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Connection arm for controlling activation/inhibition of chimeric antigen receptor T cell and application of connection arm

A technology of linking arms and cells, applied in the field of cellular immunotherapy, can solve the problems of inability to shut down CAR-T cells, fail to achieve CRS inhibition effect, fail to achieve CAR-T cell control effect, etc., and achieve rapid and flexible conversion Effect

Active Publication Date: 2019-01-04
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, although this method can effectively control the activation of CAR-T cells, it cannot immediately and effectively shut down CAR-T cells; compared with the above two methods, antibody-based control methods are more popular in clinical practice
However, the CRS effect caused by CAR-T cells is caused by the co-release of multiple factors, so single control of one factor cannot achieve a systemic CRS inhibitory effect
In summary, although the existing research can solve the problem of CAR-T control to a certain extent, they have only improved one aspect (shutdown or activation), and have not yet achieved effective, immediate and effective control of CAR-T cells. reversible control effect
At present, there is also a lack of technical and theoretical methods that can effectively achieve the above-mentioned regulatory effects on CAR-T cells.

Method used

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  • Connection arm for controlling activation/inhibition of chimeric antigen receptor T cell and application of connection arm
  • Connection arm for controlling activation/inhibition of chimeric antigen receptor T cell and application of connection arm
  • Connection arm for controlling activation/inhibition of chimeric antigen receptor T cell and application of connection arm

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Synthesized a bifunctional tether (Folate-NBE-FITC) based on FITC, folic acid, and o-nitrobenzyl ester with controlled breakage, which mainly includes three parts: (1) FITC as a linking ligand, and specific recognition CAR-T cell binding of FITC. As a fluorescent molecule, FITC has been proved to have good bio-orthogonality and biocompatibility in vivo; (2) folic acid is used as the target part to target tumor cells with high folic acid expression. The existing results show that α-type folate receptor is fully expressed on about 50% of tumor cells, but rarely expressed in normal tissues, so folic acid molecule can be used as an ideal target for model verification; (3) UV Photocontrollable cleavage of o-nitrobenzyl ester (ortho-nitrobenzylic ester group, NBE). This group can undergo rapid and efficient cleavage under biocompatible ultraviolet light conditions (UV365nm), which is convenient for model verification.

[0033] Such as figure 1 As shown, take a 200ml round ...

Embodiment 2

[0044] A CAR-T cell (anti-FITC-CAR-T) that recognizes FITC molecules was constructed. By fusing the single-chain antibody gene sequence that recognizes FITC molecules with the hinge region and transmembrane region of CD8, 4-1BB and CD3ζ region (CAR structure), and constructing the constructed gene fragment into a lentiviral vector ( image 3 -A). And the packaged lentiviral vectors are used to infect human CD3+ primary T cells, so that CD3+ T cells express the CAR structure, and then obtain CAR-T cells that specifically recognize FITC molecules and can be specifically activated by them. We used APC-labeled anti-mouse-Fab antibody to detect the expression of the CAR structure on the cell surface ( image 3 -B), and through the method of double labeling with small molecules and Anti-mouse-Fab-APC antibodies, it was confirmed that the Anti-FITC-scFv was correctly folded on the surface of T cells, and the specific recognition of Folate-NBE-FITC was successfully prepared CAR-T ce...

Embodiment 3

[0049] By co-incubating CAR-T cells with KB cells, by adding different concentrations of Folate-NBE-FITC, it was verified that the cytotoxicity of CAR-T cells can be effectively activated by Folate-NBE-FITC, and it has a dose-dependent effect, EC50 approximately equal to 85pM ( Figure 7 -A); T cell activation surface markers CD25 and CD69 were also significantly increased with the addition of Folate-NBE-FITC ( Figure 7 -B); The release level of cytokines is consistent with the cytotoxic effect, which can be significantly activated by Folate-NBE-FITC, and presents a dose-dependent effect ( Figure 7 -C); In addition, through a microscope, it can be clearly observed that only the experimental group in which CAR-T cells were co-incubated with KB cells showed an obvious T cell aggregation effect, while the rest of the control group had no obvious phenomenon. This protocol proves that Folate-NBE-FITC can mediate the effective activation of CAR-T cells and has a dose-dependent ef...

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Abstract

The invention discloses a connection arm for controlling activation / inhibition of CAR-T. One end of the connection arm contains a target molecule capable of identifying a target cell, the other end ofthe connection arm contains a part, capable of being identified by the specificity CAR-T cell, with biological orthogonality, and the middle is coupled by a chemical group capable of being broken through biological orthogonality. According to the connection arm, the CAR-T cell can be quickly and flexibly switched from the activated state to the static state, and the connection arm has the systematicness and high efficiency on inhibition of the CAR-T cell. The connection arm has adjustment reversibility, an existing CAR-T cell is not injured or killed, the CAR-T cell can be flexibly switched from the activated state to the closed state to the activated state, and the integral effect of treatment is not influenced.

Description

technical field [0001] The invention belongs to the field of cellular immunotherapy, and relates to a technology and an application method for regulating chimeric antigen receptor T cells (CAR-T) in a "switchable" mode. Background technique [0002] CAR-T technology has become a key technology in cellular immunotherapy, and has broad application prospects in tumors, autoimmune diseases, and antiviral treatments. In normal physiology, the recognition of tumor cells by T cells first requires MHC-I molecules to present specific markers on the surface of tumor cells as the first signal, and then requires co-stimulation such as CD28 as the second signal to effectively activate T cells to kill tumor cells. off. This condition often cannot effectively activate immune T cells, which can easily cause immune escape of tumor cells. CAR-T cell technology combines an antibody that can recognize a certain tumor antigen with the CD3ζ chain or the intracellular part of FcεRIγ to form a fu...

Claims

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Application Information

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IPC IPC(8): C07D475/04A61K31/519A61P35/00
CPCA61P35/00A61K31/519C07D475/04
Inventor 张博张烜李凌君周德敏王妍黄申龙
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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