1 Steven A. Feldman, Ph.D. Surgery Branch, NCI MEASUREMENT CHALLENGES FOR CAR-T BIOMANUFACTURING January 29, 2016 Adoptive Cell Therapy for the Treatment of Cancer: The CAR-T Cell Experience

Disclosures No financial disclosures This work is supported, in part, through a CRADA with KITE Pharma 2

Outline Background CD19 CAR-T process development CD19 CAR-T clinical experience CAR-T therapy for solid cancers 3

1.Non-specific stimulation of immune reactions Stimulate effector cells (IL-2, IL-12) Inhibit regulatory factors (PD-1, CTLA-4) 2.Active immunizations to enhance anti-tumor reactions Cancer vaccines 3.Passively transfer activated immune cells with anti-tumor activity Adoptive cell transfer Three Main Approaches to Cancer Immunotherapy 4

Gene Therapy Approach for Adoptive Cell Transfer 5 Morgan RA, et al. Science 2006; 314(5796): 68-9.

1.High avidity anti-tumor T cell receptors (TCR) and/or chimeric antigen receptors (CAR) can be identified and cloned using in vitro assays. 2.Peripheral blood lymphocytes can be genetically modified to express these high avidity TCRs/CARs. 3.Large numbers of tumor-specific lymphocytes can be grown in vitro. 4.The host can be manipulated to provide a favorable tumor microenvironment prior to administering the cells. 5.ACT can mediate tumor regressions. Advantages of Cell Transfer Therapy Using Genetically Engineered Cells 6

A Critical Challenge Confronting the Development of Human Cancer Immunotherapy is the Identification of Antigens to Target 1.Differentiation antigens overexpressed on cancers compared to normal tissue (MART-1, gp100, CEA, Her-2, Mesothelin) 2.Antigens expressed on cancers and on non-essential normal tissues (CD19, thyroglobulin) 3.Shared antigens unique to cancer (cancer-testes antigens, NY-ESO-1, MAGE-A) 4.Critical components of the tumor stroma (VEGFR2, FAP) 5.Mutations unique to each cancer (EGFRvIII) 7

Chimeric Antigen Receptors (CARs) 8 Antibody Producing Hybridoma Ig Genes Step 2 Step 1 Step 3 Step 4 Step 5 Chimeric Antigen Receptor (CAR) Ig scFv Linker/TM T cell signaling CD28 CD3 zeta LTR  sd sa CD8 4-1BB CD28 CD3 zeta Anti-tumor Ag-scFv LTR  sd sa Anti-tumor Ag-scFv Transduce PBL

More than 20,000 people die of B-cell malignancies annually in the U.S. CD19 is expressed by more than 90% of B-cell malignancies. CD19 is expressed by mature B cells, B-cell precursors and plasma cells but not any other normal tissues. B cells are considered a “non-essential” tissue.  Anti-CD19 CAR 3’ LTR CD28FMC63 scFvCD3-zeta 5’ LTR 9 CAR-T Program for the Treatment of CD19+ Hematologic Malignancies

Process Development Objectives: Develop a closed process Production time of 6 days Cryopreserved cell product GMP-compliant A Rapid Cell Expansion Process for the Production of CD19 CAR-T Cells 10

Development of a CAR-T Cell Closed Production Process 11

Optimization Parameters Process Steps: Cell culture medium Serum replacement Bag comparison Cell density at OKT3 stimulation Post-stimulation wash Transduction  Retronectin concentration  Vector dilution  1 vs 2 transductions  Bag flip  Cell density Sepax  Apheresis  OKT3 wash  Final product wash Cryopreservation Analytical Assays: FACS – CAR+ cells – Phenotype Co-culture ELISA qPCR – Copy number – Persistence 12

13 Sepax 2 can Efficiently Process Apheresis Products Sepax Technologies, Inc.

Optimizer-TCSR Medium can Support T Cell Growth 14

Optimization of Cell Concentration During Transduction 15 6 well (9.5 cm 2 ):PL240 (540 cm 2 ): 2e6 cell/well2e8 cell/bag 10 ng/ml RN10 ng/ml RN 4ml (1:1 vector)400ml (1:1 vector) 0.5e6 cell/ml0.5e6 cell/ml 1e5 cell/cm 2 3.7e5 cell/cm 2

6 Day Closed Production Process – Engineering Runs 16 5 engineering runs at scale and compared to standard open process Evaluated Transduction efficiency Function (IFN  secretion) Cell expansion Phenotype

A Comparison of the Standard Open and 6 Day Closed Production Process 17 PL240 Bag

A Comparison of the Standard Open and 6 Day Closed Production Process 18 Naïve:CD45RA+/CCR7+ Tcm:CD45RA-/CCR7+ Tem:CD45RA-/CCR7- Temra:CD45RA+/CCR7-

Development of a CAR-T Cell Closed Production Process 19

CD19 Before treatment 3 months after treatment CD19 CD20 Efficacy of CD19 CAR-T Cells in CLL Kochenderfer et al. Blood

CD19 CAR-T cells can Mediate Tumor regression 21

CD19 CAR-T Cells Eliminate normal B cells and Show Minimal Persistence in Vivo 22

CAR-T Cell Production Process for Solid Cancers 23 REP: 6000 IU/ml IL-2 200:1 irradiated feeder cells OKT3 Gene Modification

CAR-T Program for the Treatment of Solid Cancers ReceptorGenerationCancersStatus ERBB2 (Her2/neu)3rdAll Her2+ CancersClosed VEGFR23rdAll cancersClosed EGFRvIII3rdGlioblastomaAccruing Mesothelin2ndPancreatic/Mesothelioma/OvarianAccruing 24 Receptor Patients Treated Cells InfusedORR ERBB2 (Her2/neu)11e100/1 VEGFR2231e6 – 3e10 1/23 (4%) PR (2 months) EGFRvIII161e7 – 3e100/16 Mesothelin141e6 – 1e8 0/13 (Pt. 14, too early)

Summary CD19 CAR-T cell therapy effectively treats a variety of B cell malignancies We have developed a closed cell production process sufficient for current CD19 CAR-T cell demands – Closed system – Serum-free – 6 days – Cryopreserved cell product Currently no effective CAR-T therapies for the treatment of solid cancers Considerations – How to handle increased cell numbers – Increase transduction efficiency – RCR testing beyond 6 days – Direct detection of CD19 CAR+ T cells (other CARs ???) – Patient variability – How to extend CAR T therapies to solid cancers 25