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(12) Patent Application Publication (10) Pub. No.: US 2010/0239583 A1 MURTHY Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2010/0239583 A1 MURTHY Et Al US 2010O239583A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0239583 A1 MURTHY et al. (43) Pub. Date: Sep. 23, 2010 (54) ANTIBODIES AGAINST FLAGELLIN AND C07K I6/46 (2006.01) USES THEREOF CI2N 5/16 (2006.01) C7H 2L/04 (2006.01) (75) Inventors: KannegantiMURTHY, Stoneham, CI2N 5/3 (2006.01) MA (US); Andrew L. SALZMAN, AOIN 63/02 (2006.01) Herzliya (IL) AOIP I/00 (2006.01) A6IP3L/04 (2006.01) Correspondence Address: A6IP3L/08 (2006.01) LAHIVE & COCKFIELD, LLP A6IP3L/06 (2006.01) FLOOR 30, SUITE 3000 A6IPI3/02 (2006.01) ONE POST OFFICE SQUARE A6IP 7/00 (2006.01) BOSTON, MA 02109 (US) GOIN 33/53 (2006.01) (73) Assignee: NOTEKPHARMACEUTICALS CORPORATION, Beverly, MA (52) U.S. Cl. ................ 424/136.1; 530/388.4:530/389.5; (US) 530/387.9; 530/387.3; 530/388.15:435/340: 530/3917; 424/150.1; 424/164.1; 424/178.1; (21) Appl. No.: 12/717,351 536/23.53; 435/320.1; 435/7.1 (22) Filed: Mar. 4, 2010 (57) ABSTRACT Related U.S. Application Data The present invention provides a novel class of monoclonal (60) Provisional application No. 61/209,189, filed on Mar. antibodies which have a high affinity, broad spectrum neu 4, 2009. tralizing reactivity to flagellin from various Gram-negative O O bacteria including, but not limited to, E. coli, Salmonella, Publication Classification Serratia, Proteus, Enterobacter Citrobacter, Campylobacter (51) Int. Cl. and Pseudomonas. The present invention further provides A6K 39/395 (2006.01) methods of treating infections and diseases using anti-flagel C07K 6/12 (2006.01) lin antibodies in humans, other animals and birds. Patent Application Publication Sep. 23, 2010 Sheet 1 of 4 US 2010/02395.83 A1 mAb 743 anti-flagellin monoclonal antibody reacts strongly with Pseudomonas aeruginosa type A flagellins and Weakly with type B 1. PAKwt (type A) 2. PAK (flagellin mutant) 3. PAO wt (type B) - 4. PAO (flagellin mutant) M 4 3 2 1 M Fig. 1 Patent Application Publication Sep. 23, 2010 Sheet 2 of 4 US 2010/02395.83 A1 mAb 743 antibody cross-reacts with flagellins from various gram-negative bacteria O.800 | O.7OO- -- Proteus Vulgaris -(- Non Pathogenic E. Coli O600- -- Citobacter S. 0.500- -- Serratia marCeSCens s 0.400- -A - Pseudomonas aeruginosa 12691 g - A - Salmonella typhimurium 0.300- -O- Proteus mirabilis 0.2OO- -O- Enteropathogenic E. Coli 0.100 0000-- -T- 0.01 uglml 0.1 uglml 1 ugliml Fig. 2 Patent Application Publication Sep. 23, 2010 Sheet 3 of 4 US 2010/02395.83 A1 g.314 ?|N\/IV/OSOV/V/O,CIXIV/SN|?715)ST>ee Patent Application Publication Sep. 23, 2010 Sheet 4 of 4 US 2010/0239583 A1 mAb 743 inhibits flagellin activity in NO production assay 80 : FLAGELLIN FLAGELLIN+ 2 FLAGELLIN + Non mAb 743 Specific Mab 4060 2 y % % 2 O 2 32 % % 3 FLA (0.5 ng/ml) FLA (0.5 ng/ml) FLA (2.5 ng/ml) FLA (2.5 ng/ml) + 1 UAb + 5UAb + 1 ul Ab + 5u Ab F LA ( 9 | m Fig. 4 US 2010/02395.83 A1 Sep. 23, 2010 ANTIBODIES AGAINST FLAGELLIN AND including, but not limited to, gram-negative bacteria, Such as USES THEREOF E. coli, Salmonella, Serratia, Proteus, Enterobacter; Citro bacter; Campylobacter and Pseudomonas. Accordingly, the RELATED APPLICATION antibodies of the present invention can be used to treat, pre 0001. This application claims the benefit of U.S. provi vent and diagnose a variety of bacterial diseases associated sional application No. 61/209,189, filed Mar. 4, 2009, which with flagellin, including both infectious and non-infectious is incorporated by reference herein in its entirety. diseases in humans, other animals and birds. 0008 Antibodies of the invention generally are character GOVERNMENT FUNDING ized as having one or more of the following properties: (i) neutralization (i.e., inhibition) of bacterial flagellin, (includ 0002 This invention was made with government support ing flagellin bound to bacteria or “free', circulating flagellin under National Institutes of Health Grant it's R01 GM57407, in the systemic circulation); (ii) cross-reactivity with flagellin R43AI48249, R29GM54773 and R01 GM60699. The United from a broad spectrum of bacteria; (iii) inhibition of bacterial States government has certain rights in the invention. invasion into Susceptible epithelial cells; (iv) binding to flagellin with an affinity of at least 10'M', (v) reduction or BACKGROUND OF THE INVENTION prevention of flagellin-induced tissue injury; (vi) reduction or 0003. The human intestine is colonized by a large and prevention of flagellin-stimulated neutrophil infiltration; (vii) diverse population of commensal bacteria and, on occasion, is reduction or prevention of colonic mucosal congestion, ero exposed to potentially pathogenic bacteria. One particular sion and/or hemorrhagic ulcerations associated with IBD; subset of intestinal bacteria have flagella, which are whip-like and (viii) reduction or prevention of cytokine production, organelles that attach to a rotatory motor embedded in the including MDA, IL-1 B, TNFC, MIP-1, MIP-2, IL-6 and IL-8, bacterial cell wall. Flagella provide bacteria with motility and and pro-inflammatory free radical synthesizing enzymes, enable these microbes to reach, adhere and eventually invade such as the inducible nitric-oxide synthases; (ix) ability to or colonize a particular niche in their host. An individual opSonize bacteria; and (X) ability to promote macrophage flagellum is composed of approximately 20,000 subunits of ingestion of bacteria. U.S. patent application Ser. No. 12/231, the monomeric protein flagellin. Due to physical constraints 777, U.S. patent application Ser. No. 12/231,797, PCT Appli by its function, flagellin has a relatively conserved structure cation No.: PCT/US2008/075387, and PCT Application No. among widely diverse bacterial species (Steiner, T. S. Infect PCT/US2008/075383, teach anti-flagellin antibodies and all Immun. 2007 February: 75(2):545-52). are incorporated herein by reference in their entireties. 0004 Flagellin is highly antigenic and is a major immu 0009 From a structural standpoint, particular representa noglobulin target in a variety of infectious events (Sitaraman tive antibodies of the invention include a heavy chain variable et al., Am J Physiol Gastrointest Liver Physiol. 2005 Febru region comprising an amino acid sequence which is at least ary; 288(2):G403-6). As such, it is a potent and direct activa 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99%) identi tor of the innate immune system. From the perspective of the cal to the heavy chain variable region amino acid sequence set host, flagellin is a microbial-associated molecular pattern forth in SEQ ID NO:1. Other particular antibodies of the (MAMP) i.e., a microbial-associated determinant that can be present invention include a light chain variable region com perceived by the innate immune system, typically by pattern prising an amino acid sequence which is at least 80% (e.g., recognition receptors. Flagellin therefore serves as a danger 85%, 90%. 95%, 96%, 97%, 98% or 99%) identical to the signal across a wide variety of eukaryotes and is a potent light chain variable region amino acid sequence set forth in inducer of inflammatory effector responses in the mammalian SEQ ID NO:2. The antibodies may include a heavy chain gut (Neish, A. S., Am J Physiol Gastrointest Liver Physiol. only, a light chain only, or they may also include both of the 2007 February; 292(2):G462-6). aforementioned heavy chain and light chain variable regions. 0005 Specifically, upon detection of miniscule levels of 0010 Specifically, in one embodiment, the invention pro the monomeric protein flagellin, the mammalian germline vides an isolated monoclonal antibody that binds to flagellin encoded cell surface receptor Toll-like receptor 5 (TRL5) can comprising a heavy chain variable region comprising an directly promote a mucosal inflammatory response and trig amino acid sequence set forth in SEQID NO:1 (or an amino ger a massive induction of host gene expression designed to acid sequence at least 80% identical thereto). In another arm and protect the host against the invading microbe. The embodiment, the invention provides an isolated monoclonal resulting inflammatory cascade triggered by flagellin can be antibody that binds to flagellin comprising a light chain vari profound, causing clinical manifestations and tissue damage able region comprising an amino acid sequence set forth in (Gewirtz, A. T., Am J Physiol Gastrointest Liver Physiol. SEQID NO:2 (or an amino acid sequence at least 80% iden 2007 March; 292(3):G706-10). tical thereto). Inafurther embodiment, the invention provides 0006 Current treatments to offset the deleterious effects an isolated monoclonal antibody that binds to flagellin com of an inflammatory cascade stimulated by the flagellin prising a heavy and light chain variable region comprising the include anti-inflammatory drugs, immune system Suppres amino acid sequences set forth in SEQ ID NOs: 1 and 2. sors and other over-the-counter (OTC) drugs. These thera respectively (or amino acid sequences at least 80% identical pies, however, have clear drawbacks in that they are associ thereto). In yet another embodiment, the invention provides, ated with undesirable side effects and are merely palliative in an isolated monoclonal antibody that binds to flagellin and nature. Accordingly, improved agents and therapeutic treat comprises a heavy chain variable region comprising SEQID ments would be beneficial. NO:1, a light chain variable region comprising SEQID NO:2. or a combination thereof. SUMMARY OF THE INVENTION 0011. The variable heavy and light chain regions of the 0007. The present invention provides antibodies that bind antibodies typically include one or more complementarity to flagellin and neutralize a broad spectrum of bacteria determining regions (CDRS).
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