Non-clinical evaluation of NT-175, an autologous T cell product engineered to express an HLA-A*02:01-restricted TCR targeting TP53 R175H and resistant to TGF-b inhibition.
Authors: Tubb Vanessa , Ma Jennifer , Buchner Karen , Eshuis Sander , Mojadidi Michelle , Kong Xiangjun , Kroon Paula , Sareen Aastha , Duwal Rojina , Bies Laura , Walker Julia , Spaapen Robbert , Stringer Bas , Krijgsman Oscar , Paz-Chang Helicia , Shin Yoonjung , Kuilman Thomas , van Heijst Jeroen , Bendle Gavin , Perez Arianne 
Published: June 2024

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Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in tumor suppressor genes such as TP53. TP53 is the most commonly mutated gene across all cancer types, with the R175H mutation being the most prevalent across different indications. NT-175 is an autologous engineered T-cell product expressing an HLA-A*02:01-restricted TCR that specifically targets the TP53 R175H mutation. NT-175 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-175, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-A*02:01-restricted TP53 R175H neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-175 product functionality was evaluated in vivo models and in vitro. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. The benefit of TGFBR2 knock-out (KO) in the presence of TGF-b was evaluated by measuring phosphorylation of SMAD2/3 proteins, the impact on cell viability and serial killing of target cells. For determination of NT-175 product safety, cross-reactivity and HLA-specificity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-175. Results: High reactivity of the NT-175 TCR against TP53 R175H and HLA-A*02:01 expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against TP53 WT, minimal cross-reactivity against antigens with up to 4 mismatches to the minimal TP53 R175H encoding epitope recognized by the NT-175 TCR, and lack of T-cell activation in the absence of HLA-A*02:01. In the presence of TGF-b, TGFBR2 KO TCR-edited T-cells displayed inhibition of SMAD2/3 phosphorylation, increased cell viability and increased cytotoxicity and proliferation in serial stimulation assays. In vivo, NT-175 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-175 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-175 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant TP53-targeted cancer immunotherapy.