Search

BackgroundOvarian cancer is a leading cause of cancer-related deaths among women due to the development of therapeutic resistance. Natural killer (NK) cells are cytotoxic lymphocytes that can kill neoplastic cells without prior sensitization. A key anti-tumor function of human NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC), mediated exclusively by the IgG Fcy (FcyR) receptor CD16A. The mechanism of action of several clinically successful antitumor therapeutic monoclonal antibodies (mAbs) involves ADCC; however, their therapeutic efficacy is reduced due to regulatory checkpoints of CD16A, which include its low IgG binding affinity and rapid downregulation upon NK cell activation by the membrane metalloprotease ADAM17.1–3 CD64, the highest affinity FcyR, is expressed on myeloid-derived cells and not lymphocytes and is also not cleaved by ADAM17. To enhance ADCC, we generated CD64/16A, a novel recombinant FcyR consisting of extracellular CD64 and intracellular and transmembrane CD16A to mediate high affinity IgG binding and potent cell signaling.4 5MethodsEngineered NK cells expressing CD64/16A were derived from human induced pluripotent stem-cells (iPSCs), referred to here as iNKs, which are clonal and clinically scalable NK cells. ADCC and natural cytotoxicity of three ovarian cancer cell lines were measured in vitro using Delfia EuTDA and IncuCyte cytotoxicity assays, and production of anti-tumor cytokines was determined via flow cytometry. Finally, tumor cell killing was assessed in vivo using a human xenograft mouse model of peritoneal metastatic ovarian cancer.ResultsOur data show that iNK-CD64/16A cells uniquely facilitate mAb absorption, robustly produce IFN-y and TNF-alpha, and kill several ovarian cancer cell lines in the presence of the therapeutic mAbs trastuzumab or cetuximab, which target HER2 or EGFR, respectively. We found that iNK-CD64/16A cells can capture soluble mAbs and retain antibody arming and ADCC efficacy after cryopreservation. Additionally, iNK-CD64/16A cells robustly mediate ADCC and reduce overall tumor burden of HER2+ tumor cells in vivo in the described metastatic ovarian cancer xenograft model.ConclusionsOur findings provide new insights into using high affinity Fc receptor-based adoptive NK cell therapies and lay the preclinical foundation necessary for developing an ‘off-the-shelf’ cellular therapy that can be combined with therapeutic tumor-targeting mAbs for the treatment of ovarian cancer. Importantly, iNK-CD64/16A cells can serve as a docking platform for therapeutic antibodies that can be switched and mixed for universal tumor antigen targeting to treat multiple malignancies.AcknowledgementsGrant supportNIH R01CA203348, HHMI Medical Research Fellows Program/Burroughs Wellcome FundReferencesJing Y, Ni Z, Wu J, et al. Identification of an ADAM17 cleavage region in human CD16 (FcgammaRIII) and the engineering of a non-cleavable version of the receptor in NK cells. PLoS One 2015;10(3):e0121788.Wu J, Mishra HK, Walcheck B. Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy. J Leukoc Biol 2019;105(6):1297–303.Dixon KJ, Wu J, Walcheck B. Engineering Anti-Tumor Monoclonal Antibodies and Fc Receptors to Enhance ADCC by Human NK Cells. Cancers (Basel) 2021;13(2).Snyder KM, Hullsiek R, Mishra HK, et al. Expression of a Recombinant High Affinity IgG Fc Receptor by Engineered NK Cells as a Docking Platform for Therapeutic mAbs to Target Cancer Cells. Front Immunol 2018;9:2873.Walcheck B, Wu J. iNK-CD64/16A cells: a promising approach for ADCC? Expert Opin Biol Ther 2019;19(12):1229–32.Ethics ApprovalThis study was approved by the University of Minnesota’s Institutional Animal Care and Use Committee, protocol number 1902–36768A.