Optimization of T cell expansion in a perfusion bioreactor Clive Glover PhD Product Leader, Cell Bioprocessing

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Chimeric Antigen Receptor T cells- CARTs TH TH TH TC TC Antibody variable region T cell Receptor intracellular signalling component

Clinical Trials Results Approach # patients CR PR CAR T cells (anti CD19) 8 4 (50%) 2 (25%) Rosenberg et.al. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood; 119(12) March, 2012 3 2 (66%) 1 (33%) June et.al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med.; 10(3) Aug, 2011

Lentiviral – expressing Chimeric Antigen Receptor CART – Chimeric Antigen Receptor T cells + T cells Lentiviral – expressing Chimeric Antigen Receptor Cell Infusion into Patient Cell Harvest & Concentration CAR T cells

Typical cell dose = 1x108/kg 20 kg patient = 2 x 109 cells 100 kg patient = 1 x 1010 cells

Factory Scale Cell Separation Cell Collection Cell Selection Cell Activation & Expansion Cell Harvest & Concentration Cell Infusion into Patient Cell Separation Cell Collection Cell Selection Cell Activation & Expansion Cell Harvest & Concentration Cell Infusion into Patient 5000 patients Process time = 10 days Number of patients in parallel = 140

Key Requirements of Cell Therapy Manufacturing Processes Scalable. Sample contained in 1 vessel Easy to scale out to make most efficient use of manufacturing space Automatable to minimize the chance of human error Single Use and Traceable to eliminate cross contamination with other patient cells Closed system to eliminate chance of contamination with adventitious agents due to handling Robust and Compliant. To ensure consistency of product and satisfaction of regulatory requirements

WAVE 2/10 Closed. Automated. Single-use

Growth kinetics Total Cell No. Day of Culture

Optimization Studies Objective: Maximize the expansion of viable T cells in a 10 day period 2,2 2,6 2,9 Angle Rocking Speed 10,2 10,9 10,6 18,2 18,9 18,6 Speed (rpm) 2 10 18 Angle (º) 6 9 # of expts 3 1 5

Experimental Design 5 6 7 8 9 10 Daily monitoring of: Day of culture 5 6 7 8 9 10 1 2 3 4 Culture to 1L Perfuse 500mls Perfuse 750mls Daily monitoring of: Cell proliferation/viability Glucose/Lactate/Ammonia Perfuse 1L

Experimental Design 5 10 Phenotype monitoring of: CD4/CD8 ratio QC analysis 5 10 1 2 3 4 6 7 8 9 Phenotype monitoring of: CD4/CD8 ratio CD27/CD28 expression to assess differentiation state CD57 expression to assess the presence of senescent cells CD62L expression to assess migratory ability

Results No significant effects of angle or rpm on cell health

Results Significant effect of rocking speed on cell expansion

Optimization Optimized speed and angle: 15.02 rpm, 5.625 º Fold expansion sum Optimized speed and angle: 15.02 rpm, 5.625 º

Optimization Cell count (106/ mL) Day

Summary Autologous cellular immunotherapies have unique scalability requirements WAVE systems provide robust and reliable expansion of functional T cells 10% increase in cell yield using optimized bioreactor settings Higher cell densities and a closed and automated system make them ideal for therapeutic use