Patent Application: US-59815305-A

Abstract:
a method of assessing the titre of fertility - relevant autoantibodies of a mammalian female , wherein said method comprises : the presentation of a defined single molecule capable of binding to the idiotype of the fertility - relevant autoantibody , and determining the amount of autoantibody being bound by said molecule from a sample of a body fluid of said mammalian female .

Description:
surprisingly , the problem underlying the invention is solved by methods , peptides , diagnostics , medicaments and kits of claims 1 to 19 . peptides , which are able to bind autoantibodies being present in the blood of patients with reproductive problems , are obtainable by the following method : a large number of biotinylated combinatorial peptides was synthesized by solid phase synthesis . these peptides were tested against plasma probes of patients with well - defined clinical symptomotology as well as against control samples . peptides with specific recognition of patient samples ( recognition defined by increased binding of human antibodies to the peptide as compared to the control samples ) were tested in a larger population of samples . sequence variations of each positive peptide were tested to optimise a positive sequence . peptide sequences were used to check databases for sequence - related proteins harbouring the autoantibody target . surprisingly , so far unknown peptides could be identified , which matched on fertility - relevant proteins . the medicament and the diagnostic of the invention are useful for the treatment and diagnosis of fertility disorders or pregnancy complications . subject of the invention are also pharmaceutical compositions comprising the peptides of the invention , including variants such as derivatives or multimers , preferably in a suitable formulation for contraception or the induction of sterility in a patient , caused by the generation of an antibody response to the respective peptide . more than thousand random 12 - mer peptide sequences were synthesized on an automated synthesis roboter and purified on an automated lc / ms - system , which enables the purification and analysis in a one - step approach . all sequences were foreseen with an additional terminal glycine residue coupled to a biotin molecule . per peptide , an amount of 5 - 10 mg — the final yield per specific peptide depending on the losses due to purification and problematic synthesis — were synthesized . a typical synthesis protocol is given as an example of the invention . plasma samples were obtained from patients with clinically well - documented reproductive problems . samples from healthy female blood donors were used as reference . non - immunological causes ( anatomical variations , endocrine problems , fertility problem of the male , etc .) of the respective reproductive disorders present in a given patient , were excluded . moreover , all plasma samples were checked for the presence of anti - phospholipid antibodies . aps is an autoimmune syndrome , which is frequently associated with coagulation disorders , but also with fertility problems . samples with anti - phospholipid antibody titres above the clinically relevant threshold were excluded from the first step of the peptide screen . plasma samples were grouped according to the most common reproductive disorders : unexplained infertility in patients with repetitive ( at least 2 times ) failure in conventional in vitro - fertilization procedures . habitual abortion , characterized by at least 3 consecutive abortions during the 11 trimester of pregnancy . late pregnancy problems , this group being heterogenous and composed of plasma samples from patients with preeclampsia and / or severe intrauterine growth restriction of the fetus without maternal preeclamptic complications . in an elisa - protocol , the serum and plasma samples were tested against peptides from the available peptide pool . basically , the elisa was designed to detect antibodies being present in the patients blood , which bind to the peptides presented to the sample . technically , the elisa followed the protocol given in example 2 and comprised the following basic steps : incubation of the wells and appropriate control wells ( without peptide ) with the serum samples . in a first step , all peptides were tested against a reduced set of samples including pooled sample preparations in order to identify promising candidate peptides for large scale analysis . in a second step , promising candidate peptides were tested against all samples to confirm the results obtained in the 1 st screen . using such elisa - based protocols , it is easily possible to examine large numbers of blood samples and to test peptides against such samples . we could identify the following peptides , which showed clearly increased binding of patient &# 39 ; s antibodies as compared to control antibodies : based on these peptides , it is easily possible to deduce sequence variants , which also have the ability to bind antibodies occurring in the tested samples . for the skilled person in the field it is evident that conservative exchange of amino acids in the above mentioned peptide sequences will also lead to binding peptides , if the exchange does not affect the physicochemical or structural properties of the peptide sequence to a large extent . in further embodiments of the invention oligopeptides are used which are derivatives , fragments and / or homologues of the oligopeptides of seq . id no . 1 to 8 . the oligopeptide amino acid sequence is extended at the amino - terminus and / or the carboxy - terminus by either up to 5 amino acids per terminus , preferably by up to 3 or 2 amino acids and / or in the homologous oligopeptides up to 3 , preferably 2 or 1 amino acids are substituted by other amino acids , and / or the fragments lack 1 or 2 amino acids at the n - and / or c - terminus , and / or the oligopeptides are derivatized for detection by modifications including biotinylation , labelling by fluorochromes or radiolabelling . the oligopeptides of the invention comprise retro - inverso derivatives of these peptides , derivatives , fragments or homologues . in a further embodiment of the invention , the oligopeptides are fragments of the sequences of pregnancy - associated plasma protein a ( papp - a ; gi : 38045915 ) or from adam - ts 13 ( gi : 21265049 ) comprising 8 to 50 , preferably 10 to 40 or 10 to 20 amino acids . an especially preferred way of intensifying the reactivity of the above mentioned peptides with autoantibodies is dimerisation or even multimerization . surprisingly , we could demonstrate that biotinylated homodimers of these peptides , connected by a disulfide bridge after elongation with a cysteine residue , strongly increase the signals which can be detected . increased reactivity of dimers as compared with the monomeric peptides is also shown in fig2 . dimerisation is thus proven to effectively increase the signal intensity , and it is obvious that other technically suitable ways of generating di - or multimeric peptides based on said peptides as reactive moieties also lead to increased signal intensity . thus , dimers , trimers or multimers of the above mentioned peptides are subject of the invention . using the peptide sequences as search sequences in b last - algorithms as they are offered e . g . by the ncbi or embl web portals ended up with proteins , which harbour sequences similar or identical to the peptides 1 - 3 . suprisingly good results were obtained with sequences 1 and 2 with pregnancy - associated plasma protein a ( papp - a ). both peptides show varying degrees of homology with this protein sequence . peptide sequence 3 shows homology with the protein adam - ts 13 ( a disintegrin and a metalloproteinase and thrombopondin - 13 ). peptide sequence 1 shows a very good correlation with a sequence occurring in pregnancy - associated plasma protein a ( papp - a ). peptide sequence 2 shows homologies with another region in papp - a . amino acids described in this invention can be of the naturally occurring l stereoisomer form as well as the enantiomeric d form . the one - letter code refers to the accepted standard polypeptide nomenclature , but can mean alternatively a d - or l - amino acid : the protocol below describes the general setup for synthesis of the specific sequences 1 - 8 described above . for proper usage in the elisa - protocol on streptavidin - coated microtitre plates , these sequences are all extended n - terminally by one glycine moiety . the n - term of this glycine moiety is coupled to biotin in the last step of synthesis . in order to prepare dimeric peptides , the sequences are n - terminally extended by a , preferably the sequence gcg , with the last glycine moiety either being biotinylated n - terminally or not . equimolar amounts of biotinylated and not - biotinylated compounds are then mixed together and oxidized in order to achieve dimerisation by closure of disulfide bridges . purification by hplc - ms then is used to isolate those dimers , which harbour one biotinylated and one not - biotinylated peptide moiety . such monovalently biotinylated dimers are then used for the assay as their mono - valently biotinylated monomeric analogs . unless stated otherwise a washing step is conducted by adding the solvent to the resin , shaking the mixture , and removing the solvent by vacuum filtration . at all steps it must be ensured that each resin bead is immersed in the reaction solution . 1 g 2 - chlorotrityl resin ( 1 . 0 - 2 . 0 mmol / g capacity ) is suspended in 8 ml dichloromethane ( dcm ), shaken for 5 minutes at room temperature , and the solvent is removed by vacuum filtration . a solution of 2 mmol of the fmoc protected amino acid and 5 mmol ( 0 . 850 ml ) diisopropylethylamine ( dipea ) in 8 ml dcm is added and the reaction is shaken for 1 hour at room temperature . after removing the reaction solution the resin is washed three times with 20 ml dimethylformamide ( dmf ) each . 20 ml of a mixture of dcm / methanol / dipea 80 : 15 : 5 ( v / v / v ) is added , shaken for 15 to 30 minutes , the solution removed , and this step is repeated once . the resin is washed four times with 20 ml dmf each . the fmoc group is removed by adding 20 ml of 25 vol -% piperidine in dmf , shaking for 3 minutes , removing the solvent , adding another 20 ml of 25 vol -% piperidine in dmf , shaking for 30 minutes , and removing the solution by vacuum filtration . the resin is washed six times with 20 ml dmf each . in this state the resin can be stored overnight . for this purpose it has to be washed two times with 20 ml dcm each , and dried in vacuo . should a second amino acid be coupled the procedure can be directly continued at step two instead of washing with dcm . in the case that the resin has been stored overnight it has to be swollen by filling the reaction vessel completely with dmf . after 20 minutes the dmf is removed . a solution of 5 mmol of the fmoc protected amino acid , that will be introduced , 7 . 5 mmol 1 - hydroxybenzotriazole ( hobt ), and 1 ml of dipea in 20 ml dmf ( eventually up to 30 ml in the case that the amino acid derivative is not dissolved completely ) is added to the resin . the suspension is vortexed for 5 minutes and 5 mmol of benzotriazole - 1 - yl - oxy - trispyrrolidinophosphonium hexafluorophosphate ( pybop )- is - added as a solid as well as another ml of dipea . after vortexing for 60 to 90 minutes the reaction solution is filtered off , and the resin is washed 6 times with 30 ml dmf each . the resin can be stored in this state ( after washing twice with dcm and drying in vacuo ). further amino acids are coupled by removing the fmoc group with 25 % piperidine in dmf ( as described above ) and repeating step 2 . biotin is introduced by repeating step 2 using biotin instead of an amino acid derivative . due to the poor solubility of biotin the coupling time was four times as long as for a normal amino acid . the resin which is loaded with the fmoc deprotected peptide is washed 6 times with 20 ml dmf and twice with 20 ml dcm each . 40 ml 2 , 2 , 2 - trifluoroethanol / dcm 2 : 8 ( v : v ) is added . the reaction mixture is shaken from time to time and otherwise left standing for 60 minutes . the resin is filtered off and the filtrate co - evaporated several times with dcm . 40 ml of a mixture of trifluoroacetic acid / water / triisopropylsilane ( tis ) 95 : 5 : 5 ( v / v / v ) is added . in the case that the solution is still coloured yellow after about 1 minute several drops of tis are added . the mixture is left standing for about 60 minutes . afterwards the cleaving mixture is removed by coevapourating several times with dcm . the product is dissolved in water ( eventually adding a minimal amount of methanol ) and lyophilisated . the crude peptide is purified by preparative hplc . two peptides with the same amino acid sequence but different derivatisation of the n - termini are oxidized to form a heterodimer . for that purpose 10 mg each of the biotinylated and the non - biotinylated peptide ( hplc purified ) are dissolved in 200 ml acetonitrile / water ( 1 : 1 ). 10 ml dimethylsulfoxide are added and the reaction solution is vortexed for 10 hours at room temperature . the solvent is removed as far as possible from the reaction mixture by vacuum distillation , the remaining solution is lyophilisated and the residue is purified by preparative hplc to isolate the heterodimer . shaking at all incubation and washing steps was done on a laboratory shaking platform at 150 rpm dilute apl peptides from ( dimethylsulfoxide ) dmso stock ( 10 mg / ml ) into washing buffer to 1 μg / ml ( for this dilution , pipette : 1 μl in 10 ml ) dilute stock of reactolab solution 1 : 1000 in washing buffer + 2 % milk powder + 1 % fcs , then : for wells a 1 , 2 : pipette 100 μl washing buffer + 2 % milk powder + 1 % fcs into each well . for wells a 3 , 4 : pipette 4 μl of this solution into 2 ml washing buffer + 20 % milk powder + 1 % fcs , then pipette 100 μl of this solution into each well . for wells a 5 , 6 : pipette 1011 of this solution into 2 ml washing buffer + 2 % milk powder + 1 % fcs , then pipette 100 μl of this solution into each well . for wells a 7 , 8 : pipette 20 μl of this solution into 2 ml washing buffer + 2 % milk powder + 1 % fcs , then pipette 100 μl of this solution into each well . for wells a 9 , 10 : pipette 30 μl of this solution into 2 ml washing buffer + 2 % milk powder + 1 % fcs , then pipette 100 μl of this solution into each well . for wells a 11 , 12 : pipette 40 μl of this solution into 2 ml washing buffer + 2 % milk powder + 1 % fcs , then pipette 100 μl of this solution into each well . step standard ( row a ) peptide and lw ( b - h ) 1 equilibrate all wells in a equilibrate all wells ( b - h ) with 100 μl ( calibration curve *) with 100 μl washing buffer ( 10 min ., room washing buffer + 2 % milk temperature ) powder + 1 % fcs ( 10 min ., room temperature ) 2 pipette 100 μl of the pipette 100 μl of the diluted apl peptides appropriately diluted standard ( 100 ng total amount ) in the wells , last solution into the respective wells row ( h , containing blank values ) without and incubate 1 hour at rt . peptide ( only washing buffer ), incubate 1 hour at rt 3 wash 3x 5 min . with washing buffer 4 add 100 μl washing buffer per add 1 % human serum or plasma diluted well , incubate 1h rt in blocking buffer , 100 μl per well , incubate 1h rt 5 wash 3x 10 min . with washing buffer 6 add 100 μl anti - hu iga , g , m - hrp1 : 10000 ( stock : 22 mg / ml ) diluted in blocking buffer to all wells , incubate 1 hour at rt 7 wash 3x 5 min . with washing buffer 8 add 100 μl opd substrate to each well : 1 tablet opd ( 20 mg ) in 33 ml citrate - phosphate buffer + 17 μl 30 % h2o2 ( suffices for 3 plates ) 9 incubate opd for 10 min and stop reaction with 100 μl 1n hcl to each well 10 measure at measuring wl : 492 with reference wl : 620 in elisa reader 1 2 3 4 5 6 7 8 9 10 11 12 standards a 0 0 10 ng 10 ng 25 ng 25 ng 50 ng 50 ng 75 ng 75 ng 100 ng 100 ng peptides b pep1 pep1 pep1 pep1 pep1 pep1 pep1 pep1 pep1 pep1 pep1 pep1 c pep2 pep2 pep2 pep2 pep2 pep2 pep2 pep2 pep2 pep2 pep2 pep2 d pep3 pep3 pep3 pep3 pep3 pep3 pep3 pep3 pep3 pep3 pep3 pep3 e pep4 pep4 pep4 pep4 pep4 pep4 pep4 pep4 pep4 pep4 pep4 pep4 f pep5 pep5 pep5 pep5 pep5 pep5 pep5 pep5 pep5 pep5 pep5 pep5 g pep6 pep6 pep6 pep6 pep6 pep6 pep6 pep6 pep6 pep6 pep6 pep6 blanks h { acute over (∅)} pep { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} { acute over (∅)} serum serum serum serum serum serum serum serum serum serum serum serum 1 2 3 4 5 6 7 8 9 10 11 12 in the following tables , the diagnostic potency of the peptides for pregnancy complications and fertility diagnostics is illustrated exemplary in tables . the groups of patients , which were analysed comprise : patients presenting the first time for ivf in an ivf - facility . these patients did not show any endocrine or morphological reason for infertility . there was no male infertility involved in the fertility problem of the couple . no other autoantibody diseases such as apl - syndrome or lupus erythematodes were known or detected . patients after repetitive ivf - failure ( two cycles without pregnancy ). not autoimmune disease known and nor endocrine , morphological or male reasons known for infertility . patients with an anamnesis of habitual abortion , wherein these patients had at least two consecutive abortions in the 1 st trimester of pregnancy . patients with an anamnesis of preeclampsia in at least one preceding pregnancy . for optimal diagnostic sensitivity and specificity , the peptides can be combined to a small panel of diagnostic peptides . abbreviations are as follows : mw ( mean ); ci ( confidence interval ) c - off ( cutoff value , defined as 1 . 1 * ( mw + ci )). the peptide shown in table 1 exemplifies a peptide with a strong diagnostic profile in pregnancy complications , while infertility is detected much less sensitive . the peptide shown in table 2 has profile , which shows no strong preference of one of the patient groups , but gives a relatively constant , relatively low sensitivity profile through all groups . the peptide in table 3 shows a strong profile in the field of infertile patients , while no diagnostic potency is present in the field of pregnancy complications ( habitual abortion and preeclampsia ). fig1 shows an example of an assay with sera from female blood bank donors , which form a reference group for the general female population . it is shown that two of the peptides demonstrate increased reactivity in patients with ivf ( in vitro fertilization ) failure and patients with habitual abortion . fig2 shows that dimerisation of peptides using the protocol described in this text and using these dimeric peptides clearly increases the titers , which can be obtained in an elisa - protocol . beer a e , kwak - kim j y , beaman k d , gilman - sachs a ( 1998 ) clinical utility of antiphospholipid antibodies ? a negative study with power ! fertility and sterility 69 : 166 - 168 bermas b l , schur p h , kaplan m , rose bd ( 1996a ) prognosis and therapy of the antiphospholipid antibody syndrome . uptodate in medicine 800 : 998 - 6374 bermas b l , schur p h , rose bd ( 1996b ) clinical characteristics of the antiphospholipid antibody syndrome . uptodate in medicine 800 bermas b l , schur p h , rose bd ( 1996c ) pathogenesis of the antiphospholipid antibody syndrome . uptodate in medicine 800 bick r l , baker w f ( 1999 ) antiphospholipid syndrome and thrombosis . seminars in thrombosis and hemostasis 25 : 333 - 350 birdsall m a , lockwood g m , ledger w l , johnson p m , chamley l w ( 1996 ) antiphospholipid antibodies in women having in - vitro fertilization . human reproduction 11 : 1185 - 1189 check j h ( 1998 ) a negative study with power ? fertility and sterility 70 : 599 chilcott i t , margara r , cohen h , rai r , skull j , pickering w , regan l ( 2000 ) pregnancy outcome is not affected by antiphospholipid antibody status in women referred for in vitro fertilization . fertility and sterility 73 : 526 - 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