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Blocking antibody and application thereof in preparation of CAR-T cell of targeting T cell expression antigen

A technology of blocking antibodies and cells, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, animal cells, antibody medical components, etc., to increase the proportion of CD8+ T cells, reduce expression, and improve anti-tumor Active and persistent effects

Pending Publication Date: 2021-11-05
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Png, Y.T., et al., Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies. Blood Adv, 2017.1(25): p.2348-2360), obtained CD7-negative T cells, but this The two technologies will introduce uncontrollable risk factors such as "off-target", and whether the deletion of CD7 molecules on the surface of T lymphocyte membranes will affect the unexplored biological functions is still unknown

Method used

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  • Blocking antibody and application thereof in preparation of CAR-T cell of targeting T cell expression antigen
  • Blocking antibody and application thereof in preparation of CAR-T cell of targeting T cell expression antigen
  • Blocking antibody and application thereof in preparation of CAR-T cell of targeting T cell expression antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Preparation of recombinant lentiviral vector

[0075] This example relates to the construction of anti-CD7 CAR and anti-CD19 CAR recombinant lentiviral vectors.

[0076] 1. Construction of recombinant plasmid loaded with anti-CD7 CAR gene and recombinant plasmid loaded with anti-CD19 CAR gene

[0077] (1) Enzyme digestion: The pUT-Bacbone plasmid was mixed with EcoR I and BamH I restriction endonucleases according to Table 1.

[0078] (2) Recovering linear plasmids: After performing gel electrophoresis (120 V, 20 min), a 7000 bp linear plasmid was recovered and purified using an agarose gel DNA recovery kit.

[0079] Table 1: Enzyme digestion reaction system (50 μL)

[0080]

[0081] (3) Synthesis of the target gene: Design primers CART-F (SEQ ID NO.19) and CART-R (SEQ ID NO.20), add them into the synthesis system of anti-CD7 CAR gene (or anti-CD19 CAR gene), and by polymerizing The target fragment of about 1500bp was amplified by enzyme chain reaction (...

Embodiment 2

[0143] Example 2 Preparation and Expansion of CAR-T Cells

[0144] 1. Isolation of T cells

[0145] (1) Separation of peripheral blood mononuclear cells (PBMC)

[0146] 1) Prepare a 50mL centrifuge tube, and mix blood and PBS at a volume ratio of 1:2 to achieve the effect of blood dilution.

[0147] 2) Slowly add the diluted blood along the tube wall of the centrifuge tube into the mononuclear cell separation solution of 1.5 times the blood volume.

[0148] 3) Slowly transfer the centrifuge tube to a centrifuge at 800G for 20 minutes.

[0149] 4) Pipette the middle buffy coat layer, add 30mL PBS, mix well, and centrifuge at 1500rpm for 5min.

[0150] 5) Discard the supernatant, add 1mL PBS to resuspend, and use the hemocytometer method to count.

[0151] (2) CD4 + T cells and CD8 + T cell isolation

[0152] 1) After counting, add 3mL PBS to resuspend, and centrifuge at 1500rpm for 5min.

[0153] 2) Discard the supernatant, follow every 1x10 7 Add 10 μL of CD4 separati...

Embodiment 3

[0184] Example 3 Preparation and Characterization of Anti-CD7 Antibody

[0185] 1. Construction and extraction of anti-CD7 recombinant antibody plasmid

[0186] The method is the same as the plasmid construction and extraction in Example 1, but the target fragment is replaced by the nucleic acid fragment encoding the anti-CD7 antibody.

[0187] 2. Expression of anti-CD7 antibody

[0188] (1) Preparation of transfection reagent

[0189] 1) Prepare a 15mL centrifuge tube, add 2mL CaCl 2 , 58 μg of the target plasmid, add bacterial endotoxin test water to 4mL.

[0190] 2) Add 4mL HBS while vortexing, and then continue to shake for 20s after adding, and set aside.

[0191] (2) Perform transfection according to (2)-(4) in the four-plasmid expression system recombinant lentiviral vector packaging in Example 1.

[0192] 3. Antibody affinity chromatography purification

[0193] (1) Install the His-tagged protein purification prepacked column.

[0194] (2) Equilibrium chromatogr...

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Abstract

The invention provides a blocking antibody and application thereof in preparation of a CAR-T cell of a targeting T cell expression antigen. A heavy chain variable region and a light chain variable region of the blocking antibody are respectively the same as a heavy chain variable region and a light chain variable region of scFv of the CAR-T cell to be blocked; and the scFv of the CAR-T cell to be blocked targets the T cell expression antigen. The invention also provides a pharmaceutical composition containing the blocking antibody and the CAR-T cell to be blocked, the CAR-T cell combined with the blocking antibody and targeting the T cell expression antigen, and a preparation method of the CAR-T cell. According to the invention, a free antibody having the same binding domain as a chimeric antigen receptor (CAR) is adopted to block (close) the binding of the CAR-T cell and the T cell surface antigen so as to overcome the self-killing problem of the CAR-T cell of the targeting T cell expression antigen. The method provided by the invention is simple, safe and effective, and provides a new idea for clinical treatment of tumours.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, and in particular relates to a blocking antibody and its application in preparing CAR-T cells targeting T cells to express antigens. Background technique: [0002] Acute lymphoblastic leukemia (ALL) is a common malignant hematological disease. The imbalance of proliferation and differentiation ability of hematopoietic stem cells leads to abnormal lymphocyte maturation and function, among which the incidence of children accounts for 15%. T-acute lymphoblastic leukemia (T-ALL) is highly aggressive, 30% of patients will progress to relapsed or refractory T-ALL (r / rT-ALL), and relapsed patients with three-year event-free survival less than 15%. Generally speaking, the malignant degree of T-cell malignancies is higher than that of B-cell malignancies, which also brings greater challenges to the treatment of T-ALL. Nelarabine is currently the only FDA-approved drug for r / rT-ALL, but clinical data show tha...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N5/10A61P35/00A61K39/00A61K47/68
CPCC07K16/2803C12N5/0636A61K39/001111A61P35/00A61K47/6803C07K2317/56C07K2317/622C12N2510/00C12N2501/599
Inventor 余宙叶晶徐南谭静雯康立清
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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