FATE NK-100: A novel NK cell-mediated cancer therapy

1 RESULTSFATE NK-100: A novel NK cell-mediated cancer therapyFrank Cichocki1, BahramValamehr2, Ryan Bjordahl2, Bin Zhang1, Betsy Rezner2, Paul Rogers2, Svetlana Gaidarova2, Stacey Moreno2, Katie Tuininga1, Phillip Dougherty1, Valarie McCullar1, Peter Howard1, DhifafSarhan1, Emily Taras1, Heinrich Schlums3, Stewart Abbot2, Daniel Shoemaker2, YenanT. Bryceson3, Bruce R. Blazar4, Scott Wolchko2, Sarah Cooley1, Jeffrey S. Miller1; 1 Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota Masonic cancer Center, Minneapolis, MN. 2 Fate Therapeutics, San Diego, CA, USA, 3 Centre for Hematology and Regenerative Medicine, Department of Medicine, KarolinskaInstitutet, KarolinskaUniversity Hospital Huddinge, Stockholm, Sweden, 4 Department of Pediatrics, University of Minnesota cancer Center, Minneapolis, MN.

2 Correspondence (A)ShownisfoldNKcellexpansionafter7daysi neachcultureconditionrelativetoday0(n=15 ).(B)RepresentativeFACS scatterplotsofCD57andNKG2 CexpressionongatedCD3-CD56+NKcellsfrombe foreandafterculture.(C)Cumulative(n=15)d atashowingthepercentages(leftpanel)ofCD5 7+NKcellsandthemeanfluorescenceintensity ofCD57gatedonCD57+NKcells(rightpanel)wit hinthetotalCD3-CD56+ (D)Cumulative(n=15)datashowingthepercent agesofadaptiveCD57+NKG2C+NKcellsbeforean dafterculture.(E)Cumulative(n=6)datashow ingtherelativemeanfluorescenceintensityf oreachindicatedreceptororcytotoxicgranul ecomponentongatedCD3-CD56+NKcellsfromGSK 3iculturesrelativetoDMSO controls.(F)CD3-CD56dimCD57-NKcellsandCD 3-CD56dimCD57+NKcellsweresortedfromdonor PBMCs(n=4)byFACS,labeledwithCellTracedye andculturedfor7daysata2:1ratiowithsorted ,autologousCD14+monocyteswith10ng/mlIL-1 5andeitherDMSOor5 , SEM,andpairedStudent sttestswereusedforstatisticalanalyses.

3 *p ,**p ,**p ABSTRACTSKOV-3-NLR AloneO/N IL-15-Primed7-Day IL-15 + DMSO7-Day IL-15 + GSK3i0 Hours1 Hour3 Hours24 Hours48 HoursNK Cell Culture ,labeledwithCellTraceproliferationdye,an dculturedfor7dayswith10 +CD57-(leftpanel)andCD3-CD56+CD57+NKcell s(rightpanel). ,for7dayswith10ng/mlIL-15andDMSO orfor7dayswith10 :1ration,andintracellularFACS wasusedtomeasurethepercentageofCD3-CD56+ NKcellspositiveforTNF(leftpanel)orIFN- (rightpanel)(n=7).Resultsarefrom4indepen dentexperimentsandareshownasmean sttestswereusedforstatisticalanalyses.*p ,**p ,for7dayswith10ng/mlIL-15andDMSO orfor7dayswith10 , ,for7dayswith10ng/mlIL-15andDMSO orfor7dayswith10 (A)A549cells,(B)SKOV-3cellsand(C)PANC-1c ellsexpressingNucLightRedfluorescentprot einata3 (n=3)culturedintheconditionsabovewereper formedwith(D)A549cells,(E)SKOV-3cellsand (F)PANC-1cellsexpressingNucLightRedfluor escentproteinata3 smultiplecomparisonstestswereusedtodeter minesignificance.

4 *p ,**p ,**p (n=17),treatedwithHerceptinalone(n=20),t reatedwithovernightIL-15-primedNKcellspl usHerceptin(n=15),treatedwithNKcellscult uredfor7dayswith10ng/mlIL-15andDMSO plusHerceptin(n=10)ortreatedwithNKcellsc ulturedfor7dayswith10ng/mlIL-15andGSK3ip lusHerceptin(n=16).AlldonorswereCMVserop ositive,andcultureswereperformedusingCD3 (A)Bioluminescenceimagesofmicefromeachgr oupattheindicatedintervalspost-adoptiveN Kcelltransferfromarepresentativeexperime ntareshown.(B) ,anddataisshownasmean sMultipleComparisonsTestswereusedtodeter minesignificance.*p ,wedemonstratethatadditionofasmallmolecu leinhibitorofglycogensynthasekinase(GSK) 3toperipheralbloodNKcellsexpandedexvivow ithinterleukin(IL) , productioninresponsetoK562myeloidleukemi acellsandhighernaturalcytotoxicityandADC C againstA549lungcarcinomacells, (FATENK-100)hasbeensuccessfullyscaled-up undercGMPconditions, (n=9).

5 CD3-CD56+NKcellswerethenisolatedfromeach cultureconditionandanalyzedbyqRT-PCRfore xpressionofTBX21,ZEB2,PRDM1A, SEM,andpairedStudent sttestswereusedforstatisticalanalyses.*p ,**p BTNFIFN-gMaturation during ex vivo expansion (CD57, KIR, NKG2A, TIGIT) Natural cytotoxicity against solid tumortargetsAntibody-dependent cellular cytotoxicityagainst solid tumor targetsTumor necrosis factor (TNF) and interferon(IFN)-g control in a xenogeneic model ofovarian cancerEnrichment of CD57+NKG2C+ adaptive NKcells from CMV+ ex vivoCD57, KIR,NfCD57+NKG2CV+ IL-15 + DMSO7-Day IL-15 + IL-15 + GSK3i7-Day IL-15 + DMSOFold NK Cell CD57+ NK cells **CD57 MFI (CD57+ NK cells )**010203040 Pre-culture7-Day IL-15 + DMSO7-Day IL-15 + GSK3i% CD57+NKG2C+ NK cells **CDE050010001500200025000500010000 1500020000050001000015000010002000300002 0004000600080000100020003000010020030040 0010000200003000040000500000500010000150 0020000020000400006000080000050000100000 1500002000000100020003000050010001500 MFI04000800012000 NKG2 AKIRCD16 NKp30 NKp46 NKp80 NKp44 MFIDNAM-1 LFA-12B4PD1 TIGITP erforinGranzyme b7-Day IL-15 + DMSO7-Day IL-15 + GSK3i** * ** ** mRNA Relative Fold ExpressionIL-15 +DMSOIL-15 +GSK3i**ZEB2 mRNA Relative Fold Expression**PRDM1A mRNA Relative Fold Expression*PRDM1B mRNA Relative Fold Expression**IL-15 +DMSOIL-15 +GSK3iIL-15 +DMSOIL-15 +GSK3iIL-15 +DMSOIL-15

6 +GSK3iCellTraceSorted CD3-CD56+CD57- NK cells7-Day IL-15 + DMSO7-Day IL-15 + GSKiCD57CD62 LCellTraceCD57CD62 LSorted CD3-CD56+CD57+ NK cellsF% cells % cells % CellsCellTraceCD3-CD56+CD57-NK cells % CellsCellTraceCD3-CD56+CD57+NK Cells7-day DMSO7-day GSK3i (low conc.)7-day GSK3i (intermediate conc.)7-day 5 mM (high conc.)0102030 Effectors AloneEffectors + K562% TNF-Positive NK Cells0102030O/N Primed NK7-Day DMSO NK Cells7-Day GSK3i NK CellsEffectors AloneEffectors + K562% IFN-g-Positive NK cells * ** **A1007550250 Normalized A549 Count (% of A549 Alive)0612182430364248 Time (hours)A549 Alone (EGFR-high, Her-2-low, CD20-neg)A549 + O/N IL-15-Primed + Control AbA549 + 7-Day IL-15 + DMSO + Control AbA549 + 7-Day IL-15 + GSK3i + Control Ab1007550250 Normalized SKOV3 Count (% of SKOV3 Alive)0612182430364248 Time (hours)SKOV3 Alone (EGFR-high, Her-2-high, CD20-neg)

7 SKOV3 + O/N IL-15-Primed + Control AbSKOV3 + 7-Day IL-15 + DMSO + Control AbSKOV3 + 7-Day IL-15 + GSK3i + Control Ab1007550250 Normalized PANC1 Count (% of PANC1 Alive)0612182430364248 Time (hours)PANC1 Alone (EGFR-high, Her-2-low, CD20-neg)PANC1 + O/N IL-15-Primed + Control AbPANC1 + 7-Day IL-15 + DMSO + Control AbPANC1 + 7-Day IL-15 + GSK3i + Control AbBC**A549 Alone (EGFR-high, Her-2-low, CD20-neg)A549 + O/N IL-15-Primed + anti-Her-2 AbA549 + O/N IL-15-Primed + anti-EGFR Ab1007550250 Normalized A549 Count (% of A549 Alive)0612182430364248 Time (hours)A549 + 7-Day IL-15 + DMSO + anti-Her-2 AbA549 + 7-Day IL-15 + DMSO + anti-EGFR AbA549 + 7-Day IL-15 + GSK3i + anti-Her-2 AbA549 + 7-Day IL-15 + GSK3i + anti-EGFR AbSKOV3 Alone (EGFR-high, Her-2-high, CD20-neg)SKOV3 + O/N IL-15-Primed + anti-Her-2 AbSKOV3 + O/N IL-15-Primed + anti-EGFR Ab1007550250 Normalized SKOV3 Count (% of A549 Alive)0612182430364248 Time (hours)SKOV3 + 7-Day IL-15 + DMSO + anti-Her-2 AbSKOV3 + 7-Day IL-15 + DMSO + anti-EGFR AbSKOV3 + 7-Day IL-15 + GSK3i + anti-Her-2 AbSKOV3 + 7-Day IL-15 + GSK3i + anti-EGFR AbPANC1 Alone (EGFR-high, Her-2-low, CD20-neg)

8 PANC1 + O/N IL-15-Primed + anti-Her-2 AbPANC1 + O/N IL-15-Primed + anti-EGFR Ab1007550250 Normalized PANC1 Count (% of A549 Alive)0612182430364248 Time (hours)PANC1 + 7-Day IL-15 + DMSO + anti-Her-2 AbPANC1 + 7-Day IL-15 + DMSO + anti-EGFR AbPANC1 + 7-Day IL-15 + GSK3i + anti-Her-2 AbPANC1 + 7-Day IL-15 + GSK3i + anti-EGFR AbDEF**IVIS ControlSKOV-3 AloneSKOV-3 + HerceptinSKOV-3 + 7-Day IL-15 + DMSO NK cells + HerceptinSKOV-3 + O/N IL-15-Primed NK cells + HerceptinSKOV-3 + 7-Day IL-15 + GSK3i NK cells + Herceptin010203040 Days post tumor inoculationTotal Flux [ p / s ]1x10e051x10e061x10e071x10e081x10e091x10 e101x10e111x10e12**SKOV-3 AloneSKOV-3 + HerceptinSKOV-3 + O/N Primed NK cells + HerceptinSKOV-3 + 7-Day DMSO NK cells + HerceptinSKOV-3 + 7-Day GSK3i NK cells + HerceptinDay 4 Day 7 Day 10 Day 17 Day 40 ABEx Vivo Modulation with Unique Small Molecules