US 2005O23917OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0239170 A1 Hedley et al. (43) Pub. Date: Oct. 27, 2005 (54) ALPHA-MSH RELATED COMPOUNDS AND filed on Aug. 18, 2000. Provisional application No. METHODS OF USE 60/238,380, filed on Oct. 6, 2000. Provisional appli cation No. 60/258,764, filed on Dec. 29, 2000. Pro (76) Inventors: Mary Lynne Hedley, Lexington, MA visional application No. 60/298,317, filed on Jun. 14, (US); Robert G. Urban, Lexington, 2001. MA (US); Nazneen Aziz, Lexington, MA (US); Hongmin Chen, Publication Classification Framingham, MA (US); Bijan Etemad-Moghadam, Jamaica Plain, (51) Int. Cl." ....................... C07K 14/685; CO7H 21/04; MA (US); Peng Yin, Newton, MA (US) C12N 15/09; A61K 38/27 (52) U.S. Cl. ..................... 435/69.4; 435/320.1; 435/325; Correspondence Address: 530/399; 514/12; 536/23.5 FISH & RICHARDSON PC P.O. BOX 1022 ABSTRACT MINNEAPOLIS, MN 55440-1022 (US) (57) Appl. No.: 09/906,206 The invention provides polypeptides containing C-MSH that (21) can be used to treat diseases characterized by inflammation Filed: Jul. 16, 2001 and/or autoimmunity. Also included in the invention are (22) C-MSH analogs and nucleic acids encoding polypeptides Related U.S. Application Data containing C-MSH and C-MSH analogs optionally linked to heterologous Sequences. Also included in the invention are (60) Provisional application No. 60/218,381, filed on Jul. methods of delivering C-MSH containing peptides, C-MSH 14, 2000. Provisional application No. 60/226,382, analogs, an DNA encoding C-MSH and C-MSH analogs. Patent Application Publication Oct. 27, 2005 Sheet 1 of 29 US 2005/0239170 A1 IHuegVVL IHSS{H *IIIpUIH Patent Application Publication Oct. 27, 2005 Sheet 7 of 29 US 2005/0239170 A1 SE Y s Sa D 4. C g s s Patent Application Publication Oct. 27, 2005 Sheet 8 of 29 D-]©CIILIGJ‘IVNOIS Patent Application Publication Oct. 27, 2005 Sheet 9 of 29 US 2005/0239170 A1 9 OIH`S Patent Application Publication Oct. 27, 2005 Sheet 10 of 29 US 2005/0239170 A1 000L00090009000;000€000Z000l() (TW10J)HSWWHATW 9'OIH 3d |() S(){} Patent Application Publication Oct. 27, 2005 Sheet 11 of 29 US 2005/0239170 A1 M LIHLOVAWOd O C 9|-|HLOV-AWOd e > 35 2. N 22 ti-IHLov-AWod & CD 2. 2 2 t g OWOdzulu-AWOd 2 21 s ld ROS-AWOd 3 / < t g/ lf C r O Patent Application Publication Oct. 27, 2005 Sheet 12 of 29 US 2005/0239170 A1 Li-IHLOV-AWOd s 9| EHLOW-ANOd vi-IHLOW-AWOd Se s SSSS S sesse Nassa,SSSS as a creasNessa Patent Application Publication Oct. 27, 2005 Sheet 13 of 29 US 2005/0239170 A1 XZ-SEINIG HSWSS () ---- HSWSS-AWOdHSWSS-OXZd| SLOÎTRILSN00 Patent Application Publication Oct. 27, 2005 Sheet 14 of 29 US 2005/0239170 A1 |tuffd000ç<(6H)()ITH 0['OIH Patent Application Publication Oct. 27, 2005 Sheet 15 of 29 US 2005/0239170 A1 HSWSS XC-Sglid HSWSS-OAZd HSWSS-AWOd ld ROSAWOd (6n01) HSW WHdTV r NITWS Patent Application Publication Oct. 27, 2005 Sheet 16 of 29 US 2005/0239170 A1 I I HSWSSHSWSS-OXZd XC-S3Rd HSWSS-AWOd ld ROSAWOd (6nO) HSW WHdTW 5 NITWS Patent Application Publication Oct. 27, 2005 Sheet 17 of 29 US 2005/0239170 A1 (6H)OTH S6 HSd OWO UW Patent Application Publication Oct. 27, 2005 Sheet 18 of 29 US 2005/0239170 A1 K. - Patent Application Publication Oct. 27, 2005 Sheet 19 of 29 US 2005/0239170 A1 L - IHLOW 9- HE) W 7- HW YC w Ca C Patent Application Publication Oct. 27, 2005 Sheet 20 of 29 US 2005/0239170 A1 Patent Application Publication Oct. 27, 2005 Sheet 21 of 29 US 2005/0239170 A1 -- CP S is 29 s () Qu > N A. lf wu ... - O CD S d t 29 2. < A4 5. rir r (a wry C Patent Application Publication Oct. 27, 2005 Sheet 22 of 29 US 2005/0239170 A1 5 s a r ym 2 C 5 Patent Application Publication Oct. 27, 2005 Sheet 23 of 29 US 2005/0239170 A1 3C aha r -la. r ra SF Sir c n CNru-1 re- S. we' s Patent Application Publication Oct. 27, 2005 Sheet 24 of 29 US 2005/0239170 A1 SAWO WAWO I 4 8. WWC i at M CE AWO | RWCI O AWO 6C AWO 8&AWO LCAWO 9C KWO SAWO 7. KWO AWO KWO KWO 0. KWO 6. KWO 8 KWO L. KWC 9 Avg St AWO 7. KWO 9 KWC AWO AWO 8 KWC s Patent Application Publication Oct. 27, 2005 Sheet 25 of 29 US 2005/0239170 A1 c H O <C | | || NCG t - Q-9 e A II. st O O vu aC A. C e t w D d 2. aC C/ vu 7 R CN N. r Patent Application Publication Oct. 27, 2005 Sheet 27 of 29 US 2005/0239170 A1 HSWESTHId\,001'0IH(§§){HLI Patent Application Publication Oct. 27, 2005 Sheet 28 of 29 US 2005/0239170 A1 | H s PC S2 st s US 2005/0239170 A1 Oct. 27, 2005 ALPHA-MSH RELATED COMPOUNDS AND 11-13 inhibits LPS-mediated brain inflammation by prevent METHODS OF USE ing inactivation of I-KB and subsequent activation of NF-kB (Ichiyama et al. (1999) Brain Res. 836:31). NF-kB is a CROSS REFERENCE TO RELATED transcription factor that is necessary for the transcription of APPLICATIONS proinflammatory cytokines, including yIFN (Baeuerle et al. 0001. This application claims priority from U.S. Provi (1994) Ann Rev Immunol. 12:141). sional Application No. 60/218,381, filed Jul 14, 2000, U.S. SUMMARY OF THE INVENTION Provisional Application No. 60/226,382, filed Aug. 18, 0006 The invention is based on the discovery that fusion 2000, U.S. Provisional Application No. 60/238,380, filed polypeptides containing C-MSH can be used to elicit a Oct. 6, 2000, U.S. Provisional Application No. 60/258,764, variety of biological responses, in vitro and in Vivo. filed Dec. 29, 2000, and U.S. Provisional Application No. 60/298,317, filed Jun. 14, 2001. These applications are 0007. In one aspect, the invention features a polypeptide, incorporated herein by reference in their entirety. or a nucleic acid Sequence encoding a polypeptide, wherein the polypeptide contains an C-MSH concatamer. The FIELD OF THE INVENTION polypeptide can further include a trafficking Sequence, e.g., a signal Sequence. In one embodiment, the Signal Sequence 0002 The invention relates to alpha-MSH compounds contains the pro-opiomelanocortin (POMC) signal Sequence and methods of use. or a portion thereof that directs the Secretion of the polypep tide when expressed in a mammalian cell. Alternatively, the BACKGROUND trafficking Sequence can direct the C-MSH concatamer to an 0003) Pro-opiomelanocortin (POMC) is a precursor of endoSome, nucleus, or a lysosome. In another embodiment, various bioactive peptides, including adrenocorticotropic the trafficking sequence can direct secretion of the C-MSH hormone (ACTH) and alpha MSH (C-MSH). C-MSH and COncatamer. ACTH are members of the melanocortin family, which also 0008. A polypeptide containing an C-MSH concatamer includes -MSH and Y-MSH. POMC contains eight pairs of can further include a linker between two C-MSH units of the basic amino acids and one Sequence of four basic amino C-MSH concatamer. The linker can optionally include a acids, which are the Sites of cleavage for the enzymes protease cleavage Site, e.g., a protease cleavage Site Specific prohormone convertase 1 (PC1) and prohormone convertase for a cell associated protease or a Serum protease. 2 (PC2). Specifically, PC1 cleaves the POMC polypeptide to yield ACTH(1-39), which is in turn cleaved by PC2 to yield 0009. A polypeptide containing an C-MSH concatamer ACTH(1-17), which is further cleaved by PC2 to result in can further include a membrane Sequence. The membrane ACTH(1-14). A 13 amino acid C-MSH peptide is generated Sequence can include the membrane domain or a portion of by the action of peptidylglycine alpha-amidating monooxy the membrane domain of a naturally occurring protein, e.g., genase, which results in a C-terminally amidated C-MSH a human protein, e.g., the transferrin receptor. The polypep peptide. ACTH(1-17) contains the consensus amidation Sig tide optionally includes a linker between the C-MSH con nal of Gly-Lys-Lys immediately carboxy the valine residue catamer and the membrane Sequence. The linker can include at position 13. This valine residue is Subject to amidation in a protease cleavage Site. The polypeptide can further include C-MSH and ACTH peptides. Further enzymatic modifica a signal Sequence and/or a cytoplasmic domain. tions including N-alpha-acetylation by opiomelanotropin 0010) A polypeptide containing an C-MSH concatamer acetyltransferase can occur during or after proteolytic pro can further include a glycosylphosphatidylinositol (GPI) cessing. C-MSH and ACTH can be produced in amidated or attachment Signal peptide. The polypeptide optionally non-amidated forms, as well as des-acetylated, mono-acety includes a linker between two C-MSH units of the C-MSH lated or di-acetylated forms. C-terminal amidation and/or concatamer. The linker can contain a protease cleavage Site. N-terminal acetylation may contribute to the biological 0011. In one embodiment, a polypeptide containing an activity of C-MSH and/or ACTH peptides. C-MSH concatamer can further include a therapeutic 0004 Under normal conditions the level of C-MSH is polypeptide. Examples of therapeutic polypeptides include tightly regulated, having a half-life in the circulation on the C-gliadin, basement membrane collagen, collagen, albumin, order of a few minutes (Lipton et al.