PROJECT SUMMARY Glioblastoma (GBM) is the most lethal form of brain cancer and more than 12,000 people are diagnosed with this disease every year. Current standard of care involved surgery where possible, followed by a combination of chemotherapy and radiotherapy. Unfortunately, the outcome of treatment remains poor with only 15% of patients surviving beyond 5 years. Recent advances in immunotherapy, such as CAR-T cell therapy, have been promising with other cancers but numerous studies have identified several hurdles in applying these methods effectively to glioblastoma. Particularly, GBM cells have high heterogeneity allowing them to escape immune detection, and they present an immunosuppressive microenvironment which reduces the efficacy of immunotherapies. Moreover, the engineered T cells are challenging to source as they elicit a strong graft-vs- host disease response. Monon Bioventures, in collaboration with Dr. Matosevic at Purdue University, is developing an immunotherapy that addresses the limitations of previous approaches. We have designed a novel triple-specific CAR-NK cell capable of overcoming these hurdles through multiple specific mechanisms – improved tumor infiltration and locally-responsive targeted activity while overcoming multiple mechanisms of immunosuppression. Additionally, NK cells do not require stringent donor-recipient matching and can be obtained off-the-shelf. Preliminary studies with the triple-specific CAR-NK cells show efficient killing of GBM cells in vitro. Further, in vivo studies with GBM xenografted mice show increased tumor infiltration, reduction in tumor growth and improved survival. Monon Bioventures is bringing this promising therapy from the lab to the clinic. To achieve this goal, we propose to develop a scalable method to produce the CAR-NK cells using NK cells sourced from blood and demonstrate the efficacy of the therapeutic in a GBM xenograft model. The following Specific Aims are planned: Aim 1. Engineer and characterize NK cells isolated from cord blood. This will be carried out through two sub-tasks: Aim 1 A. Recreate the novel lentiviral vector designed by Dr. Matosevic in a GMP-capable facility, validating vector production through scalable methods that are consistent with cGMP standards. Aim 1B. Isolate and transduce NK cells from cord blood; determine transduction efficiency and characterize functionality of the CAR-NK cells. Aim 2. Demonstrate efficacy of CAR-NK in a patient-derived GBM xenograft mouse model. This project will generate the results needed for a Type B Pre-IND meeting with the FDA. With feedback from the FDA, Phase II funding will go to support the IND package necessary to begin the Phase I trial. Once developed, our novel CAR-NK based therapy has the potential to establish a much-needed new standard of care for those suffering from GBM and could significantly improve the outcome of the disease.