T Cell TransAct™ Efficient T cell activation and expansion Your new solution for T cell activation What is T Cell TransAct™ ? This ready-to-use reagent provides an innovative method for physiological activation and expansion of human T cells. T Cell TransAct is a colloidal polymeric nanomatrix conjugated to humanized recombinant CD3 and CD28 agonists ensuring successful activation of resting T cells from hematological cell populations (e.g. PBMCs or enriched T cell populations) without the involvement of CD4 or CD8. MACS® GMP T Cell TransAct is manufactured and controlled under ISO 13485 requirements. It is designed following the recommendations of USP <1043> on ancillary materials. In the US, a master file is held with the FDA for IND applications with Product Quality Certificate available via our website. Figure 1: T Cell TransAct is in suspension when added to cell culture for polyclonal T cell stimulation. T cell activation made simple and convenient Practical application • Volumetric dosage • Ready-to-use • Removal by simple washing Robust stimulation • Highest cell viability • Physiological and stable stimulation Convenient compatibility • Available for research and GMP T cell workflows GMP • Optimized for CAR T cell production on the CliniMACS Prodigy® • Can be sterile filtered 22 Efficient T cell activation and expansion Effective stimulation Excellent proliferation T Cell TransAct™ enables optimal T cell activation with Proliferation of T cells is observed after stimulation a polymeric nanomatrix. The activation efficiency is with T Cell TransAct. Equal amounts of proliferation comparable to larger activation beads. T cell activation are observed when compared to bead-based made simple and convenient. stimulation methods. 100 100 75 75 cells (%) cells + 50 50 CD25 + 25 (%) Proliferation 25 CD69 0 0 Negative control T Cell TransAct Competitor product Negative control T Cell TransAct Competitor product Figure 2: Comparison of activation efficiency at day two between Figure 4: After seven days, proliferation of T Cell TransAct-activated T Cell TransAct and competitor product according to the activation cells is equal to bead-activated cells. T cells were cultivated markers CD69 and CD25. After two days, T Cell TransAct-activated cells in TexMACS™ Medium supplemented with IL-7 and IL-15. are comparable to bead-activated cells. High expansion T cell phenotype When striving for robust and reliable T cell Generating CAR T cells requires a stable T cell proliferation, T Cell TransAct enables serum-free phenotype. T cells activated with T cell TransAct T Cell cultivation while maintaining consistently and subsequently expanded with IL-7 and IL-15, high cell expansion. display a phenotype of early differentiated T cells. 150 100 125 80 100 75 60 50 40 Expansion (x-fold) 25 0 20 0 2 4 6 8 10 12 14 Frequency of cell subset (%) Time (days) 0 Phenotype of activated T cells Negative control T Cell TransAct Competitor product Central memory T cells/ Effector T cells/ Naive T cells Figure 3: Comparable results of T cell expansion after stimulation with Stem memory T cells Effector memory T Cell TransAct or competitor product in TexMACS Medium without T cells human AB serum supplementation. Figure 5: T cells were activated with T Cell TransAct and expanded for 14 days in TexMACS Medium supplemented with IL-7 and IL-15. More than 85% were stem memory T cells and central memory T cells. 4 Setting the stage for automated production of engineered T cells Optimal design for CliniMACS Prodigy® Cell expansion in serum-free media MACS® GMP T Cell TransAct™ is tailor made for the Clinical-scale expansion of transduced T cells is CliniMACS Prodigy. effective under cultivation conditions with or without human AB serum (fig. 7A and B). The synergy between • Maximum activation capacity for up to 1×10⁸ cells MACS GMP T Cell TransAct, TexMACS™ Medium • 1 vial of MACS GMP T Cell TransAct per T cell and our MACS GMP Cytokines delivers an optimal transduction (TCT) production run final engineerd cell product independent of serum addition (fig. 7C). MACS GMP T Cell TransAct allows potent polyclonal T cell activation prior gene modification without the need for feeder cells. A B 80 100 Control Gene-modified 80 ) 8 60 A B Lentivirus 10³ 10³ 60 40 10² 10² 40 Viability (%) 10¹ 10¹ Cell count (×10 20 20 1 1 CD3-APC-Vio700 CD3-APC-Vio700 CD3-APC-Vio770-A 0 CD3-APC-Vio770-A 0 -1 -1 0 0 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ 2 4 6 8 10 12 14 2 4 6 8 10 12 14 GFP-AGFP GFP-AGFP Time (days) Time (days) C D Retrovirus C Final cell product 10³ 10³ 100 10² 10² 80 10¹ 10¹ 60 1 1 CD3-APC-Vio700 CD3-APC-Vio700 CD3-APC-Vio700 CD3-APC-Vio770-A 0 CD3-APC-Vio770-A 0 40 -1 -1 -1 0 1 10¹ 10² 10³ -1 0 1 10¹ 10² 10³ percentage Cell 20 GFP-AGFP GFP-AGFP Figure 6: Isolated T cells were activated with MACS GMP T Cell TransAct 0 and transduced with lentivirus (B) or retrovirus (D). Transduction Starting Enriched With serum Serum-free of T cells with GFP vector resulted in strong GFP expression eleven days material population TCT TCT after gene modification (B, D). Untransduced T cells show no expression of GFP (A, C). Serum-free With serum CD4+ T cells NK cells B cells CD8+ T cells Monocytes Granulocytes NKT cells Figure 7: Enriched CD4+ and CD8+ T cells were stimulated with MACS GMP T Cell TransAct and expanded in a TCT process with 3% human AB serum or serum-free. Cell count (A) and viability (B) of cultured cells were measured at different time points. Cellular composition was determined in starting material, enriched population and in the final expanded product (C). 5 MACS® GMP T Cell TransAct™- Large Scale A B 2.5 100 2.0 80 ) 10 1.5 60 1.0 40 Viability (%) Cell count (×10 0.5 20 0 0 2 4 6 8 10 12 2 4 6 8 10 12 Time (days) Time (days) C Final cell product 100 80 Scale-up your T cell expansion 60 40 MACS GMP T Cell TransAct - Large Scale is optimized for the activation of high cell numbers. It is tailor- (%) Frequencies 20 made for the application on the CliniMACS Prodigy® 0 T T T T T T in combination with the tubing set including the large N SCM EM N SCM EM TCM TEFF TCM TEFF cultivation chamber. Standard-scale chamber Large-scale chamber • Efficient T cell activation and expansion Standard-scale chamber Large-scale chamber for high cell numbers Figure 8: Enriched CD4+/CD8+ T cells were automatically expanded • Optimized to activate and expand up to on the CliniMACS Prodigy after polyclonal stimulation with MACS 4×10⁸ enriched T cells GMP T Cell TransAct, standard or Large Scale. Either the standard-scale chamber (black, n=4) or the large-scale chamber (orange, n=6) was • One vial of MACS GMP T Cell TransAct - Large Scale used for culture. Cell cultivation was monitored at different time points to determine cell number (A) and viability (B). On average, a total cell is sufficient for one T cell transduction number of 2.1×101⁰ cells was reached using the large-scale chamber large-scale production run and MACS GMP T Cell TransAct - Large Scale in comparison to 6.5×10⁹ cells expanded in the standard-scale chamber. The cellular composition of the enriched fraction was analyzed by flow cytometry on the MACSQuant® Analyzer 10. Frequencies of T cell phenotypes among viable CD45+ cells were determined for the final cell product (C). 6 Translational solutions for T cell activation Research ACTIVATION Clinical T Cell TransAct™ MACS® GMP T Cell TransAct • Easy-to-use • GMP-grade • Robust T cell activation • Compatible with the • Colloidal polymeric CliniMACS Prodigy® nanomatrix • Can be sterile filtered STIMULATION MACS Cytokines MACS GMP Cytokines • High grade quality • S uperior cytokine purity • Reliable, high biological • High specific activity activity • Free of animal- and • Flexible: customized fillings human-derived materials EXPANSION TexMACS™ Medium MACS GMP TexMACS • Serum-free T cell medium • I deal for GMP-compliant • S pecifically designed for T cell expansion T cell expansion • Serum- and xeno-component free • Excellent quality • Available with or without phenol red VIDEO See the CliniMACS Prodigy Process in action! Generate gene-modified T cells in a simple and automated fashion. Easy to use, this unique process will surely change the way you work. miltenyibiotec.com/tct 777 miltenyibiotec.com Germany/Austria Benelux Italy Singapore Switzerland Miltenyi Biotec B.V. & Co. KG Miltenyi Biotec B.V. Miltenyi Biotec S.r.l. Miltenyi Biotec Asia Pacific Pte Ltd Miltenyi Biotec Swiss AG Friedrich-Ebert-Straße 68 Sandifortdreef 17 Via Paolo Nanni Costa, 30 438B Alexandra Road, Block B Gibelinstrasse 27 51429 Bergisch Gladbach 2333 ZZ Leiden 40133 Bologna Alexandra Technopark 4500 Solothurn Germany The Netherlands Italy #06-01 Switzerland Phone +49 2204 8306-0 [email protected] Phone +39 051 6 460 411 Singapore 119968 Phone +41 32 623 08 47 Fax +49 2204 85197 Customer service Fax +39 051 6 460 499 Phone +65 6238 8183 Fax +49 2204 85197 [email protected] The Netherlands [email protected] Fax +65 6238 0302 [email protected] Phone 0800 4020120 [email protected] USA/Canada Fax 0800 4020100 Japan United Kingdom Miltenyi Biotec Inc. 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