title: Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy.
abstract: BACKGROUND
  PROSTVAC®, an active immunotherapy currently studied for the treatment of metastatic castration-resistant prostate cancer (mCRPC), consists of a heterologous prime-boost regimen with two different poxvirus-based vectors to provoke productive immune responses against prostate specific antigen (PSA) as the target tumor antigen. A Phase 2 study of PROSTVAC immunotherapy showed significantly improved median overall survival by 8.5 months and is currently being validated in a global Phase 3 study (PROSPECT; NCT01322490). Here, preclinical models were explored to investigate the mechanism of action and immune signatures of anti-tumor efficacy with PROSTVAC immunotherapy with the goal to identify potential immune correlates of clinical benefit.
  
  
  METHODS
  PROSTVAC-induced immune responses and anti-tumor efficacy were studied in male BALB/c mice. Functionality of the induced T cell response was characterized by interferon-gamma (IFNγ) ELISPOT, cytotoxic degranulation, multi-cytokine intracellular staining, and in vivo T cell depletion. Tumor infiltrating lymphocytes (TILs) were evaluated phenotypically by flow cytometry.
  
  
  RESULTS
  The heterologous prime-boost regimen of the two PROSTVAC vectors significantly enhanced the magnitude and quality of activated PSA-specific CD4 and CD8 T cell responses compared to homologous, single vector regimens. PROSTVAC-activated CD4 and CD8 T cells were highly functional as evidenced by expression of activation markers, production of multiple cytokines, and amplified cytotoxic T cell activity. Importantly, PROSTVAC immunotherapy resulted in significant anti-tumor efficacy in a transplantable prostate cancer mouse model. Antigen-spreading occurred in PROSTVAC-treated animals that rejected PSA-expressing tumors, as shown by subsequent rejection of PSA-negative tumors. In vivo CD4 and CD8 depletion revealed that both T cell subsets contributed to anti-tumor efficacy. Characterization of TILs demonstrated that PROSTVAC immunotherapy greatly increased the intra-tumoral ratio of activated effector to regulatory T cells.
  
  
  CONCLUSIONS
  PROSTVAC immunotherapy activates broad, highly functional T cell immunity to PSA and to endogenous tumor antigens via immune-mediated antigen spreading. These preclinical results further elucidate the mode of action of PROSTVAC immunotherapy and its potential causal relationship to extended overall survival as observed in the PROSTVAC Phase 2 study. The clinical validation is ongoing in the PROSPECT Phase 3 clinical study.
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Stefanie J
authors.initials: SJ
authors.lastname: Mandl
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Ryan B
authors.initials: RB
authors.lastname: Rountree
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Tracy B
authors.initials: TB
authors.lastname: Dela Cruz
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Foy
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Cote
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Gordon
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Trent
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA ; ExoThera LLC, 675 Olive Street, Menlo Park, CA 94025 USA.
authors.forename: Alain
authors.initials: A
authors.lastname: Delcayre
authors.affiliation: Bavarian Nordic, Inc, 2425 Garcia Ave, Mountain View, CA 94043 USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Franzusoff
keywords: Active immunotherapy; Heterologous prime-boost; Immune correlates; Mechanism of action; PROSTVAC; Prostate cancer; Teff :Treg ratio