ENGINEERING CYTOLYTIC EFFECTOR CELLS FOR GLIOMA IMMUNOTHERAPY USING GENE INSERTION AND ZINC FINGER NUCLEASE GENOMIC EDITING

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1 ENGINEERING CYTOLYTIC EFFECTOR CELLS FOR GLIOMA IMMUNOTHERAPY USING GENE INSERTION AND ZINC FINGER NUCLEASE GENOMIC EDITING 22 ND ANNUAL isbtc MEETING BOSTON DIVISION OF CANCER IMMUNOTHERAPEUTICS & TUMOR IMMUNOLOGY BECKMAN RESEARCH INSTITUTE

2 TARGET POPULATION: ESITMATED PROPORTION SURVIVING YEARS AFTER DIAGNOSIS PILOCYTIC ASTROCYTOMA GLIOBLASTOMA SEER DATA REPORTED BY DAVIS et al J. Neurosurg 88:1-10, 1998 Median survival duration following GBM relapse is 36-weeks

3 GLIOMA TROPISM OF ADOPTIVELY TRANSFERRED EX VIVO PROPAGATED T-CELLS: fluc+ T-Cells INTRAPARECHYMAL HOMING OF T-CELLS TO ORTHOTOPIC CNS GLIOMA XENOGRAFTS SubQ GBM T-CELLS USE WHITE MATTER TRACKS

4 PATIENT-DERIVED RESECTION CAVITY CSF CHEMOKINE CONTENT AND CHEMOTACTIC BIOACTIVITY TNF-α TNF-β EGF IGF-I MCP-1 MCP-2 MCP-3 MCSF IL-2 IL-3 IL-4 IL-5 Pos Pos Neg Neg Ang MDC IL-6 ENA-78 OSM MIG IL-7 GCSF Tpo MIP-1δ IL-8 GM-CSF VEGF PDGF-β Leptin RANTESIL-10 SCF IL-12 SDF-1&2IL-13 GRO GRO-α I-309 % Chemotaxis IL-8 IP-10 Neg Pos TARC TGF-β IL-15 IFN-γ IL-1a IL-1b Glioma CSF Hydroceph. CSF 0-5 glioma CSF hydrocephalus CSF MCP-1 MIG RANTES Cytokine [pg/ml]

5 GLIOMA-DERIVED CCL2/MCP-1 DRIVES T-CELL TUMOR TROPISM MCP-1 Quantitation (CBA Analysis) Chemotaxis Assay rhmcp-1 (ng/ml) rhmcp-1 U251T CM CM + anti-mcp-1 CM + anti-il-8 CM + anti-mcp-1 + rhmcp-1 T98 U87o % Chemotaxis rhmcp-1 U251T T98 U87o

6 TUMOR SECRETED MCP-1 IS SUFFICIENT FOR IN VIVO HOMING OF ADOPTIVELY TRANSFERRED T-CELLS DAUDI-p DAUDI-MCP1 2.5 Quantification of T-cell Infiltration T-cells per mm Daudi-Parental Daudi-MCP1 Intracranial Tumor Group

7 TUMOR HOMING CYTOLYTIC T-CELLS: NANOSURGEONS IN THE BRAIN

8 EQUIPPING CTLs FOR TUMOR RECOGNITION BY GENE INSERTION

9 SELECTION OF A GLIOMA-SPECIFIC TARGET ANTIGEN TARGET: NORMAL BRAIN GLIOMA T-CELLS TnfR +/ EGFR EGFRvIII (<30%) - IL-13Rα (>90%) -

10 THE IL13-ZETAKINE CHIMERIC ANTIGEN RECEPTOR FOR RE-DIRECTING CTL EFFECTOR FUNCTION TO GBM IL13-ZETAKINE CHIMERIC ANTIGEN RECEPTOR huil-13(e13y) RE-DIRECTED HLA UNRESTRICTED ZETAKINE-REGULATED GLIOMA KILLING huigg4 hinge-fc hucd4 TM hucd3-ζ cyto

11 U87 GLIOBLASTOMA (ffluczeo/il-2) DAY 0: 2x10 5 tumor cells i.c AntiCD19-CAR + CD8 + CTL Clone E8 DAY +5: 10x10 5 CD8 + CTL i.c. IL13-zetakine + CD8 + CTL Clone 2D

12 GLIOMA STEM-LIKE TUMOR PROGENITOR CELLS CD133 CD133+/GFAP- CD133/GFAP+ SOCS2 EGF/FGF/LIF/NO SERUM DMEM 10%FCS

13 PROTOCOL UPN-033: TARGET VALIDATION/T-CELL KILLING OF AUTOLOGOUS TUMOR/TUMOR STEM CELLS GFAP + /CD133 - DIFFERENTIATED TUMOR S8D Count IL13Ra2 GFAP - /CD133 + TUMOR PROGENITORS % Specific Lysis 120 PBT015 NS 100 PBT015 Adh 80 Daudi :1 5:1 1:1 E:T ratio IL13Ra

14 CLINICAL APPLICATIONS

15 PLASMID EXPRESSION VECTOR IL13 ZETAKINE/HyTK-pMG bgh pa hef1p HyTK IL13zetakine/HyTK-pMG 6785 bp IL13zetakine late SV40pAN h CMV-1Aprom SpAn PacI (3240) ori ColE1

16 SCHEMA FOR T-CELL GENETIC MODIFICATION, CLONING, AND EX VIVO EXPANSION Plasmid DNA Vector Day 0 Polyclonal Activation of T Cells with OKT3 Day 3 Electroporation of OKT3- Activated PBMC Day 5 Addition of Selection Drug Day 28 Cloning of Surviving T Cells Day 42 Expansion of Drug- Resistant Clones Day 56 Large Scale Expansion of Zetakine+ Clones for Re-Infusion

17 PROCESS DEVELOPMENT T-CELL BIOREACTORS CLOSED SYSTEM CELL PROCESSING IMMUNOMAGNETIC SELECTION ENGINEERED FEEDER CELLS OPTIMIZED CYTOKINE COMBINATIONS

18 THE CENTER FOR BIOMEDICINE AND GENETICS AT CITY OF HOPE cgmp-manufacturing FACILITY: PLASMID DNA, VIRAL VECTORS, MONOCLONAL ANTIBODIES, AUTOLOGOUS CELL PRODUCTS

19 Glioma-Stimulated Cytokine Production IL-2 IFN-gamma IL-4 IL-10 IL-5 TNF-alpha Cytokine (pg/ml) CD3 CD45RO CD27 CD28 CD3-FITC CD45 RO-FITC CD27-PE CD28-PE CXCR3 CXCR4 CCR2 CCR5 CXCR3-P CXCR4-P CCR2-PE CCR5-PE

20 COHNMC PROTOCOL IRB#01020: (BB-IND #10109) A PILOT FEASIBILITY/SAFETY STUDY OF CELLULAR IMMUNOTHERAPY FOR RECURRENT/REFRACTORY MALIGNANT GLIOMA USING GENETICALLY MODIFIED AUTOLOGOUS CD8 + T-CELL CLONES

21 INTRACAVITARY CYCLE: WEEK: DAY: CELL DOSE: x PROTOCOL # CELL DOSING SCHEDULE 3 REST/RESTAGING REST/RESTAGING

22 PLACEMENT OF RICKHAM SHUNT VAD IN RESECTION CAVITY

23 PRE-ADOPTIVE TRANSFER POST-COURSE #1 POST-COURSE #2 4-WKS POST RX T1-GADOLINIUM MRI PULSE SEQUENCE

24 PROTOCOL SAFETY DATA Cell Dose Level Number of Doses Observed Side-Effects Grade 3 or higher Attributable to Cell Doses Intra-cavitary None 5x None Headache 2x only on UPN 028 Intra-parenchyma 25 x None 50 x WBC, Headache, Pain localized to scalp/catheter site Hypoxia, Headache (3x), Fatigue Cumulative Cell Dose UPN x 10 8 cells over 21-days (Intra-cavitary) UPN x 10 8 cells over 21-days (Intra-cavitary) x 10 8 cells over 5-days (Intra-parenchymal) Best Response to therapy UPN-028 Radiographic CR UPN-033 Radiographic CR Survival Duration post Relapse: 12 months Survival Duration post Relapse: 11 Months

25 PROTOCOL UPN-033: RADIOGRAPHIC COMPLETE RESPONSE FOLLOWING ADOPTIVE THERAPY PRE-RX 14-WEEKS POST-RX 5 T-CELL 10e8 Bx. Proven Progressive GBM Spectroscopy=Necrosis FDG PET = Cold

26 INITIAL CLINICAL IMAGING EXPERIENCE USING [ 18 F]-FHBG PET ON COHNMC PROTOCOL #01020

27 PET-BASED MOLECULAR IMAGING OF HyTK EXPRESSING T-CELLS HSV TK- HSV TK+ [ 18 F] FHBG* P-FHBG* P-FHBG* P-FHBG*

28 UPN-033: Correlative Imaging of CNS Changes Observed Following Intracranial Adoptive Transfer Of Autologous IL13-zetakine + /HyTK + CD8 + CTL Clone Pre-Adoptive Rx Post-Adoptive Rx Ax Flair irfse Ax Flair irfse 18 FHBG PET Tumor Progression Versus T-Cell Mediated Inflammation FHBG Signal From TK Reporter Expressed in T-Cells

29 UPN-033: Correlative Imaging of CNS Changes Observed Following Intracranial Adoptive Transfer Of Autologous IL13-zetakine + /HyTK + CD8 + CTL Clone Ax Flair irfse 18 FHBG PET Tumor Progression Versus T-Cell Mediated Inflammation FHBG CD8+ T CELL PET SIGNAL INFILTRATE

30 CHALLENGES TO BROADER CLINICAL UTILITY: PATIENTS FREQUENTLY NEED TO TAKE GLUCOCORTICOIDS (DEXAMETHASONE) TO MANAGE CERBRAL EDEMA TIME TO PRODUCE AUTOLOGOUS PRODUCT NOT COMPATIBLE WITH RECURRENT DISEASE PROGRESSION KINETICS

31 Potential Advantages of Steroid-Resistant Alloclone: READY-TO-USE PRODUCT AVAILABLE AT TIME OF NEED FUNCTIONAL IN PATIENTS RECEIVING DEXAMETHASONE CONCURRENT USE OF DEXAMETHASONE LIMITS CNS INFLAMMATORY REACTION TO ADOPTIVE THERAPY/ PROLONGS SURVIVAL OF T-CELL ALLOGRAFT BY INHIBITION OF REJECTION RESPONSE

32 INTRAPARENCHYMAL CNS LYMPHOID ALLOGRAFTS ARE NOT REJECTED Grp.A 10^6 NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK s.c. Grp.B 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c. FLANK FLANK Flux (photons/sec) Day post tumor injection Grp.C 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c. & 1x10^6 s.c. - Flank only Flux (photons/sec) Day post tumor injection + Flux (photons/sec) Day post tumor injection Grp.C 25,000 cells NSO IL2(2)_ffluc-Hytk-pMG,GFP-DHFRdm-pEK i.c. & 1x10^6 s.c. - Head only Flux (photons/sec) Day post tumor injection BRAIN BRAIN

33 ZFP-Fok I Fusion Proteins

34 ZFN-Mediated Targeted Gene Disruption (TGD) 1. Endogenous gene targeted for disruption 2. x ZFNs dimerize and introduce a double stranded DNA break in the gene x Break repaired by nonhomologous end-joining (NHEJ) resulting in loss of genetic information Gene disrupted

35 Western Blot Shows Reduction of GR Protein Levels in GR-ZFN Treated CD8+ T-cells GFP ZFN CD8 CTL Clone IL-13 ZK Pool 10A1 GR (N-terminus) TFIIB

36 RT-PCR Analysis Shows Loss of GR Target Gene Transcriptional Regulation in ZFN Modified T-Cells GILZ/GAPDH IFNg/GAPDH GILZ Expression un dex un dex un dex un dex IL-13ZK Pool GFP 30 ZFN A1 IFNg Expression un dex un dex un dex un dex IL-13ZK Pool GFP 30 ZFN A1

37 ZFN-MEDIATED GLUCOCORTICOID DISRUPTION OF ZETAKINE + CTLs RESULTS IN RESISTANCE TO DEXAMETHASONE-TRIGGERED APOPTOSIS % VIABILITY WT GCR WT GCR KO GCR KO GCR ADENO MOI: NO DEX DEX

38 GR-KO CTLs RETAIN ZETAKINE-REGULATED LYTIC ACTIVITY GR-ZFN Adeno Treated MOI 100 % Chromium Release Cytotoxicity: cj04274 UPN ZFN, MOI 100, DEX U251(T) 50:1 25:1 5:1 1:1 Effector:Target Daudi-IL13Ra2 Daudi TM-LCL-OKT3hygro GFP Adeno Control MOI 100 Dex 10e-4M % Chromium Release Cytotoxicity: cj04271 UPN GFP, MOI 100, DEX U251(T) 50:1 25:1 5:1 1:1 Effector:Target Daudi-IL13Ra2 Daudi TM-LCL-OKT3hygro

39 GR- ZETAKINE+ CTL ARE DEX RESISTANT IN VIVO 0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 0 PBS control i.c. Day 5 & x10^6 U87O-ffluczeo-IL2 i.c. Day 0 PBS control i.c. Day 5 & 8 4mg/kg Dex Day 5 Flux (photons/sec) Day post tumor injection 0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 0 2x10^6 UPN IL13-zeta_ GR MOI 100 i.c. Day 5 & 8 Flux (photons/sec) Day post tumor injection 0.2 x10^6 U87O-ffluczeo-IL2 i.c. Day 0 2x10^6 UPN IL13-zeta_ GR MOI 100 i.c. Day 5 & 8 4mg/kg Dex Day 5 Flux (photons/sec) Day post tumor injection Flux (photons/sec) Day post tumor injection

40 GR-KO IL13-ZETAKINE ALLOCLONE CLINICAL TRIAL UNRESECTABLE TUMORS DECADRON DEPENDENT PATIENTS INTRAPARENCHYMAL T-CELL INFUSIONS CONVECTION ENHANCED rhuil-2 DELIVERY

41 Jensen Lab/Translational Research Team Reena Vishwanath COH Grad. Student Tumor Tropism Project COH Collaborators- Behnam Badie, MD David DiGiusto, PhD Andrew Raubitschek, MD Stephen Forman, MD Michael Kalos, PhD Collaborators- Stanley Riddell FHCRC Sam Gambhir Stanford Sangamo Biosciences