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Wo 2009/023270 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date PCT 19 February 2009 (19.02.2009) WO 2009/023270 A2 (51) International Patent Classification: 94083 (US). WANG, Chia-wei [-/US]; 3444 El Camino C12P 21/00 (2006.01) A61K 38/28 (2006.01) Real #307, Santa Clara, CA 95051 (US). GEETHING, C12N 15/09 (2006.01) A61K 38/21 (2006.01) Nathan, C. [US/US]; 2009 Garzoni Place, Santa Clara, C12N 1/00 (2006.01) A61P 43/00 (2006.01) CA 95054 (US). C07H 21/00 (2006.01) C07K 14/00 (2006.01) A61K 38/16 (2006.01) C 0 40/70 (2006.01) (74) Agents: WRONG, Karen, K. et al.; Wilson Sonsini Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA (21) International Application Number: 94304-1050 (US). PCT/US2008/009787 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: 15 August 2008 (15.08.2008) AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, (25) Filing Language: English EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, (26) Publication Language: English LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, (30) Priority Data: RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TJ, 15 August 2007 (15.08.2007) US 60/956,109 TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, 60/981,073 18 October 2007 (18.10.2007) US ZW 60/986,569 8 November 2007 (08. 11.2007) US (84) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): AMU- kind of regional protection available): ARIPO (BW, GH, NIX, INC. [US/US]; 1455 Adams Drive, Suite 1120, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, Menlo Park, CA 94025 (US). ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, (72) Inventors; and FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, (75) Inventors/Applicants (for US only): BOGIN, Oren NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, [US/US]; 1231 Manet Dr., Sunnyvale, CA 94087 (US). CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). STEMMER, Willem, P. [NL/US]; 108 Kathy Court, Los Gatos, CA 95030 (US). SCHELLENBERGER, Volker Published: [DE/US]; 914 Moreno Avenue, Palo Alto, CA 94303 (US). — without international search report and to be republished YIN, Yong [US/US]; 107 Sunset Dr., Sunnyvale, CA upon receipt of that report (54) Title: COMPOSITIONS AND METHODS FOR MODIFYING PROPERTIES OF BIOLOGICALLY ACTIVE POLYPEP TIDES (57) Abstract: The present invention relates to biologically active polypeptides linked to one or more accessory polypeptides. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applica- tions. POSITIONS AND METHODS FOR MODIFYING PROPERTIES O F BIOLOGICALLY ACTIVE POLYPEPTIDES CROSS-REFERENCE TO RELATED APPLICATIONS [0001 ] This application claims the priority benefit of U S Provisional Application Serial Nos 60/956, 109 filed on August 15, 2007, 60/98 1,073, filed October 18, 2007 and 60/986,569, filed November 8, 2007, pending, which are hereby incorporated herein by reference in their entirety BACKGROUND O F THE INVENTION [0002] Recombinant proteins have become very attractive candidates for the development of novel therapeutics However, production of protein pharmaceuticals requires significant optimization of processes to obtain sufficient yields of specific biologically active polypeptides It is well established that the expression of recombinant proteins in the cytoplasm of Escherichia coli, in particular mammalian recombinant proteins, frequently results in the formation of insoluble aggregates known as inclusion bodies High cell density fermentation and purification of the recombinant protein from inclusion bodies of E coli are two major bottlenecks for the cost effective production of therapeutic proteins (Panda, A K, 2003, Adv Biochem Eng Biotechnol , 85, 43) Similarly, for research purposes, where hundreds of proteins may need to be screened for various activities, the expression of soluble, active protein is desirable, thereby avoiding the step of first purifying inclusion bodies and then having to denature and refold protein each separately [0003] Examples of the many pharmaceutically important proteins that form insoluble inclusion bodies when expressed in the cytoplasmic space of E coli include human Growth Hormone (hGH) (Patra, A K et al , 2000, Protein Expr Puπf, 18, 182, Khan, R H, et al , 1998, Biotechnol Prog , 14, 722), human Granulocyte-Colony Stimulating Factor (G-CSF)( Zaveckas, M et al 2007, J Chromatogr B Analyt Technol Biomed Life Sci 852, 409, Lee, A Y et al , 2003, Biotechnol Lett , 25, 205,) and Interferon alpha (IFN-alpha, Valente, C A et al , 2006, Protein Expr Puπf 45, 226) Furthermore, the immunoglobulin domains of antibodies and their fragments, including domain antibody fragments (dAb), Fv fragments, single-chain Fv fragments (scFv), Fab fragments, Fab'2 fragments, and many non-antibody proteins (such as FnIII domains) generally form inclusion bodies upon expression in the cytoplasm of bacterial hosts (Kou, G , et al , 2007, Protein Expr Purif 52, 131, Cao, P , et al 2006, Appl Microbiol Biotechnol , 73, 15 1, Chen, L H et al , 2006, Protein Expr Pun f , 46, 495 ) [0004] Human proteins typically fold using a hydrophobic core comprising a large number of hydrophobic amino acids Research has shown that proteins can aggregate and form inclusion bodies, especially when genes from one organism are expressed in another expression host, such that the protein's native binding partners are absent, so that folding help is unavailable and hydrophobic patches remain exposed This is especially true when large evolutionary distances are crossed a cDNA isolated from a eukaryote for example, when expressed as a recombinant gene in a prokaryote, has a high risk of aggregating and forming an inclusion body While the cDNA may properly code for a translatable mRNA, the protein that results will emerge in a foreign microenvironment This often results in misfolded, inactive protein that generally accumulates as aggregates if the concentration is high enough Other effectors, such as the internal microenvironment of a prokaryotic cell (pH, osmolaπty) may differ from that of the original source of the gene and affect protein folding Mechanisms for folding a protein may also be host-dependent and thus be absent in a heterologous host, and hydrophobic residues that normally would remain buried as part of the hydrophobic core instead remain exposed and available for interaction with hydrophobic sites on other proteins. Processing systems for the cleavage and removal of internal peptides of the expressed protein may also be absent in bacteria. In addition, the fine controls that may keep the concentration of a protein low will also be missing in a prokaryotic cell, and over-expression can result in filling a cell with protein that, even if it were properly folded, would precipitate by saturating its environment. [0005] The recovery of biologically active products from the aggregated state found in inclusion bodies is typically accomplished by unfolding with chaotropic agents or acids, followed by dilution or dialysis into optimized refolding buffers. However, many polypeptides (especially structurally complex oligomeric proteins and those containing multiple disulfide bonds) do not easily adopt an active conformation following chemical denaturation. [0006] Small changes in primary structure can affect solubility, presumably by altering folding pathways (Mitraki, A. et al. (1989) Bio/Technology 7, 690; Baneyx, F, et. al. 2004 Nat Biotechnol, 22, 1399; Ventura, S. 2005 Microb Cell Fact, 4, 11). In order to reduce the formation of insoluble aggregates during high-density fermentation, some groups have linked heterologous fusion proteins to the protein of interest. Examples of such fusion sequences are Glutathione-S-Transferase (GST), Protein Disulfide Isomerase (PDI), Thioredoxin (TRX), Maltose Binding Protein (MBP), His6 tag, Chitin Binding Domain (CBD) and Cellulose Binding Domain (CBD) (Sahadev, S. et al. 2007, MoI. Cell. Biochem.; Dysom, M.R. et al. 2004, BMC Biotechnol, 14, 32). In summary, these approaches were found to be protein-specific, as they do not work for all proteins. [0007] While various fusion proteins have been designed to improve folding, chemical PEGylation of proteins has also been reported to enhance protein solubility, reduce aggregation, reduce immunogenicity, and reduce proteolysis. Nonetheless, the proper folding of overproduced polypeptides remains problematic within the highly concentrated and viscous environment of the cell cytoplasm, where aggregation occurs in a concentration-dependent manner. Another approach for the expression of mammalian proteins in bacterial hosts avoids leader peptides and expresses the active protein directly in the cytoplasm of the host. However, this process tends to result in aggregation and inclusion body formation. [0008] One widely used approach for the expression of mammalian proteins in active form in bacteria is to direct the protein into the non-reducing environment of the periplasmic space of bacterial hosts such as E.coli, typically using signal- or leader-peptides to direct secretion. Secretion into the periplasm (and rarely into the media) appears to mimic the native eukaryotic process of protein secretion, folding and disulfide formation and often results in active protein.
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