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Bifunctional angiogenesis inhibitor and use thereof

An angiogenesis-inhibiting, dual-function technology, used in epidermal growth factors, cardiovascular system diseases, medical preparations containing active ingredients, etc.

Pending Publication Date: 2020-06-23
REMEGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the course of the research, it was found that the activity of the prepared VF28 was unstable with the prolongation of the storage time. , 1 month, 3 months, and 6 months, the binding activity (ELISA method) analysis was carried out, and the data showed that at each monitoring time point of 1 month, 3 months, and 6 months, there were 1 batch and 2 batches respectively 、The binding activity test results of 3 batches of VF28 stock solution did not meet the requirements of the evaluation standard (caused by the non-compliance of the binding activity of the FGF end); in the accelerated test condition stability test of 3 batches of VF28 finished products, they were placed under the accelerated test conditions of 25°C±2°C 1 month, for cell viability analysis
The results showed that one of the three batches of VF28 finished cell viability test results exceeded the requirements of the evaluation standards and did not meet the requirements of the evaluation standards.

Method used

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  • Bifunctional angiogenesis inhibitor and use thereof
  • Bifunctional angiogenesis inhibitor and use thereof
  • Bifunctional angiogenesis inhibitor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Selection of host cells

[0047] Dihydrofolate reductase (DHFR) gene-deficient Chinese hamster ovary cells (Chinese Hamster OvaryCell, CHO / dhfr-) were purchased from ATCC, USA, catalog number CRL-9096, lot number (Lot number) 3691620. CHO / dhfr-cells were cultured in IMDM complete medium supplemented with hypoxanthin and thymidine (HT) and 10% fetal bovine serum (FBS). The cells were polygonal and adhered to the wall. Nitrogen freezer. In order to obtain CHO / dhfr-cells adapted to serum-free suspension culture, one of the frozen cells was taken, thawed in a water bath at 37°C, and the cells were suspended in Ex-Cell 302CHO medium (Sigma) containing 10% FBS and HT. Let the cells adhere to the wall and grow in the cell culture flask. After the cells grow well, suspend the cells in 30 mL of Ex-Cell 302 CHO medium containing 5% fetal bovine serum, and culture in shake flasks. When the cells grow to 1-2×10 6 / mL, transferred to Ex-Cell 302 CHO cell culture medium...

Embodiment 2

[0048] Example 2: Target gene

[0049] The RC28-05 fusion protein is a fusion protein with bifunctional angiogenesis inhibitory activity, which is composed of partial amino acid sequences of VEGFR and FGFR, fused with the Fc fragment of human immunoglobulin, and its amino acid sequence is as follows (such as SEQ ID NO: 1 shown):

[0050]

[0051] The nucleotide sequence of RC28-05 is as follows (1923bp) (as shown in SEQ ID NO:3):

[0052]

[0053]

[0054] After introducing double restriction sites at both ends of the RC28-05 target gene sequence and inserting a conventional expression vector, the general method was used to transfect CHO cells, and the RC28-05 expression cell line was screened out to express the RC28-05 fusion protein.

Embodiment 3

[0055] Embodiment 3: the affinity experiment of fusion protein

[0056] Use ForteBio Octet (PALL company) to detect the affinity of VF28 and RC28-05, add PBS (pH7.4) to each well of the first column of the test plate A-E row as the balance solution, soak the probe for 10min to activate; dilute RC28 with PBS respectively -05 and VF28 to a concentration of 50nM, add the 2nd column of the A-E row of the 96-well assay plate, set the program as Loading, 300s; add PBS to each well of the 3rd column of the test plate A-E row as the balance solution, set the program as Baseline, 180s ; Using PBS as the diluent, dilute rhVEGF (R&D) and rhFGF (R&D) to concentrations of 500nM and 400nM, respectively, and then serially dilute 3 gradients (total of four gradients) at 1:2 to concentrations of 62.5nM and 50nM , add the serially diluted samples to the 4th column of row A-D of the 96-well plate, and add the diluted solution to the 4th column of E row as a negative control, set the program to A...

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Abstract

The present invention relates to a bifunctional angiogenesis inhibitor having VEGF inhibitory activities and FGF inhibitory activities and capable of inhibiting VEGF and FGF dual factors or high sugar-induced cell proliferation, cell migration and / or lumen formation. The present invention also relates to use of the bifunctional vascular inhibitor for inhibiting retinal angiogenesis, such as diabetic retinopathy, age-related macular degeneration.

Description

technical field [0001] The present invention relates to a bifunctional angiogenesis inhibitor and its use, in particular to a fusion protein with VEGF inhibitory activity and FGF inhibitory activity and its ability to inhibit cell proliferation, cell migration and / or use in lumen formation, such as inhibition of retinal angiogenesis, angiogenesis-related eye diseases, diabetic retinopathy, age-related macular degeneration, etc. Background technique [0002] Fundus neovascularization is a serious complication of various eye diseases. Neovascularization can occur in almost all tissues in the eye, such as the cornea, iris, ciliary body, choroid, retina, macula, and optic disc. It can cause tissue hemorrhage in these parts, A series of pathological changes such as exudation and hyperplasia, thus causing damage to the structure and function of the eyeball, and seriously impairing visual function. This series of fundus diseases include diabetic retinopathy (diabetic retinopathy,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K38/18A61P9/10A61P27/02
CPCC07K14/71C12N15/85A61K38/1825A61K38/1866A61P9/10A61P27/02C07K2319/30A61P27/00C07K14/50C07K14/485C07K2319/00A61K38/00C12N15/62A61K38/179C07K19/00A61K8/64
Inventor 姜静房健民栾雪晶姚雪静王凌
Owner REMEGEN CO LTD
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