A Bispecific Chimeric Antigen Receptor Molecule Enhances T cell Activation Through Dual Immunologycal Synapse Formation and Offsets Antigen Escape in Glioblastoma Meenakshi Hegde, Zakaria Grada, Antonella Pignata, Amanda Wakefield, Kristen Fousek, Kevin Bielawowicz, Kevin Chow, Vita S Brawley Tiara T Byrd, Simone Krebs, Stephen Gottschalk, Malini Mukherjee, Winfried S. Wels, Matthew L Baker, Gianpietro Dotti, Jordan Orange and Nabil Ahmed
Glioma Antigens 60 HER2 IL13Rα2 EphA2 EGFRv3 Survivin MAGE-I Cell Number % Lysis % Lysis 40 20 0 60 40 20 0 60 1:40 1:20 1:10 1:5 1:40 1:20 1:10 1:5 MART-I % Lysis 40 20 0 1:40 1:20 1:10 1:5 target : effector Ahmed et al. Clin Can Res 2010; Krebs et al. Mol Ther 2013; Chow KH et al. Mol Ther 2013
CART cells vs. GBM day 6 day 9 day 12 day 15 Ahmed and Salsman et al. Clin Can Res 2009 Autologous GBM HER2.CD28.ζ T cells NT T cells Tumor only
Antigen Escape DAPI HER2 IL13Rα2 Meena Hegde HER2 CAR T cell IL13Rα2 CAR T cell Day 1 3 7 14 HER2 IL13Rα2 Hegde et al. Mol Ther 2013; Bielamowicz et al. Frontiers in Oncology 2014
GBM: intratumoral heterogeneity IL13Rα2 ~ 70% ~ 93% ~ 98% p<0.0001 P>0.05 20 GBM (surgical excision) - Flowcytometry on 100,000 cells - IFC on 5+ cuts; 6+ hpf; 200+ cells/ hpf Hegde et al. Mol Ther 2013
Hypothesis + + Possible advantages: Circumvent tumor escape - Heterogenous expression of target - Down regulation of target - Antigen loss variants Improve T cell activation Hegde et al. Mol Ther 2013
Multiplex T cell Products tumor CAR.pool bi.car Tan.CAR Hegde et al. Mol Ther 2013; Grada and Hegde et al. Mol Ther NA 2013
Offsetting Antigen Escape IL13Rα2 positive 100 HER2 positive 80 HER2+IL13Rα2 positive positive HER2+IL13Rα2 negative negative % viable tumor cells 60 40 20 0 initial HER2 HER2+IL13Rα2 HER2IL-13Rα2 NT tumor only + Hegde et al. Mol Ther 2013
Meena Hegde 280 aa 65 A Zakaria Grada IL13Rα2 632 aa 125 A HER2 TanCAR Grada and Hegde et al. Mol Ther NA 2013
Meena Hegde 280 aa 65 A 632 aa 125 A Zakaria Grada HER2 IL13 m IL13Rα2 TanCAR Hegde et al. in review
Matthew Baker Tan.CAR Docking IL13Rα2 TUMOR HER2 IL13 mutein TanCAR FRP5 T cell Grada and Hegde et et al. al. in Mol review Ther NA 2013
HER2 IL13Rα2 TanCAR 13 F EagI F 5'LTR SrfI AMP 6000 8000 HIL TanCAR psfg SexAI AgeI DraIII AccI BsaAI PmlI NcoI Leader IL-13 mutein HER2.Fc 53% EcoRI 3'LTR 4000 Hinge Linker HER2 scfv IL13Rα2.Fc IL13Rα2.Fc 51% MluI ClaI AccIII Zeta CD28 Hegde et al. in review
Enhanced Cytolysis 60 UPN 2 UPN 3 60 60 U373-GBM Percent 51 Cr Release 40 20 40 20 40 20 0 40:1 20:1 10:1 5:1 0 40:1 20:1 10:1 5:1 0 10:1 5:1 2.5:1 1:1 effector : target HIL TanCAR T cells HER2 CAR T cells IL13Rα2 CAR T cells NT T cells Hegde et al. in review
HIL TanCAR Synapse HER2 IL13R2 OVERLAP Bright field T Cell HER2 CAR GBM T Cell IL13Rα2 CAR GBM T Cell HIL TanCAR GBM T Cell NT GBM Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
Larger HIL TanCAR Synapse Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
Dynamics of Activation: IFNγ 700 600-700 500-600 IFN-γ release pg/ml 600 500 400 300 200 400-500 300-400 200-300 100-200 0-100 1.0 100 0 10 8 6 4 IL13Rα2.Fc (ug/ml) 2 0 0.2 0.4 0.6 0.8 HER2.Fc (ug/ml) OKT3=550 GD2 anti--idiotype=5 Hegde et al. in review
Can TanCAR Co-engage Targets? PHASE CONFOCAL STED GBM T cell Her 2 AF555, IL13Ra2 AF488 Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
13 Simultaneous Engagement? Tan.CAR T cells # of STED clusters Her2 IL13Rα2 Doubles Synapse 1 2 3 4 5 6 7 8 9 10 Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
Unimodal Synapse bi.car T cells # of STED clusters Her2 IL13Rα2 Doubles ChartTte Synapse 1 2 3 4 5 6 7 8 9 10 Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
1.E+07 Anti-glioma in vivo Activity 1.E+06 photons/cm 2 /sec/sr 1.E+05 1.E+04 1.E+03 Tumor only NT T cells IL13Rα2 T cells HER2 T cells HER2 and IL13Rα2 bicar T cells HER2/IL13Rα2 TanCAR T cells 1 5 6 8 9 12 15 19 22 26 29 33 36 40 days Hegde et al. in review
Improved TTP Hegde et al. in review Percent Probability
Mitigating Antigen Escape HIL Tan CAR DAPI HER2 IL13Rα2 DAPI HER2 IL13R HIL bi CAR NT Tumor HER2 CAR IL13Rα2 CAR Hegde et al. in review
Conclusion: TanCAR T cells CAR.pool bi.car Tan.CAR offset antigen escape offset antigen escape + enhance T cell activation offset antigen escape + enhance T cell activation + selective synergy Hegde et al. Mol Ther 2013; Grada and Hegde et al. Mol Ther NA 2013; Byrd and Hegde
Future TanCAR Molecules CIS: HER2/GD2 Tumor Cell TRANS: TAA/TEM8 Target the tumor PROFILE Custom-made vs. Universal CART Develop an RNA platform HIL TanCAR in a Phase I trial.. Tumor Vessel Hegde et al. Mol Ther 2013; Grada and Hegde et al. Mol Ther NA 2013; Byrd and Hegde
Kristen Fousek Amanda Corder NOSTROPOLIS 2015 Tiara Byrd Vita Brawley Meena Hegde Daniel Landi Antonella Pignata Kevin B present past
THE PATIENTS Acknowledgments Center for Cell and Gene Therapy Baylor College of Medicine Stephen Gottschalk Baylor College of Medicine Texas Children s Hospital The Methodist The Methodist Research Institute (Y. Kew, R. Grossman, SZ Powell, D Baskin, J Zhang) Georg Speyer Haus (J Koch; W Wels)