Chimeric antigen receptor targeting HIV infected cells, CAR-T cell and application

Abstract

The invention discloses a chimeric antigen receptor targeting an HIV infected cell, a CAR-T cell and application. The chimeric antigen receptor comprises an antigen binding domain, a chimeric receptor hinge region, a transmembrane domain, a costimulatory signal transduction domain and a signal transduction domain, the antigen binding structural domain is an anti-HIV monoclonal antibody JSR771 and is composed of a heavy chain VH and a light chain VL, the amino acid sequence of the heavy chain VH is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain VL is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the anti-HIV monoclonal antibody JSR771. By optimizing the separation, activation, transduction and culture conditions of the T cells and strictly controlling the quality control standard, the prepared anti-HIV-1CAR-T cells have high motility, high specificity and strong killing ability, and a new hope is provided for clinical treatment of HIV infection.

Description

Technical Field

[0001] The present invention belongs to the field of cellular immunity technology, and specifically relates to a chimeric antigen receptor targeting HIV-infected cells, a CAR-T cell and applications thereof. Background Art

[0002] Immunotherapy is a treatment method that combats disease by regulating or enhancing the body's immune system. It has shown great potential in the treatment of cancer, autoimmune diseases, and infectious diseases. Common immunotherapy approaches include monoclonal antibody therapy, vaccine therapy, cell therapy, and immune checkpoint inhibitors. In recent years, chimeric antigen receptor T cell (CAR-T) therapy, as an innovative cell therapy, has achieved remarkable success in the treatment of certain hematological malignancies, such as B-cell lymphoma and acute lymphoblastic leukemia.

[0003] CAR-T therapy uses genetic engineering technology to transform the patient's own T cells into cells that can recognize and attack specific antigens. The basic steps include T cell isolation, T cell activation and expansion, gene transduction, in vitro expansion, quality control testing, and re-infusion into the patient. As a serious public health problem worldwide, HIV-1 (human immunodeficiency virus type 1) infection is the main cause of acquired immunodeficiency syndrome (AIDS). In the treatment of HIV-1 infection, CAR-T cell therapy has also shown great potential. HIV-1 virus mainly infects CD4 + In order to achieve a functional cure for HIV-1 infection, researchers have developed CAR-T cells that can recognize and kill HIV-1 infected cells.

[0004] However, despite significant progress in cancer treatment, CAR-T cell therapy faces numerous challenges in HIV-1 treatment, including viral escape, CAR-T cell infectivity, and accessibility to cryptic reservoirs. These challenges limit the widespread application and effectiveness of CAR-T cell therapy in HIV-1 treatment. Specifically, the following issues need to be addressed urgently:

[0005] Target selection specificity and stability: When HIV-1 infects cells, it integrates into the host cell genome, forming a latent reservoir. Ideally, a CAR-T cell target must be able to specifically recognize and eliminate all infected cells, including those in the latent phase. This requires a target with high specificity and stability, and one that is not susceptible to viral mutations.

[0006] CAR-T cell persistence and function: Effective CAR-T cell therapy requires the ability of T cells to survive and function long-term in vivo. However, the complex immune environment of HIV-1-infected individuals may affect the persistence and efficacy of CAR-T cells. Therefore, optimization of CAR structure and improvement of T cell culture methods are needed to enhance the persistence and anti-HIV-1 function of CAR-T cells.

[0007] Safety and Side Effects: CAR-T cell therapy can cause serious side effects, such as cytokine release syndrome (CRS) and neurotoxicity. Ensuring the safety of CAR-T cell therapy is equally important in HIV-1 treatment, requiring rational design and strict quality control to minimize side effects.

[0008] Production Process and Quality Control: The CAR-T cell production process is complex, involving multiple steps, including T cell isolation, gene transduction, in vitro expansion, and quality control testing. Optimization and standardization of each step are crucial to ensuring the quality and consistency of the final product. Especially in the treatment of HIV-1 infection, the production process must consider the specificity of the virus and individual patient differences, and comprehensive quality control standards must be established.

[0009] Therefore, developing a method for efficiently preparing anti-HIV-1 chimeric antigen receptor T cells and establishing strict quality control standards to ensure their safety and effectiveness in clinical applications have important practical significance and application value. Summary of the Invention

[0010] In view of the shortcomings and deficiencies in the prior art, the present invention aims to provide a chimeric antigen receptor, CAR-T cells and applications targeting HIV-infected cells.

[0011] The first aspect of the present invention provides a chimeric antigen receptor targeting HIV-infected cells, wherein the chimeric antigen receptor comprises an antigen binding domain, a chimeric receptor hinge region, a transmembrane domain, a co-stimulatory signaling domain, and a signaling domain;

[0012] The antigen binding domain is the anti-HIV monoclonal antibody JSR771, which is composed of a heavy chain VH and a light chain VL. The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.1, and the amino acid sequence of the light chain VL is shown in SEQ ID NO.2.

[0013] Furthermore, the chimeric antigen receptor includes an antigen binding domain, a chimeric receptor hinge region, a transmembrane domain, a co-stimulatory signal transduction domain, a signal transduction domain, a 2A peptide and a fluorescent marker protein connected in series.

[0014] Furthermore, the chimeric receptor hinge region is CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO.3;

[0015] The transmembrane domain is CD8TM, and its amino acid sequence is shown in SEQ ID NO.4;

[0016] The co-stimulatory signal transduction domain is 41BB, and its amino acid sequence is shown in SEQ ID NO.5;

[0017] The signal transduction domain is CD3zeta, and its amino acid sequence is shown in SEQ ID NO.6;

[0018] The amino acid sequence of the 2A peptide is shown in SEQ ID NO.7;

[0019] The fluorescent marker protein is EGFP, and its amino acid sequence is shown in SEQ ID NO.8.

[0020] Furthermore, the chimeric antigen receptor is scFv-JSR771VH-JSR771VL-CD8 hinge region-CD8TM-41BB-CD3zeta-2A-EGFP, and its amino acid sequence is composed of SEQ ID NO.1 to SEQ ID NO.8 connected in series.

[0021] The second aspect of the present invention provides a nucleic acid molecule comprising nucleotides encoding the chimeric antigen receptor targeting HIV-infected cells according to the first aspect of the present invention.

[0022] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule provided by the second aspect of the present invention.

[0023] The fourth aspect of the present invention provides a CAR-T cell, wherein the CAR-T cell expresses the chimeric antigen receptor targeting HIV-infected cells provided in the first aspect of the present invention.

[0024] The fifth aspect of the present invention provides a method for preparing the CAR-T cells according to the fourth aspect of the present invention, comprising the following steps:

[0025] Step 1: Constructing lentiviral vectors and preparing viruses

[0026] Adding both ends of the chimeric antigen receptor to a lentiviral vector and co-transfecting the lentiviral packaging plasmid to obtain a virus;

[0027] Step 2: Prepare CAR-T cells

[0028] Mononuclear cells were isolated from the peripheral blood of HIV-1 patients;

[0029] Isolation and activation of CD8 from peripheral blood mononuclear cells using magnetic bead separation + T cells;

[0030] Transduction of CD8 cells using lentiviral vectors + T cells, causing them to express the chimeric antigen receptor;

[0031] The obtained T cells were subjected to quality control tests to ensure that they met the following standards: no visible foreign matter, osmotic pressure 302 ± 10 mOsmol / kg, cell viability > 90%, and total cell count > 2 × 10 8 、CD8 + T cell ratio>98%, CD4 + T cell ratio <0.2%, CD8 + CAR + The proportion of T cells is >20%, the copy number of CAR in T cells is 2-5 copies / cell, and the ability of CAR-T cells to kill target cells is >50%.

[0032] If CAR-T cells are made into preparations, the preparations must meet safety indicators such as sterility, mycoplasma negativity, endotoxin <3EU / mL, no HIV-1 detection, no RCL detection, IL-2 <200pg / mL, and IL-21 <10pg / mL.

[0033] The sixth aspect of the present invention provides the use of the chimeric antigen receptor targeting HIV-infected cells described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention, or the CAR-T cell described in the fourth aspect of the present invention in the preparation of drugs for preventing and / or treating HIV.

[0034] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the chimeric antigen receptor targeting HIV-infected cells described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant expression vector described in the third aspect of the present invention or the CAR-T cell described in the fourth aspect of the present invention, and one or more pharmaceutically acceptable carriers.

[0035] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0036] (1) This invention is the first to use the JSR771 anti-HIV antibody sequence in the construction of CAR-T, which improves the efficacy of CAR-T cells and has better anti-HIV efficacy than the current clinical CAR-T technology.

[0037] (2) By optimizing the isolation, activation, transduction and culture conditions of T cells and strictly controlling the quality control standards, the anti-HIV-1 CAR-T cells prepared by the present invention have high viability, high specificity and strong killing ability, providing new hope for the clinical treatment of HIV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 This is a schematic diagram of the transfection efficiency measured after transfection of 293T cells with the constructed scFv-JSR771VH-JSR771VL-CD8 hinge region-CD8TM-41BB-CD3zeta-2A-EGFP plasmid packaged with lentivirus in Example 1.

[0040] Figure 2 Schematic diagram of the test results of the CAR-T cell positivity rate in Example 1. DETAILED DESCRIPTION

[0041] The embodiments of the present invention will be clearly and completely described below with reference to the examples. The examples described are only some of the embodiments of the present invention, rather than all of them. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field based on the examples in the present invention without making any creative work are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the instruments and materials used in the following examples are all commonly used laboratory equipment, and the technical solutions are all conventional techniques in the art.

[0043] Example 1

[0044] 1. Construction of the Lentiviral Vector ABpCCLsin-EF1-hAXL-5(HL)-BBz-P2A-EGFP and Virus Production

[0045] The scFv-JSR771VH-JSR771VL (SEQ ID NO.1 and SEQ ID NO.2) was cloned into the ABpCCLsin-EF1 vector constructed based on a conventional lentiviral vector by adding the 2A sequence (SEQ ID NO.7) in the middle through overlap PCR, and NehI and MluI restriction sites were added at both ends. The clone (scFv-JSR771VH-JSR771VL-CD8 hingeregion-CD8TM-41BB-CD3zeta-2A-EGFP, whose amino acid sequence is concatenated from SEQ ID NO.1 to SEQ ID NO.8) with correct sequencing was extracted without endotoxin and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag / Pol, RSV-REV) into 293T cells. The positive rate of transfected cells reached more than 95%. Figure 1 The supernatant was collected after 48 and 72 hours, filtered through 0.45 μM, and concentrated by centrifugation at 25,000 rpm for 2 hours using a Beckman ultracentrifuge and an SW28 rotor. This concentrated virus produced the scFv-JSR771VH-JSR771VL-CD8hinge region-CD8TM-41BB-CD3zeta-2A-EGFP virus for subsequent CAR-T cell production.

[0046] 2. Preparation of CAR-T Cells

[0047] After sorting T cells from PBMC of HIV patients, CD4 + T cells, and then use CD8 negative selection magnetic beads to separate CD8 + T cells were then activated with CD8 T cells using antibodies against CD3 and CD28 + The obtained cells were inoculated into 6-well culture plates. After 2 days, they were infected with scFv-JSR771VH-JSR771VL-CD8 hinge region-CD8TM-41BB-CD3zeta-2A-EGFP virus at an MOI of 10-20. After 1 day, the medium was changed and the cells were cultured. Artificial antigen presenting cells or anti-hCD3 / 28 were used for stimulation every 6 days. After 2 rounds of stimulation, the obtained cells were CAR-T cells. The screening results were as follows: Figure 2 As shown in the figure, the obtained cells were CAR positive, with a positive rate of 12.9%.

[0048] Example 2

[0049] 1. Obtaining Mononuclear Cells (PBMCs)

[0050] (1) HIV-1 patient screening and enrollment

[0051] Strict clinical trial inclusion and exclusion criteria have been established. Clinical units select clinical subjects based on the corresponding criteria. HIV-1 patients who meet the inclusion criteria will initiate the corresponding clinical trial under the supervision of CRO after signing the informed consent form for the clinical trial.

[0052] (2) Peripheral blood collection

[0053] HIV-1 patients enrolled in the anti-HIV-1 chimeric antigen receptor T cell therapy underwent blood sampling according to standard operating procedures (World Health Organization Guidelines for Blood Collection: Best Practices for Venous Blood Collection). The amount of blood drawn depended on the absolute lymphocyte count (generally 50-100 mL of peripheral blood, ensuring that the lymphocyte count exceeded 1.5 × 10 8 that’s it).

[0054] (3) Isolation of peripheral blood mononuclear cells

[0055] Peripheral blood mononuclear cells are isolated from the sample using a lymphocyte separation medium (density gradient centrifugation). The lymphocyte separation medium is of medical grade. Except for the centrifugation in a centrifuge with a Class B background, the rest of the process is performed in a Class A centrifuge with a Class B background.

[0056] 1) Blood samples from HIV-1 patients who meet treatment criteria were transferred from anticoagulant blood collection tubes into 50 mL centrifuge tubes and centrifuged at 600 g for 15 min (room temperature, 5% ramp-up, 5% ramp-down).

[0057] 2) Separate the sample into layers: plasma on top and blood cells on the bottom. Transfer the plasma to a new 50 mL centrifuge tube and place in a preheated 56°C water bath for 30 minutes to inactivate plasma complement.

[0058] 3) Remove the inactivated plasma and centrifuge at 600g for 15 minutes (room temperature, 5°C ramp, 5°C ramp). Transfer the supernatant to a new 50mL centrifuge tube and store in a refrigerator at 4°C. (If the cells are in poor condition or a secondary infection is required, autologous nutrients can be added to improve cell health.)

[0059] 4) Dilute the lower blood cell layer with sodium chloride injection to a total of approximately 100 mL in two 50 mL tubes and mix well with a Pasteur pipette.

[0060] 5) Divide the diluted sample equally and slowly drip equal amounts along the tube wall in a 1:1 ratio into a 50 mL centrifuge tube containing 22 mL of human peripheral blood lymphocyte separation medium. Note: Use a Pasteur pipette to add the blood diluent to the lymphocyte separation medium at a rate of 2 drops / second. Be sure not to disrupt the separation medium surface, otherwise the separation effect will be affected.

[0061] 6) Centrifuge the tube at 600 g for 30 min (room temperature, speed increase 5, speed decrease 0). At this point, the cells in the tube are divided into four layers from top to bottom. The first layer is the diluent layer (containing platelets); the second layer is the ring mononuclear cell layer (containing monocytes and lymphocytes); the third layer is the separation fluid layer; and the fourth layer is the red blood cell layer (containing a large number of neutrophils).

[0062] 7) Collect the second layer of mononuclear cells (containing monocytes and lymphocytes) from each tube into a new 50 mL centrifuge tube, add sodium chloride injection to 50 mL, mix thoroughly, and centrifuge at 500 g for 20 min (room temperature, 5% speed, 5% speed).

[0063] 8) Discard the supernatant, resuspend the pellet in 50 mL of sodium chloride injection, and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0064] 9) Discard the supernatant and resuspend the pellet in ImmunoCult-XF T Cell Exp Medium (serum-free medium). Transfer 20 μL of the cell suspension to a 1.5 mL centrifuge tube, dilute with 1% trypan blue solution, and test the activity and number of PBMCs.

[0065] 10) Add culture medium to maintain the cell density at 1×10 6 cells / mL, add Xingjier (recombinant human interleukin-2 (135Ala) injection, working concentration 130 IU / mL) and mix thoroughly.

[0066] 11) Transfer the cells to a suitable cell culture flask and place them in a 37°C, 5% CO2 incubator for 24 hours before isolating CD8 + T cells.

[0067] 2. CD8 + T cell acquisition and activation

[0068] Isolation of CD8 from peripheral blood mononuclear cells + T cells were separated by magnetic beads, because the patient's CD4 + T cells may integrate HIV-1, and CD8 + Residual CD4 T cells + T cells, this step uses positive selection to remove CD4 + T cells, and then use CD8 negative selection magnetic beads to separate CD8 + For the T cell protocol, except for the centrifugation in this step which is performed in a centrifuge with a Class B background, all other process operations are performed in a Class A environment with a Class B background.

[0069] (1) Positive selection magnetic beads to remove CD4 in PBMC + T cells

[0070] 1) Transfer PBMCs to a 50 mL centrifuge tube and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0071] 2) Discard the supernatant, resuspend the cells in sodium chloride injection to 50 mL, and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0072] 3) Discard the supernatant, add sodium chloride injection to resuspend the cells to 50 mL, take 20 μL for cell counting and record the data, and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0073] 4) Remove the supernatant and add 1×10 6 cells, add BD IMag™ anti-human CD4 Particles in a volume of 5 μL, mix well and incubate at room temperature for 30 minutes.

[0074] 5) Add sodium chloride injection and resuspend the cells to 1×10 7 cells / mL, transfer the cell suspension to a 5mL flow cytometry tube, place it in a magnetic rack and let it stand at room temperature for 8 minutes (note that the amount of cell suspension added to each flow cytometry tube should not exceed 3mL, otherwise the upper layer of suspension will not be able to absorb Streptavidin Particles Plus-DM by magnetic force if the liquid level is too high).

[0075] 6) At this point, Streptavidin Particles Plus-DM is adsorbed to the side of the magnetic rack. The clear solution is aspirated and transferred to a new 50 mL centrifuge tube. Sodium chloride injection is added to resuspend the cells to 50 mL. Cell count is performed and the tube is centrifuged at 500 g for 5 minutes (room temperature, 5% acceleration, 5% deceleration).

[0076] (2) Negative selection of magnetic beads to obtain CD8 + T cells

[0077] 1) Discard the supernatant and 6 cells, add 5 μL of Biotin Human CD8 TLymphocyte Enrichment Cocktail, mix well and incubate at room temperature for 15 minutes.

[0078] 2) Resuspend the cells in sodium chloride injection to 50 mL and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0079] 3) According to every 1×10 6cells, add 5 μL of Streptavidin Particles Plus-DM, mix well, and incubate at room temperature for 30 minutes (Note: Streptavidin Particles Plus-DM must be thoroughly shaken before aspirating).

[0080] 4) Add sodium chloride injection and resuspend the cells to 1×10 7 Transfer the cell suspension to a 5 mL flow cytometry tube and place it in a magnetic stand at room temperature for 8 minutes (note that the amount of cell suspension added to each flow cytometry tube should not exceed 3 mL. If the liquid level is too high, the upper layer of suspension will not be able to attract Streptavidin Particles Plus-DM by magnetic force).

[0081] 5) At this time, Streptavidin Particles Plus-DM is adsorbed on the side of the magnetic rack. The clear solution is aspirated and transferred to a new 50mL centrifuge tube. Repeat step 4 to increase the CD8 + Purity of T cells.

[0082] 6) Add sodium chloride injection to resuspend the cells to 10 mL, take 100 μL for cell counting and flow cytometry staining, and detect CD8 + T cells and CD4 + The proportion of T cells was detected by diluting with 1% trypan blue solution. + T cell count and activity. Determine the total number of cells > 4 × 10 6 , and CD8 + T>98%, CD4 + T cells <0.2%.

[0083] 7) Centrifuge the cells at 500 g for 5 min (room temperature, 5°C for 5 min, 5°C for 5 min). Discard the supernatant and resuspend the cells in ImmunoCult-XF T CellExp Medium (serum-free medium) to maintain a cell density of 1×10 6 cells / mL.

[0084] (3) CD3 & CD28 antibodies activate CD8 + T cells

[0085] Previous studies have found that anti-HIV-1 chimeric antigen receptor infection activates CD8 +The infection rates of T cells are different. Studies have found that the positive rates of the two markers CD25 and CD69 show a certain linear relationship with the infection rate of anti-HIV-1 chimeric antigen receptor T cells. At the same time, optimizing the density of cell culture after infection and the concentration of cytokines can obtain faster amplification efficiency. Except for centrifugation in a Class B environment, all other process operations are performed in a Class B background and a Class A environment.

[0086] 1) Take 4×10 6 CD8 + Add Shiner (recombinant human interleukin-2 (135Ala) injection, working concentration 130 IU / mL) and CD3 & CD28 activation antibodies (concentration 10 μg / mL) to the T cell culture medium, mix thoroughly, and transfer to a T25 cell culture flask and culture in an incubator at 37°C and 5% CO2. Continue to culture the remaining cells for later use.

[0087] 2) In CD8 + After 24 hours of T cell activation, activated CD8 + 100 μL of T cells were subjected to flow cytometry analysis and labeled with CD8, CD25, and CD69 flow cytometry antibodies. + CD69 + ) in CD8 + More than 70% of T cells were infected with lentivirus.

[0088] 3. Preparation of anti-HIV-1 chimeric antigen receptor T cells

[0089] (1) Anti-HIV-1 chimeric antigen receptor lentivirus infection

[0090] 1) Ensure that the cells have reached the infection standard and centrifuge CD8 + T cells (speed 5, speed 5, centrifugation at 500g for 5 minutes), according to the cell count results, anti-HIV-1 chimeric antigen receptor lentivirus (same as Example 1) was added and the cells were resuspended in a 6-well cell culture plate (infected at an MOI of 10) and mixed by gently blowing.

[0091] 2) Centrifuge the 6-well cell culture plate at 500 g for 60 min (room temperature, 5% acceleration, 5% deceleration).

[0092] 3) Place the 6-well cell culture plate back into an incubator at 37°C and 5% CO2 for 8-10 hours.

[0093] 4) Transfer the cell culture medium to a 1.5 mL centrifuge tube and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0094] 5) Remove the supernatant, add sodium chloride injection to 1 mL and resuspend, and centrifuge at 500 g for 5 min (room temperature, 5% acceleration, 5% deceleration).

[0095] 6) Discard the supernatant and resuspend the cells in ImmunoCult-XF T Cell Exp Medium (serum-free medium). Measure the cell density, take 20 μL of the diluted aliquot, count the cells, and record the data.

[0096] 7) Maintain the cell density at 1×10 6 cells / mL, add Xingjier (recombinant human interleukin-2 (135Ala) injection, working concentration 130 IU / mL) and IL-21 (working concentration 10 ng / mL) and mix thoroughly, and culture in an incubator at 37°C and 5% CO2 concentration.

[0097] (2) Expansion and culture of anti-HIV-1 chimeric antigen receptor T cells

[0098] 1) Take out the cell culture flask every 2 days and observe the cell status under a microscope. The cell density is evaluated by the supplementation of culture medium and cytokines (IL-2 & IL-21). The cell density is maintained at 1×10 6 cells / mL (when the culture volume reached 20 mL, it was replaced with a T75 culture flask; when the culture volume reached 60 mL, it was replaced with a T225 culture flask). The changes in the osmotic pressure of the culture medium were simultaneously detected and the osmotic pressure of the culture medium was adjusted to 302±10 mOsmol / kg.

[0099] 2) 3 days after infection, 100 μL of cells were collected to detect the positive rate of anti-HIV-1 chimeric antigen receptor T cells and confirm the CD8 + T>98%, CD4 + T cells <0.2%.

[0100] 3) If the anti-HIV-1 CAR T cell positivity rate is >20%, continue to replenish culture medium and cytokines every two days. If the anti-HIV-1 CAR T cell positivity rate is <20%, resuspend the cells in a new anti-HIV-1 CAR lentivirus in a 6-well cell culture plate (re-infect the T cells at an MOI of 10) and gently pipette to mix. The procedure is the same as above.

[0101] IV. Quality Control of Anti-HIV-1 Chimeric Antigen Receptor T Cells

[0102] Seven days after infection, 15 mL of cell culture medium was collected to conduct a series of tests on the proportion, safety, and effectiveness of anti-HIV-1 chimeric antigen receptor T cells, including: appearance, osmotic pressure, cell viability, total cell count, CD4 + T cell ratio, CD8+ T cell ratio, CD8 + CAR + The proportion of T cells, the number of CAR copies in T cells, the ability of anti-HIV-1 chimeric antigen receptor T cells to kill target cells, sterility, mycoplasma, endotoxin, HIV-1, and RCL. This step uses rapid testing protocols for sterility, mycoplasma testing, and RCL testing.

[0103] The quality control items, methods and standards for the anti-HIV-1 chimeric antigen receptor T cells in this example are shown in the following table:

[0104] Control Project method standard Appearance inspection Visible foreign matter method No visible foreign matter Osmolality testing Freezing point osmotic pressure test 302±10mOsmol / kg Cell viability assay Trypan blue staining >90％ Total cell count detection Blood cell counting plate test <![CDATA[>2×10 7 ]]> CD8+ T cell ratio detection Flow cytometry >98％ CD4+ T cell ratio detection Flow cytometry <0.2％ Detection of CD8+CAR+T cell ratio Flow cytometry >20％ Vector copy number detection Real-Time PCR 2-5 copies / cell Cell functional testing LDH (detection of target cell killing) >50% (E:T=8:1) Sterility testing PCR Negative Mycoplasma testing PCR method Negative Endotoxin testing Chromogenic substrate method <3EU / mL RCL detection Real-Time PCR (release standard) No detection HIV-1 ELISA No detection

[0105] In summary, the present invention utilizes the JSR771 anti-HIV antibody sequence for the construction of CAR-T cells for the first time, improving the efficacy of CAR-T cells compared to currently used clinical CAR-T technology and demonstrating superior anti-HIV efficacy. By optimizing T cell isolation, activation, transduction, and culture conditions, and strictly controlling quality control standards, the anti-HIV-1 CAR-T cells prepared by the present invention exhibit high viability, high specificity, and potent killing ability, offering new hope for the clinical treatment of HIV infection.

[0106] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A chimeric antigen receptor targeting HIV-infected cells, characterized in that: The chimeric antigen receptor comprises an antigen binding domain, a chimeric receptor hinge region, a transmembrane domain, a co-stimulatory signaling domain, and a signaling domain; The antigen binding domain is the anti-HIV monoclonal antibody JSR771, which is composed of a heavy chain VH and a light chain VL. The amino acid sequence of the heavy chain VH is shown in SEQ ID NO.1, and the amino acid sequence of the light chain VL is shown in SEQ ID NO.

2.

2. The chimeric antigen receptor targeting HIV-infected cells according to claim 1, characterized in that: The chimeric antigen receptor comprises an antigen binding domain, a chimeric receptor hinge region, a transmembrane domain, a co-stimulatory signal transduction domain, a signal transduction domain, a 2A peptide and a fluorescent marker protein which are connected in series.

3. The chimeric antigen receptor targeting HIV-infected cells according to claim 2, characterized in that: The chimeric receptor hinge region is CD8 hinge region, and its amino acid sequence is shown in SEQ ID NO.3; The transmembrane domain is CD8TM, and its amino acid sequence is shown in SEQ ID NO.4; The co-stimulatory signal transduction domain is 41BB, and its amino acid sequence is shown in SEQ ID NO.5; The signal transduction domain is CD3zeta, and its amino acid sequence is shown in SEQ ID NO.6; The amino acid sequence of the 2A peptide is shown in SEQ ID NO.7; The fluorescent marker protein is EGFP, and its amino acid sequence is shown in SEQ ID NO.

8.

4. The chimeric antigen receptor targeting HIV-infected cells according to claim 3, characterized in that: The chimeric antigen receptor is scFv-JSR771VH-JSR771VL-CD8 hinge region-CD8TM-41BB-CD3zeta-2A-EGFP, and its amino acid sequence is composed of SEQ ID NO.1 to SEQ ID NO.8 connected in series in sequence.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises nucleotides encoding the chimeric antigen receptor targeting HIV-infected cells according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 5.

7. A CAR-T cell, characterized in that: The CAR-T cells express the chimeric antigen receptor targeting HIV-infected cells as described in any one of claims 1-4.

8. The method for preparing CAR-T cells according to claim 7, characterized in that: The steps include: Step 1: Construction of lentiviral vector and preparation of virus Adding both ends of the chimeric antigen receptor to a lentiviral vector, and co-transfecting with a lentiviral packaging plasmid to obtain a virus; Step 2: Preparation of CAR-T cells Mononuclear cells were isolated from peripheral blood of HIV-1 patients; Isolation and activation of CD8 from peripheral blood mononuclear cells using magnetic bead separation + T cells; Transduction of CD8 with lentiviral vectors + T cells, causing them to express the chimeric antigen receptor; The obtained T cells were subjected to quality control tests to ensure that they met the following standards: no visible foreign matter, osmotic pressure 302±10mOsmol / kg, cell viability>90%, total cell mass>2×10 8 、CD8 + T cell ratio>98%, CD4 + T cell ratio <0.2%, CD8 + CAR + The proportion of T cells is >20%, the copy number of CAR in T cells is 2-5 copies / cell, and the ability of CAR-T cells to kill target cells is >50%.

9. Use of the chimeric antigen receptor targeting HIV-infected cells according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant expression vector according to claim 6 or the CAR-T cell according to claim 7 in the preparation of drugs for preventing and / or treating HIV.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor targeting HIV-infected cells according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the recombinant expression vector according to claim 6 or the CAR-T cell according to claim 7, and one or more pharmaceutically acceptable carriers.

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