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Vectors for Expression of Antimicrobial Peptides in Mammary Gland

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Vectors for Expression of Antimicrobial Peptides in Mammary Gland (19) TZZ _T (11) EP 2 257 569 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/47 (2006.01) A61P 1/00 (2006.01) 01.10.2014 Bulletin 2014/40 A61P 31/04 (2006.01) C12N 15/85 (2006.01) A01K 67/027 (2006.01) A61P 11/00 (2006.01) (2006.01) (2006.01) (21) Application number: 09720751.8 A61P 31/10 C12N 15/86 A23C 3/00 (2006.01) A61P 15/14 (2006.01) C07K 19/00 (2006.01) C12N 15/861 (2006.01) (22) Date of filing: 13.03.2009 A23C 19/097 (2006.01) A61P 27/16 (2006.01) C12N 15/63 (2006.01) C12N 15/869 (2006.01) A61K 38/17 (2006.01) A61P 31/00 (2006.01) C12N 15/82 (2006.01) (86) International application number: PCT/AU2009/000301 (87) International publication number: WO 2009/111838 (17.09.2009 Gazette 2009/38) (54) Vectors for expression of antimicrobial peptides in mammary gland Vektoren zur Expression von antimikrobiellen Peptiden in der Milchdrüsse Vecteurs pour l’expression de peptides antimicrobiennes dans la glande mammaire (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A1-95/30751 WO-A1-96/32482 HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL WO-A1-98/08534 WO-A1-03/091437 PT RO SE SI SK TR WO-A1-2007/106951 WO-A2-2005/033274 WO-A2-2005/033281 WO-A2-2005/077046 (30) Priority: 13.03.2008 AU 2008901249 WO-A2-2007/142542 (43) Date of publication of application: • DONOVAN D M ET AL: "Peptidoglycan hydrolase 08.12.2010 Bulletin 2010/49 fusions maintain their parental specificities", APPLIED AND ENVIRONMENTAL (73) Proprietor: Agriculture Victoria Services PTY MICROBIOLOGY, AMERICAN SOCIETY FOR Limited MICROBIOLOGY, US, vol. 72, no. 4, 1 April 2006 Attwood, VIC 3049 (AU) (2006-04-01), pages 2988-2996, XP002582772, ISSN: 0099-2240, DOI: DOI:10.1128/AEM. (72) Inventors: 72.4.2988-2996.2006 • COCKS, Benjamin • BROADBENT J R ET AL: "NISIN INHIBITS Viewbank, Victoria 3084 (AU) SEVERAL GRAM-POSITIVE MASTITIS- CAUSING • SPANGENBERG, German PATHOGENS", JOURNAL OF DAIRY SCIENCE, Bundoora, Victoria 3083 (AU) AMERICAN DAIRY SCIENCE ASSOCIATION, US, • WANG, Jianghui vol. 72,no. 12,1 January 1989 (1989-01-01), pages Bundoora, Victoria 3083 (AU) 3342-3345, XP009149724, ISSN: 0022-0302 (74) Representative: Marshall, Cameron John Carpmaels & Ransford LLP One Southampton Row London WC1B 5HA (GB) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 257 569 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 257 569 B1 • HOEBEN D ET AL: "Effect of Bovine • RECIO I ET AL: "Protective effect of milk Somatotropin on Neutrophil Functions and peptides: Antibacterial and antitumor Clinical Symptoms During Streptococcus uberis properties", ADVANCES IN EXPERIMENTAL Mastitis", JOURNAL OF DAIRY SCIENCE, MEDICINE AND BIOLOGY, SPRINGER, US, vol. AMERICAN DAIRY SCIENCE ASSOCIATION, US, 606, 1 January 2008 (2008-01-01), pages 271-293, vol. 82, no. 7, 1 July 1999 (1999-07-01), pages XP009149736, ISSN: 0065-2598 1465-1481, XP026993631, ISSN: 0022-0302 • DONOVAN, D. M. ET AL.: ’Peptidoglycan [retrieved on 1999-07-01] hydrolase fusions maintain their parental • SPARO M D ET AL: "In vitro efficacy of the novel specificities’ APPLIED AND ENVIRONMENTAL peptide CECT7121 against bacteria isolated from MICROBIOLOGY vol. 72, no. 4, 2006, pages 2988 mastitic dairy cattle", LETTERS IN APPLIED - 2996 MICROBIOLOGY, WILEY-BLACKWELL • BROADBENT, J. R. ET AL.: ’Nisin inhibits several PUBLISHING LTD, GB, vol. 48, no. 2, 1 January gram-positive, mastitis-causing pathogens’ 2009 (2009-01-01), pages 187-193, XP007918966, JOURNAL OF DAIRY SCIENCE vol. 72, no. 12, ISSN: 0266-8254, DOI: DOI:10.1111/J.1472-765X. 1989, pages 3342 - 3345 2008.02507.X [retrieved on 2009-01-05] • HOEBEN, D. ET AL.: ’Effect of bovine • ROBERT J WALL ET AL: "Genetically enhanced somatotropinon neutrophil functions and clinical cows resist intramammary Staphylococcus symptoms during Streptococcus uberis mastitis’ aureus infection", NATURE BIOTECHNOLOGY, JOURNALOF DAIRY SCIENCE vol. 82, no. 7, 1999, NATURE PUBLISHING GROUP, NEW YORK, NY, pages 1465 - 1481 US, vol. 23, no. 4, 1 July 2005 (2005-07-01), pages • SPARO, M. D. ET AL.: ’In vitro efficacy of the novel 445-451,897, XP007918965, ISSN: 1087-0156, peptide CECT7121 against bacteria isolated from DOI: DOI:10.1038/NBT1078 [retrieved on mastitic dairy cattle’ LETTERS IN APPLIED 2005-04-03] MICROBIOLOGY vol. 48, no. 2, 2009, pages 187 - • DALYET AL: "Identification, characterization and 192 expression of cathelicidin in the pouch young of • WALL, R.J. ET AL.: ’Genetically enhanced cows tammar wallaby (Macropus eugenii)", resist intramammary Staphylococcus aureus COMPARATIVE BIOCHEMISTRY AND infection’ NAT. BIOTECHNOL. vol. 23, no. 4, 2005, PHYSIOLOGY. PART B, BIOCHEMISTRYAND pages 445 - 451 MOLECULAR BIOLOGY, ELSEVIER,OXFORD, GB, vol. 149, no. 3, 11 January 2008 (2008-01-11), pages 524-533, XP022453792, ISSN: 1096-4959, DOI: DOI:10.1016/J.CBPB.2007.12.002 • WANG J ET AL: "Mammary gland and innate immunity of the tammar wallaby", TISSUE ANTIGENS, MUNKSGAARD, COPENHAGEN, DK, vol. 66, no. 5, 1 January 2005 (2005-01-01), page 583, XP003023789, ISSN: 0001-2815 2 EP 2 257 569 B1 Description Field of the invention 5 [0001] The present invention relates to antibacterial peptide reagents and methods employing same for the treatment of microbial disease(s), in particular microbial disease(s) mediated in part of whole by one or more bacteria and/or fungi. Background of the invention 10 [0002] Human and animal health are valuable commercial sectors and bacterial and fungal pathogenic infections in humans, livestock and domestic pets represent a high cost to these sectors in terms of lost productivity and existing treatment costs. Many bacterial and fungal pathogens of diseases in humans, livestock animals and domestic pets are also recalcitrant to treatment with existing antibiotics, further exacerbating these adverse consequences of infection. [0003] For example, the economic value of the dairy industry worldwide is significant. For example, the International 15 Dairy Foods Association estimates that sales of cow milk in USA alone in 2006 was USD 23.9 billion. This value will be significantly increased when considered on a worldwide scale, and expanded to include all dairy products, e.g., cheese and butter, and to include sales of products from all major animal producers of dairy products, e.g., goats, sheep, camels and buffalo. The pharmaceutical and biotechnology industries have also developed an interest in dairy mammals as suitable bioreactors for the production of biological agents, particularly peptides and proteins. In this respect, the com- 20 bination of large daily protein output, post-translational processing capabilities, ease of access to recombinant protein by milking and low capital cost of production plants, i.e., farms compared to high volume industrial fermenters makes dairy mammals excellent candidates for the production of recombinant peptides and proteins (Echelard, Curr. Opin. Biotechnol., 7: 536-540, 1996). [0004] Mastitis is currently the most economically important disease of dairy mammals (Pyörälä Reprod. Dom Anim., 25 37: 211-216, 2002 and Bergonier et al., 34: 689-716, 2003). The annual costs of mastitis in dairy cattle alone is estimated to be about 10% of the total sales of farm milk, i.e., about USD 2 billion in USA alone(Radostits et al.,: Veterinary Medicine: A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, Ninth Ed., Elsevier Health Sciences, 2000). In this respect, the costs of mastitis extend beyond treatment and prevention costs and include losses in milk production, labor costs and loss of animals due to culling. Other effects of mastitis include the impact of agricultural use 30 of antibiotics that are used to treat mastitis on the development of antibiotic resistant human pathogens (Smith et al., Proc. Natl. Acad. Sci., USA 99: 6434-6439, 2002) and the effect of residues of these antibiotics on human health (Clement Anim. Pharm., 407: 22-23, 1998). Moreover, the limited milk production resulting from mastitis limits the utility of mammals as bioreactors for producing pharmaceutical agents. [0005] Mastitis is an inflammatory reaction of the mammary gland, usually to microbial infection. This condition is 35 characterized by an influx of somatic cells, primarily polymorphonuclear neutrophils (PMN), into the mammary gland, and by an increase in milk protease content (Verdi et al., J. Dairy Sci., 70: 230-242, 1987). Mastitis is generally classified into clinical infections and non-clinical infections. Clinical infections are diagnosed by red, swollen appearance of a mammary gland and flakes or clots (protein aggregates) in the milk. Sub-clinical infections show no obvious symptoms. The majority of cases of mastitis are caused by one or more ofStaphylococcus aureus, Streptococcus dysgalactiae, 40 Streptococcus agalactiae, Streptococcus uberis or Escherichia coli. In this respect, S. aureus, S. dysgalactiae and S. agalactiae have a contagious route of transmission, whereas S. uberis and E. coli are environmental pathogens (Kerr and Wellnitz, J. Anim. Sci., 81: 38-47, 2003). [0006] Susceptibility of a mammal to an intra-mammary infection leading to mastitis dramatically increases during early involution and during the periparturient period (Nickerson J. Dairy Sci., 72: 1665-1678, 1989; and Oliver and Sordillo, 45 J. Dairy Sci., 71: 2584-2606, 1988). These infections are often associated with clinical mastitis during early lactation, and can have a marked detrimental effect on subsequent milk yield and/or quality.
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