US 2005O175581A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0175581A1 Haupts et al. (43) Pub. Date: Aug. 11, 2005

(54) BIOLOGICAL ENTITIES AND THE (60) Provisional application No. 60/524.960, filed on Nov. PHARMACEUTICAL AND DIAGNOSTIC USE 25, 2003. Provisional application No. 60/543,518, THEREOF filed on Feb. 11, 2004. (76) Inventors: Ulrich Haupts, Koln (DE); Andre (30) Foreign Application Priority Data Koltermann, Koln (DE); Andreas Scheidig, Koln (DE); Christian Jun. 18, 2003 (EP)...... O3O13819 Votsmeier, Koln (DE); Ulrich Kettling, Nov. 10, 2003 (EP). ... O3O25851 Koln (DE); Wayne Michael Coco, Nov. 11, 2003 (EP). ... O3O25871 Koln (DE) Feb. 11, 2004 (EP)...... O4003058 Correspondence Address: Publication Classification NEEDLE & ROSENBERG, PC. SUTE 1000 (51) Int. Cl." ...... A61K 38/19; A61K 38/48 999 PEACHTREE STREET (52) U.S. Cl...... 424/85.1; 424/94.63 ATLANTA, GA 30309-3915 (US) (57) ABSTRACT (21) Appl. No.: 11/021,951 The present invention provides method for the treatment of a disease by applying a medicament comprising a protease (22) Filed: Dec. 22, 2004 with a defined Specificity is capable to hydrolyze specific Related U.S. Application Data peptide bonds within a target Substrate related to Such disease. The proteases with Such a defined specificity can (63) Continuation-in-part of application No. 10/872,198, further be used for related therapeutic or diagnostic pur filed on Jun. 18, 2004. pOSes. Patent Application Publication Aug. 11, 2005 Sheet 1 of 18 US 2005/0175581 A1

Fig. 1 Patent Application Publication Aug. 11, 2005 Sheet 2 of 18 US 2005/0175581 A1

Trypsin IVGGYNCEENSVPYQVSL---NSGYHF- CGGSLINEQWVVSAGHCY - - - - a-Thrombin IVEGSDAEIGMSPWQVMLFRKSPQELL - CGASLISDRWVLTAAHCLLYPP Enteropeptidase WGGSNAKEGAWPWVVGL - - - YYGGRLLCGASLVSSDWLVSAAHCWYGRN t

Trypsin - ar. KSRIOVRLGEH---NIEVLEGN-EOFINAAKIIRHPQYD-RKTL a-Thrombin WDKNFTENDLLVRIGKH---SRTRYERNIEKISMLEKIYIHPRYNWRENL Enteropeptidase LE- - - - PSKWTAILGLHMKSNLTSPOTV-PRLID. --EIVINPHYN-RRRK - - - - -

Trypsin NNDIMLIKLSSRAVINARVSTISLPTA- - - - PPAT- - - - - GTKCISGWG a-Thrombin DRDIALMKLKKPVAFSDYIHPVCLPDR- - - - ETAASLLOAGYKGRVTGWG Enteropeptidase DNDIAMMHLEFKWNYTDYIOPICLPEENOVFPP------GRNCSIAGWG ------2 ------

Trypsin N- - - - - TASSGADYPDELOCLDAPVLSOAKCEASYPG-KITSNMFCWGFL a-Thrombin NLKETWTANVGKGOPSVLOVVNLPIVERPVCKDSTRI-RITDNMFCAGYK Enteropeptidase T- - - - - VVYOGTT-ANILOEADVPLLSNERCOOOMPEYNITENIMICAGYE - -3 - -

Trypsin -EGGK--DSCOGDSGGPWVCNGO- - - - LQ------GVVSWGDGCAQKNKP a-Thrombin PDEGKRGDACEGDSGGPFWMKSP- - - - FNNRWYQMGIWSWGEGCDRDGKY Enteropeptidase -EGGI- - DSCOGDSGGPLMCOENNRWFLA------GVTSFGYKCALPNRP ...... 4 - - - - -

Trypsin GVYTKWYNYWKWIKNTIAANS a-Thrombin GFYTHVFRLKKWIOKVIDOFGE Enteropeptidase GVYARWSRFTEWIQSFLH- - - -

Fig. 2 Patent Application Publication Aug. 11, 2005 Sheet 3 of 18 US 2005/0175581 A1

Patent Application Publication Aug. 11, 2005 Sheet 4 of 18 US 2005/0175581 A1

sub IAHEYAOSV-- PY------GISO--IKAPALHSQGY ------furin VAKRRAKRD-- VYOEPTDPKFPQQWYLSGVTQRDLNVKEAWAQGF------PC SK1 EKERSKRSALRDS------ALNL--FNDPMWNOOWYLODTRMTAALPKLDL PC SK5 NTHPCO------SD--MNEGAWKRGY ------2 - -

sub ------TGSNWKVAWIDSGDSSHPDL. --NVRGGAS- - FWPSETN------P- - - furin ------TGHGIWWSILDDGIEKNHPDLAGNYDPGAS--FDVNDOD------PDPO PC SK1 HVIPVWQKGITGKGVVITVLDDGLEWNHTDIYANYDPEASYDFNDNDHD------P- - - PC SK5 ------TGKNIWVTILDDGIERTHPDL. --MONYDA- -LASCDVNGNDLDPMP- - - as a or a lar a k ------3 ------

sub - - - - YO- - - - - DGSS- - -HGTHVAGTIA- -A-NNSIGWGVSPSASLYAVKVLDS - - - - furin PRYTOM- - - - - NDNR - - - HGTRCAGEVA- -AWANNGWCGVGWAYNARGGVRMLD - - - - - PC SK1 - - - - FPRYDPTNENK---HGTRCAGEIAMOAN-NHKCGV-GVAYNSKVGGIRMLDG - - - - PC SK5 - - - -RY - - - - - DASNENKHGTRCAGEVA- -AAANNSHCTWGIAFNAKIGGWRMLDGDWTD 4 ------r a r r - air

sub -TGSGOYSWIINGIE-WAISNNMDVINMSLG ------GP - - GSTA ------LKT- - furin - - - - GEVTDAWEARS-LGLNPNHIHIYSASW ------GPEDDGKVDGPARIAEE - - PC SK1 - IVTDAIEASSIGFN- - - - PGHVDIYSASWGPNDDGKTVEGP- --GRIA ------OKAFE PC SK5 MVEAKSVSFNPOHVHIYSASWGPDDDGKTVD------GPA - - PLT------RQ- - - S ------6------7------8 -

sub - -WWDKAWSSG - - - - - WVAAAAGNEGSS ------GSTSTWGYPAKYPSTIAVGAV- - - furin --AFFRGVSOGRGGLGSIFVWASGNGGREHDSCNCDGYTNSI-YTLSISSATOFGNV- - - PC SK1 YGVKOGROGKG - - - - - SIFVWASGNGGRO ------GDNCDCD- - - GYTDSIYTISI - - - PC SK5 --AFENGWRMGRRGLGSVFWWASGNGGRSKDHCSCDGYTNSI-YTISSSTAESGKKPWY - - - - 8------9 ------

Sub --N------SSNOR------ASFSSAG-SELDVMAPGVSIOSTLPGGTYGAY furin --PWYSEACSSTLA------TTYSSGNONEKOIVTTDLROKCT------ESH PC SKl --S------SASQOGLSPWYAEKCSSTLATSYSSG-DYTDQRITSADLHNDCT- - - - ETH PC SK5 LEE------CSSTL ------ATTYSSG-ESYDKKI - - - - ITTDLRQRCTDNH ------O ------11 - - -

sub NGTSMATPHVAGAAALIL--SKHP- - TWTNAOVRDRLESTATY--LG-NSFYYGKGLINV furin TGTSASAPLAAGIIALTLEANKNL--TWRDMOHLVVOTSKPAH--LN-ADDWATNGWGRK PC SKl TGTSASAPLAAGIFALAL- -EANP--NLTWRDMQHLVVWTSEYDPLA-NNPGWKKNGAGL PC SK5 TGTSASAPMAAGIIALAL- -EANPFLTWRDVOHVIVRTSRAGH--LNANDWKTNAAGFKV - - - r Fig. 4 Patent Application Publication Aug. 11, 2005 Sheet 5 of 18 US 2005/0175581 A1

Fig.5 Patent Application Publication Aug. 11, 2005 Sheet 6 of 18 US 2005/0175581 A1

Peps. TLVDEOP- - - - LENYLDMEYFGTIGIGTPAODFTWWFDTGSSNLWVPSVYCSSL--ACTN Secr. EMWDN ------LRGKSGOGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFL------Cath. PAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWI t - - - - 1 a a - - - - - 2- -

Peps. HNRFNPEDSSTYQSTSETVSITYGTGSMTGILGYDTVOV------G - - - GISDTN Secr. HRYYOROLSSTYRDLRKGVYVPYTOGKWEGELGTDLVSI ------PHGPNWTVRA Cath. HHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSODTVSWPCOSASSASALG---GVKVER - - - - - 3 ------4. ------w x

Peps. QIFGLSETEPGSFLYYAPFDGILGLAYPSIS--SSGATPVFDNIWNOGLVSQDLFSVYLS Sec. NIAAITESDK-FFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVP-NLFSLQLC Cath. OVFGEATKOPGITFIAAKFDGILGMAYPRIS--VNNVLPVFDNLMQQKLVDONIFSFYLS - - - - 5 - - - - it it ------6 -

Peps. ADD------KS-- GSWWIFGGIDSSYYTGSLNWVPVTVEGYWOITVDSITMNGETI Secr. GAGFPLNOSEVLASV-- GGSMIIGGIDHSLYTGSLWYTPIRREWYYEWIIWRVEINGODL Cath. RDP------DAOPGGEL.MLGGTDSKYYKGSLSYLNVTRKAYWOVHLDOVEVASGLT ------7------8 - - - - -

Peps. A- - CAEGC--OAIVDTGTSLLTGPTSPIANIOSDIGASENSD------GDMVVSCSAI Secr. KMDCKEYNYDKSIVDSGTTNLRLPKKWFEAAVKSIKAASSTEKFPDGFWLGEOLV-CWOA Cath. L--CKEGC--EAIVDTGTSLMWGPWDEVRELOKAIGAVPLIO------GEYMPCEKV ------9 ------t

Peps. SSLPDIWFT ------NGVCYPVPPSAYILOSEGS - - - - CISGFOGMNVP-TESG Sec. GTTPWNIFPVISLYLMGEWTNOSFRETILPOOYLRPVEDV - - - -ATSODDCYKFAISQSS Cath. STLPATLK ------GGKGYKLSPEDYTLKVSOAGKTLCLSGFMGMDIP-PPSG ------O - - - - s F ------1. O - -

Peps. ELWILGDWFIROYFTVFDRANNOVGLAPVA Secr. TGTWMGAVIMEGFYWWFDRARKRIGFAVSA Cath. PLWILGDVFIGRYYTVFDRDNNRWGFAEAA s

Fig. 6 Patent Application Publication Aug. 11, 2005 Sheet 7 of 18 US 2005/0175581 A1

Fig. 7 Patent Application Publication Aug. 11, 2005 Sheet 8 of 18 US 2005/0175581 A1

Ol MLEADDQGCI EEOGVEDSAN EDSWDAKPDR SSFVPSLFSK KKKNWTMRSI KTTRDRVPTY

61 OYNMNFEKLG KCIIINNKNF DKVTGMGWRN GTDKDAEALF KCFRSLGFDV IWYNDCSCAK ------

121 MODLLKKASE EDHTNAACFA CILLSHGEEN VIYGKDGVTP IKDLTAHFRG DRSKTLLEKP a - - - - 2------

181 KLFFIOACRG TELDDGIOAD SGPINDTDAN PRYKIPWEAD FLFAYSTVPG YYSWRSPGRG - - - - - 3 ------4 - - - -

241 SWFVQALCSI LEEHGKDLEI MOILTRVNDR VARHFESQSD DPHFHEKKQI PCVVSMLTKE ------5------

301 LYFSQ

Fig. 8 Patent Application Publication Aug. 11, 2005 Sheet 9 of 18 US 2005/0175581 A1

Protein Scaffod Protease A Protease B

1 2 3 Candidate SDR insertion sites

Insertion of random SDRs into protein scaffold

H- - -

N-N-1 SpecificitySelection for increased

NBE with intended specificity SDR1 SDR2 SDR3 Fig. 9 Patent Application Publication Aug. 11, 2005 Sheet 10 of 18 US 2005/0175581 A1

54 kDa 48 kDa 35 kDa

24 kDa

Fig. 10 Patent Application Publication Aug. 11, 2005 Sheet 11 of 18 US 2005/0175581 A1

-- expressing cells - A - negative control

O 1O 20 30 40 50 60 time (min)

Fig. 11

Patent Application Publication Aug. 11, 2005 Sheet 13 of 18 US 2005/0175581 A1

trypsin SDR2 SDR1 & SDR2

Fig. 13 Patent Application Publication Aug. 11, 2005 Sheet 14 of 18 US 2005/0175581 A1

Fig. 14 Patent Application Publication Aug. 11, 2005 Sheet 15 of 18 US 2005/0175581 A1

4. O

3 5 22 O5

1 O live Cells dead Cells Varianti variantii variant iii

Fig. 15 Patent Application Publication Aug. 11, 2005 Sheet 16 of 18 US 2005/0175581 A1 CNCN LOO

15

10

5

O

19 16 Patent Application Publication Aug. 11, 2005 Sheet 17 of 18 US 2005/0175581 A1 proteolytic digestion of TNF

-ol-variantx -O- variantxi -A-variantxii -v-Trypsin -0-negative

O 50 100 150 200 250 time min) Fig. 17a proteolytic digestion of serum protein

1 O

0. 8

0. 6

0. 4 -- Variantx -O-Variantxi -A-variantxi 0. 2 -v-Trypsin -0-negative 0. O O 50 100 150 200 250 300 350 400 time (min) Fig. 17b Patent Application Publication Aug. 11, 2005 Sheet 18 of 18 US 2005/0175581 A1

O 50 100 150 200 250 time (min)

Fig. 18 US 2005/0175581 A1 Aug. 11, 2005

BIOLOGICAL ENTITIES AND THE mechanisms contribute to the vast repertoire of antibody PHARMACEUTICAL AND DIAGNOSTIC USE Specificity and occur at different Stages of immune response THEREOF generation and antibody maturation (Janeway, C et al. (1999) Immunobiology, Elsevier Science Ltd., Garland Pub 0001. This application is a continuation-in-part of U.S. lishing, New York). Specifically, antibodies contain comple application Ser. No. 10/872,198 filed Jun. 18, 2004 which mentarity determining regions (CDRS) which interact with claims the priority benefit of European Application No. the antigen in a highly specific manner and allow discrimi 03013819, filed Jun. 18, 2003; European Application No. nation even between very similar epitopes. The light as well 03025851, filed Nov. 10, 2003; European Application No. as the heavy chain of the antibody each contribute three 03025871, filed Nov. 11, 2003; U.S. Provisional Application CDRS to the binding domain. Nature uses recombination of No. 60/524.960, filed Nov. 25, 2003; European Application various gene Segments combined with further mutagenesis No. 04003058, filed Feb. 11, 2004; and U.S. Provisional in the generation of CDRS. AS a result, the Sequences of the Application No. 60/543,518, filed Feb. 11, 2004, which Six CDR loops are highly variable in composition and length applications are incorporated herein fully by this reference. and this forms the basis for the diversity of binding speci 0002 The present invention provides methods for the ficities in antibodies. A similar principle for the generation treatment of a disease by applying a medicament comprising of a diversity of catalytic Specificities is not known from a protease with a defined Specificity is capable to hydrolyze nature. Specific peptide bonds within a target Substrate related to 0006 Catalysis, i.e. the increase of the rate of a specific Such disease. The proteases with Such a defined specificity chemical reaction, is besides binding the most important can further be used for related therapeutic or diagnostic protein function. Catalytic proteins, i.e. enzymes, are clas purposes. sified according to the chemical reaction they catalyze. BACKGROUND 0007 Transferases are enzymes transferring a group, for example, the methyl group or a glycosyl group, from one 0.003 Academic and industrial research continuously compound (generally regarded as donor) to another com Searches for functional proteins to be used as therapeutic, pound (generally regarded as acceptor). For example, gly research, diagnostic, nutritional, personal care or industrial cosyltransferases (EC 2.4) transfer glycosyl residues from a agents. Today, Such functional proteins can be classified donor to an acceptor molecule. Some of the glycosyltrans mainly into two categories: natural proteins and engineered ferases also catalyze hydrolysis, which can be regarded as proteins. Natural proteins, on the one hand, are discovered from nature, e.g. by Screening natural isolates or by Sequenc transfer of a glycosyl group from the donor to water. The ing genomes from diverse species. Engineered proteins, on subclass is further subdivided into hexosyltransferases (EC the other hand, are typically based on known proteins and 2.4.1), pentosyltransferases (EC 2.4.2) and those transfer are altered in order to acquire modified functionalities. The ring other glycosyl groups (EC 2.4.99, Nomenclature Com present invention discloses engineered proteins with novel mittee of the International Union of Biochemistry and functions as compared to the Starting components. Such Molecular Biology (NC-IUBMB)). proteins are called NBEs (New Biologic Entities). The 0008 Oxidoreductases catalyze oxido-reductions. The NBES disclosed in the present invention are engineered Substrate that is oxidized is regarded as hydrogen or electron enzymes with novel Substrate specificities or fusion proteins donor. Oxidoreductases are classified as dehydrogenases, of Such engineered enzymes with other functional compo oxidases, mono- and dioxygenases. Dehydrogenases trans nentS. fer hydrogen from a hydrogen donor to a hydrogen acceptor molecule. OxidaseS react with molecular oxygen as hydro 0004 Specificity is an essential element of enzyme func gen acceptor and produce oxidized products as well as either tion. A cell consists of thousands of different, highly reactive hydrogen peroxide or water. Monooxygenases transfer one catalysts. Yet the cell is able to maintain a coordinated metabolism and a highly organized three-dimensional Struc oxygen atom from molecular oxygen to the Substrate and ture. This is due in part to the Specificity of enzymes, i.e. the one is reduced to water. In contrast, dioxygenases catalyze Selective conversion of their respective Substrates. Specific the insert of both oxygen atoms from molecular oxygen into ity is a qualitative and a quantitative property: the Specificity the Substrate. of a particular enzyme can vary widely, ranging from just 0009 Lyases calalyze elimination reactions and thereby one particular type of target molecules to all molecular types generate double bonds or, in the reverse direction, catalyze with certain chemical Substructures. In nature, the Specificity the additions at double bonds. Isomerases catalyze intramo of an organism's enzymes has been evolved to the particular lecular rearrangements. Ligases catalyze the formation of needs of the organism. Arbitrary Specificities with high value chemical bonds at the expense of ATP consumption. for therapeutic, research, diagnostic, nutritional or industrial 0010 Finally, hydrolases are enzymes that catalyze the applications are unlikely to be found in any organism’s hydrolysis of chemical bonds like C-O or C-N. The E.C. enzymatic repertoire due to the large Space of possible classification for these enzymes generally classifies them by Specificities. The only realistic way of obtaining Such speci the nature of the bond hydrolysed and by the nature of the ficities is their generation de novo. Substrate. Hydrolases Such as lipases and proteases play an 0005. When comparing enzymes with binders, a para important role in nature as well in technical applications of digm of Specificity is given by antibodies recognizing indi biocatalysts. Proteases hydrolyse a peptide bond within the vidual epitopes as Small distinct Structures within large context of an oligo- or polypeptide. Depending on the molecules. The naturally occurring vast range of antibody catalytic mechanism proteases are grouped into aspartic, Specificities is attributed to the diversity generated by the Serin, cysteine, metallo- and threonine proteases (Handbook immune System combined with natural Selection. Several of proteolytic enzymes. (1998) Eds: Barret, A.; Rawling, N.; US 2005/0175581 A1 Aug. 11, 2005

Woessner, J.; Academic Press, London). This classification were available. However, the use of Such enzymes with high, is based on the amino acid Side chains that are responsible low or any defined Specificity is currently limited to those for catalysis and which are typically presented in the active which can be isolated from natural Sources. The field of Site in Very similar orientation to each other. The Scissile application for these enzymes varies from therapeutic, bond of the substrate is brought into register with the research, diagnostic, nutritional to personal care and indus catalytic residues due to Specific interactions between the trial purposes. amino acid Side chains of the Substrate and complementary 0014 Enzyme additives in detergents have come to con regions of the protease (Perona, J. & Craik, C (1995) Protein Stitute nearly a third of the whole industrial enzyme market. Science, 4,337-360). The residues on the N- and C-terminal Detergent enzymes include proteinases for removing Side of the Scissile bond are usually called P, P., P. etc and organic Stains, lipases for removing greasy Stains, amylases P', P, P and the binding pockets complementary to the for removing residues of Starchy foods and cellulases for Substrate S, S, S and S', S', S', respectively (nomen restoring of smooth surface of the fiber. The best-known clature according to Schlechter & Berger, Biochem. Bio detergent enzyme is probably the nonspecific proteinase phys. Res. Commun. 27 (1967) 157-162). The selectivity of proteases can vary widely from being virtually nonselec Subtilisin, isolated from various Bacillus Species. tive-e.g. the Subtilisins-over a strict preference at the P. 0015 Starch enzymes, such as amylases, occupy the position-e.g. Trypsin Selectively cutting on the C-terminal majority of those used in food processing. While Starch Side of arginine or lysine residues-to highly Specific pro enzymes include products that are important for textile teases—e.g. human tissue-type plasminogen activator (t-PA) desizing, alcohol fermentation, paper and pulp processing, cleaving at the C-terminal Side of the arginine in the and laundry detergent additives, the largest application is for sequence CPGRVVG (Ding, Let al. (1995) Proc. Natl. the production of high fructose corn Syrup. The production Acad. Sci. USA92, 7627-7631; Coombs, Get al. (1996).J. of corn Syrup from Starch by means of industrial enzymes Biol. Chem. 271, 4461–4467). was a Successful alternative to acid hydrolysis. 0.011 The specificity of proteases, i.e. their ability to 0016 Apart from Starch processing, enzymes are used for recognize and hydrolyze preferentially certain peptide Sub an increasing range of applications in food. Enzymes in food Strates, can be expressed qualitatively and quantitatively. can improve texture, appearance and nutritional value or Qualitative Specificity refers to the kind of amino acid may generate desirable flavours and aromas. Currently used residues that are accepted by a protease at certain positions food enzymes in bakery are amylase, amyloglycosidases, of the peptide Substrate. For example, trypsin and t-PA are pentosanases for breakdown of pentosan and reduced gluten related with respect to their qualitative Specificity, Since both production or glucose oxidases to increase the Stability of of them require at the P position an arginine or a similar dough. Common enzymes for dairy are rennet (protease) as residue. On the other hand, quantitative specificity refers to coagulant in cheese production, lactase for hydrolysis of the relative number of peptide Substrates that are accepted as lactose, protease for hydrolysis of whey proteins or catalase Substrates by the protease, or more precisely, to the relative for the removel of hydrogen peroxides. Enzymes used in k/kM ratioS of the protease for the different peptides that brewing proceSS are the above named amylases, but also are accepted by the protease. Proteases that accept only a cellulases or proteases to clarify the beer from Suspended Small portion of all possible peptides have a high Specificity, proteins. In wines and fruit juices, cloudineSS is more whereas the Specificity of proteases that, as an extreme, commenly caused by Starch and pectins So that amylases and cleave any peptide Substrate would theoretically be Zero. pectinases increase yield and clarification. Papain and other proteinases are used for meat tenderizing. 0012 Comparison of the primary, secondary as well as the tertiary structure of proteases (Fersht, A., Enzyme Struc 0017 Enzymes have also been developed to aid animals ture and Mechanism, W. H. Freeman and Company, New in the digestion of feed. In the Western hemisphere, corn is York, 1995) allows identification of classes showing a high a major Source of food for cattle, Swine, and poultry. In order degree of conservation (Rawlings, N. D. & Barrett, A. J. to improve the bioavailability of phosphate from corn, (1997) In: Proteolysis in Cell Functions Eds. Hopsu-Havu, phytase is commonly added (Wyss, M. et al., Biochemical V. K.; Jarvinen, M., Kirschke, H., pp. 13-21, IOS Press, characterization of fungal phytases (myo-inositol hexak Amsterdam). A widely accepted Scheme for protease clas isphosphate phosphohydrolases); Catalytic properties. sification has been proposed by Rawlings & Barrett (Hand Applied & Environmental Microbiology 65, 367-373 book of proteolytic enzymes. (1998) Eds: Barret, A.; Raw (1999)). Moreover, phytate hydrolysis has been shown to ling, N.; Woessner, J.; Academic Press, London). For bring about improvements in digestibility of protein and example, the Serine proteases family can be Subdivided into absorption of minerals such as calcium (Bedford, M. R. & Structural classes with chymotrypsin (class S1), Subtilisin Schulze, H., Exogenous Enzymes for Pigs and Poultry (class S8) and carboxypeptidase (class SC) folds, each of Review). Nutrition Research Reviews 11, 91-114 (1998)). which includes nonspecific as well as Specific proteases Another major feed enzyme is Xylanase. This enzyme is (Rawlings, N. D. & Barrett, A.J. (1994) Methods Enzymol. particularly useful as a Supplement for feeding Stuff com 244, 19-61). This applies to other protease families analo prising more than about 10% of wheat barley or rye, because gously. An additional distinction can be made according to of their relatively high soluble fiber content. Xylanases the relative location of the cleaved bond in the Substrate. cause two important actions: reduction of Viscosity of the Carboxy- and aminopeptidases cleave amino acids from the intestinal contents by hydrolyzing the gel-like high molecu C- and N-terminus, respectively, while endopeptidases cut lar weight arabinoxylans in feed (Murphy, Tet al., Effect of anywhere along the oligopeptide. range of new Xylanases on in vitro Viscosity and on perfor mance of broiler diets. British Pultry Science 44, S16-S18 0013 Many applications would be conceivable if (2003)) and break down of polymers in cell walls which enzymes with a basically unlimited Spectrum of Specificities improve the bioavailability of protein and starch. US 2005/0175581 A1 Aug. 11, 2005

0.018 Biotech research and development laboratories 0022 Monoclonal antibodies represent another important routinely use Special enzymes in Small quantities along with biological class of Substances with therapeutic capabilities. many other reagents. These enzymes create a significant One of the main antibody targets are tumor necrosis factors market for various enzymes. Enzymes like alkaline phos (TNFs) which belong to the family of cytokines. TNFs play phatase, horseradish peroxidase and luciferase are only a major role in the inflammation process. AS homotrimers Some examples. Thermostable DNA polymerases like Taq they could bind to receptors of nearly every cell. They polymerase or restriction endonucleaseS revolutionized activate a multiplicity of cellular genes, multiple signal laboratory work. transduction mechanisms, kinases and transcription factors. 0019. The use of enzymes in the diagnosis of disease is The most important TNFs are TNF-alpha and TNF-beta. another important benefit derived from the intensive TNF-alpha is produced by macrophages, monocytes and research in biochemistry. Within the recent past few years other cells. TNF-alpha is an inflammation mediator. There that interest in diagnostic enzymology has increased and fore, research of the last decade has been focused on there are still large areas of medical research in which the TNF-alpha inhibitors like monoclonal antibodies as possible diagnostic potential of enzyme reactions has not been therapeutics for different therapeutic indications like Rheu explored at all. Common enzymes used for clinical diagno matoid Arthritis, Crohn's disease or Psoriasis (Hamilton et sis are acid phosphatase, alanine aminotransferase, alkaline al. (2000) Expert Opin Pharmacother, 1 (5): 1041-1052). phosphatase, amylase, angiotensin converting enzymes, One of the major disadvantages of monoclonal antibodies aspartate aminotransferase, cholinesterase, creatinine are their high costs, So that new biological alternatives are of kinase, gamma glutamyltransferase, lactate dehydrogena great importance. Seor rennin. 0023 There are a lot of examples for engineered enzymes 0020. Therapeutic enzymes are a particular class of in literature. Fulani et al. (Fulani F. et al. (2003) Protein drugs, categorized by the FDA as biologicals, with a lot of Engineering 16, 515-519) describe a rhodanase (thiosulfat advantages compared to other, especially non-biological :cyanide sulfurtransferase) from Azotobacter vinelandii pharmaceuticals. Examples for Successful therapeutic which has a catalytic domain Structurally related to catalytic enzymes are human clotting factors like factor VIII and subunit of Cdc25 phosphatase enzymes. The difference in factor IX for human treatment. In addition, digestive catalytic mechanism depends on the different size of the enzymes are used for various deficiencies in human diges active site. Both rhodanase and phosphatase are highly tive processes. Other examples are t-PA and Streptokinase specific on different Substrates (Sulfate vs. phosphate). The for the treatment of cardiovascular disease, beta-glucocer catalytic mechanism of the rhodanase could be shifted ebrosidase for the treatment of Type I Gaucher disease, towards Serine/threonine phosphatase by Single-residue L-asparaginase for the the treatment of acute lymphoblastic insertion. Therefore, Fulani et al. give a Single example for leukemia and DNASe for the treatment of cystic fibrosis. An the change of a catalytic mechanism by Structural compari important issue in the application of proteins as therapeutics Son and Sequence alignment of naturally known enzymes is their potential immunogenicity. To reduce this risk, one from different enzyme classes but lack an indication of how would prefer enzymes of human origin, which narrows to generate a user-definable Substrate Specificity while keep down the set of available enzymes. The provision of ing the same catalytic mechanism. designed enzymes, preferably of human origin, with novel, tailor-made Specificities would allow the Specific modifica 0024. The thioredoxin reductase described by Briggs et tion of target Substrates at will, while minimizing the risk of al. (WO 02/090300 A2) has an altered cofactor specificity immunogenicity. A further advantage of highly specific which preferably binds NADPH compared to NADH. Thus, enzymes as therapeutics would be their lower risk of Side both enzymes, the Starting point as well as the resulting effects. Due to the limited possibility of specific interactions engineered enzyme are highly specific towards different between a Small molecule and a protein, binding to non Substrates. The methods to achieve Such an altered Substrate target proteins and therefore Side effects are quite common Specificity are either computational processing methods or and often cause termination of an otherwise promising lead Sequence alignments of related proteins to define variable compound. Specific enzymes, on the other hand, provide and conserved residues. They all have in common that they many more contact Sites and mechanisms for Substrate are based on the comparison of Structures and Sequences of discrimination and therefore enable a higher specificity and proteins with known specificities followed by the transfer of thereby leSS Side activities. the same to another backbone. 0021 Proteases represent an important class of therapeu 0025 There are other examples of specificity-engineered tic agents (Drugs of today, 33, 641-648 (1997)). However, enzymes and, in particular, of proteases which have been currently the therapeutic protease is usually a Substitute for published in the literature. None of these examples, how insufficient acitivity of the body's own proteases. For ever, provides a means for generating novel Specificites example, factor VII can be administered in certain cases of compared to the Specificity of the Starting material used coagulation deficiencies of bleeders or during Surgery within the described methods. The methods range from (Heuer L.; Blumenberg D. (2002) Anaesthesist 51:388). Structure-directed single point mutations (Kurth, T. et al. Tissue-type plasminogen activator (t-PA) is applied in acute (1998) Biochemistry 37, 11434-11440; Ballinger, M et al. cardiac infarction, initializing the dissolution of fibrin clots (1996) Biochemistry, 35:13579-13585), exchange of surface through specific cleavage and activation of plasminogen loops between two specific proteases (Horrevoets et al. (Verstraete, M. et al. (1995) Drugs, 50, 29-41). So far a (1993).J. Biol. Chem. 268,779-782), to random mutagenesis protease with taylor-made specificity is generated to provide either regio-Selectively or acroSS the whole gene combined a therapeutic agent that Specifically activates or inactivates with in-vitro or in-vivo selection (Sices, H. & Kristie, T. a disease related target protein. (1998) Proc. Natl. Acad. Sci. USA, 95, 2828-2833). US 2005/0175581 A1 Aug. 11, 2005

0026. The rational design of protease specificity is lim 0029. A method has been described that aims at the ited to very few examples. This approach is Severely limited generation of new catalytic activities and Specificities within by the insufficient understanding of the complexities that the C/B-barrel proteins (WO 01/42432; Fershtetal, Methods govern folding and dynamics as well as Structure-function of producing novel enzymes, Altamirano et al. (2000) relationships in proteins (Corey, M. J. & Corey, E. (1996) Nature 403, 617-622). The C/B-barrel proteins comprise a Proc. Natl. Acad. Sci. USA, 93:11428-11434). It is therefore large Superfamily of proteins accounting for a large fraction difficult to alter the primary amino acid Sequence of a of all known enzymes. The Structure of the proteins is made protease in order to change its activity or Specificity in a from C/B-barrel Surrounded by C.-helices. The loops con predictive way. In a Successful example, Kurth et al. engi necting B-Strands and helices comprise the So-called lid neered trypsin to show a preference for a dibasic motive Structure including the acitve site residues. The method is (Kurth, T. et al. (1998) Biochemistry, 37:11434-11440). In based on the classification of C/B-barrel proteins into two another example, Hedstrom et al. converted the S. Substrate classes based on the catalytic lid structure. An extensive Specificity of trypsin to that of chymotrypsin (Hedstrom, L. comparison of C/B-barrel protein Structures led the authors et al. (1992) Science, 255:1249-1253). This is an example to the conclusion that the Substrate binding and Specificity is where a known property was transferred from one backbone primarily defined by the barrel structure while the specificity to another. of the chemical reaction resides within the loops. It is 0027 Ballinger et al. (WO 96/27671) describe subtilisin Suggested that barrels and lid Structures from different variants with combination mutations (N62D/G166D, and enzymes can be combined to generate new enzymatic activi optionally Y104D) having a shift of substrate specificity ties and to provide a starting point to fine tune the properties towards peptide or polypeptide Substrates with basic amino by targeted or randomized mutagenesis and Selection. The acids at the P1, P2 and P4 positions of the substrate. Suitable method does not provide for the generation of user-defined substrates of the variant subtilisin were revealed by sorting Specificity. a library of phage particles (Substrate phage) containing five 0030. In summary, it is clear that there are many possible contiguous randomized residues. These Subtilisin variants applications in the fields of therapeutics, research and diag are useful for cleaving fusion proteins with basic Substrate nostics, industrial enzymes, food and feed processing, cos linkers and processing hormones or other proteins (in vitro metics and other areas that would become possible by the or in vivo) that contain basic cleavage Sites. The problems availability of enzymes with a novel Substrate Specificity. asSociated with rational redesign of enzymes can partially be However, only a limited number of Specific enzymes has overcome by directed evolution (as disclosed in PCT/EP03/ been identified from natural sources so far. Methods of 04864). These studies can be classified by their expression rational design to modify, alter, convert or transfer Sequence and Selection Systems. Genetic Selection means to produce Specificity as well as random approaches described above inside an organism an enzyme, e.g. a protease, which is able did not enable the generation of a novel and user-definable to cleave a precursor protein which in turn results in an Specificity that was not present in the employed Starting alteration of the growth behavior of the producing organism. material. From a population of organisms with different proteases those can be selected which have an altered growth behavior. 0031. Therefore, none of the currently available methods This principle was for example reported by Davis et al. (U.S. can provide enzymes with a novel and user-defined Pat. No. 5,258,289, WO 96/21009). The production of a Sequence Specificity. Such enzymes were disclosed in appli phage System is dependent on the cleavage of a phage cant's yet unpublished applications PCT/EP2004/051172, protein which only can be activated in the presence of a PCT/EP2004/051173; U.S. Ser. No. 10/872,197 and U.S. proteolytic enzyme which is able to cleave the phage pro Ser. No. 10/872,198. The current invention provides further tein. Other approaches use a reporter System which allows a Such enzymes as well as methods for generating them. Selection by Screening instead of a genetic Selection, but also cannot overcome the intrinsic insufficiency of the intracel SUMMARY OF THE INVENTION lular characterization of enzymes. 0032. The objective of the present invention is to provide 0028 Systems to generate enzymes with altered a method for the treatment of a disease by applying a Sequence Specificities with Self-Secreting enzymes are also medicament comprising a protease. Further the present reported. Duffet al. (WO 98/11237) describe an expression invention provides engineered proteins with novel functions System for a Self-Secreting protease. An essential element of that do not exist in the components used for the engineering the experimental design is that the catalytic reaction acts on of Such proteins. In particular, the invention provides the protease itself by an autoproteolytic processing of the enzymes with user-definable specificities. User-definable membrane-bound precursor molecule to release the matured Specificity means that enzymes are provided with Specifici protease from the cellular membrane into the extracellular ties that do not exist in the components used for the environment. Therefore, a fusion protein must be con engineering of Such enzymes. The Specificities can be cho Structed where the target peptide Sequence replaces the Sen by the user So that one or more intended target Substrates natural cleavage Site for autoproteolysis. Limitations of Such are preferentially recognised and converted by the enzymes. a System are that positively identified proteases will have the Furthermore, the invention provides enzymes that possess ability to cleave a certain amino acid Sequence but they also essentially identical Sequences to human proteins but have may cleave many other peptide Sequences. Therefore, high different Specificities. In a particular embodiment, the inven Substrate Specificity cannot be achieved. Additionally, Such tion provides proteases with user-definable specificities. a System is not able to control that Selected proteases cleave at a specific position in a defined amino acid Sequence and 0033. Furthermore, the present invention is directed to it does not allow a precise characterization of the kinetic engineered enzymes which are fused to one or more further constants of the Selected proteases (kat, KM). functional components. These further components can be US 2005/0175581 A1 Aug. 11, 2005 proteinacious components which preferably have binding Substrates, Such specificities not being present in the indi properties and are of the group consisting of Substrate vidual starting components, comprising at least the follow binding domains, antibodies, receptorS or fragments thereof. ing steps: In a particular aspect, the engineered proteases are fused to proteins or peptide Sequences that bind to marker molecules 0043 (a) providing a protein scaffold which catalyzes at that are only present or over-expressed in Specific tissues, least one chemical reaction on at least one Substrate, Specific organs, Specific cell types, Specific diseases or a 0044 (b) generating a library of engineered enzymes by combination thereof, thereby increasing the half-life of the combining the protein Scaffold from Step engineered proteases or increasing the local concentration in the respective tissues, organs or diseased areas of the body. 0045 (a) with fully or partially random peptide In another aspect the engineered proteases are fused to Sequences at Sites in the protein Scaffold that enable the proteins or peptide Sequences that bind to the target mol resulting engineered enzyme to discriminate between at least ecule of the engineered protease, thereby increasing the one target Substrate and one or more different Substrates, and interaction between protease and target. In another aspect of 0046 (c) selecting out of the library of engineered the invention the engineered proteases are fused to proteins enzymes generated in step (b) one or more enzymes that or peptide Sequences that reduce the rate of clearance from have specificities towards at least one target Substrate; the Serum after i.v. administration. In another aspect of the 0047 (5) a fusion protein which is comprised of at least invention the engineered proteases are fused to proteins or one engineered enzyme as defined in (2) above and at least peptide Sequences that trigger the import of the protease into one further component, preferably the at least one further target cells or the transport of the proteases acroSS the blood component having binding properties and more preferably brain barrier. being Selected from the group consisting of antibodies, 0034) Furthermore, the above further components can be binding domains, receptors, and fragments thereof; further functional components, preferably being Selected 0048 (6) a composition or pharmaceutical composition from the group consisting of polyethylenglycols, carbohy comprising one or more engineered enzymes as defined in drates, lipids, fatty acids, nucleic acids, metals, metal che (2) above or a fusion protein as defined in (5) above, said lates, and fragments or derivatives thereof. The resulting pharmaceutical composition may optionally comprise an fusion proteins are understood as enzymes with user-defin acceptable carrier, excipient and/or auxiliary agent; able Specificities within the present invention. 0049 (7) a DNA encoding the engineered enzyme as 0.035 Besides, the invention is directed to the application of Such enzymes with novel, user-definable Specificities for defined in (2) above; therapeutic, research, diagnostic, nutritional, personal care 0050 (8) a vector comprising the DNA as defined in (7) or industrial purposes. Moreover, the invention is directed to above; a method for generating engineered enzymes with user 0051 (9) a host cell or transgenic organism being trans definable specificities. In particular, the invention is directed formed/transfected with a vector as defined in (8) above to generate enzymes that possess essentially identical and/or containing the DNA as defined in (7) above; and Sequences to human enzymes but have different Specificities. 0.052 (10) a method for producing the engineered 0036) This problem has been solved by the embodiments enzyme of (2) above comprising culturing a cell or organism of the invention specified in the description below and in the as defined in (8) above and isolating the enzyme from the claims. The present invention is thus directed to culture broth. 0037 (1) the use of a protease with defined specificity for a target Substrate for preparing a medicament for the treat BRIEF DESCRIPTION OF THE FIGURES ment of a specific disease related to Said target Substrate, 0053. The following figures are provided in order to 0038 (2) an engineered enzyme with defined specificity explain further the present invention in Supplement to the characterized by the combination of the following compo detailed description: nentS,. 0054 FIG. 1 illustrates the three-dimensional structure of human trypsin I with the active site residues shown in 0039 (a) a protein scaffold which catalyzes at least one "ball-and-Stick” representation and with the marked regions chemical reaction on at least one Substrate, and indicating potential SDR insertion Sites. 0040 (b) one or more specificity determining regions 0055 FIG. 2 shows the alignment of the primary amino (SDRs) located at sites in the protein scaffold that enable the acid Sequence of three members of the Serine protease class resulting engineered protein to discriminate between at least S1 family: human trypsin I, human alpha-thrombin and one target Substrate and one or more different Substrates, and wherein the SDRs are essentially synthetic peptide human enteropeptidase (see also SEQ ID NOs: 1, 5 and 6). Sequences, 0056 FIG. 3 illustrates the three-dimensional structure of subtilisin with the active site residues being shown in 0041 (3) the use of an engineered enzyme as defined in “ball-and-stick” representation and with the numbered (2) above for therapeutic, research, diagnostic, nutritional, regions indicating potential SDR insertion Sites. personal care or industrial purposes, preferably for the use as 0057 FIG. 4 shows the alignment of the primary amino defined in (1) above; acid Sequences of four members of the Serine protease class 0.042 (4) a method for generating engineered enzymes as S8 family: subtilisin E, furin, PC1 and PC5 (see also SEQID defined in (2) above having specificities towards target NOs: 7-10). US 2005/0175581 A1 Aug. 11, 2005

0.058 FIG. 5 illustrates the three-dimensional structure naturally-occurring or artificial proteolytic enzymes, as well of pepsin with the active site residues being shown in as variants thereof obtained by Site-directed or random “ball-and-stick” representation and with the numbered mutagenesis or any other protein engineering method, any regions indicating potential SDR insertion Sites. active fragment of a proteolytic enzyme, or any molecular 0059 FIG. 6 shows the alignment of the primary amino complex or fusion protein comprising one of the aforemen acid Sequences of three members of the A1 aspartic acid tioned proteins. A “chimera of proteases' means a fusion protease family: pepsin, ?-Secretase and cathepsin D (See protein of two or more fragments derived from different also SEQ ID NOs: 11-13). parent proteases. 0060 FIG. 7: illustrates the three-dimensional structure 0074 The term “substrate” means any molecule that can of caspase 7 with the active Site residues being shown in be converted catalytically by an enzyme. The term "peptide “ball-and-stick” representation and with the numbered Substrate” means any peptide, oligopeptide, or protein mol regions indicating potential SDR insertion Sites. ecule of any amino acid composition, Sequence or length, that contains a peptide bond that can be hydrolyzed cata 0061 FIG. 8: shows the primary amino acid sequence of lytically by a protease. The peptide bond that is hydrolyzed caspase 7 as a member of the cysteine protease class C14 is referred to as the “cleavage Site”. Numbering of positions family (see also SEQ ID NO: 14). in the Substrate is done according to the System introduced 0.062 FIG. 9 depicts schematically the third aspect of the by Schlechter & Berger (Biochem. Biophys. Res. Commun. invention. 27 (1967) 157-162). Amino acid residues adjacent N-termi nal to the cleavage site are numbered P, P., P., etc., 0063 FIG. 10 shows a Western blot analysis of a culture whereas residues adjacent C-terminal to the cleavage Site are Supernatant of cells expressing variants of human trypsin I with SDR1 and SDR2, compared to negative controls. numbered P', P, P, etc. 0075. The term “target substrate” describes a user-de 0.064 FIG. 11 shows the time course of the proteolytic fined Substrate which is specifically recognized and con cleavage of a target Substrate by human trypsin I. verted by an enzyme according to the invention. The term 0065 FIG. 12 shows the relative activities of three “target peptide Substrate' describes a user-defined peptide variants of inventive engineered proteolytic enzymes in Substrate. The term “target specificity' describes the quali comparison with human trypsin I on two different peptide tative and quantitative Specificity of an enzyme that is Substrates. capable of recognizing and converting a target Substrate. 0.066 FIG. 13 shows the relative specificities of human 0076 Catalytic properties of enzymes are expressed trypsin I and variants of inventive engineered proteolytic using the kinetic parameters “KM or “Michaelis Menten enzymes with one or two SDRs, respectively. constant”, “k” or “catalytic rate constant, and “k/K” 0067 FIG. 14: shows the relative specificities of human or “catalytic efficiency', according to the definitions of trypsin I and of variants of inventive engineered proteolytic Michaelis and Menten (Fersht, A., Enzyme Structure and enzymes being specific for human TNF-alpha with this Mechanism, W. H. Freeman and Company, New York, Scaffold on peptides with a target Sequence of human TNF 1995). The term “catalytic activity” describes quantitatively alpha. the conversion of a given Substrate under defined reaction conditions. 0068 FIG. 15: shows the reduction of cytotoxicity induced by TNF-alpha when incubating the TNF-alpha with 0077. The term “specificity” means the ability of an concentrated Supernatant from cultures expressing the enzyme to recognize and convert preferentially certain Sub inventive engineered proteolytic enzymes being Specific for Strates. Specificity can be expressed qualitatively and quan human TNF-alpha. titatively. “Oualitative specificity” refers to the chemical nature of the Substrate residues that are recognized by an 0069 FIG. 16: shows the reduction of cytotoxicity enzyme. “Quantitative specificity” refers to the number of induced by TNF-alpha when incubating the TNF-alpha with Substrates that are accepted as Substrates. Quantitative Speci purified inventive engineered proteolytic enzyme being spe ficity can be expressed by the terms, which is defined as the cific for human TNF-alpha. negative logarithm of the number of all accepted Substrates 0070 FIG. 17: compares the activity of inventive engi divided by the number of all possible substrates. Proteases, neered proteolytic enzymes being Specific for human TNF for example, that accept preferentially a Small portion of all alpha with the activity of human trypsin I on two protein possible peptide substrates have a “high specificity”. Pro substrates: (a) human TNF-alpha; (b) mixture of human teases that accept almost any peptide Substrate have a “low Serum proteins. specificity'. Definitions are made in accordance to WO 03/095670 which is therefore incorporated by reference. 0071 FIG. 18: showes the specific activity of an inven Proteases with very low specificity are also referred to as tive engineered proteolytic enzyme with Specificity for “unspecific proteases”. The term “defined specificity” refers human VEGF to a certain type of Specificity, i.e. to a certain target Subtrate or a set of certain target Substrates that are preferentially DEFINITIONS converted versus other Substrates. 0.072 In the framework of the present invention the 0078. The term “engineered” in combination with the following terms and definitions are used. term “enzyme” describes an enzyme that is comprised of 0073. The term “protease” means any protein molecule different components and that has features not being con that is capable of hydrolysing peptide bonds. This includes ferred by the individual components alone. US 2005/0175581 A1 Aug. 11, 2005

007.9 The term “protein scaffold” or “scaffold protein' refers to a variety of primary, Secondary and tertiary TABLE 1. polypeptide Structures. 0080. The term “peptide sequence” indicates any peptide Amino acid abbreviations Sequence used for insertion or Substitution into or combi Abbreviations Amino acid nation with a protein Scaffold. Peptide Sequences are usually A. Ala Alanine obtained by expression from DNA sequences which can be C Cys Cysteine Synthesized according to well-established techniqueS or can D Asp Aspartic acid be obtained from natural Sources. Insertion, Substitution or E Glu Glutamic acid F Phe Phenylalanine combination of peptide Sequences with the protein Scaffold G Gly Glycine are generated by insertion, Substitution or combination of H His Histidine oligonucleotides into or with a polynucleotide encoding the I Ile Isoleucine protein scaffold. The term “synthetic' in combination with K Lys Lysine L Leu Leucine the term "peptide Sequence” refers to peptide Sequences that M Met Methionine are not present in the protein Scaffold in which the peptide N Asn Asparagine Sequences are inserted or Substituted or with which they are P Pro Proline combined. O Glin Glutamine R Arg Arginine 0081. The term “components” in combination with the S Ser Serine term “engineered enzyme” refers to peptide or polypeptide T Thr Threonine Sequences that are combined in the engineering of Such V Val Valine enzymes. Such components may among others comprise one W Trp Tryptophane or more protein Scaffolds and one or more Synthetic peptide Y Tyr Tyrosine Sequences. The term “library of engineered enzymes' describes a mixture of engineered enzymes, whereby every Single engineered enzyme is encoded by a different poly DETAILED DESCRIPTION OF THE nucleotide Sequence. The term “gene library' indicates a INVENTION library of polynucleotides that encodes the library of engi 0086 The present invention provides engineered proteins neered enzymes. The term “SDR' or “Specificity determin with novel functions. In particular, the invention provides ing region” refers to a synthetic peptide sequence that enzymes with user-definable specificities. In a particular provides the defined specificity when combined with the embodiment, the invention provides proteases with user protein Scaffold at Sites that enable the resulting enzymes to definable Specificities. Besides, the invention provides appli discriminate between the target Substrate and one or more cations of Such enzymes with novel, user-definable Speci other Substrates. Such sites are termed "SDR sites”. ficities for therapeutic, research, diagnostic, nutritional, personal care or industrial purposes. Moreover, the inven 0082 The terms “tertiary structure similar to the structure tion provides a method for generating enzymes with Speci of and “similar tertiary structure” in combination with the ficities that are not present in the components used for the terms “enzyme” or “protein” refer to proteins in which the engineering of Such enzymes. In particular, the invention is type, Sequence, connectivity and relative orientation of the directed to the generation of enzymes that have Sequences typical Secondary Structural elements of a protein, e.g. that are essentially identical to mammalian, especially alpha-helices, beta-sheets, beta-turns and loops, are similar human enzymes but have different specificities. Moreover, and the proteins are therefore grouped into the same Struc the invention provides libraries of Specific engineered tural or topological class or fold. This includes proteins that enzymes with corresponding Specificities encoded geneti have altered, additional or deleted Structural elements of any cally, a method for the generation of libraries of Specific type but otherwise unchanged topology. Examples of Such engineered enzymes with corresponding Specificities structural classes are the TNF Superfamily, the S1 fold or the encoded genetically, and the application of Such libraries for S8 fold within the serine proteases, the GPCRs, or the technical, diagnostic, nutritional, personal care or research C/B-barrel fold. purposes. 0.083. The term “positions that correspond structurally' indicates amino acids in proteins of Similar tertiary Structure 0087. A first aspect of the invention is directed to the that correspond Structurally to each other, i.e. they are application of engineered enzymes with Specificities for usually located within the same Structural or topological therapeutic, research, diagnostic, nutritional, personal care element of the structure. Within the structural element they or industrial purposes. The application comprises at least the possess the same relative positions with respect to beginning following Steps: and end of the Structural element. If, e.g. the topological 0088 (a) identification of a target peptide substrate comparison of two proteins reveals two structurally corre whose hydrolysis has a positive effect in connection sponding Sequences of different length, then amino acids with the intended purpose, Such as curing a disease, within, e.g. 20% and 40% of the respective region lengths, diagnosing a disease, processing of ingredients for correspond to each other structurally. human or animal nutrition, or other technical pro 0084. The term “library of engineered enzymes” of the CeSSeS, present invention refers to a multiplicity of enzymes or 0089 (b) provision of an engineered enzyme, the enzyme variants, which may exist as a mixture or in isolated enzyme being Specific for the target peptide identi form. fied in Step (a); and 0085 Amino acids residues are abbreviated according to 0090 (c) use of the enzyme as provided in step (b) the following Table 1 either in one- or in three-letter code. for the intended purpose. US 2005/0175581 A1 Aug. 11, 2005

0.091 In a first variant of this aspect of the invention, the 0097. In a second embodiment of this variant the enzyme engineered enzyme is used as a therapeutic means to inac is a protease and is capable of hydrolysing human Tumor tivate a disease-related target Substrate. This application necrosis factor ligand Superfamily member 5 (hCD40-L). comprises at least the following Steps: The enzymes or the fusion protein can thus be used for preparing medicaments for the treatment of diseases, Such 0092 (a) identification of a target substrate whose as, but not limited to, Systemic lupus erythematosus and function is connected to a disease and whose inac classical Hodgkin's Lymphoma (cHL), as well as other tivation has a positive effect in connection with the diseases connected with hCD40-L. Preferably, said enzyme disease, and determination of a target Site within the or said fusion protein is capable of Specifically inactivating target Substrate characterized by the fact that modi hCD40-L (SEQ ID NO:143). More preferably said enzyme fication at the target Site leads to the inactivation of or said fusion protein is capable of hydrolysing the peptide the target Substrate; bonds between positions 133/134, 145/146, 165/166, 200/ 0093 (b) provision of an engineered enzyme, the 201, 201/202, 207/208 and/or 216/217 (most preferred enzyme being specific for the target Site identified in between positions 133/134, 165/166, 201/202 and/or 216/ Step (a); and 217) in hCD40-L, or a peptide bond in proximity to these positions in hCD40-L, or peptide bonds in protein targets 0094) (c) use of the enzyme for the inactivation of related to hCD40-L at positions having structural homology the target Substrate inside or outside the human body. or Sequence homology to these positions. 0.095 Preferably, the scaffold is a protease and the modi 0098. In a third embodiment of this variant the enzyme is fication is hydrolysis of a target Site in a protein target. a protease and is capable of hydrolysing human Macrophage Preferably, the hydrolysis leads to the activation or inacti migration inhibitory factor (hMIF). The enzymes or the Vation of the peptide or protein target. Potential peptide or fusion protein can thus be used for preparing medicaments protein targets include Soluble proteins, in particular cytok for the treatment of diseases, Such as, but not limited to, ines, Such as proteins of the TNF-Superfamily, interleukines, inflammatory diseases, as well as other diseases connected interferons, chemokines and growth factors, hormones, tox with hMIF. Preferably, said enzyme or said fusion protein is ins; enzymes, Such as oxidoreductases, transferases, hydro capable of specifically inactivating hMIF (SEQID NO:109). lases, lyases, isomerases and ligases, Structural proteins, More preferably Said engineered or said fusion protein is Such as collagen; immunoglobulins, activity modulating capable of hydrolysing the peptide bonds between positions proteins and DNA binding proteins, or membrane associated 16/17, 44/45, 66/67, 73/74, 77/78, 88/89, 92/93 and/or proteins, in particular Single pass transmembrane proteins, 100/101 (most preferred between positions 16/17 and/or multipass transmembrane proteins, Such as G-protein 92/93) in hMIF, or a peptide bond in proximity to these coupled receptors, ion channels and transporters, lipid positions in hMIF, or peptide bonds in protein targets related anchored membrane proteins and GPI-anchored membrane to hMIF at positions having Structural homology or proteins. Sequence homology to these positions. 0096. In a first embodiment of this variant the engineered enzyme is a protease and is capable of hydrolysing human 0099. In a fourth embodiment of this variant the enzyme tumor necrosis factor-alpha (hTNF-C). The enzymes or the is a protease and is capable of hydrolysing human Interleu fusion protein can thus be used for preparing medicaments kin-1beta precursor (hIL-1 beta). The enzymes or the fusion for the treatment of diseases, Such as, but not limited to, protein can thus be used for preparing medicaments for the rheumatoid arthritis, inflammatory bowel diseases, psoria treatment of diseases, Such as, but not limited to, diabetes, sis, Crohn's disease, Ulcerative colitis, diabetes type II, brain inflammation in cancer, arthritis, autoimmune and classical Hodgkin's Lymphoma (cHL), Grave's disease, inflammatory diseases, as well as other diseases connected Hashimoto's thyroiditis, Sjogren's Syndrome, Systemic with hiL-1 beta. Preferably, said enzyme or said fusion lupus erythematosus, multiple Sclerosis, Systemic inflam protein is capable of Specifically inactivating hL-1 beta matory response syndrome (SIRS) which leads to distant (SEQ ID NO:112). More preferably said enzyme or said organ damage and multiple organ dysfunction Syndrome fusion protein is capable of hydrolysing the peptide bonds (MODS), eosinophilia, neurodegenerative disease, Stroke, between positions 24/25, 35/36, 46/47, 54/55, 74/75, 75/76, closed head injury, encephalitis, CNS disorders, asthma, 76/77, 77/78, 86/87, 88/89, 93/94, 94/95, 97/98 and/or rheumatoid arthritis, Sepsis, vasodilation, intravascular 150/151 (most preferred between positions 35/36, 75/76, coagulation and multiple organ failure, as well as other 76/77,88/89,93/94, 94/95 and/or 150/151) in hiL-1 beta, or diseases connected with hTNF-C. Preferably, said enzyme a peptide bond in proximity to these positions in hD-1beta, or said fusion protein is capable of Specifically inactivating or peptide bonds in protein targets related to hill-1 beta at hTNF-C. (SEQ ID NO:96). More preferably said enzyme or positions having structural homology or Sequence homology Said fusion protein is capable of hydrolysing the peptide to these positions. bonds between positions 31/32, 32/33, 44/45, 45/46, 87/88, 0100. In a fifth embodiment of this variant the enzyme is 128/129, 130/131, 140/141 and/or 141/142 (most preferred a protease and is capable of hydrolysing human Interleukin between positions 31/32,32/33 and/or 45/46) in hTNF-D, or 2 (hIL-2). The enzymes or the fusion protein can thus be a peptide bond in proximity to these positions in hTNF-D, used for preparing medicaments for the treatment of dis or peptide bonds in protein targets related to hTNF eases, Such as, but not limited to, T-cell leukemia and hairy between positions having Structural homology or Sequence cell leukemia, Crohn's disease, Ulcerative colitis, Grave's homology to these positions. In this embodiment it is most disease, Hashimoto's thyroiditis, Sjogren's Syndrome, SyS preferred that the protease has the a sequence shown in SEQ temic lupus erythematosus, multiple Sclerosis, asthma and ID NO:74, SEQ ID NO:75 and is capable of hydrolysing chronic obstructive pulmonary and classical Hodgkin’s hTNF-C. at positions 31/32 and/or 32/33. Lymphoma (cHL), as well as other diseases connected with US 2005/0175581 A1 Aug. 11, 2005 hIL-2. Preferably, said enzyme or said fusion protein is protein targets related to h-5 at positions having Structural capable of specifically inactivating hIL-2 (SEQ ID NO:99). homology or Sequence homology to these positions. More preferably Said enzyme or Said fusion protein is 0104. In a ninth embodiment of this variant the enzyme capable of hydrolysing the peptide bonds between positions is a protease and is capable of hydrolysing human Interleu 20/21, 32/33, 38/39, 43/44, 45/46 48/49, 49/50, 54/55, kin-6 (hIL-6). The enzymes or the fusion protein can thus be 64/65, 76/77, 83/84, 84/85, 107/108, 109/110 and/or 120/ used for preparing medicaments for the treatment of dis 121 (most preferred between positions 109/110) in hiL-2, or eases, Such as, but not limited to, classical Hodgkin’s a peptide bond in proximity to these positions in hi-2, or Lymphoma (cHL), breast cancer, renal cell carcinoma, mul peptide bonds in protein targets related to hi-2 at positions tiple myeloma, lymphoma, leukemia, Grave's disease, having Structural homology or Sequence homology to these Hashimoto's thyroiditis, Sjogren's Syndrome, Systemic positions. lupus erythematosus, Systemic inflammatory response Syn 0101. In a sixth embodiment of this variant the enzyme is drome (SIRS) which leads to distant organ damage and a protease and is capable of hydrolysing human Interleukin multpile organ dysfunction syndrome (MODS), chronic 3 (hIL-3). The enzymes or the fusion protein can thus be obstructive pulmonary disease (COPD), Castleman's dis used for preparing medicaments for the treatment of dis eases, inflammatory bowel diseases, Crohn's disease, as eases, Such as, but not limited to, classical Hodgkin’s well as other diseases connected with hL-6. Preferably, said Lymphoma (cHL) and eosinophilia, as well as other diseases enzyme or Said fusion protein is capable of Specifically connected withh IL-3. Preferably, said enzyme or said fusion inactivating hiL-6 (SEQ ID NO:134). More preferably said protein is capable of specifically inactivating hiL-3 (SEQ ID enzyme or Said fusion protein is capable of hydrolysing the NO:148). More preferably said enzyme or said fusion pro peptide bonds between positions 32/33,35/36,55/56, 71/72, tein is capable of hydrolysing the peptide bonds between 129/130, 130/131, 132/133, 135/136, 141/142, 161/162, positions 21/22, 28/29, 36/37, 44/45, 46/47, 51/52, 63/64, 180/181 and/or 183/184 (most preferred between positions 66/67, 79/80, 94/95, 101/102,108/109 and/or 109/110 (most 135/136 and/or 141/142) in hiL-6, or a peptide bond in preferred between positions 21/22, 28/29, 46/47, 63/64, proximity to these positions in hD-6, or peptide bonds in 66/67, 79/80 and/or 101/102) in hIL-3, or a peptide bond in protein targets related to hi-6 at positions having Structural proximity to these positions in hi-3, or peptide bonds in homology or Sequence homology to these positions. protein targets related to hi-3 at positions having Structural 0105. In a tenth embodiment of this variant the enzyme is homology or Sequence homology to these positions. a protease and is capable of hydrolysing human Interleukin 0102) In a seventh embodiment of this variant the enzyme 8 (hIL-8). The enzymes or the fusion protein can thus be is a protease and is capable of hydrolysing human Interleu used for preparing medicaments for the treatment of dis kin 4 (hlL-4). The enzymes or the fusion protein can thus be eases, Such as, but not limited to, Crohn's disease, Ulcer used for preparing medicaments for the treatment of dis ative colitis, classical Hodgkin’s Lymphoma (cHL), Sys eases, Such as, but not limited to, classical Hodgkin’s temic inflammatory response syndrome (SIRS) which leads Lymphoma (cHL), Grave's disease, Hashimoto's thyroidi to distant organ damage and multiple organ dysfunction tis, Sjogren's Syndrome, Asthma, chronic obstructive pull syndrome (MODS), chronic obstructive pulmonary disease monary disease and allergic inflammatory reactions, as well (COPD), endometriosis, psoriasis and atherosclerotic as other diseases connected with hiL-4. Preferably, said lesions, as well as other diseases connected with hL-8. enzyme or Said fusion protein is capable of Specifically Preferably, Said enzyme or Said fusion protein is capable of inactivating hIL-4 (SEQ ID NO:118). More preferably said specifically inactivating hIL-8 (SEQ ID NO:100). More enzyme or Said fusion protein is capable of hydrolysing the preferably Said enzyme or Said fusion protein is capable of peptide bonds between positions 4/5, 12/13, 31/32, 37/38, hydrolysing the peptide bonds between positions 1/12, 61/62, 62/63, 64/65, 91/92, 102/103, 121/122 and/or 126/ 15/16, 45/46, 47/48, 52/53, 54/55, 60/61, 64/65 and/or 67/68 127 (most preferred between positions 4/5, 61/62, 62/63, (most preferred between positions 45/46) in hIL-8, or a 64/65 and/or 121/122) in hiL-4, or a peptide bond in peptide bond in proximity to these positions in hL-8, or proximity to these positions in hD-4, or peptide bonds in peptide bonds in protein targets related to hL-8 at positions protein targets related to hi-4 at positions having Structural having Structural homology or Sequence homology to these homology or Sequence homology to these positions. positions. 0103) In a eighth embodiment of this variant the enzyme 0106. In a eleventh embodiment of this variant the is a protease and is capable of hydrolysing human Interleu enzyme is a protease and is capable of hydrolysing human kin-5 (hIL-5). The enzymes or the fusion protein can thus be Interleukin-10 (hlL-10). The enzymes or the fusion protein used for preparing medicaments for the treatment of dis can thus be used for preparing medicaments for the treat eases, Such as, but not limited to, classical Hodgkin’s ment of diseases, Such as, but not limited to, classical Lymphoma (cHL), asthma, chronic obstructive pulmonary Hodgkin's Lymphoma (cHL) and diseases related to the disease, eosinophilia, allergic inflammatory diseases, as well Suppression of cytotoxic T-cells, as well as other diseases as other diseases connected with hiL-5. Preferably, said connected with hiL-10. Preferably, said enzyme or said enzyme or Said fusion protein is capable of Specifically fusion protein is capable of Specifically inactivating hill-10 inactivating hIL-5 (SEQ ID NO:133). More preferably said (SEQ ID NO:135). More preferably said enzyme or said enzyme or Said fusion protein is capable of hydrolysing the fusion protein is capable of hydrolysing the peptide bonds peptide bonds between positions 12/13,32/33, 67/68, 76/77, between positions 24/25, 25/26, 27/28, 28/29, 40/41,44/45, 77/78, 80/81, 83/84, 84/85, 85/86, 90/91, 91/92, 92/93 49/50, 57/58, 59/60, 84/85, 86/87, 106/107, 107/108, 110/ and/or 98/99 (most preferred between positions 90/91, 111, 130/131, 134/135, 137/138, 138/139 and/or 144/145 91/92, 92/93 and/or 98/99) in hIL-5, or a peptide bond in (most preferred between positions 24/25, 27/28, 44/45, proximity to these positions in h-5, or peptide bonds in 49/50, 86/87, 137/138 and/or 144/145) in hiL-10, or a US 2005/0175581 A1 Aug. 11, 2005

peptide bond in proximity to these positions in hill-10, or fusion protein can thus be used for preparing medicaments peptide bonds in protein targets related to hill-10 at positions for the treatment of diseases, Such as, but not limited to, having Structural homology or Sequence homology to these classical Hodgkin's Lymphoma (cHL), Crohn's disease and positions. type I diabetes, as well as other diseases connected with hIFN-gamma. Preferably, said enzyme or said fusion protein 0107. In a twelfth embodiment of this variant the enzyme is capable of specifically inactivating hIFN-gamma (SEQID is a protease and is capable of hydrolysing human Interleu NO:137). More preferably said enzyme or said fusion pro kin 12 beta chain (hIL-12B). The enzymes or the fusion tein is capable of hydrolysing the peptide bonds between protein can thus be used for preparing medicaments for the positions 2/3, 6/7, 13/14, 21/22, 24/25, 34/35, 36/37, 37/38, treatment of diseases, Such as, but not limited to, Crohn's 62/63, 68/69, 83/84, 86/87, 90/91, 102/103, 107/108 and/or disease and classical Hodgkin's Lymphoma (cHL), as well 108/109 (most preferred between positions 13/14, 24/25, as other diseases connected with hiL-12B. Preferably, said 37/38, 62/63, 68/69, 102/103 and/or 107/108) in hiFN enzyme or Said fusion protein is capable of Specifically gamma, or a peptide bond in proximity to these positions in inactivating hIL-12B (SEQID NO:97). More preferably said hIFN-gamma, or peptide bonds in protein targets related to enzyme or Said fusion protein is capable of hydrolysing the hIFN-gamma at positions having structural homology or peptide bonds between positions 14/15, 18/19, 29/30, 34/35, Sequence homology to these positions. 87/88, 99/100, 102/103, 104/105, 161/162, 174/175, 222/ 223, 225/226, 228/229, 238/239, 268/269 and/or 293/294 0111. In a sixteenth embodiment of this variant the (most preferred between positions 18/19, 34/35, 87/88 and/ enzyme is a protease and is capable of hydrolysing human or 161/162) in hiL-12B, or a peptide bond in proximity to Small inducible cytokine A2 (hCCL2). The enzymes or the these positions in hill-12?, or peptide bonds in protein fusion protein can thus be used for preparing medicaments targets related to hL-12B at positions having structural for the treatment of diseases, Such as, but not limited to, homology or Sequence homology to these positions. Crohn's disease and Ulcerative colitis, as well as other diseases connected with hCCL2. Preferably, said enzyme or 0108. In a thirteenth embodiment of this variant the Said fusion protein is capable of Specifically inactivating enzyme is a protease and is capable of hydrolysing human hCCL2 (SEQ ID NO:102). More preferably said enzyme or Interleukin 13 (hIL-13). The enzymes or the fusion protein Said fusion protein is capable of hydrolysing the peptide can thus be used for preparing medicaments for the treat bonds between positions 3/4, 13/14, 18/19, 19/20, 24/25, ment of cancer, Such as, but not limited to, classical 29/30, 38/39, 54/55, 56/57, 58/59, 62/63, 65/66 and/or 68/69 Hodgkin's Lymphoma (cHL), eosinophilia, asthma, chronic (most preferred between positions 19/20, 29/30, 38/39, obstructive pulmonary disease, fibrosis, psoriasis, atopic 54/55 and/or 62/63) in hCCL2, or a peptide bond in prox dermatitis and Ulcerative colitis, as well as other diseases imity to these positions in hCCL2, or peptide bonds in connected with hiL-13. Preferably, said enzyme or said protein targets related to hCCL2 at positions having Struc fusion protein is capable of Specifically inactivating hL-13 tural homology or Sequence homology to these positions. (SEQ ID NO:119). More preferably said enzyme or said fusion protein is capable of hydrolysing the peptide bonds 0112 In a seventeenth embodiment of this variant the between positions 25/26, 62/63, 65/66, 86/87, 87/88,98/99, enzyme is a protease and is capable of hydrolysing human 108/109 and/or 111/112 (most preferred between positions Eotaxin (hCCL11). The enzymes or the fusion protein can 87/88) in hIL-13, or a peptide bond in proximity to these thus be used for preparing medicaments for the treatment of positions in h-13, or peptide bonds in protein targets diseases, Such as, but not limited to, Crohn's disease and related to hD-13 at positions having Structural homology or Ulcerative colitis, classical Hodgkin's Lymphoma (cHL), Sequence homology to these positions. chronic pathophysiologic dysfunction, characterized by an influx mainly of Th2 cells, and eosinophilia, as well as other 0109. In a fourteenth embodiment of this variant the diseases connected with hCCL11. Preferably, said enzyme enzyme is a protease and is capable of hydrolysing human or said fusion protein is capable of Specifically inactivating Interleukin 18 (hIL-18). The enzymes or the fusion protein hCCL11 (SEQ ID NO:101). More preferably said enzyme or can thus be used for preparing medicaments for the treat Said fusion protein is capable of hydrolysing the peptide ment of diseases, Such as, but not limited to, Crohn's bonds between positions 11/12, 16/17, 17/18, 22/23, 27/28, disease, inflammation liver injuries, pulmonary tuberculosis, 33/34, 44/45, 47/48, 48/49, 52/53, 54/55, 56/57, 60/61, plural tuberculosis and rheumatoid arthritis, as well as other 66/67 and/or 73/74 (most preferred between positions 48/49 diseases connected with hL-18. Preferably, said enzyme or and/or 66/67) in hCCL11, or a peptide bond in proximity to Said fusion protein is capable of Specifically inactivating these positions in hCCL11, or peptide bonds in protein hIL-18 (SEQ ID NO: 98). More preferably said enzyme or targets related to hCCL11 at positions having Structural Said fusion protein is capable of hydrolysing the peptide homology or Sequence homology to these positions. bonds between positions 17/18, 32/33, 37/38, 39/40, 40/41, 53/54, 58/59, 79/80, 90/91, 93/94, 98/99, 110/111, 120/121, 0113. In an eighteenth embodiment of this variant the 123/124, 131/132, 132/133, 142/143, 147/148 and/or 157/ enzyme is a protease and is capable of hydrolysing human 158 (most preferred between positions 37/38, 132/133, Vascular endothelial growth factor (hVEGF). The enzymes 142/143 and/or 157/158) in hIL-18, or a peptide bond in or the fusion protein can thus be used for preparing medi proximity to these positions in hL-18, or peptide bonds in caments for the treatment of diseases, Such as, but not protein targets related to hL-18 at positions having Struc limited to, all Solid tumors and metastatic Solid tumors, tural homology or Sequence homology to these positions. inflammatory breast cancer, as well as other diseases con nected with hVEGF. Preferably, said enzyme or said fusion 0110. In a fifteenth embodiment of this variant the protein is capable of specifically inactivating hVEGF (SEQ enzyme is a protease and is capable of hydrolysing human ID NO:103). More preferably said enzyme or said fusion Interferon-gamma (hIFN-gamma). The enzymes or the protein is capable of hydrolysing the peptide bonds between US 2005/0175581 A1 Aug. 11, 2005

positions 16/17, 19/20, 23/24, 34/35, 41/42, 56/57, 62/63, 0117. In a twenty-second embodiment of this variant the 63/64, 64/65, 65/66, 82/83, and/or 84/85 (most preferred enzyme is a protease and is capable of hydrolysing human between positions 23/24, 41/42, 63/64, 82/83 and/or 84/85) Hepatocyte growth factor (hPIGF). The enzymes or the in hVEGF, or a peptide bond in proximity to these positions fusion protein can thus be used for preparing medicaments in hVEGF, or peptide bonds in protein targets related to for the treatment of diseases, Such as, but not limited to, hVEGF at positions having Structural homology or Sequence angiogenic disorders and hepatocellular carcinoma, as well homology to these positions. as other diseases connected with hHGF. Preferably, said 0114. In an ninteenth embodiment of this variant the enzyme or Said fusion protein is capable of Specifically enzyme is a protease and is capable of hydrolysing human inactivating hHGF (SEQ ID NO:120). More preferably said Transforming growth factor beta 1 (hTGF-f1). The enzymes enzyme or Said fusion protein is capable of hydrolysing the or the fusion protein can thus be used for preparing medi peptide bonds between positions 54/55, 60/61, 62/63, 63/64, caments for the treatment of diseases, Such as, but not 68/69, 76/77, 112/113, 123/124, 134/135, 168/169, 198/199 limited to, a variety of cancers, including breast cancer, and/or 202/203 (most preferred between positions 63/64, colorectal cancer and classical Hodgkin's Lymphoma 68/69, 76/77, 168/169 and/or 202/203) in hHGF, or a peptide (cHL), fibrosis, Suppression of cell-mediated immunity, bond in proximity to these positions in hHGF, or peptide glaucoma, diffuse Systemic Sclerosis as well as other dis bonds in protein targets related to hCGF at positions having eases connected with hTGF-Y1. Preferably, said enzyme or Structural homology or Sequence homology to these posi Said fusion protein is capable of Specifically inactivating tions. hTGF-?31. (SEQ ID NO:104). More preferably said enzyme 0118. In a twenty-third embodiment of this variant the or Said fusion protein is capable of hydrolysing the peptide enzyme is a protease and is capable of hydrolysing human bonds between positions 23/24, 25/26, 26/27, 27/28, 37/38, hInsulin (hInsulin). The enzymes or the fusion protein can 55/56 and/or 94/95 (most preferred between positions 25/26, thus be used for preparing medicaments for the treatment of 55/56 and/or 94/95) in hTGF-31, or a peptide bond in diseases, Such as, but not limited to, insulin overdosage, as proximity to these positions in hTGF-31, or peptide bonds well as other diseases connected with hinsulin. Preferably, in protein targets related to hTGF-B1 at positions having Said enzyme or Said fusion protein is capable of Specifically Structural homology or Sequence homology to these posi inactivating hInsulin B chain (SEQ ID NO:105) and/or tions. hInsulin A chain (SEQ ID NO:106). More preferably said enzyme or Said fusion protein is capable of hydrolysing the 0115) In a twentieth embodiment of this variant the enzyme is a protease and is capable of hydrolysing human peptide bonds between positions 16/17 and/or 22/23 in Somatotropin (human Growth hormone; hoH). The hInsulin B and/or between position 14/15 in Insulin A, or a enzymes or the fusion protein can thus be used for preparing peptide bond in proximity to these positions in hinsulin A or medicaments for the treatment of diseases, Such as, but not B, or peptide bonds in protein targets related to hinsulin A limited to, acromegaly, diabetes and diabetic kidney disease or B at positions having Structural homology or Sequence including renal hypertrophy and glomerular enlargement homology to these positions. and cardiovascular disorders, as well as other diseases 0119). In a twenty-fourth embodiment of this variant the connected with hCH. Preferably, said enzyme or said fusion enzyme is a protease and is capable of hydrolysing human protein is capable of specifically inactivating hCGH (SEQ ID hGhrelin (hGhrelin). The enzymes or the fusion protein can NO:121). More preferably said enzyme or said fusion pro thus be used for preparing medicaments for the treatment of tein is capable of hydrolysing the peptide bonds between diseases, Such as, but not limited to, obesity, as well as other positions 8/9, 16/17, 19/20, 26/27, 33/34, 38/39, 41/42, diseases connected with hChrelin. Preferably, said enzyme 70/71, 77/78, 94/95, 103/104, 112/113, 115/116, 116/117, or said fusion protein is capable of Specifically inactivating 130/131, 147/148, 154/155 and/or 178/179 (most preferred hChrelin (SEQ ID NO:107). More preferably said enzyme between positions 112/113, 147/148 and/or 154/155) in or said fusion protein is capable of hydrolysing the peptide hGH, or a peptide bond in proximity to these positions in bonds between positions 1/2, 2/3, 3/4 and/or 4/5 in hChrelin, hGH, or peptide bonds in protein targets related to hCGH at or a peptide bond in proximity to these positions in hChrelin, positions having structural homology or Sequence homology or peptide bonds in protein targets related to hChrelin at to these positions. positions having structural homology or Sequence homology 0116. In a twenty-first embodiment of this variant the to these positions. enzyme is a protease and is capable of hydrolysing human 0120 In a twenty-fifth embodiment of this variant the Insulin-like growth factor II (hIGF-II). The enzymes or the enzyme is a protease and is capable of hydrolysing human fusion protein can thus be used for preparing medicaments angiotensinogen (angiotensin). The enzymes or the fusion for the treatment of diseases, Such as, but not limited to, protein can thus be used for preparing medicaments for the diabetes and diabetic kidney disease, as well as other dis treatment of diseases, Such as, but not limited to, essential eases connected with hiGF-II. Preferably, said enzyme or hypertension, as well as other diseases connected with Said fusion protein is capable of Specifically inactivating angiotensin. Preferably, Said enzyme or Said fusion protein hIGF-II (SEQ ID NO:122). More preferably said enzyme or is capable of specifically inactivating angiotensin (SEQ ID Said fusion protein is capable of hydrolysing the peptide NO:108). More preferably said enzyme or said fusion pro bonds between positions 15/16, 23/24, 24/25, 34/35, 37/38, tein is capable of hydrolysing the peptide bonds between 38/39, 48/49 and/or 49/50 (most preferred between positions positions 1/2, 3/4 and/or 7/8 (most preferred between posi 23/24) in hiGF-II, or a peptide bond in proximity to these tions 3/4) in angiotensin, or a peptide bond in proximity to positions in hCF-II, or peptide bonds in protein targets these positions in angiotensin, or peptide bonds in protein related to hCF-II at positions having Structural homology or targets related to angiotensin at positions having Structural Sequence homology to these positions. homology or Sequence homology to these positions. US 2005/0175581 A1 Aug. 11, 2005

0121. In a twenty-sixth embodiment of this variant the targets related to thrombin at positions having Structural enzyme is a protease and is capable of hydrolysing human homology or Sequence homology to these positions leptin precursor (leptin). The enzymes or the fusion protein 0.125. In a thirty embodiment of this variant the enzyme can thus be used for preparing medicaments for the treat is a protease and is capable of hydrolysing human beta ment of diseases, Such as, but not limited to, obesity, as well Secretase. The enzymes or the fusion protein can thus be as other diseases connected with leptin. Preferably, said used for preparing medicaments for the treatment of dis enzyme or Said fusion protein is capable of Specifically eases, Such as, but not limited to, Alzheimer, as well as other inactivating leptin (SEQ ID NO: 127). More preferably said diseases connected with human beta-Secretase precursor. enzyme or Said fusion protein is capable of hydrolysing the Preferably, Said enzyme or Said fusion protein is capable of peptide bonds between positions 8/9, 9/10, 15/16, 23/24, Specifically inactivating human beta-Secretase precursor 40/41, 53/54, 71/72, 85/86, 94/95, 108/109 and/or 141/142 (SEQ ID NO:139). More preferably said enzyme or said (most preferred between positions 9/10, 40/41, 71/72, 94/95 fusion protein is capable of hydrolysing the peptide bonds and/or 108/109) in leptin, or a peptide bond in proximity to between positions 61/62, 64/65, 159/160,238/239,239/240, these positions in leptin, or peptide bonds in protein targets 246/247, 256/257, 330/331 and/or 365/366 (most preferred related to leptin at positions having structural homology or between positions 61/62, 246/247 and/or 365/366) in human Sequence homology to these positions. beta-Secretase precursor, or a peptide bond in proximity to these positions in human beta-Secretase precursor, or peptide 0122) In a twenty-seventh embodiment of this variant the bonds in protein targets related to human beta-Secretase enzyme is a protease and is capable of hydrolysing Protec precursor at positions having Structural homology or tive antigen (PA-83). The enzymes or the fusion protein can Sequence homology to these positions. thus be used for preparing medicaments for the treatment of diseases, Such as, but not limited to, anthrax infection, as 0.126 In a thirty-first embodiment of this variant the well as other diseases connected with PA-83. Preferably, enzyme is a protease and is capable of hydrolysing human Said enzyme or Said fusion protein is capable of Specifically matrix metalloproteinase-2 (hMMP-2). The enzymes or the inactivating PA-83 (SEQ ID NO:123). More preferably said fusion protein can thus be used for preparing medicaments enzyme or Said fusion protein is capable of hydrolysing the for the treatment of diseases, Such as, but not limited to, a peptide bonds between positions 72/73, 73/74,92/93, 93/94, variety of cancers including bladder cancer, breast tumor 131/132, 149/150, 178/179, 213/214, 214/215, 387/388, cancer, gastric cancer and lung cancer, as well as other 425/426, 426/427, 427/428, 453/454, 520/521, 608/609, diseases connected with hMMP-2. Preferably, said enzyme 617/618, 671/672, 679/680, 680/681, 683/684 and/or 684/ or said fusion protein is capable of Specifically inactivating 685 (most preferred between positions 72/73, 73/74,93/94, hMMP-2 (SEQ ID NO:131). More preferably said enzyme 149/150, 387/388, 425/426, 427/428 and/or 683/684) in or said fusion protein is capable of hydrolysing the peptide PA-83, or a peptide bond in proximity to these positions in bonds between positions 62/63, 68/69, 75/76, 76/77, 79/80, PA-83, or peptide bonds in protein targets related to PA-83 88/89, 110/111, 112/113, 115/116, 120/121, 164/165, 254/ at positions having Structural homology or Sequence homol 255, 267/268, 296/297, 324/325, 325/326, 382/383,383/ ogy to these positions. 384, 470/471, 500/501, 550/551, 564/565, 595/596, 597/ 598, 608/609, 646/647, 649/650 and/or 650/651 (most 0123. In a twenty-eighth embodiment of this variant the preferred between positions 68/69, 115/116, 120/121, 164/ enzyme is a protease and is capable of hydrolysing human 165,325/326,383/384,470/471,500/501, 595/596,608/609 plasminogen (plasminogen). The enzymes or the fusion and/or 650/651) in hMMP-2, or a peptide bond in proximity protein can thus be used for preparing medicaments for the to these positions in hMMP-2, or peptide bonds in protein treatment of diseases, Such as, but not limited to, thrombosis, targets related to hMMP-2 at positions having structural as well as other diseases connected with plasminogen. homology or Sequence homology to these positions. Preferably, Said enzyme or Said fusion protein is capable of specifically inactivating plasminogen (SEQ ID NO:140). 0127. In a thirty-second embodiment of this variant the More preferably Said enzyme or Said fusion protein is enzyme is a protease and is capable of hydrolysing human capable of hydrolysing the peptide bond between position matrix metalloproteinase-9 (hMMP-9). The enzymes or the 580/581 in plasminogen, or a peptide bond in proximity to fusion protein can thus be used for preparing medicaments this position in plasminogen, or peptide bonds in protein for the treatment of diseases, Such as, but not limited to, a targets related to plasminogen at positions having structural variety of cancers including bladder cancer, breast tumor homology or Sequence homology to these positions. cancer, gastric cancer and lung cancer, as well as other diseases connected with hMMP-9. Preferably, said enzyme 0.124. In a twenty-ninth embodiment of this variant the or said fusion protein is capable of Specifically inactivating enzyme is a protease and is capable of hydrolysing human hMMP-9 (SEQ ID NO:132). More preferably said enzyme Prothrombin (thrombin). The enzymes or the fusion protein or said fusion protein is capable of hydrolysing the peptide can thus be used for preparing medicaments for the treat bonds between positions 41/42.42/43, 106/107, 113/114, ment of diseases, Such as, but not limited to, bleeding, as 134/135, 160/161, 162/163, 163/164, 222/223, 226/227, well as other diseases connected with thrombin. Preferably, 265/266, 266/267, 267/268, 284/285, 309/310, 321/322, Said enzyme or Said fusion protein is capable of Specifically 322/323, 324/325, 356/357, 380/381, 433/434 and/or 440/ inactivating thrombin (SEQ ID NO:149). More preferably 441 (most preferred between positions 160/161, 163/164, Said enzyme or Said fusion protein is capable of hydrolysing 226/227, 284/285, 321/322, 322/323 and/or 433/434) in the peptide bonds between positions 198/199, 327/328, hMMP-9, or a peptide bond in proximity to these positions 363/364 (most preferred between positions 327/328 and/or in hMMP-9, or peptide bonds in protein targets related to 363/364) in thrombin, or a peptide bond in proximity to hMMP-9 at positions having structural homology or these positions in thrombin, or peptide bonds in protein Sequence homology to these positions. US 2005/0175581 A1 Aug. 11, 2005

0128. In a thirty-third embodiment of this variant the as, but not limited to, Solid tumors and metastatic Solid enzyme is a protease and is capable of hydrolysing HIV tumors, astrocytic brain tumors, pancreatic cancer, meta membrane glycoprotein (GP120). The enzymes or the fusion Static renal cancer, metastatic Solid tumors, as well as other protein can thus be used for preparing medicaments for the diseases connected with hVEGFR 1. Preferably, said treatment of diseases, Such as, but not limited to, AIDS or enzyme or Said fusion protein is capable of Specifically HIV infection, as well as other diseases connected with inactivating hVEGFR 1 (SEQID NO: 114). More preferably GP120 or HIV infection. Preferably, said enzyme or said Said enzyme or Said fusion protein is capable of hydrolysing fusion protein is capable of specifically inactivating GP120 the peptide bonds between positions 189/190, 190/191, (SEQ ID NO:124). More preferably said enzyme or said 224/225 and/or 331/332 (most preferred between positions fusion protein is capable of hydrolysing the peptide bonds 189/190 and/or 331/332) in hVEGFR1, or a peptide bond in between positions 97/98, 99/100, 107/108, 113/114, 117/ proximity to these positions in hVEGFR 1, or peptide bonds 118, 227/228, 231/233, 279/280, 335/336, 337/338, 368/ in protein targets related to hVEGFR 1 at positions having 369, 412/413, 419/420, 429/430, 444/445, 457/458, 474/ Structural homology or Sequence homology to these posi 475, 476/477, 477/478, 485/486 and/or 490/491 (most tions. preferred between positions 99/100,368/369, 412/413,419/ 0.132. In a thirty-seventh embodiment of this variant the 420, 444/445 and/or 490/491) in GP120, or a peptide bond enzyme is a protease and is capable of hydrolysing human in proximity to these positions in GP120, or peptide bonds Vascular endothelial growth factor receptor 2 (hVEGFR 2). in protein targets related to GP120 at positions having The enzymes or the fusion protein can thus be used for Structural homology or Sequence homology to these posi preparing medicaments for the treatment of diseases, Such tions. as, but not limited to, Solid tumors and metatstatic Solid 0129. In a thirty-fourth embodiment of this variant the tumors, pancreatic cancer, metastatic renal cancer, meta enzyme is a protease and is capable of hydrolysing human Static CRC, as well as other diseases connected with Cytotoxic T-lymphocyte protein 4 (hCTLA-4). The enzymes hVEGFR 2. Preferably, said enzyme or said fusion protein or the fusion protein can thus be used for preparing medi is capable of specifically inactivating hVEGFR 2 (SEQ ID caments for the treatment of diseases, Such as, but not NO:115). More preferably said enzyme or said fusion pro limited to, breast cancer, as well as other diseases connected tein is capable of hydrolysing the peptide bonds between with hCTLA-4. Preferably, said enzyme or said fusion positions 214/215, and/or 323/324 (most preferred between protein is capable of Specifically inactivating hCTLA-4 position 214/215) in hVEGFR 2, or a peptide bond in (SEQ ID NO: 144). More preferably said enzyme or said proximity to these positions in hVEGFR 2, or peptide bonds fusion protein is capable of hydrolysing the peptide bonds in protein targets related to hVEGFR 2 at positions having between positions 14/15, 28/29, 33/34, 38/39, 41/42, 62/63, Structural homology or Sequence homology to these posi 72/73, 85/86, 95/96, 100/101, 105/106, 119/120, 125/126 tions. and/or 127/128 (most preferred between positions 14/15, 28/29, 38/39, 41/42, 62/63 and/or 85/86) in hCTLA-4, or a 0133. In a thirty-eighth embodiment of this variant the peptide bond in proximity to these positions in hCTLA-4, or enzyme is a protease and is capable of hydrolysing human peptide bonds in protein targets related to hCTLA-4 at Epidermal growth factor receptor (hEGFr). The enzymes or positions having structural homology or Sequence homology the fusion protein can thus be used for preparing medica to these positions. ments for the treatment of diseSaes, Such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colorectal 0130. In a thirty-fifth embodiment of this variant the cancer, endometrial cancer, oesophageal cancer, head and enzyme is a protease and is capable of hydrolysing human neck cancer, gastric cancer, non-Small-cell lung carcinoma Integrin alpha-2 (hVLA-2). The enzymes or the fusion and ovarian cancer, as well as other diseases connected with protein can thus be used for preparing medicaments for the hEGFr. Preferably, said enzyme or said fusion protein is treatment of diseases, Such as, but not limited to, renal capable of specifically inactivating hEGFr (SEQ ID tumors, uveal melanomas and gastrointestinal tumors, as NO:116). More preferably said enzyme or said fusion pro well as other diseases connected with hVLA-2. Preferably, tein is capable of hydrolysing the peptide bonds between Said enzyme or Said fusion protein is capable of Specifically positions 20/21, 29/30, 48/49, 74/75, 165/166, 202/203, inactivating hVLA-2 (SEQ ID NO:147). More preferably 220/221, 246/247, 251/252, 269/270, 270/271, 304/305, Said enzyme or Said fusion protein is capable of hydrolysing 305/306, 357/358, 430/431, 443/444, 454/455, 455/456, the peptide bonds between positions 160/161, 174/175, 463/464, 465/466, 476/477, 507/508 and/or 509/510 in 201/202, 219/220, 231/232, 232/233, 233/234, 243/244, hEGFr, or a peptide bond in proximity to these positions in 259/260, 264/265, 268/269, 288/289, 292/293, 294/295, hEGFr, or peptide bonds in protein targets related to hEGFr 298/299, 301/302,310/311 and/or 317/318 (most preferred at positions having Structural homology or Sequence homol between positions 160/161, 174/175, 201/202, 219/220, ogy to these positions. 243/244, 264/265, 292/293 and/or 294/295) in hVLA-2, or a peptide bond in proximity to these positions in hVLA-2, or 0.134. In a thirty-ninth embodiment of this variant the peptide bonds in protein targets related to hVLA-2 at posi enzyme is a protease and is capable of hydrolysing human tions having Structural homology or Sequence homology to Epithelial cell adhesion molecule (hEp-CAM). The enzymes these positions. or the fusion protein can thus be used for preparing medi caments for the treatment of diseases, Such as, but not 0131. In a thirty-sixth embodiment of this variant the limited to, colorectal cancer, as well as other diseases enzyme is a protease and is capable of hydrolysing human connected with hEp-CAM. Preferably, said enzyme or said Vascular endothelial growth factor receptor 1 (hVEGFR 1). fusion protein is capable of Specifically inactivating hEp The enzymes or the fusion protein can thus be used for CAM (SEQ ID NO:125). More preferably said enzyme or preparing medicaments for the treatment of diseases, Such Said fusion protein is capable of hydrolysing the peptide US 2005/0175581 A1 Aug. 11, 2005

bonds between positions 14/15, 19/20, 25/26, 30/31, 33/34, Integrin alpha-L (hCD11a). The enzymes or the fusion 55/56 an/or 70/71 (most preferred between positions 14/15, protein can thus be used for preparing medicaments for the 30/31 and/or 70/71) in hEp-CAM, or a peptide bond in treatment of diseases, Such as, but not limited to, pSoriasis as proximity to these positions in hEp-CAM, or peptide bonds well as other diseases connected with hCD11a. Preferably, in protein targets related to hEp-CAM at positions having Said enzyme or Said fusion protein is capable of Specifically Structural homology or Sequence homology to these posi inactivating hCD11a (SEQ ID NO: 130). More preferably tions. Said enzyme or Said fusion protein is capable of hydrolysing the peptide bonds between positions 145/146, 152/153, 0135) In a forty embodiment of this variant the enzyme is 156/157, 159/160, 160/161, 177/178, 178/179, 189/190, a protease and is capable of hydrolysing human Insulin-like 190/191, 191/192, 193/194, 197/198, 200/201, 221/222, growth factor I receptor (hIGF-1r). The enzymes or the 229/230, 249/250, 253/254, 268/269, 290/291, 297/298, fusion protein can thus be used for preparing medicaments 304/305 and/or 305/306 (most preferred between positions for the treatment of diseases, Such as, but not limited to, a 145/146, 159/160, 160/161, 189/190, 229/230, 249/250, variety of cancers including breast cancer, as well as other 268/269,297/298,304/305 and/or 305/306) in hCD11a, or a diseases connected with hiGF-1 r. Preferably, said enzyme or peptide bond in proximity to these positions in hCD11a, or Said fusion protein is capable of Specifically inactivating peptide bonds in protein targets related to hCD11a at posi hIGF-1r (SEQ ID NO:126). More preferably said enzyme or tions having Structural homology or Sequence homology to Said fusion protein is capable of hydrolysing the peptide these positions. bonds between positions 59/60, 115/116, 146/147, 171/172, 191/192, 290/291, 306/307, 307/308, 335/336, 336/337, 0.139. In a forty-fourth embodiment of this variant the 455/456 and/or 470/471 (most preferred between positions enzyme is a protease and is capable of hydrolysing human 306/307, 307/308,335/336 and/or 470/471) in hiGF-1r, or a Interferon-gamma receptor alpha chain (hIFN-gamma-R1). peptide bond in proximity to these positions in hCGF-1r, or The enzymes or the fusion protein can thus be used for peptide bonds in protein targets related to hCGF-1r at posi preparing medicaments for the treatment of diseases, Such tions having Structural homology or Sequence homology to as, but not limited to, classical Hodgkin's Lymphoma (cHL) these positions. and type I diabetes, as well as other diseases connected with hIFN-gamma-R1. Preferably, said enzyme or said fusion 0136. In a forty-first embodiment of this variant the protein is capable of Specifically inactivating hEN-gamma enzyme is a protease and is capable of hydrolysing human R1 (SEQ ID NO:136). More preferably said enzyme or said T-cell Surface antigen CD2 precursor (hCD2). The enzymes fusion protein is capable of hydrolysing the peptide bonds or the fusion protein can thus be used for preparing medi between positions 49/50, 52/53, 62/63, 106/107, 122/123, caments for the treatment of diseases, Such as, but not 174/175, 215/216 and/or 222/223 (most preferred between limited to, pSoriasis, as well as other diseases connected with positions 49/50, 122/123, 174/175 and/or 215/216) in hiFN hCD2. Preferably, said enzyme or said fusion protein is gamma-R1, or a peptide bond in proximity to these positions capable of specifically inactivating hCD2 (SEQ ID in hEN-gamma-R1, or peptide bonds in protein targets NO:128). More preferably said enzyme or said fusion pro related to hEN-gamma-R1 at positions having Structural tein is capable of hydrolysing the peptide bonds between homology or Sequence homology to these positions. positions 42/43, 43/44, 48/49, 49/50.51/52, 54/55, 63/64, 69/70, 89/90 and/or 91/92 (most preferred between positions 0140. In a forty-fifth embodiment of this variant the 43/44, 51/52 and/or 89/90) in hCD2, or a peptide bond in enzyme is a protease and is capable of hydrolysing human proximity to these positions in hCD2, or peptide bonds in Platelet membrane glycoprotein IIb/IIIa (hGPIb/IIIa). The protein targets related to hCD2 at positions having Structural enzymes or the fusion protein can thus be used for preparing homology or Sequence homology to these positions. medicaments for the treatment of diseases, Such as, but not limited to, unstable angina, carotid Stenting, ischemic Stroke, 0.137 In a forty-second embodiment of this variant the peripheral vascular diseases, angiogenesis-related diseases enzyme is a protease and is capable of hydrolysing human and disseminating tumors, as well as other diseases con T-cell surface glycoprotein CD4 (hCD4). The enzymes or nected with hCGPIb/IIIa. Preferably, said enzyme or said the fusion protein can thus be used for preparing medica fusion protein is capable of specifically inactivating hCGPIIb/ ments for the treatment of diseases, Such as, but not limited IIIa (SEQID NO:141). More preferably said enzyme or said to, pSoriasis, transplant rejection, graft-Versus-host colitis, fusion protein is capable of hydrolysing the peptide bonds autoimmune disorders and rheumatoid arthritis, as well as between positions 67/68,91/92, 129/130, 143/144, 144/145, other diseases connected with hCD4. Preferably, said 181/182, 208/209, 209/210, 216/217, 239/240, 261/262, enzyme or Said fusion protein is capable of Specifically 410/411,532/533,556/557,557/558,597/598,650/651 and/ inactivating hCD4 (SEQ ID NO:129). More preferably said or 689/690 (most preferred between positions 67/68, 261/ enzyme or Said fusion protein is capable of hydrolysing the 262, 410/411, 650/651 and/or 689/690) in hoPIIb/IIIa, or a peptide bonds between positions 166/167, 167/168, 206/ peptide bond in proximity to these positions in hCGPIb/IIIa, 207, 219/220, 224/225, 226/227, 251/252, 252/253, 322/ or peptide bonds in protein targets related to hCGPIb/IIIa at 323,329/330 and/or 334/335 (most preferred between posi positions having structural homology or Sequence homology tions 206/207,219/220, 251/252 and/or 252/253) in hCD4, to these positions. or a peptide bond in proximity to these positions in hCD4, or peptide bonds in protein targets related to hCD4 at 0.141. In a forty-sixth embodiment of this variant the positions having structural homology or Sequence homology enzyme is a protease and is capable of hydrolysing human to these positions. Intercellular adhesion molecule-1 (hICAM-1). The enzymes or the fusion protein can thus be used for preparing medi 0.138. In a forty-third embodiment of this variant the caments for the treatment of diseases, Such as, but not enzyme is a protease and is capable of hydrolysing human limited to, Crohn's disease, as well as other diseases con US 2005/0175581 A1 Aug. 11, 2005 nected with hiCAM-1. Preferably, said enzyme or said 0145. In a fifth embodiment of this variant the enzyme is fusion protein is capable of Specifically inactivating a protease and is capable of hydrolysing human Protease hICAM-1 (SEQ ID NO: 142). More preferably said enzyme activated receptor 2 (hPAR2). The enzymes or the fusion or Said fusion protein is capable of hydrolysing the peptide protein can thus be used for preparing medicaments for the bonds between positions 40/41, 88/89, 97/98, 102/103, treatment of diseases, Such as, but not limited to, Crohn's 128/129, 131/132, 132/133, 149/150, 150/151,160/161 and/ disease, Ulcerative colitis and Inflammatory bowel disease, or 166/167 (most preferred between positions 88/89, 102/ asthma, inflammation associated pain and arthritis, as well as other diseases connected with hPAR2. Preferably, said 103, 150/151, 160/161 and/or 166/167) in hICAM-1, or a enzyme or Said fusion protein is capable of Specifically peptide bond in proximity to these positions in hiCAM-1, or inactivating hPAR2 (SEQ ID NO:111). More preferably said peptide bonds in protein targets related to hiCAM-1 at enzyme or Said fusion protein is capable of hydrolysing the positions having structural homology or Sequence homology peptide bonds between positions 41/42, 51/52 and/or 59/60 to these positions. in hPAR2, or a peptide bond in proximity to these positions 0142. In a forty-seventh embodiment of this variant the in hPAR2, or peptide bonds in protein targets related to enzyme is a protease and is capable of hydrolysing human hPAR2 at positions having Structural homology or Sequence TGF-beta receptor type II (hTGF-beta RII). The enzymes or homology to these positions. the fusion protein can thus be used for preparing medica 0146 In a fifty-first embodiment of this variant the ments for the treatment of diseases, Such as, but not limited enzyme is a protease and is capable of hydrolysing human to, diffuse Systemic Sclerosis, as well as other diseases Protease activated receptor 4 (hPAR4). The enzymes or the connected with hTGF-beta RII. Preferably, said enzyme or fusion protein can thus be used for preparing medicaments Said fusion protein is capable of Specifically inactivating for the treatment of diseases, Such as, but not limited to, hTGF-beta RII (SEQ ID NO:145). More preferably said thrombosis, as well as other diseases connected with hPAR4. enzyme or Said fusion protein is capable of hydrolysing the Preferably, Said enzyme or Said fusion protein is capable of peptide bonds between positions 32/33,34/35,35/36, 66/67, specifically inactivating hPAR4 (SEQ ID NO:113). More 67/68, 69/70, 82/83, 103/104, 104/105, 105/106, 118/119, preferably Said enzyme or Said fusion protein is capable of 122/123 and/or 130/131 (most preferred between positions hydrolysing the peptide bonds between positions 68/69, 32/33, 34/35, 66/67, 69/70, 104/105, 122/123 and/or 130/ 131) in hTGF-beta RII, or a peptide bond in proximity to 74/75 and/or 78/79 in hPAR4, or a peptide bond in proximity these positions in hTGF-beta RII, or peptide bonds in protein to these positions in hPAR4, or peptide bonds in protein targets related to hTGF-beta RII at positions having struc targets related to hPAR4 at positions having structural tural homology or Sequence homology to these positions. homology or Sequence homology to these positions. 0147 In a fifty-second embodiment of this variant the 0143. In a forty-eighth embodiment of this variant the enzyme is a protease and is capable of hydrolysing human enzyme is a protease and is capable of hydrolysing human 5-hydroxytryptamine 1A receptor (h5-HT-1A). The Membrane cofactor protein (hMCP). The enzymes or the enzymes or the fusion protein can thus be used for preparing fusion protein can thus be used for preparing medicaments medicaments for the treatment of diseases, Such as, but not for the treatment of diseases, Such as, but not limited to, limited to, irritable bowel syndrome, as well as other dis renal tumors, uveal melanomas and gastrointestinal tumors, eases connected with hi5-HT-1A. Preferably, said enzyme or as well as other diseases connected with hMCP. Preferably, Said fusion protein is capable of Specifically inactivating Said enzyme or Said fusion protein is capable of Specifically h5-HT1A (SEQ ID NO:117). More preferably said enzyme inactivating hMCP (SEQ ID NO:146). More preferably said or said fusion protein is capable of hydrolysing the peptide enzyme or Said fusion protein is capable of hydrolysing the bonds between positions 101/102, 102/103, 181/182 and/or peptide bonds between positions 15/16, 17/18, 25/26, 31/32, 370/371 in hi5-HT1A a peptide bond in proximity to these 32/33, 35/36, 48/49, 67/68, 69/70, 110/111, 119/120 and/or positions in hi5-HT-1A, or peptide bonds in protein targets 125/126 (most preferred between positions 15/16, 32/33, related to hi5-HT-1A at positions having Structural homology 48/49, 119/120 and/or 125/126) 130/131) in hMCP, or a or Sequence homology to these positions. peptide bond in proximity to these positions in hMCP, or peptide bonds in protein targets related to hMCP at positions 0.148. In a fifty-third embodiment of this variant the having Structural homology or Sequence homology to these enzyme is a protease and is capable of hydrolysing human positions. carcinoembryonic antigen (hCEA). The enzymes or the fusion protein can thus be used for preparing medicaments 0144. In a forty-ninth embodiment of this variant the for the treatment of diseases, Such as, but not limited to, enzyme is a protease and is capable of hydrolysing human colon cancer, as well as other diseases connected with Protease activated receptor 1 (hPAR1). The enzymes or the hCEA. Preferably, said enzyme or said fusion protein is fusion protein can thus be used for preparing medicaments capable of specifically inactivating hCEA (SEQ ID for the treatment of diseases, Such as, but not limited to, NO:138). More preferably said enzyme or said fusion pro thrombosis, as well as other diseases connected with hPAR1. tein is capable of hydrolysing the peptide bonds between Preferably, Said enzyme or Said fusion protein is capable of positions 17/18, 69/70, 71/72, 74/75, 77/78,98/99, 116/117, specifically inactivating hPAR1 (SEQ ID NO:110). More 126/127 and/or 128/129 in hCEA, or a peptide bond in preferably Said enzyme or Said fusion protein is capable of proximity to these positions in hCEA, or peptide bonds in hydrolysing the peptide bonds between positions 46/47, protein targets related to hCEA at positions having Structural 51/52 and/or 52/53 in PAR1, or a peptide bond in proximity homology or Sequence homology to these positions. to these positions in hPAR1, or peptide bonds in protein targets related to hPAR1 at positions having structural 0149. In a fifty-fourth embodiment of this variant the homology or Sequence homology to these positions. enzyme is a protease and is capable of hydrolyzing human US 2005/0175581 A1 Aug. 11, 2005 interleukin-1 receptor type 1 (hIL-1R). The enzymes or the 0152. In a fifty-seventh embodiment of this variant the fusion proteins can thus be used for preparing medicaments enzyme is a protease and is capable of hydrolyzing human for the treatment of diseases, Such as, but not limited to, tumor necrosis factor receptor (hTNFR). The enzymes or the asthma, inflammation, rheumatic disorders, as well as other fusion proteins can thus be used for preparing medicaments diseases connected with hL-1R. Preferably, said enzyme or for the treatment of diseases, Such as, but not limited to, Said fusion protein is capable of Specifically inactivating asthma, Crohn's disease, HIV infection, inflammation, pSo hIL-1R (SEQ ID NO:150). More preferably said enzyme or riasis, rheumatoid arthritis, as well as other diseases con nected with hTNFR. Preferably, said enzyme or said fusion Said fusion protein is capable of hydrolysing the peptide protein is capable of specifically inactivating hTNFR (SEQ bonds between positions 35/36, 42/43, 43/44, 44/45, 46/47, ID NO:153). More preferably said enzyme or said fusion 56/57, 61/62, 72/73, 82/83, 98/99, 132/133, 137/138, 140/ protein is capable of hydrolysing the peptide bonds between 141, 145/146, 146/147, 148/149, 153/154, 171/172, 172/ positions 40/41, 49/50, 51/52, 53/54, 54/55, 56/57, 68/69, 173, 190/191, 202/203, 203/204, 205/206, 242/243, 245/ 75/76, 77/78, 78/79, 79/80, 84/85, 91/92, 99/100, 100/101, 246, 251/252, 252/253, 253/254, 254/255, 261/262, 262/ 107/108, 109/110, 131/132, 132/133, 147/148, 149/150, 263, 265/266, 271/272, 272/273, 283/284, 285/286, 287/ 157/158, 158/159 and/or 161/162 (most preferred between 288, 290/291 and/or 298/299 (most preferred between positions 40/41, 49/50, 54/55, 78/79, 84/85, 99/100, 107/ positions 44/45, 46/47, 52/53, 61/62, 137/138, 148/149, 108, 109/110, 132/133, 149/150 and/or 157/158) in hTNFR 153/154, 171/172, 172/173, 203/204, 252/253, 253/254, or a peptide bond in proximity to these positions in hTNFR, 261/262,271/272 and/or 290/291) in hIL-1R, or a peptide or peptide bonds in protein targets related to hTNFR at bond in proximity to these positions hi-1R, or peptide positions having structural homology or Sequence homology bonds in protein targets related to hD-1R at positions having to these positions. Structural homology or Sequence homology to these posi 0153. In a fifty-eighth embodiment of this variant the tions. enzyme is a protease and is capable of hydrolyzing human 0150. In a fifty-fifth embodiment of this variant the C-X-C chemokine receptor type 5 (hCCR5). The enzyme is a protease and is capable of hydrolyzing human enzymes or the fusion proteins can thus be used for prepar interleukin-2 receptor beta chain (hlL-2Rb). The enzymes or ing medicaments for the treatment of diseases, Such as, but the fusion proteins can thus be used for preparing medica not limited to, HIV infection, as well as other diseases ments for the treatment of diseases, Such as, but not limited connected with hCCR5. Preferably, said enzyme or said to, acute myeloid leukemia, inflammation, psoriasis, as well fusion protein is capable of specifically inactivating hCCR5 as other diseases connected with hiL-2Rb. Preferably, said (SEQ ID NO: 154). More preferably said enzyme or said enzyme or Said fusion protein is capable of Specifically fusion protein is capable of hydrolysing the peptide bonds inactivating hIL-2Rb (SEQ ID NO:151). More preferably between positions 8/9, 10/11, 12/13, 16/17, 19/20, 22/23, Said enzyme or Said fusion protein is capable of hydrolysing 23/24, 25/26, 27/28, 29/30, 34/35, 42/43, 50/51, 110/111, the peptide bonds between positions 38/39, 40/41, 41/42, 115/116, 120/121, 123/124, 189/190, 201/202, 204/205, 42/43, 43/44, 49/50, 71/72, 76/77, 81/82, 85/86, 86/87, 207/208, 211/212, 215/216, 216/217, 219/220, 281/282, 89/90,91/92, 102/103, 105/106, 118/119, 134/135, 152/153, 285/286, 287/288, 290/291 and/or 294/295 in hCCR5 or a 153/154, 154/155, 161/162, 163/164, 165/166, 175/176, peptide bond in proximity to these positions in hCCR5, or 194/195 and/or 197/198 (most preferred between positions peptide bonds in protein targets related to hCCR5 at posi 38/39, 43/44, 81/82, 118/119, 134/135, 153/154, 161/162, tions having Structural homology or Sequence homology to 165/166 and/or 194/195) in hIL-2Rb or a peptide bond in these positions. proximity to these positions in hL-2Rb, or peptide bonds in 0154) In a fifty-ninth embodiment of this variant the protein targets related to hD-2Rb at positions having Struc enzyme is a protease and is capable of hydrolyzing human tural homology or Sequence homology to these positions. C-X-C chemokine receptor type 3 (hCXCR3). The enzymes or the fusion proteins can thus be used for prepar 0151. In a fifty-sixth embodiment of this variant the ing medicaments for the treatment of diseases, Such as, but enzyme is a protease and is capable of hydrolyzing human not limited to, multiple Sclerosis, rheumatoid arthritis, as interleukin-4 receptor alpha chain (hIL-4Ra). The enzymes well as other diseases connected with hCXCR3. Preferably, or the fusion proteins can thus be used for preparing medi Said enzyme or Said fusion protein is capable of Specifically caments for the treatment of diseases, Such as, but not inactivating hCXCR3 (SEQ ID NO:155). More preferably limited to, asthma and allergy, as well as other diseases Said enzyme or Said fusion protein is capable of hydrolysing connected with hL-4Ra. Preferably, said enzyme or said the peptide bonds between positions 4/5, 7/8, 13/14, 21/22, fusion protein is capable of Specifically inactivating hL-4Ra 23/24, 27/28, 28/29, 29/30, 35/36, 46/47, 47/48, 52/53, (SEQ ID NO: 152). More preferably said enzyme or said 53/54, 112/113, 117/118, 119/120, 125/126, 195/196, 197/ fusion protein is capable of hydrolysing the peptide bonds 198, 205/206, 207/208, 212/213, 278/279, 282/283, 288/ between positions 22/23, 32/33, 45/46, 52/53, 66/67, 67/68, 289,292/293,293/294, 295/296 and/or 297/298 in hCXCR3 87/88, 112/113, 125/126, 127/128, 129/130, 141/142, 143/ or a peptide bond in proximity to these positions in 144, 148/149, 150/151, 154/155, 156/157, 160/161, 167/ hCXCR3, or peptide bonds in protein targets related to 168, 173/174, 175/176, 177/178, 183/184 and/or 189/190 hCXCR3 at positions having structural homology or (most preferred between positions 52/53, 66/67, 112/113, Sequence homology to these positions. 125/126, 143/144, 154/155 and/or 160/161) in hIL-4Ra or a peptide bond in proximity to these positions in hD-4Ra, or O155 In a sixty embodiment of this variant the enzyme is peptide bonds in protein targets related to hL-4Ra at posi a protease and is capable of hydrolyzing human epidermal tions having Structural homology or Sequence homology to growth factor (hEGF). The enzymes or the fusion proteins these positions. can thus be used for preparing medicaments for the treat US 2005/0175581 A1 Aug. 11, 2005

ment of diseases, Such as, but not limited to, carcinomas, bonds between positions 9/10, 10/11, 14/15, 17/18, 18/19, Solid cancers like breast, colon or Stomach cancer, as well as 19/20, 32/33, 44/45, 50/51, 51/52, 53/54, 55/56, 62/63, other diseases connected with hEGF. Preferably, said 90/91, 91/92, 104/105,105/106, 109/110, 127/128, 135/136, enzyme or Said fusion protein is capable of Specifically 149/150, 150/151, 175/176, 176/177, 177/178, 182/183, inactivating hEGF (SEQ ID NO: 156). More preferably said 189/190, 190/191 and/or 210/211 (most preferred between enzyme or Said fusion protein is capable of hydrolysing the positions 14/15, 19/20, 53/54, 55/56, 91/92, 105/106, 149/ peptide bonds between positions 11/12, 13/14, 17/18, 22/23, 150, 150/151, 175/176, 176/177, 182/183, 189/190 and/or 27/28, 28/29, 40/41, 41/42, 44/45, 45/46, 46/47, 49/50 210/211) in hFGFR-2 or a peptide bond in proximity to these and/or 50/51 (most preferred between positions 11/12, positions in hEGFR-2, or peptide bonds in protein targets 17/18, 44/45 an/or 49/50) in hEGF or a peptide bond in related to hEGFR-2 at positions having structural homology proximity to these positions in hEGF, or peptide bonds in or Sequence homology to these positions. protein targets related to hEGF at positions having Structural 0159. In a sixty-fourth embodiment of this variant the homology or Sequence homology to these positions. enzyme is a protease and is capable of hydrolyzing human 0156. In a sixty-first embodiment of this variant the C-C chemokine receptor type 1 (hCCR1). The enzymes or enzyme is a protease and is capable of hydrolyzing human the fusion proteins can thus be used for preparing medica fibroblast growth factor (hPGF-1). The enzymes or the ments for the treatment of diseases, Such as, but not limited fusion proteins can thus be used for preparing medicaments to, multiple Sclerosis, as well as other diseases connected for the treatment of diseases, Such as, but not limited to, with hCCR1. Preferably, said enzyme or said fusion protein cancer, angiogenesis, as well as other diseases connected is capable of specifically inactivating hCCR1 (SEQ ID NO: with hEGF-1. Preferably, said enzyme or said fusion protein 160). More preferably said enzyme or said fusion protein is is capable of specifically inactivating hFGF-1 (SEQ ID capable of hydrolysing the peptide bonds between positions NO:157). More preferably said enzyme or said fusion pro 2/3, 8/9, 9/10, 10/11, 11/12, 15/16, 17/18, 18/19, 26/27, tein is capable of hydrolysing the peptide bonds between 29/30, 30/31, 32/33, 92/93, 93/94, 94/95, 96/97, 97/98, positions 28/29, 35/36, 36/37, 37/38, 39/40, 49/50, 60/61, 98/99, 99/100, 101/102, 103/104, 107/108, 173/174, 176/ 70/71, 74/75, 81/82, 94/95, 100/101, 101/102, 104/105, 177, 177/178, 178/179, 187/188, 190/191, 193/194, 194/ 10.5/106, 112/113, 113/114, 119/120, 122/123, 125/126 and/ 195, 195/196, 196/197,266/267,272/273,274/275,277/278 or 128/129 (most preferred between positions 28/29, 35/36, and/or 280/281 in hCCR1 or a peptide bond in proximity to 70/71, 81/82, 100/101,104/105, 113/114 and/or 122/123) in these positions in hCCR1, or peptide bonds in protein targets hFGF-1 or a peptide bond in proximity to these positions in related to hCCR1 at positions having structural homology or hFGF-1, or peptide bonds in protein targets related to Sequence homology to these positions. hFGF-1 at positions having Structural homology or Sequence 0160 In a sixty-fifth embodiment of this variant the homology to these positions. enzyme is a protease and is capable of hydrolyzing human O157. In a sixty-second embodiment of this variant the C-C chemokine receptor type 2 (hCCR2). The enzymes or enzyme is a protease and is capable of hydrolyzing human the fusion proteins can thus be used for preparing medica fibroblast growth factor receptor 1 (hFGFR-1). The enzymes ments for the treatment of diseases, Such as, but not limited or the fusion proteins can thus be used for preparing medi to, multiple Sclerosis, rheumatoid arthritis, as well as other caments for the treatment of diseases, Such as, but not diseases connected with hCCR2. Preferably, said enzyme or limited to, cancer, angiogenesis, as well as other diseases Said fusion protein is capable of Specifically inactivating connected with hEGFR-1. Preferably, said enzyme or said hCCR2 (SEQ ID NO: 161). More preferably said enzyme or fusion protein is capable of Specifically inactivating Said fusion protein is capable of hydrolysing the peptide hFGFR-1 (SEQ ID NO:158). More preferably said enzyme bonds between positions 6/7, 8/9, 9/10, 11/12, 15/16, 18/19, or Said fusion protein is capable of hydrolysing the peptide 19/20, 23/24, 25/26, 27/28, 34/35, 36/37, 38/39, 105/106, bonds between positions 15/16, 19/20, 22/23, 23/24, 24/25, 106/107, 108/109, 114/115, 180/181, 183/184, 184/185, 32/33, 49/50, 55/56, 56/57, 58/59, 60/61, 69/70, 70/71, 185/186, 188/189, 193/194, 194/195, 196/197, 198/199, 93/94, 95/96, 96/97, 110/111, 114/115, 134/135, 181/182, 201/202,206/207,270/271,271/272,272/273,278/279 and/ 182/183, 189/190, 194/195, 195/196 and/or 215/216 (most or 284/285 in hCCR2 or a peptide bond in proximity to these preferred between positions 19/20, 24/25, 49/50, 55/56, positions in hCCR2, or peptide bonds in protein targets 58/59, 60/61, 95/96, 96/97, 110/111, 181/182, 189/190, related to hCCR2 at positions having Structural homology or 195/196 and/or 215/216) in hFGFR-1 or a peptide bond in Sequence homology to these positions. proximity to these positions in hEGFR-1, or peptide bonds 0.161 In a sixty-sixth embodiment of this variant the in protein targets related to hEGFR-1 at positions having enzyme is a protease and is capable of hydrolyzing human Structural homology or Sequence homology to these posi tyrosine protein kinase (hSrc). The enzymes or the fusion tions. proteins can thus be used for preparing medicaments for the 0158. In a sixty-third embodiment of this variant the treatment of diseases, Such as, but not limited to, cancer, enzyme is a protease and is capable of hydrolyzing human Osteoporosis, as well as other diseases connected with hSrc. fibroblast growth factor receptor 2 (hFGFR-2). The enzymes Preferably, Said enzyme or Said fusion protein is capable of or the fusion proteins can thus be used for preparing medi specifically inactivating hSrc (SEQ ID NO: 162). More caments for the treatment of diseases, Such as, but not preferably Said enzyme or Said fusion protein is capable of limited to, cancers like astrocytomas, as well as other hydrolysing the peptide bonds between positions 15/16, diseases connected with hEGFR-2. Preferably, said enzyme 22/23, 25/26, 59/60, 65/66, 87/88, 94/95, 99/100, 102/103, or said fusion protein is capable of Specifically inactivating 123/124, 124/125, 126/127, 135/136, 147/148, 150/151, hFGFR-2 (SEQ ID NO: 159). More preferably said enzyme 153/154, 158/159, 176/177, 178/179, 182/183, 200/201, or Said fusion protein is capable of hydrolysing the peptide 216/217, 223/224, 234/235, 249/250, 261/262, 266/267, US 2005/0175581 A1 Aug. 11, 2005

271/272, 275/276, 277/278, 297/298, 327/328, 331/332, inactivating Bradykinin (SEQID NO:165). More preferably 333/334, 337/338, 354/355, 356/357, 378/379, 387/388, Said enzyme or Said fusion protein is capable of hydrolysing 397/398, 391/392, 395/396, 398/399, 407/408, 411/412, the peptide bonds between positions 1/2, 5/6 and/or 8/9 in 418/419, 41.9/420, 420/421, 422/423, 423/424 and/or 436/ Bradykinin or a peptide bond in proximity to these positions 437 (most preferred between positions 59/60, 123/124, in Bradykinin, or peptide bonds in protein targets related to 126/127, 135/136, 176/177, 182/183, 200/201, 275/276, Bradykinin at positions having Structural homology or 277/278, 331/332, 354/355, 387/388, 391/392, 395/396, Sequence homology to these positions. 418/419 and/or 423/424) in hSrc or a peptide bond in 0.165. In a seventy embodiment of this variant the enzyme proximity to these positions in hSrc, or peptide bonds in is a protease and is capable of hydrolyzing human Coagul protein targets related to hSrc at positions having Structural lation factor IX (Factor IX). The enzymes or the fusion homology or Sequence homology to these positions. proteins can thus be used for preparing medicaments for the 0162. In a sixty-seventh embodiment of this variant the treatment of diseases, Such as, but not limited to, hamophilia enzyme is a protease and is capable of hydrolyzing human B, as well as other diseases connected with Factor IX. RAC-beta serine/threonine protein kinase (hakt-2). The Preferably, Said enzyme or Said fusion protein is capable of enzymes or the fusion proteins can thus be used for prepar specifically inactivating Factor IX (SEQ ID NO: 166). More ing medicaments for the treatment of diseases, Such as, but preferably Said enzyme or Said fusion protein is capable of not limited to, cancer, as well as other diseases connected hydrolysing the peptide bonds between positions 21/22, with hAkt-2. Preferably, said enzyme or said fusion protein 23/24, 36/37, 38/39, 59/60, 63/64, 74/75, 75/76, 87/88, is capable of specifically inactivating hAkt-2 (SEQ ID NO: 111/112, 112/113, 119/120, 127/128, 128/129, 129/130, 130/ 163). More preferably said enzyme or said fusion protein is 131, 136/137, 137/138, 149/150,151/152, 153/154, 162/163, capable of hydrolysing the peptide bonds between positions 167/168, 173/174, 176/177, 190/191, 209/210, 222/223, 9/10, 15/16, 25/26, 26/27, 27/28, 39/40, 63/64, 71/72, 72/73, 223/224 and/or 227/228 (most preferred between positions 78/79, 79/80, 98/99, 99/100, 100/101, 104/105, 105/106, 63/64, 127/128, 136/137, 149/150, 151/152, 173/174, 176/ 106/107, 120/121, 124/125, 125/126, 141/142, 170/171, 177 and/or 227/228) in Factor IX or a peptide bond in 178/179, 179/180, 181/182, 182/183, 184/185, 206/207, proximity to these positions in Factor IX, or peptide bonds 209/210, 211/212, 212/213, 220/221, 221/222, 223/224, in protein targets related to Factor IX at positions having 226/227, 242/243, 243/244, 245/246, 256/257, 260/261, Structural homology or Sequence homology to these posi 262/263,275/276,276/277,282/283, and/or 292/293 (most tions. preferred between positions 27/28, 39/40, 41/42, 72/73, 78/79, 100/101, 105/106, 106/107, 125/126, 179/180, 184/ 0166 In a seventy-first embodiment of this variant the 185,209/210, 223/224, 245/246,262/263 and/or 282/283) in enzyme is a protease and is capable of hydrolyzing human hAkt-2 or a peptide bond in proximity to these positions in glycogen Synthase kinase-3-beta (hGSK-3). The enzymes or hAkt-2, or peptide bonds in protein targets related to hakt-2 the fusion proteins can thus be used for preparing medica at positions having Structural homology or Sequence homol ments for the treatment of diseases, Such as, but not limited to, diabetes, as well as other diseases connected with hCSK ogy to these positions. 3. Preferably, Said enzyme or Said fusion protein is capable 0163. In a sixty-eighth embodiment of this variant the of specifically inactivating hCSK-3 (SEQ ID NO: 167). enzyme is a protease and is capable of hydrolyzing human More preferably Said enzyme or Said fusion protein is substance P (substance P). The enzymes or the fusion capable of hydrolysing the peptide bonds between positions proteins can thus be used for preparing medicaments for the 16/17, 19/20, 24/25, 26/27, 43/44, 52/53, 57/58, 60/61, treatment of diseases, Such as, but not limited to, cancers like 62/63, 68/69, 69/70, 87/88, 88/89, 89/90, 90/91, 91/92, Small cell lung cancer, colorectal cancer, astrocytic/glial 107/108, 110/111, 112/113, 114/115, 116/117, 156/157, 158/ brain tumors, as well as other diseases connected with 159, 175/176, 177/178, 182/183, 186/187, 187/188, 189/ substance P. Preferably, said enzyme or said fusion protein 190, 226/227, 230/231, 234/235, 244/245, 245/246, 248/ is capable of specifically inactivating substance P (SEQ ID 249, 249/250, 254/255, 256/257, 263/264, 269/270, 272/ NO: 164). More preferably said enzyme or said fusion 273, 274/275, 307/308, 308/309, 311/312, 315/316 and/or protein is capable of hydrolysing the peptide bonds between 321/322 (most preferred between positions 57/58, 87/88, positions 1/2, 3/4, 7/8 and/or 8/9 in substance P or a peptide 88/89, 89/90, 90/91, 114/115, 116/117, 158/159, 175/176, bond in proximity to these positions in Substance P, or 182/183, 230/231, 244/245, 248/249, 254/255, 256/257, peptide bonds in protein targets related to Substance P at 274/275 and/or 321/322) in hCSK-3 or a peptide bond in positions having structural homology or Sequence homology proximity to these positions in hCGSK-3, or peptide bonds in to these positions. protein targets related to hCGSK-3 at positions having Struc 0164. In a sixty-ninth embodiment of this variant the tural homology or Sequence homology to these positions. enzyme is a protease and is capable of hydrolyzing human 0167. In a seventy-second embodiment of this variant the Bradykinin (Bradykinin). The enzymes or the fusion pro enzyme is a protease and is capable of hydrolyzing human teins can thus be used for preparing medicaments for the cyclin-dependent protein kinase-2 (hcdk-2). The enzymes or treatment of diseases, Such as, but not limited to, Vascular the fusion proteins can thus be used for preparing medica and neuro-glial pathology in diabetic retinopathy, cerebral ments for the treatment of diseases, Such as, but not limited ischemia and trauma, hyperalgesia, inflammatory diseases to, cancer, as well as other diseases connected with hcdk-2. or conditions, asthma and cancer, pain, pathological vascular Preferably, Said enzyme or Said fusion protein is capable of leakage or vasodilation, pathological contraction of various specifically inactivating hcdk-2 (SEQ ID NO: 168). More Smooth muscles, pathological cell proliferation, as well as preferably Said enzyme or Said fusion protein is capable of other diseases connected with Bradykinin. Preferably, said hydrolysing the peptide bonds between positions 8/9, 9/10, enzyme or Said fusion protein is capable of Specifically 12/13, 15/16, 19/20, 22/23, 24/25, 34/35, 50/51, 57/58, US 2005/0175581 A1 Aug. 11, 2005

68/69, 73/74, 75/76, 88/89,89/90, 92/93, 122/123, 138/139, bonds in protein targets related to caspase-3 at positions 162/163, 178/179, 179/180, 180/181, 199/200, 200/201, having Structural homology or Sequence homology to these 206/207, 208/209, 210/211, 217/218, 223/224, 224/225, positions. 237/238,242/243,245/246, 247/248,250/251, 273/274 and/ 0170 In a seventy-fifth embodiment of this variant the or 291/292 (most preferred between positions 12/13, 50/51, enzyme is a protease and is capable of hydrolyzing human 57/58, 73/74, 138/139, 180/181, 200/201, 206/207,223/224, caspase-7 (caspase-7). The enzymes or the fusion proteins 242/243, 247/248 and/or 273/274) in hcdk-2 or a peptide can thus be used for preparing medicaments for the treat bond in proximity to these positions in hcdk-2, or peptide ment of diseases, Such as, but not limited to, apoptosis bonds in protein targets related to hcdk-2 at positions having associated disorders like immunodeficiency diseases (AIDS/ Structural homology or Sequence homology to these posi HIV), Alzheimers, Senescence, degenerative disorders like tions. neurodegenerative diseases, ischemic acell death, reperfu 0.168. In a seventy-third embodiment of this variant the Sion cell death, acute ischemic injury, infertility, wounds, enzyme is a protease and is capable of hydrolyzing human toxin cell death induced by cytolethal distending toxin caspase-2 (caspase-2). The enzymes or the fusion proteins (CDT), acute lymphoblast leukemia, as well as other dis can thus be used for preparing medicaments for the treat eases connected with caspase-7. Preferably, Said enzyme or ment of diseases, Such as, but not limited to, apoptosis Said fusion protein is capable of Specifically inactivating associated disorders like immunodeficiency diseases (AIDS/ caspase-7 (SEQ ID NO: 171). More preferably said enzyme HIV), Alzheimers, Senescence, degenerative disorders like or said fusion protein is capable of hydrolysing the peptide neurodegenerative diseases, pathological ischemic cell bonds between positions 35/36, 42/43, 43/44, 56/57, 57/58, death, pathological reperfusion cell death, pathological reti 68/69, 72/73, 76/77, 79/80, 84/85, 88/89, 101/102,102/103, nal neuronal cell death, pathological apoptosis initiated by 105/106, 106/107, 107/108, 149/150, 188/189, 189/190, beta-amyloid toxicity or by trophic factor deprivation, dis 227/228, 228/229, 231/232, 232/233, 251/252, 255/256, eases with mitochondrial permeabilization components, 256/257 and/or 277/278 (most preferred between positions toxin cell death induced by cytolethal distending toxin 57/58, 79/80, 84/85, 102/103, 107/108, 228/229, 231/232, (CDT), acute ischemic injury, infertility, wounds, as well as 232/233 and/or 255/256) in caspase-7 or a peptide bond in other diseases connected with caspase-2. Preferably, Said proximity to these positions in caspase-7, or peptide bonds enzyme or Said fusion protein is capable of Specifically in protein targets related to caspase-7 at positions having inactivating caspase-2 (SEQ ID NO: 169). More preferably Structural homology or Sequence homology to these posi Said enzyme or said fusion protein is capable of hydrolysing tions. the peptide bonds between positions 13/14, 14/15, 24/25, 28/29, 44/45, 45/46, 46/47, 48/49, 54/55, 65/66, 70/71, 0171 In a seventy-sixth embodiment of this variant the 81/82, 84/85, 96/97, 118/119, 120/121, 126/127, 154/155, enzyme is a protease and is capable of hydrolyzing human 155/156, 186/187, 188/189, 212/213, 213/214, 227/228, caspase-9 (caspase-9). The enzymes or the fusion proteins 228/229, 247/248, 249/250, 251/252, 259/260 and/or 272/ can thus be used for preparing medicaments for the treat 273 (most preferred between positions 48/49, 81/82, 154/ ment of diseases, Such as, but not limited to, apoptosis 155, 186/187, 213/214, 228/229 and/or 251/252) in associated disorders like immunodeficiency diseases (AIDS/ caspase-2 or a peptide bond in proximity to these positions HIV), Alzheimers, Senescence, degenerative disorders like in caspase-2, or peptide bonds in protein targets related to neurodegenerative diseases, ischemic acell death, reperfu caspase-2 at positions having Structural homology or Sion cell death, acute ischemic injury, infertility, wounds, Sequence homology to these positions. neurological diseases like Stroke, neurodegenerative dis eases, brain injury caused by hypoxia, Parkinson's, amyo 0169. In a seventy-fourth embodiment of this variant the trophic lateral Sclerosis (ALS), as well as other diseases enzyme is a protease and is capable of hydrolyzing human connected with caspase-9. Preferably, Said enzyme or Said caspase-3 (caspase-3). The enzymes or the fusion proteins fusion protein is capable of Specifically inactivating can thus be used for preparing medicaments for the treat caspase-9 (SEQ ID NO: 172). More preferably said enzyme ment of diseases, Such as, but not limited to, apoptosis or said fusion protein is capable of hydrolysing the peptide associated disorders like immunodeficiency diseases (AIDS/ bonds between positions 4/5, 19/20, 34/35, 35/36, 39/40, HIV), Alzheimers, Senescence, degenerative disorders like 49/50, 52/53, 53/54, 54/55, 63/64, 67/68, 71/72, 72/73, neurodegenerative diseases, ischemic acell death, reperfu 79/80, 84/85, 112/113, 123/124, 151/152, 158/159, 215/216, Sion cell death, acute ischemic injury, infertility, wounds, 216/217, 217/218, 219/220, 223/224, 226/227, 229/230, neural degeneration in amyotrophic lateral Sclerosis, Hun 230/231, 233/234, 235/236 and/or 258/259 (most preferred tington, Infection with vesicular stomatitis virus (VSV), as between positions 19/20, 35/36, 34/35, 52/53, 53/54, 71/72, well as other diseases connected with caspase-3. Preferably, 79/80, 219/220 and/or 230/231) in caspase-9 or a peptide Said enzyme or Said fusion protein is capable of Specifically bond in proximity to these positions in caspase-9, or peptide inactivating caspase-3 (SEQ ID NO: 170). More preferably bonds in protein targets related to caspase-9 at positions Said enzyme or Said fusion protein is capable of hydrolysing having Structural homology or Sequence homology to these the peptide bonds between positions 12/13, 25/26, 29/30, positions. 40/41, 47/48, 48/49, 51/52, 54/55, 56/57, 58/59, 66/67, 67/68, 73/74, 74/75, 77/78, 78/79, 79/80, 82/83, 83/84, 0172 In a seventy-seventh embodiment of this variant 110/111, 139/140, 151/152, 152/153, 153/154, 158/159, the enzyme is a protease and is capable of hydrolyzing 196/197, 198/199, 200/201, 201/202, 218/219, 220/221, human apoptotic protease activating factor 1 (hapaf-1). The 225/226 and/or 248/249 (most preferred between positions enzymes or the fusion proteins can thus be used for prepar 29/30, 40/41, 51/52, 56/57, 67/68, 73/74, 79/80, 83/84, ing medicaments for the treatment of diseases, Such as, but 153/154, 218/219 and/or 225/226) in caspase-3 or a peptide not limited to, apoptosis associated disorders like immuno bond in proximity to these positions in caspase-3, or peptide deficiency diseases (AIDS/HIV), Alzheimers, senescence, US 2005/0175581 A1 Aug. 11, 2005 degenerative disorders like neurodegenerative diseases, related to hPARP at positions having structural homology or ischemic acell death, reperfusion cell death, acute ischemic Sequence homology to these positions. injury, infertility, wounds, as well as other diseases con 0.175. In an eighty embodiment of this variant the enzyme nected with hApaf-1. Preferably, said enzyme or said fusion is a protease and is capable of hydrolyzing human Tumor protein is capable of specifically inactivating hapaf-1 (SEQ protein p53 (hp53). The enzymes or the fusion proteins can ID NO: 173). More preferably said enzyme or said fusion thus be used for preparing medicaments for the treatment of protein is capable of hydrolysing the peptide bonds between diseases, Such as, but not limited to, apoptosis associated positions 6/7, 13/14, 14/15, 17/18, 18/19, 19/20, 24/25, disorders like immunodeficiency diseases (AIDS/HIV), 27/28, 32/33, 39/40, 40/41, 41/42, 44/45, 46/47, 62/63, Alzheimers, Senescence, any degenerative disorders (neuro 63/64, 64/65, 66/67, 80/81, 81/82 and/or 82/83 (most pre degenerative diseases), ischemic and reperfusion cell death, ferred between positions 13/14, 14/15, 18/19, 41/42, 62/63 acute ischemic injury, infertility, wounds, as well as other and/or 64/65) in hapaf-1 or a peptide bond in proximity to diseases connected with hp53. Preferably, said enzyme or these positions in hapaf-1, or peptide bonds in protein Said fusion protein is capable of Specifically inactivating targets related to hapaf-1 at positions having structural hp53 (SEQ ID NO: 176). More preferably said enzyme or homology or Sequence homology to these positions. Said fusion protein is capable of hydrolysing the peptide 0173. In a seventy-eighth embodiment of this variant the bonds between positions 8/9, 10/11, 14/15, 17/18, 27/28, enzyme is a protease and is capable of hydrolyzing human 33/34, 53/54, 55/56, 63/64, 78/79, 81/82, 87/88, 88/89, BH3 interacting domain death agonist (hBID). The enzymes 93/94, 10.5/106, 109/110, 112/113, 114/115, 115/116, 116/ or the fusion proteins can thus be used for preparing medi 117, 131/132, 135/136, 155/156, 156/157, 166/167, 180/ caments for the treatment of diseases, Such as, but not 181, 187/188, 189/190, 190/191, 192/193, 193/194 and/or limited to, apoptosis associated disorders like immunodefi 194/195 (most preferred between positions 14/15, 27/28, ciency diseases (AIDS/HIV), Alzheimers, Senescence, 53/54, 88/89, 114/115, 116/117, 131/132, 155/156, 190/191 degenerative disorders like neurodegenerative diseases, and/or 194/195) in hp53 or a peptide bond in proximity to ischemic acell death, reperfusion cell death, acute ischemic these positions in hp53, or peptide bonds in protein targets injury, infertility, wounds, as well as other diseases con related to hp53 at positions having Structural homology or nected with hEBID. Preferably, said enzyme or said fusion Sequence homology to these positions. protein is capable of specifically inactivating hBID (SEQ ID 0176). In an eighty-first embodiment of this variant the NO: 174). More preferably said enzyme or said fusion enzyme is a protease and is capable of hydrolyzing human protein is capable of hydrolysing the peptide bonds between P-selectin (hP-selectin). The enzymes or the fusion proteins positions 6/7, 15/16, 32/33, 36/37, 37/38, 38/39, 53/54, can thus be used for preparing medicaments for the treat 54/55, 56/57, 57/58, 58/59, 62/63, 65/66, 73/74, 77/78, ment of diseases, Such as, but not limited to, inflammation, 79/80, 101/102, 116/117, 120/121, 122/123, 123/124, 124/ as well as other diseases connected with hP-selectin. Pref 125, 134/135, 140/141, 142/143, 143/144, 145/146 and/or erably, Said enzyme or said fusion protein is capable of 170/171 (most preferred between positions 36/37, 53/54, specifically inactivating hP-selectin (SEQ ID NO: 177). 57/58, 62/63, 65/66, 73/74 and/or 79/80) in hBID or a More preferably Said enzyme or Said fusion protein is peptide bond in proximity to these positions in hEBID, or capable of hydrolysing the peptide bonds between positions peptide bonds in protein targets related to hEBID at positions 8/9, 16/17, 17/18, 18/19, 22/23, 23/24, 36/37, 37/38, 40/41, having Structural homology or Sequence homology to these 44/45, 45/46, 54/55, 55/56, 66/67, 67/68, 72/73, 74/75, positions. 78/79, 80/81, 84/85, 85/86, 88/89, 92/93, 94/95, 96/97, 106/107,107/108, 111/112, 112/113, 124/125, 129/130, 140/ 0.174. In a seventy-ninth embodiment of this variant the 141, 152/153 and/or 154/155 (most preferred between posi enzyme is a protease and is capable of hydrolyzing human tions 17/18, 22/23, 44/45, 55/56, 72/73, 78/79, 84/85, 85/86, poly (ADP-ribose) polymerase-1 (hPARP). The enzymes or 107/108, 112/113, 152/153 and/or 154/155) in hp-selectin or the fusion proteins can thus be used for preparing medica a peptide bond in proximity to these positions in hP-Selectin, ments for the treatment of diseases, Such as, but not limited or peptide bonds in protein targets related to hP-Selectin at to, apoptosis associated disorders like immunodeficiency positions having structural homology or Sequence homology diseases (AIDS/HIV), Alzheimers, Senescence, degenerative disorders like neurodegenerative diseases, ischemic acell to these positions. death, reperfusion cell death, acute ischemic injury, infer 0177. In an eighty-second embodiment of this variant the tility, wounds, as well as other diseases connected with enzyme is a protease and is capable of hydrolyzing human hPARP. Preferably, said enzyme or said fusion protein is Oncostatin M (hCSM). The enzymes or the fusion proteins capable of specifically inactivating hPARP (SEQ ID NO: can thus be used for preparing medicaments for the treat 175). More preferably said enzyme or said fusion protein is ment of diseases, Such as, but not limited to, cancer like capable of hydrolysing the peptide bonds between positions prostate cancer, as well as other diseases connected with 13/14, 19/20, 22/23, 23/24, 27/28, 29/30, 34/35, 39/40, hOSM. Preferably, said enzyme or said fusion protein is 42/43, 43/44, 65/66, 70/71, 74/75, 82/83, 86/87, 87/88, capable of specifically inactivating hOSM (SEQ ID NO: 114/115, 118/119, 122/123, 126/127, 133/134, 134/135, 178). More preferably said enzyme or said fusion protein is 141/142, 144/145, 145/146, 146/147, 148/149, 149/150, capable of hydrolysing the peptide bonds between positions 158/159, 179/180, 181/182, 188/189, 196/197, 222/223, 11/12, 19/20, 22/23, 26/27, 32/33, 36/37, 41/42, 44/45, 270/271, 272/273, 282/283, 304/305, 307/308 and/or 320/ 46/47, 47/48, 50/51, 52/53, 59/60, 60/61, 67/68, 68/69, 321 (most preferred between positions 22/23, 43/44, 118/ 84/85, 97/98, 99/100, 100/101, 106/107, 107/108, 109/110, 119, 122/123, 145/146, 146/147, 148/149, 179/180 and/or 122/123, 126/127, 133/134, 158/159, 162/163, 163/164 and/ 272/273) in hPARP or a peptide bond in proximity to these or 175/176 (most preferred between positions 19/20, 44/45, positions in hPARP, or peptide bonds in protein targets 47/48, 60/61, 67/68, 97/98, 100/101, 109/110, 126/127, US 2005/0175581 A1 Aug. 11, 2005

133/134, 162/163 and/or 175/176) in hCSM or a peptide 87/88, 95/96, 96/97, 99/100, 103/104, 104/105, 114/115, bond in proximity to these positions in hCSM, or peptide 117/118, 120/121, 124/125, 141/142, 148/149, 155/156, bonds in protein targets related to h0SM at positions having 159/160, 160/161, 182/183, 189/190, 193/194, 198/199, Structural homology or Sequence homology to these posi 191/192, 192/193, 205/206 and/or 206/207 (most preferred tions. between positions 40/41, 87/88, 95/96, 103/104, 120/121, 141/142, 155/156, 159/160, 192/193 and/or 206/207) in 0178. In an eighty-third embodiment of this variant the cathepsin L or a peptide bond in proximity to these positions enzyme is a protease and is capable of hydrolyzing human in cathepsin L, or peptide bonds in protein targets related to cathepsin B (cathepsin B). The enzymes or the fusion cathepsin L at positions having Structural homology or proteins can thus be used for preparing medicaments for the Sequence homology to these positions. treatment of diseases, Such as, but not limited to, inflam matory bowel disease, Crohn's disease, colitis ulcerosa, as 0181. In an eighty-sixth embodiment of this variant the well as other diseases connected with cathepsin B. Prefer enzyme is a protease and is capable of hydrolyzing human ably, Said enzyme or said fusion protein is capable of Galectin-3 (hGalectin-3). The enzymes or the fusion pro specifically inactivating cathepsin B (SEQ ID NO: 179). teins can thus be used for preparing medicaments for the More preferably Said enzyme or Said fusion protein is treatment of diseases, Such as, but not limited to, inflam capable of hydrolysing the peptide bonds between positions matory bowel disease, Crohn's disease, colitis ulcerosa, as 8/9, 9/10, 18/19, 53/54, 69/70, 75/76, 78/79, 85/86, 86/87, well as other diseases connected with hCalectin-3. Prefer 94/95, 95/96, 124/125, 127/128, 130/131, 141/142, 146/147, ably, Said enzyme or said fusion protein is capable of 148/149, 151/152, 158/159, 159/160, 165/166, 166/167, specifically inactivating hCGalectin-3 (SEQ ID NO: 182). 184/185, 194/195, 224/225, 227/228, 238/239, 245/246 and/ More preferably Said enzyme or Said fusion protein is or 252/253 (most preferred between positions 75/76, 85/86, capable of hydrolysing the peptide bonds between positions 95/96, 124/125, 130/131, 141/142, 148/149, 158/159 and/or 5/6, 16/17, 26/27, 31/32, 35/36, 52/53, 55/56, 56/57, 65/66, 194/195) in cathepsin B or a peptide bond in proximity to 68/69, 70/71, 71/72, 72/73, 73/74, 80/81, 83/84, 92/93, these positions in cathepsin B, or peptide bonds in protein 97/98, 102/103, 111/112, 113/114, 114/115, 126/127, 128/ targets related to cathepsin B at positions having structural 129 and/or 134/135 (most preferred between positions homology or Sequence homology to these positions. 55/56, 65/66, 70/71, 102/103, 113/114 and/or 128/129) in hGalectin-3 or a peptide bond in proximity to these positions 0179. In an eighty-fourth embodiment of this variant the in hCialectin-3, or peptide bonds in protein targets related to enzyme is a protease and is capable of hydrolyzing human hCialectin-3 at positions having structural homology or cathepsin D (cathepsin D). The enzymes or the fusion Sequence homology to these positions. proteins can thus be used for preparing medicaments for the treatment of diseases, Such as, but not limited to, inflam 0182. In an eighty-seventh embodiment of this variant the matory bowel disease, Crohn's disease, colitis ulcerosa, as enzyme is a protease and is capable of hydrolyzing human well as other diseases connected with cathepsin D. Prefer receptor tyrosine-protein kinase erbB-2 (hHER2). The ably, Said enzyme or said fusion protein is capable of enzymes or the fusion proteins can thus be used for prepar specifically inactivating cathepsin D (SEQ ID NO: 180). ing medicaments for the treatment of diseases, Such as, but More preferably Said enzyme or Said fusion protein is not limited to, cancer, as well as other diseases connected capable of hydrolysing the peptide bonds between positions with hHER2. Preferably, said enzyme or said fusion protein 10/11, 18/19, 47/48, 54/55, 58/59, 62/63, 63/64, 67/68, is capable of specifically inactivating hHER2 (SEQ ID NO: 69/70, 75/76, 86/87, 111/112, 112/113, 141/142, 158/159, 183). More preferably said enzyme or said fusion protein is 161/162, 172/173, 174/175, 189/190, 191/192, 192/193, capable of hydrolysing the peptide bonds between positions 197/198, 202/203, 203/204, 214/215, 223/224, 224/225, 18/19, 70/71, 87/88, 88/89, 99/100, 116/117, 135/136, 143/ 227/228, 242/243, 243/244, 245/246, 246/247, 249/250, 144, 153/154, 163/164, 168/169, 188/189, 206/207, 216/ 266/267, 281/282, 283/284, 284/285, 288/289, 289/290, 217, 226/227, 252/253, 255/256, 258/259, 264/265, 266/ 293/294, 299/300, 310/311 and/or 336/337 (most preferred 267, 279/280, 311/312, 314/315, 318/319, 326/327, 330/ between positions 54/55, 62/63, 63/64, 112/113, 158/159, 331, 332/333, 357/358, 360/361, 373/374, 395/396, 477/ 174/175, 189/190, 197/198, 224/225, 242/243, 245/246, 478, 479/480, 480/481, 481/482, 485/486, 495/496, 514/ 266/267, 281/282, 288/289 and/or 299/300) in cathepsin D 515, 520/521, 521/522, 523/524, 530/531, 532/533, 536/ or a peptide bond in proximity to these positions in cathepsin 537,558/559, 560/561,568/569,577/578,592/593,597/598 D, or peptide bonds in protein targets related to cathepsin D and/or 598/599 (most preferred between positions 88/89, at positions having Structural homology or Sequence homol 135/136, 143/144, 226/227, 252/253, 255/256, 258/259, ogy to these positions. 314/315, 318/319, 360/361, 480/481, 485/486, 495/496, 520/521, 523/524,560/561, 568/569, 577/578 and/or 592/ 0180. In an eighty-fifth embodiment of this variant the 593) in hHER2 or a peptide bond in proximity to these enzyme is a protease and is capable of hydrolyzing human positions in hER2, or peptide bonds in protein targets cathepsin L (cathepsin L). The enzymes or the fusion related to hER2 at positions having Structural homology or proteins can thus be used for preparing medicaments for the Sequence homology to these positions. treatment of diseases, Such as, but not limited to, inflam matory bowel disease, Crohn's disease, colitis ulcerosa, as 0183 In an eighty-eighth embodiment of this variant the well as other diseases connected with cathepsin L. Prefer enzyme is a protease and is capable of hydrolyzing human ably, Said enzyme or said fusion protein is capable of matrix metalloproteinase-7 (hMMP-7). The enzymes or the specifically inactivating cathepsin L (SEQ ID NO: 181). fusion proteins can thus be used for preparing medicaments More preferably Said enzyme or Said fusion protein is for the treatment of diseases, Such as, but not limited to, capable of hydrolysing the peptide bonds between positions cancer, as well as other diseases connected with hMMP-7. 9/10, 10/11, 40/41, 41/42, 44/45, 72/73, 76/77, 79/80, 86/87, Preferably, Said enzyme or Said fusion protein is capable of US 2005/0175581 A1 Aug. 11, 2005 22 specifically inactivating hMMP-7 (SEQ ID NO: 184). More connected with hp38-kinase. Preferably, said enzyme or said preferably Said enzyme or Said fusion protein is capable of fusion protein is capable of Specifically inactivating hp38 hydrolysing the peptide bonds between positions 13/14, kinase (SEQ ID NO: 187). More preferably said enzyme or 22/23, 24/25, 25/26, 33/34, 37/38, 44/45, 45/46, 51/52, Said fusion protein is capable of hydrolysing the peptide 52/53, 55/56, 66/67, 73/74, 76/77, 100/101, 101/102, 102/ bonds between positions 8/9, 11/12, 14/15, 21/22, 48/49, 103, 103/104, 106/107, 133/134, 146/147, 151/152, 155/ 53/54, 56/57, 66/67, 93/94, 96/97,97/98, 117/118, 120/121, 156, 162/163 and/or 166/167 (most preferred between posi 123/124, 124/125, 159/160, 160/161, 162/163, 172/173, tions 24/25, 33/34, 51/52, 55/56, 73/74, 76/77, 101/102, 175/176, 176/177, 177/178, 181/182, 199/200, 219/220, 133/134 and/or 146/147) in hMMP-7 or a peptide bond in 229/230, 232/233, 236/237, 244/245, 247/248, 248/249, proximity to these positions in hMMP-7, or peptide bonds in 252/253, 255/256, 257/258, 285/286, 286/287, 293/294, protein targets related to hMMP-7 at positions having struc 294/295, 310/311, 312/313, 314/315, 315/316, 316/317, tural homology or Sequence homology to these positions. 320/321, 323/324, 329/330, 330/331, 334/335, 335/336, 0184. In an eighty-ninth embodiment of this variant the 341/342, 342/343 and/or 343/344 (most preferred between enzyme is a protease and is capable of hydrolyzing human positions 48/49, 56/57, 93/94, 96/97, 123/124, 160/161, matrix metalloproteinase-14 (hMMP-14). The enzymes or 175/176, 236/237, 247/248, 255/256, 257/258, 294/295, the fusion proteins can thus be used for preparing medica 314/315, 315/316, 329/330, 330/331, 334/335 and/or 342/ ments for the treatment of diseases, Such as, but not limited 343) in hp38-kinase or a peptide bond in proximity to these to, cancer, as well as other diseases connected with hMMP positions in hp38-kinase, or peptide bonds in protein targets 14. Preferably, Said enzyme or Said fusion protein is capable related to hp38-kinase at positions having Structural homol of specifically inactivating hMMP-14 (SEQ ID NO: 185). ogy or Sequence homology to these positions. More preferably Said enzyme or Said fusion protein is 0187. In a ninety-second embodiment of this variant the capable of hydrolysing the peptide bonds between positions enzyme is a protease and is capable of hydrolyzing human 12/13, 20/21, 26/27, 27/28, 34/35, 35/36, 38/39, 47/48, Stress-activated protein kinase JNK3 (h.JNK3-kinase). The 49/50, 57/58, 50/51, 53/54, 55/56, 58/59, 62/63, 72/73, enzymes or the fusion proteins can thus be used for prepar 82/83, 84/85, 113/114, 115/116, 116/117, 141/142, 152/153, ing medicaments for the treatment of diseases, Such as, but 154/155, 156/157, 165/166 and/or 166/167 (most preferred not limited to, intimal hyperplasia, Vascular remodeling between positions 27/28, 38/39, 55/56, 57/58, 58/59, 82/83, upon blood vessel injury, as well as other diseases connected 113/114, 116/117, 141/142 and/or 152/153) in hMMP-14 or with hNK3-kinase. Preferably, said enzyme or said fusion a peptide bond in proximity to these positions in hMMP-14, protein is capable of Specifically inactivating hJNK3-kinase or peptide bonds in protein targets related to hMMP-14 at (SEQ ID NO: 188). More preferably said enzyme or said positions having structural homology or Sequence homology fusion protein is capable of hydrolysing the peptide bonds to these positions. between positions 11/12, 19/20, 38/39, 43/44, 44/45, 53/54, 0185. In a ninety embodiment of this variant the enzyme 65/66, 72/73, 90/91, 93/94, 94/95, 106/107, 116/117, 118/ is a protease and is capable of hydrolyzing human Vascular 119, 120/121, 124/125, 134/135, 179/180, 196/197, 197/ endothelial growth factor receptor 2 (hVEGFR-2). The 198, 198/199, 214/215, 216/217, 222/223, 223/224, 233/ enzymes or the fusion proteins can thus be used for prepar 234, 244/245, 245/246, 251/252, 253/254, 255/256, 259/ ing medicaments for the treatment of diseases, Such as, but 260, 267/268, 271/272, 277/278, 279/280, 282/283, 302/ not limited to, cancer, as well as other diseases connected 303, 307/308, 308/309, 318/319, 319/320, 325/326, 338/ with hVEGFR-2. Preferably, said enzyme or said fusion 339, 339/340, 344/345 and/or 345/346 (most preferred protein is capable of specifically inactivating hVEGFR-2 between positions 38/39, 90/91, 93/94, 116/117, 179/180, (SEQ ID NO: 186). More preferably said enzyme or said 216/217, 222/223, 255/256, 259/260, 267/268, 271/272, fusion protein is capable of hydrolysing the peptide bonds 277/278, 279/280, 318/319, 319/320 and/or 339/340) in between positions 13/14, 16/17, 19/20, 23/24, 33/34, 39/40, hNK3-kinase or a peptide bond in proximity to these 53/54, 61/62, 88/89, 110/111, 112/113, 113/114, 115/116, positions in hNK3-kinase, or peptide bonds in protein 119/120, 120/121, 179/180, 184/185, 198/199, 203/204, targets related to hJNK3-kinase at positions having Struc 204/205, 208/209, 220/221, 245/246, 256/257, 260/261, tural homology or Sequence homology to these positions. 261/262, 291/292, 293/294, 294/295, 295/296, 298/299, 0188 In a ninety-third embodiment of this variant the 299/300, 301/302, 302/303, 307/308, 310/311, 311/312, enzyme is a protease and is capable of hydrolyzing human 317/318,322/323, 327/328,336/337 and/or 339/340 (most C-X-C chemokine receptor type 4 (hCCR-4). The preferred between positions 39/40, 53/54, 61/62, 88/89, enzymes or the fusion proteins can thus be used for prepar 119/120, 120/121, 204/205, 260/261, 293/294, 298/299, ing medicaments for the treatment of diseases, Such as, but 299/300, 302/303 and/or 336/337) in hVEGFR-2 or a pep not limited to, inflammation, as well as other diseases tide bond in proximity to these positions in hVEGFR-2, or connected with hCCR-4. Preferably, said enzyme or said peptide bonds in protein targets related to hVEGFR-2 at fusion protein is capable of Specifically inactivating hCCR-4 positions having structural homology or Sequence homology (SEQ ID NO: 189). More preferably said enzyme or said to these positions. fusion protein is capable of hydrolysing the peptide bonds 0186. In a ninety-first embodiment of this variant the between positions 7/8, 10/11, 12/13, 14/15, 15/16, 20/21, enzyme is a protease and is capable of hydrolyzing human 21/22, 22/23, 25/26, 26/27, 30/31, 31/32, 32/33, 36/37, Mitogen-activated protein kinase p38-alpha (hp38-kinase). 38/39, 102/103, 103/104, 104/105, 107/108, 110/111, 181/ The enzymes or the fusion proteins can thus be used for 182, 182/183, 183/184, 184/185, 187/188, 188/189, 190/ preparing medicaments for the treatment of diseases, Such 191, 193/194, 195/196, 262/263, 268/269, 271/272, 275/ as, but not limited to, intimal hyperplasia, Vascular remod 276, 277/278, 282/283 and/or 283/284 in hCCR-4 or a eling upon blood vessel injury, as well as other diseases peptide bond in proximity to these positions in hCCR-4, or US 2005/0175581 A1 Aug. 11, 2005 23 peptide bonds in protein targets related to hCCR-4 at posi eases, Such as, but not limited to, inflammatory bowel tions having Structural homology or Sequence homology to disease, Crohn's disease, colitis ulcerosa, as well as other these positions. diseases connected with hvemA. Preferably, Said enzyme or 0189 In a ninety-fourth embodiment of this variant the Said fusion protein is capable of Specifically inactivating enzyme is a protease and is capable of hydrolyzing human hvemA (SEQ ID NO: 191). More preferably said enzyme or beta-amyloid (hbeta-amyloid). The enzymes or the fusion Said fusion protein is capable of hydrolysing the peptide proteins can thus be used for preparing medicaments for the bonds between positions 14/15, 18/19, 23/24, 24/25, 26/27, treatment of diseases, Such as, but not limited to, Alzheimer, 31/32, 54/55, 62/63, 68/69, 71/72, 75/76, 95/96, 101/102 as well as other diseases connected with hbeta-amyloid. and/or 103/104 (most preferred between positions 23/24, Preferably, Said enzyme or Said fusion protein is capable of 26/27, 62/63, 68/69, 95/96, 101/102 and/or 103/104) in specifically inactivating hbeta-amyloid (SEQ ID NO: 190). hvemA or a peptide bond in proximity to these positions in More preferably Said enzyme or Said fusion protein is hvemA, or peptide bonds in protein targets related to hvemA capable of hydrolysing the peptide bonds between positions at positions having Structural homology or Sequence homol 1/2, 3/4, 5/6, 7/8, 10/11, 11/12, 16/17, 19/20, 20/21, 22/23, ogy to these positions. 23/24 and/or 28/29 (most preferred between positions 7/8, 10/11, 11/12, 16/17 and/or 23/24) in hbeta-amyloid or a 0191 In Some examples, the enzyme is a protease and is peptide bond in proximity to these positions in hbeta capable of hydrolyzing a target given in Table 1a to treat a amyloid, or peptide bonds in protein targets related to pathology or disease associated with that protein. hbeta-amyloid at positions having Structural homology or 0.192 It is obvious to someone skilled in the art that also Sequence homology to these positions. polymorphisms of all target Sequences referred to are 0190. In a ninety-fifth embodiment of this variant the included. The expression “proximity to these positions' in enzyme is a protease and is capable of hydrolyzing human all embodiments above refer to positions of peptide bonds Tumor necrosis factor receptor Superfamily member 14 that are between 10 and 5 Angström and/or 5 amino acids, (hvemA). The enzymes or the fusion proteins can thus be preferably 3 amino acids, next to the positions of the peptide used for preparing medicaments for the treatment of dis bonds

TABLE 1 a Target for NBE cleavage Disease or condition to be improved a5B1 (VLA-5) cancer cell migration and adhesion of several cancers including lung cancers and myelomas ADAM-12-S Cancer ADAM-9 Cancer Adiponectin (also called GBP-28, apM1, chronic renal failure, type I diabetes, AdipoC) and Acrp30) anorexia nervosa ADP receptors (e.g., ADP receptor P2Y (12), trombosis and platelet diseases ADP receptor P2T(AC), ADP receptor P2Y(1)) advanced glycation endproducts receptor diabetes (RAGE) Aldose reductase diabetes, including autoimmune diabetes angiotensin-converting enzyme (ACE) diabetes mellitus Anthrax: EF: Edema Factor Anthrax Anthrax: LF: Leathal Factor Anthrax Anthrax: PA, Protective Antigen Anthrax AP-1 intimal hyperplasia - vascular remodeling upon blood vessel injury B7-1, B7-2, CD28 Graft-v.-host disorder, rheumatoid arthritis, transplant rejection, diabetes mellitus 14 BAD apoptosis associated disorders, immunodeficiency diseases (AIDS/HIV), senescence, degenerative disorders (neurodegenerative diseases), ischemic and reperfusion cell death, acute ischemic injury, infertility, infectious colitis, inflammatory bowel disease (IBD), in particular in Crohn's disease, improved heart function after heart attack, cell death in Sainonelia infections 15 BAX apoptosis associated disorders, immunodeficiency diseases (AIDS/HIV), senescence, degenerative disorders (neurodegenerative diseases), ischemic and reperfusion cell death, acute ischemic injury, infertility, infectious colitis, inflammatory bowel disease (IBD), in particular in Crohn's disease, improved US 2005/0175581 A1 Aug. 11, 2005 24

TABLE 1a-continued Target for NBE cleavage Disease or condition to be improved heart function after heart attack, cell death in Sainonelia infections 16 Bcl-2 CaCe 17 BCR-Abl CaCe 18 beta-catenin cancer (e.g., metastasis) 19 beta-lactamases from bacteria (e.g., Infections including cystic fibrosis and Pseudomonas aeruginosa, Moraxeia chronic lung infection, pneumonia or (Branhamella) catarrhalis) bronchitis 2O BLyS Systemic lupus erythematosus 21 Bovine, swine, sheep, human and other animal prion diseases animal glycophosphatidylinositol (GPI)- anchored protein PrP(C) or their isoforms, PrP(Sc) 22 Asthma; Coronary artery bypass graft (CABG) surgery, Acute Pancreatitis, Inflamm. 23 Carbohydrate sulfotransferases (e.g., Nod H inflammation, viral infection and cancer sulfotransferase, UDP glucuronosyltransferase, Heparan sulfate 3 O-sulfotransferase isoform 3, human estrogen sulfotransferase, phenol sulfotransferase SULT1A1 (ST1A3), human GalCer sulfotransferase) 24 caspase-6 apoptosis associated disorders, immunodeficiency diseases (AIDS/HIV), senescence, degenerative disorders (neurodegenerative diseases), ischemic and reperfusion cell death, acute ischemic injury, infertility, infectious colitis, inflammatory bowel disease (IBD), in particular in Crohn's disease, improved heart function after heart attack, cell death in Sainonelia infections 25 Caspase-1 (IL1beta converting enzyme infectious colitis, inflammatory bowel (ICE) disease (IBD), in particular in Crohn's disease, improved heart function after heart attack or ischaemiafreperfusion, cell death in Sainonella infections 26 caspase-8 Parkinson's disease. 27 CCR8 Asthma, COPD, Chr Bronchitis 28 CD18 inflammation 29 CD2O NHL 3O CD22 NHL 31 CD22 NHL 32 CD25, IL-2 receptor Transplant rejection 33 CD3 Graft-versus-host disease, Transplant rejection and rejection prophylaxis; Type I diabetis, 34 CD3OL receptor Cancer, malignant lymphoma, classical Hodgkin's Lymphoma 35 CD3OL classical Hodgkin's Lymphoma (cHL) 36 CD33 AML, acute myelogenous leukemia 37 CD35 RA, Transplant rejection 38 CD4 HIV, psoriasis, transplant rejection and graft-versus-host colitis and autoimmune disorders; rheumatoid arthritis 39 CD40 classical Hodgkin's Lymphoma (cHL); Graft-V.-host disorder, transplant rejection, psoriasis 40 Acute Pancreatitis, systemic lupus erythematosus, classical Hodgkin's Lymphoma (cHL) 41 CD46 (MCP) renal tumors, uveal melanomas, gastrointestinal tumours and other forms of cancer 42 CD52 B-CLL 43 CD55 (DAF) renal tumors, gastric and other forms of CaCe 44 CD59 renal tumors and other forms of cancer 45 cdk-4 CaCe 46 chitin from fungal pathogens Fungal infections 47 CINC/GRO-alpha Acute Pancreatitis, Inflamm. 48 c-Jun intimal hyperplasia - vascular remodeling upon blood vessel injury US 2005/0175581 A1 Aug. 11, 2005 25

TABLE 1a-continued Target for NBE cleavage Disease or condition to be improved 49 ClfA Staph. aureus infections 50 c-met (Hepatocyte growth factor receptor) angiogenic growth factor in cancers 51 CO-029 or other human tetraspanin proteins Cancer 52 Corticotropin-releasing hormone (CRH) Modulation of arousal, modulation of reproductive behavior and function, Modulation of behavior in feeding, cerebrospinal hypercortisolemia, anxiety and affective disorders, melancholic depression as well as post-traumatic stress disorder (PTSD) 53 CTLA-4 (CD152) Breast cancer 54 CXCR1 Asthma, COPD, Chr Bronchitis 55 Asthma, COPD, Chr Bronchitis 56 cyclo-oxygenase (COX) thrombosis 57 cytocrome C (same as caspase) 58 diacylglycerol acyltransferase (DGAT) diet induced obesity and diabetes 59 ErbB3 (Her-3), ErbB4 (Her4) CaCe 60 EGFR endodomain (intracellular) metastatic adenocarcinoma (cancer) of the colon or rectum or stage III colon cancer or metastatic epidermal growth factor receptor-positive colorectal cancer or Cancer of the Oropharynx, Hypopharynx, or Larynx or Head and Neck Cancer 61 eotaxin classical Hodgkin's Lymphoma (cHL) and eosinophilia-mediated inflammation 62 EPA1 (e.g., from Candida glabrata and other fungemia, mucosal infection fungal pathogens) 63 Ep-CAM (epithelial cell adhesion molecule) colorectal cancer EGP-2 64 ERK intimal hyperplasia - vascular remodeling upon blood vessel injury 65 E-Selectin Prostate Cancer 66 exfoliative toxin (e.g., from Staphylococcus scalded skin syndrome (SSS) aureus) 67 exorphins (e.g., gluten exorphin A5, B4, B5 Autism and schizophrenia and C; alpha-casein exorphin on CA1) 68 F protein (e.g., from RSV) RSV 69 factor Xa trombosis including deep vein thrombosis 70 fibrinogen cardiovascular disorders 71 G(q/11) trombosis and G(q)-mediated diseases 72 gangliosides (GT3, GD3 and especially autoimmune diabetes GM-1 and the islet-specific monosialo ganglioside GM2-1) 73 glycogen phosphorylase (GP) type 2 diabetes 74 cystic fibrosis, Lung Inflammation, classical Hodgkin's Lymphoma (cHL) and eosinophilia-mediated inflammation 75 gp41 HIV infection 76 Hag (e.g., from Moraxella (Branhamella) pneumonia or bronchitis catarrhalis) 77 hemaglutinin influenza infection 78 Heme Oxygenase CF, Lung Inflammation 79 HIFI CaCe 8O Histone deacetylase Cancer 81 IgE/IgER Graft-V.-host disorder, transplant rejection 82 IGF breast cancer 83 IL-12/IL-12 receptor Crohn's disease, inflammatory bowel disease, classical Hodgkin's Lymphoma (cHL), multiple sclerosis 84 IL-13R asthma, fibrosis, psoriasis and atopic dermatitis, classical Hodgkin's Lymphoma (cHL). 85 IL-15/IL-15R psoriasis, acute myeloid leukemia, rheumatoid arthritis, inflammation or inflammatory bowel disease and in diseases associated with the retrovirus HTLV-I (human T-cell lymphotropic virus I) 86 IL-18 receptor (“IL-1- related protein, IL inflammation, organ and graft rejection 1Rp) 87 IL-27 asthma, inflammation, rheumatic disorders 88 IL-2R alpha and beta autoimmune disorders, Graft-v.-host disorders, rheumatoid arthritis, T-cell leukemia/lymphoma US 2005/0175581 A1 Aug. 11, 2005 26

TABLE 1a-continued Target for NBE cleavage Disease or condition to be improved 89 IL-31 asthma, inflammation, rheumatic disorders 90 IL-SR asthma, classical Hodgkin's Lymphoma (cHL), and eosinophilia-mediated inflammation 91 IL-7 classical Hodgkin's Lymphoma (cHL) 92 IL-9 Chronic Obstructive Pulmonary Disease, classical Hodgkin's Lymphoma (cHL), airway inflammation and asthma 93 inner layer protein p24 (e.g., from HIV) AIDS 94 Integrin a(4) b1) multiple sclerosis; Crohn's disease, inflammatory bowel disease 95 Integrin a(4) b(7) Crohn's 96 Integrin a(v) b(3) Melanoma 97 Integrin b(1) Cron's disease, inflammatory bowel disease 98 Integrin b(7) Cron's disease, inflammatory bowel disease 99 interleukin 11: Cron's disease, inflammatory bowel disease IP-10, Mig, MIP-1 alpha classical Hodgkin's Lymphoma (cHL) IRAK-1 Inflammation, Sepsis, and Autoimmunity IRAK-4 Inflammation, Sepsis, and Autoimmunity Jun N-terminal kinase (JNK) intimal hyperplasia - vascular remodeling upon blood vessel injury and diabetes Kallikrein hereditary angioedema (HAE) eukocyte function-associated antigen-1 organ and graft rejection eukotriene B(4) Chronic Obstructive Pulmonary Disease, inflammation eukotriene D (LTD) Chronic Obstructive Pulmonary Disease, inflammation eukotriene receptor Cys-LT Chronic Obstructive Pulmonary Disease, inflammation eukotriene receptor Cys-LT, Chronic Obstructive Pulmonary Disease, inflammation eukotriene receptor LTB4-1, LTB4-2 Chronic Obstructive Pulmonary Disease, inflammation 11 eukotriene receptors Chronic Obstructive Pulmonary Disease, inflammation 12 Lewis y/b antigen Cancer; Lung cancer 13 ipoprotein(a) cardiovascular disorders 14 LT-alpha classical Hodgkin's Lymphoma (cHL) 15 yphotoxin beta colitis, diabetes, arthritis, infammation 16 matrix metalloprotease-1 (MMP-1) emphysema 17 mcaP adherence protein (e.g., from pneumonia or bronchitis Moraxella (Branhamella) catarrhalis) 18 MCP-1 Acute Pancreatitis, Inflamm. 19 classical Hodgkin's Lymphoma (cHL) 2O MDC classical Hodgkin's Lymphoma (cHL) and eosinophilia-mediated inflammation 21 MHC class II receptors lymphomas and other cancers including non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma and hairy cell leukemia. 22 MID (e.g., from Moraxella (Branhamella) pneumonia or bronchitis catarrhalis) 23 MMP-12 emphysema 24 MMP-13 CaCe 25 MN antigen Liver cancer 26 muscarinic receptor, M1 and M3 Lung diseases, e.g., Chronic Obstructive Pulmonary Disease 27 NAD(P)H oxidase vascular complications associated with diabetes and other diseases related to reactive oxygen species (ROS) 28 neutrophil elastase Chronic Obstructive Pulmonary Disease 29 NF-kappaB Chronic Obstructive Pulmonary Disease, atherosclerosis and thrombosis 3O nucleocapsid p17 (e.g., from HIV) AIDS 31 p10 protease (e.g., from HIV) AIDS 32 p115-RhoGEF A-site cancer (e.g., metastasis) 33 p32 integrase (e.g., from HIV) AIDS 34 p64 Reverse transcriptase (e.g., from HIV) AIDS 35 PAF Acute Pancreatitis, Inflamm. 36 parathyroid hormone chronic renal failure, Cardiovascular disease US 2005/0175581 A1 Aug. 11, 2005 27

TABLE 1a-continued Target for NBE cleavage Disease or condition to be improved 37 parathyroid hormone-related peptide chronic renal failure, Cardiovascular (PTHrP) receptor disease 38 PDE3A, Platelet cyclic adenosine thrombosis monophosphate (cAMP) phosphodiesterase 39 phosphodiesterase 4 Chronic Obstructive Pulmonary Disease 40 Polymorphic epithelial mucin (PEM) Cancer (Solid tumors), Ovarian cancer (MUC-1) 41 porin F (OprF) (e.g., from Pseudomonas human alveolar epithelial adhesin aeruginosa) 42 Proteasome subunits trombosis including arterial thrombosis 43 Protein-Tyrosine Phosphatase PTPase 1B Diabetes and Related States of Insulin (PTP1B) Resistance 44 PTH receptor chronic renal failure, Cardiovascular disease 45 RANK classical Hodgkin's Lymphoma (cHL) 46 RANKL classical Hodgkin's Lymphoma (cHL) 47 Rip2 bacteria-induced inflammation 48 RSV (respiratory syncytium virus) fusion RSV infection protein 49 Sortase (e.g., from Streptococcus mutans) Caries 50 Sre-Homology Inositol Phosphatase-2 type 2 diabetes mellitus. (SHIP2) 51 T1/ST2 Inflammation, for example, eosinophilic inflammation of the airways 52 TARC classical Hodgkin's Lymphoma (cHL) and eosinophilia-mediated inflammation 53 TGFbeta-1, 2, 3, 4 Glaucoma, suppression of cell-mediated immunity 54 TGF-betaRI diffuse systemic sclerosis 55 thrombin blood clotting 56 tissue factor?factor VIIa trombosis including venous thrombosis 57 Toll-like Receptors (TLRs) 1-10 CF, Lung Inflammation 58 transmembrane PTPase leukocyte antigen Diabetes and Related States of Insulin related (LAR) Resistance 59 triggering receptor expressed on myeloid CF, Lung Inflammation, septic shock, cells (TREM)-1 cancer, acute pancreatitis 60 UspA1(e.g., from Moraxella (Branhamella) pneumonia or bronchitis catarrhalis) 61 VAP-1 (Vascular adhesion protein-1) inflammation 62 VEGFR-3 CaCe 63 Wnt proteins 2, 3, 4, and 7B Cancer (e.g., breast cancer) 64 OSM receptor inflammation, rheumatoid arthritis, inflammatory bowel disease 65 IL-6 receptor alpha chain inflammation, rheumatoid arthritis, inflammatory bowel disease 66 IL-6 receptor beta chain inflammation, rheumatoid arthritis, inflammatory bowel disease 67 lymphotoxin beta receptor inflammation, rheumatoid arthritis, inflammatory bowel disease 68 leukemia inhibitory factor receptor inflammation, rheumatoid arthritis, inflammatory bowel disease

0193 Preferably, in this variant the scaffold of the engi Vated. Examples of Such pro-drugs are pro-proteins Such as neered enzyme provided in step (c) is of human origin in the inactivated forms of coagulations factors. In another order to avoid or reduce immunogenicity or allergenic particular variant, the engineered enzyme is an oxidoreduc effects associated with the application of the enzyme in the tase and the target Substrate is a chemical that can be human body. activated by oxidation. 0194 Alternatively, immunogenicity and allergenicity 0196. In a second variant of this aspect of the invention, can be reduced by deimmunization of the engineered the engineered enzyme is used for diagnostic puposes. In a enzyme. Deimmunization in this context refers to the particular embodiment of this variant, the engineered removal or exchange of those amino acid residues that enzyme is target-Specific protease. Such diagnostic purposes confer immunogenicity or allergenicity to the engineered comprise but are not limited to applications with the aim of enzyme. diagnosing diseases, testing genetic predispositions or moni toring disease progression during therapy. 0.195. In further embodiment of this variant, the target Substrate is a pro-drug which is activated by the engineered 0197). In a particular embodiment, the diagnosis is based enzyme. In a particular embodiment of this variant, the on the testing for the presence or absence of a disease engineered enzyme has proteolytic activity and the target Specific marker protein or a disease-specific variant of a Substrate is a protein target which is proteolytically acti human protein in test Samples Such as human tissue Samples, US 2005/0175581 A1 Aug. 11, 2005 28 blood Samples or other Samples taken from patients. The engineered enzyme Specifically oxidizes or reduces one or testing employs a protease with Specificity for a particular, more industrially, technically or nutrionally relevant chemi disease-related target protein. The testing is done by anal cal Substrates. ySing the proteolytic degradation of Such protein in the test 0200. A second aspect of the invention discloses engi Sample. neered enzymes with defined Specificities. These engineered 0198 In a preferred embodiment the aim of the diagnos enzymes are characterized by the following components: tic test is to detect and/or quantify a disease-specific variant 0201 (a) a protein scaffold capable of catalyzing at least of a native human protein. Such a diagnostic test employs a one chemical reaction on a Substrate, and protease that is specific for the disease-related protein vari ant, i.e. it has significantly higher proteolytic activity on the 0202 (b) one or more specificity determining regions disease-related protein variant compared to the native (SDRs) located at sites in the protein scaffold that enable the human protein. The disease-related protein variant is there resulting engineered protein to discriminate between at least fore detected and/or quantified by detecting and/or quanti one target Substrate and one or more different Substrates, fying the activity of the target-Specific protease. Such detec wherein the SDRs are essentially synthetic peptide tion and/or quantification is done by directly measuring the Sequences. degradation products of the target protein or indirectly by 0203 Preferably, such defined specificity of the engi measuring the influence of the target protein on the activity neered enzymes is not conferred by the protein Scaffold. of the target-Specific protease by a competition assay. In another preferred embodiment the aim of the diagnostic test 0204. In principle, the protein scaffold can have a variety is to detect and/or quantify a protein that is specific for an of primary, Secondary and tertiary Structures. The primary infection by an infectious agent Such as a virus or a bacte Structure, i.e. the amino acid Sequence, can be an engineered rium. Such a diagnostic test employs a protease that is Sequence or can be derived from any viral, prokaryotic or Specific for a protein Specifically expressed upon infection eukaryotic origin. For human therapeutic use, however, the by the infectious agent, i.e. it has significantly higher pro protein Scaffold is preferably of mammalian origin, and teolytic activity on a particular infection-indicating protein more preferably, of human origin. Furthermore, the protein compared to any other native human protein. The infection Scaffold is capable to catalyze one or more chemical reac indicating protein is therefore detected and/or quantified by tions and has preferably only a low specificity. detecting and/or quantifying the proteolytic activity of the 0205 Preferably, derivatives of the protein scaffold are target-Specific protease. Such detection and/or quantification used that have modified amino acid Sequences that confer is done by directly measuring the degradation products of improved characteristics for the applicability as protein the infection-indicating protein or indirectly by measuring Scaffolds. Such improved characteristics comprise, but are the influence of the infection-indicating protein on the not limited to, Stability; expression or Secretion yield; fold activity of the target-Specific protease by a competition ing, in particular after combination of the protein Scaffold asSay. with SDRs, increased or decreased Sensitivity to regulators 0199. In a third variant of this aspect of the invention, the Such as activators or inhibitors, immunogenicity; catalytic engineered enzyme is used as a technical means in order to rate; kM or substrate affinity. catalyze an industrially or nutritionally relevant reaction 0206. The engineered enzymes reveal their quantitative with defined specificity. In a particular embodiment of this Specificity from the peptide Sequences that are combined variant the engineered enzyme has proteolytic activity, the with the protein Scaffold. Therefore, the engineered peptide catalyzed reaction is a proteolytic processing, and the engi Sequences are acting as Specificity Determining Regions or neered enzyme specifically hydrolyses one or more indus SDRs. The number, the length and the positions of Such trially or nutrionally relevant protein Substrates. In a pre SDRs can vary over a wide range. The number of SDRs ferred embodiment of this variant the engineered enzyme within the Scaffold is at least one, preferably more than one, hydrolyses one or more industrially or nutrionally relevant more preferably between two and eleven, most preferably protein Substrates at Specific Sites, thereby leading to indus between two and six. The SDRs have a length between one trially or nutrionally desired product properties Such as and 50 amino acid residues, preferably a length between one texture, taste or precipitation characteristics. In a further and 15 amino acid residues, more preferably a length particular embodiment of this variant, the engineered between one and Six amino acid residues. Alternatively, the enzyme catalyzes the hydrolysis of glycosidic bonds (gly SDRs have a length between two and 20 amino acid resi cosidase or glycosylases activity). Then, preferably, the dues, preferably a length between two and ten amino acid catalyzed reaction is a polysaccharide processing, and the residues, more preferably a length between three and eight engineered enzyme specifically hydrolyses one or more amino acid residues. industrially, technically or nutrionally relevant polysaccha ride substrates. In a further particular embodiment of this 0207. The inventive engineered enzymes can further be variant, the engineered enzyme catalyzes the hydrolysis of desribed as antibody-like protein molecules comprising con triglyceride esters or lipids (lipase activity). Then, prefer Stant and variable regions, but having a non-immunoglogu ably, the catalyzed reaction is a lipid processing Step, and the lin backbone and having an active site (catalytic activity) in engineered enzyme specifically hydrolyses one or more the constant region, whereby the Substrate Specificity of the industrially, technically or nutrionally relevant lipid Sub active site is modulated by the variable region. Preferably, as Strates. In a further particular variant of this embodiment, the in the immunoglobulin Structure, the variable regions are engineered enzyme catalyzes the oxidation or reduction of loops of variable length and composition that interact with Substrates (oxidoreductase activity). Then, preferably, the a target molecule. US 2005/0175581 A1 Aug. 11, 2005 29

0208. In a particular variant of the invention, the engi turally or by amino acid Sequence homology to the regions neered enzymes have hydrolase activity. In a preferred 18-25, 38-48, 54-63, 73-86, 122-130, 148-156, 165-171 and variant, the engineered enzymes have proteolytic activity. 194-204 in human trypsin I, and more preferably at one or Particularly preferred protein scaffolds for this variant are more positions from the group of positions that correspond unspecific proteases or are parts from unspecific proteases or Structurally or by amino acid Sequence homology to the are otherwise derived from unspecific proteases. The expres regions 20-23, 41-45, 57-60, 76-83, 125-128, 150-153, sions “derived from' or “a derivative thereof in this respect 167-169 and 197-201 (numbering of amino acids according and in the following variants and embodiments refer to derivatives of proteins that are mutated at one or more amino to SEQID NO:1). The number of SDRs to be combined with acid positions and/or have a homology of at least 70%, this type of protein scaffold is preferably between 1 and 10, preferably 90%, more preferably 95% and most preferably and more preferably between 2 and 4. Preferably, the protein 99% to the original protein, and/or that are proteolytically Scaffold is equal to or is a derivative or homologue of one or processed, and/or that have an altered glycosylation pattern, more of the following proteins: chymotrypsin, granzyme, and/or that are covalently linked to non-protein Substances, kallikrein, trypsin, mesotrypsin, neutrophil elastase, pancre and/or that are fused with further protein domains, and/or atic elastase, enteropeptidase, cathepsin, thrombin, ancrod, that have C-terminal and/or N-terminal truncations, and/or coagulation factor IXa, coagulation factor VIIa, coagulation that have specific insertions, Substitutions and/or deletions. factor Xa, activated protein C, urokinase, tissue-type plas Alternatively, “derived from may refer to derivatives that minogen activator, plasmin, Desmodus-type plasminogen are combinations or chimeras of two or more fragments activator. More preferably, the protein Scaffold is trypsin or from two or more proteins, each of which optionally com thrombin or is a derivative or homologue from trypsin or prises any or all of the aforementioned modifications. The thrombin. For the use as a human therapeutic, the trypsin or tertiary Structure of the protein Scaffold can be of any type. thrombin scaffold is most preferably of human origin in Preferably, however, the tertiary structure belongs to one of order to minimize the risk of an immune response or an the following structural classes: class S1 (chymotrypsin fold allergenic reaction. of the serine proteases family), class S8 (subtilisin fold of 0211 Preferably, derivatives with improved characteris the Serine proteases family), class SC (carboxypeptidase tics derived from human trypsin I or from proteins with fold of the Serine proteases family), class A1 (pepsin A fold Similar tertiary Structure are used. Preferred examples of of the aspartic proteases), or class C14 (caspase-1 fold of the such derivatives are derived from human trypsin I (SEQ ID cysteine proteases). Examples of proteases that can serve as NO:1) and comprise one or more of the following amino the protein Scaffold of engineered proteolytic enzymes for acid Substitutions E56G, R78W: Y131F: A146T, C183R. the use as human therapeutics are or are derived from human trypsin, human thrombin, human chymotrypsin, human pep 0212. It is preferred that at least one of two SDRs are Sin, human endothiapepsin, human caspases 1 to 14, and/or inserted into human trypsin I, or a derivative thereof, human furin. between residues 42 and 43 (SDR 1) and between 123 and 0209 The defined specificity of the engineered pro 124 (SDR 2), respectively (numbering of amino acids teolytic enzymes is a measure of their ability to discriminate according to SEQ ID NO:1). In addition the SDR 1 has a between at least one target peptide or protein Substrates and preferred length of 6 and the SDR 2 has a preferred length one or more further peptide or protein Substrates. Preferably, of 5 amino acids, respectively. In a preferred variant of this the defined specificity refers to the ability to discriminate embodiment, the SDR1 and SDR 2 sequences comprise one peptide or protein Substrates that differ in other positions of the amino acid Sequences listed in table 2. Such engi than the P1 site, more preferably, the defined specificity neered proteolytic enzymes have specificity for the target refers to the ability to discriminate peptide or protein Sub substrate B as exemplified in example IV. strates that differ in other positions than the P1 site and the 0213. In a further embodiment the protein scaffold P1" site. Most preferably, the engineered proteolytic belongs to the S8 Structural Subclass of Serine proteases enzymes distinguish target peptid or protein Substrates at as and/or has a tertiary Structure Similar to Subtilisin E from many Sites as is necessary to preferentially hydrolyse the Bacillus subtilis and/or has at least 70% identity on the target Substrate versus other proteins. AS an example, a amino acid level to a protein of the S8 structural Subclass of therapeutically useful engineered proteolytic enzyme serine proteases. Preferably, the scaffold belongs to the applied intravenously in the human body should be suffi Subtilisin family or the human pro-protein convertases. It is ciently Specific to discriminate between the target Substrate preferred that SDRs are inserted into the protein scaffold at and any other protein in the human Serum. Preferably, Such one or more positions from the group of positions that an engineered proteolytic enzyme recognizes and discrimi correspond structurally or by amino acid Sequence homol nates peptide Substrates at three or more amino acid posi ogy to the regions 6-17, 25-29, 47-55, 59-69, 101-111, tions, more preferably at four or more positions, and even 117-125, 129-137, 139-154, 158-169, 185-195 and 204-225 more preferably at five or more amino acid positions. These in subtilisin E from Bacillus Subtilis, and more preferably at positions may either be adjacent or non-adjacent. one or more positions from the group of positions that 0210. In a first embodiment, the protein scaffold has a correspond structurally or by amino acid Sequence homol tertiary Structure or fold equal or Similar to the tertiary ogy to the regions 59-69, 101-111, 129-137, 158-169 and structure or fold of the S1 structural Subclass of serine 204-225 (numbering of amino acids according to SEQ ID proteases, i. e. the chymotrypsin fold, and/or has at least NO:7). It is preferred that the protein scaffold is equal to or 70% identity on the amino acid level to a protein of the S1 is a derivative or homologue of one or more of the following Structural Subclass of Serine proteases. It is preferred that proteins: Subtilisin Carlsberg, B. Subtilis Subtilisin E; Sub SDRs are inserted into the protein scaffold at one or more tilisin BPN"; B. licheniformis Subtilisin; B. lenius Subtilisin; positions from the group of positions that correspond struc Bacillus alcalophilus alkaline protease; proteinase K., keXin; US 2005/0175581 A1 Aug. 11, 2005 30 human pro-protein convertase; human furin. In a preferred Similar to assemblin from human cytomegalovirus. It is variant, subtilisin BPN' or one of the proteins SPC 1 to 7 is preferred that SDRs are inserted into the protein scaffold at used as the protein Scaffold. one or more positions from the group of positions that 0214. In a further embodiment the protein scaffold correspond structurally or by amino acid Sequence homol belongs to the family of aspartic proteases and/or has a ogy to the regions 25-33, 64-69, 134-155, 162-169 and tertiary Structure Similar to human pepsin. Preferably, the 217-244 in assemblin from human cytomegalovirus, and Scaffold belongs to the A1 class of proteases and/or has at more preferably at one or more positions from the group of least 70% identity on the amino acid level to a protein of the positions that correspond structurally or by amino acid A1 class of proteases. It is preferred that SDRs are inserted Sequence homology to the regions 27-31, 164-168 and into the protein Scaffold at one or more positions from the 222-239 (numbering of amino acids according to SEQ ID group of positions that correspond structurally or by amino NO:16). It is preferred that the assemblin from human acid sequence homology to the regions 6-18, 49-55, 74-83, cytomegalovirus or a derivative or homologue thereof is 91-97, 112-120, 126-137, 159-164, 184-194, 242-247, 262 used as the Scaffold. 267 and 277-300 in human pepsin, and more preferably at one or more positions from the group of positions that 0218. In a further embodiment the protein scaffold correspond structurally or by amino acid Sequence homol belongs to the S26 class of Serine proteases or has at least ogy to the regions 10-15, 75-80, 114-118, 130-134, 186-191 70% identity on the amino acid level to a protein of the S26 and 280–296 (numbering of amino acids according to SEQ class of Serine proteases and/or has a tertiary Structure ID NO:11). It is preferred that the protein scaffold is equal Similar to the Signal peptidase from Escherichia coli. It is to or is a derivative or homologue of one or more of the preferred that SDRs are inserted into the protein scaffold at following proteins: pepsin, chymosin, renin, cathepsin, one or more positions from the group of positions that yapsin. Preferably, pepsin or endothiopepsin or a derivative correspond structurally or by amino acid Sequence homol or homologue thereof is used as the protein Scaffold. ogy to the regions 8-14, 57-68, 125-134, 239-254, 200-211 0215. In a further embodiment the protein scaffold and 228-239 in signal peptidase from Escherichia coli, and belongs to the cysteine protease family and/or has a tertiary more preferably at one or more positions from the group of structure similar to human caspase 7. Preferably the scaffold positions that correspond structurally or by amino acid belongs to the C14 class of cysteine proteases or has at least sequence homology to the regions 9-13, 60-67, 127-132 and 70% identity on the amino acid level to a protein of the C14 203-209 (numbering of amino acids according to SEQ ID class of cysteine proteases. It is preferred that SDRS are NO:17). It is preferred that the signal peptidase from inserted into the protein Scaffold at one or more positions Escherichia coli or a derivative or homologue thereof is from the group of positions that correspond Structurally or used as the Scaffold. by amino acid Sequence homology to the regions 78-91, 144-160, 186-198, 226-243 and 271-291 in human caspase 0219. In an further embodiment the protein scaffold 7, and more preferably at one or more positions from the belongs to the S33 class of Serine proteases or has at least group of positions that correspond structurally or by amino 70% identity on the amino acid level to a protein of the S33 acid sequence homology to the regions 80-86, 149-157, class of Serine proteases and/or has a tertiary Structure 190-194 and 233-238 (numbering of amino acids according Similar to the prolyl aminopeptidase from Serratia marce to SEQ ID NO:14). It is preferred that the protein scaffold Scens. It is preferred that SDRs are inserted into the protein is equal to or is a derivative or homologue of one of the Scaffold at one or more positions from the group of positions caspases 1 to 9. that correspond Structurally or by amino acid Sequence homology to the regions 47-54, 152-160, 203-212 and 0216) In a further embodiment the protein scaffold 297-302 in prolyl aminopeptidase from Serratia marce belongs to the S11 class of Serine proteases or has at least Scens, and more preferably at one or more positions from the 70% identity on the amino acid level to a protein of the S11 group of positions that correspond structurally or by amino class of Serine proteases and/or has a tertiary Structure acid sequence homology to the regions 50-53, 154-158 and Similar to D-alanyl-D-alanine transpeptidase from Strepto 206-210 (numbering of amino acids according to SEQ ID myces species K15. It is preferred that SDRs are inserted NO:18). It is preferred that the prolyl aminopeptidase from into the protein Scaffold at one or more positions from the Serratia marceScens or a derivative or homologue thereof is group of positions that correspond structurally or by amino used as the Scaffold. acid sequence homology to the regions 67-79, 137-150, 191-206, 212-222 and 241-251 in D-alanyl-D-alanine 0220. In a further embodiment the protein scaffold transpeptidase from Streptomyces Species K15, and more belongs to the S51 class of Serine proteases or has at least preferably at one or more positions from the group of 70% identity on the amino acid level to a protein of the S51 positions that correspond structurally or by amino acid class of Serine proteases and/or has a tertiary Structure sequence homology to the regions 70-75, 141-147, 195-202 similar to aspartyl dipeptidase from Escherichia coli. It is and 216-220 (numbering of amino acids according to SEQ preferred that SDRs are inserted into the protein scaffold at ID NO:15). It is preferred that the D-alanyl-D-alanine one or more positions from the group of positions that transpeptidase from Streptomyces Species K15 or a deriva correspond structurally or by amino acid Sequence homol tive or homologue thereof is used as the Scaffold. ogy to the regions 8-16, 38-46, 85-92, 132-140, 159-170 and 205-211 in aspartyl dipeptidase from Escherichia coli, and 0217. In a further embodiment the protein scaffold more preferably at one or more positions from the group of belongs to the S21 class of Serine proteases or has at least positions that correspond structurally or by amino acid 70% identity on the amino acid level to a protein of the S21 sequence homology to the regions 10-14, 87-90, 134-138 class of Serine proteases and/or has a tertiary Structure and 160-165 (numbering of amino acids according to SEQ US 2005/0175581 A1 Aug. 11, 2005 31

ID NO:19). It is preferred that the aspartyl dipeptidase from the group of positions that correspond structurally or by Escherichia coli or a derivative or homologue thereof is amino acid Sequence homology to the regions 92-101, used as the Scaffold. 245-250 and 287-291 (numbering of amino acids according to SEQ ID NO:23). It is preferred that the human calpain-2 0221) In a further embodiment the protein scaffold belongs to the A2 class of aspartic proteases or has at least or a derivative or homologue thereof is used as the Scaffold. 70% identity on the amino acid level to a protein of the A2 0225. In a further embodiment the protein scaffold class of aspartic proteases and/or has a tertiary Structure belongs to the C4 class of cysteine proteases or has at least Similar to the protease from human immunodeficiency virus. 70% identity on the amino acid level to a protein of the C4 It is preferred that SDRs are inserted into the protein scaffold class of cysteine proteases and/or has a tertiary Structure at one or more positions from the group of positions that similar to NIa protease from tobacco etch virus. It is correspond structurally or by amino acid Sequence homol preferred that SDRs are inserted into the protein scaffold at ogy to the regions 5-12, 17-23, 27-30, 33-38 and 77-83 in one or more positions from the group of positions that protease from human immunodeficiency virus, and more correspond structurally or by amino acid Sequence homol preferably at one or more positions from the group of ogy to the regions 23-31, 112-120, 144-150, 168-176 and positions that correspond structurally or by amino acid 205-218 in NIa protease from tobacco etch virus, and more sequence homology to the regions 7-10, 18-21, 34-37 and preferably at one or more positions from the group of 79-82 (numbering of amino acids according to SEQ ID positions that correspond structurally or by amino acid NO:20). It is preferred that the protease from human immu sequence homology to the regions 145-149, 169-174 and nodeficiency virus, preferably HIV-1 protease, or a deriva 212-218 (numbering of amino acids according to SEQ ID tive or homologue thereof is used as the Scaffold. NO:24). It is preferred that the NIa protease from tobacco etch virus (TEV protease) or a derivative or homologue 0222. In an further embodiment the protein scaffold thereof is used as the Scaffold. belongs to the A26 class of aspartic proteases or has at least 70% identity on the amino acid level to a protein of the A26 0226. In a further embodiment the protein scaffold class of aspartic proteases and/or has a tertiary Structure belongs to the C10 class of cysteine proteases or has at least similar to the omptin from Escherichia coli. It is preferred 70% identity on the amino acid level to a protein of the C10 that SDRs are inserted into the protein scaffold at one or class of cysteine proteases and/or has a tertiary Structure more positions from the group of positions that correspond Similar to the Streptopain from StreptococcuS pyogenes. It is Structurally or by amino acid sequence homology to the preferred that SDRS are inserted into the protein Scaffold at regions 28-40, 86-98, 150-168, 213–219 and 267-278 in one or more positions from the group of positions that omptin from Escherichia coli, and more preferably at one or correspond structurally or by amino acid Sequence homol more positions from the group of positions that correspond ogy to the regions 81-90, 133-140, 150-164, 191-199,219 Structurally or by amino acid Sequence homology to the 229, 246-256, 306–312 and 330-337 in streptopain from regions 33-38, 161-168 and 273-277 (numbering of amino StreptococcuS pyogenes, and more preferably at one or more acids according to SEQ ID NO:21). It is preferred that the positions from the group of positions that correspond struc omptin from Escherichia coli or a derivative or homologue turally or by amino acid Sequence homology to the regions thereof is used as the Scaffold. 82-87, 134-138, 250-254 and 331-335 (numbering of amino acids according to SEQ ID NO:25). It is preferred that the 0223) In a further embodiment the protein scaffold Streptopain from StreptococcuS pyogenes or a derivative or belongs to the C1 class of cysteine proteases or has at least homologue thereof is used as the Scaffold. 70% identity on the amino acid level to a protein of the C1 class of cysteine proteases and/or has a tertiary Structure 0227. In a further embodiment the protein scaffold Similar to the papain from Carica papaya. It is preferred that belongs to the C19 class of cysteine proteases or has at least SDRs are inserted into the protein scaffold at one or more 70% identity on the amino acid level to a protein of the C19 positions from the group of positions that correspond struc class of cysteine proteases and/or has a tertiary Structure turally or by amino acid Sequence homology to the regions Similar to human ubiquitin Specific protease 7. It is preferred 17-24, 61-68, 88-95, 135-142, 153-158 and 176-184 in that SDRs are inserted into the protein scaffold at one or papain from Carica papaya, and more preferably at one or more positions from the group of positions that correspond more positions from the group of positions that correspond Structurally or by amino acid Sequence homology to the Structurally or by amino acid Sequence homology to the regions 3-15, 63-70, 80-86, 248-256, 272-283 and 292-304 regions 63-66, 136-139 and 177-181 (numbering of amino in human ubiquitin Specific protease 7, and more preferably acids according to SEQ ID NO:22). It is preferred that the at one or more positions from the group of positions that papain from Carica papaya or a derivative or homologue correspond structurally or by amino acid Sequence homol thereof is used as the Scaffold. ogy to the regions 10-15, 251-255, 277-281 and 298-304 (numbering of amino acids according to SEQ ID NO:26). It 0224. In a further embodiment the protein scaffold is preferred that the human ubiquitin Specific protease 7 or belongs to the C2 class of cysteine proteases or has at least a derivative or homologue thereof is used as the Scaffold. 70% identity on the amino acid level to a protein of the C2 class of cysteine proteases and/or has a tertiary Structure 0228. In a further embodiment the protein scaffold similar to human calpain-2. It is preferred that SDRs are belongs to the C47 class of cysteine proteases or has at least inserted into the protein Scaffold at one or more positions 70% identity on the amino acid level to a protein of the C47 from the group of positions that correspond Structurally or class of cysteine proteases and/or has a tertiary Structure by amino acid Sequence homology to the regions 90-103, Similar to the Staphopain from StaphylococcuS aureus. It is 160-172, 193-199,243-260,286-294 and 316-322 in human preferred that SDRs are inserted into the protein scaffold at calpain-2, and more preferably at one or more positions from one or more positions from the group of positions that US 2005/0175581 A1 Aug. 11, 2005 32 correspond structurally or by amino acid Sequence homol 70% identity on the amino acid level to a protein of the M10 ogy to the regions 15-23, 57-66, 108-119, 142-149 and class of metallo proteases and/or has a tertiary Structure 157-164 in Staphopain from StaphylococcuS aureus, and Similar to human collagenase. It is preferred that SDRS are more preferably at one or more positions from the group of inserted into the protein Scaffold at one or more positions positions that correspond structurally or by amino acid from the group of positions that correspond Structurally or sequence homology to the regions 17-22, 111-117, 143-147 by amino acid Sequence homology to the regions 2-7, 68-79, and 159-163 (numbering of amino acids according to SEQ 85-90, 107-111 and 135-141 in human collagenase, and ID NO:27). It is preferred that the staphopain from Staphy more preferably at one or more positions from the group of lococcuS aureuS or a derivative or homologue thereof is used positions that correspond structurally or by amino acid as the Scaffold. sequence homology to the regions 3-6, 71-78 and 136-140 0229. In an further embodiment the protein scaffold (numbering of amino acids according to SEQ ID NO:31). It belongs to the C48 class of cysteine proteases or has at least is preferred that human collagenase or a derivative or 70% identity on the amino acid level to a protein of the C48 homologue thereof is used as the Scaffold. class of cysteine proteases and/or has a tertiary Structure 0233. It is further preferred that the engineered enzymes Similar to the Ulp1 endopeptidase from Saccharomyces have glycosidase activity. A particularly Suited protein Scaf cerevisiae. It is preferred that SDRs are inserted into the fold for this variant is a glycosylase or is derived from a protein Scaffold at one or more positions from the group of glycosylase. Preferably, the tertiary Structure belongs to one positions that correspond structurally or by amino acid of the following structural classes: class GH13, GH7, GH12, sequence homology to the regions 40-51, 108-115, 132-141, GH11, GH10, GH28, GH26, and GH18 (beta/alpha)8 barrel. 173-179 and 597-605 in Ulp1 endopeptidase from Saccha romyces cerevisiae, and more preferably at one or more 0234. In a first embodiment the protein scaffold belongs positions from the group of positions that correspond struc to the GH13 class of glycosylases or has at least 70% turally or by amino acid Sequence homology to the regions identity on the amino acid level to a protein of the GH13 43-49, 110-113, 133-137 and 175-178 (numbering of amino class of glycosylases and/or has a tertiary Structure Similar to acids according to SEQ ID NO:28). It is preferred that the human pancreatic alpha-amylase. It is preferred that SDRS Ulp1 endopeptidase from Saccharomyces cerevisiae or a are inserted into the protein Scaffold at one or more positions derivative or homologue thereof is used as the Scaffold. from the group of positions that correspond Structurally or 0230. In a further embodiment the protein scaffold by amino acid Sequence homology to the regions 50-60, belongs to the C56 class of cysteine proteases or has at least 100-110, 148-167, 235-244, 302-310 and 346-359 in human 70% identity on the amino acid level to a protein of the C56 pancreatic alpha-amylase, and more preferably at one or class of cysteine proteases and/or has a tertiary Structure more positions from the group of positions that correspond similar to the Pfp1 endopeptidase from Pyrococcus horiko Structurally or by amino acid Sequence homology to the Shii. It is preferred that SDRs are inserted into the protein regions 51-58, 148-155 and 303-309 (numbering of amino Scaffold at one or more positions from the group of positions acids according to SEQ ID NO:32). It is preferred that that correspond Structurally or by amino acid Sequence human pancreatic alpha-amylase or a derivative or homo homology to the regions 8-16, 40-47, 66-73, 118-125 and logue thereof is used as the Scaffold. 147-153 in Pfp1 endopeptidase from Pyrococcus horikoshii, 0235. In a further embodiment the protein scaffold and more preferably at one or more positions from the group belongs to the GH7 class of glycosylases or has at least 70% of positions that correspond structurally or by amino acid identity on the amino acid level to a protein of the GH7 class sequence homology to the regions 9-14, 68-71, 120-123 and of glycosylases and/or has a tertiary Structure Similar to 148-151 (numbering of amino acids according to SEQ ID cellulase from Trichoderma reesei. It is preferred that SDRs NO:29). It is preferred that the Pfp1 endopeptidase from are inserted into the protein Scaffold at one or more positions Pyrococcus horikoshii or a derivative or homologue thereof from the group of positions that correspond Structurally or is used as the Scaffold. by amino acid Sequence homology to the regions 47-56, 0231. In a further embodiment the protein scaffold 93-104, 173-182, 215-223, 229-236 and 322-334 in cellu belongs to the M4 class of metallo proteases or has at least lase from Trichoderma reesei, and more preferably at one or 70% identity on the amino acid level to a protein of the M4 more positions from the group of positions that correspond class of metallo proteases and/or has a tertiary Structure Structurally or by amino acid Sequence homology to the similar to thermolysin from Bacillus thermoproteolyticus. It regions 175-180, 218-222 and 324-332 (numbering of is preferred that SDRs are inserted into the protein scaffold amino acids according to SEQID NO:33). It is preferred that at one or more positions from the group of positions that cellulase from Trichoderma reesei or a derivative or homo correspond structurally or by amino acid Sequence homol logue thereof is used as the Scaffold. ogy to the regions 106-118, 125-130, 152-160, 197-204, 0236. In a further embodiment the protein scaffold 210-213 and 221-229 in thermolysin from Bacillus thermo belongs to the GH12 class of glycosylases or has at least proteolyticus, and more preferably at one or more positions 70% identity on the amino acid level to a protein of the from the group of positions that correspond Structurally or GH12 class of glycosylases and/or has a tertiary Structure by amino acid Sequence homology to the regions 108-115, similar to cellulase from Aspergillus niger. It is preferred that 126-129, 199-203 and 223-227 (numbering of amino acids SDRs are inserted into the protein scaffold at one or more according to SEQ ID NO:30). It is preferred that the positions from the group of positions that correspond struc thermolysin from Bacillus thermoproteolyticus or a deriva turally or by amino acid Sequence homology to the regions tive or homologue thereof is used as the Scaffold. 18-28, 55-60, 106-113, 126-132 and 149-159 in cellulase 0232. In a further embodiment the protein scaffold from Aspergillus niger, and more preferably at one or more belongs to the M10 class of metallo proteases or has at least positions from the group of positions that correspond struc US 2005/0175581 A1 Aug. 11, 2005 33 turally or by amino acid Sequence homology to the regions ogy to the regions 75-83, 113-125, 174-182, 217-224, 247 20-26, 56-59, 108-112 and 151-156 (numbering of amino 254,324-332 and 325-340 in mannanase from Pseudomonas acids according to SEQ ID NO:34). It is preferred that cellulosa, and more preferably at one or more positions from cellulase from Aspergillus niger or a derivative or homo the group of positions that correspond structurally or by logue thereof is used as the Scaffold. amino acid Sequence homology to the regions 115-123, 176-180, 286-291 and 328-337 (numbering of amino acids 0237. In a further embodiment the protein scaffold according to SEQ ID NO:38). It is preferred that mannanase belongs to the GH11 class of glycosylases or has at least from Pseudomonas cellulosa or a derivative or homologue 70% identity on the amino acid level to a protein of the thereof is used as the Scaffold. GH11 class of glycosylases and/or has a tertiary Structure Similar to Xylanase from Aspergillus niger. It is preferred that 0241. In an further embodiment the protein scaffold SDRs are inserted into the protein scaffold at one or more belongs to the GH18 (beta/alpha)8 barrel class of glycosy positions from the group of positions that correspond struc lases or has at least 70% identity on the amino acid level to turally or by amino acid Sequence homology to the regions a protein of the GH18 class of glycosylases and/or has a 7-14, 33-39, 88-97, 114-126 and 158-167 in Xylanase from tertiary Structure Similar to chitinase from Bacillus circu Aspergillus niger, and more preferably at one or more lans. It is preferred that SDRs are inserted into the protein positions from the group of positions that correspond struc Scaffold at one or more positions from the group of positions turally or by amino acid Sequence homology to the regions that correspond Structurally or by amino acid Sequence 20-26, 56-59, 108-112 and 151-156 (numbering of amino homology to the regions 21-29, 57-65, 130-136, 176-183, acids according to SEQ ID NO:35). It is preferred that 221-229, 249-257 and 327-337 in chitinase from Bacillus Xylanase from Aspergillus niger or a derivative or homo circulans, and more preferably at one or more positions from logue thereof is used as the Scaffold. the group of positions that correspond structurally or by amino acid Sequence homology to the regions 59-63, 178 0238. In a further embodiment the protein scaffold 181, 250-254 and 330-336 (numbering of amino acids belongs to the GH10 class of glycosylases or has at least according to SEQ ID NO:39). It is preferred that chitinase 70% identity on the amino acid level to a protein of the from Bacillus circulans or a derivative or homologue thereof GH10 class of glycosylases and/or has a tertiary Structure is used as the Scaffold. Similar to Xylanase from Streptomyces lividans. It is pre ferred that SDRs are inserted into the protein scaffold at one 0242. It is further preferred that the engineered enzymes or more positions from the group of positions that corre have esterhydrolase activity. Preferably, the protein Scaffold spond Structurally or by amino acid Sequence homology to for this variant have lipase, phosphatase, phytase, or phos the regions 21-29, 42-50, 84-92, 130-136, 206-217 and phodiesterase activity. 269-278 in Xylanase from Streptomyces lividans, and more preferably at one or more positions from the group of 0243 In a first embodiment the protein scaffold belongs positions that correspond structurally or by amino acid to the GX class of esterases or has at least 70% identity on sequence homology to the regions 43–49, 86-90, 208-213 the amino acid level to a protein of the GX class of esterases and 271-276 (numbering of amino acids according to SEQ and/or has a tertiary Structure Similar to the Structure of the ID NO:36). It is preferred that xylanase from Streptomyces lipase B from Candida antarctica. Preferably, the scaffold lividans or a derivative or homologue thereof is used as the has lipase activity. It is preferred that SDRs are inserted into Scaffold. the protein Scaffold at one or more positions from the group of positions that correspond structurally or by amino acid 0239). In a further embodiment the protein scaffold sequence homology to the regions 139-148, 188-195, 216 belongs to the GH28 class of glycosylases or has at least 224, 256-266, 272-287 in lipase B from Candida antarctica, 70% identity on the amino acid level to a protein of the and more preferably at one or more positions from the group GH28 class of glycosylases and/or has a tertiary Structure of positions that correspond structurally or by amino acid Similar to pectinase from Aspergillus niger. It is preferred sequence homology to the regions 141-146, 218-222, 259 that SDRs are inserted into the protein scaffold at one or 263 and 275-283 (numbering of amino acids according to more positions from the group of positions that correspond SEQ ID NO:40). It is preferred that lipase B from Candida Structurally or by amino acid Sequence homology to the antarctica or a derivative or homologue thereof is used as regions 82-88, 118-126, 171-178, 228-236, 256-264 and the scaffold. 289-299 in pectinase from Aspergillus niger, and more preferably at one or more positions from the group of 0244. In a further embodiment the protein scaffold positions that correspond structurally or by amino acid belongs to the GX class of esterases or has at least 70% Sequence homology to the regions 116-124, 174-178 and identity on the amino acid level to a protein of the GX class 291-296 (numbering of amino acids according to SEQ ID of esterases and/or has a tertiary Structure Similar to the NO:37). It is preferred that pectinase from Aspergillus niger pancreatic lipase from guinea pig. Preferably, the Scaffold has lipase activity. It is preferred that SDRs are inserted into or a derivative or homologue thereof is used as the Scaffold. the protein Scaffold at one or more positions from the group 0240. In a further embodiment the protein scaffold of positions that correspond structurally or by amino acid belongs to the GH26 class of glycosylases or has at least sequence homology to the regions 78-90, 91-100, 112-120, 70% identity on the amino acid level to a protein of the 179-186, 207-218, 238-247 and 248-260 in pancreatic lipase GH26 class of glycosylases and/or has a tertiary Structure from guinea pig, and more preferably at one or more Similar to mannanase from Pseudomonas cellulosa. It is positions from the group of positions that correspond struc preferred that SDRs are inserted into the protein scaffold at turally or by amino acid Sequence homology to the regions one or more positions from the group of positions that 80-87, 114-118, 209-215 and 239-246 (numbering of amino correspond structurally or by amino acid Sequence homol acids according to SEQ ID NO:41). It is preferred that US 2005/0175581 A1 Aug. 11, 2005 34 pancreatic lipase from guinea pig or a derivative or homo trin glucanotransferase from Bacillus circulans, and more logue thereof is used as the Scaffold. preferably at one or more positions from the group of positions that correspond structurally or by amino acid 0245. In a further embodiment the protein scaffold has a sequence homology to the regions 87-92, 180-185,261-264 tertiary Structure Similar to the Structure of the alkaline and 269-275 (numbering of amino acids according to SEQ phosphatase from Escherichia coli or has at least 70% ID NO:44). It is preferred that cyclomaltodextrin glucan identity on the amino acid level to a protein that has a tertiary otransferase from Bacillus circulans or a derivative or Structure Similar to the Structure of the alkaline phosphatase homologue thereof is used as the Scaffold. from Escherichia coli. Preferably, the scaffold has phos phatase activity. It is preferred that SDRs are inserted into 0249. In a further embodiment the protein scaffold the protein Scaffold at one or more positions from the group belongs to the GT1 class of tranferases or has at least 70% of positions that correspond structurally or by amino acid identity on the amino acid level to a protein of the GT1 class sequence homology to the regions 110-122, 187-142, 170 of transferases and/or has a tertiary Structure Similar to the 175, 186-193,280-287 and 425-435 in alkaline phosphatase Structure of the glycosyltransferase from Amycolatopsis from Escherichia coli, and more preferably at one or more Orientalis A82846. Preferably the scaffold has transferase positions from the group of positions that correspond struc activity, and more preferably glycosyltransferase activity. It turally or by amino acid Sequence homology to the regions is preferred that SDRs are inserted into the protein scaffold 171-174, 187-191, 282-286 and 426-433 (numbering of at one or more positions from the group of positions that amino acids according to SEQID NO:42). It is preferred that correspond structurally or by amino acid Sequence homol alkaline phosphatase from Escherichia coli or a derivative or ogy to the regions 58-74, 130-138, 185-193, 228-236 and homologue thereof is used as the Scaffold. 314-323 in glycosyltransferase from Amycolatopsis Orien talis A82846, and more preferably at one or more positions 0246. In a further embodiment the protein scaffold has a from the group of positions that correspond Structurally or tertiary structure similar to the structure of the bovine by amino acid Sequence homology to the regions 61-71, pancreatic desoxyribonuclease I or has at least 70% identity 230-234 and 316-321 (numbering of amino acids according on the amino acid level to a protein that has a tertiary to SEQ ID NO:45). It is preferred that the glycosyltrans Structure Similar to the Structure of the bovine pancreatic desoxyribonuclease I. Preferably, the scaffold has phos ferase from Amycolatopsis Orientalis A82846 or a derivative phodiesterase activity. More preferably, a nuclease, and or homologue thereof is used as the Scaffold. most preferably, an unspecific endonuclease or a derivative 0250. It is further preferred that the engineered enzymes thereof is used as the scaffold. It is preferred that SDRs are have oxidoreductase activity. A particularly Suited protein inserted into the protein Scaffold at one or more positions Scaffold for this variant is a monooxygenase, a dioxygenase from the group of positions that correspond Structurally or or a alcohol dehydrogenase, or a derivative thereof. The by amino acid Sequence homology to the regions 14-21, tertiary Structure of the protein Scaffold can be of any type. 41-47, 72-77, 97-111, 135-143, 171-178, 202-209 and 242 251 in bovine pancreatic desoxyribonuclease I, and more 0251. In a first embodiment the protein scaffold has a preferably at one or more positions from the group of tertiary Structure Similar to the Structure of the 2,3-diphy positions that correspond structurally or by amino acid droxybiphenyl dioxygenase from Pseudomonas sp. or has at Sequence homology to the regions 16-19, 42-46, 136-141 least 70% identity on the amino acid level to a protein that and 172-176 (numbering of amino acids according to SEQ has a tertiary Structure Similar to the Structure of the 2,3- ID NO:43). It is preferred that bovine pancreatic desoxyri diphydroxybiphenyl dioxygenase from Pseudomonas sp. bonuclease I or human desoxyribonuclease I or a derivative Preferably, the Scaffold has dioxygenase activity. It is pre ferred that SDRs are inserted into the protein scaffold at one or homologue thereof is used as the Scaffold. or more positions from the group of positions that corre 0247. It is further preferred that the engineered enzyme spond Structurally or by amino acid Sequence homology to has transferase activity. Aparticularly Suited protein Scaffold the regions 172-185, 198-206, 231-237, 250-259 and 282 for this variant is a glycosyl-, a phospho- or a methyltrans 287 in 2,3-diphydroxybiphenyl dioxygenase from ferase, or is a derivative thereof. Particularly preferred Pseudomonas sp., and more preferably at one or more protein Scaffolds for this variant are glycosyltransferases or positions from the group of positions that correspond struc are derived from glycosyltransferases. The tertiary Structure turally or by amino acid Sequence homology to the regions of the protein scaffold can be of any type. Preferably, 175-182, 200-204, 252-257 and 284-287 (numbering of however, the tertiary structure belongs to one of the follow amino acids according to SEQID NO:46). It is preferred that ing structural classes: GH13 and GT1. the 2,3-diphydroxybiphenyl dioxygenase from Pseudomo nas Sp or a derivative or homologue thereof is used as the 0248. In a first embodiment the protein scaffold belongs Scaffold. to the GH13 class of transferases or has at least 70% identity on the amino acid level to a protein of the GH13 class of 0252) In a further embodiment the protein scaffold has a transferases and/or has a tertiary Structure Similar to the tertiary Structure Similar to the Structure of the catechol Structure of the cyclomaltodextrin glucanotransferase from dioxygenase from Acinetobacter sp. or has at least 70% Bacillus circulans. Preferably, the scaffold has transferase identity on the amino acid level to a protein that has a tertiary activity, and more preferably a glycosyltransferase is used as Structure Similar to the Structure of the catechol dioxygenase the scaffold. It is preferred that SDRs are inserted into the from Acinetobacter sp. Preferably, the scaffold has dioxy protein Scaffold at one or more positions from the group of genase activity, and more preferably catechol dioxygenase positions that correspond structurally or by amino acid activity. It is preferred that SDRs are inserted into the protein sequence homology to the regions 38-48, 85-94, 142-154, Scaffold at one or more positions from the group of positions 178-186, 259-266,331-340 and 367-377 in cyclomaltodex that correspond Structurally or by amino acid Sequence US 2005/0175581 A1 Aug. 11, 2005 35 homology to the regions 66-72, 105-112, 156-171 and from the group of positions that correspond Structurally or 198-207 in catechol dioxygenase from Acinetobacter sp., by amino acid Sequence homology to the regions 45-55, and more preferably at one or more positions from the group 78-87, 105-113, 137-146, 164-171, 187-193, 205-210, 244 of positions that correspond structurally or by amino acid 255 and 269-276 in N-acetyl-d-neuramic acid aldolase from sequence homology to the regions 107-110, 161-171 and Escherichia coli, and more preferably at one or more posi 201-205 (numbering of amino acids according to SEQ ID tions from the group of positions that correspond structurally NO:47). It is preferred that the catechol dioxygenase from or by amino acid Sequence homology to the regions 45-52, Acinetobacter Sp or a derivative or homologue thereof is 138-144, 189-192, 247-253 and 271-275 (numbering of used as the Scaffold. amino acids according to SEQID NO:50). It is preferred that the N-acetyl-d-neuramic acid aldolase from Escherichia coli 0253) In a further embodiment the protein scaffold has a or a derivative or homologue thereof is used as the Scaffold. tertiary Structure Similar to the Structure of the camphor-5- monooxygenase from Pseudomonas putida or has at least 0257. In a further embodiment the protein scaffold has a 70% identity on the amino acid level to a protein that has a tertiary Structure Similar to the Structure of the tryptophan tertiary Structure Similar to the Structure of the camphor-5- synthase from Salmonella typhimurium or has at least 70% monooxygenase from Pseudomonas putida. Preferably, the identity on the amino acid level to a protein that has a tertiary Scaffold has monooxygenase activity, and more preferably Structure Similar to the Structure of the tryptophan Synthase camphor monooxygenase activity. It is preferred that SDRS from Salmonella typhimurium. Preferably, the scaffold has are inserted into the protein Scaffold at one or more positions synthase activity. It is preferred that SDRs are inserted into from the group of positions that correspond Structurally or the protein Scaffold at one or more positions from the group by amino acid Sequence homology to the regions 26-31, of positions that correspond structurally or by amino acid 57-63, 84-98, 182-191, 242-256, 292-299 and 392–399 in sequence homology to the regions 56-63, 127-134, 154-161, camphor-5-monooxygenase from Pseudomonas putida, and 175-193, 209-216 and 230-240 in tryptophan synthase from more preferably at one or more positions from the group of Salmonella typhimurium, and more preferably at one or positions that correspond structurally or by amino acid more positions from the group of positions that correspond sequence homology to the regions 85-96, 183-188,244-253, Structurally or by amino acid Sequence homology to the 293–298 and 393-398 (numbering of amino acids according regions 57-62, 155-160, 178-190 and 210-215 (numbering to SEQ ID NO:48). It is preferred that the camphor-5- of amino acids according to SEQ ID NO:51). It is preferred monooxygenase from Pseudomonas putida or a derivative that the tryptophan Synthase from SalmOnella typhimurium or homologue thereof is used as the Scaffold. or a derivative or homologue thereof is used as the scaffold. 0254. In a further embodiment the protein scaffold has a 0258. It is further preferred that the engineered enzymes tertiary Structure Similar to the Structure of the alcohol have isomerase activity. A particularly Suited protein Scaf dehydrogenase from Equus callabus or has at least 70% fold for this variant is a converting aldose or a converting identity on the amino acid level to a protein that has a tertiary ketose, or is a derivative thereof. Structure Similar to the Structure of the alcohol dehydroge 0259. In a first embodiment, the protein scaffold has a nase from Equus callabus. Preferably, the scaffold has tertiary Structure Similar to the Structure of the Xylose alcohol dehydrogenase activity. It is preferred that SDRs are isomerase from Actinoplanes missOuriensis or has at least inserted into the protein Scaffold at one or more positions 70% identity on the amino acid level to a protein that has a from the group of positions that correspond Structurally or tertiary Structure Similar to the Structure of the Xylose by amino acid Sequence homology to the regions 49-63, isomerase from Actinoplanes missOuriensis. It is preferred 111-112, 294-301 and 361-369 in alcohol dehydrogenase that SDRs are inserted into the protein scaffold at one or from Equus callabus, and more preferably at one or more more positions from the group of positions that correspond positions from the group of positions that correspond struc Structurally or by amino acid Sequence homology to the turally or by amino acid Sequence homology to the regions regions 18-31, 92-103, 136-147, 178-188 and 250-257 in 51-61 and 295-299 (numbering of amino acids according to Xylose isomerase from Actinoplanes missOuriensis, and SEQ ID NO:49). It is preferred that the alcohol dehydroge more preferably at one or more positions from the group of nase from Equus callabus or a derivative or homologue positions that correspond structurally or by amino acid thereof is used as the Scaffold. sequence homology to the regions 20-27, 92-99 and 180-186 0255. It is further preferred that the engineered enzymes (numbering of amino acids according to SEQ ID NO:52). It have lyase activity. A particularly Suited protein Scaffold for is preferred that the Xylose isomerase from Actinoplanes this variant is a oxoacid lyase or is a derivative thereof. missOuriensis or a derivative or homologue thereof is used Particularly preferred protein scaffolds for this variant are as the Scaffold. aldolases or Synthases, or are derived thereof. The tertiary 0260. It is further preferred that the engineered enzymes Structure of the protein Scaffold can be of any type, but a have ligase activity. Aparticularly Suited protein Scaffold for (beta/alpha)8 barrel structure is preferred. this variant is a DNA ligase, or is a derivative thereof. 0256 In a first embodiment the protein scaffold has a 0261. In a first embodiment, the protein scaffold has a tertiary structure similar to the structure of the N-acetyl-d- tertiary structure similar to the structure of the DNA ligase neuramic acid aldolase from Escherichia coli or has at least from Bacteriophage T7 or has at least 70% identity on the 70% identity on the amino acid level to a protein that has a amino acid level to a protein that has a tertiary Structure tertiary structure similar to the structure of the N-acetyl-d- similar to the structure of the DNA-ligase from Bacterioph neuramic acid aldolase from Escherichia coli. Preferably, age T7. It is preferred that SDRs are inserted into the protein the scaffold has aldolase activity. It is preferred that SDRs Scaffold at one or more positions from the group of positions are inserted into the protein Scaffold at one or more positions that correspond Structurally or by amino acid Sequence US 2005/0175581 A1 Aug. 11, 2005 36 homology to the regions 52-60, 94-108, 119-131, 241-248, natively, the length is between two and 20 amino acid 255-263 and 302-318 in DNAligase from Bacteriophage T7, residues, preferably between two and ten amino acid resi and more preferably at one or more positions from the group dues, more preferably between three and eight amino acid of positions that correspond structurally or by amino acid residues. sequence homology to the regions 96-106, 121-129, 256 0272 Preferably such insertions or other combinations 262 and 304-316 (numbering of amino acids according to are performed on the DNA level, using polynucleotides SEQ ID NO:53). It is preferred that the DNA ligase from encoding Such protein Scaffolds and polynucleotides or Bacteriophage T7 or a derivative or homologue thereof is oligonucleotides encoding Such fully or partially random used as the Scaffold. peptide Sequences. 0262. A third aspect of the invention is directed to a 0273 Optionally, steps (a) to (c) are repeated cyclically, method for generating engineered enzymes with Specificities whereby enzymes Selected in step (c) serve as the protein that are qualitatively and/or quantitatively novel in combi Scaffold in Step (a) of a further cycle, and randomized nation with the protein scaffold. The inventive method peptide Sequences are either inserted or, alternatively, Sub comprises at least the following Steps: Stituted for peptide Sequences that have been inserted in 0263 (a) providing a protein scaffold capable to former cycles. Thereby, the number of inserted peptide catalyze at least one chemical reaction on at least one Sequences is either constant or increases over the cycles. The target Substrate, cycles are repeated until one or more enzymes with the 0264 (b) generating a library of engineered intended Specificities are generated. enzymes or isolated engineered enzymes by com 0274) Moreover, during or after one or more rounds of bining the protein Scaffold from step (a) with one or Steps (a) to (c), the Scaffold may be mutated at one or more more fully or partially random peptide Sequences at positions in order to make the Scaffold more acceptable for Sites in the protein Scaffold that enable the resulting the combination with SDR Sequences, and/or to increase engineered enzyme to discriminate between at least catalytic activity at a specific pH and temperature, and/or to one target Substrate and one or more different Sub change the glycosylation pattern, and/or to decrease Sensi Strates and tivity towards enzyme inhibitors, and/or to change enzyme stability. 0265 (c) selecting out of the library of engineered enzymes generated in step (b) one or more enzymes 0275. In a second variant of this aspect of the invention, that have defined specificities towards at least one the inventive method comprises at least the following Steps: target Substrate. 0276 (a) providing a first protein scaffold fragment, 0266. In a first variant of this aspect of the invention, the 0277 (b) connecting said protein scaffold fragment via a inventive method comprises at least the following Steps: peptide linkage with a first SDR, and optionally 0267 (a) providing a protein scaffold capable to 0278 (c) connecting the product of step (b) via a peptide catalyze at least one chemical reaction on at least one linkage with a further SDR peptide or with a further protein target Substrate, Scaffold fragment, and optionally 0268 (b) generating a library of engineered 0279 (d) repeating Step (c) for as many cycles as neces enzymes or isolated engineered enzymes by insert Sary in order to generate a Sufficiently specific enzyme, and ing into the protein Scaffold from step (a) one or more fully or partially random peptide Sequences at 0280 (e) selecting out of the population generated in Sites in the protein Scaffold that enable the resulting Steps (a)-(d) one or more enzymes that have the desired engineered enzyme to discriminate between at least Specificities toward the one or more target Substrates. one target Substrate and one or more different Sub 0281 Protein scaffold fragment means a part of the Strates and Sequence of a protein Scaffold. A protein Scaffold is com 0269 (c) selecting out of the library of engineered prised of at least two protein Scaffold fragments. enzymes generated in step (b) one or more enzymes 0282. In a third variant of this aspect of the invention, the that have defined specificities towards at least one protein Scaffold, the SDRS and the engineered enzyme are target Substrate. encoded by a DNA sequence and an expression System is 0270 Preferably, the positions at which the one or more used in order to produce the protein. In an alternative fully or partially random peptide Sequences are combined variant, the protein scaffold, the SDRs and/or the engineered with or inserted into the protein scaffold are identified prior enzyme are chemically Synthesized from peptide building to the combination or insertion. blocks. 0271 The number of insertions or other combinations of 0283. In a fourth variant of this aspect of the invention, fully or partially random peptide Sequences as well as their the inventive method comprises at least the following Steps: length may vary over a wide range. The number is at least one, preferably more than one, more preferably between two 0284 (a) providing a polynucleotide encoding a protein and eleven, most preferably between two and Six. The length Scaffold capable of catalyzing one or more chemical reac of Such fully or partially random peptide Sequences is tions on one or more target Substrates, usually less than 50 amino acid residues. Preferably, the 0285) (b) combining one or more fully or partially ran length is between one and 15 amino acid residues, more dom oligonucleotide Sequence with the polynucleotide preferably between one and Six amino acid residues. Alter encoding the protein Scaffold, the fully or partially random US 2005/0175581 A1 Aug. 11, 2005 37 oligonucleotide Sequences being located at Sites in the Sites can be identified by Several approaches. Preferably, polynucleotide that enable the encoded engineered enzyme insertion Sites are identified by analysis of the three-dimen to discriminate between the one or more target Substrates Sional Structure of the protein Scaffolds, by comparative and one or more other Substrates, and analysis of the primary Sequences of the protein Scaffold 0286 (c) Selecting out of the population generated in Step with other enzymes having different quantitative Specifici (b) one or more polynucleotides that encode enzymes that ties, or experimentally by techniqueS Such as alanine Scan have the defined specificities toward the one or more target ning, random mutagenesis, or random deletion, or by any Substrates. combination thereof. 0287 Any enzyme can serve as the protein scaffold in 0293. A first approach to identify insertion sites for SDRs Step (a). It can be a naturally occurring enzyme, a variant or bases on the three-dimensional Structure of the protein a truncated derivate therefore, or an engineered enzyme. For Scaffold as it can be obtained by X-ray crystallography or by human therapeutic use, the protein Scaffold is preferably a nuclear magnetic resonance Studies. Structural alignment of mammalian enzyme, and more preferably a human enzyme. the protein Scaffold in comparison with other enzymes of the In that aspect, the invention is directed to a method for the Same Structural class but having different quantitative Speci generation of essentially mammalian, especially of essen ficities reveals regions of high Structural Similarity and tially human enzymes with Specificities that are different regions with low Structural Similarity. Such an analysis can from Specificities of any enzyme encoded in mammalian for example be done using public Software Such as Swiss genomes or in the human genome, respectively. PDB viewer (Guex, N. and Peitsch, M. C. (1997) Electro phoresis 18, 2714-2723). Regions of low structural similar 0288 According to the invention, the protein scaffold ity are preferred SDR insertion sites. provided in Step (a) of this aspect requires to be capable of catalyzing one or more chemical reactions on a target 0294. In a second approach to identify insertion sites for substrate. Therefore, a protein scaffold is selected from the SDRs, three-dimensional structures of the scaffold protein in group of potential protein Scaffolds by its activity on the complex with competitive inhibitors or Substrate analogs are target Substrate. analysed. It is assumed that the binding site of a competitive inhibitor significantly overlaps with the binding site of the 0289. In a preferred variant of this aspect of the inven Substrate. In that case, atoms of the protein that are within a tion, a protein Scaffold with hydrolase activity is used. certain distance of atoms of the inhibitor are likely to be in Preferably, a protein scaffold with proteolytic activity is a similar distance to the Substrate as well. Choosing a short used, and more preferably, a protease with very low speci distance, e.g. <5 A, will result in an ensemble of protein ficity having basic activity on the target Substrate is used as atoms that are in close contact with the Substrate. These the protein Scaffold. Examples of proteases from different residues would constitute the first shell contacts and are Structural classes with low Substrate Specificity are Papain, therefore preferred insertion sites for SDRs. Once first shell Trypsin, Chymotrypsin, Subtilisin, SET (trypsin-like serine contacts have been identified, Second shell contacts can be protease from Streptomyces erythraeus), Elastase, Cathepsin found by repeating the distance analysis Starting from first G or Chymase. Before being employed as the protein shell atoms. In yet another alternative of the invention the Scaffold, the amino acid Sequence of the protease may be distance analysis described above is performed Starting from modified in order to change protein properties other than the active site residues. Specificity, e.g. catalytic activity, Stability, inhibitor Sensitiv ity, or expression yield, essentially as described in WO 0295). In third approach to identify insertion sites for 92/18645, or in order to change Specificity, essentially as SDRS, the primary Sequence of the Scaffold protein is described in EP O2O2O576.3 and PCT/EPO3/04864. aligned with other enzymes of the same Structural class but having different quantitative Specificities using an alignment 0290 Another option for a feasible protein scaffold are algorithm. Examples of Such alignment algorithms are pub lipases. Hepatic lipase, lipoprotein lipase and pancreatic lished (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & lipase belong to the "lipoprotein lipase Superfamily', which Lipman, D.J. (1990).J. Mol. Biol. 215:403-410; “Statistical in turn is an example of the GX-class of lipases (M. Fischer, methods in Bioinformatics: an introduction” by Ewens, W. J. Pleiss (2003), Nucl. Acid. Res., 31, 319-321). The sub & Grant, G. R. 2001, Springer, New York). Such an align Strate Specificity of lipases can be characterized by their ment may reveal conserved and non-conserved regions with relative activity towards triglycerol esters of fatty acids and varying Sequence homology, and, in particular, additional phospholipids, bearing a charged head group. Alternatively, Sequence elements in one or more enzymes compared to the other hydrolaseS Such as esterases, glycosylases, amidases, Scaffold protein. Conserved regions of are more likely to or nitrilases may be used as Scaffolds. contribute to phenotypes shared among the different pro 0291 Transferases are also feasible protein scaffolds. teins, e.g. Stabilizing the three-dimensional fold. Non-con Glycoslytransferases are involved in many biological Syn Served regions and, in particular, additional Sequences in thesis involving a variety of donors and acceptors. Alterna enzymes with quantitatively higher specificity (Turner, R. et tively, the protein Scaffold may have ligase, lyase, oxi al. (2002) J. Biol. Chem., 277, 33068-33074) are preferred doreductase, or isomerase activity. insertion sites for SDRs. 0292. In a first embodiment, the one or more fully or 0296 For proteases currently five families are known, partially random peptide Sequences are inserted at Specific namely aspartic-, cysteine-, Serine-, metallo- and threonine Sites in the protein Scaffold. These insertion sites are char proteases. Each family includes groups of proteases that acterized by the fact that the inserted peptide Sequences can share a similar fold. Crystallographic structures of members act as discriminators between different Substrates, i.e. as of these groups have been Solved and are accessible through Specificity Determining Regions or SDRs. Such insertion public databases, e.g. the Brookhaven protein database (H. US 2005/0175581 A1 Aug. 11, 2005 38

M. Berman et al. Nucleic Acids Research, 28 pp. 235-242 Enzymol. 248, 105-120; Chitpinityol, S. & Crabbe, M J. (2000)). Such databases also include structural homologs in (1998), Food Chemistry, 61,395-418). Examples for the A1 other enzyme classes and nonenzymatically active proteins Structural class of aspartic proteases are pepsin with a low as of each class. Several tools are available to Search public well as beta-secretase (Grüninger-Leitch, F., et al. (2002) J. databases for structural homologues: SCOP-a structural Biol. Chem. 277,4687-4693) and renin (Wang, W. & Liang, classification of proteins database for the investigation of T C. (1994) Biochemistry, 33, 14636-14641) with relatively sequences and structures. (Murzin A. G. et al. (1995).J. Mol. high Substrate Specificities. Retroviral proteases also belong Biol. 247, 536-540); CATH-Class, Architecture, Topology to this class, although the active enzyme is a dimer of two and Homologous Superfamily: a hierarchical classification identical Subunits. The viral proteases are essential for the of protein domain structures (Orengo et al. (1997) Structure correct processing of the polyprotein precursor to generate 5(8) 1093-1108); FSSP-Fold classification based on struc functional proteins which requires a high Substrate Specific ture-structure alignment of proteins (Holm and Sander ity in each case (Wu, J. et al. (1998) Biochemistry, 37, (1998) Nucl. Acids Res. 26 316-319); or VAST Vector 4518-4526; Pettit, S. et al. (1991) J. Biol. Chem., 266, alignment search tool (Gibrat, Madej and Bryant (1996) 14539-14547). Pepsin is the type protease for this class and Current Opinion in Structural Biology 6, 377-385). represents an unspecific protease (Kageyama, T. (2002) Cell. Mol. Life Sci. 59, 288-306). B-secretase and Cathepsin D 0297. In the above described approaches, members of (Aguilar, C. F. et al. (1995) Adv. Exp. Med. Biol. 362, Structural classes are compared in order to identify insertion 155-166) are proteases of the same structural class and have Sites for SDRs. a high Substrate Specificity. In a preferred variant of the 0298. In a preferred variant of these approaches serine approach alignments of the primary amino acids Sequences proteases of the Structural class S1 are compared with each (FIG. 6) are used to identify six sequence Stretches longer other. Trypsin represents a member with low substrate than three amino acids which are inserted in the Specific Specificity, as it requires only an arginine or lysine residue at proteases compared to pepsin and are therefore potential the P. position. On the other hand, thrombin, tissue-type Specificity determining regions. In a further variant of the plasminogen activator or enterokinase all have a high Speci approach information from the three-dimensional Structure ficity towards their substrate sequences, i.e. (L/I/V/F)XPR of b-Secretase can be used in order to further narrow down NA, CPGRVVGG and DDDK, respectively (Perona, J. the Selection. Out of the Six inserted Sequence Stretches, & Craik, C. (1997) J. Biol. Chem..., 272, 29987-29990; three are especially close to the active site residues, namely Perona, J. & Craik, C (1995) Protein Science, 4, 337-360). Stretch number 1, 3 and 4 which are insertions in cathepsin An alignment of the amino acid Sequences of these proteases D and beta-secretase, respectively (FIG. 5). In a preferred is described in example 1 (FIG. 2) along with the identifi variant of the approach, one or Several amino acid stretches cation of SDRs. of variable length and composition can be inserted into the pepsin Sequence at one or Several of the Six positions. In a 0299. A further example within the family of serine more preferred embodiment of the invention the insertion is proteases is given by members of the Structural class S8 performed at the positions 1, 3 or 4 or any combination (Subtilisin fold). Subtilisin is the type protease for this class thereof. In another preferred embodiment of the invention and represents an unspecific protease (Ottesen, M. & Svend protease Scaffolds other than pepsin are used. sen, A. (1998) Methods Enzymol. 19, 199-215). Furin, PC1 and PC5 are proteases of the same structural class involved 0301 There are cases where a certain structural class in the processing of propeptides and have a high Substrate does not include known members of low and high Specific specificity (Seidah, N. & Chretien, M. (1997) Curr. Opin. ity. This is exemplified by the C14 class of caspases which Biotech., 8: 602-607; Bergeron, F. et al. (2000) J. Mol. belong to the cysteine protease family (Rawlings, N. D. & Endocrin., 24:1-22). In a preferred variant of the approach Barrett, A.J. (1994) Methods Enzymol. 244, 461-486) and alignments of the primary amino acids Sequences (FIG. 4) which all show high specificity for P to P. positions. For are used to identify eleven Sequence Stretches longer than example, caspase-1, caspase-3 and caspase-9 recognize the three amino acids which specific proteases have in addition sequences YVAD, DEVD or LEHD, respectively. Iden compared to Subtilisin and are therefore potential Specificity tification of the regions that differ between the caspases will determining regions. In a further variant of the approach include the regions responsible for the differences in Sub information from the three-dimensional structure of Subtili strate specificity (FIGS. 7 and 8). Sin can be used in order to further narrow down the Selection 0302 Finally, non-enzymatic proteins of the same fold as (FIG.3). Out of the eleven inserted sequence stretches, three the enzyme Scaffold may also contribute to the identification are especially close to the active Site residues, namely stretch of insertion sites for SDRs. For example, haptoglobin (Arco number 7, 8 and 11 which are insertions in PC5, PC1 and all leo, J. & Greer, J.; (1982) J. Biol. Chem. 257, 10063-10068) three specific proteases, respectively (FIG.3). In a preferred and azurocidin (Almeida, R. et al. (1991) Biochem. Bio variant, one or Several amino acid stretches of variable phys. Res. Commun. 177, 688-695) share the same chymot length and composition can be inserted into the Subtilisin rypsin-like fold with all S1 proteases. Due to substitutions in Sequence at one or Several of the eleven positions. In a more the active Site residues these proteins do not posses any preferred variant of the approach the insertion is performed proteolytic function, yet they show high homology with at regions 7, 8 or 11 or any combination thereof. In another active proteases. Differences between these proteins and preferred variant of the approach protease Scaffolds other Specific proteases include regions that can Serve as insertion than Subtilisin from the structural class S8 are used. Sites for SDRs. 0300. In a further preferred variant of this approach, 0303. In a fourth approach, insertion sites for SDRs are aspartic acid proteases of the Structural class A1 are ana identified experimentally by techniqueS Such as alanine lyzed (Rawlings, N. D. & Barrett, A. J. (1995). Methods Scanning, random mutagenesis, random insertion or random US 2005/0175581 A1 Aug. 11, 2005 39 deletion. In contrast to the approach disclosed above, this alternative, after intracellular expression of the enzyme approach does not require detailed knowledge about the variants, or Secretion into the periplasmatic space using three-dimensional Structure of the Scaffold protein. In one signal sequences such as DsbA, PhoA, PelB, Ompa, OmpT preferred variant of this approach, random mutagenesis of or g|II for Escherichia coli, a permeabilisation or lysis step enzymes with relatively high Specificity from the same releases the enzyme variants into the Supernatant. The Structural class as the protein Scaffold and Screening for loSS destruction of the membrane barrier can be forced by the use or change of Specificity can be used to identify insertion Sites of mechanical means Such as ultraSonic, French press, or the for SDRs in the protein scaffold. use of membrane-digesting enzymes Such as lysozyme. AS 0304 Random mutagenesis, alanine Scanning, random another, further alternative, the genes encoding the enzyme insertion or random deletion are all done on the level of the variants are expressed cell-free by the use of a Suitable polynucleotides encoding the enzymes. There are a variety cell-free expression System. For example, the S30 extract of protocols known in the literature (e.g. Sambrook, J. F.; from Escherichia coli cells is used for this purpose as Fritsch, E. F.; Maniatis, T.; Cold Spring Harbor Laboratory described by Lesly et al. (Methods in Molecular Biology 37 Press, Second Edition, 1989, New York). For example, (1995) 265-278). random mutagenesis can be achieved by the use of a polymerase as described in patent WO9218645. According 0307 The ensemble of gene variants generated and to this patent, the one or more genes encoding the one or expressed by any of the above methods are analyzed with more proteases are amplified by use of a DNA polymerase respect to their affinity, Substrate Specificity or activity by with a high error rate or under conditions that increase the appropriate assay and Screening methods as described in rate of misincorporations. For example the method of Cad detail for example in patent application PCT/EP03/04864. well and Joyce can be employed (Cadwell, R. C. and Joyce, Genes from catalytically active variants having reduced G. F., PCR methods. Appl. 2 (1992) 28-33). Other methods Specificity in comparison to the original enzyme are ana of random mutagenesis Such as, but not limited to, the use lyzed by Sequencing. Sites at which mutations and/or inser of mutator Stains, chemical mutagens or UV-radiation can be tions and/or deletions occurred are preferred insertion Sites employed as well. at which SDRs can be inserted site-specifically. 0305 Alternatively, oligonucleotides can be used for 0308. In a second embodiment, the one or more fully or mutagenesis that Substitute randomly distributed amino acid partially random peptide Sequences are inserted at random residues with an alanine. This method is generally referred sites in the protein scaffold. This modification is usually to as alanine Scanning mutagenesis (Fersht, A. R. Biochem done on the polynucleotide level, i.e. by inserting nucleotide istry (1989) 8031-8036). As a further alternative, modifica Sequences into the gene that encodes the protein Scaffold. tions of the alanine Scanning mutagenesis Such as binominal Several methods are available that enable the random inser mutagenesis (Gregoret, L. M. and Sauer, R.T. PNAS (1993) tion of nucleotide Sequences. Systems that can be used for 4246-4250) or combinatorial alanine scanning (Weiss et al., random insertion are for example ligation based Systems PNAS (2000) 8950-8954) can be employed. (Murakami et al. Nature Biotechnology 20 (2002) 76-81), Systems based on DNA polymerisation and transposon 0306 In order to express engineered enzymes, the DNA based systems (e.g. GPS-MTM mutagenesis system, NEB encoding Such engineered proteins is ligated into a Suitable Biolabs; MGSTM mutation generation system, Finnzymes). expression vector by Standard molecular cloning techniques The transposon-based methods employ a transposase-medi (e.g. Sambrook, J. F.; Fritsch, E. F.; Maniatis, T.; Cold Spring ated insertion of a Selectable marker gene that contains at its Harbor Laboratory Press, Second Edition, 1989, New York). termini recognition Sequences for the transposase as well as The vector is introduced in a Suitable expression host cell, two Sites for a rare cutting restriction endonuclease. Using which expresses the corresponding engineered enzyme vari the latter endonuclease one usually releases the Selection ant. Particularly Suitable expression hosts are bacterial marker and after religation obtains an insertion. Instead of expression hosts such as Escherichia coli or Bacillus Sub performing the religation one can alternatively insert a tilis, or yeast expression hosts Such as Saccharomyces fragment that has terminal recognition Sequences for one or cerevisae or Pichia pastoris, or mammalian expression hosts two outside cutting restriction endonuclease as well as a such as Chinese Hamster Ovary (CHO) or Baby Hamster Selectable marker. After ligation, one releases this fragment Kidney (BHK) cell lines, or viral expression Systems Such as using the one or two outside cutting endonucleases. After bacteriophages like M13 or Lambda, or viruses Such as the creating blunt ends by Standard methods one inserts blunt Baculovirus expression System. As a further alternative, ended random fragments at random positions into the gene. Systems for in Vitro protein expression can be used. Typi cally, the DNA is ligated into an expression vector behind a 0309. In a further preferred embodiment, methods for Suitable signal Sequence that leads to Secretion of the homologous in-vitro recombination are used to combine the enzyme variants into the extracellular space, thereby allow mutations introduced by the above mentioned methods to ing direct detection of protease activity in the cell Superna generate enzyme populations. Examples of methods that can tant. Particularly Suitable Signal Sequences for Escherichia be applied are the Recombination Chain Reaction (RCR) coli are Hly A, for Bacillus Subtilis AprE, NprB, Mpr, according to patent application WO 0134835, the DNA Amy A, AmyE, Blac, SacB, and for S. cerevisiae Bar1, Suc2, Shuffling method according to the patent application WO Mato, Inu1 A, Ggp1p. Alternatively, the enzyme variants are 9522625, the Staggered Extension method according to expressed intracellularly and the Substrates are expressed patent WO9842728, or the Random Priming recombination also intracellularly. Preferably, this is done essentially as according to patent application WO9842728. Furthermore, described in patent application WO 0212543, using a fusion also methods for non-homologous recombination Such as the peptide Substrate comprising two auto-fluorescent proteins Itchy method can be applied (Ostermeier, M. et al. Nature linked by the Substrate amino-acid Sequence. As a further Biotechnology 17 (1999) 1205-1209). US 2005/0175581 A1 Aug. 11, 2005 40

0310. Upon random insertion of a nucleotide sequence 0314. In a preferred alternative, the sequences selected in into the protein scaffold one obtains a library of different one round are analysed and randomized oligonucleotides are genes encoding enzyme variants. The polynucleotide library generated based on these Sequences. This can, for example, is Subsequently transferred to an appropriate expression be achieved by using in addition to the original nucleotide vector. Upon expression in a Suitable host or by use of an in with a certain percentage mixtures of the other three nucle Vitro expression System, a library of enzymes containing otides monomers at each position in the oligonucleotide randomly inserted Stretches of amino acids is obtained. synthesis. If, for example, in a first round an SDRs is identified that has the amino acid Sequence ARLT, e.g. 0311. According to step (b) of this third aspect of the encoded by the nucleotide sequence GCGCGC CTTACC, invention, one or more fully or partially random peptide a random peptide Sequence inserted in this SDR Site could Sequences are inserted into the protein Scaffold. The actual be encoded by an oligonucleotide with 70% G, 10% A, 10% number of Such inserted SDRs is determined by the intended Tand 10% Cat the first position, 70% C, 10% G, 10% Tand quantitative Specificity following the relation: the higher the 10% A at the Second position, etc. This leads at each position intended specificity is, the more SDRs are inserted. Whereas approximately in 1 of 3 cases to the template amino acid and a single SDR enables the generation of moderately specific in 2 of 3 cases to another amino acid. enzymes, two SDRS enable already the generation of Sig 0315. In another preferred alternative, the sequences nificantly Specific enzymes. However, up to Six and more Selected in one round are analyzed and a consensus library SDRs can be inserted into a protein scaffold. A similar is generated based on these Sequences. This can, for relation is valid for the length of the SDRs: the higher the example, be achieved by using defined mixtures of nucle intended specificity is, the longer are the SDRs that are to be otides at each position in the oligonucleotide Synthesis in a inserted. SDRS can be as short as one to four amino acid way that leads to mixtures of the amino acid residues that residues. They can, however, also be as long as 50 amino were identified at each position of the SDR selected in the acid residues. Significant Specificity can already be gener previous round. If, for example, in a first round two SDRs ated by the use of SDRs of a length of four to six amino acid are identified that have the amino acid Sequences ARLT and residues. VPGS, a consensus library inserted in this SDR site in the 0312 The peptid sequences that are inserted can be fully following round could be encoded by an oligonucleotide or partially random. In this context, fully random means that with the sequence G(C/T)G C(G/C)C (G/T)(G/T)G a Set of Sequences are inserted in parallel that includes (A/T)CC. This would correspond to the random peptide Sequences that differ from each other in each and every sequence (A/V)(R/P)(L/G/V/W)(T/S), thereby allowing all position. Partially random means that a Set of Sequences are combinations of the amino acid residues identified in the inserted in parallel that includes Sequences that differ from first round, and, due to the degeneracy of the genetic code, each other in at least one position. This difference can be allowing in addition to a lower degree alternative amino acid either pair-wise or with respect to a single Sequence. For residues at Some positions. example, when regarding an insertion of the length of four 0316. In another preferred alternative, the sequences amino acids, partial random could be a set (i) that includes Selected in one round are, without previous analysis, recom AGGG, GVGG, GGLG, GGGI, or (ii) that includes AGGG, bined using methods for the in vitro recombination of VGGG, LGGG and IGGG. Alternatively, random sequences polynucleotides, such as the methods described in WO also comprises Sequences that differ from each other in 01/34835 (the following also provides details of the eighth length. Randomization of the peptide Sequences is achieved and ninth aspect of the invention). by randomization of the nucleotide Sequences that are inserted into the gene at the respective Sites. Thereby, 0317. After insertion of the partially or fully random randomization can be achieved by employing mixtures of Sequences into the gene encoding the Scaffold protein, and nucleobases as monomers during chemical Synthesis of the eventually ligation of the resulting gene into a Suitable oligonucleotides. A particularly preferred mixture of mono expression vector using Standard molecular cloning tech merS for a fully random codon that in addition minimizes the niques (Sambrook, J. F.; Fritsch, E. F.; Maniatis, T.; Cold probability of stop codons is NN(GTC). Alternatively, ran Spring Harbor Laboratory Press, Second Edition, 1989, New dom oligonucleotides can be obtained by fragmentation of York), the vector is introduced in a Suitable expression host DNA into short fragments that are inserted into the gene at cell which expresses the corresponding enzyme variant. the respective sites. The source of the DNA to be fragmented Particularly Suitable expression hosts are bacterial expres may be a Synthetic oligonucleotide but alternatively may Sion hosts such as Escherichia coli or Bacillus Subtilis, or originate from cloned genes, cDNAS, or genomic DNA. yeast expression hosts Such as Saccharomyces cerevisae or Preferably, the DNA is a gene encoding an enzyme. The Pichia pastoris, or mammalian expression hosts Such as fragmentation can, for example, be achieved by random Chinese Hamster Ovary (CHO) or Baby Hamster Kidney endonucleolytic digestion of DNA. Preferably, an unspecific (BHK) cell lines, or viral expression Systems Such as bac endonuclease Such as DNASe I (e.g. from bovine pancreas) teriophages like M13 T7 phage or Lambda, or viruses such is employed for the endonucleolytic digestion. as the Baculovirus expression System. As a further alterna tive, Systems for in vitro protein expression can be used. 0313) If steps (a)-(c) of the inventive method are repeated Typically, the DNA is ligated into an expression vector cyclically, there are different alternatives for obtaining ran behind a Suitable signal Sequence that leads to Secretion of dom peptide Sequences that are inserted in consecutive the enzyme variants into the extracellular space, thereby rounds. Preferably, SDRs that were identified in one round allowing direct detection of enzyme activity in the cell as leading to increased Specificity of enzyme are used as Supernatant. Particularly Suitable signal Sequences for templates for the random peptide Sequences that are inserted Escherichia coli are ompa, pelB, Hly A, for Bacillus Subtilis in the following round. AprE, NprB, Mpr, Amy A, AmyE, Blac, SacB, and for S. US 2005/0175581 A1 Aug. 11, 2005

cerevisiae Bar1, Suc2, Mato, Inu1A, Ggp1p. Alternatively, and the other peptide Substrate having an amino-acid the enzyme variants are expressed intracellularly and the Sequence identical to or resembling the intended target Substrates are expressed also intracellularly. According to Substrate Sequence thereby enabling to monitor the activity protease variants this is done essentially as described in on the target Substrate. Especially preferably, these two patent application WO 0212543, using a fusion peptide peptide Substrates are linked to fluorescent marker mol Substrate comprising two auto-fluorescent proteins linked by ecules, and the fluorescent properties of the two peptide the Substrate amino-acid Sequence. As a further alternative, Substrates are Sufficiently different in order to distinguish after intracellular expression of the enzyme variants, or both activities when measured consecutively or in parallel. Secretion into the periplasmatic Space using Signal Sequences For example, a fusion protein comprising a first autofluo such as DsbA, PhoA, PelB, Ompa, OmpT or g|II for rescent protein, a peptide, and a Second autofluorescent Escherichia coli, a permeabilisation or lysis Step releases the protein according to patent application WO 0212543 can be enzyme variants into the Supernatant. The destruction of the used for this purpose. Alternatively, fluorophores Such as membrane barrier can be forced by the use of mechanical rhodamines are linked chemically to the peptide Substrates. means Such as ultraSonic, French preSS, or the use of 0321) As a third alternative, enzymes that recognize and membrane-digesting enzymes Such as lysozyme. AS another, convert the target Substrate preferably are identified by further alternative, the genes encoding the enzyme variants employing one or more Substrates resembling the target are expressed cell-free by the use of a Suitable cell-free Substrate together with competing Substrates in high excess. expression system. For example, the S30 extract from Screening with respect to activity on the Substrates resem Escherichia coli cells is used for this purpose as described bling the target Substrate is then done in the presence of the by Lesly et al. (Methods in Molecular Biology 37 (1995) competing Substrates. Enzymes having a specificity which 265-278). corresponds qualitatively to the target Specificity, but having 0318. After introduction of the vector into host cells, only a low quantitative Specificity are identified as negative these cells are Screened for the expression of enzymes with Samples in Such a Screen. Whereas enzymes having a Specificity for the intended target Substrate. Such Screening Specificity which corresponds qualitatively and quantita is typically done by Separating the cells from each other, in tively to the target specificity are identified positively. Pref order to enable the correlation of genotype and phenotype, erably, the one or more Substrates resembling the target and assaying the activity of each cell clone after a growth Substrate are linked to marker molecules, thereby enabling and expression period. Such Separation can for example be the detection of their modifications, whereas the competing done by distribution of the cells into the compartments of Substrates do not carry marker molecules. The competing sample carriers, e.g. as described in WO 01/24933. Alter Substrates have arbitrarily chosen or random amino-acid natively, the cells are Separated by Streaking on agar plates, Sequences, thereby acting as competitive inhibitors for the by enclosing in a polymer Such as agarose, by filling into hydrolysis of the marker-carrying Substrates. For example, capillaries, or by Similar methods. Identification of variants protein hydrolysates Such as Trypton can Serve as competing with the intended specificity can be done by different Substrates for engineered proteolytic enzymes according to approaches. In the case of proteases, preferably assays using the invention. As a fourth alternative, enzymes that recog peptide substrates essentially as described in PCT/EPO3/ nize and convert the target Substrate preferably are identified 04864 are employed. and Selected by an amplification-coupled or growth-coupled 0319 Regardless of the expression format, selection of Selection Step. Furthermore, the activity can be measured enzyme variants is done under conditions that allow iden intracellularily and the Selection can be done by a cell Sorter, tification of enzymes that recognize and convert the target Such as a fluorescence-activated cell Sorter. Sequence preferably. As a first alternative, enzymes that 0322. As a further alternative, enzymes that recognize recognize and convert the target Sequence preferably are and convert the target Substrate are identified by first Select identified by Screening for enzymes with a high affinity for ing enzymes that preferentially bind to the target Substrate, the target Substrate Sequence. High affinity corresponds to a and Secondly Selecting out of this Subgroup of enzyme low KM which is Selected by Screening at target Substrate variants those enzymes that convert the target Substrate. concentrations Substantially below the KM of the first Selection for enzymes that preferentially bind the target enzyme. Preferably, the substrates that are used are linked to substrate can be either done by selection of binders to the one or more fluorophores that enable the detection of the target Substrate or by counter-Selection of enzymes that bind modification of the substrate at concentrations below 10 uM, to other Substrates. Methods for the selection of binders or preferably below 1 uM, more preferably below 100 nM, and for the counter-Selection of non-binderS is known in the art. most preferably below 10 nM. Such methods typically require phenotype-genotype cou 0320 AS a Second alternative, enzymes that recognize pling which can be Solved by using Surface display expres and convert the target Substrate preferably are identified by Sion methods. Such methods include, for example, phage or employing two or more Substrates in the assay and Screening Viral display, cell Surface display and in vitro display. Phage for activity on these two or more Substrates in comparison. or viral display typically involves fusion of the protein of Preferably, the two or more substrates employed are linked interest to a viral/phage protein. Cell Surface display, i.e. to different marker molecules, thereby enabling the detec either bacterial or eukaryotic cell display, typically involves tion of the modification of the two or more Substrates fusion of the protein of interest to a peptide or protein that consecutively or in parallel. In the case of proteases, par is located at the cell Surface. In in-vitro display, the protein ticularly preferably two peptide Substrates are employed, is typically made in vitro and linked directly or indirectly to one peptide Substrate having an arbitrarily chosen or even the mRNA encoding the protein (DE 19646372). partially or fully random amino-acid Sequence thereby 0323 The invention also provides for a composition or enabling to monitor the activity on an arbitrary Substrate, pharmaceutical composition comprising one or more engi US 2005/0175581 A1 Aug. 11, 2005 42 neered enzymes according to the first aspect of the invention substrate binding site. These regions therefore fullfil two as defined herein before. The composition may optionally criteria to be selected as candidates for SDRs: firstly, they comprise an acceptable carrier, excipient and/or auxiliary represent insertions in the Specific proteases compared to the agent. unspecific one and, Secondly, they are close to the Substrate 0324 Pharmaceutical compositions according to the binding site. A representation of the three-dimensional Struc invention may optionally comprise a pharmaceutically ture is given in FIG. 3. acceptable carrier. Pharmaceutical formulations are well 0330 FIG. 3: Three-dimensional structure of subtilisin known and pharmaceutical compositions may be routinely with the active site residues being shown in “ball-and-stick” formulated by one having ordinary skill in the art. The representation and with the numbered regions indicating composition can be formulated as a Solution, Suspension, potential SDR insertion sites. emulsion, or lyophilized powderin association with a 0331 FIG. 4: Alignment of the primary amino acid pharma-ceutically acceptable vehicle. Examples of Such sequences of subtilisin E, furin, PC1 and PC5 all of which vehicles are water, Saline, Ringer's Solution, dextrose Solu belong to the Structural class S8 of the Serine protease tion, and human Serum albumin. Liposomes and nonaaque family. Subtilisin E represents an unspecific protease of this ous vehicles Such as fixed oils may also be used. The vehicle structural class, while furin, PC1 and PC5 are proteases with or lyophilized powder may contain additives that maintain high Substrate Specificity. Compared to Subtilisin Several isotonicity (e.g. Sodium chloride, mannitol) and chemical regions of insertions of three or more amino acids into the Stability (e.g. buffers and preservatives). The composition is primary sequence of furin, PC1 and PC5 are seen. The Sterilized by commonly used techniques. regions marked with (-4-), (-5-), (-7-), (-9-) and (-11-) are 0325 The pharmaceutical composition of the present preferred SDR insertion sites. These regions stretches fulfill invention may be administrated by any means that enables two criteria to be selected as candidates for SDRs: firstly, the active agent to reach the agent's Site of action in the body they represent insertions in the Specific proteases compared of a mammal. Pharmaceutical compositions may be admin to the unspecific one and, Secondly, they are close to the istered parentally, i.e. intravenous (i.v.), Subcutaneous (s.c.), active Site residues. intramuscular. 0332 FIG. 5: Three-dimensional structure of beta-secre 0326 Dosage varies depending upon known factors Such tase with the active site residues being shown in “ball-and as the pharmacodynamic characteristics of the particular Stick” representation and with the numbered regions indi agent, and its mode and route of administration; age, health, cating potential SDR insertion sites. and weight of the recipient; nature and extent of Symptoms, 0333 FIG. 6: Alignment of the primary amino acid kind of concurrent treatment, frequency of treatment, and Sequences of pepsin, b-Secretase and cathepsin D, all of the effect desired. which belong to the Structural class A1 of the aspartic 0327 Non-pharmaceutical compositions as defined protease family. Pepsin represents an unspecific protease of herein are research composition, nutritional composition, this structural class, while b-Secretase and cathepsin D are cleaning composition, desinfection composition, cosmetic proteases with high Substrate Specificity. Compared to pep composition or composition for personal care. Moreover, Sin Several regions of insertions of three or more amino acids DNA sequences coding for the engineered enzyme as into the primary Sequence of b-Secretase and cathepsin Dare defined herein before and vectors containing said DNA Seen. The regions marked with -1- to -11- correspond to Sequences are also provided. Finally, transformed host cells possible SDR combining sites and are also marked in FIG. (prokaryotic or eukaryotic) or transgenic organisms contain 5. ing Such DNA sequences and/or vectors, as well as a method 0334 FIG. 7: illustrates the three-dimensional structure utilizing Such host cells or transgenic animals for producing of caspase 7 with the active site residues being shown in the engineered enzyme of the first aspect of the invention are “ball-and-stick” representation and with the numbered also contemplated. regions indicating potential SDR insertion Sites. DETAILED DESCRIPTION OF THE FIGURES 0335 FIG. 8: shows the primary amino acid sequence of caspase 7 as a member of the cysteine protease class C14 0328 FIG. 1: Three-dimensional structure of human family (see also SEQ ID NO: 14). trypsin I with the active site residues shown in “ball-and Stick” representation and with the marked regions indicating 0336 FIG. 9: Schematic representation of method potential SDR insertion sites. according to the third aspect of the invention. 0329 FIG. 2: Alignment of the primary amino acid 0337 FIG. 10: Western blot analysis of trypsin expres Sequences of the human proteases trypsin I, alpha-thrombin Sion. Supernatant of cell cultures expressing variants of and enteropeptidase all of which belong to the Structural trypsin are compared to negative controls. Lane 1: molecular class S1 of the Serine protease family. Trypsin represents an weight Standard; lane 2: negative control; lane 3: Supernatant unspecific protease of this Structural class, while alpha of variant a, lane 4: negative control; lane 5: Supernatant of thrombin and enteropeptidase are proteases with high Sub variant b. A primary antibody Specific to the expressed Strate Specificity. Compared to trypsin Several regions of protein and a Secondary antibody for generation of the Signal insertions of three or more amino acids into the primary were used. Sequence of a-thrombin and enterokinase are seen. The 0338 FIG. 11: Time course of the proteolytic cleavage of region marked with (-1-) and the region marked with (-3-) a target Substrate. Supernatant of cells containing the Vector are preferred SDR insertion sites. In the tertiary structure of with the gene for human trypsin and that of cells containing alpha-thrombin both regions are in the vicinity of the the vector without the gene was incubated with the peptide US 2005/0175581 A1 Aug. 11, 2005 43

Substrate described in the text. Cleavage of the peptide EXAMPLES results in a decreased read out value. Proteolytic activity is 0346. In the following examples, materials and methods confirmed for the positive clone. of the present invention are provided including the deter 0339 FIG. 12: Relative activity of three engineered mination of catalytic properties of enzymes obtained by the proteolytic enzymes in comparison with human trypsin I on method. It should be understood that these examples are for two different peptide Substrates. A time course of the pro illustrative purpose only and are not to be construed as teolytic digestion of the two Substrates was performed and limiting this invention in any manner. All publications, evaluated. Substrate B was used for Screening and Substrate patents, and patent applications cited herein are hereby A is a closely related Sequence. Relative activity of the three incorporated by reference in their entirety for all purposes. variants was normalized to the activity of human trypsin I. Variant 1 and 2 clearly show increased specificity towards 0347 In the experimental examples described below, the target Substrate. Variant 3, on the other hand, Serves as Standard techniques of recombinant DNA technology were a negative control with Similar activities as the human used that were described in various publications, e.g. Sam trypsin I. brook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, or Ausubel et al. 0340 FIG. 13: Relative specificities of trypsin and vari (1987), Current Protocols in Molecular Biology 1987-1988, ants of engineered proteolytic enzymes with one or two Wiley Interscience. Unless otherwise indicated, restriction SDRs, respectively. Activity of the proteases was deter enzymes, polymerases and other enzymes as well as DNA mined in the presence and absence of competitor Substrate, purification kits were used according to the manufacturers i.e. peptone at a concentration of 10 mg/ml. Time courses for Specifications. the proteolytic cleavage were recorded and the time con Stants k determined. The ratios between the time constants Example I with and without competitor were formed and represent a quantitative measure for the Specificity of the protease. The ratios were normalized to trypsin. The Specificity of the Identification of SDR Sites in Human Trypsin variant containing two SDRs is 2.5 fold higher than that of 0348. Insertion sites for SDRs have been identified in the the variant with SDR2 alone. Serine protease human trypsin I (Structural class S1) by 0341 FIG. 14: Shows the relative specificities of pro comparison with members of the same Structural class tease variants in absence and presence of competitor Sub having a higher Sequence Specificity. Trypsin represents a Strate. The protease variants containig two inserts with member with low Substrate Specificity, as it requires only an different Sequences and the non-modified Scaffold human arginine or lysine residue at the P position. On the other trypsin I were expressed in a Suitable host. Activity of the hand, thrombin, tissue-type plasminogen activator or enter protease variants was determined as the cleavage rate of a okinase all have a high Specificity towards their Substrate peptide with the desired target sequence of TNF-alpha in the sequences, i.e. (L/I/V/F)XPRNA, CPGRVVGG and absence and presence of competitor Substrate. Specificity is DDDK, respectively. The primary sequences and tertiary expressed as the ratio of cleavage rates in the presence and Structures of these and further S1 Serine proteases have been absence of competitor. aligned in order to determine regions of low and high Sequence and structure homology and especially regions that 0342 FIG. 15: The figure shows the reduction of cyto correspond to insertions in the Sequences of the more toxicity induced by human TNF-alpha when incubating the Specific proteases (FIG. 2). Several regions of insertions human TNF-alpha with concentrated Supernatant from cul equal or longer than 3 amino acids representing potential tures expressing the inventive engineered proteolytic SDR sites have been identified as indicated in FIG. 1. These enzymes being specific for human TNF-alpha. This indicates regions were chosen as target Sites for the insertion of SDRS the efficacy of the inventive engineered proteolytic enzymes. in the examples below, e.g. SDR1 (region one in FIG. 2, 0343 FIG. 16: The figure shows the reduction of cyto after amino acid 42 according to SEQ ID NO:1) with a toxicity induced by human TNF-alpha when incubating the length of six and SDR2 (region three in FIG. 2, after amino human TNF-alpha with different concentrations of purified acid 123 according to SEQ ID NO:1) with a length of five inventive engineered proteolytic enzyme being Specific for amino acids, respectively. human TNF-alpha. Variant g comprises SEQ ID NO:72 as SDR1 and SEO ID NO:73 as SDR2. This indicates the Example II efficacy of the inventive engineered proteolytic enzymes. 0344 FIG. 17: The figure compares the activity of inven Molecular Cloning of the Human Trypsin I Gene to tive engineered proteolytic enzymes being Specific for be Used as Scaffold Protein and Expression of the human TNF-alpha with the activity of human trypsin I on Mature Protease in B. Subtilis two protein substrates: (a) human TNF-alpha; (b) mixture of 0349 The gene encoding the unspecific protease human human Serum proteins. This indicates the Safety of the trypsinogen I was cloned into the vector puC18. Cloning inventive engineered proteolytic enzymes. Variant X corre was done as follows: the coding Sequence of the protein was sponds to Seq ID No. 75 comprising the SDRs according to amplified by PCR using primers that introduced a Kipni site Seq ID No. 89 (SDR1) and 95 (SDR2). Variants xi and xii at the 5' end and a Bam-HI site at the 3' end. This PCR correspond to derivatives thereof comprising the same SDR fragment was cloned into the appropriate Sites of the Vector Sequences. pUC18. Identity was confirmed by Sequencing. After 0345 FIG. 18: Specific hydrolysis of human VEGF by Sequencing the coding Sequence of the mature protein was an engineered proteolytic enzyme derived from human amplified by PCR using primers that introduced different trypsin. Bgll sites at the 5' end and the 3' end. US 2005/0175581 A1 Aug. 11, 2005 44

0350. This PCR fragment was cloned into the appropriate fication Kit following the Suppliers instructions. Resulting sites of an E. coli-B. Subtilis shuttle vector. The vector plasmids were transformed into B. Subtilis cells. contains a pMB1 origin for amplification in E. coli, a neomycin resistance marker for Selection in E. coli, as well 0355 Alternatively, or in addition to random mutagen as a P43 promoter for the constitutive expression in B. esis, variants of the gene were Statistically recombined at Subtilis. A87 bp fragment that contains the leader Sequence homologous positions by use of the Recombination Chain encoding the Signal peptide from the SacB gene of B. Subtilis Reaction, essentially as described in WO 0134835. PCR was introduced behind the P43 promoter. Different BglI products of the genes encoding the protease variants were restriction sites Serve as insertion sites for heterologous purified using the QIAquick PCR Purification Kit following genes to be expressed. the Suppliers instructions, checked for correct size by 0351 Expression of human trypsin I was confirmed by agarose gel electrophoresis and mixed together in equimolar measurement of the proteolytic aciticity in Supernatant of amounts. 80 ug of this PCR mix in 150 mM TrishCl pH 7.6, cells containing the vector with the gene in comparison to a 6.6 mM MgCl2 were heated for 5 min at 94 C. and negative control. A peptide including an arginine cleavage Subsequently cooled down to 37 C. at 0.05 C./s in order to Site was chosen as a Substrate. The peptide was N-terminally re-anneal Strands and thereby produce heteroduplices in a biotinylated and labeled with a fluorophore at the C-termi stochastic manner. Then, 2.5 U Exonuclease III per ug DNA nus. After incubation of the peptide with culture Supernatant were added and incubated for 20, 40 or 60 min at 37 C. in Streptavidin was added. Uncleaved peptide associate with order to digest different lengths from both 3' ends of the Streptavidin and lead to a high read out value while cleavage heteroduplices. The partly digested PCR products were results in low read out values. FIG. 11 shows the time course refilled with 0.6 U Pfu polymerase per ug DNA by incubat of a proteolytic digestion of B. Subtilis cells containing the ing for 15 min at 72° C. in 0.17 mM dNTPs and Pfu vector with the trypsin I gene in comparison to B. Subtilis polymerase buffer according to the Suppliers instructions. After performing a single PCR cycle, the resulting DNA was cells containing the vector without the trypsin I gene (nega purified using the QIAquick PCR Purification Kit following tive control). the Suppliers instructions, digested with BglI and ligated 0352. As a further confirmation of expression of the into the linearized vector. The ligation products were trans protease, Supernatants of cells containing the vector with the formed into E. coli, amplified in LB containing amplicillin as gene and control cells were analyzed by polyacrylamid gel marker, and the plasmids were purified using the Qiagen electrophoreses and Subsequent western blot using an anti Plasmid Purification Kit following the suppliers instruc body Specific to the target protease. The procedure was tions. Resulting plasmids were transformed into B. Subtilis performed according to Standard methods (Sambrook, J. F.; cells. Fritsch, E. F.; Maniatis, T.; Cold Spring Harbor Laboratory Press, Second Edition, 1989, New York). FIG. 8 confirms Example IV expression of the protein only in the cells harbouring the vector with the gene for trypsin. Insertion of SDRS Into the Protein Scaffold of Human Trypsin I and Generation of an Engineered Example III Proteolytic Enzyme with Specificity for a Peptide Substrate Having the Sequence Providing a Scaffold Protein KKWLGRVPGGPV 0353. In this example, human trypsin I was used as the 0356. In order to create insertion sites for SDRs in human Scaffold protein. The gene was either used in its natural trypsin I, two pairs of different restriction sites were intro form, or, alternatively, was modified to result in a Scaffold duced into the gene at Sites that were identified as potential protein with increased catalytic activity or further improved SDR sites (see Example I above) without changing the characteristics. amino acid Sequence. The insertion of the restriction Sites 0354) The modification was done by random modifica was done by overlap extension PCR. Primers restr1 and tion of the gene, followed by expression of the enzyme and restr2 were used for the introduction of SacII and BamHI Subsequent Selection for increased activity. First, the gene restriction sites, restr3 and restr4 were used for the intro was PCR amplified under error-prone conditions, essentially duction of KpnI and Nhe restriction sites. The sequences of as described by Cadwell, R. C and Joyce, G. F. (PCR the primers were as follows: Methods Appl. 2 (1992) 28-33). Error-prone PCR was done 0357 Binding site for restr1 and restr2 and the corre using 30 pmol of each primer, 20 nmol dGTP and dATP, 100 sponding amino acid sequence (SEQ ID NO:54): nmol dCTP and dTTP, 20 fmol template, and 5 U Taq DNA polymerase in 10 mM Tris HCl pH 7.6, 50 mM KC1, 7 mM MgCl2, 0.5 mM MnC12, 0.01% gelatin for 20 cycles of 1 Binding site for restr1 and restr2 and the min at 94 C., 1 min at 65° C. and 1 min at 72° C. The corresponding amino acid sequence: (SEQ ID NO:54) resulting DNA library was purified using the Qiaquick PCR 5'-GGTGGTATCAGCAGGCCACTGCTACAAGTCCCGCATCCAGGT-3' Purification Kit following the suppliers instructions. The W W S A G H C Y K S R I, Q PCR product was digested with the restriction enzyme BglI and purified. Afterwards, the PCR product was ligated into Forward primer restr1 : the E. coli-B. Subtilis shuttle vector described above which (SEQ ID NO:56) was digested with BglI and dephosphorylated. The ligation 5'-GGTGGTATCCGCGGGCCACTGCTACAAGTCCCGGATCCAGGT-3' products were transformed into E. coli, amplified in LB, and Reverse primer restr2: the plasmids were purified using the Qiagen Plasmid Puri US 2005/0175581 A1 Aug. 11, 2005 45

0362. The restriction sites generated thereby were sub -continued Sequently used to insert defined or random oligonucleotides (SEQ ID NO:57) 5'-ACCTGGATCCGGGACTTGTAGCAGTGGCCCGCGGATACCACC-3' into the SDR1 and SDR2 insertion sites by standard restric tion and ligation methods. Typically, two complementary 0358 Binding site for restr3 and restra and the corre Synthetic 5'-phosphorylated oligonucleotides were annealed sponding amino acid sequence (SEQ ID NO:58): and ligated into a vector carrying the modified human

Binding site for restr3 and restr A and the corresponding amino acid sequence: 5'-CCACTGGCACGAAGTGCCTCATCTCTGGCTGGGGCAACACTGCGAGCTCT-3' (SEQ ID NO:58) T G T K C L I S. G. W. G. N. T. A. S. S

0359 Forward primer restr3 (SEQ ID NO:60): trypsin I gene that was cleaved with the respective restric tion enzymes. Oligonucleotides encoding SDR1 were inserted via the SacII/BamHI sites whereas oligonucleotides Forward primer restr3: (SEQ ID NO: 60) encoding SDR2 were inserted via the KpnI/Nhe sites. For 5'-CCACTGGCACGAAGTGCCTCATCTCTGGCTGGGGCAACACT each insertion an oligonucleotide pair according to the following general Sequences was used (P) indicating GCGAGCTCT-3' 5'-phosphorylation, N and X indicating any nucleotide or 0360 Reverse primer restr4 (SEQ ID NO:61): amino acid residue, respectively): 0363) oligox-SDR1f (SEQ ID NO:64): Reverse primer restr A: (SEQ ID NO: 61) 5'-AGAGCTAGCAGTGTTGCCCCAGCCAGAGATGAGGCACTTGGTACC

0361). In a first overlap extension PCR, the SacII/BamHI oligox-SDR1 f : Sites were introduced, enabling to insert SDR1, and in a (SEQ ID NO: 64) second overlap extension PCR the KpnI/NeI sites, enabling 5'-P-GGGCCACTGCTACNNNNNNNNNNNNNNNNNNAAGTCCCG-3' the insertion of SDR2. The product of the overlap extension PCR was amplified using primers puC-forward and puC reverse. The sequences of puC-forward and puC-reverse 0364) oligox-SDR1r (SEQ ID NO:66): are as follows:

oligox-SDR1r: 3'-CGCCCGGTGACGATGNNNNNNNNNNNNNNNNNNTTCAGGGCCTAG-P-5' (SEQ ID NO: 66) G. H. C. Y X X X X X X K. S

0365) oligox-SDR2f (SEQ ID NO:67):

oligox-SDR2f: 5'-P-CAAGTGCCTCATCTCTGGCTGGGGCAACNNNNNNNNNNNNNNNACTG-3' (SEQ ID NO: 69) oligox-SDR2r: 3'-CATGGTTCACGGAGTAGAGACCGACCCCGTTGNNNNNNNNNNNNNNNTGACGATC-P-5' (SEQ ID NO: 69) K C L I S. G. W. G. N. X X X X X T

0366 As an alternative to the above method, a PCR based method was used for the integration of random-Sequences into the SDR1 and SDR2 insertion sites in the modified pUC-forward: human trypsin I. For each SDR, one primer was used where (SEQ ID NO: 62) the SDR region is fully randomized. Sequences of the 5'-GGGGTACCCCACCACCATGAATCCACTCCT-3' primers were as follows (N=A/C/G/T, B=C/G/T, V=A/C/G): 0367 Primer SDR1-mutnnb-forward (SEQ ID NO:70): pUC-reverse :

(SEQ ID NO: 63) Primer SDR1-mutninb-forward: 5'-CGGGATCCGGTATAGAGACTGAAGAGATAC-3' (SEQ ID NO: 70) US 2005/0175581 A1 Aug. 11, 2005 46

0374 Variants selected in the screening procedure were -continued further evaluated for their specificity towards substrate B 5'-TGGTATCCGCGGGCCACTGCTACNNBNNBNNBNNBNNBNNBAAGTCC and closely related Substrate Aby measuring time courses of the proteolytic digestion and determining the rate constants CGGATCCAGGTG-3' which are proportional to the ki/KM values. Clearly, com pared to the human trypsin that was used as Scaffold protein, 0368 Primer SDR2-mutnnb-reverse (SEQ ID NO:71): the specific activity of variants 1 and 2 is shifted (SEQ ID NOs: 2 and 3, respectively) towards substrate B. Variant 3 Primer SDR2-mutninb-reverse: (SEQ ID NO:4), on the other hand, serves as a negative (SEQ ID NO: 71.) control with Similar activities as the human trypsin I. 5'-GGCGCCAGAGCTAGCAGTWNNWNNWNNWNNWNNGTTGCCCCAGCC Sequencing of the genes of the three variants revealed the AGAGATG-3' following amino acid Sequences in the SDRS. 0369. The codon NNB, or VNN in the reverse strand, TABLE 2 allows all 20 amino acids to made, but reduces the prob Sequences of the two SDRs in three different variants selected ability of encoding a stop codon from 0.047 to 0.021. for specific hydrolysis of substrate B (SEQ ID NOS: 78–83).

0370. As a further alternative, after identification of SDR 1 SDR 2 SDRs that lead to increased specificity, these SDRs were used as templates for further randomization. Thereby, ran Trypsin Variant 1 D A W G R D T T N S dom peptide Sequences were inserted that were partially Variant 2 N G R D L E V R G T W randomized at each position and partially identical at each Variant 3 G F W M F N R S P L T position to the original Sequence. 0371 AS an example, random peptide sequences that have in approximately 1 of 3 cases the template amino acid 0375. In a further experiment a pool of variants contain residue and in approximately 2 of 3 cases any other amino ing different numbers of SDRs per gene were screened for acid residue at each position were inserted into the two SDR increased specificity using a mixture of the defined Substrate insertion sites of the modified human trypsin I. For this and pepton as a competing Substrate. Variants containing one purpose, primers that contain at each nucleotide position of or two SDRs per gene have been analyzed further. As a the SDR approximately 70% of the template bases and 30% measure for the Specificity the activity in the peptide cleav of a mixture of the three other bases were used. age assay was compared with and without the presence of the competing Substrate. The concentration of the competing 0372 With each primer pair a PCR was performed under Substrate was 10 mg/ml. Under these conditions, unspecific Standard conditions using the human trypsin I gene as proteases show, compared to specific proteases, a Stronger template. The resulting DNA was purified using the decrease in activity with increasing competitor concentra QIAquick PCR Purification Kit following the Suppliers tions (range between 0 and 100 mg/ml). The ratio of instructions and digested with SacII and Nhe. After diges proteolytic activity with and without Substrate is a quanti tion the DNA was purified and ligated into the SacII and tative measure for the specificity of the proteases. FIG. 9 Nhe digested and dephosphorylayted vector. The ligation shows the relative activities with and without competing products were transformed into E. coli, amplified in LB Substrate. Human trypsin I that was used as the Scaffold containing the respective marker, and the plasmids were protein and two variants, one containing only. SDR2, and purified using the Qiagen Plasmid Purification Kit following one containing both SDRS, were compared. The Specificity the Suppliers instructions. Resulting plasmids were trans of the variant with both SDRs is by a factor of 2.5 higher formed into B. Subtilis cells. These cells were then separated than that of the variant with SDR2 only, confirming that to Single cells, grown to clones, and after expression of the there is a direct relation between the number of SDRs and protease gene Screened for proteolytic activity. The follow the quantitative Specificity of resulting engineered pro ing Substrates were employed for Screening for proteolytic teolytic enzymes. activity (SEQ ID NOS:76 and 77): EXAMPLE V substrate A L. L W L G R V V G G P V Generation of an Engineered Proteolytic Enzyme substrate B K K W L G R V P G G P V that Specifically Inactivates Human TNF-alpha 0376 Human trypsin alpha I or a derivative comprising 0373) Protease variants were screened on Substrate B at one or more of the following amino acid substitutions E56G, complexities of 10° variants by confocal fluorescence spec R78W: Y131F; A146T, C183R was used as protein scaffold troScopy. The Substrate was a peptide biotinylated at the for the generation of an engineered proteolytic enzyme with N-terminus and fluorescently labeled at the C-terminus. high specificity towards human TNF-alpha. The identifica After incubation of the peptide with Supernatant of cells tion of SDR sites in human trypsin I or derivatives thereof expressing different variants of the protease, Streptavidin is was done as described above. Two insertion sites within the added and the Samples are analysed by confocal fluorimetry. scaffold were choosen for SDRs. The protease variants The low concentration of the peptide (20 nM) leads to a containing two inserts with different Sequences and also the preferential cleavage by proteases with a high ki/KM value, human trypsin I itself with no inserts were expressed in a i.e. proteases with high Specificity towards the target Bacillus Subtilis cells. The variant protease cells were Sepa Sequence. rated to Single cell clones and the protease expressing US 2005/0175581 A1 Aug. 11, 2005 47 variants were Screened for proteolytic activity on peptides 0378. To demonstrate the specificity of the inventive with the desired target sequence of TNF-alpha. The activity protease variants, proteins from human blood Serum or of the protease variants was determined as the cleavage rate purified human TNF-alpha have been incubated with human of a peptide with the desired target Sequence of TNF-alpha trypsin I or the inventive engineered proteolytic enzyme in the absence and presence of competitor Substrate. The variants, respectively. Here, variant X corresponds to Seq ID Specificity is expressed as the ratio of cleavage rates in the No. 75 comprising the same SDRs as variant f, i.e. SDRs presence and absence of competitor (FIG. 14). according to Seq ID No. 89 (SDR1) and 95 (SDR2). Variants Xi and Xii correspond to derivatives thereof comprising the TABLE 3 Same SDR Sequences. Remaining intact protein was was determined as a function of time. While the variants as well Relative specificity of variants of engineered proteolytic enzymes with different SDR sequences in absence and presence as human trypsin I digest human TNF-alpha, only trypsin of competitor Substrate (SEQ ID NOS: 84-95). shows activity on serum protein (FIGS. 17a and b). This demonstrates the high TNF-alpha specificity of the inventive k with comp.f proteolytic enzymes and indicates their Safety and accord k without comp. Seq. of SDR 1 Seq. of SDR 2 ingly their low Side effects for therapeutic use. scaffold (no SDRs) O.O92 variant a O.130 RPWDPS WHPTS EXAMPLE VI variant b O.187 GFVMFN RSPLT variant c O.235 EANRE RGART variant d O.310 KAVVGT RTPS Generation of an Engineered Proteolytic Enzyme variant e O.374 VNIMAA TTARK that Specifically Hydrolysis Human VEGF variant f O.487 AAFNGD RKDFW 0379 Human trypsin I was used as protein scaffold for the generation of an engineered proteolytic enzyme with 0377 The antagonistic effect of three inventive protease high specificity towards human VEGF. The identification of variants on human TNF-alpha is shown in FIG. 15. By the SDR sites in human trypsin I was done as described above. use of the variants, the induction of apoptosis is almost Two insertion sites within the scaffold were choosen for completely eliminated indicating the anti-inflammatory effi SDRs. The protease variants containing two inserts with cacy of the inventive proteases to initiate TNF-alpha break different Sequences were expressed in Bacillus Subtilis cells. down. TNF-alpha has been incubated with concentrated The variant protease cells were separated to single cell Supernatant from cultures expressing the variants i to iii for clones and the protease expressing variants were Screened as 2 hours. The resulting TNF-alpha has been incubated with described above. The activity of the protease variants was non-modified cells for 4 hours. The effect of the remaining determined as the rate of VEGF cleavage. 4 ug of recom TNF-alpha activity was determined as the extent of apop binant human VEGF165 was incubated with 0.18 lug of tosis induction by detection of activated caspase-3 as marker purified protease in PBS/pH 7.4 at room temperature. Ali for apoptotic cells. For the controls either no protease was quots were taken at the indicated time points and analysed added with the human TNF-alpha (dead cells) or buffer on a polyacrylamide gel. The extend of cleavage was instead of human TNF-alpha (live cells) was used, respec quantified by densitometric analysis of the bands. The tively. An analogous experiment is shown in FIG. 16 using activity is plotted over incubation time in FIG. 18. Specific purified variant xiii. TNF-alpha was incubated with different cleavage was controlled by further SDS polyacrylamide gel concentrations of the purified inventive protease variant. analyses.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 191 <210> SEQ ID NO 1 <211& LENGTH: 224 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 1 Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Glin Wall 1 5 10 15

Ser Lieu. Asn. Ser Gly Tyr His Phe Cys Gly Gly Ser Lieu Ile Asn Glu 2O 25 30

Gln Trp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Glin Wall 35 40 45

Arg Lieu Gly Glu His Asn. Ile Glu Val Lieu Glu Gly Asn Glu Glin Phe 5 O 55 60 Ile Asn Ala Ala Lys Ile Ile Arg His Pro Glin Tyr Asp Arg Lys Thr US 2005/0175581 A1 Aug. 11, 2005 48

-continued

65 70 75 8O Lieu. Asn. Asn Asp Ile Met Lieu. Ile Lys Lieu Ser Ser Arg Ala Val Ile 85 90 95 Asn Ala Arg Val Ser Thr Ile Ser Leu Pro Thr Ala Pro Pro Ala Thr 100 105 110 Gly Thr Lys Cys Lieu. Ile Ser Gly Trp Gly Asn. Thr Ala Ser Ser Gly 115 120 125 Ala Asp Tyr Pro Asp Glu Lieu Gln Cys Lieu. Asp Ala Pro Val Lieu Ser 130 135 1 4 0 Glin Ala Lys Cys Glu Ala Ser Tyr Pro Gly Lys Ile Thr Ser Asn Met 145 15 O 155 160 Phe Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Glin Gly Asp 1.65 170 175 Ser Gly Gly Pro Val Val Cys Asn Gly Glin Leu Gln Gly Val Val Ser 18O 185 19 O Trp Gly Asp Gly Cys Ala Glin Lys Asn Lys Pro Gly Val Tyr Thr Lys 195 200 2O5 Val Tyr Asn Tyr Val Lys Trp Ile Lys Asn. Thir Ile Ala Ala Asn. Ser 210 215 220

<210> SEQ ID NO 2 &2 11s LENGTH 2.35 &212> TYPE PRT <213> ORGANISM: artificial sequence &220s FEATURE <223> OTHER INFORMATION: trypsin variant 1 <400 SEQUENCE: 2 Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Tyr Glin Val 1 5 10 15 Ser Lieu. Asn. Ser Gly Tyr His Phe Cys Gly Gly Ser Lieu. Ile Asn. Glu 2O 25 30 Glin Trp Val Val Ser Ala Gly His Cys Tyr Asp Ala Val Gly Arg Asp 35 40 45 Lys Ser Arg Ile Glin Val Arg Lieu Gly Glu. His Asn. Ile Glu Val Lieu 50 55 60 Glu Gly Asn. Glu Glin Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro 65 70 75 8O Glin Tyr Asp Arg Lys Thr Lieu. Asn. Asn Asp Ile Met Lieu. Ile Lys Lieu 85 90 95 Ser Ser Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro 100 105 110 Thr Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly 115 120 125 Asn Thr Ile Thr Asn Ser Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp 130 135 1 4 0 Glu Lieu Gln Cys Lieu. Asp Ala Pro Val Lieu Ser Glin Ala Lys Cys Glu 145 15 O 155 160 Ala Ser Tyr Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe 1.65 170 175 Leu Glu Gly Gly Lys Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Wal 18O 185 19 O Val Cys Asn Gly Glin Lieu Glin Gly Val Val Ser Trp Gly Asp Gly Cys US 2005/0175581 A1 Aug. 11, 2005 49

-continued

195 200 2O5 Ala Glin Lys Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val 210 215 220 Lys Trp Ile Lys Asn. Thir Ile Ala Ala Asn. Ser 225 230 235

<210> SEQ ID NO 3 &2 11s LENGTH 2.35 &212> TYPE PRT <213> ORGANISM: artificial sequence &220s FEATURE <223> OTHER INFORMATION: trypsin variant 2 <400 SEQUENCE: 3 Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Tyr Glin Val 1 5 10 15 Ser Lieu. Asn. Ser Gly Tyr His Phe Cys Gly Gly Ser Lieu. Ile Asn. Glu 2O 25 30 Glin Trp Val Val Ser Ala Gly His Cys Tyr Asn Gly Arg Asp Leu Glu 35 40 45 Lys Ser Arg Ile Glin Val Arg Lieu Gly Glu. His Asn. Ile Glu Val Lieu 50 55 60 Glu Gly Asn. Glu Glin Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro 65 70 75 8O Glin Tyr Asp Arg Lys Thr Lieu. Asn. Asn Asp Ile Met Lieu. Ile Lys Lieu 85 90 95 Ser Ser Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro 100 105 110 Thr Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly 115 120 125 Asn Val Arg Gly Thr Trp Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp 130 135 1 4 0 Glu Lieu Gln Cys Lieu. Asp Ala Pro Val Lieu Ser Glin Ala Lys Cys Glu 145 15 O 155 160 Ala Ser Tyr Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe 1.65 170 175 Leu Glu Gly Gly Lys Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Wal 18O 185 19 O Val Cys Asn Gly Glin Lieu Glin Gly Val Val Ser Trp Gly Asp Gly Cys 195 200 2O5 Ala Glin Lys Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val 210 215 220 Lys Trp Ile Lys Asn. Thir Ile Ala Ala Asn. Ser 225 230 235

<210> SEQ ID NO 4 &2 11s LENGTH 2.35 &212> TYPE PRT <213> ORGANISM: artificial sequence &220s FEATURE <223> OTHER INFORMATION: trypsin variant 3 <400 SEQUENCE: 4 Ile Val Gly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Tyr Glin Val 1 5 10 15 US 2005/0175581 A1 Aug. 11, 2005 50

-continued Ser Lieu. Asn. Ser Gly Tyr His Phe Cys Gly Gly Ser Lieu. Ile Asn. Glu 2O 25 30 Glin Trp Val Val Ser Ala Gly His Cys Tyr Ala Ala Thr Asn Gly Asp 35 40 45 Lys Ser Arg Ile Glin Val Arg Lieu Gly Glu. His Asn. Ile Glu Val Lieu 50 55 60 Glu Gly Asn. Glu Glin Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro 65 70 75 8O Glin Tyr Asp Arg Lys Thr Lieu. Asn. Asn Asp Ile Met Lieu. Ile Lys Lieu 85 90 95 Ser Ser Arg Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro 100 105 110 Thr Ala Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly 115 120 125 Asn Arg Lys Asp Phe Trp Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp 130 135 1 4 0 Glu Lieu Gln Cys Lieu. Asp Ala Pro Val Lieu Ser Glin Ala Lys Cys Glu 145 15 O 155 160 Ala Ser Tyr Pro Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe 1.65 170 175 Leu Glu Gly Gly Lys Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Wal 18O 185 19 O Val Cys Asn Gly Glin Lieu Glin Gly Val Val Ser Trp Gly Asp Gly Cys 195 200 2O5 Ala Glin Lys Asn Lys Pro Gly Val Tyr Thr Lys Val Tyr Asn Tyr Val 210 215 220 Lys Trp Ile Lys Asn. Thir Ile Ala Ala Asn. Ser 225 230 235

<210 SEQ ID NO 5 &2 11s LENGTH 259 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 5 Ile Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser Pro Trp Glin Val 1 5 10 15 Met Leu Phe Arg Lys Ser Pro Glin Glu Lieu Lieu. Cys Gly Ala Ser Lieu 2O 25 30 Ile Ser Asp Arg Trp Val Lieu. Thir Ala Ala His Cys Lieu Lleu Tyr Pro 35 40 45 Pro Trp Asp Lys Asn. Phe Thr Glu Asn Asp Leu Lleu Val Arg Ile Gly 50 55 60 Lys His Ser Arg Thr Arg Tyr Glu Arg Asn. Ile Glu Lys Ile Ser Met 65 70 75 8O Leu Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Lieu 85 90 95 Asp Arg Asp Ile Ala Lieu Met Lys Lieu Lys Llys Pro Wall Ala Phe Ser 100 105 110 Asp Tyr Ile His Pro Val Cys Lieu Pro Asp Arg Glu Thir Ala Ala Ser 115 120 125 Leu Lieu Glin Ala Gly Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Lieu 130 135 1 4 0 US 2005/0175581 A1 Aug. 11, 2005 51

-continued

Lys Glu Thir Trp Thr Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu 145 15 O 155 160 Glin Val Val Asn Lieu Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser 1.65 170 175 Thr Arg Ile Arg Ile Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro 18O 185 19 O Asp Glu Gly Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro 195 200 2O5 Phe Val Met Lys Ser Pro Phe Asin Asn Arg Trp Tyr Gln Met Gly Ile 210 215 220 Val Ser Trp Gly Glu Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr 225 230 235 240 Thr His Val Phe Arg Lieu Lys Lys Trp Ile Glin Lys Val Ile Asp Glin 245 250 255 Phe Gly Glu

<210> SEQ ID NO 6 &2 11s LENGTH 2.35 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 6 Ile Val Gly Gly Ser Asn Ala Lys Glu Gly Ala Trp Pro Trp Val Val 1 5 10 15 Gly Lieu. Tyr Tyr Gly Gly Arg Lieu Lieu. Cys Gly Ala Ser Lieu Val Ser 2O 25 30 Ser Asp Trp Lieu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Lieu 35 40 45 Glu Pro Ser Lys Trp Thr Ala Ile Leu Gly Lieu. His Met Lys Ser Asn 50 55 60 Leu Thir Ser Pro Gln Thr Val Pro Arg Leu Ile Asp Glu Ile Val Ile 65 70 75 8O Asn Pro His Tyr Asn Arg Arg Arg Lys Asp Asn Asp Ile Ala Met Met 85 90 95 His Leu Glu Phe Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys 100 105 110 Leu Pro Glu Glu Asn Glin Val Phe Pro Pro Gly Arg Asn Cys Ser Ile 115 120 125 Ala Gly Trp Gly Thr Val Val Tyr Glin Gly. Thir Thr Ala Asn Ile Leu 130 135 1 4 0 Glin Glu Ala Asp Val Pro Leu Lleu Ser Asn. Glu Arg Cys Glin Glin Glin 145 15 O 155 160 Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu 1.65 170 175 Glu Gly Gly Ile Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Leu Met 18O 185 19 O Cys Glin Glu Asn Asn Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly 195 200 2O5 Tyr Lys Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Ser 210 215 220 Arg Phe Thr Glu Trp Ile Glin Ser Phe Leu. His 225 230 235 US 2005/0175581 A1 Aug. 11, 2005 52

-continued

<210 SEQ ID NO 7 &2 11s LENGTH 2.75 &212> TYPE PRT <213> ORGANISM: Bacillus subtilis

<400 SEQUENCE: 7 Ile Ala His Glu Tyr Ala Glin Ser Val Pro Tyr Gly Ile Ser Glin Ile 1 5 10 15 Lys Ala Pro Ala Lieu. His Ser Glin Gly Tyr Thr Gly Ser Asn. Wall Lys 2O 25 30 Val Ala Val Ile Asp Ser Gly Ile Asp Ser Ser His Pro Asp Lieu. Asn 35 40 45 Val Arg Gly Gly Ala Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Glin 50 55 60 Asp Gly Ser Ser His Gly Thr His Val Ala Gly. Thir Ile Ala Ala Leu 65 70 75 8O Asn Asn Ser Ile Gly Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr 85 90 95 Ala Val Lys Val Leu Asp Ser Thr Gly Ser Gly Glin Tyr Ser Trp Ile 100 105 110 Ile Asin Gly Ile Glu Trp Ala Ile Ser Asn. Asn Met Asp Val Ile Asn 115 120 125 Met Ser Leu Gly Gly Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val 130 135 1 4 0 Asp Lys Ala Val Ser Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn 145 15 O 155 160 Glu Gly Ser Ser Gly Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr 1.65 170 175 Pro Ser Thr Ile Ala Val Gly Ala Val Asn Ser Ser Asn Glin Arg Ala 18O 185 19 O Ser Phe Ser Ser Ala Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val 195 200 2O5 Ser Ile Glin Ser Thr Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly 210 215 220 Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu 225 230 235 240 Ser Lys His Pro Thr Trp Thr Asn Ala Glin Val Arg Asp Arg Lieu Glu 245 250 255 Ser Thr Ala Thr Tyr Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu 260 265 27 O

Ile Asn. Wall 275

<210 SEQ ID NO 8 &2 11s LENGTH 320 &212> TYPE PRT <213> ORGANISM: Murinae gen. sp. <400 SEQUENCE: 8 Val Ala Lys Arg Arg Ala Lys Arg Asp Val Tyr Glin Glu Pro Thr Asp 1 5 10 15 Pro Llys Phe Pro Gln Gln Trp Tyr Leu Ser Gly Val Thr Glin Arg Asp 2O 25 30 US 2005/0175581 A1 Aug. 11, 2005 53

-continued

Lieu. Asn. Wall Lys Glu Ala Trp Ala Glin Gly Phe Thr Gly His Gly Ile 35 40 45 Val Val Ser Ile Leu Asp Asp Gly Ile Glu Lys Asn His Pro Asp Lieu 50 55 60 Ala Gly Asn Tyr Asp Pro Gly Ala Ser Phe Asp Wall Asn Asp Glin Asp 65 70 75 8O Pro Asp Pro Glin Pro Arg Tyr Thr Gln Met Asn Asp Asn Arg His Gly 85 90 95 Thr Arg Cys Ala Gly Glu Val Ala Ala Wall Ala Asn. Asn Gly Val Cys 100 105 110 Gly Val Gly Val Ala Tyr Asn Ala Arg Ile Gly Gly Val Arg Met Lieu 115 120 125 Asp Gly Glu Val Thr Asp Ala Val Glu Ala Arg Ser Leu Gly Lieu. Asn 130 135 1 4 0 Pro Asn His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro Glu Asp Asp 145 15 O 155 160 Gly Lys Thr Val Asp Gly Pro Ala Arg Lieu Ala Glu Glu Ala Phe Phe 1.65 170 175 Arg Gly Val Ser Glin Gly Arg Gly Gly Lieu Gly Ser Ile Phe Val Trp 18O 185 19 O Ala Ser Gly Asn Gly Gly Arg Glu His Asp Ser Cys Asn. Cys Asp Gly 195 200 2O5 Tyr Thr Asn Ser Ile Tyr Thr Leu Ser Ile Ser Ser Ala Thr Glin Phe 210 215 220 Gly Asin Val Pro Trp Tyr Ser Glu Ala Cys Ser Ser Thr Leu Ala Thr 225 230 235 240 Thr Tyr Ser Ser Gly Asn Gln Asn Glu Lys Glin Ile Val Thr Thr Asp 245 250 255 Leu Arg Gln Lys Cys Thr Glu Ser His Thr Gly Thr Ser Ala Ser Ala 260 265 27 O Pro Leu Ala Ala Gly Ile Ile Ala Lieu. Thir Lieu Glu Ala Asn Lys Asn 275 280 285 Leu Thir Trp Arg Asp Met Gln His Leu Val Val Glin Thr Ser Lys Pro 29 O 295 3OO Ala His Lieu. Asn Ala Asp Asp Trp Ala Thr Asn Gly Val Gly Arg Lys 305 310 315 320

<210 SEQ ID NO 9 &2 11s LENGTH 330 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 9 Glu Lys Glu Arg Ser Lys Arg Ser Ala Lieu Arg Asp Ser Ala Lieu. Asn 1 5 10 15 Leu Phe Asin Asp Pro Met Trp Asin Glin Glin Trp Tyr Leu Glin Asp Thr 2O 25 30 Arg Met Thr Ala Ala Lieu Pro Llys Lieu. Asp Lieu. His Val Ile Pro Val 35 40 45 Trp Gln Lys Gly Ile Thr Gly Lys Gly Val Val Ile Thr Val Leu Asp 50 55 60 Asp Gly Lieu Glu Trp Asn His Thr Asp Ile Tyr Ala Asn Tyr Asp Pro US 2005/0175581 A1 Aug. 11, 2005 54

-continued

65 70 75 8O Glu Ala Ser Tyr Asp Phe Asn Asp Asn Asp His Asp Pro Phe Pro Arg 85 90 95 Tyr Asp Pro Thr Asn. Glu Asn Lys His Gly Thr Arg Cys Ala Gly Glu 100 105 110 Ile Ala Met Glin Ala Asn. Asn His Lys Cys Gly Val Gly Val Ala Tyr 115 120 125 Asn Ser Lys Val Gly Gly Ile Arg Met Lieu. Asp Gly Ile Val Thr Asp 130 135 1 4 0 Ala Ile Glu Ala Ser Ser Ile Gly Phe Asin Pro Gly. His Val Asp Ile 145 15 O 155 160 Tyr Ser Ala Ser Trp Gly Pro Asn Asp Asp Gly Lys Thr Val Glu Gly 1.65 170 175 Pro Gly Arg Lieu Ala Glin Lys Ala Phe Glu Tyr Gly Wall Lys Glin Gly 18O 185 19 O Arg Glin Gly Lys Gly Ser Ile Phe Val Trp Ala Ser Gly Asn Gly Gly 195 200 2O5 Arg Glin Gly Asp Asn. Cys Asp Cys Asp Gly Tyr Thr Asp Ser Ile Tyr 210 215 220 Thir Ile Ser Ile Ser Ser Ala Ser Glin Glin Gly Leu Ser Pro Trp Tyr 225 230 235 240 Ala Glu Lys Cys Ser Ser Thr Leu Ala Thr Ser Tyr Ser Ser Gly Asp 245 250 255 Tyr Thr Asp Glin Arg Ile Thr Ser Ala Asp Lieu. His Asn Asp Cys Thr 260 265 27 O Glu Thr His Thr Gly Thr Ser Ala Ser Ala Pro Leu Ala Ala Gly Ile 275 280 285 Phe Ala Lieu Ala Leu Glu Ala Asn Pro Asn Lieu. Thir Trp Arg Asp Met 29 O 295 3OO Gln His Leu Val Val Trp Thir Ser Glu Tyr Asp Pro Leu Ala Asn Asn 305 310 315 320 Pro Gly Trp Llys Lys Asn Gly Ala Gly Lieu 325 330

<210> SEQ ID NO 10 &2 11s LENGTH 297 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 10 Asn Thr His Pro Cys Glin Ser Asp Met Asn. Ile Glu Gly Ala Trp Lys 1 5 10 15 Arg Gly Tyr Thr Gly Lys Asn. Ile Val Val Thr Ile Leu Asp Asp Gly 2O 25 30 Ile Glu Arg Thr His Pro Asp Leu Met Glin Asn Tyr Asp Ala Lieu Ala 35 40 45 Ser Cys Asp Wall Asn Gly Asn Asp Lieu. Asp Pro Met Pro Arg Tyr Asp 50 55 60 Ala Ser Asn. Glu Asn Lys His Gly. Thir Arg Cys Ala Gly Glu Val Ala 65 70 75 8O Ala Ala Ala Asn. Asn. Ser His Cys Thr Val Gly Ile Ala Phe Asn Ala 85 90 95 US 2005/0175581 A1 Aug. 11, 2005 55

-continued Lys Ile Gly Gly Val Arg Met Lieu. Asp Gly Asp Val Thr Asp Met Val 100 105 110 Glu Ala Lys Ser Val Ser Phe Asin Pro Gln His Val His Ile Tyr Ser 115 120 125 Ala Ser Trp Gly Pro Asp Asp Asp Gly Lys Thr Val Asp Gly Pro Ala 130 135 1 4 0 Pro Leu Thir Arg Glin Ala Phe Glu Asn Gly Val Arg Met Gly Arg Arg 145 15 O 155 160 Gly Lieu Gly Ser Val Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Ser 1.65 170 175 Lys Asp His Cys Ser Cys Asp Gly Tyr Thr Asn. Ser Ile Tyr Thr Ile 18O 185 19 O Ser Ile Ser Ser Thr Ala Glu Ser Gly Lys Llys Pro Trp Tyr Leu Glu 195 200 2O5 Glu Cys Ser Ser Thr Leu Ala Thr Thr Tyr Ser Ser Gly Glu Ser Tyr 210 215 220 Asp Llys Lys Ile Ile Thir Thr Asp Leu Arg Glin Arg Cys Thr Asp Asn 225 230 235 240 His Thr Gly Thr Ser Ala Ser Ala Pro Met Ala Ala Gly Ile Ile Ala 245 250 255 Leu Ala Lieu Glu Ala Asn Pro Phe Lieu. Thir Trp Arg Asp Val Glin His 260 265 27 O Val Ile Val Arg Thr Ser Arg Ala Gly His Lieu. Asn Ala Asn Asp Trip 275 280 285 Lys Thr Asn Ala Ala Gly Phe Lys Val 29 O 295

<210> SEQ ID NO 11 &2 11s LENGTH 328 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 11 Thr Lieu Val Asp Glu Gln Pro Leu Glu Asn Tyr Lieu. Asp Met Glu Tyr 1 5 10 15 Phe Gly. Thir Ile Gly Ile Gly Thr Pro Ala Glin Asp Phe Thr Val Val 2O 25 30 Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Val Tyr Cys Ser 35 40 45 Ser Lieu Ala Cys Thr Asn His Asn Arg Phe Asin Pro Glu Asp Ser Ser 50 55 60 Thr Tyr Glin Ser Thr Ser Glu Thr Val Ser Ile Thr Tyr Gly Thr Gly 65 70 75 8O Ser Met Thr Gly Ile Leu Gly Tyr Asp Thr Val Glin Val Gly Gly Ile 85 90 95 Ser Asp Thr Asn Glin Ile Phe Gly Leu Ser Glu Thr Glu Pro Gly Ser 100 105 110 Phe Leu Tyr Tyr Ala Pro Phe Asp Gly Ile Leu Gly Leu Ala Tyr Pro 115 120 125 Ser Ile Ser Ser Ser Gly Ala Thr Pro Val Phe Asp Asn Ile Trp Asn 130 135 1 4 0 Glin Gly Lieu Val Ser Glin Asp Leu Phe Ser Val Tyr Lieu Ser Ala Asp 145 15 O 155 160 US 2005/0175581 A1 Aug. 11, 2005 56

-continued

Asp Llys Ser Gly Ser Val Val Ile Phe Gly Gly Ile Asp Ser Ser Tyr 1.65 170 175 Tyr Thr Gly Ser Leu Asn Trp Val Pro Val Thr Val Glu Gly Tyr Trp 18O 185 19 O Glin Ile Thr Val Asp Ser Ile Thr Met Asin Gly Glu Thir Ile Ala Cys 195 200 2O5 Ala Glu Gly Cys Glin Ala Ile Val Asp Thr Gly. Thir Ser Leu Leu Thr 210 215 220 Gly Pro Thr Ser Pro Ile Ala Asn Ile Glin Ser Asp Ile Gly Ala Ser 225 230 235 240 Glu Asn. Ser Asp Gly Asp Met Val Val Ser Cys Ser Ala Ile Ser Ser 245 250 255 Leu Pro Asp Ile Val Phe Thr Ile Asin Gly Val Glin Tyr Pro Val Pro 260 265 27 O Pro Ser Ala Tyr Ile Leu Gln Ser Glu Gly Ser Cys Ile Ser Gly Phe 275 280 285 Gln Gly Met Asn Val Pro Thr Glu Ser Gly Glu Leu Trp Ile Leu Gly 29 O 295 3OO Asp Val Phe Ile Arg Glin Tyr Phe Thr Val Phe Asp Arg Ala Asn Asn 305 310 315 320 Glin Val Gly Leu Ala Pro Val Ala 325

<210> SEQ ID NO 12 &2 11s LENGTH 358 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 12 Glu Met Val Asp Asn Lieu Arg Gly Lys Ser Gly Glin Gly Tyr Tyr Val 1 5 10 15 Glu Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp 2O 25 30 Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu 35 40 45 His Arg Tyr Tyr Glin Arg Glin Leu Ser Ser Thr Tyr Arg Asp Leu Arg 50 55 60 Lys Gly Val Tyr Val Pro Tyr Thr Glin Gly Lys Trp Glu Gly Glu Leu 65 70 75 8O Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg 85 90 95 Ala Asn. Ile Ala Ala Ile Thr Glu Ser Asp Llys Phe Phe Ile Asn Gly 100 105 110 Ser Asn Trp Glu Gly Ile Leu Gly Lieu Ala Tyr Ala Glu Ile Ala Arg 115 120 125 Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Lieu Val Lys Glin Thr 130 135 1 4 0 His Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro 145 15 O 155 160 Leu Asin Glin Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile 1.65 170 175 Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro US 2005/0175581 A1 Aug. 11, 2005 57

-continued

18O 185 19 O

Ile Arg Arg Glu Trp Tyr Tyr Glu Wall Ile Ile Wall Arg Wall Glu Ile 195 200 2O5

Asn Gly Glin Asp Teu Lys Met Asp Cys Glu Tyr Asn Asp 210 215 220

Ser Ile Wall Asp Ser Gly Thr Thr Asn Telu Arg Teu Pro Wall 225 230 235 240

Phe Glu Ala Ala Wall Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu 245 250 255

Phe Pro Asp Gly Phe Trp Teu Gly Glu Glin Teu Wall Trp Glin Ala 260 265 27 O

Gly Thr Thr Pro Trp Asn Ile Phe Pro Wall Ile Ser Teu Telu Met 275 280 285

Gly Glu Wall Thr Asn Glin Ser Phe Ile Thr Ile Teu Pro Glin Glin 29 O 295

Tyr Telu Arg Pro Wall Glu Asp Wall Ala Thr Ser Glin Asp Asp Tyr 305 310 315 320

Phe Ala Ile Ser Glin Ser Ser Thr Gly Thr Wall Met Gly Ala Wall 325 330 335

Ile Met Glu Gly Phe Tyr Wall Wall Phe Asp Arg Ala Arg Lys Arg Ile 340 345 35 O

Gly Phe Ala Wall Ser Ala 355

SEQ ID NO 13 LENGTH 351 TYPE PRT ORGANISM: Homo sapiens <400 SEQUENCE: 13

Pro Ala Wall Thr Glu Gly Pro Ile Pro Glu Wall Teu Asn Tyr Met 1 5 10 15

Asp Ala Glin Tyr Tyr Gly Glu Ile Gly Ile Gly Thr Pro Pro Glin 25 30

Phe Thr Wall Wall Phe Asp Thr Gly Ser Ser Asn Teu Trp Wall Pro Ser 35 40 45

Ile His Lys Teu Teu Asp Ile Ala Trp Ile His His 50 55 60

Asn Ser Lys Ser Ser Thr Tyr Wall Asn Gly Thr Ser Phe Asp 65 70 75 8O

Ile His Gly Ser Gly Ser Telu Ser Gly Tyr Teu Ser Glin Asp Thr 85 90 95

Wall Ser Wall Pro Cys Glin Ser Ala Ser Ser Ala Ser Ala Telu Gly Gly 100 105 110

Wall Wall Glu Glin Wall Phe Gly Glu Ala Thr Lys Glin Pro Gly 115 120 125

Ile Thr Phe Ile Ala Ala Lys Phe Gly Ile Teu Gly Met Ala 130 135 1 4 0

Pro Arg Ile Ser Wall Asn Asn Wall Telu Pro Wall Phe Asp Asn Telu Met 145 15 O 155 160

Glin Glin Lys Telu Wall Asp Glin Asn Ile Phe Ser Phe Telu Ser Arg 1.65 170 175 US 2005/0175581 A1 Aug. 11, 2005 58

-continued Asp Pro Asp Ala Glin Pro Gly Gly Glu Lieu Met Leu Gly Gly. Thir Asp 18O 185 19 O Ser Lys Tyr Tyr Lys Gly Ser Lieu Ser Tyr Lieu. Asn Val Thr Arg Lys 195 200 2O5 Ala Tyr Trp Glin Val His Leu Asp Glin Val Glu Val Ala Ser Gly Lieu 210 215 220 Thr Lieu. Cys Lys Glu Gly Cys Glu Ala Ile Val Asp Thr Gly Thr Ser 225 230 235 240 Leu Met Val Gly Pro Val Asp Glu Val Arg Glu Lieu Gln Lys Ala Ile 245 250 255 Gly Ala Val Pro Leu Ile Glin Gly Glu Tyr Met Ile Pro Cys Glu Lys 260 265 27 O Val Ser Thr Lieu Pro Ala Ile Thr Lieu Lys Lieu Gly Gly Lys Gly Tyr 275 280 285 Lys Lieu Ser Pro Glu Asp Tyr Thr Lieu Lys Val Ser Glin Ala Gly Lys 29 O 295 3OO Thr Leu Cys Leu Ser Gly Phe Met Gly Met Asp Ile Pro Pro Pro Ser 305 310 315 320 Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Ile Gly Arg Tyr Tyr Thr 325 330 335 Val Phe Asp Arg Asp Asn. Asn Arg Val Gly Phe Ala Glu Ala Ala 340 345 35 O

<210> SEQ ID NO 14 &2 11s LENGTH 305 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 14 Met Leu Glu Ala Asp Asp Glin Gly Cys Ile Glu Glu Glin Gly Val Glu 1 5 10 15 Asp Ser Ala Asn. Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser 2O 25 30 Phe Val Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg 35 40 45 Ser Ile Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Glin Tyr Asn Met 50 55 60 Asn Phe Glu Lys Lieu Gly Lys Cys Ile Ile Ile Asn. Asn Lys Asn. Phe 65 70 75 8O Asp Llys Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala 85 90 95 Glu Ala Lieu Phe Lys Cys Phe Arg Ser Lieu Gly Phe Asp Val Ile Val 100 105 110 Tyr Asn Asp Cys Ser Cys Ala Lys Met Glin Asp Leu Lleu Lys Lys Ala 115 120 125 Ser Glu Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Lieu 130 135 1 4 0 Ser His Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro 145 15 O 155 160 Ile Lys Asp Lieu. Thir Ala His Phe Arg Gly Asp Arg Ser Lys Thr Lieu 1.65 170 175 Leu Glu Lys Pro Lys Lieu Phe Phe Ile Glin Ala Cys Arg Gly Thr Glu 18O 185 19 O US 2005/0175581 A1 Aug. 11, 2005 59

-continued

Leu Asp Asp Gly Ile Glin Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp 195 200 2O5 Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala 210 215 220 Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly 225 230 235 240 Ser Trp Phe Val Glin Ala Lieu. Cys Ser Ile Leu Glu Glu His Gly Lys 245 250 255 Asp Leu Glu Ile Met Glin Ile Lieu. Thir Arg Val Asn Asp Arg Val Ala 260 265 27 O Arg His Phe Glu Ser Glin Ser Asp Asp Pro His Phe His Glu Lys Lys 275 280 285 Glin Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser 29 O 295 3OO

Glin 305

<210 SEQ ID NO 15 <211& LENGTH 262 &212> TYPE PRT <213> ORGANISM: Streptomyces sp. K15 <400 SEQUENCE: 15 Val Thr Lys Pro Thr Ile Ala Ala Val Gly Gly Tyr Ala Met Asn Asn 1 5 10 15 Gly Thr Gly Thr Thr Leu Tyr Thr Lys Ala Ala Asp Thr Arg Arg Ser 2O 25 30 Thr Gly Ser Thr Thr Lys Ile Met Thr Ala Lys Val Val Leu Ala Glin 35 40 45 Ser Asn Lieu. Asn Lieu. Asp Ala Lys Val Thir Ile Glin Lys Ala Tyr Ser 50 55 60 Asp Tyr Val Val Ala Asn. Asn Ala Ser Glin Ala His Lieu. Ile Val Gly 65 70 75 8O Asp Llys Val Thr Val Arg Glin Leu Lleu Tyr Gly Lieu Met Leu Pro Ser 85 90 95 Gly Cys Asp Ala Ala Tyr Ala Lieu Ala Asp Llys Tyr Gly Ser Gly Ser 100 105 110 Thr Arg Ala Ala Arg Val Lys Ser Phe Ile Gly Lys Met Asn. Thir Ala 115 120 125 Ala Thr Asn Lieu Gly Lieu. His Asn. Thir His Phe Asp Ser Phe Asp Gly 130 135 1 4 0 Ile Gly Asn Gly Ala Asn Tyr Ser Thr Pro Arg Asp Lieu. Thir Lys Ile 145 15 O 155 160 Ala Ser Ser Ala Met Lys Asn Ser Thr Phe Arg Thr Val Val Lys Thr 1.65 170 175 Lys Ala Tyr Thr Ala Lys Thr Val Thr Lys Thr Gly Ser Ile Arg Thr 18O 185 19 O Met Asp Thir Trp Lys Asn Thr Asn Gly Lieu Lleu Ser Ser Tyr Ser Gly 195 200 2O5 Ala Ile Gly Val Lys Thr Gly Ser Gly Pro Glu Ala Lys Tyr Cys Lieu 210 215 220 Val Phe Ala Ala Thr Arg Gly Gly Lys Thr Val Ile Gly Thr Val Leu US 2005/0175581 A1 Aug. 11, 2005 60

-continued

225 230 235 240 Ala Ser Thr Ser Ile Pro Ala Arg Glu Ser Asp Ala Thr Lys Ile Met 245 250 255 Asn Tyr Gly Phe Ala Lieu 260

<210> SEQ ID NO 16 &2 11s LENGTH 256 &212> TYPE PRT <213> ORGANISM: Human cytomegalovirus <400 SEQUENCE: 16 Met Thr Met Asp Glu Gln Gln Ser Glin Ala Val Ala Pro Val Tyr Val 1 5 10 15 Gly Gly Phe Leu Ala Arg Tyr Asp Glin Ser Pro Asp Glu Ala Glu Lieu 2O 25 30 Leu Lleu Pro Arg Asp Val Val Glu His Trp Lieu. His Ala Glin Gly Glin 35 40 45 Gly Glin Pro Ser Leu Ser Val Ala Lieu Pro Leu Asn. Ile Asn His Asp 50 55 60 Asp Thr Ala Val Val Gly His Val Ala Ala Met Gln Ser Val Arg Asp 65 70 75 8O Gly Leu Phe Cys Leu Gly Cys Val Thr Ser Pro Arg Phe Leu Glu Ile 85 90 95 Val Arg Arg Ala Ser Glu Lys Ser Glu Lieu Val Ser Arg Gly Pro Val 100 105 110 Ser Pro Leu Gln Pro Asp Lys Val Val Glu Phe Leu Ser Gly Ser Tyr 115 120 125 Ala Gly Lieu Ser Leu Ser Ser Arg Arg Cys Asp Asp Val Glu Glin Ala 130 135 1 4 0 Thr Ser Leu Ser Gly Ser Glu Thir Thr Pro Phe Lys His Val Ala Leu 145 15 O 155 160 Cys Ser Val Gly Arg Arg Arg Gly Thr Lieu Ala Val Tyr Gly Arg Asp 1.65 170 175 Pro Glu Trp Val Thr Glin Arg Phe Pro Asp Leu Thr Ala Ala Asp Arg 18O 185 19 O Asp Gly Lieu Arg Ala Glin Trp Glin Arg Cys Gly Ser Thr Ala Val Asp 195 200 2O5 Ala Ser Gly Asp Pro Phe Arg Ser Asp Ser Tyr Gly Lieu Lieu Gly Asn 210 215 220 Ser Val Asp Ala Leu Tyr Ile Arg Glu Arg Lieu Pro Lys Lieu Arg Tyr 225 230 235 240 Asp Lys Glin Leu Val Gly Val Thr Glu Arg Glu Ser Tyr Val Lys Ala 245 250 255

<210 SEQ ID NO 17 <211& LENGTH 248 &212> TYPE PRT <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 17 Val Arg Ser Phe Ile Tyr Glu Pro Phe Glin Ile Pro Ser Gly Ser Met 1 5 10 15 Met Pro Thr Leu Leu Ile Gly Asp Phe Ile Leu Val Glu Lys Phe Ala US 2005/0175581 A1 Aug. 11, 2005 61

-continued

2O 25 30 Tyr Gly Ile Lys Asp Pro Ile Tyr Gln Lys Thr Leu Ile Glu Thr Gly 35 40 45 His Pro Lys Arg Gly Asp Ile Val Val Phe Lys Tyr Pro Glu Asp Pro 50 55 60 Lys Lieu. Asp Tyr Ile Lys Arg Ala Val Gly Lieu Pro Gly Asp Llys Val 65 70 75 8O Thr Tyr Asp Pro Val Ser Lys Glu Leu Thir Ile Gln Pro Gly Cys Ser 85 90 95 Ser Gly Glin Ala Cys Glu Asn Ala Leu Pro Val Thr Tyr Ser Asn Val 100 105 110 Glu Pro Ser Asp Phe Val Glin Thr Phe Ser Arg Arg Asn Gly Gly Glu 115 120 125 Ala Thr Ser Gly Phe Phe Glu Val Pro Lys Asn Glu Thir Lys Glu Asn 130 135 1 4 0 Gly Ile Arg Lieu Ser Glu Arg Lys Glu Thir Lieu Gly Asp Val Thr His 145 15 O 155 160 Arg Ile Leu Thr Val Pro Ile Ala Glin Asp Glin Val Gly Met Tyr Tyr 1.65 170 175 Gln Glin Pro Gly Glin Gln Leu Ala Thr Trp Ile Val Pro Pro Gly Glin 18O 185 19 O Tyr Phe Met Met Gly Asp Asn Arg Asp Asn Ser Ala Asp Ser Arg Tyr 195 200 2O5 Trp Gly Phe Val Pro Glu Ala Asn Leu Val Gly Arg Ala Thr Ala Ile 210 215 220 Trp Met Ser Phe Asp Lys Glin Glu Gly Glu Trp Pro Thr Gly Leu Arg 225 230 235 240 Leu Ser Arg Ile Gly Gly Ile His 245

<210> SEQ ID NO 18 &2 11s LENGTH 317 &212> TYPE PRT <213> ORGANISM: Serratia marce scens

<400 SEQUENCE: 18 Met Glu Gln Leu Arg Gly Lieu. Tyr Pro Pro Leu Ala Ala Tyr Asp Ser 1 5 10 15 Gly Trp Lieu. Asp Thr Gly Asp Gly His Arg Ile Tyr Trp Glu Lieu Ser 2O 25 30 Gly Asn Pro Asn Gly Lys Pro Ala Val Phe Ile His Gly Gly Pro Gly 35 40 45 Gly Gly Ile Ser Pro His His Arg Glin Leu Phe Asp Pro Glu Arg Tyr 50 55 60 Lys Val Lieu Lleu Phe Asp Glin Arg Gly Cys Gly Arg Ser Arg Pro His 65 70 75 8O Ala Ser Lieu. Asp Asn. Asn. Thir Thr Trp His Lieu Val Ala Asp Ile Glu 85 90 95 Arg Lieu Arg Glu Met Ala Gly Val Glu Gln Trp Lieu Val Phe Gly Gly 100 105 110 Ser Trp Gly Ser Thr Leu Ala Leu Ala Tyr Ala Gln Thr His Pro Glu 115 120 125 US 2005/0175581 A1 Aug. 11, 2005 62

-continued Arg Val Ser Glu Met Val Lieu Arg Gly Ile Phe Thr Lieu Arg Lys Glin 130 135 1 4 0 Arg Leu. His Trp Tyr Tyr Glin Asp Gly Ala Ser Arg Phe Phe Pro Glu 145 15 O 155 160 Lys Trp Glu Arg Val Lieu Ser Ile Leu Ser Asp Asp Glu Arg Lys Asp 1.65 170 175 Val Ile Ala Ala Tyr Arg Glin Arg Lieu. Thir Ser Ala Asp Pro Glin Val 18O 185 19 O Glin Leu Glu Ala Ala Lys Lieu Trp Ser Val Trp Glu Gly Glu Thr Val 195 200 2O5 Thr Lieu Lieu Pro Ser Arg Glu Ser Ala Ser Phe Gly Glu Asp Asp Phe 210 215 220 Ala Lieu Ala Phe Ala Arg Ile Glu Asn His Tyr Phe Thr His Leu Gly 225 230 235 240 Phe Leu Glu Ser Asp Asp Gln Leu Lieu Arg Asn Val Pro Lieu. Ile Arg 245 250 255 His Ile Pro Ala Val Ile Val His Gly Arg Tyr Asp Met Ala Cys Glin 260 265 27 O Val Glin Asn Ala Trp Asp Leu Ala Lys Ala Trp Pro Glu Ala Glu Lieu 275 280 285 His Ile Val Glu Gly Ala Gly His Ser Tyr Asp Glu Pro Gly Ile Leu 29 O 295 3OO His Glin Leu Met Ile Ala Thr Asp Arg Phe Ala Gly Lys 305 310 315

<210 SEQ ID NO 19 &2 11s LENGTH 229 &212> TYPE PRT <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 19 Met Glu Lieu Lleu Lleu Lleu Ser Asn. Ser Thr Lieu Pro Gly Lys Ala Trp 1 5 10 15 Leu Glu His Ala Leu Pro Leu. Ile Ala Asn Glin Lieu. Asn Gly Arg Arg 2O 25 30 Ser Ala Val Phe Ile Pro Phe Ala Gly Val Thr Gln Thr Trp Asp Glu 35 40 45 Tyr Thr Asp Llys Thr Ala Glu Val Leu Ala Pro Leu Gly Val Asin Val 50 55 60 Thr Gly Ile His Arg Val Ala Asp Pro Leu Ala Ala Ile Glu Lys Ala 65 70 75 8O Glu Ile Ile Ile Val Gly Gly Gly Asn. Thir Phe Glin Lieu Lleu Lys Glu 85 90 95 Ser Arg Glu Arg Gly Lieu Lleu Ala Pro Met Ala Asp Arg Wall Lys Arg 100 105 110 Gly Ala Leu Tyr Ile Gly Trp Ser Ala Gly Ala Asn Lieu Ala Cys Pro 115 120 125 Thir Ile Arg Thr Thr Asn Asp Met Pro Ile Val Asp Pro Asn Gly Phe 130 135 1 4 0 Asp Ala Lieu. Asp Leu Phe Pro Leu Glin Ile Asn Pro His Phe Thr Asn 145 15 O 155 160 Ala Lieu Pro Glu Gly. His Lys Gly Glu Thir Arg Glu Glin Arg Ile Arg 1.65 170 175 US 2005/0175581 A1 Aug. 11, 2005 63

-continued

Glu Leu Leu Val Val Ala Pro Glu Leu Thr Val Ile Gly Leu Pro Glu 18O 185 19 O Gly Asn Trp Ile Glin Val Ser Asn Gly Glin Ala Val Lieu Gly Gly Pro 195 200 2O5 Asn Thr Thr Trp Val Phe Lys Ala Gly Glu Glu Ala Val Ala Leu Glu 210 215 220 Ala Gly His Arg Phe 225

<210> SEQ ID NO 20 &2 11s LENGTH 99 &212> TYPE PRT <213> ORGANISM: Human immunodeficiency virus <400 SEQUENCE: 20 Pro Glin Ile Thr Leu Trp Glin Arg Pro Leu Val Thr Val Lys Ile Gly 1 5 10 15 Gly Glin Leu Arg Glu Ala Leu Lieu. Asp Thr Gly Ala Asp Asp Thr Val 2O 25 30 Leu Glu Asp Ile Asn Lieu Pro Gly Lys Trp Llys Pro Lys Met Ile Gly 35 40 45 Gly Ile Gly Gly Phe Ile Lys Val Arg Glin Tyr Asp Glin Ile Lieu. Ile 50 55 60 Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr 65 70 75 8O Pro Val Asin Ile Ile Gly Arg Asn Met Leu Thr Glin Ile Gly Cys Thr 85 90 95

Lieu. Asn. Phe

<210> SEQ ID NO 21 &2 11s LENGTH 297 &212> TYPE PRT <213> ORGANISM: Escherichia coli

<400 SEQUENCE: 21 Ser Thr Glu Thr Leu Ser Phe Thr Pro Asp Asn Ile Asn Ala Asp Ile 1 5 10 15 Ser Lieu Gly. Thir Lieu Ser Gly Lys Thr Lys Glu Arg Val Tyr Lieu Ala 2O 25 30 Glu Glu Gly Gly Arg Lys Wal Ser Glin Lieu. Asp Trp Llys Phe Asn. Asn 35 40 45 Ala Ala Ile Ile Lys Gly Ala Ile Asn Trp Asp Leu Met Pro Glin Ile 50 55 60 Ser Ile Gly Ala Ala Gly Trp Thir Thr Lieu Gly Ser Arg Gly Gly Asn 65 70 75 8O Met Val Asp Glin Asp Trp Met Asp Ser Ser Asn Pro Gly Thr Trp Thr 85 90 95 Asp Glu Ala Arg His Pro Asp Thr Glin Lieu. Asn Tyr Ala Asn. Glu Phe 100 105 110 Asp Lieu. Asn. Ile Lys Gly Trp Lieu Lieu. Asn. Glu Pro Asn Tyr Arg Lieu 115 120 125 Gly Leu Met Ala Gly Tyr Glin Glu Ser Arg Tyr Ser Phe Thr Ala Arg 130 135 1 4 0 US 2005/0175581 A1 Aug. 11, 2005 64

-continued Gly Gly Ser Tyr Ile Tyr Ser Ser Glu Glu Gly Phe Arg Asp Asp Ile 145 15 O 155 160 Gly Ser Phe Pro Asn Gly Glu Arg Ala Ile Gly Tyr Lys Glin Arg Phe 1.65 170 175 Lys Met Pro Tyr Ile Gly Leu Thr Gly Ser Tyr Arg Tyr Glu Asp Phe 18O 185 19 O Glu Leu Gly Gly Thr Phe Lys Tyr Ser Gly Trp Val Glu Ser Ser Asp 195 200 2O5 Asn Asp Glu. His Tyr Asp Pro Lys Gly Arg Ile Thr Tyr Arg Ser Lys 210 215 220 Val Lys Asp Glin Asn Tyr Tyr Ser Val Ala Val Asn Ala Gly Tyr Tyr 225 230 235 240 Val Thr Pro Asn Ala Lys Val Tyr Val Glu Gly Ala Trp Asn Arg Val 245 250 255 Thr Asn Lys Lys Gly Asn. Thir Ser Leu Tyr Asp His Asn. Asn. Asn Thr 260 265 27 O Ser Asp Tyr Ser Lys Asn Gly Ala Gly Ile Glu Asn Tyr Asn. Phe Ile 275 280 285 Thir Thr Ala Gly Leu Lys Tyr Thr Phe 29 O 295

<210> SEQ ID NO 22 <211& LENGTH 212 &212> TYPE PRT <213> ORGANISM: Carica papaya <400 SEQUENCE: 22 Ile Pro Glu Tyr Val Asp Trp Arg Glin Lys Gly Ala Val Thr Pro Val 1 5 10 15 Lys Asn Glin Gly Ser Cys Gly Ser Cys Trp Ala Phe Ser Ala Val Val 2O 25 30 Thir Ile Glu Gly Ile Ile Lys Ile Arg Thr Gly Asn Lieu. Asn Glin Tyr 35 40 45 Ser Glu Glin Glu Lieu Lleu. Asp Cys Asp Arg Arg Ser Tyr Gly Cys Asn 50 55 60 Gly Gly Tyr Pro Trp Ser Ala Leu Gln Leu Val Ala Glin Tyr Gly Ile 65 70 75 8O His Tyr Arg Asn Thr Tyr Pro Tyr Glu Gly Val Glin Arg Tyr Cys Arg 85 90 95 Ser Arg Glu Lys Gly Pro Tyr Ala Ala Lys Thr Asp Gly Val Arg Glin 100 105 110 Val Glin Pro Tyr Asn Glin Gly Ala Lieu Lleu Tyr Ser Ile Ala Asn Glin 115 120 125 Pro Wal Ser Val Val Lieu Glin Ala Ala Gly Lys Asp Phe Glin Leu Tyr 130 135 1 4 0 Arg Gly Gly Ile Phe Val Gly Pro Cys Gly Asn Lys Wall Asp His Ala 145 15 O 155 160 Val Ala Ala Val Gly Tyr Gly Pro Asn Tyr Ile Lieu. Ile Lys Asn. Ser 1.65 170 175 Trp Gly Thr Gly Trp Gly Glu Asn Gly Tyr Ile Arg Ile Lys Arg Gly 18O 185 19 O Thr Gly Asn Ser Tyr Gly Val Cys Gly Leu Tyr Thr Ser Ser Phe Tyr 195 200 2O5 US 2005/0175581 A1 Aug. 11, 2005 65

-continued

Pro Wall Lys Asn 210

<210> SEQ ID NO 23 &2 11s LENGTH 699 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 23 Ala Gly Ile Ala Ala Lys Lieu Ala Lys Asp Arg Glu Ala Ala Glu Gly 1 5 10 15 Leu Gly Ser His Glu Arg Ala Ile Lys Tyr Lieu. Asn Glin Asp Tyr Glu 2O 25 30 Ala Lieu Arg Asn. Glu Cys Lieu Glu Ala Gly Thr Lieu Phe Glin Asp Pro 35 40 45 Ser Phe Pro Ala Ile Pro Ser Ala Leu Gly Phe Lys Glu Leu Gly Pro 50 55 60 Tyr Ser Ser Lys Thr Arg Gly Met Arg Trp Lys Arg Pro Thr Glu Ile 65 70 75 8O Cys Ala Asp Pro Glin Phe Ile Ile Gly Gly Ala Thr Arg Thr Asp Ile 85 90 95 Cys Glin Gly Ala Leu Gly Asp Cys Trp Lieu Lieu Ala Ala Ile Ala Ser 100 105 110 Lieu. Thir Lieu. Asn. Glu Glu Ile Leu Ala Arg Val Val Pro Leu Asn Glin 115 120 125 Ser Phe Glin Glu Asn Tyr Ala Gly Ile Phe His Phe Glin Phe Trp Gln 130 135 1 4 0 Tyr Gly Glu Trp Val Glu Val Val Val Asp Asp Arg Leu Pro Thr Lys 145 15 O 155 160 Asp Gly Glu Lieu Lleu Phe Val His Ser Ala Glu Gly Ser Glu Phe Trp 1.65 170 175 Ser Ala Lieu Lieu Glu Lys Ala Tyr Ala Lys Ile Asn Gly Cys Tyr Glu 18O 185 19 O Ala Leu Ser Gly Gly Ala Thir Thr Glu Gly Phe Glu Asp Phe Thr Gly 195 200 2O5 Gly Ile Ala Glu Trp Tyr Glu Lieu Lys Lys Pro Pro Pro Asn Lieu Phe 210 215 220 Lys Ile Ile Glin Lys Ala Leu Glin Lys Gly Ser Lieu Lleu Gly Cys Ser 225 230 235 240 Ile Asp Ile Thir Ser Ala Ala Asp Ser Glu Ala Ile Thr Phe Glin Lys 245 250 255 Leu Val Lys Gly His Ala Tyr Ser Val Thr Gly Ala Glu Glu Val Glu 260 265 27 O Ser Asn Gly Ser Leu Gln Lys Lieu. Ile Arg Ile Arg Asn Pro Trp Gly 275 280 285 Glu Val Glu Trp Thr Gly Arg Trp Asn Asp Asn. Cys Pro Ser Trp Asn 29 O 295 3OO Thir Ile Asp Pro Glu G Arg Glu Arg Lieu. Thr Arg Arg His Glu Asp 305 3 315 320 Gly Glu Phe Trp Met Ser Phe Ser Asp Phe Leu Arg His Tyr Ser Arg 325 330 335 Leu Glu Ile Cys Asn Leu Thr Pro Asp Thr Leu Thir Ser Asp Thr Tyr US 2005/0175581 A1 Aug. 11, 2005 66

-continued

340 345 35 O Lys Lys Trp Llys Lieu. Thir Lys Met Asp Gly Asn Trp Arg Arg Gly Ser 355 360 365 Thr Ala Gly Gly Cys Arg Asn Tyr Pro Asn Thr Phe Trp Met Asin Pro 370 375 38O Glin Tyr Lieu. Ile Lys Lieu Glu Glu Glu Asp Glu Asp Glu Glu Asp Gly 385 390 395 400 Glu Ser Gly Cys Thr Phe Leu Val Gly Lieu. Ile Glin Lys His Arg Arg 405 410 415 Arg Glin Arg Lys Met Gly Glu Asp Met His Thr Ile Gly Phe Gly Ile 420 425 43 O Tyr Glu Val Pro Glu Glu Leu Ser Gly Glin Thr Asn Ile His Leu Ser 435 4 40 4 45 Lys Asn. Phe Phe Lieu. Thr Asn Arg Ala Arg Glu Arg Ser Asp Thr Phe 450 455 460 Ile Asn Lieu Arg Glu Val Lieu. Asn Arg Phe Lys Lieu Pro Pro Gly Glu 465 470 475 480 Tyr Ile Leu Val Pro Ser Thr Phe Glu Pro Asn Lys Asp Gly Asp Phe 485 490 495 Cys Ile Arg Val Phe Ser Glu Lys Lys Ala Asp Tyr Glin Ala Val Asp 5 OO 505 51O. Asp Glu Ile Glu Ala Asn Leu Glu Glu Phe Asp Ile Ser Glu Asp Asp 515 52O 525 Ile Asp Asp Gly Val Arg Arg Lieu Phe Ala Glin Leu Ala Gly Glu Asp 530 535 540 Ala Glu Ile Ser Ala Phe Glu Lieu Glin Thir Ile Leu Arg Arg Val Lieu 545 550 555 560 Ala Lys Arg Glin Asp Ile Lys Ser Asp Gly Phe Ser Ile Glu Thir Cys 565 570 575 Lys Ile Met Val Asp Met Leu Asp Ser Asp Gly Ser Gly Lys Lieu Gly 58O 585 59 O Leu Lys Glu Phe Tyr Ile Leu Trp Thr Lys Ile Glin Lys Tyr Glin Lys 595 600 605 Ile Tyr Arg Glu Ile Asp Wall Asp Arg Ser Gly Thr Met Asn. Ser Tyr 610 615 62O Glu Met Arg Lys Ala Lieu Glu Glu Ala Gly Phe Lys Met Pro Cys Glin 625 630 635 640 Lieu. His Glin Val Ile Val Ala Arg Phe Ala Asp Asp Glin Lieu. Ile Ile 645 650 655 Asp Phe Asp Asn. Phe Val Arg Cys Lieu Val Arg Lieu Glu Thir Lieu Phe 660 665 67 O Lys Ile Phe Lys Glin Lieu. Asp Pro Glu Asn Thr Gly Thir Ile Glu Lieu 675 680 685 Asp Lieu. Ile Ser Trp Lieu. Cys Phe Ser Val Lieu 69 O. 695

<210> SEQ ID NO 24 <211& LENGTH: 221 &212> TYPE PRT <213> ORGANISM: Tobacco etch virus

<400 SEQUENCE: 24 US 2005/0175581 A1 Aug. 11, 2005 67

-continued Gly Glu Ser Lieu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile Ser Ser 1 5 10 15 Thir Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu 2O 25 30 Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His Leu Phe 35 40 45 Arg Arg Asn. Asn Gly. Thir Lieu Lieu Val Glin Ser Lieu. His Gly Val Phe 50 55 60 Lys Wall Lys Asn. Thir Thr Thr Lieu Glin Gln His Lieu. Ile Asp Gly Arg 65 70 75 8O Asp Met Ile Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Glin 85 90 95 Lys Lieu Lys Phe Arg Glu Pro Glin Arg Glu Glu Arg Ile Cys Lieu Val 100 105 110 Thir Thr Asn Phe Gln Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr 115 120 125 Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp Lys His Trp Ile 130 135 1 4 0 Glin Thr Lys Asp Gly Glin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp 145 15 O 155 160 Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr Asn 1.65 170 175 Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn 18O 185 19 O Glin Glu Ala Glin Glin Trp Val Ser Gly Trp Arg Lieu. Asn Ala Asp Ser 195 200 2O5 Val Leu Trp Gly Gly His Lys Val Phe Met Asp Llys Pro 210 215 220

<210> SEQ ID NO 25 &2 11s LENGTH 371 &212> TYPE PRT <213> ORGANISM: Streptococcus pyogenes <400 SEQUENCE: 25 Asp Glin Asn. Phe Ala Arg Asn. Glu Lys Glu Ala Lys Asp Ser Ala Ile 1 5 10 15 Thr Phe Ile Glin Lys Ser Ala Ala Ile Lys Ala Gly Ala Arg Ser Ala 2O 25 30 Glu Asp Ile Lys Lieu. Asp Llys Val Asn Lieu Gly Gly Glu Lieu Ser Gly 35 40 45 Ser Asn Met Tyr Val Tyr Asn Ile Ser Thr Gly Gly Phe Val Ile Val 50 55 60 Ser Gly Asp Lys Arg Ser Pro Glu Ile Leu Gly Tyr Ser Thir Ser Gly 65 70 75 8O Ser Phe Asp Wall Asn Gly Lys Glu Asn. Ile Ala Ser Phe Met Glu Ser 85 90 95 Tyr Val Glu Glin Ile Lys Glu Asn Lys Lys Lieu. Asp Ser Thr Tyr Ala 100 105 110 Gly Thr Ala Glu Ile Lys Glin Pro Val Val Lys Ser Lieu Lieu. Asp Ser 115 120 125 Lys Gly Ile His Tyr Asn Glin Gly Asn Pro Tyr Asn Lieu Lieu. Thr Pro 130 135 1 4 0 US 2005/0175581 A1 Aug. 11, 2005 68

-continued

Val Ile Glu Lys Val Lys Pro Gly Glu Glin Ser Phe Val Gly Glin His 145 15 O 155 160 Ala Ala Thr Gly Ser Val Ala Thr Ala Thr Ala Glin Ile Met Lys Tyr 1.65 170 175 His Asn Tyr Pro Asn Lys Gly Lieu Lys Asp Tyr Thr Tyr Thr Lieu Ser 18O 185 19 O Ser Asn. Asn Pro Tyr Phe Asn His Pro Lys Asn Lieu Phe Ala Ala Ile 195 200 2O5 Ser Thr Arg Glin Tyr Asn Trp Asn Asn Ile Leu Pro Thr Tyr Ser Gly 210 215 220 Arg Glu Ser Asn Val Glin Lys Met Ala Ile Ser Glu Lieu Met Ala Asp 225 230 235 240 Val Gly Ile Ser Val Asp Met Asp Tyr Gly Pro Ser Ser Gly Ser Ala 245 250 255 Gly Ser Ser Arg Val Glin Arg Ala Lieu Lys Glu Asn. Phe Gly Tyr Asn 260 265 27 O Glin Ser Wal His Glin Ile Asn Arg Gly Asp Phe Ser Lys Glin Asp Trp 275 280 285 Glu Ala Glin Ile Asp Lys Glu Lieu Ser Glin Asn. Glin Pro Val Tyr Tyr 29 O 295 3OO Glin Gly Val Gly Lys Val Gly Gly His Ala Phe Val Ile Asp Gly Ala 305 310 315 320 Asp Gly Arg Asn. Phe Tyr His Val Asn Trp Gly Trp Gly Gly Val Ser 325 330 335 Asp Gly Phe Phe Arg Lieu. Asp Ala Lieu. Asn. Pro Ser Ala Leu Gly Thr 340 345 35 O Gly Gly Gly Ala Gly Gly Phe Asn Gly Tyr Glin Ser Ala Val Val Gly 355 360 365 Ile Llys Pro 370

<210> SEQ ID NO 26 &2 11s LENGTH 353 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 26 Lys Lys His Thr Gly Tyr Val Gly Lieu Lys Asn. Glin Gly Ala Thr Cys 1 5 10 15 Tyr Met Asn Ser Leu Leu Gln Thr Leu Phe Phe Thr Asn Glin Leu Arg 2O 25 30 Lys Ala Val Tyr Met Met Pro Thr Glu Gly Asp Asp Ser Ser Lys Ser 35 40 45 Val Pro Leu Ala Leu Glin Arg Val Phe Tyr Glu Lieu Gln His Ser Asp 50 55 60 Lys Pro Val Gly Thr Lys Lys Leu Thr Lys Ser Phe Gly Trp Glu Thr 65 70 75 8O Leu Asp Ser Phe Met Gln His Asp Val Glin Glu Lieu. Cys Arg Val Lieu 85 90 95 Leu Asp Asn Val Glu Asn Lys Met Lys Gly Thr Cys Val Glu Gly Thr 100 105 110 Ile Pro Llys Lieu Phe Arg Gly Lys Met Val Ser Tyr Ile Glin Cys Lys US 2005/0175581 A1 Aug. 11, 2005 69

-continued

115 120 125 Glu Val Asp Tyr Arg Ser Asp Arg Arg Glu Asp Tyr Tyr Asp Ile Glin 130 135 1 4 0 Leu Ser Ile Lys Gly Lys Lys Asn. Ile Phe Glu Ser Phe Val Asp Tyr 145 15 O 155 160 Val Ala Val Glu Gln Lieu. Asp Gly Asp Asn Lys Tyr Asp Ala Gly Glu 1.65 170 175 His Gly Lieu Glin Glu Ala Glu Lys Gly Wall Lys Phe Lieu. Thir Lieu Pro 18O 185 19 O Pro Val Leu. His Leu Gln Leu Met Arg Phe Met Tyr Asp Pro Glin Thr 195 200 2O5 Asp Glin Asn. Ile Lys Ile Asn Asp Arg Phe Glu Phe Pro Glu Gln Leu 210 215 220 Pro Leu Asp Glu Phe Leu Gln Lys Thr Asp Pro Lys Asp Pro Ala Asn 225 230 235 240 Tyr Ile Lieu. His Ala Val Lieu Val His Ser Gly Asp Asn His Gly Gly 245 250 255 His Tyr Val Val Tyr Lieu. Asn Pro Lys Gly Asp Gly Lys Trp Cys Lys 260 265 27 O Phe Asp Asp Asp Val Val Ser Arg Cys Thr Lys Glu Glu Ala Ile Glu 275 280 285 His Asn Tyr Gly Gly His Asp Asp Asp Leu Ser Val Arg His Cys Thr 29 O 295 3OO Asn Ala Tyr Met Leu Val Tyr Ile Arg Glu Ser Lys Lieu Ser Glu Val 305 310 315 320 Leu Glin Ala Val Thr Asp His Asp Ile Pro Glin Glin Lieu Val Glu Arg 325 330 335 Leu Glin Glu Glu Lys Arg Ile Glu Ala Glin Lys Arg Lys Glu Arg Glin 340 345 35 O

Glu

<210 SEQ ID NO 27 &2 11s LENGTH 174 &212> TYPE PRT <213> ORGANISM: Staphylococcus aureus <400 SEQUENCE: 27 Tyr Asn. Glu Glin Tyr Val Asn Lys Lieu Glu Asn. Phe Lys Ile Arg Glu 1 5 10 15 Thr Glin Gly Asn Asn Gly Trp Cys Ala Gly Tyr Thr Met Ser Ala Leu 2O 25 30 Leu Asn Ala Thr Tyr Asn Thr Asn Lys Tyr His Ala Glu Ala Val Met 35 40 45 Arg Phe Leu. His Pro Asn Leu Gln Gly Glin Glin Phe Glin Phe Thr Gly 50 55 60 Leu Thr Pro Arg Glu Met Ile Tyr Phe Gly Glin Thr Glin Gly Arg Ser 65 70 75 8O Pro Glin Lieu Lieu. Asn Arg Met Thir Thr Tyr Asn. Glu Val Asp Asn Lieu 85 90 95 Thr Lys Asn. Asn Lys Gly Ile Ala Ile Leu Gly Ser Arg Val Glu Ser 100 105 110 Arg Asn Gly Met His Ala Gly His Ala Met Ala Val Val Gly Asn Ala US 2005/0175581 A1 Aug. 11, 2005 70

-continued

115 120 125 Lys Lieu. Asn. Asn Gly Glin Glu Val Ile Ile Ile Trp Asn Pro Trp Asp 130 135 1 4 0 Asn Gly Phe Met Thr Glin Asp Ala Lys Asn Asn Val Ile Pro Val Ser 145 15 O 155 160 Asn Gly Asp His Tyr Gln Trp Tyr Ser Ser Ile Tyr Gly Tyr 1.65 170

<210> SEQ ID NO 28 <211& LENGTH: 221 &212> TYPE PRT <213> ORGANISM: Saccharomyces cerevisiae <400 SEQUENCE: 28 Gly Ser Lieu Val Pro Glu Lieu. Asn. Glu Lys Asp Asp Asp Glin Val Glin 1 5 10 15 Lys Ala Lieu Ala Ser Arg Glu Asn Thr Glin Leu Met Asn Arg Asp Asn 2O 25 30 Ile Glu Ile Thr Val Arg Asp Phe Lys Thr Lieu Ala Pro Arg Arg Trp 35 40 45 Leu Asn Asp Thr Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser 50 55 60 Thr Pro Asn Thr Val Ala Phe Asin Ser Phe Phe Tyr Thr Asn Leu Ser 65 70 75 8O Glu Arg Gly Tyr Glin Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr 85 90 95 Glin Ile Asp Llys Lieu. Asp Lys Ile Phe Thr Pro Ile Asn Lieu. Asn Glin 100 105 110 Ser His Trp Ala Leu Gly Ile Ile Asp Lieu Lys Lys Lys Thir Ile Gly 115 120 125 Tyr Val Asp Ser Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile 130 135 1 4 0 Leu Thr Asp Leu Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile 145 15 O 155 160 Gly Glu Asp Phe Asp Lieu. Ile His Lieu. Asp Cys Pro Glin Glin Pro Asn 1.65 170 175 Gly Tyr Asp Cys Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser 18O 185 19 O Ala Asp Ala Pro Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg 195 200 2O5 Arg Phe Ile Ala His Lieu. Ile Lieu. Thir Asp Ala Lieu Lys 210 215 220

<210 SEQ ID NO 29 &2 11s LENGTH 166 &212> TYPE PRT <213> ORGANISM: Pyrococcus horikoshii <400 SEQUENCE: 29 Met Lys Wall Leu Phe Lieu. Thr Ala Asn. Glu Phe Glu Asp Val Glu Lieu 1 5 10 15 Ile Tyr Pro Tyr His Arg Leu Lys Glu Glu Gly His Glu Val Tyr Ile 2O 25 30 Ala Ser Phe Glu Arg Gly. Thir Ile Thr Gly Lys His Gly Tyr Ser Val US 2005/0175581 A1 Aug. 11, 2005 71

-continued

35 40 45 Lys Val Asp Lieu. Thr Phe Asp Llys Val Asn Pro Glu Glu Phe Asp Ala 50 55 60 Leu Val Lieu Pro Gly Gly Arg Ala Pro Glu Arg Val Arg Lieu. Asn. Glu 65 70 75 8O Lys Ala Val Ser Ile Ala Arg Lys Met Phe Ser Glu Gly Lys Pro Wal 85 90 95 Ala Ser Ile Cys His Gly Pro Glin Ile Lieu. Ile Ser Ala Gly Val Lieu 100 105 110 Arg Gly Arg Lys Gly. Thir Ser Tyr Pro Gly Ile Lys Asp Asp Met Ile 115 120 125 Asn Ala Gly Val Glu Trp Val Asp Ala Glu Val Val Val Asp Gly Asn 130 135 1 4 0 Trp Val Ser Ser Arg Val Pro Ala Asp Leu Tyr Ala Trp Met Arg Glu 145 15 O 155 160 Phe Wall Lys Lieu Lleu Lys 1.65

<210 SEQ ID NO 30 &2 11s LENGTH 316 &212> TYPE PRT <213> ORGANISM: Bacillus thermoproteolyticus <400> SEQUENCE: 30 Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly Asp 1 5 10 15 Gln Lys Asn Ile Asn Thr Thr Tyr Ser Thr Tyr Tyr Tyr Leu Glin Asp 2O 25 30 Asn Thr Arg Gly Asp Gly Ile Phe Thr Tyr Asp Ala Lys Tyr Arg Thr 35 40 45 Thr Lieu Pro Gly Ser Leu Trp Ala Asp Ala Asp Asn Glin Phe Phe Ala 50 55 60 Ser Tyr Asp Ala Pro Ala Val Asp Ala His Tyr Tyr Ala Gly Val Thr 65 70 75 8O Tyr Asp Tyr Tyr Lys Asn. Wal His Asn Arg Lieu Ser Tyr Asp Gly Asn 85 90 95 Asn Ala Ala Ile Arg Ser Ser Wal His Tyr Ser Glin Gly Tyr Asn. Asn 100 105 110 Ala Phe Trp Asn Gly Ser Glu Met Val Tyr Gly Asp Gly Asp Gly Glin 115 120 125 Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Val Val Ala His Glu Leu 130 135 1 4 0 Thr His Ala Val Thr Asp Tyr Thr Ala Gly Leu Ile Tyr Glin Asin Glu 145 15 O 155 160 Ser Gly Ala Ile Asn. Glu Ala Ile Ser Asp Ile Phe Gly. Thir Lieu Val 1.65 170 175 Glu Phe Tyr Ala Asn Lys Asn Pro Asp Trp Glu Ile Gly Glu Asp Wal 18O 185 19 O Tyr Thr Pro Gly Ile Ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro 195 200 2O5 Ala Lys Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr 210 215 220 US 2005/0175581 A1 Aug. 11, 2005 72

-continued Glin Asp Asn Gly Gly Val His Ile Asn. Ser Gly Ile Ile Asn Lys Ala 225 230 235 240 Ala Tyr Leu Ile Ser Glin Gly Gly Thr His Tyr Gly Val Ser Val Val 245 250 255 Gly Ile Gly Arg Asp Lys Lieu Gly Lys Ile Phe Tyr Arg Ala Lieu. Thr 260 265 27 O Gln Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gln Leu Arg Ala Ala Ala 275 280 285 Val Glin Ser Ala Thr Asp Leu Tyr Gly Ser Thr Ser Glin Glu Val Ala 29 O 295 3OO Ser Val Lys Glin Ala Phe Asp Ala Val Gly Val Lys 305 310 315

<210> SEQ ID NO 31 &2 11s LENGTH 169 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 31 Val Leu Thr Glu Gly Asn Pro Arg Trp Glu Gln Thr His Leu Thr Tyr 1 5 10 15 Arg Ile Glu Asn Tyr Thr Pro Asp Leu Pro Arg Ala Asp Val Asp His 2O 25 30 Ala Ile Glu Lys Ala Phe Gln Leu Trp Ser Asn Val Thr Pro Leu Thr 35 40 45 Phe Thr Lys Val Ser Glu Gly Glin Ala Asp Ile Met Ile Ser Phe Val 50 55 60 Arg Gly Asp His Arg Asp Asn. Ser Pro Phe Asp Gly Pro Gly Gly Asn 65 70 75 8O Leu Ala His Ala Phe Glin Pro Gly Pro Gly Ile Gly Gly Asp Ala His 85 90 95 Phe Asp Glu Asp Glu Arg Trp Thr Asn. Asn. Phe Arg Glu Tyr Asn Lieu 100 105 110 His Arg Val Ala Ala His Glu Lieu Gly. His Ser Leu Gly Lieu Ser His 115 120 125 Ser Thr Asp Ile Gly Ala Leu Met Tyr Pro Ser Tyr Thr Phe Ser Gly 130 135 1 4 0 Asp Val Glin Leu Ala Glin Asp Asp Ile Asp Gly Ile Glin Ala Ile Tyr 145 15 O 155 160 Gly Arg Ser Glin Asn Pro Val Glin Pro 1.65

<210> SEQ ID NO 32 &2 11s LENGTH 496 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 32 Gln Tyr Ser Pro Asn Thr Glin Glin Gly Arg Thr Ser Ile Val His Leu 1 5 10 15 Phe Glu Trp Arg Trp Val Asp Ile Ala Leu Glu Cys Glu Arg Tyr Lieu 2O 25 30 Ala Pro Lys Gly Phe Gly Gly Val Glin Val Ser Pro Pro Asn Glu Asn 35 40 45 US 2005/0175581 A1 Aug. 11, 2005 73

-continued Val Ala Ile Tyr Asn Pro Phe Arg Pro Trp Trp Glu Arg Tyr Glin Pro 50 55 60 Val Ser Tyr Lys Lieu. Cys Thr Arg Ser Gly Asn. Glu Asp Glu Phe Arg 65 70 75 8O Asn Met Val Thr Arg Cys Asn Asn Val Gly Val Arg Ile Tyr Val Asp 85 90 95 Ala Val Ile Asn His Met Cys Gly Asn Ala Val Ser Ala Gly Thr Ser 100 105 110 Ser Thr Cys Gly Ser Tyr Phe Asin Pro Gly Ser Arg Asp Phe Pro Ala 115 120 125 Val Pro Tyr Ser Gly Trp Asp Phe Asn Asp Gly Lys Cys Lys Thr Gly 130 135 1 4 0 Ser Gly Asp Ile Glu Asn Tyr Asn Asp Ala Thr Glin Val Arg Asp Cys 145 15 O 155 160 Arg Lieu. Thr Gly Lieu Lieu. Asp Leu Ala Leu Glu Lys Asp Tyr Val Arg 1.65 170 175 Ser Lys Ile Ala Glu Tyr Met Asn His Lieu. Ile Asp Ile Gly Val Ala 18O 185 19 O Gly Phe Arg Lieu. Asp Ala Ser Lys His Met Trp Pro Gly Asp Ile Lys 195 200 2O5 Ala Ile Leu Asp Lys Lieu. His Asn Lieu. Asn. Ser Asn Trp Phe Pro Ala 210 215 220 Gly Ser Lys Pro Phe Ile Tyr Glin Glu Val Ile Asp Lieu Gly Gly Glu 225 230 235 240 Pro Ile Lys Ser Ser Asp Tyr Phe Gly Asn Gly Arg Val Thr Glu Phe 245 250 255 Lys Tyr Gly Ala Lys Lieu Gly Thr Val Ile Arg Lys Trp Asin Gly Glu 260 265 27 O Lys Met Ser Tyr Leu Lys Asn Trp Gly Glu Gly Trp Gly Phe Val Pro 275 280 285 Ser Asp Arg Ala Leu Val Phe Val Asp Asn His Asp Asn Glin Arg Gly 29 O 295 3OO His Gly Ala Gly Gly Ala Ser Ile Lieu. Thir Phe Trp Asp Ala Arg Lieu 305 310 315 320 Tyr Lys Met Ala Val Gly Phe Met Leu Ala His Pro Tyr Gly Phe Thr 325 330 335 Arg Val Met Ser Ser Tyr Arg Trp Pro Arg Glin Phe Glin Asn Gly Asn 340 345 35 O Asp Wall Asn Asp Trp Val Gly Pro Pro Asn. Asn. Asn Gly Val Ile Lys 355 360 365 Glu Val Thr Ile Asn Pro Asp Thr Thr Cys Gly Asn Asp Trp Val Cys 370 375 38O Glu His Arg Trp Arg Glin Ile Arg Asn Met Val Ile Phe Arg Asn. Wal 385 390 395 400 Val Asp Gly Glin Pro Phe Thr Asn Trp Tyr Asp Asn Gly Ser Asn Glin 405 410 415 Val Ala Phe Gly Arg Gly Asn Arg Gly Phe Ile Val Phe Asn. Asn Asp 420 425 43 O Asp Trp Ser Phe Ser Leu Thir Leu Gln Thr Gly Leu Pro Ala Gly Thr 435 4 40 4 45 Tyr Cys Asp Val Ile Ser Gly Asp Lys Ile Asn Gly Asn. Cys Thr Gly US 2005/0175581 A1 Aug. 11, 2005 74

-continued

450 455 460

Ile Lys Ile Tyr Val Ser Asp Asp Gly Lys Ala His Phe Ser Ile Ser 465 470 475 480

Asn Ser Ala Glu Asp Pro Phe Ile Ala Ile His Ala Glu Ser Lys Telu 485 490 495

SEQ ID NO 33 LENGTH 370 TYPE PRT ORGANISM: Trichoderma reesei

<400 SEQUENCE: 33

Gln Pro Gly Thr Ser Thr Pro Glu Wall His Pro Teu Thr Thr 1 5 10 15

Lys Cys Thr Lys Ser Gly Gly Cys Wall Ala Glin Asp Thr Ser Wall 25 30

Teu Asp Trp Asn Tyr Trp Met His Asp Ala Asn Tyr Asn Ser 35 40 45

Thr Wall Asn Gly Gly Wall Asn Thr Thr Telu Pro Asp Glu Ala Thr 50 55 60

Cys Gly Lys Asn Cys Phe Ile Glu Gly Wall Asp Ala Ala Ser Gly 65 70 75

Wall Thr Thr Ser Gly Ser Ser Telu Thr Met Asn Glin Met Pro Ser 85 90 95

Ser Ser Gly Gly Tyr Ser Ser Wall Ser Pro Arg Teu Telu Telu Asp 100 105 110

Ser Asp Gly Glu Tyr Wall Met Telu Lys Telu Asn Gly Glin Glu Telu Ser 115 120 125

Phe Asp Wall Asp Teu Ser Ala Telu Pro Cys Gly Glu Asn Gly Ser Telu 130 135 1 4 0

Tyr Telu Ser Glin Met Asp Glu Asn Gly Gly Ala Asn Asn Thr 145 15 O 155 160

Ala Gly Ala Asn Tyr Gly Ser Gly Tyr Cys Asp Ala Pro Wall 1.65 170 175

Glin Thr Trp Arg Asn Gly Thr Telu Asn Thr Ser His Gly Phe 18O 185 19 O

Asn Glu Met Asp Ile Teu Glu Gly Asn Ser Arg Asn Ala Telu 195 200

Thr Pro His Ser Cys Thr Ala Thr Ala Cys Asp Ser Gly Gly 210 215 220

Phe Asn Pro Gly Ser Gly Tyr Lys Ser Tyr Pro Gly Asp 225 230 235 240

Thr Wall Asp Thr Ser Lys Thr Phe Thr Ile Ile Thr Phe Asn Thr 245 250 255

Asp Asn Gly Ser Pro Ser Gly Asn Telu Wall Ser Ile Thr Arg 260 265 27 O

Glin Glin Asn Gly Wall Asp Ile Pro Ser Ala Glin Pro Gly Gly Asp Thr 275 280 285

Ile Ser Ser Pro Ser Ala Ser Ala Tyr Gly Gly Teu Ala Thr Met 29 O 295

Gly Ala Telu Ser Ser Gly Met Wall Telu Wall Phe Ser Ile Trp Asn 305 310 315 320 US 2005/0175581 A1 Aug. 11, 2005 75

-continued Asp Asn. Ser Glin Tyr Met Asn Trp Lieu. Asp Ser Gly Asn Ala Gly Pro 325 330 335 Cys Ser Ser Thr Glu Gly Asn Pro Ser Asn. Ile Leu Ala Asn. Asn Pro 340 345 35 O Asn Thr His Val Val Phe Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser 355 360 365

Thir Thr 370

<210> SEQ ID NO 34 <211& LENGTH 223 &212> TYPE PRT <213> ORGANISM: Aspergillus niger <400 SEQUENCE: 34 Gln Thr Met Cys Ser Glin Tyr Asp Ser Ala Ser Ser Pro Pro Tyr Ser 1 5 10 15 Val Asn Glin Asn Lieu Trp Gly Glu Tyr Glin Gly Thr Gly Ser Glin Cys 2O 25 30 Val Tyr Val Asp Lys Leu Ser Ser Ser Gly Ala Ser Trp His Thr Glu 35 40 45 Trp Thr Trp Ser Gly Gly Glu Gly Thr Val Lys Ser Tyr Ser Asn Ser 50 55 60 Gly Val Thr Phe Asn Lys Lys Leu Val Ser Asp Val Ser Ser Ile Pro 65 70 75 8O Thir Ser Val Glu Trp Lys Glin Asp Asn. Thir Asn. Wall Asn Ala Asp Wal 85 90 95 Ala Tyr Asp Leu Phe Thr Ala Ala Asn Val Asp His Ala Thr Ser Ser 100 105 110 Gly Asp Tyr Glu Lieu Met Ile Trp Lieu Ala Arg Tyr Gly Asn. Ile Glin 115 120 125 Pro Ile Gly Lys Glin Ile Ala Thr Ala Thr Val Gly Gly Lys Ser Trp 130 135 1 4 0 Glu Val Trp Tyr Gly Ser Thr Thr Glin Ala Gly Ala Glu Glin Arg Thr 145 15 O 155 160 Tyr Ser Phe Val Ser Glu Ser Pro Ile Asin Ser Tyr Ser Gly Asp Ile 1.65 170 175 Asn Ala Phe Phe Ser Tyr Leu Thr Glin Asn Gln Gly Phe Pro Ala Ser 18O 185 19 O Ser Glin Tyr Leu Ile Asn Leu Glin Phe Gly. Thr Glu Ala Phe Thr Gly 195 200 2O5 Gly Pro Ala Thr Phe Thr Val Asp Asn Trp Thr Ala Ser Val Asn 210 215 220

<210 SEQ ID NO 35 &2 11s LENGTH 184 &212> TYPE PRT <213> ORGANISM: Aspergillus niger <400 SEQUENCE: 35 Ser Ala Gly Ile Asn Tyr Val Glin Asn Tyr Asn Gly Asn Lieu Gly Asp 1 5 10 15 Phe Thr Tyr Asp Glu Ser Ala Gly Thr Phe Ser Met Tyr Trp Glu Asp 2O 25 30 US 2005/0175581 A1 Aug. 11, 2005 76

-continued Gly Val Ser Ser Asp Phe Val Val Gly Leu Gly Trp Thr Thr Gly Ser 35 40 45 Ser Asn Ala Ile Thr Tyr Ser Ala Glu Tyr Ser Ala Ser Gly Ser Ala 50 55 60 Ser Tyr Leu Ala Val Tyr Gly Trp Val Asn Tyr Pro Glin Ala Glu Tyr 65 70 75 8O Tyr Ile Val Glu Asp Tyr Gly Asp Tyr Asn Pro Cys Ser Ser Ala Thr 85 90 95 Ser Leu Gly Thr Val Tyr Ser Asp Gly Ser Thr Tyr Glin Val Cys Thr 100 105 110 Asp Thr Arg Thr Asn Glu Pro Ser Ile Thr Gly. Thir Ser Thr Phe Thr 115 120 125 Gln Tyr Phe Ser Val Arg Glu Ser Thr Arg Thr Ser Gly Thr Val Thr 130 135 1 4 0 Val Ala Asn His Phe Asn Phe Trp Ala His His Gly Phe Gly Asn Ser 145 15 O 155 160 Asp Phe Asn Tyr Glin Val Val Ala Val Glu Ala Trp Ser Gly Ala Gly 1.65 170 175

Ser Ala Ser Wall Thir Ile Ser Ser 18O

<210 SEQ ID NO 36 &2 11s LENGTH 313 &212> TYPE PRT <213> ORGANISM: Streptomyces lividans <400 SEQUENCE: 36 Ala Glu Ser Thr Lieu Gly Ala Ala Ala Ala Glin Ser Gly Arg Tyr Phe 1 5 10 15 Gly Thr Ala Ile Ala Ser Gly Arg Leu Ser Asp Ser Thr Tyr Thr Ser 2O 25 30 Ile Ala Gly Arg Glu Phe Asn Met Val Thr Ala Glu Asn. Glu Met Lys 35 40 45 Ile Asp Ala Thr Glu Pro Glin Arg Gly Glin Phe Asn Phe Ser Ser Ala 50 55 60 Asp Arg Val Tyr Asn Trp Ala Val Glin Asn Gly Lys Glin Val Arg Gly 65 70 75 8O His Thr Leu Ala Trp His Ser Glin Gln Pro Gly Trp Met Glin Ser Leu 85 90 95 Ser Gly Ser Ala Leu Arg Glin Ala Met Ile Asp His Ile Asin Gly Val 100 105 110 Met Ala His Tyr Lys Gly Lys Ile Val Glin Trp Asp Val Val Asn. Glu 115 120 125 Ala Phe Ala Asp Gly Ser Ser Gly Ala Arg Arg Asp Ser Asn Lieu Glin 130 135 1 4 0 Arg Ser Gly Asn Asp Trp Ile Glu Val Ala Phe Arg Thr Ala Arg Ala 145 15 O 155 160 Ala Asp Pro Ser Ala Lys Lieu. Cys Tyr Asn Asp Tyr Asn Val Glu Asn 1.65 170 175 Trp Thr Trp Ala Lys Thr Glin Ala Met Tyr Asn Met Val Arg Asp Phe 18O 185 19 O Lys Glin Arg Gly Val Pro Ile Asp Cys Val Gly Phe Glin Ser His Phe 195 200 2O5 US 2005/0175581 A1 Aug. 11, 2005 77

-continued

Asn Ser Gly Ser Pro Tyr Asn Ser Asn Phe Arg Thr Thr Leu Glin Asn 210 215 220 Phe Ala Ala Leu Gly Val Asp Val Ala Ile Thr Glu Lieu. Asp Ile Glin 225 230 235 240 Gly Ala Pro Ala Ser Thr Tyr Ala Asn Val Thr Asn Asp Cys Lieu Ala 245 250 255 Val Ser Arg Cys Lieu Gly Ile Thr Val Trp Gly Val Arg Asp Ser Asp 260 265 27 O Ser Trp Arg Ser Glu Glin Thr Pro Leu Lieu Phe Asn. Asn Asp Gly Ser 275 280 285 Lys Lys Ala Ala Tyr Thr Ala Val Lieu. Asp Ala Lieu. Asn Gly Gly Ala 29 O 295 3OO Ser Ser Glu Pro Pro Ala Asp Gly Gly 305 310

<210 SEQ ID NO 37 &2 11s LENGTH 362 &212> TYPE PRT <213> ORGANISM: Aspergillus niger <400 SEQUENCE: 37 Met His Ser Phe Ala Ser Lieu Lieu Ala Tyr Gly Lieu Val Ala Gly Ala 1 5 10 15 Thr Phe Ala Ser Ala Ser Pro Ile Glu Ala Arg Asp Ser Cys Thr Phe 2O 25 30 Thir Thr Ala Ala Ala Ala Lys Ala Gly Lys Ala Lys Cys Ser Thr Ile 35 40 45 Thr Leu Asn Asn Ile Glu Val Pro Ala Gly. Thir Thr Leu Asp Leu Thr 50 55 60 Gly Leu Thir Ser Gly Thr Lys Val Ile Phe Glu Gly Thr Thr Thr Phe 65 70 75 8O Gln Tyr Glu Glu Trp Ala Gly Pro Leu Ile Ser Met Ser Gly Glu His 85 90 95 Ile Thr Val Thr Gly Ala Ser Gly His Lieu. Ile Asn. Cys Asp Gly Ala 100 105 110 Arg Trp Trp Asp Gly Lys Gly Thr Ser Gly Lys Lys Lys Pro Llys Phe 115 120 125 Phe Tyr Ala His Gly Lieu. Asp Ser Ser Ser Ile Thr Gly Lieu. Asn. Ile 130 135 1 4 0 Lys Asn Thr Pro Leu Met Ala Phe Ser Val Glin Ala Asn Asp Ile Thr 145 15 O 155 160 Phe Thr Asp Val Thr Ile Asn. Asn Ala Asp Gly Asp Thr Glin Gly Gly 1.65 170 175 His Asn. Thir Asp Ala Phe Asp Val Gly Asn. Ser Val Gly Val Asn. Ile 18O 185 19 O Ile Llys Pro Trp Wal His Asn Glin Asp Asp Cys Lieu Ala Val Asn. Ser 195 200 2O5 Gly Glu Asn Ile Trp Phe Thr Gly Gly Thr Cys Ile Gly Gly His Gly 210 215 220 Leu Ser Ile Gly Ser Val Gly Asp Arg Ser Asn. Asn Val Val Lys Asn 225 230 235 240 Val Thr Ile Glu His Ser Thr Val Ser Asn Ser Glu Asn Ala Val Arg US 2005/0175581 A1 Aug. 11, 2005 78

-continued

245 250 255 Ile Lys Thr Ile Ser Gly Ala Thr Gly Ser Val Ser Glu Ile Thr Tyr 260 265 27 O Ser Asn Ile Val Met Ser Gly Ile Ser Asp Tyr Gly Val Val Ile Glin 275 280 285 Glin Asp Tyr Glu Asp Gly Lys Pro Thr Gly Lys Pro Thr Asn Gly Val 29 O 295 3OO Thir Ile Glin Asp Val Lys Leu Glu Ser Val Thr Gly Ser Val Asp Ser 305 310 315 320 Gly Ala Thr Glu Ile Tyr Lieu Lieu. Cys Gly Ser Gly Ser Cys Ser Asp 325 330 335 Trp. Thir Trp Asp Asp Wall Lys Val Thr Gly Gly Lys Lys Ser Thr Ala 340 345 35 O Cys Lys Asn Phe Pro Ser Val Ala Ser Cys 355 360

<210 SEQ ID NO 38 &2 11s LENGTH 383 &212> TYPE PRT <213> ORGANISM: Pseudomonas cellulosa

<400 SEQUENCE: 38 Arg Ala Asp Wall Lys Pro Val Thr Val Lys Lieu Val Asp Ser Glin Ala 1 5 10 15 Thr Met Glu Thr Arg Ser Leu Phe Ala Phe Met Gln Glu Glin Arg Arg 2O 25 30 His Ser Ile Met Phe Gly. His Gln His Glu Thir Thr Glin Gly Leu Thr 35 40 45 Ile Thr Arg Thr Asp Gly Thr Glin Ser Asp Thr Phe Asn Ala Val Gly 50 55 60 Asp Phe Ala Ala Val Tyr Gly Trp Asp Thr Leu Ser Ile Val Ala Pro 65 70 75 8O Lys Ala Glu Gly Asp Ile Val Ala Glin Wall Lys Lys Ala Tyr Ala Arg 85 90 95 Gly Gly Ile Ile Thr Val Ser Ser His Phe Asp Asn Pro Llys Thr Asp 100 105 110 Thr Glin Lys Gly Val Trp Pro Val Gly Thr Ser Trp Asp Glin Thr Pro 115 120 125 Ala Val Val Asp Ser Lieu Pro Gly Gly Ala Tyr Asn Pro Val Lieu. Asn 130 135 1 4 0 Gly Tyr Lieu. Asp Glin Val Ala Glu Trp Ala Asn. Asn Lieu Lys Asp Glu 145 15 O 155 160 Gln Gly Arg Leu Ile Pro Val Ile Phe Arg Leu Tyr His Ala Asn Thr 1.65 170 175 Gly Ser Trp Phe Trp Trp Gly Asp Lys Glin Ser Thr Pro Glu Glin Tyr 18O 185 19 O Lys Glin Leu Phe Arg Tyr Ser Val Glu Tyr Lieu Arg Asp Wall Lys Gly 195 200 2O5 Val Arg Asin Phe Leu Tyr Ala Tyr Ser Pro Asn Asn Phe Trp Asp Val 210 215 220 Thr Glu Ala Asn Tyr Lieu Glu Arg Tyr Pro Gly Asp Glu Trp Val Asp 225 230 235 240 US 2005/0175581 A1 Aug. 11, 2005 79

-continued Val Lieu Gly Phe Asp Thr Tyr Gly Pro Val Ala Asp Asn Ala Asp Trip 245 250 255 Phe Arg Asn Val Val Ala Asn Ala Ala Lieu Val Ala Arg Met Ala Glu 260 265 27 O Ala Arg Gly Lys Ile Pro Val Ile Ser Glu Ile Gly Ile Arg Ala Pro 275 280 285 Asp Ile Glu Ala Gly Lieu. Tyr Asp Asn Glin Trp Tyr Arg Lys Lieu. Ile 29 O 295 3OO Ser Gly Lieu Lys Ala Asp Pro Asp Ala Arg Glu Ile Ala Phe Lieu Lieu 305 310 315 320 Val Trp Arg Asn Ala Pro Gln Gly Val Pro Gly Pro Asn Gly Thr Glin 325 330 335 Val Pro His Tyr Trp Val Pro Ala Asn Arg Pro Glu Asn Ile Asin Asn 340 345 35 O Gly Thr Leu Glu Asp Phe Glin Ala Phe Tyr Ala Asp Glu Phe Thr Ala 355 360 365 Phe Asin Arg Asp Ile Glu Glin Val Tyr Glin Arg Pro Thr Leu Ile 370 375 38O

<210 SEQ ID NO 39 &2 11s LENGTH 419 &212> TYPE PRT <213> ORGANISM: Bacillus circulans

<400 SEQUENCE: 39 Leu Gln Pro Ala Thr Ala Glu Ala Ala Asp Ser Tyr Lys Ile Val Gly 1 5 10 15 Tyr Tyr Pro Ser Trp Ala Ala Tyr Gly Arg Asn Tyr Asn. Wall Ala Asp 2O 25 30 Ile Asp Pro Thr Lys Val Thr His Ile Asn Tyr Ala Phe Ala Asp Ile 35 40 45 Cys Trp Asn Gly Ile His Gly Asn Pro Asp Pro Ser Gly Pro Asn Pro 50 55 60 Val Thr Trp Thr Cys Glin Asn Glu Lys Ser Gln Thr Ile Asin Val Pro 65 70 75 8O Asn Gly Thir Ile Val Lieu Gly Asp Pro Trp Ile Asp Thr Gly Lys Thr 85 90 95 Phe Ala Gly Asp Thr Trp Asp Glin Pro Ile Ala Gly Asn. Ile Asn Glin 100 105 110 Lieu. Asn Lys Lieu Lys Glin Thr Asn Pro Asn Lieu Lys Thir Ile Ile Ser 115 120 125 Val Gly Gly Trp Thr Trp Ser Asn Arg Phe Ser Asp Val Ala Ala Thr 130 135 1 4 0 Ala Ala Thr Arg Glu Val Phe Ala Asn. Ser Ala Val Asp Phe Lieu Arg 145 15 O 155 160 Lys Tyr Asn. Phe Asp Gly Val Asp Lieu. Asp Trp Glu Tyr Pro Val Ser 1.65 170 175 Gly Gly Lieu. Asp Gly Asn. Ser Lys Arg Pro Glu Asp Lys Glin Asn Tyr 18O 185 19 O Thr Lieu Lleu Lleu Ser Lys Ile Arg Glu Lys Lieu. Asp Ala Ala Gly Ala 195 200 2O5 Val Asp Gly Lys Lys Tyr Lieu Lieu. Thir Ile Ala Ser Gly Ala Ser Ala 210 215 220 US 2005/0175581 A1 Aug. 11, 2005 8O

-continued

Thr Tyr Ala Ala Asn Thr Glu Lieu Ala Lys Ile Ala Ala Ile Val Asp 225 230 235 240 Trp Ile Asin Ile Met Thr Tyr Asp Phe Asin Gly Ala Trp Glin Lys Ile 245 250 255 Ser Ala His Asn Ala Pro Leu Asn Tyr Asp Pro Ala Ala Ser Ala Ala 260 265 27 O Gly Val Pro Asp Ala Asn. Thir Phe Asn. Wall Ala Ala Gly Ala Glin Gly 275 280 285 His Lieu. Asp Ala Gly Val Pro Ala Ala Lys Lieu Val Lieu Gly Val Pro 29 O 295 3OO Phe Tyr Gly Arg Gly Trp Asp Gly Cys Ala Glin Ala Gly Asn Gly Glin 305 310 315 320 Tyr Glin Thr Cys Thr Gly Gly Ser Ser Val Gly Thr Trp Glu Ala Gly 325 330 335 Ser Phe Asp Phe Tyr Asp Lieu Glu Ala Asn Tyr Ile Asn Lys Asn Gly 340 345 35 O Tyr Thr Arg Tyr Trp Asn Asp Thr Ala Lys Val Pro Tyr Leu Tyr Asn 355 360 365 Ala Ser Asn Lys Arg Phe Ile Ser Tyr Asp Asp Ala Glu Ser Val Gly 370 375 38O Tyr Lys Thr Ala Tyr Ile Lys Ser Lys Gly Lieu Gly Gly Ala Met Phe 385 390 395 400 Trp Glu Lieu Ser Gly Asp Arg Asn Lys Thr Lieu Glin Asn Lys Lieu Lys 405 410 415 Ala Asp Lieu

<210> SEQ ID NO 40 &2 11s LENGTH 317 &212> TYPE PRT <213> ORGANISM: Candida antarctica

<400 SEQUENCE: 40 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu 1 5 10 15 Asp Ala Gly Lieu. Thr Cys Glin Gly Ala Ser Pro Ser Ser Val Ser Lys 2O 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Glin Ser Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Thr Gln Leu Gly Tyr Thr Pro Cys 50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Glin Val Asn Thr 65 70 75 8O Glu Tyr Met Val Asn Ala Ile Thr Ala Leu Tyr Ala Gly Ser Gly Asn 85 90 95 Asn Lys Lieu Pro Val Lieu. Thir Trp Ser Glin Gly Gly Lieu Val Ala Glin 100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu 115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu 130 135 1 4 0 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 15 O 155 160 US 2005/0175581 A1 Aug. 11, 2005 81

-continued

Ser Ala Lieu. Thir Thr Ala Lieu Arg Asn Ala Gly Gly Lieu. Thr Glin Ile 1.65 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Glin Pro 18O 185 19 O Glin Val Ser Asn. Ser Pro Leu Asp Ser Ser Tyr Lieu Phe Asin Gly Lys 195 200 2O5 Asn Val Glin Ala Glin Ala Val Cys Gly Pro Leu Phe Val Ile Asp His 210 215 220 Ala Gly Ser Leu Thir Ser Glin Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Glin Ala Arg Ser Ala Asp Tyr Gly Ile Thr 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Lieu. Thr Pro Glu Glin Lys Val 260 265 27 O Ala Ala Ala Ala Leu Lleu Ala Pro Ala Ala Ala Ala Ile Val Ala Gly 275 280 285 Pro Lys Glin Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe 29 O 295 3OO Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315

<210> SEQ ID NO 41 <211& LENGTH 434 &212> TYPE PRT <213> ORGANISM: artificial sequence &220s FEATURE <223> OTHER INFORMATION: chimera of guinea pig and homo sapiens (human= approx. last 30 amino acids) <400 SEQUENCE: 41 Ala Glu Val Cys Tyr Ser His Leu Gly Cys Phe Ser Asp Glu Lys Pro 1 5 10 15 Trp Ala Gly Thr Ser Glin Arg Pro Ile Llys Ser Leu Pro Ser Asp Pro 2O 25 30 Lys Lys Ile Asn. Thr Arg Phe Lieu Lleu Tyr Thr Asn. Glu Asn Glin Asn 35 40 45 Ser Tyr Gln Leu Ile Thr Ala Thr Asp Ile Ala Thr Ile Lys Ala Ser 50 55 60 Asn Phe Asn Leu Asn Arg Lys Thr Arg Phe Ile Ile His Gly Phe Thr 65 70 75 8O Asp Ser Gly Glu Asn. Ser Trp Leu Ser Asp Met Cys Lys Asn Met Phe 85 90 95 Glin Val Glu Lys Val Asn. Cys Ile Cys Val Asp Trp Lys Gly Gly Ser 100 105 110 Lys Ala Glin Tyr Ser Glin Ala Ser Glin Asn. Ile Arg Val Val Gly Ala 115 120 125 Glu Val Ala Tyr Leu Val Glin Val Leu Ser Thr Ser Leu Asn Tyr Ala 130 135 1 4 0 Pro Glu Asn Val His Ile Ile Gly His Ser Leu Gly Ala His Thr Ala 145 15 O 155 160 Gly Glu Ala Gly Lys Arg Lieu. Asn Gly Lieu Val Gly Arg Ile Thr Gly 1.65 170 175 Leu Asp Pro Ala Glu Pro Tyr Phe Glin Asp Thr Pro Glu Glu Val Arg