1111111111111111 IIIIII IIIII 111111111111111 111111111111111 IIIII IIIII IIIII 1111111111 11111111 US 20190241665Al c19) United States 02) Patent Application Publication (10) Pub. No.: US 2019/0241665 Al KREEGER et al. (43) Pub. Date: Aug. 8, 2019 (54) METHODS OF INHIBITING METASTASIS IN C07K 16/24 (2006.01) CANCER C12N 15/113 (2006.01) A61K 31/439 (2006.01) (71) Applicant: WISCONSIN ALUMNI RESEARCH (52) U.S. Cl. FOUNDATION, Madison, WI (US) CPC .......... C07K 1612854 (2013.01); A61P 35/04 (2018.01); C07K 16/24 (2013.01); Cl2N (72) Inventors: PAMELA KAY KREEGER, 2310/14 (2013.01); C12N 15/1138 (2013.01); MIDDLETON, WI (US); MOLLY A61K 31/439 (2013.01); C07K 2317/76 JANE CARROLL, MADISON, WI (2013.01); C07K 16/2866 (2013.01) (US); KAITLIN C. FOGG, FITCHBURG, WI (US) (57) ABSTRACT (21) Appl. No.: 16/256,065 As described herein, a method of inhibiting metastasis in (22) Filed: Jan. 24, 2019 cancer includes administering to a human subject diagnosed with a cancer of an organ of the peritoneal cavity a thera­ Related U.S. Application Data peutically effective amount of an inhibitor of CCR5 or P-selectin. Preferably the subject has a tumor positive for a (60) Provisional application No. 62/621,769, filed on Jan. ligand of P-selectin such as a CD24+ or PSGL-1 + tumor. 25, 2018. Analysis of samples from HGSOC patients confirmed increased MIP-1 fJ and P-selectin, suggesting that this novel Publication Classification multi-cellular mechanism can be targeted to slow or stop (51) Int. Cl. metastasis in cancers such as high-grade serous ovarian C07K 16/28 (2006.01) cancer, for example by using anti-CCR5 and P-selectin A61P 35/04 (2006.01) therapies developed for other indications. Patent Application Publication Aug. 8, 2019 Sheet 1 of 36 US 2019/0241665 Al AltemativelyNactivated macrophage Ovarian cancer cell SELP Mesothelial cell ~ FIGURE 1 Pour and cure 1 PDMS 11 mm 14 mm FIGURE 2A Patent Application Publication Aug. 8, 2019 Sheet 2 of 36 US 2019/0241665 Al Seed mesothelial Differentiate Meso/MM cells AAMs co-culture (24 h) Adhesion (3 h) Ovarian cancer cell Cross-Section -I AAM Mesothelial Cell 11 mm FIGURE2B Patent Application Publication Aug. 8, 2019 Sheet 3 of 36 US 2019/0241665 Al Cl) -Q) (..) (/) -(l) (.) rn Q) ...,J::, 0 Cl) Q) 2 FIGURE 3A -MMS +MMS FIGURE 3B Patent Application Publication Aug. 8, 2019 Sheet 4 of 36 US 2019/0241665 Al * OV90 OVCAR5 EE1 -MMs • +AAMs FIGURE 3C Direct Indirect signaling signaling FIGURE 3D Patent Application Publication Aug. 8, 2019 Sheet 5 of 36 US 2019/0241665 Al 100 ~ Q) L. * Q) ..c -0 <( ~ 0 CaOV3 OV90 OVCARS C -AAMs a Direct • +AAMs m Indirect FIGURE3E 2 01 -0 ., -6------- -6 2 log(-AAMs) FIGURE 3F Patent Application Publication Aug. 8, 2019 Sheet 6 of 36 US 2019/0241665 Al * . .c () -0 -0 LL 0 + AAMs FIGURE 3G 10 * OV90 OVCARS mm lsotype ii P~Sel FIGURE 3H Patent Application Publication Aug. 8, 2019 Sheet 7 of 36 US 2019/0241665 Al 100 * * -0 (1) \._ Cl) .r::. "O <( '#. + + ,.. + CaOV3 OV90 OVCAR5 /• lsotype ii/ P--selectin Ab FIGURE 31 Patent Application Publication Aug. 8, 2019 Sheet 8 of 36 US 2019/0241665 Al -AAMs +AAMs ~o~~o5t.() LO 0°'>000'>>cu > ro > 0 0 O O 0 0 PDGFBB IL•1~ MIP-1a. MMP~12 GCSF RANTES Eotaxin TNF<x. IFNy 1Lw9 GM~CSF IL-8 Basic FGF IP-10 IL-17a IL~5 IL-4 IL-12p70 IL-10 IL-7 VEGF IL-6 MCP-1 IL .. 13 1L.. 1ra MIP .. 1f1 MMP-7 z-score -2 2 FIGURE4A Patent Application Publication Aug. 8, 2019 Sheet 9 of 36 US 2019/0241665 Al 100 -0 (],)c (],) Cl) .0 0 0 a 100 Predicted - + AAMs e CaOV3 A OV90 ■ OVCAR5 FIGURE4B ,,,i . MIP .. 1f3 .,,,, IL•13 11 % Adhered :::::, r--::::r,-· -+-----ii-----i PC 1 PC2 FIGURE4C VIP 0 N IL-1 ra IL-13 MIP-1p RANTES IL-8 IL-5 PDGFBB IL-17a Eotaxin IL'"9 IFNv a~ MMP--1 ~ TNFa rn .i;;:. GCSF u IL-10 ~ :::::::::::::::1 IP-10 Basic FGF MM~1it : IL-1p MIP-1a ILM+12p70 ILM06 ~, -<-<-xi MCP ... 1 IL "' 7 F~b ·.:~::::::::::"""""""'::::::::<I~·:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.: .. VEGF IV S99lt'Z0/6JOZ Sil 9£ JO OJ J.l.lQS 6JOZ '8 ·~nv UOffBJ!{qlld UO!JBJ!{ddy JU.lfBd Patent Application Publication Aug. 8, 2019 Sheet 11 of 36 US 2019/0241665 Al * * * lso IL 13 PDGF MlP ra -AAMs • + AAMs FIGURE4E 100 -0 * * 0) * '- (l) .c 1J <( ';$!.0 0 Isa IL 13 PDGF MIP li3 -AAMs • +AAMs FIGURE4F Patent Application Publication Aug. 8, 2019 Sheet 12 of 36 US 2019/0241665 Al A B 5 1 E]J R2Y • a2v N>- a PC1 ~ N 6 0:: -5 1 2 Component PC2 Ca0V3 OVCAR5 C 100 100 * * * "O (1) ~ (1) .c "O <( * * * 0~ lso IL13PDGF MIP lso IL13PDGF MIP IEJ -AAMs Ill +AAMs D E 100 :J' "O 60!400 .€0) ~ * 0.. (1) ---20 .c ~ 5 "O ~ <( I a. -::R.0 ~ CaOV3 OV90 OVCAR5 li3 Veh fl;! MIP-1~ +AAMs FIGURES Patent Application Publication Aug. 8, 2019 Sheet 13 of 36 US 2019/0241665 Al ct.12 * aj Cl) . ..c: (.) -0 0 LL. 0 -AAMs + AAMs 1• lsotype B;/a.J MIP-1f3 Ab FIGURE6A Patent Application Publication Aug. 8, 2019 Sheet 14 of 36 US 2019/0241665 Al 1.5 * 0 -AAMs +AAMs .,. lsotype I MIP-.1 p Ab FIGURE6B Patent Application Publication Aug. 8, 2019 Sheet 15 of 36 US 2019/0241665 Al 20 o~-~~~~~"·--~~~~ 0 1 2 3 1() 10 . 10 10 10 BL 1-H (CD62p) FIGURE 6C A 3 a.. * ~ .c () 32 0 LL 0 0 1 10 100 MIP-1 p (ng/mL) Patent Application Publication Aug. 8, 2019 Sheet 16 of 36 US 2019/0241665 Al FIGURE6D 100 * 1J (D "- (l) .c * 1J <{ 0~ 0 ..........-.. ..... MIP-1 f~ - + + + CaOV3 OV90 OVCAR5 /lll DMSO Bl- KF38789 FIGURE6E Cl 3 iD * Cf) ..c• A t) "O -0 LL. O lsotYpe._,.,-CCR5 Ab Eil Veh - MIP-1~ Patent Application Publication Aug. 8, 2019 Sheet 17 of 36 US 2019/0241665 Al FIGURE6F * ,. !: () "O -0 LL 0 DMSO Maraviroc II Veh fll MIP~1f1 FIGURE6G 5 min 20 min 60 min 240 min FIGURE6H Patent Application Publication Aug. 8, 2019 Sheet 18 of 36 US 2019/0241665 Al ct4 * GJ (/) . ..c t) A. "O A -0 0 LL oMSo LY PD & Veh rmaEGI M*Pf -JJ1A FIGURE 61 ~"-= -=('D ~- 'e -(') ~-· -0=-· "-= =-0- Thr202/Tyr204 Ser473 Thr308 -(') ~-· 0.8 Thr185ffyr187 7 0.3 * -0-· ~ * = a:: ~ ~ w ~ ~ I, !* ___ .,.-.,,.-~--! ~ _.,,.~,,,#-" t ~ r " j..... ·---.... _j ~ t ct: ~ ~ ~ <( *~--- ;;& _______________, __ I w w--::~---~,..JB N a., ---~'%"'., - " - ,. ;!I! ~ a.. ...,.-'3! 0 ,».-.$(.,_- ·i~•·---~--"-·'-'"---111 o__ o "°- Time (min) 240 Time (min) 240 Time (min) 240 r,;_ ('D=­ ('D --ffll---· Veh - -• - MIP-113 - "°-0 t.H FIGURE 6J -O'I cr,;_ N 0 "°-0 -N ~ O'I -O'I u. -> Patent Application Publication Aug. 8, 2019 Sheet 20 of 36 US 2019/0241665 Al A lsotype MIP-1p Ab B 3 (/1 2: Q oj * ~ Cl) .c () '"O 0 fl) LL ~ ~ + lilllll Veh F2J' MIP•1 ~ C LP-9 HUVEC 100 : lsotype eo ""'''"' Veh -IL-4 ao 40 20 ------ BL 1·H (CD62p)------ FIGURE7 Patent Application Publication Aug. 8, 2019 Sheet 21 of 36 US 2019/0241665 Al CaOV3 OV90 OVCAR5 : t1\~ ' 1n1j' ·I()(< ·1 . ·•' lsolype ~ 1\ ~ -CD162 H I i ~ J\ l l ,!t t i \ I,,I \ © I \ D i I lj i \ j f 1 * I \ I \ ~ i \ .1 I ~1 ! l I 1\ 1 / \ ,, !j ., ' ;:, I ~ :< ., ~~ ·, j(,. 1:1 H) J!"J 11/ HJ 1:) .. ,;. H:: ·:rt 1U 1/ ·::/ H} HJ 1 ~i ::": 1/' FIGURE SA CaOV3 OV90 OVCAR5 (/) LO ~ Cl t.) FIGURE 8B Patent Application Publication Aug. 8, 2019 Sheet 22 of 36 US 2019/0241665 Al 4 © R2=0. 9321 C)C: 3 CaOV3,, .cco ,. ,.,,,,. .... "" t-'\ 2 OV90,,,,.,.. V QVCAR5...,., ..... "' 9 OVCAR3 32 ' OVCAH4 0 1 OVCAR8 LL 0 o 500 1000 1500 Norm. Median Fl FIGURE 8C 100 * "O Cl) \... Cl) .c ~ 'cf2. Veh MIP-1f3 Ill siC mtlB siCD24 FIGURE 8D Patent Application Publication Aug. 8, 2019 Sheet 23 of 36 US 2019/0241665 Al OA --0-- Veh er(]) -a- M!P~1f3 I... IJ.. (]) 0:: fa'.'J 0.0 0 2000 Speed (i.tmfrnin) FIGURE SE * FIGURE 8F Patent Application Publication Aug. 8, 2019 Sheet 24 of 36 US 2019/0241665 Al (1) Q. "¢ oa>,N Cl) (..) Ill I ..-... 'q" 'q" N ,❖ Cl 0:: (:, <( -u.._.. (.) J: > I 0 _,~ ca 8 '""" peJe4p\:f % <( Ill Patent Application Publication Aug. 8, 2019 Sheet 25 of 36 US 2019/0241665 Al A B CD24 DAPI AF488 OAP! ('<') > 0 8 0 0 0) OJ > 6 0 If) tr) n:: 0:: <( <( 0> 0 ~ FIGURE 10 Relative Frequency Distribution at 25 ~tUmin 0 -C 0.25 .Q ** ...... vc 0 -....,ttf... 0.20 + M!P18 200 400 Speed (J.tm/sec) FIGURE 11 Patent Application Publication Aug. 8, 2019 Sheet 26 of 36 US 2019/0241665 Al ...... vc .,. MIP1B 200 400 600 800 Speed (µm/sec) FIGURE 12 5 .s(l.) * Cl) .c (.) "O 0 u... 0 Peritoneal Wall Mesentery EE Vehicle li3 MIP-1 p FIGURE 13A Patent Application Publication Aug. 8, 2019 Sheet 27 of 36 US 2019/0241665 Al Peritoneal Wall Omentum Mesentery FIGURE 13B Patent Application Publication Aug. 8, 2019 Sheet 28 of 36 US 2019/0241665 Al u -C'f'i Patent Application Publication Aug. 8, 2019 Sheet 29 of 36 US 2019/0241665 Al FIGURE 14A Vehicle MIP-1~ 500 * N E .......0 (/) 0) (.) VehiclMIP-1 f) FIGURE 14B Patent Application Publication Aug.