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Method for preparing chimeric antigen receptor-T (CAR-T) cells, and prepared CAR-T cells and application thereof

A cell and mononuclear cell technology, applied in the fields of immunology and molecular biology, can solve the problems of missed patient treatment, low T cell activity, unsuitable blood collection, etc., achieving high safety, simple method, and good effectiveness Effect

Active Publication Date: 2016-12-14
BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] CAR-T cells have attractive prospects in tumor immunotherapy. At present, most of the cell sources of CAR-T cell clinical trials are obtained from the patient's own peripheral blood T cells, which will lead to the following three very significant problems 1) It takes 10-14 days after the patient’s peripheral blood is collected to complete the CAR-T reinfusion, which may miss the best time for the patient’s treatment; The activity of T cells will be too low, and the amount of CAR-T reinfusion cannot be guaranteed; 3) The patient may not be suitable for blood collection
This short board seriously restricts the application and marketing of CAR-T cells for individualized treatment

Method used

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  • Method for preparing chimeric antigen receptor-T (CAR-T) cells, and prepared CAR-T cells and application thereof
  • Method for preparing chimeric antigen receptor-T (CAR-T) cells, and prepared CAR-T cells and application thereof
  • Method for preparing chimeric antigen receptor-T (CAR-T) cells, and prepared CAR-T cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Collection of cord blood

[0052] After the severed fetal umbilical cord is cleaned, blood is collected, and the wall of the blood collection tube is fully covered with anticoagulant before blood collection;

[0053] Then place the blood collection bag at a position lower than the umbilical cord, insert the blood collection needle into the lower end of the umbilical vein filling, and use gravity and extrusion to make the umbilical cord blood enter the blood collection bag;

[0054] Gently shake the blood collection bag while collecting cord blood to fully mix the cord blood and anticoagulant;

[0055] Cord blood collection can be terminated when the umbilical vein collapses, turns white, or the cord blood in the collection bag stops flowing;

[0056] The collected umbilical cord blood is temporarily stored in a refrigerator at 4°C, and transported to a GMP laboratory for separation and culture within 24 hours via a transfer car equipped with constant temperature equipm...

Embodiment 2

[0058] Preparation of umbilical cord blood mononuclear cells

[0059] Use a pipette to draw DPBS or saline into the collected or commercially purchased cord blood (volume ratio 1:1) to dilute the cord blood cells;

[0060] Slowly add the cord blood cell dilution into a centrifuge tube filled with lymphocyte separation medium (Ficoll or Histopaque-1077), centrifuge at 800g for 20-30 minutes, and absorb the buffy coat cells above the lymphocyte separation medium;

[0061] The aspirated buffy coat cells were transferred to a new centrifuge tube, and Lonza x-vivo 15 serum-free cell culture medium was added, the supernatant was discarded after centrifugation, and the cell pellet at the bottom of the centrifuge tube was kept to obtain umbilical cord blood mononuclear cells.

Embodiment 3

[0063] Enrichment of CD3 Positive T Cells from Cord Blood Mononuclear Cells

[0064] Count the umbilical cord blood mononuclear cells obtained in Example 2, add magnetic beads coupled with CD3 / CD28 antibody at a ratio of 1:1, shake gently for 20 minutes, and use a magnetic stand to obtain CD3-positive T cells .

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Abstract

The invention relates to the fields of immunology and molecular biology, in particular to a method for preparing chimeric antigen receptor-T (CAR-T) cells, and the prepared CAR-T cells and application thereof. According to the method for preparing the CAR-T cells, the CAR-T cells are obtained by enabling CD3 positive T cells to be infected by CAR-containing virus. The invention also discloses the prepared CAR-T cells and a composition containing the CAR-T cells. The method for preparing the CAR-T cells enables the CD3 positive T cells to be infected by the CAR-containing virus so as to obtain the CAR-T cells, thus being capable of realizing the industrial production and preparation of the CAR-T cells used for individualized treatment; the prepared CAR-T cells eliminate the repellency between individuals; the CAR-T cells prepared by the method are better in effectiveness and higher in safety. The invention also provides the application of the CAR-T cells in preparation of medicine for treating or preventing cancers.

Description

technical field [0001] The present invention relates to the fields of immunology and molecular biology, in particular, to a method for preparing CAR-T cells, the prepared CAR-T cells and applications thereof. Background technique [0002] Most patients with B-cell malignancies, including chronic lymphocytic leukemia (CLL), have a poor cure rate and prognosis with chemotherapy and targeted therapy. One approach to treating these patients is to genetically modify T cells to target antigens expressed on tumor cells through the expression of Chimeric Antigen Receptors (CARs). CARs are antigen receptors designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts to treat these types of patients with CAR-expressing genetically modified T cells have met with some success (Molecular Therapy, 2010, 18:4, 666-668; Blood, 2008, 112:2261-2271). [0003] With the continuous development of Chimeric Antigen Receptor-T cell (CAR-T) technology, CAR...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 何霆鲁薪安尤亚南
Owner BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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