Abstract SY05-01: Non-immunogenic PET reporter imaging of HSC transplants and T cell immunotherapies
Authors: Witte Owen N. 
Published: August 2015

Abstract
Melissa N. McCracken, Eric H. Gschweng, Dimitrios N. Vatakis, Dhaval Dixit, Jami McLaughlin, Aaron R. Cooper, Donald B. Kohn, Jerome A. Zack, Owen N. Witte
Transfused tumor reactive T cells have reduced tumor burden, but in rare cases, on-target/off-tumor effects have been lethal in clinical settings. As novel cellular immunotherapies are developed, there is an essential need for a non-invasive method to track the location of engineered cells with high sensitivity and resolution identifying the correct cell homing or dangerous off-target locations in pre-clinical and future clinical applications. Positron emission tomography (PET) allows for non-invasive and repetitive in vivo visualization of positron-emitting probes directly translatable from pre-clinical to clinical applications. Here we use a human PET reporter gene to detect only the specific engineered immune cell populations.
Human deoxycytidine kinase triple mutant (hdCK3mut), a new non-immunogenic PET reporter, is an effective tool for whole-body monitoring of hematopoiesis. hdCK3mut contains three point mutations within the active site and is used in combination with the PET probe [18F]-L-FMAU (1-(2-deoxy-2-18 fluoro-β-L-arabinofuranosyl)-5-methyluracil). Comparison of hdCK3mut and sr39TK established that hdCK3mut was approximately two fold more sensitive for in vivo detection. In mice, both human and mouse hematopoietic stem cells expressing hdCK3mut retained equivalent engraftment, differentiation, expansion and longevity in vivo.
We then engineered hdCK3mut to be co-expressed with the anti-melanoma T cell receptor F5 in human CD34 cells or PBMCs to model engineered immunity. In CD34 cells the expression of hdCK3mut allowed for visualization of engrafted stem/progenitor cells within the bone marrow. T cell homing was detected non-invasively by the accumulation of [18F]-L-FMAU in hdCK3mut expressing T cells located intra-tumorally. Coexpression of hdCK3mut with the F5 anti-melanoma TCR had demonstrably higher signals in HLA matched tumors when compared to animals solely expressing hdCK3mut. In vivo engineered T cells coexpressing hdCK3mut and F5 caused HLA/antigen matched tumor cytotoxicity. In vitro no observable difference in IFN- production, cytotoxicity, or T cell activation after antigen presentation was seen in comparing F5 to F5/hdCK3mut. These results demonstrate that expression of hdCK3mut is inert, and engineered cells retain their cytotoxic function. hdCK3mut can be used to simultaneously monitor both engraftment and tumor infiltration without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.
Citation Format: Owen N. Witte. Non-immunogenic PET reporter imaging of HSC transplants and T cell immunotherapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY05-01. doi:10.1158/1538-7445.AM2015-SY05-01