Patent Application: US-86017497-A

Abstract:
a bispecific or bivalent double head antibody fragment , which is composed of a binding complex containing two polypeptide chains , whereby one polypeptide chain has two times a variable domain of a heavy chain in series and the other polypeptide chain has two times a variable domain of a light chain in series , and the binding complex contains two pairs of variable domains , wherein said double head antibody fragments have binfunctional antigen binding activity . a process for producing such an antibody fragment is disclosed . an immunoassay is also provided for , wherein the improvement is the inclusion of a bispecific or bivalent double head antibody fragment , which is composed of a binding complex containing two polypeptide chains , whereby one polypeptide chain has two times a variable domain of a heavy chain in series and the other polypeptide chain has two times a variable domain of a light chain in series , as a binding means .

Description:
in this specification the construction of an antibody fragment analogue consisting of a two chain protein complex is described , in which one of the chains consists of two heavy chain v - domains and the other chain consists of the two corresponding light chain v - domains in either order . the variable domains are linked either directly or through a polypeptide linker . subsequent molecular modelling of this combination suggested that the protein chains could fold such that both binding sites are fully accessible , provided that the connecting linkers are kept long enough to span 30 to 35 å . whereas in patent application wo 93 / 11161 it is explicitly described that for the above described bispecific complexes two flexible polypeptide linkers in the self assembling complex are required , the present invention illustrated here describes in particular the construction of a two chain protein complex containing only one linker or no linkers at all . the latter antibody fragment analogue thus consists of a two chain protein complex containing one polypeptide chain comprising heavy chain v - domains fused directly together and another polypeptide chain comprising the corresponding light chain v - domains fused together , both fusions in the absence of linkers . but also two chain protein complexes in which each chain comprises a linker between the two variable domains can be used as antibody fragment analogues according to the invention as described below with construct pgosa . e . however , the two chain complexes containing only one linker or no linker at all are preferred . the abbreviation gosa used in this specification relates to a combination of glucose oxidase and streptococcus sanguis . in this specification evidence is provided that these antibody fragment analogues (&# 34 ; double heads &# 34 ;) contain both antigen binding specificities of the fv &# 39 ; s used to generate these bispecific antibody fragments . it is exemplified that these type of constructs according to the invention can be used to target the enzyme glucose oxidase to whole bacteria , using antibody fragments derived from hybridomas expressing antibodies directed against these antigens . the present invention is now described by reference to some specific examples , which are included for purposes of illustration only and are not intended to limit the scope of the invention . all cloning steps were performed in e . coli jm109 ( enda1 , reca1 , gyra96 , thi , hsdr17 ( r k , m k + ), rela1 , supe44 , δ ( lac - proab ), [ f &# 39 ;, trad36 , proab , laci q zδm15 ]. e . coli cultures were grown in 2 × ty medium ( 16 g tryptone , 10 g yeast extract , 5 g nacl per liter h 2 o ), where indicated supplemented with 2 % glucose and / or 100 μg / ml ampicillin . transformations were plated out on sobag plates ( 20 g tryptone , 5 g yeast extract , 15 g agar , 0 . 5 g nacl per liter h 2 o plus 10 mm mgcl 2 , 2 % glucose , 100 μg / ml ampicillin ) the expression vectors used are derivatives of puc19 . the oligonucleotide primers used in the pcr reactions were synthesized on an applied biosystems 381a dna synthesiser by the phosphoramidite method . colonies from freshly transformed jm109 plated onto sobag plates were used to inoculate 2 × ty medium supplemented with 100 μg / ml ampicillin , 2 % glucose . cultures were shaken at 37 ° c . to an od 600 in the range of 0 . 5 to 1 . 0 . cells were pelleted by centrifugation and the supernatant was removed . the pelleted cells were resuspended in 2 × ty medium with 100 μg / ml ampicillin , 1 mm iptg , and grown for a further 18 hours at 25 ° c . cells were pelleted by centrifugation and the supernatant , containing the secreted chains , used directly in an elisa . the proteins in the periplasm of the pelleted cells were extracted by resuspending the cell pellet in 1 / 20 of the original culture volume of lysis buffer ( 20 % sucrose , 200 mm tris - hcl ph 7 . 5 , 1 mm edta , 500 μg / ml lysozyme ). after incubation at 25 ° c . for 20 minutes an equal volume of h 2 o was added and the incubation was continued for another 20 minutes . the suspension was spun at 10 . 000 g for 15 minutes and the supernatant containing the periplasmic proteins was used directly in an elisa . 96 well elisa plates ( greiner hc plates ) were activated overnight at 37 ° c . with 200 μl / well of an 1 / 10 dilution of an over night culture of streptococcus cells in 0 . 05 m sodium carbonate buffer at ph = 9 . 5 . following one wash with pbst , the antigen sensitised plates were pre - blocked for 1 hour at 37 ° c . with 200 μl / well blocking buffer ( 2 % bsa , 0 . 15 % tween in pbs ). samples containing 50 μl blocking buffer plus 50 μl culture supernatants or periplasmic cell extracts ( neat or diluted with pbs ) were added to the streptococcus sensitised plate and incubated for 2 hours at 37 ° c . following 4 washes with pbs - t , 100 μl of blocking buffer containing glucose oxidase ( 50 μg / ml ) was added to every well . after incubation at 37 ° c . for 1 hour unbound glucose oxidase was removed by 4 washes with pbs - t . bound glucose oxidase was detected by adding 100 μl substrate to each well ( 70 mm na - citrate , 320 mm na - phosphate , 27 mg / ml glucose , 0 . 5 μg / ml hrp , 100 μg / ml tmb ). the colour reaction was stopped after 1 hour by the addition of 35 μl 2 m hcl and the a450 was measured ( compare fig1 / 15 ). gosa . e , gosa . v , gosa . s and gosa . t were partially purified by affinity chromatography . 100 ml periplasmic extract of each of these constructs was loaded onto a glucose - oxidase - sepharose column ( cnbr - sepharose , pharmacia ) prepared according to the manufacturer &# 39 ; s instructions . after extensive washes with pbs the bound gosa antibody fragments were eluted in 0 . 1m glycine buffer at ph = 2 . 8 . the fractions were neutralised with tris and analysed by polyacrylamide gel electrophoresis followed by silver staining and tested for the presence of double head activity . in this example the construction of a two chain protein complex is described , in which one of the chains consists of two heavy chain v - domains and the other chain consists of the two corresponding light chain v - domains . the variable domains are linked either directly or through a polypeptide linker . the expression vectors used are derivatives of a puc19 derived plasmid containing a hindiii - ecori fragment that in the case of plasmid scfv . 4715 - myc contains a dna fragment encoding one pelb signal sequence fused to the n - terminus of the v h that is directly linked to the corresponding v l of the antibody through a connecting flexible peptide linker , ( gly 4 ser ) 3 ( present in seq id no : 2 as amino acids 109 - 123 and in seq id no : 10 as amino acids 121 - 135 ), thus generating a single - chain molecule ( see fig7 ). in the dual - chain fv and the pgosa expression vectors , the dna fragments encoding both the v h and v l of the antibody are preceded by a ribosome binding site and a dna sequence encoding the pelb signal sequence in an artificial dicistronic operon under the control of a single inducible promoter ( see fig5 and 8a and 8b ). expression of these constructs is driven by the inducible lacz promoter . the nucleotide sequence of the hindiii - ecori inserts of the plasmids pur . 4124 , fv . 3418 , fv . 4715 - myc and scfv . 4715 - myc constructs used for the generation of the bispecific antibody fragments are given in fig4 - 7 , respectively . moreover , a culture of e . coli cells harbouring plasmid scfv . 4715 - myc and a culture of e . coli cells harbouring plasmid fv . 3418 were deposited under the budapest treaty at the national collection of type cultures ( central public health laboratory ) in london ( united kingdom ) with deposition numbers nctc 12916 and nctc 12915 , respectively . in agreement with rule 28 ( 4 ) epc , or a similar arrangement for a state not being a contracting state of the epc , it is hereby requested that a sample of such deposit , when requested , will be submitted to an expert only . the construction of pgosa . e ( see fig8 a and 8b for the hindill - ecori insert of puc19 ) involved several cloning steps . the appropriate restriction sites in the various domains were introduced by pcr directed mutagenesis using the oligonucleotides listed in table 1 below . the pgosa . e derivatives pgosa . v , pgosa . s and pgosa . t with only one or no linker sequence are derived from the pgosa . e construct by removing the linker sequences by means of pcr directed mutagenesis with oligonucleotides listed in table 1 below . table 1__________________________________________________________________________dbl . 15 &# 39 ;- cac cat ctc cag aga caa tgg caa g - 3 &# 39 ; (= seq id no : 14 ) dbl . 25 &# 39 ;- gag cgc gag ctc ggc cga acc ggc c . sup . 1 ga tcc gcc (= seq id no : 15 ) acc gcc aga gcc - 3 &# 39 ; dbl . 35 &# 39 ;- cag gat ccg gcc ggt tcg gcc . sup . 1 cag gtc cag ctg (= seq id no : 16 ) caa cag tca gga - 3 &# 39 ; dbl . 45 &# 39 ;- cta cat gaa ttc . sup . 2 gct agc . sup . 3 tta tta tga gga gac (= seq id no : 17 ) ggt gac ggt ggt ccc ttg gc - 3 &# 39 ; dbl . 55 &# 39 ;- taa taa gct agc . sup . 3 gga gct gca tgc aaa ttc tat (= seq id no : 18 ) ttc - 3 &# 39 ; dbl . 65 &# 39 ;- acc aag ctc gag . sup . 4 atc aaa cgg gg - 3 &# 39 ; (= seq id no : 19 ) dbl . 75 &# 39 ;- aat gtc gaa ttc . sup . 2 gtc gac . sup . 5 tcc gcc acc gcc aga (= seq id no : 20 ) gcc - 3 &# 39 ; dbl . 85 &# 39 ;- att gga gtc gac . sup . 5 atc gaa ctc act cag tct cca (= seq id no : 21 ) ttc tcc - 3 &# 39 ; dbl . 95 &# 39 ;- tga agt gaa ttc . sup . 2 gcg gcc gc . sup . 6 t tat tac cgt ttg (= seq id no : 22 ) att tcg agc ttg gtc cc - 3 &# 39 ; dbl . 105 &# 39 ;- cga att cgg tca cc . sup . 8 g tct cct cac agg tcc agt (= seq id no : 23 ) tgc aac ag - 3 &# 39 ; dbl . 115 &# 39 ;- cga att ctc gag . sup . 4 atc aaa cgg gac atc gaa ctc (= seq id no : 24 ) act cag tct cc - 3 &# 39 ; dbl . 125 &# 39 ;- cga att cgg tca cc . sup . 8 g tct cct cac agg tgc agt (= seq id no : 25 ) tgc agg ag - 3 &# 39 ; pcr . 515 &# 39 ;- agg t ( c / g )( a / c ) a ( c / a ) c tgc ag . sup . 7 ( c / g ) agt c ( a / t ) g (= seq id no : 26 ) g - 3 &# 39 ; pcr . 895 &# 39 ;- tga gga gac ggt gac c . sup . 8 gt ggt ccc ttg gcc cc - 3 &# 39 ; (= seq id no : 27 ) pcr . 905 &# 39 ;- gac att gag ctc . sup . 9 acc cag tct cca - 3 &# 39 ; (= seq id no : 28 ) pcr . 1165 &# 39 ;- gtt aga tct cga g . sup . 4 ct tgg tcc c - 3 &# 39 ; (= seq id no : 29 ) __________________________________________________________________________ 1 = sfii , 2 = ecori , 3 = nhei , 4 = xhoi , 5 = sali , 6 = noti , 7 = psti , 8 = bsteii , 9 = saci these three constructs lack some of the restriction sites at the new joining points . the v h a - v h b gene fragment without a linker lacks the 5 &# 39 ; v h b sfii site . the v l b - v l a gene fragment without a linker lacks the 5 &# 39 ; v l a sali site . the position of the oligonucleotides in the pgosa constructs given in table 1 are shown in fig9 . the pgosa expression vectors and the oligonucleotides in table 1 have been designed to enable most specificities to be cloned into the pgosa constructs . fig1 shows the amino acid sequence of the junctions between the v h a - v h b and v l b - v l a fragments encoded by dna present in pgosa . e , pgosa . v , pgosa . s and pgosa . t . a more detailed description of the preparation of pgosa . e , pgosa . v , pgosa . s and pgosa . t is given in example 5 . in this example we provide evidence that the above described molecules (&# 34 ; double heads &# 34 ;), i . e . the two chain protein complexes , contain both antigen binding specificities of the fv &# 39 ; s used to generate these multi - functional antibody fragment analogues . fig1 - 15 show that gosa . e , gosa . v , gosa . s and gosa . t can be used to specifically target the enzyme glucose oxidase to several streptococcus sanguis strains using antibody fragments derived from hybridoma &# 39 ; s expressing antibodies directed against these antigens . comparison of the binding specificity of the gosa constructs ( see fig1 - 15 ) and the binding specificity of the scfv . 4715 - myc ( see fig1 ) shows that the fine specificity of the anti - streptococcus sanguis scfv . 4715 is preserved in the gosa &# 34 ; double heads &# 34 ;. partially purified gosa . e , gosa . v , gosa . s and gosa . t samples ( estimated to be 50 - 80 % pure by polyacrylamide gel electrophoresis ) were analysed on a pharmacia fplc superose 12 column . the analysis was performed using pbs at a flow rate of 0 . 3 ml / minute . eluate was monitored at 280 nm and 0 . 3 ml fractions were collected and analysed by elisa . usually gosa . e , gosa . v , gosa . s and gosa . t samples only gave one gosa double head activity peak as determined by elisa ( see fig1 - 18 ). the position of this peak in the elution pattern indicated that the molecular weight of the gosa double head is 40 - 50 kd . since this molecular weight corresponds to the expected molecular weight of the v h 2 + v l 2 double head dimer , the conclusion is justified that gosa . e , gosa . v , gosa . s and gosa . t are primarily produced as dimeric molecules . occasionally an activity peak with an apparent molecular weight of ≈ 200 kd was observed ( see fig1 ). the presence of glucose oxidase activity in these fractions ( data not shown ) indicate that these fractions contain gosa double head complexed with glucose oxidase that was eluted with the gosa sample from the glucose oxidase - sepharose affinity matrix . the methods described in the previous examples were used to produce other double heads , which also appeared to be active against the antigens for which they were developed . these other double heads had the following specificities : detailed description of the preparation of intermediate constructs pgosa . a , pgosa . b pgosa . c and pgosa . d and their use for the preparation of plasmid pgosa . e and its derivatives pgosa . v , pgosa . s and pgosa . t the primary structures of the oligonucleotide primers used in the construction of the bispecific ` pgosa ` constructs are shown in table 1 above . reaction mixture used for amplification of dna fragments were 10 mm tris - hcl , ph 8 . 3 , 2 . 5 mm mgcl 2 , 50 mm kcl , 0 . 01 % gelatin ( w / v ), 0 . 1 % triton x - 100 , 400 mm of each dntp , 5 . 0 units of vent dna polymerase ( new england biolabs ), 100 ng of template dna , and 500 ng of each primer ( for 100 μl reactions ). reaction conditions were : 94 ° c . for 4 minutes , followed by 33 cycles of each 1 minute at 94 ° c ., 1 minute at 55 ° c ., and 1 minute 72 ° c . plasmid dna was prepared using the ` qiagen p - 100 midi - dna preparation ` system . vectors and inserts were prepared by digestion of 10 μg ( for vector preparation ) or 20 μg ( for insert preparation ) with the specified restriction endonucleases under appropriate conditions ( buffers and temperatures as specified by suppliers ). modification of the dna ends with klenow dna polymerase and dephosphorylation with calf intestine phosphorylase were performed according to the manufacturers instructions . vector dnas and inserts were separated through agarose gel electrophoresis and purified with deae - membranes na45 ( schleicher & amp ; schuell ) as described by maniatis et al . ligations were performed in 20 μl volumes containing 30 mm tris - hcl ph 7 . 8 , 10 mm mgcl 2 , 10 mm dtt , 1 mm atp , 300 - 400 ng vector dna , 100 - 200 ng insert dna and 1 weiss unit t 4 dna ligase . after ligation for 2 - 4 h at room temperature , cacl 2 competent e . coli jm109 ( maniatis ) were transformed using 7 . 5 μl ligation reaction . the transformation mixtures were plated onto sobag plates and grown overnight at 37 ° c . correct clones were identified by restriction analysis and verified by automated dideoxy sequencing ( applied biosystems ). following amplification each reaction was checked for the presence of a band of the appropriate size by agarose gel electrophoresis . one or two 100 μl pcr reaction mixtures of each of the pcr reactions pcr . i - pcr . x ( fig2 - 29 ), together containing approximately 2 - 4 μg dna product were subjected to phenol - chloroform extraction , chloroform extraction and ethanol precipitation . the dna pellets were washed twice with 70 % ethanol and allowed to dry . next , the pcr products were digested overnight ( 18 h ) in the presence of excess restriction enzyme in the following mixes at the specified temperatures and volumes . 50 mm tris - hcl ph 8 . 0 , 10 mm mgcl 2 , 50 mm nacl , 4 mm spermidine , 0 . 4 μg / ml bsa , 4 μl (= 40 u ) saci , 4 μl (= 40 u ) bsteii , in 100 μl total volume at 37 ° c . 10 mm tris - acetate ph 7 . 5 , 10 mm mgac 2 , 50 mm kac ( 1x &# 34 ; one - phor - all &# 34 ; buffer ex pharmacia ), 4 μl (= 48 u ) sfii , in 50 μl total volume at 50 ° c . under mineral oil . after overnight digestion , pcr . ii - sfii was digested with ecori ( overnight at 37 ° c .) by the addition of 16 μl h 2 o , 30 μl 10x &# 34 ; one - phor - all &# 34 ; buffer ( phanmacia ) ( 100 mm tris - acetate ph 7 . 5 , 100 mm mgac 2 , 500 mm kac ) and 4 μl (= 40 u ) ecori . 10 mm tris - acetate ph 7 . 5 , 10 mm mgac 2 , 50 mm kac ( 1x &# 34 ; one - phor - all &# 34 ; buffer { pharmacia }), 4 μl (= 40 u ) nhei , 4 μl (= 40 u ) saci , in 100 μl total volume at 37 ° c . 20 mm tris - acetate ph 7 . 5 , 20 mm mgac 2 , 100 mm kac ( 2x &# 34 ; one - phor - all &# 34 ; buffer { pharmacia }), 4μl (= 40 u ) xhoi , 4 μl (= 40 u ) ecori , in 100 μl total volume at 37 ° c . 20 nm tris - acetate ph 7 . 5 , 20 mm mgac 2 , 100 mm kac ( 2x &# 34 ; one - phor - all &# 34 ; buffer { pharmacia }), 4 μl (= 40 u ) sali , 4 μl (= 40 u ) ecori , in 100 μl total volume at 37 ° c . 10 mm tris - acetate ph 7 . 5 , 10 mm mgac 2 , 50 mm kac ( 1x &# 34 ; one - phor - all &# 34 ; buffer { pharmacia }), 4 μl (= 48 u ) sfii , in 50 μl total volume at 50 ° c . under mineral oil . after overnight digestion , pcr . vi - sfii was digested with nhei ( overnight at 37 ° c .) by the addition of 41 μl h 2 o , 5 μl 10x &# 34 ; one - phor - all &# 34 ; buffer ( pharmacia ) ( 100 mm tris - acetate ph 7 . 5 , 100 mm mgac 2 , 500 mm kac ) and 4 μl (= 40 u ) nhei . 50 mm tris - hcl , ph 8 . 0 , 10 mm mgcl 2 , 50 mm nacl , 4 mm spermidine , 0 . 4 μg / ml bsa , 4 μl (= 40 u ) nhei , 4 μl (= 40 u ) bsteii , in 100 μl total volume at 37 ° c . 20 mm tris - acetate ph 7 . 5 , 20 mm mgac 2 , 100 mm kac ( 2x &# 34 ; one - phor - all &# 34 ; buffer { pharmacia }), 4 μl (= 40 u ) ecori , in 50 μl total volume at 37 ° c . after overnight digestion , pcr . viii - ecori was digested with xhoi ( overnight at 37 ° ) by the addition of 46 μl h 2 o and 4 μl (= 40 u ) xhoi . 25 mm tris - acetate , ph 7 . 8 , 100 mm kac , 10 mm mgac , 1 mm dtt ( 1x &# 34 ; multi - core &# 34 ; buffer { promega }), 4 mm spermidine , 0 . 4 μg / ml bsa , 4 μl (= 40 u ) nhei , 4 μl (= 40 u ) bsteii , in 100 μl total volume at 37 ° c . 50 mm tris - hcl , ph 8 . 0 , 10 mm mgcl 2 , 50 mm nacl , 4 mm permidine , 0 . 4 μg / ml bsa , 4 μl (= 40 u ) psti , 4 μl (= 40 u ) ecori , in 100 μl total volume at 37 ° c . ______________________________________the digested pcr fragments______________________________________pcr . i - saci / bsteii , pcr . ii - sfii / ecori , pcr . iii - nhei / saci , pcr . iv - xhoi / ecori , pcr . v - sali / ecori , pcr . vi - sfii / nhei , pcr . vii - bsteii / nhei , pcr . viii - xhoi / ecori , pcr . ix - bsteii / nhei , and pcr . x - psti / ecori______________________________________ were purified on an 1 . 2 % agarose gel using deae - membranes na45 ( schleicher & amp ; schuell ) as described by maniatis et al . the purified fragments were dissolved in h 2 o at a concentration of 100 - 150 ng / μl . the construction of pgosa . e ( see fig8 a and 8b ) involved several cloning steps that produced 4 intermediate constructs pgosa . a to pgosa . d ( see fig2 - 33 ). the final expression vector pgosa . e and the oligonucleotides in table 1 above have been designed to enable most specificities to be cloned into the final pgosa . e construct ( fig9 ). the upstream v h domain can be replaced by any psti - bsteii v h gene fragment obtained with oligonucleotides pcr . 51 and pcr . 89 ( see table 1 above ). the oligonucleotides dbl . 3 and dbl . 4 ( see table 1 above ) were designed to introduce sfii and nhei restriction sites in the v h gene fragments thus allowing cloning of those v h gene fragments into the sfil - nhei sites as the downstream v h domain . all v l gene fragments obtained with oligonucleotides pcr . 116 and pcr . 90 ( see table 1 above ) can be cloned into the position of the v l . 3418 gene fragment as a saci - xhoi fragment . a complication here however is the presence of an internal saci site in the v h . 3418 gene fragment . oligonucleotides dbl . 8 and dbl . 9 ( see table 1 above ) are designed to allow cloning of v l gene fragments into the position of the v l . 4715 gene fragment - as a sali - noti fragment . the pgosa . e derivatives pgosa . v , pgosa . s and pgosa . t with only one or no linker sequences contain some aberrant restriction sites at the new joining points . the v h a - v h b construct without a linker lacks the 5 &# 39 ; v h b sfii site . the v h b fragment is cloned into these constructs as a bsteii / nhei fragment using oligonucleotides dbl . 10 or dbl . 11 and dbl . 4 ( see table 1 above ). the v l b - v l a construct without a linker lacks the 5 &# 39 ; v l a sali site . the v l a fragment is cloned into these constructs as a xhoi / ecori fragment using oligonucleotides dbl . 11 and dbl . 9 ( see table 1 above ). in the following part of the description the following linkers are mentioned which are also present in the sequence listing : the ( gly 4 ser ) 3 linker , present in seq id no : 2 as amino acids 109 - 123 and seq id no : 10 as amino acids 121 - 135 , the ( gly 4 ser ) 3 alaglyserala linker (= linkera ), present in seq id no : 12 as amino acids 121 - 139 , and the ( gly 4 ser ) 2 gly 4 val linker (= linkerv ), present in seq id no : 13 as amino acids 108 - 122 . this plasmid is derived from both the fv . 4715 - myc construct and the scfv . 4715 - myc construct . an sfii restriction site was introduced between the dna sequence encoding the ( gly 4 ser ) 3 linker and the gene fragment encoding the v l of the scfv . 4715 - myc construct ( see fig3 ). this was achieved by replacing the bsteii - saci fragment of the latter construct by the fragment pcr - i bsteii / saci ( fig1 ) that contains an sfii site between the dna encoding the ( gly 4 ser ) 3 linker and the v l . 4715 gene fragment . the introduction of the sfii site also introduced 4 additional amino acids ( alaglyserala ) between the ( gly 4 ser ) 3 linker and v l . 4715 resulting in a ( gly 4 ser ) 3 alaglyserala linker ( linkera ). the oligonucleotides used to produce pcr - i ( dbl . 1 and dbl . 2 , see table 1 above ) were designed to match the sequence of the framework - 3 region of v h . 4715 and to prime at the junction of the dna encoding the ( gly 4 ser ) 3 linker and the v l . 4715 gene fragment , respectively . thus pgosa . a can be indicated as : pelb - v h 4715 - linkera - ( sfii ) - v l 4715 - myc . this plasmid is derived from plasmid fv . 3418 ( see fig3 ). the xhoi - ecori fragment of plasmid fv . 3418 comprising the 3 &# 39 ; end of dna encoding framework - 4 of the v l including the stop codon was removed and replaced by the fragment pcr - iv xhoi / ecori ( fig2 ). the oligonucleotides used to produce pcr - iv ( dbl . 6 and dbl . 7 , see table 1 above ) were designed to match the sequence at the junction of the v l and the ( gly 4 ser ) 3 linker perfectly ( dbl . 6 ), and to be able to prime at the junction of the ( gly 4 ser ) 3 linker and the v h in pur . 4124 ( dbl . 7 ). dbl . 7 removed the psti site in the v h ( silent mutation ) and introduced a sali restriction site at the junction of the ( gly 4 ser ) 3 linker and the v h , thereby replacing the last ser of the linker by a val residue resulting in a ( gly 4 ser ) 2 gly 4 val linker ( linkerv ). thus pgosa . b can be indicated as : this plasmid contains dna encoding v h . 4715 linked by the ( gly 4 ser ) 3 alaglyserala linker to v h . 3418 ( see fig3 ), thus : pelb - v h 4715 - linkera - v h 3418 . this construct was obtained by replacing the sfii - ecori fragment from pgosa . a encoding v l . 4715 by the fragment pcr - ii sfii / ecori containing the v h . 3418 gene ( see fig2 ). the oligonucleotides used to produce pcr - ii ( dbl . 3 and dbl . 4 , see table 1 above ) hybridize in the framework - 1 and framework - 4 region of the gene encoding v h . 3418 , respectively . dbl . 3 was designed to remove the psti restriction site ( silent mutation ) and to introduce an sfii restriction site upstream of the v h gene . dbl . 4 destroys the bsteii restriction site in the framework - 4 region and introduces an nhei restriction site downstream of the stopcodon . this plasmid contains a dicistronic operon comprising the v h . 3418 gene and dna encoding v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 ( see fig3 ), thus : this construct was obtained by digesting plasmid pgosa . b with sali - ecori and inserting the fragment pcr - v sali / ecori ( fig2 ) containing the v l . 4715 gene . the oligonucleotides used to obtain pcr - v ( dbl . 8 and dbl . 9 , see table 1 above ) were designed to match the nucleotide sequence of the framework - 1 and framework - 4 regions of the v l . 4715 gene , respectively . dbl . 8 removed the saci site from the framework - 1 region ( silent mutation ) and introduced a sali restriction site upstream of the v l . 4715 gene . dbl . 9 destroyed the xhoi restriction site in the framework - 4 region of the v l . 4715 gene ( silent mutation ) and introduced a noti and an ecori restriction site downstream of the stop codon . this plasmid contains a dicistronic operon comprising dna encoding v h . 4715 linked by the ( gly4ser ) 3 alaglyserala linker to v h . 3418 plus dna encoding v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 ( see fig3 ), thus : both translational units are preceded by a ribosome binding site and dna encoding a pelb leader sequence . this plasmid was obtained by a three - point ligation by mixing the vector resulting from pgosa . d after removal of the v h 3418 - encoding psti - saci insert with the psti - nhei pgosa . c insert containing v h . 4715 linked to v h . 3418 and the pcr - iii nhei / saci fragment ( see fig2 ). the remaining psti - saci pgosa . d vector contains the 5 &# 39 ; end of the framework - 1 region of v h 3418 up to the psti restriction site and v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 starting from the saci restriction site in v l . 3418 . the psti - nhei pgosa . c insert contains v h . 4715 linked by the ( gly 4 ser ) 3 - alaglyserala linker to v h . 3418 , starting from the psti restriction site in the framework - 1 region in v h . 4715 . the nhei - saci pcr - iii fragment provides the ribosome binding site and dna encoding the pelb leader sequence for the v l . 3418 -( gly 4 ser ) 2 gly 4 val - v l . 4715 construct . the oligo - nucleotides dbl . 5 and pcr . 116 ( see table 1 above ) used to generate pcr - iii were designed to match the sequence upstream of the ribosome binding site of v l . 4715 in fv . 4715 and to introduce an nhei restriction site ( dbl . 5 ), and to match the framework - 4 region of v l . 3418 ( pcr . 116 ). this plasmid is derived from pgosa . e ( see fig3 ) from which the bsteii / nhei fragment containing dna encoding linkera - v h . 3418 was excised and replaced by the fragment pcr - vii bsteii / nhei containing the v h . 3418 gene ( see fig2 ). the resulting plasmid pgosa . v contains v h . 3418 linked directly to the framework - 4 region of v h . 4715 , plus v l . 4715 linked by the ( gly 4 ser ) 2 gly 4 val linker to the framework - 4 region of v l . 3418 , thus : this plasmid is derived from pgosa . e ( see fig3 ) from which the ( gly 4 ser ) 2 gly 4 val - v l 4715 xhoi / ecori fragment was excised and replaced by the fragment pcr - viii xhoi / ecori which contains v l . 4715 ( see fig2 ). the resulting plasmid pgosa . s contains v h . 4715 linked by the ( gly 4 ser ) 3 - alaglyserala linker to v h . 3418 plus v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 , thus : this plasmid contains a dicistronic operon consisting of v h . 3418 directly to the framework - 4 region of v h . 4715 plus v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 ( see fig3 ). both transcriptional units are preceded by a ribosome binding site and a pelb leader sequence , thus : this construct was obtained by inserting the nhei / ecori fragment of pgosa . s which contains v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 , into the vector pgosa . v from which the nhei / ecori fragment containing v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 was removed . detailed description of the preparation of other dicistronic constructs pgosa . g , and pgosa . j , pgosa . z , pgosa . aa and pgosa . ab this plasmid is an intermediate for the synthesis of pgosa . j . it is derived from pgosa . e from which the v h 4715 psti / bsteii fragment has been excised and replaced by the v h 3418 psti / bsteii fragment ( excised from fv . 3418 ). the resulting plasmid pgosa . g ( see fig3 a and 37b ) contains two copies of v h . 3418 linked by the ( gly 4 ser ) 3 alaglyserala linker , plus v l . 4715 linked by the ( gly 4 ser ) 2 gly 4 val linker to the framework - 4 region of v l . 3418 , thus : this plasmid contains a dicistronic operon consisting of v h . 3418 linked by the ( gly 4 ser ) 3 alaglyserala linker to v h . 4715 plus v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 . both transcriptional units are preceded by a ribosome binding site - and a pelb leader sequence ( see fig3 ), thus : this construct was obtained by inserting the fragment pcr - vi sfii / nhei which contains v h 4715 ( fig2 ), into the vector pgosa . g from which the sfii / nhei v h 3418 fragment was removed . this plasmid is derived from pgosa . g from which the ( gly 4 ser ) 3 alaglyserala linker - v h 3418 bsteii / nhei fragment was excised and replaced by the fragment pcr - ix bsteii / nhei which contains v h . 4715 ( fig2 ). the resulting plasmid pgosa . z ( see fig3 ) contains v h . 3418 linked directly to the framework - 1 region of v h . 4715 , plus v l . 4715 linked by the ( gly 4 ser ) 2 gly 4 val linker to the framework - 4 region of v l . 3418 , thus : this plasmid contains a dicistronic operon consisting of the v h . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v h . 4715 plus v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 . both transcriptional units are preceded by a ribosome binding site and a pelb leader sequence ( see fig4 ). this construct was obtained by inserting the nhei / ecori fragment of pgosa . t which contains v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 , into the vector pgosa . z from which the nhei / ecori fragment containing v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to v l . 4715 was removed , thus : this plasmid is derived from pgosa . j by a three point ligation reaction ( see fig4 ). the saci / ecori insert , containing part of v h . 3418 and the full ( gly4ser ) 3 alaglyserala linker - v h . 4715 and the v l . 3418 -( gly 4 ser ) 2 gly 4 val - v l . 4715 encoding sequences , was removed and replaced by the saci / saci pgosa . j fragment containing the same part of v h . 3418 and the full ( gly 4 ser ) 3 alaglyserala linker - v h . 4715 and the saci / ecori pgosa . t fragment containing v l . 3418 linked directly to the framework - 1 region of v l . 4715 ( see fig3 ). the resulting plasmid contains v h . 3418 linked by the ( gly 4 ser ) 3 alaglyserala linker to the 5 &# 39 ; end of the framework - 1 region of v h . 4715 plus v l . 3418 linked directly to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 , thus : detailed description of the preparation of monocistronic constructs pgosa . l and pgosa . y , and pgosa . c , pgosa . x . pgosa . ac and pgosa . ad this plasmid is derived from pgosa . e from which the hindlil / nhei fragment containing dna encoding v h . 4715 -( gly 4 ser ) 3 alaglyserala - v h . 3418 was removed ( see fig4 ). the dna ends of the vector were made blunt - end using klenow dna polymerase and ligated . the resulting plasmid pgosa . l contains v l . 3418 linked by the ( gly 4 ser ) 2 gly 4 val linker to the 5 &# 39 ; end of the framework - 1 region of v l . 4715 , thus : this plasmid is derived from pgosa . t from which the hindiii / nhei fragment containing dna encoding v h . 4715 - v h . 3418 was removed ( see fig4 ). the dna ends of the vector were made blunt - end using klenow dna polymerase and ligated . the resulting plasmid pgosa . y contains v l . 3418 linked directly to 5 &# 39 ; end of the framework - 1 region of v l . 4715 , thus : the preparation of pgosa . c was given in example 5 above ; it can be indicated with : pelb - v h . 4715 - linkera - v h . 3418 . this plasmid is derived from pgosa . t from which the rhei / ecori fragment containing dna encoding v l . 3418 - v l . 4715 was removed . the dna ends of the vector were made blunt - end using klenow dna polymerase and ligated . the resulting plasmid pgosa . x ( see fig4 ) contains v h . 4715 linked directly to 5 &# 39 ; end of the framework - 1 region of v h . 3418 , thus : pelb - v h . 4715 * v h . 3418 . this plasmid is derived from pgosa . z from which the nhei / ecori fragment containing dna encoding v l . 3418 -( gly 4 ser ) 2 gly 4 val - v l . 4715 was removed ( see fig4 ). the dna ends of the vector were made blunt - end using klenow dna polymerase and ligated . the resulting plasmid pgosa . ac contains v h . 3418 linked directly to 5 &# 39 ; end of the framework - 1 region of v h . 4715 , thus : this plasmid was obtained by inserting the psti / ecori pcr . x . fragment containing dna encoding v h . 3418 - ( gly 4 ser ) 3 alagly - serala - v h . 4715 ( see fig2 ) into the fv . 4715 - myc vector from which the psti / ecori fv . 4715 - myc insert was removed ( see fig4 ), thus : pelb - v h . 3418 - linkera - v h . 4715 . these monocistronic constructs can be used to transform the same host with two different plasmids or to transform two different hosts , so that the two v h &# 39 ; s in series can be produced separately from the two v l &# 39 ; s in series . in this specification the construction of a two chain protein complex is described , in which one of the chains consists of two heavy chain v - domains and the other chain consists of the two corresponding light chain v - domains . the variable domains are linked either directly or through a polypeptide linker . in this specification evidence is provided that these type of molecules (&# 34 ; double heads &# 34 ;) contain both antigen binding specificities of the fv &# 39 ; s used to generate these multi - functional antibody fragments . fig1 shows that gosa . e can be used to specifically target the enzyme glucose oxidase to several streptococcus sanguis strains , using antibody fragments derived from hybridomas expressing antibodies directed against these antigens . fig1 further shows that the fine specificity of the anti - streptococcus sanguis scfv 4715 is preserved in the gosa . e double head . effect of linkers and relative position of v - domains on double head activity after it was shown that the &# 34 ; cross - over double - head &# 34 ; approach ( v h a - v h b + v l b - v l a ) yields active bispecific molecules , the importance of the relative position of the v - domains in these constructs was investigated . both possible positional orientations ( gosa . e = v h a - linkera - v h b + v l b - linkerv - v l a and gosa . j = v h b - linkera - v h a + v l b - linkerv - v l a ) were constructed and tested for bispecific activity , despite the suggestion obtained by molecular modelling that the binding site formed by the second ( downstream / c - terminal ) v - domains in the configuration v h b - v h a + v l b - v l a ( gosa . j ) was in an unfavourable position for binding to large protein antigens on the surface of cells . surprisingly however , it was found experimentally that the downstream binding site is in fact accessible . although the relative position of the heavy chains and the light chains was found to have an effect on the observed reactivity both tested combinations show bispecific activity with the &# 34 ; cross - over &# 34 ; combination ( gosa . e = v h a - v h b + v l b - v l a ) exhibiting a higher level of reactivity compared to the combination v h b - v l a + v l b - v l (= gosa . j ) as demonstrated for a = anti - strep and b = anti - gox . molecular modelling of the v h b - v h a + v l b - v l a (= gosa . j ) configuration further suggested that , only when the connecting linkers are kept long enough ( to span 30 to 35 å ), the protein chains could fold such that both binding sites are fully accessible . the &# 34 ; cross - over &# 34 ; configuration : v h a - v h b + v l b - v l a ( gosa . e ) wherein linker length was not critical , was predicted to result in a complex with both binding sites facing in opposite directions , without the restraints suggested for the configuration v h b - v h a + v l b - v l a ( gosa . j ). removing the flexible polypeptide linker from the v h a - v h b chain only had a minimal effect on the ability of the double head in the &# 34 ; cross - over &# 34 ; configuration ( gosa . v = v h a * v h b + v l b - v l a ) to bind both s . sanguis and glucose oxidase . however , removing the flexible polypeptide linker from the v h b - v h a chain from the molecule in the v h b - v h a + v l b - v l a configuration ( gosa . z = v h b * v h a + v l b - v l a ) resulted in a dramatic reduction of its ability to bind both s . sanguis and glucose oxidase . in contrast with the double head in the &# 34 ; cross - over &# 34 ; configuration without the flexible polypeptide linker between the two heavy chain domains ( gosa . v ), where molecular modelling predicted the resulting molecule to be active , removal of the flexible linker from the v l b - v l a chain could not be modelled such that both binding sites were fully accessible . elisa results confirm that the double head in the v h b - v h a + v l b - v l a configuration without a linker between the two light chain domains ( gosa . ab ) exhibits only minimal s . sanguis and glucose oxidase binding activity . surprisingly , deletion of the flexible linker from the v l b - v l a chain from the double head in the &# 34 ; cross - over &# 34 ; configuration ( gosa . s ) only had a small effect on the bispecific activity of the resulting molecule . as expected from the molecular modelling results from the double heads without a flexible linker between the two light chain domains , removal of both the flexible polypeptide linkers from the double head molecules , could not be modelled such that both binding sites were fully accessible . in agreement with the elisa results obtained with the gosa . ab construct , the double head in the v h b - v h a + v l b - v l a configuration without any linkers ( gosa . aa ) only exhibits minimal if any s . sanguis and glucose oxidase binding activity . surprisingly , the double head in the &# 34 ; cross - over &# 34 ; configuration without any linkers ( gosa . t = v h a * v h b + v l b * v l a ) still exhibited 25 - 50 % of s . sanguis and glucose oxidase bispecific binding activity when compared to the activity of the double head in the &# 34 ; cross - over &# 34 ; configuration with two linkers ( gosa . e ). thus the conclusion of this work is that modelling can give some indications , but that the computer programmes cannot predict what is possible and what not . several deviations from the modelling expectations were found . with a paraphrase on an old saying : theories are nice but the experiment is the ultimate proof . using an elisa format it was shown that the sensitivity of the gosa . e double head is as least as a sensitive as an igg - glucose oxidase conjugate , as determined by the lowest concentration of streptococcus sanguis antigen immobilised on a solid phase that is still detectable . fplc analysis of partially affinity - purified gosa . e , gosa . v , gosa . s and gosa . t samples usually gave only one gosa double head activity peak as determined by elisa ( fig1 - 18 ). the position of this peak in the elution pattern indicated that the molecular weight of the gosa double head is 40 - 50 kd . since this molecular weight corresponds to the expected molecular weight of the v h 2 + v l 2 double head dimer , it was concluded that gosa . e , gosa . v , gosa . s and gosa . t are primarily produced as dimeric molecules . occasionally an activity peak with an apparent molecular weight of ≈ 200 kd was observed ( fig1 ). the presence of glucose oxidase activity in these fractions indicate that these fractions contain gosa double head complexed with glucose oxidase . it was shown that bifunctionally active dimeric gosa molecules together in one cell can be produced by translation from one dicistronic messenger ( gosa . e , gosa . s , gosa . t , gosa . v , gosa . j , gosa . ab , gosa . aa and gosa . z ). in addition high levels of s . sanguis and glucose oxidase bispecific binding activity is formed when supernatants of cultures producing the separate gosa subunits are mixed ( see example 7 ). the effects of linkers and the relative position of the individual v h - domains on the s . sanquis and glucose oxidase bispecific binding activity observed in these mixing experiments are comparable to the dicistronic constructs . ( lia ) stands for the v h -- v h linker ( gly 4 ser ) 3 alaglyserala (= linkera ) ( liv ) stands for the v l -- v l linker ( gly 4 ser ) 2 gly 4 val (= linkerv ) table 2______________________________________table 2agosa . a : v . sub . h . 4715 - lia -( sfii )- v . sub . l . 4715 - mycgosa . b : v . sub . h . 3418 - liv - v . sub . l . 3418 -( sali / ecori ) gosa . d : v . sub . h . 3418 + v . sub . l . 3418 - liv - v . sub . l . 4715gosa . g : v . sub . h . 3418 - lia - v . sub . h . 3418 + v . sub . l . 3418 - liv - v . sub . l . 4715table 2bgosa . e : v . sub . h . 4715 - lia - v . sub . h . 3418 + v . sub . l . 3418 - liv - v . sub . l . 4715gosa . s : v . sub . h . 4715 - lia - v . sub . h . 3418 + v . sub . l . 3418 * v . sub . l . 4715gosa . t : v . sub . h . 4715 * v . sub . h . 3418 + v . sub . l . 3418 * v . sub . l . 4715gosa . v : v . sub . h . 4715 * v . sub . h . 3418 + v . sub . l . 3418 - liv - v . sub . l . 4715gosa . j : v . sub . h . 3418 - lia - v . sub . h . 4715 + v . sub . l . 3418 - liv - v . sub . l . 4715gosa . ab : v . sub . h . 3418 - lia - v . sub . h . 4715 + v . sub . l . 3418 * v . sub . l . 4715gosa . aa : v . sub . h . 3418 * v . sub . h . 4715 + v . sub . l . 3418 * v . sub . l . 4715gosa . z : v . sub . h . 3418 * v . sub . h . 4715 + v . sub . l . 3418 - liv - v . sub . l . 4715table 2cgosa . l : v . sub . l . 3418 - liv - v . sub . l . 4715gosa . y : v . sub . l . 3418 * v . sub . l . 4715gosa . ad : v . sub . h . 3418 - lia - v . sub . h . 4715gosa . ac : v . sub . h . 3418 * v . sub . h . 4715gosa . c : v . sub . h . 4715 - lia - v . sub . h . 3418gosa . x : v . sub . h . 4715 * v . sub . h . 3418______________________________________ (*) indicates that the two heavy chain domains or the two light chain domains are fused together without a connecting linker . ep - 0281604 ; genex / enzon ; single polypeptide chain binding molecules ; priority date sep . 2 , 1986 . wo 93 / 11161 ; enzon , inc ; multivalent antigen - binding proteins ; priority date nov . 25 , 19991 . wo 94 / 09131 ; scotgen limited ; recombinant specific binding protein ; priority date oct . 15 , 19992 ). wo 94 / 13804 ( cambridge antibody technology / medical research council ; multivalent and multispecific binding proteins , their manufacture and use ; first priority date dec . 4 , 1992 . wo 94 / 13806 ; the dow chemical company ; multivalent single chain antibodies ; priority date dec . 11 , 19992 . wo 94 / 25591 ; unilever n . v ., unilever plc , hamers , r ., hamers - 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emmons , t ., cohen , j . a ., myers , j . n ., weiner , d . b . and greene , m . i . ; proc . natl . acad . sci . usa . 86 ( 1989 ) 5537 - 5541 ; development of biologically active peptides based on antibody structure . __________________________________________________________________________ # sequence listing - ( 1 ) general information :- ( iii ) number of sequences : 31 - ( 2 ) information for seq id no : 1 :- ( i ) sequence characteristics :# pairs ( a ) length : 737 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; cdna domains with syntheticc linker ( s )&# 34 ;- ( vii ) immediate source :# insert of pur4124e : ecori - hindiii - ( ix ) feature : ( a ) name / key : cds ( b ) location : 11 .. 730 ( d ) other information :/ pro - # duct = &# 34 ; vllys - gs - vhlys &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 11 .. 334 ( d ) other information :/ pro - # duct = &# 34 ; vllys &# 34 ;- ( ix ) feature : ( a ) name / key : misc . sub .-- - # rna ( b ) location : 335 .. 379 ( d ) other information :/ pro - # duct = &# 34 ;( gly4ser ) 3 linker &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 380 .. 727 ( d ) other information :/ pro - # duct = &# 34 ; vhlys &# 34 ;# 1 : ( xi ) sequence description : seq id no :- gaattcggcc gac atc gag ctc acc cag tct cca gc - # c tcc ctt tct gcg 49 # thr gln ser pro ala ser leu ser ala # 10 - tct gtg gga gaa act gtc acc atc aca tgt cg - # a gca agt ggg aat att 97ser val gly glu thr val thr ile thr cys ar - # g ala ser gly asn ile # 25 - cac aat tat tta gca tgg tat cag cag aaa ca - # g gga aaa tct cct cag 145his asn tyr leu ala trp tyr gln gln lys gl - # n gly lys ser pro gln # 45 - ctc ctg gtc tat tat aca aca acc tta gca ga - # t ggt gtg cca tca agg 193leu leu val tyr tyr thr thr thr leu ala as - # p gly val pro ser arg # 60 - ttc agt ggc agt gga tca gga aca caa tat tc - # t ctc aag atc aac agc 241phe ser gly ser gly ser gly thr gln tyr se - # r leu lys ile asn ser # 75 - ctg caa cct gaa gat ttt ggg agt tat tac tg - # t caa cat ttt tgg agt 289leu gln pro glu asp phe gly ser tyr tyr cy - # s gln his phe trp ser # 90 - act cct cgg acg ttc ggt gga ggg acc aag ct - # c gag atc aaa cgg ggt 337thr pro arg thr phe gly gly gly thr lys le - # u glu ile lys arg gly # 105 - gga ggc ggt tca ggc gga ggt ggc tct ggc gg - # t ggc gga tcg cag gtg 385gly gly gly ser gly gly gly gly ser gly gl - # y gly gly ser gln val110 1 - # 15 1 - # 20 1 -# 25 - cag ctg cag gag tca gga cct ggc ctg gtg gc - # g ccc tca cag agc ctg 433gln leu gln glu ser gly pro gly leu val al - # a pro ser gln ser leu # 140 - tcc atc aca tgc acc gtc tca ggg ttc tca tt - # a acc ggc tat ggt gta 481ser ile thr cys thr val ser gly phe ser le - # u thr gly tyr gly val # 155 - aac tgg gtt cgc cag cct cca gga aag ggt ct - # g gag tgg ctg gga atg 529asn trp val arg gln pro pro gly lys gly le - # u glu trp leu gly met # 170 - att tgg ggt gat gga aac aca gac tat aat tc - # a gct ctc aaa tcc aga 577ile trp gly asp gly asn thr asp tyr asn se - # r ala leu lys ser arg # 185 - ctg agc atc agc aag gac aac tcc aag agc ca - # a gtt ttc tta aaa atg 625leu ser ile ser lys asp asn ser lys ser gl - # n val phe leu lys met190 1 - # 95 2 - # 00 2 -# 05 - aac agt ctg cac act gat gac aca gcc agg ta - # c tac tgt gcc aga gag 673asn ser leu his thr asp asp thr ala arg ty - # r tyr cys ala arg glu # 220 - aga gat tat agg ctt gac tac tgg ggc caa gg - # g acc acg gtc acc gtc 721arg asp tyr arg leu asp tyr trp gly gln gl - # y thr thr val thr val # 235 # 737 ttser ser - ( 2 ) information for seq id no : 2 :- ( i ) sequence characteristics :# acids ( a ) length : 239 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 2 : ( xi ) sequence description : seq id no :- asp ile glu leu thr gln ser pro ala ser le - # u ser ala ser val gly # 15 - glu thr val thr ile thr cys arg ala ser gl - # y asn ile his asn tyr # 30 - leu ala trp tyr gln gln lys gln gly lys se - # r pro gln leu leu val # 45 - tyr tyr thr thr thr leu ala asp gly val pr - # o ser arg phe ser gly # 60 - ser gly ser gly thr gln tyr ser leu lys il - # e asn ser leu gln pro # 80 - glu asp phe gly ser tyr tyr cys gln his ph - # e trp ser thr pro arg # 95 - thr phe gly gly gly thr lys leu glu ile ly - # s arg gly gly gly gly # 110 - ser gly gly gly gly ser gly gly gly gly se - # r gln val gln leu gln # 125 - glu ser gly pro gly leu val ala pro ser gl - # n ser leu ser ile thr # 140 - cys thr val ser gly phe ser leu thr gly ty - # r gly val asn trp val145 1 - # 50 1 - # 55 1 -# 60 - arg gln pro pro gly lys gly leu glu trp le - # u gly met ile trp gly # 175 - asp gly asn thr asp tyr asn ser ala leu ly - # s ser arg leu ser ile # 190 - ser lys asp asn ser lys ser gln val phe le - # u lys met asn ser leu # 205 - his thr asp asp thr ala arg tyr tyr cys al - # a arg glu arg asp tyr # 220 - arg leu asp tyr trp gly gln gly thr thr va - # l thr val ser ser225 2 - # 30 2 - # 35 - ( 2 ) information for seq id no : 3 :- ( i ) sequence characteristics :# pairs ( a ) length : 920 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; cdna domains with syntheticc linker ( s )&# 34 ;- ( vii ) immediate source :# insert fv . 3418lone : hindiii - ecori - ( ix ) feature : ( a ) name / key : cds ( b ) location : 36 .. 443 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vh3418 &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 36 .. 101 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 102 .. 440 ( d ) other information :/ pro - # duct = &# 34 ; vh3418 &# 34 ;- ( ix ) feature : ( a ) name / key : cds ( b ) location : 495 .. 884 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vl4318 &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 495 .. 560 #/ product = &# 34 ; pectate lyase &# 34 ; ation :- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 561 .. 881 ( d ) other information :/ pro - # duct = &# 34 ; vl3418 &# 34 ;# 3 : ( xi ) sequence description : seq id no :# cta ttg cct 53c aaggagacag tcata atg aaa tac # met lys tyr leu leu pro20 - acg gca gcc gct gga ttg tta tta ctc gct gc - # c caa cca gcg atg gcc 101thr ala ala ala gly leu leu leu leu ala al - # a gln pro ala met ala - cag gtg cag ctg cag cag tca gga cct gag ct - # g gta aag cct ggg gct 149gln val gln leu gln gln ser gly pro glu le - # u val lys pro gly ala # 15 - tca gtg aag atg tcc tgc aag gct tct gga ta - # c aca ttc act agc tat 197ser val lys met ser cys lys ala ser gly ty - # r thr phe thr ser tyr # 30 - gtt atg cac tgg gtg aaa cag aag cct ggg ca - # g ggc ctt gag tgg att 245val met his trp val lys gln lys pro gly gl - # n gly leu glu trp ile # 45 - gga tat att tat cct tac aat gat ggt act aa - # g tac aat gag aag ttc 293gly tyr ile tyr pro tyr asn asp gly thr ly - # s tyr asn glu lys phe # 60 - aaa ggc aag gcc aca ctg act tca gac aaa tc - # c tcc agc aca gcc tac 341lys gly lys ala thr leu thr ser asp lys se - # r ser ser thr ala tyr # 80 - atg gag ctc agc agc ctg acc tct gag gac tc - # t gcg gtc tat tac tgt 389met glu leu ser ser leu thr ser glu asp se - # r ala val tyr tyr cys # 95 - tca aga cgc ttt gac tac tgg ggc caa ggg ac - # c acg gtc acc gtc tcc 437ser arg arg phe asp tyr trp gly gln gly th - # r thr val thr val ser # 110 - tca taa taagagctat gggagcttgc atgcaaattc tatttcaagg ag - # acagtcat 493ser # gga ttg tta tta ctc 539 gca gcc gct met lys tyr leu leu pro thr ala ala a - # la gly leu leu leu leu10 - gct gcc caa cca gcg atg gcc gac atc gag ct - # c acc cag tct cca tct 587ala ala gln pro ala met ala asp ile glu le - # u thr gln ser pro ser # 5 1 - tcc atg tat gca tct cta gga gag aga atc ac - # t atc act tgc aag gcg 635ser met tyr ala ser leu gly glu arg ile th - # r ile thr cys lys ala # 25 - agt cag gac att aat acc tat tta acc tgg tt - # c cag cag aaa cca ggg 683ser gln asp ile asn thr tyr leu thr trp ph - # e gln gln lys pro gly # 40 - aaa tct ccc aag acc ctg atc tat cgt gca aa - # c aga ttg cta gat ggg 731lys ser pro lys thr leu ile tyr arg ala as - # n arg leu leu asp gly # 55 - gtc cca tca agg ttc agt ggc agt gga tct gg - # g caa gat tat tct ctc 779val pro ser arg phe ser gly ser gly ser gl - # y gln asp tyr ser leu # 70 - acc atc agc agc ctg gac tat gaa gat atg gg - # a att tat tat tgt cta 827thr ile ser ser leu asp tyr glu asp met gl - # y ile tyr tyr cys leu # 85 - caa tat gat gag ttg tac acg ttc gga ggg gg - # g acc aag ctc gag atc 875gln tyr asp glu leu tyr thr phe gly gly gl - # y thr lys leu glu ile # 105 # 920caa acggtataag gatccagctc gaattclys arg - ( 2 ) information for seq id no : 4 :- ( i ) sequence characteristics :# acids ( a ) length : 135 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 4 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala gln val gln leu gl - # n gln ser gly pro glu # 10 - leu val lys pro gly ala ser val lys met se - # r cys lys ala ser gly # 25 - tyr thr phe thr ser tyr val met his trp va - # l lys gln lys pro gly # 40 - gln gly leu glu trp ile gly tyr ile tyr pr - # o tyr asn asp gly thr # 55 - lys tyr asn glu lys phe lys gly lys ala th - # r leu thr ser asp lys # 70 - ser ser ser thr ala tyr met glu leu ser se - # r leu thr ser glu asp # 90 - ser ala val tyr tyr cys ser arg arg phe as - # p tyr trp gly gln gly # 105 - thr thr val thr val ser ser 110 - ( 2 ) information for seq id no : 5 :- ( i ) sequence characteristics :# acids ( a ) length : 129 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 5 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala asp ile glu leu th - # r gln ser pro ser ser # 10 - met tyr ala ser leu gly glu arg ile thr il - # e thr cys lys ala ser # 25 - gln asp ile asn thr tyr leu thr trp phe gl - # n gln lys pro gly lys # 40 - ser pro lys thr leu ile tyr arg ala asn ar - # g leu leu asp gly val # 55 - pro ser arg phe ser gly ser gly ser gly gl - # n asp tyr ser leu thr # 70 - ile ser ser leu asp tyr glu asp met gly il - # e tyr tyr cys leu gln # 90 - tyr asp glu leu tyr thr phe gly gly gly th - # r lys leu glu ile lys # 105 - arg - ( 2 ) information for seq id no : 6 :- ( i ) sequence characteristics :# pairs ( a ) length : 999 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; cdna domains with syntheticc linker ( s )&# 34 ;- ( vii ) immediate source :# insert of fv . 4715 - mycindiii - ecori - ( ix ) feature : ( a ) name / key : cds ( b ) location : 40 .. 468 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vh4715 &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 40 .. 105 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 106 .. 465 ( d ) other information :/ pro - # duct = &# 34 ; vh4715 &# 34 ;- ( ix ) feature : ( a ) name / key : cds ( b ) location : 520 .. 963 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vl4715 - myc &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 520 .. 585 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 586 .. 927 ( d ) other information :/ pro - # duct = &# 34 ; vl4715 &# 34 ;- ( ix ) feature : ( a ) name / key : misc rna ( b ) location : 928 .. 960 ( d ) other information :/ pro - # duct = &# 34 ; myc - tag &# 34 ;# 6 : ( xi ) sequence description : seq id no :- aagcttgcat gcaaattcta tttcaaggag acagtcata atg aaa tac - # cta ttg 54 # met lys tyr leu leu20 - cct acg gca gcc gct gga ttg tta tta ctc gc - # t gcc caa cca gcg atg 102pro thr ala ala ala gly leu leu leu leu al - # a ala gln pro ala met5 - gcc cag gtg cag ctg cag gag tca ggg gga ga - # c tta gtg aag cct gga 150ala gln val gln leu gln glu ser gly gly as - # p leu val lys pro gly # 15 - ggg tcc ctg aca ctc tcc tgt gca acc tct gg - # a ttc act ttc agt agt 198gly ser leu thr leu ser cys ala thr ser gl - # y phe thr phe ser ser # 30 - tat gcc ttt tct tgg gtc cgc cag acc tca ga - # c aag agt ctg gag tgg 246tyr ala phe ser trp val arg gln thr ser as - # p lys ser leu glu trp # 45 - gtc gca acc atc agt agt act gat act tat ac - # c tat tat tca gac aat 294val ala thr ile ser ser thr asp thr tyr th - # r tyr tyr ser asp asn # 60 - gtg aag ggg cgc ttc acc atc tcc aga gac aa - # t ggc aag aac acc ctg 342val lys gly arg phe thr ile ser arg asp as - # n gly lys asn thr leu # 75 - tac ctg caa atg agc agt ctg aag tct gag ga - # c aca gcc gtg tat tac 390tyr leu gln met ser ser leu lys ser glu as - # p thr ala val tyr tyr # 95 - tgt gca aga cat ggg tac tat ggt aaa ggc ta - # t ttt gac tac tgg ggc 438cys ala arg his gly tyr tyr gly lys gly ty - # r phe asp tyr trp gly # 110 - caa ggg acc acg gtc acc gtc tcc tca taa ta - # agagctat gggagcttgc 488gln gly thr thr val thr val ser ser # 120 # ttg cct acg 540g agacagtcat a atg aaa tac cta # met - # lys tyr leu leu pro thr20 - #- gca gcc gct gga ttg tta tta ctc gct gcc ca - # a cca gcg atg gcc gac 588ala ala ala gly leu leu leu leu ala ala gl - # n pro ala met ala asp # 1 # 5 - atc gag ctc act cag tct cca ttc tcc ctg ac - # t gtg aca gca gga gag 636ile glu leu thr gln ser pro phe ser leu th - # r val thr ala gly glu # 15 - aag gtc act atg aat tgc aag tcc ggt cag ag - # t ctg tta aac agt gta 684lys val thr met asn cys lys ser gly gln se - # r leu leu asn ser val # 30 - aat cag agg aac tac ttg acc tgg tac cag ca - # g aag cca ggg cag cct 732asn gln arg asn tyr leu thr trp tyr gln gl - # n lys pro gly gln pro # 45 - cct aaa ctg ttg atc tac tgg gca tcc act ag - # g gaa tct gga gtc cct 780pro lys leu leu ile tyr trp ala ser thr ar - # g glu ser gly val pro # 65 - gat cgc ttc aca gcc agt gga tct gga aca ga - # t ttc act ctc acc atc 828asp arg phe thr ala ser gly ser gly thr as - # p phe thr leu thr ile # 80 - agc agt gtg cag gct gaa gac ctg gca gtt ta - # t tac tgt cag aat gat 876ser ser val gln ala glu asp leu ala val ty - # r tyr cys gln asn asp # 95 - tat act tat ccg ttc acg ttc gga ggg ggg ac - # c aag ctc gag atc aaa 924tyr thr tyr pro phe thr phe gly gly gly th - # r lys leu glu ile lys # 110 - cgg gaa caa aaa ctc atc tca gaa gag gat ct - # g aat taa taagatcaaa 973arg glu gln lys leu ile ser glu glu asp le - # u asn # 125 # 999 gctc gaattc - ( 2 ) information for seq id no : 7 :- ( i ) sequence characteristics :# acids ( a ) length : 142 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 7 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala gln val gln leu gl - # n glu ser gly gly asp # 10 - leu val lys pro gly gly ser leu thr leu se - # r cys ala thr ser gly # 25 - phe thr phe ser ser tyr ala phe ser trp va - # l arg gln thr ser asp # 40 - lys ser leu glu trp val ala thr ile ser se - # r thr asp thr tyr thr # 55 - tyr tyr ser asp asn val lys gly arg phe th - # r ile ser arg asp asn # 70 - gly lys asn thr leu tyr leu gln met ser se - # r leu lys ser glu asp # 90 - thr ala val tyr tyr cys ala arg his gly ty - # r tyr gly lys gly tyr # 105 - phe asp tyr trp gly gln gly thr thr val th - # r val ser ser # 120 - ( 2 ) information for seq id no : 8 :- ( i ) sequence characteristics :# acids ( a ) length : 147 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 8 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala asp ile glu leu th - # r gln ser pro phe ser # 10 - leu thr val thr ala gly glu lys val thr me - # t asn cys lys ser gly # 25 - gln ser leu leu asn ser val asn gln arg as - # n tyr leu thr trp tyr # 40 - gln gln lys pro gly gln pro pro lys leu le - # u ile tyr trp ala ser # 55 - thr arg glu ser gly val pro asp arg phe th - # r ala ser gly ser gly # 70 - thr asp phe thr leu thr ile ser ser val gl - # n ala glu asp leu ala # 90 - val tyr tyr cys gln asn asp tyr thr tyr pr - # o phe thr phe gly gly # 105 - gly thr lys leu glu ile lys arg glu gln ly - # s leu ile ser glu glu # 120 - asp leu asn 125 - ( 2 ) information for seq id no : 9 :- ( i ) sequence characteristics :# pairs ( a ) length : 924 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; cdna domains with syntheticc linker ( s )&# 34 ;- ( vii ) immediate source :# insert of scfv . 4715 - mycdiii - ecori - ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 40 .. 105 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 106 .. 465 ( d ) other information :/ pro - # duct = &# 34 ; vh4715 &# 34 ;- ( ix ) feature : ( a ) name / key : misc . sub .-- - # rna ( b ) location : 466 .. 510 ( d ) other information :/ pro - # duct = &# 34 ;( gly4ser ) 3 - linker &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 511 .. 852 ( d ) other information :/ pro - # duct = &# 34 ; vl4715 &# 34 ;- ( ix ) feature : ( a ) name / key : misc . sub .-- - # rna ( b ) location : 853 .. 885 ( d ) other information :/ pro - # duct = &# 34 ; myc - tag &# 34 ;- ( ix ) feature : ( a ) name / key : cds ( b ) location : 40 .. 888 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vh4715 -( gly4ser ) 3 - vl4715 - myc &# 34 ;# 9 : ( xi ) sequence description : seq id no :- aagcttgcat gcaaattcta tttcaaggag acagtcata atg aaa tac - # cta ttg 54 # met lys tyr leu leu20 - cct acg gca gcc gct gga ttg tta tta ctc gc - # t gcc caa cca gcg atg 102pro thr ala ala ala gly leu leu leu leu al - # a ala gln pro ala met5 - gcc cag gtg cag ctg cag gag tca ggg gga ga - # c tta gtg aag cct gga 150ala gln val gln leu gln glu ser gly gly as - # p leu val lys pro gly # 15 - ggg tcc ctg aca ctc tcc tgt gca acc tct gg - # a ttc act ttc agt agt 198gly ser leu thr leu ser cys ala thr ser gl - # y phe thr phe ser ser # 30 - tat gcc ttt tct tgg gtc cgc cag acc tca ga - # c aag agt ctg gag tgg 246tyr ala phe ser trp val arg gln thr ser as - # p lys ser leu glu trp # 45 - gtc gca acc atc agt agt act gat act tat ac - # c tat tat tca gac aat 294val ala thr ile ser ser thr asp thr tyr th - # r tyr tyr ser asp asn # 60 - gtg aag ggg cgc ttc acc atc tcc aga gac aa - # t ggc aag aac acc ctg 342val lys gly arg phe thr ile ser arg asp as - # n gly lys asn thr leu # 75 - tac ctg caa atg agc agt ctg aag tct gag ga - # c aca gcc gtg tat tac 390tyr leu gln met ser ser leu lys ser glu as - # p thr ala val tyr tyr # 95 - tgt gca aga cat ggg tac tat ggt aaa ggc ta - # t ttt gac tac tgg ggc 438cys ala arg his gly tyr tyr gly lys gly ty - # r phe asp tyr trp gly # 110 - caa ggg acc acg gtc acc gtc tcc tca ggt gg - # a ggc ggt tca ggc gga 486gln gly thr thr val thr val ser ser gly gl - # y gly gly ser gly gly # 125 - ggt ggc tct ggc ggt ggc gga tcg gac atc ga - # g ctc act cag tct cca 534gly gly ser gly gly gly gly ser asp ile gl - # u leu thr gln ser pro # 140 - ttc tcc ctg act gtg aca gca gga gag aag gt - # c act atg aat tgc aag 582phe ser leu thr val thr ala gly glu lys va - # l thr met asn cys lys # 155 - tcc ggt cag agt ctg tta aac agt gta aat ca - # g agg aac tac ttg acc 630ser gly gln ser leu leu asn ser val asn gl - # n arg asn tyr leu thr160 1 - # 65 1 - # 70 1 -# 75 - tgg tac cag cag aag cca ggg cag cct cct aa - # a ctg ttg atc tac tgg 678trp tyr gln gln lys pro gly gln pro pro ly - # s leu leu ile tyr trp # 190 - gca tcc act agg gaa tct gga gtc cct gat cg - # c ttc aca gcc agt gga 726ala ser thr arg glu ser gly val pro asp ar - # g phe thr ala ser gly # 205 - tct gga aca gat ttc act ctc acc atc agc ag - # t gtg cag gct gaa gac 774ser gly thr asp phe thr leu thr ile ser se - # r val gln ala glu asp # 220 - ctg gca gtt tat tac tgt cag aat gat tat ac - # t tat ccg ttc acg ttc 822leu ala val tyr tyr cys gln asn asp tyr th - # r tyr pro phe thr phe # 235 - gga ggg ggg acc aag ctc gag atc aaa cgg ga - # a caa aaa ctc atc tca 870gly gly gly thr lys leu glu ile lys arg gl - # u gln lys leu ile ser240 2 - # 45 2 - # 50 2 -# 55 - gaa gag gat ctg aat taa taagatcaaa cggtaataag ga - # tccagctc gaattc 924glu glu asp leu asn 260 - ( 2 ) information for seq id no : 10 :- ( i ) sequence characteristics :# acids ( a ) length : 282 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 10 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala gln val gln leu gl - # n glu ser gly gly asp # 10 - leu val lys pro gly gly ser leu thr leu se - # r cys ala thr ser gly # 25 - phe thr phe ser ser tyr ala phe ser trp va - # l arg gln thr ser asp # 40 - lys ser leu glu trp val ala thr ile ser se - # r thr asp thr tyr thr # 55 - tyr tyr ser asp asn val lys gly arg phe th - # r ile ser arg asp asn # 70 - gly lys asn thr leu tyr leu gln met ser se - # r leu lys ser glu asp # 90 - thr ala val tyr tyr cys ala arg his gly ty - # r tyr gly lys gly tyr # 105 - phe asp tyr trp gly gln gly thr thr val th - # r val ser ser gly gly # 120 - gly gly ser gly gly gly gly ser gly gly gl - # y gly ser asp ile glu # 135 - leu thr gln ser pro phe ser leu thr val th - # r ala gly glu lys val # 150 - thr met asn cys lys ser gly gln ser leu le - # u asn ser val asn gln155 1 - # 60 1 - # 65 1 -# 70 - arg asn tyr leu thr trp tyr gln gln lys pr - # o gly gln pro pro lys # 185 - leu leu ile tyr trp ala ser thr arg glu se - # r gly val pro asp arg # 200 - phe thr ala ser gly ser gly thr asp phe th - # r leu thr ile ser ser # 215 - val gln ala glu asp leu ala val tyr tyr cy - # s gln asn asp tyr thr # 230 - tyr pro phe thr phe gly gly gly thr lys le - # u glu ile lys arg glu235 2 - # 40 2 - # 45 2 -# 50 - gln lys leu ile ser glu glu asp leu asn # 260 - ( 2 ) information for seq id no : 11 :- ( i ) sequence characteristics :# pairs ( a ) length : 1706 base ( b ) type : nucleic acid ( c ) strandedness : double ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; cdna domains with syntheticc linker ( s )&# 34 ;- ( vii ) immediate source :# insert of pgosa . ee : hindiii - ecori - ( ix ) feature : ( a ) name / key : cds ( b ) location : 40 .. 864 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vh4715 - lia - vh3418 &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 40 .. 105 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 106 .. 465 ( d ) other information :/ pro - # duct = &# 34 ; vh4715 &# 34 ;- ( ix ) feature : ( a ) name / key : misc . sub .-- - # rna ( b ) location : 466 .. 522 ( d ) other information :/ pro - # duct = &# 34 ; linkera ( gly4ser ) 3alaglyseral - # a &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 523 .. 861 ( d ) other information :/ pro - # duct = &# 34 ; vh3418 &# 34 ;- ( ix ) feature : ( a ) name / key : cds ( b ) location : 913 .. 1689 ( d ) other information :/ pro - # duct = &# 34 ; pelb - vl3418 - liv - vl4715 &# 34 ;- ( ix ) feature : ( a ) name / key : sig . sub .-- - # peptide ( b ) location : 913 .. 978 ( d ) other information :/ pro - # duct = &# 34 ; pectate lyase &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 979 .. 1299 ( d ) other information :/ pro - # duct = &# 34 ; vl3418 &# 34 ;- ( ix ) feature : ( a ) name / key : misc . sub .-- - # rna ( b ) location : 1300 .. 1344 ( d ) other information :/ pro - # duct = &# 34 ; linker v ( gly4ser ) 2gly4val &# 34 ;- ( ix ) feature : ( a ) name / key : mat . sub .-- - # peptide ( b ) location : 1345 .. 1686 ( d ) other information :/ pro - # duct = &# 34 ; vl4715 &# 34 ;# 11 : ( xi ) sequence description : seq id no :- aagcttgcat ggaaattcta tttcaaggag acagtcata atg aaa tac - # cta ttg 54 # met lys tyr leu leu20 - cct acg gca gcc gct gga ttg tta tta ctc gc - # t gcc caa cca gcg atg 102pro thr ala ala ala gly leu leu leu leu al - # a ala gln pro ala met5 - gcc cag gtg cag ctg cag gag tca ggg gga ga - # c tta gtg aag cct gga 150ala gln val gln leu gln glu ser gly gly as - # p leu val lys pro gly # 15 - ggg tcc ctg aca ctc tcc tgt gca acc tct gg - # a ttc act ttc agt agt 198gly ser leu thr leu ser cys ala thr ser gl - # y phe thr phe ser ser # 30 - tat gcc ttt tct tgg gtc cgc cag acc tca ga - # c aag agt ctg gag tgg 246tyr ala phe ser trp val arg gln thr ser as - # p lys ser leu glu trp # 45 - gtc gca acc atc agt agt act gat act tat ac - # c tat tat tca gac aat 294val ala thr ile ser ser thr asp thr tyr th - # r tyr tyr ser asp asn # 60 - gtg aag ggg cgc ttc acc atc tcc aga gac aa - # t ggc aag aac acc ctg 342val lys gly arg phe thr ile ser arg asp as - # n gly lys asn thr leu # 75 - tac ctg caa atg agc agt ctg aag tct gag ga - # c aca gcc gtg tat tac 390tyr leu gln met ser ser leu lys ser glu as - # p thr ala val tyr tyr # 95 - tgt gca aga cat ggg tac tat ggt aaa ggc ta - # t ttt gac tac tgg ggc 438cys ala arg his gly tyr tyr gly lys gly ty - # r phe asp tyr trp gly # 110 - caa ggg acc acg gtc acc gtc tcc tca ggt gg - # a ggc ggt tca ggc gga 486gln gly thr thr val thr val ser ser gly gl - # y gly gly ser gly gly # 125 - ggt ggc tct ggc ggt ggc gga tcg gcc ggt tc - # g gcc cag gtc cag ctg 534gly gly ser gly gly gly gly ser ala gly se - # r ala gln val gln leu # 140 - caa cag tca gga cct gag ctg gta aag cct gg - # g gct tca gtg aag atg 582gln gln ser gly pro glu leu val lys pro gl - # y ala ser val lys met # 155 - tcc tgc aag gct tct gga tac aca ttc act ag - # c tat gtt atg cac tgg 630ser cys lys ala ser gly tyr thr phe thr se - # r tyr val met his trp160 1 - # 65 1 - # 70 1 -# 75 - gtg aaa cag aag cct ggg cag ggc ctt gag tg - # g att gga tat att tat 678val lys gln lys pro gly gln gly leu glu tr - # p ile gly tyr ile tyr # 190 - cct tac aat gat ggt act aag tac aat gag aa - # g ttc aaa ggc aag gcc 726pro tyr asn asp gly thr lys tyr asn glu ly - # s phe lys gly lys ala # 205 - aca ctg act tca gac aaa tcc tcc agc aca gc - # c tac atg gag ctc agc 774thr leu thr ser asp lys ser ser ser thr al - # a tyr met glu leu ser # 220 - agc ctg acc tct gag gac tct gcg gtc tat ta - # c tgt tca aga cgc ttt 822ser leu thr ser glu asp ser ala val tyr ty - # r cys ser arg arg phe # 235 - gac tac tgg ggc caa ggg acc acc gtc acc gt - # c tcc tca taa # 864asp tyr trp gly gln gly thr thr val thr va - # l ser ser240 2 - # 45 2 - # 50 # aaa tac 921ctgcatg caaattctat ttcaaggaga cagtcata atg # met - # lys tyr20 - #- cta ttg cct acg gca gcc gct gga ttg tta tt - # a ctc gct gcc caa cca 969leu leu pro thr ala ala ala gly leu leu le - # u leu ala ala gln pro5 - gcg atg gcc gac atc gag ctc acc cag tct cc - # a tct tcc atg tat gca1017ala met ala asp ile glu leu thr gln ser pr - # o ser ser met tyr ala # 10 - tct cta gga gag aga atc act atc act tgc aa - # g gcg agt cag gac att1065ser leu gly glu arg ile thr ile thr cys ly - # s ala ser gln asp ile # 25 - aat acc tat tta acc tgg ttc cag cag aaa cc - # a ggg aaa tct ccc aag1113asn thr tyr leu thr trp phe gln gln lys pr - # o gly lys ser pro lys # 45 - acc ctg atc tat cgt gca aac aga ttg cta ga - # t ggg gtc cca tca agg1161thr leu ile tyr arg ala asn arg leu leu as - # p gly val pro ser arg # 60 - ttc agt ggc agt gga tct ggg caa gat tat tc - # t ctc acc atc agc agc1209phe ser gly ser gly ser gly gln asp tyr se - # r leu thr ile ser ser # 75 - ctg gac tat gaa gat atg gga att tat tat tg - # t cta caa tat gat gag1257leu asp tyr glu asp met gly ile tyr tyr cy - # s leu gln tyr asp glu # 90 - ttg tac acg ttc gga ggg ggg acc aag ctc ga - # g atc aaa cgg ggt gga1305leu tyr thr phe gly gly gly thr lys leu gl - # u ile lys arg gly gly # 105 - ggc ggt tca ggc gga ggt ggc tct ggc ggt gg - # c gga gtc gac atc gaa1353gly gly ser gly gly gly gly ser gly gly gl - # y gly val asp ile glu110 1 - # 15 1 - # 20 1 -# 25 - ctc act cag tct cca ttc tcc ctg act gtg ac - # a gca gga gag aag gtc1401leu thr gln ser pro phe ser leu thr val th - # r ala gly glu lys val # 140 - act atg aat tgc aag tcc ggt cag agt ctg tt - # a aac agt gta aat cag1449thr met asn cys lys ser gly gln ser leu le - # u asn ser val asn gln # 155 - agg aac tac ttg acc tgg tac cag cag aag cc - # a ggg cag cct cct aaa1497arg asn tyr leu thr trp tyr gln gln lys pr - # o gly gln pro pro lys # 170 - ctg ttg atc tac tgg gca tcc act agg gaa tc - # t gga gtc cct gat cgc1545leu leu ile tyr trp ala ser thr arg glu se - # r gly val pro asp arg # 185 - ttc aca gcc agt gga tct gga aca gat ttc ac - # t ctc acc atc agc agt1593phe thr ala ser gly ser gly thr asp phe th - # r leu thr ile ser ser190 1 - # 95 2 - # 00 2 -# 05 - gtg cag gct gaa gac ctg gca gtt tat tac tg - # t cag aat gat tat act1641val gln ala glu asp leu ala val tyr tyr cy - # s gln asn asp tyr thr # 220 - tat ccg ttc acg ttc gga ggg ggg acc aag ct - # c gaa atc aaa cgg taa1689tyr pro phe thr phe gly gly gly thr lys le - # u glu ile lys arg # 235 # 1706 c - ( 2 ) information for seq id no : 12 :- ( i ) sequence characteristics :# acids ( a ) length : 274 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 12 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala gln val gln leu gl - # n glu ser gly gly asp # 10 - leu val lys pro gly gly ser leu thr leu se - # r cys ala thr ser gly # 25 - phe thr phe ser ser tyr ala phe ser trp va - # l arg gln thr ser asp # 40 - lys ser leu glu trp val ala thr ile ser se - # r thr asp thr tyr thr # 55 - tyr tyr ser asp asn val lys gly arg phe th - # r ile ser arg asp asn # 70 - gly lys asn thr leu tyr leu gln met ser se - # r leu lys ser glu asp # 90 - thr ala val tyr tyr cys ala arg his gly ty - # r tyr gly lys gly tyr # 105 - phe asp tyr trp gly gln gly thr thr val th - # r val ser ser gly gly # 120 - gly gly ser gly gly gly gly ser gly gly gl - # y gly ser ala gly ser # 135 - ala gln val gln leu gln gln ser gly pro gl - # u leu val lys pro gly # 150 - ala ser val lys met ser cys lys ala ser gl - # y tyr thr phe thr ser155 1 - # 60 1 - # 65 1 -# 70 - tyr val met his trp val lys gln lys pro gl - # y gln gly leu glu trp # 185 - ile gly tyr ile tyr pro tyr asn asp gly th - # r lys tyr asn glu lys # 200 - phe lys gly lys ala thr leu thr ser asp ly - # s ser ser ser thr ala # 215 - tyr met glu leu ser ser leu thr ser glu as - # p ser ala val tyr tyr # 230 - cys ser arg arg phe asp tyr trp gly gln gl - # y thr thr val thr val235 2 - # 40 2 - # 45 2 -# 50 - ser ser - ( 2 ) information for seq id no : 13 :- ( i ) sequence characteristics :# acids ( a ) length : 258 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 13 : ( xi ) sequence description : seq id no :- met lys tyr leu leu pro thr ala ala ala gl - # y leu leu leu leu ala10 - ala gln pro ala met ala asp ile glu leu th - # r gln ser pro ser ser # 10 - met tyr ala ser leu gly glu arg ile thr il - # e thr cys lys ala ser # 25 - gln asp ile asn thr tyr leu thr trp phe gl - # n gln lys pro gly lys # 40 - ser pro lys thr leu ile tyr arg ala asn ar - # g leu leu asp gly val # 55 - pro ser arg phe ser gly ser gly ser gly gl - # n asp tyr ser leu thr # 70 - ile ser ser leu asp tyr glu asp met gly il - # e tyr tyr cys leu gln # 90 - tyr asp glu leu tyr thr phe gly gly gly th - # r lys leu glu ile lys # 105 - arg gly gly gly gly ser gly gly gly gly se - # r gly gly gly gly val # 120 - asp ile glu leu thr gln ser pro phe ser le - # u thr val thr ala gly # 135 - glu lys val thr met asn cys lys ser gly gl - # n ser leu leu asn ser # 150 - val asn gln arg asn tyr leu thr trp tyr gl - # n gln lys pro gly gln155 1 - # 60 1 - # 65 1 -# 70 - pro pro lys leu leu ile tyr trp ala ser th - # r arg glu ser gly val # 185 - pro asp arg phe thr ala ser gly ser gly th - # r asp phe thr leu thr # 200 - ile ser ser val gln ala glu asp leu ala va - # l tyr tyr cys gln asn # 215 - asp tyr thr tyr pro phe thr phe gly gly gl - # y thr lys leu glu ile # 230 - lys arg235 - ( 2 ) information for seq id no : 14 :- ( i ) sequence characteristics :# pairs ( a ) length : 25 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 1 # 14 : ( xi ) sequence description : seq id no :# 25 aatg gcaag - ( 2 ) information for seq id no : 15 :- ( i ) sequence characteristics :# pairs ( a ) length : 45 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 2 # 15 : ( xi ) sequence description : seq id no :# 45 gaac cggccgatcc gccaccgcca gagcc - ( 2 ) information for seq id no : 16 :- ( i ) sequence characteristics :# pairs ( a ) length : 45 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 3 # 16 : ( xi ) sequence description : seq id no :# 45 cggc ccaggtccag ctgcaacagt cagga - ( 2 ) information for seq id no : 17 :- ( i ) sequence characteristics :# pairs ( a ) length : 53 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 4 # 17 : ( xi ) sequence description : seq id no :- ctacatgaat tcgctagctt attatgagga gacggtgacg gtggtccctt gg - # c 53 - ( 2 ) information for seq id no : 18 :- ( i ) sequence characteristics :# pairs ( a ) length : 36 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 5 # 18 : ( xi ) sequence description : seq id no :# 36 ctgc atgcaaattc tatttc - ( 2 ) information for seq id no : 19 :- ( i ) sequence characteristics :# pairs ( a ) length : 23 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 6 # 19 : ( xi ) sequence description : seq id no :# 23aacg ggg - ( 2 ) information for seq id no : 20 :- ( i ) sequence characteristics :# pairs ( a ) length : 36 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 7 # 20 : ( xi ) sequence description : seq id no :# 36 actc cgccaccgcc agagcc - ( 2 ) information for seq id no : 21 :- ( i ) sequence characteristics :# pairs ( a ) length : 39 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= synthetic dna &# 34 ; cription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 8 # 21 : ( xi ) sequence description : seq id no :# 39 aact cactcagtct ccattctcc - ( 2 ) information for seq id no : 22 :- ( i ) sequence characteristics :# pairs ( a ) length : 50 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 9 # 22 : ( xi ) sequence description : seq id no :# 50ggccgc ttattaccgt ttgatttcga gcttggtccc - ( 2 ) information for seq id no : 23 :- ( i ) sequence characteristics :# pairs ( a ) length : 41 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 1 - # 0 # 23 : ( xi ) sequence description : seq id no :# 41 ctcc tcacaggtcc agttgcaaca g - ( 2 ) information for seq id no : 24 :- ( i ) sequence characteristics :# pairs ( a ) length : 44 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 1 - # 1 # 24 : ( xi ) sequence description : seq id no :# 44 aacg ggacatcgaa ctcactcagt ctcc - ( 2 ) information for seq id no : 25 :- ( i ) sequence characteristics :# pairs ( a ) length : 41 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer dbl . 1 - # 2 # 25 : ( xi ) sequence description : seq id no :# 41 ctcc tcacaggtgc agttgcagga g - ( 2 ) information for seq id no : 26 :- ( i ) sequence characteristics :# pairs ( a ) length : 22 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer pcr . 5 - # 1 # 26 : ( xi ) sequence description : seq id no :# 22tcw gg - ( 2 ) information for seq id no : 27 :- ( i ) sequence characteristics :# pairs ( a ) length : 32 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer pcr . 8 - # 9 # 27 : ( xi ) sequence description : seq id no :# 32 gtgg tcccttggcc cc - ( 2 ) information for seq id no : 28 :- ( i ) sequence characteristics :# pairs ( a ) length : 24 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer pcr . 9 - # 0 # 28 : ( xi ) sequence description : seq id no :# 24agtc tcca - ( 2 ) information for seq id no : 29 :- ( i ) sequence characteristics :# pairs ( a ) length : 22 base ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear - ( ii ) molecule type : other nucleic acid #= &# 34 ; synthetic dna &# 34 ; ription : / desc - ( vii ) immediate source : ( b ) clone : primer pcr . 1 - # 16 # 29 : ( xi ) sequence description : seq id no :# 22gtc cc - ( 2 ) information for seq id no : 30 :- ( i ) sequence characteristics :# acids ( a ) length : 13 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 30 : ( xi ) sequence description : seq id no :- thr thr val thr val ser ser gln val gln le - # u gln gln # 10 - ( 2 ) information for seq id no : 31 :- ( i ) sequence characteristics :# acids ( a ) length : 12 amino ( b ) type : amino acid ( d ) topology : linear - ( ii ) molecule type : protein # 31 : ( xi ) sequence description : seq id no :- lys leu glu ile lys arg asp ile glu leu th - # r gln # 10__________________________________________________________________________