Featured Poster Abstract 84: CD8 T cell-mediated immune responses are critical to the increased efficacy of Doxil in BRCA1 deficient tumors Gina Mantia-Smaldone, Victoria Gamerman, Phyllis Gimotty, Regina Loomis, Lukas Ronner, Christopher Morse, Sandra Orsulic, Stephen Rubin, George Coukos, Sarah Adams

BRCA-deficient tumors are vulnerable to the immunomodulatory effects of Doxil 56% of women with recurrent BRCA-associated ovarian cancer demonstrated a response to Doxil 1 In addition to causing DNA damage, Doxil increases the vulnerability of ovarian cancer cells to T cell attack Immunophenotypic changes in response to Doxil are more pronounced in BRCA-/- cells BRCA-/- tumors recruit higher numbers of T cells after Doxil exposure We hypothesized that the immunogenicity of BRCA-/- tumors contributes to the improved clinical response to Doxil among women with hereditary ovarian cancer The role of T cells in tumor clearance following exposure to Doxil was tested using a murine BRCA1-/- ovarian cancer model 1 Adams SF et al, Gynecol Oncol 2011 Evaluated in a cohort of 23 women with BRCA mutations and 41 women with sporadic cancer treated at three institutions between for recurrent cancer. The response to Doxil was associated with a significant improval in overall survival Expression of Fas is associated with increased Fas-FasL mediated T cell cytotoxicity; MHC I expression, which is higher in BRCA-/- cells, is required for a CD8 T cell response

Doxil increased intratumoral T cell recruitment and overall survival in mice with BRCA1- tumors Doxil therapy significantly prolonged the survival of animals with BRCA1- tumors Doxil exposure increased cytotoxic CD8 T cells and reduced suppressive FoxP3 T cells Both CD4 and CD8 T cell populations were present in BRCA1- tumors and ascites Survival measured from the time of tumor inoculation until animals reached 30g due to ascites accumulation Survival (Days) P= Control Doxil Flow cytometry was performed on tumor, ascites, spleen and peripheral blood mononuclear cells (PBMCs). Illustrated are the portion of CD45+/CD3+ T cells present in representative tumor or ascites samples for placebo or Doxil treated mice.

The survival advantage associated with Doxil treatment persisted in the absence of CD4 T cells An immunodepleting antibody successfully eliminated CD4 T cells from BRCA1- tumors and ascites In the absence of CD4 T cells, the proportion of CD8 T cells was higher in Doxil-treated animals After treatment with anti-CD4 antibody, a population of CD4-/CD8- cells persisted Doxil significantly prolonged the survival of CD4-depleted animals with BRCA1- tumors Survival measured from the time of tumor inoculation until animals reached 30g due to ascites accumulation Survival (Days) * * p< 0.05 P= Doxil Control

There was no statistically significant difference in survival after Doxil treatment in the absence of CD8 T cells An immunodepleting antibody successfully eliminated all CD8 T cells from BRCA1- tumors and ascites In mice lacking CD8 T cells, the proportion of mature CD4 T cells, but not suppressive FoxP3 T cells, was higher after Doxil treatment The survival among animals depleted of CD8 cells was not improved with Doxil treatment After treatment with anti-CD8 antibody, a population of CD4-/CD8- cells persisted Survival measured from the time of tumor inoculation until animals reached 30g due to ascites accumulation * p< 0.05 P= Doxil Control Survival (Days) *

Doxil improved survival in a BRCA1- model of ovarian cancer This effect persisted in the absence of CD4 T cells but is diminished in the absence of CD8 T cells Cytotoxic CD8 T cells are necessary for the enhanced response to Doxil observed in BRCA1- tumor bearing mice BRCA1- deficient ovarian cancers may be more susceptible to immunotherapeutic strategies Limitations: These results represent a small pilot study and need to be confirmed in larger cohorts Unfortunately the national Doxil shortage has limited our ability to conduct planned experiments This work was supported by a grant from the Conclusions