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A kind of chimeric antigen receptor and its application

A chimeric antigen receptor and antigen recognition technology, applied in the direction of antibody medical components, receptors/cell surface antigens/cell surface determinants, hybrid peptides, etc., to prolong the period of complete remission, reduce the probability of occurrence, and enhance immune monitoring effect of ability

Active Publication Date: 2022-06-17
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the widespread application of CAR-T therapy is also encountering a series of difficulties and bottlenecks, among which the immune rejection of CAR molecules mediated by the host immune system, including T cells and antibodies, may limit the persistence and long-term efficacy of CAR-T cells in vivo One of the key obstacles and one of the important factors affecting its application

Method used

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  • A kind of chimeric antigen receptor and its application
  • A kind of chimeric antigen receptor and its application
  • A kind of chimeric antigen receptor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Preparation and identification of hCD19 protein

[0070] Prepare Hcd19 protein according to the following steps, as follows:

[0071] hCD19-fc: Human CD19 recombinant protein (Accession #AAH06338) was expressed in HEK293 cells. The sequence of Pro20-Lys291 in the coding region of human CD19 gene (the C-terminus was tagged with Fc) was selected for transfection. The purified protein was identified by SDS-PAGE gel.

[0072] hCD19-his: Human CD19 recombinant protein (Accession #P15391-1) was expressed in HEK293 cells. The human CD19 gene coding region Pro20-Lys291 sequence (His-tagged at the C-terminus) was selected for transfection. The purified protein was identified by SDS-PAGE gel.

Embodiment 2

[0073] Example 2 Antibody Preparation

[0074] (I) Antigen Conjugation and Immunization

[0075] (1) The hCD19-Fc recombinant protein was immunoconjugated with various MabSpace immune-enhancing peptides, and the conjugated protein was detected by SDS-PAGE gel;

[0076] (2) Freund's complete adjuvant (Pierce, Cat#77140) was added to the above conjugated hCD19-Fc protein at a ratio of 1:1 for emulsification, and then injected into H2L2 mice by subcutaneous and intraperitoneal injection respectively for immunization. H2L2 mice are produced by HarbourBioMed and carry genes for human variable regions and rat constant regions, and do not contain endogenous mouse antibody genes. Additional immunizations were performed using CpG (cytosine-phosphosulfoyl-guanine) and alum to preserve the native structure of the protein. After the first immunization and after the immunization (at least once every 2 weeks), the mouse serum was obtained, and the anti-hCD19 titer in the antiserum was a...

Embodiment 3

[0081] Example 3 Subcloning acquisition of positive hybridoma cells and small-scale antibody production

[0082] (1) positive hybridoma cell subclone

[0083] (1) Carry out gradient dilution of ELISA-positive hybridoma cells on a 96-well plate, and select cells with ideal affinity and blocking activity; after culturing for 7 days, form a cell clone group, collect the supernatant, and use the examples according to the antigen binding ability. 2 to further filter;

[0084] (2) Select the clone with the highest antigen affinity according to the screening result, and expand and cultivate in the hybridoma growth medium. After 7 days, the antigen-binding ability of the cultured supernatant of the hybridoma cells after screening was tested again; the subclone screening test was performed at least twice, until at least 90 wells (96-well plate);

[0085] (3) When more than 90 wells showed a positive binding signal, identify the two clones with the highest antigen-binding activity a...

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Abstract

The present invention relates to a chimeric antigen receptor, its encoding nucleic acid and cells expressing it, as well as its use in the preparation of drugs for treating tumors. The chimeric antigen receptor of the present invention comprises at least one extracellular domain, an optional transmembrane domain and at least one intracellular co-stimulatory signaling domain, wherein the extracellular domain comprises a CD19 antigen recognition and binding structure area. The chimeric antigen receptor of the present invention undergoes humanization transformation, and the survival period in the body is longer, and the complete remission period of the patient can also be extended correspondingly.

Description

technical field [0001] The present invention relates to the technical field of cellular immunotherapy of tumors, in particular, to a chimeric antigen receptor, a nucleic acid encoding the same, cells expressing the same, and use thereof in the preparation of a medicament for treating tumors. Background technique [0002] Chimeric Antigen Receptor (CAR, Chimeric Antigen Receptors) T cells are T cells that express chimeric receptors that recognize specific antigens and transmit signals. CAR T cells play an important role in anti-tumor by expressing chimeric antigen receptor (CAR) molecules. CAR molecules usually include an extracellular segment, a transmembrane region, and an intracellular segment: the extracellular segment is composed of antibody heavy and light chains. A single-chain variable region (ScFv) formed by connecting the variable regions by a peptide segment; the intracellular segment is an intracellular segment chimera of various signaling molecules, including CD3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C07K16/30C07K16/2803C07K2317/622C07K2319/02C07K14/7051C07K2319/03C07K2319/33C12N2510/00C07K2317/56A61K39/001112A61K2039/5156C12N15/86C12N5/0636C12N5/0638C07K2317/24A61K38/00A61K45/06C07K14/70521
Inventor 不公告发明人
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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