WO2023039968A1

WO2023039968A1 - Multifunctional anti-hiv-1 car-t cell, construction method therefor and application thereof

Info

Publication number: WO2023039968A1
Application number: PCT/CN2021/123408
Authority: WO
Inventors: 徐建青; 张晓燕; 毛蕴玉
Original assignee: 复旦大学
Priority date: 2021-09-14
Filing date: 2021-10-13
Publication date: 2023-03-23
Also published as: CN113943709A

Abstract

Provided are a multifunctional anti-HIV-1 CAR-T cell, a construction method therefor and an application thereof. The CAR-T cell can simultaneously express HIV-1 specific CAR molecules and secrete an anti-HIV-1 broad-spectrum neutralizing antibody. According to the cell, a broad-spectrum neutralizing antibody is modified in the CAR-T cell by means of lentivirus, so that the cell neutralizes free viruses while killing cells infected by HIV-1, the CAR-T cell and uninfected normal cells are protected, and the therapeutic effect of CAR-T immunotherapy on HIV/AIDS is thus improved.

Description

A kind of multifunctional anti-HIV-1 CAR-T cell and its construction method and application

technical field

The invention relates to the technical field of immunotherapy, in particular to a multifunctional anti-HIV-1 CAR-T cell and its construction method and application.

Background technique

Highly active anti-retroviral therapy (HAART) can inhibit the virus replication level in HIV-1 infected patients, control the viral load in the peripheral blood of AIDS patients below the detection line, and effectively delay AIDS disease However, due to the serious side effects of this therapy and the limitation of life-long medication, it is easy to lead to poor patient compliance. Not only that, the HIV-1 virus integrates with the host genome in the body to establish a latent infection, forming a virus latent pool that cannot be cleared by HAART, which constitutes the most important obstacle to the cure of HIV/AIDS.

One of the HIV functional cure strategies, the principle of the "activation and clearance" (Shock and kill) strategy is to use latent reversal drugs to activate the latent HIV-1 virus, so that the activated infected cells are recognized and killed by the immune system , thereby reducing/controlling the virus latent library. At present, anti-HIV latent small-molecule drugs and transcriptional activation systems modified based on gene editing technology can only "awaken" latent HIV-1 virus and cannot be eliminated. Finding and effectively eradicating these concealed and escaped HIV-1 viruses is the key to achieving a functional cure for AIDS.

Chimeric antigen receptor T cells (CAR-Ts) can specifically target HIV-1 virus and kill infected cells by combining single-chain antibody technology and T cell activation motifs. cells, so it can be used as a powerful weapon to kill reactivated cells in the "shock and kill" strategy. As early as 1994, Roberts et al. selected the natural receptor of HIV-1 virus—CD4 molecule as the extracellular recognition domain of CAR-T cells, and prepared the first generation of CAR-T cells targeting HIV-1 virus ( CD4-CD3ζ CAR-T). Subsequent studies have found that although CD4-CD3ζ CAR-T cells can specifically target and kill cells expressing HIV-1 envelope protein Env or infected by HIV-1 virus in vitro, their killing ability is not maintained well in vivo. At the same time, because the extracellular recognition domain of CD4-CD3ζ CAR-T cells directly copied the sequence of CD4 molecules, CAR-T cells themselves became target cells infected by HIV-1 virus, which greatly affected the survival of CAR-T cells in vivo capability and lethality. In recent years, with the deepening understanding of the signaling mechanism of T cell activation, researchers have introduced co-stimulatory signals such as CD28 and 4-1BB on the basis of the first-generation CAR-T, and designed HIV-1 virus-targeted The second-generation (only one co-stimulatory signal), the third-generation (two or more co-stimulatory signals at the same time) CAR-T, significantly improved the activation level, proliferation ability and survival time of CAR-T cells . On the other hand, the design of the extracellular recognition domain of CAR-T cells is also constantly innovating. In 2017, Mustafa et al. coupled the D1D2 domain of the CD4 molecule with the sugar recognition domain of human C-type lectin, and targeted the HIV-1 viral envelope at the same time. The highly conserved CD4 binding site on the membrane protein and the mannose-oligosaccharide plaques presented on the surface of the protein can kill target cells well, limit the production of virus particles and induce the secretion of IFN-γ at the same time. Liu et al. constructed a variety of single-chain antibodies based on HIV-1 broad-spectrum neutralizing antibodies, and found that using them as the extracellular recognition domain of CAR-T cells can specifically target and kill cells infected by HIV-1. Scholler et al. followed up patients in three clinical trials from 1998 to 2005 and found that HIV-1-targeted CAR-T cells can survive stably for at least 11 years in infected patients, showing the strong potential of CAR-T in the field of AIDS treatment.

Unfortunately, the reported CAR-T can only recognize the antigen presented on the target cell membrane, but cannot recognize the free HIV-1 virus in the infected person, so that it can escape the killing of CAR-T cells and even further infect CAR-T cells and their surrounding normal cells. Aiming at this deficiency, the present invention provides a novel multifunctional anti-HIV-1 CAR-T cell and its construction method and application, which can kill cells infected by HIV-1 while neutralizing free viruses and protect the CAR-T cells themselves Compared with uninfected normal cells, it can improve the therapeutic effect of CAR-T immunotherapy on HIV/AIDS.

Contents of the invention

The technical problem to be solved by the present invention is to provide a new type of multifunctional anti-HIV-1 CAR-T cell, which modifies a broad-spectrum neutralizing antibody on the CAR-T cell through a lentivirus. -In T cells, it can neutralize the free virus while killing cells infected by HIV-1, protect CAR-T cells themselves and uninfected normal cells, and improve the therapeutic effect of CAR-T immunotherapy on HIV/AIDS .

In order to solve the above technical problems, the present invention provides a multifunctional anti-HIV-1 CAR-T cell, which can simultaneously express HIV-1-specific CAR molecules and secrete a broad-spectrum anti-HIV-1 Neutralizing antibodies.

Preferably, the anti-HIV-1 CAR-T cells are multi-target HIV-1-specific CAR-T cells that simultaneously target multiple highly conserved regions of the virus.

Preferably, the anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody;

Preferably, the anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody derived from 10E8.

Preferably, the HIV-1-specific CAR molecule expressed by the CAR-T cells can simultaneously target the CD4 binding site and co-receptor binding site of HIV-1 viral envelope protein gp120.

Preferably, the CAR-T cell genome comprises gene coding sequences of HIV-1-specific CAR molecules and anti-HIV-1 broad-spectrum neutralizing antibodies, wherein the gene coding sequences of HIV-1-specific CAR molecules are as SEQ ID NO .1, the amino acid sequence is shown in SEQ NO.2; the anti-HIV-1 broad-spectrum neutralizing antibody gene coding sequence is shown in SEQ ID No.3, and the amino acid sequence is shown in SEQ No.4.

A method for preparing the multifunctional anti-HIV-1 CAR-T cell according to claim 1, the method comprising the steps of: combining the anti-HIV-1 chimeric antigen receptor coding gene and the anti-HIV-1 broad-spectrum neutralizing antibody The coding gene is connected to the vector pKL, packaged to obtain lentiviral particles, and the lentivirus is used to infect T cells to obtain anti-HIV-1 chimeric antigen receptor modified T lymphocytes that can secrete broad-spectrum neutralizing antibodies against HIV-1 .

Further, the specific steps of the preparation method for preparing multifunctional HIV-1 CAR cells are as follows:

(1) Synthesize the MD CAR gene (SEQ ID No.1) and the 10E8 single-chain antibody gene (SEQ ID No.3) respectively, link the two genes through the linker peptide P2A (SEQ ID No.5), and clone into the blank Lentiviral expression plasmid (pKL) Obtain pKL-M10 recombinant lentiviral expression plasmid;

(2) Packaging of lentivirus: Culture HEK293T cells, mix lentivirus expression plasmid (pKL-M10): packaging plasmid psPAX2 and envelope plasmid PMD2.G to prepare transfection mixture, add TurboFect transfection reagent, add to Culture in HEK293T cell culture dish, collect supernatant and carry out virus concentration;

(3) Infection and expansion of T cells: culture peripheral blood mononuclear cells activated by immunomagnetic beads in advance, add the packaged and concentrated lentiviral vector in step (2), add protamine sulfate, culture after centrifugation, and add regularly Fresh T cell growth medium, remove the immunomagnetic beads of activated T cells after 6-7 days after infection, and continue to culture.

Preferably, the multifunctional anti-HIV-1 CAR-T cells can express the chemokine receptor CXCR5 receptor while expressing the HIV-1 specific CAR molecule and secreting an anti-HIV-1 broad-spectrum neutralizing antibody. In vivo, CXCR5 ⁺ M10 CAR-T cells were obtained, and homing receptors were further increased to promote the homing of cells to the latent pool of HIV-1 virus.

Preferably, the multifunctional anti-HIV-1 CAR-T cell genome also includes a CXCR5 receptor gene sequence, so that the multifunctional anti-HIV-1 CAR-T cell can express the chemokine receptor CXCR5.

The application of the above-mentioned multifunctional anti-HIV-1 CAR-T cells in the treatment or prevention of AIDS drugs, wherein the multifunctional anti-HIV-1 CAR-T cells can be used in combination with cytokines to enhance the curative effect, wherein the cytokines For IL-7, IL-21, IL-15.

The beneficial effects of the present invention are:

(1) To overcome the defect that currently reported CAR-T cells can only recognize HIV-1-related antigens presented on the surface of target cell membranes, and provide a novel multifunctional anti-HIV-1 CAR-T cell and its preparation method and In application, the CAR-T cells can effectively neutralize the free HIV-1 virus while killing the infected cells, and avoid the release of the free HIV-1 virus caused by the killing of the target cells.

(2) Multifunctional anti-HIV-1 CAR-T cells constitutively and continuously secrete broad-spectrum neutralizing antibodies, which can protect CAR-T cells themselves and surrounding uninfected normal cells from being infected by HIV-1 virus, and enhance anti-HIV- 1 Safety of CAR-T immunotherapy.

(3) Multifunctional anti-HIV-1 CAR-T cells based on CAR-T immunotherapy and broad-spectrum neutralizing antibodies, compared with single anti-HIV-1 CAR-T cells, broaden the range of targeted virus strains, Reduce the chance of virus immune escape in the body, accelerate the clearance of HIV-1 infected cells, and improve the therapeutic effect of CAR-T immunotherapy on HIV/AIDS.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 is a schematic diagram of the structure of the recombinant lentiviral vector of M10 CAR-T, wherein P2A is the connecting peptide.

Figure 2 shows the expression of anti-HIV-1 chimeric antigen receptor and the secretion level of anti-HIV-1 broad-spectrum neutralizing antibody of M10 CAR-T, in which UTD is T cells not transduced with lentivirus, and GAPDH is an internal reference.

Figure 3 shows that M10 CAR-T specifically recognizes target cell MT4-gp145 and secretes cytokines IFN-γ and IL-2, in which UTD is T cells that have not been transduced with lentivirus.

Figure 4 shows the killing efficiency of gp145 overexpressed cells by M10 CAR-T cells, where A is the monitoring result of real-time label-free cell analyzer, B is the statistical result of killing efficiency at 9 hours, and UTD is T cells not transduced with lentivirus .

Figure 5 shows the inhibition of virus secretion by M10 CAR-T cells on HIV-1-infected cells, where UTD is T cells that have not been transduced with lentivirus.

Figure 6 shows the neutralization effect of M10 CAR-T cell culture supernatant on HIV-1 virus, in which UTD is T cells not transduced with lentivirus.

Figure 7 shows that the combination of M10 CAR-T cells and chemokine receptor CXCR5 enables the multifunctional anti-HIV-1 CAR-T to further have the chemotaxis ability of HIV-1 virus latent pool, where UTD is the T that has not been transduced with lentivirus Cell control.

Detailed ways

A multifunctional anti-HIV-1 CAR-T cell provided by the present invention is an engineered cell constructed on the basis of T cells, which can simultaneously express HIV-1 specific CAR molecules and secrete a A broad-spectrum neutralizing antibody. For the convenience of illustration, the HIV-1 specific CAR molecule selected in the following examples is an MD CAR molecule, which can simultaneously target the CD4 binding site and the co-receptor binding site of the HIV-1 viral envelope protein gp120, Its nucleotide and amino acid sequences are shown in SEQ ID No.1 and SEQ No.2 respectively; the broad-spectrum neutralizing antibody selected is a single-chain antibody derived from 10E8, and its nucleotide and amino acid sequences are shown in SEQ ID No. 3 and shown in SEQ No.4. The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention.

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 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. 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.

Fetal bovine serum was purchased from BI Company.

TurboFect transfection kit was purchased from Thermo Fisher Scientific.

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.

Real-time label-free cell function analyzer (RTCA) was purchased from Shanghai Youzhe Biotechnology Co., Ltd.

Example 1 Construction of lentiviral expression plasmid

The MD CAR gene (SEQ ID No.1) and the 10E8 single-chain antibody gene (SEQ ID No.3) were synthesized by Shanghai Jierui Bioengineering Co., Ltd. respectively, and the two genes were linked by the connecting peptide P2A (SEQ ID No.5). , and cloned into a blank lentiviral expression plasmid (pKL) to obtain the pKL-M10 recombinant lentiviral expression plasmid. The structure of the recombinant lentiviral vector is shown in Figure 1.

Packaging, concentration and titer determination of embodiment 2 lentivirus

1.1 Packaging of lentivirus

HEK293T cell pretreatment: 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 store the cells in 10 mL of complete DMEM medium Grow in medium, culture at 37°C, 5% CO2 for 18-24 hours, and transfection can be carried out when the cell density reaches 70-90%.

HEK293T cell transfection: add 1mL basal DMEM medium to a 15mL centrifuge tube, and prepare transfection according to the mass ratio of lentiviral expression plasmid (pKL-M10):packaging plasmid psPAX2:enveloping plasmid PMD2.G=1:3:1 Mixed solution, the total amount of plasmid is 15μg/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 at 37°C, 5% CO2 for cell culture After culturing in the box for 48 hours, the cell supernatant was collected, centrifuged at 1000×g, 4°C for 10 minutes, and the supernatant was collected.

1.2 Enrichment of lentivirus

Filter the virus supernatant collected above through a 0.45 μm filter, add 1/4 of the volume of the virus supernatant Lenti-X lentivirus concentration reagent, invert and mix several times, incubate overnight at 4°C, centrifuge at 1500×g, 4°C for 30 minutes , the white precipitate at the bottom of the centrifuge tube is the virus. Carefully discard the supernatant, resuspend the white precipitate with 1/100 volume of the original virus supernatant in RPMI1640 medium, aliquot and freeze at -80°C for future use.

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, and replace the supernatant with fresh RPMI after overnight incubation Culture medium (containing 10% fetal bovine serum) was continued for 48 hours. The proportion of fluorescent 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 fluorescent positive cells/100×1000×corresponding dilution factor.

Example 3 Infection and Expansion of T Cells

Spread 1× ¹⁰⁷ peripheral blood mononuclear cells in a 6-well plate at a density of 2× ¹⁰⁶ /mL, add immunomagnetic beads pre-coated with CD3/CD28 antibody at a ratio of 1:1, and culture for 72 hours to complete Preactivation of peripheral blood mononuclear cells. In a 48-well flat-bottomed cell culture plate (containing 1×10 ⁶ pre-activated peripheral blood mononuclear cells), add the packaged and concentrated lentiviral vector (LV-M10) in Example 2 (MOI=5～10), add Protamine sulfate 10μg/mL, 1000×g, centrifuge at 32°C for 90 minutes and culture overnight. 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. After 6-7 days after infection, the immunomagnetic beads of activated T cells were removed, and the culture was continued. The cell surface chimeric antigen receptor expression and anti-HIV-1 broad-spectrum neutralizing antibody secretion of M10 CAR-T were detected by flow cytometry and western blot respectively. The results are shown in Figure 2. After lentiviral transduction, the expression of HIV-1-specific CAR molecules can be detected on the surface of M10 CAR-T (transduction positive rate was 22.2%), and the broad-spectrum anti-HIV-1 CAR-T can be secreted. and antibodies.

Example 4 Activation and cytokine secretion of M10 CAR-T

1×10 ⁵ wild-type MT4 cells or MT4 cells overexpressing gp145 protein (MT4-gp145) were plated in a U-bottom 96-well cell culture plate, and untransduced lentivirus T cells (UTD) or M10 CAR- T was plated in the test wells with target cells according to the ratio of effector cells: target cells at 1:1, 200uL medium per well. After 24 hours of co-cultivation, 100 uL of supernatant was collected, and the secretion of cytokines IFN-γ and IL-2 in the supernatant was detected by ELISA method.

The results are shown in Figure 3. M10 CAR-T secretes high levels of cytokines IFN-γ and IL-2 only when co-cultured with MT4-gp145, indicating that M10 CAR-T can specifically recognize the HIV envelope protein and is activated.

Example 5 Effect of M10 CAR-T on killing gp145 overexpressed cells

Target cell killing efficiency was detected by real-time label-free cell analysis (RTCA, Real Time Cellular Analysis). First, inoculate 100 μL of A549 cells (5×10 ⁴ cells/well) 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 M10 CAR-T to the wells containing the target cells at a ratio of effector cells: target cells = 1:1, record the measurement results every 15 minutes, and record continuously for 24 hours.

The results are shown in Figure 4. M10 CAR-T can rapidly decrease the growth curve of target cells and effectively kill target cells overexpressing gp145 protein. Among them, the killing rate after co-incubation for 9 hours is as high as 75.16%.

Example 6 M10 CAR-T cells kill HIV-1 infected CD4+ T cells and inhibit virus secretion

CD4 ⁺ T cells were cultured in a 6-well plate at a density of 1×10 ⁶ cells/mL (3ml/well), and were stimulated by adding magnetic beads coated with anti-CD3/CD28 antibody at 1:1 for 48 hours and then removed Magnetic beads, add 1 mL of cell culture suspension containing HIV-1 virus and infect for 48 hours. After washing the HIV-1-infected CD4 ⁺ T cells twice with medium, spread them in 48-well plates at 5× ¹⁰⁴ cells/well, and spread them into non-transduced T cells or M10 CAR-T, then collected 1/10 of the culture supernatant every 24 hours to monitor the release of HIV-1 virus particles.

As shown in Figure 5, compared with the blank control group and the non-transduced T cell group, M10 CAR-T can effectively kill CD4 ⁺ T cells infected by HIV-1 and inhibit the secretion of viral particles.

Example 7 M10 CAR-T cell culture supernatant can effectively neutralize HIV-1 virus and protect CD4+ T cells from infection

Untransduced T cells or M10 CAR-T were plated in a 96-well plate at 1×10 ⁶ cells/well (200uL/well), and magnetic beads coated with anti-CD3/CD28 antibody were added at a ratio of 1:1 for stimulation. The supernatant was collected after 96 hours. Take 400uL of culture supernatant and 100uL of HIV-1 virus suspension and incubate at room temperature for 30 minutes, add 5× ¹⁰⁴ activated CD4 ⁺ T cells to each well, discard the supernatant after overnight culture, add fresh medium, every 24 Collect 1/10 of the culture supernatant every hour to monitor the release of HIV-1 virus particles.

As shown in Figure 6, the culture supernatant of M10 CAR-T cells can still effectively neutralize HIV-1 virus and protect CD4 ⁺ T cells from infection until the 8th day.

Example 8 The combination of M10 CAR-T and chemokine receptor CXCR5 enables multifunctional anti-HIV-1 CAR-T cells to further have HIV-1 virus latent library chemotactic ability

On the basis of M10 CAR-T, the chemokine receptor CXCR5 (SEQ ID No.6) was further overexpressed to obtain CXCR5+M10 CAR-T. Spread 1×10 ⁵ UTD cells or M10 CAR-T or CXCR5+M10 CAR-T (100uL/well) on the upper layer of a Transwell plate with a pore size of 5.0um, and add 600uL to the lower layer of the well plate with a final concentration of 1ug/mL chemotactic RPMI1640 medium (containing 10% FBS, 1% PS) of factor CXCL13, let it stand still. After 2 hours, 4 hours, and 6 hours, 10uL of the lower layer solution was taken to calculate the cell concentration, so as to calculate the number and chemotaxis efficiency of cells from the upper layer to the lower layer in each experimental group, as shown in Figure 7. Almost no cells in the UTD control group and M10 CAR-T group were chemoattracted to the lower layer solution, while CXCR5+M10 CAR-T could be chemoattracted to the lower layer under the action of 1ug/mL CXCL13, and the chemotaxis efficiency was 69.3% in 6 hours. Chemokine CXCL13 is mainly produced in B cell lymphoid follicles, which is also one of the main latent reservoirs of HIV-1 virus. Therefore, CXCR5+M10 CAR-T cells have chemotactic ability of HIV-1 virus latent library.

The basic principles, main features and 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

A multifunctional anti-HIV-1 CAR-T cell, characterized in that the CAR-T cell can simultaneously express HIV-1-specific CAR molecules and secrete a broad-spectrum anti-HIV-1 neutralizing antibody.
The multifunctional anti-HIV-1 CAR-T cell according to claim 1, wherein the anti-HIV-1

CAR-T cells are multi-target HIV-1-specific CAR-T cells that simultaneously target multiple highly conserved regions of the virus.
The multifunctional anti-HIV-1 CAR-T cell according to claim 1, wherein the anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody.
The multifunctional anti-HIV-1 CAR-T cell according to claim 2, wherein the anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody derived from 10E8.
A multifunctional anti-HIV-1 CAR-T cell according to claim 1, wherein the HIV-1-specific CAR molecule expressed by the CAR-T cell can simultaneously target HIV-1 virus envelope protein CD4 binding site and co-receptor binding site of gp120.
A multifunctional anti-HIV-1 CAR-T cell according to claim 1, characterized in that the CAR-T cell genome comprises HIV-1-specific CAR molecules and anti-HIV-1 broad-spectrum neutralizing antibodies Gene coding sequence, wherein, the gene coding sequence of HIV-1 specific CAR molecule is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ NO.2; the gene coding sequence of anti-HIV-1 broad-spectrum neutralizing antibody is shown in SEQ ID NO.1 Shown in ID No.3, the amino acid sequence is shown in SEQ No.4.
A method for preparing multifunctional anti-HIV-1 CAR-T cells according to claim 1, characterized in that the method comprises the following steps: combining the anti-HIV-1 chimeric antigen receptor coding gene and the anti-HIV-1 broad-spectrum The spectrum neutralizing antibody coding gene is linked to the carrier pKL, packaged to obtain lentiviral particles, and the lentivirus is used to infect T cells to obtain anti-HIV-1 chimeric antigen receptor modification and secrete broad-spectrum neutralizing antibody against HIV-1 of T lymphocytes.
A method for preparing multifunctional HIV-1 CAR cells according to claim 7, wherein the specific steps are as follows:

(1) Synthesize the MD CAR gene (SEQ ID No.1) and the 10E8 single-chain antibody gene (SEQ ID No.3) respectively, link the two genes through the linker peptide P2A (SEQ ID No.5), and clone into the blank Lentiviral expression plasmid (pKL) Obtain pKL-M10 recombinant lentiviral expression plasmid;

(2) Packaging of lentivirus: Culture HEK293T cells, mix lentivirus expression plasmid (pKL-M10): packaging plasmid psPAX2 and envelope plasmid PMD2.G to prepare transfection mixture, add TurboFect transfection reagent, add to Culture in HEK293T cell culture dish, collect supernatant and carry out virus concentration;

(3) Infection and expansion of T cells: culture peripheral blood mononuclear cells activated by immunomagnetic beads in advance, add the packaged and concentrated lentiviral vector in step (2), add protamine sulfate, culture after centrifugation, and add regularly Fresh T cell growth medium, remove the immunomagnetic beads of activated T cells after 6-7 days after infection, and continue to culture.
The multifunctional anti-HIV-1 CAR-T cell according to claim 1, wherein the multifunctional anti-HIV-1 CAR-T cell expresses HIV-1 specific CAR molecules and secretes an anti-HIV-1 At the same time as the broad-spectrum neutralizing antibody, it can also express the chemokine receptor CXCR5 receptor, obtain CXCR5 + M10 CAR-T cells, and further increase the homing receptor to promote the homing of cells to the latent pool of HIV-1 virus.
A kind of multifunctional anti-HIV-1 CAR-T cell according to claim 6, is characterized in that, also comprises CXCR5 receptor gene sequence in the genome of described multifunctional anti-HIV-1 CAR-T cell, makes described multifunctional Functional anti-HIV-1 CAR-T cells can express the chemokine receptor CXCR5.
An application of the multifunctional anti-HIV-1 CAR-T cell described in claim 1 in the treatment or prevention of AIDS drugs.
According to the application of the multifunctional anti-HIV-1 CAR-T cells in the treatment or prevention of AIDS drugs according to claim 1, it is characterized in that the multifunctional anti-HIV-1 CAR-T cells can be combined with cytokines to enhance Curative effect, wherein, cytokines are IL-7, IL-21, IL-15.