Development of a truncated EGFR marker as a safeguard for adoptive T cell therapy (2014)

Adoptive transfer of T cells that are engineered to carry defined antigen-specific receptors, both T cell receptors (TCRs) and chimeric antigen receptors (CARs), has shown to be effective in different clinical settings, especially for the treatment of B cell malignancies.Optimized receptor design and increased persistence of transferred T cells have led to better clinical outcomes, but unfortunately, several incidents of serious on-and off-target toxicities were also reported.Thus, key issues still need to be addressed to improve the quality and safety of gene-modified cell products for therapy.These include: 1) optimized selection protocols to obtain a highly pure cell population that expresses the introduced antigen receptor, and 2) a safety mechanism to facilitate elimination of transferred cells in the event of adverse effects.A promising strategy is to co-express the transgenic receptor with a truncated cell surface marker derived from the human epidermal growth factor receptor (EGFRt).The aim of this PhD thesis was to evaluate and further develop the EGFRt for cell selection, in vivo tracking and cell ablation.For validating the EGFRt as a tracking marker, we genetically modified T cells with different CAR/EGFRt constructs and confirmed stable expression of the EGFRt in vitro and in vivo over long time periods.For obtaining highly pure cell products, we developed cell selection protocols based on the Streptamer technology.Therefore, we generated a reversible EGFR-specific Fab fragment that could be multimerized on magnetic nanobeads to enrich EGFRt + T cells with high purities and good yields.As reversible Streptamer selection reagents can be completely removed from the cell surface after the selection procedure, the purified cell product can directly be transferred into the patient in future applications.For in vivo depletion of engineered cells, the EGFRt marker is targeted by the clinically approved anti-EGFR antibody Cetuximab, which mediates antibody-dependent cellular cytotoxicity.To study the feasibility and efficiency of this strategy, we established a mouse model that allowed for efficient transfer of GFP/EGFRt + T cells, reliable in vivo tracking using GFP as an additional marker and sensitive analysis of the extent of the depletion by using ovalbumin-specific (OT-I) cells that can be expanded in vivo by vaccination.We found that T cells with high expression levels of the EGFRt were completely eliminated by Cetuximab. VWe also addressed the question whether T cell depletion via the EGFRt safeguard can counteract T cell-mediated toxicities in clinically relevant mouse models.In a syngeneic mouse model, we transferred functional anti-mouse CD19-CAR/EGFRt-transduced T cells that suppressed the growth of endogenous B cells.However, if T cells were eliminated using Cetuximab, the B cell population recovered within few weeks.In HBVtransgenic mice, the function of HBV-specific T cells can be analyzed by monitoring the levels of alanine aminotransferase (ALT) as a measure for T cell killing of HBV + hepatocytes.We could show that Cetuximab-mediated depletion of functional HBVspecific, EGFRt + T cells prevented liver toxicity as indicated by unchanged ALT levels.In addition, we evaluated the EGFRt-based depletion strategy in a pilot study in nonhuman primates.Therefore, we transduced macaque T cells with an EGFRt lentiviral construct and subsequently purified the cells using EGFR Fab Streptamers.Importantly, both the transfer of EGFRt + T cells and Cetuximab infusions appeared to be safe and we observed a decrease in EGFRt + T cells after Cetuximab treatment.In summary, these data demonstrate that the EGFRt marker is a safe and valuable tool for clinical cell purification, in vivo tracking and effective cell depletion.The approach can be readily made available for clinical application and can be transferred to other types of cell-based therapies.The co-expression of EGFRt therefore represents a promising concept to improve the safety of therapies based on gene-engineered cells. VI