Abstract 2898: HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC)
Authors: Gil Veronica , Guo Christina , Seed George , Neeb Antje , Buroni Lorenzo , Gurel Bora , Figueiredo Ines , Hemmati Golzar , Aaron Wade , de Bono Johann S. 
Published: June 2022

Abstract
Background: Advanced prostate cancers (PC) are largely insensitive to existing immunotherapies. T-cell-engaging antibodies, linking tumor cells to T cells, are being developed to treat malignancies. HPN424 is a Tri-specific T-cell Activating Construct (TriTAC), binding PSMA, CD3ε, and albumin to improve drug half-life. We evaluated the activity of HPN424 to induce T-cell-mediated cytotoxicity against organoids generated from patient-derived xenograft (PDX-Os) established from human mCRPC biopsies.
Design: PDX-Os from three models were evaluated; their membranous PSMA immunohistochemical expression was quantified using H-scores (HS). PDX-Os were co-cultured with: i) healthy donor allogeneic T cells, ii) healthy donor allogeneic T cells combined with HPN424 (1pM, 10pM or 100pM), or iii) healthy donor allogeneic T cells combined with PL711, a non-targeting TriTAC control (1pM, 10pM, 100 pM). Cytotoxicity was determined by LDH release and visualisation of T cell/PDO co-localisation and PDO disruption using confocal microscopy. Early and mid-late T cell activation was determined by FACS for CD69 and CD25 expression, respectively. Cytolytic (CTL) and CD4 T cell cytokine release (IFNγ, TNF∝, IL-1β/2/4/6/8/10) was evaluated using the AlphaLISA kit (MSD). FACS studies were performed after 48- and 72-hours, while microscopy, LDH and cytokine assays were performed after 24, 48, and 72-hours, of co-culture respectively.
Results: In the highest PSMA-expressing PDX-Os (CP50c, HS: 225; CP142c, HS: 165), co-culture of resting healthy donor T cells with CRPC PDX-Os led to dose-dependent increases in LDH release at 48-hours (p&lt;0.0001) and 72-hours (p&lt;0.0001), in the presence of HPN424 but not PL711. Correspondingly, in these models, confocal microscopy showed T cells co-localising with and disrupting the PDX-Os by 24-hours across all 3 HPN424 concentrations tested. HPN424 induced T-cell expression of CD25 and CD69 within 48-hours of co-culture across all 3 concentrations in both models. When compared with PL711, HPN424 induced dose-dependent upregulation of IFNγ, IL-1β, IL-2, IL-4, IL-10, and TNF∝ in the PSMA-high PDX-Os, CP50c and CP142c (all p&lt;0.0001), indicating T cell activation and induction of CTL and Th1/Th2/Treg cytokines by HPN424. Interestingly, in co-cultures of T cells and CP89c PDX-Os, which have lower PSMA expression (HS of 25), HPN424 did not significantly increase LDH release when compared with PL711; in this model CD25/CD69 induction and the upregulation of IFNγ, IL-1β/2, and TNF∝ was only observed at the highest concentration of HPN424 (100pM).
Conclusions: HPN424, a PSMA-targeting TriTAC antibody construct, induced T cell activation and elicited T-cell mediated cytotoxicity against PSMA expressing mCRPC PDX-Os. Our data support further evaluation of this agent in advanced PC with membranous PSMA expression.
Citation Format: Veronica Gil, Christina Guo, George Seed, Antje Neeb, Lorenzo Buroni, Bora Gurel, Ines Figueiredo, Golzar Hemmati, Wade Aaron, Johann S. de Bono. HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2898.