Patent Application: US-65817900-A

Abstract:
the present invention is related to the field of biotechnology and genetic engineering techniques , particularly to a method for obtaining mutants obtain from streptokinase , to the molecules obtained from this method , as well as the expression vectors and microorganisms for recombinant obtaining . the object of the present invention is to achieve streptokinase mutants from modifications of skc - 2 gene coding for streptokinase skc - 2 , such that the obtained mutants conserve their capacity for plasminogen activator complex formation having reduced antigenicity , that could constitute preferred alternatives to native streptokinase for thrombolytic therapy . the molecules obtained from present invention can be used in the treatment of disorders as myocardial infarct , pulmonary thromboembolism , surgical complications and other cases of thrombosis .

Description:
the present invention relates to the mapping of antigenic regions located on skc - 2 using cellulose - bound peptide scans and human total sera from patients treated with heberkinase ®. the present invention also relates to the immunological features of a synthetic 42 amino acids peptide resembling amino acids 373 - 414 from the skc - 2 c - terminal region using a panel of sera collected from patients before and after heberkinase ® therapy and tested in anti - skc - 2 ( 373 - 414 ) peptide elisa and skc - 2 ( 373 - 414 ) direct binding assay . the present invention relates to a method for the cloning and expression of skc - 2 mutants corresponding to the fragments 40 - 1245 and 1 - 1119 from the skc - 2 gene , which codes for skc - 2 , previously described in the european pat . no . ep 0 489 201 b1 , which products are proteins presenting : a deletion of the first 13 amino acid residues at the n - terminal region , called skc - 2 - n13 , which sequence corresponds to the seq . ident . no . 1 . a deletion of the first 13 amino acid residues at the n - terminal region with asp - ile - val - asp - gly - gly - 6xhis tail fused at the c - terminus of the protein , called skc - 2 - n13 - asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ) which sequence corresponds to the seq . ident . no . 2 . a deletion of the last 42 amino acid residues at the c - terminal region from position 373 to 414 , called skc - 2 - c42 , which sequence corresponds to the seq . ident . no . 3 . a deletion of the last 42 amino acid residues at the c - terminal region from position 373 to 414 with asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ) tail fused at the c - terminus of the protein , called skc - 2 - c42 - asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ), which sequence corresponds to the seq . ident . no . 4 . the present invention also relates to these mutant proteins , which molecular weight is approximately 46 , 000 dalton for skc - 2 - n13 , 47 , 000 dalton for skc - 2 - n13 - asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ), 42 , 000 dalton for skc - 2 - c42 and 43 , 000 dalton for skc - 2 - c42 - asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ), which amino acid sequences corresponds to the seq . ident . no . 1 - 4 . the fragments of nucleotide sequence from skc - 2 gene were obtained from pekg3 plasmid ( european pat . no . . ep 0 489 201 b1 ), by genetic amplification using the polymerase chain reaction ( pcr ) with 6 synthetic oligonucleotides denominated sk1 , sk2 , sk3 , sk4 , sk5 and sk6 , having sequences identified with the seq . ident . no . 5 - 10 . the present invention also relates to recombinant dna including the nucleotide fragments 40 - 1245 and 1 - 1119 from skc - 2 gene , such as vectors pemi - 1 ( fig2 ), pskh - 1 ( fig3 ), pij - 4 ( fig4 ) and pmc - 8 ( fig5 ) for the expression of these fragments in bacteria . for expression in e . coli these fragments were cloned under the tryptophan promoter and with the transcription termination signal from phage t4 . pskh - 11 and pmc8 vectors also having a coding sequence for the asp - ile - val - asp - gly - gly - 6x his ( seq id no : 15 ) amino acids fused at the 3 ′ end from the respective dna fragments and translation termination codon taa . the present invention relates to the microorganisms resulting from transformation of e . coli strain w 3110 with vectors pemi - 1 , pskh - 11 , pu - 4 and pmc8 . the transformants e . coli clones were called wsk - n13 , wsk - n13 - h , wsk - c42 and wsk - c42 - h respectively . another aspect of this method is the possibility to express the dna fragments 40 - 1245 and 1 - 1119 from skc - 2 gene in bacteria , reaching high levels of expression ; around 350 mg / l from both mutant proteins , which were called mut - n13 and mut - c42 , respectively . the method described in the present invention , given the expression levels obtained for these products , makes it possible to reach optimum purity thereof for its administration to human beings and animals , without the need to develop a complex and expensive purification process . the present invention relates to biological activity of mutant protein mut - n13 , which showed a dramatically diminution of their activity and of mut - c42 , which conserved similar activity as native protein . the present invention also relates to the mutant proteins mut - n13 and mut - c42 , which present reduced antigenicity with respect to the native skc - 2 protein . these mutant proteins were subjected to evaluation of their antigenicity in a direct binding assay and competition experiment between mutant and native proteins , using human sera collected from patients after heberkinase ® treatment . the present invention also relates to the mut - c42 activity which , when compared to the skc - 2 activity , was less affected by skc - 2 neutralizing antibodies present in sera from 15 patients treated with heberkinase ®, which was evidenced by “ in vitro ” neutralizing assay . the present invention relates to the slightly lower anti - skc - 2 antibodies generation in monkeys treated with mut - c42 in comparison with those treated with the native protein skc - 2 . the present invention relates to the neutralizing capacity developed by monkeys treated with skc - 2 , which was significantly higher against skc - 2 than against mut - c42 , indicating that the 42 c - terminal residues of skc - 2 contain one or more important epitopes for induction of neutralizing antibodies . to identify the regions of skc - 2 involved in anti - skc - 2 antibodies binding , the peptide spot synthesis approach as previously described by frank , r . ( 1992 ) tetrahedron 48 , 9217 - 9232 was used . a cellulose - bound set of 41 overlapping 20 - mer peptides ( 10 overlapping amino acids ) spanning the primary sequence of skc - 2 ( amino acids 1 - 414 ) ( european pat . no . ep 0 489 201 b1 ; estrada et al ( 1992 ) biotechnology 10 , 1138 - 1142 ) was elaborated . the cellulose sheet was probed with human sera collected from ten patients at 10 days after heberkinase ® therapy . cellulose sheet was soaked in ethanol to prevent possible hydrophobic interactions between the peptides . ethanol was exchanged against tris - buffered saline ( tbs ) ( 10 mm tris , ph 7 . 6 , 150 mm nacl ) by sequential washing , and nonspecific binding was blocked by incubating overnight in 10 ml of t - tbs blocking buffer ( 0 . 05 % tween 20 in tbs ). the sheet was subsequently incubated for 3 h at room temperature with serum samples obtained from ten patients 10 days after heberkinase ® therapy , diluted in 10 ml of t - tbs blocking buffer . serum samples were diluted according to the predetermined anti - skc - 2 ab titers . sera with 5 × 10 5 , 10 5 and 5 × 10 4 ab titers , were diluted 1 : 1000 , 1 : 500 and 1 : 300 , respectively . cellulose sheets were washed three times with t - tbs . then , an alkaline phosphatase - conjugated anti - human ab ( sigma ) was added at 1 : 2500 dilution in t - tbs blocking buffer for 2h . sheets were washed three times with t - tbs . detection of bound anti - skc - 2 abs was achieved by incubating the sheets with 0 . 3 mg / ml 5 - bromo 4 - chloro 3 - indolyl phosphate ( bcip ) ( sigma ), 4 . 5 mg / ml 3 -( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyl - tetrazolium bromide ( mtt ) ( sigma ) in substrate buffer ( 100 mm tris , ph 8 . 9 , 100 mm nacl , 2 mm mgcl 2 ). positive spots developed a violet color . washing with pbs stopped staining . cellulose sheets carrying the peptides were finally regenerated for the next test . several distinct binding areas were observed for the ten tested sera ( fig1 ). however , there are in the skc - 2 molecule binding sequences that are common for most of the patients . eight out of ten sera recognized spot 14 comprising amino acids 130 - 149 . seventy percent of patients recognized spot 18 comprising residues 170 - 189 . six out of ten samples bound at spot 1 comprising amino acids 1 - 20 of the skc - 2 n - terminal region . other six patients recognized spot 39 comprising residues 380 - 399 . fifty percent of tested sera recognized spot 40 comprising amino acids 390 - 409 within the c - terminal region . the simultaneous recognition of the spots 6 and 7 indicates the presence of a continuous epitope comprised between residues 60 - 69 ( skpfatdsga ( seq id no : 16 )). likewise , for spots 39 and 40 , the existence of a continuous epitope comprising residues 390 - 399 ( teeerevysy ( seq id no : 17 )) was delineated . the recognition of spots 27 , 28 and 29 indicated the presence of one or more continuous epitopes comprised between residues 270 - 289 ( isekyyvlkkgekpydpfdr ( seq id no : 48 )). spots showing isolated positive signals , without recognition of adjacent positions , suggested the existence of continuous epitopes including more than ten amino acids . human total sera collected from 64 patients in different hospitals in havana , cuba , before ( a ) and ten days after ( b ) heberkinase ® therapy were tested in an anti - skc - 2 elisa . samples before therapy showed anti - skc - 2 ab titers between 1 : 10 and 1 : 10 4 , while after therapy ab titer range increased to 1 : 10 3 − 1 : 5 × 10 5 . these samples were assayed in an anti - skc - 2 ( 373 - 414 ) peptide elisa in order to assess the recognition rate for the c - terminal region of skc - 2 . in order to know the immunodominance of skc - 2 c - terminus , a peptide corresponding to the sequence 373 - 414 of skc - 2 , containing 42 amino acid residues ( pegenasyhlaydkdryteeerevysylrytg tpipdnpndk ( seq id no : 18 )) was synthesized . polyvinyl plates ( high binding , costar , cambridge , mass ., u . s . a .). plates were coated with 1 g / ml skc - 2 ( 373 - 414 ) peptide , and incubated overnight at 4 ° c . after washing three times with pbs - tween , plates were blocked using 2 % bovine serum albumin ( bsa ) ( sigma ), and 100 μl of 1 : 50 dilution of each human serum were added . the binding of human abs to skc - 2 ( 373 - 414 ) peptide was measured using a horseradish peroxidase - conjugated anti - human ab ( sigma ). the reaction was developed using 100 μl per well of 1 mg / ml o - phenylenediamine ( sigma ), 0 . 03 % h 2 o 2 in substrate buffer ( 0 . 1m citric acid , 0 . 2m na 2 hpo 4 , ph 5 . 0 ). after 30 min , the reaction was stopped with 50 μl of 4m h 2 so 0 4 . results were measured on a multiskan system ( titertek , helsinki , finland ) at 492 nm . each sample was tested by duplicated . different degrees were considered for positive samples according to the sample / background ratio : samples showing absorbance values two , three and four or more times higher than the background were classified as +, ++ and +++, respectively . the results are shown in the table 1 . before therapy ( a ), 39 % of patients recognized the skc - 2 ( 373 - 414 ) peptide . as it was expected , the recognition increased to 64 % after therapy ( b ). this increase was not only due to a larger number of positive samples , but also to higher intensity of these positive signals . in order to assess the proportion of the anti - skc - 2 ( 373414 ) recognition with respect to the total anti - skc - 2 ab response , a direct binding assay was performed with 21 out of 64 patient sera obtained after heberkinase ® therapy . experimental conditions were determined by titration of samples against native skc - 2 and skc - 2 ( 373 - 414 ) peptide in order to select those dilution conditions ( dln . 1 for skc - 2 ( 373 - 414 ) and dln . 2 for skc - 2 ) in which there was not an excess of ab directed to each molecule . polyvinyl plates ( high binding , costar , cambridge , mass ., u . s . a .) plates were divided in two sections and coated with 10 μg / ml skc - 2 and 1 μg / ml skc - 2 ( 373 - 414 ) peptide , respectively . after washing three times with pbs - tween , plates were blocked with 2 % bsa . one hundred μl of human sera collected from patients ten days after heberkinase ® therapy were added at previously determined optimal dilutions . after incubation for 1 h at 37 ° c ., the binding of human anti - skc - 2 abs to molecules on solid phase was measured using a horseradish peroxidase - conjugated anti - human ab ( sigma ). the reaction was developed using 100 μl per well of 1 mg / ml o - phenylenediamine ( sigma ), 0 . 03 % h 2 o 2 in substrate buffer ( 0 . 1m citric acid , 0 . 2m na 2 hpo 4 , ph 5 . 0 ). after 30 min , the reaction was stopped with 50 μl of 4m h 2 so 4 . results were measured on a multiskan system ( titertek , helsinki , finland ) at 492 nm . each sample was tested by duplicated . percent direct binding of human anti - skc - 2 abs to skc - 2 ( 373 - 414 ) peptide was determined from the following formula : 100 × ( absorbance   binding   to   skc  -  2  ( 373  – 414 ) ) ×  ln  . 1 ( absorbance   binding   to   skc  -  2 ) ×  ln  . 2 percent ab binding to skc - 2 ( 373 - 414 ) ranged between 0 . 14 and 10 . 68 % with respect to anti - skc - 2 ab recognition ( table 2 ). the mean value from 21 samples was 2 . 96 % ( st . dev .= 3 . 30 ). antibodies directed against streptokinase are found in most individuals as a result of recurrent streptococcal infections . regarding the immunodominance of skc - 2 c - terminal region , part of this antibody response is likely to direct against amino acids 373 - 414 from the c - terminus of the molecule . in order to assess the proportion of this recognition in normal population , 1008 normal donor sera were tested using an anti - skc - 2 ( 373 - 414 ) peptide ultra - micro - elisa . plates ( greiner , frankfurt , germany ) were coated with 15μ l per well of 2μ g / ml skc - 2 ( 373 - 414 ) in coating buffer ( 50 mn na 2 co 3 , 50 mm nahco 3 , co 3 , ph 9 . 6 ), and incubated at 37 ° c . for 4 h . after washing with tris - buffered saline , 0 . 05 % tween 20 ( tbs - tween ), plates were blocked with 2 % bsa ( sigma ) at room temperature , overnight . blocking solution was removed and plates were dried at 37 ° c . for 1 h . ten μ l of 1 : 20 dilution of each human serum in tbs , 0 . 05 % tween 20 , 1 % bsa were added . plates were incubated at 37 ° c . for 30 min and washed four times . binding of human abs to skc - 2 ( 373 - 414 ) peptide was measured using 1 : 5000 dilution of an alkaline phosphatase - conjugated anti - human iggab ( sigma ). plates were incubated at 37 ° c . for 30 min and washed four times . reactions were developed by addition of 10 μ l per well of substrate solution ( 0 . 13 mg / ml 4 - methylumbelliferyl phosphate in 3m diethanolamine - hcl buffer , ph 9 . 8 ) and plates were incubated at room temperature for 30 min . fluorescence was measured using an ultra - micro - elisa plates reader pr - 521 ( suma technology , havana , cuba ). each sample was tested by duplicated . the experiment was validated by positive ,. negative and blank controls . in order to homogenize the results , the sample / positive control ratio was determined for each tested serum using the following formula : sample / positive = ( sample   fluorescence ) - ( blank   fluorescence ) ( positive   control   fluorescence ) - ( blank   fluorescence ) sample / positive ratio of the 1008 tested samples ranged between 0 . 005 and 1 . 970 . the mean value was 0 . 369 ( st . dev . 0 . 499 ). a frequency distribution was made according to 40 classes defined by sample / positive ratio ( fig2 ). in order to determine the cut off for the assay an auxiliary experiment was performed . inhibition of anti - skc - 2 ( 373 - 414 ) ab binding by previous adsorption of samples with the same peptide was studied . this experiment was performed with 140 samples randomly selected from 1008 previously tested . a 1 : 4 dilution of each sample was mixed with skc - 2 ( 373 - 414 ) peptide at a final concentration of 5 μ g / ml and incubated at room temperature with agitation , overnight . samples were centrifuged at 12000 rpm for 10 min in order to precipitate immunocomplexes . plates were coated with 2 μ g / ml skc - 2 ( 373 - 414 ), as described above . adsorbed samples were diluted 1 : 5 to reach 1 : 20 final dilution . each one was accompanied by 1 : 20 dilution of intact serum as a control . plates were incubated at 37 ° c . for 30min and washed four times . next steps were performed as described above . each sample was tested by duplicated . the proportion of each adsorbed sample with respect to its intact control ( adsorbed / intact ) was determined . positive sample was considered when adsorbed / intact ratio was no higher than 0 . 6 . fig3 shows plots of adsorbed / intact ratio versus sample / positive ratio . for small sample / positive ratio values there is a high concentration of samples over 0 . 6 . however , as this ratio increases , negative samples decrease . based on these results a sample / positive ratio value of 0 . 3 was selected as cut off because it assures to take the highest number of positive individuals with a minimum unspecificity . regarding a cut off of 0 . 3 , the analysis of the results showed that 306 out of 1008 tested samples recognize skc - 2 ( 373 - 414 ) peptide , representing 30 . 36 % from total ( fig4 ). for subcloning of skc - 2 mutants in bacteria , dna from plasmid pekg - 3 ( containing skc - 2 gene ; european pat . no . ep 0 489 201 b1 ; estrada et al ( 1992 ) biotechnology 10 , 1138 - 1142 ) was taken and the fragments were amplified by pcr using oligonucleotides sk1 , sk2 , sk3 , sk4 , sk5 and sk6 . oligonucleotides sk1and sk4 have an ecori restriction site , oligonucleotides sk2 and sk5 have a bamhi restriction site , and oligonucleotides sk3 and sk6 have an ecorv restriction site . oligonucleotides sk1and sk4 have an atg codon for the translation initiation . oligonucleotides sk2 and sk5have a taa codon for translation termination . one μ g of pekg - 3 was taken and the gene coding for skc - 2 was amplified by pcr ( dagert , m ., and erlich , s . l . ( 1974 ) gene 6 : 23 - 28 ) using the oligonucleotides sk1 , sk2 and sk3 for cloning the mutant gene cloning with a 39 bp deletion at the 5 ′ end , corresponding to the nucleotide fragment 40 - 1245 from skc - 2 gene ; and oligonucleotides sk4 , sk5 and sk6 for the cloning mutant gene with a 126 bp deletion at the 3 ′ end , corresponding to the nucleotide fragment 1 - 1119 from skc - 2 gene . for each reaction 100 pmol of each oligonucleotide , 2 units of taq polymerase ( enzibiot ) and 200 μ mol of each dntp were used . reactions were performed in 10 mm mgcl 2 , 100 mm dtt , 10 mm nacl and 100 μ l mineral oil . twenty five amplification cycles were performed , wherein each one the reaction was incubated at 95 ° c . for 1 minute for denaturisation , at 50 ° c . for 45 seconds for oligonucleotide anneling at 70 ° c . for 80 seconds for dna chains extension . an amplification efficacy higher than 5 % was obtained . for cloning in bacteria ( e . coli ), a genetic construct containing the trytophan promoter of e . coli and the termination signal of bacteriophage t4 terminator was used . fragments amplified by pcr using combinations of primer - oligonucleotides sk1 - sk2 and sk4 - sk5 were digested with ecori and bamhi , and ligated with the ecori - bamhi digsted vector . fragments amplified by pcr using combinations of primer - oligonucleotides sk1 - sk3 and sk4 - sk6 were digested with ecori and ecorv , and ligated with the ecori - ecorv digested vector containing a coding sequence for the amino acid tail asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ) that was fused to the 3 ′ end of both fragments . these constructions were transformed into a preparation of competent cells (( hanahan , d . ( 1983 ) j . mol . biol . 166 , 557 - 580 ) of e . coli strain mc1061 ( f ara d 139 ( ara - leu ) 7696 ( lac ) x74 gal u galk hsd r2 ( rk − mk + ) mcrb1 rpsl ( str r )), having a frequency higher than 10 7 transformants per dna μ g . resultant colonies were applied to lb plates ( 10 gr / l trypton , 5 gr / l yeast extract , 10 gr / l nacl and 50 82 g / ml ampicillin ), and subjected to hybridization ( maniatis , t . ; frisch , e . f . and sambrook , j . ( 1982 ) cold spring harbor laboratory , usa ), using the fragment resulting from pcr amplification as a probe , labelled with datp 32 ( amersham , r . u .) and the klenow fragment of dna - polymerase i of e . coli for 30 minutes at 37 ° c . the reaction was stopped by edta and heat . the hybridization was performed in whatman 541 filters , 8 % of the colonies were positive clones , which were examined by restriction analysis and had the same pattern of digestion with more than 10 restriction enzymes . moreover , positive clones were checked by double chain dna sequencing ( sanger , f . ; nickler , s . and coulson , a . k . ( 1977 ) proc . natl . acad . sci . usa 74 , 5463 - 5467 ), using an oligonucleotide of 17 bases ( 5 atcatcgaactagtaa 3 ′( seq id no : 19 )) which anneals at the 3 ′ promoter end , corroborating that 39 bp deletion at the 5 ′ end of the gene and joining to the promoter were correct ; and an oligonucleotide of 22 bases ( 5 ′ ggtcattcaaaaggtcatccac 3 ′( seq id no : 20 )) which anneals at the 5 ′ end of the t4 terminator , corroborating that 126 bp deletion of at the 3 ′ end of the gene and fusion to the coding sequence for asp - lie - val - asp - gly - gly - 6xhis ( seq id no : 15 ) tail was correct . the selected clones ( fig5 , 7 and 8 ) were called pemi - 1 ( mutant with 39 bp deletion the at the 5 ′ end having the skc - 2 gene fragment 40 - 1245 ), pskh - 11 ( mutant with 39 bp deletion at the 5 ′ end having the skc - 2 gene fragment 40 - 1245 fused to the coding sequence for asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ) tail at the 3 ′ end ), pij4 ( mutant with 126 bp deletion at the 3 ′ end having the skc - 2 gene fragment 1 - 1119 ) and pmc - 8 ( mutant with 126 bp deletion at the 3 ′ end having the skc - 2 gene fragment 1 - 1119 fused to the coding sequence for asp - ile - val - asp - gly - gly - 6xhis ( seq id no : 15 ) tail at the 3 ′ end ). these clones were transformed in the e . coli strain w3110 and were subjected to a fermentation process , wherein stable expression levels higher than 10 % of the total protein content of the cells were obtained , and 150 - 200 mg of skc - 2 mutants mut - n13 and mut - c42 per liter of culture medium were obtained . e . coli cells were suspended in the disruption buffer that containing 50 mm tris - hcl , 0 . 5 m nacl , 3 mm edta , ph 7 . 0 at a concentration of 40 % ( w / v ) and mechanically disrupted by using a french pressure ( ohtake , japan ). cells were passed twice through the french pressure in order to achieve an optimal cell disruption . disrupted cells were homogenized and centrifuged at 15000 rpm for 1 h at 4 ° c . by using a rpr 20 - 2 rotor ( hitachi , japan ). the supernatant , containing the recombinant streptokinase , was collected for protein purification . the supernatant was loaded into a sephadex g - 25 gel filtration column ( 2 . 6 × 27 . 5 ; i . d .× l ., in cm ) ( pharmacia , sweden ) which was previously equilibrated with 0 . 02 m tris - hcl ph 6 . 0 at a flow rate of 5 ml / min . proteins eluted from the gel filtration support were loaded into a q - sepharose fast flow anion exchange column ( 2 . 6 × 5 . 5 ; i . d .× l ., in cm ) ( pharmacia , sweden ), previously equilibrated with 0 . 02 m tris - hcl ph 6 . 0 at a flow rate of 10 m / min . the non - bound protein was washed from the column with this equilibrium buffer . elution of proteins was carried out with a linear gradient of increasing nacl concentrations , which was produced by using an fplc system ( pharmacia , sweden ). the recombinant streptokinase was eluted at 0 . 12 m nacl in the equilibrium buffer . the ph of the eluate from the ion exchange support was increased from 6 . 0 to 8 . 0 by adding a 1 m tris solution . ammonium sulfate was added to this sample up to 10 % saturation of this salt . this sample was loaded into a column ( 1 . 6 × 5 ; i . d .× l ., in cm ) containing a tsk - butyl ( tosohaas technical center , usa ) hydrophobic interaction chromatography support . this column was equilibrated with 0 . 02 m tris - hcl , ammonium sulfate at 10 % saturation , ph 8 . 0 , at a flow rate of 4 ml / min . after washing the non - bound protein with equilibrium buffer , the recombinant streptokinase was eluted by using an fplc system ( pharmacia , sweden ) which produced a linear gradient of decreasing ammonium sulfate concentration in the equilibrated buffer . the recombinant streptokinase was eluted at a concentration of ammonium sulfate of 3 % saturation . the material obtained was sterilized by filtration through a 0 . 22 μm millipore filter . the in vitro biological activity of mutant proteins mut - n13 and mut - c42 was determined by agarose - fibrin plates assay ( astrup , t . and mullertz , s . ( 1952 ) arch . biochem . biophys 40 , 346 - 351 ), chromogenic substrate ( fiberger , p . ( 1982 ) j . clin . lab . invest . 42 , suppl . 162 , 49 - 54 ) and clot lysis ( westtund , l . e . and anderson , l . o . ( 1985 ) thrombosis research 37 , 213 - 223 ). mut - c42 showed a specific activity of 50000 - 100000 iu / mg similar to that obtained for native skc - 2 , and mut - n 13 showed a dramatic diminution of its specific activity with values of 2000 - 4000 lu / mg . mut - c42 in vivo fibrinolytic activity was verified in clinical test on animals , wherein there was success in dissolving clots in the femoral arteries of rabbits and coronary arteries of dogs . blood parameters maintained similar to those obtained for native skc - 2 and those reported in the literature for this type of product . example 6 : immunological characterization of skc - 2 mutant proteins . in vitro assays a ) mut - n13 and mut - c42 direct binding assay by human anti - skc - 2 antibodies a direct binding assay was performed in order to compare mut - n13 and mut - c42 mutant proteins with native skc - 2 regarding their capacity for binding human anti - skc - 2 abs present in sera from patients after heberkinasa ® therapy . polyviline plates ( medium binding , costar , cambridge , mass ., u . s . a ) were divided in three sections and coated with 10 g / ml of full length skc - 2 , mut - n13 and mut - c42 , respectively . then , plates were washed three times with pbs - tween . one hundred μl of human sera collected from eight patients ten days after heberkinase ® therapy were added at a previously determined optimal dilution . samples were diluted according to the predetermined anti - skc - 2 ab titers . for sera with 5 × 10 5 , 10 5 and 5 × 10 4 ab titers , dilutions were of 1 : 3 . 2 × 10 4 , 1 : 1 . 6 × 10 4 and 1 : 2x10 3 , respectively . after incubation for 1 h at 37 ° c ., the binding of human anti - skc - 2 abs to molecules on solid phase was measured using a horseradish peroxidase - conjugated anti - human ab ( sigma ). the reaction was developed using 100 μl per well of 1 mg / ml o - phenylenediamine ( sigma ), 0 . 03 % h202 in substrate buffer ( 0 . 1m citric acid , 0 . 2m na 2 hpo 4 , ph 5 . 0 ). after 30 min , the reaction was stopped with 50 μl of 4m h 2 so 4 . each sample was tested by duplicated . percent direct binding of human anti - skc - 2 abs to deletion mutants ( table 3 ) was determined from the following formula : all eight tested sera showed a similar binding pattern . binding of human anti - skc - 2 abs to mut - n13 was 89 . 92 % ( p = 0 . 00012 ) and to mut - c42 was 50 . 73 % ( p = 1 . 52 × 10 − 9 ) of their binding to native skc - 2 ( fig9 ). similar results were obtained from the same eight samples using a competition assay in which native and mutant proteins mut - n13 and mut - c42 competed with a biotinylated skc - 2 for binding human anti - skc - 2 abs . plates ( costar ) were coated with 5 μg / ml of goat anti - human abs in coating buffer . after washing three times with pbs - tween , plates were blocked using 2 % bsa ( sigma ). one hundred μl of human sera collected from eight patients ten days after heberkinase ® therapy were added at a previously determined optimal dilution . samples were diluted according to the predetermined anti - skc - 2 ab titers . for sera with 5 × 10 5 , 10 5 and 5 × 10 4 ab titers , dilutions were of 1 : 10 4 , 1 : 5 × 10 3 and 1 : 10 3 , respectively . this way , human anti - skc - 2 abs were immobilized on the coated plates . after washing , 100 μl of a solution of 1 μg / ml of biotinylated skc - 2 mixed with different concentrations of non - labeled full length skc - 2 or deletion mutants ( 4 - 0 . 25 μg / ml , two - fold dilutions ) were added . the binding of biotinylated skc - 2 to human anti - skc - 2 abs , after competition with non - labeled molecules , was measured using horseradish peroxidase - conjugated streptavidin . the reaction was developped using 100 μl per well of 1 mg / ml o - phenylenediamine ( sigma ), 0 . 03 % h 2 o 2 in substrate buffer ( 0 . 1m citric acid , 0 . 2m na 2 hpo 4 , ph 5 . 0 ). after 30 min , the reaction was stopped with 50 μl of 4m h 2 so 4 . each sample was tested by duplicate . the effective dose 50 % ( ed50 ) values for mutant and native proteins were determined from plots of absorbance versus concentration of non - labeled molecules using a probit transformation in order to obtain 50 % inhibition ( table 4 ; fig1 and 11 ). statistical significance of differences was determined by student &# 39 ; s t test ( tables 5 and 6 ) for paired values , evidencing the existence of significan differences between each mutant and native protein ( p = 0 . 0036 for mutn13 and p = 0 . 0036 for mut - c42 ). mut - n13 and mut - c - 42 ed50 values were expressed in terms of percent with respect to skc - 2 . binding of mut - n13 and mut - c42 to human anti - skc - 2 abs was 80 . 57 % ( p = 0 . 0036 ) and 67 . 57 % ( p = 0 . 0001 ) of reactivity to native skc - 2 , respectively ( table 7 ; fig1 ). neutralizing activity titers ( nat ) against mut - c42 and native skc - 2 proteins were determined for 15 patients , ten days after heberkinase ® therapy . the chromogenic substrate ( s - 2251 ) reaction was performed in ployvinyl plates ( costar , cambridge , mass ., u . s . a .). serial dilutions of skc - 2 and mut - c42 ( 128 - 2 iu , two - fold dilutions in 20 mm tris - hcl ph8 / 0 . 5 m nacl ) were prepared in a volume of 25 μ . curves were mixed with 25 82 l of 1 : 10 dilutions of each patient serum , and a negative control consisting of a human serum having low anti - skc - 2 ab titer and preabsorbed with skc - 2 . fifty μl of 25 μg / ml human plg were added and allowed to mix for 10 min at room temperature . the reaction was developed by addition of 50 82 l of chromogenic substrate s - 2251 ( chromogenix , antwerp , belgium ). after incubation for 30 min , the reaction was stopped with 25 μl of 20 % acetic acid . results were measured on a multiskan system ( titertek , helsinki , finland ) at 450 nm . the experiment was validated by a standard curve of each protein . all samples were tested by duplicated . the activity required to obtain an absorbance of 0 . 7 was determined from plots of absorbance versus activity . neutralizing activity titer ( nat ) was determined as the difference between tested serum and negative control values and was expressed as microgrammes of protein neutralized per milliliter of tested serum ( fig1 ). results were statistically analyzed by the student &# 39 ; s t test for paired values ( table 8 ). nat values ranged between 61 . 63 and 428 . 27 μg of protein neutralized per ml of tested serum . for most of the individuals mut - c42 - nat decreased with respect to skc - 2 - nat , ranging from 30 to 91 % of the native protein value ( p = 0 . 0013 ). example 7 : immunological characterization of mut - c42 compared to skc - 2 . animals study fourteen monkeys ( cercopithecus aethiops ) of either sex , between two to three years old , weighing 1 . 8 - 2 . 5 kg , were selected for the study . sera from these monkeys were tested in an anti - skc - 2 elisa and animals were divided in two groups according to the results : group a : eight monkeys without previous anti - skc - 2 ab titers group b : six monkeys with previous anti - skc - 2 ab titers , probably due to previous contact with streptococcus . the comparative antigenicity of mutant protein mut - c42 versus native skc - 2 was studied after 850 μ g ( 425 μ g / kg of corporal weight ) subcutaneous administrations in groups a and b . in each group half of monkeys were treated with mut - c42 and the other half with skc - 2 . humoral response was quantified at week 8 after 4 administrations , for group a ; and at week 2 after one administration , for group b . titration was performed by an anti - skc - 2 elisa . polyvinyl plates ( costar , cambridge , mass ., u . s . a .) were coated with 10 μg / ml skc - 2 in coating buffer ( 0 . m na 2 co 3 , 0 . 1m nahco 3 , ph 9 . 6 ), and incubated overnight at 4 ° c . then , plates were washed three times with 0 . 05 % tween 20 in pbs ( pbs - tween ). one hundred μl of serial dilutions ( 1 : 2 - 1 : 4096 , two - fold dilutions in 3 % fat - free milk , pbs , 0 . 05 % tween 20 ) of each monkey serum were added . after incubation for 1 h at 37 ° c ., plates were incubated with a biotinylated protein a solution at 1 : 3000 dilution . after incubation for 1 h at 37 ° c ., the binding of monkey abs to skc - 2 was measured using a horseradish peroxidase - conjugated streptavidin ( sigma ) the reaction was developed using 100 μl per well of 1 mg / ml o - phenylenediamine ( sigma ), 0 . 03 % h 2 o in substrate buffer ( 0 . m citric acid , 0 . 2m na 2 hpo 4 , ph 5 . 0 ). after 30 min , the reaction was stopped with 50 μl of 4m h 2 so 4 . results were measured on a multiskan system ( titertek , helsinki , finland ) at 492 nm . the anti - skc - 2 ab titer was determined as the maximal dilution in which positive signal was obtained . positive signal was considered when the value was at least two - fold the background . anti - skc - 2 ab titers rose post - treatment , but animals from group b developed titers notably higher than those from group a ( table 9 ). ab titers from group a were slightly lower for monkeys treated with mut - c42 compared with those treated with skc - 2 . there are two particular monkeys ( 33 and 85 ) showing very low ab titers . ab titers generated by animals from group b showed no differences between treatments . serial dilutions of skc - 2 and mut - c42 ( 128 - 2 iu , two - fold dilutions in 20 mm tris - hcl ph8 / 0 . 5 m nacl ) were prepared in a volume of 25 μl in polyvinyl plates ( costar , cambridge , mass ., usa ). skc - 2 and mut - c42 curves were mixed with 25m 1 of each monkey diluted serum and a negative control . for monkeys without previous anti - skc - 2 ab titer a 1 : 2 dilution was used , and sera from monkeys with previous anti - skc - 2 ab titer were diluted 1 : 5 . the negative control was a monkey serum without anti - skc - 2 ab titer . fifty μl of 25 μg / ml human pasminogen were added and allowed to mix for 10 min at room temperature . the reaction was developed by addition of 50 μl of chromogenic substrate s - 2251 ( chromogenix , antwerp , belgium ). after incubation for 30 min , the reaction was stopped with 25 μl of 20 % acetic acid . results were measured on a multiskan system ( titeriek , helsinki , finland ) at 405 nm . the experiment was validated by a standard curve of each protein . all samples were tested by duplicate . the activity required to obtain an absorbance of 0 . 87 for group a and 0 . 37 for group b , was determined from plots of absorbance versus activity . the neutralizing activity titer ( nat ) was determined as the difference between the tested serum and negative control values and was expressed as microgrammes of protein neutralized per milliliter of tested serum ( table 10 ; fig1 ). abs from most of the the monkeys inhibited the formation of skc - 2 - plg and mut - c42 - plg activator complexes in vitro . skc - 2 - nat developed by monkeys from group a were considerably lower than skc2 neutralizing capacity exhibited by group b . however , mut - c42 - nat values were similar for both groups . monkeys from group a treated with skc - 2 showed nat values ranging between 35 . 43 and 54 . 17 μg ( 45 . 3 ± 8 . 33 ) of skc - 2 and between 0 and 19 . 3 μg ( 9 . 13 ± 8 . 47 ) of mut - c42 moiety neutralized per ml of tested serum . sera from monkeys treated with mut - c42 elicited nat values ranging between 6 . 79 and 44 μg ( 24 . 3 ± 20 ) of skc - 2 and between 0 and 14 . 12 μg ( 7 . 5 ± 8 . 69 ) of mut - c - 42 moiety neutralized per ml of tested serum . interestingly , animals 33 and 85 , showing low anti - skc - 2 ab titers , exhibited insignificant or none nat against both proteins . monkeys from group b showed a considerable increase in ab titers after one administration of the proteins . however , there were no differences in anti - skc - 2 ab titers between monkeys treated with native and mutant proteins . these animals showed no skc - 2 neutralizing capacity before the treatment . after only one administration of the studied proteins , abs from most of the monkeys inhibited the formation of skc - 2 - plg and mut - c42 - plg activator complexes in vitro . monkeys treated with skc - 2 showed nat values ranging between 89 . 25 and 241 . 9 μg ( 160 . 75 ± 76 . 77 ) of skc - 2 and between 0 and 21 . 7 μg ( 9 . 42 ± 11 . 13 ) of mut - c42 moiety neutralized per ml of tested serum . sera from monkeys treated with mut - c42 elicited nat values ranging between 184 . 29 and 374 . 11 μg ( 290 . 67 ± 96 . 96 ) of skc - 2 and between 15 . 55 and 33 . 9 μg ( 24 . 6 ± 9 . 17 ) of mut - c - 42 moiety neutralized per ml of tested serum . statistical analyses supported the following results : ( a ) mut - c42 was significantly less affected than skc - 2 by neutralizing abs from monkeys treated with the native protein ( p = 0 . 0042 for group a and p = 0 . 0369 for group b ), ( b ) the same result was obtained for group b animals treated with mut - c42 ( p = 0 . 0394 ), ( c ) in contrast , monkeys from group a receiving mut - c42 treatment showed no significant differences between skc - 2 - and mut - c42 - neutralizing activities ( p = 0 . 0621 ), and ( d ) within each group , no statistical significance was obtained from comparison between skc - 2 and mut - c42 treatments . statistical significance of the differences for neutralizing activities in monkeys treated with skc - 2 was determined using the student &# 39 ; 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