Tonic CAR signaling in T cells:

Transcription

1 Tonic CAR signaling in T cells: toxicities and ways to manage Max Mamonkin, PhD CAR-TCR Summit 9/6/217 Boston, MA

2 CAR signaling Antige n Target cell CAR Signal 2 T cell Signal 1 Clustering Signaling

3 Tonic CAR signaling 1 2 No tonic signaling Ligand-independent tonic signaling Ligand-driven tonic signaling GD2 CAR, CD19 CAR CD5 CAR, CD7 CAR

4 Tonic CAR signaling 1 2 Ligand-independent tonic signaling Short-lived effectors Exhaustion Long-lived Central memory Reversal of exhaustion Long et al. Nat Med 215

5 Tonic CAR signaling 1 2 Ligand-independent tonic signaling Ligand-driven tonic signaling Is tonic CAR-derived 4-1BB signaling in T cells universally beneficial?

6 Modeling CAR-derived tonic 4-1BB signaling CD19 CAR Expression in gammaretroviral vectors CAR expression FMC63 HIGH 5 LTR CAR 3 LTR IRES CD8a LOW 5 LTR IRES CAR 3 LTR IRES CD19 CAR 4-1BB IRES zeta p-cd3ζ Tonic CAR signaling GAPDH

7 Modeling CAR-derived tonic 4-1BB signaling % positive cells CD19 CAR FMC63 6 Naive Central Memory 4 CD8a 2 4-1BB zeta

8 Cell count, x1^6 Cell count, x1^6 Reduced expansion of CD19 CAR T cells % apoptotic cells Cell expansion Apoptosis IRES NTR IRES IRES ** Annexin V 6 ** * Days post-transduction Days post-transduction 2 IRES

9 Reduced anti-tumor activity of CD19 CAR T cells % CAR T in blood Percent survival.4 ** NALM6 GFP-FFluc CD19 CAR T CAR T expansion in blood. IRES 3d 1 IRES 5 ** Survival Days post-engraftement

10 Cell death is dependent on TRAF2 signaling % apoptotic cells 4-1BB endodomain signaling mutants TRAF2 TRAF2 TRAF1 TRAF2 TRAF3 TRAF1 TRAF2 TRAF3 4-1BB 4-1BB mut 1 4-1BB mut 2 ** 6 ** ** 4 Apoptosis 2 eta eta mut1 mut 2

11 Tonic 4-1BB signaling is associated with increased CAR expression CAR expression CAR expression Tonic signaling mut 1 mut 2 NT mut 1 mut CD3ζ (total CAR) p-cd3ζ (tonic CAR signaling) GAPDH CD19 CAR Does 4-1BB signaling increase CAR expression?

12 Tonic 4-1BB signaling activates the NF-kB pathway and increases LTR activity Normalized CD19 CAR mrna TRAF2 NT mut 1 mut 2 4-1BB p-ikkα/β NF-kB pathway TRAF1 TRAF2 TRAF3 GAPDH 8 * * ** P IKK 6 4 LTR promoter activity 2 NF-kB 5 LTR CAR LTR 3 mut1 LTR promoter activity = mut2 IRES # of CD19 CAR mrna #of RV proviruses per 1ng gdna

13 Tonic 4-1BB signaling promotes positive feedback loop in retroviral vectors PROBLEM CAR Tonic 4-1eta CAR signaling TRAF2 1. Activates the NF-kB pathway and enhances CAR expression from the LTR promoter. P IKK Selfamplification + CELL DEATH 1. Promotes T cell death and impairs anti-tumor function. NF-kB LTR CAR LTR

14 MFI x Volume Fas MFI Fas MFI Fas MFI Fas MFI MFI x Volume Fas MFI Fas MFI Fas MFI Fas MFI Tonic signaling from CAR enhances expression of proapoptotic receptors Fas NF-kB target genes Fas FasL TRAIL CAR-Em Total Surface * * Fas FasL TRAIL 2 mut 1 mut 2 Fas FasL BB B mut1 mut1mut2 m Total FasL Surface * Overlay 1 1 B mut1 mut1mut2 m

15 % Annexin V+ MFI x Volume Fas MFI Fas MFI Fas MFI Fas MFI MFI x Volume Fas MFI Fas MFI Fas MFI Fas MFI Fas-dependent apoptosis contributes to the toxicity in CAR T cells CD19 CAR Fas Fas Fas 48% 88% 52% Fas KO CAR-Em Total Surface * * 1 CAR Fas Fas BB B mut1 mut1mut2 m 6 4 ** Apoptosis FasL Total FasL Surface * 2 Overlay 1 Fas+ Fas- 1 B mut1 mut1mut2 m

16 Cell count, x1^6 Cell count, x1^6 Cell count, x1^6 Cell count, x1^6 Cell count, x1^6 Cell count, x1^6 Similar toxicities in other CAR T cells CD19 CAR IRES NTR5 1 4 IRES GD2 CAR 28.OX4.z IRES NTR IRES kappa CAR IRES NTR NTR IRES IRES Days post-transduction Days post-transduction Days post-transduction Days post-transduction Days post-transduction

17 Tonic CAR signaling 1 2 No tonic signaling Ligand-independent tonic signaling Ligand-driven tonic signaling GD2 CAR, CD19 CAR CD5 CAR

18 Cell count, x1^6 Expression of CD5 CAR impairs T-cell expansion % apoptotic cells CAR T expansion Apoptosis Cell count x transduction mut1 mut2 CD8a IRES Days post-transduction Days post-transduction *** ** ** ** mut1 mut2

19 High Fas levels and formation of fratricidal immune synapses in CD5 CAR T cells Fas MFI Fas expression Fratricidal immune synapses BF F-Actin Fas ** mut 1 mut 2 mut1 mut2 Fas

20 High ICAM-1 levels promote fratricidal immune synapses in CD5 CAR T cells Annexin V (%) mut1 ICAM-1 expression mut 1 mut 2 F-actin ICAM-1 2 ICAM-1 blockade αicam-1 6 ICAM * 4 3 Annexin V 2 αicam-1

21 Toxicity from tonic signaling in 4-1eta CARs PROBLEM CAR Fas ICAM-1 Tonic 4-1eta CAR signaling TRAF2 1. Activates the NF-kB pathway and enhances CAR expression from the LTR promoter. P IKK Selfamplification + CELL DEATH 1. Promotes T cell death and impairs anti-tumor function. NF-kB LTR CAR LTR 2. Augments Fas-dependent apoptosis (CD19 CAR) and strengthens fratricidal immune synapses (CD5 CAR).

22 Toxicity from tonic signaling in 4-1eta CARs PROBLEM SOLUTIONS CAR Fas ICAM-1 Reduce tonic signaling by: TRAF2 1. Changing CAR configuration. P IKK Selfamplification + CELL DEATH NF-kB LTR CAR LTR

23 Replacing spacer and TM regions may alleviate the toxicity and improve function Percent survival % apoptotic cells CD19 CAR Apoptosis Day 7 * NTR (CD8a) IRES (CD8a) (IgG4 sh) CD8a stalk CD8a TM IgG4 hinge CD28 TM In vivo 1 5 ** NT CD19 CAR CD8a CD19 CAR IgG4 sh 4-1BB zeta Days post-engraftement

24 Disrupting the positive feedback loop by choosing a different expression system PROBLEM SOLUTIONS CAR Fas ICAM-1 Reduce tonic signaling by: TRAF2 1. Changing CAR configuration - spacer, TM, scfv P IKK Selfamplification + CELL DEATH 2. Replacing vector/promoter NF-kB LTR CAR LTR

25 CD19 CAR expression from gammaretrovs SIN lentiviral vectors CAR expression Retroviral 5 LTR CAR 3 LTR LV Lentiviral (SIN) 5 ΔLTR EF1α CAR 3 ΔLTR LV CD19 CAR

26 Reduced Fas/FasL levels and restored expansion of lentivirally transduced T cells % apoptotic cells Fold expansion Fas FasL Apoptosis CAR T expansion 5 * 4 ** LV Fas FasL LV LV

27 Improved in vivo antitumor function of lentivirally transduced T cells % CAR T in blood Percent survival.8 *.6.4 CAR T expansion in blood NALM6 GFP-FFluc CD19 CAR T.2. LV 3d 1 5 ** LV Survival Days post-engraftement

28 Fold expansion Similar effect in GD2 CAR T cells GD2 CAR expression CAR T expansion *** *** 1 LV 5 28.OX4.z GD2 CAR 28. OX4.z LV

29 Expression of CD5 CAR from an LV vector improves expansion but ablates cytotoxicity % live cells CD5 CAR expression Viability 1 *** *** AlgParm-1: Fc1 CD5 CAR LV T RV LV T cells CD5 CAR T LV CD5 CAR T Day 4 Day 18

30 Overcoming tonic signaling by regulating CAR expression PROBLEM SOLUTIONS CAR Fas ICAM-1 Reduce tonic signaling by: TRAF2 1. Changing CAR configuration - spacer, TM, scfv P IKK Selfamplification + CELL DEATH 2. Replacing vector/promoter - Non-LTR (SIN) lentivirus NF-kB 3. Controlling CAR expression LTR CAR LTR

31 Controlling CAR expression to minimize fratricide activation expansion infusion T cells CD5 CAR viral transduction INHIBIT CAR expression RESTORE CAR expression

32 Controlling CAR expression to minimize fratricide Cell count, x1^6 Regulated (Tet-OFF) CAR expression system CAR T expansion 5 ΔLTR tta tta TRE + DOX DOX CD5 CAR tta EF1a tta 3 ΔLTR 4 3 +DOX 2 1 DOX +DOX Days post-transduction CD5 CAR

33 GFP (Jurkat) Controlling CAR expression and T cell function with doxycycline CAR re-expression Jurkat Anti-tumor activity CD5 CAR CD5 CAR DOX 96h 72h 48h DOX 99 24h +DOX CD5 CAR 7-AAD CAR inhibition 72h 48h +DOX 24h - DOX CD5 CAR

34 hcd45+ % CAR T cells T in in blood (%) Percent survival Percent survival % CAR T in blood Tet-OFF CD5 CAR T cells have superior antitumor activity in vivo In vivo function CAR T expansion Survival Jurkat GFP-FFluc CAR T ** Tet-OFF **** Tet-OFF Tet-OFF Tet-OFF CAR expression.2 N.D Days post-car T injection Days Days post-car T injection T CD5 CAR Tet-OFF.2 Days post-car T injection Day 7 CD5 CAR Tet-OFF +DOX Day 18 Day 25

35 SUMMARY PROBLEM SOLUTIONS CAR Fas ICAM-1 Reduce tonic signaling by: TRAF2 1. Changing CAR configuration - Spacer, TM, scfv P IKK Selfamplification + CELL DEATH 2. Replacing vector/promoter - Non-LTR (SIN) lentivirus NF-kB LTR CAR LTR 3. Controlling CAR expression - Tet-OFF regulated system

36 ACKNOWLEDGEMENTS Malcolm Brenner Diogo Gomes-Silva Madhuwanti Srinivasan Feiyan Mo Clio Rooney Sandhya Sharma Stephen Gottschalk Giedre Krenciute Malini Mukherjee Jordan Orange Carlos Ramos Helen Heslop Funding Sources: ASH Scholar Award (PI) SPORE in Lymphoma (Co-PI) SPORE in Lymphoma DRP (PI) Gloria Levin Fund (PI)

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38 Enhanced cell aggregation correlates with high ICAM-1 expression in CAR T cells ICAM1 MFI mut1 mut2 ICAM-1 expression mut 1 mut * * * ICAM-1 NT mut1 mut2