Natural killer (NK)-cell related anti-tumor surveillance is limited by the ability of the tumor to escape killing. Previously, we constructed a bispecific NK-cell engager (BiKE) consisting of two scFV against CD16 (FcγRIII) on NK cells and EpCAM on tumor cells (EpCAM16). Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein with prevalent expression on carcinomas making it a valuable marker for cancer targeting. This BiKE facilitated ADCC and tumor elimination, but did not account for the cellular expansion required for the success of T CARs. To improve this, we incorporated a modified interleukin (IL)-15 crosslinker to create a trispecific construct (TriKE) to enhance activation, proliferation, and to prolong survival of NK-cells. IL-15 was chosen since it is an established immunostimulatory cytokine with known effects on NK cells and is recognized as a promising cancer cure drug in NIH guided review. TriKE was assembled, expressed in E.coli, extracted, refolded, and purified to >90% with a molecular weight of 68,860 daltons. To determine the functional activity of 1615EpCAM, its killing ability was measured in standard Cr-51 release assays with EpCAM+ HT-29 colorectal cancer cells. The 1615EpCAM TriKE induced the highest level of killing compared to BiKE and other controls. To determine if the effect of IL-15 in the drug correlated with NK-cell levels, donors were selected with different naturally occurring NK cell levels. Freshly isolated PBMCs were added to HT-29 cells at E:T ratios of 20:1, 6.6:1, and 2:1. Donors showed increasingly higher levels of Cr-51 kill with TriKE, but not with BiKE indicating that greater the presence of NK-cells, the greater the TriKE effect. In order to study lytic degranulation as a function of NK-cell activity, CD107a expression was measured. Cells treated with TriKE showed significantly elevated degranulation when co-cultivated with targets (p<0.001) compared to BiKE. IL-15 alone did not enhance lytic degranulation. When PBMC were exposed to TriKE, only NK-cells, but not T-cells showed a proliferation specific pattern. Direct comparison of the BiKE and the TriKE showed that the TriKE had the ability to induce proliferation and expansion, the BiKE did not. Only with TriKE or IL-15 exposure was there a significantly enhanced expansion index. IFN-γ production from the same CD56+/CD3- NK-cell population was enhanced by 1615EpCAM, but levels did not approach levels seen with the IL12/IL18 combination that is known to stimulate cytokine production at supraphysiologic levels. We believe that this is a platform technology since anti-CD133 also can be substituted for anti-EpCAM to create functional anti-cancer stem cell TriKEs. These results indicate that we have successfully developed an immune engager that simultaneously mediates ADCC and also provides a self-sustaining costimulatory signal inducing NK effector cell expansion.

Citation Format: Daniel A. Vallera, Joerg U. Schmohl, Martin Felices, Jeffrey S. Miller. Improvement of the bispecific antibody ADCC platform by genetic insertion of IL-15 as a cross-linker to create NK cell reactive TriKEs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1493.