New Methods of Expanding T Cells. Mark E. Dudley, Ph.D. Surgery Branch National Cancer Institute

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1 New Methods of Expanding T Cells Mark E. Dudley, Ph.D. Surgery Branch National Cancer Institute

2 Autologous T Cell and Gene Therapies Service Product New tools for T cell expansion

3 Early clinical trials with autologous T cells: focus on safety, efficacy complex processes, unreliable methods, exotic reagents Walter et al, Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T cell clones from the donor, 1995, N Engl J Med 333: Heslop et al, Long term restoration of immunity against Epstein Barr virus infection by adoptive transfer of gene modified virus specific T lymphocytes, 1996, Nat Med 2:551 5 Dudley et al, Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes, 2002, Science 298:850 4 Morgan et al, Cancer regression in patients after transfer of genetically engineered lymphocytes, 2006, Science 314: 126 9

4 TIL Expansion Strategies Protocol TIL Initiation Rapid Expansion Selected TIL Young TIL Fragments Select for tumor reactivity Digest No selection for tumor reactivity, CD8+ enrichment Rapid expansion T175 Bags Rapid expansion T175 Bags T175 WAVE GRex flasks

5 Method Initiate multiple independent TIL cultures Expand microcultures in 24 well plates Screen for tumor recognition (IFNg release) Rapid Expansion Protocol Anti CD3 (OKT3), IL 2, irradiated PBMC feeders 48x T175 Flasks, 50L process volume

6 Coculture and IFNg ELISA (pg/ml IFNg) 1200 TBI patient; Complete Responder Melanoma Cell Line A2- A2+ Auto TC None A1,24 A1,24 A2,3 A2,3 A2 Controls None AK JKF6 (A2/MART) >21120 >23110 >2794 L2D8 (A2/g209) >24320 >20870 >2529 FRAGMENTS F6 d F8 d DIGESTS W1 d W2 d W3 d W4 d Complex assay with multiple effector and target controls Assay demonstrated variation between microculture activities Recognition of shared and autologous only antigens

7 Coculture and IFNg ELISA (pg/ml IFNg) 1200 TBI patient; Non responder Melanoma Cell Line FrTu A2- A2+ Auto control1 control2 None A1,24 A1,24 A2,3 A2,3 A0201,25 A24 A33,66 Controls None AK JKF >20690 >27790 > JR6C >15250 >18080 > Bonita 2744 W1 d W2 d W3 d W4 d F1 d F2 d F3 d F4 d F5 d F6 d F7 d F8 d Variation between microculture activities Recognition of shared and auto only antigens Tumor recognition does not predict response

8 Method Enzymatic digestion or mechanical disaggregation to single cell suspension Initiate cultures in 24 well plates Develop bulk TIL with minimum time in culture, don t screen (CD8+ enrich) REP: Flasks Bags Flasks WAVE GRex

9 CD8+ enriched Young TIL Method Standard TIL Young TIL

10 Eliminating tumor selection improves manufacturing reliability and patient accrual Selected TIL Young TIL Accrual Period 7/2002 7/2007 9/2008 4/2011 Months of accrual Accessions to lab Excluded (NED, Alt. Prot, etc) Eligible Patients No Growth or not active 183 (52%) 51 (24%) Progressed/clinically excluded 62 (18%) 39 (18%) Treated 107 (30%) 122 (57%) Goff SL, et al 2010, J Immunother. 33:840-7 Dudley ME, et al 2010, Clin. Cancer Res. 16:

11 Adoptive Cell Therapy for patients with cancer 11

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14 Treatments using selected TIL vs young TIL Selected TIL Young TIL Tumor reactivity 100% Objective response (RECIST) 56% (20% CR) 36% (7% CR) Accrual 30% 57% Selected TIL: Logistically challenging, lower accrual, exclusion of potential responders, high response rate Young TIL: Reliable, quantifiable, high accrual, extensive clinical effort, lower response rate

15 Autologous T Cell and Gene Therapies Service Product Two TIL manufacturing strategies emphasize the trade off between quality and quantity New tools for T cell expansion

16 New Tools for T cell expansion Biologicals (ECCE) Bioreactors

17 TIL cultures don t grow from all melanoma suspension digests 100% Lymphocytes in initial suspension 75% 50% 25% p2=5e-8 0% No Growth Rx TIL

18 Engineered Cells for Costimulation Enhancement (ECCE) T cells require activation for growth TCR stimulation Gamma chain cytokine Costimulation 4 1BBL provides potent costimulation to human CD8+ T cells Function in trans of TCR signal K562 was transfected to express 4 1BBL A MCB was made under GMP and aliquots prepared for clinical use

19 ECCE expand human CD8+ TIL in vitro 100 Minimally Cultured TIL d11 Fold Expansion 10 p= IL-2 only IL-2 and ECCE Friedman KM, et al 2011, J Immunother 34:651-61

20 Biological Tools for T cell expansion ECCE Artificial Antigen Presenting Cells Soluble costimulatory molecules/blockade CD28, anti PD 1 Cytokines IL 7, IL 12, IL 15, IL 21 Lymphocyte subset selection Genetic enhancement

21 Bioreactor Process Development Day 0 Day 7 n >100 Fold Expansion FACS Coculture Data >100 >100 > Jin J, et al, 2011, submitted Somerville RPT, et al, 2011, submitted

22 Similar TIL expansion in different bioreactors p=0.25 p=0.23 p=0.07 n=27 n=14 n=25

23 Similar TIL function in different bioreactors p=0.28 p=0.35 p=0.46 n=25 n=14 n=25

24 Similar TIL composition in different bioreactors p=0.17 p=0.50 p=0.20 p=<0.05 p=0.16 p=<0.05 n=27 n=14 n=25 n=27 n=14 n=25

25 Different bioreactors produce TIL that mediate similar clinical results p=0.3 p=0.1 p=0.2 n=27 n=14 n=25 Clinical Responses OR Bags 8 (30%) WAVE 6 (24%) GRex 4 (29%) NR 19 (79%) 19 (74%) 10 (71%)

26 New Tools for T cell expansion Preclinical process/reagent development cgmp reagent qualification/production Safety data, first in man clinical trials Efficacy data

27 Autologous T Cell and Gene Therapies Service Product Two TIL manufacturing strategies emphasize the trade off between quality and quantity New tools for T cell expansion Powerful new solutions to technical problems of scale up/scale out

28 John Wunderlich Mary Ganges Rob Somerville Linda Parker Azam Nahvi Laura Devillier Tom Shelton Kate Hogan Michelle Langhan Colin Gross Marcos Garcia Kevin Friedman Russ Langan Thank You Immunotherapy Fellows Immunotherapy Nurses Don White Carrie Laurencot Protocol Support Office David Stroncek Jianjian Jin Marianna Sabatino Steve Feldman Marybeth Hughes Udai Kammula Richard Morgan Maria Parkhurst Giao Phan Nick Restifo Paul Robbins Richard Sherry Jim Yang Dr. Steven A. Rosenberg