T Cell Therapy Reagents T Cell Isolation, Activation and Expansion From Bench to Bedside: T Cell Therapy Reagents Take your T cell therapy research from bench to bedside. Our collaboration with GE Healthcare aims to give researchers the confidence of a path to the clinic with cGMP-grade T cell reagents. The currently available products developed by STEMCELL Technologies are designed for cell therapy research applications following the recommendations of USP<1043> on Ancillary Materials, and we can currently work with you to qualify these reagents under an approved investigational new drug (IND) or clinical trial application (CTA). T cell products that aim to be developed as cGMP-grade reagents: PRODUCT CATALOG # DESCRIPTION • Isolate up to 99% pure CD3+ cells free of EasySep™ Release Human CD3 magnetic particles in under 30 minutes 17751 Positive Selection Kit • Obtain highly viable, functional cells Isolation without the need for columns ImmunoCult™ Human CD3/CD28 10971 • Soluble reagents for robust activation and T Cell Activator expansion without magnetic beads ImmunoCult™ Human CD3/CD28/CD2 • Not exclusively licensed by certain 10970 T Cell Activator institutions Activation • Formulated for rapid expansion without ImmunoCult™-XF T Cell Expansion the use of serum 10981 Medium • Consistent expansion of functional human T cells Expansion Learn more at www.stemcell.com/t-cell-therapy Scientists Helping Scientists™ | WWW.STEMCELL.COM DOCUMENT #DX20367 VERSION 2.1.0 MAY 2017 TOLL FREE PHONE 1 800 667 0322 • PHONE +1 604 877 0713 • [email protected] • [email protected] FOR GLOBAL CONTACT DETAILS VISIT OUR WEBSITE Isolation Activation Expansion 1 Start your T cell research workflow with highly purified T cells isolated from Leuko Paks or PBMCs using EasySep™ Start Isolated Why Use EasySep™ Release Human CD3 Positive Selection Kit? PARTICLE-FREE. Culture and analyze your positively selected cells without magnetic particles. FAST AND EASY. Obtain highly purified cells free of magnetic particles in under 30 minutes. CD3 PE CD3 PE COLUMN-FREE. With no columns, the simple EasySep™ pour-off is gentle on your cells. Figure 1. T cells are highly purified when isolated with EasySep™ HIGH PURITY. Achieve purities of up to 99%. Release Human CD3 Positive Selection Kit Starting with human PBMCs, the CD3+ cell content of the isolated fraction, using the EasySep™ Release CD3 Positive Selection Kit (Catalog #17751) is typically 98.7 ± 0.9% (mean ± SD using the purple EasySep™ Magnet). Isolation Activation Expansion 2 Activate isolated T cells with ImmunoCult™ Human CD3/CD28 or CD3/CD28/CD2 T Cell Activator in ImmunoCult™-XF T Cell Expansion Medium A B C A B Figure 2. T cells are activated when stimulated with ImmunoCult™ Figure 3. T cells exhibit an activated morphology when activated with Human CD3/CD28 or CD3/CD28/CD2 T Cell Activator ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator EasySep™ isolated T cells were cultured on day 0 with either ImmunoCult™ Images of EasySep™-isolated T cells activated with (A) soluble ImmunoCult™ Human CD3/CD28 T Cell Activator (Catalog #10971) or ImmunoCult™ Human Human CD3/CD28/CD2 T Cell Activator and (B) the competitor’s bead-based CD3/CD28/CD2 T Cell Activator (Catalog #10970) in ImmunoCult™-XF activation reagent. T Cell Expansion Medium (Catalog #10981). Cells were gated on CD4+ T cells and CD8+ T cells and T cell activation was assessed by CD25+ expression on day 0 and day 3. At the start of culture, the CD25+ cell population was (A) 5.63 ± 2.4% (mean ± SD). After three days of activation, the CD25+ cell population was (B) 75.4 ± 13.8% (mean ± SD) when activated with ImmunoCult™ Human CD3/CD28 T Cell Activator and (C) 88.8 ± 3.2% (mean ± SD) when activated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator. 2 T Cell Therapy Reagents Isolation Activation Expansion 3 Expand activated T cells by culturing in ImmunoCult™-XF T Cell Expansion Medium ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator ImmunoCult™ Human CD3/CD28 T Cell Activator ImmunoCult™ Human CD3/CD28 T Cell Activator 5000 Competitor’s bead-based activation reagentImmunoCult™-XF Competitor’s bead-based activation reagent 4000 Competitor 1 Competitor 2 n o i s 3000 n Competitor 3 a p x Competitor 4 E d l 2000 o FigureF 4. T cells show robust expansion when stimulated with Figure 5. T cells are highly viable when stimulated with ImmunoCult™ ImmunoCult™1000 Human T Cell Activators in ImmunoCult™-XF T Cell Human T Cell Activators in ImmunoCult™-XF T Cell Expansion Expansion Medium Medium 0 T cells were0 expanded5 over 1410 days with15 ImmunoCult™20 Human25 CD3/CD28 T Cell T cells were expanded over 14 days with ImmunoCult™ Human CD3/CD28 T Cell Activator, ImmunoCult™ HumanCulture lengthCD3/CD28/CD2 (days) T Cell Activator or competitor’s Activator, ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator or competitor’s bead-based activation reagent in ImmunoCult™-XF T Cell Expansion Medium bead-based activation reagent in ImmunoCult™-XF T Cell Expansion Medium supplemented with rhIL-2. Fold expansion was determined between 0 to 14 supplemented with rhIL-2. % viability was determined between 0 to 14 days. days. (Note that T cells were not reactivated during the course of expansion.) (Note that T cells were not reactivated during the course of expansion.) ™ AA BB Why Use ImmunoCult T Cell 10000 10000 Activation and Expansion n 1000 n o o i 1000 i Reagents? s s n n a 100 a p p x 100 x OPTIMIZED. Robust activation and rapid E E 10 d d l l o o 10 expansion without the use of magnetic beads. F F 1 0.1 1 DEFINED FORMULATION. Consistent expansion F 1 2 3 4 5 6 F 1 2 3 4 X r r r r r r X r r r r - to to to to to to - to to to to ™ ti ti ti ti ti ti ™ ti ti ti ti lt e e e e e e lt e e e e without the need to add serum. u u C mp mp mp mp mp mp C mp mp mp mp o o o o o o o o o o o o n C C C C C C n C C C C FREEDOM TO USE. Not exclusively licensed for Immu Immu use in certain institutions. Figure 6. ImmunoCult™-XF T Cell Expansion Medium supports greater T cell expansion than other serum-free and serum-supplemented media T cells were activated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator, and cultured in (A) ImmunoCult™-XF T Cell Expansion Medium or serum-free competitor media with rhIL-2 in three replicate cultures per donor, or cultured in (B) ImmunoCult™-XF T Cell Expansion Medium or serum-supplemented competitor mediaA with rhIL-2 in three replicate cultures perB donor. T cells were stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator on day 0 and every 7 to 8 days for the duration of the culture. T cells were analyzed on day 21 for fold expansion relative to the initial cell seeding density. (A) Compared to all serum-free competitor CD4+CD8- in CD3+ Gate CD4-CD8+ in CD3+ Gate media tested, ImmunoCult™-XF T Cell Expansion Medium showed significantly higher expansion of total T cells. Competitors 1 to 6 represent serum-free competitor media. 1Each00 column with error bars represents the100 mean ± S.E.M. (p <5x10-13 for ImmunoCult™-XF T Cell Expansion Medium versus all other serum-free media, tested using the linear mixed effect model with linear regression, n = 4 to 19 donors). (B) Compared to all serum-supplemented competitor media tested, ImmunoCult™-XF T Cell Expansion80 Medium showed similar or significantly80 higher expansion of total T cells. Competitors 1 to 4 represent serum-supplemented competitor media. Each column with error bars represents the mean ± S.E.M. (p<0.0006 for ImmunoCult™-XF T Cell Expansion Medium versus all other serum-supplemented media except for (%) (%) competitor 60 4, tested using the linear mixed effect 6 0model with linear regression, n = 1 to 19 donors). e e g g a a t t n n e e c c r 40 r 40 e e P P 20 20 0 0 Day 0 Day 21 Day 0 Day 21 3 100 IFN-gamma+ IL-4+ 80 ) 60 % ( e g a t n e c r e 40 P 20 0 - + 8 + 3 D 8 D C D C + -C 4 4 D D C C + + 3 3 D D C C T Cell Therapy Reagents Isolation, Activation and Expansion Manufacture Chimeric Antigen Receptor (CAR) T Cells Using EasySep™ Release and ImmunoCult™ Reagents Cells Releasable RapidSpheres™ T cell isolation TAC B T cell activation using ImmunoCult™ Human T Cell A Activator and genetic C modification to express CAR. T cell isolation using CAR T cell expansion EasySep™ Release. using ImmunoCult™-XF T Cell Expansion Medium. Leuko Pak from patient. = T cells = Releasable RapidSpheres™ = ImmunoCult™ Human T Cell Activator = TAC = Activated T cells = Retrovirus = CAR Figure 7. Integrated workflow for the manufacturing of CAR T cells T cell isolation using (A) EasySep™ Release Human CD3 Positive Selection Kit; (B) T cell activation using soluble ImmunoCult™ Human T Cell Activator and gene modification to express the CAR construct; and (C) expansion of CAR T cells in xeno-free and serum-free ImmunoCult™-XF T Cell Expansion Medium. Why Use STEMCELL’s Reagents for Cell Therapy Research Applications? DOCUMENTATION. Traceability documentation including CoAs and CoOs to help reduce time in preparing IND submissions or clinical trial applications. CONSISTENCY.