Abstract C117: Preclinical characterization of allogeneic CAR γδ T Cell therapy for prostate cancer targeting a novel dimeric epitope on PSMA

Abstract

Abstract Introduction: PSMA is a transmembrane glycosylated homodimer overexpressed in >80% of prostate cancers, with increased expression in advanced stages of the disease. Formation of homodimeric PSMA is necessary for enzymatic function and displays epitopes that are potentially more distinct from alternative PSMA-like proteins. Clinically, autologous anti-PSMA αβ CAR T cells have shown initial efficacy with limited therapeutic index. Compared to αβ T cells and other innate cells, γδ T cells are capable of multifunctional innate and adaptive targeting and infiltrate into tumor-associated tissues, including associated tropism for prostate tumor tissues. Additionally, γδ CAR T cells have demonstrated enhanced tumoricidal activity and activation-induced cytokine profiles that may decrease CRS-associated toxicities. We characterized γδ T cells modified from a set of novel scFv-based CARs targeting PSMA for prostate cancer and mapped unique epitopes for binders associated with the most potent CARs. Methods: Phage panning was used to identify anti-PSMA scFv sequences, which were reformatted into IgGs and characterized for binding to both cells expressing endogenous PSMA and recombinant PSMA. Binders with favorable profiles were reformatted into CARs in VH-VL and VL-VH orientations and transduced into Vδ1 T cells, a primarily tissue-resident subset, activated from healthy donor PBMCs. We identified lead CARs based on anti-tumor efficacy both in vitro in coculture assays and in vivo in tumor xenograft models and compared their activity to Vδ1 CAR T cells transduced with a clinically validated benchmark, J591. Epitope mapping of binders in the lead CARs was performed using a funnel of molecular assays including dot blots, competition assays, ELISAs on recombinant PSMA from different species and cross-linking mass spectrometry (XL-MS). Results: High-affinity PSMA binders yielded potent CARs. Lead PSMA CARs demonstrated robust in vitro cytotoxicity and antigen-specific binding to PSMA-expressing cell lines in a manner comparable to, or greater than the J591-based CAR T cells. Additionally, lead PSMA CARs showed anti-tumor control in 22Rv1 human prostate cancer xenografts, a highly resistant model with heterogenous PSMA expression. Epitope mapping studies on binders in lead PSMA CARs identified conformational, membrane-distal epitopes that are distinct from the predicted linear epitope of J591 and relevant comparators. Conclusion: The epitope for a lead PSMA antibody includes residues in a helix involved in PSMA dimer formation. The γδ CAR T construct derived from this binder demonstrates enhanced preclinical performance when compared to the J591 reference CAR T. Combining the features of this novel target and the potential of the γδ-based CAR T approach, the lead PSMA γδ CAR T cells described here are candidates for further clinical development in the context of additional armoring technologies and CAR architecture modifications designed to complement therapeutic potential. Citation Format: Nitya S. Ramadoss, Elizabeth Speltz, Tonya Capillo, Erika Meaddough, Katherine Wang, Natalia Friedland, Jonathan Wong, Alexander G Teague, Aruna Azameera, Smitha Rao Yelaguli Gundurao, Ramandeep Kaur, Morgan Smith-Boeck, Michael Salum, Yvan Chanthery, Shon Green, Marissa Herrman, Kevin P Nishimoto, Blake T Aftab, Arun Bhat. Preclinical characterization of allogeneic CAR γδ T Cell therapy for prostate cancer targeting a novel dimeric epitope on PSMA [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C117.