A myeloid leukemia car-t therapeutic vector based on octs technology and its construction method and application

A technology of myeloid leukemia and carrier, which is applied in the treatment of myeloid leukemia CAR-T carrier. The construction method and application field of the carrier can solve the problems that have not been overcome, so as to save economic expenditure, reduce the probability of relapse, transduce high efficiency effect

Active Publication Date: 2020-02-07
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there are no relevant reports on CAR-T therapy targeting CD33 and CD123 antigens to overcome the above shortcomings

Method used

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  • A myeloid leukemia car-t therapeutic vector based on octs technology and its construction method and application
  • A myeloid leukemia car-t therapeutic vector based on octs technology and its construction method and application
  • A myeloid leukemia car-t therapeutic vector based on octs technology and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Construction of OCTS-CAR-T cells

[0096] 1. Construction, purification and detection methods of recombinant lentiviral vectors lvOCTS12333s and lvOCTS12333t.

[0097] see image 3 , the construction method of the recombinant lentiviral vector of the present invention is as follows:

[0098] 1. Human EF1α promoter (SEQ ID NO.14), OCTS structure [OCTS12333s, OCTS12333t] (CD8leader chimeric receptor signal peptide (SEQ ID NO.15), CD33 single-chain antibody light chain VL (SEQ ID NO. 16), CD33 single-chain antibody heavy chain VH (SEQ ID NO.17), CD123 single-chain antibody light chain VL (SEQ ID NO.18), CD123 single-chain antibody heavy chain VH (SEQ ID NO.19), intrabody Hinge Inner-Linker (SEQ ID NO.20), single-chain antibody inter-hinge Inter-Linker (SEQ ID NO.21), CD8Hinge chimeric receptor hinge (SEQ ID NO.22), CD8Transmembrane chimeric receptor transmembrane region (SEQ ID NO.23), CD28 chimeric receptor co-stimulator (SEQ ID NO.24), CD134 chimeric recepto...

Embodiment 2

[0207] OCTS-CAR-T cell pathogen detection and expression detection.

[0208] 1. Endotoxin detection;

[0209](1), endotoxin working standard is 15EU / branch;

[0210] (2), Limulus reagent sensitivity λ=0.25EU / ml, 0.5ml / tube

[0211] (3) Dilution of endotoxin standard substance: Take one endotoxin standard substance, dilute it with BET water in proportion to dissolve into 4λ and 2λ respectively, seal with parafilm, shake and dissolve for 15min; each step of dilution should be mixed in the vortex Mix on the mixer for 30s;

[0212] (4) Adding samples: Take several LAL reagents, add 0.5 ml of BET water to each tube to dissolve, and distribute to several endotoxin-free test tubes, each tube has 0.1 ml. Two of them are negative control tubes, add 0.1ml of BET water;

[0213] Two are positive control tubes, add 0.1ml of endotoxin working standard solution with 2λ concentration;

[0214] 2 tubes are sample positive control tubes, add 0.1ml sample solution containing 2λ endotoxin s...

Embodiment 3

[0244] Example 3 Functional detection of OCTS-CAR-T cells.

[0245] 1. Evaluation of target cell killing effect.

[0246] (1) Culture target cells separately [CD33 + K562, CD123 + K562, CD33 + CD123 + K562, K562 cells] and effector cells [OCTS-CAR-T cells];

[0247] (2) Collect target cells 4x10 5 cells and OCTS-CAR-T cells 2.8x10 6 cells, 800g, centrifuge for 6min, discard the supernatant;

[0248] (3) Resuspend the target cells and effector cells in 1ml D-PBS(-) solution, centrifuge at 800g for 6min, discard the supernatant;

[0249] (4) Repeat step 3 once;

[0250] (5) Resuspend effector cells with 700ul medium (AIM-V medium + 1-10% FBS), and resuspend target cells with 2ml medium (AIM-V medium + 1-10% FBS);

[0251] (6) Experimental wells with effect-to-target ratios of 1:1, 5:1, and 10:1 were set, and the grouping of effector cells co-incubated with single-target cells and double-target cells was shown in Table 6, and a control group ( K562 cells), 3 replicate w...

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Abstract

Provided is an OCTS technology-based myeloid leukemia chimeric antigen receptor T-cell immunotherapy (CAR-T) therapeutic vector, which comprises: a lentiviral backbone plasmid, a human elongation factor 1 alpha (EF1α) promoter, an OCTS chimeric receptor area and a programmed death-ligand 1 (PDL1) single chain antibody; the OCTS chimeric receptor area comprises: a cluster of differentiation 8 (CD8) leader chimeric receptor signal peptide, a CD33 single-chain antibody light chain VL and heavy chain VH, a CD123 single chain antibody light chain VL and heavy chain VH, an intra-antibody hinge Inner-Linker, an inter-single chain antibody hinge Inter-Linker, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane region, a T cell receptor (TCR) chimeric receptor T cell activation area, and a chimeric receptor co-stimulatory factor zone. Further provided are a method for constructing the vector and an application thereof in the preparation of a medicament for treating myeloid leukemia.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a carrier, in particular to a CAR-T treatment carrier for myeloid leukemia based on OCTS technology. In addition, the present invention also relates to the construction method and application of the carrier. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the 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 an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/62C12N5/10A61K35/17A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/2827C07K16/3069C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2740/15043
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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