Fusion protein with immunoregulation function, pharmaceutical composition, cell and application thereof

A fusion protein and immune regulation technology, which can be used in drug combinations, medical preparations containing active ingredients, and cells modified by introducing foreign genetic material, etc., and can solve the problem of insufficient response rate and high

Active Publication Date: 2019-12-13
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 immunoregulation function, pharmaceutical composition, cell and application thereof
  • Fusion protein with immunoregulation function, pharmaceutical composition, cell and application thereof
  • Fusion protein with immunoregulation function, pharmaceutical composition, cell and application thereof

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 Applications(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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