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Novel NEUROPILIN/growth factor binding and use thereof

A NP-1, molecular technology, applied in the direction of growth factors/inducing factors, growth factors/growth regulators, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as unresearched effects

Inactive Publication Date: 2001-11-28
LUDWIG INST FOR CANCER RES LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of VEGFR-3 in the development of lymphatic vessels in these mice could not be investigated because the embryo died before the lymphatic system was formed

Method used

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  • Novel NEUROPILIN/growth factor binding and use thereof
  • Novel NEUROPILIN/growth factor binding and use thereof
  • Novel NEUROPILIN/growth factor binding and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Cloning of soluble Neuropilin-1-Ig fusion constructs

[0088] The extracellular domain of murine NP-1 (248-2914 bp of murine Neuropilin cDNA, accession number D50086) was cloned into pIgplus (Ingenius). The 3' part (2738-2914bp) of this extracellular domain was first ligated into pIgplus as an EcoRV-BamHI fragment, and the 5' part (248-2738bp) was then ligated into the pIgplus as an EcoRV fragment (the 5' EcoRV site was derived from the pBluescript KSII vector). carrier. The sequence of the extracellular domain was thus cloned in frame with the pIgplus vector sequence encoding the Fc part of human IgG to generate a fusion protein that could be precipitated from the medium of transfected 293T cells by protein A sepharose, Then SDS-PAGE and silver staining were performed. The results are shown in figure 1 . NP-pIgplus or pIgplus were transfected into cells, and serum-free conditioned medium was collected between 24-32 hours after transfection. Proteins wer...

Embodiment 2

[0089] Example 2: Transfection, immunoprecipitation and soluble receptor binding

[0090] By calcium phosphate precipitation with the encoding soluble receptor Ig-fusion protein VEGFR-1-Ig (Olofsson et al., Proc. The 1-Ig plasmid was transfected into 293-T cells. VEGFR-3 was constructed by amplifying the seven Ig loops of R3, plus placing the Fc portion of human Ig in the vector pREP7.

[0091] 293-T cells were cultured for 24 hours after transfection, washed with Dulbecco's minimal essential medium (DMEM) containing 0.2% bovine serum albumin (BSA), and starved for 24-32 hours. The medium was collected and clarified by centrifugation, and the fusion protein was precipitated with protein A sepharose.

[0092] encoded with hVEGF 165 ,mVEGF-B 167 ,mVEGF-B 186 , hVEGF-C, hVEGF-D, PlGF-1 or PlGF-2 plasmid similarly transfected 293-T or 293EBNA cells, and 24 hours after transfection with 100μCi / ml Pro-mix TML- 35 S (Amersham) metabolically labeled transfected cells for 6-7 hou...

Embodiment 3

[0095] Example 3: Binding of VEGF family members to specific antibodies, NP-1, Flt-1 and Flt-4

[0096] express VEGF-B with 167 ,VEGF-B 186 ,mVEGF-B kEx1-5 (produced by expressing a construct containing exons 1-5 of murine VEGF-B), VEGF 165 , VEGF-C, VEGF-C△N△C (a construct consisting of amino acids 102-225 of VEGF-C constructed as described in Joukov et al., EMBO Journal, 16:3898-911 (1997), VEGF- D, and VEGF-D ΔN ΔC (construct composed of amino acids 93-201 of VEGF-D constructed as described in Achen et al., Proceedings of the American Academy of Sciences 95:548-53 (1998)) cell conditions Medium The general procedure of Example 2 was repeated. The results are shown in Figure 5-8 .

[0097] Figure 5 Ligand VEGF-B 167 and VEGF-B 186 Binding analysis with NP-1-Ig, Flt-1-Ig and Flt-4-Ig, Figure 6 shows ligand mVEGF-B kEx1-5 and VEGF 165 Binding assays to NP-1-Ig, Flt-1-Ig and Flt-4-Ig. In the figure, IP refers to immunoprecipitation of ligand with antibody precipit...

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Abstract

The invention discloses selected from VEGF-B 167 , VEGF-C, VEGF-D, processed VEGF-B 186 Complexes of a protein and its analogues with neuropilin-1 (NP-1) receptors, its extracellular domains or ligand-binding fragments, or analogues thereof. The invention also discloses that the complex is detected with VEGF-B 167 , VEGF-C, VEGF-D, processed VEGF-B 186 Use in the assay of growth factor proteins having substantially the same binding affinity to cell surface receptors, and / or the role of said complex in promoting or antagonizing 167 , VEGF-C, VEGF-D and / or processed VEGF-B 186 mediated cellular responses. The invention also discloses specific binding partners of such complexes, such as antibodies.

Description

Background of the invention [0001] The present invention relates to complexes of Neuropilin (NP-1) receptors and growth factors selected from the group consisting of, and methods of using such complexes to induce or antagonize cellular responses mediated by one or more of said growth factors, and isolated binding partners, such as antibodies that bind to complexes of one or more growth factors of the group consisting of: vascular endothelial growth factor-B and NP-1 167 (VEGF-B 167 ) vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor-D (VEGF-D) and processed forms of vascular endothelial growth factor-B 186 (processed VEGF-B 186 ). [0002] During embryonic development, the primary vascular network is established by in situ differentiation of mesoderm cells in a process known as angiogenesis. All subsequent processes involving embryonic neovascularization and adult neovascularization are believed to be influenced by the initiation and divisio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K38/22A61K47/48A61P9/00A61P17/06A61P27/02A61P35/00A61P43/00C07K14/475C07K14/52C07K14/71C07K14/715C07K16/28C07K19/00C12N5/07C12N5/0783C12P21/08C12Q1/02
CPCC07K14/71C07K2319/00C07K14/52A61P17/06A61P27/02A61P35/00A61P43/00A61P9/00
Inventor 卡里·阿利塔洛乌尔夫·埃里克松布里吉塔·奥洛夫松泰贾·马基嫩
Owner LUDWIG INST FOR CANCER RES LTD
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