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Chimeric receptor and application thereof

A chimeric receptor and receptor technology, applied in the field of biomedicine, can solve the problems of weakening antibody-dependent cell-mediated cytotoxicity and low affinity, and achieve the effect of enhancing ADCC effect, high affinity, and avoiding functional impairment.

Pending Publication Date: 2021-11-02
SHANGHAI SINOBAY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Studies have found that there are genetic polymorphisms in CD16 molecules, and the affinity of wild-type CD16 molecules is usually low, which weakens antibody-dependent cell-mediated cytotoxicity

Method used

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  • Chimeric receptor and application thereof
  • Chimeric receptor and application thereof
  • Chimeric receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] The wild-type CD16.BBz, F176V mutant CD16.BBz, S197P mutant CD16.BBz and hrCD16.BBz genes (respectively shown in SEQ ID NO: 13-16) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., and cloned into blank slow Virus expression plasmids (pXW-EF1α-MCS-P2A-EGFP) were obtained pXW-EF1α-CD16.BBz-P2A-EGFP, pXW-EF1α-CD16F176V.BBz-P2A-EGFP, pXW-EF1α-CD16S197P.BBz-P2A- EGFP and pXW-EF1α-hrCD16.BBz-P2A-EGFP recombinant lentiviral expression plasmid, the plasmid map is as follows figure 1 shown.

Embodiment 2

[0115] Example 2 Lentiviral expression plasmid transfection of HEK 293T cells

[0116] The day before the experiment, seed HEK293T cells on a 12-well flat-bottomed cell culture plate, 4×10 5cells / 2mL / well. On the next day, the pXW-CD16.BBz and XW-hrCD16.BBz lentiviral expression plasmids were transfected with TurboFect transfection reagent, and the total amount of plasmids was 2 μg / well. Add 4 μL of TurboFect transfection reagent at a ratio of plasmid amount (μg): transfection reagent (μL) = 1:2, and add the freshly prepared plasmid transfection complex to the above cell culture plate after incubating at room temperature for 15-20 min. Place at 37°C, 5% CO 2 Continue to culture under the conditions for 48 hours, centrifuge at 500×g at room temperature for 5 minutes, discard the supernatant, and collect the cells for later use.

Embodiment 3

[0117] Example 3 hrCD16 chimeric receptor efficiently binds antibody Fc fragment

[0118] HEK293T cells expressing wild-type CD16 chimeric receptor (CD16.BBz) and hrCD16 chimeric receptor (hrCD16.BBz) were respectively prepared by the method described in Example 2.

[0119] Take 1×10 respectively 5 A wild-type CD16 chimeric receptor and hrCD16 chimeric receptor-modified HEK293T cells were placed in a 1.5mL EP tube, and EGFR-targeting antibody labeled with fluorescent dye AF647 was added, and the working concentration of the antibody was adjusted to 0.5, 1, and 5 μg / mL , incubated at 4° C. in the dark for 30 min, and then eluted twice with FACS buffer (1×PBS containing 2% FBS), resuspended in 200-300 μL of FACS buffer and detected by flow cytometry.

[0120] The result is as follows: figure 2 A is the flow diagram of HEK293T cells expressing wild-type CD16 chimeric receptor and hrCD16 chimeric receptor bound to the Fc fragment of EGFR-targeting antibody respectively. At t...

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Abstract

The invention provides a chimeric receptor. The chimeric receptor comprises (1) an extracellular recognition domain of an Fc fragment; (2) an extracellular spacer region; (3) a transmembrane area; (4) an intracellular signaling domain; optionally, the chimeric receptor further comprises (5) one or more co-stimulatory signaling domains and / or (6) one or more cytokine receptor signaling domains. The invention also provides an immune cell expressing the chimeric receptor. The invention also provides a combination comprising the immune cell and a tumor antigen targeting antibody or a viral antigen targeting antibody. The brand-new Fc fragment high-affinity chimeric receptor provided by the invention has higher antibody Fc fragment affinity, and can remarkably enhance ADCC effect when being used in combination with a tumor antigen targeting antibody or a virus antigen targeting antibody.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. In particular, the invention relates to a chimeric receptor. The present invention also relates to said chimeric receptor and its use. Background technique [0002] 2013 was an important turning point in cancer treatment. Cancer immunotherapy based on reversing the body's tumor immunosuppressive microenvironment won the top ten scientific breakthroughs in 2013 published by Science. Subsequently, Chimeric antigen receptor T-cell immunotherapy (CAR-T) has shown remarkable efficacy in the treatment of hematological malignancies. CAR-T cell therapy uses viral or non-viral exogenous gene delivery technology to express fusion proteins that recognize tumor-specific / associated antigen recognition domains and T cell activation-related sequences on the surface of T cells, making them antigen-dependent but not MHC-dependent The restricted method specifically binds to tumor antigens, initiates activat...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C07K16/283C12N5/0636A61P35/00C07K2319/30C07K2319/02C07K2319/03C12N2510/00A61K39/4611A61K2239/38A61K2239/55A61K39/4631A61K2239/31A61K39/464838A61K39/464406A61K39/464411C07K19/00C12N5/10C12N15/62C12N15/85
Inventor 徐建青张晓燕廖启彬丁相卿
Owner SHANGHAI SINOBAY BIOTECH CO LTD
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