Utilizing pharmacodynamic immunohistochemistry (PD-IHC) for in situ & quantitative measures, we explored the mechanism of action of a Dual-Affinity Re-Targeting (DART®) bispecific recombinant antibody engineered with enhanced pharmacokinetic properties to extend in vivo half-life. This bispecific, designated P-cadherin LP-DART, is designed to engage and activate polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. Following administration in mice bearing established human tumors and implanted with human T-cells we examined the localization of P-cadherin LP-DART within the tumor xenografts, P-cadherin expression, quantitation and immunophenotyping of tumor infiltrating lymphocytes (TILs), downstream biomarkers of T-cell effector function and immunoregulatory mechanisms. Cell surface P-cadherin expression was maintained on the established tumor xenografts after the administration of single and multiple doses of the bispecific molecule. Furthermore, we detected P-cadherin LP-DART in the tumors more than one week after administration. Pan lymphocyte IHC and digital image analysis demonstrated P-cadherin LP-DART mediated CD3+ T-cell infiltration, resulting in nearly half of the viable cells in the tumor being TILs. Conversely, we did not detect infiltrating CD3+ human T-cells in normal organs, confirming a specific target mediated T-cell response at the tumor site. Elevated proximal and downstream mediators of drug action (granzyme B and cleaved caspase 3) further support that P-cadherin LP-DART localized within the tumor induces T-cell mediated growth inhibition and sustained regression. Additionally, to study the the tumor response to redirected T-cell mediated killing we measured the up-regulation of critical immune check point pathways after treatment P-cadherin LP-DART. In the in vivo tumor models examined, tumor cells acutely and robustly induced expression of immunoregulatory pathways in response to effector T-cell activity. Taken together, we demonstrate the utility of in situ kinetic PD-IHC methodologies to demonstrate target expression, drug localization, downstream biomarkers of drug action, and provide insights into potential immunoregulatory mechanisms in response to T-cell mediated bispecific immunotherapy.
Citation Format: Justin Lucas, Andrea T. Hooper, Jonathon Golas, Bryan Peano, Alan Opsahl, Leslie Obert, Maria Gavriil, Timothy Fisher, Anton Xavier, Michael Cinque, Roger Conant, Judy Lucas, Adam Root, Lioudmila Tchistiakova, Hans Peter Gerber, Chad May. Pharmacodynamics and mechanisms of drug action for bispecific redirected T cell immunotherapy against P-cadherin. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B010.
- ©2016 American Association for Cancer Research.