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Wo 2007/038619 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 5 April 2007 (05.04.2007) WO 2007/038619 A2 (51) International Patent Classification: (74) Agents: WONG, Karen, K. et al.; WILSON SONSINI C40B 40/10 (2006.01) GOODRICH & ROSATI, 650 Page Mill Road, Palo Alto, CA 94304-1050 (US). (21) International Application Number: PCT/US2006/037713 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AT,AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, 27 September 2006 (27.09.2006) CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, HN, HR, HU, ID, IL, IN, IS, JP, (25) Filing Language: English KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LV,LY,MA, MD, MG, MK, MN, MW, MX, MY, MZ, (26) Publication Language: English NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, TR, (30) Priority Data: TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW 60/721,270 27 September 2005 (27.09.2005) US 60/721,188 27 September 2005 (27.09.2005) US (84) Designated States (unless otherwise indicated, for every 60/743,622 21 March 2006 (2 1.03.2006) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (71) Applicant (for all designated States except US): AMU- ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), NIX, INC. [US/US]; 500 Ellis Street - Suite B, Mountain European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, View, CA 94043 (US). FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, (72) Inventors; and GN, GQ, GW, ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): STEMMER, Willem, P.C. [NIVUS]; 108 Kathy Court, Los Gatos, CA Published: 95030 (US). SCHELLENBERGER, Volker [DE/US]; — without international search report and to be republished 914 Moreno Avenue, Palo Alto, CA 94303 (US). BADER, upon receipt of that report Martin [DE/US]; 600 Rainbow Drive, Apt 214, Mountain View, CA 94041 (US). SCHOLLE, Michael [US/US]; For two-letter codes and other abbreviations, refer to the "G uid 100 North Whitman Road, #3613, Mountain View, CA ance Notes on Codes and Abbreviations" appearing at the beg in 94043 (US). ning of each regular issue of the PCT Gazette. (54) Title: PROTEINACEOUS PHARMACEUTICALS AND USES THEREOF (57) Abstract: The present invention provides cysteine-containing scaffolds and/or proteins, expression vectors, host cell and dis- play systems harboring and/or expressing such cysteine-containing products. The present invention also provides methods of de- signing libraries of such products, methods of screening such libraries to yield entities exhibiting binding specificities towards a taraget molecule. Further provided by the invention are pharmaceutical compositions comprising the cysteine-containing products of the present invention. PROTEINACEOUS PHARMACEUTICALS AND USES THEREOF CROSS-REFERENCE [0001] This application claims priority to U.S. Provisional Application Nos. 60/721,270 and 60/721,188, both filed on September 27, 2005, and U.S. Provisional Application No. 60/743,622 filed on March 21, 2006, all which are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION [0002] One of the fundamental concepts of molecular biology is that each natural protein adopts a single 'native' structure or fold. Adoption of any fold other than the native fold is regarded as 'misfolding'. Few or no examples exist of natural proteins adopting multiple native, functional folds. Misfolding is a serious problem, exemplified by the infectious nature of prions, whose 'wrong' fold causes other prion proteins to misfold in a catalytic manner and leads to brain disease and certain death. Almost any protein, when denatured, can misfold to form fibrillar polymers, which appear to be involved in a number of degenerative diseases. An example are the beta-amyloid fibrils involved in Alzheimer's disease. Misfolding of proteins generally results in the irreversible formation of insoluble aggregates, but denatured proteins can also occur as molten globules. From a molten globule state, which explores a huge diversity of unstable structures, the protein is thought to follow a funnel-shaped pathway, gradually reducing the diversity of folding intermediates until a single, stably folded native structure is achieved. The native protein can be altered structurally by allosteric regulation, lid/flap-type movements of one domain relative to other domains, induced fit upon binding to a ligand, or by crystallization forces, but these alterations generally involve movement in hinge-like structures rather than fundamental change in the basic fold. All of the available examples support the notion that natural proteins have evolved to adopt a single stable fold to effect their biological function, and that deviation from this native structure is deleterious. [0003] There have been a few examples of the same protein sequence (excluding variants created by alternative splicing, glycosylation or proteolytic processing) existing naturally in more than one form, but the second form is usually simply an inactive by-product which has lost a disulfide bond (Schulz et al, 2005; Petersen et al, 2003; Lauber et al, 2003). In the microprotein family, which include small proteins with high disulfide density (mostly toxins and receptor-domains), examples have been found of closely related sequences adopting a different structure due to fully formed (not simply defective) but alternative disulfide bonding pattern. Examples are Somatomedin (Kamikubo et al, 2004) and Maurotoxin (Fajloun et al, 2000). [0004] Protein display libraries have traditionally used a single fixed protein fold, like immunoglobulin domains of various species, Interferons, Protein A, Ankyrins, A-domains, T-cell receptors, Fibronectin III, gamma-Crystallin, Ubiquitin and many others, as reviewed in Binz, A. et al. (2005) Nature Biotechnology 23: 1257. In some cases, like immunoglobulin libraries derived from the human immune repertoire, a single library uses many different V-region sequences as scaffolds, but they all share the basic immunoglobulin fold. A different type of library is the random peptide or cyclic peptide library, but these are not considered proteins since they do not have any defined fold and do not adopt a single stable structure. [0005] There remains a considerable need for the design of novel protein structures that are amenable to rational selection via, e.g., directed evolution to create therapeutics that exhibit one or more desirable properties. Such '"" """' ! enhanced stability or half life, multispecificity, multivalency, and high target binding affinity. SUMMARY OF THE INVENTION [0006] One aspect of the present invention is the design of novel protein structures exhibiting high disulfide 5 density. The protein structures are particularly amenable to rational design and selection via, e.g., directed evolution to create therapeutics that exhibit one or more desirable properties. Such desired properties include but are not limited to high target binding affinity and/or avidity, reduced molecular weight and improved tissue penetration, enhanced thermal and protease stability, enhanced shelflife, enhanced hydrophilicity, enhanced formulation (esp. high concentration), and reduced immunogenicity. 10 [0007] In one embodiment, the present invention provides various protein structures in form of, e.g. scaffolds, and libraries of such protein structures. In one aspect, the scaffolds exhibit a diversity of folds or other non-primary structures. In another aspect, the scaffolds have defined topologies to effect the biological functions. In another embodiment, the present invention provides methods of constructing libraries of such protein structures, methods of displaying such libraries on genetic vehicles or packages (e.g., viral packages such as phages or the like, and non- 15 viral packages (such as yeast display, E. coli surface display, ribosome display, or CIS (DNA-linked) display), as well as methods of screening such libraries to yield therapeutics or candidate therapeutics. The present invention further provides vectors, host cells and other in vitro systems expressing or utilizing the subject protein structures. [0008] In another embodiment, the present invention privides a non-naturally occurring cysteine (C)-containing scaffold exhibiting a binding specificity towards a target molecule, wherein the non-naturally occurring cysteine 20 (C)-containing scaffold comprise intra-scaffold cysteines according to a pattern selected from the group of , permutations represented by the formula Error! Objects cannot be created from editing field codes., wherein n equals to the predicted number of disulfide bonds formed by the cysteine residues, and wherein Error! Objects cannot be created from editing field codes.represents the product of (2i-l), where i is a positive integer ranging from 1 up to n. 25 [0009] In another embodiment, the present invention provides a non-naturally occurring cysteine (C)-containing protein comprising a polypeptide having no more than 35 amino acids, in which at least 10% of the amino acids in the polypeptide are cysteines, at least two disulfide bonds are formed by pairing intra-scaffold cysteines, and
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