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Chimeric antigen receptor targeting NK activated receptor

A technology of chimeric antigen receptors and activating receptors, applied in the direction of carriers, fusion polypeptides, anti-infective drugs, etc., can solve CAR-T cell killing, affect CAR-T cell proliferation and survival, and affect CAR-T cell Efficacy and other issues to achieve the effect of improving the treatment effect and enhancing survival

Active Publication Date: 2020-12-04
NANJING BIOHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this inhibition or knockout will activate NK cells in the host, leading to the killing of CAR-T cells by host NK cells, seriously affecting the proliferation and survival of CAR-T cells in vivo, and further affecting the efficacy of CAR-T cells
[0005] Therefore, there is a need to develop a novel chimeric antigen receptor to address the killing of therapeutic CAR cells by NK cells

Method used

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  • Chimeric antigen receptor targeting NK activated receptor
  • Chimeric antigen receptor targeting NK activated receptor
  • Chimeric antigen receptor targeting NK activated receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1. Preparation of CAR T cells containing different scFvs and verification of their functions

[0129] 1.1 Preparation of CAR T cells

[0130] Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein / PlasmidLibrary (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv, CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), 4-1BB intracellular region (SEQ ID NO: 9), CD3ζ intracellular signaling domain (SEQ ID NO: 11), and by sequencing Confirm correct insertion of the target sequence. Wherein, the amino acid sequence of the anti-NKG2D scFv contained in NKG2D-1CAR is shown in SEQ ID NO: 27; the amino acid sequence of the anti-NKG2D scFv contained in NKG2D-2CAR is shown in SEQ ID NO: 29; the anti-NKG2D scFv contained in NKG2D-3CAR The amino acid sequence of the anti-NKG2D scFv contained in NKG2D-4CAR is shown in SEQ ID NO: 31; the amino acid sequence of the anti-NKG...

Embodiment 2

[0137] Example 2: Preparation of CAR T cells and its killing effect on NK92 target cells and release of cytokines

[0138] 2.1 Preparation of CAR T cells

[0139] Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein / PlasmidLibrary (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 17), anti-NKG2D scFv (SEQ ID NO: 27) , CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 3), costimulatory domain, CD3ζ intracellular signaling domain (SEQ ID NO: 11), wherein the costimulatory domain includes CD28 intracellular region (SEQ ID NO: 7), or 4-1BB intracellular region (SEQ ID NO: 9) + DAP10 intracellular region (SEQ ID NO: 45), to obtain 2D28z-CAR and 2Dbb10z-CAR, and pass Sequencing confirms correct insertion of the target sequence. figure 1 The CAR structure constructed in this example is shown.

[0140] The preparation of CAR T cells was completed according to the method described in Example 1, ...

Embodiment 3

[0157] Example 3. Preparation of CAR-T cells in which targeted NK-activating receptors are knocked out

[0158] Knockout endogenous NKG2D in NT, 2Dbbz-CAR T cells, 2D28z-CAR T and 2Dbb10z-CAR T cells by CRISP / Cas9 system, and use PE mouse anti-human NKG2D (biolegend product number 302806) by flow cytometry The expression level of NKG2D in CAR T cells after knockout was detected (Table 1), and the proportion of CD8+ T cells in CAR-T cells was detected with APC-anti human CD8 (BDPharmingen, 555369) ( Figure 8 ). Non-knockout NT cells served as controls.

[0159] Table 1. NKG2D expression levels in CAR-T cells

[0160] gene name 2DbbzKO-CAR 2Dbb10zKO-CAR 2D28zKO-CAR NT NK2D 5.00％ 6.20％ 6.50％ 39.40％

[0161] The results in Table 1 show that NKG2D was effectively knocked out in each CAR-T cell.

[0162] From Figure 8 It can be seen that when the CAR targeting NKG2D is expressed, further knocking out endogenous NKG2D can significantly increase t...

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Abstract

The invention relates to a novel chimeric antigen receptor targeting an NK activated receptor. The chimeric antigen receptor can treat malignant tumors caused by NK cytopathy and can also prevent NK cells in a patient from killing introduced therapeutic CAR cells under the background of universal CAR cells. The invention also relates to an engineered immune cell expressing the novel chimeric antigen receptor, a composition containing the engineered immune cell and application of the engineered immune cell in treatment of diseases.

Description

technical field [0001] The invention belongs to the field of immunotherapy. More specifically, the present invention relates to chimeric antigen receptors targeting NK-activating receptors, engineered immune cells expressing such chimeric antigen receptors, and uses thereof. Background technique [0002] Chimeric antigen receptor (CAR) cell therapy, as a new type of precise targeted therapy targeting tumors, has proved its good effect in tumor treatment, especially in the treatment of hematological tumors in recent years. Its basic principle is to load exogenous chimeric antigen receptors through genetic engineering in immune cells (such as commonly used T cells, NK cells, etc.), and after in vitro expansion, the modified immune cells are reinfused into Human body, so as to kill the targeted tumor cells. Chimeric antigen receptors generally consist of an antigen-binding region, a transmembrane region, and an intracellular signal transduction region. The signal is transmit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K35/17A61P35/00A61P37/02A61P31/00A61P33/00
CPCC07K16/2851C07K16/2803C07K14/70517C07K14/70578C07K14/7051C12N15/86C12N5/0636A61K39/001112A61K39/001111A61K39/001102A61K35/17A61P35/00A61P37/02A61P31/00A61P33/00C07K2317/565C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156
Inventor 周亚丽陈功姜小燕任江涛贺小宏王延宾韩露
Owner NANJING BIOHENG BIOTECH CO LTD
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