Use of T cell comprising chimeric antigen receptor (CAR) modification for preparing cell drug

A chimeric antigen receptor and antigen technology, applied in the field of biomedicine, can solve the problems of HIV amplification, high cost, inability to target or eliminate latent HIV virus, etc.

Active Publication Date: 2019-10-08
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) The cost of this therapy is very high, and it has resulted in the existence of a larger number of HIV carriers, who are completely dependent on antiretroviral drugs and have to bear the serious side effects and economic burden brought by it;
[0007] (2) The therapy is ineffective for some patients, and HIV in some patients still cannot be suppressed but amplified in large numbers;
[0008] (3) The therapy cannot target or eliminate the latent HIV virus present in the resting CD4+ central memory T cells in the patient's body. These latently infected cells are very stable and continue to increase in the body, which will cause the virus to rebound immediately after stopping the drug

Method used

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  • Use of T cell comprising chimeric antigen receptor (CAR) modification for preparing cell drug
  • Use of T cell comprising chimeric antigen receptor (CAR) modification for preparing cell drug
  • Use of T cell comprising chimeric antigen receptor (CAR) modification for preparing cell drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1, Construction of 3BNC117-41BBCD3ξ-CAR

[0066] Prepare a variety of 3BNC117scFv, and generate CAR based on these scFv, the specific process is as follows:

[0067] (1) Preparation of various 3BNC117scFv

[0068] 1. Based on the nucleotide sequence of 3BNC117, synthesize antibody heavy chain (VH) and light chain (VL) sequences, and design Overlap PCR primers based on the sequences.

[0069] 2. Use DNA polymerase to perform PCR amplification to amplify the VL fragment and VH fragment of 3BNC117. After completion, perform PCR product recovery and DNA agarose gel electrophoresis to verify whether the PCR band size is correct.

[0070] 3. Using the correctly identified 3BNC117 VL and VH fragments as templates, Overlap PCR primers were used to perform overlap PCR amplification. After completion, DNA agarose gel electrophoresis was performed to verify the correct bands, and the correct bands were cut and recovered to obtain 3BNC117scFv;

[0071] 4. The 3BNC117scFv ...

Embodiment 2

[0109] Example 2. Preparation of CAR-T cells (exemplarily using phage-3BNC117scFv-CAR as described in SEQ ID NO: 14)

[0110] (1) Lentivirus packaging, concentration and titer detection

[0111] 1.1 Resuscitate and adjust the state of 293T cells, re-plating about 24 hours before transfection, and it is advisable to pack and transfect cells with a confluence of more than 70%;

[0112] 1.2 Plasmids psPAX2, pMD2.G and CAR-expressing lentiviral plasmid phage-3BNC117scFv-CAR required for the transfection virus packaging system. Before transfection, replace the medium with serum-free medium. For a 15cm cell culture dish, proceed as follows operate:

[0113] Add 60μl PEI dropwise to 1ml DMEM, let stand for 5 minutes,

[0114] Take another tube to join

[0115] phage-3BNC117scFv-CAR 15μg

[0116] psPAX2 10 μg

[0117] pMD2.G 5 μg

[0118] Add DMEM to 1ml,

[0119] Add the PEI mixture dropwise and mix it into the packaging plasmid mixture, and let it stand for 20 minutes;

[01...

Embodiment 3

[0131]Example 3, HIV envelope protein and 3BNC117-41BBCD3ζ-CAR specific recognition and binding verification experiments

[0132] In this experiment, to study whether the HIV envelope protein specifically recognizes and binds to 3BNC117-41BBCD3ζ-CAR, 3BNC117-41BB CD3ζ-CAR-GFP was first transfected into 293T cells, and after 24 hours, the HIV envelope with his tag Protein gp120 was incubated with anti-his PE-labeled antibody, and the combination of HIV envelope protein by CAR-GFP positive cells was observed by flow cytometry.

[0133] (1) Cells and reagents

[0134] 1. Experimental cells: 293T cells, purchased from ATCC, and cultured in the cell experiment platform of Tsinghua University.

[0135] 2. HIV envelope protein for experiment: The HIV envelope protein gene (CNE54) from Chinese HIV-infected patients was cloned into a eukaryotic vector, transferred into 293f cell culture for production, and purified by the AKTA protein purification system.

[0136] 3. The anti-his PE-...

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PUM

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Abstract

The invention discloses a T cell comprising a chimeric antigen receptor (CAR) modification, a preparation or a medicine prepared from the T cells, the use of the T cells for preparing a cell drug, andthe preparation or the drug for treating and removing a cell storage bank of human immunodeficiency virus (HIV) in a HIV infected patient. The invention further discloses a cell immunotherapy using gp120 antibody CART, and specifically using 3BNC117 scFv-CART, which achieves MHC independent recognition and killing specific for HIV latent cells, so that the goal of eradicating AIDS is achieved.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the use of T cells modified with a chimeric antigen receptor (CAR) in the preparation of cell medicines. Background technique [0002] Human immunodeficiency virus (Human immunodeficiency virus, HIV), that is, acquired immunodeficiency syndrome (AIDS, AIDS) virus, is a lentivirus (Lentivirus) that infects cells of the human immune system, and induces human immunodeficiency syndrome. The chief culprit of AIDS. [0003] Because HIV directly invades the immune system of the human body and destroys the cellular immunity and humoral immunity of the human body, HIV-infected patients are often accompanied by various complications, such as meningitis, tuberculosis, cancer, hepatitis, etc. According to statistics from the United Nations AIDS Program (UNAIDS), since the spread of HIV, about 76.1 million (65.2 million-88 million) people around the world have been infected with the virus. In 201...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/62A61P31/18G01N33/569
CPCA61K35/17A61P31/18C07K14/7051C07K16/1045C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N2510/00G01N33/56988G01N2333/16G01N2469/10
Inventor 赵学强张林琦林欣丁志超
Owner TSINGHUA UNIV
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