ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA-24-029. Bispecific T cell engagers (BTE) connect cancer cells to endogenous host T cells and induce T cell expansion, cytokine production, and T cell-mediated cancer cell death independently of TCR-MHC-I interactions. The FDA has approved blinatumomab (BlincytoR) for adult and pediatric pre-B cell leukemia and amivantamab (RybrevantR)) for non-small cell lung cancer, and over 80 other BTEs are currently in clinical development with 190 clinical trials underway (clinicaltrials.gov). However, little is known about how T cell responses are regulated during BTE treatment of solid tumors, and a deeper mechanistic understanding is critical to mitigate T cell exhaustion and promote efficacious tumor control and durable protection from relapse in the long-term. Leveraging our team’s collective expertise in BTE therapy, T cell exhaustion and regulatory T cells, the specific goal of this supplemental project is to understand how Treg cells impact effector and memory-like T cell responses, and efficacy and safety of BTE therapy in the immunosuppressive neuroblastoma (NB) microenvironment. Based on the classic immunoregulatory role of Treg cells in infections, cancers and autoimmunity, we propose that Tregs exert a dual role in BTE immunotherapy – (i) by suppressing effector cytotoxic T lymphocyte (CTL) responses Tregs may limit the therapeutic efficacy of BTE in solid tumors. (ii) Concomitantly, Tregs may exert a beneficial role by mitigating the systemic toxicity associated with T cell activation. Hence, we hypothesize that transient ablation of Treg cells during BTE treatment will lead to enhanced tumor control by driving vigorous effector CTL expansion, function and chemoattraction of new T cells to the cold tumor microenvironment. We further propose that key immunoregulatory pathways in the Treg/effector T cell axis may be leveraged to selectively augment effector and stem-like memory anti-tumor responses while mitigating toxicity. This hypothesis is bedrocked in promising new data from our group showing enhanced effector differentiation and improved tumor control upon ablation of Tregs, with significantly tempered toxicity when Treg function is partially replaced through agonist CTLA-4 signaling. Successful completion of these new collaborative supplemental studies will synergize with the overarching goal of our U01 award to improve therapeutic outcomes for aggressive pediatric cancers such as neuroblastomas (NB) and diffuse midline gliomas (DMG) through bispecific T cell engagers (BTE) by identifying potential new targets in the Treg/effector T cell axis that may be leveraged in combination treatments of pediatric NBs, DMGs, melanomas and sarcomas as well.

NARRATIVE This supplemental project seeks to advance our understanding of how Treg cells modulate effector, exhausted and memory T cell responses during BTE immunotherapy. This new knowledge gain will fuel newer combination regimens aimed at augmenting the therapeutic efficacy of BTEs by promoting anti-tumor effector and memory T cell responses in immunologically cold tumors characterized by paucity of T cells, while simultaneously mitigating bystander toxicity. These studies are of high significance to treatment of several pediatric and adult cold tumors such as neuroblastomas, gliomas, sarcomas and melanomas.