Title: A PSMA-targeting CD3 bispecific antibody induces antitumor responses that are enhanced by 4-1BB costimulation Running Title: Enhancing PSMAxCD3 antitumor efficacy with costimulation

Patients with hematological cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells towards cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor–specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by Immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced Tcell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell on June 12, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 17, 2020; DOI: 10.1158/2326-6066.CIR-19-0518