Patent Abstract:
contiguous overlapping peptides for the treatment of allergic patients by specific immunotherapy are provided from the sequence of the major allergens of house dust mites der p 1 and der p 2 . such peptides while providing all potential t cell epitopes are devoid of the three dimensional structure of the original allergen , therefore reducing their ability to bind ige . as a result increased amounts of cops can be administered per injection , therefore reducing both the number of injections and the length of the immunotherapy treatment .

Detailed Description:
the invention is described below by way of examples with reference to the following experimental procedures and results . purified natural der p 1 ( na - dp1 - 2 ) and natural der p 2 ( na - dp2 - 2 ) were obtained from indoor biotechnologies ltd , uk . the aim was to prevent the formation of stable tertiary structures of b cell epitopes , while presenting all t cell epitopes present within the der p 1 and der p 2 protein sequences presented below as seq id no : 27 ( der p 1 ) swiss - prot sequence p08176 . 2 and seq id no : 28 ( der p2 ) genbank sequence afj68067 . 1 set out below . it should be noted that der p 2 exists in multiple isotypes with swiss prot p49278 . 1 ( seq id no : 29 ) described in previous patent application wo 2004 - 081028 ( a2 ) the disclosure of which is incorporated by reference herein . there are four amino acid differences between the genbank sequence afj68067 . 1 and the mature protein ( without signal peptide ) swiss prot p49278 . 1 sequence . the sequences also differ with the swiss prot p49278 . 1 sequence being shorter due to a single peptide being cleaved off its n - terminal end . all cops were synthesized by solid phase fmoc chemistry at research scale to allow determination of ige binding . preparative hplc was used to obtain over 90 % pure peptides which were lyophilized . peptides were resuspended either in water at 2 mg / ml or at 10 to 20 mg per ml in dmso ( see table 1 ) and frozen in aliquots . purified der p 1 or der p 2 at 1 . 0 μg / ml were coated overnight on 96 - well nunc maxisorp ® immunoplates ( thermo fisher scientific inc ., wohlen , switzerland ). after blocking with 1 % bsa , either twenty - fold , thirty - fold or forty - fold up to 200 - fold dilutions of patient serum were added depending on specific ige content . biotin mouse anti - human mab ige at 5 μg / ml ( biolegend , san diego , calif .) were then added and antibodies were revealed with streptavidin hrp ( bd - biosciences , san diego , calif .) and the substrate tmb . sera of allergic patients were obtained from us and europe ( plasmalab international , everett , wash ., usa and biomnis , lyon , france ) respectively ) selected for positive cap for d . pteronyssinus and clear signal over background and used to test for competition with the peptides . serial dilutions of each set of cops , namely for der p 1 set a , b , c or d and for der p 2 mixes d , e or f at concentrations ranging from 10 − 5 to 10 − 10 m were pre - incubated with selected serums for 15 min . on ice . serums were then incubated on nder p 1 or nder p 2 coated 96 - well plates and residual ige binding was determined as described above . nder p 1 and nder p 2 dilutions were used as control for inhibition . degranulation assay was performed as described in [ 37 ]. rbl - 703 / 21 cells transfected with human fc epsilon ri receptor were plated in 96 - well tissue culture plates ( 105 cells / well ) overnight . passive sensitization of rbl - 703 / 21 cells was carried out with sera from patients with house dust mite allergy at 1 : 20 overnight . unbound antibodies were removed by washing the cell layer twice in hanks &# 39 ; balanced salt solution ( gibco , life technologies ) the next day . degranulation of rbl cells was induced for 1 h at 37 ° c . by adding nder p1 , nder p 2 or individual cops and mixes at indicated concentrations diluted in tyrode &# 39 ; s buffer ( 130 mm nacl , 5 mm kcl , 1 . 4 mm cacl2 , 1 mm mgcl2 , 5 . 6 mm glucose , 10 mm hepes ph7 . 4 , 0 . 1 % bsa ). β - hexosaminidase activity was analyzed by incubating 30 μl of cell supernatant with 50 μl of 1 . 3 mg / ml 4 - nitrophenyl n - acetyl - β - d - glucosaminide ( sigma ) in citrate buffer ( 0 . 1m , ph 4 . 5 ) for 1 h at 37 ° c . the reaction was stopped by addition of 100 μl glycine buffer ( 0 . 2m glycine , 0 . 2m nacl , ph 10 . 7 ) and optical densities ( od ) were measured at 405 nm . balb / c mice were sensitized by repeated injections of low doses ( 1 μg ) of an allergen mix of equimolar der p 1 and der p 2 by 3 subcutaneous injections at 14 days interval . mice were challenged with high doses of either mix of der p 1 and der p 2 ( 30 μg / animal ) or allerdm cops ( 30 μg / animal ) 7 days after the last injection . anaphylactic symptoms were scored using a scale of 6 grades ( 0 , no symptoms to 5 , death ) adapted from sade et al . j investig allergol clin immunol ; 17 ( 6 ): 379 - 385 ( 2007 ). rectal temperature was monitored at 15 minutes intervals for 60 minutes after challenge using a digital thermometer . pbs was used as control for challenge leading . der p 1 , der p 2 allergens and individual allerdm cops were injected i . p . in balb / c mice . the allergens and peptides were given together with complete freund &# 39 ; s adjuvant . injections were repeated twice at a one month interval with incomplete freund &# 39 ; s adjuvant . blood was collected from the retroorbital sinus 15 days after the last injection and serum prepared by standard method . results are expressed as μg / ml specific igg . in order to select for products with lowered ige binding , four sets of long ( 30 - 90 amino acids ) contiguous overlapping peptides ( cop ) were devised encompassing the entire der p 1 allergen ( swiss - prot p08176 . 2 , thus providing all possible t cell epitopes . a first set of three cops , derp1_set a composed of allerdm1 . 1 , 1 . 2 and 1 . 3 , seq id 1 , seq id2 and seq id 3 respectively , had already been proposed patent deposit “ allergen peptide fragments and use thereof ” w02004 - 081028 ( a2 ). derp1_set a cops were synthesized , purified and assayed for ige binding using competition elisa . residual ige binding was observed with different sera from patients allergic to house dust mites to allerdm1 . 2 and allerdm1 . 3 but not allerdm1 . 1 . a second set ( derp1_set b ) was devised taking into account potential ige binding regions from the literature and tested for ige binding by competition elisa . derp1_set b , a mix of allerdm1 . 4 , 1 . 5 , 1 . 6 , 1 . 7 , respectively seq id 4 , 5 , 6 and 7 showed residual ige reactivity . a . when using individual peptides , allerdm1 . 4 and 1 . 7 showed no detectable ige binding , whereas allerdm1 . 5 and 1 . 6 were found to be responsible for the reactivity previously observed with allerdm1 . 2 and 1 . 3 or complete derp1_set b . allerdm1 . 5 included two cysteines which might reform a disulfide bridge and recreate an ige epitope . thus , the c - terminal cysteine was removed , leading to allerdm1 . 52 ( seq id 9 ) which unfortunately still bound ige . for allerdm1 . 6 , shortening the c - terminal part had also no influence on ige binding ( allerdm1 . 62 , seq id 10 ). since the n - terminal end of allerdm1 . 52 and the c - terminal end of allerdm1 . 62 were modified , alternative peptides to allerdm1 . 4 and 1 . 7 had to be devised in order to provide sufficient overlaps to ensure the presence of possible t cell terminal epitopes . allerdm1 . 42 and 1 . 72 received 4 and 3 additional amino acids in the overlapping region ( seq id 8 and 11 respectively ). the candidate derp1_set c including allerdm1 . 52 and 1 . 62 showing residual ige binding was thus discarded . after modeling the 3d structure of der p 1 and the synthesized peptides up to now , a further set , namely derp1_setd was prepared in which each of the ige binding peptides allerdm1 . 52 and 1 . 62 were further split . allerdm1 . 52 was split in two peptides , namely allerdm1 . 53 and 1 . 5a ( seq id 12 and 26 respectively ) and allerdm1 . 62 was split in allerdm1 . 63 and 1 . 64 ( seq id 14 and 15 ) ( fig1 ). these peptides finally did not show any detectable ige binding , indicating that all possible ige epitopes had been removed . since 1 . 53 and 1 . 5a overlapped by only 5 amino acids , an extended version allerdm1 . 54 ( seq id 13 ), overlapping by 8 amino acids , was synthesized and showed the same ige non - binding properties as allerdm1 . 5a . this finding indicates that varying the cops end by 3 amino acids has no effect on ige binding capacity . having to split allerdm1 . 62 in two pieces was unexpected regarding current knowledge , since the region r248 to r254 had not been previously retained as potentially binding ige ( see background of the invention ). the final set of cops issued from der p 1 with no detectable ige binding includes six peptides , namely allerdm1 . 42 , 1 . 53 , 1 . 54 , 1 . 63 , 1 . 64 and 1 . 72 . ( seq id 8 , 12 , 13 , 14 , 15 and 11 respectively ). the first set of cops was derived from previous patent application w02004 - 081028 ( a2 ) ( der p 2 isotype p49278 . 1 ) ( seq id 29 ) and contained two peptides of equal length allerdm2 . 1 and 2 . 2 ( seq id 16 and 17 ). this set of cops ( derp2_seta ) showed low but detectable ige binding in competition elisa and allerdm2 . 2 was found to bind ige in direct elisa . derp2_setb , composed of allerdm2 . 3 , 2 . 4 and 2 . 5 ( seq id 18 , 19 and 20 ) was devised in order to prevent disulfide bridges responsible for two short loops known to play a role in the maintenance of the 3d structure of der p 2 . in parallel an alternative set ( derp2_setc ) was synthesized , where disulfide bridges were kept , namely allerdm2 . 6 , 2 . 7 and 2 . 8 ( seq id 21 , 22 and 23 ). both sets b and c , showed residual ige binding in competition elisa . in setb ige binding was restricted to allerdm2 . 5 as confirmed by both direct and competition elisa . ige binding to individual cops from setc proved to be multiple . allerdm2 . 7 , which covers the central part of der p 2 , showed residual ige binding in competition elisa , whereas allerdm2 . 8 ( c - terminal part of der p 2 ) was found to bind ige non - specifically ige from sera from allergic and non - allergic donors . in addition , derp2_setc elicited weak but detectable response in rbl degranulation assay and was therefore discarded from further allerdm development . surprisingly , derp2_setb ( allerdm2 . 3 , 2 . 4 and 2 . 5 ), was able to induce basophil degranulation in an rbl assays whereas derp2_seta ( aller dm 2 . 1 and 2 . 2 ) did not ( fig2 ). furthermore , allerdm2 . 4 and 2 . 5 in combination resulted in degranulation , whereas each individual peptide did not ( fig2 , panel c ). the same phenomenon was observed when combining allerdm2 . 1 with allerdm2 . 5 . since allerdm2 . 1 and allerdm2 . 4 did not bind ige when tested alone in competitive elisa , and since two ige epitopes are required in addition to trigger degranulation , these results raised the possibility that some interaction between the two peptides allerdm2 . 4 and 2 . 5 takes place in solution leading to partial reconstitution of the original 3d structure of the allergen , thus re - creating a second ige epitope . analyzing der p 2 crystal structures in view of the results above lead to propose that allerdm2 . 4 and 2 . 5 may partly refold in solution due to the presence of antiparallel beta sheets and combine to re - create a second ige binding site . since the combination allerdm 2 . 2 in combination with either 2 . 1 or 2 . 4 did not induce degranulation , s57 to c73 was added at the n - terminus of allerdm2 . 5 in the next set of cops leading to allerdm2 . 10 ( seq id 24 ). in order to further reduce ige binding potential , allerdm2 . 10 was trimmed at its c - terminus to d113 . to maintain a minimal overlap ( 5 amino acids ) with allerdm2 . 10 , allerdm2 . 9 ( seq id 25 ) had to be synthesized starting at k109 , in order to include the complete der p 2 sequence . derp2_setd ( allerdm2 . 3 , 2 . 4 , 2 . 10 and 2 . 9 ) and its individual components showed no residual ige binding in competition elisa and were not able to trigger basophil degranulation anymore . the final set of cops issued from der p 2 thus includes four peptides , namely allerdm2 . 3 , 2 . 4 , 2 . 10 and 2 . 9 ( seq id 18 , 19 , 24 and 25 respectively ). a further finding indicated that ige binding and basophil degranulation is related to partial reconstitution of original 3d structure in solution came from the use of denaturing salts . adding denaturing salts , including guanidinium chloride or urea , strongly diminished ige binding to allerdm1 . 52 , 1 . 62 and 2 . 2 ( fig3 ). this indicates an alternative for product formulation to decrease potential immediate allergic reactions upon injection of allergen fragments . in order to further verify the absence of ige binding the final mix of ten peptides derived from both der p 1 and der p 2 , now called allerdm , namely composed of combination of allerdm1 . 42 , 1 . 53 , 1 . 54 , 1 . 63 , 1 . 64 and 1 . 72 . ( seq id 8 , 12 , 13 , 14 , 15 and 11 respectively ) with allerdm2 . 3 , 2 . 4 , 2 . 10 and 2 . 9 ( seq id 18 , 19 , 24 and 25 respectively ) was used in competition elisa and rbl degranulation tests . as seen in fig4 , allerdm showed no detectable reactivity up to a concentration of 10 − 5 m with a panel of 21 sera from patients allergic to house dust mites from both europe and us . controls using either der p 1 or der p 2 natural allergens showed expected competition in the range of 10 − 7 to 10 − 9 m . reduced ige binding allows to consider administering a higher dose of cops in human compared to traditional sit treatments . rbl cells transfected with human fc epsilon receptor i were found to bind human ige and degranulate in presence of allergens [ 37 ]. allerdm , up to 10 − 5 m again , was unable to trigger basophil degranulation when pre - loading rbl cells with a panel of 18 different human serums of patients allergic to house dust mites ( fig5 a and b ). on the contrary , der p 1 and der p 2 were able to induce degranulation in combination with the same serums . the absence of degranulation of basophils indicates a potentially diminished risk of immediate allergic reaction upon application in human . from these experiments it can be concluded that a preferred product composed of ten cops to be called allerdm will contain a combination of soluble peptides derived from both der p 1 and der p 2 proteins . the preferred product candidate will be composed of seq id : 8 , seq id no : 12 , seq id no : 13 , seq id no : 14 , seq id no : 15 , seq id no : 11 , seq id no : 18 , seq id no : 19 , seq id no : 24 and seq id no : 25 . a further improvement of the invention includes splitting cops in parts with improved purification and solubility properties . also contemplated are homologs of the cops allerdm1 . 42 to 1 . 72 and allerdm2 . 3 to 2 . 10 , by amino acid changes within each peptide to produce homologs thereof wherein the reactivity of the homologs to ige antibodies of patients who are allergic to hdm is eliminated while that with the t lymphocytes is still retained . further contemplated are homologs of the cops by shifting the limits of the cops within the house dust mites allergens der p 1 and der p 2 . such homologs will result in products with equivalent profiles of non - detectable ige binding and t lymphocyte activity . such products will present the same potential for safety and efficacy in human as allerdm and can be considered as equivalent in terms of chances for treatment , unless shown otherwise . suitable homologs characterized by no reactivity to anti house dust mites ige antibodies while maintaining reactivity to t lymphocytes may be identified by the methods described herein balb / c mice were sensitized by repeated subcutaneous injections of a mix of der p 1 and der p 2 allergens ( 1 μg / ml ). 7 days after the last injection mice were challenged with a single massive dose of allergens ( der p 1 and der p 2 at 30 μg / ml ) or allerdm ( 30 μg / ml ). anaphylactic shock in mice has been associated with body temperature drop . controls and allerdm challenges resulted in essentially no temperature drop , whereas the mix der p 1 / der p 2 induced a marked decrease in rectal temperature ( fig6 ). these results show that allerdm differs from its parent allergens by a lack of challenging activity , indicating that allerdm does not trigger an anaphylactic shock in der p 1 / der p 2 sensitized mice . allerdm and der p 1 / der p 2 were injected in balb / c mice with freund &# 39 ; s adjuvant . igg levels were measured after intraperitoneal injection ( fig7 ). iggs raised by the allerdm mix recognize natural der p 1 and der p 2 to almost the same extend as the original allergens themselves ( fig7 , panel a ). in separate experiments , the presence of iggs against each individual cop was detected in sera from mice immunized with allerdm ( fig7 , panel b ) showing that each cop can contribute to the overall immunogenicity , albeit to different extent . also contemplated are homologs of the allerdm1 . 42 to dm2 . 10 cops , by amino acid changes within each peptide to produce homologs thereof wherein the reactivity of the homologs to ige antibodies of patients who are allergic to house dust mite allergens is eliminated while that with the t lymphocytes is still retained . further contemplated are homologs of the cops by shifting the limits of the cops within the house dust mite allergens der p 1 and der p 2 . such homologs will result in products with equivalent profiles of non - detectable ige binding and t lymphocyte activity . such products will present the same potential for safety and efficacy in human as allerdm and can be considered as equivalent in terms of chances for treatment , unless shown otherwise . suitable homologs characterized by no reactivity to anti house dust mite ige antibodies while maintaining reactivity to t lymphocytes may be identified by the methods described herein . also contemplated are sets of cops with reduced or eliminated ige reactivity which retain t lymphocyte reactivity but which are also soluble and / or which show improved synthesis and purification . numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the presently preferred embodiments thereof . consequently , the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims . settipane r a . demographics and epidemiology of allergic and nonallergic rhinitis . allergy asthma proc . 2001 ; 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