OCTS (One CAR (Chimeric Antigen Receptor) with two ScFvs (Single-chain variable Fragments)) technique based CAR-T (Chimeric Antigen Receptor-T cell immunotherapy) therapeutic vector for glioblastoma and construction method and application thereof

A malignant glioma, carrier technology, applied in the field of medical biology, can solve the problems that have not been overcome, and achieve the effects of improving curative effect, inhibiting immune escape, and ensuring reliable protection

Active Publication Date: 2017-10-20
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are no reports on CAR-T therapy targeti

Method used

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  • OCTS (One CAR (Chimeric Antigen Receptor) with two ScFvs (Single-chain variable Fragments)) technique based CAR-T (Chimeric Antigen Receptor-T cell immunotherapy) therapeutic vector for glioblastoma and construction method and application thereof
  • OCTS (One CAR (Chimeric Antigen Receptor) with two ScFvs (Single-chain variable Fragments)) technique based CAR-T (Chimeric Antigen Receptor-T cell immunotherapy) therapeutic vector for glioblastoma and construction method and application thereof
  • OCTS (One CAR (Chimeric Antigen Receptor) with two ScFvs (Single-chain variable Fragments)) technique based CAR-T (Chimeric Antigen Receptor-T cell immunotherapy) therapeutic vector for glioblastoma and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 OCTS-CAR-T cell construction

[0095] 1. Construction, purification and detection methods of recombinant lentiviral vector lvOCTS-PEvIIIs and lvOCTS-PEvIIIt.

[0096] See image 3 The method for constructing the recombinant lentiviral vector of the present invention is as follows:

[0097] 1. The human EF1α promoter (SEQ ID NO.14), OCTS structure [OCTS-PEvIIIs, OCTS-PEvIIIt] (CD8leader chimeric receptor signal peptide (SEQ ID NO.15), PDL1 single chain antibody light chain VL ( SEQ ID NO.16), PDL1 single chain antibody heavy chain VH (SEQ ID NO.17), EGFRvIII single chain antibody light chain VL (SEQ ID NO.18), EGFRvIII single chain antibody heavy chain VH (SEQ ID NO.19) ), antibody inner hinge Inner-Linker (SEQ ID NO.20), single-chain antibody hinge Inter-Linker (SEQ ID NO.21), CD8Hinge chimeric receptor hinge (SEQ ID NO.22), CD8Transmembrane chimeric receptor Somatic transmembrane region (SEQ ID NO.23), CD28 chimeric receptor costimulatory factor (SEQ ID NO.24), CD...

Embodiment 2

[0204] OCTS-CAR-T cell pathogen detection and expression detection.

[0205] 1. Endotoxin detection;

[0206] (1) The standard product of endotoxin is 15EU / piece;

[0207] (2) Limulus reagent sensitivity λ = 0.25EU / ml, 0.5ml / tube

[0208] (3). Dilution of endotoxin standard: take one endotoxin standard and dilute it with BET water to 4λ and 2λ respectively. Seal with parafilm and shake to dissolve for 15min; each dilution step should be mixed in a vortex during dilution. Mix on the device for 30s;

[0209] (4) Sample addition: Take several limulus reagents, add 0.5ml of BET water to dissolve each one, and distribute them into several endotoxin-free test tubes, each 0.1ml. Two of them are negative control tubes, add 0.1ml of BET water;

[0210] 2 positive control tubes, add 0.1ml endotoxin working standard solution with 2λ concentration;

[0211] 2 positive control tubes for samples, add 0.1ml sample solution containing 2λ endotoxin standard (1ml of the sample to be tested diluted 20 tim...

Embodiment 3

[0241] Example 3 Functional detection of OCTS-CAR-T cells.

[0242] 1. Evaluation of target cell killing effect.

[0243] (1) Culture target cells separately [PDL1 + K562, EGFRvIII + K562, PDL1 + EGFRvIII + K562, K562 cells] and effector cells [OCTS-CAR-T cells];

[0244] (2) Collect target cells 4x10 5 cells and OCTS-CAR-T cells 2.8x10 6 Cells, 800g, 6min centrifugation, discard the supernatant;

[0245] (3) Resuspend target cells and effector cells with 1ml D-PBS(-) solution, centrifuge at 800g for 6min, discard the supernatant;

[0246] (4) Repeat step 3 once;

[0247] (5) Resuspend the effector cells with 700ul medium (AIM-V medium+1-10% FBS), and resuspend the target cells with 2ml medium (AIM-V medium+1-10% FBS);

[0248] (6) Set the experimental wells with the ratio of effect to target of 1:1, 5:1, 10:1, and the grouping situation of effector cells co-incubated with single target cells and dual target cells are shown in Table 6, and set up a control group ( K562 cells), 3 replicat...

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Abstract

The invention discloses an OCTS (One CAR (Chimeric Antigen Receptor) with two ScFvs (Single-chain variable Fragments)) technique based CAR-T (Chimeric Antigen Receptor-T cell immunotherapy) therapeutic vector for glioblastoma. The OCTS technique based CAR-T therapeutic vector comprises a lentiviral skeleton plasmid, a human EF1 [alpha] promoter (SEQ ID NO. 14), an OCTS chimeric receptor structural domain and a PDL1 single-chain antibody, wherein the OCTS chimeric receptor structural domain comprises a CD8 leader chimeric receptor signal peptide (SEQ ID NO. 15), a PDL1 single-chain antibody light chain VL (SEQ ID NO. 16), a PDL1 single-chain antibody heavy chain VH (SEQ ID NO. 17), an EGFRvIII single-chain antibody light chain VL (SEQ ID NO. 18), an EGFRvIII single-chain antibody heavy chain VH (SEQ ID NO. 19), an antibody Inner-Linker (SEQ ID NO. 20), a single-chain antibody Inter-Linker (SEQ ID NO. 21), a CD8 Hinge chimeric receptor linker (SEQ ID NO. 22), a CD8 Transmembrane chimeric receptor transmembrane domain (SEQ ID NO. 23), a TCR (T Cell Receptor) chimeric receptor T cell activation domain (SEQ ID NO. 26) and a chimeric receptor co-stimulator domain. In addition, the invention also discloses a construction method of the vector and application of the vector to the preparation of a medicine for treating the glioblastoma.

Description

Technical field [0001] The invention belongs to the field of medical biology, and specifically relates to a carrier, in particular to a malignant glioma CAR-T treatment carrier based on OCTS technology. In addition, the present invention also relates to the construction method and application of the vector. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-related antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas proposed the "immune surveillance" theory, which believed that mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, laying a theoretical foundation for tumor immunotherapy [Burnet FM.Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumor immunotherapies including cytokine therapy, monoclonal antibody therapy, adoptive i...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61K35/17C12N5/0636C12N15/86C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K2319/03C07K2319/02C07K2317/622C07K2319/33C12N2740/15043C07K16/2863C07K16/30C07K2317/24C12N2510/00C12N2501/51C12N2501/515A61P35/00C12N2740/16043A61K38/00C07K2317/53C07K2319/30C12N7/00C12N2730/10144C12N2800/10
Inventor 祁伟俞磊康立清林高武余宙
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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