US009074015B2 (12) United States Patent (10) Patent No.: US 9,074,015 B2 Lancaster et al. (45) Date of Patent: Jul. 7, 2015 (54) RECOMBINANTLY EXPRESSED INSULIN 5,922,675 A 7, 1999 Baker et al. POLYPEPTIDES AND USES THEREOF 33. A E. s al. SO (75) Inventors: Thomas M. Lancaster, Stoneham, MA 6,034,054 A 3/2000 DeFelippis et al. (US); Sylaja Murikipudi, Medford, MA 1932 R 38: SE tal (US); Rassol Laleau, Woburn, MA 6,323,3114. r. I B1 1 1/2001 Liujeldsen et al. et al. (US); Todd C. Zion, Marblehead, MA 6,342,225 B1 1/2002 Jones et al. (US) 6.410,053 B1 6/2002 Taylor (73) Assignee: SMARTCELLS, INC., Whitehouse 39SR. 339; its at Station, NJ (US) 6,551992 B1 4/2003 DeFelippis et al. (*) Notice: Subject to any disclaimer, the term of this 6,777.207 B2 8/2004 Kjeldsen et al. patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 13/811,910 (22) PCT Filed: Jul. 22, 2011 CN 1O1273961 10, 2008 EP O09842 4f1980 (86). PCT No.: PCT/US2O11AO4SOO8 (Continued) S371 (c)(1), (2), (4) Date: Jan. 24, 2013 OTHER PUBLICATIONS UniProtKB/Swiss-Prot Accession No. PO1314 (accessed Aug. 20, (87) PCT Pub. No.: WO2012/015692 2014 at URL uniprot.org/uniprot/P01314).* PCT Pub. Date: Feb. 2, 2012 UniProtKB/Swiss-Prot Accession No. C9JNR5 (accessed Aug. 20. O O 2014 at URL uniprot.org/uniprot/C9JNR5).* (65) Prior Publication Data UniProtKB/Swiss-Prot Accession No. P30410 (accessed Aug. 20, US 2013/O190476A1 Jul 25, 2013 2014 at URL uniprot.org/uniprot/P30410).* • 1-a-s Zion, Bringing the Cure Home, Slideshare.net, PowerPoint presen tation-2-1319001 (Apr. 6, 2009). Related U.S. Application Data Chu, Smart Insulin, MIT Technology Review, p. 2. (Oct. 30, 2008). (60) Provisional application No. 61/368,607, filed on Jul. Baudys, et al., “Physical Stabilization of Insulin by Glycosylation'J 28, 2010. Pharma Sci (1995) 64:28-33. Brownlee & Cerami, "A Glucose-Controlled-Insulin-Delivery-Sys (51) Skuo (2006.01) tem: Semisynthetic Insulin Bound to Lectin' Diabetes (1983) 32:499-504. A61 K38/28 (2006.01) (Continued) (52) U.S. Cl. CPC C07K 14/62 (2013.01); A6 IK 38/28 (2013.01) (58) Field of Classification Search Primary Examiner — Julie Ha CPC .......... ... SES 66. SS ,% Assistant Examiner — Kristina M Hellman See application file for complete search history (74) Attorney, Agent, or Firm — Immac J. Thampoe; John David Reilly (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 3.591,574 A 7, 1971 Fenichel et al. The present disclosure provides recombinantly expressed 3,684,791 A 8/1972 Geiger et al. insulin polypeptides that comprise an N-linked glycan motif. 3,847,890 A 11/1974 Green et al. The N-linked glycan motif is not present in wild-type insulins 4,348,387 A 9, 1982 Brownlee et al. and enables the recombinant expression of glycosylated insu 4,372,948 A 2f1983 Yoshikumi et al. lin polypeptides (e.g., in yeast cells). Based on results 4,377.567 A 3, 1983 Geho 4,444,683 A 4, 1984 Kim et al. obtained with synthetic glycosylated insulin conjugates we 4,603,044 A 7, 1986 Geho et al. predict that when these recombinant glycosylated insulin 4,863,896 A 9, 1989 Geho et al. polypeptides are administered to a mammal, at least one 5,239,062 A 8, 1993 Battler et al. pharmacokinetic or pharmacodynamic property of the glyco 5,395,924 A 3, 1995 Battler et al. 5,461,031 A 10/1995 De Felippis Sylated insulin polypeptide will be sensitive to serum concen 5,478,575 A 12/1995 Miyazaki et al. trations of glucose (or an exogenous saccharide Such as alpha 5,527,524. A 6, 1996 Tomalia et al. methyl mannose). Exemplary insulin polypeptides, 5,563,056 A 10, 1996 Swan et al. polynucleotides encoding these insulin polypeptides, glyco 5,723,589 A 3/1998 Miljkovic et al. Sylated insulin polypeptides, pharmaceutical formulations 5,830,506 A 1 1/1998 Taylor 5,843,886 A 12/1998 Weiner et al. and Sustained release formulations are provided in addition to 5,854.208 A 12/1998 Jones et al. methods of use and preparation. 5,866.538 A 2/1999 Norup et al. 5,902,607 A 5/1999 Taylor 5,905,140 A 5, 1999 Hansen 7 Claims, 50 Drawing Sheets US 9,074.015 B2 Page 2 (56) References Cited WO WO2006/082184 8, 2006 WO WO2006/088473 8, 2006 U.S. PATENT DOCUMENTS WO WO2006,102762 10, 2006 WO WO2007/042470 4/2007 6,844,166 Bl 1/2005 Wolf WO WO2007/043050 4/2007 6,869,930 B1 3, 2005 Havelund et al. WO WO2008/O12440 1, 2008 RE39,055 E 4, 2006 Jones et al. WO WO2008/O12528 1, 2008 7,063,863 B2 6, 2006 Taylor WO WO2008/036147 3, 2008 7,087.408 B2 8, 2006 Kjeldsen et al. 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No. 13/145.532 dated Sep. 15, 2014. WO WOO3/0489.15 6, 2003 Response to Office Action for U.S. Appl. No. 13/145.532 dated Dec. WO WOO3,O74087 9, 2003 4, 2014. WO WO2004/057 002 T 2004 WO WO2006/0O8238 1, 2006 * cited by examiner U.S. Patent Jul. 7, 2015 Sheet 1 of 50 US 9,074,015 B2 o GlcNAc: N-Acetylglucosamine NANA: N-Acetylneuraminic Acid OH HO OH HO-20 "S-HO NH OH l?o Os, H0 OH DMon: Monnose (2 Gal: Galactose O Glo: Glucose OH OH HO-a--0 OH HO OH HO s OH HO l OH HO OOH FIG. 1A GlcNAc2Mans GlcNAc2Mang GlcNAc2Man5(GlcNAc2 -- -- GlcNAc)Man5 -: GlcNAc2Man5(GlcNAc20al -- GlcNAC2 Mong-100 GlcNAc)Man5 -- . GlcNAc2Man5(GlcNAc20al?NANA2 GlcNAc2Mon3 ". GlcNAc2Man5(GlcNAc GlcNAc2Mang Glc3 --- -- GlcNAc2Man5(GlcNAc -- -- FIG. 1B U.S. Patent Jul. 7, 2015 Sheet 3 Of 50 US 9,074,015 B2 Endoplasmic Reticulum Golgi GlcNAc2MangGlc3 GlcNAc)Mang -- O-E. Glucosidose J. 1,6-MnT Glucosidase I GlcNAc2Mang GlcNAc2Mang N -- Series of 1,6-MnT and Transport to GlcNAC2 Mong-100 1,2-MnT Golgi 6. in S. cerevisiae, not in P. pastoris FIG.3 U.S. Patent Jul. 7, 2015 Sheet 4 of 50 US 9,074,015 B2 A0ch 1p Gene Endoplasmic Reticulum GlcNAc2MangGlc3 Glucosidose Glucosidase I GlcNAc2ManB GlcNAc2Man5 P. pastoris does not have an a-1,3-MnS FIG.4A U.S.