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 geneediting -- relies on different biologic approaches, however, they all require some type of cellengineering for therapeutic manufacturing. MaxCyte developed a non-viral, electroporation-basedcell engineering technology that has the performance, flexibility, safety and scalability for use in celltherapy development through to manufacturing for patient treatment. In this poster, we presentcapabilities of MaxCyte scalable electroporation, a platform of cGMP-compliant, CE-markedinstruments with an FDA Master File. Data for high performance electroporation of a variety of celltypes commonly used in cellular therapeutics, including adherent and suspension cells as well as celllines and primary cells, are summarized. Use of MaxCyte electroporation for a breadth of real worldapplications are highlighted including lentivirus and AAV production, engineering of primary T-cells forthe 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 thatenables 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; jcarmen@maxcyte.com MaxCyte, Inc., Tel: (301) 944-1700 info@maxcyte.com, www.maxcyte.com

MaxCyte STX®

5E5 Cells in SecondsUp 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 minOptimized 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-ScaleProduction Using the MaxCyte Platform. Suspension-adapted HEK 293FT cells weresuspended in MaxCyte’s electroporation (EP) buffer at 1E8 cells/mL. A mixture of plasmidsencoding lentiviral vector components was added to the cells (0.4µg of DNA/1E6 cells), andcells were transferred to sterile OC-400, CL-2 and VLXD processing assemblies. Cells in theOC-400 and CL-2 were transfected by static and flow EP, respectively, using the STXinstrument; cells in the VLXD were transfected by flow EP on the VLX. Lentiviral titers weremeasured after 24-48 hrs in culture. Normalized titer data show seamless scalability of theMaxCyte transfection process.

MaxCyte STX to VLX Instrument Scale-up4 Plasmid Lentiviral System Production in Suspension Cells

Table 1. Large-scale Lentiviral Vector Production. Suspension-adapted HEK 293FT cellswere harvested, resuspended at 1E8 cells/mL, and co-transfected with 4 plasmids (HIV-based lentivector system) using flow electroporation (CL-2 processing assembly). Cellswere cultured post EP in 10-L Cellbag in a Wave Bioreactor System in a final volume of 2.1to 2.3 L. 48 hours post EP, media was collected and infectious units measured as well asp24 concentrations*. Results for three independent production runs are shown. Thesedata demonstrate the reproducibility of MaxCyte flow electroporation enabling large-scale, quality lentivirus production. *See Human Gene Therapy (2012) 23:243-249 fordetailed methodology.

Consistent Large-scale Lentivirus ManufacturingSuspension Cells Facilitate Scalability of Vector Production

High Titer AAV Production in HEK Cells3 Plasmid Co-transfection Yields High Efficiency & Robust Titers

Figure 2: Production of AAV in HEK Cells. (A). Adherent HEK cells were transfected withthree plasmids encoding AAV vector components (GFP transgene) via staticelectroporation using the MaxCyte STX. (B). Nearly 100% of the transfected cellsexhibited robust transgene expression 48 hours post electroporation. (C). High AAV titerswere 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 inAAVS1 site of CD34+ hematopoietic stem cells. Cells were either nottransfected (-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 withoutCel-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 and170 basepairs, and the parental band is 468 base pairs. The editing rate wascalculated as (density of digested bands)/[(density of digested bands +density of parental band). HSCs transfected with mRNA encoding Cas9 andguide RNA exhibited 43, 60, 54, and 52% editing in three differentexperiments.

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 theviability (A), and proliferation (B) of HSC transfected by mRNA-Cas9/gRNA andmRNA-GFP. High viability and proliferation rate relative to control cells wereachieved 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.) inNOD/scid/γc(−/−) (NSG) mice (n = 6). Sixty-six days after tumorinoculation, mice were randomized to equalize tumor burden andtreated with meso-CAR RNA-electroporated T-cells. The T-cells (1E7 to1.5 E7) were injected intratumorally every 4 days for a total of fourinjections using the same healthy donor; mice treated with saline servedas controls (n = 3). Tumor size was measured weekly. Adoptive transferof these mesothelin-targeted CAR T- cells via mouse subcutaneousinjection was safe without overt evidence of off-tumor on-target toxicityagainst normal tissues. Additionally, meso-CAR RNA electroporated T-cells injected in mice with established mesothelin-positive tumors wereable to reduce tumor size, whereas progressive tumor growth wasobserved in the control group of mice. (B). Tumors were established inNSG mice (n=6 per group) by i.p. injection with 8x106 M108-Luc cells.Beginning on day 58, RNA CAR-electroporated T-cells (1E7) expressingss1-bbz were injected twice weekly for 2 weeks. RNA CAR T-cellsexpressing CD19-bbz RNA CAR or saline were injected as controls. Onday 78 the luminescence signal was significantly decreased in the ss1-bbz mice compared with the CD19-bbz mice (P<0.01). The above studiesindicate that biweekly injections of RNA CAR T cells can control advancedflank 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 inMice Treated with RNA CAR T-cells. (A). Stimulated T-cells wereelectroporated with clinical-grade, in vitro transcribed RNA (10 μgRNA/100 μL T-cells) encoding anti-mesothelin CAR that includes both theCD3-ζ and 4-1BB co-stimulatory domains using the MaxCyte GT andtransgene expression analyzed via FACS daily. Electroporated cellsexpressed the transgene for as long as 7 days post transfection