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V. Wiebking et al.
TCR complex on the cell surface.38 TRAC is advantageous over TRBC because it only exists once per haploid genome. Although 4-1BB co-stimulation has been shown to lead to enhanced persistence of CAR T cells due to decreased exhaustion,39 we chose CD28 as the co-stimu- latory domain in the CAR in order to account for the expected low number of target cells in the setting after HSCT. In the presence of minimal disease burden and absent or low numbers of B cells immediately after HSCT, the stronger effector signaling from CD28 co- stimulation could lead to enhanced activation and prolif- eration of the CAR T cells. It was recently shown that the method of targeted integration of a CAR into the TRAC locus with expression from the endogenous promotor can preserve functionality of cells with CD28 co- stimulation,37 which are otherwise prone to exhaustion.
We used an sgRNA (termed TRAC-1) that had previ- ously been shown to have high on-target activity and no detectable off-target activity.40 Potential off-target sites across the human genome were predicted by the COS- MID algorithm34 and comparison to other possible sgRNA (Online Supplementary Figure S1A, B) confirmed that this sgRNA was among the most specific in exon 1 of TRAC with no highly similar off-targets. The most similar pre- dicted off-target site had three mismatched nucleotides, suggesting a low probability of Cas9 cleavage activity (see below).
To investigate whether genome editing with TCR knockout and targeted integration of a CAR is feasible in TCRaβ+ T cells removed from the graft during aβ haplo- HSCT, we cultured and stimulated these cells and electro- porated them with an RNP complex consisting of a high- fidelity version of the Cas9 protein41 complexed with chemically-modified sgRNA,42 immediately followed by transduction of a DNA repair template by a non-integrat- ing rAAV6. Following this process, on average 95.7% of the cells lost TCR expression (95% CI: 94.2-97.3) and 79.4% (95% CI: 73.5-85.3) of bulk cells and 81.4% (95% CI: 75.7-87.1) of TCR- cells expressed tNGFR (a truncated non-signaling cell surface form of the nerve growth factor receptor which has been used safely in clinical immunotherapy trials24) (Figure 2B and C). These unprecedented efficiencies of targeted integration of a large gene expression cassette (2.7 kb) in primary T cells was reproduced in cells from 11 different donors with similar efficiencies (Figure 2C). Importantly, this proves that cellular double-strand break repair can efficiently be skewed toward homologous recombination to the level at which it constitutes the predominant repair pathway and targeted integration becomes more frequent than insertion/deletion formation by non-homologous end- joining. Notably, the starting cells had been processed at two different GMP facilities (5 at the University of California, San Francisco and 6 at the Stanford University Laboratory for Cell and Gene Medicine), but the outcome after gene editing was highly reproducible (Figure 2C). To confirm co-expression of the CAR in the NGFR+ cells, we stained the cells with an antibody that detects the CAR, which confirmed that both genes of the bicistronic expression cassette are translated (Figure 2D).
Efficient depletion of potentially alloreactive TCR+ cells and optimization of editing methods
Despite the efficiency of the genome editing process, a small fraction of cells (<8%) retained expression of their
TCR. Prior studies have suggested that the frequency of GvHD occurrence for allogeneic CAR T cells with CD28 co-stimulation is low,43 supposedly due to exhaustion and clonal deletion of alloreactive cells44 stimulated through both the CAR and their TCR. Despite these promising results, this is not guaranteed to be universally true, espe- cially since our method creates CAR expression levels dif- ferent from virally transduced CAR T cells and could lead to different biological properties. The residual TCR+ cells – being HLA-haploidentical to the recipient - carry the potential for alloreactivity, and their further depletion from the cell product could decrease the probability of GvHD and allow higher doses of cells to be administered. We therefore evaluated the depletion of residual TCRaβ+ cells from the expanded cell population by magnetic bead activated cell sorting using reagents for which GMP-com- patible counterparts are available. We were able to achieve efficient depletion with a maximum of 0.03% TCRaβ+ cells remaining in the resulting cell product (a depletion efficiency of 2-3 orders of magnitude) (Figure 2E and F), a higher efficiency than in prior studies that created TCR– CAR T cells.45,46 We termed the resulting cell product after genome editing, expansion and TCRaβ+ depletion “aβTCR-CD19 CAR-T”.
The cells rapidly expanded following genome editing (over 60-fold in 7 days), with no negative effect of RNP electroporation on cell yields, an 11% decrease in expan- sion after AAV transduction, and a decrease of 27% for cells electroporated with RNP and transduced with AAV (Online Supplementary Figure S2A). This suggested that AAV transduction is the main factor affecting cell expan- sion, which led us to determine the optimal AAV dose for maximal gene targeting efficiency. Interestingly, we found that a change in the multiplicity of infection beyond 2,500 vector genomes (vg)/cell only led to a minor change in targeting outcomes with saturation at 5,000 vg/cell (Online Supplementary Figure S2B). Instead, the duration of time during which the cells were kept at a high concentration for AAV transduction (>5x106 cells/mL) directly after electroporation and before dilu- tion to the target cell density influenced gene targeting outcomes to a greater extent (Online Supplementary Figure S2C). Using a multiplicity of infection of 5,000 vg/cell and a prolonged transduction time at high density (>12 h), we observed that the cells expanded on average 103-fold within the 7 days following gene editing (Figure 2G). With these conditions for gene editing, the expansion rate of the cells was primarily dependent on the culture densi- ty, reaching the threshold of >100-fold expansion in 7 days if cultured at 0.125x106 cells/mL or within 10 days if cultured at 0.5x106 cells/mL (Figure 2H). This confirms that the CAR T cells are able to expand rapidly without further TCR stimulation after gene editing despite the manipulation during the gene editing process. This will aid in the development of a cell product at clinically rele- vant scale.
To summarize, we achieved efficient disruption of the TCR and high frequencies of CAR expression in T cells derived from the otherwise discarded TCRaβ+ T cells, while allowing for rapid expansion of the resulting cells after the editing process when using an optimized protocol.
Phenotype and in vitro efficacy
To measure in vitro cytokine production and cytotoxic
activity, we used the CD19+ lymphoblastic cell lines
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haematologica | 2021; 106(3)