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Immune cell for expressing CD3 antibody receptor complex and application thereof

A technology of immune cells and complexes, applied in the field of immune cells, which can solve problems such as cytotoxicity, B cell and immunoglobulin deficiency

Pending Publication Date: 2021-01-08
CURE GENETICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-term CAR-T cells may lead to the loss of normal B cells and immunoglobulins, and CAR-T targeting solid tumors has safety issues such as target but off-tumor cytotoxicity

Method used

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  • Immune cell for expressing CD3 antibody receptor complex and application thereof
  • Immune cell for expressing CD3 antibody receptor complex and application thereof
  • Immune cell for expressing CD3 antibody receptor complex and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0242] Example 1 Design of CD3 Antibody Receptor Complex Molecules and Construction of Plasmids

[0243] (1) Design molecules

[0244]The gene sequence information (Table 1) was searched from the NCBI website database (https: / / www.ncbi.nlm.nih.gov / ), and the gene CG-UST-1 (SEQ ID NO. 13), CG-UST-1 comprises two parts, is the tandem of two CD3 recombinant protein genes, and the first CD3 recombinant protein comprises CD3 epsilon ectodomain, CD28 transmembrane region, CD28 intracellular domain and CD3 zeta intracellular domain (SEQ ID NO:10). The second CD3 recombinant protein comprises CD3 gamma extracellular domain, CD28 transmembrane region and CD3 gamma intracellular domain (SEQ ID NO: 11). The two CD3 recombinant proteins are connected through the linker T2A gene (SEQ ID NO.16).

[0245] The CD3 gamma intracellular domain of the gene encoding the second CD3 recombinant protein of CG-UST-1 was replaced with a gene encoding a short peptide to obtain CG-UST-2 (SEQ ID NO.14)...

Embodiment 2

[0248] Example 2 Preparation of lentivirus

[0249] (1) Extract plasmid

[0250] The lentiviral vector plasmid constructed above was re-transformed into Escherichia coli. Pick a single clone from the transformed plate and put it into a 3ml shaking tube of liquid LB medium containing ampicillin, rotate at 220rpm, and culture it on a shaking table for 8h; draw 500μl from the activated bacterial solution and inoculate it into 250ml containing ammonia Bian penicillin liquid LB culture medium, 220rpm, shaker shake culture 12-16h. Use the Qiagen HiSpeed ​​Plasmid Maxi Kit kit (Catalog No.: 12662) for plasmid extraction according to the experimental procedure provided by the kit. After extracting the plasmid, Nanodrop (Thermo Fisher Scientific) was used to detect the plasmid concentration and the supercoiled plasmid content was detected by DNA agarose gel.

[0251] (2) Culture 293T cells

[0252] After the frozen 293T cells (ATCC) were taken out from the liquid nitrogen, they wer...

Embodiment 3

[0257] Example 3 Preparation of general-purpose T cells expressing CD3 antibody-receptor complex

[0258] PMBCs from peripheral blood of healthy donors (purchased from Miaotong Biotech) were isolated using an apheresis machine. Dilute PBMCs to 2×106. T cells were activated using CD3 / CD28 magnetic beads (Thermo Fisher Scientific) at a cell to bead ratio of 1:3 with the addition of IL-2 (PeproTech; 200-02). On the 3rd day after activation, the concentrated lentivirus was added to the T cell culture flask to transfect T cells. On day 5 after T cell activation, TCR and B2M in T cells were knocked out using CRISPR / Cas9 to construct universal T cells. The used gRNA sequence and operation process refer to Example 3 of patent WO2019 / 011118.

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Abstract

An engineered immune cell that does not express a T cell receptor (TCR) and comprises a CD3 antibody receptor complex. The invention also relates to a pharmaceutical composition containing the modified immune cell and a bispecific antibody, and an application of the pharmaceutical composition in preparation of medicines.

Description

technical field [0001] The present application relates to the field of biomedicine, in particular to an immune cell expressing a CD3 antibody-receptor complex, and the expression of the CD3 antibody-receptor complex may be independent of TCR. Background technique [0002] In recent years, due to the obvious clinical efficacy in the field of cancer treatment, the immunotherapy method of chimeric antigen receptor T cell (CAR-T) therapy has gradually become a research hotspot worldwide. At present, the source of fever in the clinical preparation of CAR-T cells is generally the patient himself, so they are all autologous CAR-T cells. However, autologous CAR-T has many problems in clinical application, such as long preparation period and high preparation cost. When the patient's immune function status is relatively poor, CAR-T cells cannot be successfully prepared. [0003] There is also a method of using gene editing tools such as CRISPR / Cas9 nucleases to specifically knock ou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867C07K19/00A61K35/17A61K39/395A61P35/00A61P35/02
CPCA61K35/17A61K2039/505A61P35/00A61P35/02C07K14/7051C07K14/70539C07K2319/02C07K2319/03C12N15/86C12N2740/15043
Inventor 王文博郭佩佩冯爱华林彦妮
Owner CURE GENETICS CO LTD
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