Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies.

Purpose: Hormad1 is a novel cancer/testis antigen (CTA) that is highly expressed in several solid tumor types such as lung cancer, esophageal carcinoma, as well as head and neck cancer. High expression of Hormad1 is correlated with elevated mutational burden and reduced survival in lung adenocarcinoma, indicating that Hormad1 could be a potential target for immunotherapy. The aims of this study are to identify HLA-A0201-restricted epitope of Hormad1 and generate antigen-specific T cells and T-cell receptor (TCR)-engineered T cells (TCR-T) for adoptive cell therapy (ACT) to treat patients with Hormad1-expressing solid tumors. Methods: Candidate HLA-A0201 restricted epitopes of Hormad1 were identified by reverse-immunology and MHC class I stabilization assay. Mature HLA-A0201+ dendritic cells (mDC) were pulsed with the candidate epitope peptides and used to stimulate autologous CD25 depleted T cells. Tetramer guided sorting was used to enrich for Hormad1-specific T cells from which CTL cell lines and clones were generated. The alpha and beta TCR chains were sequenced from HORMAD1-specific, tumor-reactive CTL and cloned into a retroviral vector construct to introduce the TCR to allogeneic PBMC to generate functional TCR-T cells. Results: From 6 candidate peptides, peptide 4, which showed highest HLA-A2 binding characters, was chosen for T-cell generation. After stimulation with peptide pulsed mDC, functional CTL cell line and clones were generated from two healthy donor PBMC. Both the CTL cell line and clone selectively recognize tumor targets expressing HLA-A0201 and Hormad1. Specificity of the CTL for the HORMAD1 epitope presented by HLA-A0201 was confirmed using lines coexpressing HLA-A0201 and/or HORMAD1. Cold target inhibition assays further confirmed that the Hormad1 peptide 4 was naturally processed and presented by tumor targets. After transduction with the recombinant retroviral vector expressing the alpha and beta TCR chains of the functional CTL cell line, the resultant TCR-T cells displayed high affinity, specific tumor target killing at E:T of 1: 1.25. Moreover, these TCR-T cells specifically enhanced CD137, CD69, IFN-γ, and TNF-α expression in response to HLA-A0201-positive and Hormad1-positive tumor target cells. Conclusion: The HLA-A0201 restricted Hormad1 epitope identified in this study is a novel candidate peptide for antigen-specific vaccine development and endogenous T-cell therapy. A Hormad1-specific TCR-encoding vector was also created that conferred tumor reactivity and antigen specificity among TCR-T cells, offering a high-value product for use in adoptive T-cell therapy for patients with Hormad1-expressing solid tumor malignancies. Citation Format: Ke Pan, Yulun Chiu, Farah Hasan, Natalia Miroballi, Angelique Whitehurst, Cassian Yee. Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A69.