A Simplified Method for Transduction and Expansion of T Cells for Clinical Application
Authors: Khalil Mariam , Foster Aaron , Gagliardi Christine 
Published: October 2019

Abstract
Introduction: Genetically modified T cells are being investigated to treat a variety of disorders and have been particularly successful in treating B cell cancers. As more effort is poured into new targets, molecular switches, and various other modifications, development of processes to quickly manufacture new products must keep up. Current manufacturing processes often require highly skilled operators and specialized equipment. Here, we demonstrate a simplified, novel method for transduction of T cells, followed by robust expansion in the G-Rex bioreactor with no need for intervention until harvest. A scaled-up, closed-version of the same process, including a closed harvest step with the GatheRex is currently under evaluation.
Methods: Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors were used as starting material. PBMCs were thawed, washed, and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags (32-C, Saint-Gobain Cell Therapy) or in G-Rex bioreactors (Wilson Wolf Corporation). Cells were cultured in TexMACS GMP medium (Miltenyi Biotec) with IL-7 and IL-15 throughout. For the transduction step, activated PMBCs and retroviral supernatant were incubated in cell culture bags coated with Retronectin (Takara) or in G-Rex bioreactors with vectofusin-1 (Miltenyi Biotec). Viral constructs contained either a CD34 or CD19 marker detectable by antibody staining. For transduction in the G-Rex, various cell densities, volumes, constructs, and multiplicity of infection (MOIs) were tested. Where applicable, the GatheRex device (Wilson Wolf Corporation) was used for volume reduction and harvest. Transduction efficiency and T cell phenotype were measured by flow cytometry. Cell count and viability were assessed with the NC-3000 (Chemometic). Glucose and lactate concentrations were checked daily for in-processing monitoring.
Results: Overall transduction efficiency ranged from 30-90% depending on the experimental conditions. Incubating 1x107 activated PBMCs in 10 ml of medium in a 10-cm2 G-Rex (1.0 ml/cm2) with retrovirus at an MOI of 1 resulted in 3% transduced cells. Addition of vectofusin-1 to the same condition yielded transduction efficiency of 44%. Increasing the MOI to 10 lead to 86% transduced cells. Decreasing the transduction volume from 1.0 ml/cm2 to 0.4 ml/cm2 increased transduction efficiency from 34% to 55%. Reducing the volume further to 0.2 ml/cm2 did not improve efficiency, and rather had a negative impact compared to the 0.4 ml/cm2 condition (38%). 16-24 hrs after transduction, the volume of medium was increased to 10.0 ml/cm2 without a wash step. The dilution in place of a wash step had no negative impact on cell viability. 10.0 ml/cm2 medium supported high viability (>90%) and expansion (30-50 fold) over an additional 9 days without operator intervention. The phenotype of cells expanded in the G-Rex contained a mixed population of CD45RO+ and CD45RA+ cells, with a similar distribution of naive and memory cell subsets in G-Rex and bag cultures. Harvest of cells with the GatheRex was efficient; a 1L volume was reduced 10-fold in 5 minutes, and 95% of cells were recovered.
Summary: T cells can be transduced with retroviral vectors in the G-Rex bioreactor. Clinically relevant levels of transgene expression can be achieved by combining reagents in the G-Rex, without complicated coating steps or time-consuming spinning steps. This simplified procedure reduces the hands-on time of the T cell transduction to minutes rather than hours. Transgenic cells can be expanded 30-50-fold in the G-Rex with limited operator intervention and without specialized equipment.

Disclosures
Khalil: Bellicum Pharmaceuticals: Employment. Foster:Bellicum: Employment, Equity Ownership. Gagliardi:Bellicum Pharmaceuticals: Employment.