Patent Application: US-25465508-A

Abstract:
the present invention relates to truncated egf receptor molecules that exhibit increased binding affinities for egfr ligands such as egf and tgf1 . the present invention also relates to methods of screening for egf receptor ligands and methods of treatment which involve the use of these molecules .

Description:
when used herein the term “ egfr ” is intended to encompass members of the egf receptor family such as erbb1 ( also known as the egf receptor ), erbb2 , erbb3 and erbb4 . in general , egf receptor family molecules show similar domain arrangements and share significant sequence identity , preferably at least 40 % identity . when used herein , the phrase “ full length egfr ectodomain ” refers to the ectodomain consisting of residues 1 - 621 of erbb1 or equivalent residues of other members of the egf receptor family . the amino acid sequence of the full length ectodomain has been previously described ( 13 ). the full length ectodomain contains four sub - domains , referred to as l1 , cr1 , l2 and cr2 , where l and cr are acronyms for large and cys - rich respectively . fig1 a and 1b show a sequence alignment of the full length ectodomains of erbb1 , erbb2 , erbb3 and erbb4 . the cr2 sub - domain of erbb1 , erbb3 and erbb4 consists of the following seven modules joined by linkers of 2 or 3 amino acid residues and bounded by cysteine residues as follows : the results presented herein show that deletions in the cr2 region of the egfr ectodomain unexpectedly increase binding affinity of the ectodomain for egf and / or tgf - α . in light of this information , a person skilled in the art would be able to readily generate a number of candidate truncated ectodomains and screen these candidates for increased ligand affinity and for therapeutic potential . for example , truncated ectodomains may be prepared by recombinant dna technology as described herein or as described previously ( 8 ). alternatively , truncated ectodomains may be prepared by subjecting the full length ectodomain or full length receptor to limited proteolysis as described previously ( 9 ). binding affinity and inhibitor potency may be measured for candidate truncated ectodomains using biosensor technology . truncated egfr ectodomains of the invention may be in a substantially isolated form . it will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated . a truncated ectodomain of the invention may also be in a substantially purified form , in which case it will generally comprise the protein in a preparation in which more than 90 %, e . g . 95 %, 98 % or 99 % of the protein in the preparation is a protein of the invention . in the context of the present invention , the amino acid sequence of the truncated egfr ectodomain may be modified provided that the modification does not adversely affect the binding affinity of the truncated ectodomain for at least one egfr ligand . for example , modified ectodomains may be constructed by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for binding activity . generally , substitutions should be made conservatively ; i . e ., the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced . similarly , when a deletion or insertion strategy is adopted , the potential effect of the deletion or insertion on biological activity should be considered . in order to preserve the biological activity of the truncated ectodomains , deletions and substitutions will preferably result in homologous or conservatively substituted sequences , meaning that a given residue is replaced by a biologically similar residue . examples of conservative substitutions include substitution of one aliphatic residue for another , such as ile , val , leu , met or ala for one another , or substitutions of one polar residue for another , such as between lys and arg ; glu and asp ; or gln and asn . other such conservative substitutions , for example , substitutions of entire regions having similar hydrophobicity characteristics , are well known . moreover , particular amino acid differences between human , murine and other mammalian egfrs is suggestive of additional conservative substitutions that may be made without altering the essential biological characteristics of the truncated egfr ectodomains . modifications encompassed by the present invention also include various structural forms of the primary protein which retain binding affinities . due to the presence of ionizable amino and carboxyl groups , for example , a truncated ectodomain may be in the form of acidic or basic salts , or may be in neutral form . individual amino acid residues may also be modified by oxidation or reduction . the primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties , such as glycosyl groups , lipids , phosphate , acetyl groups and the like , or by creating amino acid sequence mutants . other modifications within the scope of this invention include covalent or aggregative conjugates of the truncated ectodomain with other proteins or polypeptides , such as by synthesis in recombinant culture as n - terminal or c - terminal fusions . for example , the conjugated polypeptide may be a signal ( or leader ) polypeptide sequence at the n - terminal region of the protein which co - translationally or post - translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall ( e . g ., the yeast i - factor leader ). truncated egfr ectodomain fusions may comprise peptides added to facilitate purification or identification of the truncated ectodomain ( e . g ., poly - his ) or to enhance stability or delivery of the ectodomain in vivo . the truncated egfr ectodomains of the present invention may also be fused to the constant domain of an immunoglobulin molecule . for example , a recombinant chimeric antibody molecule may be produced having truncated egfr ectodomain sequences substituted for the variable domains of either or both of the immunoglobulin molecule heavy and light chains and having unmodified constant region domains . this may result in a single chimeric antibody molecule having truncated egfr ectodomains displayed bivalently . such polyvalent forms of the truncated egfr ectodomain may have enhanced binding affinity for egfr ligands . details relating to the construction of such chimeric antibody molecules are disclosed in wo 89 / 09622 and ep 315062 . as tgfα exists as a membrane bound form , the truncated ectodomains of the present invention may be used to target compounds to cancer cells . accordingly , truncated egfr ectodomain fusions may comprise compounds useful in the diagnosis or treatment of cancer cells such as drugs , isotopes or toxins . truncated egfr ectodomain derivatives may also be obtained by cross - linking agents , such as m - maleimidobenzoyl succinimide ester and n - hydroxysuccinimide , at cysteine and lysine residues . the truncated ectodomains may also be covalently bound through reactive side groups to various insoluble substrates , such as cyanogen bromide - activated , bisoxirane - activated , carbonyldiimidazole - activated or tosyl - activated agarose structures , or by adsorbing to polyolefin surfaces ( with or without glutaraldehyde cross - linking ). once bound to a substrate , the truncated ectodomain may be used to selectively bind ( for purpose of assay or purification ) anti - egfr antibodies or egf . it may also be desirable to use derivatives of the ectodomains of the invention that are conformationally constrained . conformational constraint refers to the stability and preferred conformation of the three - dimensional shape assumed by a peptide . conformational constraints include local constraints , involving restricting the conformational mobility of a single residue in a peptide ; regional constraints , involving restricting the conformational mobility of a group of residues , which residues may form some secondary structural unit ; and global constraints , involving the entire peptide structure . it will be appreciated that the truncated egfr ectodomains of the present invention may be used as immunogens , reagents in receptor - based immunoassays , or as binding agents for affinity purification procedures of egf or other binding ligands . truncated egfr ectodomains may be tested for their ability to modulate receptor activity using a cell - based assay incorporating a stably transfected , egf - responsive reporter gene ( 10 ). the assay addresses the ability of egf to activate the reporter gene in the presence of novel ligands . it offers a rapid ( results within 6 - 8 hours of hormone exposure ), high - throughput ( assay can be conducted in a 96 - well format for automated counting ) analysis using an extremely sensitive detection system ( chemiluminescence ). once candidate compounds have been identified , their ability to antagonise signal transduction via the egf - r can be assessed using a number of routine in vitro cellular assays such as inhibition of egf - mediated cell proliferation . ultimately , the efficiency of truncated egfr ectodomains as tumour therapeutics may be tested in vitro in animals bearing tumour isografts and xenografts as described ( 11 , 12 ). truncated egfr ectodomains of the invention ( and substances identified by the assay methods of the invention ) may preferably be combined with various components to produce compositions of the invention . preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition ( which may be for human or animal use ). suitable carriers and diluents include isotonic saline solutions , for example phosphate - buffered saline . the compositions may be formulated , for example , for parenteral , intramuscular , intravenous , subcutaneous , intraocular , oral or transdermal administration . typically , each protein may be administered at a dose of from 0 . 01 to 30 mg / kg body weight , preferably from 0 . 1 to 10 mg / kg , more preferably from 0 . 1 to 1 mg / kg body weight . the routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition . in view of the ability of the truncated egfr ectodomains of the present invention to bind strongly to egfr ligands , the truncated ectodomains will be useful in diagnostic assays for egfr ligands , as well as in raising antibodies to the egfr for use in diagnosis and therapy . in addition , purified truncated egfr ectodomains may be used directly in therapy to bind or scavenge egfr ligands , thereby providing means for regulating the activities of these ligands . in particular , truncated egfr ectodomains of the present invention may be administered for the purpose of inhibiting egf - dependent responses . throughout this specification the word “ comprise ”, or variations such as “ comprises ” or “ comprising ”, will be understood to imply the inclusion of a stated element , integer or step , or group of elements , integers or steps , but not the exclusion of any other element , integer or step , or group of elements , integers or steps . throughout this specification , the term “ consisting essentially of ” is intended to exclude elements which would materially affect the properties of the claimed composition . the invention will now be described in further detail with reference to the following non - limiting examples . construction of plasmids used for the expression of truncated forms of herbb1 ectodomain . the plasmid perbb1 , used in the construction of truncated herbb1 cdnas , comprises nucleotides 167 - 3970 of herbb1 ( 13 ) in the multiple cloning site of plasmid puc18 . coding is in the opposite sense to the lacz α peptide , and the insertion is downstream of the xbai site of puc18 . this plasmid was used later in excision of the truncated constructs for insertion into the mammalian expression vector pee14 ( 14 ). construction of perbb1476 . an initial plasmid containing nucleotides 167 - 3150 of herbb1 was constructed by ligation of a xbai / nsii fragment from pegfr and xbai / psti - cut pbluescript ks +. from this plasmid , a 4 kbp fragment bbsi / bglii fragment ( containing all of pbluescript ks + and nucleotides 167 - 1150 and 2951 - 3150 of egfr ) and a 528 bp bbsi / pvuii fragment ( nucleotides 1151 - 1679 ) were ligated with a 70 bp pcr - derived pvuii and bglii fragment , encoding amino acids 474 - 476 of hegfr , an enterokinase cleavage site and a c - myc epitope tag to facilitate purification . the 70 bp pcr cassette was produced using a similar previous construct ( 15 ) as template . a plasmid for mammalian cell transfection , perbb1476 , was constructed from this plasmid by ligation of a 1 . 6 kbp xbai / ecorv fragment with xbai / smai - cut pee14 . construction of perbb1501 and perbb1513 . in each construction pcr was used with three oligonucleotides to produce a fragment of hegfr cdna ( nucleotides 1121 to 1760 or 1121 to 1797 respectively ), followed by a sequence encoding an enterokinase cleavage site , a c - myc epitope tag and a termination codon . the upstream primer in pcr corresponded to an arbitrary choice of nucleotides 1121 - 1140 of hegfr cdna , while two overlapping downstream primers were used to construct additional sequence adjacent to nucleotide 1760 or 1797 respectively . the pcr products were cloned using the pcr - script vector ( stratagene ). in each case this allowed an apai fragment harbouring the newly constructed sequence beginning at nucleotide 1738 of herbb1 , to be excised for subsequent insertion into the large apai fragment of pegfr ( which included the entire puc18 sequence with herbb1 cdna to nucleotide 1737 ), in order to prepare a plasmid encoding a truncated hegfr with xbai restriction sites adjacent to the coding sequence . from these puc18 - based plasmids the fragments harbouring the truncated herbb1 cdnas were excised by xbai digestion , and inserted into plasmid pee14 at the xbai site to prepare plasmids perbb1501 and perbb1513 respectively for mammalian cell transfection . mutagenesis . the 1 . 7 kbp fragment harbouring the truncated herbb1 cdna of pegfr501 was introduced into m13 mp18 ( 16 ) for mutagenesis . oligonucleotide - directed in vitro mutagenesis , using the usb - t7 gen ™ in vitro mutagenesis kit , was employed to produce three single site mutants of the truncated human serbb1501 ectodomain , with residues glu367 , gly441 and glu472 respectively mutated to lys to match the corresponding residues in chicken erbb1 ( 4 ). clones incorporating the mutations were identified by colony hybridisation ( 17 ) using 32 p - labelled mutagenic oligonucleotide as a probe , and the mutations were confirmed by dna sequence analysis ( 18 ). vehicles for mammalian cell expression were generated for each mutant by excising the 1 . 7 kbp fragment harbouring the mutated serbb1501 cdna from m13 rf - dna by xbai digestion and inserting it into plasmid pee14 ( 16 ) at the xbai site . vector assembly for production of erbb1 , erbb3 and erbb4 fc fusion proteins . dna templates encoding a number of full - length and truncated ectodomain fragments for erbb1 , erbb3 and erbb4 , fused in frame with a genomic fragment coding for the human igg1 fc region together with an n - terminal spacer peptide and a c - terminal epitope tag , were generated using standard molecular techniques . dna templates were assembled in plasmid expression vectors , enabling transcription and the subsequent translation of the encoded fusion protein following transfection into mammalian cells . cell culture , dna transfection and protein analysis . for transient transfection assays , human 293t fibroblasts maintained in dmem plus 10 % fetal calf serum ( fcs ) were transfected with plasmid dna using fugene ( roche molecular biochemicals , sydney , nsw ) according to the manufacturer &# 39 ; s instructions . supernatants were harvested 48 h after transfection , and cell lysates were prepared in np - 40 lysis buffer . to characterise secreted egfr mutants , aliquots of supernatant and lysate were immunoprecipitated with a monoclonal antibody ( 9e10 ) to the c - myc tag , or with mab 225 ( hb - 8508 , american type culture collection ), a conformationally dependent monoclonal antibody recognising the extracellular domain of the herbb1 ( 19 ). immune complexes were collected on protein a - sepharose beads ( zymed laboratories , bioscientific pty . ltd ., gymea , nsw ), fractionated by sds polyacrylamide gel electrophoresis ( 10 % gel ) and transferred to nitrocellulose membranes . truncated hegfr ectodomains and mutants were identified by probing membranes with horseradish peroxidase ( hrp )- conjugated mab9e10 ( roche ), followed by chemilumiscent detection with pierce super signal substrate . stable cell lines expressing erbb1501 were established in the lec8 mutant cell line from chok ( 7 ) using glutamine synthetase as a selectable marker ( 15 ). supernatants from methionine sulfoximine ( msx )- resistant cell colonies were screened for secreted receptor by biosensor analysis ( see below ) or by dot blotting onto nitrocellulose and probing with hrp - mab9e10 . a single cell line was selected for cloning by limiting dilution . lec8 cells expressing erbb1501 protein were cultured in a celligen plus bioreactor ( new brunswick scientific , new jersey , usa ) using 70 g fibra - cell disks carriers with 1 . 7 l working volume . continuous perfusion culture using glutamine - free dmem / f12 medium supplemented with non - essential amino acids , nucleosides and 10 % fcs was maintained for 6 weeks . selection pressure was maintained with 25 μm msx for the duration of the fermentation . perfusion rate was adjusted as required to ensure a residual glucose level of 1 . 0 - 1 . 5 g / l , with a corresponding lactate concentration of 2 . 0 - 2 . 3 g / l . hek 293t cells were transfected with plasmid dna encoding erbb1 , erbb3 and erbb4 fc fusion proteins complexed with fugene ( boehringer ) 24 hours after seeding into six - well plates . twenty four hours later , the culture medium was replaced with serum - free medium , and supernatant harvested 24 - 48 hours later . purification of truncated egfr ectodomains . for biosensor and auc analyses , conditioned medium containing the segfr truncated proteins ( 4 l ) was adjusted to ph 8 . 0 with tris - hcl ( sigma ) containing sodium azide ( 0 . 02 % ( w / v )) ( tbsa ), and particulates removed by centrifugation prior to recovery of c - myc - tagged protein by affinity purification at 4 ° c . on a column of monoclonal antibody 9e10 covalently - bound to agarose , using peptide elution ( 15 ). eluted protein was further purified by size exclusion chromatography on superdex 200 ( hr10 / 30 , amersham pharmacia biotech ) at room temperature using tbsa buffer at a flow rate of 0 . 8 ml / min . protein was detected by absorbance at 280 nm . biacore binding assays . protein - protein interactions were monitored in real time on an instrumental optical biosensor using surface plasmon resonance detection ( biacore 2000 or 3000 , biacore , uppsala , sweden ). recombinant hegf or htgf - i ( gropep , adelaide , australia ) were purified immediately prior to immobilisation by micropreparative rp - hplc using a smart system ( amersham pharmacia biotech ) as described previously ( 20 ). the proteins were immobilised onto the biosensor surface using amine coupling chemistry ( n - hydroxysuccinimide and n - ethyl - n ′- dimethylaminopropyl - carbodiimide ) at a flow rate of 4 μl / min . typically 100 - 200 ru were immobilised equivalent to 0 . 1 - 0 . 2 ng / mm 2 ( 20 ). automated targeting of immobilisation levels was achieved using the biacore 3 . 1 control software ( 21 ). prior to analysis , erbb1621 ( 23 ), erbb1501 and the erbb1501 mutant samples were characterised by micropreparative size exclusion chromatography ( superose 12 3 . 2 / 30 , amersham pharmacia biotech ) to ensure size homogeneity ( 20 ) and pooled fractions were diluted in biacore buffer ( hbs : 10 mm hepes ph 7 . 4 containing 3 . 4 mm edta , 0 . 15 mm nacl and 0 . 005 % ( v / v ) tween 20 ) to the appropriate concentration . typically , samples ( 30 μl ) at concentrations of 10 - 1000 nm were injected sequentially over the sensor surfaces at a flow rate of 5 or 10 μl / min . following completion of the injection phase , dissociation was monitored in biacore buffer at the same flow rate . the sensor surface and sample blocks were maintained at 25 ° c . bound receptor was eluted , and the surface regenerated between injections , using 40 μl of 10 mm hcl . this treatment did not denature hegf or htgf - α immobilised onto the sensor surface , as shown by equivalent signals on re - injection of receptor . kinetic rate constants ( ka , kd ) were determined using the biaevaluation 3 . 02 software ( biacore , http // www . biacore . com / products / eval3 . html ) as described previously ( 22 ), or by global analysis using clamp ( 23 , 24 ). equilibrium binding constants ( ka , kd ) were determined by direct non - linear least squares analysis of the binding data using an equation defining steady state equilibrium ( ka * conc * rmax /( ka * conc * n ); biaevaluation 3 . 1 ). the data was also plotted in scatchard format ( req / nc versus req , where req is the biosensor response at equilibrium , n is the valency and c is the concentration ) ( 25 ). analytical ultracentrifugation . experiments were performed using a beckman xl - a analytical ultracentrifuge ( beckman coulter , inc ., fullerton , calif .) equipped with absorption optics , using an an60 - ti rotor with cells containing quartz windows , as described previously ( 23 ). centrifugation experiments were conducted at 20 ° c . using a sample volume of 100 μl . equilibrium sedimentation distributions obtained at 12 , 000 and 20 , 000 rpm , were monitored at 280 or 290 nm and analysed using the program sedeq1b ( 26 ). the partial specific volume of egf was taken as 0 . 71 ml / g ( 23 ). chemical cross linking . chemically cross - linked erbb1501 dimers were generated by the incubation of segfr501 ( 5 μm ) with megf ( 20 μm ) in 20 mm hepes ph7 . 4 containing 150 mm nacl for 1 h at room temperature followed by the addition of bis ( sulfosuccinimidyl ) suberate ( bs3 , pierce , rockford , ill ., usa ) to a final concentration of 0 . 5 mm and incubation for a further 30 min . the reaction was terminated by the addition of tris - hcl buffer ( ph 7 . 5 ) to a final concentration of 10 mm . monomer - dimer separation was achieved on novex non - reducing sds - page gels ( 10 %). proteins were transferred onto poly ( vinyl difluoride ) ( pvdf ) membranes ( bio - rad , hercules , calif ., usa ) and identified by incubation with anti - egfr mab528 ( 19 ) ( 0 . 5 μg / ml ) followed by horseradish - peroxidase labelled goat anti - mouse igg ( bio - rad ) and ecl detection ( amersham pharmacia biotech ). cell proliferation assays . baf / 3erx cells , a cell line derived from baf / 3 cells transfected with human egfr ( obtained from ludwig institute for cancer research , melbourne ) were washed three times to remove residual il - 3 and resuspended in rpmi 1640 + 10 % fcs . cells were seeded into 96 well plates using a biomek 2000 robotic autosampler ( beckman ) at 2 × 10 4 cells per 200 μl and incubated for 4 h at 37 ° c . in 10 % co 2 . appropriate concentrations of erbb1501 or erbb1621 or the anti - egfr monoclonal antibody mab528 , were added to the first titration point and titrated in two - fold dilutions across the 96 well plate in duplicate with or without a constant amount of megf ( 207 pm ). 3 h - thymidine ( 0 . 5 μci / well ) was added and the plates were incubated for 20 h at 37 ° c . in 5 % co 2 . the cells were then lysed in 0 . 5 m naoh at room temperature for 30 min before harvesting onto nitrocellulose filter mats using an automatic harvester ( tomtec , conn ., usa ). the mats were dried in a microwave , placed in a plastic counting bag and scintillant ( 10 ml ) was added . 3 h - thymidine incorporation was determined using an automated beta counter ( 1205 betaplate , wallac , finland ). ligand binding assays . the wells of a 96 - well lumitrac 600 plate ( greiner ) were coated with protein g ( 2 ug / ml in 10 mm sodium citrate buffer , ph 9 . 6 ; sigma ). the wells were subsequently blocked with 0 . 5 % ovalbumin / tris - buffered saline ( tbs ). culture supernatant from cell transfectants was added to the wells and incubated overnight at 4 ° c . to allow the binding of fc fusion proteins to protein g . wells were washed and a cocktail of a fixed concentration of europium ( eu )- labelled ligand ( egf or hrgβ , depending on the fusion protein ), together with varying concentrations of unlabelled competitor ligand , added to each well in ligand - binding buffer . after incubation at room temperature , wells were aspirated , washed in tbs plus 0 . 05 % tween - 20 , and enhancement solution ( perkin - elmer ) added to each well for 20 minutes . samples were then assayed for time - resolved fluorescence ( trf ) using a wallac victor2 1420 multilabel counter . preliminary analysis of conditioned media from cells transiently expressing erbb1476 , erbb1501 and erbb1513 showed that only the latter two truncated receptors gave detectable binding to hegf immobilised on the biacore biosensor . stably transfected lec8 cells expressing erbb1501 , were generated and used to produce truncated receptor protein at a yield of ˜ 1 . 8 mg / l of fermentation medium for physical - chemical characterisation . erbb1501 purified from a mab9e10 anti - c - myc peptide affinity column using peptide elution showed a single symmetrical peak on size exclusion chromatography ( apparent molecular mass of ˜ 80 kda ) and migrated as a single band of ˜ 70 kda on sds - page under reducing conditions . erbb1501 gave a unique expected sequence , leekkvxqgt ( 13 ) on n - terminal amino acid sequence analysis , the x at cycle 7 being due to the presence of a disulphide - bonded cysteine residue at that position . the apparent molecular mass of approximately 70 kda on sds - page is due to the residual glycosylation reported for the glycosylation defective lec 8 cells ( 33 ) since the calculated mass of human erbb1501 apo - protein is ˜ 57 . 5 kda . there are eight potential n - linked glycosylation sites in erbb1501 ( 13 ) and incubation of erbb1501 with peptide - n - glycosidase ( png &# 39 ; ase ) at 37 ° c . resulted in the generation of a major band migrating on sds - page with an apparent molecular mass of 57 - 58 kda ( data not shown ). we have shown previously using biacore analysis that removal of carbohydrate using pngase does not affect binding of segfr621 to the immobilised ligand , in agreement with the concept that glycosylation is required for correct processing but not for biological activity . all subsequent experiments were carried out using the ˜ 70 kda erbb1501 . the biacore biosensor was used to determine both the rate and equilibrium binding constants for the interaction between erbb1501 and hegf or htgf - α . full length ectodomain ( erbb1621 ) was used as a positive control for the surface reactivity , since this interaction has been studied in detail previously ( 23 , 27 ). representative sensorgrams for the interaction between erbb1501 or erbb1621 and hegf or tgf - α are shown in fig2 . visual inspection revealed that the curves approached equilibrium over the concentration ranges tested . additionally , the htgf - α sensorgrams appeared to show more rapid , and virtually complete , dissociation . thermodynamic analysis of the equilibrium binding data in scatchard format ( fig3 ) indicated kd values of 30 and 47 nm ( correlation coefficient r = 0 . 993 and 0 . 999 respectively ) for the interactions between erbb1501 and immobilised hegf or htgf - α and 412 and 961 nm ( r = 0 . 997 and 0 . 999 respectively ) for the corresponding interactions with erbb1621 . the values obtained by scatchard transformation were also confirmed by direct non - linear least squares analysis of the binding data ( data not shown ) using an equation defining steady state equilibrium ( ka * conc * rmax /( ka * conc * n ); biaevaluation 3 . 1 ). using this analysis , kd values of 32 and 46 nm were calculated for the interaction between erbb1501 and immobilised hegf and htgf - α respectively and 570 and 959 nm for the interaction between full - length ectodomain ( erbb1621 ) and immobilised hegf and htgf - α . the values obtained with erbb1621 were in good agreement with those reported previously ( 23 ), confirming the surface viability . the individual rate constants were determined from those parts of the curves where first order kinetics appeared to be operative ( 27 , 28 ), and the corresponding dissociation constants calculated ( table 1 ). again , there was good agreement between the kd values calculated in this manner and those obtained from the equilibrium binding data . it is interesting to note that the binding curves obtained with both erbb1501 and erbb1621 for htgf - α appeared to be better fitted to a 1 : 1 model than the corresponding data for the hegf surface ( as suggested by the virtually complete dissociation ). the observation that erbb1501 bound egf with high affinity prompted us to test whether erbb1501 would act as a competitive inhibitor for the mitogenic stimulation of egfr in a cell - based assay using the baf / 3erx cell line . this cell line responds to megf with an ec50 of approximately 30 pm ( fig4 a ). the competition assay ( fig4 b ) used a constant concentration of megf ( 207 pm ), which causes maximal stimulation ( fig4 a ), and varying levels ( 0 . 00045 - 0 . 5 μm ) of erbb1501 , erbb1621 or the neutralising anti - egfr monoclonal antibody mab528 raised against epidermal growth factor receptors on a human epidermoid carcinoma cell line , a431 ( 19 ). this antibody has been shown to prevent the growth of a431 cell xenografts , bearing high numbers of egf receptors , in nude mice . the erbb1501 ( ic50 = 0 . 02 μm ) was almost 10 fold more potent than the full - length ectodomain ( ic50 = 0 . 15 μm ) and approximately 3 - fold more potent than the mab528 anti - egfr monoclonal antibody ( ic50 = 0 . 06 μm ). chemical cross - linking revealed that erbb1501 formed dimeric complexes in the presence of ligand . in the presence of 20 μm megf , a single high molecular weight species ( apparent mr 180 , 000 da ) was formed after chemical cross - linking which was not detectable when the cross - linking was attempted in the absence of ligand ( fig5 ). western blotting was employed to confirm the authenticity of the bands observed , but similar data were obtained with silver or coomassie blue staining . in addition , size exclusion chromatographic analysis of the reaction mixture , using a tsk g2000sw column developed with a mobile phase of pbs at a flow rate of 0 . 25 ml / min , showed a peak of apparent mr 158 , 000 which corresponded to dimer ( data not shown ). similar results have previously been obtained with erbb1621 . analytical ultracentrifugation showed that the egf binding sites on erbb1501 were saturated at an equimolar ratio of ligand and receptor leading to the formation of a 2 : 2 egf / erbb1501 complex ( fig6 ). the data for 20 ™ egf alone ( fig6 a ) indicate a single solute of molecular weight 5 , 980 da , in good agreement with the value calculated from the amino acid composition ( 6 , 040 da ). the molecular weight ( 65 , 600 da ) and partial specific volume ( 0 . 71 ml / g ) determined for 10 ™ erbb1501 alone was calculated from the sedimentation equilibrium distribution ( fig6 a ) and is based on the known amino acid composition and a calculated value of 12 % ( w / w ) for the carbohydrate composition . sedimentation equilibrium data for a mixture of egf ( 20 μm ) and erbb1501 ( 10 μm ) was analyzed assuming two species ( fig6 a ). the molecular weight of the first species was fixed at the value obtained for free egf ( 6 , 000 da ) with the molecular weight and weight fraction of the second species used as fitting parameters . under these conditions the molecular weight of the second species provides a good approximation to the weight - average molecular weight of erbb1501 and its complexes . the best - fit value showed a complex of weight - average mw 106 , 400 da , higher than predicted for a 1 : 1 complex ( 71 , 600 da ) and more consistent with the formation of a significant proportion of dimeric 2 : 2 egf / erbb1501 complex ( see below ). high - speed meniscus depletion experiments were performed to determine the molar ratio required for saturation of erbb1501 with egf ( fig6 b ). a solution of erbb1501 ( 5 μm ) was titrated with egf to determine the molar ratio at which free egf is detectable at the meniscus . the results show that this occurs above 5 μm egf , implying an equimolar ratio is required for saturation of the egf binding site ( s ) on erbb1501 . these data , taken together with the observed weight average molecular weight of the egf / erbb1501 complex obtained from the equilibrium analysis ( fig6 a ), confirm that the stoichiometry of the egf / erbb1501 dimeric species is 2 : 2 not 2 : 1 . sedimentation equilibrium was used for the analysis of data obtained for erbb1501 ( 5 μm ) in the presence of a range of egf concentrations ( fig6 c ). the weight average molecular weight obtained for the “ second ” species increases rapidly as the ratio of egf / erbb1501 is increased to 1 : 1 and then tends to plateau around approximately 108 , 000 at ratios above 2 : 1 ( fig6 c ). the data in fig6 a could also be fitted assuming a mixture of 1 : 1 and 2 : 2 complexes with weight fractions of the monomeric and dimeric erbb1501 complexes of 57 % and 31 % respectively . similar data was obtained with erbb1621 ( 23 ). biosensor analysis was also used to analyse the binding of the transiently expressed segfr501 mutants to both immobilised hegf and htgf - α surfaces . the presence of the mutant proteins in culture supernatants from transfected cell lines was demonstrated by both immunoblotting with the anti - egfr monoclonal antibody , mab 528 , and biosensor analysis using mab 528 immobilised on the surface . culture supernatants from all cell lines showed demonstrable binding to the mab surface ( 441 & gt ; 472 = wt & gt ; 367 ). in preliminary experiments , the glu367lys mutant and the glu472lys mutant showed similar binding characteristics to segfr501 when passed over the hegf sensor surface . the gly441 lys mutant showed much reduced binding , even though the mab528 surface had indicated that the gly441 lys mutant was present at higher concentrations than segfr501 . interestingly , when the same samples were passed over the parallel htgf - α sensor surface the gly441 lys mutant now showed the highest binding , whilst the binding of the glu367lys mutant the glu472lys mutant and wild type erbb1501 were again similar but lower . for full biosensor analysis the mutant proteins present in the conditioned media from transient transfected 293t fibroblasts were concentrated and purified by a combination of affinity purification using the 9e10 monoclonal antibody and size exclusion chromatography on superdex 200 and superose 12 . the sensorgrams obtained with the immobilised hegf and htgf - α surfaces ( 160 and 132 ru immobilised respectively ) are shown in fig7 a , 7 b . as we had observed in the preliminary experiments , whilst the binding characteristics of the glu367lys and glu472lys mutants were essentially undistinguishable from those of erbb1501 shown in fig2 the gly441lys mutant again showed preferential binding to the htgf - α surface ( fig7 a , 7 b ). scatchard analysis of the equilibrium binding data ( fig7 c , 7 d ) indicated that whilst binding to the tgf - α surface was similar to that observed with erbb1501 ( kd = 77 nm , correlation coefficient r = 0 . 999 ), the reactivity of the gly441lys mutant towards the egf surface was now considerably reduced ( kd = 455 nm , r = 0 . 995 ). similar values ( 78 nm and 469 nm ) were obtained by direct non - linear least squares analysis of the binding data using the equation defining steady state equilibrium . kinetic analysis of the binding data ( table 2 ) indicated that the interaction with the immobilised tgf - α could be described by an association rate constant ( ka ) of 5 . 2 - 6 . 9 × 10 - 5m - 1 s - 1 and a dissociation rate constant ( kd ) of 0 . 025s - 1 giving a kd = kd / ka of 36 - 44 nm . the corresponding interaction with egf was described by a ka of 1 . 9 - 2 . 3 × 10 - 5m - 1 s - 1 and a significantly faster kd of 0 . 103 s - 1 giving a kd = kd / ka of 442 - 545 nm , in good agreement with the results observed from the thermodynamic analysis . a previous published study had demonstrated that the full - length extracellular domain of erbb1 , expressed as an fc fusion protein homodimer , bound a number of egf ligands with ic50 values ranging from 1 - 10 nm ( 29 ), while the corresponding heterodimer with erbb2 showed at best a two - fold increase in affinity . using a monolabelled eu - egf ( wallac ) as trace , we show that the truncated form of the extracellular domain of the egf receptor , expressed as an fc fusion homodimer , binds egf ligand with 10 - fold higher affinity than that reported for the full - length erbb1 ectodomain - fc fusion protein ( 30 ) ( see fig8 ). erbb3 and erbb4 fc constructs , incorporating the corresponding egf receptor domains used in erbb1 501 . fc , were assembled and analysed following transient transfection . in preliminary studies , the full - length erbb4 ectodomain fc fusion protein , expressed as a homodimer and with erbb2 ectodomain fc fusion protein as a heterodimer , was used to validate the data of fitzpatrick et . al . ( 30 ). as demonstrated in fig9 , the full - length receptor fc homodimer exhibits an ic 50 value for heregulin β ( hrgβ ) of 18 nm , which is in good agreement with that previously reported ( 5 . 1 nm ; ( 29 )) allowing for differences in trace ligand preparation . we also find that co - expression of erbb2 has dramatic affect on the ic 50 value for ligand , resulting in an increase by two orders of magnitude . jones et . al . ( 29 ) report an increase in ic 50 of 200 - fold using the heterodimer receptors ; again , this probably reflects differences in assay format . the ic 50 values obtained using the truncated forms of erbb3 and erbb4 as homodimer fc fusion proteins are shown in fig1 . while erbb3 500 . fc has an ic 50 of 0 . 49 nm for hrgβ , the value obtained for erbb4 497 . fc ( 0 . 057 nm ) approaches that obtained for the full - length ectodomain erbb2 / 4 fc heterodimer reported by genentech ( 0 . 025 nm ; ( 30 )). the characteristics of truncated versions of egfr ectodomains ( erbb1 501 , erbb3 500 and erbb4 497 ) that bind hegf , tgf - α ( erbb1 501 ) and hrgβ ( erbb3 500 and erbb4 497 ) with high affinity are described herein . the kd values of 13 - 21 nm for hegf binding to erbb1501 are similar to those , ( 15 - 30 nm ), seen with chemically cross - linked dimers of full - length egfr ectodomain and are 10 to 25 - fold higher than the values generally reported for soluble , full - length egfr ectodomain derived from either a431 tumour cells , transfected sf9 insect cells or cho cells . erbb1501 , which lacks most of ( 6 of 7 modules of ) cr2 , exhibits ligand - induced receptor dimerisation indicating that the regions responsible for dimerisation are unlikely to include cr2 . it also confirms that membrane anchoring is not required for the generation of high affinity dimers in contrast to the situation with erbb2 / erbb3 heterodimers and neuregulin . the ultracentrifugation analyses showed that the binding sites on erbb1 501 were saturated , and the extent of dimerisation began to plateau , at molar ratios greater than of 1 : 1 ( fig5 c ), even at the relatively low concentration of erbb1 501 of 5m ( 320 μg / ml ). this compares favourably with the small angle x - ray scattering data and our previous analytical ultracentrifugation analyses that showed that erbb1 621 dimerisation , induced by egf or tgf - α binding , reached a maximum when the ratio of egf / erbb1 was 1 : 1 . it is envisaged that the truncated constructs of the present invention will have therapeutic potential given their high affinity for ligand and their ability to competitively inhibit egf - induced proliferation responses in a model cell system . the inhibition shown by erbb1 501 , for example , was greater than that achieved in the same assay with a neutralising monoclonal antibody raised against the receptor ( mab528 ), chimeric forms of which ( c225 ) are currently in clinical trials . erbb1 501 was also employed to investigate the residue responsible for the differential binding between htgf - α and hegf observed with chicken egfr ( 9 ). these data demonstrate that the lys442 in chicken egfr , which corresponds to gly441 in hegfr , is the residue responsible for discriminating between htgf - α and hegf binding . a number of studies have described the ligand - binding characteristics of isoforms of the ectodomains of different egf receptor family members , either alone or as fc fusion proteins . full - length ectodomain homodimers of erbb3 and erbb4 , fused to the human igg1 fc domain , bind their respective ligands with a range of affinities ( ic 50 values 1 - 1000 nm ;). however , heterodimerisation with the corresponding erbb2 ectodomain fc fusion protein is required before very high affinity binding (& lt ; 1 nm ) of the sort observed for cell - surface heterodimers is achieved ( 30 ). our present studies indicate that truncated ectodomain fragments of erbb1 , 3 and 4 , expressed as fc fusion protein homodimers , bind ligand with very high affinity (& lt ; 1 nm ). in the example of erbb4 , the affinity approaches that reported for the heterodimer of the full length ectodomain fc fusion proteins of erbb2 and erbb4 ( 30 ). various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . indeed , various modifications of the described modes for carrying out the invention which are apparent to those skilled in molecular biology or related fields are intended to be within the scope of the invention . 1 . burgess a w and thumwood c m . 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