Patent Application: US-201013138372-A

Abstract:
the invention relates to the fields of protein chemistry , biology and medicine . more specifically , it relates to the design and preparation of proteinmimics of members of the cystine - knot growth factor superfamily . further , the invention relates to the use of these proteinmimics as a medicament or prophylactic agent . the invention provides proteinmimics of members of the cystine - knot growth factor superfamily , preferably for use in immunogenic and / or therapeutic compositions .

Description:
amino acids are indicated by the single - letter codes ; “ ac ” refers to n - terminal acetylation ; “#” indicates c - terminal amidation ; cysteines ( c1 - c6 ) in boldface indicate cysteines involved in formation of the cystine - knot fold ; alanines in boldface indicate native cysteines that were replaced by ala . i . direct synthesis ( fmoc ) of full - length peptide ; only used for humvegf 26 - 104 . ii . peptide - thioester synthesis using fmoc - chemistry . subsequent native chemical ligation ( ncl ) of peptide fragments humvegf 26 - 67 ( thioester )+ humvegf 68 - 104 ( free n - terminal cysteine ) for humvegf 26 - 104 , humvegf 25 - 67 ( thioester )+ humvegf 68 - 107 ( free n - terminal cysteine ) for humvegf 25 - 107 , and humvegf 25 - 67 ( thioester )+ humvegf 68 - 109 ( free n - terminal cysteine ) for humvegf 25 - 109 . iii . peptide - thioester synthesis using boc - chemistry . subsequent native chemical ligation ( ncl ) of peptide fragments humvegf 25 - 67 ( thioester )+ humvegf 68 - 107 ( free n - terminal cysteine ) for humvegf 25 - 107 and humvegf 26 - 67 ( thioester )+ humvegf 68 - 104 ( free n - terminal cysteine ) or humvegf 26 - 60 ( thioester )+ humvegf 61 - 104 ( free n - terminal cysteine ) for humvegf 26 - 104 . peptides were synthesized on solid - phase using a 4 ( 2 ′, 4 ′- dimethoxyphenyl - fmoc - aminomethyl )- phenoxy ( rinkamide ) resin ( bachem , germany ) on a symphony ( protein technologies inc ., usa ), voyager ( cem gmbh , germany ), or syroii ( multisyntech , germany ) synthesizer . all fmoc - amino acids were purchased from biosolve ( netherlands ) or bachem gmbh ( germany ) with side - chain functionalities protected as n - t - boc ( kw ), o - t - bu ( desty ), n - trt ( hnq ), s - trt ( c ), or n - pbf ( r ) groups . a coupling protocol using a five - fold excess of hbtu / hobt / amino acid / dipea ( 1 : 1 : 1 : 2 ) in nmp with a 20 - minute activation time using double couplings was employed for every amino acid coupling step . acetylation ( ac ) of the peptide was performed by reacting the resin with nmp / ac 2 o / diea ( 10 : 1 : 0 . 1 , v / v / v ) for 30 minutes at room temperature . the acetylated peptide was cleaved from the resin by reaction with tfa ( 40 ml / mmol resin ) containing 13 . 3 % ( w ) phenol , 5 % ( v ) thioanisole , 2 . 5 % ( v ) 1 , 2 - ethanedithiol , and 5 % ( v ) milliq - h 2 o for 2 hours at room temperature , unless indicated otherwise . precipitation with ice - cold et 2 o + lyophilization of the precipitated material afforded the crude peptide . humvegf 26 - 104 was synthesized in one step following this procedure ( resin - loading 0 . 88 mmol / g ) on a symphony synthesizer ( protein technologies inc ., usa ). in the first coupling step , a 4 : 1 ( w / w ) mixture of ac - cys ( trt )- oh and fmoc - cys ( trt )- oh was used . the acylated peptide was cleaved from the resin by reaction with a slightly different mixture : tfa ( 40 ml / mmol resin ) containing 5 % ( v ) tes , 2 . 5 % ( v ) 1 , 2 - ethanedithiol , and 2 . 5 % ( v ) milliq - h 2 o . finally , the peptide was purified by hplc and folded by oxidation following procedure g . the fragment peptides humvegf 68 - 104 , humvegf 68 - 107 , and humvegf 68 - 109 ( free n - terminal cysteine for ncl ; see procedure ii ) were also synthesized following this procedure as described above for humvegf 26 - 104 on a rink - made resin ( loading 0 . 5 mmol / g ) using a liberty - synthesizer ( cem gmbh , germany ). the fragment peptides humvegf 25 - 67 and humvegf 26 - 67 ( free c - terminus ) were synthesized on a sasrin - resin ( loading 0 . 5 mmol / g ; bachem gmbh , germany ) following the general procedure for fmoc - synthesis of peptides as described in procedure i . the peptides were cleaved from the resin by repetitive treatment ( 20 cycles ) with 1 % tfa ( 40 ml / mmol resin ) in dcm . the combined fractions were neutralized with pyridine , whereafter dcm was removed by evaporation under reduced pressure . finally , the peptides were precipitated by addition of excess of h 2 o , followed by centrifugation and lyophilization . the crude lyophilized peptides were dissolved in dcm ( 2 . 0 mm ), twelve equivalents of 4 - acetamidothiophenol in dcm ( 0 . 334 mg / ml , 2 . 0 mm ), three equivalents of pybop in dcm ( 1 . 040 mg / ml , 2 . 0 mm ), and 2 . 6 equivalents of dipea in dcm ( 1 vol %) were subsequently added and the mixture was stirred at room temperature for six hours . then , another twelve equivalents of 4 - acetamidothiophenol in dcm ( 0 . 334 mg / ml , 2 . 0 mm ) were added and the mixture was stirred overnight at room temperature . finally , the mixture was neutralized with ˜ 2 . 6 equivalents of tfa and dcm was removed by evaporation under reduced pressure . the crude fragment peptide thioesters were then deprotected and purified by rp - hplc following general procedures . condensation of fragment peptides humvegf 68 - 104 , humvegf 68 - 107 , or humvegf 68 - 109 ( a ) with either fragment peptide thioesters humvegf 25 - 67 or humvegf 26 - 67 ( b ) by native chemical ligation was performed by mixing almost equimolar ( 1 : 1 . 2 ) solutions of a ( 10 mg / ml ; ˜ 2 . 0 mm ) and b ( 10 mg / ml ; ˜ 2 . 0 mm ) in working buffer ( 6 m guanhcl / 20 mm tcep / 200 mm mpaa in 0 . 2 m phosphate buffer ph 8 . 0 ) and overnight stirring at room temperature . after mixing of the solutions ( acidic ! ), the ph was adjusted to 6 . 5 by addition of 10 m naoh ( μl of naoh is roughly equal to mg of mpaa used ). excess of mpaa was removed by amicon filtration using working buffer ( without mpaa !!) in the washing steps . finally , the crude humvegf 26 - 104 , humvegf 25 - 107 , or humvegf 25 - 109 in reduced form were purified by rp / hplc following the standard procedure . fully reduced red - humvegf 26 - 104 , red - humvegf 25 - 107 , or red - humvegf 25 - 109 were dissolved in 0 . 1 m tris - buffer ( ph 8 . 0 ), with or without 1 m guanidine . hcl , containing 1 . 0 mm cystine ( ss - form ) and 8 . 0 mm cysteine ( sh - form ) in a final concentration of 0 . 1 mg / ml and stirred at room temperature . immediately , a sharp peak appears at a lower retention time ( more polar ) in addition to some broad peaks that correspond to incomplete or incorrectly folded peptide . when hplc - analysis showed no further change in peak intensities ( usually after ˜ 4 hours ), the mixture was loaded onto a preparative rp / c 18 column and purified following our standard procedure ( see below ). fragment peptides were prepared by manual solid phase peptide synthesis ( spps ) typically on a 0 . 25 mmol scale using the in situ neutralization / hbtu activation procedure for boc chemistry as previously described . each synthetic cycle consisted of nα - boc - removal by a one - to two - minute treatment with neat tfa , a one - minute dmf - flow wash , a ten - to twenty - minute coupling time with 1 . 0 mmol preactivated boc - amino acid in the presence of excess diea , followed by a second dmf - flow wash . nα - boc amino acids ( 1 . 1 mmol ) were preactivated for 3 minutes with 1 . 0 mmol hbtu ( 0 . 5 m in dmf ) in the presence of excess diea ( 3 mmol ). after coupling of gln residues , a dcm flow wash was used before and after deprotection using tfa , to prevent possible high - temperature ( tfa / dmf )- catalyzed pyrrolidonecarboxylic acid formation . side - chain protected amino acids were : boc - arg ( p - toluenesulfonyl )- oh , boc - asn ( xanthyl )- oh , boc - asp ( o - cyclohexyl )- oh , boc - cys ( 4 - methylbenzyl )- oh , boc - glu ( o - cyclohexyl )- oh , boc - his ( dinitrophenyl )- oh , boc - lys ( 2 - cl — z )— oh , boc - ser ( benzyl )- oh , boc - thr ( benzyl )- oh , and boc - tyr ( 2 - br — z )— oh . other amino acids were used without side - chain protection . nα - acetylation of peptides was performed by treatment with acetic anhydride ( 0 . 1 m )/ pyridine ( 0 . 1 m ) in dmf for 2 × 2 minutes ). after chain assembly was completed , the peptides were deprotected and cleaved from the resin by treatment with anhydrous hf for one hour at 0 ° c . with 4 % p - cresol as a scavenger . in all cases , the imidazole side chain - dinitrophenyl ( dnp ) protecting groups remained on his residues because the dnp - removal procedure is incompatible with c - terminal thioester groups . however , dnp is gradually removed by thiols during the ligation reaction yielding unprotected his . after cleavage , the peptide fragments were precipitated with ice - cold diethylether , dissolved in aqueous acetonitrile and lyophilized . 1 . 1 mmol nα - boc leu was activated with 1 mmol hbtu in the presence of 3 mmol diea and coupled for 10 minutes to 0 . 25 mmol mbha resin . next , 1 . 1 mmol s - trityl mercaptopropionic acid was activated with 1 mmol hbtu in the presence of 3 mmol diea and coupled for 30 minutes to leu - mbha resin . the resulting trityl - mercaptopropionic acid - leucine resin can be used as a starting resin for polypeptide chain assembly following removal of the trityl protecting group with 2 × 1 - minute treatments with 2 . 5 % triisopropylsilane and 2 . 5 % h 2 o in tfa . the thioester bond was formed with the desired amino acid using standard peptide coupling protocols . treatment of the final peptide with anhydrous hf yielded the c - terminal activated mercaptopropionic acid - leucine ( mpal ) thioester (- cosr ) peptides for participation in the native chemical ligation reaction . the ligation of fully deprotected fragment peptide thioesters humvegf 26 - 60 , humveg 26 - 67 , and humvegf 25 - 67 with either the fragment peptides humvegf 61 - 104 , humvegf 68 - 104 , or humvegf 68 - 107 was performed as follows : peptide fragments were dissolved in a ˜ 1 : 1 molar ratio at 10 mg / ml in 0 . 1 m tris buffer , ph 8 . 0 , containing 6 m guanidine . benzylmercaptan and thiophenol were added to 2 % ( v / v ) resulting in a final peptide concentration of 1 - 3 mm at a ph ˜ 7 ( lowered due to addition of thiols and tfa from the lyophilized peptide ). the ligation reaction was performed in a heating block at 37 ° c . and was vortexed periodically to equilibrate the thiol additives . the reaction was monitored by hplc and esi - ms until completion . respective ncls ( humvegf 26 - 60 + humvegf 61 - 104 ; humvegf 26 - 67 + humvegf 68 - 104 ) yielded reduced vegf 26 - 104 with identical hplc and esi - ms specifications . fully reduced red - humvegf 26 - 104 and red - humvegf 25 - 107 were dissolved in 0 . 1 m tris - buffer ( ph 8 . 0 ), with or without 1 m guanidin . hcl , containing 1 . 0 mm cystine ( ss - form ) and 8 . 0 mm cysteine ( sh - form ) in a final concentration of 0 . 1 mg / ml and stirred at room temperature . immediately , a sharp peak appears at a lower retention time ( more polar ) corresponding to the correctly folded cysknot structure , in addition to some broad peaks that correspond to incomplete or incorrectly folded peptide . when hplc - analysis showed no further change in peak intensities ( usually after ˜ 4 hours ), the mixture was loaded onto a preparative rp / c 18 column and purified following our standard procedure ( see below ). crude peptides were purified by reversed - phase high - performance liquid chromatography ( rp - hplc ), either on a “ deltapack ” ( 25 × 100 or 40 × 210 mm inner diameter , 15 μm particle size , 100 å pore size ; waters , usa ) or on a “ atlantis ” ( 10 × 100 mm inner diameter , 5 μm particle size ( waters , usa ) rp - 18 preparative c 18 column with a linear ab gradient of 1 - 2 % b / minute where solvent a was 0 . 05 % tfa in water and solvent b was 0 . 05 % tfa in acn . alternatively , analytical reversed - phase hplc was performed on a varian prostar system using vydac c - 18 columns ( 5 μm , 0 . 46 × 15 cm ) and preparative reversed - phase hplc was performed on a waters system using vydac c - 18 columns ( 10 μm , 1 . 0 / 2 . 5 × 25 cm ). linear gradients of acetonitrile in water / 0 . 1 % tfa were used to elute bound peptides . the flow rates used were 1 ml / minute ( analytical ), and 5 / 10 ml / minute ( preparative ). analysis of the purified peptide was performed by reversed - phase high - performance liquid chromatography ( rp - hplc ) on an “ acquity ” uplc ( waters , usa ) using a rp - 18 preparative “ beh ” column ( 2 . 1 × 50 inner diameter , 1 . 7 mm particle size , waters , usa ) with a linear ab gradient ( 5 - 55 % b , 25 % b / minute ), where solvent a was 0 . 05 % tfa in water and solvent b was 0 . 05 % tfa in acn . the primary ion molecular weight of the peptides was determined by electron - spray ionization mass spectrometry . electrospray ionization mass spectrometry ( esi - ms ) of hplc samples was performed on an api - 150 single quadrupole mass spectrometer ( applied biosystems ). peptide masses were calculated from the experimental mass to charge ( m / z ) ratios from all the observed protonation states of a peptide using analysis software . oxidation state retention mw mw peptide ( red / ox ) (% acn ) calculated experimental red - humvegf 26 - 104 red 48 . 5 9065 . 6 9064 . 4 oxid - humvegf 26 - 104 ox 42 . 5 9059 . 6 9058 . 5 red - humvegf 25 - 107 red 45 . 8 9569 . 1 9566 . 4 ( boc ) oxid - humvegf 25 - 107 ox 40 . 5 9563 . 1 9560 . 7 ( boc ) red - humvegf 25 - 107 red 45 . 8 9569 . 1 9568 . 8 ( fmoc ) oxid - humvegf 25 - 107 ox 40 . 5 9563 . 1 9561 . 7 ( fmoc ) red - humvegf 25 - 109 red 43 . 8 9869 . 5 9869 . 6 oxid - humvegf 25 - 109 ox 38 . 2 9863 . 5 9863 . 8 these data and fig1 show that the various forms of humvegf trunc can be synthesized in various different ways with identical outcomes . inhibitory activity of oxid - humvegf 26 - 104 in avastin ™- binding to surface - immobilized oxid - humvegf 1 - 165 binding elisa : binding of various mabs ( avastin ™, mab 293 , pdl - antibody ) to oxid - humvegf 26 - 104 and humvegf 1 - 165 was determined in elisa . therefore , polystyrene 96 - well plates ( greiner , germany ) were treated with 100 μl / well of 0 . 2 % glutaric dialdehyde in phosphate - buffer ( 0 . 1 m , ph = 5 ) for four hours at room temperature while shaking , following by washing ( 3 × 10 minutes ) with phosphate - buffer ( 0 . 1 m , ph = 8 ). then , the wells were coated with 100 μl / well of a 1 μg / ml solution of oxid - humvegf 26 - 104 / humvegf 1 - 165 in phosphate - buffer ( 0 . 1 m , ph = 8 ) for three hours at 37 ° c ., followed by overnight standing at room temperature . after washing with 1 % tween ®- 80 ( 3 ×), the plates were incubated with the antibody at various different dilutions in horse serum ( 4 % in pbs / 1 % tween ®- 80 / 3 % nacl ), starting with 1 / 10 dilution in the first well and three - fold dilution steps in subsequent wells . incubation was performed for one hour at 37 ° c ., followed by washing with 1 % tween ®- 80 ( 3 ×). then , the plates were incubated with 100 μl / well of peroxidase - labeled goat - anti - rat serum ( 1 / 1000 dilution in 4 % horse serum , see above ) for one hour at 25 ° c ., followed by washing with 1 % tween ®- 80 ( 4 ×). finally , the plates were incubated with a 0 . 5 μg / ml solution of abts ( 2 , 2 ′- azine - di ( ethylbenzthiazoline sulfonate )) containing 0 . 006 % h 2 o 2 in citric acid / phosphate - buffer ( 0 . 1 m each , ph = 4 ). od 405 nm - values were measured after 45 minutes standing at room temperature in the dark . competition elisa : elisa binding competition studies were carried out largely following the procedure as described for binding in elisa ( see above ). incubation with antibody was carried out at one fixed antibody - concentration ( 10 ng / ml of avastin ™; od 405 nm between 1 . 0 - 1 . 5 ) in the presence of decreasing amounts of oxid - humvegf 26 - 104 ( start at 5 μm ; 1 / 5 dilution steps ) and humvegf 1 - 165 ( positive control ; start at 500 nm ; 1 / 5 dilution steps ). the data in fig2 show that oxid - humvegf 26 - 104 binds with less than five - fold difference in affinity ( as compared to humvegf 1 - 165 ) to avastin ™, while the ( cyclic ) peptide - mimic derived from the beta3 - loop of humvegf is & gt ; 10 , 000 - fold less active in binding to avastin ™. this illustrates the big step forward in reconstruction of the discontinuous avastin ™ binding site on humvegf using this novel technology of the present invention . use of oxid - humvegf 26 - 104 for generating vegf - neutralizing antibodies and sera in rats and mice immunization experiments using oxid - humvegf 26 - 104 ( not - conjugated to a carrier protein !!) were carried out both in female wistar rats and female balb / c mice . the antisera were analyzed for : a ) binding to surface - immobilized humvegf 1 - 165 ( titer determination ) b ) ability to inhibit the binding of avastin ™ to surface - immobilized humvegf 1 - 165 c ) neutralizing activity for humvegf 1 - 165 in a baf3 - cell proliferation assay the results of these studies are shown below and in fig3 - 6 . wistar rats : female wistar rats were immunized with anti - humvegf 26 - 104 at day 0 with 400 μl ( intramuscular + subcutaneous , 200 μl each ) of a 375 μg / ml solution of humvegf 26 - 104 in pbs / covaccine 1 : 1 ( v / v ) ( pbs = phosphate - buffered saline ), followed by a booster ( same quantity and concentration ) at two and four weeks . subsequently , the rats were bled after six weeks and the antisera collected . anti - vegf titers were determined as described as below . balb / c mice : immunization with oxid - humvegf 26 - 104 was performed in female balb / c mice , using two different formulations , i . e ., with a cfa / ifa adjuvant ( group 1 : two animals ), and with a covaccine adjuvant ( group 2 : three animals ). the animals ( 2 ) in group 1 were immunized intraperitoneal ( i . p .) at day 0 with 250 μl of a 1 . 0 mg / ml solution of oxid - humvegf 26 - 104 in pbs / cfa 2 : 3 ( v / v ) ( pbs = phosphate - buffered saline , cfa = complete freund &# 39 ; s adjuvance ), followed by a booster ( same quantity , method and concentration ; incomplete freund &# 39 ; s adjuvance ( ifa ) instead of cfa ) at four weeks . the animals ( 3 ) in group 2 were immunized at day 0 with 210 μl ( intramuscular + subcutaneous , 105 μl each ) of a 1 . 25 mg / ml solution of vegf 26 - 104 in pbs / covaccin 1 : 1 ( v / v ) ( pbs = phosphate - buffered saline ), followed by a booster ( same quantity , method and concentration ) at two and four weeks . subsequently , all five mice were bled after six weeks and the antisera collected . anti - vegf titers were determined as described as below . titers were calculated by determining the serum dilution for which od 405 nm is equal to 4 × od 405 nm that of a buffer solution ( see “ elisa - binding studies , example 1b ”). the titer defines the negative 10 log - value of the dilution factor ( 1 / 10 = 1 , 1 / 100 = 2 , 1 / 1000 = 3 , 1 / 10000 = 4 , etc .). humvegf 1 - 165 humvegf 1 - 165 titer 0 wpv titer 6 wpv animal 50 . 49 ( wistar rat 1 ; covaccine ) & lt ;& lt ; 2 4 . 8 50 . 67 ( wistar rat 2 ; covaccine ) & lt ;& lt ; 2 5 . 4 59 . 01 ( balb / c mouse 1 , cfa / ifa ) & lt ;& lt ; 2 5 . 3 59 . 02 ( balb / c mouse 2 , cfa / ifa ) & lt ;& lt ; 2 5 . 2 59 . 03 ( balb / c mouse 3 , covaccine ) & lt ;& lt ; 2 5 . 4 59 . 04 ( balb / c mouse 4 , covaccine ) & lt ;& lt ; 2 † 59 . 05 ( balb / c mouse 5 , covaccine ) & lt ;& lt ; 2 5 . 3 control abs avastin ™ ( 500 ng / ml start ) — 4 . 4 biovision ™ ( 5000 ng / ml ) — 4 . 2 elisa binding competition studies were carried out largely following the procedure as described for binding in elisa ( see above ). incubation with antibody was carried out at a fixed avastin ™- concentration ( 10 ng / ml ; od 405 nm between 1 . 0 - 1 . 5 ) in the presence of decreasing amounts of rat antisera ( start at 1 / 5 ; further 1 / 3 dilution steps ). the cells that are used in the assay are murine pre - b lymphocytes stable expressing human ( h ) humvegf - receptor 2 ( makinen et al ., 2001 ). these recombinant cells survive / proliferate only in the presence of il - 3 ( natural cytokine required for the survival of the parental cells ) or humvegf . for the experiment , il - 3 has to be washed off the medium so that proliferation capability in dependence of humvegf can be tested . ba / f3 r2 cells were grown in dmem ( gibco # 31885 ) containing 10 % fetal bovine serum ( perbio # ch30160 . 03 ), 2 mm l - glutamine ( sigma # g7513 ), 2 ng / ml mil - 3 ( calbiochem # 407631 ) and 500 μg / ml zeocin ( invitrogen # 450430 ). cells were grown at 37 ° c . in a humidified incubator with an atmosphere of 5 % co2 / 95 % air . differently concentrated humvegf (+ humvegf ) or medium (− humvegf ) was either added directly to the cells ( to test the proliferation efficiency ) or pre - incubated for one hour with different concentrations of avastin ™ ( positive control ), different concentrations of rat or mouse sera and then added to the cells ( in case of inhibition experiments ). two days later , cell proliferation was measured by adding wst - 1 ( roche # 1644807 ). see fig9 for a graphical representation of the assay . the wst - 1 assay is based on the measurement of the mitochondrial succinate dehydrogenase activity . to function correctly , this enzyme requires the integrity of this organelle and is a good indicator of the number of proliferating cells present in the culture . a tetrazolium salt ( wst - 1 ) is used as substrate since it generates a soluble dark metabolic ( formazon ) through the action of the enzyme , which can then be quantified by measuring the absorbance ( 450 nm ) in an elisa reader . the higher the absorbance measured in the assay , the stronger the proliferation . absorbance is positively correlated with proliferation . experiments were repeated three times in triplicate showing overall similar results . the data obtained proves that high levels of antibodies were successfully generated via immunization with oxid - humvegf 26 - 104 ( not - conjugated to a carrier protein !! ), both in female wistar rats and female balb / c mice . the antisera generated in this way exhibit strong neutralizing activity for humvegf 1 - 165 in a baf3 - cell proliferation assay ( fig3 - 6 ), and the ability to inhibit binding of avastin ™ to humvegf ( fig7 ). in order to check whether oxid - humvegf 26 - 104 , the truncated form of humvegf 1 - 165 , is also able to induce baf3 - cell proliferation , we measured cell proliferation in the presence of varying amounts of oxid - humvegf 26 - 104 ( 0 . 01 - 20 ng / ml ). in order to check if oxid - humvegf 26 - 104 was able to enhance or inhibit the proliferative capacity of humvegf 1 - 165 , itself , the experiments with varying amounts of oxid - humvegf 26 - 104 were also run in the presence of humvegf 1 - 165 = 1 . 2 ng / ml . the results shown in fig8 clearly demonstrate no activity for oxid - humvegf 26 - 104 in baf3 - cell proliferation nor any affect on the proliferating ability of humvegf 1 - 165 . passive immunization study with anti - humvegf 26 - 104 rat - antisera in swiss nu / nu mice inoculated with human ls174t tumor cells : in vivo proof of principle of the tumor - reducing potential of anti - humvegf 26 - 104 antisera in order to demonstrate the tumor - reducing potential of anti - humvegf 26 - 104 antisera , the following immunization experiment was carried out in 30 male swiss nu / nu mice ( charles river ), six weeks of age at the beginning of the study . the animals were divided in the following three treatment groups : group 1 : pbs ( n = 10 ; negative control group ): intraperitoneal ( i . p .) pbs injections ( 500 μl ) after tumor cell inoculation . group 2 : oxid - humvegf 26 - 104 ( n = 10 ): i . p . injections ( 500 μl ) with igg - purified anti - vegf peptide rat - antiserum after tumor cell inoculation . group 3 : avastin ™ ( n = 10 ; positive control group ): i . p . injections ( 500 μl ) with anti - humvegf mab avastin ™ following tumor cell inoculation . on day 1 of the study , all 30 mice were injected subcutaneously ( right flank ) with 10 million human ls174t tumor cells suspended in a 100 μl solution . tumor - take was ˜ 100 %. subsequently , the mice were given on days 1 , 8 , and 15 , i . p . injections ( 500 μl ) with either a ) pbs ( group 1 ), b ) anti - oxid - humvegf 26 - 104 rat - antiserum ( 5 × conc . rat serum ; group 2 ), and c ) avastin ™ ( group 3 ). anti - oxid - humvegf 26 - 104 rat serum was obtained by immunizing a total number of 20 male whistar rats in a separate experiment 4 × with 250 - microgram doses of humvegf 26 - 104 using covaccine adjuvant ( inoculations at days 0 , 14 , 28 , and 49 ; bled on day 63 ). the resulting rat sera were purified by affinity chromatography ( protg - column ) and concentrated 5 ×. the ten most potent antisera ( based on in vitro neutralization data in baf3 assay ; see previous example ) of these were pooled and used for inoculation of the ten mice in treatment group 2 . lengths and breadths of the tumors were measured every other day , starting on the first day after tumor cell inoculation . tumor volumes were estimated using the formula ( breadth2 × length )/ 2 . ( ref 6 ) the data are shown in fig1 . 1 . anti - oxid - humveg f 26 - 104 antisera have the ability to strongly reduce tumor growth in mice . 2 . in this experimental setting , the observed effect of treatment with anti - oxid - humvegf 26 - 104 antisera was visibly more pronounced than that for avastin ™. 3 . treatment of nude mice with anti - oxid - humvegf 26 - 104 antibodies was received well by all animals and is thus not toxic ! solid - phase synthesis of ratvegf 26 - 104 . ratvegf 26 - 104 was synthesized by normal solid - phase synthesis on a rink - amide resin ( downloaded to 0 . 1 mmol / g ) following standard procedures as described for humvegf 26 - 104 ( see example 1 ). subsequent oxidative refolding was carried out exactly as described for humvegf 26 - 104 . purification of both red - ratvegf 26 - 104 and oxid - ratvegf 26 - 104 was carried out by preparative high performance liquid chromatography ( hplc ). characterization of both peptides was carried out by analytical hplc and electrospray ionization mass spectrometry ( esi - ms ). the successful refolding of red - ratvegf 26 - 104 was evidenced by the characteristic shift to lower rf - values ( from 48 . 5 % to 41 . 3 % acn , see table below ), normally observed when proteins or fragments thereof are oxidative refolded . the characteristic narrow shape of the new peak at lower r f - value provides evidence that an intact cystine - knot structure is indeed formed upon oxidative refolding of red - ratvegf 26 - 104 . also , the esi - ms spectrum undergoes a significant change upon oxidative refolding . first of all , the overall mass goes down by six mass units ( formation of three disulfide bonds releases a total of 6h ). moreover , there is a very characteristic shift of ms - signals to higher m / z - values . for example , the ms - spectrum for red - ratvegf 26 - 104 gives the most intense signals for the m 9 + and m 10 + charged species , whereas these signals disappear and a much weaker signal at m 5 + remains ( see fig1 ) that is much less intense . also , this shift is characteristic for folding of proteins into their oxidized native structure and shows that oxidative refolding of red - ratvegf 26 - 104 has been successful . the reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges . in contrast to this , the flexible and extended structure of the reduced protein is able to accommodate many more charges . this example describes the results of an immunization study in male whistar rats with both oxid - hum - vegf 26 - 104 and oxid - ratvegf 26 - 104 with an intact cystine - knot fold ( oxid - form ). the data unequivocally show that oxid - ratvegf 26 - 104 is equally immunogenic and potent as compared to oxid - humvegf 26 - 104 in generating antibodies in rats . the use of truncated vegf as described in this patent can thus be used to bypass immune tolerance to “ self proteins ,” like , for example , the full - length homodimeric vegf protein in this particular case . a total of four wistar rats ( 2 × 2 ) were immunized on day 0 with 250 micrograms each of either oxid - ratvegf 26 - 104 ( two rats ) or oxid - humvegf 26 - 104 ( two rats ) using covaccine as adjuvant , followed by booster inoculations at day 14 , 28 , and 42 . the rats were finally bled at day 56 , and the sera were analyzed for antibody titers against ratvegf 1 - 165 , humvegf 1 - 165 , oxid - ratvegf 26 - 104 , and oxid - humvegf 26 - 104 . ( part of ) the antibody - binding data are shown in table 1 and fig1 . the data in table 1 and fig1 do not show any detectable difference in binding between antisera elicited with oxid - ratvegf 26 - 104 and those elicited with oxid - humvegf 26 - 104 in rats , which strongly suggests that oxid - ratvegf 26 - 104 is equally immunogenic in rats ( homologous species ) as compared to oxid - humvegf 26 - 104 ( heterologous species ), and is able to elicit comparable amounts of antibodies that even show cross - reactivity with the homodimeric vegf 1 - 165 protein ( table 1c ). furthermore , the experiment provides a very strong basis for the fact that oxid - humvegf 26 - 104 can be used to elicit anti - vegf in humans , and that oxid - humvegf 26 - 104 will not suffer from lack of immunogenicity as a result of immune tolerance to self proteins . solid - phase synthesis of red - plgf 34 - 112 . red - plgf 34 - 112 was synthesized by normal solid - phase synthesis on a rink - amide resin ( downloaded to 0 . 1 mmol / g ) following standard procedures as described for red - humvegf 26 - 104 ( see example 1e ). subsequent oxidative refolding was carried out exactly as described for oxid - humvegf 26 - 104 . purification of both red - humplgf 34 - 112 and oxid - humplgf 34 - 112 was carried out by preparative high performance liquid chromatography ( hplc ). characterization of both red - humplgf 34 - 112 and oxid - humplgf 34 - 112 was carried out by analytical hplc and electrospray ionization mass spectrometry ( esi - ms ). the successful refolding of red - humplgf 34 - 112 was evidenced by the characteristic shift to lower rf - values ( from 49 % to 38 . 3 % acn , see table below ) that is normally observed when proteins or fragments thereof are oxidative refolded . the characteristic narrow shape of the new peak at lower rf - value provides evidence that an intact cystine - knot structure is indeed formed upon oxidative refolding of red - humplgf 34 - 112 . also the esi - ms spectrum undergoes a significant change upon oxidative refolding . first of all , the overall mass goes down by six mass units ( formation of three disulfide bonds releases a total of 6h ). moreover , there is a very characteristic shift of ms - signals to higher m / z - values . for example , the ms - spectrum for red - humplgf 34 - 112 gives clear signals for the m 6 + to m 10 + charged species , whereas these signals disappear and a much weaker signal at m 5 + remains ( see fig1 ) that is much less intense . also this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of red - humplgf 34 - 112 was successful . the reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges . in contrast to this , the flexible and extended structure of the reduced protein is able to accommodate many more charges . synthesis of red - humsost 57 - 144 could not be performed directly on solid - phase on a downloaded resin , as described for humvegf 26 - 104 . therefore , the shorter fragments humsost - f1 / 3 were synthesized and subsequently ligated by native chemical ligation ( ncl ) as described below . also , the subsequent oxidative refolding of fully red - humsost 57 - 144 was carried out as described below . solid - phase synthesis of the fragments humsost - f1 / 3 was carried out following standard procedures as described for humvegf 26 - 104 . fragment condensation of humsost - f1 / 3 by ncl to give red - humsost 57 - 144 ( for a schematic overview see fig1 ) first , humsost - f2 and humsost - f3 were dissolved ( 2 mg / ml ) in ncl reaction mixture ( 6 m guanidine , 20 mm tcep , 200 mm mpaa , 0 . 2 m disodium hydrogenphosphate , adjusted with 10 m sodium hydroxide to ph 6 . 5 ) in a 1 . 2 : 1 ratio , and reacted for 24 hours at room temperature . the thiaproline - protected humsost - f2 / 3 was obtained in 66 . 5 % yield after reversed phase hplc purification . subsequently , the thiaproline was deprotected with 0 . 02 m methoxyamine in ncl buffer at ph 4 . 0 for 60 hours . then , the ph was adjusted to 6 . 5 and 1 . 2 equivalents of humsost - f1 was added and reacted for 1 . 5 day . the reaction was monitored by rplc / ms and each day 40 mm tcep was added to completely reduce all reagents . after completion of the reaction , crude red - humsost 57 - 144 was purified using ion exchange chromatography , and subsequently by reversed phase hplc giving pure red - humsost 57 - 144 in 24 . 2 % yield ( overall 16 . 1 %). structure of peptide fragments used for the fragment condensation of reduced sost 67 - 144 name peptide sequence humsost 57 - 144 biotine - ggg c relhftryvtdgp c rsakpvtelv c sgq c gparllpnaigrgkwwrpsgpdfr c ipdryr aqrvqll c pggeaprarkvrlvas c k c ( seq id no : 31 )- amide humsost - f1 biotine - ggg c relhftryvtdgp c rsakpvtelv c sgq ( seq id no : 32 )- thioester humsost - f2 bocnh - c ( thz ) gparllpnaigrgkwwrpsgpdfr ( seq id no : 33 )- thioester humsost - f3 amine - c ipdryraqrvqll c pggeaprarkvrlva s c k c ( seq id no : 34 )- amide c = cysteines involved in cystine - knot formation ; c = cysteines forming ss - bond between loop - 1 and loop - 3 of humsost subsequently , red - humsost 57 - 144 was natively refolded by dissolving the peptide ( 2 mg / ml ) in a ph 8 . 0 buffer solution , containing 55 mm tris - hcl , 21 mm sodium chloride , 0 . 88 mm potassium chloride , 0 . 48 l - arginine , 20 mm glutathion - sh , and 4 mm glutathion - ss . the peptide was oxidized over time and yielded 10 . 2 % of oxid - humsost 57 - 144 after 3 . 5 days at 4 ° c . ( see fig1 ). purification of both red - humsost 57 - 144 and oxid - humsost 57 - 144 was carried out by preparative high performance liquid chromatography ( hplc ). characterization of both compounds was carried out by analytical hplc and electrospray ionization mass spectrometry ( esi - ms ; see below ). the successful refolding of humsost 57 - 144 was evidenced by the characteristic shift to lower rf - values ( from 35 % to 30 % acn , see table below ) that is normally observed when proteins or fragments thereof are oxidative refolded . the characteristic narrow shape of the new peak at lower rf - value provides evidence that an intact cystine - knot structure is indeed formed upon oxidative refolding . also , the esi - ms spectrum undergoes a significant change upon oxidative refolding . first of all , the overall mass goes down by eight mass units ( formation of four disulfide bonds releases a total of 8h ). moreover , there is a very characteristic shift of ms - signals to higher m / z - values . for example , the ms - spectrum for the red - humsost 57 - 144 gives clear signals for the m 8 + to m 12 + charged species , whereas these signals disappear and a much weaker signal at m 6 + and m 7 + remains ( see fig1 d ) that is much less intense . also , this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of red - humsost 57 - 144 was successful . the reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges . in contrast to this , the flexible and extended structure of the reduced protein is able to accommodate many more charges . in order to prove further that oxid - humsost 57 - 144 adopts a native cystine - knot fold , we present binding data of a series of three mabs that were selected from phage - display libraries using oxid - humsost 57 - 144 . it was shown that all three anti - oxid - humsost 57 - 144 antibodies : bind strongly to oxid - humsost 57 - 144 in elisa . bind strongly to recombinant full length humsost / sclerostin in elisa . do not bind at all to aa 8 - humsost 57 - 144 in elisa . do not bind at all to three other , non - related proteins in elisa . altogether , these data show that oxid - humsost 57 - 144 can be used instead of full - length humsost / sclerostin to select antibodies from phage - display libraries ( pdls ), that show full selectivity and specificity to full - length humsost / sclerostin with respect to non - related proteins , and that oxid - humsost 57 - 144 can , therefore , be used as an “ easy - available ” protein mimic of full - length humsost / sclerostin for purposes of antibody generation and selection . in this example , we demonstrate the synthesis of the truncated protein mimic of oxid - humtgfb2 15 - 111 , in which the beta2 - loop ( 28 amino acids long ; x3 in general sequence ) was replaced by the humvegf beta2 - loop ( aa 62 - 67 ). the successful synthesis and oxidative ( cystine - knot ) folding of this tgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 mainly serves as an example to demonstrate that interchange of beta2 - loop sequences amongst different cystine - knot proteins in general leads to chimeric peptides that retain the ability to form an intact cystine - knot fold , just like that observed for the fully homologous trunc - peptides ( see other examples ). solid - phase synthesis of red - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 . red - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 was synthesized by normal solid - phase synthesis on a rink - amide resin ( downloaded to 0 . 1 mmol / g ) following standard procedures as described for humvegf 26 - 104 ( see example 1 ). subsequent oxidative refolding was carried out exactly as described for humvegf 26 - 104 . purification of both red - and oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 was carried out by preparative high performance liquid chromatography ( hplc ). characterization of both the red - and oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 was carried out by analytical hplc and electrospray ionization mass spectrometry ( esi - ms ). the successful refolding of red - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 was evidenced by the characteristic shift to lower rf - values upon oxidative refolding ( from 46 . 8 % to 42 . 0 % acn , see table below ) ( see other examples ). the characteristic narrow shape of the new peak at lower rf - value provides evidence that an intact cystine - knot structure is indeed formed . also , the esi - ms spectrum undergoes a significant change upon oxidative refolding . first of all , the overall mass goes down by six mass units ( formation of three disulfide bonds releases a total of 6h ). moreover , there is a very characteristic shift of ms - signals to higher m / z - values . for example , the ms - spectrum for the red - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 gives clear signals for the m 6 + to m 11 + charged species , whereas these signals completely disappear and a much weaker signal at m 5 + remains ( see fig2 ) that is much less intense . also this shift is characteristic for folding of proteins into their oxidized native structure and shows that refolding of humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 was successful . the reason is that the protein or protein fragment adopts a more condensed structure that is no longer able to pick up so many charges . in contrast to this , the flexible and extended structure of red humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 is able to accommodate many more charges . in order to prove that oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 can be used to generate anti - tgf - b2 antibodies via immunization , we carried out an immunization experiment in two rats . each animal received four inoculations ( 0 , 2 , 4 , and 7 . 5 wks ) with 2 × 450 + 2 × 130 microgram of oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 . analysis of the nine weeks post vaccination ( wpv ) antisera ( fig2 ) showed strong binding in elisa to full - length tgf - b2 ( titers 3 . 8 and 4 . 1 ) compared to those of the pre - immune sera (≦ 2 . 1 ) indicating that antibodies specific for tgf - b2 were generated upon immunization . moreover , it was observed that the majority of antibodies in the sera were directed towards the tgfb2 - part of the peptide in oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 rather that to the vegf - part ( humvegf 62 - 67 ). this indicates the humvegf 62 - 67 sequence is a good substitute for the much longer b2 - loop of humtgfb2 ( 28 amino acids ), but that it does not disturb the making of humtgf - b2 - specific antibodies , nor the oxidative refolding of red - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 into oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 . these data prove that oxid - humtgfb2 15 - 111 / δ49 - 77 - humvegf 62 - 67 can be used as a substitute for tgf - b2 for eliciting anti - humtgfb2 antibodies that are fully cross - reactive with the native protein humtgf - b2 . 1 . vitt u . a ., y . h . sheau , and a . j . w . hsueh , “ evolution and classification of cystine knot - containing hormones and related extracellular signaling molecules ,” mol . endocrin . 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