Abstract B57: Effect of ADAM17 inhibition on the release of IFN-y during natural killer cell-mediated antibody dependent cellular cytotoxicity in cancer
Authors: Mishra Hemant K. , Jianming Wu , Pore Nabendu , Walcheck Bruce 
Published: February 2019

Introduction: Antibody-dependent cell cytotoxicity (ADCC) by NK cells is a key mechanism in the anti-cancer effects of therapeutic antibodies. CD16a is an IgG Fc receptor (FcRIII) present on the surface of NK cells and plays essential role in recognizing tumor-bound antibodies to initiate ADCC. NK cells are triggered to release cytokines like IFN-y during ADCC which has been reported to have a number of antitumor activities. A disintegrin and metalloproteinase 17 (ADAM17) plays key role in downregulating CD16a in the tumor microenvironment and facilitate tumor induced anergy through the inactivation of cell cytotoxic function. Downregulation of CD16a on NK cells and decreased levels of IFN-y have been reported in ovarian cancer patients. We hypothesize that by blocking the function of ADAM17 we can enhance IFN-y release during ADCC by maintaining higher CD16a levels on the NK cells. To test this hypothesis, we used ADAM17 function blocking antibody MEDI3622 (MedImmune), HER2 receptor positive ovarian (SKOV-3 and MA148) and CD20 receptor positive lymphoblastoid (Raji) cancer cell lines. To further study the role of CD16a in this process we used monoclonal function block anti-CD16 antibody (clone: 3G8) and cleavage resistant CD16a NK92 cells (CD16a/S197P) where we substituted serine with proline at the location of 197 in the membrane proximal region.

Description: NK cells derived from peripheral blood (PBNK) were isolated through negative selection EasySepTM (StemCell, Cat: 19055). These PBNK cells were incubated with target cells in the presence of a therapeutic antibody to initiate ADCC with or without blocking ADAM17 by MEDI3622. The samples were incubated at 37°C in CO2 incubator and the supernatant were collected after 4 hrs of incubation by briefly spinning them at 1500 rpm for 5 minutes. IFN-y was measured through CBA (BD Bioscience, Cat: 560111).

Summary: The blocking of ADAM17 by MEDI3622 in the presence of herceptin showed an increase in the IFN-y release in comparison to herceptin alone group during ADCC with two ovarian cancer cell lines i.e. SKOV-3 (p&lt;0.0001) and MA148 (p&lt;0.0001). Furthermore, this increase was consistent when we performed the ADCC assay with a different lymphoblastoid related cancer cell line (Raji) with a dissimilar target (CD20 receptor) and therapeutic antibody (rituximab) where we again found a significant increase in IFN-y release in MEDI3622 with rituximab group in comparison to rituximab alone group (p&lt;0.0001).

Next we investigated the role of CD16a receptor in the release of IFN-y. We blocked the CD16a receptor with a monoclonal antibody (clone: 3G8) during herceptin mediated ADCC by PBNK cells in SKOV-3 cancer cell line with or without MEDI3622 and found a substantial decrease in the IFN-y release (P&lt;0.0001) in both the groups (herceptin+MEDI3622 and herceptin alone groups).

In another experiment, where we expressed a cleavage resistant CD16a (CD16a/S197P) in human NK cells line (NK92), we compared the IFN-y release by CD16a/S197P to CD16a/WT NK92 cells during herceptin mediated ADCC in SKOV-3 tumor. In this experiment, NK92a/S197P showed higher release of IFN-y in comparison to NK92aWT (p&lt;0.0001). Upon blocking ADAM17 on CD16a/WT there was a significant increase in the IFN-y (p&lt;0.0003) release. However, NK92a/S197P did not show any further increase in the presence of MEDI3622 (p=ns) signifying the key role of CD16a in this process.

Conclusion: Our results indicate that the inhibition of ADAM17 enhances the IFN-y release by NK cells during ADCC by blocking the shedding of CD16a. Based on these results, ADAM17 may be considered a checkpoint target to improve tumor killing and its utility can be exploited in cancer immunotherapy.

Citation Format: Hemant K. Mishra, Wu Jianming, Nabendu Pore, Bruce Walcheck. Effect of ADAM17 inhibition on the release of IFN-y during natural killer cell-mediated antibody dependent cellular cytotoxicity in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B57.