Despite progress in the development of curative therapies, cancer remains the leading cause of death from disease in childhood, and most survivors have treatment-associated late effects. Consequently, new therapies are needed to increase cure rates and decrease side effects. Most “molecularly targeted” drugs have activity in specific and often small subsets of cancer. This proposal represents a completely new approach to the treatment of cancer that may be effective against a wide array of malignancies in children and adults. Natural killer (NK) cells, especially when “activated”, mediate direct cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) against cancer cells.
We focus on the therapeutic potential of extracellular vesicles (EVs), which include exosomes, released by activated human NK (aNK) cells in combination with antibodies that target them to human acute lymphoblastic leukemia (ALL) or neuroblastoma (NB) cells. Only one report of NK cell EVs, which was limited to in vitro studies with five cell lines (1), has been published before our recent publication (2). We showed for the first time that large quantities of EVs are released by aNK cells propagated ex vivo with K562-mbIL21 artificial antigen-presenting cells (aAPCs) and that they are cytotoxic in vitro for ALL and NB cells. These EVs retain aNK receptors that could mediate binding to target cells and contain proteins and miRNAs that could cause target cell cytotoxicity.
Further, we discovered that EV cytotoxicity can be increased in vitro by a monoclonal antibody (mAb) directed against disialoganglioside (GD2) expressed by NB cells. Finally, initial experiments using xenograft models of NB or ALL in immune deficient NSG mice demonstrate anti-NB and anti-ALL activity of aNK EVs. A critical barrier to the translation of EV-based therapies to the clinic is the inability to purify them in large quantities. In the proposed work, we will develop scalable continuous-flow methodologies for the purification of clinically active EVs.
We hypothesize that continuous flow purification will provide aNK cell EVs that are cytotoxic against cell lines and primary malignant cells from patients and that targeting anti-CD19 and anti-GD2 mAbs will increase EV-mediated cytotoxicity against ALL and NB cells. Our specific aims are to develop a new, automated, large-scale device for purifying aNK EVs and characterize their ability to kill human ALL and NB cell lines in vitro and cell lines and patient derived xenografts (PDXs) in NSG mice. This project will culminate in good manufacturing practice (GMP) production of NK cell EVs for human investigation through our early phase clinical trial groups, the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) and the New Approaches to Neuroblastoma Therapy (NANT) consortium.
