Abstract
We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection.
Highlights
A subgroup of patients with Ewing’s sarcoma family of tumors (ESFT) is still threatened by a poor long term prognosis
As the extent of cell antigen receptor (CAR) internalization is strongly determined by the target antigen density [31,32], we investigated the effect of interaction with ESFT cell lines characterized by different levels of NKG2D-L expression
In confirmation and extension of Verhoeven et al.’s data, we found NKG2D-Ls consistently expressed in all of 14 tested ESFT cell lines (Fig. 1)
Summary
A subgroup of patients with Ewing’s sarcoma family of tumors (ESFT) is still threatened by a poor long term prognosis. Despite modern multimodal therapy (chemotherapy, radiation, and surgery) ESFT relapse in about 30% of patients with localized disease. Long-term survival of those who relapsed and of patients with metastatic disease at diagnosis is currently below 30% [1,2,3]. The infusion of NK cells has emerged as a promising new treatment strategy for malignant tumors in general and has been suggested for the treatment of ESFT [4,5]. CD8pos T cells, on the other hand, are characterized by the capacity to differentiate into effector cells or long term memory cells, and have been adopted in the past with a broad range of new antigenic specificities by receptor transfer (for review see [8])
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