Methods for Characterization of CL-001 Derived Exosomes as a Stimulating Agent for NK Cells for Immunotherapy

The immune system is a complex set of cells in the body that work to prevent disease or infection by recognizing and eliminating foreign material or unhealthy cells. One essential type of immune cell that is part of the innate immune response are Natural Killer (NK) cells. These cytotoxic effector lymphocytes can detect certain unhealthy cells, such as cancer cells, that normal B and T cells would miss. For example, many tumor cells have evolved to bypass immune system surveillance by not expressing major histocompatibility complex class I molecules (MHC class I), on their surface, reducing recognition by cytotoxic T cells. However, NK cells are activated when they detect low to no MHC class I on cells and lyse them by secreting toxic perforins and granzymes. NK cells are currently being developed as an adoptive cell therapy for treatment of cancer. However, because these cells only make up 5-10% of the lymphocytes, novel methods are being developed to expand these NK cells outside the body while also enhancing their cytotoxic properties. One method for NK expansion has been developed by the Copik Lab utilizing plasma membrane particles. These particles are derived from a K562 Leukemia cell line that expresses IL21 and 41BBL on the plasma membrane (CSTX-002) that have been found to enhance NK cell proliferation while maintaining their cytotoxic abilities. The Copik Lab is now developing a novel methodology to expand and/or modify NK cells using engineered exosomes. Exosomes are nanovesicles secreted by many types of cells, such as cancer cells, to transport genetic information and to communicate with other neighboring cells. Preliminary studies have shown that exosomes secreted from the CSTX-002 cells can expand NK cells. This study will develop methods to characterize exosomes from a new cell line engineered in the Copik lab, CL-001, that specifically target IL21 and 41BBL to the exosome surface and determine if they can expand functional NK cells. Methods for characterizing tThe exosomes will be characterized for size, quantity, and protein expression will be developed. Their ability to expand functional NK cells will be determined by culturing NK cells in the presence of the exosome and testing the cytotoxicity of the exosome-expanded NK cells. This project will provide the foundation for new studies in the Copik lab developing novel methods for ex vivo expansion and/or modification of NK cells using exosomes