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quadrant C in panel vi

quadrant C in panel vi. by NK cells was reversed by a function-blocking CD16A mAb. In addition, NK92 cells, a human NK cell line that lacks endogenous FcRs, expressing a recombinant non-cleavable version of CD16A released significantly higher levels of IFN than NK92 cells expressing equivalent levels of wildtype CD16A. Taken together, our data shows that MEDI3622 enhances the release of IFN by NK cells engaging antibody-bound tumor cells by blocking the shedding of CD16A. These findings support ADAM17 as a dynamic inhibitory checkpoint of the potent activating receptor CD16A, which can be targeted by MEDI3622 to potentially increase the efficacy of anti-tumor therapeutic antibodies. manner at a specific location proximal to the cell membrane [7, 8]. Therapeutic antibodies have been generated against a variety Tideglusib of tumor antigens and tested in clinical trials for assorted malignancies [9]. Several clinically successful tumor-targeting antibodies, such as trastuzumab Tideglusib (anti-HER2) and rituximab (anti-CD20), utilize FcR recognition as a mechanism of action [2, 10]. A limitation of therapeutic antibodies is the development of resistance in patients and the non-responsiveness of some malignancies [11, 12]. Modifying the Fc region of these antibodies to improve their therapeutic efficacy has been a major focus [9, 13]; however, if CD16A is downregulated in expression, this strategy may have limited effectiveness. Indeed, CD16A downregulation has been reported to occur in the tumor environment of patients, in individuals receiving therapeutics antibodies, and during the expansion of NK cells for adoptive transfer into cancer patients [14C18]. There have been extensive efforts to develop ADAM17 inhibitors [19]. A primary focus has been on targeting its activity in tumor cells where ADAM17 facilitates the release of various growth factors and adhesion molecules [20C23]. Initial pharmacological inhibitors of ADAM17 were small-molecule antagonists [19]. However, to overcome issues of specificity and half-life, recent efforts have focused on function-blocking antibodies of ADAM17 [24C29]. MEDI3622 is a human mAb generated through screening scFv phage libraries using ADAM17. Its epitope is distinct from other ADAM17 mAbs and has been mapped to a surface loop unique to the metalloprotease catalytic domain of ADAM17, resulting in high specificity and a potent inhibitory activity [30]. MEDI3622 has been reported to directly inhibit the growth of human head and neck as well as colorectal tumor cells and in a mouse xenograft model [28, 29]. We investigated for the first time the effects of blocking ADAM17 with MEDI3622 on NK cell activation induced by therapeutic antibody-bound tumor cells. Cytokine production by NK cells is a key effector function and in particular they are major producers of IFN, which has broad anti-cancer activity. This includes crosstalk with leukocytes of the innate and adaptive immunity, induction of ICAM-1 and MHC surface expression on tumor cells that promote leukocyte attachment and stimulation, and inhibition Tideglusib of cell proliferation and angiogenesis in developing and established tumors [31C34]. We show that combining MEDI3622 with a tumor antigentargeting antibody greatly augments the production of IFN by NK cells and that this is due to blocking CD16A shedding. Materials and Methods. Antibodies. The anti-human mAbs PE-conjugated anti-CD107a (LAMP-1), unconjugated and allophycocyanin-(APC) conjugated anti-CD16 (3G8), PE/Cy7-conjugated anti-CD56 (HCD56), PerCP-conjugated anti-CD3 (UCHT1), and isotype-matched negative control mAbs were purchased from BioLegend (San Diego, CA). APC-conjugated anti-CD62L (L-selectin) was purchased from Ancell (Bayport, MN). APC-conjugated F(ab)2 donkey anti-human IgG (H+L) was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The anti-ADAM17 mAb MEDI3622 was generated from a human phage display library displaying scFv Tideglusib and converted into an IgG1, as previously described [28]. Trastuzumab and rituximab, human IgG1 mAbs, were manufactured by Genentech (South San Francisco, CA). An isotype-matched negative control human IgG1 antibody was obtained from Sigma (Saint Louis, MO). Cells. Peripheral blood was obtained from mice housed in a specified pathogen free facility. Mice used in this study were (mice (B6.Cg-Tg(Vav1-cre)A2Kio/J). The and mice were crossed to the C57BL/6J genetic background (both 98.4%) and then crossed together to generate mice and littermate mice, as we have previously described [35, 36]. mice and mice are referred to below as conditional ADAM17 knockout and control mice, respectively. Total leukocytes were obtained from peripheral blood by red blood cell lysis using 0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2 solution. Fresh human peripheral blood leukocytes from plateletpheresis were obtained from Innovative Blood Resources (St. Paul, MN). PBMCs were further enriched on a Mouse monoclonal to KRT13 Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient and then NK cells were purified by negative depletion using an EasySep human NK cell kit (StemCell Technologies, Cambridge, MA), as per the manufacturers instructions, with 95% viability and 90% enrichment of CD56+ CD3? lymphocytes..