Abstract
Anti-tumor mAbs are the most widely used and characterized cancer immunotherapy. Despite having a significant impact on some malignancies, most cancer patients respond poorly or develop resistance to this therapy. A known mechanism of action of these therapeutic mAbs is antibody-dependent cell-mediated cytotoxicity (ADCC), a key effector function of human NK cells. CD16A on human NK cells has an exclusive role in binding to tumor-bound IgG antibodies. Though CD16A is a potent activating receptor, it is also a low affinity IgG Fc receptor (FcγR) that undergoes a rapid downregulation in expression by a proteolytic process involving ADAM17 upon NK cell activation. These regulatory processes are likely to limit the efficacy of tumor-targeting therapeutic mAbs in the tumor environment. We sought to enhance NK cell binding to anti-tumor mAbs by engineering these cells with a recombinant FcγR consisting of the extracellular region of CD64, the highest affinity FcγR expressed by leukocytes, and the transmembrane and cytoplasmic regions of CD16A. This novel recombinant FcγR (CD64/16A) was expressed in the human NK cell line NK92 and in induced pluripotent stem cells from which primary NK cells were derived. CD64/16A lacked the ADAM17 cleavage region in CD16A and it was not rapidly downregulated in expression following NK cell activation during ADCC. CD64/16A on NK cells facilitated conjugation to antibody-treated tumor cells, ADCC, and cytokine production, demonstrating functional activity by its two components. Unlike NK cells expressing CD16A, CD64/16A captured soluble therapeutic mAbs and the modified NK cells mediated tumor cell killing. Hence, CD64/16A could potentially be used as a docking platform on engineered NK cells for therapeutic mAbs and IgG Fc chimeric proteins, allowing for switchable targeting elements and a novel cancer cellular therapy.
Highlights
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that target stressed, infected, and neoplastic cells [1]
We generated the novel recombinant receptor CD64/16A that consists of the extracellular region of human CD64 for high affinity antibody binding, and the transmembrane and intracellular regions of human CD16A for mediating NK cell signal transduction
We found that CD16A underwent a >50% decrease in expression upon NK92 stimulation by antibody-dependent cell-mediated cytotoxicity (ADCC), whereas CD64/16A demonstrated little to no downregulation (Figure 1C)
Summary
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that target stressed, infected, and neoplastic cells [1]. In contrast to the diverse array of receptors involved in natural cytotoxicity, human NK cells mediate ADCC exclusively through the IgG Fc receptor CD16A (FcγRIIIA) [2,3,4]. This is a potent activating receptor and its signal transduction involves the association of the transmembrane and cytoplasmic regions of CD16A with FcRγ and/or CD3ζ [4,5,6,7,8,9]. A disintegrin and metalloproteinase-17 (ADAM17) expressed by NK cells plays a key role in its downregulation by cleaving CD16A in a cis manner at a specific location proximal to the cell membrane upon NK cell activation [13, 14, 20]
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