Abstract 2831: High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies

Abstract

Abstract Adoptive T cell therapy with CD8+ T cells engineered to express T cell receptors (TCRs) that recognize hematopoietically-restricted minor histocompatibility antigens (miHAs) is an attractive approach for improving the efficacy of allogeneic hematopoietic stem cell transplantation. We developed a mouse model to determine how to optimally implement anti-miHA T cell therapy, focusing on the key characteristics of anti-miHA TCRs that correlate with in vivo efficacy. We immunized B6 mice to the Kb-restricted miHA H60 and used a high throughput approach to amplify TCR α/β chains from single CD8+ MHC Multimer-H60+ (TetH60+) cells. Based on β chain sequencing of 472 single T cells, a broad repertoire was engaged by H60-vaccination, without a clear β chain preference. We randomly chose 178 unique TCRs, cloned them into retroviral backbones and expressed them in a TCR-CD8+ NFAT-reporter reporter cell lines and screened for H60-reactivity by NFAT-induction after stimulation with H60-pulsed antigen presenting cells (APCs). We chose 22 unique TCRs that had clear H60-specific NFAT-induction for deeper characterization. In similarly-transduced 4G4 reporter cells, these TCRs spanned a range of surface expression levels, affinities for H60 as measured by TetH60 binding, and EC50s in response to H60-pulsed APCs (between 1 and 12 μM peptide concentration). We focused initial functional studies on 2 high avidity TCRs (TCR 61 and TCR 167) with EC50s of 1.25 and 2.5 µM, respectively. These were cloned into an optimized retroviral vector and transduced into B6 CD8 cells concomitant with efficient CRISPR-knock out of the endogenous TCRα/β genes. Engineered T cells bound H60 loaded MHC multimers and killed H60+ targets in vitro. The mean TCR expression of transduced cells was 16-30% of that in unmanipulated T cells, though some had TCR expression similar to or greater than that in unmanipulated T cells. We used TCR 61 and 167 transduced T cell products to treat H60+ blast crisis leukemia cells (BC-CML). TCR 61 and 167 T cell products expanded and were equally effective at reducing BC-CML numbers in spleen and bone marrow (BM). Despite having a greater EC50 and equivalent surface expression in reporter cells, when recovered post-transplant, TCR167 cells had higher TetH60 binding, and produced more IFN-γ. Ongoing studies are testing these and additional TCRs alone and in competition to better elucidate TCR properties that predict for in vivo efficacy. Citation Format: Alexander M. Rowe, Dominic Didiano, Wenzhong Wei, Sawa Ito, Mark Shlomchik, Warren Shlomchik. High-throughput T cell receptor cloning allows optimization of anti-miHA T cell therapies [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 2831.