Patent Application: US-8138598-A

Abstract:
the present invention relates to methods of regulating tnf receptor releasing enzyme activity . composition altering trre activity , including a family of proteins and the genes encoding these proteins having trre activity , are provided . these proteins , rna products , or dna sequences can be administered to individuals suffering from a disease characterized by abnormal trre activity . in the case of diseases associated with elevated levels of tnf , such as rheumatoid arthritis , an inhibitor of trre is administered to the disease site to decrease the local levels of tnf . methods of isolating other compositons which increase or decrease trre activity are also provided .

Description:
the invention encompasses factors which modulate tumor necrosis factor receptor ( tnfr ) releasing enzymatic ( trre ) activity . the invention encompasses factors which increase or decrease trre activity . effective amounts of the compositions of the present invention are those that alter trre by at least about 10 %, more preferably by at least about 25 %, more preferably by at about 50 %, and even more preferably by at least about 75 %. the invention encompasses nucleic acid sequences that act as templates for rnas or encode proteins that that substantially alter trre in a cell , and methods of use thereof , and methods of screening thereof . tnf is a major proinflammatory and immunomodulatory cytokine produced during immune responses . tnf also regulates the expression of il - 2r leading to enhanced t cell responses mediated by il - 2 and appears to be required for generating proliferative responses in mixed lymphocyte cultures . additional studies have shown that cd8 + , ctl and lymphokine activated killer cells are optimally induced with tnf , in combination with il - 2 , suggesting the importance of this cytokine in regulating cytotoxic effector function . as discussed in detail above , tnf mediates its activity by binding to a tnf - r . soluble tnf - rs inhibit tnf activity by two methods : they decrease the available binding sites on a cell and bind to soluble tnf to decrease the local concentration . the present invention encompasses compositions and methods for modulating the level of soluble tnf - r by modulating the cleavage of tnf - r from the cell surface and thus indirectly modulating the effect of tnf . nucleic acid sequences of clones capable of enhancing trre activity are presented in seq id nos : 1 to 10 . the corresponding polypeptide sequences thereof are presented in seq id nos : 147 to 154 . these sequences were generated from clones designated 2 - 8 , 2 - 9 , 2 - 14 , 2 - 15 , p2 - 2 , p2 - 10 , p2 - 13 , p2 - 14 , and p2 - 15 , each of which enhances 130 %, as shown in fig6 . the clones were prepared from a library ( stratagene , la jolla , calif .) of jurkat cells , which have a high trre activity , transformed into cos - 1 cells , which normally lack trre activity , as described in example 5 . jurkat library clones which produced high trre activity in cos - 1 cells were isolated and sequenced . this method can also be used to obtain additional genes which enhance trre activity . in addition , in a method of obtain clones which reduce trre activity , a library of cells with reduced trre activity can be introduced into a cell with relatively higher trre activity . those clones which reduce trre activity can be thus identified . the sequences of seq id nos : 1 to 10 were analyzed by a blast ( basic local alignment search tool ) sequence analysis to determine if they were similar or identical to known genes . all these sequences were found to be novel , except that of clone 2 - 8 ( sequence designation aim3t3 , seq id no : 2 ), which is highly similarly to the m . musculus 45s pre - rrna gene , clone 2 - 14 ( sequence designation aim4 , seq id no : 4 ), which is highly similar to human arfaptin 2 , and clone p2 - 10 ( sequence designation aim7 , seq id no : 7 ), which is highly similar to the human insulin - like growth factor ii receptor . in addition , the sequence of clone 2 - 15 ( sequence designation aim5 , seq id no : 5 ) is novel but has some similar to human eif - 5a transcription factor . none of these known genes has previously been linked to modulating trre activity . in addition to using the jurkat library ( or similar library from a cell expressing high trre activity ), an in vitro trre activity can be used to identify genes which enhance trre activity . briefly , in this assay ( described in detail in example 1 ), a gene encoding a membrane - bound tnf receptor ( tnf - r ) is transformed into a cell which normally lacks this gene . these cells and controls are incubated with medium to be tested for trre activity . the supernatant is then collected and tested for solubilized tnf - r by elisa . mutants , variants , and derivatives of the polypeptides disclosed herein can be assayed for trre activity with this assay . in another embodiment , nucleic acids thought to encode proteins or rnas that affect trre activity can be transformed into cells in this assay and tested for their effect on trre activity . this invention therefore encompasses polypeptides and genes identified by methods of obtaining polypeptides and genes that enhance trre activity . this in vitro trre activity assay can also be used to identify factors which inhibit trre activity . antibodies to proteins which enhance trre activity can be introduced into the cellular medium along with such proteins to determine if the antibodies block trre activity . anti - sense rnas to nucleic acids encoding trre activity can also be tested in this assay . the terms “ polypeptide ”, “ peptide ” and “ protein ” are used interchangeably herein to refer to polymers of amino acid residues of any length . the polymer can be linear or branched , it can comprise modified amino acids or amino acid analogs , and it can be interrupted or modified by chemical moieties other than amino acids . the terms also encompass an amino acid polymer that has been modified naturally or by chemical intervention ; for example , disulfide bond formation , glycosylation , lipidation , acetylation , phosphorylation , or any other manipulation or modification , such as conjugation with a labeling or bioactive component . unless stated or implied otherwise , the term trre includes any polypeptide monomer or polymer with trre enzymatic specificity , including the intact trre , and smaller and larger functionally equivalent polypeptides , as described herein . the present invention encompasses polypeptides encoded by at least 5 , preferably at least 10 , more preferably at least 15 , contiguous amino acids encoded by any of seq id nos : 1 to 10 . the invention further encompasses polypeptides represented in seq id nos : 147 to 154 , or functional fragments , variants and derivatives thereof capable of modulating trre activity in a cell . a “ fusion polypeptide ” is a polypeptide comprising regions in a different position in the sequence than occurs in nature . the regions can normally exist in separate proteins and are brought together in the fusion polypeptide ; they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide ; or they can be synthetically arranged . for instance , as described below , the invention encompasses recombinant proteins that are comprised of a functional portion of trre and an antibody . methods of making these fusion proteins are known in the art and are described , for instance , in wo93 / 07286 . a “ functionally equivalent fragment ” of a trre polypeptide varies from the native sequence by addition ( s ), deletion ( s ), or substitution ( s ), or any combination thereof , while preserving at least one functional property of the fragment relevant to the context in which it is being used . a functionally equivalent fragment of a trre polypeptide typically has the ability to bind membrane bound tnf - r and enzymatically cleave tnf - r to provide soluble tnf - r . amino acid substitutions , if present , are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide . groups of functionally related amino acids within which conservative substitutions can be made are glycine / alanine ; valine / isoleucine / leucine ; asparagine / glutamine ; aspartic acid / glutamic acid ; serine / threonine / methionine ; lysine / arginine ; and phenylalanine / tyrosine / tryptophan . polypeptides of this invention can be in glycosylated or unglycosylated form , can be modified post - translationally ( e . g ., removal of signal peptide , transmembrane or cytoplasmic regions , acetylation , and phosphorylation ) or can be modified synthetically ( e . g ., by a labeling group ). effective amounts of a polypeptides of the present invention can be administered to a subject or cell in order to modulate trre activity in the subject or cell . in addition , variants , mutants and derivatives of the polypeptides described herein can be tested for trre activity in an in vitro assay . those which display high activity can then be administered to subjects . alternatively , polynucleotides encoding these polypeptides , variants , mutants or derivatives can be introduced . in the case of trre genes in which the gene product is an rna ( e . g ., an rrna ), it is preferable to administer a nucleic acid which is a dna . administration can be performed locally , parenterally , subcutaneously , intramuscularly , intraperitoneally , intracavity , intrathecally , and intravenously , or via any method known in the art . the preparation of pharmaceutical compositions that contain a polynucleotide or polypeptide as an active ingredient is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations . see , for example , remington &# 39 ; s pharmaceutical sciences 18 th edition ( 1990 ), e . w . martin ed ., mack publishing co ., pa . depending on the intended use and mode of administration , it may be desirable to process the active ingredient further in the preparation of pharmaceutical compositions . appropriate processing may include sterilizing , mixing with appropriate non - toxic and non - interfering components , dividing into dose units , and enclosing in a delivery device . various methods of delivering proteins and nucleic acids into cells and individuals are known in the art . in addition , the polypeptides and polynucleotides disclosed herein can be used to inhibit or decrease trre activity levels in a cell or subject , particularly a subject suffering from an indication characterized by excessive tnf activity . such inhibitors include metalloprotease inhibitors , an antibody which blocks the effective interaction between tnf receptor and trre or a polynucleotide encoding such an antibody , an antisense oligonucleotide specific for a trre , and a ribozyme specific for a gene encoding trre . antisense nucleic acids ( e . g ., antisense rnas ) include those complementary to the sequences of seq id nos : 1 to 10 . these can bind to the nucleic acids in a cell and prevent their expression . alternatively , antisense nucleic acids can be constructed to bind to mrnas encoded by these sequences to prevent their translation . furthermore , the polypeptides described in seq id nos : 147 to 154 can be used to generate antibodies . administration of an effective amount of these antibodies to a cell or subject can reduce trre activity in that cell or subject . in addition to an inhibitor of trre activity , a subject can be treated with a cytokine such as il - 2 , - 4 , gm - csf , or gsf and / or a chemotherapeutic agent such as radioisotopes , vinca alkaloids , adriamycin , bleomycin sulfate , carboplatin , cisplatin , cyclophosphamide , cytarabine , dacarbazine , dactinomycin , duanorubicin hydrochloride , doxorubicin hydrochloride , etoposide , fluorouracil , lomustine , mechlororethamine hydrochloride , melphalan , mercaptopurine , methotrexate , mitomycin , mitotane , pentostatin , pipobroman , procarbaze hydrochloride , streptozotocin , taxol , thioguanine , and uracil mustard . methods of administering these various agents are known in the art . an “ effective amount ” in treatment is an amount sufficient to effect beneficial or desired clinical results . an effective amount can be administered in one or more administrations . for purposes of this invention , an effective amount of an adenoviral vector is an amount that is sufficient to palliate , ameliorate , stabilize , reverse , slow or delay the progression of the disease state . subjects including those who are suspected of being at risk of a pathological effect of any neoplasia , particularly carcinoma , are suitable for treatment with the pharmaceutical compositions of this invention . those with a history of cancer are especially suitable . suitable subjects for therapy comprise two groups , which may be distinguished by clinical criteria . patients with “ advanced disease ” or “ high tumor burden ” are those who bear a clinically measurable tumor . a clinically measurable tumor is one that can be detected on the basis of tumor mass ( e . g ., by palpation , cat scan , or x - ray ; positive biochemical or histopathological markers on their own are insufficient to identify this population ). a pharmaceutical composition embodied in this invention is administered to these patients to elicit an anti - tumor response , with the objective of palliating their condition . ideally , reduction in tumor mass occurs as a result , but any clinical improvement constitutes a benefit . clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor . a second group of suitable subjects is known in the art as the “ adjuvant group ”. these are individuals who have had a history of cancer , but have been responsive to another mode of therapy . the prior therapy may have included ( but is not restricted to ) surgical resection , radiotherapy , and traditional chemotherapy . as a result , these individuals have no clinically measurable tumor . however , they are suspected of being at risk for progression of the disease , either near the original tumor site , or by metastases . this adjuvant group can be further subdivided into high - risk and low - risk individuals . the subdivision is made on the basis of features observed before or after the initial treatment . these features are known in the clinical arts , and are suitably defined for each different cancer . features typical of high risk subgroups are those in which the tumor has invaded neighboring tissues , or involvement of lymph nodes . another suitable group of subjects is those with a genetic predisposition to cancer but who have not yet evidenced clinical signs of cancer . for instance , women testing positive for a genetic mutation associated with breast cancer , but still of childbearing age , may wish to receive trre inhibitor treatment prophylactically to prevent the occurrence of cancer until it is suitable to perform preventive surgery . of course , crossovers between these two patient groups occur , and the pharmaceutical compositions of this invention can be administered at any time that is appropriate . for example , therapy can be conducted before or during traditional therapy of a patient with high tumor burden , and continued after the tumor becomes clinically undetectable . therapy can be continued in a patient who initially fell in the adjuvant group , but is showing signs of recurrence . the attending physician can determine how or when the compositions of this invention are to be used . as provided herein , treatment , diagnosis and monitoring of cancers includes any cancers known in the art . these include , but are not limited to , glioblastoma , melanoma , neuroblastoma , adenocarcinoma , soft tissue sarcoma , leukemias , lymphomas and carcinoma . the invention is particularly useful for treatment , diagnosis and monitoring of carcinomas . carcinomas include , but are not limited to , astrocytoma , oligodendroglioma , ependymoma , medulloblastoma , primitive neural ectodermal tumor , pancreatic ductal adenocarcinoma , small and large cell lung adenocarcinomas , squamous cell carcinoma , bronchoalveolarcarcinoma , epithelial adenocarcinoma and liver metastases thereof , hepatoma , cholangiocarcinoma , ductal and lobular adenocarcinoma , squamous and adenocarcinomas of the uterine cervix , uterine and ovarian epithelial carcinomas , prostatic adenocarcinomas , transitional squamous cell bladder carcinoma , b and t cell lymphomas ( nodular and diffuse ), plasmacytoma , acute and chronic leukemias , malignant melanoma , soft tissue sarcomas and leiomyosarcomas . embodied in this invention are compositions comprising polynucleotides with a therapeutically relevant genetic sequence as an active ingredient . the polynucleotides can comprise a portion of a sequence shown in any seq id nos : 1 to 10 and / or a portion of any sequence encoding at least 5 contiguous amino acids , preferably at least 10 , more preferably at least 15 , even more preferably 20 , of any of the amino acid sequences of seq id nos : 147 to 154 . this portion can comprise at least 10 , preferably at least 15 , more preferably at least 20 , and even more preferably at least 30 contiguous nucleotides of any of the sequences of seq id nos : 1 to 10 , or the complementary strand thereof , or any nucleotide which can encode at least 10 contiguous amino acids of any of seq id nos : 147 to 154 . the polynucleotide can be administered , for example , to augment or attenuate the natural level of expression of trre within a target cell . a polynucleotide for enhancing or attenuating trre expression can be introduced into cells as part of any suitable delivery vehicle known in the art . the polynucleotide can be administered to cells or injected into a tissue site as naked dna , preferably in a supercoiled configuration . it is generally preferred to administer the polynucleotide as part of a composition that enhances expression in the target cell . components of the composition can include those that protect the polynucleotide until delivery to the cell , enhance binding to or localization near target cells , enhance uptake or endocytosis into cells , promote translocation of the polynucleotide across the membrane into the cytoplasm , or enhance transport of the polynucleotide inside the cell to the site of action . in one example , the composition comprises one half of a ligand - receptor binding pair , the other of which is present on the surface of the target cell . this can promote localization near the cell surface , endocytosis into the cell , or homing to the cell in vivo , or any combination thereof . suitable components for including in the composition include , but are not limited to , antibodies or antibody fragments specific for the target tissue ( for example , a tumor - associated antigen ), integrins and integrin ligands optionally specific for the target tissue , and ligands for cytokine receptors on the target tissue . where the object is to decrease tnf - r levels on the target cell by enhancing trre expression , a particularly preferred ligand is tnf itself . in this way , the composition will be focused towards cells with the phenotype to be treated , in preference to other cell types and cells already treated effectively . in another example , the composition comprises a delivery vehicle that protects the polynucleotide and enhances its delivery into the cell . one type of suitable vehicle is a liposome that either encapsulates the polynucleotide , or ( in the case of cationic liposomes ) binds it by charge association . another type of suitable vehicle is the capsid or envelope of a virus , defective viral particle , or synthetic viral particle , encapsidating or enveloping the polynucleotide . preferred amongst such virally related particles are those that are tropic for the target tissue type , and comprise polypeptides ( such as the influenza hemagglutinin ) that promote fusion and delivery of the polynucleotide . the composition can also optionally comprise genetic elements of a virus that promotes replication of the therapeutic polynucleotide and / or integration into the genome of the target cell . suitable viral systems for use with this invention include adenovirus vectors , retroviral vectors , adeno - associated viral vectors , sindbis virus vectors , and the like . preferred are vectors that comprise viral genetic elements required in cis for packaging , the genetic elements required for replication or integration of the therapeutic polynucleotide , but not other viral genetic elements . such vectors can be produced by packaging systems in which viral elements required only in trans are supplied by a host cell or second virus . see , e . g ., flotte et al . wo 95 / 13365 . it is often preferable to combine several such components and strategies into the composition with the therapeutic polynucleotide . for example , a polynucleotide can be enveloped in an adenovirus vector that expresses a targeting molecule like tnf as part of the viral package . the vector might alternatively express a coupling molecule , such as an avidin binding site , that can then be coupled with biotin - tnf for purposes of targeting to the target cell . the following examples are meant to illustrate , but not limit , the claimed invention . the objective of this study was to establish an assay system that measures trre activity on the human tnf - r in its native conformation integrated into the cell surface membrane . the transfected cos - 1 cell line was chosen for the assay system since no background of endogenous p75 tnf - r was observed . attempts to study and characterize the enzyme responsible for stnf - r release have been difficult because the presence of an active form of the proteolytic enzyme is indicated only indirectly by the generation of soluble receptors . studies of release of other membrane bound proteins as well as tnf - r have been carried out by measuring the levels of soluble counterparts by elisa or by facs analysis for the presence or absence of the surface antigens . therefore , the level of the enzyme itself has not yet been quantitated . we therefore devised a novel assay system to detect and quantitate trre . it was found that the level of soluble forms released into the medium depends on the level of expression of surface antigens on the membrane and the rate at which the cells can synthesize more and express these proteins on the membrane . in some studies , the enzyme levels and the kinetics of active enzyme formed have been correlated with the levels of soluble forms released and the kinetics of their release . we have now devised a more defined assay system to detect and also quantitate trre specifically and enzymes that cleave membrane receptor proteins in general . membrane - associated tnf - r was chosen as the substrate for trre instead of the recombinant tnf - r molecule , because the membrane - associated tnf - r simulates a more physiological microenvironment and substrate for the evaluation of trre activity . membrane - associated tnf - r can also assist in alleviating nonspecific cleavage by other proteases which can occur in nonmembrane - associated forms . since most human cells express only extremely low levels of both tnf - rs , human p75 tnf - r - overexpressing cells were constructed by cdna transfection into monkey cos - 1 cells which do not express either tnf - rs . the cdna of the human p75 tnf - r was cloned from a λgt10 cdna library derived from human monocytic u - 937 cells ( clontech laboratories , palo alto , calif .). the cdna was then subcloned into the ecori site of the mammalian expression vector pcdna3 ( invitrogen , san diego , calif .) which contains the neomycin - resistance gene for the selection of transfected cells in the presence of g418 . this construct was transfected into cos - 1 cells using the calcium phosphate - dna precipitation method described by chen and okayama . 24 hours post transfection , the transfected cells were placed in 600 μg / ml g418 ( gibco brl life technologies , gaithersburg , md .) for the selection of neomycin - resistant clones . the resistant cells were pooled and named c75r . these cells expressed approximately 70 , 000 receptors / cell by scatchard analysis . the first 300 bp on both 5 ′ and 3 ′ ends of the cloned fragment was sequenced and compared to the reported cdna sequence of human p75 tnf - r . the cloned sequence was a 2 . 3 kb fragment covering positions 58 - 2380 of the reported p75 tnf - r sequence , which encompasses the full length of the p75 tnf - r - coding sequence from positions 90 - 1475 . the 2 . 3 kb p75 tnf - r cdna was then subcloned into the multiple cloning site of the pcdna3 eukaryotic expression vector . the orientation of the p75 tnf - r cdna was verified by restriction endonuclease mapping . the final 7 . 7 kb construct , pcdtr2 , carried the neomycin - resistance gene for the selection of transfected cells in g418 , and the expression of the p75 tnf - r was driven by the cytomegalovirus promoter ( fig1 ). the pcdtr2 was then transfected into monkey kidney cos - 1 cells using the calcium phosphate - dna precipitation method . the selected clone in g418 medium , termed c75r , was identified and subcultured . 125 i was purchased from icn pharmaceuticals , inc . ( costa mesa , calif .) and the human recombinant tnf was radiolabeled using the chloramine - t method . to determine the level of p75 tnf - r expression on c75r cells , 2 × 10 5 cells / well were plated into a 24 - well culture plate and incubated for 12 to 16 hours in 5 % co 2 at 37 ° c . they were then incubated with 2 - 30 ng 125 i radiolabeled human recombinant tnf in the presence or absence of 100 - fold excess of unlabeled human tnf at 4 ° c . for 2 hours . after three washes with ice - cold pbs , cells were lysed with 0 . 1n naoh and radioactivity was determined in a pharmacia clinigamma counter ( uppsala , sweden ). to determine the effect of trre on the surface levels of p75 tnf - r , cells were incubated with or without the trre - containing supernatant for 30 min at 37 ° c ., and then the medium was aspirated before incubation with radiolabeled tnf . soluble p75 tnf - r was generated from c75r cells by incubation with trre - containing supernatant . after a 30 min incubation , the supernatant was collected and centrifugally concentrated with centriprep - 10 filter ( 10 , 000 mw cut - off membrane ) ( amicon , beverly , mass .) and applied to 10 % acrylamide sds - page . the proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane ( immobilon ) ( millipore , bedford , mass .). immunostaining was performed using the biotin - streptavidin system ( amersham , amersham , uk ) and the peroxidase substrate kit dab ( vector laboratories , burlingame , calif .). the results obtained are shown in fig2 c75r had a very high level of specific binding of radiolabeled 125 i - tnf , while parental cos - 1 cells did not . the number of tnf - r expressed on c75r was determined to be 60 , 000 - 70 , 000 receptors / cell by scatchard analysis ( fig2 inset ). the level of tnf - r expression in this clone was 40 to 50 times higher than that of thp - 1 cells . the kd value calculated from the tnf binding assay of c75r was 5 . 6 × 10 − 10 m . this kd value was in close agreement to the values previously reported for native p75 tnf - r . thus , transfected cos - 1 cells expressed high levels of human p75 tnf - r in a form that appeared to be similar to native tnf - r . in order to measure the effect of trre on membrane - bound tnf - r , the following experiment was performed . c75r cells were seeded at a density of 2 × 10 5 cells / well in a 24 - well cell culture plate and incubated for 12 to 16 hours at 37 ° c . in 5 % co 2 . the medium in the wells was aspirated , replaced with fresh medium alone or with trre medium , and incubated for 30 min at 37 ° c . throughout the examples , the “ trre - medium ” was that collected by stimulation of thp - 1 cells with pma followed by incubation of the cells in fresh medium for 2 hours as described . after this incubation , the medium was replaced with fresh medium containing 30 ng / ml 125 i - labeled tnf . after 2 hours at 4 ° c ., the cells were lysed with 0 . 1 n naoh and the level of bound radioactivity was measured . the level of specific binding of c75r by 125 i - tnf was significantly decreased after incubation with trre . the radioactive count was 1 , 393 cpm on the cells incubated with trre compared to 10 , 567 cpm on the cells not treated with trre , a loss of 87 % of binding capacity . in order to determine the size of the p75 tnf - r cleared from c75r by trre , the following experiment was performed . 15 × 10 6 c75r cells were seeded in a 150 mm cell culture plate and incubated at 37 ° c . in 5 % co 2 for 12 to 16 hours . trre medium was incubated with c75r cells in the 150 mm plate for 30 min and the resulting supernatant was collected and centrifuged . the concentrated sample was applied to 10 % acrylamide sds - page and electrophoretically transferred to a polyvinylidene difluoride membrane ( immobilon ). immunostaining resulted in a single band of 40 kda , similar to the size found in biological fluids ( fig3 ). the following method and assay were used throughout the examples to measure trre activity . c75r cells and cos - 1 cells were seeded into 24 - well culture plates at a density of 2 . 5 × 10 5 cells / ml / well and incubated overnight ( for 12 to 16 hours ) in 5 % co 2 at 37 ° c . after aspirating the medium in the well , 300 μl of trre medium was incubated in each well of both the c75r and cos - 1 plates for 30 min in 5 % co 2 at 37 ° c . ( corresponding to a and c mentioned below , respectively ). simultaneously , c75r cells in 24 - well plates were also incubated with 300 μl of fresh medium or buffer ( corresponding to b mentioned below ). the supernatants were collected , centrifuged , and then assayed for the concentration of soluble p75 tnf - r by elisa as described above . the following values were assigned and calculations made . a =( amount of soluble p75 tnf - r in a c75r plate treated with the trre containing sample ); i . e . the total amount of stnf - r in a c75r plate . b =( amount of soluble p75 tnf - r spontaneously released in a c75r plate treated with only medium or buffer containing the same reagent as the corresponding samples but without exogenous trre ); i . e . the spontaneous release of stnf - r from c75r cells . c =( amount of soluble p75 tnf - r in a cos - 1 plate treated with the trre sample or the background level of soluble p75 tnf - r released by thp - 1 . ); i . e . the degraded value of transferred ( pre - existing ) stnf - r in the trre sample during 30 min incubation in a cos - 1 plate . this corresponds to the background level of stnf - r degraded in a c75r plate . the net release of soluble p75 tnf - r produced only by trre activity existing in the initial sample is calculated as follows : ( net release of soluble p75 tnf - r only by trre )= a − b − c . we assigned the net release value of soluble p75 tnf - r as the amount of trre activity and defined 1 pg of soluble p75 tnf - r net release ( a − b − c ) as one unit ( u ) of trre activity . once the trre assay was devised , the time course of receptor shedding was assayed by the following method . trre - medium was incubated with c75r and cos - 1 cells for varying lengths of time between 5 and 90 min . the supernatants were then collected and assayed for the level of soluble p75 tnf - r by elisa and the net trre activity was calculated as described above . detectable levels of soluble receptor were released by trre within 5 min and increased up to 30 min ( fig4 a ). subsequent experiments with longer incubation times showed that the level of trre remained relatively constant after 30 min , presumably from the depletion of substrates ( fig4 b ). therefore , 30 min was determined to be the optimal incubation time for this assay system . the binding assay clearly showed that the parental cos - 1 cells did not bind human 125 i - tnf , whereas the transfected c75r cells showed strong specific binding . scatchard analysis indicated receptor levels of 70 , 000 per cell which were 40 to 50 times higher than that typically found on other cell lines . this higher level of substrate allowed detection of trre activity with much more sensitivity than with other cell lines . the kd value calculated from scatchard analysis was 5 . 6 × 10 − 10 m , similar to the values previously reported for the native human p75 tnf - r . thus , the transfected cells provided the membrane form of the receptor in its native configuration , resulting in an excellent source of substrate . when c75r cells were incubated with trre medium , soluble p75 tnf - r was released into the supernatant which was measurable by elisa . the amount of receptors released corresponded to level of trre activity . as c75r cells were incubated with trre medium , another well of c75r cells was simultaneously incubated with medium or buffer alone to measure the level of spontaneous release by c75r . the spontaneous release can be due to an endogenous source of proteolytic enzyme , a homolog of the human trre of monkey origin . in addition , trre medium was incubated with the parent cos - 1 cells to detect the level of soluble receptors that was pre - existing in the sample . for this purpose , rather than directly measuring the level of soluble receptors in the supernatant by elisa , we incubated the sample with cos - 1 cells because we found that after incubation for 30 min with cos - 1 cells , significant degradation of the soluble receptors was observed . the level of initial soluble receptors in the supernatant may decrease up to 50 % after a 30 min incubation with cos - 1 cells . incorporating these two sources of background soluble receptors was the most accurate way to calculate the net trre activity . the premise that increase in the level of soluble receptors in the supernatant was due to the proteolytic cleavage of membrane bound receptors was also supported by the loss of binding of 125 i - labeled tnf to c75r cells after incubation with trre . since the receptor generated by trre was similar in size to that found in biological fluids , this reinforced the finding that trre generates stnf - r in vivo . the induction patterns of trre and known mmps by pma stimulation are quite different . in order to induce mmps , monocytic u - 937 cells , fibrosarcoma ht - 1080 cells , or peritoneal exudate macrophages ( pem ) usually have to be stimulated for one to three days with lps or pma . on the other hand , as compared with this prolonged induction , trre is released very quickly in culture supernatant following 30 min of pma - stimulation . as disclosed in example 2 , trre is stored in the cell very close to the cell membrane to be secreted immediately by pma - stimulation , and trre is synthesized very quickly within 2 hours also by pma - stimulation . therefore , judging from zymography gel data and the different induction patterns by pma , trre cannot be classified into one of the pre - existing mmp families , despite their resemblance regarding metal - requirement and involvement of serine proteases in their activation . soluble tnf - r has been shown to bind to tnf or lt and form a complex consisting of 3 stnf - r with 3 tnf or lt . banner et al . ( 1993 ). according to gel filtration analysis presented above , the profile of trre and soluble p75 tnf - r was quite similar , with both peaks approximately at 150 kda . since the molecular size of soluble p75 tnf - r was reported to be 40 kda , this suggests that stnf - r exist as a complex formed with trre or tnf , or otherwise as homo oligomers . the hypothesis that trre and stnf - r form a complex in vitro was confirmed by the experiment that 25 % trre activity was recovered from soluble p75 tnf - r affinity column . this means that free trre has the ability to bind to its catalytic product , stnf - r . the remaining 75 % which did not combine to the affinity column may already be bound to stnf - r or may not have enough affinity to bind to stnf - r even though it is in a free form . although a considerable amount of enzyme product ( ep ) complex is thought to exist in the reacting solution , trre retained 86 % of its activity after treated once with excessive substrate , suggesting that this complex can be easily separated when it meets new substrate . this ep complex does not seem to inhibit the enzymatic reaction of trre significantly . while stnf - r is a potent inhibitor against the biological activities of tnf and lt , it was also shown that stnf - r has another role in stabilizing tnf activity in vitro . aderka et al . ( 1992 ) j . exp . med . 175 : 323 - 329 . thus stnf - r might act as a stabilizer not only for tnf , but also for trre by composing complex formation . this ep complex between trre and stnf - r may be formed presumably under in vitro conditions , however it is possible that trre , stnf - r and tnf make up several types of complexes in vivo as well as in vitro , and therefore may have physiological significance . in this example , the effect and biological significance of trre is investigated , including ( a ) substrate specificity and ( b ) function in vitro . fluorescein isothiocyanate ( fitc )- conjugated anti - cd54 , fitc - conjugated goat anti - rabbit and mouse antibodies , mouse monoclonal anti - cd30 , anti - cd11b and anti - il - 1r ( serotec , washington d . c .) were utilized in this study . rabbit polyclonal anti - p55 and p75 tnf - r were constructed according to the method described by yamamoto et al . ( 1978 ) cell immunol . 38 : 403 - 416 . thp - 1 cells were treated for 30 min with 1 , 000 and / or 5 , 000 u / ml of trre eluted from the deae - sephadex column and transferred to 12 × 75 mm polystyrene tubes ( fischer scientific , pittsburgh , pa .) at 1 × 10 5 cells / 100 μl / tube . the cells were then pelleted by centrifugation at 350 × g for 5 min at 4 ° c . and stained directly with 10 μl fitc - conjugated anti - cd54 ( diluted in cold pbs / 0 . 5 % sodium aside ), indirectly with fitc - conjugated anti - mouse antibody after treatment of mouse monoclonal anti - cd11b , il - 1r and cd30 and also indirectly with fitc - conjugated anti - rabbit antibody after treatment of rabbit polyclonal anti - p55 and p75 tnf - r . thp - 1 cells stained with each of the antibodies without treatment of trre were utilized as negative controls . the tubes were incubated for 45 min at 4 ° c ., agitated every 15 min , washed twice with pbs / 2 % fcs , repelleted and then resuspended in 200 μl of 1 % paraformaldehyde . these labeled thp - 1 cells were analyzed using a fluorescence activated cell sorter ( facs ) ( becton - dickinson , san jose , calif .) with a 15 mw argon laser with an excitation of 488 nm . fluorescent signals were gated on the basis of forward and right angle light scattering to eliminate dead cells and aggregates from analysis . gated signals ( 10 4 ) were detected at 585 bp filter and analyzed using lysis ii software . values were expressed as percentage of positive cells , which was calculated by dividing mean channel fluorescence intensity ( mfi ) of stained thp - 1 cells treated with trre by the mfi of the cells without trre treatment ( negative control cells ). in order to test the in vitro tnf cytolytic assay by trre treatment the l929 cytolytic assay was performed according to the method described by gatanaga et al . ( 1990b ). briefly , l929 cells , an adherent murine fibroblast cell line , were plated ( 70 , 000 cells / 0 . 1 ml / well in a 96 - well plate ) overnight . monolayered l929 cells were pretreated for 30 min with 100 , 500 or 2 , 500 u / mi of partially - purified trre and then exposed to serial dilutions of recombinant human tnf for 1 hour . after washing the plate with rpmi - 1640 with 10 % fcs to remove the trre and tnf , the cells were incubated for 18 hours in rpmi - 1640 with 10 % fcs containing 1 μg / ml actinomycin d at 37 ° c . in 5 % co 2 . culture supernatants were then aspirated and 50 μl of 1 % crystal violet solution was added to each well . the plates were incubated for 15 min at room temperature . after the plates were washed with tap water and air - dried , the cells stained with crystal violet were lysed by 100 μl per well of 100 mm hcl in methanol . the absorbance at 550 nm was measured using an ear 400 at plate reader ( slt - labinstruments , salzburg , austria ). trre was originally defined as a protease which truncated the human p75 tnf - r that was overexpressed on cdna - transduced cos - 1 cells ( c75r ). to investigate whether trre may truncate not only p75 but also p55 tnf - r on human cells , partially - purified trre from human thp - 1 cells was applied to thp - 1 cells which express low levels of both p55 and p75 tnf - r ( approximately 1 , 500 receptors / cell by scatchard analysis , data not shown ). trre eluate from the deae - sephadex column was added to thp - 1 cells ( 5 × 10 6 cells / ml ) at a final trre concentration of 1 , 000 u / ml for 30 min . the concentration of soluble p55 and p75 tnf - r in that supernatant was measured by soluble p55 and p75 tnf - r elisa . trre was found to truncate both human p55 and p75 tnf - r on thp - 1 cells and released 2 , 382 and 1 , 662 pg / ml soluble p55 and p75 tnf - r , respectively ( fig4 ). therefore , trre was capable of truncating human p75 tnf - r on c75r cells and both human p55 and p75 tnf - r on thp - 1 cells . the following protocol was followed to test the effects of trre in preventing mortality in test animals which were treated with lipopolysaccharides ( lps ) to induce sepsis and septic shock . generally , mice were injected with lethal or sublethal levels of lps , and then with a control buffer or trre . samples of peripheral blood were then collected at intervals to establish if trre blocked tnf - induced production of other cytokines in the bloodstream . animals were assessed grossly for the ability of trre to block the clinical effects of shock and then euthanized and tissues examined by histopathological methods . more specifically , adult balb / c mice , the traditional animal model for septic shock studies [ see , for example , mack et al . ( 1997 ) j . surg . res . 69 : 399 - 407 ; and seljelid et al . ( 1997 ) scand . j . immunol . 45 : 683 - 7 ], were placed in a restraining device and injected intravenously via the tail vein with a 0 . 1 ml solution containing 10 ng to 10 mg of lps in phosphate buffer saline ( pbs ). these levels of lps induce mild to lethal levels of shock in this strain of mice . shock results from changes in vascular permeability , fluid loss , and dehydration , and is often accompanied by symptoms including lethargy , a hunched , stationary position , rumpled fur , cessation of eating , cyanosis , and , in serious cases , death within 12 to 24 hours . control mice received an injection of pbs . different amounts ( 2 , 000 or 4 , 000 u ) of purified human trre were injected iv in a 0 . 1 ml volume within an hour prior to or after lps injection . serum ( 0 . 1 ml ) was collected with a 27 gauge needle and 1 ml syringe iv from the tail vein at 30 , 60 and 90 minutes after lps injection . this serum was heparinized and stored frozen at − 20 ° c . samples from multiple experiments were tested by elisa for the presence of stnf - r , tnf , il - 8 and il - 6 . animals were monitored over the next 12 hours for the clinical effects of shock . selected animals were euthanized at periods from 3 to 12 hours after treatment , autopsied and various organs and tissues fixed in formalin , imbedded in paraffin , sectioned and stained by hematoxalin - eosin ( h and e ). tissue sections were subjected to histopathologic and immunopathologic examination . as shown in fig5 mice injected with lps alone or lps and a control buffer demonstrated rapid mortality . 50 % of the test animals were dead after 8 hours ( lps ) or 9 hours ( lps plus control buffer ), and 100 % of the animals were dead at 15 hours . in contrast , when injections of lps were accompanied by injections of a 2 , 000 u of trre , death was delayed and death rates were lower . only 40 % of the animals were dead at 24 hours . when 4 , 000 u of trre was injected along with lps , all of the animals had survived at 24 hours . thus , trre is able to counteract the mortality induced by lps in test animals . effect of trre on the necrotizing activity of human tnf in vivo the following protocol was followed to test the effects of trre in affecting tumor necrosis in test animals in which tumors were produced , and in which tnf was subsequently injected . generally , on day 0 , cutaneous meth a tumors were produced on the abdominal wall of fifteen balb / c mice by intradermal injection of 2 × 20 5 meth a tumor cells . on day 7 , the mice were divided into three groups of five mice each and treated as follows : group 2 : injected intravenously with tnf ( 1 μg / mouse ) and injected intratumorally with trre ( 400 units / mouse , 6 , 12 hours after tnf injection ). group 3 : injected intravenously with tnf ( 1 μg / mouse ) and injected intratumorally with control medium ( 6 , 12 hours after tnf injection ). on day 8 , tumor necrosis was measured with the following results : therefore , injections of trre greatly reduced the ability of tnf to induce necrosis in meth a tumors in balb / c mice . nine clones involved in trre activity were obtained and designated clones 2 - 8 , 2 - 9 , 2 - 14 , 2 - 15 , p2 - 2 , p2 - 10 , p2 - 13 , p2 - 14 , and p2 - 15 ; the dna sequences represented by seq id nos : 1 to 10 , with partial sequences of clone 2 - 8 represented by seq id nos : 2 and 3 . while not wishing to be bound by any particular theory , the inventors suggest that none of these clones encodes the trre itself , but these clones may be templates for rnas or encode proteins involved in trre expression or function . clones 2 - 8 , 2 - 9 , 2 - 14 , 2 - 15 , p2 - 2 , p2 - 10 , p2 - 13 , p2 - 14 , and p2 - 15 ( represented by seq id nos : 1 to 10 ) were selected from a library of 10 6 jurkat t cell ( atcc # tib - 152 ) cdna inserts in the zap express ™/ ecori vector ( catalog no . 938201 , stratagene , la jolla , calif .). jurkat cells have a high trre activity ( 850 trre u / ml at 10 − 7 m pma ). the library was divided into 48 groups of dna and transformed into cos - 1 cells , which normally lack trre activity . once these cells were grown out , the trre assay ( described above ) was performed , and five positive groups selected . dna from each of these five groups and transfected into e . coli , with 15 plates per group . dna was prepared from these cells and then transfected again into cos - 1 cells . again , once the cells were grown out , the trre activity was tested . two positive groups were selected and transfected into e . coli , yielding 98 colonies . dna was prepared from 96 of these colonies and transfected into cos - 1 cells . the trre assay was performed again , and nine positive clones selected that substantially increased trre activity . these clones were designated 2 - 8 , 2 - 9 , 2 - 14 , 2 - 15 , p2 - 2 , p2 - 10 , p2 - 13 , p2 - 14 , and p2 - 15 . the production of trre activity from these clones is demonstrated in fig6 . this figure shows that each clone is able to substantially increase ( by 85 % to 130 %) trre activity compared to the control . these nine clones were then sequenced . the strategy used to sequence the inserts in the clones included a combination of procedures which are summarized below : 1 . plasmid dna was prepared using a modified alkaline lysis procedure . 2 . dna sequencing was performed using dyedeoxy termination reactions ( abi ). base - specific fluorescent dyes were used as labels . 3 . sequencing reactions were analyzed on 5 . 75 % long ranger ™ gels by an abi 373a - s or on 5 . 0 % long ranger ™ m gels by an abi 377 automated sequencer . 5 . standard primers t7x , t3x , - 40 , - 48 reverse , and bk reverse ( bkr ) were used in sequencing reactions . for each clone , several additional internal sequencing primers ( listed below ) were synthesized . the sequence alignment printout reports generated using sequencher ™ 3 . 0 software and edited by hand are presented below . ncbi blast ( basic local alignment search tool ) sequence analysis [ altschul et al . ( 1990 ) j . mol . biol . 215 : 403 - 410 ] was performed to determine if any known sequences were significantly similar to these sequences . both the dna sequences of the clones and the corresponding orfs ( if any ) were compared to sequences available in databases . * clone 2 - 8 ( aim3 ) was only partially sequenced , generating two partial sequences of 739 and 233 bp , designated aim3t3 and aim3t7 , respectively . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the sequence of aim2 is presented in seq id no : 1 . the complementary strand of the aim2 sequence is seq id no : 147 . the longest orf in the aim2 sequence is 474 aa long and represented in seq id no : 148 . the blast search did not reveal any sequences with significant similarity to the aim2 sequence . of all the clones obtained , only this clone was not sequenced in its entirety . two partial sequences of length 739 and 233 were obtained and designated aim3t3 and aim3t7 . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the sequences of aim3t3 and aim3t7 are presented in seq id nos : 2 and 3 , respectively . the blast search revealed that the aim3t3 sequence may be homologous to the mouse ( m . musculus ) 28s ribosomal rna [ hassouna et al . ( 1984 ) nucleic acids res . 12 : 3563 - 3583 ] and the m . musculus 45s pre - rrna genes [ accession no . x82564 , goegel et al ., chromosoma , in press ]. the complementary sequence of the aim3t3 sequence showed 99 % similarity over 408 bp beginning with nt 221 of seq id no : 2 to the former and 97 % similarity over the same span to the latter . the blast search did not reveal any known sequence homologous to the aim3t7 sequence . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the complementary strand of the aim4 sequence is seq id no : 149 . the longest orf in the aim4 sequence is 236 aa long and represented in seq id no : 150 . the blast search revealed that this clone may be homologous or identical to the human arfaptin 2 , putative target protein of adp - ribosylation factor ( genbank locus hsu52522 , accession u52522 ). the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the blast search revealed that the aim5 sequence is novel . however , it displays some similarity , but not complete similarity , to human initiation factor 5a ( eif - 5a ) [ koettnitz et al . ( 1995 ) gene 159 : 283 - 284 ] and human initiation factor 4d ( eif 4d ) [ smit - mcbride et al . ( 1989 ) j . biol . chem . 264 : 1578 - 1583 ]. the internal sequencing primers synthesized and used to obtain the sequence of this clone were : sequence analysis of the aim6 clone sequence revealed the orf represented in seq id no : 151 . the sequence of aim6 is presented in seq id no : 6 . the longest orf in the aim6 sequence is 1038 aa long and represented in seq id no : 151 . the blast search did not reveal any sequences of known function with significant similarity to the aim6 sequence . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the sequence of aim7 is presented as seq id no : 7 . the longest orf in the aim7 sequence is 849 aa long and represented in seq id no : 152 . the blast search revealed that this clone may be the human insulin - like growth factor ii receptor [ morgan et al . ( 1987 ) nature 329 : 301 - 307 ] or the human cation - independent mannose 6 - phosphate receptor mrna [ oshima et al . ( 1988 ) j . biol . chem . 263 : 2553 - 2562 ]. the aim7 sequence showed 99 % identity to both sequences over 2520 nucleotides beginning with nt 12 of seq id no : 7 and 99 % similarity to the latter over the same span . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the longest orf in the aim8 sequence is 852 aa long and represented in seq id no : 153 . the blast search did not reveal significant similarity of the aim8 sequence to any sequence in the database . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the sequence of aim9 is presented as seq id no : 9 . no orfs longer than 149 aa long were found in the aim9 sequence . the blast search did not reveal any sequences which had significant similarity to the aim9 sequence . the internal sequencing primers synthesized and used to obtain the sequence of this clone were : the sequence of aim10 is presented as seq id no : 10 . the longest orf in the aim10 sequence is 693 aa long and represented in seq id no : 154 . the blast search did not reveal any sequences with significant similarity to the aim10 sequence . thus , cloning the trre gene yielded nine clones , each of which encoded a protein having trre activity . these clones were designated 2 - 8 , 2 - 9 , 2 - 14 , 2 - 15 , p2 - 2 , p2 - 10 , p2 - 13 , p2 - 14 , and p2 - 15 , which encode sequences designated aim2 , aim3t3 / aim3t7 , aim4 , aim5 , aim6 , aim7 , aim8 , aim9 , and aim10 , and shown in seq id nos : 1 to 10 . each clone increases trre activity of cos - 1 cells in vitro . sequence analysis of these clones indicated that aim3 may be homologous to m musculus 45s pre - rrna gene ; aim5 , human eif - 5a transcription factor ; and aim7 , human insulin - like growth factor ii receptor . without wishing to be bound by any particular theory , the inventors suggest that some or all of these clones may be templates for rnas or encode proteins which are involved in transcription and / or translation of trre . alternatively , some or all of these factors may be involved in increasing the activity of trre ( e . g ., acting as an accessory protein ). clonal dna may be directly injected into test animals in order to test the ability of these nucleic acids to induce trre activity , counteract septic shock and / or affect tumor necrosis , as is described in detail in examples 3 and 4 . alternatively , proteins or rna can be generated from the clonal dna and similarly tested in animals . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding , it will be apparent to those skilled in the art that certain changes and modifications can be practiced . therefore , the description and examples should not be construed as limiting the scope of the invention which is delineated by the appended claims . cattccagaa attcagg atg aat ggg gat atg ccc cat gtc ccc att act 410 act ctt gcg ggg att gct agt ctc aca gac ctc ctg aac cag ctg cct 458 ctt cca tct cct tta cct gct aca act aca aag agc ctt ctc ttt aat 506 gca cga ata gca gaa gag gtg aac tgc ctt ttg gct tgt agg gat gac 554 aat ttg gtt tca cag ctt gtc cat agc ctc aac cag gta tca aca gat 602 cac ata gag ttg aaa gat aac ctt ggc agt gat gac cca gaa ggt gac 650 ata cca gtc ttg ttg cag gcc gtc ctg gca agg agt cct aat gtt ttc 698 agg gag aaa agc atg cag aac aga tat gta caa agt gga atg atg atg 746 tct cag tat aaa ctt tct cag aat tcc atg cac agt agt cct gca tct 794 tcc aat tat caa caa acc act atc tca cat agc ccc tcc agc cgg ttt 842 gtg cca cca cag aca agc tct ggg aac aga ttt atg cca cag caa aat 890 agc cca gtg cct agt cca tac gcc cca caa agc cct gca gga tac atg 938 cca tat tcc cat cct tca agt tac aca aca cat cca cag atg caa caa 986 gca tcg gta tca agt ccc att gtt gca ggt ggt ttg aga aac ata cat 1034 gat aat aaa gtt tct ggt ccg ttg tct ggc aat tca gct aat cat cat 1082 gct gat aat cct aga cat ggt tca agt gag gac tac cta cac atg gtg 1130 ala asp asn pro arg his gly ser ser glu asp tyr leu his met val cac agg cta agt agt gac gat gga gat tct tca aca atg agg aat gct 1178 gca tct ttt ccc ttg aga tct cca cag cca gta tgc tcc cct gct gga 1226 agt gaa gga act cct aaa ggc tca aga cca cct tta atc cta caa tct 1274 cag tct cta cct tgt tca tca cct cga gat gtt cca cca gat atc ttg 1322 cta gat tct cca gaa aga aaa caa aag aag cag aag aaa atg aaa tta 1370 ggc aag gat gaa aaa gag cag agt gag aaa gcg gca atg tat gat ata 1418 att agt tct cca tcc aag gac tct act aaa ctt aca tta aga ctt tct 1466 cgt gta agg tct tca gac atg gac cag caa gag gat atg att tct ggt 1514 gtg gaa aat agc aat gtt tca gaa aat gat att cct ttt aat gtg cag 1562 tac cca gga cag act tca aaa aca ccc att act cca caa gat ata aac 1610 cgc cca cta aat gct gct caa tgt ttg tcg cag caa gaa caa aca gca 1658 ttc ctt cca gca aat caa gtg cct gtt tta caa cag aac act tca gtt 1706 gct gca aaa caa ccc cag acc aat agt cac aaa acc ttg gtg cag cct 1754 gga aca ggc ata gag gtc tca gca gag ctg ccc aag gac aag acc taaga 1804 gly thr gly ile glu val ser ala glu leu pro lys asp lys thr met asn gly asp met pro his val pro ile thr thr leu ala gly ile pro ala thr thr thr lys ser leu leu phe asn ala arg ile ala glu leu val his ser leu asn gln val ser thr asp his ile glu leu lys gln ala val leu ala arg ser pro asn val phe arg glu lys ser met pro ile val ala gly gly leu arg asn ile his asp asn lys val ser asp asp gly asp ser ser thr met arg asn ala ala ser phe pro leu val ser glu asn asp ile pro phe asn val gln tyr pro gly gln thr ser lys thr pro ile thr pro gln asp ile asn arg pro leu asn ala gln thr asn ser his lys thr leu val gln pro gly thr gly ile glu aagctttttg aattcggcac gagat gct aca cag gct ata ttt gaa ata ctg 52 gag aaa tcc tgg ttg ccc cag aat tgt aca ctg gtt gat atg aag att 100 glu lys ser trp leu pro gln asn cys thr leu val asp met lys ile gaa ttt ggt gtt gat gta acc acc aaa gaa att gtt ctt gct gat gtt 148 att gac aat gat tcc tgg aga ctc tgg cca tca gga gat cga agc caa 196 cag aaa gac aaa cag tct tat cgg gac ctc aaa gaa gta act cct gaa 244 gln lys asp lys gln ser tyr arg asp leu lys glu val thr pro glu ggg ctc caa atg gta aag aaa aac ttt gag tgg gtt gca gag aga gta 292 gly leu gln met val lys lys asn phe glu trp val ala glu arg val gag ttg ctt ttg aaa tca gaa agt cag tgc agg gtt gta gtg ttg atg 340 ggc tct act tct gat ctt ggt cac tgt gaa aaa atc aag aag gcc tgt 388 gly ser thr ser asp leu gly his cys glu lys ile lys lys ala cys gga aat ttt ggc att cca tgt gaa ctt cga gta aca tct gcg cat aaa 436 gly asn phe gly ile pro cys glu leu arg val thr ser ala his lys gga cca gat gaa act ctg agg att aaa gct gag tat gaa ggg gat ggc 484 gly pro asp glu thr leu arg ile lys ala glu tyr glu gly asp gly att cct act gta ttt gtg gca gtg gca ggc aga agt aat ggt ttg gga 532 ile pro thr val phe val ala val ala gly arg ser asn gly leu gly cca gtg atg tct ggg aac act gca tat cca gtt atc agc tgt cct ccc 580 pro val met ser gly asn thr ala tyr pro val ile ser cys pro pro ctc aca cca gac tgg gga gtt cag gat gtg tgg tct tct ctt cga cta 628 ccc agt ggt ctt ggc tgt tca acc gta ctt tct cca gaa gga tca gct 676 caa ttt gct gct cag ata ttt ggg tta agc aac cat ttg gta tgg agc 724 gln phe ala ala gln ile phe gly leu ser asn his leu val trp ser aaa ctg cga gca agc att ttg aac aca tgg att tcc ttg aag cag gct 772 gac aag aaa atc aga gaa tgt aat tta taagaaagaa tgccattgaa tttttta 826 ala thr gln ala ile phe glu ile leu glu lys ser trp leu pro gln asn cys thr leu val asp met lys ile glu phe gly val asp val thr thr lys glu ile val leu ala asp val ile asp asn asp ser trp arg arg asp leu lys glu val thr pro glu gly leu gln met val lys lys glu leu arg val thr ser ala his lys gly pro asp glu thr leu arg ile lys ala glu tyr glu gly asp gly ile pro thr val phe val ala ala tyr pro val ile ser cys pro pro leu thr pro asp trp gly val thr val leu ser pro glu gly ser ala gln phe ala ala gln ile phe gly leu ser asn his leu val trp ser lys leu arg ala ser ile leu asn thr trp ile ser leu lys gln ala asp lys lys ile arg glu cys ile gln arg phe gly thr ser gly his ile met asn leu gln ala gln pro lys ala gln asn lys arg lys arg cys leu phe gly gly gln glu ile arg val lys glu glu gln tyr leu gly his glu gly pro gly gly ala leu leu asn ser val val tyr gly pro glu arg thr ser ala ala asn cys his ser leu ser leu tyr ser ala thr lys gly ser pro his pro gly val gly val pro thr tyr tyr asn his pro glu ala leu lys arg glu lys ala gly gly pro gln leu asp arg tyr val arg pro met gly val glu phe ser glu pro ser leu ala thr lys arg ala arg glu gly lys gly leu glu gln asn pro ala glu his lys pro ser val ile gln ala glu asp met asn val lys leu glu gly glu pro ser val arg val arg glu gly ser gly leu tyr phe asn ala ile ile ser thr ser met gly glu ala thr pro val ser ile glu pro arg ile asn val gly ser arg phe gln ala glu ile pro leu met arg asp arg ala leu ala ala ala cys ser ser ile phe pro gly ala gly thr asn gln glu leu ala leu his cys leu his glu ser arg gly asp ile leu glu thr leu ala thr tyr his tyr thr gly ser asp gln trp lys met ala glu arg val glu val asp ile lys thr ser gln lys phe pro arg val pro leu ile arg his glu val ser phe leu trp asn thr glu ala ala cys pro thr met pro val cys gly thr ile leu gly lys pro ala ser gly cys pro val gly ile glu lys ser leu gln leu ser thr glu gly phe ile phe ile val arg phe val cys asn asp asp val tyr ser gly pro leu lys phe leu his gln asp ile asp ser gly gln gly ile arg asn thr tyr phe glu phe glu thr ala leu ala cys val pro ser pro val asp cys gln val thr asp leu ala gly asn glu tyr asp leu thr gly leu arg lys arg thr phe tyr leu ser val cys asn pro leu pro tyr ile asn gly ser leu ser ile met tyr val asn gly asp lys cys gly asn ser pro ala phe gln leu gln asp gly cys glu tyr val phe ile trp glu val lys asp pro arg his gly asn leu tyr asp leu lys pro leu gly leu asn asp thr ile val ser ala gly glu tyr thr tyr tyr phe his lys val ala gly leu leu thr gln lys leu thr tyr glu asn gly leu leu lys 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