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Long-acting human endothelium chalone containing immune globulin Fc segment

A technology of immunoglobulin and endostatin, applied in the direction of peptide/protein components, medical preparations containing active ingredients, medical preparations with non-active ingredients, etc.

Inactive Publication Date: 2013-06-26
BEIJING HANMI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as described in WO 2005 / 047337, the resulting recombinant protein or polypeptide drug only retains about 50% of its in vitro cytological activity at most

Method used

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  • Long-acting human endothelium chalone containing immune globulin Fc segment
  • Long-acting human endothelium chalone containing immune globulin Fc segment
  • Long-acting human endothelium chalone containing immune globulin Fc segment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1.rhES-PEG

[0055] 3.4kDa polyethylene glycol (ALD-PEG-ALD) with aldehyde groups at both ends and recombinant human endostatin rhES (from Jiangsu Xiansheng Mai De Jin Biopharmaceutical Co., Ltd., product batch number 20100305, the sequence is as follows figure 2 shown) was dissolved in 2M sodium acetate buffer at a molar ratio of 15:1, and the reducing agent sodium cyanoborohydride with a final concentration of 20 mM was added to the mixture, and reacted at 4 ° C for 1 h under gentle stirring to make PEG and The N-terminal amino group of rhES is linked.

[0056] The final product of the reaction was purified using Source S (purchased from GE Healthcare, Source 15S, Cat. No. 17-0944-03). The above reaction mixture was diluted 20-fold with 50 mM, pH 6.4 4-morpholineethanesulfonic acid (MES) buffer and loaded. The elution buffer is 50mM MES buffer containing 1M NaCl, and the gradient concentration is used for elution. The gradient is that...

Embodiment 2

[0057] Example 2. Preparation of rhES-PEG-Fc

[0058] The mono-PEGylated rhES purified in Example 1 above was further coupled to the N-terminus of the Fc fragment of the immunoglobulin.

[0059] The Fc fragment (non-glycosylated human IgG4 Fc fragment expressed by Escherichia coli BL21 (DE3), NCBI Gene ID: 3503. Obtained from Beijing Hanmei Pharmaceutical Co., Ltd.) and the rhES-PEG at a molar ratio of 15:1 Dissolve in 50mM, pH5.5 sodium acetate buffer, add the reducing agent sodium cyanoborohydride at a final concentration of 20mM to this mixture, and react at 4°C for 16h under gentle stirring to make the rhES-PEG and Fc fragment The N-terminal coupling.

[0060]The final product of the reaction was purified using Source S (purchased from GE Healthcare, Source 15S, Cat. No. 17-0944-03). The above reaction mixture was diluted 20-fold with 50 mM sodium acetate buffer, pH 5.5, and then loaded. The elution buffer was 50 mM sodium acetate buffer containing 1 M NaCl. Elute with...

Embodiment 3

[0064] Example 3. Determination of anti-vascular endothelial cell migration activity of rhES-PEG-Fc in vitro

[0065] In this example, the anti-migration activity of rhES-PEG-Fc of the present invention on human microvascular endothelial cells (HMVEC) was tested.

[0066] HMVEC cells in the logarithmic growth phase (purchased from ATCC, product number CRL-4025) were starved overnight, and inoculated with 8×10 4 The cells were placed in a culture dish Transwell (purchased from Corning, Cat. No. 3422) for detecting cell migration. The Transwell culture dish is a culture chamber with a permeable support, and the bottom of the chamber is a permeable membrane, that is, the permeable support. In this embodiment, a Transwell culture dish with a PC membrane with a pore size of 8.0 μm is used.

[0067] Establish negative control group (N.C. group, serum-free medium DMEM), positive control group (P.C. group, add 2% fetal bovine serum FBS and 10ng / ml vascular endothelial growth factor ...

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Abstract

The invention relates to a long-acting human endothelium chalone. The long-acting human endothelium chalone is a human endothelium chalone molecule covalently connected with an immune globulin Fc segment by polyethylene glycol. The invention also discloses a preparation and purification method of the long-acting human endothelium chalone and an application of the long-acting human endothelium chalone in preparation of a drug used for treating cancer.

Description

technical field [0001] The invention belongs to the technical field of biological product pharmacy, and relates to a preparation method of long-acting recombinant human endostatin. Background technique [0002] Since Folkman proposed in 1971 that "tumor growth and metastasis are dependent on the formation of new blood vessels" [Folkman J.N Engl J Med 1971; 285: 1182-1186], people have taken the inhibition of tumor angiogenesis as a research object . Endostatin was first discovered in 1997 by O'Reilly et al. in the United States. O'Reilly et al. found that the cell culture medium of murine hemangioendothelioma (EOMA) cultured in vitro could inhibit the proliferation of bovine capillary endothelial cells in vitro, and they purified a new protein from this culture medium. And named endostatin (Endostatin, ES). It has been identified that mouse endostatin is composed of 184 amino acid fragments in the C-terminal region of collagen XVIII [O'Reily M S. et al. US 005854205A]. X...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/08A61K38/39A61K47/48A61P35/00
Inventor 张世奇刘家望杜伯雨马玉芬杨亚平李永平周筠连忠辉文圣焕
Owner BEIJING HANMI PHARMA CO LTD
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