Truncated Cell-Surface CD19 As a Conditional Suicide Switch For Adoptive T Cell Immunotherapy

• Published in: Blood 2013-11, Vol.122 (21), p.1660-1660
• Main Authors: Budde, Lihua Elizabeth, Mardiros, Armen, Chang, Wen-Chung, Wang, Xiuli, Berger, Carolina, Brown, Christine, Riddell, Stanley R, Press, Oliver W., Forman, Stephen J.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.1660.1660

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. 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. 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