Chimeric antigen receptor of target CD19 and application of chimeric antigen receptor

A technology of antigen and single-chain antibody, which is applied in the direction of antibody medical components, antibody mimics/scaffolds, and medical preparations containing active ingredients, etc., which can solve problems such as unsatisfactory curative effect, apoptosis, and short survival period that cannot stimulate anti-tumor effects

Active Publication Date: 2017-12-08
HRAIN BIOTECHNOLOGY CO LTD
View PDF5 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the first-generation CAR can see some specific cytotoxicity, when it was summarized in clinical trials in 2006, it was found that the curative effect was not satisfactory.
The reason is that the first generation of CAR-T cells will soon be exhausted in the patient's body, and their persistence is so poor that the CAR-T cells have not yet had time to contact a large number of tumor cells before they die up
This kind of CAR-T cells can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively small, but their survival period in vivo is short and cannot stimulate long-lasting anti-tumor effects [Zhang T et al., Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Cancer Res 2007, 67(22):11029-11036〕

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Chimeric antigen receptor of target CD19 and application of chimeric antigen receptor
  • Chimeric antigen receptor of target CD19 and application of chimeric antigen receptor
  • Chimeric antigen receptor of target CD19 and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Determination of CD8 Leader Sequence-mCD19scFv-CD8α-CD28-CD3ζ Gene Sequence and Construction of Retroviral Vector

[0101] The gene sequence information of human CD8α hinge region, human CD28 transmembrane region, human CD28 intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-CD19 single-chain antibody clone number is FMC63. These sequences are available on the website Codon optimization is performed on http: / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression without changing the encoded amino acid sequence.

[0102] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD19 scFv, human CD8α hinge region gene, human CD28 transmembrane region gene, human CD28 intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence. sites to form a comp...

Embodiment 2

[0109] Example 2: Retroviral packaging

[0110] The retroviral vector purified and obtained in Example 1 was transfected into 293T cells by the calcium phosphate method to carry out the retroviral packaging experiment, and the specific steps were as follows:

[0111] Day 1: Select 293T cells less than 20 passages and not overly congested, and use 0.6×10 6 Cells / ml were plated, 10ml DMEM medium was added to a 10cm dish, the cells were thoroughly mixed, and cultured overnight at 37 degrees.

[0112] Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18h after plating); prepare the plasmid complex, the amount of various plasmids is: the MSCV backbone vector prepared in Example 1 12.5ug, Gag -pol 10ug, VSVg6.25ug, CaCl 2 250ul,H 2 O 1ml, the total volume is 1.25ml; add HBSS (Hank’s Balanced Salt Buffer) equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to...

Embodiment 3

[0114] Example 3: Retrovirus infection of human T cells

[0115] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate and obtain relatively pure CD3+ T cells, and use X-VIVO (LONZA) medium containing 5% AB serum to adjust the cell density to 1×10 6 / mL. Inoculate cells at 1ml / well onto cell culture plates pre-coated with anti-human 50ng / ml CD3 antibody (Beijing Tongli Haiyuan) and 50ng / ml CD28 antibody (Beijing Tongli Haiyuan), and then add 100IU / ml Interleukin 2 (Beijing Shuanglu), stimulated and cultured for 48 hours before virus infection.

[0116] 2. The next day after T cell activation culture, PBS was diluted to a final concentration of 15 μg / ml, and Retronectin (Takara) was used to coat a non-tissue-treated culture plate, 250 μl per well of a 24-well plate. Protected from light, overnight at 4°C for later use.

[0117] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was discarded, and HBSS c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a chimeric antigen receptor of target CD19 and application of the chimeric antigen receptor. Specifically, the invention provides a fusion protein, and the fusion protein is selected from the followings: (1) a fusion protein containing leader peptide of CD8 antigen, an anti-CD19 single-chain antibody, a human CD8 alpha chain region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3 zeta intracellular region which are connected in sequence; and (2) a (1) derived fusion protein retaining activated T cell activity by replacing, losing or adding one or more amino acids in a (1) limited amino acid sequence. The invention also provides a coding sequence of the fusion protein, a carrier containing the coding sequence, and application of the fusion protein, the coding sequence and the carrier.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to CD19-targeting chimeric antigen receptors and uses thereof. Background technique [0002] Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted manner by MHC after genetic modification, and continuously activate and expand T cells. In 2012, the annual meeting of the International Society for Cell Therapy pointed out that biological immune cell therapy has become the fourth means of tumor treatment besides surgery, radiotherapy, and chemotherapy, and will become a must-have means for future tumor treatment. CAR-T cell reinfusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61P35/02A61K35/17C12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70521C07K16/2803C07K16/30C07K16/3061C12N2510/00C12N2740/10043C12N2740/15043C12N2800/107A61K2039/5158A61K2039/5156C07K2319/74C07K2317/515C07K2317/51C07K2319/00C07K2319/02C07K2319/03C07K2317/622C07K2319/33
Inventor 李启靖金涛何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap