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T cell enriching method and application thereof to adoptive T cell therapy

A cell and enrichment technology, applied in the direction of cell culture active agents, receptors/cell surface antigens/cell surface determinants, animal cells, etc., can solve the problem of affecting cell activity, low T cell purity, and reduced T cell yield, etc. problems, to achieve the effect of increasing CAR expression rate, high T cell yield, and reducing operational risk

Inactive Publication Date: 2020-11-10
BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of T cells isolated by the first two methods is low, which affects the activation efficiency of T cells, the expression rate of CAR and the efficiency of cell proliferation. The sorting after activation in the third method affects the activity of cells, and the yield of T cells is reduced. Low controllability of cell number
In addition, the T cells isolated by the existing technology still have problems such as low expression rate of the target protein during the subsequent virus infection process

Method used

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  • T cell enriching method and application thereof to adoptive T cell therapy
  • T cell enriching method and application thereof to adoptive T cell therapy
  • T cell enriching method and application thereof to adoptive T cell therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The isolation of the patient's autologous PBMCs of embodiment 1 different sources

[0069] The T cells used in the autologous CAR-T culture process are mainly derived from the patient itself, but in some cases, due to the patient's condition or the patient's own conditions, the content of T cells in PBMCs is low or the activity of T cells is insufficient, which increases the T cells in the process of CAR-T production. Difficulty in cell isolation and proliferation. For such patients, peripheral blood stem cells stored in a healthy state, PBMCs or highly active PBMCs stored in the early stage of patient onset can be used for CAR-T production, which can effectively avoid culture failure caused by low T cell activity.

[0070] Preparation of PBMCs from different sources

[0071] Scheme 1: PBMCs were obtained by separating autologous single blood using Ficoll separation fluid.

[0072] The experimental steps are as follows:

[0073] 1. The blood sample is diluted accordi...

Embodiment 2

[0092] Example 2 Obtaining the patient's own T cells

[0093] During the T cell sorting process, the isolated high-purity T cells can improve the activation efficiency of T cells, the proliferation multiple of T cells and the expression rate of CAR. However, in the process of CAR-T production, due to the patient's own reasons, PBMCs contain a large number of monocytes, which leads to a low content of T cells, and it is difficult to increase the isolation of high-purity T cells.

[0094] In this protocol, PBMCs are adhered to the wall, monocytes in PBMCs are removed, suspension cells are collected, and T cells are sorted by adding Dynabeads CD3 / CD28.

[0095] T cell isolation steps:

[0096] 1. Put the isolated PBMCs in 30mL / bottle (total 1×10 8 cells) into T175 culture flasks, placed in a carbon dioxide incubator (37°C, 5% CO 2 )nourish.

[0097] Take it out after 2.2 hours, absorb the suspended cells, and centrifuge (400g / 6min / liter 8 to 8).

[0098] 3. After resuspensio...

Embodiment 3

[0108] Virus infection steps:

[0109] 1. Collect T cells after 24-48 hours of activation, according to 1.5×10 6 cells / mL spread to the culture flask.

[0110] 2. Calculate the amount of virus needed according to the multiplicity of infection (MOI).

[0111] 3. Aspirate the required volume of virus, add it to the cell suspension, mix gently, and place in a carbon dioxide incubator (37°C, 5% CO 2 )nourish.

[0112] In this scheme, the inventors found that the use of viral infection cofactors had no significant effect on the expression rate of CAR (Table 2 and Figure 4 ). Therefore the present invention does not add viral infection cofactors.

[0113] Different infection times had a significant effect on the CAR expression rate (Table 3), among which 48 hours after activation was better than 24 hours after virus infection. To meet the needs of clinical production, virus infection was carried out 24 to 48 hours after T activation.

[0114] From the influence of the number ...

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Abstract

The invention relates to the technical field of adoptive T cell immunotherapy, in particular to a T cell enriching method and an application thereof to adoptive T cell therapy. The enriching method comprises the following steps: culturing peripheral blood mononuclear cells (PBMCs) for 1-4 hours, then removing adherent cells, and then separating T cells from the remaining cells by using magnetic beads. The method can effectively improve the purity of the T cells, the yield of the T cells is high, and the operation is simple. According to the method disclosed by the invention, the expression rate of a chimeric antigen receptor (CAR) is improved by changing a virus infection method, and the operation risk is reduced.

Description

technical field [0001] The present invention relates to the technical field of adoptive T cell therapy, in particular to a method for enriching T cells and the application of the method in preparing T cells for adoptive cell therapy. Background technique [0002] Tumor immunotherapy has received extensive attention and application in recent years. This therapy controls and eliminates malignant tumor cells by activating the body's anti-tumor immune response (Sanmamed MF, et al. Cell. 2019, 176:677). Cancer cells are transformed from normal cells through gene mutation or epigenetic changes, which express many abnormal tumor antigens. Under normal circumstances, the human innate immune system can specifically recognize and kill cancer cells by recognizing tumor antigens. However, cancer tissue adopts various mechanisms to build an immunosuppressive microenvironment, which enables immune cells such as T and B lymphocytes to produce immune tolerance to cancer cells, thus leading...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/10C12N7/01A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C12N5/0636C12N7/00C12N2501/2302C12N2740/15021
Inventor 鲁薪安何霆齐菲菲
Owner BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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