Webinar Series. Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research

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1 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Participating experts Bruce Levine, Ph.D. University of Pennsylvania Philadelphia, PA Laurence Cooper, M.D., Ph.D. MD Anderson Cancer Center, Houston, TX ZIOPHARM Oncology, Boston MA Sponsored by Chris Ramsborg, Ph.D. Juno Therapeutics Seattle, WA

2 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Instructions for viewers Change the size of any window by dragging the lower right corner. Use controls in top right corner to close or maximize each window shows the video screen opens the Ask a Question box download slides and more info shows slide window shows speaker bios search Wikipedia Sponsored by Facebook login Twitter login (#ScienceWebinar) LinkedIn login if you need help

3 Bench to Bedside Translation of Redirected Immunity to Treat Relapsed/Refractory Cancers Bruce Levine, Ph.D. Barbara and Edward Netter Professor in Cancer Gene Therapy Department of Pathology and Laboratory Medicine University of Pennsylvania

4 Conflict of Interest Statement Declaration of financial interest due to intellectual property and patents in the field of cell and gene therapy. Funding support for trials: ACGT, LLS, NCI, Lustgarten Foundation and Novartis Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight

5 Cancer Immunotherapy Problem 1: The enemy is ourselves Problem 2: Cancer-specific immune cells are very rare, if present at all Problem 3: Cancer induced immune suppression and immune evasion

6 Overcoming the Scarcity of Tumor Specific Immunity and Tumor Suppression: Creation of Re-directed T cells TCR heterodimer CAR a molecular chimera - off the shelf - MHC independent Chimeric Protein Tumor binding domain Extracellular Intracellular

7 Using Synthetic Biology to Overcome Tolerance First Generation CD4 / CD8z CARs First Generation scfv CARs Second Generation scfv CD28z CARs Second Generation scfv BBz CARs Second Generation scfv CD27z CARs scfv ICOSz CARs Extracellular V H V L V L V H V H V L V L V H V H V L V L V H V H V L V L V H Intracellular CD28 4-1BB CD27 ICOS ζ ζ ζ ζ ζ ζ ζ ζ ζ Irving & Weiss, 1991 Letourneur, 1991 Romeo, 1991 Kuwana, 1987 Eshhar, 1993 Roberts, 1995 Finney, 1998 Maher, 2002 Finney, 2003 Imai, 2004 Milone, 2009 Carpenito, 2009 Song, 2012 Guedan, 2014 Duong, 2013 Evolution of Chimeric Antigen Receptor Design

8 Bead Based in vitro T Cell Culture System Artificial APC: Bead Anti-CD3 Anti-CD28 TcR/CD4 Signal 1 CD28 CTLA4 + Growth J Immunol 1997; 159: 5921 Science 1997; 276: 273 Immunol. Rev. 1997; 160: 43

9 Gene Delivery For Permanent CAR Expression Lentiviral (HIV-1 based) vectors- advantages over other vector types including murine leukemia viruses: Efficiently transduce primary cells Long term stable gene expression without gene silencing that occurs at high levels in oncoretroviral vectors Favorable safety profile Levine, BL, PNAS 2006;103(46):17372 McGarrity, GJ, J Gene Med 2013; 15: 78

10 Gene Delivery For Temporary CAR Expression Currently much faster and cheaper than manufacture of viral vectors In some settings, repeated infusion of temporary CAR s may be sufficient to treat disease Currently primarily a tool for assessing new tumor targets, CAR designs, trial designs CAR Expression via RNA Zhao, Y, Cancer Res. 2010;70(22):9053 Beatty, GL,, Cancer Immunol Res. 2014;2(2):112

11 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Native TCR T cell CD19 CAR, chimeric antigen receptor TCR, T-cell receptor. Tumor cell

12 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Lentiviral vector Native TCR T cell CD19 CAR, chimeric antigen receptor TCR, T-cell receptor. Tumor cell

13 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Lentiviral vector Native TCR T cell CD19 CAR, chimeric antigen receptor TCR, T-cell receptor. Tumor cell

14 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Native TCR T cell CD19 CAR, chimeric antigen receptor TCR, T-cell receptor. Tumor cell

15 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Anti-CD19 CAR construct CAR, chimeric antigen receptor TCR, T-cell receptor.

16 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity Anti-CD19 CAR construct CAR, chimeric antigen receptor TCR, T-cell receptor.

17 Targeting CD19+ CLL with CAR-Modified T cells CARs combine an antigen recognition domain of antibody with intracellular signaling domains into a single chimeric protein Gene transfer (lentiviral vector) to stably express CAR on T cells confers novel antigen specificity CTL019 cell Anti-CD19 CAR construct Dead tumor cell CAR, chimeric antigen receptor TCR, T-cell receptor.

18 Robust CAR T Cell Production Process

19 Complete & Partial Responses in Relapsed/Refractory CLL New Engl J Med 365:725, 2011, Science Translational Med 3:95ra73, 2011

20 CLL is an indolent disease Can CAR T cells work in rapidly growing malignancies?

21 April, 2012 Pediatric ALL 1 st Patient Karyotype: high risk Dx 5/2010, R1: 10/2011,R2: 2/2012 3/2012: high dose cytoxan/clofaribine: persistent ALL Marrow 4/16/2012: 60% blasts w/kidney, liver, spleen lesions CAR T cells infused with no additional chemotherapy

22 April, 2012 May, 2015

23 May, 2015

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25 Center For Advanced Cell Therapy

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28 Developing a Clinical Algorithm: Management of CAR T Cell Toxicities B cell aplasia observed in all responding patients to date managed with IV immune globulin replacement therapy Tumor Lysis syndrome Cytokine release syndrome (CRS) reversible, on-target toxicity Controlled with anti-il-6 therapy (tocilizumab) Grupp, SA et al. N Engl J Med Apr 18;368(16):1509 Lee, DW et al. Blood Jul 10;124(2):188 Severity related to tumor burden

29 The First CAR Assembly Lines

30 Modern CAR Assembly Lines

31 Penn Platform Technology: Allows design and targeting against other tumors

32 Chimeric Antigen Receptor T Cell Translation: Key Points (I) Engineered CAR T cells are a dividing drug and persist for years providing functional immunity Sci Transl Med Sep 2 CTL019 CARs have potent activity in refractory ALL, CLL, DLBCL, FL, myeloma Sci Transl Med. 2011, NEJM 2011, NEJM 2013, NEJM 2014, NEJM 2015 CARs targeting antigens in solid tumors are promising in early stage trials

33 Chimeric Antigen Receptor T Cell Translation: Key Points (II) Tech transfer from academia (Penn) to industry (Novartis) accomplished Novartis now manufacturing for the pediatric r/r ALL global clinical trial and the DLBCL global clinical trial in the US Will be expanded into other countries in the second half of 2015.

34 CAR Clinical Trials in Philadelphia Lentiviral vector CAR s Adult Acute Lymphoblastic Leukemia NCT Chronic Lymphocytic Leukemia NCT Adult Lymphomas: NCT Myeloma: NCT (CART19), NCT (BCMA) Pediatric Leukemia and Lymphoma: NCT Glioblastoma: NCT Mesothelin expressing cancers: NCT (CARTmeso), NCT (CARTmeso19) Multi-center CTL019 trials 8 sites enrolling NCT RNA CAR s Mesothelioma: NCT Pancreatic Cancer: NCT Breast Cancer: NCT Hodgkin s Lymphoma: NCT AML: pending Neuroblastoma: pending

35 CAR Clinical Trials in Philadelphia Lentiviral vector CAR s Adult Acute Lymphoblastic Leukemia NCT Chronic Lymphocytic Leukemia NCT Adult Lymphomas: NCT Myeloma: NCT (CART19), NCT (BCMA) Pediatric Leukemia and Lymphoma: NCT Glioblastoma: NCT Mesothelin expressing cancers: NCT (CARTmeso), NCT (CARTmeso19) Multi-center CTL019 trials 8 sites enrolling NCT RNA CAR s Mesothelioma: NCT Pancreatic Cancer: NCT Breast Cancer: NCT Hodgkin s Lymphoma: NCT AML: pending Neuroblastoma: pending To Date >200 CTL019 Patients >40 other CAR s

36 Abramson Cancer Center Center for Cellular Immunotherapies Director, Carl June Clinical Cell and Vaccine Production Facility Process Development and Correlative Studies Laboratory Department of Medicine Department of Pathology and Laboratory Medicine Division of Transfusion Medicine and Therapeutic Pathology Study Participants

37 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Participating experts Bruce Levine, Ph.D. University of Pennsylvania Philadelphia, PA Laurence Cooper, M.D., Ph.D. MD Anderson Cancer Center, Houston, TX ZIOPHARM Oncology, Boston MA Sponsored by Chris Ramsborg, Ph.D. Juno Therapeutics Seattle, WA

38 Cost-effective T-cell-based immunotherapies for cancer October 14, 2015 Webinar Title: Don't get lost in translation: How smart design and technology are enabling bench-to-bedside in translational research Science/AAAS

39 Some of technology described was advanced through research conducted at the MD Anderson Cancer Center by Laurence Cooper, M.D., Ph.D. Both MD Anderson Cancer Center and Dr. Cooper have a financial interest in ZIOPHARM Oncology, Inc., and Intrexon Corporation. On May 7, 2015, Dr. Cooper was appointed as the Chief Executive Officer at ZIOPHARM. Dr. Cooper is now a Visiting Scientist at MD Anderson where he will continue to supervise the development of this technology.

40 T cells genetically modified with tumor-specific CAR or TCR Clinical & Translational Immunology (2014) 3, e16;

41 Organization Immunology Correlative studies Regulatory Bioprocessing Clinical conduct Manufacturing Bench Bedside

42 Approaches to manufacture and distribution of engineered T cells

43 Approaches to manufacture and distribution of engineered T cells

44 The 3 P s Procurement Process Product

45 The 3 P s Avoid technology that relies on single source vendors Procurement Process Product

46 The 3 P s Avoid technology that relies on single source vendors Innovation versus continuation Procurement Process Product

47 The 3 P s Avoid technology that relies on single source vendors Innovation versus continuation Scalable Procurement Process Product

48 Procurement Tumor infiltrating lymphocytes Venipuncture Apheresis Select/sort T-cell starting population

49 Process #1: Gene transfer Viral Retrovirus Lentivirus Non viral DNA mrna

50 Process #2: Propagation Non-specific Cross-link CD3 Cross-link co-stimulatory molecules Antigen-specific Tumor-associated antigen CAR Cell-free or cell-based Beads K-562

51 K-562-derived activating and propagating cells (AaPC) gd T cells NK cells Geneticallymodified K-562 cells T reg cells NKT cells ab T cells (TIL) T H17 cells

52 Criteria to produce a master cell bank of aapc Parameter Endotoxin LAL Sterility Viruses Mycoplasma Identity Immunophenotyping (gated flow cytometry) Test <5 EU/mL Direct inoculation method Replication-competent lentivirus In vitro and in vivo culturing Bovine 9CFR in vitro assay PCR for human viruses and Bovine polyoma virus Quantitative product enhanced reverse transcriptase (Q-PERT) assay for retroviruses Test for presence of agar-cultivable and non-agar cultivable mycoplasma Transmission electron microscopic examination Isoenzyme Analysis DNA fingerprinting Viability 70% Introduced cell surface antigen(s) 40% CD32 75% Immunol Rev Jan;257(1):181-90

53 Product Donor Autologous Recipient Allogeneic Donor 3 rd party T cells Recipients

54 Patient Specific Dual distribution approaches for T-cell immunotherapy Genetically modified T cells One patient Multiple Patients Autologous T cells Near real time Allogeneic T cells In advance of need Off-the-shelf (OTS) Disease cure Point-of-care (POC) Disease control Centralized

55 Eliminating TCR on CAR + T cells Patient CAR TCRαβ Blood Aug 22;122(8): Blood Jun 14;119(24):

56 Eliminating TCR on CAR + T cells Patient CAR TCRαβ HLAs Blood Aug 22;122(8): Blood Jun 14;119(24): Normal cells

57 Eliminating TCR on CAR + T cells Patient Intended response CD19 TCRαβ CAR HLAs B-cell leukemia/lymphoma HLAs Blood Aug 22;122(8): Blood Jun 14;119(24): Normal cells

58 Eliminating TCR on CAR + T cells Patient Intended response CD19 TCRαβ CAR HLAs B-cell leukemia/lymphoma Blood Aug 22;122(8): Blood Jun 14;119(24):

59 Eliminating TCR on CAR + T cells Gene insertion: Retrovirus/lentivirus Sleeping Beauty mrna Patient Intended response CD19 TCRαβ CAR HLAs B-cell leukemia/lymphoma Artificial nuclease Blood Aug 22;122(8): Blood Jun 14;119(24):

60 Personalized Therapy for Disease and Patient Intra-tumor Heterogeneity of tumor-associated antigen (TAA) Inter-tumor Infuse T cells with more than one specificity Personalized for the disease Infuse T cells with one or more specificity Personalized for the patient N=1 trial paradigm

61 Number of patients Power-law curve The new industrialization of T cells Number of trials

62 Number of patients Power-law curve The new industrialization of T cells Traditional Med Centers Number of trials

63 Number of patients Power-law curve The new industrialization of T cells Traditional Med Centers Number of trials Immuno-oncology at Med Centers

64 Number of patients Power-law curve The new industrialization of T cells Traditional Med Centers Cost of distribution Immuno-oncology at Med Centers Number of trials

65 Number of patients Power-law curve The new industrialization of T cells Traditional Med Centers Cost of distribution 1 Immuno-oncology at Med Centers Number of trials

66 Points to consider No-single source failure Keep-it-simple Non viral Keep it simple Put in un graphic

67 Acknowledgements

68 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Participating experts Bruce Levine, Ph.D. University of Pennsylvania Philadelphia, PA Laurence Cooper, M.D., Ph.D. MD Anderson Cancer Center, Houston, TX ZIOPHARM Oncology, Boston MA Sponsored by Chris Ramsborg, Ph.D. Juno Therapeutics Seattle, WA

69 How product knowledge enables process scale-up/out. Juno Therapeutics Proprietary Materials September 2015

70 Forward-Looking Statements This presentation and the accompanying oral commentary contain forward-looking statements that involve risks, uncertainties, and assumptions. If the risks or uncertainties ever materialize or the assumptions prove incorrect, our results may differ materially from those expressed or implied by such forward-looking statements. All statements other than statements of historical fact could be deemed forward-looking, including, but not limited to, any expectations regarding investment returns; any projections of financial information; any statements about historical results that may suggest trends for our business; any statements of the plans, strategies, and objectives of management for future operations, including our manufacturing and process development; any statements of expectation or belief regarding future events, potential markets or market size, technology developments, our product pipeline, clinical data, enforceability of our intellectual property rights, competitive strengths or our position within the industry; any statements regarding the anticipated benefits of our Celgene collaboration or other strategic transactions; and any statements of assumptions underlying any of the items mentioned. These statements are based on estimates and information available to us at the time of this presentation and are not guarantees of future performance. Actual results could differ materially from our current expectations as a result of many risks and uncertainties, including but not limited to, risks associated with: the success, cost, and timing of our product development activities and clinical trials; our ability to obtain regulatory approval for and to commercialize our product candidates; our ability to establish a commercially-viable manufacturing process and manufacturing infrastructure; regulatory requirements and regulatory developments; the effects of competition and technological advances; our dependence on third-party collaborators and other contractors in our research and development activities, including for the conduct of clinical trials and the manufacture of our product candidates; our dependence on Celgene for the development and commercialization outside of North America of product candidates for which Celgene exercises an option; our ability to obtain, maintain, or protect intellectual property rights related to our product candidates; among others. For a further description of the risks and uncertainties that could cause actual results to differ from those expressed in these forward-looking statements, as well as risks relating to our business in general, see our Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission on August 14, 2015 and our other periodic reports filed from time to time with the Securities and Exchange Commission. Except as required by law, we assume no obligation and do not intend to update these forward-looking statements or to conform these statements to actual results or to changes in our expectations. Juno Therapeutics Proprietary Materials 70

71 Translating Research into Clinical and Commercial Products FOUNDING INSTITUTIONS ACADEMIC PARTNERS Juno Therapeutics has a network of founding institutions and academic partners. This network provides insights into the process of translating novel research into clinical products (including clinical manufacturing) My expertise is in product and process development. Research -> Early Phase Early Phase -> Late Phase Late Phase -> Commercial Juno Therapeutics Proprietary Materials 71

72 Scaling-Up/Out Requires Product and Process Knowledge Product Knowledge: Which product attributes are important for biological activity (safety and efficacy) of product. Process Knowledge: Understanding the combination of process parameters and material attributes required to manufacture the desired product. Moving any bioprocess (cell therapy or otherwise) into a multiproduct manufacturing setting requires the product/process knowledge to establish a process robust enough that it can be repeatably executed (process control). Building process knowledge and a process control strategy to enable transfer to a moderately-sized multi-product facility is resource intensive. In most cases it is not feasible or cost effective to build this amount of process knowledge prior to demonstrating efficacy in the clinic. Juno Therapeutics Proprietary Materials 72

73 Recommendations for institutions and companies in early-phase development 1. Focus on building product knowledge. 2. Invest is translational infrastructure and knowledge management. Centralized databases to store and compare clinical, translational and manufacturing data. Organizational structures to enable cross-functional collaboration between scientific and non-scientific functions. 3. Document, Document, Document Manufacturing Batch Records Store analytical data (manufacturing, translational) in a thoughtful way. Know and document source of all raw materials (e.g. cell lines, plasmids). Juno Therapeutics Proprietary Materials 73

74 Engineered T-Cell Product Knowledge Adoptive immunotherapy for cancer: harnessing the T cell response Nicholas P. Restifo, Mark E. Dudley & Steven A. Rosenberg Nature Reviews Immunology 12, (April 2012) doi: /nri3191 Product Knowledge can be developed via a product characterization effort. Phenotype In Vitro Function In Vivo Function Juno Therapeutics Proprietary Materials 74

75 Invest is translational infrastructure and knowledge management Data for JCAR014 in Adult R/R ALL CR = 17/17 MRD negative CR = 16/17 Median DFS for patients in CR: Cy±VP16 = 150 days Flu/Cy, not reached DFS = disease-free survival; DL2 = dose level 2; Flu/Cy = fludarabine & cyclophosphamide; CR = complete response; MRD = minimal residual disease Juno Therapeutics Proprietary Materials 75

76 Recommendations for institutions and companies in early-phase development 4. Invest in translational sample inventory system Take multiple drug product, in process, raw material, translational (Clinical) samples from all products. Aliquot and store samples in multiple formats (Cell-based assays, transcriptional analysis (e.g. NGS), mass spectroscopy). Run small proof of concept studies to demonstrate sample suitability and stability. 5. Use stored samples to help elucidate mechanisms and attributes that discriminate response and safety in products demonstrating activity. Product Knowledge can be developed after clinical proofof-concept has been established using sample inventory. Juno Therapeutics Proprietary Materials 76

77 Clinical evidence and product knowledge will justify investment in process knowledge ALL Patient Before CD19 CAR T cells Day 37 After CD19 CAR T Cells Our early experience in leukemia trials demonstrates 87%- 93% complete remission rates NHL Patient Before CD19 CAR T cells Day 28 After CD19 CAR T Cells Resolu' on)of)advanced)lymphoma)with)cd19)car)t)cells)) Resolu' on)of)advanced)lymphoma)with)cd19)car)t)cells)) Our early experience in lymphoma demonstrates 56% overall response rate (1) Pre)CD)19)CAR?T)cell)infusion) Pre)CD)19)CAR?T)cell)infusion) 28)Days)Post)CD)19)CAR?T)cell)infusion) 28)Days)Post)CD)19)CAR?T)cell)infusion) (1) ORR of 62% for patients treated following cyclosphosphamide / fludarabine conditioning regimen.. Juno Therapeutics Proprietary Materials 77

78 Ease of Scale-Up/Out scales with Product Knowledge Building Product Knowledge early enables process changes between early- and late-phase trials as well as building process knowledge. Process changes allow the deployment of new technologies that are suitable for a multi-product large-scale manufacturing environment. Closed Systems Fully-Defined Raw Materials Wait until clinical proof-of-concept has been established before investing in process knowledge, unless a platform process has been established for product class. Juno Therapeutics Proprietary Materials 78

79 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Participating experts Bruce Levine, Ph.D. University of Pennsylvania Philadelphia, PA Sponsored by Laurence Cooper, M.D., Ph.D. MDACC, Houston, TX ZIOPHARM Oncology, Boston MA Chris Ramsborg, Ph.D. Juno Therapeutics Seattle, WA To ask a question, type it into the text box and click Submit

80 Don't get lost in translation: How smart design and technology are enabling benchto-bedside in translational research October 14, 2015 Webinar Series Brought to you by the Science/ AAAS Custom Publishing Office Look out for more webinars in the series at: webinar.sciencemag.org To provide feedback on this webinar, please your comments to Sponsored by For related information on this webinar topic, go to: gelifesciences.com/xuri