Background

Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe Methods for removing transferred cells after treatment an important consideration. In addition, there is a lack of effective commercially available agents which allow for monitoring of CAR expression, tracking, isolating, and eliminating CAR- transduced cells. Therefore, adoptive T cell immunotherapy would benefit from a molecule which is stably expressed on the cell surface, of human origin, easily detected on transduced cells, lacking active biological function at baseline and capable of effectively ablating transduced cells on demand. Truncated CD19 (CD19t) harbors excellent features to be such a molecule. Its truncation shortens the intracytoplasmic domain to only 19 amino acids with removal of all conserved tyrosine residues that mediate known intracellular signaling transduction. It has been used successfully to mark transduced CAR T cells by several research groups. In this study, we set out to evaluate the activity of this truncated CD19 as a conditional suicide switch.

Methods

Lentiviral constructs containing a CD20 CAR and CD19t were used to transduce Jurkat T cells and primary human T cells to generate cells that express both molecules on the cell surface. CD19-mediated selection was carried out using PE conjugated anti-CD19 antibody. Internalization experiments were performed using transduced Jurkat cells that were kept at 4°C or 37°C. Surface CD19 expression was determined by flow cytometric analysis at 0 hour, 1 hour, 2 hours, and 4 hours after initial primary anti-CD19 antibody staining. NIH3T3 cells with truncated CD19 expressed on the surface (NIH3T3-19t) were generated and used for in vitro ablation experiments. Cells were left untreated or incubated withincreasing concentrations of CD19-ETA’, an anti-CD19 Pseudomonas toxin conjugate. The viability of NIH3T3-CD19t was determined by trypan blue exclusion at various time points.

Results

Using flow cytometry, we confirmed the expression of CAR and truncated CD19 on the transduced cell surface. Truncated CD19 was able to enrich transduced cells to more than 90% purity when used as a selectable marker. For CD19t to function as a conditional suicide switch, it needs to retain its ability to mediate antigen-antibody conjugate internalization. As expected, only up to 10% of CD19t remained on the surface of transduced cells after 4-hour period at 37°C. On the contrary, surface CD19t expression level remained largely unchanged when the cells were incubated at 4°C for 4 hours. CD19t also mediated robust ablation of transduced cells in vitro. More than 90% of transduced cells were ablated after 72-hour incubation with 10ug/ml CD19-ETA’. Mouse xenograft experiments are currently ongoing to test the in vivo tracking ability of CD19t and its ablation effect of transferred T cells upon administration of anti-CD19-drug conjugates.

Conclusions

These in vitro Results suggest that CD19t retains the ability to mediate antibody internalization upon its engagement. When exposed to an anti-CD19-drug conjugate, cells expressing truncated CD19 are effectively ablated in a dose dependent manner. We therefore predict that CD19t will be an excellent molecule to mark, select, track and eliminate modified T cells in vivo and it will be a useful tool for detection of engineered T cells and improvement of the safety of adoptive T cell immunotherapy.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.