Glioma Antigens 60. HER2 IL13Rα2 EphA2 EGFRv3 Survivin MAGE-I MART-I
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1 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
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3 Glioma Antigens 60 HER2 IL13Rα2 EphA2 EGFRv3 Survivin MAGE-I Cell Number % Lysis % Lysis :40 1:20 1:10 1:5 1:40 1:20 1:10 1:5 MART-I % Lysis :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
4 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
5 Antigen Escape DAPI HER2 IL13Rα2 Meena Hegde HER2 CAR T cell IL13Rα2 CAR T cell Day HER2 IL13Rα2 Hegde et al. Mol Ther 2013; Bielamowicz et al. Frontiers in Oncology 2014
6 GBM: intratumoral heterogeneity IL13Rα2 ~ 70% ~ 93% ~ 98% p< P> GBM (surgical excision) - Flowcytometry on 100,000 cells - IFC on 5+ cuts; 6+ hpf; 200+ cells/ hpf Hegde et al. Mol Ther 2013
7 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
8 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
9 Offsetting Antigen Escape IL13Rα2 positive 100 HER2 positive 80 HER2+IL13Rα2 positive positive HER2+IL13Rα2 negative negative % viable tumor cells initial HER2 HER2+IL13Rα2 HER2IL-13Rα2 NT tumor only + Hegde et al. Mol Ther 2013
10 Meena Hegde 280 aa 65 A Zakaria Grada IL13Rα2 632 aa 125 A HER2 TanCAR Grada and Hegde et al. Mol Ther NA 2013
11 Meena Hegde 280 aa 65 A 632 aa 125 A Zakaria Grada HER2 IL13 m IL13Rα2 TanCAR Hegde et al. in review
12 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
13 HER2 IL13Rα2 TanCAR 13 F EagI F 5'LTR SrfI AMP 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
14 Enhanced Cytolysis 60 UPN 2 UPN U373-GBM Percent 51 Cr Release :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
15 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
16 Larger HIL TanCAR Synapse Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
17 Dynamics of Activation: IFNγ IFN-γ release pg/ml IL13Rα2.Fc (ug/ml) HER2.Fc (ug/ml) OKT3=550 GD2 anti--idiotype=5 Hegde et al. in review
18 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
19 13 Simultaneous Engagement? Tan.CAR T cells # of STED clusters Her2 IL13Rα2 Doubles Synapse Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
20 Unimodal Synapse bi.car T cells # of STED clusters Her2 IL13Rα2 Doubles ChartTte Synapse Mukherjee and Orange; Center for Immunobiology, Texas Children s Hospital
21 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 days Hegde et al. in review
22 Improved TTP Hegde et al. in review Percent Probability
23 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
24 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
25 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
26 Kristen Fousek Amanda Corder NOSTROPOLIS 2015 Tiara Byrd Vita Brawley Meena Hegde Daniel Landi Antonella Pignata Kevin B present past
27 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)
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