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Novel fusion protein, pharmaceutical composition and preparation method therefor and use thereof

A technology of fusion protein and composition, which is applied in the field of fusion protein to achieve the effects of alleviating the pain of drug administration, significant tumor growth, and reducing the number of times

Active Publication Date: 2015-12-02
DONGGUAN YUNJING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problem that VEGF-Trap and PD-1 antibodies require multiple injections per day in clinical application in the prior art, and provide a VEGF-Trap and PD-1 antibody with both physiological activity and long-term function. A novel fusion protein, and discloses a pharmaceutical composition composed of the novel fusion protein, a preparation method and an application of the novel fusion protein

Method used

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  • Novel fusion protein, pharmaceutical composition and preparation method therefor and use thereof
  • Novel fusion protein, pharmaceutical composition and preparation method therefor and use thereof
  • Novel fusion protein, pharmaceutical composition and preparation method therefor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 protein preparation

[0028] A novel fusion protein, the general structural formula of which is as follows:

[0029] X-linked peptide-Y-linked peptide-IgGFc fragment; wherein, X is VEGF-Trap and its derivatives; Y is a PD-1 antibody fragment and its derivatives or a PD-1 antagonist fragment and its derivatives.

[0030] In this embodiment, X is VEGF-Trap, and its nucleotide sequence is shown in SEQ ID NO: 4; Y is a PD-1 antibody fragment, and its nucleotide sequence is shown in SEQ ID NO: 6; the connecting peptide is the formula ( The amino acid sequence shown by Gly-Gly-Gly-Gly-Ser) n, n in the connecting peptide is 3; the nucleotide sequence of the IgGFc fragment is shown in SEQ ID NO: 16; that is, the gene for synthesizing the fusion protein in this embodiment The nucleotide sequence of the fragment is shown in SEQ ID NO:18.

[0031] The specific synthetic route of the above-mentioned novel fusion protein in this embodiment is as follows:

[0032] 1. Con...

Embodiment 2

[0049] Example 2. VNI Binding Kinetic Analysis

[0050] (1) Binding kinetic analysis of VNI and VEGF-A

[0051] Bind 25 μg / ml Biotin-hVEGF-A to SAsensor through Loading; set the concentration range according to the pre-experiment: 10000, 5000, 1000, 500, 100 nM respectively, and use SampleDiluentbuffer as blank control. Kinetic analysis on the OctetQK detection platform was used to detect the kinetic parameters of hVEGF-A, VNI, VEGF-Trap and PD1 antibodies. The test steps are set as shown in Table 1:

[0052] Detection steps the solution step type detection time (s) 1 Sample Diluent buffer Baseline 120 2 Biotin-hVEGF-A Loading 360 3 Sample Diluent buffer Baseline 120 4 sample Association 720 5 Sample Diluent buffer Dissociation 1800

[0053] The experimental results obtained through the above test steps are shown in Table 2:

[0054] 　 KD (M) kon(1 / Ms) kon Error kdis(1 / s) kdis Error Full R...

Embodiment 3

[0063] Example 3. In vitro biological activity detection of VNI.

[0064] (1) Comparative study on the effect on HUVEC cell proliferation

[0065] HUVEC cells in the logarithmic growth phase were seeded in 96-well culture plates, 3×10 3 cells / well, 100μl / well, 37℃, 5%CO 2 Cultivate for 20 hours; prepare different molar concentrations of VNI, PD-1 antibody and VEGF-Trap (9.7-10000ng / ml) in Base medium, mix with 40ng / ml VEGF respectively, and incubate at 37°C for 2 hours; then inoculate with HUVEC cells 96-well plate, 100 μl / well, 3 replicate wells for each group, 37°C, 5% CO 2 Continue to incubate for 96 hours, add Base medium and complete medium respectively to the negative control group; add CCK-8 reagent, 20 μl / well, incubate at 37°C in the dark for 2 hours, detect the absorbance at 450nm, and the EC50 value curve of the effect of VNI on HUVEC cell proliferation Schematic such as figure 1 As shown, the experimental results are shown in Table 5.

[0066] sampl...

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Abstract

The invention discloses a novel fusion protein, a pharmaceutical composition formed by the novel fusion protein, a preparation method for the novel fusion protein and use of the novel fusion protein. According to novel fusion protein, the structural general formula is X-connecting peptide-Y-connecting peptide-IgG Fc fragment or Y-connecting peptide-X-connecting peptide-IgG Fc fragment, wherein X is VEGF-Trap and derivatives thereof, and Y is a PD-1 antibody fragment and derivatives thereof or a PD-1 antagonist fragment and derivatives thereof. The fusion protein plays roles in suppressing regenerated blood vessels and activating T cellular immunity and plays a role in coordinating and promoting two functions, so that the function of inhibiting tumor growth is more remarkable.

Description

technical field [0001] The present invention relates to a fusion protein, in particular to a novel fusion protein and pharmaceutical composition for inhibiting tumor growth, as well as a preparation method and application of the novel fusion protein. Background technique [0002] Immune escape is a very important step in the progression of tumor progression. Tumor cells grow rapidly by avoiding the killing effect of the human immune system, thereby promoting the occurrence of cancer. The important negative regulatory molecules PD-1, CTLA-4 and TIM-3 on the surface of T cells can all inhibit the immune killing effect of T cells in the process of cancer development. At present, there are clinically available drugs targeting PD-1 and CTLA-4, and these drugs have very significant clinical therapeutic effects. [0003] Programmed cell death 1 (PD-1) receptor is mainly expressed in activated T cells and B cells, and its main function is to inhibit the activation of immune system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/395A61K47/48A61P35/00
Inventor 雷霞张仕琼
Owner DONGGUAN YUNJING BIOTECHNOLOGY CO LTD
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