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CAR-T cell for treating AIDS-associated lymphoma, and preparation method and application thereof

A tumor cell and cell technology, applied in the direction of genetically modified cells, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of missed treatment timing, long process, low tolerance, etc., to reduce the risk of GVHD , strong amplification and differentiation capabilities, simple collection process

Pending Publication Date: 2018-04-13
SHANGHAI LONGYAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] (1) The blood of AIDS patients has strict requirements on the operating environment and conditions due to the HIV virus, and the company's existing conditions cannot meet the requirements. Although there are very few institutions that have the qualifications to process blood carrying infectious viruses, they cannot meet the clinical needs. Therefore, it is urgent to seek blood sources other than the autologous blood of AIDS patients as the source of T cells;
[0010] (2) AIDS patients are in weak physical condition, less resistant to cytokine storm and tumor lysis syndrome caused by CAR-T treatment, autologous T cells cannot be used, and allogeneic donated T cells need to be matched to be haploidentical and Only the above can be used, and the risk of graft-versus-host reaction (GVHD) may still occur. Therefore, it is urgent to seek T cells with low immunogenicity as the source of T cells for the preparation of CAR-T to minimize the risk of GVHD.
[0011] (3) The CAR-T prepared by the patient's own cells can only be used by himself. Due to individual differences, some patients' cell status is poor, and the prepared dose cannot reach the effective dose, resulting in poor reinfusion effect. If donated by a healthy person, then Need to find a qualified match, the process is long and easy to miss the opportunity for treatment
[0012] (4) Since CD4+ CAR-T has the risk of being infected by HIV, and it is uncertain whether the following options can be adopted: whether CD8+ T cells can be used to produce CAR-T for AIDS patients, or CD4+ T can be removed by gene editing In order to reduce the risk of the imported CAR-T being infected by HIV in the body and causing the disease to aggravate

Method used

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  • CAR-T cell for treating AIDS-associated lymphoma, and preparation method and application thereof
  • CAR-T cell for treating AIDS-associated lymphoma, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This embodiment is a preparation step of CAR-T cells targeting CD19, such as figure 1 and figure 2 As shown, the specific steps are as follows:

[0047] 1) Immunotype the patient's tumor cells to confirm the expression of the surface molecule CD19, which can be treated by CD19-CAR-T;

[0048] 2) Obtain umbilical cord blood mononuclear cells by leukapheresis, the specific steps are as follows:

[0049] a) Collecting and screening cord blood from volunteers, wherein the inner wall of the test tube for collecting cord blood is provided with an anticoagulant to prevent cord blood from coagulating, and the screening and testing items include carrying virus or genetic family history;

[0050] b) Centrifuge the collected umbilical cord blood at 1200rmp for 25min to obtain plasma, and add DPBS or normal saline at a volume ratio of 1:1 for dilution;

[0051] c) Add a commercially available lymphocyte separation solution (such as Ficoll separation solution) to the diluted pla...

Embodiment 2

[0061] The CAR-T cells made from umbilical cord blood in Example 1 were compared with the CAR-T cells made from peripheral blood in vitro. The specific experimental steps are as follows:

[0062] The first step: Calcein-AM labeling target cells;

[0063] Dilute Calcein-AM with DMSO to 1 mg / mL; resuspend the target cells with full medium to 1×10 6 Density / mL; add 15 μM Calcein-AM, 37°C, 5% CO 2 Incubate for 30min, mix gently every 10min; centrifuge at 1500rpm, remove supernatant, resuspend with full medium, repeat twice;

[0064] The second step: killing;

[0065] Resuspend the labeled target cells at a density of 5,000-50,000 / mL, take 100 μL and add them to a 96-well plate, add 100 μL of effector cells according to the appropriate ET ratio, and each group has 3 parallels; at the same time, there is a separate group A 6 parallels, only target cells (spontaneous release); 6 parallels in separate group B, only target cells + 2% Triton X-100 (maximum release);

[0066] third s...

experiment example 3

[0070] This example is to verify the safety and effectiveness of the prepared CAR-T cells in vivo.

[0071] First, use AIDS model mice to verify the effectiveness of CAR-T cells. The specific steps are as follows:

[0072] 1) Construction of a humanized mouse AIDS model: After the humanized mouse Scid-hu-Thy / Liv mice were anesthetized, the renal capsule graft was exposed, and HIV virus was directly inoculated into the graft to replicate HIV-1 infection model, and then screen for concurrent lymphoma (volume 100mm 3 Left and right) mice were used as test subjects.

[0073] 2) cell culture;

[0074] T cell culture:

[0075] Count and determine the viability of T cells, resuspend with normal saline after centrifugation, and adjust its concentration to 5×10 5 pc / mL, total up to 6×10 5 indivual.

[0076] CAR-T cell culture:

[0077] Determine the CAR content percentage of CAR-T cells, count and measure the activity rate, resuspend with normal saline after centrifugation, adju...

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Abstract

The invention discloses a preparation method of a CAR-T cell for treating AIDS-related lymphoma. A CD8+ T cell is used to produce the CAR-T cell, and the CD8-T cell derives from a cord blood T cell. The method includes the following steps: preparing cord blood mononuclear cells from cord blood, removing tumors and other cells by using a human T cell purification kit to obtain T cells, and carryingout separation by using magnetic beads to obtain the CD8+ T cell; activating the CD8+ T cell by using an appropriate medium and appropriate stimulation conditions; transferring CAR to the CD8+ T cellby using lentivirus to prepare the CAR-T cell; and amplifying the CAR-T cell in vitro by using cytokines and activating stimulators to achieve the desired effective dose. The invention also relates to the CAR-T cell prepared by the method, and an application thereof. The CAR-T cell prepared in the invention has a good cell activity and a high proliferation speed, and reduces the attack risk of GVHD; and only the CD8+T cell of the umbilical blood T cells of AIDS patients is used to prepare the CAR-T cell for, so the risk of input CAR-T infected with in-vivo HIV is reduced.

Description

technical field [0001] The present invention relates to the technical field of cellular immunotherapy, in particular to a CAR-T cell for treating AIDS combined with lymphoma and its preparation method and application. A method for preparing complex antigen receptor T cells (CAR-T) and cell preparations thereof. Background technique [0002] The use of immunological treatment to overcome tumors has always been an important direction for the application of immunology in translational medicine. With the development of various omics (genomics, proteomics, etc.), the immunogenicity of tumor cells due to mutations has been widely recognized, which has laid a theoretical foundation for tumor immunotherapy. At the same time, with the accumulation of tumor immunology research itself, tumor immunotherapy has made great progress recently, and a series of new immunotherapy methods have gradually entered the clinic. The current tumor immunology research has established the central posi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00A61P31/18
CPCA61K35/17C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 杨选明
Owner SHANGHAI LONGYAO BIOTECH CO LTD
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