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A preparation method of CTL targeting multiple epitopes of glioblastoma

An antigenic epitope and glioma technology, applied in the field of biotechnology development and application research, can solve the problems of not being able to effectively kill tumor cells, the effect is unsatisfactory, and tumor cell immune escape

Active Publication Date: 2021-09-17
上海尚泰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the current adoptive CTL therapy for glioma, most of the immune attacks are not enough to effectively kill tumor cells, but will induce immune escape of tumor cells, and the effect is still unsatisfactory.

Method used

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  • A preparation method of CTL targeting multiple epitopes of glioblastoma
  • A preparation method of CTL targeting multiple epitopes of glioblastoma
  • A preparation method of CTL targeting multiple epitopes of glioblastoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1: Construction of lentiviral expression vector containing coding antigen sequence

[0134] Construction of lentiviral expression vector: The schematic diagram of the construction of the lentiviral expression vector carrying the antigen coding sequence is attached figure 1 As shown, first by adopting the Linker sequence (as shown in SEQ ID NO: 1), the epitope sequences of 3 GAA (EGFR-VIII, CD133, CMV-pp65) are connected in series (preferably the series combination sequence is CMV-pp65— EGFR-VIII-CD133), the start and stop codons were added at the beginning and end of the tandem sequence, respectively, to create a new artificial antigen coding sequence (as shown in SEQ ID NO: 2; referred to as PEC), the sequence size was 858bp; then the The BshT1 and Mlu1 restriction enzyme cleavage site sequences were added to both ends of the new artificial antigen coding sequence and gene synthesis was performed; finally, the synthesized antigen coding sequence fragments and t...

Embodiment 2

[0135] Example 2: Packaging, concentration and titer detection of lentiviral particles

[0136] 1) Virus packaging and concentration: P10-P12 293FT (human embryonic kidney cells) cells were grown in 10 cm dishes at 8 × 10 6 amount of cells plated. Plasmid transfection by calcium phosphate-DNA co-precipitation method: The constructed pLVX-PEC lentiviral expression vector and packaging vectors pLP1, pLP2, pLP / VSVG were added to 2.5M CaCl 2 in solution with ddH 2 O make up the volume to 500ul, shake well, let stand for 5 minutes, slowly mix the plasmid and CaCl on a vortex shaker 2 The mixed solution was added dropwise to an equal volume of 500ul pH7.0 2×HBS solution at a constant speed. After the dropwise addition, the bubbles were blown five times, and 1 ml of the mixture was added to the adherent 293FT petri dish and placed in an incubator. After overnight, the old medium was discarded and 10-15 mL of fresh medium was added to continue the culture. After 48 hours of transfe...

Embodiment 3

[0138] Example 3: Transfection, maturation induction and preparation of specific CTLs of DCs

[0139] 1) DC transfection and maturation induction: Collect the peripheral blood of volunteers, and separate autologous plasma (inactivated at 56°C for 30 minutes, stored at 4°C) and peripheral blood single blood by Ficoll-Hypaque (Ficoll-Hypaque) density gradient separation method. Nucleated cell PBMC, this PBMC was treated with serum-free RPMI-1640 at 5-7 x 10 7 The cells were plated in T75 culture flasks, and the suspension cells were collected after 1 h to separate CD3+ T lymphocytes. The adherent cells were replaced with IL-4 (100ng / mL), GM-CSF (100ng / mL) and autologous plasma (2 %) of 1640 medium stimulation induced monocytes to differentiate into DCs. On the 5th day, the concentrated lentiviral particles were used to infect and collect immature DCs suspended in a 24-well plate, and the required volume of lentivirus was calculated according to the optimal MOI value of the pre-ex...

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Abstract

The invention relates to the field of biotechnology development and application research. Specifically, the present invention provides a method for preparing cytotoxic T lymphocytes (CTL) targeting multiple glioblastoma antigen epitopes. In the method of the present invention, a new artificially encoded antigen sequence synthesized by tandem combination of three glioma-associated antigen (GAA) epitope sequences is cloned into a lentiviral expression vector, and the virus is packaged and transfected into dendritic cells, and then separated from Chosen CD3 + T cells are co-cultured to stimulate the proliferation and differentiation of antigen-specific T cells, so that the prepared CTLs have the specificity of targeting the three GAA antigens. The GAA antigen-specific CTL prepared by the invention has the advantages of strong targeting specificity, no side effects, and is not easy to cause immune escape, and has great application prospects in the treatment of GAA-expressing glioblastoma.

Description

technical field [0001] The invention relates to the field of biotechnology development and application research, in particular to a method for preparing cytotoxic T lymphocytes targeting multiple antigenic epitopes of glioblastoma. Background technique [0002] Glioma is one of the most common intracranial tumors, with an incidence of about 40%-50% of all intracranial tumors, among which glioblastoma (GBM) is the most common and malignant tumor. For plasmoma, the 5-year survival rate is only 5.1%. Now the most effective treatment for GBM is the comprehensive treatment of surgery, radiotherapy and chemotherapy. The combination of chemotherapy drug temozolomide and radiotherapy has only improved the median survival time of radiotherapy alone. 3 months, and the median survival is still less than 15 months after aggressive treatment. [0003] With the continuous improvement of genetic engineering technology and cell engineering technology, the clinical research of immunotherapy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
Inventor 李晨蔚邓婷婷
Owner 上海尚泰生物技术有限公司
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