Abstract
BackgroundThe design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133.ResultsProliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release.MethodsAssembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity.Conclusion1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs.
Highlights
Immune engagers show promising results in cancer treatment and already demonstrate some clinical success
We show that our tetraspecific killer engager (TetraKE) is highly specific against epithelial cell adhesion molecule (EpCAM) and CD133 bearing cells, is capable of both NK cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and NK cell expansion, and represents a promising new therapeutic modality
To evaluate specificity of our drug, flow cytometry based blocking assays were performed with HT-29 (EpCAM+, CD133-) and Caco-2 (EpCAM+, CD133+) colon carcinoma cell lines
Summary
Immune engagers show promising results in cancer treatment and already demonstrate some clinical success. In order to engage NK cells, bispecific NK cell engagers (BiKEs) were synthesized forming an immune synapse between NK cells and cancer cells [4,5,6,7,8]. A problem is that all of these BiKEs are limited by an inability to facilitate NK cell expansion, as CD16 ligation does not trigger proliferation To resolve this issue we incorporated modified IL-15 as a cross-linker between the scFv to form an IL-15 TetraKE that could induce expansion and proliferation. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. An Interleukin (IL)-15crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133
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