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Gastric cancer pharmaceutical composition and application thereof

A composition and drug technology, applied in the field of immunology, can solve the problem of patients' response rate reduction to previous therapy, and achieve significant cytotoxicity and effective tumor control

Active Publication Date: 2020-05-05
GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, as gastric cancer is often diagnosed at an advanced stage, patients have a reduced response rate to previous therapies

Method used

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  • Gastric cancer pharmaceutical composition and application thereof
  • Gastric cancer pharmaceutical composition and application thereof
  • Gastric cancer pharmaceutical composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Preparation of anti-PSCA CAR-T cells

[0077] (1) Construction of CAR molecular lentiviral vector

[0078] Gene synthesis of a CAR molecule composed of GM-CSF signal peptide, anti-PSCA single chain variable fragment (single chain variable fragment, scFv), hinge region, CD28, CD3ζ and DAP10, the nucleic acid sequence is shown in SEQ ID NO: 8-13, and A recognition site for the restriction endonuclease Pme1 is added to the C-terminus of the CAR molecule, a recognition site for the restriction endonuclease Spe1 is added to the N-terminus, and the synthetic gene sequence is temporarily connected to the PUC57 vector;

[0079] The linearized CAR molecule and the Pwpxld-eGFP sequence were respectively obtained by double enzyme digestion with Pme1 and Spe1, and the above fragments were recovered after agarose gel electrophoresis;

[0080] Using TAKARA's solution 1, connect the linearized CAR molecule with Pwpxld-eGFP to obtain a circular plasmid;

[0081] Transform the...

Embodiment 2

[0096] Example 2 In vitro detection of the killing ability of anti-PSCA CAR-T cells on gastric tumor cells

[0097] (1) Anti-PSCA CAR-T cells and GFP-T cells prepared in Example 1 were co-incubated with target cells BGC-823-GL, KATO-Ⅲ-GL and MKN28-GL at different ratios for 18-24 hours , using a luciferase substrate to detect the ratio of living cells, the effect-to-target ratio of CAR-T cells and tumor cells is 2:1, 1:1, 1:2 or 1:4, and the amount of target cells is 1×10 4 per well (96-well plate), the incubation volume is 200 μL;

[0098] (2) After the co-incubation is completed, take 100 μL of the incubation supernatant and temporarily store it at -20°C to detect the secretion of cytokines;

[0099] (3) After diluting 100× fluorescein sodium salt to 2× with PBS, add 100 μL per well to the remaining cell solution in step (2), mix well while avoiding the generation of air bubbles, and incubate at 37°C for 5-10 minutes. The RLU (relative light unit) was detected by a fluores...

Embodiment 3

[0103] Example 3 In vitro detection of cytokine secretion ability of anti-PSCA CAR-T and GFP-T

[0104] (1) Take out the culture supernatant temporarily stored at -20°C in step (2) of Example 2, and dilute the corresponding ratio after thawing;

[0105] (2) The contents of IL-2, IFN-γ, GM-CSF and TNF-α in the supernatant were detected by cytokine detection kit (ebioscience).

[0106] The result is as figure 2 As shown, anti-PSCA CAR-T has significantly improved secretion capacity of IL-2, IFN-γ, GM-CSF and TNF-α compared with GFP-T.

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Abstract

The invention provides a gastric cancer pharmaceutical composition and an application thereof. The pharmaceutical composition comprises anti-PSCA CAR-T cells. The anti-PSCA CAR-T cells are used to target gastric tumor cells, the killing effect on different types of gastric tumor cells is realized in vivo and in vivo, and the tumor growth in an xenotransplantation gastric cancer mouse model is effectively controlled, so that a new treatment idea is provided for treatment failure due to tumor heterogeneity in gastric cancer treatment.

Description

technical field [0001] The invention belongs to the technical field of immunology and the technical field of tumor cell immunotherapy, and relates to a gastric cancer pharmaceutical composition and its application. Background technique [0002] Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium. There are obvious regional differences in the incidence. The northwest and eastern coastal areas of my country are high-incidence areas of gastric cancer. Gastric cancer is most common in people over the age of 50, with a male-to-female ratio of about 2:1. Due to dietary structure, work pressure, and Helicobacter pylori infection, gastric cancer has shown a younger trend in recent years. Gastric cancer can occur in any part of the stomach, and more than half of them occur in the antrum, greater curvature, lesser curvature, and the anterior and posterior walls. The vast majority of gastric cancer is adenocarcinoma, with no obvious symptoms in the ear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61P35/00A61P1/00C12N5/10C12N15/62C12N15/867C12N7/01
CPCA61K39/001193A61P35/00A61P1/00C07K16/3069C07K14/7051C12N15/86C12N7/00C12N5/0636C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15021C12N2740/15043A61K2039/828A61K2039/5156
Inventor 不公告发明人
Owner GUANGDONG ZHAOTAI INVIVO BIOMEDICINE CO LTD
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