Patent Application: US-55163604-A

Abstract:
the present invention relates to a peptide comprising a symmetrical dimethylated arginine , and constitute an immunologic determinant of antibodies present in sera from patients with systemic lupus erythematosus , and wherein the methylation is a prerequisite for reacting with said antibodies . the invention also relates to the use of said peptide for diagnosis of sle and the differentiation between sle and mctd .

Description:
sera ( n = 628 ) were collected from patients suffering from systemic lupus erytlhrematosus ( sle ; n = 176 ), rheumatoid arthritis ( ra , n = 86 ), sjögren syndrome ( ss , n = 24 ); mixed connective tissue disease ( mctd , n = 26 ), scleroderma ( ssc , n = 26 ) and polymyositis / dermatomyositis ( pm / dm , n = 13 ). all patients were classified according to the acr - criteria for each disease ( tan et al ., 1982 ; arnett et al ., 1988 ). to further assess the assay specificity , we analyzed a group of sera from patients with infection diseases ( n = 77 ) including hepatitis - c ( hcv ; n = 30 ), cytomegalo ( cmv ; n = 22 ) and epstein - barr virus ( ebv ; n = 25 ) as well as from 192 healthy blood donors . all sera were stored at − 80 ° c . until use . for epitope - mapping a panel of five sera containing anti - sm antibodies was used . as negative controls autoimmune sera with other antibody specificities than anti - sm were selected . serological characterization of randomly selected sle patient sera . all autoimmune patient sera were tested for autoantibodies to histones , dsdna and the sm - complex using quantitative varelisa ® s ( pharmacia diagnostics , freiburg , germany ). sle sera and samples , which demonstrated unexpected results were also measured in the semi quantitative ana - split elisa research kit ( pharmacia , freiburg , germany ). the latter assay contains the autoantigens u1 - 68 kda , u1 - a , u1 - c , smbb ′, smd , ro - 52 , ro - 60 and la . all elisas were performed according to the instructions of use . the published sequences of smd1 , p13641 , ( rokeach et al ., 1988 ) and smd3 , p43331 , ( lehmeier et al ., 1994 ) were used to synthesize overlapping 15 mer peptides with a pipetting robot according to the protocol described by gausepohl and behn ( 2002 ). the c - terminal extensions of both polypeptides were synthesized with an offset of 2 amino acids ( 13 amino acids overlap ). each arginine containing peptide was synthesized as three variants , with natural arginine , with sdma or with asymmetrical dimethylarginine ( asdma ) at the respective positions . later on , a highly reactive peptide of smd3 was synthesized with certain combinations of natural arginine and sdma . following completion of the peptide synthesis non - specific binding sites were blocked by over - night incubation of the membranes in blocking buffer ( bb ) at room temperature ( rt ). after one washing step membranes were incubated with serum samples at a dilution of 1 : 100 in bb for 2 h at rt . unbound antibodies were removed by three washing steps . for detection peroxidase conjugated goat - anti - human igg antibody was diluted 1 : 5000 in bb and incubated for 75 min ( rt ). superfluous secondary antibodies were removed by three washing steps . finally , bound antibodies were visualized using the enhanced chemoluminiscence ( ecl ) detection - system . assay conditions were used under which negative sera showed no reactivity . preparation of elisa - plates . the lyophilized s33 peptide was used to prepare a stock solution of 10 μg / μl , which was stored in aliquots at − 20 ° c . until use . binding of the peptide to elisa plates was carried out using 2 . 5 μg / ml of the peptide in coating buffer in a final volume of 120 μl per well . the coating procedure was carried out at 15 ° c . for 20 h . unspecific binding sites were blocked with blocking solution . after discarding the blocking solution solid phases were dried at 37 ° c . for 2 h and sealed . the assay was performed according to the general protocol of the varelisa ® system ( pharmacia diagnostics , freiburg ). blood donors demonstrated a reactivity range of 0 . 4 - 11 . 5 u / ml resulting in a mean value of 2 . 2 u / ml and a sd of 1 . 2 u / ml . the cut - off was technically set to 13 u / ml after roc - analysis . ppvs and npvs were calculated at different cut - off values . precision and reproducibility . measurements of imprecision ( inter - and intra - assay variability ) were performed with 4 and 6 replicates , respectively . to assess precision of the anti - s33 peptide elisa suitable anti - sm sera , a low value sample ( l ); a medium value sample ( m ) and a high value sample ( h ) were assayed in five independent runs on one day ( inter - assay ), or in a single run ( intra - assay ). for within - run precision l , m and h were measured in six replicates on one solid phase . the precision data was calculated using anova analysis . linearity . the linearity was analyzed by testing dilutions ( 1 : 1 ; 2 : 3 ; 1 : 2 ; 1 : 4 ; 1 : 8 ; 1 : 16 ; 1 : 32 ) of the highest standard point ( s6 ) and of the high value sample from the precision analysis ( h ). for each dilution point , a ratio of the measured reactivity to the expected value was calculated , and 1 was subtracted from this quotient . randomly selected sle sera ( n = 50 ) and various controls ( n = 100 ) were tested using the commercially available anti - sm antibody tests from different suppliers ( sm test a — sm test d ) and the results were compared to the findings of the anti - s33 elisa test . a male sle patient was clinically and serologically observed over a time period of 18 month ( 6 serum samples ; see fig3 ). the patient was tested for antibodies to the rnp / sm complex , to the sm antigen , to the isolated u1 - rnp complex , to histones , to dsdna and to the s33 peptide using the respective test kits from pharmacia diagnostics . epitope fine - mapping of the c - terminal extensions of smd1 and d3 . to evaluate the effect of arginine - dimethylation on the antigenicity of smd1 and smd3 and to map relevant epitopes on both polypeptides a panel of anti - sm sera was tested with peptide arrays ( 15 mer , 2 offset ) covering the c - terminal region of smd1 ( p13641 ) and smd3 ( p43331 ). the results show that dimethylation of arginine residues affects the binding of anti - sm antibodies to c - terminal smd1 - and d3 polypeptides , significantly ( see fig1 ). all anti - smd sera (# 36 , # 37 , # 31 , # 84 , # sm ) demonstrated an increased binding to smd1 peptides containing the symmetrical form of dimethylarginine ( sdma ). especially the peptides that consist of glycine and dma repeats , exclusively showed a strong reactivity with the antibodies ( peptide no . 9 , 10 ). nevertheless , smd1 polypeptides containing dma represent a rather unspecific substrate for anti - sm antibodies since they were also target of anti - centromere antibodies ( aca ; # serum cen ( centromer )). interestingly , those aca bound also to peptides containing the asymmetrical form of dma . binding experiments with peptides derived from smd3 showed similar results . only smd3 peptides containing sdma reacted with anti - sm antibodies confirming the importance of the symmetric methylation of arginine residues ( see fig1 b ). in contrast to smd1 , no control serum ( e . g . cen ) demonstrated antibody binding to smd3 derived peptides reflecting a high specificity . one particular peptide ( no . 77 , 108 aasdrgsdrgsdrgmgsdrgnif 122 ) ( seq id no : 3 ) was strongly recognized by three out of five anti - sm sera . using a mutational analysis in which arginine residues of 108 aargrgrgmgrgnf 122 ( seq id no : 4 ) were successively replaced by sdma we were able to show that a mimotope peptide with a single dimethylated arginine residue at position 112 displayed immunoreactivity with all of the five anti - sm sera (# 36 , # 37 , # 31 , # 84 , # sm ) but not with the controls ( e . g . cen ; see fig1 c .). thus , by introducing only one sdma and at a defined position ( amino acid 112 ) of smd3 , it was possible to increase the sensitivity of this peptide ( 108 aargsdrgrgmgrgnif 122 ; s33 ) ( seq id no : 1 ) without a loss in specificity . this candidate peptide was subsequently synthesized as soluble antigen and used as substrate in elisa . immunoserologic characterization of the sle patient group . to evaluate if our sle patient cohort represents a representative sle serum panel approximately 100 sle samples were randomly selected and tested for u1 - 68kd , u1 - a , u1 - c , smbb ′, smd , ro - 52 / ss - a , ro - 60 / ss - a , la / ss - b , histone dsdna and β2 - glycoprotein reactivity ( split ana - profil research assay , pharmacia diagnostics , freiburg , germany ). the prevalence of the different autoantibodies was found in a good agreement to previous studies ( jaekel et al ., 2001 ). thus , with regard to their autoantibody profiles , the sle cohort seems to be a representative sle population . results of the measurements of the sle panel are summarized in table 1 . a 15 amino acid soluble peptide displaying highest sensitivity and specificity in the spot - assay ( 108 aargsdrgrgmgrgnif 122 ) ( seq id no : 1 ) was synthesized for technical reasons with an additional cys at the c - terminus . this peptide was subsequently used to develop an elisa system based on the general protocol of the varelisa ® tests ( pharmacia , freiburg , germany ). assay performance characteristic . to evaluate the assay performance characteristics precision , reproducibility and linearity were analyzed . the intra - and interassay variability ( cv %) of three samples were found ranging from 1 . 82 to 6 . 52 % and from 2 . 27 to 7 . 42 %, respectively . dilution series of two samples demonstrated a linear range on five subsequent dilutions (& gt ; 20 % deviation ). for the cut - off definition a receiver operating characteristic ( roc )- analysis was performed with sl1 and control sera . the assay performance characteristics of the new anti - s33 - test including intra - and interassay variability ( a . ), linearity ( b . ), roc - analysis , ppv , npv and efficiency ( c .) are summarized in fig2 ( a .- c .). for the evaluation of the diagnostic relevance of the new test a technical cut - off of 13 u / ml was used to combine high specificity with moderate sensitivity . sera from 176 sle patients , from 181 autoimmune patients diagnosed differently than sle , from 77 patients with infection diseases and from 192 human normal donors were analyzed in the new elisa system . 28 sle patients ( 15 . 9 %) were tested positive for anti - s33 antibodies displaying a significantly increased reactivity of up to 952 u / ml with a mean value of 43 u / ml ( sd = 160 . 2 u / ml ). patients from related disorders demonstrated a significant reduced reactivity in the new elisa system ( mean 3 . 36 u / ml ). only one patient of the ra group was assayed positive ( 24 . 6 u / ml ). none of the remaining controls including patients suffering from ssc ( n = 26 ), pm / dm ( n = 13 ), mctd ( n = 126 ) or infection diseases ( n = 77 ) showed reactivity to the s33 peptide . the serum samples from patients with infectious diseases demonstrated a reduced reactivity ( mean 0 . 67 u / ml ; top value 3 . 3 u / ml ), even when compared to the healthy donors ( mean 2 . 21 u / ml ; top value 11 . 5 u / ml ). the top value of the infectious disease sera was found in the ebv group . results are summarized in table 2 . in summary , 15 samples of the sle group ( n = 176 ) and only one serum of the controls ( n = 449 , 0 . 2 %) was tested positive resulting in a diagnostic specificity of 99 . 8 % and a sensitivity of 15 . 9 %. ppv and npv , as well as the diagnostic efficiency was calculated at 96 . 6 %, 75 . 3 % and 76 . 3 %, respectively ( see fig2 c .). these data indicate that anti - s33 antibodies appear to be exclusively present in sera from sle patients . apart from the anti - s33 peptide reactivity the false positive ra sample contains high titers of antibodies to the u1 - rnps - 68 kda ( ratio 4 . 5 ), u1 - c ( ratio 9 . 4 ) and histones ( 133 . 8 u / ml ) ( see table 3 ). anti - smbb ′ and anti - smd titers as determined by elisa were elevated when compared to the controls , but still below the cut - off values ( see table 3 ). correlation to other autoantibodies . with regard to possible existing correlations between anti - s33 antibodies and other autoantibody species , a statistic evaluation was performed using the sle panel of approximately 100 randomly selected sera . significant correlations were to u1 - 68kda ( p = 0 . 0335 ), u1 - a ( p & lt ; 0 . 0001 ), u1 - c ( p & lt ; 0 . 0001 ) smbb ′ ( p & lt ; 0 . 0001 ), smd ( p & lt ; 0 . 0001 ), dsdna ( p & lt ; 0 . 0001 ) and histone ( p & lt ; 0 . 0001 ), but not to ro - 52 ( p = 0 . 2192 ), ro - 60 ( p = 0 . 2212 ) and la ( p = 0 . 8785 ) ( see table 4 ). looking at the reactivity towards the sm - complex , five samples of the randomly selected sle patients ( n = 101 ) reacted with the purified smd antigen , but not with the s33 peptide . the remaining 11 smd positive sera ( 68 . 8 %) were also tested positive in the new anti - s33 peptide elisa . interestingly , among the anti - s33 positive samples , 4 patients (# 89 , # 92 , # 20627 , # 9811 ) were found , all anti - smd negative showing anti - s33 peptide reactivities of 15 . 4 , 21 . 3 , 41 . 3 and 13 . 9 units , respectively . to evaluate correlations to commercially available anti - sm antibody tests from different suppliers 50 randomly selected sle sera from the sle patient group and 100 controls were tested using the anti - sm antibody tests from different suppliers . 6 out of 50 sle sera ( 12 %) and none of the controls ( 0 %) were positive in the anti - s33 antibody test resulting in a sensitivity of 12 % and a specificity of 100 %. in contrast the anti - sm assay from different suppliers sm test a , b and c accessed only 5 sle samples ( 10 %) and between 6 ( sm test a , c ) and 12 ( sm test d ) patients from the control group . the majority of false positive results were found within the group of mctd patients ( see table 5 ). follow - up study of a sle patient . a male sle patient was clinically and serologically observed over a time period of 18 month ( 6 serum samples ; see fig3 ). at the beginning of the follow - up study the patient displayed a strong immunoresponse towards the rnp / sm complex ( ratio of 18 ), to the sm antigen ( ratio of 6 ), to the new sm antigen ( 337 . 5 u / ml ) and a moderate response to the isolated u1 - rnp complex ( ratio of 2 ) as well as to histones ( 59 . 5 u / ml ). no reactivity to dsdna could be found ( 19 . 1 u / ml ; cut - off 55 u / ml ). at that time point the medical record reported an inactive phase of disease . later on the antibody titer towards the new sm antigen significantly increased reaching its peak in the third serum sample withdrawn in august 1999 . in contrast a decreasing anti - rnp / sm titer could be observed between the second and the fourth blood sampling followed by another strong increase in the fifths sample . at that time point the titer against the new sm antigen ( s33 ) was lower than before and the disease status was reported as inactive according to the medical record . no significant alterations could be observed in the anti - dsdna and anti - histone titer during the observation time of the patient . in the presented examples the anti - sm immune response have been analyzed towards the sm antigens d1 and d3 , which are considered to be the sle specific polypeptides ( van venrooji et al ., 1991 ; hoch et al ., 1999 ). using immobilized peptides it has been shown that symmetric dimethylation of arginine residues plays an important role in the formation of the major b - cell epitopes on both autoantigens . this observation was found in a good agreement to the result of brahms et al . ( 2000 ) and thus contradictory to the findings of riemekasten and colleagues ( 1998 ). interestingly and in addition to previous investigations , it was found that with peptides as previously described the specificity of smd3 peptides was higher than of those derived from smd1 . mcclain and colleagues ( 2002 ) described four antigenic regions on smd3 of which antigenic region 4 covers the area 104 - 126 . in this invention peptides synthesized on pins were subjected to analysis but without using the modified form of arginine . in the present invention reactivity within this region was only found in case natural arginine was replaced by sdma . these contradictory results might be explained by the use of different sera , methology and / or by the varying peptide length . three out of five sera specifically recognized the peptide 108 aasdrgsdrgsdrgmgsdrgnif 122 ( seq id no : 3 ) of this example . interestingly , the dimethylation of only one arginine and at a defined position ( aa 112 ) could further increase the sensitivity of this particular mimotope peptide without a loss in specificity . based on this data a candidate peptide was used ( 108 aargsdrgrgmgrgnif 122 ) ( seq id no : 1 ) to develop an elisa system . the new anti - sm assay ( anti - s33 ) demonstrated a sensitivity of 14 . 9 % and a specificity of 99 . 7 % for lupus resulting in a high positive ( ppv ; 93 . 7 %) and negative predictive value ( npv ; 80 . 2 %) and thus a high diagnostic efficiency ( 80 . 7 %). therefore this test offers new opportunities for the diagnosis of systemic lupus erythrematosus , especially for the differentiation between sle and mctd as revealed by the correlation study . looking at the biochemical properties of the identified sm - epitopes reveals that the pi can be regarded as predictor of antigenicity on the sm - complex . on u1 - rnp - a , smb ′ and d1 , the average pi of antigenic regions was 10 . 4 ( nonantigenic 6 . 0 ) and on smd2 and d3 more than pis 9 . 0 ( mcclain et al ., 2002 ). these inventive findings fit well to the high pi of the s33 peptide (& gt ; 12 . 88 ). whether the basic character simply increases the probability of surface exposure of these regions and thus the accessibility to antibodies has to be further investigated . ebv , ebna and anti - smd antibodies . epitope - mapping studies on smd1 have identified an epitope - motif ( aa 95 - 119 ) that cross - reacts with a homologue sequence 35 - 58 of the epstein - barr virus nuclear antigen 1 ( ebna - 1 ) ( sabbatini et al ., 1993 ; sabbatini et al ., 1993 ; marchini et al ., 1994 ). a more recent study has shown that this epitope also cross - reacts with a homologue region of smd3 containing glycine arginine repeats ( rgrgrgmgr ) ( seq id no : 5 ) ( mcclain et al ., 2002 ). moreover it became evident that gprr ( aa 114 - 119 on smd1 ) represents a common cross - reactive autoepitope motif , which is present not only on ebna - 1 , but also on a variety of autoantigens including cenp - a , b , c , smbb ′, smd1 and ro - 52 , to term only a few ( mahler et al ., 2001 ). thus patients suffering from infectious mononucleosis or sle related disorders might be tested false positive in elisas using the c - terminal extensions of smd1 or smd3 . furthermore , several studies have suggested an influence of ebv on the development of lupus - like conditions ( james et al ., 1997 ). therefore , it is considered that the use of ebv positive sera as controls is an important finding towards a highly specific and reliable anti - smd immunoassay . among the 25 ebv disease controls presented , no false positive sample was found confirming the suggested high specificity of the anti - s33 - abs assay . unfortunately , riemekasten and colleagues ( 1998 ) did not include this patient group in the evaluation of their test . correlations to other autoantibody species . overlapping reactivity between dna and sm antigens has been reported in several publications ( bloom et al ., 1993 ; reichlin et al ., 1994 ; zhang et al ., 1995 ). while in these studies full - length smd was used , in present invention , there was also a correlation of the anti - dsdna and anti - s33 reactivity ( p & lt ; 0 . 0001 ). apart from dna the present invention also shows a positive correlation of anti - s33 to u1 - 68 ( p & lt ; 0 . 0001 ), u1 - a ( p & lt ; 0 . 0001 ), u1 - c ( p & lt ; 0 . 0001 ), smbb ′ ( p & lt ; 0 . 0001 ), sm ( p & lt ; 0 . 0001 ) and smd ( p & lt ; 0 . 0001 ), but not to histones ( p = 0 . 0259 ), la ( p = 0 . 8747 ), ro - 52 ( p = 0 . 4034 ) and ro - 60 ( p = 0 . 0143 ). whether the observed associations are caused by cross - reactivity or by different autoantibody species that often occur simultaneously , remains unclear . further studies have to be addressed to shed more light on this issue . riemekasten vs brahms . the obvious conflict between the results of riemekasten et al . and brahms et al . might be explained by the existence of different epitopes on the c - terminal extensions of smd1 . the peptide aa 83 - 119 ( riemekasten et al ., 1998 ) may form a conformational epitope , whereas the shorter peptides used in the second study contain linear , sdma dependent binding sites ( brahms et al ., 2000 ). furthermore , the reduced reactivity against the full - length smd1 ( riemekasten et al ., 1998 ), compared to smd1 83 - 119 peptide , suggests that this peptide epitope represents a cryptic structure . this observation raises the question , which epitopes are “ seen ” in vivo and which ones play the central role in the pathogenesis of sle . in a recent study it became evident , that the injection of smd1 83 - 119 fused to a carrier protein is able to accelerate the pathogenic process of lupus - prone mice ( riemekasten et al ., 2001 ). ” rhupus ”- syndrom . rheumatoid arthritis ( ra ) and systemic lupus erythremtosus ( sle ) are related disorders with an autoimmune etiology . both diseases are accompanied by the occurrence of self - reactive antibodies to defined structures . several studies have reported overlap syndromes between ra and lupus , which were therefore sometimes called the “ rhupus ”- syndrom ( miyachi and tan , 1979 ; panush et al ., 1988 ; brand et al ., 1992 ). in the presented examples one patient was found within the ra group who demonstrated anti - s33 reactivity ( 24 . 6 u / ml ). whether this result reflects a false positive testing or wether autoantibodies to the s33 peptide represent a precursor of lupus - like conditions remains unclear and has to be investigated . abuaf n , johanet c , chretien p , absalon b i , homberg j c , buri j f . detection of autoantibodies to sm antigen in systemic lupus erythematosus by immunodiffusion , elisa and immunoblotting : variability of incidence related to assays and ethnic origin of patients . eur j clin invest . august 1990 ; 20 ( 4 ): 354 - 9 . arbuckle m r , reichlin m , harley j b , james j a . shared early autoantibody recognition events in the development of anti - sm b / b ′ in human lupus . scand j immunol . november 1999 ; 50 ( 5 ): 447 - 55 . arnett f c , edworthy s m , bloch d a , mcshane d j , fries j f , cooper n s , healey l a , kaplan s r , liang m h , luthra h s , et al . the american rheumatism association 1987 revised criteria for the classification of rheumatoid arritis . arthritis rheum . march 1988 ; 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