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Chimeric antigen receptor and application thereof

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

Active Publication Date: 2019-05-10
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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  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof
  • Chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Preparation and identification of embodiment 1 hCD19 protein

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

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

[0072] hCD19-his: Human CD19 recombinant protein (Accession#P15391-1) expressed in HEK293 cells. The human CD19 gene coding region Pro20-Lys291 sequence (His tag used at the C-terminal) 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) Immunoconjugate the hCD19-Fc recombinant protein with various MabSpace immunoenhancing peptides, and complete the control of the conjugated protein by SDS-PAGE gel detection;

[0076] (2) The above conjugated hCD19-Fc protein was emulsified by adding Freund's complete adjuvant (Pierce, Cat#77140) at a ratio of 1:1, and then injected subcutaneously and intraperitoneally into H2L2 mice for immunization. H2L2 mice, produced by HarbourBioMed, carry genes for human variable regions and rat constant regions, and do not contain endogenous mouse antibody genes. Additional immunizations were performed with CpG (cytosine-phosphoryl-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 analyzed by ELISA method....

Embodiment 3

[0081] Example 3 Positive hybridoma cell subcloning acquisition and small-scale antibody production

[0082] (I) Positive hybridoma cell subcloning

[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 7 days of culture, cell clones are formed, and the supernatant is collected. 2 for further screening;

[0084] (2) Select the clone with the highest antigen affinity based on the screening results, and expand and culture it in the hybridoma growth medium. After 7 days, the antigen-binding ability of the hybridoma cell culture supernatant after screening was detected again; the subcloning screening test was performed at least twice, until at least 90 wells (96-well plate);

[0085] (3) When more than 90 wells showed positive binding signals, two clones with the highest antigen-binding activity were identified and transferred to 24-well plate culture medium and allowed to...

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Abstract

The invention relates to a chimeric antigen receptor, nucleic acid encoding the same, cells expressing the chimeric antigen receptor, and application of the chimeric antigen receptor in preparing drugs for treating tumors. The chimeric antigen receptor comprises at least one extracellular structural domain, an optional membrane spanning structural domain and at least one intracellular co-stimulation signal transduction structural domain, wherein the extracellular structural domains involve CD19 antigen recognition binding structural domains. The chimeric antigen receptor has a longer in-vivo survival period after being subjected to humanized transformation, and the complete remission period of a patient can also be prolonged accordingly.

Description

technical field [0001] The present invention relates to the technical field of tumor cell immunotherapy, in particular to a chimeric antigen receptor, a nucleic acid encoding it, a cell expressing it, and its use in preparing a drug for treating tumors. Background technique [0002] Chimeric antigen receptor (CAR, Chimeric Antigen Receptors) T cells are T cells that express chimeric receptors on the surface that recognize specific antigens and can 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 linking variable regions through a peptide segment; the intracellular segment is a chimera of intracellular segments of various signaling molecules, including CD3zeta, CD28, OX-40, 4...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/85C12N5/10A61K35/17A61P35/00
CPCA61P35/00C07K16/30C07K16/2803C07K2317/622C07K2319/02C07K14/7051C07K2319/03C07K2319/33C12N2510/00C07K2317/56C12N15/86C12N5/0636C12N5/0638C07K2317/24A61K39/4631A61K39/464412A61K39/4611A61K38/00A61K45/06C07K14/70521
Inventor 不公告发明人
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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