Abstract CN01-03: T cell receptor gene therapy for a public neoantigen derived from mutated PIK3CA, a dominant driver oncogene in breast and endometrial cancers
Authors: Chandran Smita , Ma Jiaqi , Klatt Martin G. , Dündar Friederike , Zumbo Paul , Femia Matthew R. , Betel Doron , Scheinberg David A. , Baker Brian M. , Klebanoff Christopher A. 
Published: March 2020

Abstract
Background: “Public” neoantigens represent immunogenic epitopes encompassing hotspot mutations in driver oncogenes that are also restricted by common HLA alleles. In contrast with patient-specific “private” neoantigens, public neoantigens are conceptually attractive because they are tumor-specific, clonally conserved, and shared across patients. Whether PIK3CA, the most common driver oncogene in breast and endometrial cancer, can yield public neoepitopes that may be exploited for cancer immunotherapy is unknown. Methods: We have developed a high-throughput, single-cell functional assay for the discovery and retrieval of TCR a/b gene sequences that confer specific recognition of endogenously processed and presented public neoantigens and not the corresponding wild type (WT) sequence. In this approach, donor-derived T cells are sensitized with autologous antigen presenting cells (APCs) electroporated with RNA encoding PIK3CA hotspot mutations. Expanded T cells are subsequently divided into paired daughter wells for short-term co-culture with APCs electroporated with minigenes containing either mutant or WT PIK3CA sequences. Acutely re-stimulated T cells from paired wells are subject to single-cell a/b TCR VDJ and RNA sequencing. TCR a/b gene sequences associated with selective upregulation of TCR signaling transcripts to mutant but not WT PIK3CA stimulation are subsequently cloned into retroviral vectors to confirm reactivity. Results: Using this method, we have retrieved multiple TCRs that confer specific recognition of a public neoepitope derived from a PIK3CA hotspot mutation (PIK3CAH1047L). These TCRs are restricted by HLA-A*03:01, an allele present in 20.5% of the North American population. Immune-precipitation/tandem mass spectrometry analysis determined that the endogenously processed and presented public neoepitope is a 9 amino acid sequence containing a HisàLeu substitution at position 2. To understand the mechanistic basis for the immunogenicity of this public neoepitope, we generated x-ray crystallography structures of mutant and WT epitopes bound to HLA-A*03:01 at ~2Å resolution. These studies revealed significant topologic overlap in the bound peptides. By contrast, the thermal and kinetic stability of the mutant peptide/HLA-A*03:01 complex was significantly enhanced relative to the WT complex, as measured by differential scanning fluorimetry and fluorescence anisotropy assays. Peripheral blood T cells genetically engineered with PIK3CA public neoantigen-specific TCRs cytolytically cleared target cells in a HLA/mutation-specific manner, leaving HLA-mismatched or WT target cells unperturbed. Conclusions: These findings reveal for the first time the existence of an endogenously processed and presented public neoantigen derived from a PIK3CA hotspot mutation. These results open the possibility of targeting this common driver oncogene using adoptively transferred and genetically redirected T cells.
Citation Format: Smita Chandran, Jiaqi Ma, Martin G. Klatt, Friederike Dündar, Paul Zumbo, Matthew R. Femia, Doron Betel, David A. Scheinberg, Brian M. Baker, Christopher A. Klebanoff. T cell receptor gene therapy for a public neoantigen derived from mutated PIK3CA, a dominant driver oncogene in breast and endometrial cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN01-03. doi:10.1158/1535-7163.TARG-19-CN01-03