Checkpoint Antibody Receptor Modified ARMed CAR T circumvents the suppressive immunome in GBM

Danielle R Cook^1,2*Alina C Boesteanu¹Yibo Yin¹Reiss Reid¹Laura Roccograndi³Nadia Dahmane³Maria Martinez-Lage^3,4Donald M O'rourke³Carl H June^1,5Laura A Johnson^1,4*

• ¹Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
• ²University of Virginia, Charlottesville, Virginia, United States
• ³Glioblastoma Translational Center of Excellence, Abramson Cancer Center, Perelman School of Medicine, Phillidephia, United States
• ⁴Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States
• ⁵Division of Hematopathology, Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States

Glioblastoma (GBM) is a deadly cancer with non-curative upfront treatment consisting of radiation, resection, and chemotherapy. Not only has the standard of care for GBM patients not improved significantly over the past decade, life expectancy is less than 15 months, with no standard second-line therapy. We recently developed a 2nd generation 4-1BB co-stimulated chimeric antigen receptor (CAR) targeting tumor-specific variant of the epidermal growth factor receptor (EGFRvIII) for treating patients with GBM. This CAR T was used in Phase 1 clinical trials, and demonstrated CAR T cells rapidly trafficked to tumor and activated upon encountering EGFRvIII-bearing tumor cells. However, the CAR T soon lost function, becoming exhausted, with no durable objective tumor responses. Here, we evaluated the GBM immune environment in a syngeneic implantable GL261 murine model over time. Prior to tumor implantation, brain-resident immune cells were mostly absent. Following tumor engraftment, there was a pronounced increase in immune cell infiltration with increasing duration of tumor engraftment and GBM size. Immune infiltrates shifted from early-arriving lymphocytes including T, NK, and B cells, later this shifted towards increased presence of M2 macrophages and myeloid-derived suppressor cells. Evaluating both fresh and archival GBM samples from patients, we found similarly high levels of infiltrating immune cells, and PDL1 expression on both tumor and immune cells. PD1/PDL1-antibody (Ab) mediated checkpoint inhibition has been transformative at treating multiple types of solid tumors, however the location of GBM behind the blood-brain barrier limits access of Abs, and failed to treat GBM. To deliver PD1/PDL1 checkpoint Ab for patients with GBM, we engineered our EGFRvIII-targeted CAR T cells to function as bio-factories, producing secreted anti-PD1 mini-Abs in situ within GBM. These Ab receptor-modified (ARMed) T cells produced PD1 minibodies that were functional in vitro, and homed to GBM xenografts in vivo in NOD-Scid gammaC-null (NSG) mice. Delivered intravenously both soluble Ab plus CAR T, and ARMed CAR T cells improved GBM treatment over CAR T alone, while orthotopic GBM treatment was only improved with ARMed CAR T therapy.