NTLA5001, a T Cell Product Candidate with CRISPR-Based Targeted Insertion of a High-Avidity, Natural, WT1-Specific TCR, Shows Efficacy in In Vivo Models of AML and ALL

Adoptive cell therapy using T cells expressing transgenic (tg) tumor antigen-targeting T cell receptors (TCRs) has become an attractive modality to treat hematological and solid cancers due to a broader array of accessible targets relative to CAR-T cell therapies. However, high-avidity TCRs specific for shared oncogenic antigens are difficult to identify. In addition, manufacturing of TCR-redirected T cells with single TCR specificity is desired to avoid mispairings and competition with endogenous chains, which can negatively impact T cell specificity and TCR expression levels. This can be achieved with CRISPR/Cas9-mediated replacement of the endogenous TCR α and β chains, by knocking out the TRAC and TRBC genes and inserting the tgTCR into the TRAC locus. While CRISPR/Cas9 genome editing has been demonstrated to be highly efficient, simultaneous edits in different loci could result in increased translocations, potentially impairing the quality and safety of the cell product. Moreover, existing cell engineering technology negatively impacts T cell quality and yield. Here, we focused on engineering T cells with specificity for Wilms' Tumor 1 (WT1), a transcription factor overexpressed by a wide range of hematological and solid tumors, that has both, restricted expression on healthy tissues and a strong correlation with oncogenesis. By applying rapid isolation technologies of WT1-specific T cells from healthy donors, we identified a lead TCR to the WT137-45 epitope, restricted to the common human leukocyte antigen, HLA-A*02:01. T cells expressing this tgTCR showed nM avidity and killed leukemia cell lines and primary acute myeloid leukemia (AML) blasts at low effector-to-target cell ratios. Epitope specificity evaluation by alanine scanning suggested that the minimal peptide recognition sequence for this TCR is restricted to WT1. Further, the lead TCR was able to activate CD8+ and CD4+ T cells, which may be beneficial for T cell persistence. By developing an improved T cell engineering process, we have achieved multiple sequential gene edits in primary human T cells, leading to knockout of the endogenous TCR with up to 99% efficiency and insertion of tgTCRs into 55-80% of the cells. This cell engineering process is scalable, adaptable to a closed system, and results in marked improvements in T cell expansion, yield, stem cell memory phenotype and T cell polyfunctionality, such as cytotoxicity, cytokine release and proliferation in response to WT1+ target cel