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ROR1 specific chimeric antigen receptor and application thereof

A chimeric antigen receptor, specific technology, used in applications, antibody mimics/scaffolds, targeted for specific cell fusion, etc., can solve the problems of T cells killing normal B cells, releasing a large number of cytokines and other side effects

Active Publication Date: 2016-09-07
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty of CAR-T cell therapy is that it requires very high specificity of target expression on tumor cells, otherwise it is easy to cause continuous activation of T cells to kill normal B cells, or release a large number of cytokines and cause serious side effects

Method used

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  • ROR1 specific chimeric antigen receptor and application thereof
  • ROR1 specific chimeric antigen receptor and application thereof
  • ROR1 specific chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Construction of hROR1 scFv-hinge-TM-CD8α-CD28-CD3ζ expression plasmid

[0047] 1. Determination of hROR1 scFv-hinge-TM-CD8α-CD28-CD3ζ gene sequence

[0048] From the U.S. National Library of Medicine website (http: / / www.ncbi.nlm.nih.gov / entrez) GenBank database searched human CD8α hinge region and transmembrane region genes, human CD28 transmembrane region and intracellular region genes, Human CD3ζ intracellular region gene sequence information, and anti-human ROR1 scFv gene sequence, and codon optimization to ensure that it is more suitable for human cell expression under the condition that the encoded amino acid sequence remains unchanged.

[0049] Wherein, the nucleotide sequence of the anti-human ROR1 scFv gene is shown in sequence 6 in the sequence listing. The nucleotide sequence of human CD8α hinge region and transmembrane region gene is shown as sequence 7 in the sequence list. The nucleotide sequence of human CD28 transmembrane region and intracell...

Embodiment 2

[0077] Example 2. Preparation and Identification of Chimeric Antigen Receptor hROR1scFv-hinge-TM-CD8α-CD28-CD3ζ Lentiviral Modified T Cells

[0078] 1. Preparation of chimeric antigen receptor hROR1scFv-hinge-TM-CD8α-CD28-CD3ζ lentiviral modified T cells

[0079] 1. The pCDH-CAR plasmid constructed in Example 1 and the packaging plasmids pSPAX2 and pMD2.G were transfected into 293T cells with polyethyleneimine transfection reagent (Sigma Company) at a ratio of 4:2:1. For details, see PEI transfection Reagent instructions. The virus supernatant was collected 72 hours after transfection, centrifuged at 4°C, 3000rpm for 10 minutes, filtered through a 0.45μm filter, and concentrated 10 times after ultracentrifugation at 50000g at 4°C for 3h, and the collected virus concentrate was transferred to - Store at 80°C.

[0080] 2. Preparation of T cells

[0081] Take fresh peripheral blood from healthy donors, and use Ficoll separation solution and human T cell enrichment antibody mix...

Embodiment 3

[0087] Example 3. In vitro tumoricidal activity detection of T cells modified by chimeric antigen receptor hROR1 scFv-hinge-TM-CD8α-CD28-CD3ζ lentivirus

[0088] 1. ROR1 expression level in various tumor cell lines

[0089] Tested tumor cell lines: Raji, K562, K562 / ROR1 (K562 cells transfected with ROR1), MDA-MB-231.

[0090] After each test tumor cell line was cultured separately, 5×10 5 After washing twice with PBS, APC-labeled anti-human ROR1 monoclonal antibody (eBioscience company) was added to the cell suspension of two cells, and labeled APC-isotype (eBioscience company product) was used as the control group, incubated on ice for 30min, and analyzed by flow cytometry. The expression level of ROR1 in various cell lines was detected by technique. The experiment was repeated three times, and the results were averaged.

[0091] The results showed that the percentages of Raji, K562 / ROR1 and MDA-MB-231 cell lines expressing ROR1 were 69%, 90% and 95%, respectively; while K...

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Abstract

The invention discloses an ROR1 specific chimeric antigen receptor and an application thereof. The ROR1 specific chimeric antigen receptor is a protein formed through serially connecting scFv of anti-human ROR1, the hinge region and the transmembrane region of CD8alpha, the transmembrane region and the intracellular region of CD28 and the intracellular region of CD3zeta from the amino end to the carboxyl end. T cells modified with the chimeric antigen receptor have a specific killing effect on tumor cells, and are expected to be in widely used in treatment of all kinds of solid tumor and blood tumors.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, and relates to a ROR1-specific chimeric antigen receptor and its application. Background technique [0002] With the development of tumor immunology theory and technology, adoptive cell immunotherapy has made great progress in recent years, and the achievement of tumor-targeted immunotherapy represented by chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cells is particularly important. Outstanding, in vitro and clinical trials showed good targeting, lethality and persistence, showing great application potential and development prospects. Even so, there are not many tumor-specific target antigens to choose from, the killing effect is not significant, and there are safety issues such as "cytokine storm" and "off-target effects". Therefore, a new generation of broad-spectrum, efficient and safe tumor Targeted immunotherapy is the future development trend of cellul...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K16/2857C07K16/30C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 卢戌刘静维杨照敏邓丽娟刘雪松李京坡黄彩庭
Owner BEIJING BIOHEALTHCARE BIOTECH
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