Chimeric antigen receptor containing truncated CD20 molecules and lentiviral vector as well as application

A technology of chimeric antigen receptor and lentivirus, which is applied in the field of chimeric antigen receptor and tumor immunotherapy, can solve the problems of side effects, cells and toxicity, and achieve the effect of ensuring safety

Active Publication Date: 2018-07-13
山东省齐鲁细胞治疗工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Coexisting with the effectiveness of CAR-T therapy are its side effects and cytotoxicity

Method used

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  • Chimeric antigen receptor containing truncated CD20 molecules and lentiviral vector as well as application
  • Chimeric antigen receptor containing truncated CD20 molecules and lentiviral vector as well as application
  • Chimeric antigen receptor containing truncated CD20 molecules and lentiviral vector as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Preparation of Lv-anti-HER-2CAR lentiviral expression vector

[0065] Such as figure 1 As shown in A, the CD8 transmembrane signal peptide, anti-HER-2Scfv, CD8 transmembrane region, 4-1BB co-stimulatory signal region, and CD3Zeta TCR activation region were sequentially cloned into the lentiviral backbone plasmid pHR to obtain the following figure 2 The pHR-antiHER-2CAR plasmid containing the tCD2 tag shown in A (the nucleotide sequence of the plasmid is shown in SEQ ID NO.1, which expresses a chimeric antigen receptor comprising a truncated CD20 molecule, and its amino acid sequence is shown in SEQ ID NO.1 shown in ID NO.2). Such as figure 1 As shown in B, the CD8 transmembrane signal peptide, myc tag, anti-HER-2Scfv, CD8 transmembrane region, 4-1BB co-stimulatory signal region, and CD3Zeta TCR activation region were sequentially cloned into the lentiviral backbone plasmid pHR to obtain the following: figure 2 The pHR-antiHER-2 CAR plasmid containing myc ...

Embodiment 2

[0067] Example 2 Determination of virus titer

[0068] A cell infection method: 1 × 10 per well in 24 wells 5 For each K562 cell, take the concentrated virus and dilute it 10 times, add 1 μL, 3 μL, 10 μL, 30 μL virus respectively, and store at 37°C in 5% CO 2 After culturing for 48 hours, 200 μL of cell liquid was taken from each well for flow cytometric detection. Add 1 μL myc-tag primary antibody (CST, cat: 2276) to each sample, incubate at room temperature in the dark for 15 minutes, add 2 μL PEanti-mouse Ig light chain lambda (Biolegend, cat: 407308) secondary antibody to each sample, and incubate at room temperature in the dark for 15 minutes , centrifuged at 400 g for 5 min, and the pellet was resuspended in 200 μL of PBS and tested on a machine (Millipore guava easyCyte HT). The result is as image 3 As shown in A, as the amount of virus added increases, the proportion of positive cells gradually increases. When the positive rate is less than or equal to 10%, the nu...

Embodiment 3

[0070] Example 3 Preparation of CAR-T cells

[0071] Take 50 mL of fresh blood, and conduct density gradient centrifugation with lymphocyte separation medium (Tianjin Haoyang) to separate mononuclear cells. Divide mononuclear cells into 1-2 x 10 6 / mL resuspended into X-VIVO 15 medium (Lonza), and at the same time add CD3 monoclonal antibody (Ebioscience, cat: 160037) 50ng / mL and CD28 monoclonal antibody (Novoprotein, cat: GMP-A063) 50ng / mL to activate T Lymphocytes, 37°C 5% CO 2 Incubate for 48 hours.

[0072] Take 2×10 6 Dilute the aliquoted virus with the same medium according to MOI=5, add IL-2 (Quangang) and 4ug / mL polybrene (Sigma) at a final concentration of 500U / mL at the same time, mix well, and store at 37°C in 5% CO 2 Cultivate for 6-8 hours, centrifuge at 300g for 5min to change the medium into fresh X-VIVO 15 medium (containing 500U / mL IL-2).

[0073] Add fresh X-VIVO 15 medium (containing 500U / mL IL-2) every 2-3 days to maintain the cell density at 1×10 6 / ...

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Abstract

The invention discloses a chimeric antigen receptor containing truncated CD20 molecules. The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 2, and the nucleotide sequenceencoding the chimeric antigen receptor is shown in SEQ ID NO. 1; the chimeric antigen receptor comprises leader peptides, ScFv, a hinge region and a transmembrane region, immunoreceptor tyrosine activation motifs, an internal ribosome entry site (IRES) and tCD20. The invention also discloses a lentiviral vector, which comprises the nucleotide sequence encoding the chimeric antigen receptor. The invention further discloses immune cells capable of expressing the chimeric antigen receptor. The chimeric antigen receptor containing the truncated CD20 molecules can effectively kill antigen-positivetumor cells and does not have toxic and side effects on the negative cells after being expressed in the immune cells; the tCD20 carried by the chimeric antigen receptor not only can be used as tag peptide for detecting CAR expression, but also can be fed with a scavenging antibody such as rituximab after tumor cells are killed, therefore, CAR-T cells are removed, and the clinical use safety is guaranteed.

Description

technical field [0001] The invention relates to a chimeric antigen receptor comprising a truncated CD20 molecule, a lentiviral vector and its application, and belongs to the tumor immunotherapy technology in the field of genetic engineering. Background technique [0002] According to WHO statistics, cancer has become the second largest killer threatening human health. Statistics show that there will be 14.068 million new cases worldwide each year, and 8.201 million deaths from cancer each year. In the case that surgery, chemotherapy, radiotherapy and other techniques cannot achieve good therapeutic effects, people are gradually focusing on the field of biological therapy. [0003] Cell therapy is an important branch in the field of biological therapy. Chimeric antigen receptors (chimeric antigenreceptors, CAR) T cells are considered to be the rising stars in the field of cell therapy, because of their outstanding performance in the treatment of acute leukemia and non-Hodgki...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70578C07K14/70596C07K16/32C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 谭毅梁晨
Owner 山东省齐鲁细胞治疗工程技术有限公司
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