Modified immunologic effector cell and preparation method thereof

A technology of immune effector cells and cells, applied in genetically modified cells, cells modified by introducing foreign genetic material, and other methods of inserting foreign genetic material, etc., can solve the problem of rapid rejection by the host immune system

Active Publication Date: 2021-07-02
宁波茂行生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main design principle of universal CAR T cells is to generate tumor antigen-specific T cells from allogeneic healthy donors, and the expansion efficiency and viability of CAR T cells obtained in this way are enhanced. However, the endogenous TCR of allogeneic T cells can Alloantigen recognition of the recipient can lead to graft-versus-host disease (GVHD), while the expression of HLA on the surface of allogeneic T cells can lead to rapid rejection by the host immune system (HVGR)

Method used

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  • Modified immunologic effector cell and preparation method thereof
  • Modified immunologic effector cell and preparation method thereof
  • Modified immunologic effector cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0275] 1. A modified immune effector cell, wherein the expression and / or activity of the TRAC gene and the HLA-A gene are down-regulated compared with the expression and / or activity of the corresponding gene in the corresponding cell without said modification, and the expression and / or activity of the B2M gene Expression and / or activity is not downregulated, and expression and / or activity of the CIITA gene is not downregulated.

[0276] 2. The immune effector cell according to embodiment 1, wherein the modification results in down-regulation of the expression and / or activity of two genes, wherein the two genes consist of the TRAC gene and the HLA-A gene.

[0277] 3. The immune effector cell according to any one of embodiments 1-2, wherein compared with the corresponding wild-type cells, the expression and / or activity of the TRAC gene and the HLA-A gene are down-regulated, and the expression and / or activity of the B2M gene are down-regulated. / or activity is not down-regulated,...

Embodiment 1

[0325] Example 1 Design guide RNA

[0326] Through the website https: / / www.ncbi.nlm.nih.gov / , find and download the corresponding gene sequence (as shown in SEQ ID No.55-63), and use the SnapGene software to open the gene sequence, which can be used in different exosomes of the target gene Design sgRNA on sub. The non-limiting design principle of the sgRNA of the CRISPR / Cas9 system used in this example is: 5'-NNN(20)-NGG-3', NGG is called protospacer adjacent motif (PAM), wherein , N represents A, T, C or G. Since many sgRNAs can be designed on the same exon, and sgRNAs consisting of 20 nucleotide sequences may appear repeatedly in the genome, use the website http: / / crispr.cos.uni-heidelberg.de to Carry out the design and evaluation of sgRNA, paste the exon sequence to this website, the website designs sgRNA and conducts prediction evaluation, the higher the score in the evaluation, it means that there may be higher editing efficiency and lower off-target risk, from which S...

Embodiment 2

[0327] Example 2: CD3 + T cell preparation

[0328] (1) Isolation of PBMCs from peripheral blood

[0329] Peripheral blood was collected from healthy donors and diluted 1:1 with PBS buffer. Into a new 50ml centrifuge tube, first add cell separation solution (Ficoll) with 1 / 3 of the diluted blood volume, then add the blood cell dilution solution very slowly along the tube wall, and centrifuge at 800g for 20min at room temperature (the centrifuge is set to speed up 1, speed down 0). After centrifugation, the liquid in the centrifuge tube is divided into PBS and serum layer, white blood cell layer, lymphocyte separation solution, and red blood cell layer from top to bottom. Remove the PBS and serum layer, move the white blood cell layer to a new 50ml centrifuge tube, add PBS to 40ml to wash the cells, and centrifuge at 450g for 10min. After centrifugation, the supernatant was discarded to obtain peripheral blood mononuclear cells. Cell counts were performed after the cells w...

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Abstract

The invention relates to a modified immunologic effector cell. Compared with the expression and / or activity of corresponding genes in corresponding cells that are not modified, the expression and / or activity of a TRAC gene and an HLA-A gene in the invention is down-regulated, the expression and / or activity of a B2M gene is not down-regulated, and the expression and / or activity of the CIITA gene is not down-regulated. The invention also relates to a method for preparing the modified immunologic effector cell.

Description

technical field [0001] The present application relates to the field of biomedicine, in particular to a modified immune effector cell and a preparation method thereof. Background technique [0002] Antitumor immunotherapy elicits durable and robust responses in a variety of malignancies and can be used to treat many different types of cancer, showing broad potential. Current anti-tumor immunotherapy mainly includes two types of immunotherapy: immune cell-targeting monoclonal antibody (mAb) therapy and adoptive cell therapy (ACT). Among them, ACT refers to the reinfusion of autologous or allogeneic lymphocytes stimulated and expanded in vitro into the human body to achieve anti-tumor effects, but this therapy is relatively effective only in patients with consistent MHC polymorphisms. CAR-T is a new type of effective MHC-independent adoptive cell therapy. CAR, also known as chimeric antigen receptor, is an artificial receptor that simulates the function of TCR. It can specific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/87C12N15/867C12N15/55C12N15/12C12N15/62A61K39/00A61P35/00
CPCC12N5/0636C12N15/87C12N15/86C12N9/22C07K14/70539C07K14/7051C07K16/2803C07K16/2887C07K16/2878A61K39/001112A61K39/001124A61K39/001117A61P35/00C12N2510/00C12N2740/15043C12N2800/107C07K2319/33C07K2319/74A61K2039/5156A61K39/00C12N5/10C12N9/14C12N15/62C12N15/867A61K35/17A61P31/00A61P37/02A61P37/06A61P37/08A61P35/02C07K14/4702C07K2319/03A61K48/005A01K2227/105A01K2267/0331C12N15/113A61P37/00C07K14/435C12N15/85
Inventor 尚小云蒋海娟王丹沈慧马丽辛雨徐凡丽李甲璐马少文赵丹
Owner 宁波茂行生物医药科技有限公司
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