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Chimeric antigen recipient cell targeting human CD33 and NKG2DL as well as preparation method and application of chimeric antigen recipient cell

A chimeric antigen receptor, targeted binding technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low killing efficiency and low tumor cell specificity. , to achieve the effect of improving killing efficiency, high killing rate and simple steps

Active Publication Date: 2022-07-05
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the above-mentioned problems and / or other problems of related technologies, the purpose of the present invention is to overcome the effector cell killing in the intra-tumor environment faced in the existing AML clinical technology The problem of low specificity and low killing efficiency of tumor cells, CD33 is widely expressed in AML, but targeting CD33 and NKG2DL surface antigens at the same time may bring two obvious benefits

Method used

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  • Chimeric antigen recipient cell targeting human CD33 and NKG2DL as well as preparation method and application of chimeric antigen recipient cell
  • Chimeric antigen recipient cell targeting human CD33 and NKG2DL as well as preparation method and application of chimeric antigen recipient cell
  • Chimeric antigen recipient cell targeting human CD33 and NKG2DL as well as preparation method and application of chimeric antigen recipient cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1 Expression plasmid of bispecific chimeric antigen receptor targeting human CD33 and NKG2DL

[0126] The overall design is as follows:

[0127] 1. Determination of the amino acid sequence of the bispecific chimeric antigen receptor targeting CD33 and NKG2DL

[0128] First, the full-length amino acid sequence (NP_031386.2) of human NKG2D was searched from the Genbank database of the National Library of Medicine (NCBI), and the full-length amino acid sequence number of CD33 was: (NP_001076087). CD33 uses nanobodies. Compared with ordinary antibodies, nanobodies have small molecular weight, simple structure, easy genetic modification, small size, good antigen specificity, strong tissue penetration, and high stability. Broad application prospects. Camelids are found to have a single heavy chain variable domain (VHH) with high affinity for antigen without the help of a light chain. The VHH was constructed on the lenti-hIgG1-Fc2 eukaryotic expression vector, and t...

Embodiment 2

[0150] Example 2: Preparation of viral liquid of lentiviral vector

[0151] The recombinant plasmid (KD-347 expression plasmid) of the bispecific chimeric antigen receptor targeting human CD33 and NKG2DL obtained in Example 1 and the packaging vectors pol / gag, Rev and VSVG were used according to 12:10:5:6 ratio with Lipofectamine TM 6000 transfection reagent (purchased from Biyuntian, product model C0526) was co-transfected into 293T cells (see the transfection instructions for the specific transfection procedure), and replaced with complete medium (purchased from hyclone) 4-6 hours after transfection Company, product model SH30243.01), after 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain bispecific targeting human CD33. and KD-347 virus fluid of lentiviral vector of chimeric antigen receptor of NKG2DL (hereinafter referred to as KD-...

Embodiment 3

[0152] Example 3: Isolation and culture of T cells

[0153] Take fresh peripheral blood from healthy donors, and separate fresh peripheral blood mononuclear cells by density gradient centrifugation; then use paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, Product Information for Human T-Activator CD3 / CD28) to enrich CD3+ T cells, specifically, peripheral blood mononuclear cells were diluted to a concentration of (10-30) × 10 6 A single cell / ml, then the magnetic beads and cells were mixed in a petri dish at a ratio of 3:1, and incubated for 2-3 hours at room temperature. A magnetic particle collector (MPC for short, purchased from Invitrogen, USA) was used. Company) enriched for CD3+ T cells. Finally, the enriched CD3+ T cells were resuspended in the medium (purchased from Life Technologies, USA, product information is OpTmizer TM T-Cell Expansion SFM), adjust the cell concentration to 1 × 10 6 pcs / ml, finally at ...

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Abstract

The invention belongs to the field of chimeric antigen recipient cells, and relates to a chimeric antigen recipient cell targeting human CD33 and NKG2DL as well as a preparation method and application thereof, and the chimeric antigen recipient cell contains amino acid sequences targeting binding human CD33 and targeting binding human NKG2DL. The immune response cell modified by the bispecific chimeric antigen receptor targeting CD33 and NKG2DL can enhance the combination with tumor cells, and has obvious anti-tumor activity in tumors expressing human CD33 / NKG2DL antigens, such as acute myelogenous leukemia (AML).

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptor cells, and relates to an amino acid coding sequence of a bispecific chimeric antigen receptor targeting human CD33 and NKG2DL, a modified immune response cell thereof, and a preparation method thereof and application in pharmaceutical preparation. Background technique [0002] With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune-junction blockade therapy. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become a star that research institutions and pharmaceutical companies are vying for. [0003] Acute Myeloid Leukemia (AML) is a clonal hematopoietic stem cell disease with a high incidence. Abnormal blasts in the bone marrow proliferate during the onset, not only inhibiting the no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P31/04A61P31/12A61P35/00A61P35/02A61P37/00
CPCC07K16/2803C07K16/30C12N15/86C12N5/0636C12N5/0646A61K39/001102A61K39/001111A61P35/00A61P31/04A61P31/12A61P37/00A61P35/02C07K2319/02C07K2319/03C12N2740/10043C12N2510/00A61K2039/5156
Inventor 代红久徐慧朱靓婧刘霜
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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