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Targeting chimeric antigen receptor modified immune cell as well as preparation method and application thereof

A technology of chimeric antigen receptors and immune cells, which is applied to genetically modified cells, cells modified by introducing foreign genetic material, and targeted specific cell fusion. High cost and other issues, to achieve the effect of expanding the target range, lasting tumor killing effect, and low cost

Inactive Publication Date: 2016-08-31
WUHAN HAMILTON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for better precision on specific areas within an organism's own tissue or blood vessels through introducing two types of molecules called chaperones (Carpons) into them. These molecules help keep these parts from being overexpressed during inflammatory responses when they fight against foreign substances like bacteria. By adding genes involved with this mechanism, it becomes possible to make sure there will be enough healthy lymphocytes at risk of relapse if needed without affecting their ability to attack normal ones. Overall, this innovative approach provides greater accuracy than current methods but also reduces costs compared to traditional treatments.

Problems solved by technology

Technological Problem addressed in this patents includes improving the selective induction of specific types of immune responses against tumour cells while minimizing negative impacts caused by non-targeted agents like drugs used in treatments. Additionally, reducing the complexity involved in preparation processes needed for effective use of CART cells without compromising their efficacy when applied to other diseases also needs improvement.

Method used

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  • Targeting chimeric antigen receptor modified immune cell as well as preparation method and application thereof
  • Targeting chimeric antigen receptor modified immune cell as well as preparation method and application thereof
  • Targeting chimeric antigen receptor modified immune cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Preparation of T cells

[0074] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by using lymphocyte separation medium and density gradient centrifugation.

[0075] (2) PBMCs were washed three times, and the final concentration of cells was adjusted to 2×10 by T cell culture medium (X-VIVO 15 serum-free medium) containing 5% autologous serum. 6 cell / mL; the cells were inoculated in a 75 cm cell culture flask coated with a final concentration of 5 ug / mL LCD3 monoclonal antibody and a final concentration of 10 ug / mL of retronectin (purchased from TAKORA). Add recombinant human protein interferon-γ with a final concentration of 1000U / mL and recombinant human interleukin-2 at a final concentration of 1000U / mL to the culture medium, and store at 37°C and saturated humidity of 5% CO 2 cultured in an incubator.

[0076] (3) On the fourth day, 100 ml of T cell culture medium containing 0.6% autologous serum by...

Embodiment 2

[0077] Example 2: Construction of chimeric antigen receptor (pCDH-mCD19ScFv-CD28-41BB-CD3ζ) lentiviral expression vector

[0078] (1) Human CD8α signal peptide gene, human CD8α hinge region gene, and human CD28 transmembrane gene were searched from the GennBank database of the National Library of Medicine (htp: / / www.ncbi.nlm.nih.gov / entroz) region and intracellular region genes, human 41BB intracellular region and human CD3ζ intracellular region gene sequences. The mouse-derived anti-CD19 single-chain antibody (anti-CD19ScFv) gene sequence comes from a patent (patent number: ZL200610015650.9), and its codons are optimized to ensure that it is more suitable for human cell expression without changing the encoded amino acid sequence. The DNA sequence of CD19scFV is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4; the DNA sequence of CD8 is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6; the DNA sequence of CD28 ...

Embodiment 3

[0106] Example 3 Preparation of Chimeric Antigen Receptor pCDH-CD19ScFv-CD28-41BB-CD3ζ Modified T Cells

[0107] (1) Packaging and concentration of lentivirus

[0108] The lentiviral expression plasmid pCDH-CD19scFv-CD28-41BB-CD3ζ and the helper plasmids pCMV-G, pRSV-Rev and pMDLG-pRRE were respectively extracted with the plasmid extraction kit to measure the concentration of the plasmids. The four plasmids were divided into 3:1: 293T cells were co-transfected with calcium phosphate transfection reagent at a mass ratio of 2:2. Collect the virus supernatant in 15ml tubes at 72 hours after transfection, centrifuge at 500G for 10min at 4°C, transfer the supernatant to a new 15ml tube, filter the virus supernatant with a 0.45um filter; filter the virus supernatant with 5×PEG6000- Mix NaCI according to the volume ratio of 4:1, shake it at 4°C overnight, then centrifuge at 10,000G for 60 minutes at 4°C, discard the supernatant, and dissolve the precipitate in X-VIVO 15 serum-free m...

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Abstract

The invention provides a targeting chimeric antigen receptor. The chimeric antigen receptor is formed by serially connecting a CD8 alpha signal peptide, CD19ScFv, a CD8 alpha hinge region, a CD28 transmembrane domain and an intracellular signal structural domain, an intracellular signal structural domain of 41BB and an intracellular signal structural domain of CD3 zeta,. The invention further provides a targeting chimeric antigen receptor modified immune cell and a preparation method thereof, as well as application of the targeting chimeric antigen receptor modified immune cell in preparation of antitumor related drugs. A gene sequence for promoting proliferation and activation of T cells is added in a targeting chimeric antigen receptor structure, so that the condition that the T cells can proliferate after entering a human body can be guaranteed, and a more lasting tumor killing effect, more accurate treatment, more accurate targeting and wider tumor killing range are realized; meanwhile, the targeting chimeric antigen receptor modified immune cell is lower in cost and easy to popularize.

Description

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Claims

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Application Information

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Owner WUHAN HAMILTON BIOTECH
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