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Angiogenesis-inhibiting chimeric proteins and the use

a chimeric protein and angiogenesis technology, applied in the field of gene engineering technology, can solve the problems of short serum half-life, inability to achieve effective serum concentration, short serum half-life, etc., and achieve the effect of substantiating the anti-chimeric effect, effectively inhibiting tumor growth, and prolonging animal li

Inactive Publication Date: 2010-08-26
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The chimeric proteins demonstrate high affinity for VEGF, effectively blocking its activity and inhibiting vascular endothelial cell proliferation, leading to significant anti-tumor activity and prolonged animal survival in cancer models.

Problems solved by technology

In addition, rapid growth of tumor cells often leads to hypoxia inside the tumor, and hypoxia further induces expression of VEGF.
The soluble FLT-1 could effectively inhibit growth of vascular endothelial cells in vitro, but it has a short serum half-life and can not reach effective serum concentration.
Similarly, the soluble KDR was also able to inhibit growth of vascular endothelial cells in vitro, but its anti-tumor activity in animal models was not satisfactory (Yoko Hasumi: Soluble FLT-1 Expression Suppresses Carcinomatous Ascites in Nude Mice Bearing Ovarian Cancer.

Method used

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  • Angiogenesis-inhibiting chimeric proteins and the use
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  • Angiogenesis-inhibiting chimeric proteins and the use

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

Cloning of the DNA Sequences Encoding the Chimeric Proteins and Construction of the Recombinant Vectors

[0047]Other than the immunoglobulin Fc coding DNA sequence, coding DNA sequences of the various chimeric proteins of the invention come from cDNAs of FLT-1 and KDR. Since FLT-1 and KDR are mainly expressed in vascular endothelial cells, the total RNA were extracted from human umbilical vein endothelial cells (HUVEC) using a RNA purification kit (QIAGEN); then cDNAs were synthesized from the RNA using AMV Reverse Transcriptese (Promega); then various FLT-1 and KDR fragments were PCR amplified with different primers; finally the sequences from FLT-1, KDR, and human immunoglobulin Fc (IgG1 Fc) were fused together by PCR to construct recombinant DNA sequences encoding various chimeric proteins. Structures of all six chimeric proteins (including FP1′ of the prior art) are shown in FIG. 1.

example 1

Construction of FP3′ Coding Sequence and Recombinant Vector

[0048]HUVEC cells (Clonetics) were cultured with EGM-2 media (Clonetics) in T-175 flasks. 1×107 cells were collected and subjected to the total RNA extraction using the RNA purification kit from Qiagen, and then cDNA was synthesized using the Invitrogen cDNA kit. The cDNA product was stored at −80° C. until usage.

[0049]PCR utilizing specific primers were used to amplify various FLT-1 and KDR domains from the HUVEC cDNA. Likewise, PCR utilizing specific primers from Lymph Nodes cDNA (BD Clontech) were used to amplify human IgG1 Fc was PCR.

[0050]Primers:

for FLT-1 d2 forward:5′-cctttcgtagagatgtacagtga-3′;(SEQ ID NO: 8)for FLT-1 d2 reverse:5′-tatgattgtattggtttgtccat-3′;(SEQ ID NO: 9)for KDR d3-4 forward:5′-gatgtggttctgagtccgtctca-3′;(SEQ ID NO: 10)for KDR d3-4 reverse:5′-cggtgggacatacacaaccaga-3′;(SEQ ID NO: 11)for human IgG1 Fc forward:5′-gacaaaactcacacatgcccact-3′;(SEQ ID NO: 12)andfor human IgG1 Fc reverse:5′-tcatttacccggagac...

example 2

Construction of FP1′ Gene and Recombinant Vector

[0053]FP1′ was constructed similarly as in Example 1. The only difference was that the targeted recombinant DNA was constructed by fusing together the 2nd Ig-like domain of FLT-1, the 3rd Ig-like domain of KDR, and the same human IgG1 Fc as in Example 1.

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Abstract

The present invention is directed to DNA sequence encoding angionenesis-inhibiting recombinant chimeric protein, the chimeric protein per se, the pharmaceutical use of the chimeric protein, and to the pharmaceutical composition containing the recombinant protein and the formulation thereof.

Description

[0001]This application is a divisional application of U.S. patent application Ser. No. 11 / 628,735 filed Dec. 7, 2006, which was filed under 35 U.S.C. 371 as a national stage of PCT / CN2005 / 000802, with an international filing date of Jun. 8, 2005, and claims priority to patent application CN 200410044965.7 filed Jun. 8, 2004, the content of each of which is hereby incorporated by reference in its entirety.FIELD OF INVENTION[0002]The present invention relates to gene engineering technology, more specifically to DNA sequences encoding angiogenesis-inhibiting recombinant chimeric proteins, the encoded chimeric proteins herein, therapeutic applications thereof, medical composition and formulation containing the chimeric proteins.BACKGROUND OF INVENTION[0003]Angiogenesis is a process of growing new blood vessels from existing blood vessels. Most adult vascular system is quiescence, angiogenesis only occurs in some physiological and pathological mechanisms, such as tumor, diabetic retinopa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K14/475C07H21/04A61P3/10A61P7/06A61P19/02A61P27/02A61P35/00C12N15/63C12N5/10C12N1/21C12N1/19A61K38/00A61K38/18C07K14/47C07K14/705C07K14/71C12N15/09C12N15/11
CPCA61K38/00C07K14/4703C07K2319/30C07K2319/00C07K14/705A61P15/08A61P19/02A61P27/02A61P3/10A61P35/00A61P43/00A61P7/06A61P9/10C07K14/71A61K38/18C12N5/10
Inventor FANG, JIANMINLIU, ZHENGYU, DECHAO
Owner CHENGDU KANGHONG BIOTECH
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