Patent Application: US-84752401-A

Abstract:
a method for activating only the vascular endothelial growth factor receptor - 3 has been created . the method comprises administration of a composition comprising a polypeptide to an animal wherein the composition has the ability to stimulate one or more lymphatic vessel endothelial cells to proliferate , differentiate , migrate , or survive . methods are also provided to selectively activate a vegf receptor - 3 , to screen for neoplastic disease characterized by an increase in lymph vessel endothelial cells , and to identify lymph vessel endothelial cells .

Description:
to test the receptor binding properties of mouse vegf - d , a plasmid was constructed for expression of the vegf homology domain ( vhd ) of mouse vegf - d . a dna fragment encoding amino acid residues 92 to 201 ( seq id no : 6 ) of full - length mouse vegf - d2 ( seq id no : 4 ) was amplified by polymerase chain reaction ( pcr ) with pfu dna polymerase , using as template a plasmid comprising full - length mouse vegf - d cdna ( seq id nos : 1 or 3 ). 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 ). the pefbossflag vector contains dna encoding the signal sequence for protein secretion from the murine interleukin - 3 ( il - 3 ) gene and the flag ® octapeptide ( ibi / kodak ). the flag ® octapeptide can be recognized by commercially available antibodies such as the m2 monoclonal antibody ( ibi / kodak ). the vegf - d pcr fragment was inserted into the vector such that the il - 3 signal sequence was immediately upstream from the flag ® octapeptide , which was in turn immediately upstream from the truncated vegf - d sequence . all three sequences were in the same reading frame , so that translation of mrna resulting from transfection of pefbossflag - mousevegf - dδnδc into mammalian cells would give rise to a protein which would have the il - 3 signal sequence at its n - terminus , followed by the flag ® octapeptide and the truncated vegf - d sequence . cleavage of the signal sequence and subsequent secretion of the protein from the cell would give rise to a vegf - d polypeptide which is tagged with the flag ® octapeptide adjacent to the n - terminus . this protein was designated mouse vegf - dδnδc . the expression cassette encoding the flag - tagged truncated vegf - d construct was subcloned into the papex - 3 expression vector and then transiently expressed in 293ebna - 1 cells using fugene ( boehringer mannheim ) mediated transfection . after seven days of incubation , the conditioned medium was collected ( approximately 150 ml ) and subjected to affinity chromatography using the m2 ( anti - flag ) beads ( ibi / kodak ) according to the manufacturer . mouse vegf - dδnδc arising from affinity chromatography and purified human vegf - dδnδc ( for comparison purposes ) were analyzed by western blotting . about 50 ng of each protein was separately combined with sds - page sample buffer under reducing ( 2 % - mercaptoethanol ) conditions , boiled and resolved by sds - page . the proteins were transferred to nitrocellulose and blotted with m2 antibody . as seen in fig2 both human and mouse vegf - dδnδc subunits have the expected molecular weight of 22 kda . the capacity of mouse vegf - dδnδc to bind and cross - link vegfr - 2 and vegfr - 3 was tested in bioassays . fig3 and 4 shows the results of analysis of mouse vegf - dδnδc protein using a vegfr - 2 /- 3 bioassay , respectively . the bioassay was performed using ba / f3 cells which express a chimeric receptor consisting of the extracellular domain of mouse vegfr - 2 or human vegfr - 3 and the transmembrane and cytoplasmic domains of the mouse erythropoietin receptor ( epor ). these cell lines die in the absence of il - 3 , unless they are supplied with ligands that cross - link the chimeric receptors . cross - linking of the vegfr / epor chimeric receptors induces signaling from the epor cytoplasmic domains that stimulates cell survival and proliferation . the cells were maintained in dulbecco &# 39 ; s modified eagle medium ( dmem ) containing 10 % fetal bovine serum ( fbs ), 50 mm l - glutamine , 50 μg / ml gentamicin and 10 % of the walter and eliza hall institute of medical research ( wehi )- 3d - conditioned medium as a source of interleukin - 3 ( il - 3 ) cells expressing the vegfr - 2 - epor or vegfr - 3 - epor chimeric receptor were washed three times in phosphate buffered saline ( pbs ), and once in complete medium lacking il - 3 . cells ( 10 4 ) were aliquoted into 96 - well microtiter plates containing dilutions of the test reagent or medium alone . cells were incubated for 48 hours at 37 ° c . in a humidified atmosphere of 5 % co 2 . cell proliferation was quantified by the addition of 1 μci of 3 h - thymidine for four hours prior to harvesting . incorporation of 3 h - thymidine was determined using a cell harvester and β - counting . as mentioned above , activation of the chimeric receptor rescues the cells from their dependence on il - 3 and causes the cells to proliferate in the absence of il - 3 . human vegf - dδnδc which is a ligand for both vegfr - 2 and vegfr - 3 , stimulates growth of these cell lines in a specific and dose - dependent fashion ( see fig3 and 4 , respectively ). mouse vegf - dδnδc was able to simulate growth of vegfr - 3 / epor cell line in a specific and dose - dependent fashion , but had no significant effect on the vegfr - 2 / epor cell line even at a concentration as high as 4 μg / ml ( see fig4 and 3 , respectively ). assays were carried out in duplicate and error bars denote a standard deviation of +/− 1 . 0 . this unexpected finding demonstrates that mouse vegf - dδnδc is not an activating ligand for vegfr - 2 . note that the vegfr - 2 extracellular domain in the chimeric vegfr - 2 / epor receptor expressed in the ba / f3 - vegfr - 2 - epor cell line was derived from mouse vegfr - 2 . therefore the inability of mouse vegf - dδnδc to induce survival and proliferation of these cells was not due to a species difference between this ligand and the extracellular domain of the chimeric receptor . the foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting . since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art , the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof . tagacattta aaatattcaa a atg tat gga gaa tgg gga atg ggg aat atc 111 ctc atg atg ttc cat gtg tac ttg gtg cag ggc ttc agg agc gaa cat 159 leu met met phe his val tyr leu val gln gly phe arg ser glu his gga cca gtg aag gat ttt tct ttt gag cga tca tcc cgg tcc atg ttg 207 gly pro val lys asp phe ser phe glu arg ser ser arg ser met leu gaa cga tct gaa caa cag atc cga gca gct tct agt ttg gag gag ttg 255 ctg caa atc gcg cac tct gag gac tgg aag ctg tgg cga tgc cgg ttg 303 leu gln ile ala his ser glu asp trp lys leu trp arg cys arg leu aag ctc aaa agt ctt gcc agt atg gac tca cgc tca gca tcc cat cgc 351 tcc acc aga ttt gcg gca act ttc tat gac act gaa aca cta aaa gtt 399 ser thr arg phe ala ala thr phe tyr asp thr glu thr leu lys val ata gat gaa gaa tgg cag agg acc caa tgc agc cct aga gag aca tgc 447 gta gaa gtc gcc agt gag ctg ggg aag aca acc aac aca ttc 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