Invariant natural killer T cells (iNKTs) are innate-type lipid-reactive T lymphocytes that directly kill tumor cells as well as exhibit robust capacity to trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells. Based on their dual functions as potent cytokine producers and tumor killers, iNKTs serve as a powerful tool for use in cell-based immunotherapies for cancer. In most cases, iNKT cell functions are T cell receptor (TCR)-dependent and require engagement of the invariant T cell receptor (iTCR) by lipids such as the potent iNKT agonist α-galactosyl ceramide (αGC). However, transformed cells often down-regulate CD1d expression and as a result, they become invisible to iNKT cell attack. To circumvent this critical barrier, which limits our ability to capitalize on the therapeutic potential of iNKTs, we recently characterized the iNKT stimulatory properties of a novel monoclonal antibody (NKTT320), which is specific for the human iTCR. NKTT320 is a recombinant DNA derived humanized monoclonal antibody that binds selectively and with high affinity to the CDR3 loop of the alpha chain within the α-β hetero-dimer of the extracellular domain of the human iTCR. For these studies, we have developed methods to successfully expand human iNKTs up to 1000-2000 fold and purify them from human peripheral blood mononuclear cells. To assess whether the NKTT320 antibody induces human iNKT cell functions, highly purified human iNKTs were cultured with or without varying concentrations (0.01-10.0 μg/ml) of plate-bound NKTT320 antibody. At varying times, culture supernatants were collected and cells were harvested, counted and viability assessed. iNKT cell activation (by examining for upregulation of activation markers [CD25, CD69], degranulation (measured by exposure of CD107 on the cell surface) and proliferation (by measuring CFSE dilution) was assessed by flow cytometry. Cell culture supernatants were evaluated for the secretion of a wide array of cytokines and chemokines using Luminex. Strikingly, immobilized NKTT320 antibody induced a robust dose-dependent iNKT cell activation, proliferation and degranulation. Additionally, iNKTs stimulated by the plate-bound antibody secreted increased levels of Th1 (IL-1β, IFN-γ, TNF-α, IL-2, IL-6) and Th2 (IL-4, IL-5, IL-10) cytokines as well as chemokines in a dose-dependent manner. Our in vitro studies are consistent with in vivo data in Vα24 transgenic mice, which express the human iTCR alpha chain. Dosing of NKTT320 in these animals led to iNKT cell activation (upregulation of activation markers and intracellular IFN-γ production), as well as incorporation of BRDU indicating in vivo iNKT cell proliferation. These studies provide a framework by which human iNKT cell functions could be enhanced for therapeutic purposes in cancer.
Citation Format: Rupali Das, Felix Scheuplein, Nishant P. Patel, Peng Guan, Robert G. Schaub, Kim E. Nichols. NKTT320, a novel monoclonal antibody activates the immuno-stimulatory functions of human invariant natural killer T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2014-653