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Composite capable of strengthening anti-tumor immune response

An anti-tumor immune and complex technology, applied in the field of biomedicine, can solve the problems of limitation, poor specificity, and low application efficiency, and achieve the effect of up-regulating the anti-tumor immune response ability.

Active Publication Date: 2011-07-06
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] Design and screening of embodiment 1Foxp3siRNA

[0031] Search the sequence through NCBI and use the design software of Ambion to design siRNA. The siRNA with the designed sequence was obtained by chemical synthesis, and 4 Foxp3-specific siRNAs were synthesized, and the Foxp3 siRNA with stable expression and good interference effect was screened out: the inventor designed and synthesized image 3 Foxp3siRNA shown, and these Foxp3siRNA were transfected into Treg separated by magnetic beads (stemcell company, Canada) by liposomes (lipo2000, invitrogen company, USA) in vitro [9，21] , Degradation of Foxp3 mRNA detected by real-time quantitative PCR and flow cytometry [9，21] , from which a Foxp3 siRNA (siRNA3, Figure 4 A). After the synthetic 4-segment Foxp3-specific siRNA was transfected into Treg, RT-PCR and realtime-PCR were used to evaluate the specific degradation of Foxp3 mRNA by Foxp3 siRNA. It was found that the degradation efficiency of Foxp3 by siRNA3 in a serie...

Embodiment 2

[0049] Example 2 Evaluation of Foxp3siRNA transfection efficiency through liposomes and analysis of Treg cell apoptosis after transfection:

[0050] Foxp3 siRNA at different concentrations (5nM, 10nM, 20nM) was transfected into Treg cells via liposomes (lipo2000, Invitrogen, USA), and the transfection efficiency was evaluated with FITC fluorescently labeled siRNA. BD Company, U.S.A.) analyzed the effect of different concentrations of Foxp3siRNA transfection on Treg cell apoptosis, and found that the Foxp3siRNA transfection efficiency can reach 41.7% at 20nM concentration ( Figure 5 B), at the same time about 15% of the cells showed early apoptosis or death ( Figure 5 A), while the transfection efficiency of 5 nM or 10 nM Foxp3 siRNA is only 17% and 25%, in view of the above results, the inventors of the present application adopted 20 nM as the optimal transfection concentration of Foxp3 siRNA in subsequent studies.

Embodiment 3

[0051] Example 3 Preparation of Foxp3siRNA-protamine-anti-CD25 antibody complex

[0052] Anti-CD25-scFv monoclonal antibody with His tag and scFv-protamine fragment (amino acid 8-29, scFv-P) were expressed by baculovirus expression system, and purified by high performance liquid chromatography to obtain purified protein 5mg (entrusted to Beijing Yixing Industry Company to synthesize). The synthesized Foxp3siRNA was mixed with anti-CD25-scFv monoclonal antibody and scFv-protamine fragment in PBS buffer at a molar ratio of 6:1:1, and after 30 minutes at 4°C, the Foxp3siRNA-fish Binding of protamine-anti-CD25 antibody complexes.

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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 invention relates to the field of biomedicine, in particular to a drug for targeting and inhibiting regulatory T cells of tumor patients through Foxp3siRNA to enhance anti-tumor immune response. Background technique [0002] Primary hepatocellular carcinoma (HCC) is a malignant tumor with high degree of malignancy, low cure rate and poor prognosis. Its incidence rate ranks second among all malignant tumors in my country. Traditional treatment methods such as chemotherapy, radiotherapy, and surgery have been proven to be difficult to solve the problem of tumor metastasis and recurrence for decades, and chemotherapy and radiotherapy have serious side effects, which often make liver cancer patients unbearable. Therefore, researchers have been exploring more effective biological therapies based on human immune mobilization that can kill tumor cells to the maximum extent without harming normal cells. Through the deepening understanding of the immune an...

Claims

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Application Information

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