MaxCyte Scalable Electroporation: A Universal Cell Engineering Platform for Development of Cell-based Medicines from R&D to Clinic. Payal Roychoudhury, Jessica Carmen, Linhong Li, Pachai Natarajan, Krista Steger, and Madhusudan Peshwa. MaxCyte, Gaithersburg, MD, USA. Each cell-based therapeutic modality – from viral vectors to immune cell engineering and in situ gene editing -- relies on different biologic approaches, however, they all require some type of cell engineering for therapeutic manufacturing. MaxCyte developed a non-viral, electroporation-based cell engineering technology that has the performance, flexibility, safety and scalability for use in cell therapy development through to manufacturing for patient treatment. In this poster, we present capabilities of MaxCyte scalable electroporation, a platform of cGMP-compliant, CE-marked instruments with an FDA Master File. Data for high performance electroporation of a variety of cell types commonly used in cellular therapeutics, including adherent and suspension cells as well as cell lines and primary cells, are summarized. Use of MaxCyte electroporation for a breadth of real world applications are highlighted including lentivirus and AAV production, engineering of primary T-cells for the expression of an anti-mesothelin CAR molecule, and CRISPR-mediate gene editing of stem cells. These data will directly illustrate the scalability and consistency of MaxCyte electroporation that enables the use of this single cell engineering technology from early R&D to patient dosing of cell- based biotherapeutics. Abstract Summary • MaxCyte electroporation is a universal cell engineering technology that supports the development and manufacturing of viral vectors and T-cell therapies, including gene editing- mediated cell modification. • MaxCyte cell engineering technology efficiently transfects a variety of cell types, including historically difficult-to-transfect cells such as primary cells, with low levels of cell cytotoxicity. • MaxCyte flow electroporation has the safety, efficiency and scalability to support cell therapy and gene editing development from early R&D through patient treatment. • Production scale-up from the MaxCyte STX to the MaxCyte VLX is seamless – high cell viability and transfection efficiencies are maintained without the need for reoptimization. • MaxCyte electroporation has the reproducibility, and scalability for use in biomanufacturing. • MaxCyte electroporation offers a non-viral means of engineering T-cells that have in vitro and in vivo anti-tumor activity. • MaxCyte instruments are closed, computer-controlled, cGMP- compliant systems with a Master file with the US FDA & Health Canada enabling simplified migration to the clinic. MaxCyte, MaxCyte STX, MaxCyte GT and MaxCyte VLX are registered trademarks of MaxCyte, Inc. ©2016 MaxCyte, Inc. All Rights Reserved. Corresponding Author: Jessica Carmen; [email protected] MaxCyte, Inc., Tel: (301) 944-1700 [email protected] , www.maxcyte.com MaxCyte STX ® 5E5 Cells in Seconds Up to 2E10 Cells in <30 min MaxCyte VLX ® Up to 2E11 Cells in <30 min MaxCyte Transient Transfection Platform MaxCyte GT ® Up to 2E11 Cells in <30 min Optimized for clinical use • Broad cell compatibility • True scalability requiring no re-optimization • High efficiency & high cell viability The MaxCyte STX ® , MaxCyte VLX ® , and MaxCyte GT® Transient Transfection Systems use fully scalable flow electroporation for rapid, highly efficient transfection. • Single use processing assemblies • Master file with US FDA & Health Canada • Closed, computer-controlled instruments • cGMP-compliant & CE-marked Viral Vector Production: Lentivirus & AAV Figure 1: Scale Up of Lentiviral Vector Production from Small-Scale to Large-Scale Production Using the MaxCyte Platform. Suspension-adapted HEK 293FT cells were suspended in MaxCyte’s electroporation (EP) buffer at 1E8 cells/mL. A mixture of plasmids encoding lentiviral vector components was added to the cells (0.4µg of DNA/1E6 cells), and cells were transferred to sterile OC-400, CL-2 and VLXD processing assemblies. Cells in the OC-400 and CL-2 were transfected by static and flow EP, respectively, using the STX instrument; cells in the VLXD were transfected by flow EP on the VLX. Lentiviral titers were measured after 24-48 hrs in culture. Normalized titer data show seamless scalability of the MaxCyte transfection process. MaxCyte STX to VLX Instrument Scale-up 4 Plasmid Lentiviral System Production in Suspension Cells Table 1. Large-scale Lentiviral Vector Production. Suspension-adapted HEK 293FT cells were harvested, resuspended at 1E8 cells/mL, and co-transfected with 4 plasmids (HIV- based lentivector system) using flow electroporation (CL-2 processing assembly). Cells were cultured post EP in 10-L Cellbag in a Wave Bioreactor System in a final volume of 2.1 to 2.3 L. 48 hours post EP, media was collected and infectious units measured as well as p24 concentrations*. Results for three independent production runs are shown. These data demonstrate the reproducibility of MaxCyte flow electroporation enabling large- scale, quality lentivirus production. *See Human Gene Therapy (2012) 23:243-249 for detailed methodology. Consistent Large-scale Lentivirus Manufacturing Suspension Cells Facilitate Scalability of Vector Production High Titer AAV Production in HEK Cells 3 Plasmid Co-transfection Yields High Efficiency & Robust Titers Figure 2: Production of AAV in HEK Cells. (A). Adherent HEK cells were transfected with three plasmids encoding AAV vector components (GFP transgene) via static electroporation using the MaxCyte STX. (B). Nearly 100% of the transfected cells exhibited robust transgene expression 48 hours post electroporation. (C). High AAV titers were detected in cell pellets via qPCR analysis. Gene Editing: CRISPR -EP C+G Marker +Cel1 -Cel1 +Cel1-1 -Cel1-1 +Cel1-2 -Cel1-2 +Cel1-3 -Cel1-3 0% 47% 50% 42% 300 100 200 500 468 298 170 Figure 6: mRNA-CRISPR transfection induced genomic DNA editing in AAVS1 site of CD34+ hematopoietic stem cells. Cells were either not transfected (-EP), or transfected mRNA-CRISPR with 3 repeats (C+G 1,2,3). The samples of the electrophoresis gel were loaded as follows: 1) Marker; 2) –EP with Cel-1; 3) –EP without cel-1; 4) C+G-1 with Cel-1; 5) C+G-1 without Cel-1; 6) C+G-2 with Cel-1; 7) C+G-2 without cel-1; 8) C+G-3 with Cel-1; 9) C+G without cel-1. The cut products of an edited AAVS-1 site are 298 and 170 basepairs, and the parental band is 468 base pairs. The editing rate was calculated as (density of digested bands)/[(density of digested bands + density of parental band). HSCs transfected with mRNA encoding Cas9 and guide RNA exhibited 43, 60, 54, and 52% editing in three different experiments. Figure 5: Transfection of HSC with mRNA-Cas9/gRNA has low cytotoxicity. HSC cells were electroporated two days after thawing. Shown in FIG. 5 is the viability (A), and proliferation (B) of HSC transfected by mRNA-Cas9/gRNA and mRNA-GFP. High viability and proliferation rate relative to control cells were achieved for HSC by using MaxCyte GT system. Strong Cell Health & Viability Post Transfection Low Cytotoxicity in Primary Cells Efficient Gene Editing Following MaxCyte Electroporation Transfection of CD34+ Stem Cells T-cell Engineering: mRNA CAR Figure 4: Regression of Tumors in Mice Treated with RNA CAR T-cells. (A). Flank tumors were established by M108 injection (s.c.) in NOD/scid/γc(−/−) (NSG) mice (n = 6). Sixty-six days after tumor inoculation, mice were randomized to equalize tumor burden and treated with meso-CAR RNA-electroporated T-cells. The T-cells (1E7 to 1.5 E7) were injected intratumorally every 4 days for a total of four injections using the same healthy donor; mice treated with saline served as controls (n = 3). Tumor size was measured weekly. Adoptive transfer of these mesothelin-targeted CAR T- cells via mouse subcutaneous injection was safe without overt evidence of off-tumor on-target toxicity against normal tissues. Additionally, meso-CAR RNA electroporated T- cells injected in mice with established mesothelin-positive tumors were able to reduce tumor size, whereas progressive tumor growth was observed in the control group of mice. (B). Tumors were established in NSG mice (n=6 per group) by i.p. injection with 8x10 6 M108-Luc cells. Beginning on day 58, RNA CAR-electroporated T-cells (1E7) expressing ss1-bbz were injected twice weekly for 2 weeks. RNA CAR T-cells expressing CD19-bbz RNA CAR or saline were injected as controls. On day 78 the luminescence signal was significantly decreased in the ss1- bbz mice compared with the CD19-bbz mice (P<0.01). The above studies indicate that biweekly injections of RNA CAR T cells can control advanced flank and i.p. tumors. Cancer Res. 70(22), 2010, p9053. Development of an α-mesothelin CAR mRNA T-cells for Solid Tumors Multiple Injection of Electroporated T-cells Expressing CAR Mediate Tumor Regression Figure 3: Sustained RNA CAR expression and Regression of Tumors in Mice Treated with RNA CAR T-cells. (A). Stimulated T-cells were electroporated with clinical-grade, in vitro transcribed RNA (10 μg RNA/100 μL T-cells) encoding anti-mesothelin CAR that includes both the CD3-ζ and 4-1BB co-stimulatory domains using the MaxCyte GT and transgene expression analyzed via FACS daily. Electroporated cells expressed the transgene for as long as 7 days post transfection