Molecular Characterization of Anti-Tumor Activity Mediated by Extracellular Vesicles Derived from Natural Killer Cells

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Multiple myeloma (MM) is the second most common hematological cancer in the U.S and increasing in frequency. Veterans who served in Vietnam where herbicides like Agent Orange were sprayed may have increased risk of developing MM. Early-stage disease is often asymptomatic, so patients are diagnosed late, with bone pain, kidney dysfunction and infections. Although there has been progress in developing new therapies for MM, it remains incurable. Patients initially achieve remission but ultimately relapse. The disease returns more quickly, tumor cells become more resistant to treatment and the patient’s quality of life declines. Natural killer (NK) cells kill MM cells in vitro and in vivo. Clinical trials using NK cell-based immunotherapy are ongoing but not widely available. Several drugs for MM (bortezomib, carfilzomib, lenalidomide) sensitize MM cells to NK-mediated lysis and/or enhance NK killing activity. However, challenges remain. Therefore, new treatments are needed to extend survival and increase durability of remission in relapsed/refractory patients and those ineligible for front-line therapy. My laboratory developed NK3.3, the only normal human NK cell line. It was cloned from peripheral blood NK cells and kills an array of tumor cells. As NK3.3 cells grow in culture, they release small membrane-bound extracellular vesicles (EVs). We demonstrated that purified NK3.3 EVs kill MM cell lines and primary patient samples, without harming normal cells. NK3.3 EVs also kill drug-resistant and cancer stem cells (CSC). There are many advantages to using NK3.3 EVs for cancer treatment. They can be generated in large quantities, are stable, and can be frozen and thawed without loss of function. EVs are resistant to the hypoxic tumor microenvironment and unlike cellular therapy, do not induce a detrimental cytokine storm. NK3.3-derived EVs may provide the advantages of NK cell therapy without the challenges of expanding cells and side effects. The goal of these studies is to establish the feasibility of using NK3.3-derived EVs for MM treatment. We developed a murine xenograft model of minimal residual disease in MM, which approximates a human clinical condition. After intravenous injection of RPM1-8226 MM cells into immunodeficient mice, tumor cells disseminate, infiltrate bones, and induce osteolytic lesions, characteristics of MM. We will test the ability of NK3.3 EVs to prevent MM recurrence after chemotherapy treatment. Aim 1: Characterize NK3.3 EVs and establish best practices for production. We will develop optimal NK3.3 culture conditions for EV production. Proteomic and lipidomic analysis will be performed on EV preparations. NK3.3 EVs will be evaluated for killing MM cell lines and patient samples and for lack of toxicity against healthy bone marrow, peripheral blood lymphocytes and fibroblasts. Aim 2: Identify the mechanism(s) of NK EV-mediated killing. NK EVs induce caspase-mediated apoptosis. However, like NK cells, NK EVs likely kill via multiple mechanisms. We will evaluate caspase- dependent and independent killing pathways in EV-treated tumor cells. We will inhibit killing pathways by gene knockdown and chemical treatment. MM cell lines and primary tumor cells will be used to determine whether drug-resistant and CSC-like MM cells are sensitive to killing by NK3.3 EVs. Aim 3: Test the in vivo efficacy of NK3.3 EVs in preventing/delaying tumor recurrence in a murine model of minimal residual disease in MM. Different concentrations of NK EVs will be administered intravenously. Tumor dissemination will be monitored by bioluminescence imaging; bone lesions by X-ray and CT scanning. Toxicity will be assessed by analysis of body and organ weights and blood chemistry. Normal and tumor-bearing mice will be infused with labeled NK EVs to monitor biodistribution and half-life. These studies are the first step towards developing NK3.3 EVs as a treatment for MM patients who desperately need new options to improve their quality of life and prolong their survival.