BiTE-secreting T cells rationally combine with PD-1 blockade and promote remodeling of the local immune landscape to enhance response durability in ovarian cancer

scRNA-seq (5' GEX) was used to examine the peritoneal TME in IE9-mp1-hFRa cells bearing animals treated with FRB-T cell + anti-PD1 therapies. We observed unique TME composition associated with acute and durable responses to combination therapy that was disrupted in progressive disease. Further we found that providing an AD-OVA immunization boost along the course of therapy enhanced favorable TME composition and overall tumor control. Overall design: 5x106 IE9-mp1-hFRa cells (IP in 500µl PBS) were injected to establish tumors, with adoptive T cell transfer (ACT) delivered 5 days later. Mice received 1.0x106 FR-B T cells or an equivalent number of Control T cells delivered by loco-regional IP infusion, followed by 2 doses of aPD1 or IgG control. On Day 12 (7 days post ACT), mice were injected IP with 5ml PBS and the peritoneal wash (corresponding to the OC TME) collected from 4 mice/group. Recovered cells were washed in PBS and immediately stained with Zombie NIR, prepared in PBS for sorting of viable cells. Stained cells were sorted using a BD FACSAria II, collecting 30,000 live cells per mouse, which were then pooled within cohorts (120,000 live cells/group). For later time points, mice receiving FR-B T cell + anti-PD-1 treated mice were categorized as having Progressive Disease (PD; defined as animals with ascites accumulation and abdominal circumference = 8.0 cm) or Durable Response (DR; defined as mice having no ascites accumulation) on Day 38. Ascites and/or peritoneal washes were collected from PD and DR animals (n=4/group), subjected to ACK lysis to remove red blood cells, counted and pooled equally within each cohort (50,000 cells/mouse) to reach 200,000 total live cells/group. Similarly, ascites or peritoneal wash from mice treated with FR-B T cells + ?PD1 or FR-B T cells+ ?PD1+ Ad-OVA Vax were collected 14 days post vaccination (day 28) were collected from 4 mice/group, subjected to ACK lysis, counted, and equally pooled to reach 120,000 live cells/group. Immediately following preparation, cells were provided to the Roswell Park Genomics Shared Resource (GSR) for further processing and single cell sequencing (10x Genomics).