgaacg acagggttga cagccggcgg 240
agcctgtggg accagggaaa cttcccactg atcatcaaga acctgaagcc agaggacagc 300
gacacctaca tctgcgaggt ggaggaccag aaggaggagg tgcagctggt agtggtaggc 360
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcacaggt gcagctggtg 420
cagtctgggg gaggcttggt acagcctgga gggtccctga gactctcctg tgcagcctct 480
gctttcgatt tctctgatta tgaaatgagc tgggtccgcg aggctccagg gaaggggctg 540
gagtggattg gggaaatcaa tgatagtgga aacaccattt acaatccgtc cctcaagagt 600
cgagtcacca tctccagaga caattccaag aacacactgt atctgcaaat gaacaccctg 660
agagccgagg acacagccat atattactgt gcgatatatg gtggtaactc cgggggagag 720
tactggggcc agggcaccct ggtcaccgtc tcctcagcta gcatcgaggt gatgtaccct 780
cccccttacc tggacaacga gaagagcaac ggcaccatca tccacgtgaa gggcaagcac 840
ctgtgcccta gccccctgtt ccccggacct agcaagccct tttgggtgct ggtggtggtg 900
ggcggcgtgc tggcctgtta ctccctgctg gtgaccgtgg ccttcattat cttctgggtg 960
aggagcaaga ggagcaggct gctgcacagc gactacatga acatgacacc caggagacct 1020
ggccccacca gaaagcacta ccagccctat gcccccccca gagactttgc cgcctacaga 1080
agcaggttca gcgtggtgaa gaggggcagg aagaagctgc tgtacatctt caagcagccc 1140
ttcatgaggc ccgtgcagac cacccaggag gaggacggct gcagctgcag gttccccgag 1200
gaggaggaag gcggatgcga gctgagagtg aagttctcca gaagcgctga cgcccctgcc 1260
taccagcagg gacagaacca gctgtataac gagctgaacc tgggcaggag agaggagtac 1320
gatgtcctgg acaagaggag aggacgtgat cctgagatgg gcggcaagcc ccaaaggaga 1380
aagaaccccc aggagggact gtacaatgag ctgcagaagg acaagatggc cgaggcctac 1440
tccgaaatcg gcatgaaagg cgagaggaga aggggcaaag gccacgatgg cctgtaccag 1500
ggcctgagca cagccaccaa agacacatac gacgccctgc acatgcaggc cctgccccct 1560
aggtga 1566

Claims (6)

1. A bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein is characterized in that the bispecific chimeric antigen receptor sequentially comprises a signal peptide CD8, a recognition unit single domain antibody m36.4 targeting HIV-1gp120 protein co-receptor binding site and a connecting peptide 3 XG from N end to C end 4 S, recognition unit mutant C targeting other binding sites of HIV-1gp120D4 D1 domain, hinge and transmembrane region CD28, one or more costimulatory signal domains CD28 and 4-1BB, and intracellular signaling domain cd3ζ; the nucleotide sequence of the gene for encoding the bispecific chimeric antigen receptor is shown as SEQ ID NO.8, and the amino acid sequence of the bispecific chimeric antigen receptor is shown as SEQ ID NO. 7.
2. A vector capable of expressing the bispecific chimeric antigen receptor targeted to the envelope protein of HIV-1 virus of claim 1, comprising the nucleotide sequence set forth in SEQ ID No. 8.
3. A genetically engineered T lymphocyte, wherein the T lymphocyte
A bispecific chimeric antigen receptor comprising the vector of claim 2 and capable of expressing a target HIV-1 viral envelope protein.
4. A method of preparing genetically engineered T-lymphocytes according to claim 3, comprising the steps of: the bispecific chimeric antigen receptor coding sequence of the HIV-1 virus-targeted envelope protein according to claim 1 is linked to a vector, packaged to obtain lentiviral particles, and T lymphocytes are infected with the lentivirus to obtain bispecific chimeric antigen receptor modified T lymphocytes.
5. The use of a bispecific chimeric antigen receptor targeting HIV-1 viral envelope protein according to claim 1 for the preparation of a medicament for the treatment or prophylaxis of aids.
6. A method for the preparation of a bispecific chimeric antigen receptor targeting HIV-1 viral envelope proteins according to claim 1, comprising the specific steps of: the nucleotide sequences respectively encoding the recognition unit targeting the HIV-1gp120 protein co-receptor binding site, the connecting peptide, the recognition unit targeting other HIV-1gp120 binding sites, the hinge and transmembrane region, the co-stimulatory signaling domain and the intracellular signaling domain are sequentially connected in series in the 5 'to 3' direction and cloned onto a blank lentiviral expression vector pKL by gene synthesis, thereby obtaining the lentiviral plasmid expressing the bispecific chimeric antigen receptor.
CN202111312730.1A 2021-11-08 2021-11-08 Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof Active CN114163534B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
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
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

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
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 (2)

Publication Number Publication Date
CN114163534A CN114163534A (en) 2022-03-11
CN114163534B true CN114163534B (en) 2023-07-18

Family

ID=80478205

Family Applications (1)

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

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3957651A1 (en) * 2010-05-21 2022-02-23 The United States of America, as Represented By the Secretary, Department of Health and Human Services High-affinity fully functional soluble single-domain human cd4, antibodies, and related fusion proteins
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
AU2018388997A1 (en) * 2017-12-20 2020-07-09 Lentigen Technology, Inc. Compositions and methods for treating HIV/AIDS with immunotherapy
SG11202012043RA (en) * 2018-07-03 2021-01-28 Gilead Sciences Inc Antibodies that target hiv gp120 and methods of use
CN111196858B (en) * 2020-02-05 2021-02-09 武汉科技大学 Bispecific chimeric antigen receptor for treating hematological tumor complicated with HIV infection, gene, construction 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
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

Also Published As

Publication number Publication date
CN114163534A (en) 2022-03-11
WO2023077943A1 (en) 2023-05-11

Similar Documents

Publication Publication Date Title
KR102460173B1 (en) Culturing method of natural killer cells using transformed T cells
KR20240046644A (en) Chimeric antigen receptor for solid cancer and t cells expressing chimeric antigen receptor
CN109111525B (en) HLA-G chimeric antigen receptor, coding sequence, expression vector and application
CN112029729B (en) CD19 and CD22 double-target chimeric antigen receptor NK (natural killer) cell and application thereof
US20220354890A1 (en) Chimeric antigen receptor targeting cll1 and use thereof
CN113248616B (en) Chimeric antigen receptor targeting GPC3 and uses thereof
JP2022526372A (en) CAR for use in the treatment of HvG disease
CN111057158A (en) Chimeric antigen receptor targeting human GPC3 and application thereof
CN107987173B (en) Double-target-point chimeric antigen receptor, coding gene thereof, plasmid with gene, immune T effector cell and HIV-1 application
CN114874335B (en) Three-target-point regulatable CAR immune cell composition and application thereof
CN114163534B (en) Bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and preparation method and application thereof
CN109517798B (en) NK (natural killer) cell of chimeric CEA antigen receptor as well as preparation method and application of NK cell
CN111171160A (en) chimeric antigen receptor based on TGF- β modification and modified immune cell thereof
KR102338957B1 (en) Method for culturing natural killer cells derived from cord bloods with genetically engineered t cells
WO2023039968A1 (en) Multifunctional anti-hiv-1 car-t cell, construction method therefor and application thereof
CN115595310A (en) NK cell trophoblast, preparation method and application thereof
CN113735981A (en) CD19-CAR-T cell and preparation method thereof
CN110964112B (en) Humanized antibody for enhancing activity of anti-PSCA chimeric antigen receptor and application thereof
CN111286512A (en) Chimeric antigen receptor targeting humanized tyrosine kinase orphan receptor 1 and uses thereof
CN115197330B (en) Chimeric antigen receptor for simultaneously targeting CLL1 and CD33 and application thereof
CN112521515B (en) CD19 and CD10 double-target chimeric antigen receptor and application thereof
CN114149978B (en) Method for producing CAR-T cells
CN114560948B (en) Chimeric antigen receptor, CAR-T cell and application thereof
WO2022228429A1 (en) Bcma-targeting chimeric antigen receptor and use thereof
CN117343191A (en) EGFRvIII CAR-T cell secreting PD1 and CTLA4 double-single-domain antibodies, and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information

Inventor after: Xu Jianqing

Inventor after: Zhang Xiaoyan

Inventor after: Ying Tianlei

Inventor after: Mao Yunyu

Inventor after: Liao Qibin

Inventor before: Xu Jianqing

Inventor before: Zhang Xiaoyan

Inventor before: Ying Tianlei

CB03 Change of inventor or designer information
GR01 Patent grant
GR01 Patent grant