Disclosure Information Malcolm K Brenner

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2 Disclosure Information Malcolm K Brenner I have the following financial relationships to disclose: Consultant for: Grant/Research support from: Celgene Stockholder/Patent licensee; bluebirdbio,tessa Therapeutics, Viracyte Cell Medica Scientific Advisory Board member; Nankwest; Neon I will discuss the following off label use and/or investigational use in my presentation: Cytotoxic T cells for the treatment of virus infections and of cancer

3 T cell Mediated Cancer Therapies Where are we now? Most striking successes with B cell tumors High complete and sustained response rates using Native T cell receptors (TCRs) Chimeric Antigen receptors (CARs)

4 T Cell Mediated Cancer Therapies; Where do we want to be? High sustained complete response rate in most solid tumors

5 Epstein Barr Virus Infects >90% population

6 Epstein Barr Virus Infects >90% population Acute infection is followed by life-long latency Expression of limited array of viral latency proteins Usually benign

7 Epstein Barr Virus Can Cause Tumors in Immunocompetent Hosts Infects >90% population Acute infection is followed by life-long latency Expression of limited array of viral latency proteins Usually benign Latent virus can produce malignant transformation in B/T lymphocytes (40% of HD, NHL), and epithelial cells (90% of Nasopharyngeal cancer)

8 EBV Commonly causes Tumors in Immunocompromised Host Up to 25% of T cell depleted stem cell transplant recipients developed (fatal) immunoblastic lymphoma Associated with EBV reactivation If put back functional EB-Virus specific T cells (EB-VSTs) would this cure the cancer?

9 EB-VSTs can prevent and treat Lymphoma after Stem cell transplant Gene Marked so could track in vivo Extensive (>3 logs) in vivo expansion Long term (>10 years) persistence) No disease in >120 high risk patients receiving CTL prophylaxis versus 12% of controls Complete and sustained resolution of tumor in 11/13 patients with resistant lymphoma

10 How well do EB-VSTs work in immunocompetent patients with Lymphoma?

11 EB-VSTs eliminate relapsed EBV+ Hodgkin and NH-Lymphoma in immunocompetent host Relapsed Disease Arm (n=23) No toxicity 12 CR 2 very good partial responses (up to 36 months) 9 no response or progressive disease Patients with disease at CTL infusion PR CR n =23

12 Studies with EBV Lymphoma/NPC Target T cell Patients Enrolled Virus specific T cells post transplant EBV LCL-induced EBVST 108 EBV-ADV-CMV Ad5/35pp65 DC and LCL VST 86 EBV-ADV-CMV DC transfected with plasmid VST 11 EBV-ADV-CMV Peptide induced VST 11 EBV specific T cells in lymphoma # of Trials EBV LCL-induced EBVST 42 LMP Ad5/35pp65 DC and LCL VST 72 LMP Peptide induced VST 5 TAA Peptide induced VST 5 EBV specific T cells in NPC EBV LCL-induced EBVST 37 LMP Ad5/35pp65 DC and LCL VST 23 TOTAL 400+

13 Why are EB-VSTs so Safe and Effective? Strong and unique (viral) antigens

14 Why are EB-VSTs so Safe and Effective? Strong and unique antigens presented with ample co-stimulation

15 Interactions between T cell and tumor cell ICOS CD28 CD4, CD8 LICOS? B7-2? B7-1? OX-40R 4-1BB LFA-1 LFA-2 B7-H3? OX-40? 4-1BBL? ICAM-1? CD58? CD59? Tumor cell

16 Why are EB-VSTs so Safe and Effective? Strong and unique antigens presented with ample co-stimulation produce sustained immune response which can evolve with the tumor

17 120 MAGEA4 60 Survivin PRAME pre-infusion post-infusion 0 pre-infusion post-infusion 0 pre-infusion post-infusion Epitope Spreading in Responders to LMP1/2 CTLs for EBV Lymphoma

18 Functional Advantages of Antibodies Over T cells Can target Tumor Associated Antigens derived from carbohydrates and glycolipids MHC independent/unrestricted

19 Chimeric Antigen Receptor (CAR) Monoclonal Antibody γ ε α β ε δ ζ ζ Tumor Antigen Tumor Linker TCR complex CAR v H v L Spacer T Cell T cell Intracytoplasmic

20 Advantages of CAR-modified T cells Retain desired properties of T cells Trafficking Expansion/persistence Effector Function MHC independent/unrestricted Can target carbohydrates and glycolipids or non-processed surface proteins

21 Incomplete activation of 1 st generation Incomplete activation of T cells CAR-directed T cells CD28 B7 Tumor T cell T cell ζ 1 st gen CAR Limited Killing of tumor cells

22 Improved activation of 2nd generation CARdirected T cells Incomplete activation of T cells CD28 T cell T cell ζ B7 1 st gen CAR Tumor CD28/41BBζ 2 nd gen CAR?Improved T cell activation and proliferation T cell Killing of tumor cells

23 CD19 CAR T cells for ALL Penn Outcome After Lymphodepleting Conditioning : Active Disease 45/48 CR; MSKCC 12/14 CR NCI Uppsala/BC M 6/8 CR 3/4 CR TOTAL CR >85%

24 8/20/15 10/12//5

25 Why are CARs targeting B cells so effective? Strong and unique ( to B cells) antigen presented with ample co-stimulation

26 How do we get where we want to be? High sustained complete response rate in solid tumors

27 Why are EB-VSTs so Safe and Effective? Strong, unique and stable antigens presented with ample co-stimulation produce sustained and evolving immune response Solid tumors have few antigens that are strong and unique and consistently/stably expressed (evolving patterns). Lack Costimulation

28 Detection of 1 st Generation GD2 CAR T cells in patients with NBL Mean % infused cells detected Day 1 1 week 2 weeks 4 weeks 6 weeks

29 Third Generation GD2 CAR Construct map ψ 2A 5 LTR 3 LTR icaspase9 GD2 CAR (14g2a.scFv) CD28 signaling domain OX40 signaling domain CD3ζ

30 Rationale for Combining Costimulatory Signaling endodomains in 3G CAR APC CD80/CD86 APC 4-1BBL/OX40L Early CD28 Late 4-1BB/OX40 T cell PI3K T cell TRAF2

31 Detection of 3rd Generation CAR GD2 T cells in NBL patients Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 4 Reinfusion Patient 7 reinfusion CAR transgene copy / 1 g DNA Pre-Infusion 4 hrs 1 week 2 weeks 4 weeks 6 weeks 8 weeks 3 months 6 months 9 months

32 Use T lymphocytes whose native receptors are Virus specific as a platform for CARs. Thereby provide physiological co-stimulation in trans.

33 Virus-Specific T-Lymphocytes as CAR Platform Costimulation for CTL activation and expansion B7 CD28 APC expressing viral Ags Native αβ TCR ζ T CD8+ cell VST CAR Tumor Tumor Improved killing CD4+ T cell Cognate help for CTL activation and expansion Rossig et al., Blood 2002 Savoldo et al., Blood 2007

34 GD2-CAR-VSTs First Generation (no costimulation) GD2 CAR-VSTs administered to patients with neuroblastoma

35 Detection of 1 st Generation GD2 CAR T cells Patients with NBL Mean % infused cells detected Day 1 1 week 2 weeks 4 weeks 6 weeks

36 GD2.CAR copies per g PBMC DNA # #2 Weeks after infusion

37 MIBG (Neuroblastoma) Response to 1 st Gen GD2.CAR-VSTs Pre-treatment 9 weeks post Anterior Posterior

38 Response to 1Gen. GD2 CAR VST Infusion In 12 Patients with Neuroblastoma 3 CR (25%) Bone marrow resolution Resolution of spinal lesion Resolution of skull lesion PD CR 2 PR (17%) PD 1 SD (9%) 2 tumor necrosis (17%) Scapular lesion Partial Necrosis SD PR Liver lesion

39 CAR-Virus Specific T cells CAR VST expansion and persistence is influenced by exposure to viral antigen (native receptor stimulation) Substitute a planned local vaccination for a random systemic viral infection?

40 CAR-Virus Specific T cells CAR VST expansion and persistence is influenced by exposure to viral antigen (native receptor stimulation) Substitute a planned local vaccination for a random systemic viral infection? Most viral vaccines have unknown effects (e.g. EBV) or stimulate Th2/Ab (e.g. flu ) VZV vaccine is widely used and safe live vaccine for young and old

41 This image cannot currently be displayed. Augment CAR VSTs by Native T cell Receptor Stimulation/Co-stimulation VZV specific T cell transduced with GD2 CAR VZV vaccination Peptide induced VZVspecific T cells In vivo expansion PBMCs

42 Approved IND Study: GD2 CAR-VZ-VST to Treat GD2 + Tumors Make GD2-CAR-VST Administer to patients with GD2+ Tumors Vaccinate with VZV to stimulate GD2-CAR VSTs via native receptors

43 VZ-VSTs and GD2.CAR transgene increase after vaccination VZV SFC per 3x10 5 PBMC Pre-vacc. Day 4 week1 week Transgene copy # / g DNA

44 Approved IND Study: GD2 CAR-VZ-VST to Treat GD2 + Tumors Make GD2-CAR-VST Administer to patients with GD2+ Tumors Vaccinate with VZV to stimulate GD2-CAR VSTs via native receptors Next step is combine CAR-VSTs with Oncolytic Viruses: Provide potent TCR stimulation

45 Tumor Microenvironment Inappropriate co-stimulation modulation of MHC B7-H1 Tumor battle field Tumor microenvironment Fas FasL proapoptotic molecules T cells Tumor cells CCL120 T reg TGF-β IL-10 IDO IL-4 IL-13 immunosuppressive molecules Regulatory CD recruitment of regulatory T cells

46 Incorporate Proinflammatory mediators into Oncolytic Viruses Recruitment Activation Checkpoint Chemokine Cytokine Blockade

47 Incorporate Proinflammatory mediators into Oncolytic Viruses Recruitment Activation Checkpoint Chemokine Cytokine Blockade Multiple inserts reduce oncolytic Titers

48 Construct all in one HDAd vector Recruitment Activation Modification Chemokine Cytokine Blockade Rescue with co-administered Oncolytic Ad

49 Construct all in one HDAd vector Recruitment Activation Modification Chemokine Cytokine Blockade Stuffer hmip-1α pa hil-2 pa ahpd1 pa Rescue with co-administered Oncolytic Ad Infuse (tumor directed) Ad-VSTs

50 Phase II onwards: Biotech/Pharma Support Broad based collaboration - Celgene/bluebird bio (CAR-T cells) Narrow Pivotal trials Cell Medica (EBV lymphoma) Bellicum (ic9 safety system) Tessa Therapeutics (NPC/HPV head and neck) In house development Viracyte (OTS T cells for infection)

51 SUMMARY Promise and Perils T cell therapies (TCR/CAR) successful for viraltumors and hematological malignancy Equivalent outcomes for solid tumors by creating a hot environment (?with oncolytic viruses) to favor a response that co-evolves with the tumor Avoid Valley of Death by long term and bidirectional industry/academic partnership

52 Acknowledgements CAGT Cliona Rooney Helen Heslop Robert Krance Clinical BMT Service Kathy Leung Caridad Martinez George Carrum Rammurthi Kamble Carlos Ramos Clinical Research Bambi Grilley Yu-Feng Lin Hao Liu Jesse Wu CAGT ic9 project Xiaoou Zhou Shigeki Yagyu Antonio Stasi David Spencer Pietro Dotti Barbara Savoldo Boolean Logic Stephen Gottschalk Nabil Ahmed Max Mamonkin CAGT VST project Ann Leen Juan Vera GMP Laboratory Adrian Gee Carlos Lee Crystal Silva-Lentz Zhuyong Mei April Durett/Flow lab Deborah Lyon/QC Lab VST GMP Laboratory Tessie Lopez Joyce Ku Huimin Zhang Liu Weili

53 Complex Biological Therapies Gene Modified Organoids Allogeneic Cell Therapy Autologous Cell Therapy ips Organoid Therapy Gene-Modified Autologous/Allogenei c Cell Therapy Viral Vectors Non-Viral Vectors

54 CBT s not the Pharmaceutical Model Not pharmaceutical since: Often individualized Complexity requires Iterative studies Lab-> Phase I->Lab->Phase I Complex IP High Cost of (poorly scalable) Goods

55 Development of Standard Drugs Pre-clinical Discovery Pre-clinical validation Lead Compound Phase I Clinical Phase II Clinical Phase III Clinical Source of Funding Scientist/Academic Academic, Biotech, Pharma Biotech, Pharma, Academic Biotech, Pharma,?Academic Pharma, Biotech

56 Development of CBTs Pre-clinical Discovery Pre-clinical validation Optimization Phase I Clinical Phase II Clinical Phase III Clinical Into the Valley of Death

57 CAGT: Components Basic science research Translational research GMP manufacturing facilities IND cell therapy studies Clinical cell therapy (incl. HSCT)

58 Quality Control Cell Storage Gowning Quality Control Freezer Storage Cell Processing Flow Cytometry Released Supplies Gowning Vector Production Materials Management Traffic Pattern Flow Cell Processing Supplies Quality Assurance Administration Pass-through Offices & Administration QA Materials Management Supplies Gowning Swing Suites Vector Production QA