CRISPR-based gene disruption coupled to targeted integration of novel, high-avidity, natural T cell receptors to WT1 for acute leukemia

To better characterize and identify the candidate TCR among the receptors that we have isolated, we dissected the gene expression profiles and biological pathways of HD1- and HD3-TCR edited T cells by single cell sequencing. When challenged with target cells (either WT137-45- pulsed T2 cell line or primary AML blasts), results highlighted an immune response gene signature in each stimulated T cell culture, compared to resting counterparts. Interestingly, upregulation of genes mediating IFNa and g response, T cell proliferation and CD4+ T cell activation was observed in HD1- but not in HD3-TCR T cells stimulated with primary leukemic blasts. T cells harvested from 3 independent donors (DN1, DN2, DN3) were genetically engineered with the TCR gene editing protocol and transduced with the HD1- or HD3-TCR. 14 days upon stimulation with the anti-CD3 and anti-CD28 magnetic beads (ClinExVivo CD3/CD28; Invitrogen), engineered T cells from each donor were co-cultured with WT137-45- pulsed HLA-A*02:01+ T2 cells or with primary leukemic blasts (pAML1) at an effector to target ratio of 2:1. As control we included in the experimental setting HD1- and HD3-edited T cells without further addition of target cells. 24 hours later T cells were enriched by magnetically sorting using the PE-fluorescently labeled Vb8 (for HD1-TCR cultures) and the Vb7.2 (for the HD3-TCR cultures) antibodies and the anti-PE microbeads (Miltenyi Biotec). In total, we collected T cells from 18 different conditions, 6 for each DN included in the analysis: HD1-TCR T cells and HD3-TCR T cells, each left unstimulated, co-cultured with WT137-45-pulsed T2 cells, or co-cultured with pAML1 blasts. Cell hashing (74,75) with anti-human CD45, anti-human CD3 and anti-human β2-microglobulin barcoded antibodies (TotalSeq-C, Biolegend) was performed in order to multiplex samples from each donor in an individual single cell reaction following the manufacturer’s instruction. Afterwards, 5x104 cells/sample were washed and pooled in 3 different Eppendorfs (1 for each donor) in PBS supplemented with 0.04% Bovine Serum Albumine (BSA).