US 20070134261A1 US 20070134261A1 (19) United States (12) Patent Application Publication oo) Pub. No.: US 2007/0134261 A l Hancock et al. (43 ) Pub. Date: Jun. 14,2007 (54) EFFECTORS OF INNATE IMMUNITY which is a continuation-in-part of application No. 10/308,905, filed on Dec. 2, 2002. (76) Inventors: Robert E.W. Hancock, Vancouver (CA); B. Brett Finlay,Richmond (CA); (60) Provisional application No. 60/336,632, filed on Dec. Monisha Gough Scott, Portland, OR 3, 2001. (US); Dawn Bowdish, Marston (GB); Carrie Melissa Rosenberger, Seattle, Publication Classification WA (US); Jon-Paul Steven Powers, Vancouver (CA); Jie Yu, Vancouver (51) Int. Cl. (CA); Neeloffer Mookherjee, A61K 39/02 (2006.01) Vancouver (CA) C12Q 1/68 (2006.01) (52) U.S. Cl........................... 424/185.1; 435/6; 424/234.1 Correspondence Address: DFA PIPER US FFP (57) ABSTRACT 4365 EXECUTIVE DRIVE The present invention provides a method of identifying SUITE 1100 agents that enhance innate immunity in a subject. The SAN DIEGO, CA 92121-2133 (US) invention further provides a method of selectively supress- (21) Appl. No.: 11/400,411 ing sepsis by suppressing expression of a proinflammatory gene while maintaining expression of an anti-inflammatory (22) Filed: Apr. 7, 2006 gene. Also provided are methods of identifying a polynucle­ otide or pattern of polynucleotides regulated by one or more Related U.S. Application Data sepsis or inflammatory inducing agents and inhibited by a peptide is described, methods of identifying a pattern of (63) Continuation-in-part of application No. 11/241,882, polynucleotide expression for inhibition of an inflammatory filed on Sep. 29, 2005, which is a continuation-in-part or septic response, and compounds and agents identified by of application No. 10/661,471, filed on Sep. 12, 2003, the methods of the invention. Patent Application Publication Jun. 14, 2007 Sheet I of 22 US 2007/0134261 A l 25000 20000 15000 1000 0 - Average Average CFU/ral 5000 - 2.5 mg/kg 10 mg/kg S. aureus 0.1 mg/kg SEQ ID NO: 7 SEQ ID NO: 7 0.1 mg/kg Cefepime + 2.5 Cefepime + + S. aureus + S. aureus mg/kg SEQ ID NO: 7 S. aureus + S. aureus Treatment Group - Concentration FIG. I Patent Application Publication Jun. 14, 2007 Sheet 2 of 22 US 2007/0134261 A l Pqitide ERK-P Jl-actin FIG. 2A ERK-P SOOO 0 KAnIpqrtiA 1Ie1WjFCS 4000 Intensity 50 KAnlpqrtide.Iim FCS 3000 2000 1000 0 Donortfl Donorffi Donorffi FIG. 2B P38-P O KAnlpq7AA l IOV j FCS Intensity 50 KAnlpqitiA. 10¾¾ FCS Dourfl Dcncr #2 Oconeiffi FIG. 2C Patent Application Publication Jun. 14, 2007 Sheet 3 of 22 US 2007/0134261 A l 80000 70000 +10% FCS 60000 Serum free 50000 LLJZ ^ g /m l LL-37 40000 150 μς/ΓΠΐ LL-37 Intensity 30000 20000 10000 serum free 10% FCS 10% HS FIG. 3 Patent Application Publication Jun. 14, 2007 Sheet 4 of 22 US 2007/0134261 A l 50000 40000 30000 (ϋ Donor 1 ■ Donor 2 20000 10000 50 50 50 50 DC m edia FCS IL-4 GM-CSF only 100ng/ml 100ng/ml FIG. 4 Patent Application Publication Jun. 14, 2007 Sheet 5 of 22 US 2007/0134261 A l 1000 I 800 600 0 pg/ml SEQ ID NO: 1 IL-8 IL-8 pg/ml 400 50pg/ml SEQ ID NO: 1 200 apical basolateral FIG.5A Vi "ω a XJ ω TO φ t"· CO I to(I) TO s? O C 35 O U- I MCP-1 IL-6 IL-8 MCP-3 FIG. 5B Patent Application Publication Jun. 14, 2007 Sheet 6 of 22 US 2007/0134261 Al B 2-5 2 η ZO 1.5 1 -I 0.5 0 0.0 0 0.5 1 2 5 10 20 50 LL-37 μς/πιΙ cP FIG. 6A FIG. 6B 3.5 -I 3 g 250 E 2.5 'W'C 2 a b. 1.5 Z 1 Z 100 H 0.5 ■ C L FIG. 6C FIG. 6D Patent Application Publication Jun. 14,2007 Sheet 7 of 22 US 2007/0134261 A l 0.8 0.6 0.8 - 0.4 0.6 0.4 TNF-α TNF-α (ng/ml)0.2 - 0.2 I 2 4 24 hr 1 2 4 24 h r FIG. 7A FIG. 7B % Inhibition of TNF-a 1 hr 2 hr 4 hr 24 hr A Simultaneous 90 95 97 97 B Delayed 15 54 58 80 C Pre-treatment 64 35 35 24 0.8 0.6 0.4 1 2 4 24 h r FIG. IC Patent Application Publication Jun. 14, 2007 Sheet 8 of 22 US 2007/0134261 A l 3.0 Ί i r i r 1.2 LPS LTA 2.0 0.8 JXL 0.4 1.0 0.0 0.0 12η 18η LPS LTA 1 2 0.6| Control CpG 0.8 0.4 24 24 hr 4 hr 0.4 Concentration (ng/ml) 0.2 0.0 L 0.0 LL TNF-α N/A IL-ip IL-6 IL-8 TNF-a IL-Ιβ IL-6 IL-8 TNF-a FIG. 8 Patent Application Publication Jun. 14, 2007 Sheet 9 of 22 US 2007/0134261 A l 1 hr 2 hr 4hr 24 hr S n ' + LPS m m m OiftjTf0 + LPS + LL-37 FIG. 9A + LPS 24 W fy ri O '■M <0 O) O 7 ΝΡ-κΒ-related genes 5 NF-icB-related genes 4 NF-xB-related genes I I 24 1 24 1 O ε OCD —I 11 NF-ieB-rolated genes 1 2 4 24 No. DE Total No. NF-κΒ Targets Genes No. clusters Clusters with NF k B targets BIRC2, CD80, IL6, KRT3, PTAFR1 LPS 561 15 3 BIRC3, CCL2T CD44, CD83. CFLAR, CXCL2,IER3, ILIA, NFKBIA, NFKBI, RELB, PTGS2,TNF1 TNFAIP2, TNFAIP3, FUCAl LPS + BCL2A1. BIRC3, CCL3, CCLI9, LL-37 410 S 2 CCL2, CXCL2, IER3, NFKBIA, RELB, SOD2, TNFAIP3, TRAFl FIG. 9B Patent Application Publication Jun. 14, 2007 Sheet 10 of 22 US 2007/0134261 A l 10 RELB 1 100 1,000 100 TNFAIP2 IL-10 100 100 1,000 100 IL-8 1CXCLI 100 Relative Relative fold change 100 100 TNIP3 100 NFKBIAi 10 1 2 4 24 hr 1 2 4 24 hr 1 2 4 24 hr FIG. 10 Patent Application Publication Jun. 14, 2007 Sheet 11 of 22 US 2007/0134261 A l LL-37 RELB cREL FIG. IlA neg cells + LL-37+ LPS + LPS + LPS + LL37 + Inh FIG. IlB Patent Application Publication Jun. 14, 2007 Sheet 12 of 22 US 2007/0134261 Al LL-37 TNF-a LL-37 TLR-4 Membrane m lL lL MyD88 B-4A TRAM MAPK IRAK LL-37 TRAF Ιν Ν Λ Λ Λ Λ Nucleus Deeradation I TNF-α I INFAIP2 1 ► I-------- — ► NFk BIA ---- TNFAIP3 I NFkBIA ELK-I & other TFs FIG. 12 Patent Application Publication Jun. 14, 2007 Sheet 13 of 22 US 2007/0134261 A l fCtORA · * > : ISTAT31 I du^ed paliMay; FIG. 13 Patent Application Publication Jun. 14, 2007 Sheet 14 of 22 US 2007/0134261 A l N1N2N3N* N1N2N3N4 Relative Relative Fold Change Ni he re Nt mrcrcm N1N2N3N4 Bdogjcal replicates: Monocytes isdatedfrom fdr incfvidual donors FIG. 14 Patent Application Publication Jun. 14, 2007 Sheet 15 of 22 US 2007/0134261 Al C l] Control ■ SEQ 7 NI N2 N3 N4 NI N2 N3 N4 Biological replicates: Tissue culture supernatants from PBMC of four individual donors FIG. 15 Relative Relative Fold Change (pg/ml)IL-6 NI N2 N3 N4 N1N2N3N4 N I h C W M Biological replicates: MonotytesisolatBdftqTifourincividuaI donors FIG. 16 Patent Application Publication Jun. 14, 2007 Sheet 16 of 22 US 2007/0134261 A l ■ LPS (2 ng/ml) P LPS + SEQ 7 J NI N2 N3 Biological replicates: Tissue culture supernatants from PBMC of three individual donors FIG. 17A B 900 -| 800 £ 700 Ui 600 a 500 a 400 U- 300 Z I- 200 100 0 Untreated LPS SEQ 7 LPS+SEQ 7 FIG. 17B Patent Application Publication Jun. 14, 2007 Sheet 17 of 22 US 2007/0134261 A l Total DE genes: 681 LL37 ^59719 SEQ ID NO: 7 DE and DE and m statistically statistically significant gene v^gnificant genes FIG. 18 Total-ΙκΒ α GAPDH 0 0.5 I 2 3 6 h LL-37 FIG. 19 Patent Application Publication Jun. 14, 2007 Sheet 18 of 22 US 2007/0134261 A l IL-6 release from PBMCs 2000 1600 1200 800 IL-6 IL-6 (pg/ml) 400 0 .Z f * V ΛΓ vV. * v VP sV VvV FIG. 20A MCP-3 release from PBMCs —1200 1900 Q « 600 I O 300 § 0 V o sV nV V FIG. 20B Patent Application Publication Jun. 14, 2007 Sheet 19 of 22 US 2007/0134261 A l P B M C s ΐ3 & β φ ρ-ΙκΒα (41 kDa) GAPDH (36kDa) C trl IL-1 β LL-37 comb C trl IL-1 β LL-37 com b FIG. 21A Ctrl IL-1 β LL-37 comb ctrl IL-1 P LL-37 comb I 2 *. * * 'I V ·-‘ I" p50/105 ν ύ& ά β ίφ * fi Λ ei^ 5tV i "eT THP-1 cells f* * * .··:■■ · . · '1T:. ■>··■.'. .··?', »7>·|'.;··· :· .V'j*· .·· *u I ·-..■· *·■·. ·· ' .·: \ · ; v .V. , ., · · . λ. ^ P50/105 Human PBMCs C trl IL-Ip LL-37 comb ctrl IL-1 P LL-37 comb 30 60 FIG. 21B Patent Application Publication Jun. 14, 2007 Sheet 20 of 22 US 2007/0134261 A l 0 -LY294002 MCP-3 production in PBMCs 1 +LY294002 1200 800 MCP-3 (p g/m400 l) * \V V «vV λ* FIG.