Novel Cytocompatible Encapsulation-based Vitrification Method for Natural Killer Cell Immunotherapy Summary: Use of natural killer (NK) cells as immunotherapy against hematologic and solid tumor cancers has gained much attention in the last few years. Critical advantages of NK cells include lack of risk for transfusion- associated graft-versus-host disease and decreased likelihood of tumor escape. The fact that NK cells recognize targets without needing MHC class I allows for the use of allogeneic products. At the University of Minnesota, co-I Dr. Miller and his NK cell Program team have treated over 400 cancer patients with donor NK cells and single dosing is a major limitation to fully test their anti-cancer activity. To allow for third party, off-the- shelf large- scale manufacturing needed for multidose strategies, we must be able to freeze and thaw NK cells on demand with minimal loss of viability and function. However, current cryopreservation practice heavily relies on the use of toxic cryoprotectant agents (CPAs) such as dimethyl sulfoxide (DMSO) and freeze/thaw protocols that are not optimized for NK cells. As a result, there is a) a significant decrease in post-thaw viability of the NK cells, and b) loss of NK cell cytotoxicity and cytokine production function. PI Prof. Aksan's lab has recently developed a cytocompatible encapsulation-based vitrification method that does not require cytotoxic and mutagenic CPAs. The main goal of this proposal is development and adaptation of this novel method to vitrify and preserve expanded blood and induced pluripotent stem cell (iPSC) derived NK cells. Our method avoids detrimental intra-/extracellular ice crystallization during cooling, and re-crystallization during warming, therefore eliminating freeze/thaw and cryogenic storage-induced loss of viability and post-thaw function. To reach our main goal, we will pursue the following specific aims: Specific Aim 1: Engineer the encapsulation gel and determine the cytocompatible CPA compositions that will inhibit intra-/extracellular ice crystallization and growth, devitrification, and eutectic melting, which are detrimental. Specific Aim 2: Maximize post-thaw NK cell viability and function using optimized freeze-thaw protocols. Specific Aim 3: Verify short and medium-term storage stability of NK cells at -80oC, and in liquid nitrogen.

Novel Cytocompatible Encapsulation-based Vitrification Method for Natural Killer Cell Immunotherapy The main goal of this proposal is development and adaptation of a novel preservation and storage method for natural killer (NK) cells, which are increasingly used in immunotherapy against blood cancers and solid tumor cancers. Lack of efficient and safe cryopreservation technologies for NK cells is a major roadblock for wide scale adoption of promising immunotherapies: Current cryopreservation limitations add strain on clinical practice, introduce risks for contamination, increase costs, and most importantly decrease the potential for greater access to patients, as only institutions with advanced cell therapy manufacturing capabilities are able to treat patients.