WO2023039968A1 - Lymphocyte car-t multifonctionnel anti-vih-1, son procédé de construction et son application - Google Patents

Lymphocyte car-t multifonctionnel anti-vih-1, son procédé de construction et son application Download PDF

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WO2023039968A1
WO2023039968A1 PCT/CN2021/123408 CN2021123408W WO2023039968A1 WO 2023039968 A1 WO2023039968 A1 WO 2023039968A1 CN 2021123408 W CN2021123408 W CN 2021123408W WO 2023039968 A1 WO2023039968 A1 WO 2023039968A1
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徐建青
张晓燕
毛蕴玉
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复旦大学
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Definitions

  • 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.
  • HAART Highly active anti-retroviral therapy
  • HIV-1 infected patients 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
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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-CD3 ⁇ CAR-T).
  • CD4-CD3 ⁇ 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.
  • CD4-CD3 ⁇ 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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 anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody
  • the anti-HIV-1 broad-spectrum neutralizing antibody is a single-chain antibody derived from 10E8.
  • 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.
  • 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.
  • 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 .
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ 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.
  • a multifunctional anti-HIV-1 CAR-T cell 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.
  • 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.
  • DMEM medium and RPMI1640 medium were purchased from Corning Company, and lymphocyte medium X-VIVO 15 was purchased from Lonza Company.
  • T cell growth medium consists of basal medium and cytokines
  • the basal medium is lymphocyte medium X-VIVO 15
  • the cytokines are IL-7 at a final concentration of 5ng/mL
  • IL-15 at 10ng/mL and 30ng/mL mL of IL-21.
  • cytokines IL-7 and IL-15 were purchased from R&D Company
  • IL-21 was purchased from Nearshore Protein Technology Co., Ltd.
  • Fetal bovine serum was purchased from BI Company.
  • Lenti-X lentivirus concentrated reagent was purchased from Takara Company.
  • Synthetic genes were purchased from Shanghai Jierui Bioengineering Co., Ltd.
  • the lentiviral expression plasmid pKL was provided by Kanglin Biotechnology (Hangzhou) Co., Ltd., and the packaging plasmid psPAX2 and envelope plasmid PMD2.G were purchased from Addgene.
  • Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • the endotoxin-free plasmid mini-prep kit and the endotoxin-free plasmid mid-prep kit were purchased from OMEGA and Macherey Nagel, respectively.
  • RTCA Real-time label-free cell function analyzer
  • the 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.
  • 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%.
  • the virus supernatant collected above through a 0.45 ⁇ m 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.
  • 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 ⁇ L of virus dilution to the corresponding wells, add the pro-infection reagent protamine sulfate and adjust the concentration to 10 ⁇ g/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 1 ⁇ 10 5 ⁇ proportion of fluorescent positive cells/100 ⁇ 1000 ⁇ corresponding dilution factor.
  • MT4-gp145 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- ⁇ and IL-2 in the supernatant was detected by ELISA method.
  • UTD untransduced lentivirus T cells
  • M10 CAR- T M10 CAR- T
  • M10 CAR-T secretes high levels of cytokines IFN- ⁇ 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.
  • Target cell killing efficiency was detected by real-time label-free cell analysis (RTCA, Real Time Cellular Analysis).
  • RTCA Real Time Cellular Analysis
  • A549 cells 5 ⁇ 10 4 cells/well
  • gp145 protein 5 ⁇ 10 4 cells/well
  • RTCA Real Time Cellular Analysis
  • 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.
  • 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.
  • 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
  • the chemokine receptor CXCR5 (SEQ ID No.6) was further overexpressed to obtain CXCR5+M10 CAR-T.

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Abstract

Lymphocyte CAR-T multifonctionnel anti-VIH-1, son procédé de construction et son application Le lymphocyte CAR-T peut exprimer simultanément des molécules de CAR spécifiques du VIH-1 et sécréter un anticorps neutralisant à large spectre contre le VIH-1. Selon la cellule, un anticorps neutralisant à large spectre est modifié dans le lymphocyte CAR-T au moyen d'un lentivirus, afin que la cellule neutralise les virus libres tout en tuant les cellules infectées par le VIH-1, le lymphocyte CAR-T et les cellules normales non infectées sont protégés, et l'effet thérapeutique de l'immunothérapie par CAR-T sur le VIH/SIDA est ainsi amélioré.
PCT/CN2021/123408 2021-09-14 2021-10-13 Lymphocyte car-t multifonctionnel anti-vih-1, son procédé de construction et son application WO2023039968A1 (fr)

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