![]() ![]() Almost complete tumor growth inhibition in the blood and bone marrow was noted in the primary AML model. Animals were treated subsequently with WT1-specific or control T cells. Disseminated primary AML patient derived xenograft and acute lymphoblastic leukemia (ALL) cell line models were established by intravenous injection of the tumor cells in NSG or NOG mice. T cells engineered to express the lead TCR using this process resulted in potent anti-tumor activity in vivo. Additionally, the high viability profile of the process readily allows for sequential CRISPR/Cas9 gene knockout in T cells, leading to near-complete endogenous TCR removal while limiting TRAC/ TRBC translocation to levels close to those found in untreated 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 cells. Further, the lead TCR was able to activate CD8+ and CD4+ T cells, which may be beneficial for T cell persistence.īy 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. Epitope specificity evaluation by alanine scanning suggested that the minimal peptide recognition sequence for this TCR is restricted to WT1. 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. By applying rapid isolation technologies of WT1-specific T cells from healthy donors, we identified a lead TCR to the WT1 37-45 epitope, restricted to the common human leukocyte antigen, HLA-A*02:01. ![]() 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. Moreover, existing cell engineering technology negatively impacts T cell quality and yield. 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. 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. 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. However, high-avidity TCRs specific for shared oncogenic antigens are difficult to identify. 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. ![]()
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