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Transgenic lymphocyte co-expressing anti-EGFRvIII chimeric antigen receptor and nonfunctional EGFR receptor, and uses thereof

A chimeric antigen receptor, lymphocyte technology, applied in the field of biomedicine

Inactive Publication Date: 2017-07-21
BEIJING MARINO BIOTECH PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the most aggressive treatment was used before mothers, the median survival time is still only <15 months, and less than 1% of patients can survive for 5 years with radiotherapy alone, even if the emerging radiotherapy combined with TMZ chemotherapy The combined regimens only make nearly 10% of patients survive for more than 5 years

Method used

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  • Transgenic lymphocyte co-expressing anti-EGFRvIII chimeric antigen receptor and nonfunctional EGFR receptor, and uses thereof
  • Transgenic lymphocyte co-expressing anti-EGFRvIII chimeric antigen receptor and nonfunctional EGFR receptor, and uses thereof
  • Transgenic lymphocyte co-expressing anti-EGFRvIII chimeric antigen receptor and nonfunctional EGFR receptor, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0089] Generation of lentivirus and transduction of human T lymphocytes

[0090]Replication-defective lentiviral vectors were generated and collected by centrifugation for transduction of human T lymphocytes. The following is a brief introduction to the production and collection of lentiviral vectors: 293T cells were placed in a cell culture dish with a bottom area of ​​150-cm2, and Express-In (purchased from Open Biosystems / Thermo Scientific, Waltham , MA) for viral transduction of 293T cells. Add 15 μg of lentiviral transgenic plasmid, 5 μg of pVSV-G (VSV glycoprotein expression plasmid), 10 μg of pCMVR8.74 plasmid (Gag / Pol / Tat / Rev expression plasmid) and 174 μl of Express-In (concentration of 1 μg / μl). The supernatants were collected at 24 hours and 48 hours respectively, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (centrifuge roto...

Embodiment 2

[0095] Example 2 Construction of vectors co-expressing non-functional EGFR receptors and anti-EGFRvIII chimeric antigen receptors

[0096] In this example, the inventors cloned the sequence encoding the single-chain antibody against human EGFRvIII, the ζ-chain sequence of the 4-1BB intracellular segment and the T cell receptor combination into a lentiviral vector containing the EF-1 promoter ( lentiviral vector), during the cloning process, the selected restriction enzymes were XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion. Lentiviral plasmid with antigen receptor complex (LV-EGFRvIIICAR). The sequence containing the synthetic IRES and expressing the non-functional EGFR receptor was cloned into the LV-EGFRvIII CAR vector plasmid to construct LV-EGFRvIII CAR / tEGFR. figure 1 is a schematic diagram of a lentiviral vector, including a sequence encoding an anti-EGFRvIII chimeric antigen receptor, an IRES, and a sequence encoding a non-functional ...

Embodiment 3

[0097] Example 3 Anti-EGFR Antibodies Can Effectively Kill and Eliminate T Lymphocytes Co-expressing Non-functional EGFR Receptor and Anti-EGFRvIII Chimeric Antigen Receptor

[0098] In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) in 5% CO 2 , Incubated at 37 degrees Celsius for 72 hours, the medium was added with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100U / ml of penicillin / chain RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012) with antimycin double antibody. After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on cell culture dishes covered with recombinan...

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Abstract

The present invention provides a transgenic lymphocyte, a construction product and a cancer treatment composition. According to the present invention, the transgenic lymphocyte expresses a nonfunctional EGFR receptor and a chimeric antigen receptor, can directionally kill tumor cells, and especially provides significant directional killing effect on glioblastoma having EGFRvIII mutation.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer and a lymphatic Approaches to cell therapy safety. Background technique [0002] Glioblastoma is the most malignant brain tumor, accounting for ~81% of malignant tumors in the central nervous system. The incidence of malignant glioma is 3-6 / 100,000, and the number of deaths from this disease in China is 30,000 each year. . Malignant gliomas grow infiltratingly, without obvious boundaries with normal brain tissue, and most of them are not limited to one lobe of the brain, and they destroy the brain tissue deeply in the shape of fingers, and it is impossible to completely remove it by surgery. Although the most aggressive treatment was used before mothers, the median survival time is still only <15 months, and less than 1% of patien...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N7/01C12N15/63A61K35/17A61K39/395A61P35/00
CPCC07K14/70521C07K14/70578C07K14/70596C07K14/71C07K16/2863C12N7/00C07K2319/00C07K2317/622C07K2317/56C12N2740/15021A61K39/4611A61K39/4631A61K39/464404A61K31/7088A61K35/76C12N5/10C12N15/63
Inventor 严勇朝朱益林陈思毅
Owner BEIJING MARINO BIOTECH PTY LTD
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