US010676721B2 United States Patent ( 10 ) Patent No.: US 10,676,721 B2 Collins et al. (45 ) Date of Patent : Jun . 9 , 2020 ( 54 ) BACTERIOPHAGES EXPRESSING 6,699,701 B1 3/2004 Sulakvelidze et al . ANTIMICROBIAL PEPTIDES AND USES 6,759,229 B2 * 7/2004 Schaak 435 /235.1 7,211,426 B2 5/2007 Bruessow et al. THEREOF 8,153,119 B2 4/2012 Collins et al. 8,182,804 B1 5/2012 Collins et al. ( 75 ) Inventors : James J. Collins, Newton , MA (US ) ; 2001/0026795 A1 10/2001 Merril et al . Michael Koeris , Natick , MA (US ) ; 2002/0013671 Al 1/2002 Ananthaiyer et al. 2004/0161141 A1 8/2004 Dewaele Timothy Kuan - Ta Lu , Charlestown, 2005/0004030 A1 1/2005 Fischetti et al . MA (US ) ; Tanguy My Chau , Palo 2007/0020240 A1 1/2007 Jayasheela et al. Alto , CA ( US ) ; Gregory 2007/0134207 Al 6/2007 Bruessow et al . Stephanopoulos , Winchester , MA (US ) ; 2007/0207209 A1 * 9/2007 Murphy A61K 9/06 Christopher Jongsoo Yoon , Seoul 424/484 (KR ) 2007/0248724 Al 10/2007 Sulakvelidze et al. 2008/0194000 Al 8/2008 Pasternack et al. ( 73 ) Assignees : Trustees of Boston University , Boston , 2008/0247997 Al 10/2008 Reber et al . MA (US ) ; Massachusetts Institute of 2008/0311643 A1 12/2008 Sulakvelidze et al. Technology , Cambridge, MA (US ) 2008/0318867 A1 12/2008 Loessner et al. FOREIGN PATENT DOCUMENTS ( * ) Notice: Subject to any disclaimer , the term of this patent is extended or adjusted under 35 EP 0403458 A1 12/1990 CO7K 7/10 WO 2002/034892 Al 5/2002 U.S.C. 154 ( b ) by 2166 days. WO 2004/046319 A2 6/2004 WO 2004/062677 Al 7/2004 (21 ) Appl. No .: 13 /224,776 WO 2005/009451 A1 2/2005 WO 2005/07 1088 A1 8/2005 ( 22 ) Filed : Sep. 2 , 2011 WO 2006/137847 A2 12/2006 (Under 37 CFR 1.47 ) WO 2008/110840 A1 9/2008 WO 2009/108406 A2 9/2009 (65 ) Prior Publication Data US 2015/0050717 A1 Feb. 19 , 2015 OTHER PUBLICATIONS Westwater et al. , Antimicrobial Agents and Chemotherapy, 2003 , Related U.S. Application Data vol. 47 , pp . 1301-1307 . * (63 ) Continuation of application No. Westwater et al. , Antimicrobial Agents and Chemotherapy, 2003 , PCT /US2010 /026357 , filed on Mar. 5 , 2010 . Vo147, pp . 1301-1307. * Horgan et al. , Applied and Environmental Microbiology, 2008, vol . (60 ) Provisional application No. 61/ 157,773 , filed on Mar. 75 , pp . 872-874 .* 5 , 2009 . Chap , Genbank accession No. YP_241096.1, published May 20 , 2007. * (51 ) Int. Ci. Clark et al ., Trends in Biotechnology , 2006 , vol. 24., pp . 212-218 . * C12N 7700 ( 2006.01 ) Takahara et al. , The Journal of Biological Chemistry , 1985 , vol . 260 , A61K 35/76 (2015.01 ) pp . 2670-2674 .* A61K 38/17 ( 2006.01) Sargent et al. , FEMS, 2006 , vol. 254 , pp. 198-207 .* COZK 14/435 ( 2006.01 ) (Continued ) COZK 14/46 ( 2006.01 ) C12N 9/50 ( 2006.01 ) Primary Examiner - Mindy G Brown (52 ) U.S. CI. ( 74 ) Attorney , Agent, or Firm - Nixon Peabody LLP CPC C12N 7700 (2013.01 ) ; A61K 35/76 ( 2013.01 ) ; A61K 38/1767 (2013.01 ) ; CO7K ( 57 ) ABSTRACT 14/43504 ( 2013.01 ) ; CO7K 14/43563 ( 2013.01) ; C07K 14/463 (2013.01 ) ; C12N The present invention is generally related to engineered 9/503 (2013.01 ) ; C12N 2795/10221 ( 2013.01 ) ; bacteriophages expressing antimicrobial peptides or lytic C12N 2795/10231 ( 2013.01 ) ; C12N enzymes or fragments thereof for targeting a broad spectrum 2795/10232 (2013.01 ) ; CI2N 2795/12032 of bacterial hosts , and for the long - term suppression of ( 2013.01) ; C12N 2795/12043 ( 2013.01 ) ; C12N bacterial phage resistance for reducing bacterial infections . 2795/14132 ( 2013.01) ; C12N 2795/14143 In some embodiments , bacteriophages express antimicrobial ( 2013.01 ) peptides or antimicrobial polypeptides ( e.g. phage lytic (58 ) Field of Classification Search enzymes ) which are secreted from the host bacteria , or None alternatively released upon lysis of the bacterial host cell . See application file for complete search history. Aspects of the present invention also relate to the use of the engineered bacteriophages for the reduction of bacterial (56 ) References Cited infections, both in a subject or for bioremediation purposes, in clinical settings and wound healing . U.S. PATENT DOCUMENTS 4,678,750 A 7/1987 Vandenbergh et al. 19 Claims, 30 Drawing Sheets 6,335,012 B1 1/2002 Fischetti et al. Specification includes a Sequence Listing . US 10,676,721 B2 Page 2 ( 56 ) References Cited Jensen et al. , Appl Environ Microbiol ., 64 ( 2 ) :575-80 ( 1998) . “ Preva lence of broad - host - range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa .” OTHER PUBLICATIONS Keller et al. , Nat Rev Microbiol. , 4 ( 4 ): 249-58 (2006 ). 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Huff et al. , Poultry Science , 83: 1944-1947 , (2004 ) . “ Therapeutic “ Targeted drug - carrying bacteriophages as antibacterial nanomedicines. ” efficacy of bacteriophage and Baytril ( enrofloxacin ) individually and in combination to treat colibacillosis in broilers ." * cited by examiner U.S. Patent Jun . 9 , 2020 Sheet 1 of 30 US 10,676,721 B2 17.CecP1+OmpA 9 TIMEHOURS() 1FIG. 17.CecP1-OmpA best. 782 17.Indol-OmpA 00.00( ABSORPTION U.S. Patent Jun . 9 , 2020 Sheet 2 of 30 US 10,676,721 B2 17.Cam-Omp 1wegduo- H moment IAND ........ 10 26 8 FIG.2 terhet weet wat*** SAUREUSGROWTHCURVEFOR17 ws w 0.6 U.S. Patent Jun . 9 , 2020 Sheet 3 of 30 US 10,676,721 B2 6 Co .FIG b . 2 . 1 1 ?? 1.2 U.S. Patent Jun . 9 , 2020 Sheet 4 of 30 US 10,676,721 B2 SA.1589.41.640ug/mL2 EC.J589.7.1924g/mliEC.1589.47.192ug /mL2 ECJ589.47.640ug/mL2 $4.3589.7.192ug/mL1 SA.J589.7.192ugmL2 $4,3589.41.640ugml1 EC.J589.47.640ugml1 tristewomano betoout more times en arbetet.wetter etconceitonei see ning oleei FIG.4 Z U.S.