Professor Mark W Lowdell CSO 1 "Exploring the cellular interactions between NK cells, tumor cells and the TME" Tumor - derived Inhibitory signals Activating signals Tumor - derived Inhibitory signals Activating signals T

Professor Mark W Lowdell CSO 1 "Exploring the cellular interactions between NK cells, tumor cells and the TME"

Tumor - derived Inhibitory signals Activating signals Tumor - derived Inhibitory signals Activating signals Tumor - derived Inhibitory signals Activating signals Activating signals Activating signals NK - Tumor TROGOCYTOSIS NK - NK interactions % of conjugates NK-NK cells 0 20 40 60 80 0.0 0.5 1.0 1.5 2.0 NK:K562[1:5] NK:K562[1:1] NK:K562[5:1] Time[min] % of conjugates

Balance of signals - Tumor "sensitivity" NK:tumor cell ratio NK cell subset Prior exposure? - TiM - NK - CiM - NK Inhibitory microenvironment What determines the outcome of NK - tumor ligation? CD15 - ve K562 NK resistant rNK cell X rNK lack enough activating signals to overcome tumor inhibition

E:T determines outcome Low E:T ratios = High NK activation and degranulation - Low lysis CD107a expression on NK cells K 5 6 2 I N B 1 6 0 20 40 60 80 rNK NK+Target[1:2] NK+Target[2:1] NK+Target[5:1] NK+Target[10:1] % o f e x p r e s s i o n CD69 expression on NK cells K 5 6 2 I N B 1 6 0 20 40 60 80 rNK NK+Target[1:2] NK+Target[2:1] NK+Target[5:1] NK+Target[10:1] % o f e x p r e s s i o n NK mediated lysis K 5 6 2 I N B 1 6 0 20 40 60 80 100 NK+Target[1:2] NK+Target[2:1] NK+Target[5:1] NK+Target[10:1] % o f s p e c i f i c l y s i s CD69 expression after 4h on NK subpopulations rNK NK:INB16 [1:2]NK:INB16 [2:1]NK:INB16 [5:1]NK:INB16 [10:1] 0 20 40 60 80 100 CD107a(-) with monensin CD107a(+) with monensin CD107a(-) w/o monensin CD107a(+) w/o monensin % of expression

Prior exposure to tumor cells may impair or enhance tumor killing Priming effect % of expression on NK cells TNFa IFNy Perforin GranzymeB 0 5 10 15 50 60 70 80 90 100 rNK NK+K562 [1:2] NK+INB16 [1:2] % of expression

NK - NK activation following trogocytosis C D 6 9 0 20 40 60 rNK +TpNK +IL15[1ng/mL] % r N K r e c e p t o r e x p r e s s i o n +TpNK(1:10)+IL15 rNK priming by TpNK

CD107a expression on NK cells r N K N K + [ K 5 6 2 ] N K + [ K 5 6 2 2 4 h ] N K + [ K 5 6 2 l y s a t e s ] N K + [ I N B 1 6 ] N K + [ I N B 1 6 2 4 h ] N K + [ I N B 1 6 l y s a t e s ] 0 20 40 60 80 % o f e x p r e s s i o n Killing assay for Raji cells r N K + T a r g e t N K + [ K 5 6 2 ] + T a r g e t N K + [ K 5 6 2 2 4 h ] + T a r g e t N K + [ K 5 6 2 l y s a t e s ] + T a r g e t N K + [ I N B 1 6 ] + T a r g e t N K + [ I N B 1 6 2 4 h ] + T a r g e t N K + [ I N B 1 6 l y s a t e s ] + T a r g e t 0 20 40 60 80 % o f s p e c i f i c l y s i s NK cells are activated by tumor cell membranes but do not express CD69 or degranulate Expression on NK cells C D 6 9 C D 1 6 0 20 40 60 80 100 rNK NK+[K562] NK+[K562 24h] NK+[K562 lysates] NK+[INB16] NK+[INB16 24h] NK+[INB16 lysates] % o f e x p r e s s i o n

Does tumor exposure influence NK avidity? Kushal Kumar Das - Lumicks Acoustic Node Glass 0.1mm Piezo Element Target tumor cells Primed vs Resting NK cells Ultrasound is used to exert forces completely contactless to NK cells in a microfluidic chip

CD107a expression on NK cells rNK NK+K562[1:2]NK+INB16[1:2] 0 20 40 60 80 100 % of expression Killing assay: % of specific lysis (after 4h) NK+K562 [5:1] NK+INB16 [5:1] 0 20 40 60 80 100 % of specific lysis

Data by Amanda van Vliet CAR - NK Mock - NK 90% lysis 30% lysis

CD107a expression on NK cells rNK NK+K562[1:2]NK+INB16[1:2] 0 20 40 60 80 100 % of expression Killing assay: % of specific lysis (after 4h) NK+K562 [5:1] NK+INB16 [5:1] 0 20 40 60 80 100 % of specific lysis E:T [1:1] 15min 30min 45min 60min 0 5 10 rNK TpNK LAK % of conjugates

Alive >2yrs % K562 lysis NK function only needs to be "adequate"

Effects of hypoxia may be transient and tumor cell dependent 4 6 16 0 20 40 60 80 100 NK NK (hyp) Time (h) S p e c i f i c C y t o l y s i s ( % ) K562 + rNK RAJI +LAK15 4 6 16 24 48 0 20 40 60 80 100 Time (h) S p e c i f i c C y t o l y s i s ( % ) NK+IL15(10ng/ml) NK+IL15(10ng/ml (hyp) 4 6 16 24 48 0 20 40 60 80 100 Time (h) S p e c i f i c C y t o l y s i s ( % ) NK NK (hyp) OVCAR3 + rNK OVCAR3 +LAK15 4 6 16 0 20 40 60 80 100 Time (h) S p e c i f i c C y t o l y s i s ( % ) Raji+TpNK Raji+TpNK (hyp) 4 6 16 0 20 40 60 80 100 Time (h) S p e c i f i c C y t o l y s i s ( % ) Raji+NK+IL-15 (10ng) O/N Raji+NK+IL-15 (10ng) O/N (hyp) 4 6 16 24 48 0 20 40 60 80 100 Time (h) S p e c i f i c C y t o l y s i s ( % ) TpNK TpNK (hyp) OVCAR3 + TpNK RAJI + TpNK

Effects of inhibitory cells in the TME Myeloid - derived suppressor cells Treg Suppression of MLR T r e g : T c o n v 1 : 2 T r e g : T c o n v 1 : 4 T r e g : T c o n v 1 : 8 T r e g : T c o n v 1 : 1 6 T r e g : T c o n v 1 : 3 2 0 20 40 60 80 % o f s u p p r e s s i o n S p e c i f c i c y t o l y s i s N K + R A J I N K + T r e g + R A J I N K + I L 2 1 0 0 I U + R A J I N K + T r e g + I L 2 1 0 0 I U + R A J I T p N K + R A J I T p N K + T r e g + R A J I 0 20 40 60 80 100 Treg Suppression of NK-mediated lysis K562 lysis 5:1:1 0 20 40 60 80 100 rNK rNK:CD33 (IL-6) Allo 1 rNK:CD33 (VEGF) Allo 1 rNK:CD33 (IL-6) Allo 2 rNK:CD33 (VEGF) Allo 2 rNK:CD33 (IL-6) Auto rNK:CD33 (VEGF) Auto % o f s p e c i f i c l y s i s K562 lysis 5:1:1 0 10 20 30 40 % C D 1 0 7 a + Allo 1 Allo 2 AutoNIL

Conclusions NK:tumor cell interactions are multicellular and multifactorial Degranulation is not a direct correlate of lysis and thus not of exhaustion Tumor cell conjugation may induce activation without lysis and generate a primed state associated with increased TNF - a and IFN - g and increased avidity to secondary tumor targets Classical regulatory factors in the TME can inhibit NK function in vitro but the effects are transient and may be overcome by appropriate activating signals Increased avidity enhances lysis but not conjugation duration and may be a tool to develop, screen and qualify novel NK activating agents The molecular basis of increased avidity is being dissected