Abstract 2552: Preclinical development of T-cell receptor therapy targeting the 5T4 tumor antigen on renal cell carcinoma
Authors: Xu Yuexin , Morales Alicia J. , Cargill Michael J. , Towlerton Andrea , Warren Edus H. , Tykodi Scott S. 
Published: August 2018

Abstract
Purpose: The cancer-testis tumor antigen 5T4 is expressed on ~ 95% of primary RCC tumors as well as a variety of other tumor types but not on healthy tissue. Consequently, 5T4 has been targeted by antibody and vaccine therapies in cancer patients. Our group has previously isolated high-avidity human CD8+ T-cell clones specific for 5T417-25 and HLA-A2 that are capable of recognizing RCC tumor in vitro and as a murine xenograft. These clones provide the source for T-cell receptor alpha (TRA) and beta (TRB) genes to develop engineered T cells with re-directed cytotoxic specificity for tumors expressing the 5T4 antigen.
Experimental Procedures: Targeted single-cell RNA-seq was performed on 5T417-25 specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of TRA and TRB genes. Full length 5T417-25 specific TRA and TRB sequences were then re-constructed and modified by codon optimization and introduction of transmembrane domain cys-cys covalent crosslinking to augment correct alpha and beta chain pairing. TRA and TRB genes were cloned into third generation lentiviral vectors and transduced into CD8+ T cells from healthy donors. Re-directed effector function for target cells expressing 5T4 was measured by chromium release assay and IFN-gamma ELISA. CRISPR/Cas9 gene editing techniques to augment effector activity of engineered T cells by suppressing native TCR expression and disrupting inhibitory co-receptor genes for PD-1, LAG-3, or TIM-3, are also under development.
Results: Seven unique TRA-TRB pairs were identified from a panel of 38 5T417-25 specific CD8+ T cell clones. Lentiviral mediated expression of all seven 5T417-25 specific TCR transgene constructs was detectable by 5T417-25/HLA-A2 tetramer immunostaining with transduction efficiency from 30% to 80%. 5T4-TCR transduced CD8+ T cells demonstrated re-directed cytotoxic activity to 5T4+/HLA-A2+ RCC, breast, and colorectal tumor cells, but not to 5T4-/HLA-A2+ normal cells. CRISPR/Cas9 targeting of a conserved sequence in the native TRA constant region was sufficient to suppress native TCR expression on polyclonal CD8+ T cells derived from healthy donors. Characterization of the proliferation kinetics, differentiation phenotypes and effector functions of 5T4-TCR transduced T cells modified by gene editing inhibition of native TRA or inhibitory co-receptors (PD-1, LAG-3 or TIM-3) is ongoing.
Conclusions: Transgenic T cell products can be re-directed to recognize a broadly shared epitope of the 5T4 tumor antigen and to kill 5T4+ tumor lines in vitro. Further augmentation of effector activity and specificity by CRISPR/Cas9 gene editing techniques is a developmental priority. Transgenic 5T4-reactive T cells are of interest for future testing as a therapeutic cellular immunotherapy product in patients with 5T4+ tumors.
Citation Format: Yuexin Xu, Alicia J. Morales, Michael J. Cargill, Andrea Towlerton, Edus H. Warren, Scott S. Tykodi. Preclinical development of T-cell receptor therapy targeting the 5T4 tumor antigen on renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2552.