Patent Application: US-92953707-A

Abstract:
vegf - d , a new member of the pdgf family of growth factors , which among other things stimulates endothelial cell proliferation and angiogenesis and increases vascular permeability , as well as nucleotide sequences encoding it , methods for producing it , antibodies and other antagonists to it , transfected or transformed host cells for expressing it , pharmaceutical compositions containing it , and uses thereof in medical and diagnostic applications .

Description:
the invention will now be described in detail by reference to the figures , and to the following non - limiting examples . it has been speculated that no further members of the vegf family will be found , because there are no known orphan receptors in the vegfr family . furthermore , we are not aware of any suggestion in the prior art that other such family members would exist . a computer search of nucleic acid databases was carried out incidentally to another project , using as search topics the amino acid sequences of vegf , vegf - b , vegf - c , and plgf . several cdna sequences were identified by this search . one of these sequences , genbank accession no . h24828 , encoded a polypeptide which was similar in structure to the cysteine - riched c - terminal region of vegf - c . this sequence was obtained from the database of expressed sequence tags ( dbest ), and for the purposes of this specification is designated xpt . the xpt cdna had been isolated from a human cdna library designated “ soares breast 3nbhbst ”, which was constructed using mrna from an adult human female breast tissue . as far as can be ascertained , this sample was normal breast tissue . sequencing of the xpt dna was performed pursuant to the integrated molecular analysis of genome expression consortium ( image consortium ), which solicits c dna libraries from laboratories around the world , arrays the cdna clones , and provides them to other organizations for sequencing . the xpt sequence shown in the database was 419 nucleotides long , and encoded an amino acid sequence similar to the c - terminal 100 amino acids of vegf - c , i . e ., approximately residues 250 to 350 , using the numbering system of joukov et al . ( 1996 ). similarly , cysteine - rich regions are found in other proteins , which are entirely unrelated in function to the vegf family , for example , the secreted silk - like protein sp185 synthesized in the salivary glands of the midge chironomus tentans . this protein is encoded by the gene br3 , located in a balbiani ring , a tissue specific chromosome “ puff ” found on polytene chromosomes in the midge salivary gland ( dignam and case : gene , 1990 88 133 - 140 ; paulsson et al ., j . mol . biol ., 1990 211 331 - 349 ). it is stated in joukov et al . ( 1996 ) that the sp185 - like structural motif in vegf - c may fold into an independent domain , which is thought to be at least partially cleaved off after biosynthesis , and that there is at least one cysteine motif of the sp185 type in the c - terminal region of vegf . fig3 of joukov et al . shows that the last two - thirds of the c - terminal cysteine - rich region of vegf - c do not align with vegf or plgf , and in fact could be considered a c - terminal extension of vegf - c which is not present in vegf or plgf . the sequence encoded by xpt is similar to this extension . as the xpt cdna was truncated at its 5 ′ end , it was not possible to deduce or predict any amino acid sequence for regions n - terminal to the cysteine - rich domain . thus the portion of vegf - c which is similar to the xpt - derived sequence does not extend to regions of vegf - c which are conserved among other members of the vegf family . as described above , it was not possible to predict whether the n - terminal region of the polypeptide encoded by a full - length xpt nucleic acid ( as distinct from the truncated xpt cdna reported in dbest ) would show any further homology to any member of the vegf family , in particular vegf - c , which has a further n - terminal 250 amino acids . for example , the naturally - occurring protein encoded by a full - length xpt nucleic acid could have been the human homolog of the midge salivary gland protein . alternatively , the type of cysteine - rich motif encoded by truncated xpt cdna could be widely distributed among proteins , as are many structural domains . for example , clusters of cysteine residues may be involved in metal binding , formation of intramolecular disulfide bonds to promote accurate protein folding , or format - ion of intermolecular disulfide bonds for assembly of protein subunits into complexes ( dignam and chase , 1990 ). in order to determine whether the truncated xpt cdna was derived from sequences encoding a vegf - related molecule , it was necessary to isolate a much longer cdna . a sample of the xpt cdna reported in dbest was obtained from the american type culture collection , which is a registered supplier of cdna clones obtained by the image consortium . the identity of the xpt cdna was confirmed by nucleotide sequencing , using the dideoxy chain termination method ( sanger et al ., proc . natl . acad . sci . usa , 1977 74 5463 - 5467 ). the xpt cdna was used as a hybridization probe to screen a human breast cdna library , which was obtained commercially from clontech . one positive clone was isolated , and this clone was then sequenced on both strands . the nucleotide sequence was compiled , and an open reading frame was identified . the nucleic acid sequence is set out in seq id no : 1 . the polypeptide encoded by this sequence was designated vegf - d , and its deduced amino acid sequence , designated seq id no : 3 , is set out in fig3 . in fig3 , putative sites of n - linked glycosylation , with the consensus sequence n — x — s / t in which x is any amino acid , are indicated by the boxes . the amino acid sequence of vegf - d was compared with those of human vegf - a 165 , vegf - b , vegf - c , and plgf . these comparisons are set out in fig1 a to d , respectively . the degree of sequence homology was calculated , and if gaps in sequence introduced for the purposes of alignment are not considered in the calculation , vegf - d is 31 % identical to vegf , 48 % identical to vegf - c , 28 % identical to vegf - b , and 32 % identical to plgf . thus , the most closely - related protein identified was vegf - c . computer searches of the genbank , embl and swissprot nucleic acid databases did not reveal any protein sequences identical to vegf - d . as expected from the sequence alignment referred to above , the most closely related protein found in these databases was vegf - c . searches of dbest were also performed , but did not reveal any sequences encompassing the entire coding region of vegf - d . the sequence of vegf - d is unrelated to that of tie - 2 ligand 1 as disclosed in wo 96 / 11269 . it is important to bear in mind that the only homologies detected were at the level of the amino acid sequence . thus , it would not have been possible to isolate the cdna or gdna encoding vegf - d by methods such as low - stringency hybridization with a nucleic acid sequence encoding another member of the vegf family . vegf - d appears to be most closely related to vegf - c of all the members of the vegf family . because the vegf - d amino acid sequence includes the cysteine - rich sp185 - like motif which is found in vegf - c , the polypeptide of the invention may play an import - ant functional role in lymphatic endothelia . while we do not wish to be bound by any proposed mechanism , it is thought that vegf - c and vegf - d may constitute a silk - like matrix over which endothelial cells can grow . lymphatic vessels have no basement membrane , so the silk - like matrix can form a basement membrane - like material . this may be important in promoting cell growth and / or in cell differentiation , and may be relevant to cancer , especially metastasis , drug therapy , cancer prognosis , etc . the cdna sequence of vegf - d was used to predict the deduced amino acid sequence of vegf - d , the biochemical characteristics of the encoded polypeptide , including the numbers of strongly basic , strongly acidic , hydrophobic and polar amino acids , the molecular weight , the isoelectric point , the charge at ph 7 , and the compositional analysis of the whole protein . this analysis was performed using the protean protein analysis program , version 1 . 20 ( datastar ). these results are summarized in tables 1 and 2 below . table 1 also shows the codon usage . this analysis predicts a molecular weight for the unprocessed vegf - d monomer of 37 kilodaltons ( kd ), compared to the experimentally determined values ( foe the fully processes peptides ) of 20 to 27 kd for vegf - a monomers , 21 kd for the vegf - b monomer and 23 kd for the vegf - c monomer . the original isolation of a cdna for vegf - d , described in example 2 involved hybridization screening of a human breast cdna library . as only one cdna clone for vegf - d was thus isolated , it was not possible to confirm the structure of the cdna by comparison with other independently isolated vegf - d cdnas . the work described in this example , which involved isolation of additional human vegf - d cdna clones , was carried out in order to confirm the structure of human vegf - d cdna . in addition , mouse vegf - d cdna clones were isolated . two cdna libraries which had been obtained commercially from stratagene , one for human lung and one for mouse lung ( catalogue numbers 937210 and 936307 , respectively ) were used for hybridization screening with a vegf - d cdna probe . the probe , which spanned from nucleotides 1817 to 2495 of the cdna for human vegf - d described in example 2 , was generated by polymerase chain reaction ( pcr ) using a plasmid containing the vegf - d cdna as template and the following two oligonucleotides : approximately two million recombinant bacteriophage were screened with this probe from each of the two cdna libraries . nine human and six mouse cdna clones for vegf - d were subsequently isolated . two of the nine human cdna clones for vegf - d were sequenced completely using the dideoxy chain termination method ( sanger et al . proc . natl . acad . sci . usa , 1977 74 5463 - 5467 ). the two cdnas contained the entire coding region for human vegf - d , and were identical except that one of the clones was five nucleotides longer than the other at the 5 ′- terminus . the nucleotide sequence of the shorter cdna is shown in fig4 , and is designated seq id no : 4 . the amino acid sequence for human vegf - d ( hvegf - d ) deduced from this cdna was 354 residues long , and is shown in fig5 ; this is designated seq id no : 5 . the sequences of the 5 ′ regions of five of the other human vegf - d cdna clones were also determined . for each clone , the sequence that was characterized contained more than 100 nucleotides of dna immediately downstream from the translation start site of the coding region . in all cases , the sequences of these regions were identical to corresponding regions of the human vegf - d cdna shown in fig4 . all six mouse cdna clones for vegf - d were sequenced completely . only two of the clones contained an entire coding region for vegf - d ; the other clones were truncated . the nucleotide sequences of the two clones with an entire coding region are different , and encode amino acid sequences of different sizes . the longer amino acid sequence is designated mvegf - d1 , and the shorter sequence is designated mvegf - d2 . the nucleotide sequences of the cdnas encoding mvegf - d1 and mvegf - d2 are shown in fig6 and 7 respectively . the deduced amino acid sequences for mvegf - d1 and mvegf - d2 are shown in fig8 . these sequences are respectively designated seq id no : 6 , seq id no : 7 , seq id no : 8 and seq id no : 9 . the differences between the amino acid sequences are : i ) an insertion of five amino acids ( dfsfe ) a hater residue 30 in mvegf - d1 in comparison to mvegf - d2 ; ii ) complete divergence of the c - terminal ends after residue 317 in mvegf - d1 and residue 312 in mvegf - d2 , which results in mvegf - d1 being considerably longer . three of the four truncated cdnas for mouse vegf - d encoded the c - terminal region , but not the n - terminal 50 amino acids . all three of these cdnas encoded a c - terminal end for vegf - d which is identical to that for mvegf - d2 . the other truncated cdna encoded only the n - terminal half of vegf - d . the amino acid sequence deduced from this cdna contained the five amino acids dfsfe immediately after residue 30 found in mvegf - d1 , but not in mvegf - d2 . as described above , the entire sequence off the human vegf - d cdna clone reported in this example has been validated by comparison with that for a second human clone . in addition , the sequence of the 5 ′ end off the coding region was found to be identical in five other human vegf - d cdna clones . in contrast , the sequence reported in example 2 contained most of the coding region for vegf - d , but was incorrect near the 5 ′- end of this region . this was probably because the vegf - d cdna was truncated near the 51 - end of the coding region and at that point had been ligated with another unidentified cdna , and consequently the first 30 codons of the true coding sequence for vegf - d had been deleted and replaced with a methionine residue . this methionine residue was defined as the n - terminal amino acid of the vegf - d sequence presented in example 2 . the n - terminal regions of the deduced amino acid sequences of mouse vegf - dl and vegf - d2 are very similar to that deduced for human vegf - d ( see fig9 ). this also indicates that the correct deduced amino acid sequence for human vegf - d is reported in this example . the n - terminal 25 amino acids of human vegf - d form an extremely hydrophobic region , which is consistent with the notion that part of this region may be a signal sequence for protein secretion . fig1 shows the alignment of the human vegf - d sequence with the sequences of other members of the vegf family of growth factors , namely human vegf 165 ( hvegfl65 ), human vegf - b ( hvegf - b ), human vegf - c ( hvesge - c ), and human placental growth factor ( hplgf ). when gaps in the alignments are ignored for the purposes of calculation , human vegf - d is found to be 31 % identical in amino acid sequence to human vegf 165 , 28 % identical to human vegf - b , 48 % identical to vegf - c and 32 % identical to human plgf . clearly , vegf - c is the member of this family which is most closely - related to vegf - d . the differences in sequence for mouse vegf - d1 and vegf - d2 most probably arise from differential in rna splicing . the c - terminal 41 amino acid residues of vegf - d1 are deleted in vegf - d2 , and are replaced with 9 residues which are not closely related to the vegf - dl sequence . therefore , 4 cysteine residues present near the c - terminus of vegf - d1 are deleted in vegf - d2 . this change may alter the tertiary or quaternary structures of the protein , or may affect the localization of the protein in the cell or the extracellular environment . the c - terminal end of human vegf - d resembles that of mouse vegf - d , not mouse vegf - d2 . the small 5 amino acid insertion after residue 30 in mouse vegf - d1 , which is not present in either mouse vegf - d2 or human vegf - d , may influence proteolytic processing of the protein . vegf - d is highly conserved between mouse and man . eighty - five percent of the amino acid residues of human vegf - d are identical in mouse vecf - d1 . this is likely to reflect conservation of protein function . putative functions for vegf - d have been proposed herein . although we have not found alternative forms of human vegf - d cdna , it is possible that the rna splice variation which gives rise to numerous forms of mrna for mouse vegf - d may also occur in human tissues . a fragment of the human cdna for vegf - d , spanning from nucleotide 1 to 1520 of the sequence shown in fig4 and containing the entire coding region , was inserted into the mammalian expression vector pcdna1 - amp . the vector was used to transiently transfect cos cells by the deae - dextran method as described previously ( aruffo and seed , 1987 ) and the resulting conditioned cell culture media , collected after 7 days of incubation , were concentrated using amicon concentrators ( centricon 10 with a 10 , 000 molecular weight cut off ) according to the manufacturer . the plasmids used for transfections were the expression construct for human vegf - d and , as positive control , a construct made by insertion of mouse vegf - a cdna into pcdna1 - amp . the conditioned media were tested in two different bioassays , as described below , and the results demonstrate that the cos cells did , in fact , express and secrete biologically - active vegf - d . example 7 bioassay for capacity of vegf - d to bind to vegf receptor - 2 as shown in example 5 , vegf - d is closely related in primary structure to other members of the vegf family . most members of this protein family are mitogenic and / or chemotactic for endothelial cells ( keck et al . 1989 ; leung et al ., 1989 ; joukov , et al ., 1996 ; olofsson et al ., 1996 ) in addition , vegf - a ( previously known as vegf ), the first member of the vegf family to be described ire the literature , is a potent inducer of vascular permeability ( keck et al ., 1989 ). as protein structure is an important determinant of protein function , it seemed likely that vegf - d might also be mitogenic for endothelial cells or induce vascular permeability . therefore human vegf - d was tested in a bioassay for its capacity to bind to vegf receptor - 2 ( vegfr2 ; also known as flk - 1 ), an endothelial cell - specific receptor which , when activated by vegf - a , is thought to give rise to a mitogenic signal ( strawn et al ., 1996 ). a bioassay for detection of growth factors which bind to vegfr2 has been developed in the factor - dependent cell line ba / f3 , and is described in our earlier patent application , no . pct / us95 / 16755 . these cells grow in the presence of interleukin - 3 ( il - 3 ); however , removal of this factor results in cell death within 48 hours . if another receptor capable of delivering a growth stimulus is transfected into the ba / f3 cells , the cells can be rescued by the specific growth factor which activates that receptor when the cells are grown in medium lacking il - 3 . in the specific case of receptor - type tyrosine kinases ( e . g ., vegfr2 ), chimeric receptors containing the extracellular domain of the receptor tyrosine kinase and the transmembrane and cytoplasmic domains of the erythropoietin receptor ( epor ) can be utilized . in this case stimulation with the ligand ( e . g ., vegf ), which binds to the extracellular domain of the chimeric receptor , results in signalling via the epor cytoplasmic domain and subsequent rescue of the cell line in growth medium lacking il - 3 . the construction of the chimeric receptor used in this study , consisting of the mouse vegfr2 extracellular domain and the mouse epor transmembrane and cytoplasmic domains , and the bioassay itself , are described below . to obtain a plasmid construct with which dna encoding the extracellular domain of mouse vegfr2 could easily be ligated with dna encoding other protein domains , site - directed mutagenesis was used to generate a bglii restriction enzyme site at the position of mouse vegfr2 cdna which encoded the junction of the extracellular domain and the transmembrane domain . the full - length clone of the mouse vegfr2 cdna described by oelrichs et al . ( 1993 ) was subcloned into the mammalian expression vector pcdna1 - amp , using the bstxi restriction enzyme site . single stranded utp + dna was generated using the m13 origin of replication , and this was used as a template to generate mouse vegfr2 cdna containing the bglii site at the desired position . the plasmid containing the altered vegfr2 cdna was designated pvegfr2bgl . dna fragments encoding the transmembrane and cytoplasmic domains of any receptor can be inserted at the bglii site of pvegff2bgl in order to generate chimeric vegfr2 receptors . the mouse epor cdna was subcloned into the expression vector pcdna1 - amp , and single stranded dna was generated as a template for mutagenesis . a bglii restriction enzyme site was inserted into the epor cdna at the position encoding the junction of the transmembrane and extracellular domains of the epor to allow direct ligation of this dna fragment to the modified cdna encoding the extracellular domain of vegfr2 in pvegfr2bgl . in addition , a bglii site in the cytoplasmic domain or the epor was removed by a silent single nucleotide substitution . the dna fragment encoding the transmembrane and cytoplasmic domains of epor was then used to replace the portion of pvegfr2bgl encoding the transmembrane and cytoplasmic domains of vfgfr2 . thus a single reading frame was generated which encoded the chimeric receptor consisting of the vegfr2 extracellular domain and the epor transmembrane and cytoplasmic domains . the dna fragment encoding the chimeric receptor was subcloned into the expression vector pbos , and co - transfected into the ba / f3 cell line with plasmid pgk - neo at a ratio of 1 : 20 . cells expressing the vegfr2 - epor protein were selected by flow cytometry analysis using a monoclonal antibody to the vegfr2 extracellular domain ( mab 4h3 ). this monoclonal antibody is described in australian patent application no . pm 3794 , filed 10 feb . 1994 . cell lines expressing higher levels of vegfr2 - epor were selected by growing the cells in 5 μg / ml mab 4h3 or 25 ng / ml of recombinant vegf . a cell line expressing high levels of vegfr2 - epor , designated ba / f3 - nyk - epor , was used for the bioassay . the ba / f3 - nyk - epor cells described above were washed three times in pbs to remove all il - 3 and resuspended at a concentration of 1000 cells per 13 . 5 μl of culture medium and 13 . 5 μl was aliquoted per well of a 60 - well terasaki plate . conditioned media from transfected cos cells were then diluted into the cell culture medium . cells expressing a chimeric receptor consisting of the extracellular domain of the endothelial cell receptor tie2 and the transmembrane and cytoplasmic domains of epor were used as a non - responding control cell line . cells were incubated for 48 - 96 hours , during which the cells incubated in cell culture medium alone had died and the relative survival / proliferation seen in the other wells ( i . e ., in the presence of cos cell - conditioned media ) was scored by counting the viable cells present per well . the conditioned medium from cos cells which had been transiently transfected with expression plasmids was concentrated 30 - fold and used in the vegfr2 bioassay . concentrated conditioned medium from cos cells transfected with pcdna1 - amp was used as negative control . the results are shown in fig1 , with the percentage of 30 - fold concentrated cos cell - conditioned medium in the incubation medium ( vol / vol ) plotted versus the number of viable cells in the well after 48 hours of incubation . clearly , the conditioned medium containing either vegf - a or vegf - d was capable of promoting cell survival in this assay , indicating that both proteins can bind to and activate vegfr2 . human vegf - d , prepared as in example 6 and concentrated 30 - fold , was tested in the miles vascular permeability assay ( miles and miles , 1952 ) performed in anaesthetized guinea pigs ( albino / white , 300 - 400 g ). concentrated conditioned medium for cos cells transfected with pcdna1 - amp was again used as a negative control . guinea pigs were anaesthetized with chloral - hydrate ( 3 . 6 g / 100 ml ; 0 . 1 ml per 10 g of body weight ). the backs of the animals were then carefully shaved with clippers . animals were given an intracardiac injection of evans blue dye ( 0 . 5 % in mt pbs , 0 . 5 ml ) using a 23g needle , and were then injected intra - dermally with 100 - 150 μl of concentrated cos cell - conditioned medium . after 15 - 20 min the animals were sacrificed and the layer of skin on the back excised to expose the underlying blood vessels . for quantitation , the area of each injection was excised and heated to 45 ° c . in 2 - 5 ml of formamide . the resulting supernatants , containing extravasated dye , were then examined spectrophotometrically at 620 nm . for animal 1 , the absorbance at 620 nm arising from injection of 30 - fold concentrated vegf - a conditioned medium was 0 . 178 , that for the 30 - fold concentrated vegf - d conditioned medium was 0 . 114 , and that for 30 - fold concentrated medium from cells transfected with pcdna - 1 - amp was 0 . 004 . for animal 2 , the 30 - fold concentrated media were diluted 4 - fold in cell culture medium before intradermal injection . the absorbance at 620 nm for the vegf - a conditioned sample was 0 . 141 , that for the vegf - d conditioned sample was 0 . 116 , and that for a sample matched for serum content as negative control was 0 . 017 . the enhanced extravasation of dye observed for both animals in the presence of vegf - a or vegf - d demonstrated that both of these proteins strongly induced vascular permeability . the data described here indicate that vegf - d is a secreted protein which , like vegf - a , binds to and activates vegfr2 and can induce vascular permeability . the deduced amino acid sequence for vegf - d includes a central region which is similar in sequence to all other members of the vegf family ( approximately residues 101 to 196 of the human vegf - d amino acid sequence as shown in the alignment in fig1 ). therefore , it was thought that the bioactive portion of vegf - d might reside in the conserved region . in order to test this hypothesis , the biosynthesis of vegf - d was studied , and the conserved region of human vegf - d was expressed in mammalian cells , purified , and tested in bioassays as described below . a dna fragment encoding the portion of human vegf - d from residue 93 to 201 , i . e ., with n - and c - terminal regions removed , was amplified by polymerase chain reaction with pfu dna polymerase , using as template a plasmid comprising full - length human vegf - d cdna . the amplified dna fragment , the sequence of which was confirmed by nucleotide sequencing , was then inserted into the expression vector pefbossflag to give rise to a plasmid designated pefbosvegfdδnδc . the pefbossflag vector contains dna encoding the signal sequence for protein secretion from the interleukin - 3 ( il - 3 ) gene and the flag ™ octapeptide . 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 flags sequence , which was in turn immediately upstream from the vegf - d sequence . all three sequences were in the same reading frame , so that translation of mrna resulting from transfection of pefbosvegfdδnδc 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 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 vegfdδnδc . in addition , a second plasmid was constructed , designated pefbosvegfdfullflag , in which the full - length coding sequence of human vegf - d was inserted into pefbosiflag such that the sequence for the flag ™ octapeptide was immediately downstream from , and in the same reading frame as , the coding sequence of vegf - d . the plasmid pefbosiflag lacks the il - 3 signal sequence , so secretion of the vegf - d / flag fusion protein was driven by the signal sequence of vegf - d . pefbosvegfdfullflag was designed to drive expression in mammalian cells of full - length vegf - d which was c - terminally tagged with the flag ™ octapeptide . this protein is designated vegfdfullflag , and is useful for the study of vegf - d biosynthesis . to examine whether the vegf - d polypeptide is processed to give a mature and fully active protein , pefbosvegfdfullflag was transiently transfected into cos cells ( aruffo and seed , 1987 ). expression in cos cells followed by biosynthetic labeling with 35 s - methionine / cysteine and immunoprecipitation with m2 gel has demonstrated species of approximately 43 kd ( fa ) and 25 kd ( fb ) ( fig1 a ). these bands are consistent with the notion that vegf - d is cleaved to give a c - terminal fragment ( flags tagged ) and an internal peptide ( corresponding approximately to the vegfdδnδc protein ). reduction of the immunoprecipitates ( m2 *) gives some reduction of the fa band , indicating the potential for disulphide linkage between the two fragments . plasmid pefbosvegfdδnδc was used to transiently transfect cos cells by the deae - dextran method as described previously ( aruffo and seed , 1987 ). the resulting conditioned cell culture medium ( approximately 150 ml ), collected after 7 days of incubation , was subjected to affinity chromatography using a resin to which the m2 monoclonal antibody had been coupled . in brief , the medium was run batch - wise over a 1 ml m2 antibody column for approximately 4 hours at 4 ° c . the column was then washed extensively with 10 mm tris - hcl , ph 8 . 0 , 150 my nacl before elution with free flag ™ peptide at 25 μg / ml in the same buffer . the resulting material was used for the bioassays described below . in order to detect the purified vegfdδnδc , fractions eluted from the m2 affinity column were subjected to western blot analysis . aliquots of the column fractions were combined with 2 × sds - page sample buffer , boiled , and loaded onto a 15 % sds polyacrylamide gel . the resolved fractions were transferred to nitrocellulose membrane and non - specific binding sites blocked by incubation in tris / nacl / tween 20 ( tst ) and 10 % skim milk powder ( blotto ). membranes were then incubated with monoclonal antibody m2 or control antibody at 3 μg / ml for 2 h at room temperature , followed by extensive washing in tst . membranes were then incubated with a secondary goat anti - mouse hrp - conjugated antiserum for 1 h at room temperature , followed by washing in tst buffer . detection of the protein species was achieved using a chemiluminescent reagent ( ecl , amersham ) ( fig1 b ). under non - reducing conditions a species of molecular weight approximately 23 kd ( vegfdδnδc ) was detected by the m2 antibody . this is consistent with the predicted molecular weight for this internal fragment ( 12 , 800 ) plus n - linked glycosylation ; vegfdδnδc contains two potential n - linked glycosylation sites . a species of approximately 40 kd was also detected , and may represent a non - covalent dimer of the 23 kd protein ( vegfdδnδc ). the bioassay for the capacity of polypeptides to bind to vegf receptor - 2 is described in detail in example 7 . aliquots of fractions eluted from the m2 affinity column , containing the vegfdδnδc protein , were diluted in medium and tested in the vegfr2 bioassay as previously described . fraction # 3 from the affinity column , which was shown to contain the purified vegfdδnδc protein ( fig1 b ), demonstrated a clear ability to induce proliferation of the bioassay cell line to a dilution of 1 / 100 of the purified fraction ( fig1 ). in comparison , the void volume of the affinity column ( fraction # 1 ) showed no activity , whereas the original vegfdδnδc conditioned medium gave only weak activity . the vascular permeability assay ( miles and miles , 1952 ) is described in brief in example 8 . aliquots of purified vegfdδnδc , and samples of the void volume from the m2 affinity column ( negative control ) were combined with medium and injected intradermally into the skin of guinea pigs . the regions of skin at the sites of injections were excised , and extravasated dye was eluted . the absorbance of the extravasated dye at 620 nm arising from injection of purified vegfdδnδc was 0 . 131 ± 0 . 009 . in comparison , the value for absorbance arising from injection of a sample of the void volume was 0 . 092 ± 0 . 020 . therefore , vegfdδnδc induced vascular permeability , but the effect was only marginal . due to its ability to bind to vegfr2 , and its lower induction of vascular permeability compared to full length vegf - d , vegf - dδnδc may be said to relatively decrease the induction of vascular permeability by vegf - d through competitive inhibition . in this sense , the vegf - dδnδc fragment may be thought of as an antagonist for vegf - d as regards the induction of vascular permeability . two factors have led us to explore internal fragments of vegf - d for enhanced activity . firstly , it is the central region of vegf - d which exhibits amino acid homology with all other members of the vegf family . secondly , proteolytic processing which gives rise to internal bioactive polypeptides occurs for other growth factors such as pdgf - bb . in addition , the activity seen with the full length vegf - d protein in cos cells was lower than for the corresponding conditioned medium from vegf - a transfected cos cells . it was predicted that the mature vegf - d sequence would be derived from a fragment contained within residues 92 - 205 , with cleavage at faa ̂ tfy and iirr ̂ siqi . immunoprecipitation analysis of vegf - dfullflag expressed in cos cells produced species consistent with the internal proteolytic cleavage of the vegf - d polypeptide at these sites . therefore , a truncated form of vegf - d , with the n - and c - terminal regions removed ( vegfdδnδc ), was produced and expressed in cos cells . this protein was identified and purified using the m2 antibody . the vegfdδnδc protein was also detected by the a2 antibody , which recognizes a peptide within the 92 - 205 fragment of vegf - d ( not shown ). vegfdδnδc was evaluated by the vegfr2 bioassay and the miles vascular permeability assay , and shown to bind to and activate the vegfr2 receptor in a bioassay designed to detect cross - linking of the vegfr2 extracellular domain . induction of vascular permeability by this polypeptide in a miles assay was at best marginal , in contrast to the effect of vegf - a . the human vegf - d cdna was cloned into baculovirus shuttle vectors for the production of recombinant vegf - d . in addition to baculoviral shuttle vectors , which contained the unmodified vegf - d cdna ( referred to as “ full length vegf - d ”) two baculoviral shuttle vectors were assembled , in which the vegf - d cdna was modified in the following ways . in one construct ( referred to as “ full length vegf - d - h 6 ”) a c - terminal histidine tag was added . in the other construct the putative n - and c - terminal propeptides were removed , the melittin signal peptide was fused in - frame to the n - terminus , and a histidine tag was added to the c - terminus of the remaining vegf homology domain ( referred to as “ δ nδc - melsp - vegf - d - h 6 ,”). for each of the three constructs , baculoviral clones of two or three independent transfections were amplified . the supernatant of high five ( hf ) cells was harvested 48 h post infection with high titer virus stocks . the supernatant was adjusted to ph 7 with naoh and diluted with one volume of d - mem ( 0 . 2 % fcs ). the samples were tested for their ability to stimulate tyrosine phosphorylation of vegfr - 3 ( flt - 4 receptor ) on nih3t3 cells , as described by joukov et al ., 1996 . the supernatant of uninfected cells and the supernatant of cells infected with the short splice variant of vegf - c , which does not stimulate tyrosine phosphorylation of vegfr - 3 , were used as negative controls . vegf - c modified in the same way as δnδc - melsp - vegf - d - h 6 was used as positive control . the results are shown in fig1 . the appearance of new bands at 125 and 195 kd indicates phosphorylation , and hence activation , of the receptor . modified and unmodified human vegf - d cdna was cloned into baculovirus shuttle vectors for the production of recombinant vegf - d as described in example 10 . for each of the three constructs full length vegf - d , full length vegf - d - h 6 , and δnδc - melsp - vegf - d - h 6 , baculoviral clones of two or three independent transfections were amplified . the supernatant of high five ( hf ) cells was harvested 48 hours post infection with high titer virus stocks . the supernatant was adjusted to ph 7 with naoh and diluted with one volume of d - mem ( 0 . 2 % fcs ). the supernatants conditioned with the histidine - tagged proteins were tested for their ability to stimulate tyrosine phosphorylation of the kdr receptor according to joukov et al ., 1996 . kdr is the human homolog of flkl ( vegfr - 2 ). the supernatant of uninfected cells and the supernatant of cells infected with the vegf - c 156s mutant , which does not stimulate kdr , were used as negative controls . vegf 165 and vegf - c modified in the same way as δnδc - melsp - vegf - d - h 6 were used as positive controls . the results are shown in fig1 . the appearance of a new band at approximately 210 kd indicates phosphorylation , and hence activation , of the receptor . in order to characterize the pattern of vegf - d gene expression in the human and in mouse embryos , vegf - d cdnas were used as hybridization probes for northern blot analysis of polyadenylated human rna and for in situ hybridization analysis with mouse embryos . a 1 . 1 kb fragment of the human vegf - d cdna shown in fig4 ( seq id no : 4 ) spanning from the ecorv site to the 3 ′- terminus ( nucleotides 911 to 2029 ) was labeled with [ α - 32 p ] datp using the megaprime dna labeling system ( amersham ) according to manufacturer &# 39 ; s instructions . this probe was used to screen human multiple tissue northern blots ( clontech ) by hybridization , also according to manufacturer &# 39 ; s instructions . these blots contained polyadenylated rna obtained from tissues of adult humans who were apparently free of disease . autoradiography with the labeled blots revealed that vegf - d mrna was most abundant in heart , lung , and skeletal muscle . vegf - d mrna was of intermediate abundance in spleen , ovary , small intestine , and colon , and was of low abundance in kidney , pancreas , thymus , prostate , and testis . no vegf - d mrna was detected in rna from brain , placenta , liver , or peripheral blood leukocytes . in most of the tissues where vegf - d mrna was detected the size of the transcript was 2 . 3 kb . the only exception was skeletal muscle , where two vegf - d transcripts of 2 . 3 kb and 2 . 8 kb were detected . in skeletal muscle the 2 . 3 kb transcript was more abundant than the 2 . 8 kb transcript . in order to generate an antisense rna probe for mouse vegf - d mrna , the mouse vegf - d2 cdna shown in fig7 ( seq id no : 7 ) was inserted into the transcription vector pbluescriptiiks + ( stratagene ). the resulting plasmid was digested to completion with the restriction endonuclease foki and then used as template for an in vitro transcription reaction with t3 rna polymerase . this transcription reaction gave rise to an antisense rna probe for vegf - d mrna which was complementary in sequence to the region of the vegf - d2 cdna ( fig7 ) from the 3 ′- terminus to the foki cleavage site closest to the 3 ′- terminus ( nucleotides 1135 to 700 ). this antisense rna probe was hybridized at high stringency with paraffin - embedded tissue sections generated from mouse embryos at post - coital day 15 . 5 . hybridization and washing were essentially as described previously ( achen et al ., 1995 ). after washing and drying , slides were exposed to autoradiography film for six days . development of the autoradiography film revealed that vegf - d mrna is localized in the developing lung of post - coital day 15 . 5 embryos . the signal for vegf - d mrna in the lung was strong and highly specific . control hybridizations with sense probe gave no detect able background in lung or any other tissue . the vegf - d gene is broadly expressed in the adult human , but is certainly not ubiquitously expressed . strongest expression was detected in heart , lung and skeletal muscle . in mouse embryos at post - coital day 15 . 5 , strong and specific expression of the vegf - d gene was detected in the lung . these data suggest that vegf - d may play a role in lung development , and that expression of the vegf - d gene in lung persists in the adult , at least in humans . expression of the gene in other tissues in the adult human suggests that vegf - d may fulfill other functions in other adult tissues . some members of the vegf family of proteins , namely vegf - a ( leung et al ., 1989 ) and vegf - b ( olofsson et al ., 1996 ), are mitogenic for endothelial cells . in order to test the mitogenic capacity of vegfdδnδc for endothelial cells , this protein was expressed and purified by affinity chromatography as described in example 9 . fraction # 3 , eluted from the m2 affinity column , which contained vegfdδnδc , was diluted 1 in 10 in cell culture medium containing 5 % serum and applied to bovine aortic endothelial cells ( baes ) which had been propagated in medium containing 10 % serum . the baes had been seeded in 24 - well dishes at a density of 10 , 000 cells per well the day before addition of vegfdδnδc , and 3 days after addition of this polypeptide the cells were dissociated with trypsin and counted . purified vegf - a was include d in the experiment as positive control . results are shown in fig1 . the addition of fraction # 3 to the cell culture medium led to a 2 . 4 - fold increase in the number of baes after 3 days of incubation , a result which was comparable to that obtained with vegf - a . clearly vegfdδnδc is mitogenic for endothelial cells . in order to generate hybridization probes for localization of the vegf - d gene on human chromosomes , a human genomic dna clone for vegf - d was isolated from a human genomic dna library ( clontech ). the genomic library was screened by hybridization with the human vegf - d cdna shown in fig4 , using standard methods ( sambrook et al ., 1989 ). one of the clones thus isolated was shown to contain part of the vegf - d gene by hybridization to numerous oligonucleotides which were derived in sequence from the human vebgf - d cdna . a region of the genomic clone , approximately 13 kb in size , was purified from agarose gel , labeled by nick - translation with biotin - 14 - datp and hybridized in situ at a final concentration of 20 ng / μl to metaphases from two normal human males . the fluorescence in situ hybridization ( fish ) method was modified from that previously described ( callen et al ., 1990 ) in that chromosomes were stained before analysis with propidium iodide ( as counterstain ) and dapi ( for chromosome identification ). images of metaphase preparations were captured by a cooled ccd camera , using the cytovision ultra image collection and enhancement system ( applied imaging int . ltd .). fish signals and the dapi banding pattern were merged for analysis . fifteen metaphases from the first normal male were examined for fluorescent signal . ten of the metaphases showed signal on one chromatid ( 3 cells ) or both chromatids ( 7 cells ) of the x chromosome in band p22 . 1 . there was a total of 9 non - specific background dots observed in these 15 metaphases . a similar result was obtained from hybridization of the probe to 15 metaphases from the second normal male , where signal was observed at xp22 . 1 on one chromatid in 7 cells and on both chromatids in 4 cells . in conclusion , the human vegf - d gene is located on the x chromosome in band p22 . 1 . the mouse chromosomal location of the vegf - d gene was determined by interspecific backcross analysis using progeny generated by mating ( cs7bl / 6j × mus spretus ) f1 females and cb7bl / 67 males as described previously ( copeland and jenkins , 1991 ). this interspecific backcross mapping panel has been typed for over 2401 loci that are well distributed among all the autosomes as well as the x chromosome ( copeland and jenkins , 1991 ). c57bl / 6j and m . spretus dnas were digested with several enzymes and analyzed by southern blot hybridization for informative restrict - ion fragment length polymorphisms ( rflps ) using a 1 . 3 kb mouse vegf - d cdna probe essentially as described ( jenkins et al . 1982 ). fragments of 7 . 1 , 6 . 3 , 4 . 7 , 2 . 5 and 2 . 2 kb were detected in taqi - digested c57bl / 6j dna and major fragments of 7 . 1 , 3 . 7 , 2 . 7 and 2 . 2 kb were detected in taqi - digested m . spretus dna . the presence or absence of the 3 . 7 and 2 . 7 taqi m . spretus - specific fragments , which cosegregated , was followed in backcross mice . the mapping results indicated that the vegf - d gene is located in the distal region of the mouse x chromosome linked to bik , dxpasi and ptmb4 . although 89 mice were analyzed for every marker , up to 133 mice were typed for some pairs of markers . each locus was analyzed in pairwise combinations for recombination frequencies using the additional data . the ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are : centromere - btk - 14 / 121 - dxpasi - 3 / 99 - vegf - d - 5 / 133 - ptmb4 . the recombination frequencies [ expressed as genetic distances in centimorgans ( cm )± the standard error ], calculated using map manager ( version 2 . 6 . 5 ), are - btk - 11 . 6 +/− 2 . 9 - dxpasi - 3 . 0 +/− 1 . 7 - vegf - d - 3 . 8 +/− 1 . 7 - ptmb4 . a description of the probes and rflps for the loci linked to the vegf - d gene , including btk , dxpasi and ptmb4 , has been reported previously ( hacfliger et al ., 1992 ; holloway et al ., 1997 ). we have compared our interspecific map of the x chromosome with a composite mouse linkage map that reports the map location of many uncloned mutations ( provided from mouse genome database , a computerized database maintained at the jackson library , bar harbor , me .). the vegf - d gene mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in the locus for an endothelial cell mitogen . the distal region of the mouse x - chromosome shares a region of homology with the short arm of the human x chromosomes ( mouse genome database ). the placement of the vegf - d gene in this interval in mouse suggests that the human homolog will map to xp22 . this is consistent with our fish analysis which has localized the human gene to xp22 . 1 . numerous disease states are caused by mutations in unknown genes which have been mapped to xp22 . 1 and the positions immediately surrounding this region in the human . these disease states include kallmann syndrome , ocular albinism ( nettleship - falls type ), ocular albinism and sensorineural deafness , partington syndrome , spondyloepiphyseal dysplasia ( late ), retinitis pigmentosa 15 , gonadal dysgenesis ( xy female type ), nance - horan cataract - dental syndrome , retinoschisis , charcot - marie - tooth disease , f - cell production , hypomagnesemia , keratosis follicularis spinulosa decalvans , coffin - lowry syndrome , corneal dermoids , hypophosphatemia , agammaglobulinemia , aicardi syndrome , hereditary hypophosphatemia ii , mental retardation ( non - dysmorphic ), opitz g syndrome , pigment disorder ( reticulate ), dosage - sensitive sex reversal , adrenal hypoplasia , retinitis pigmentosa - 6 , deafness 4 ( congenital sensorineural ) and wilson - turner syndrome . the positions of the genes involved in these disease states are documented in the omim gene map which is edited by dr . victor mckusick and colleagues at johns hopkins university ( usa ). other assays are conducted to evaluate whether vegf - d has similar activities to vegf in relation to endothelial cell function , angiogenesis and wound healing . further assays may also be performed , depending on the results of receptor binding distribution studies . endothelial cell growth assays are performed by methods well known in the art , e . g ., those of ferrara & amp ; henzel ( 1989 ), gospodarowicz et al . ( 1989 ), and / or claffey et al ., biochim . biophys . acta , 1995 1246 1 - 9 . the effect of vegf - d on adhesion of polmorphonuclear granulocytes to endothelial cells is tested . the standard boyden chamber chemotaxis assay is used to test the effect of vegf - d on chemotaxis . endothelial cells are tested for the effect of vegf - d on plasminogen activator and plasminogen activator inhibitor production , using the method of pepper et al . ( 1991 ). the ability of vegf - d to stimulate endothelial cells to migrate and form tubes is assayed as described in montesano et al . ( 1986 ) alternatively , the three - dimensional collagen gel assay described by joukov et al . ( 1996 ) or a gelatinized membrane in a modified boyden chamber ( glaser et al ., 1980 ) may be used . the ability of vegf - d to induce an angiogenic response in chick chorioallantoic membrane is tested as described in leung et al . ( 1989 ). alternatively the rat cornea assay of rastinejad et al . ( 1989 ) may be used ; this is an accepted method for assay of in vivo angiogenesis , and the results are readily transferrable to other in vivo systems . the ability of vegf - d to stimulate wound heal ing is tested in the most clinically relevant model available , as described in schilling et al . ( 1959 ) and utilized by hunt et al . ( 1967 ). a variety of in vitro and in vivo assays using specific cell populations of the haemopoietic system are known in the art , and are outlined below . in particular a variety of in vitro murine stem cell assays using fluorescence - activated cell sorter purified cells are particularly convenient : these are cells capable of repopulating the bone marrow of lethally irradiated mice , and have the lin − , rh h1 , ly - 6a / e + , c - kit + phenotype . vegf - d is tested on these cells either alone , or by co - incubation with other factors , followed by measurement of cellular proliferation by 3h - thymidine incorporation . these are cells that have comparatively little bone marrow repopulating ability , but can generate d13 cfu - s . these cells have the lin − , r h1 , ly - 6a / e + c - kit + phenotype . vegf - d is incubated with these cells for a period of time , injected into lethally irradiated recipients , and the number of d13 spleen colonies enumerated . these are cells that respond in vitro to single growth factors and have the lin − , rh h1 , ly - 6a / e + , c - kit + phenotype . this assay will show if vegf - d can act directly on haemopoietic progenitor cells . vegf - d is incubated with these cells in agar cultures , and the number of colonies present after 7 - 14 days is counted smooth muscle cells play a crucial role in the development or initiation of atherosclerosis , requiring a change of their phenotype from a contractile to a synthetic state . macrophages , endothelial cells , t lymphocytes and platelets all play a role in the development of atherosclerotic plaques by influencing the growth and phenotypic modulations of smooth muscle cell . an in vitro assay using a modified rose chamber in which different cell types are seeded on to opposite coverslips measures the proliferative rate and phenotypic modulations of smooth muscle cells in a multicellular environment , and is used to assess the effect of vegf - d on smooth muscle cells . the ability of vegf - d to inhibit metastasis is assayed using the lewis lung carcinoma model , for example using the method of cao et al . ( 1995 ). the effects of vegf - d on proliferation , differentiation and function of other cell types , such as liver cells , cardiac muscle and other cells , endocrine cells and osteoblasts can readily be assayed by methods known in the art , such as h - thymidine uptake by in vitro cultures . expression of vegf - d in these and other tissues can be measured by techniques such as northern blotting and hybridization or by in situ hybridization . vegf - d is a member of the pdgf family of growth factors which exhibits a high degree of homology to the other members of the pdgf family . vegf - d contains eight conserved cysteine residues which are characteristic of this family of growth factors . these conserved cystine residues form intra - chain disulfide bonds which produce the cysteine knot structure , and inter - chain disulfide bonds that form the protein dimers which are characteristic of members of the pdgf family of growth factors . vegf - d will interact with protein tyrosine kinase growth factor receptors . in contrast to proteins where little or nothing is known about the protein structure and active sites needed for receptor binding and consequent activity , the design of active mutants of vegf - d is greatly facilitated by the fact that a great deal is known about the active sites and important amino acids of the members of the pdgf family of growth factors . published articles elucidating the structure / activity relationships of members of the pdgf family of growth factors include for pdgf : oestman et al ., j . biol . chem ., 1991 266 10073 - 10077 ; andersson et al ., j . biol . chem ., 1992 267 11260 - 1266 ; oefner et al ., embo j ., 1992 11 3921 - 3926 ; flemming et al ., molecular and cell biol ., 1993 13 4066 - 4076 and andersson et al ., growth factors , 1995 12 159 - 164 ; and for vegf : kim et al ., growth factors , 1992 7 53 - 64 ; pötgens et al ., j . biol . chem ., 1994 269 32879 - 32885 and claffey et al ., biochem . biophys . acta , 1995 1246 1 - 9 . from these publications it is apparent that because of the eight conserved cysteine residues , the members of the pdgf family of growth factors exhibit a characteristic knotted folding structure and dimerization , which result in formation of three exposed loop regions at each end of the dimerized molecule , at which the active receptor binding sites can be expected to be located . based on this information , a person skilled in the biotechnology arts can design vegf - d mutants with a very high probability of retaining vegf - d activity by conserving the eight cysteine residues responsible for the knotted folding arrangement and for dimerization , and also by conserving , or making only conservative amino acid substitutions in the likely receptor sequences in the loop 1 , loop 2 and loop 3 region of the protein structure . the formation of desired mutations at specifically targeted sites in a protein structure is considered to be a standard technique in the arsenal of the protein chemist ( kunkel et al ., methods in enzymol ., 1987 154 367 - 382 ) examples of such site - directed mutagenesis with vegf can be found in pötgens et al ., j . biol . chem ., 1994 269 32879 - 32885 and claffey et al ., biochim . biophys . acta , 1995 1246 1 - 9 . indeed , site - directed mutagenesis is so common that kits are commercially available to facilitate such procedures ( e . g . promega 1994 - 1995 catalog ., pages 142 - 145 ). the endothelial cell proliferating activity of vegf - d mutants can be readily confirmed by well established screening procedures . for example , a procedure analogous to the endothelial cell mitotic assay described by claffey et al ., ( biochim . biophys . acta ., 1995 1246 1 - 9 ) can be used . similarly the effects of vegf - d on proliferation of other cell types , on cellular differentiation and on human metastasis can be tested using methods which are well known in the art . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by reference . achen , m . g ., clauss , m ., schnürch , h . and risau , w . differentiation , 1995 59 15 - 24 andersson , m ., östman , a ., bäckstrom , g ., hellman , u ., george - nascimento , c ., westermark , b . and heldin , c - h . j . biol . chem ., 1992 267 11260 - 1266 anderson , m , östman , a ., kreysing , j ., bäckström , g . van de poll , m . and heldin , c - h . growth factors , 1995 12 159 - 164 aruffo , a . and seed , b . proc . natl . acad . sci . usa ., 1987 84 8573 - 8577 callen , d . f ., baker , e ., eyre , h . j ., chemnos , j . e ., bell , j . a . and sutherland , g . r . ann . genet ., 1990 33 219 - 221 claffey , k . p ., senger , d . r ., spiegelman , b . m biochem . biophys . acta , 1995 1246 1 - 9 cao , y . chen , c ., weatherbee , j . a ., tsang , m . and folkman , j . j . exp . med ., 1995 182 2069 - 2077 copeland , n . g . and jenkins n . a . trends genet ., 1991 7 113 - 118 ferrara , n . & amp ; 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