Patent Application: US-201113819330-A

Abstract:
described is an antigen - binding protein , preferably comprising an amino acid sequence that comprises four framework regions and three complementarity - determining regions , wherein the antigen - binding protein is capable of binding a chitinous polysaccharide , and uses thereof .

Description:
colorado potato beetles ( leptinotarsa decemlineata ) were dissected , exoskeletons and wings collected , and remainders discarded . exoskeletons and wings were separately frozen in liquid nitrogen , ground with mortar and pestle , and fine powders collected . colorado potato beetle larvae , pea aphids ( acyrthosiphon pisum ), and tobacco budworm larvae ( heliothis virescens ), were frozen in liquid nitrogen , ground with mortar and pestle , and fine powders collected . collected insect materials were resuspended in pbs and total protein concentrations of suspensions were determined with bradford protein assay . approximate total protein concentrations were 4 . 2 , 0 . 3 , 4 . 2 , 2 . 7 , and 2 . 3 mg / ml for colorado potato beetle ( cpb ) exoskeletons , cpb wings , pea aphids , cpb larvae , and tobacco budworm larvae suspensions , respectively . suspensions were mixed on basis of equal total protein concentration and aliquots were prepared , stored at − 80 ° c ., and suspensions were used for immunization . two llamas , named curley sue and jean harlow , were immunized at weekly intervals with six intramuscular injections of mixed insect suspensions using freund &# 39 ; s incomplete adjuvant ( fia ). doses for immunizations were 125 μg total protein for days 0 and 6 , and 62 . 5 μg total protein for days 13 , 20 , 27 , and 34 . at day 0 and at time of pbl collection at day 38 , sera of llamas were collected . from each immunized llama a separate vhh library was made . rna was isolated from peripheral blood lymphocytes , followed by cdna synthesis using random hexamer primers and superscript iii according to the manufacturer &# 39 ; s instructions ( invitrogen ). a first pcr was performed to amplify vhh and vh dna fragments using a forward primer mix [ 1 : 1 ratio of call001 ( 5 ′- gtcctggctgctcttctacaagg - 3 ′) and call001b ( 5 ′- cctggctgctcttctacaaggtg - 3 ′)] and reverse primer call002 ( 5 ′- ggtacgtgctgttgaactgttcc - 3 ′). after separation of vh and vhh dna fragments by agarose gel electrophoresis and purification of vhh dna fragments from gel , a second pcr was performed on vhh dna fragments to introduce appropriate restriction sites for cloning using forward primer a6e ( 5 ′- gatgtgcagctgcaggagtctggrggagg - 3 ′ ( seq id no : _ ) and reverse primer 38 ( 5 ′- ggactagtgcggccgctggagacggtgacctgggt - 3 ′ ( seq id no : _ )). the pcr fragments were digested using psti and eco911 restriction enzymes ( fermentas ), and ligated upstream of the piii gene in vector pmes3 . the ligation products were ethanol precipitated according to standard protocols , resuspended in water , and electroporated into tg1 cells . library sizes were at least 1e + 08 independent clones for each library . single colony pcr on randomly picked clones from the libraries was performed to assess insert percentages of the libraries . libraries “ curley sue ” and “ jean harlow ” had ≧ 80 % insert percentages of full - length clones . libraries were numbered 44 and 45 for llamas “ curley sue ” and “ jean harlow ,” respectively . phage from each of the libraries were produced using vcsm13 helper phage according to standard procedures . for selections against chitin , practical grade chitin ( sigma ) was coated in elisa plates ( maxisorp , nunc ). chitin was dissolved at 10 mg / ml concentration in 85 % phosphoric acid by shaking on a vortex shaker for approximately 3 hours until all particles were dissolved . serial five - fold dilutions in pbs were prepared , precipitated chitin removed by centrifuging at 20 , 000 g for 5 minutes and supernatants used for coating of elisa plates ( maxisorp , nunc ). wells with 100 μl per well chitin solutions were coated at 4 ° c . overnight or over weekend . sera of llamas curley sue and jean harlow were used to determine optimum chitin concentration for coating in a serum titer elisa performed according to standard procedures . 25 - fold and 3 , 125 - fold diluted chitin solutions were used for selections . wells were washed three times with pbs / 0 . 05 %- tween ®- 20 and blocked with 5 % skimmed milk in pbs ( 5 % mpbs ). phage were suspended in 2 . 5 % mpbs and approximately 1e + 12 cfu were used for each well . after binding to the wells at room temperature for 2 hours , unbound phage were removed by extensive washing with pbs / 0 . 05 %- tween ®- 20 and pbs . bound phage were eluted at room temperature with 0 . 1 mg / ml trypsin ( sigma ) in pbs for 30 minutes . eluted phage were transferred to a polypropylene 96 - well plate ( nunc ) containing excess aebsf trypsin inhibitor ( sigma ). the titers of phage from target - coated wells were compared to titers of phage from blank wells to assess enrichments . phage were amplified using fresh tg1 cells according to standard procedures . enrichments in selection round 1 were approximately ten - fold for both libraries 44 and 45 . enrichments in selection round 2 were five - and ≧ 3e + 03 - fold for libraries 44 and 45 , respectively . fresh tg1 cells were infected with serially diluted eluted phage and plated on lb agar ; 2 % glucose ; 100 μg / ml ampicillin . single colonies were picked in 96 - well plates containing 100 μl per well 2 × ty ; 10 % glycerol ; 2 % glucose ; 100 μg / ml ampicillin . plates were incubated at 37 ° c . and stored at − 80 ° c . as master plates . from selections against chitin 16 clones were picked from 1 st round selections and 30 clones were picked from 2 nd round selections for each library 44 and 45 , in total 92 clones . a single - point binding elisa was used to identify clones binding to plant extracts . vhh - containing extracts for elisa were prepared as follows . 96 - well plates with 100 μl per well 2 × ty , 2 % glucose 100 μg / ml ampicillin were inoculated from the master plates and grown at 37 ° c . overnight . 25 μl per well of overnight culture was used to inoculate fresh 96 - well deep - well plates containing 1 ml per well 2 × ty ; 0 . 1 % glucose ; 100 μg / ml ampicillin . after growing at 37 ° c . in a shaking incubator for 3 hours , iptg was added to 1 mm final concentration and recombinant vhh were produced during an additional incubation for 4 hours . cells were spun down by centrifugation at 3 , 000 g for 20 minutes , supernatants discarded , and pellets stored at − 20 ° c . overnight . cell pellets were thawed , briefly vortexed , and 125 μl per well of room temperature pbs was added . cells were resuspended on an elisa shaker platform at room temperature for 15 minutes . plates were centrifuged at 3 , 000 g for 20 minutes and 100 μl per well of vhh - containing extract was transferred to polypropylene 96 - well plates ( nunc ) and stored at − 20 ° c . until further use . binding of clones from chitin selections was analyzed using elisa plates coated with 100 μl per well of 25 - fold diluted chitin , prepared similarly as for selections . after coating at 4 ° c . overnight and continued coating at room temperature for 1 hour on the next day , plates were washed three times with pbs / 0 . 05 %- tween ®- 20 and blocked with 5 % skimmed milk in pbs for 1 . 5 hours . plates were emptied and filled with 90 μl per well 1 % mpbs . ten μl of vhh - containing extract from each clone was added to ( an ) antigen - coated well ( s ) and a blank well . vhh were allowed to bind at room temperature for 1 hour and non - binding vhh were removed by washing three times with pbs / 0 . 05 %- tween ®- 20 . bound vhh were detected with sequential incubations with monoclonal mouse anti - histidine antibodies ( abd serotec ) in 1 % mpbs / 0 . 05 %- tween ®- 20 and rabbit anti - mouse igg whole molecule antibodies conjugated with alkaline phosphatase ( ram / ap ) ( sigma ) in 1 % mpbs / 0 . 05 %- tween ®- 20 . unbound antibodies were removed by washing three times with pbs / 0 . 05 %- tween ®- 20 . the plates were washed an additional two times with pbs and 100 μl pnpp disodium hexahydrate substrate ( sigma ) was added to each well . the absorbance at 405 nm was measured and the ratio of vhh bound to ( a ) target - coated well ( s ) and a non - target - coated well was calculated for each clone . from selections against chitin 28 of 92 ( 30 %) clones had a ratio greater than 2 and these clones were analyzed further by sequencing . single colony pcr and sequencing was performed on elisa positive clones as follows . cultures from master plate wells with elisa positive clones were diluted ten - fold in sterile water . five μl from these diluted clones were used as template for pcr using forward primer mp57 ( 5 ′- ttatgcttccggctcgtatg - 3 ′) and reverse primer giii ( 5 ′- ccacagacagccctcatag - 3 ′). pcr products were sequenced by sanger - sequencing using primer mp57 ( vib genetic service facility , university of antwerp , belgium ). from selections against chitin clones vhh 15a9 , vhh 15d1 , vhh 15e4 , and vhh 15g2 were found . clones vhh 15e4 and vhh 15g2 are variants of clone 15d1 with one and two amino acid substitutions , respectively . vhh were produced in e . coli suppressor strain tg1 or non - suppressor strain wk6 ( fritz et al ., nucleic acids research , volume 16 number 14 1988 ) according to standard procedures . briefly , colony streaks were made and overnight cultures from single colonies inoculated in 2 × ty ; 2 % glucose ; 100 μg / ml ampicillin . the overnight cultures were used to inoculate fresh cultures 1 : 100 in 2 × ty ; 0 . 1 % glucose ; 100 μg / ml ampicillin . after growing at 37 ° c . in a shaking incubator for 3 hours , iptg was added to a 1 mm final concentration and recombinant vhh were produced during an additional incubation for 4 hours . cells were spun down and resuspended in 1 / 50 th of the original culture volume of periplasmic extraction buffer ( 50 mm phosphate ph 7 ; 1 m nacl ; 1 mm edta ) and incubated with head - over - head rotation at 4 ° c . overnight . spheroplasts were spun down by centrifugation at 3 , 000 g and 4 ° c . for 20 minutes . supernatants were transferred to fresh tubes and centrifuged again at 3 , 000 g and 4 ° c . for 20 minutes . hexahistidine - tagged vhh were purified from the periplasmic extract using 1 / 15 th of the extract volume of talon metal affinity resin ( clontech ), according to the manufacturer &# 39 ; s instructions . purified vhh were concentrated and dialyzed to pbs using vivaspin 5 kda molecular weight cut - off ( mwco ) devices ( sartorius stedim ), according to the manufacturer &# 39 ; s instructions . titration of vhh was performed on elisa plates ( maxisorp , nunc ) coated with chitin . chitin was dissolved at 10 mg / ml concentration in 85 % phosphoric acid by shaking on a vortex shaker for approximately 3 hours until all particles were dissolved . dissolved chitin was diluted 25 - fold in pbs and precipitated chitin removed by centrifuging at 20 , 000 g for 5 minutes . 100 μl per well supernatant were used for coating of elisa plates ( maxisorp , nunc ). plates were coated at 4 ° c . overnight and coating was continued at room temperature for 1 hour on the next day . plates were washed three times with pbs / 0 . 05 %- tween ®- 20 and blocked with 5 % skimmed milk in pbs for 1 hour . four - fold serial dilutions of purified vhh were prepared in 1 % mpbs / 0 . 05 %- tween ®- 20 in polypropylene 96 - well plates . antibody concentrations ranged from 3 μg / ml to 12 ng / ml . antibody dilutions were transferred to the chitin - coated plates and vhh were allowed to bind for 1 hour at room temperature . bound vhh were detected with sequential incubations with monoclonal mouse anti - histidine antibodies ( abd serotec ) and rabbit anti - mouse igg whole molecule antibodies conjugated with alkaline phosphatase ( ram / ap ) ( sigma ) in 1 % mpbs / 0 . 05 %- tween ®- 20 . unbound antibodies were removed by washing three times with pbs / 0 . 05 %- tween ®- 20 after each antibody incubation . the plates were washed an additional two times with pbs and 100 μl pnpp disodium hexahydrate substrate ( sigma ) was added to each well . the absorbance at 405 nm was measured and plotted as function of antibody concentration ( see table 1 ). in order to investigate the specificity of the selected chitin - binding vhh an elisa with different coatings was used . vhh 15a9 as well as control conditions with other antibodies were tested for binding to chitin , pectin , and potato lectin . elisa plates ( maxisorp , nunc ) were coated with 100 μl per well chitin similarly as for the titration elisa , 100 μg / ml 20 - 34 % esterified pectin from citrus fruits ( sigma ), or potato lectin ( sigma ) in pbs . plates were coated at 4 ° c . overnight and coating was continued at room temperature for 1 hour on the next day . plates were washed three times with pbs / 0 . 05 %- tween ®- 20 and blocked with 5 % skimmed milk in pbs for 1 hour . purified vi - 1h 15a9 was diluted to 3 μg / ml in 1 % mpbs / 0 . 05 %- tween ® and added to the coated plate and vhh were allowed to bind for 1 hour at room temperature . bound vhh were detected with sequential incubations with monoclonal mouse anti - histidine antibodies ( abd serotec ) and rabbit anti - mouse igg whole molecule antibodies conjugated with alkaline phosphatase ( ram / ap ) ( sigma ) in 1 % mpbs / 0 . 05 %- tween ®- 20 . unbound antibodies were removed by washing three times with pbs / 0 . 05 %- tween ®- 20 after each antibody incubation . the plates were washed an additional two times with pbs and 100 μl pnpp disodium hexahydrate substrate ( sigma ) was added to each well . the absorbance at 405 nm was measured and binding obtained binding profile for vhh 15a9 and control antibodies compared ( see table 2 ). diverse and distinct binding patterns were observed for vhh 15a9 and control antibodies . vhh 15a9 showed specific binding to chitin only . anti - chitin vhh were analyzed for binding to paramagnetic chitin beads ( new england biolabs ). these beads are formed through emulsion chemistry starting with low molecular weight chitosan and encapsulation of magnetite particles during bead formation . once the beads are formed they are acetylated to ensure that they are chitin beads . beads were equilibrated by five washes with 500 mm nacl / 20 mm tris - hcl / 1 mm edta / 0 . 1 %- tween ®- 20 using a dynamag spin magnet ( invitrogen ) and removing supernatants by pipetting . equilibrated beads were dispensed and incubated with 5 μg / ml histidine - tagged anti - chitin vhh in 1 % bsa / pbs with head - over - head rotation at 4 ° c . for 2 hours . control conditions included incubations with unrelated vhh in 1 % bsa / pbs or with 1 % bsa / pbs alone . non - bound vhh were washed away by five washes with pbs and bound vhh were detected by consecutive incubations with monoclonal mouse anti - histidine antibodies ( abd serotec ) and rabbit anti - mouse igg whole molecule antibodies conjugated with alkaline phosphatase ( ram / ap ) ( sigma ). antibodies were diluted in 1 % bsa / pbs and incubated at room temperature for 1 hour . non - bound antibodies were removed by washing five times with pbs in between different incubations . after final washes and removal of supernatant pnpp disodium hexahydrate substrate ( sigma ) was added to each condition and incubated for 10 minutes . substrates were transferred to an optical plate and the absorbance at 405 nm was measured . measured absorbance was 4 . 0 ( saturated ), 4 . 0 ( saturated ), 3 . 8 , 4 . 0 ( saturated ), 0 . 23 , and 0 . 20 for vhh 15d1 , vhh 15e4 , vhh 15g2 , vhh 15a9 , unrelated vhh , and incubation without vhh , respectively . these data show that after selecting and performing primary screens on practical grade chitin vhh 15d1 , vhh 15e4 , vhh 15g2 , and vhh 15a9 are truly binding chitin . with the objective to generate vhh - functionalized polyurea microcapsules , vhh were coupled to microcapsules with a core of 1 . 5 % uvitex ob ( ciba ) in benzyl benzoate and a shell with incorporated lysine to surface - expose carboxyl groups . a core of 1 . 5 % uvitex ob in benzyl benzoate was used for fluorescent visualization of microcapsules . after production of microcapsules , microcapsules were washed with water and stored as capsule suspensions in water . before coupling of vhh , microcapsules were washed with mes / nacl buffer ( 0 . 1 m mes / 0 . 5 m nacl ph 6 ) using a 96 - well deep - well filtration plate ( millipore ) and vacuum manifold ( millipore ). a panel of vhh was dialyzed to mes / nacl buffer and added to a final concentration of 10 - 70 μm and incubated with the microcapsules for 15 - 30 minutes . 1 - ethyl - 3 -[ 3 - dimethylaminopropyl ] carbodiimide hydrochloride ( edc ) ( pierce ) was dissolved in mes / nacl buffer and promptly added to a final concentration of 50 mm . vhh were coupled by incubation with continuous mixing at room temperature for 2 hours . the coupling reactions were stopped by adding glycine or tris - buffer ph 7 . 5 to a final concentration of 100 mm and incubation at room temperature for 30 minutes . non - bound vhh were collected using the filtration plate setup using a deep - well collector plate . microcapsules were washed three times with pbs and resuspended in pbs and stored at 4 ° c . until use . an elisa - like assay setup was used to evaluate the interaction of vhh - coupled microcapsules to chitinous polysaccharides - containing surfaces . wells of a high bind half area microplate ( greiner bio - one ) were coated with chitin . coating with chitin was performed as before for the titration and specificity elisas . 100 μg / ml potato lectin in pbs was coated as control condition . the microplate was washed three times with pbs with 0 . 05 %- tween ®- 20 and blocked with 5 % skimmed milk in pbs for 2 hours . vhh - coupled microcapsules containing a fluorescent tracer were diluted to appropriate density in 1 % skimmed milk in pbs with 0 . 05 %- tween ®- 20 . microcapsules were added in serial dilution to the chitin - coated or control wells and allowed to bind for 1 hour . unbound microcapsules were removed by washing five times with pbs with 0 . 05 %- tween ®- 20 . the bottoms of elisa plate wells were analyzed for fluorescence using a multimode microplate reader ( tecan ) for bound microcapsules ( see table 3 ). binding of vhh - coupled microcapsules to chitin magnetic beads was investigated with paramagnetic chitin beads ( new england biolabs ). beads were equilibrated by five washes with 500 mm nacl / 20 mm tris - hcl / 1 mm edta / 0 . 1 %- tween ®- 20 using a dynamag spin magnet ( invitrogen ) and removing supernatants by pipetting . 1 mg quantities of beads were dispensed and the approximate concentration of beads was calculated from the diameter of the beads ( ø 50 - 70 μm ). chitin beads were incubated with a 100 - fold excess of microcapsules ( ø 10 μm ) over the number of chitin beads in 1 % bsa in pbs and binding was allowed for 1 hour with head - over - head rotation at room temperature . control conditions included incubations with blank microcapsules to which no vhh had been coupled . five washes were performed with pbs using head - over - head rotation for each wash and using the dynamag spin magnet to collect the beads in between each wash step . beads with bound microcapsules were finally resuspended in a small volume and transferred to an 18 - 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