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Wo 2008/153745 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 18 December 2008 (18.12.2008) WO 2008/153745 A2 (51) International Patent Classification: AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, C12N 15/62 (2006.01) C07K 14/435 (2006.01) CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, (21) International Application Number: IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, PCT/US2008/006593 LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, (22) International Filing Date: 22 May 2008 (22.05.2008) PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, (25) Filing Language: English SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 60/931,344 22 May 2007 (22.05.2007) US GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), (71) Applicant (for all designated States except US): AMGEN European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, INC. [US/US]; Patent Operations, M/S 28-2-C, One Am- FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, gen Center Drive, Thousand Oaks, CA 91320-1799 (US). NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, (72) Inventors; and CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): WALKER, Ken¬ neth, W. [US/US]; 175 Mesa Avenue, Newbury Park, CA Declaration under Rule 4.17: as to applicant's entitlement to apply for and be granted a 91320 (US). GEGG, Colin, V., Jr., [US/US]; 3357 Corn — ing Street, Newbury Park, CA 91320 (US). patent (Rule 4.17(U)) (74) Agent: STEINBERG, Nisan, A.; Patent Operations M/S Published: 28-2-C, One Amgen Center Drive, Thousand Oaks, CA — without international search report and to be republished 91320-1799 (US). upon receipt of that report — with sequence listing part of description published sep a (81) Designated States (unless otherwise indicated, for every rately in electronic form and available upon request from kind of national protection available): AE, AG, AL, AM, the International Bureau (54) Title: COMPOSITIONS AND METHODS FOR PRODUCING BIOACTIVE FUSION PROTEINS FIG. 12 ABC ABC (57) Abstract: Disclosed is a composition of matter involving a recombinant fusion protein comprising a a pharmacologically active protein partner, and a small pharmacologically inactive protein domain partner of human origin, such as but not limited to, a 10th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgGl, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, a villin headpiece HP76 domain, or a fragment or modification of any of these. Also disclosed are nucleic acids (e.g., DNA constructs) encoding the fusion protein, expression vectors and recombinant host cells for expression of the fusion protein, and pharmaceutical compositions containing the recombinant fusion protein and a pharmaceutically acceptable carrier, and method of producing a pharmacologically active recombinant fusion protein. COMPOSITIONS AND METHODS FOR PRODUCING BIOACTIVE FUSION PROTEINS [0001 ] This application claims the benefit of U.S. Provisional Application No. 60/931,344, filed May 22, 2007, which is hereby incorporated by reference in its entirety. [0002] Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. BACKGROUND OF THE INVENTION [0003] 1. Field of Art. [0004] The present invention relates to the biochemical arts, particularly to recombinant expression of polypeptides. [0005] 2. Discussion of Related Art. [0006] Bioactive or therapeutic peptides can be potent drugs which specifically target and modulate unique signaling and metabolic pathways. Their relatively small size and simple composition makes these peptides amenable to molecular engineering to refine and enhance desirable activities. Subtle changes to the peptide sequence can discriminate between linked activities or help prevent degradation in vivo. Similarly, well placed linker sites can permit conjugation of large molecules, such as poly(ethylene glycol) PEG, to enhance circulating half- lives. However, these same properties also present special challenges to peptide production and delivery. [0007] Artificial synthetic techniques are not cost-effective for producing many peptides, particularly the larger peptides (15-40 amino acid residues or more). As an alternative, the use of recombinant host cells is well known for recombinant production of bioactive peptides or proteins. Commonly used recombinant host cells include bacteria (such as E. coli sp.), yeast (such as Saccharomvces sp.) and other fungi, insect cells, plant cells, and mammalian cells in culture. However, recombinant expression is often difficult. One reason for the low expression of recombinant peptides or proteins is likely due to their poor refolding potential, owing to marginally stable secondary and tertiary structures in solution. [0008] To overcome this, many peptides have been expressed as chimeric fusions with proteins such as immunoglobulin Fc domains, ubiquitin, an albumin (e.g., human serum albumin (HSA)), a transthyretin (TTR), or a thyroxine-binding globulin (TBG). (See, e.g., Sullivan et al., Toxin Peptide therapeutic agents, WO 2006/1 16156 A2; Gegg et al., Modified Fc molecules,WO 2006/036834 A2; Gegg et al., Modified Fc molecules, PCT/US2006/031609; Feige et al., Modified peptides as therapeutic agents, WO 2000/024782; Rosen et al., Albumin fusion proteins, US Patent No. 6,926,898 and US 2005/005405 1; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, US 6,887,470); Walker et al., Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins, US 2003/0195154 Al; 2003/0191056 Al). Such large fusion proteins have made possible the commercial expression of therapeutic peptides and provided the added advantage of dramatically extending the circulating half-lives of their peptide partners, thereby rendering them more efficacious in vivo. [0009] While these fusion proteins often facilitate peptide expression at much higher levels, they can also present difficult refolding challenges that can affect their bioactivity. Protein recovery can be further complicated by undesirable domain-domain interactions between the fusion partners and disulphide bond isomerizations. In addition, the cost of producing a fusion protein with a large protein carrier moiety can affect the commercial viability of such a therapeutic agent. [0010] Consequently, compositions and methods for high yield recombinant expression of bioactive fusion proteins with a relatively low mass ratio of carrier component to bioactive component are desirable. These and other benefits are provided by the present invention. SUMMARY OF THE INVENTION [001 1] The present invention relates to compositions of matter involving recombinant fusion proteins. The inventive recombinant fusion protein includes: (a) a small pharmacologically inactive protein domain of human origin as described herein; and (b) a pharmacologically active protein. The present invention is also directed to nucleic acids (e.g., DNA constructs) encoding the fusion protein, and expression vectors and recombinant host cells for expression of the fusion protein. [0012] Optionally, for modulation of the pharmacokinetic profile of the inventive recombinant fusion protein molecule to fit a particular therapeutic need by attaching or conjugating covalently one or more half-life extending moieties of various masses and configurations to the fusion protein. Thus, the invention encompasses a composition of matter of the formula: [0013] (F')a-(X2)b (I) [0014] and multimers thereof, wherein: [0015] F1 is a half-life extending moiety, a is 0 or 1, and b is 1; 2 5 3 5 4 5 6 3 [0016] X is D-(L)c-(P )d-(X )e, (XV(P )d-(L)c-D, or (X ) (P )d-(L)c-D-(L)g-(P )h-(X ),, wherein c and g are each independently 0 or 1, d and h are 1, and e, f, and i are each independently is 0, 1, 2, 3, or 4; 3 7 [0017] X is -(L)j-(P ), j is 0 or 1; 4 8 [0018] X is (P )-(L)k-, k is 0 or 1; [0019] D is small pharmacologically inactive protein domain of human origin; [0020] P5, P6 , P7 and P8 are each independently a pharmacologically active protein; and [0021] L is in each instance a peptidyl linker. Within the meaning of Formula I, the pharmaceutically active protein, "P" (i.e., P5, P6 , P7 and P8), if more than one is present, can be independently the same or different from, any other P also present in the inventive composition; this includes a P7 and/or a P8, if more than one is present, which can be the same or different 7 8 from any other P and/or P . Similarly, the peptidyl linker moiety, "L" (i.e., (L)c, (L)g, (L)J; and/or (L)k), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. [0022] The present invention also provides a high efficiency method of producing a pharmacologically active fusion protein in a host cell.
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