USOO750757OB2 (12) United States Patent (10) Patent No.: US 7,507,570 B2 Prabhakar et al. (45) Date of Patent: Mar. 24, 2009 (54) RECOMBINANT CHONDROITINASE ABC I 6,184,023 B1 2/2001 Hashimoto et al. AND USES THEREOF 6,217,863 B1 4/2001 Godavarti et al. 6,597,996 B1 7/2003 Venkataraman et al. (75) Inventors: Vikas Prabhakar, Cambridge, MA 6,869,789 B2 3/2005 Liu et al. (US); Ishan Capila, Ashland, MA (US); 6,962,699 B2 * 1 1/2005 Pojasek et al. ............. 424,945 Rahul Raman, Arlington, MA (US); 29:W w- R 3. Silvaasisekharan et al. Carlos Bosques, Cambridge, MA (US); 7,105,334 B2 9/2006 Pojasek et al. Kevin Pojasek, Cambridge, MA (US); 7,110,889 B2 9/2006 Venkataraman et al. Ram Sasisekharan, Bedford, MA (US) 7,117,100 B2 10/2006 Venkataraman et al. 7,129,335 B2 10/2006 Pojasek et al. (73) Assignee: Massachusetts Institute of Technology, 7,139,666 B2 11/2006 Venkataraman et al. Cambridge, MA (US) 7,247.445 B2 7/2007 Sasisekharan et al. 7,270,815 B2 9/2007 Sasisekharan et al. (*) Notice: Subject to any disclaimer, the term of this 2002/O122793 A1 9, 2002 Liu et al. patent is extended or adjusted under 35 2002/O128225 A1 9, 2002 Liu et al. 2002fO169143 A1 11/2002 Sasisekharan et al. U.S.C. 154(b) by 0 days. 2003, OOO8820 A1 1/2003 Kwan et al. 2003.0099628 A1 5/2003 Liu et al. (21) Appl. No.: 11/078,915 2003/O191587 A1 10/2003 Venkataraman et al. 2004/0091471 A1 5/2004 Myette et al. (22) Filed: Mar 10, 2005 2004/0091472 A1 5/2004 Pojasek et al. 2004/0092037 A1 5/2004 Sasisekharan et al. (65) Prior Publication Data US 2006/0078959 A1 Apr. 13, 2006 (Continued) FOREIGN PATENT DOCUMENTS Related U.S. Application Data EP O 576 294 A2 12, 1993 (60) Provisional application No. 60/552,232, filed on Mar. EP O 613 949 A2 9, 1994 10, 2004, provisional application No. 60/578,917, JP 20000446O1 A 2, 2000 filed on Jun. 10, 2004, provisional application No. WO WO 94,25567 A1 11F1994 60/625,052, filed on Nov. 3, 2004. WO WO 96,01894 A1 1, 1996 WO WO97, 16556 A1 5, 1997 (51) Int. Cl. Continued CI2N 9/88 (2006.01) (Continued) CI2N 9/00 (2006.01) OTHER PUBLICATIONS CI2N 15/70 (2006.01) Huang et al. Crystal structure of Proteus Vulgaris chondroitin Sulfate CI2O I/68 (2006.01) ABC 1yase I at 1.9A resolution. DJ Mol Biol. May 2, CI2P 19/28 (2006.01) 2003:328(3):623-34.* CI2P 2/06 (2006.01) Michel et al. The structure of chondroitin B lyase complexed with C7H 2L/04 (2006.01) glucosaminoglycan oligosaccharides unravels a calcium-dependent (52) U.S. Cl. ....................... 435/232; 435/183; 435/200; catalytic machinery, J Biol Chem. Jul 30, 2004:279(31):32882-96. 435/6: 435/18: 435/85; 435/69. 1; 435/320.1; Epub May 21, 2004.* 536/23.2; 424/94.5 (Continued) (58) Field of Classification Search ................. 435/200, 435/18, 85, 69.1, 320.1; 536/23.2 Primary Examiner Rebecca E Prouty See application file for complete search history. Assistant Examiner Iqbal H Chowdhury (74) Attorney, Agent, or Firm Wolf, Greenfield & Sacks, (56) References Cited P.C. U.S. PATENT DOCUMENTS (57) ABSTRACT 5,198.855 A 3, 1993 Iwai 5,292,509 A 3/1994 Hageman The invention relates to chondroitinase ABC I and uses 5,496,718 A 3, 1996 Hashimoto et al. thereof. In particular, the invention relates to recombinant and 5,498,536 A 3, 1996 Khandke modified chondroitinase ABC I, their production and their 5,525,500 A 6, 1996 Khandke et al. uses. The chondroitinase ABC I enzymes of the invention are 5,578.480 A 11/1996 Khandke useful for a variety of purposes, including degrading and 5,716,617 A 2f1998 Khandke et al. analyzing polysaccharides Such as glycosaminoglycans 5,741,692 A * 4, 1998 Khandke et al. ............ 435/200 5,763,205 A 6, 1998 Hashimoto et al. (GAGs). These GAGs can include chondroitin sulfate, der 5,773,277 A 6, 1998 Hashimoto et al. matan Sulfate, unsulfated chondroitin and hyaluronan. The 5,888,798 A 3, 1999 Lotvin et al. chondroitinase ABC I enzymes can also be used in therapeu 5.997,863 A 12, 1999 Zimmermann et al. tic methods such as promoting nerve regeneration, promoting 6,001,630 A 12/1999 Ichikawa et al. stroke recovery, treating spinal cord injury, treating epithelial 6,007,810 A 12/1999 Ishikawa et al. disease, treating infections and treating cancer. 6,054,569 A 4/2000 Bennett et al. 6,093,563 A 7/2000 Bennett et al. 20 Claims, 27 Drawing Sheets US 7,507,570 B2 Page 2 U.S. PATENT DOCUMENTS No Author Listed NCBI database, Structure Summary 1DBO, Crystal Structure of Chondroitinase B, MMDB No. 12452. Deposi 2004/O197933 A1 10/2004 Venkataraman et al. tion date Nov. 3, 1999, 1 page. 2004/0204869 A1 10/2004 Venkataraman et al. No Author Listed Protein Data Bank, Structure Explorer - 1DBO, 2005/0O3.7376 A1 2/2005 Sasisekharan et al. "Crystal Structure of Chondroitinase B” http://www.rcbs.org/pdb/ 2005, 0118157 A1 6, 2005 McMahon et al. cgi/explore.cgi/ex;ore.cgi?pdb.Id=1DBO, Deposition dated Nov. 3, 2005/0227320 A1 10/2005 Pojaseket al. 1999; Release date Jan. 12, 2000; printed Feb. 23, 2005, 1 page. 2005/0233402 A1 10/2005 Liu et al. No Author Listed Structure Summary Prinout for 1dbo, http:// 2005/0233419 A1 10/2005 Pojaseket al. pdbbeta.rcsb.org/pdb/explore.do?structureId=1dbo, Deposition date 2006.0024664 A1 2, 2006 Sasisekharan et al. Nov. 3, 1999; Release date Jan. 12, 2000; printed Feb. 23, 2005, 1 2006/0057638 A1 3/2006 Bosques et al. page. 2006, OO67927 A1 3/2006 Chandrasekaran et al. No Author Listed MMDB Summary Printout for 1HM2, http:// 2006, OO67928 A1 3, 2006 Liu et al. pdbbeta.rcsb.org/pdb/explore.do?structureId=1hm2, Deposition 2006, OO78959 A1 4/2006 Prabhakar et al. date Dec. 4, 2000; Release date May 2, 2001; printed Feb. 23, 2005, 2006, 0083711 A1 4/2006 Berry et al. 1 pages. 2006, O10543.0 A1 5/2006 Sasisekharan et al. No Author Listed Protein Data Bank, Structure Explorer - 1HM2, 2006/O12795.0 A1 6/2006 Bosques et al. "Active Site of Chondroitinase Ac Lyase Revealed by The Structure 2006, O154337 A1 7/2006 Sato et al. of Enzyme-Oligosaccharide Complexes and Mutagenesis'. http:// 2006, O154894 A1 7/2006 Berry et al. www.rcsb.org/pdb/cgi/explore.cgi?pdb.Id=1HM2, Deposition date 2006/0177885 A1 8/2006 Myette et al. Dec. 4, 2000; Release date May 2, 2001; printed Feb. 23, 2005, 1 2006/017791.0 A1 8/2006 Myette et al. page. 2006/0177911 A1 8/2006 Myette et al. No Author Listed NCBI Database, Structure Summary for 1HM2, 2006, O182734 A1 8, 2006 Liu et al. Crystal Structure of Chondroitinase B, MMDB No. 16157. Deposi 2006, O183713 A1 8, 2006 Liu et al. tion date Dec. 4, 2000; 1 page. 2006, O183891 A1 8/2006 Myette et al. No Author Listed MMDB Summary Printout for 1 plu, “Pectate 2006/0233782 A1 10, 2006 Gruskin et al. Lyase CFrom Erwinia ChrysanthemiWith 1 LU+3 Ion in the Putative 2006, O292130 A1 12/2006 Sasisekharan et al. Calcium Binding Site'. http://pdbbeta.rcsb.org/pdb/explore. 2006/0292655 A1 12/2006 Sasisekharan et al. do?structureId=1plu, Deposition date Jun. 4, 1999; Release date Jul. 2006/029.2673 A1 12/2006 Sasisekharan et al. 13, 1999; printed Feb. 23, 2005, 1 page. 2007,0004012 A1 1/2007 Sasisekharan et al. No Author Listed NCBI database, Structural Summary for 1DBG, 2007/0020243 A1 1/2007 Sengupta et al. Crystal Structure of Chondroitinase B, MMDB No. 12451. Deposi 2007/0065424 A1 3/2007 Pojasek et al. tion date Nov. 2, 1999, 1 page. 2007/0065921 A1 3/2007 Sasisekharan et al. No Author Listed Structure Summary Printout for 1dbg. http:// 2007/0066769 A1 3/2007 Venkataraman et al. pdbbeta.rcsb.org/pdb/explore.do?structureId=ldbg. Deposition date 2007/O148157 A1 6, 2007 Prabhakar et al. Nov. 2, 1999; Release date Jan. 12, 2000; printed Feb. 23, 2005, 1 2007. O1481.58 A1 6/2007 Sasisekharan et al. page. 2007. O14874.0 A1 6, 2007 Prabhakar et al. No Author Listed Protein Data Bank, Structure Explorer - 1DBG, 2007/O161073 A1 7/2007 Sasisekharan et al. "Crystal Structure of Chondroitinase B'. http://www.rcsb.org/pdb/ 2007/0202563 A1 8, 2007 Prabhakar et al. cgi/explore.cgi?pdb.Id=1DBG, Deposition date Nov. 2, 1999; 2007/0224670 A1 9, 2007 Prabhakar et al. Release date Jan. 12, 2000; printed Feb. 23, 2005, 1 page. No Author Listed NCBI database, Protein 1HNOA Chain A. Crystal FOREIGN PATENT DOCUMENTS Structure of Chondroitinase Abc Lyase I From Proteus Vulgaris. At 1.9 Angstroms Resolution, gi: 30749254. BTC Oct. 1, 2007, 6 pages. WO WOOOf 12726 A2 3, 2000 No Author Listed NCBI database, "Pedobacter Heparinus WO WOOOf 65521 A2 11/2000 Chondroitinase B Precursor (cs1B) gene Complete cols'. Accession WO WOO1/35977 A2 5, 2001 No. U27584.1 GI: 1002526. BCT Jan. 6, 2000, 2 pages. WO WOO1,66772 A2 9, 2001 No Author Listed NCB database, “Sequence 2 from patent US WO WOO2,23190 A2 3, 2002 5578480”. Accession No. AAB43331; PAT Feb. 7, 1997, 1 page.