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Medicine composition and application thereof

A composition and drug technology, applied in the field of tumor cell immunotherapy, can solve the problem of undetectable nephroblastoma, etc., and achieve significant curative effect, high quality of life, and good prognosis

Active Publication Date: 2018-05-04
SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that in the prior art, GPC3 is mainly used for the detection of liver cancer, and cannot detect Wilms tumor

Method used

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  • Medicine composition and application thereof
  • Medicine composition and application thereof
  • Medicine composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction of GPC3-CAR lentiviral vector

[0066] (1) Synthesize GPC3-CD28-OX40 (abbreviation: G28Oζ, tandem human IgM signal peptide, anti-GPC3 antibody extracellular domain, human CD28 transmembrane region, human OX40 intracellular region and human CD3ζ signaling domain) through whole gene synthesis , GPC3-CD28-ICOS (abbreviation: G28Iζ, tandem human IgM signal peptide, anti-GPC3 antibody extracellular segment, human CD28 transmembrane region, human ICOS intracellular region and human CD3ζ signaling domain), GPC3-CD28-CD40 (abbreviation: G2840ζ, tandem human IgM signal peptide, anti-GPC3 antibody extracellular domain, human CD28 transmembrane region, human CD40 intracellular region and human CD3ζ signaling domain), GPC3-41BB-CD27 (abbreviation: GBB27ζ, ie Tandem human IgM signal peptide, anti-GPC3 antibody extracellular region, human 41BB transmembrane region, human CD27 intracellular region and human CD3ζ signal transduction domain), and then the whole g...

Embodiment 2

[0073] Example 2 GPC3-CAR lentivirus infection of T cells to obtain GPC3-CAR T cells

[0074] (1) Cultivate 293T cells in a 10cm culture dish, the culture medium is: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% double antibody (100 × penicillin-streptomycin mixed solution);

[0075] (2) When the 293T cell density in the 150mm culture dish reaches 80-90%, replace the medium: DMEM high glucose medium + 1% FBS + 1% double antibody;

[0076] (3) After replacing the medium and culturing for 2-6 hours, the pWPXLd-GPC3CAR-EGFP plasmids (pWPXLd-G28Oζ-2A-EGFP, pWPXLd-G28Iζ-2A-EGFP, pWPXLd-G2840ζ-2A-EGFP, pWPXLd- GBB27ζ-2A-EGFP, pWPXLd-Mock-2A-EGFP) were co-transduced into 293T cells with lentiviral packaging helper plasmids pMD2.G and psPAX2 respectively, and the reaction system was as follows:

[0077] Reagent

dose

pWPXLd-CAR-EGFP plasmid

9μg

pMD2.G helper plasmid

3μg

psPAX2

12μg

PEI

72μg

[0078] (4) 24, ...

Embodiment 3

[0084] Example 3 Detection of GPC3-CAR T cells killing nephroblastoma in vitro

[0085] The CAR GPC3T cells and CAR Mock T cells prepared in Example 2 were mixed with 1×10 4 G401 cells (Wilms tumor cell line) were mixed and added to a 96-well U-shaped plate. Three replicate wells were set up for each group, and the tumor cell group alone was set as a positive control. After centrifugation at 250g for 5min, place at 37°C Co-culture in 5% CO2 incubator for 18 hours;

[0086] Luciferase (Luciferase) killing quantitative killing efficiency: 18 hours after CAR T cells were co-cultured with tumor cells or tumor cells were cultured alone, 100 μl / well of luciferase substrate (1×) was added to a 96-well cell culture plate, The cells were resuspended and mixed, and the RLU (relative light unit) was immediately measured by a multifunctional microplate reader, and the measurement time was set to 1 second;

[0087] Calculation formula of killing ratio: 100%×(control well reading-experime...

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Abstract

The invention relates to the field of cellular immunotherapy of tumor and in particular to a medicine composition and application thereof. The medicine composition comprises a medicine specifically targeting kidney tumor GPC3, wherein the medicine specifically targeting kidney tumor GPC3 comprises any one or a combination of at least two of CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cells, an anti-GPC3 antibody, TCR-T (T-lymphocyte Cell Receptor) cells targeting GPC3, and siRNA of DC-CIK (Dendritic Cell-Cytokine-Induced Killer) cells targeting GPC3 or targeting silenced GPC3. Testsshow that GPC3 can be new targeting antigen of nephroblastoma, and by adopting the medicine composition, nephroblastoma can be effectively killed and inhibited.

Description

technical field [0001] The invention relates to the field of tumor cell immunotherapy, in particular to a pharmaceutical composition and its application. Background technique [0002] Wilms tumor is the most common primary malignant tumor of the urinary system in children. It has the characteristics of difficult early diagnosis, poor prognosis, and easy recurrence. It accounts for 6% to 7% of malignant tumors in children under the age of 15, and about 75% of them develop In children under the age of 5, the onset age of patients with congenital malformations is usually before 1 year old, the average age of onset is about 3.5 years old, and the incidence rate is 1 in 10,000. Wilms tumor is rare in adults. [0003] In recent years, with the development of a comprehensive treatment plan based on the combination of surgery, chemotherapy and radiotherapy, the survival rate has been significantly improved, and the 5-year survival rate after treatment has reached 87%. , late clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00C12Q1/6886A61P35/00
CPCA61K45/00C07K16/303C12Q1/6886C12Q2600/118
Inventor 不公告发明人
Owner SHENZHEN IN VIVO BIOMEDICINE TECH LTD
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