Expansion and Bioengineering of Peripheral Blood-Derived Anti-Cancer Gd T Cells in Serum Free Media

Conventional immunotherapy relies on expansion and, in some settings, transduction of αβ T cells. Success is dependent on several highly variable patient-specific parameters including i) cell number obtained from harvest and ex vivo expansion, ii) targeting capacity of cells to tumors, iii) ability of infused T cells to survive and function in a immune evading tumor microenvironment (e.g., down-regulation of major histocompatibility complex (MHC) on the tumor cell surface), and iv) on-target and off-target adverse events such as graft vs host disease (GvHD). Following the the early success of a few anti-leukemia products, some progress has been made on expanding the applicability to a wider variety of hematologic and solid neoplasms but rapid progress has been hindered due to lack of efficacy and significant adverse event profiles. Thus, alternative cellular immunotherapy approaches such as gd T cells are being pursued. Although gd T cells represent a small fraction (2-5%) of circulating peripheral blood mononuclear cells (PBMC), they are ideal candidates for immunotherapy applications because they i) possess intrinsic anti-tumorigenicity, ii) require no priming to mediate tumor cell lysis in an MHC-independent manner, iii) direct tumor killing via recognition of stress-responsive ligands frequently up-regulated by tumor cells, and, as we show, iv) can be expanded to clinical cell doses in cGMP serum free media (SFM). Although tremendous effort has been devoted to αβ T cell processing, little focus has been directed toward the clinical development of genetically engineered gd T cells. Herein, we describe the optimization of drug and cytokine dosing and, culture in SFM, robust expansion and genetic modification necessary for clinical applications. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs, including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, such as RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2mM L-glutamine (if not present in the commercial product), Zoledronic Acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 IU/ml added twice weekly). Of the SFM cultures, PBMC isolated from 7 of 8 donors had robust expansion only in OpTmizer (supplemented with high-dose IL-2) with a concomitant rise in gd T cell percentage to levels sufficient for clinical use. Specifically, the percentage of gd T cells increased from a mean of 3% (2.4-3.6%) to 61% (21-84%), resulting in an 88 fold expansion at 14 days. While gd T cell numbers were lower in SFM compared to serum-containing RPMI, the final product starting with PBMC harvested during a single apheresis procedure when cultured in OpTmizer can yield therapeutic doses. The expanded gd T cells were evaluated for their transduction efficiency using a 3^rd generation self-inactivating lentiviral vector (LV) to introduce a reporter gene encoding EGFP. Two different LVs were tested: HIV and SIV harboring the transgene under the control of EF1α and MSCV promoters, respectively. The timing of LV addition was optimized with respect i) gd T cell expansion, ii) LDL Receptor (LDLR) expression (a known receptor utilized by VSV-G pseudotyped LV) and iii) LV dose (3-4 consecutive transductions over 48 hours). Transductions remained consistent using approximately 30% LV concentrated stock by volume. As expected, greater transduction was achieved in cultures with the greatest expansion. Interestingly, higher transduction efficiency was observed in gd T cell populations with the highest LDLR levels. In gd T cells, an MSCV promoter provided greater GFP expression compared to an EF1α promoter. As expected a range of transduction efficiencies was observed for various donors spanning from 13-33%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and expansion levels. Therefore, an optimized method of gd T cell expansion and transduction was developed that can be tested in early phase clinical trials. The absence of MHC-restriction affords the opportunity, with appropriate elimination of the αβ T cell component, for use in the allogeneic setting with limited risk of GvHD, and the use of SFM provides clinically safer, widely applicable, and potentially more efficacious cellular immunotherapy.