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Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas

a technology of vascular endothelial growth factor and expression vector, which is applied in the direction of drug composition, extracellular fluid disorder, peptide/protein ingredient, etc., can solve the problem of unidentified ligands for ties, and achieve the effects of stimulating healing, enhancing acceptance and/or healing of skin grafts, and promoting lymphangiogenesis

Inactive Publication Date: 2011-03-10
VEGENICS PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]The polypeptides comprising conservative substitutions, insertions or deletions but which still retain the biological activity of VEGF-D are clearly to be understood to be within the scope of the invention. Persons skilled in the art will be well aware of the methods which can be readily used to generate such polypeptides, for example the use of site-directed mutagenesis, or specific enzymatic cleavage and ligation. The skilled person will also be aware that peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally occurring amino acid or an amino acid analog may retain the required aspects of the biological activity of VEGF-D. Such compounds can be readily made and tested by methods known in the art, and are also within the scope of the invention.
[0041]According to a fifth aspect, the invention provides a method of enhancing the acceptance and / or healing of a skin graft, comprising the step of stimulating angiogenesis and lymphangiogenesis with an effective dose of VEGF-D, or a fragment or analog thereof having the biological activity of VEGF-D.

Problems solved by technology

); however, the ligand for Tie has not yet been identified.

Method used

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  • Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas
  • Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas
  • Expression vectors and cell lines expressing vascular endothelial growth factor d, and method of treating melanomas

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example 1

Cell Lines Stably Expressing VEGF-D Derivatives

[0065]In order to generate cell lines constitutively expressing derivatives of VEGF-D, regions of the human VEGF-D cDNA were inserted into the mammalian expression vector Apex-3 (Evans et al, Mol. Immunol., 1995 32 1183-1195). This vector is maintained episomally when transfected into 293-EBNA human embryonal kidney cells. For expression of VEGF-DΔNΔC, A DNA fragment encoding residues 93 to 201 (SEQ ID NO:1) was amplified by polymerase chain reaction (PCR) with Pfu DNA polymerase, using as template a plasmid comprising full length VEGF-D cDNA (SEQ ID NO: 10). The amplified DNA fragment, the correctness of which was confirmed by nucleotide sequencing, was then inserted into the expression vector pEFBOSSFLAG (a gift from Dr. Clare McFarlane at the Walter and Eliza Hall Institute for Medical Research (WEHI), Melbourne, Australia, as described in Evans, et al., J. Immunol. Methods, 184:123-135 (1995)), to give rise to a plasmid designated p...

example 2

Binding of VEGF-DΔNΔC to Soluble VEGF Receptors

[0070]To further assess the interactions between VEGF-D and the VEGF receptors, VEGF-DΔNΔC was tested for its capacity to bind to soluble immunoglobulin fusion proteins comprising the extracellular domains of human VEGFR-1, human VEGFR-2 and human VEGFR-3. The corresponding fragment of VEGF-C, VEGF-CΔNΔC, was used for comparison. For binding experiments, 293T human embryonal kidney cells were transfected with plasmids encoding the soluble receptor-immunoglobulin fusion proteins VEGFR-1-Ig, VEGFR-2-Ig or VEGFR-3-Ig using the calcium-phosphate (Ca-phosphate) method. In these fusion proteins, the extracellular domain of the relevant VEGF receptor is fused to the Fc portion of human IgG.sub.1. The cells were incubated for 24 hours after transfection, washed with Dulbecco's Modified Eagle's Medium (DMEM) containing 0.2% bovine serum albumin (BSA) and starved for 24 hours. Media were then collected and clarified by centrifugation, and fusion ...

example 3

In Situ Hybridization Studies of VEGF-D Gene Expression in Mouse Embryos

[0073]The pattern of VEGF-D gene expression was studied by in situ hybridization using a radiolabeled antisense RNA probe corresponding to nucleotides 1 to 340 of the mouse VEGF-D1 cDNA, whose sequence is shown in FIG. 4. The antisense RNA was synthesized by in vitro transcription with T3 RNA polymerase and [35S] UTPas. Mouse VEGF-D is fully described in International Patent application PCT / US97 / 14696. This antisense RNA probe was hybridized to paraffin-embedded tissue sections of mouse embryos at post-coital day 15.5. The labeled sections were subjected to autoradiography for 2 days. The resulting autoradiographs for sections hybridized to the antisense RNA and to complementary sense RNA (as negative control) are shown in FIG. 5A-B. In FIG. 5A, “U′ denotes lung and “Sk” denotes skin, and the two tissue sections shown are serial sections. Strong signals for VEGF-D mRNA were detected in the developing lung and as...

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Abstract

This invention relates to expression vectors comprising VEGF-D and its biologically active derivatives, cell lines stably expressing VEGF-D and its biologically active derivatives, and to a method of making a polypeptide using these expression vectors and host cells. The invention also relates to a method for treating and alleviating melanomas or tumors expressing VEGF-D and various diseases.

Description

[0001]This application is a continuation of U.S. patent application Ser. No. 09 / 219,345, filed Dec. 23, 1998, which claims the benefit of U.S. provisional application No. 60 / 087,392, filed May 29, 1998 and also claims the benefit of Australian Patent Application No. PP1131, filed Dec. 24, 1997.BACKGROUND OF THE INVENTION[0002]This invention relates to expression vectors comprising VEGF-D and its biologically active derivatives, cell lines stably expressing VEGF-D and its biologically active derivatives, and to a method of making a polypeptide using these expression vectors and host cells. The invention also relates to a method for treating and alleviating melanomas and various diseases.[0003]Angiogenesis is a fundamental process required for normal growth and development of tissues, and involves the proliferation of new capillaries from pre-existing blood vessels. Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is al...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/18A61P17/02C12N15/09A61K38/00A61K38/19A61P7/00A61P17/00A61P17/06A61P35/00A61P43/00C07K14/52C07K16/22C12N5/10
CPCC07K14/52A61K38/1866C07K2319/00C07K16/22A61P17/00A61P17/02A61P17/06A61P35/00A61P43/00A61P7/00A61P9/00A61K38/18
Inventor ACHEN, MARC G.STACKER, STEVEN ALANALITALO, KARI
Owner VEGENICS PTY LTD
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