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Chimeric antigen receptors carrying truncated or non-truncated natural cytotoxic receptor signaling structures and applications thereof

A technology of chimeric antigen receptors and antigens, applied in receptors/cell surface antigens/cell surface determinants, genetically modified cells, for targeting specific cell fusion, etc., can solve unfavorable CAR-T cell persistence , weakening the anti-tumor function of CAR-T cells, etc., to achieve the effect of ensuring the safety of clinical application, high clinical application safety and significant curative effect

Active Publication Date: 2019-04-19
NANJING CART MEDICAL TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of effectiveness, first of all, the tumor stroma composed of solid tumor-associated fibroblasts (CAFs) provides a physical barrier for the infiltration of CAR-T cells, and CAFs also secrete extracellular matrix proteins to separate T cells from cancer cells. Second, the metabolic environment of the tumor microenvironment of solid tumors is not conducive to the persistence of traditional CAR-T cells, because once tumor formation is activated, tumor cells stop producing ATP through oxidative phosphorylation and switch to aerobic glycosylation The solution causes the tumor environment to become acidic, which is the so-called "Warburg effect". The pH value drops from 7.4 to 6.5; finally, the tumor microenvironment causes hypoxia to further produce immune suppression, and tumor cells in a hypoxic environment produce a HIF1 -ɑ molecule, which weakens the anti-tumor function of traditional CAR-T cells by attracting regulatory T cells (Tregs) to the tumor site. Tregs suppress the immune response, thus limiting the therapeutic effect of traditional CAR-T on solid tumors

Method used

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  • Chimeric antigen receptors carrying truncated or non-truncated natural cytotoxic receptor signaling structures and applications thereof
  • Chimeric antigen receptors carrying truncated or non-truncated natural cytotoxic receptor signaling structures and applications thereof
  • Chimeric antigen receptors carrying truncated or non-truncated natural cytotoxic receptor signaling structures and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1, containing DAP12-T2A-scFv-NKp44 cut Construction of the CAR lentivirus with these 4 elements

[0045] To demonstrate the presence of DAP12-NKp44 cut CAR-T cells with intracellular signaling domains have more advantages than previously reported CAR-T cells containing 4-1BB-CD3ζ, DAP12-KIRS2 and single DAP12 stimulation signals, so it is necessary to construct viral vectors containing different combinations of stimulation signals . In this example, the single-chain antibody targeting human mesothelin (MSLN) is used as a unified extracellular recognition antigen structure, and the following five chimeric antigen receptors need to be constructed respectively ( figure 1 ):

[0046] MSLN(scfv)-CD8α-4-1BB-CD3ζ (MSLN1)

[0047] DAP12-T2A-MSLN(scfv) (MSLN2)

[0048] DAP12-T2A-MSLN(scfv)-KIRS2 (MSLN3)

[0049] DAP12-T2A-MSLN(scfv)-NKp44 cut (MSLN4)

[0050] DAP12-T2A-MSLN(scfv)-NKp44 wt (MSLN5)

[0051] 1. Synthesize the gene sequence of the chimeric antige...

Embodiment 2

[0092] Embodiment 2, virus infection T cell

[0093] 1. Isolation and activation of T cells and virus infection

[0094] (1) Isolation of human peripheral blood mononuclear cells

[0095] Use a blood collection tube containing anticoagulant to collect about 10ml of peripheral blood, settle naturally at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and centrifuge the collected upper layer of plasma at 5000r / min for 10min at a volume ratio of 1:1 Add to the lymphocyte separation medium (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.), gradient centrifugation, 3000r / min, centrifugation for 30min, after centrifugation, the centrifuge tube is layered from top to bottom: the first layer is the plasma layer ; The second layer is the buffy coat layer of lymphocytes; the third layer is the transparent separation liquid layer; the fourth layer is the red blood cell layer. Aspirate the buffy coat of lymphocytes, wash twice with ...

Embodiment 3

[0100] Example 3. Effect of Virus Infection of CAR-T Cells on Cell Proliferation

[0101] After each group of viruses infected T cells, the T cells were counted every 1-2 days with a complete medium containing 5% autologous plasma + 300 IU / ml recombinant human IL-2 + KBM581. Then observe the growth of T lymphocytes, the results are as follows: image 3 shown. The results showed that after the cells were infected with CAR-expressing virus, they could still form a typical proliferative clonal group. By counting the cells and drawing the cell proliferation curve, it can be seen that the proliferation of CAR-T cells infected with MSLN4 was similar to that of MSLN1, MSLN2, MSLN3, and MSLN5 CAR-T. Compared with uninfected T cells ( image 3 Medium NTD) proliferative ability was slightly weaker.

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Abstract

Disclosed in the present invention is a chimeric antigen receptor (CAR), comprising: an antigen binding structural domain (scfv) and a signal transduction structural domain, the signal transduction structural domain comprising a first transduction structural domain and a second transduction structural domain, the antigen binding structural domain being connected in tandem between the first transduction structural domain and the second transduction structural domain. When stimulated by antigens, the CAR structure disclosed in the present invention has a very low level of secreted cytokines, better ensuring the safety of clinical application, i.e. having high clinical application safety; the capacity for killing in vitro antigen-positive tumour cells is stronger, and anti-tumour activity is better.

Description

technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to a chimeric antigen receptor carrying a truncated or non-truncated natural cytotoxicity receptor signal structure and an application thereof. Background technique [0002] Chimeric antigen receptor (CAR) is the core component of CAR-T. Using the ligand-binding domain properties, CAR can redirect the specificity and reactivity of selected immune cells, thus endowing T cells with an HLA-independent manner. The ability to recognize tumor antigens, which allows CAR-engineered T cells to recognize a wider range of targets than natural T-cell surface receptors TCR. The basic design of CAR includes a tumor-associated antigen (tumor-associated antigen, TAA) binding region (usually derived from the scFV segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signal Area. [0003] The current t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70503C07K14/7056C07K16/30C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N2510/00
Inventor 王恩秀汪晨张海武国英
Owner NANJING CART MEDICAL TECH LTD
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