Patent Application: US-38000203-A

Abstract:
the present invention relates to hypoallergenic variants of ns - ltps allergens , to pharmaceutical compositions comprising them and to the use of such variants for the preparation of medicaments suitable in the treatment and in the prevention of the allergic forms associated with an ns - ltp allergen , in particular to the allergen corresponding to the variant used .

Description:
the experimental approach that led to the present invention consisted of a mutagenesis strategy aimed at the targeted disruption of all the disulfide bridges present in all the ns - ltps . this in order to generate molecules with reduced or absent affinity to ige , yet with intact affinity to the other classes of antibodies apt to compete with the specific iges against the native allergen . the ns - ltps allergen parj1 , whose sequence and structure is reported in comparison with par j2 in fig1 , and in particular the isoform par j 1 . 0102 ( the primary sequence thereof being reported in the annexed sequence listing as seq id nos : 1 and 2 ), was taken as a molecular model , to be used for carrying out mutagenesis and the subsequent verification of the properties of the obtained mutant . in particular , recombinant dna technology was resorted to in a strategy of site - directed mutagenesis against cysteines 4 , 29 , 30 , 50 and 52 , i . e ., the cysteines constituting the disulfide bridges according to the structural model known to the art . the results of these experiments demonstrate the close relationship existing between the three - dimensional structure of the protein and the capability of forming epitopes for the iges , and in particular that the gradual disruption of the disulfide bridges causes a reduction of the serum iges binding activity thereof , whereas it does not affect the igg4 binding activity thereof . this in light of the western blot analysis described in example 3 , and in fig5 , in which lanes 2 , 3 and 4 ( see fig5 panel a ) show the reduction of the serum iges binding activity by the mutants of the present invention , which should be construed as three - dimensional mutants . in particular , the cys29 - cys30 mutant ( pja ) shows a very weak binding band ( fig5 lane 2 ) whereas the cys50 - cys52 mutant ( pjb ) is somehow still capable of binding the iges ( fig5 , lane 3 ). instead , more remarkable is the result shown by the pjc and pjd mutants ( fig5 , lanes 4 and 5 ) for which no binding to human iges can be highlighted . such results have been confirmed by elisa and ige inhibition assays ( see examples 4 and 5 ) where the pjb was the only variant still able to bind parj1 - specific ige antibodies in solution while the other variants exhibited a very low inhibition capacity . the loss of additional disulfide bridges ( pjc and pjd ) leads - to the absence of any ige recognition ( see example 4 and fig4 ). these results all together show that ns - ltp parj1 variants lacking of at least one disulfide bridge have a reduced allergenicity , which is even absent in the variants wherein the lacking bridge is localized in the aminoterminal region of the allergen at the domain alpha - helix1 - loop1 - alphahelix2 , in particular when the lacking bridges are at least two . the maintenance by these variants of an overall antigenicity that , notwithstanding the reduced or absent allergenicity , is comparable or identical to the one of the wild type allergen , has been shown by experiments wherein binding activity of wild - type allergen and its variants to antibodies different than ige , have been compared . such experiments are western blots carried out using as antibodies rabbit policlonal antibodies and igg4 of pj allergic patients , extensively described in examples 6 and 3 respectively . in these experiments the hypoallergenic variants generated by genetic engineering presented a similar behavior compared to the wild type , with a low reduction of their binding activity towards the anti - rparj1 rabbit antibodies . accordingly , a variant that lacks of at least one disulfide bridges still contains several protein domains similar to the native molecule and , although at different extent , is apt to induce the production of igg antibodies ( see example 3 and 6 ). ige production and / or ige - mediated presentation of the allergen , would be prevented by such “ blocking ” antibodies and reducing t cell proliferation and release of cytokines ( 25 ). the above data have been confirmed also in vivo in particular by skin prick test ( spt ) analysis as described in example 7 , where the pure recombinant proteins were tested on ten pj allergic patients ( the same analyzed by elisa in example 4 and fig7 ). with regard to the single mutants , pja showed a very low ige binding activity and only 3 out of 10 patients with cutaneous type i hypersensitivity and a reduced wheal area respect to that one induced by wild - type allergen . on the contrary , loss of the cys50 - cys91 and cys4 - cys52 bridges seems to have aminor effect since an ige binding activity and a positive spt are still present . the loss of additional disulfide bridges ( pjc and pjd ) leads to the absence of any cutaneous reaction ( see example 7 ). these results obtained analyzing individuals demonstrate the reliability of the above described data in vitro , and above all that while the disruption of the disulfide bridges in the amino terminal region ( in the specific case cys4 - cys52 and cys50 - cys91 ) affect even if not markedly the human ige binding capability of this mutant , the disruption of all the four bridges ( in the specific case cys4 - cys52 , cysl4 - cys29 , cys30 - cys75 and cys50 - cys91 ) have a devastating effect on the ige recognition , and therefore on the allergenic response . accordingly , concerning the development of therapeutically useful hypoallergenic molecules , variants lacking of three or four bridges , as in the specific case pjc and pjd mutants , are considered as preferred embodiments . the above was demonstrated using a pool of sera as well as a cohort of individual patients , indicating that the obtained result is representative of the immune response of the allergic population . it is pointed out that although obtained using specific variants derived by mutating the wild type allergen , these results are in fact not limited to the said specific variants , neither to the techniques used for the relevant derivation . as such results are consequent to the modification of the three - dimensional structure of the allergen , they could anyhow have been obtained by any mutagenesis allowing the disruption of the disulfide bridges . accordingly any variant obtainable by deletion , substitution and / or the insertion of one or more amino acidic residue which results in variants lacking of at least one disulfide bridge is included in the object of the invention . the strategy of point mutation has however the remarkable advantage of allowing the insertion of minimal variations at the level of the primary sequence of the native protein and therefore of generating mutants that are more likely not to interfere with the variant recognition operated by the t cells , and above all the possibility to generate proteins having a high reproducibility . variants having substantially the same length of the wild type are accordingly considered preferred . with regard to the techniques used for deriving the variants of the invention , it is not limited to the genetic engineering ones , as they are obtainable by techniques like chemical mutagenesis ( formaldhehyde and gluteraldhehyde ) which allow the disruption of disulfide bridges even in absence of mutations . the genetic mutagenesis imply however the remarkable advantage of allowing the generation of proteins having a high reproducibility , while the , chemical mutants do not ensure a denaturation pattern constant at every preparation . furthermore , as the strategy described herein is independent on the epitope sequence on itself since it is based on the modification of the three - dimensional structure of the ige determinants , the adopted mutagenesis strategy is actually independent from the primary sequence of the allergen ( and therefore from the sequence of the specific ige epitopes ). for this reason variants of all the proteins with allergenic activity belonging to the ns - ltp family ( including parj2 ), are included in the object of the invention due to the conserved structure ( cfr . e . g . s fig2 wherein par j1 sequence is reported in comparison with the sequence of other ns - ltps together with the placement of the disulfides bridges ). in particular variant of the invention is not only any other mutein of the parj1 allergen or of other ns - ltp allergens which , independently from the mutation carried out ( substitution and / or deletion of one or more amino acid residues ) and of the way in which such a mutation is carried out ( e . g ., by the above mentioned techniques ), retain a structure equivalent to that of the corresponding native allergen lacking at least one disulfide bridge . thus the disruption of the disulfide bridges in ns - ltp allergens underlies per se a limited or absent ige binding ability of patients allergic to the related variants . moreover , in particular in light to what is known in the art concerning the high conservation of the structure and the cross - reactivity that have led to the singling out of the so - called ns - ltp pan - allergen ( see above ) these data are indicative not merely of a suitability in the therapy and prevention of the allergic forms caused by the allergens corresponding to the individual variants , but also in the therapy and prevention of allergic forms caused by ns - ltp allergens other than those corresponding to the variants used . a person skilled in the art can derive on the basis of his knowledge any information suitable for deriving uses , compositions and kit described in the summary of the invention . with the aid of the following examples , a more detailed description of specific embodiments will now be given , in order to give a better understanding of the objects , characteristics , advantages and operating methods of the present invention . for the production of the major allergen of parietaria judaica par j 1 . 0102 the pqe30 prokaryotic vector ( qiagen ) was used . the latter characteristically expresses recombinant proteins fused to a short histidine tail and inducible with isopropyl - p - d - thiogalactoside ( iptg ). the histidine residues allow the purification of the recombinant protein by affinity chromatography . for this reason , 1 ng of the p5 clone containing the processed version of the par j 1 . 0102 ( embl accession number x77414 ), the sequence thereof being reported in the annexed sequence listing as seq id no : 12 , was subjected to 30 cycles of polymerase chain reaction ( pcr ) amplification at the following design : 94 ° c . for 1 min , 52 ° c . for 1 min , 72 ° c . for 1 min . the synthetic primer oligonucleotides p5 forward and ps reverse , the sequence thereof being reported in the annexed sequence listing as seq id no : 11 and seq id no : 12 , respectively , were used . the fragment thus generated was fractionated on 1 % agarose gel in i × tbe , extracted , purified and digested with bam h1 and hind iii restriction enzymes and cloned in the pqe30 vector ( quiagen ) previously digested with the same enzyme . the linearized vector and the digested fragments were incubated for 4 hours at 16 ° c . in presence of the enzyme dna ligase according to different stoichiometric ratios . the reaction mixture was then transformed in the bacterial strain mis . the recombinant clones were sequenced with the method of sanger and the nucleotide sequence thus determined demonstrated that the dna fragment inserted into the pqe30 vector was identical to that known in the art ( 10 ). pja mutant ( cys29 → ser and cys30 → ser ) was generated using the transformer site - directed mutagenesis kit ( clontech ) following the manufacturer &# 39 ; s instruction and using the oligonucleotide ps ( 29 , 30 ) reported in the sequence listing as seq id no : 13 ( mapping from nucleotide 88 to nucleotide 105 ) and the parj1 sequence as a template . pjb mutant ( cys50 → ser and cys52 → ser ) was generated by pcr using as primers the oligonucleotide p5 ( 50 - 52 ) reported in the sequence listing as seq id no : 14 ( mapping from nucleotide 91 to nucleotide 165 ) and ps reverse oligonucleotide and 1 ng of the parj1 clone as a template . the pcr fragment was digested with pst i and hind iii restriction enzymes and ligated with the pst i - hind iii linearized plasmid vector containing the parj1 sequence ( expressing the first 31 amino acids of the wild type par j 1 . 0102 allergen ). pjc mutant ( cys4 → ser , cys29 → ser and cys30 → ser ) was generated by pcr amplification using the pja variant as a template . the cysteine residue at position 4 was mutated by pcr using the oligonucleotides p5 ( triple ), the sequence thereof being reported as seq id no : 15 , and p5 reverse . after purification , pcr fragment was digested with bam hi and hind iii enzymes and cloned in the pqe30 vector previously digested with the same restriction enzymes . pjd mutant ( cys29 → ser , cys30 → ser , cys50 → ser and cys52 → ser ) was generated using the transformer site - directed mutagenesis kit ( clontech ) following the manufacturer &# 39 ; s instruction and using the synthetic oligonucleotide p5 ( 29 , 30 ) reported in the sequence listing as seq id no : 13 and the pjb variant as a template . all clones were sequenced with the method of sanger ( 24 ) and the mutations and the open reading frames confirmed ( see fig3 for details ). with this process 4 independent mutants , hereinafter designated pja ( seq id no : 3 and seq id no : 4 ); pjb ( seq id no : 5 and seq id no : 6 ), pjc ( seq id no : 7 and seq id no : 8 ), and pjd ( seq id no : 9 and seq id no : 10 ) were isolated . purification of recombinant proteins evaluation of the relevant capability of binding ige of allergic patients 10 ml o / n culture of the recombinant clones ( nm15 strain , quiagen ) were then used for an inoculation in 400 ml of 2yt broth ( bacto - tryptone 16 gr / l , bacto - yeast 10 gr / l , nacl 5 gr / l , ph 7 . 0 ) containing ampicillin and kanamycin at a final concentration of 100 μgr / ml and 10 μgr / ml , respectively . a 1 : 40 dilution was grown for 1 hour at 37 ° c . and , after that , induced with 1 mm isopropylthio - α - galactoside for 4 hours at 37 ° c . cells were harvested by centrifugation and the recombinant proteins purified by using the his trap kit ( pharmacia ) following the manufacturer &# 39 ; s instructions . recombinant proteins , binding the hitrap chelating column , were eluted using a buffer containing : 20 mm phosphate buffer ph7 . 4 . 0 . 5 m nacl , 8 m urea and 500 mm imidazole ; fractions were analysed by 16 % sds - page and coomassie brilliant blue staining . fractions containing the purified protein were then diluted 1 : 100 in a buffer containing 20 mm phosphate buffer ph7 . 4 . 0 . 5 m nacl and 20 mm imidazole to allow refolding of the protein , reloaded on the his trap column and eluted with a buffer with no denaturing agents ( 20 mm phosphate buffer ph7 . 4 . 0 . 5 m nacl and 500 mm imidazole ). recombinant proteins were then desalted using a centrifugal filter device ( centriprep , millipore ) and analysed for their capability of binding human ige from pj allergic patients by western blot as previously described ( 12 ), using a pool of sera ( n = 30 ) of pj allergic patients which did not receive any specific immunotherapy . this analysis showed that the pjb mutant was still capable of binding human ige while the pja mutant retains only a weak ige binding activity . the pjc and pjd mutants did not show any ige binding activity suggesting that the ige recognition was dependent on the three - dimensional folding of the protein ( fig4 panel a ). after that , membranes were stripped and reprobed with a his - tag specific reagent ( india ™ hisprobe - hrp , pierce , usa ) to check that the ige - allergen complex was specific for the recombinant fused proteins . the concentration of the recombinant proteins was determined by densitometric analysis of sds - page gels stained with coomassie brilliant blue ( see fig4 panel b ). as a confirmation of this experiment another western blot carried out using ige and igg4 of allergic patients . then , the proteins purified were fractionated on 16 % page - sds and transferred on nitro - cellulose thanks to a dry - blot system ( millipore ). the membrane was incubated for 12 - 14 hours with a pool of sera from pj allergic patients ( 1 : 5 dilution ) in pbs - tween . the protein - human ige and igg4 binding complexes are highlighted using respectively a secondary anti - ige and anti - igg4 antibodies conjugated to radish peroxidase . thus , the complexes are highlighted using a chemioluminescence system ( super - signal , pierce ). the relevant results are reported in fig4 . the same pool of allergic sera from non - sensitive pj allergic patients used in example 3 has been used in an elisa experiment , showing the ige binding activity of the rparj1 and its disulfide bond variants . a non allergic subject has been tested as a negative control on the elisa . the results confirm the pattern of reaction of the experiment of the example 3 ( fig4 panel a ) with the pjb variant reacting in a way comparable to the wild - type allergen . a non allergic serum is shown as a negative control ( fig6 ). the ige binding activity of the four parj1 disulfide bond variants was also tested by elisa using sera from ten monosensitive pj allergic patients . analysis of single sera showed a remarkable homogeneity of the reaction . in particular , the cys4 - cys52 and cys50 - cys91 bridges did not influence the allergenicity of the protein since this mutant ( pjb ) showed an ige binding activity comparable to the wild - type allergen . on the other hand , the cys14 - cys29 and cys30 - cys75 bridges seem to be crucial for the ige recognition . all the variants lacking those two bonds ( pja , pjc and pjd ) presented low or even absent ige binding activity . ( fig7 ) elisa detection has been performed by adding 200 μl of a solution containing 5 μg / ml of antigen in coating buffer ( sodium carbonate buffer ph 9 . 5 ) to each well of polystyrene plates overnight at room temperature . after several washing steps ( 1 × pbs , 0 . 1 % tween 20 ) plates were saturated with a solution containing 5 % bsa , 0 . 5 % tween 20 in coating buffer . after washing , 200 μl of serum ( 1 : 5 dilution ) from pj allergic patients or from a non allergic subject were incubated for 4 hours at room temperature . bound ige antibodies were detected with a goat antihuman ige - hrp conjugate ( biosource international ) diluted at a concentration of 0 . 5 ng / ml in 1 × pbs , 0 . 25 % bsa , 0 . 1 % tween 20 for 1 hour at room temperature . after several washes , calorimetric reaction was developed by adding 0 . 2 ml / well of substrate solution ( 0 . 4 mg / ml o - phenylendiamine in 0 . 1 m citrate buffer ). optical density was read at 495 nm in a bio - rad microplate reader . in order to investigate whether the disulfide bond variants were able to inhibit the binding of the ige to the rparjl , increasing amount of each recombinant mutant were incubated with a pool of sera ( n = 10 ) of pj monosensitive allergic patients . the ability of the parj1 disulfide variants to interact with ige antibodies was determined by an elisa inhibition experiment . a pool of sera ( 1 : 5 dilution ) from ten monosensitive pj allergic patients was preincubated overnight with increasing concentration of each disulfide bond variant ( 0 . 25 - 20 μg / ml of protein ). the solutions were added to the elisa wells coated with 5 mg / ml of rparj1 and the elisa steps were performed as above described . percentage of inhibition was calculated according to the formula : %= 100 − od a / od b × 100 , where od a and od b represent the optical density read with the inhibited and non - inhibited pool of sera respectively . table i inhibition of ige binding protein tested % inhibition rparj1 95 % pja 16 % pjb 85 % pjc 14 % pjd 15 % the data reported in table i suggests that all the variants lacking , at least , cys14 - cys29 and cys30 - cys75 disulfide bonds exhibit a comparable low level of inhibition ( about 15 %). on the contrary , the pjb variant ( cys50 → ser and cys52δser ) showed a high percentage of inhibition retaining a substantial ability of binding human ige ( about 85 %). rabbits were immunized by primm srl ( milan , italy ) using the rparj1 allergen . as a control , rabbit polyclonal antibodies were analysed on a western blot using a parietaria judaica crude extract detecting a band of about 14000 da corresponding to parj1 native molecular weight . elisa plates were coated at the same conditions as above described , with the wild - type parj1 and with equal amount of each recombinant disulfide bond variant , were probed with an anti - rparj1 specific polyclonal serum to analyse their binding activity . rabbit preimmune and immune sera were diluted at a concentration of 6 ng / ml and 200 μl of these solutions were incubated at room temperature for 1 hour . wells were washed three times in 1 × pbs , 0 . 1 % tween 20 and bound antibodies were detected using a donkey antirabbit ig hrp linked ( amersham ) at a 1 : 1000 dilution . colorimetric reaction and optical density were performed as above described . the data obtained suggest that the pja , pjb and pjc variants show a similar behavior exhibiting a slight reduction of their binding ability ( about 10 %) compared to the parj1 binding . the pjd variant showed a reduced binding activity ( about 20 %) while the preimmune serum did not show any reactivity towards the proteins ( fig8 ). ten patients , with a clear history of parietaria judaica allergy and with skin prick test ( spt ) monosensitivity to pj commercial extract , were analysed in this study . all the patients did not receive immunotherapy against pj pollen and were not receiving glucocorticosteroid treatment . allergens were used at 1 μg / ml concentration diluted in 0 . 9 % nacl . about 20 μl of the test solution was placed on the forearms at a distance of more than 2 . 5 cm between each prick . all tests were performed in duplicate . histamine was used as positive control and 0 . 9 % nacl solution as a negative control . reactions were measured after 20 min . by comparison with the wheal area generated by histamine ( 100 %), positive spt were divided in three classes : 4 + were assigned to spt with an area & gt ; 100 % of area induced by histamine ; 3 + were assigned to an area ≧ 80 - 100 % and 2 + to an area ≧ 50 - 80 %. two non - allergic patients ( p . c . and d . g .) were tested as negative controls . each subject was informed by the investigators and signed informed consent before the test . all patients showed a positive cutaneous reaction to the rparj1 allergen . pjb was capable of inducing type i immediate hypersensitivity in 9 out of 10 of the tested patients . pja gave positive reaction in 3 out of 10 of the patients and the wheal areas induced by prick were reduced in size respect to that ones triggered by the wild - type allergen . the pjc and pjd did not give any spt reaction . none reactions have been observed when non allergic subjects were tested as reported in the following table ii . table ii skin prick test of the rpar j and its disulfide bond variants patient no . rparj1 pja pjb pjc pjd 1 ++++ − − − − 2 +++ ++ +++ − − 3 ++++ − +++ − − 4 ++++ ++ +++ − − 6 ++++ − +++ − − 7 ++++ − ++++ − − 8 ++++ ++ +++ − − 9 +++ − ++ − − 10 +++ − ++ − − p . c . − − − − − d . g . − − − − − 1 . menegozzo , m ., d . geraci , and a . ruffilli , isolation and characterization of an allergenic fraction from parietaria officinalis pollen . immunochemistry , 1976 . 13 ( 5 ): p . 475 - 6 . 2 . geraci , d ., u . oreste , and a . ruffilli , purification and characterization of allergens from parietaria officinalis pollen . immunochemistry , 1978 . 15 ( 7 ): p . 491 - 8 . 3 . geraci , d ., et al ., immunochemical characterization of antigens of parietaria judaica pollen . identification of allergens by means of crossed radio immunoelectrophoresis . int arch allergy appl immunol , 1985 . 78 ( 4 ): p . 421 - 8 . 4 . corbi , a . l . and j . carreira , identification and characterization of parietaria judaica allergens . int arch allergy appl immunol , 1984 . 74 ( 4 ): p . 318 - 23 . 5 . corbi , a . l ., et al ., isolation of the major ige - binding protein from parietaria judaica pollen using monoclonal antibodies . mol immunol , 1985 . 22 ( 9 ): p . 1081 - 9 . 6 . corbi , a . l ., r . ayuso , and j . carreira , identification of ige binding polypeptides cross - reactive with the parietaria judaica main allergenic polypeptide . mol immunol , 1986 . 23 ( 12 ): p . 1357 - 63 . 7 . ford , s . a ., et al ., identification of parietaria judaica pollen allergens . int arch allergy appl immunol , 1986 . 79 ( 2 ): p . 120 - 6 . 8 . ayuso , r ., f . polo , and j . carreira , purification of par j i , the major allergen of parietaria judaica pollen . mol immunol , 1988 . 25 ( 1 ): p . 49 - 56 . 9 . cocchiara , r ., et al ., purification of parj i , a major allergen from parietaria , judaica pollen . int arch allergy appl immunol , 1989 . 90 ( 1 ): p . 84 - 90 . 10 . duro , g ., et al ., isolation and characterization of two cdna clones coding for isoforms of the parietaria judaica major allergen par j 1 . 0101 . int arch allergy immunol , 1997 . 112 ( 4 ): p . 348 - 55 . 11 . duro , g ., et al ., cdna cloning , sequence analysis and allergological characterization of par j 2 . 0101 , a new major allergen of the parietaria judaica pollen . febs lett , 1996 . 399 ( 3 ): p . 295 - 8 . 12 . colombo , p ., et al ., identification of an immunodominant ige epitope of the parietaria judaica major allergen . j immunol , 1998 . 160 ( 6 ): p . 2780 - 5 . 13 . rolland , j . and r . o &# 39 ; 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