Bispecific chimeric antigen receptor targeting HIV-1 (human immunodeficiency virus-1) envelope protein as well as preparation method and application of bispecific chimeric antigen receptor

A chimeric antigen receptor and HIV-1 technology, applied in the field of immunotherapy, can solve the problem of affecting the expression level of CAR molecular membrane, unfavorable control of HIV-1 infection, and weakening the ability of CAR-T cell activation to kill HIV-1 infected target cells capacity and other issues to achieve the effect of increasing clinical effectiveness, improving broad-spectrum and specificity, and improving survival

Active Publication Date: 2022-03-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after research and comparison, it was found that the above-mentioned tandem sequence not only affects the membrane expression level of CAR molecules, but also significantly weakens the activation ability of CAR-T cells and the killing ability of HIV-1 infected target cells, which is not conducive to HIV-1 infection. control

Method used

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  • Bispecific chimeric antigen receptor targeting HIV-1 (human immunodeficiency virus-1) envelope protein as well as preparation method and application of bispecific chimeric antigen receptor
  • Bispecific chimeric antigen receptor targeting HIV-1 (human immunodeficiency virus-1) envelope protein as well as preparation method and application of bispecific chimeric antigen receptor
  • Bispecific chimeric antigen receptor targeting HIV-1 (human immunodeficiency virus-1) envelope protein as well as preparation method and application of bispecific chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of lentiviral expression plasmid The M31 CAR and M13CAR genes (shown in SEQ ID No:8 and SEQ ID No:9, respectively) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and cloned into blank lentiviral expression plasmids (pKL) to obtain pKL-M31-CAR, respectively and pKL-M13-CAR recombinant lentiviral expression plasmid, bispecific chimeric antigen receptor structure such as figure 1 and 2 shown.

Embodiment 2

[0047] Packaging, concentration and titer determination of embodiment 2 lentivirus

[0048] 1.1 Packaging of lentivirus

[0049] Treatment of HEK293T cells: 24 hours before transfection, HEK293T cells in logarithmic growth phase were collected and seeded in 10 cm cell culture dishes (6-8×10 6 cells), the cells were grown in 10 mL of complete DMEM medium at 37 °C, 5% CO 2 Culture under the conditions for 18-24 hours, and the cell density can reach above 70-90%, and then transfection can be carried out.

[0050] HEK293T cell transfection: Add 1mL basal DMEM medium in a 15mL centrifuge tube, according to the mass ratio: lentiviral expression plasmid (pKL-M31-CAR or pKL-M13-CAR): packaging plasmid psPAX2: envelope plasmid PMD2.G= Prepare the transfection mixture at 1:3:1, and the total amount of plasmids is 15 μg / dish. Add 30 μL of TurboFect transfection reagent at a ratio of plasmid amount (μg):transfection reagent (μL)=1:2, incubate at room temperature for 15-20 minutes, ad...

Embodiment 3

[0054] Infection and expansion of embodiment 3 T cells

[0055] In a 48-well flat-bottomed cell culture plate (containing 1×10 6 pre-activated peripheral blood mononuclear cells), adding the packaged and concentrated lentiviral vectors (LV-M31-CAR and LV-M13-CAR) (MOI=5~10) in Example 2, adding pro-infection reagent protamine sulfate Protein 10μg / mL, 1000×g, centrifuge at 32°C for 90 minutes, add 1×10 6 Immunomagnetic beads pre-coated with αCD3 / αCD28 antibody were cultured 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. From 6 to 7 days after infection, remove the immunomagnetic beads of activated T cells, continue to culture and expand T cells modified with bispecific chimeric receptors (M31-CAR or M13-CAR), and wait for the cells to rest (remove Immunization of magnetic beads 6-7 days) before using flow cytometry to...

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Abstract

The invention provides a bispecific chimeric antigen receptor targeting an HIV-1 virus envelope protein. The chimeric antigen receptor comprises (1) a recognition unit targeting an HIV-1 gp120 protein co-receptor binding site; (2) a recognition unit targeting other binding sites of HIV-1 gp120 (including but not limited to a CD4 binding site, a V1V2 glycan region, a V3 glycan region, a gp120-gp41 interface or a proximal membrane end external region on gp41); (3) a hinge and transmembrane region; and (4) an intracellular signal transduction domain; optionally, the chimeric antigen receptor further comprises (5) one or more co-stimulatory signal domains; preferably, according to the series connection sequence of the chimeric antigen receptor extracellular recognition domain, the far membrane end is a recognition unit of a targeted HIV-1 gp120 protein co-receptor binding site, and the near membrane end is a recognition unit of targeted HIV gp120 other binding sites. The invention provides a brand-new construction method of the HIV-1 virus envelope protein bispecific chimeric antigen receptor, immune cells modified by the HIV-1 virus envelope protein bispecific chimeric antigen receptor have stronger activating and killing capabilities, HIV-1 infected cells can be specifically recognized and killed, and the HIV-1 virus envelope protein bispecific chimeric antigen receptor has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of immunotherapy, in particular to a bispecific chimeric antigen receptor targeting HIV-1 virus envelope protein and its application. Background technique [0002] AIDS is an infectious disease caused by human immunodeficiency virus type 1 (Human Immunodeficiency Virus, HIV-1) infection that threatens the safety of human life, and it is also one of the most important public health challenges facing our country. Highly active anti-retroviral therapy (HAART) is the first revolution in the history of HIV-1 / AIDS treatment. The load is lower than the detection line, thereby limiting the AIDS disease process. However, HAART still has many limitations that cannot be ignored, such as severe drug side effects and restrictions on life-long medication. Not only that, but its biggest limitation is that it cannot target or clear the latent HIV-1 virus in resting CD4+ T cells in patients. Therefore, once HAART treatmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P31/18
CPCC07K16/1063C07K14/7051C07K14/70514C12N5/0636C12N15/86A61K39/0008A61P31/18C12N2740/15043C12N2800/107C07K2319/03C07K2317/569C07K2319/02C07K2319/43C12N2510/00C07K2319/33A61K2039/5158Y02A50/30
Inventor 徐建青张晓燕应天雷
Owner FUDAN UNIV
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