Patent Application: US-62839205-A

Abstract:
angiogenesis , the formation of new blood vessels , is an integral part of normal physiological and developmental processes as well as several pathologies , ranging from tumor growth and metastasis to inflammation and ocular disease . methods and compositions are provided for controlling normal angiogenesis and for treating angiogenesis associated or mediated diseases as well as for preventing cancer progression and metastasis through the use of a prostrate secretory protein family member .

Description:
polypeptides which are members of the psp94 family include ; wild type psp94 as defined in seq id no . : 1 , a recombinant psp94 as defined in seq id no . : 2 and psp94 derivatives , fragments and analogues as defined , for example in the amino acid sequence defined in seq id no . : 3 , seq id no . : 4 , seq id no . : 5 , seq id no . : 6 and seq id no . : 7 . pck3145 ( seq id no . : 5 ) was chosen as a representative of the psp94 family based on previous encouraging results of tumor growth inhibition observed in animals . test compound . the wild type amino acid sequence of pck3145 has been disclosed , for example , in international application no . : pct / ca01 / 01463 and is defined herein in seq id no . : 5 . a pck3145 derivative has been generated by attaching an acetylaminomethyl group to the sulfur atom of each of the three cysteines of pck3145 . these groups stabilize the compound by preventing formation of peptide dimers or polymer by blocking the sulfhydryl group of cysteines . this pck3145 derivative is defined in seq id no . : 7 . the drug was manufactured by multiple peptide systems ( 3550 ) ( general atomics court , san diego , calif .) using standard solid - phase peptide chemistry and lyophilized into a powder . other type of synthesis or manufacture method may however be performed to make a peptide or polypeptide of the invention . other pck3145 derivatives , analogs and fragments ( e . g ., seq ids no : 88 , 98 , etc .) may be generated similarly . the reconstituted drug used in the present example is made from a solution containing a 20 mg / ml of pck3145 derivative ( seq id no . : 5 derivative ); seq id no . : 7 , in a phosphate buffer at ph 7 . 4 for dilution in sterile saline ( 0 . 9 % nacl , bp ) prior to intravenous administration . the solutions is filled into type 1 glass vials , stoppered with teflon ®- faced butyl stoppers , and sealed with flip - off seals . the clinical trial is a multiple ascending dose , open - label , phase iia study evaluating the safety and tolerability of pck3145 derivative ; seq id no . : 7 administered intravenously in patients with metastatic hormone resistant prostatic cancer ( hrpc ). the study is not randomized . patients have been enrolled sequentially and chronologically . patients had fulfilled the following criteria prior to receiving the first administration of the test drug : signed informed consent , have a histologically confirmed metastatic adenocarcinoma of the prostate , be characterized as a stage iv prostatic cancer , have a metastatic hormone resistant prostatic cancer ; resistance being defined as progressive disease after at least one hormonal therapy ( orchiectomy , oestrogens , lhrh therapy ). progressive disease is defined in accordance with the recommendations of the prostate specific antigen working group ( bubley , j . g ., et al ., j . of clinic . oncol . 17 : 3461 - 3467 , 1999 ) which defines progressive disease as : an increasing or development of new measurable disease or presence of new bone lesions on bone scan with a psa level greater or equal to 5 ng / ml or two consecutive increases in psa . the first increase should occur a minimum of 1 week from the reference value , and psa level should be greater or equal to 5 ng / ml , be minimally symptomatic or asymptomatic defined as patients that may require chronic opioid analgesics but have been on a stable pain management regimen for at least 4 weeks , be males of at least 18 years of age , have baseline laboratory values as specified below : aspartate aminiotransferase ( asat ) ( s . i . unit value = upper normal limit 42 u / l or & lt ; 0 . 7 kat / l ) less than or equal to 2 . 0 times the upper limit of normal and alanine aminotransferase ( alat ) ( s . i . unit value = upper normal limit & lt ; 48 u / l or ≦ 0 . 8 μkat / l ) less than or equal to 2 . 0 times the upper limit of normal bilirubin less than 1 . 8 mg / dl ( s . i . unit value =≦ 25 . 4 μmol / l ) creatinine less than 1 . 8 mg / dl ( s . i . unit value =≦ 159 μmol / l ) platelets & gt ; 100 , 000 / mm 3 ( s . i . unit value =& gt ; 100 × 10 9 / l ), have a life expectancy of at least 6 months , have a karnofsky performance status of 70 % or greater , have the ability to understand the requirements of the study , provide written informed consent , abide by the study restrictions , and agree to return for the required assessments , reliable contraception must be used throughout the study . the drug was therefore administered to patients characterized as having metastatic adenocarcinoma of the prostate , stage iv prostatic cancer and as having a metastatic hormone resistant prostatic cancer . four patients per cohort and 4 ascending doses were evaluated . the ascending doses were 5 , 20 , 40 and 80 mg / m 2 . the dose escalation decision has been based on dose - limiting toxicity ( dlt ). the 33 - day cycle of treatment consisted of a pck3145 derivative ; seq id no . : 7 administration three times per week ( day 1 , 3 and 5 ) for 26 days , followed by a 7 day post - treatment observation period . the maximum tolerated dose ( mtd ) is the dose level below the one inducing grade 3 or 4 drug related toxicity ( dlt ) in two patients from a cohort of a minimum of 4 patients . only dlt &# 39 ; s observed during the first cycle have been used for the dose escalation decision . each patient &# 39 ; s participation consisted of the following study periods : a screening period held ( between days − 14 to − 1 ), a baseline visit ( at day 1 ) and before administration of the drug , a treatment period ( from day 1 to day 26 ), a 7 days post - treatment observation period ( from day 27 to day 33 ), a 6 month follow - up period where survival status , disease status and information about the occurrence of second primary tumors are assessed and a long term follow up period where survival status is assessed . the treatment period consisted of intravenous administration of the pck3145 derivative ( seq id no . : 5 derivative ) i . e ., seq id no . : 7 , three times per week ( day 1 , 3 and 5 ) for 26 consecutive days during which patients were closely monitored and undergone regular examination . after a week of treatment break and in the absence of toxicity and disease progression , patients optionally received additional treatment cycles . biological samples were drawn during different time points of the study for the purpose of safety monitoring and have been assayed for mmp - 9 levels . plasma samples were placed on dry ice and stored frozen ( approximately − 70 ° c .) and subsequently analyzed for total mmp - 9 levels . mmp - 9 assay methodology . an elisa assay measuring total mmp - 9 , i . e ., human active and pro - mmp - 9 , ( quantikine ®), cat . no . : dmp900 , r & amp ; d systems inc .) was performed on plasma - heparin samples . plasma samples have been collected from individuals at day 1 ( before treatment ) and at day 27 of each treatment cycle . the quantikine ® mmp - 9 immunoassay is a solid phase elisa designed to measure total mmp - 9 ( 92 kda pro - and 82 kda active forms ) in serum , plasma , saliva , urine and cell culture supernatants . it is calibrated with cho - cells expressed recombinant human pro - mmp - 9 and the antibodies were raised against the recombinant factor . both antibodies also recognize recombinant human active mmp - 9 . natural human mmp - 9 showed dose - response curves that were parallel to the standard curves obtained using the recombinant quantikine ® kit standards , indicating that the quantikine ® kit may be used to determine relative mass values of natural human mmp - 9 . the assay employs the quantitative sandwich enzyme immunoassay technique . a monoclonal antibody specific for mmp - 9 has been pre - coated onto a microplate . standards and samples are added into the wells , and mmp - 9 is thus bound by the immobilized antibody . after washing away unbound substances , an enzyme - linked polyclonal antibody specific for mmp - 9 is added to the wells . following a wash to remove unbound antibody - enzyme reagent , a substrate solution is added to the wells and color develops in proportion to the amount of total mmp - 9 ( pro and / or active ) bound in the initial step . the color development is stopped and the intensity of the color is measured . zymography . zymography is a technique generally used to analyze the activity of matrix metalloproteinases ( mmps ) in biological samples . it involves the electrophoretic separation of proteins under denaturing ( sodium dodecyl sulfate ( sds )) but non - reducing conditions through a polyacrylamide gel containing gelatin ( for example , 10 % gel containing 1 mg / ml gelatin for mmp - 9 and mmp - 2 assays ). the resolved proteins are re - natured by exchanging sds with a non - ionic detergent such as triton x - 100 and the gel is incubated in an incubation buffer for activation of mmp - 2 and mmp - 9 ( for example at 37 ° c . for 18 hrs ). the gel is stained with coomassie blue and the mmp - 2 and mmp - 9 bands may be visualized as clear bands against a blue background ( i . e ., the mmps degrade the gelatin and are visualized as clear bands ; pro mmp - 2 is 68 kda and pro - mmp - 9 is 92 kda ). these bands can be quantified using densitometry . for example , prior to stimulation , quiescent huvec were serum - starved for 16 h in the presence or absence of pck3145 or pd98059 and then stimulated with vegf . the conditioned media were collected 24 h after stimulation , and clarified by centrifugation . identical volume of conditioned media were mixed with non reducing laemmli sample buffer and subjected to 7 . 5 % sds - polyacrylamide gels containing 1 mg / ml gelatin ( sigma ). the gels were then incubated for 30 min at room temperature twice in 2 . 5 % ( v / v ) triton x - 100 and rinsed five times in doubly distilled water . the gels were incubated at 37 ° c . for a further 18 h in 200 mm nacl / 5 mm cacl 2 / 0 . 02 % ( v / v ) brij - 35 / 50 mm tris / hcl buffer ( ph 7 . 6 ), then stained with 0 . 1 % coomassie brilliant blue r - 250 , followed by destaining in 10 % ( v / v ) acetic acid / 30 % ( v / v ) methanol in water . gelatinolytic activity was detected as unstained bands on a blue background . materials . cell culture media were obtained from life technologies ( burlington , ontario , canada ) and serum was purchased from hyclone laboratories ( logan , utah ). electrophoresis reagents were purchased from bio - rad ( mississauga , ontario , canada ). the polyclonal ( c - 1158 ) and monoclonal ( a3 ) antibodies , used for precipitation and detection , respectively , of vegfr - 2 , and the anti - pdgfr pab ( 958 ) were obtained from santa cruz biotechnologies ( santa cruz , calif .). antiphosphotyrosine mab py99 was also purchased from santa cruz biotechnologies . anti - phospho - erk polyclonal antibodies were from cell signaling technology ( beverly , mass .). anti - mouse and anti - rabbit horseradish peroxidase - linked secondary antibodies were purchased from jackson immunoresearch laboratories ( west grove , pa .) and enhanced chemiluminescence ( ecl ) reagents were from amersham pharmacia biotech ( baie d &# 39 ; urfé , québec , canada ). human recombinant pdgf was obtained from r & amp ; d systems ( minneapolis , minn .). micro bicinchoninic acid protein assay reagents were from pierce ( rockford , ill .). matrigel basement membrane matrix was from becton dickinson labware ( bedford , mass .). ptk787 was obtained from novartis pharmaceuticals . the mek kinase inhibitor pd98059 was from calbiochem ( la jolla , calif .). all other reagents were from sigma - aldrich canada . vegf production . vascular endothelial growth factor ( isoform 165 ) was pcr - amplified from a pblast / vegf plasmid ( invivogen , san diego , calif .) and cloned into the ptt vector ( durocher , y , et al ., nucleic acids res 2002 ; 30 : e9 ). vegf was produced following large - scale transient transfection of human 293sfe cells in serum - free medium . the recombinant protein was expressed by the transiently transfected cells and secreted into the medium . the culture was harvested five days after transfection , the medium was clarified by centrifugation at 3 , 500 g for 10 minutes and filtered through a 0 . 22 μm membrane . clarified culture medium was loaded onto a heparin - sepharose column and the bound vegf was then eluted using a nacl gradient in pbs . a buffer exchange for pbs was performed by gel filtration and the final purified material was sterile - filtered , and stored in aliquots at − 80 ° c . cell culture . human umbilical vein endothelial cells ( huvec ) and pulmonary aortic smooth muscle cells ( pasmc ) were obtained from clonetics and maintained in endothelial cell basal medium - 2 ( ebm - 2 ; clonetics ) and smooth muscle medium - 2 ( smgm - 2 ; clonetics ), respectively . cells were cultured at 37 ° c . under a humidified atmosphere containing 5 % co2 . for experimental purposes , cells were plated in 8 100 - mm plastic dishes at 5 , 000 cells / cm 2 and were grown to confluence before overnight serum starvation . cells were treated with vehicle or with a pck3145 derivative diluted in 0 . 1 n naoh , and stimulated with 50 - 100 ng / ml vegf or pdgf , with 10 ng / ml of bfgf ( basic fibroblast growth factor ) or with 1 μm s1p ( sphingosine - 1 - phosphate ). immunoprecipitation and immunoblotting procedures . after treatment , cells were washed once with phosphate - buffered saline ( pbs ) containing 1 mm sodium orthovanadate and were incubated in the same medium for 1 h at 4 ° c . the cells were solubilized on ice in lysis buffer ( 150 mm nacl , 10 mm tris - hcl , ph 7 . 4 , 1 mm edta , 1 mm egta , 0 . 5 % nonidet p - 40 , 1 % triton x - 100 ) containing 1 mm sodium orthovanadate . the cells were then scraped from the culture dishes and the resulting lysates were clarified by centrifugation at 10 , 000 g for 10 min ; protein concentrations were determined using the micro bicinchoninic acid method ( pierce ). for immunoprecipitation studies , lysates were clarified by a 1 h incubation at 4 ° c . with a mixture of protein a / protein g sepharose beads . after removal of the sepharose beads by low - speed centrifugation , identical amounts of protein ( 200 μg ) from each sample were transferred to fresh tubes and incubated in lysis buffer overnight at 4 ° c . in the presence of 2 μg / ml of specific antibodies . immunocomplexes were collected by incubating the mixture with 25 μl ( 50 % suspension ) of protein a - ( rabbit primary antibody ) or protein g - ( mouse primary antibody ) sepharose beads , for 2 h . nonspecifically - bound material was removed by washing the beads three times in 1 ml of lysis buffer containing 1 mm sodium orthovanadate , and bound material was solubilized in 25 μl of two - fold concentrated laemmli sample buffer ( 125 mm tris - hcl ( ph 6 . 8 ), 20 % glycerol , 4 % sds , 10 % β - mercaptoethanol , and 0 . 00125 % bromphenol blue ), boiled 5 min , and resolved by sds - page . the proteins were transferred onto polyvinylidene difluoride ( pvdf ) membranes , blocked 1 h at room temperature with tris - buffered saline / tween 20 ( 147 mm nacl , 20 mm tris / hcl , ph 7 . 5 , and 0 . 1 % tween 20 ) containing 2 % bovine serum albumin and incubated overnight at 4 ° c . with primary antibody . immunoreactive bands were revealed after a 1 h incubation with horseradish peroxidase - conjugated anti - mouse or anti - rabbit antibodies , and the signals were visualized by enhanced chemiluminescence ( amersham biosciences , baie d &# 39 ; urfée , qc ). the immunoreactive bands were quantified by scanning densitometry ( molecular dynamics ). cell culture . human umbilical vein endothelial cells ( huvec ) and pulmonary aortic smooth muscle cells ( pasmc ) were obtained from clonetics and maintained in endothelial cell basal medium - 2 ( ebm - 2 ; clonetics ) and smooth muscle medium - 2 ( smgm - 2 ; clonetics ), respectively . cells were cultured at 37 ° c . under a humidified atmosphere containing 5 % co2 . for experimental purposes , cells were plated in 8 100 - mm plastic dishes at 5 , 000 cells / cm 2 and were grown to confluence before overnight serum starvation . cells were treated with vehicle or with pck3145 diluted in 0 . 1 n naoh , and stimulated with 50 ng / ml vegf , pdgf or with 1 μm s1p . rat aortic ring assay . the isolated rat aorta is cut into segments that are placed in culture , in a matrix - containing environment such as matrigel . over the next 7 - 14 days , the explants are monitored for the outgrowth of endothelial ( and other ) cells as this is affected by the addition of test substances . quantification is achieved by measurement of the length and abundance of vessel - like extensions from the explant . use of endothelium - selective reagents such as fluorescein - labeled bsl - i allows quantification by pixel counts . chick aortic arch assay . aortic arches are dissected from day 12 - 14 chick embryos and cut into rings similar to those of the rat aorta . when the rings are placed on matrigel , substantial outgrowth of cells occurs within 48 h , with the formation of vessel - like structures readily apparent . test substance is added to the medium and quantification of endothelial cell outgrowth is achieved by the use of fluorescein - labeled lectins such as bsl - i and bsl - b4 or by staining of the cultures with labeled antibodies to cd31 . standard imaging techniques are used for the enumeration of endothelial cells and for delineating the total outgrowth area . cornea angiogenesis assay : a pocket is made in the cornea of a rabbit &# 39 ; s eye or mice &# 39 ; s eye and angiogenesis is stimulated by an angiogenesis inducer ( e . g . vegf ) introduced into this pocket . the inducer elicits ingrowth of new vessels from the peripheral limbal vasculature . slow - release materials such as elvax ( ethylene vinyl copolymer ), hydron or sponge may be used to introduce test substances into the corneal pocket . inhibition of angiogenesis is monitored by the effect of the inhibitor on the locally induced ( e . g ., sponge implant ) angiogenic reaction in the cornea ( e . g ., vegf ). the test inhibitor may be administered by several administration mode including , orally , systemically , the latter either by bolus injection or , for example , by use of a sustained - release method such as implantation of osmotic pumps loaded with the test inhibitor . the vascular response is monitored by direct observation throughout the course of the experiment . this may be done by using a slit lamp for the rabbit but needs only a simple stereomicroscope in mice . visualization of the mouse corneal vasculature may be achieved by injecting india ink or fluorochrome - labeled high - molecular weight dextran . methods for quantification include measuring the area of vessel penetration , the progress of vessels toward the angiogenic stimulus overtime , or in the case of fluorescence , histogram analysis or pixel counts above a specific ( background ) threshold . cam assay the cam of day 7 - 9 chick embryos is exposed by making a window in the egg shell , and tissue or organ grafts are then placed directly on the cam . the window is sealed , eggs are reincubated , and the grafts are recovered after an appropriate length of incubation time . the grafts are then scored for growth and vascularization . the angiogenic reaction may be evaluated by ranking the vascularization on a 0 to 4 basis but also using imaging techniques such as the measurement of bifurcation points in a designated area around the test material . alternatively , an entire egg contents may be used . test substances are administered by placing them on membranes or on the underside of coverslips and applied to a desired area . test compounds are assessed by their effect either on the normal development of the cam vasculature itself or on induced angiogenesis . alternatively , fertilized chick embryos are removed from their shell on day 3 or 4 , and a methylcellulose disc containing the test compound is implanted on the chorioallantoic membrane . the embryos are examined 48 hours later , if a clear a vascular zone appears around the methylcellulose disc , the diameter of that zone is measured . such avascular zone indicates a compound having an anti - angiogenic activity ( u . s . pat . no . 5 , 001 , 116 ( col . 7 , incorporated herein by reference ). matrigel endothelial cell tube formation assay matrigel ( 12 . 5 mg / ml ) was thawed at 4 ° c ., and 50 μl were quickly added to each well of a 96 - well plate and allowed to solidify for 10 min at 37 ° c . the wells were then incubated for 18 h at 37 ° c . with huvec ( 25 , 000 cells / well ). the formation of capillary - like structures was examined microscopically and pictures ( 50 ×) were taken using a retiga 1300 camera and a zeiss axiovert s100 microscope . the extent to which capillary - like structures formed in the gel was quantified by analysis of digitized images to determine the thread length of the capillary - like network , using a commercially available image analysis program ( northern eclipse ). matrigel plug assay : matrigel containing test cells or substances is injected subcutaneously , where it solidifies to form a plug . this plug is recovered after 7 - 21 days in the animal and examined histologically to determine the extent to which blood vessels have entered it . fluorescence measurement of plasma volume is achieved using fluorescein isothiocyanate ( fitc )- labeled dextran 150 . quantification may alternatively be achieved by measuring the amount of hemoglobin contained in the plug . in another alternative assay ( the sponge / matrigel assay ) matrigel alone is first introduced into the mouse . a sponge or tissue fragment is then inserted into the plug . new vessels are measured by injection of fitc . other angiogenesis assays are described , for example , in staton , c . a . et al ., ( int . j . exp . path . ( 2004 ), 85 , 233 - 248 ) the entire content of which is incorporated herein by reference . migration assays . transwells filters ( 8 - μm pore size ; costar , cambridge , mass .) were pre - coated with 0 . 5 % gelatin / pbs for 24 h at 4 ° c . the transwells were then washed with pbs and assembled in 24 - well plates . the upper chamber of each transwell was filled with 100 μl of huvec ( 1 × 10 6 cells / ml ) and cells were allowed to adhere for 1 h . cells were then treated for 2 h by adding 100 μl of 2 - fold concentrated drug solution prepared in serum - free medium into the upper chamber and 600 μl of the drug solution into the lower chamber . migration was initiated by adding vegf ( 10 ng / ml ), or s1p ( 1 μm ) to the lower chamber . the plate was placed at 37 ° c . in 5 % co 2 / 95 % air for 4 h . cells that had migrated to the lower surface of the filters were fixed with 10 % formalin phosphate and stained with 0 . 1 % crystal violet / 20 % ( v / v ) methanol . the migration was quantified using computer - assisted imaging and data are expressed as the average density of migrated cells per four fields ( magnification × 50 ). matrigel endothelial cell tube formation assay . matrigel ( 12 . 5 mg / ml ) was thawed at 4 ° c ., and 50 μl were quickly added to each well of a 96 - well plate and allowed to solidify for 10 min at 37 ° c . the wells were then incubated for 30 min at 37 ° c . in 5 % co 2 / 95 % air with 100 μl of huvec ( 20 , 000 cells / well ) containing 1 % fetal bovine serum to allow adequate adhesion to matrigel . cells were then treated for 18 h by adding 100 μl of 2 - fold concentrated pck3145 prepared in serum - free medium into the well . the formation of capillary - like structures was examined microscopically and pictures ( 50 ×) were taken using a retiga 1300 camera coupled to a zeiss axiovert s100 microscope . the extent to which capillary - like structures formed in the gel was quantified by analysis of digitized images using a commercially available image analysis software ( northern eclipse ) ( 25 ). statistical data analysis . data are representative of three or more independent experiments and are represented as means ± sem . statistical comparisons between groups were assessed using 1 - way anova followed by student &# 39 ; s unpaired t - test . biologically active psp94 family member ; fragments , derivatives and analogues may be prepared by techniques known in the art ( recombinant technology , solid phase synthesis , etc .). the biological activity of derivatives , fragments and analogues may be determined by any of the techniques described herein or known in the field to be relevant for any of the biological activity described above . for example , serum - starved quiescent endothelial cells ( huvec ) may be incubated with different doses of a putative pck3145 derivative , analog or fragment ( e . g ., any of seq id nos . : 9 to 98 , combinations ) for 24 h and then stimulated with vegf . cells may be washed with pbs containing naf / na 3 vo 4 and incubated in the same medium buffer for 1 h at 4 ° c . the cells may be scraped from the culture dishes and the resulting lysates clarified by centrifugation . sds - page ( sodium dodecyl sulfate polyacrylamide gel electrophoresis ) may be performed to separate the proteins . western blotting and immunodetection may be performed by using anti - phosphoerk and anti - erk antibodies . the bands may be quantified to determine the level of inhibition of erk phosphorylation by the putative pck3145 derivative . an inhibitory effect of vegf - induced erk phosphorylation ( or vegfr - induced erk phosphorylation ) by the putative pck3145 derivative , analog or fragment means that the derivative , analog or fragment is biologically active . in another example , a matrigel containing a putative pck3145 derivative , fragment or analog with an angiogenesis - inducer is injected subcutaneously , to an animal . this plug is recovered after 7 - 21 days from the animal and examined histologically to determine the extent to which blood vessels have entered it . quantification is performed as described above . a biologically active pck3145 derivative , fragment or analog is identified by the reduction in the number of blood vessels which have entered the matrigel plug or the extent to which blood vessels have entered it . a derivative , fragment or analog causing a diminution in the formation or propagation of blood vessel ( tubes , capillary - like structures ) in an agiogenesis assay described herein is considered to be a biologically active derivative , fragment or analog . a putative pck3145 derivative , analog or fragment which is biologically active may also be identified in one of the assay described herein where an inhibitory effect on pdgf - induced erk phosphorylation ( or pdgfr - induced erk phosphorylation ) by the putative pck3145 derivative , analog or fragment is observed or measured . the biological activity of a desired polypeptide may also be determined , for example , by contacting a cell expressing a metalloproteinase ( e . g ., mmp - 9 , mmp - 2 ) and / or pro - metalloproteinase ( e . g ., pro - mmp - 9 , pro - mmp - 2 ) with a polypeptide of the present invention ( a pss94 family member ( e . g . : original polypeptide , fragment , derivative , analogue , and / or any modified form of an original polypeptide , fragment , derivative or analogue ) and , following incubation of the polypeptide and cell , evaluating the levels ( inside the cell or in the extracellular environment ( supernatant or blood ( plasma or serum ))) of expression of the metalloproteinase by western blot or the enzymatic activity of the metalloproteinase by zymography as described herein or by any other techniques known in the art to be representative of metalloproteinase activity or expression ( e . g ., northern blot , pcr , immunochemistry methods , etc .). a modification ( e . g ., reduction or in some cases an increase ) of the level of expression or enzymatic activity of a metalloproteinase ( and / or pro - metalloproteinase ) will identify a biologically active polypeptide . the biological activity of a desired polypeptide may further be determined using migration assays . u - 87 cells are treated with a polypeptide of the present invention ( e . g ., any pck3145 derivative , fragment , analog , such as for example , any one of or combinations of seq id nos . : 9 to 98 ). the treated cells are trypsinised , counted , and seeded on ha - coated filters inserted in modified boyden chambers as described herein or in the art . cell migration is allowed to proceed for 2 hours at 37 ° c . filters are then stained for cells that have migrated through the filter . a decreased basal u - 87 cell migration observed in cells treated with a polypeptide of the present invention is indicative of a biologically active polypeptide ( i . e ., a biologically active pck3145 derivative , fragment , analog ). each putative derivative , fragment or analogue may be tested using this technique or any other techniques described herein or known in the art . results of mmp - 9 levels in patient &# 39 ; s plasma , before and after one or more treatment cycle with pck3145 derivative ; seq id no . : 7 are illustrated in table 2 . normal values of healthy volunteers were not determined in this study but lizasa et al ., has determined that the normal range of plasma mmp - 9 concentrations is about 11 . 4 to 59 . 4 ng / ml . based on theses values , patients were sub - divided into two categories ; those having normal value of mmp - 9 ( below 100 μg / l ) and those having an elevated level of mmp - 9 ( higher than 100 μg / l ) at baseline ( see column identified as d1c1 in table 2 ). in the normal value mmp - 9 category ( patients identified as e , f , g , h and i ), there was no significant decrease in mmp - 9 levels after one cycle of treatment ( column identified d27c1 ) compared to baseline levels . for patients e and g , no decrease in mmp - 9 levels was observed compared to baseline values even after 2 cycles of treatment ( column identified d27c2 ). there was still no mmp - 9 decrease even after 3 cycles of treatment for patient e ( d27c3 ). in the elevated mmp - 9 category ( patients identified as a , b , c and d ), a significant decrease was observed for each patient after only one cycle of treatment ( see column identified as d27c1 ). for example a decrease of up to 89 % in mmp - 9 levels was observed for patient a compared to baseline levels . for patient b , the decrease in mmp - 9 was 41 % after cycle 1 . for patients c and d the decrease at cycle 1 was 90 % and 34 % respectively . this decrease was maintained for patients b and c who have received more treatment cycles ( see columns identified as d27c2 , d27c3 and d27c4 ). for example , at treatment cycle 2 , patient b showed a reduction of 64 % of its baseline level of mmp - 9 . a similar reduction was also measured for patient b at treatment cycle 3 ; i . e ., a 65 % reduction , and at treatment cycle 4 ; a 75 % reduction . in the case of patient c , a reduction of 76 % in mmp - 9 levels was measured at cycle 2 . in order to support in vivo results described in example 1 , zymography assays and western blots were performed on cell lines incubated with a pck3145 derivative ( seq id no . : 7 ). in the experiment presented in fig1 , 2 . 5 × 10 5 matlylu tumor cells ( american type culture collection no . : jhu - 5 )) were seeded in t - 25 flasks containing rpmi with 10 % fetal bovine serum ( fbs ). after overnight incubation , the cells were washed once with serum free medium and treated with various concentrations of the pck3145 derivative ( 500 ug / ml and 1 mg / ml ) in the presence of 50 ug / ml collagen type - i in serum free rpmi for 72 hrs . control cells received 50 ug / ml collagen or only serum free medium . the media were collected after 72 hours of exposure to the pck3145 derivative and subjected to gelatin zymography . zymography for mmp - 2 and mmp - 9 was performed in sds - polyacrylamide gel electrophoresis ( sds - page ) ( 10 %) containing 0 . 1 % gelatin ( invitrogen ). twenty - four microliters of culture media was mixed with non - reducing sample buffer and subjected to electrophoresis without boiling . after electrophoresis , gels were soaked for 30 minutes in 2 . 5 % triton x - 100 solution with 2 - 3 washing steps . the gels were then incubated for 18 hours at 37 ° c . in buffer containing 50 mm tris / hcl , ph 7 . 6 , 50 mm nacl , 10 mm cacl 2 and 0 . 05 % brij - 35 . after incubation , the gels were stained with 0 . 2 % coomassie blue and de - stained until clear proteolytic bands appeared . gels were scanned with microtek flatbed scanner ( scanmaker 5 software ; microtek lab , redondo beach , calif .). the band intensities were determined using the image quant software ( version 5 . 0 ) from molecular dynamics . the mmp - 9 and mmp - 2 gelatinase zymography standard were purchased from chemicon ( catalogue no . cc073 ). one nanogram of purified human pro - mmp - 2 and pro - mmp - 9 standards were used in every gel run . results of this experiment are illustrated in fig1 and indicate that pck3145 derivative treatment of matlylu cells resulted in a dose - dependent reduction of mmp - 9 secreted in the cell culture media , as detected by zymography . a separate western blot experiment was performed in which matlylu cells were treated with 100 ug / ml , 500 ug / ml and 1 mg / ml of the pck3145 derivative for 72 hrs . at the end of the experiment , the media were collected and concentrated 5 times using amicon centrifugal filter devices ( 3500 molecular weight cut - off ). twenty five microliters samples were separated on sds - page gel under reducing conditions using pre - cast gels of 4 - 12 % bis - tris ( invitrogen ). following electrophoresis , the proteins were transferred on nitrocellulose membrane . non - specific binding sites were blocked using 5 % skimmed milk in 10 mm phosphate buffer saline ( pbs ) containing 0 . 05 % tween - 20 for 1 hour at room temperature . the membrane was later incubated with a primary antibody ( monoclonal , rdi - mmp - 9abm - 2a5 ) at a concentration of 1 ug / ml ( in 10 mm pbs , containing 0 . 5 % bovine serum albumin ( bsa ) and 0 . 05 % tween - 20 ) for 3 hours at room temperature . the membranes were washed three times in pbs ( 5 minutes each wash ) to remove non - specific binding and they were incubated with the secondary antibody ( rabbit anti - mouse igg horseradish peroxidase - conjugated ( dako no . 0260 )) at a dilution of 1 : 5000 for one hour . detection of specific mmp - 9 protein was made by incubating the membrane in ecl ™ reagent ( electro - chemoluminescence , roche ) and exposing to the x - ray film . results of this experiment are illustrated in fig2 and again indicate that treatment of matlylu cells pck3145 derivative resulted in a dose - dependent reduction of mmp - 9 levels . matrix metalloproteinases ( mmps ) secreted by ec seem to play a key role in the processes of matrix remodeling and ec sprouting during angiogenesis . while prommp - 9 secretion is absent or at low levels in basal conditions , prommp - 2 secretion can however be increased by vegf in huvec . the effect of the pck3145 derivative ( seq id no . : 7 ) on mmp extracellular levels was thus assessed by gelatin - zymography in the conditioned media of serum - starved huvec . after 16 hours of starvation , huvec were stimulated with vegf in the presence or not of the pck3145 derivative . a further 24 hours treatment shows that pck3145 derivative effectively downregulated by approximately 35 % the basal prommp - 2 levels in the extracellular media ( fig3 a , fig3 b ). most importantly , the effect of pck3145 derivative ( 300 μg / ml ) was also observed on vegf - induced prommp - 2 secretion as the inhibition was of approximately 50 %. when these experiments were performed in serum - free media , but in the presence of the mapk inhibitor pd98059 , vegf - induced prommp - 2 extracellular levels were also significantly decreased . these results suggest that the effect of pck3145 derivative towards mmp secretion is indeed regulated through a mapk pathway in endothelial cells . the effect of pck3145 on mmp - 9 secretion could not be observed in huvec because only very low to undetectable levels of mmp - 9 are secreted . vegf is a strong activator of erks ( extracellular - signal - regulated protein kinases ) 1 and 2 via vegf receptor 2 . in order to test the ability of the pck3145 derivative in potentially antagonizing vegf - mediated erk phosphorylation , serum - starved quiescent endothelial cells ( huvec ) were incubated with vehicle ( phosphate - buffered saline ( pbs ) ph 7 . 4 ) or pck3145 derivative ( 300 μg / ml ) for 24 h and then stimulated with vegf , bfgf ( basic fibroblast growth factor ) or s1p ( sphingosine - 1 - phosphate ). cells were washed with pbs containing naf / na 3 vo 4 and incubated in the same medium buffer for 1 h at 4 ° c . the cells were scraped from the culture dishes and the resulting lysates clarified by centrifugation . western blotting and immunodetection using anti - phosphoerk and anti - erk antibodies was then performed . the results show a specific inhibitory effect of the pck3145 derivative on erk phosphorylation induced by vegf ( fig4 a , fig4 b ) but not that induced by bfgf or s1p ( fig4 a , fig4 b ). this inhibitory effect was confirmed for two endothelial cells , huvec and baec ( not shown ). the total amount of erk in each sample of cells was unaffected by the pck3145 derivative ( fig4 a , fig4 b ). although , pck3145 derivative also seemed to stimulate erk phosphorylation induced by s1p in huvec ( fig4 a , fig4 b ), that result was found not statistically significant . moreover , a dose - response to pck3145 derivative was found to gradually inhibit the extent of erk phosphorylation by vegf ( fig4 c , fig4 d ). the effect of pck3145 derivative was found comparable to that of pd98059 , a documented pharmacological inhibitor of erk phosphorylation . the lack of effect of a scrambled peptide ( seq id no . : 99 ) is demonstrated as a negative control ( fig4 e , fig4 f ). finally , a time - course of pck3145 derivative effect is shown at 3 and 24 hrs demonstrating the necessity of a long term action of pck3145 derivative ( fig4 g , fig4 h ). this three dimensional ecm model assay provides physiologically relevant environment for studies of cell morphology , biochemical function , and gene expression in endothelial cells ( ec ) that can be modulated for instance by tumor growth factors or hypoxic culture conditions . moreover , proteomic - based approaches to monitor levels of protein expression can also be achieved . when plated on matrigel , ec have the ability to form capillary - like structures . the extent of capillary - like structures formation ( density and size of structures ) can be quantified by analysis of digitized images to determine the relative size and area covered by the tube - like network , using an image analysis software ( un - scan - it , empix imaging ). huvec were trypsinised , counted and seeded on matrigel . adhesion to matrigel was left to proceed for 30 minutes . treatment with increasing concentrations of the pck3145 derivative ( 0 - 300 μg / ml ) was then performed in serum - free media for 24 hours . the extent of capillary - like structure formation was then assessed afterwards . the results show that the pck3145 derivative negatively affects tubulogenesis ( fig5 a , fig5 b ). cells were plated onto gelatin - coated filters inserted in modified boyden chemotactic chambers . the effect of pck3145 on basal migration and on vegf - induced migration was monitored by the number of cells that had migrated comparatively to untreated control cells . huvec were dislodged from the flasks by trypsinization , washed and resuspended in serum - free media . cells were placed onto gelatin - coated filters inserted in chambers and incubated at 37 ° c ., 5 % co 2 for 30 min to allow adequate anchoring to the filters . the monolayers were then exposed to serum - free media containing pck3145 ( 300 μg / ml ) added within the upper and lower compartment of the chambers . after 2 h , vegf ( 50 ng / ml ) was added in the lower chamber as a chemoattractant . cell migration was allowed to proceed for another 3 h . filters were then fixed , stained , and the migrated cells quantified by microscopy as described in the methods section . the results show that pck3145 treatment had no significant effect on basal cell migration or on vegf - induced cell migration ( not shown ) in this particular assay . the effect of . pck on s1p - induced huvec migration was also measured , but no inhibition was observed ( not shown ) in this particular assay . the multifunctionality of vegf at the cellular level results from its ability to initiate a diverse , complex and integrated network of signaling pathways via its major receptor , vegfr - 2 . thus , the inhibitory effect of the pck3145 derivative on erk phosphorylation induced by vegf was examined to verify whether it was a consequence of an inhibition of the phosphorylation of vegfr - 2 . huvec were grown , serum - starved , pretreated with the pck3145 derivative ( 300 μg / ml ; 24 h ), and stimulated with vegf as described in gingras et al . [ biochem j 348 : 273 - 280 , ( 2000 )]. after each treatment , equal amounts of protein were immunoprecipitated with anti - vegfr - 2 polyclonal antibodies and analysed by western blotting . results of this experiment show that the pck3145 derivative inhibited the phosphorylation of vegfr - 2 induced by vegf in huvec ( fig6 a , fig6 b ). this inhibitory effect of the pck3145 derivative is also shown to be dose - dependent ( fig6 c , fig6 d ), and could be to a certain extent compared to the action of ptk787 , a known pharmacological inhibitor of the tyrosine kinase activity associated to the vegfr - 2 . finally , the lack of effect of a scrambled peptide is shown ( fig6 e , fig6 f ) and suggests the specificity of action of the pck3145 derivative . the potential inhibitory action of the pck3145 derivative towards the tyrosine kinase activity associated to the vegfr - 2 was also tested on the kinase activity associated to another receptor the pdgf receptor ( pdgfr ) in pasmc ( pulmonary aortic smooth muscle cells ). similar treatment of the pck3145 derivative as for huvec was performed . interestingly , pck3145 derivative leads to the inhibition of pdgfr phosphorylation induced by pdgf ( fig7 a , fig7 b ), as well as of the pdgf - induced erk phosphorylation ( fig7 c , fig7 d ). in order to investigate the potential intracellular pathways triggered by the pck3145 derivative , a gene - reporter assay using the seap ( secreted alkaline phosphatase ) mercury profiling kit ( clontech ) was performed in glioma cells ( u - 87 ). this assay enables the monitoring of transcription factors that are triggered by a particular experimental condition by assaying the alkaline phosphatase activity in the extracellular media . the pck3145 derivative triggers significantly two pathways : the mapk / jnk pathway ( sre ) and the nfkb pathway ( fig8 a ). the mapk pathway induction is extremely strong as compared to that of the nfkb pathway . the latter however potentially suggests the involvement of pro - apoptotic pathways that would be triggered by the pck3145 derivative . interestingly , the secretion of the constitutively expressed seap was found to be inhibited suggesting a potential effect of the pck3145 derivative on a more general constitutive secretion pathway . the induction of the mapk pathway by the pck3145 derivative is further confirmed by the rapid and transient induction of erk phosphorylation between 5 - 10 minutes ( fig8 b ) and is shown to be dose - dependent ( fig8 c ). finally , the effects of the pck3145 derivative were also compared to those of a scrambled peptide . these results show that the scrambled peptide was unable to induce erk phosphorylation comparable to that of the pck3145 derivative ( fig8 d ). finally , these results also indicate that the mapk inhibitor pd98059 antagonized the induction of erk phosphorylation by the pck3145 derivative . matrigel containing the pck3145 or its derivative ( seq id no . : 5 or seq id no . : 7 ) is injected subcutaneously to a rat . this solidified plug is recovered after 7 - 21 days in the animal and examined histologically to determine the extent to which blood vessels have entered the plugs . in another assay , fertilized chick embryos are removed from their shell on day 3 or 4 , and a methylcellulose disc containing pck3145 ( seq id no . : 5 or seq id no . : 7 ) is implanted on the chorioallantoic membrane . the embryos are examined 48 hours later and the diameter of the avascular zone is measured . the proangiogenic factor vegf is secreted by many tumors in high concentrations , and suppression of the vegf - vegfr signaling pathway is an intensively explored avenue for suppression of tumor growth through the inhibition of angiogenesis . although prostate cells of normal , benign , and of malignant phenotype have been shown to express vegf , expression of the cognate receptors vegfr - 2 is generally believed to be restricted to ec . in light of the results presented herein , two main lines of evidence suggest and support the pleiotropic molecular effects of pck3145 in ec ( pck3145 is therefore a pleiotropic factor ). pck3145 antagonizes the vegfr - 2 tyrosine kinase - associated activity as well as the subsequent intracellular transduction through the mapk pathway . moreover , pck3145 inhibited capillary - like structure formation by ec as well as mmp secretion , two cellular pre - requisite for angiogenesis to occur . the inhibitory effect on mmp is interesting since in genera , al correlation between the stage of tumor progression and level of mmp expression has been observed . collectively , these properties reflect pck3145 antiangiogenic action on ec . treatment of established human tumors might require not only prevention of further angiogenesis but also destruction of tumor blood vessels to reduce the already existing tumor mass . although interference with vegf - mediated signalling events is effective in preventing the early growth of neovessels ( blocking early - stage angiogenesis ), mature vessels from more established tumors are largely resistant to inhibitors directed against either vegf or its receptor vegfr - 2 . these mature vessels are surrounded by periendothelial cells , such as pericytes and smooth muscle cells ( smc ), and the contact between these cells stabilizes new blood vessels , promotes endothelial survival , and inhibits ec proliferation . pdgf - b / pdgfr - β system is involved in vessel stabilization , and interference with this signalling system resulting in disruption of already established endothelial / periendothelial associations and vessel destabilization . furthermore , the inhibition of both vegf and pdgf receptors , by either simultaneous exposure to receptor - specific receptor tyrosine kinase inhibitors or by an inhibitor with broad kinase specificity ( su6668 ), blocks further growth of end - stage and well - vascularized tumors , eliciting detachment of pericytes and disruption of tumor vascularity ( blocking late - stage angiogenesis ) ( e . g ., blocking late - stage angiogenesis ). as such pck3145 may inhibit angiogenesis in highly vascularized tumors . as pck3145 as been found to interfere with both vegfr and pdgfr signalling , pck3145 may be used as a therapeutic agent in strategies devised either to interrupt or inhibit one or more of the pathogenic steps involved in the process of tumor neovascularization or to directly target and destroy the tumor vasculature and therefore blocking both the early - and late - stage angiogenesis . the inhibition of both receptors function by pck3145 may confer an intrinsic advantage to the use of this peptide to inhibit angiogenesis . migration / invasion of cancer cells is a key event in tumor metastasis . in vitro , this process can be reconstituted by plating cells onto ecm - coated filters inserted in modified boyden chemotactic chambers . the effect of the pck3145 derivative can be monitored by the number of cells that had migrated comparatively to untreated control cells . in light of previous observations , the diminished migration onto hyaluronic acid ( ha ) matrice was confirmed . u - 87 cells were treated with the pck3145 derivative ( 300 ug / ml , 48 hrs ), trypsinised , counted , and seeded on ha - coated filters inserted in modified boyden chambers . cell migration was allowed to proceed for 2 hours at 37 ° c . filters were then stained for cells that have migrated through the filter . the results show that pretreatment with the pck3145 derivative decreased basal u - 87 cell migration by approximately 3 - fold ( fig9 a ). this result was performed for 3 more times with new cell preparations . ecm recognition is a crucial event in the cell adhesion processes involved in tumor progression . this process is mediated and regulated through specialized cell surface receptors or integrins . while recent evidence suggests that a potential crosstalk between soluble mmp and cell surface integrins may regulate the cell &# 39 ; s ability to recognize and adhere to its ecm environment , the pck3145 derivative was tested in its ability to downregulate u - 87 cell adhesion onto ha . u - 87 cells were treated with the pck3145 derivative ( 300 ug / ml , 48 hrs ), trypsinised , counted , and seeded on wells coated with 10 ug / ml bsa ( bovine serum albumin ) or ha . cells were allowed to adhere for 3 hours . three independent experiments were performed . results of these experiments show that adhesion of cells treated with the pck3145 derivative was significantly diminished on ha by 45 - 76 % ( fig9 b ). collectively , the inhibitory action of the pck3145 derivative on ecm recognition and cell adhesion processes suggests that the expression of specific integrins or ha cell surface receptors such as those from the cd44 family could be targeted . alternatively , such result also suggests that intracellular signalling regulating the activation states of cell surface integrins may be triggered by the pck3145 derivative . one such potential intracellular protein is the gtpase rhoa , which is likely to mediate mechanisms regulating cytoskeletal morphogenesis . decreased cell migration and adhesion on ha was observed when u - 87 cells were pretreated with the pck3145 derivative . this can be interpreted as either a potential downregulation of cd44 expression at the cell surface or by a potential cell surface shedding . the latter hypothesis was tested by incubating serum - starve u - 87 cells for 24 hours with the pck3145 derivative ( 300 ug / ml ), a concentration known to antagonize mmp secretion . the conditioned media was then tca - precipitated and westernblotting an immunodetection for a 75 kda immunoreactive protein using the anti - cd44 antibody was performed . an increased cd44 cell surface shedding was demonstrated by the strong immunoreactive band observed in the cells which had been pre - treated with pck3145 derivative ( fig1 ). this effect is also shown in parallel with mt1 - mmp - transfected cells . such effect has been already reported by many groups and is established as one of the mt1 - mmp - mediated functions in the regulation of the ecm adhesion . interestingly , a slight increase in mt1 - mmp expression in the cells treated with the pck3145 derivative was observed that may partially explain how pck may lead to cd44 shedding . this induction has subsequently been reproduced below . altogether , these observations provide a rational for the diminished cell migration / adhesion to ha . moreover , it is tempting to further suggest that this may also be a secondary regulation by the pck3145 derivative of diminished cell surface docking of mmp - 9 to cd44 . specific manipulation of the gtpase rho activity can be used to suppress or enhance the organizational behaviour of endothelial cells as well as it can restrict cancer cells proliferation . in particular , rhoa mediates cell contractility by organizing actin filaments which consequently regulates cell migration . moreover , recent evidence suggested that rhoa / cd44 / mmp - 9 colocalized at common cell surface microdomains . tests were carried out in order to determine whether the pck3145 derivative affected rhoa gene and protein expression . u - 87 cells were either treated with the pck3145 derivative ( 300 ug / ml , 48 hrs ). results of this experiment confirm that the pck3145 derivative induced endogenous rhoa protein expression in u - 87 cells as assessed by western blotting ( fig1 a , fig1 c ). finally , results show that rhoa protein expression induced by the pck3145 derivative paralleled that of its gene expression as assessed by reverse transcription - polymerase chain reaction ( rt - pcr ) ( fig1 d ). altogether , these results highlight the potential role of rhoa as being an intracellular mediator in the subsequent inhibitory activities of the pck3145 derivative . the overall effects of psp94 family members described herein make them useful for treatment of several diseases in addition to the previously disclosed utility ( inhibition of tumor cell growth and skeletal metastasis ). for example , the effect of psp94 family members on mmp - 9 and mmp - 2 makes them useful for reduction of cancer spreading and invasion of any type of cancer and not only for reduction of skeletal metastasis as disclosed and claimed in international application no . : pct / ca02 / 01737 . as such , psp94 family member are able to prevent cancer ( tumor ) progression and metastasis as well as inhibiting angiogenesis . the content of each publication , patent and patent application mentioned in the present application is incorporated herein by reference . although the present invention has been described in details herein and illustrated in the accompanying drawings , it is to be understood that the invention is not limited to the embodiments described herein and that various changes and modifications may be effected without departing 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