CAR-T cell of autocrine CD40 antibody and targeted ErbB receptor family

A chimeric antigen receptor, cell technology, applied in the field of CAR-T cells

Active Publication Date: 2019-07-05
SHANGHAI CELL THERAPY RES INST +1
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, T1E cannot bind ErbB2 or ErbB3 alone, and is still defective in targeting the ErbB receptor family

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
  • CAR-T cell of autocrine CD40 antibody and targeted ErbB receptor family
  • CAR-T cell of autocrine CD40 antibody and targeted ErbB receptor family
  • CAR-T cell of autocrine CD40 antibody and targeted ErbB receptor family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1: Construction of recombinant plasmids pS328-αCD40-wt, pS328-αCD40 and pNB328-EHCAR-EK-28TIZ

[0111] 1. Entrust Shanghai Jierui Biological Company to synthesize EHCAR-EK-28TIZ gene (nucleotide sequence shown in SEQ ID NO: 6, amino acid sequence shown in SEQ ID NO: 5), anti-CD40 gene (nucleotide sequence As shown in SEQ ID NO: 2, amino acid sequence as shown in SEQ ID NO: 1) and anti-CD40-wt gene (nucleotide sequence as shown in SEQ ID NO: 4, amino acid sequence as shown in SEQ ID NO: 3 shown), the structure pattern is as figure 1 shown. Each gene was loaded into the pNB328 and pS328 vectors which were double digested with EcoR1+SalI (see CN 201510638974.7 for the structure and sequence of pNB328, which is incorporated herein by reference in its entirety; compared with pNB328, pS328 lacks PB transposition subsequence, and other elements are the same as the pNB328 vector), construct plasmids, respectively named as pNB328-EHCAR-EK-28TIZ, pS328-αCD40 and pS328-α...

Embodiment 2

[0113] Example 2: Determination of positive rate and antibody expression of chimeric antigen receptor modified T cells constructed by pNB328-EHCAR-EK-28TIZ and pS328-αCD40 plasmids with different mass ratios

[0114] Set the amount of pNB328-EHCAR-EK-28TIZ and pS328-αCD40 plasmids to 7 ratios: 1ug+7ug, 2ug+6ug, 3ug+5ug, 4ug+4ug, 5ug+3ug, 6ug+2ug, 7ug+1ug , Car T cell construction. The build method is as follows:

[0115] Peripheral blood mononuclear cells (PBMCs) were isolated from Shanghai Cell Therapy Production Center. Cultivate PBMCs for 2-4 hours. The unattached suspension cells are initial T cells. Collect the suspension cells into a 15ml centrifuge tube, centrifuge at 1200rmp for 3min, discard the supernatant, add physiological saline, centrifuge at 1200rmp for 3min, discard the physiological saline, and repeat this step; take eight 1.5ml centrifuge tubes and add 5×10 6 Cells, numbered a, b, c, d, e, f, g and h, centrifuged at 1200rmp for 3min, discarded the supernat...

Embodiment 3

[0133] Example 3: Construction of EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-αCD40T cells and determination of positive rate and antibody expression

[0134] Take 6ug pNB328-EHCAR-EK-28TIZ plasmid for the construction of EHCAR-EK-28TIZ T cells, respectively take 5ug pNB328-EHCAR-EK-28TIZ and 3ug pS328-αCD40 plasmids for the construction of EHCAR-EK-28TIZ-αCD40T cells, construction method With embodiment 2.

[0135] The positive rate of the Mock-T cells in Example 2 and the EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-αCD40T cells constructed in this example was detected by flow cytometry, and the method was the same as in Example 2. see results Figure 3A , Self-expression of CD40 antibody does not reduce the positive rate of CART cells.

[0136] ELISA detection of Mock-T cells, EHCAR-EK-28TIZ T cells and EHCAR-EK-28TIZ-αCD40T cell antibody expression, the method is the same as in Example 2, see the results Figure 3B .

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 provides a chimeric antigen receptor T cell of self-expression CD40 activated antibody and targeted ErbB receptor family and application thereof. The T cell contains a coding sequence ofa chimeric antigen receptor for expressing the targeted ErbB receptor family and a coding sequence of a CD40 activated antibody; and/or a chimeric antigen receptor and CD40 activated antibody expressing the targeted ErbB receptor family. The T cell has better reproduction activation performance and tumor cell killing function than those of a chimeric antigen receptor T cell of single targeted ErbB receptor family.

Description

technical field [0001] The invention belongs to genetic engineering and immunology, and relates to CAR-T cells that secrete CD40 antibodies and target the ErbB receptor family and uses thereof. Background technique [0002] Cancer has now become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices to the occurrence of cancer, making the high incidence and younger trend of cancer more and more obvious. The current commonly used treatment methods are very limited in effect, and a more effective treatment method still needs to be explored to improve the survival rate and quality of life of cancer patients. [0003] As one of the important branches of tumor immunotherapy, chimeric antigen receptor T cell (CAR-T) therapy has achieved very good results in malignant hematological tumors, and the complete remission rate for relapsed and refractory B-cell leukemia exceeds 90%. %. Chimeric...

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): C12N5/10C12N15/13C12N15/62C07K19/00C07K16/28A61K39/395A61P35/00
CPCC07K16/2863C07K16/2878C07K14/7051A61K35/17C07K2319/33C07K2319/02C07K2317/622C07K2317/73A61K2039/505A61K39/395A61P35/00C07K16/28C07K19/00C12N5/10C12N15/62C12N15/85
Inventor 钱其军金华君游术梅江芏青李林芳王超崔连振
Owner SHANGHAI CELL THERAPY RES INST
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