Fusion protein with immune regulation function, pharmaceutical composition, cell and application

A fusion protein and cell technology, applied in the field of immune enhancer, cell and its application, and pharmaceutical composition, can solve the problem of insufficient response rate

Active Publication Date: 2020-06-12
启辰生生物科技(珠海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, various clinical data show that the single use of a certain immune checkpoint inhibitor is effective in various cancer types, but there is a problem that the response rate is not high enough

Method used

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  • Fusion protein with immune regulation function, pharmaceutical composition, cell and application
  • Fusion protein with immune regulation function, pharmaceutical composition, cell and application
  • Fusion protein with immune regulation function, pharmaceutical composition, cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] This example is for the preparation of DNA and mRNA encoding antigens and fusion proteins

[0069] 1. Preparation of DNA and mRNA Constructs

[0070] Construction of a DNA sequence for encoding CMV viral pp65 protein mRNA (sequence shown in SEQ ID NO: 7), DNA sequence for encoding sPD-1-Fc, sCD80-Fc and sPD-1-Fc-CD80 fusion protein mRNA , and used for subsequent in vitro transcription reactions. Constructs were prepared by codon optimization to introduce a high GC sequence to stabilize the synthesized mRNA, followed by a 3'UTR sequence derived from human β-globin, followed by a polyadenosine fragment. The specific sequence is shown in Table 1.

[0071] Table 1

[0072] name nucleic acid sequence number sPD-1 SEQ ID NO: 1 sCD80 SEQ ID NO: 2 Fc SEQ ID NO: 3 PD1-Fc-CD80 SEQ ID NO: 4

[0073] 2. In vitro transcription

[0074] The prepared corresponding DNA plasmid was firstly linearized by using speI endonuclease, and using ...

Embodiment 2

[0076] This example is used to verify the expression level of the fusion protein mRNA of the present invention in dendritic cells and its influence on the phenotype of dendritic cells.

[0077] 1. Induction culture of DC cells in vitro

[0078] Aseptically extract 50ml of human venous blood, separate peripheral blood mononuclear cells with lymphocyte separation medium in the ultra-clean workbench, add the mononuclear cells to the AIM-V medium, and place them in 37 ° C, 5% CO 2 Incubate in an incubator to allow monocytes to adhere to the wall. After 2h, the non-adherent cells were removed, and the adherent cells were added to iDC medium (GM-CSF with a final concentration of 800U / mL and IL-4 at 500U / mL were added to the AIM-V medium), and placed at 37°C for 5 %CO 2Cultured in the incubator for 6 days. Transfer half of the cell culture medium to a centrifuge tube, collect the cells by centrifugation at 500g, remove the supernatant, add an equal volume of fresh mDC medium, and ...

Embodiment 3

[0088] This example is used to study the influence of immunomodulator composition on T cell response

[0089] 1. Induction culture of DC cells in vitro

[0090] Aseptically extract 50ml of human venous blood, separate peripheral blood mononuclear cells with lymphocyte separation medium in the ultra-clean workbench, add the mononuclear cells to the AIM-V medium, and place them in 37 ° C, 5% CO 2 Incubate in an incubator to allow monocytes to adhere to the wall. After 2h, the non-adherent cells were removed, and the adherent cells were added to iDC medium (GM-CSF with a final concentration of 800U / mL and IL-4 at 500U / mL were added to the AIM-V medium), and placed at 37°C for 5 6 days in a % CO2 incubator. Transfer half of the cell culture medium to a centrifuge tube, collect the cells by centrifugation at 500g, remove the supernatant, add an equal volume of fresh mDC medium, and its formula is 1600U / mL GM-CSF and 1000U / mL IL-4, TNF-a( 5ng / ml), IL-1β(5ng / ml), IL-6(150ng / ml) an...

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Abstract

The invention discloses a fusion protein with an immunoregulation function, a pharmaceutical composition, a cell and an application thereof. The fusion protein of the present invention comprises a PD-1-derived soluble A fragment capable of binding to CD28 and a CD80-derived soluble B fragment capable of binding to CTLA-4. The fusion protein disclosed by the invention not only can be combined withPD-L1 and PD-L2, but also can be combined with CTLA-4 to block a CTLA-4 mediated inhibitory signal path; meanwhile, the polypeptide can be combined with CD28 on the surface of a T cell to provide a co-stimulation signal for activation of the T cell and induce proliferation of the T cell and secretion of cytokines.

Description

technical field [0001] The invention relates to medical products, in particular to immune enhancers, pharmaceutical compositions, cells and applications thereof. Background technique [0002] The traditional view is that tumor cells express tumor-specific antigens and are presented to the surface of tumor cells by MHC complexes, while anti-tumor T cells are not fully activated. Based on this, tumor cell immunotherapy mainly stimulates the immune system through vaccination or adoptive cell immunotherapy, thereby triggering an immune response. [0003] In recent years, it has become clear that the immune system does recognize tumor antigens, but that T cells remain quiescent despite the presence of tumor antigens. Based on this phenomenon, it has been hypothesized that there are specific inhibitory mechanisms that limit or switch off anti-tumor immune responses. When negative regulatory T cell surface molecules are upregulated in activated T cells, their activity is attenuat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K39/39A61P37/02
CPCA61K39/39A61P37/02C07K14/70521C07K14/70532C07K2319/00C07K2319/30
Inventor 蒋俊祝道成吴斐然林鑫
Owner 启辰生生物科技(珠海)有限公司
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