Previously, we constructed a bispecific NK-cell-engager (BiKE) bearing single-chain variable fragments (scFv) against CD16 on NK cells and EpCAM on tumor cells. This BiKE facilitated antigen-specific antibody-dependent cell-mediated cytotoxicity (ADCC) but did not induce NK cell expansion. We incorporated a modified interleukin-15 cross-linker to create a trispecific construct (TriKE) in order to improve activation, proliferation, and survival of NK cells. Synthesis and assembly of hybrid genes encoding the TriKE was accomplished using DNA-shuffling and DNA-ligation techniques. The TriKE was tested for specificity, efficacy, proliferative capability, and cytokine profile using functional assays. The molecular modifications improved yield without compromising binding to EpCAM + HT-29 colorectal carcinoma cells. 51 Chromium-release and degranulation assays showed better killing rates with TriKE compared to BiKE. TriKE was more active in a variety of different carcinoma cell lines. TriKE showed the ability to stimulate expansion of CD56 + CD3 - NK cells. BiKE and TriKE showed enhanced but not supraphysiologic levels of cytokine secretion. 1615EpCAM TriKE drives enhanced ADCC while significantly improving proliferation, activation, and survival of NK cell effectors. The TriKE provides a selectively delivered self-sustaining signal at the NK/tumor cell synapse. Targeted cytokine stimulation, rather than systemic cytokine administration, may impact toxicity in patients rendering the TriKE a promising new off-the-shelf carcinoma therapy.
Bibliographical noteFunding Information:
We acknowledge the excellent technical assistance of Deborah Todhunter, Andy Sicheneder, Sami Chu, and Seunguk Oh. This work was supported in part by the US Public Health Service Grants R01-CA36725, R01-CA72669, P01-CA65493, P01-CA111412 and R35 CA197292 awarded by the NCI and the NIAID, DHHS. It was also supported by an NIH Research Evaluation and Commercialization Hub (REACH) Award (U01), the Mayo Partnership Award, the Lion Fund, William Lawrence and Blanche Hughes Fund, the Randy Shaver Foundation, the Atwater Cancer Drug Development Award, a CETI translational award from the University of Minnesota Masonic Cancer Center (D.A.V.) and the Deutsche Krebshilfe (111548) (J.U.S.). D.A.V. and J.S.M. are members of the Oxis Biotech Scientific Advisory Board and hold equity in the company. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies.
© 2016 The American Society of Gene & Cell Therapy.