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Composite capable of strengthening anti-tumor immune response

An anti-tumor immunity and compound technology, applied in the field of biomedicine, can solve problems such as limitations, low application efficiency, and poor specificity, and achieve the effect of up-regulating anti-tumor immune response ability

Active Publication Date: 2009-06-17
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the methods for removing Treg or inhibiting Treg function are currently mainly based on magnetic bead separation or antibody-immunotoxin connection. [15，16] However, these schemes have factors such as low clinical in vivo application efficiency and poor specificity, which limit their further clinical application.

Method used

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  • Composite capable of strengthening anti-tumor immune response
  • Composite capable of strengthening anti-tumor immune response
  • Composite capable of strengthening anti-tumor immune response

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Design and screening of Foxp3 siRNA

[0031] Find the sequence through NCBI and design siRNA using ambion design software. The siRNA of the design sequence was obtained by chemical synthesis, and 4 segments of Foxp3 specific siRNA were synthesized, and Foxp3 siRNA with stable expression and good interference effect was screened out: the inventor designed and synthesized image 3 The Foxp3 siRNA as shown, and these Foxp3 siRNA were transfected into Treg separated by magnetic beads (stemcell, Canada) by liposome (lipo2000, invitrogen, USA) in an in vitro experiment [9，21] , Through real-time quantitative PCR and flow cytometry to detect the degradation of Foxp3 mRNA [9，21] , From which a Foxp3 siRNA (siRNA3, Figure 4 A). After transfecting 4 segments of Foxp3 specific siRNA into Treg, RT-PCR and realtime-PCR were used to evaluate the specific degradation of Foxp3 siRNA on Foxp3 mRNA. The study found that in a series of synthesized siRNA, the degradation efficiency ...

Embodiment 2

[0049] Example 2 Evaluation of Foxp3 siRNA transfection efficiency via liposome and analysis of its effect on Treg cell apoptosis:

[0050] Different concentrations (5nM, 10nM, 20nM) of Foxp3 siRNA were transfected into Treg cells by liposome (lipo2000, invitrogen, USA), and the transfection efficiency was evaluated with FITC fluorescently-labeled siRNA. At the same time, the flow cytometer (FACSCalibur , BD Company, USA) analyzed the effect of different concentrations of Foxp3 siRNA transfection on Treg cell apoptosis. The study found that the transfection efficiency of Foxp3 siRNA can reach 41.7% ( Figure 5 B). At the same time, about 15% of cells undergo early apoptosis or death ( Figure 5 A), while the transfection efficiency of 5nM or 10nM Foxp3 siRNA is only 17% and 25%. In view of the above results, the inventor of the present application used 20nM as the optimal transfection concentration of Foxp3 siRNA in subsequent studies.

Embodiment 3

[0051] Example 3 Preparation of Foxp3 siRNA-protamine-anti-CD25 antibody complex

[0052] The baculovirus expression system was used to express His-tagged anti-CD25-scFv monoclonal antibodies and scFv-protamine fragments (amino acids 8-29, scFv-P), and purified by high performance liquid chromatography to obtain purified proteins 5mg (combined by Beijing Yixinxingye Company). The synthesized Foxp3 siRNA was mixed with anti-CD25-scFv monoclonal antibody and scFv-protamine fragment in PBS buffer at a molar ratio of 6:1:1. After 30 minutes at 4 degrees Celsius, the Foxp3 siRNA was completed -Binding of protamine-anti-CD25 antibody complex.

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Abstract

The invention provides a medicine complex capable of enhancing antitumor immune response, which is a complex of Foxp3 siRNA-protamine-anti-CD25 antibody. Experiments show that the medicine can deliver the Foxp3 siRNA to a Treg cell in a target mode, enter the cell through receptor-mediated endocytosis, realize the gene silencing to Foxp3, and specifically inhibit] the function of the Treg, thereby raising the antitumor immune response capability for stimulating tumor antigens. The medicine can be widely applied to assisting the resistance of tumor.

Description

Technical field [0001] The present invention relates to the field of biomedicine, in particular to a drug for targeting and inhibiting regulatory T cells of tumor patients through Foxp3 siRNA to enhance anti-tumor immune response. Background technique [0002] Primary hepatocellular carcinoma (HCC) is a malignant tumor with a high degree of malignancy, a low cure rate, and a poor prognosis. Its incidence is the second of all malignant tumors in my country. Past traditional treatment methods such as chemotherapy, radiotherapy, and surgery have proven to be difficult to solve the problem of tumor metastasis and recurrence for decades, and the side effects of chemotherapy and radiotherapy are serious, making liver cancer patients often unbearable. Therefore, researchers have been exploring more effective biological therapies based on human immune mobilization that can kill tumor cells to the maximum without harming normal cells. In recent years, through the continuous in-depth under...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00
Inventor 陈红松张恒辉
Owner PEOPLES HOSPITAL PEKING UNIV
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