Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Bispecific CAR structure targeting CD19 and CD22 and application of bispecific CAR structure

A bispecific and targeted technology, applied in the field of bispecific chimeric antigen receptors and CAR-T cells, can solve the problems that CAR vectors are not easy to transduce T cells, different CAR transduction capabilities, and recurrence, etc. The risk of tumor immune escape, the effect of high CAR transduction efficiency and easy virus preparation

Pending Publication Date: 2021-10-12
CHONGQING PRECISION BIOTECH CO LTD +1
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells. The expressed CAR-T target antigen escapes (Robbie G. Majzner et al. (2018) Tumor AntigenEscape from CAR T-cell Therapy), resulting in poor therapeutic effect and recurrence
Therefore, it is necessary to design a CAR structure targeting multiple targets. Simply expressing two CARs with different targets at the same time involves different CAR structures with different infectivity, and will generate a variety of CAR-T cell subsets, which is not conducive to the clinical application of the product. ; If only two simple CAR structures with different targets are connected in series in one carrier, such a large CAR carrier is not easy to transduce T cells. Different structures involve different CAR carrier virus preparation capabilities and different CAR transduction capabilities. There are many factors such as different stimuli and different curative effects

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
  • Bispecific CAR structure targeting CD19 and CD22 and application of bispecific CAR structure
  • Bispecific CAR structure targeting CD19 and CD22 and application of bispecific CAR structure
  • Bispecific CAR structure targeting CD19 and CD22 and application of bispecific CAR structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Plasmid Construction

[0043] designed as figure 1 The CAR structure shown was verified by double-targeted CAR targeting CD19 and CD22, and the anti-CD19 ScFv light chain or anti-CD22 ScFv light chain was designed respectively, and the nucleic acid sequence was as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The results showed that the fragments were cut and recovered by double enzyme digestion, the gene fragments were ligated, transformed, and single clones were selected, and the obtained vector numbers were 17 and 18, respectively. The CAR structural element comprises: a nucleotide sequence such as SEQ ID NO:3, an amino acid sequence such as SEQ ID NO:11 targeting CD19 ScFv; a nucleotide sequence such as SEQ ID NO:4, and an amino acid sequence such as SEQ ID NO:12 ScFv targeting CD22; nucleotide sequence such as SEQ ID NO:5, amino acid sequence such as the hinge structure of SEQ ID NO:13; nucleotide sequence such as SEQ ID NO:6, amino acid sequence such as the s...

Embodiment 2

[0045] Example 2: Preparation of lentivirus and infection of T lymphocytes

[0046] In this example, the calcium phosphate method was used to package the lentivirus, specifically: 293T cells were cultured to a better state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and expression plasmid Add the scale to a 1.5 centrifuge tube, add CaCl2 and 2× HBS, mix well, let it stand at room temperature, then add it to the treated 293T cell culture medium, and change the medium again to 10mL DMEM containing 10% FBS after 3-5h Culture medium, after 48h or 72h, the cell supernatant was collected, the virus was purified, and the titer was determined.

[0047] Titer table:

[0048]

[0049] The prepared lentivirus was used to infect CHO cells, and the CD19CART, CD22CAR-T, double CART19 and double CART20 infected CHO cells were respectively labeled with CD19-FC, CD22-His (Acro, 2028b-81XF1-KY) flow cytometry labeling reagents and detected The expressi...

Embodiment 3

[0052] Example 3: Target cell preparation and target antigen detection

[0053] Using K562-Luc as a model cell to construct target cells with high exogenous expression of CD19, CD22, and co-expression of CD19 and CD22, and use anti-CD19 antibody and anti-CD22 antibody (Acro, 2028b-81XF1-KY) to detect the expression of antigens on the surface of target cells . see results image 3 Shown: K562-CD19 is only CD19 positive cells, K562-CD22 is only CD22 positive cells, K562-CD19-CD22 is CD19 and CD22 double positive cells.

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 belongs to the technical field of immunotherapy, and particularly relates to a bispecific chimeric antigen receptor capable of recognizing CD19 and CD22 tumor associated antigens, an expression vector and CAR-T cell of the chimeric antigen receptor, and application of the bispecific chimeric antigen receptor, expression vector and CAR-T cell. The bispecific chimeric antigen receptor can recognize double targets of CD19 and CD22, not only can be effectively expressed in T lymphocytes, but also can reduce the probability of tumor immune escape and reduce the tumor recurrence rate after CAR-T treatment.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy, and specifically relates to a bispecific chimeric antigen receptor, a carrier comprising a bispecific chimeric antigen receptor, a CAR-T cell expressing a bispecific chimeric antigen receptor and its application. Background technique [0002] CAR-T (Chimeric Antigen Receptor T) cell therapy has achieved remarkable results in the treatment of hematological malignancies. However, due to the heterogeneity of tumors, especially solid tumors, more than one target antigen is often expressed on the surface of tumor cells, and with the progress of research, researchers have also found that a small number of patients treated with CAR-T cells have down-regulated or mutated tumor cells. The expressed CAR-T target antigen escapes (Robbie G. Majzner et al. (2018) Tumor AntigenEscape from CAR T-cell Therapy), resulting in poor therapeutic effect and recurrence. Therefore, it is necessary to design a CA...

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
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2803C07K14/7051C12N15/86C12N5/0636A61K39/001112A61K39/001113A61P35/00C07K2317/31C07K2317/622C07K2319/02C07K2319/03C12N2740/15043A61K2039/5158
Inventor 陈雪娇单娟娟黄霞徐艳敏赵文旭陈军沈俊杰张巍齐亚男赵永春
Owner CHONGQING PRECISION BIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com