Construction, expression and functional evaluation of chimeric NKG2D receptor in murine NK cells
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Molecular Genetics Honors Theses; 2018
Novel target development for immune-based cancer therapies is an active area of clinical interest. NKG2D is an activating immune cell receptor highly expressed on the surface of cytolytic natural killer (NK), NKT, and T-cells, making NKG2D an excellent target for immune therapy. Our lab developed a novel bispecific killer-cell engager antibody (BiKE) targeting human NKG2D and CS1 receptors highly expressed in multiple myeloma (MM). BiKE binds to human NKG2D receptors, activating lymphocyte effector cells while binding and lysing CS1-expressing MM tumors (Fig. 1). BiKE does not bind murine NKG2D, so human lymphocytes and activating cytokines are injected to test BiKE in our orthotopic immune deficient mouse model. However, exogenous cytokines, such as IL-2 and IL-15, can also activate NK cell cytotoxicity, making it difficult to distinguish BiKE-mediated NK killing from cytokine-mediated killing. Our in vitro studies demonstrate that BiKE activates lymphocytes and lyses CS1-expressing tumors, but an immune competent mouse model with NK, NKT and T-cells expressing human NKG2D is needed to reliably assess BiKE efficacy in vivo. In humans, NKG2D triggers NK cell activation by associating with adaptor protein DAP10, initiating cell cytotoxicity and IFN-γ release. In mice, NKG2D signals through DAP10 and DAP12, controlling cell cytotoxicity and IFN-γ release, respectively. The murine NKG2D transmembrane domain conveys adaptor protein specificity, allowing association with both DAP10 and DAP12. We have shown that substituting human NKG2D for mouse NKG2D does not elicit activation signals, likely due to DAP10/12 differences between species. Consequently, we created a chimeric NKG2D construct to evaluate BiKE efficacy in murine NK cells. Overall, we hypothesize that a chimeric NKG2D construct with a human NKG2D ectodomain and mouse NKG2D transmembrane and intracellular domain will bind BiKE, initiating both cell cytotoxicity and IFN-γ release similar to human NKG2D. After creating our chimeric protein vector, we expressed it in both a murine liver NK (LNK) cell line and primary murine NK cells to test cell cytotoxicity. Our results in the LNK cell line did not show specific lysis of MM tumor cells, but the primary murine NK cells did show significant MM lysis (p=0.0039). During our testing, we determined an error in the protein binding domains of our chimeric protein and redesigned it. Using our new chimeric vector, we transiently expressed chimeric NKG2D in 293T cells, showing binding with anti-human NKG2D antibodies and BiKE. In this study, we show that a chimeric NKG2D receptor can bind human NKG2D ligands and initiate a cytotoxic tumor lysis response. While there is still testing to be done, we are confident moving forward that our redesigned chimeric NKG2D receptor will show functionality with BiKE and can be used to design our mouse model.
Academic Major: Molecular Genetics
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