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Genome editing of donor-derived aβ+ T cells
have been the stimulus for the excellent T-cell expansion we observed in the TCR- cells during the manufacturing process.
We next phenotyped the resulting CAR T cells after the editing process and found that the ratio of CD4 to CD8 cells in the resulting CAR T cell product was about 0.8:1 (Figure 3D and F). The majority of CD4+ cells were of naïve and central memory phenotype, while in the CD8+ subpopulation the majority of cells showed a naïve phe- notype (Figure 3E and G). This confirms that the CAR T cells had a balanced CD4:CD8 ratio, and that despite the TCR stimulation before genome editing and the transient activation through the CAR mediated by B-cell cytotoxi- city, both subpopulations had a high fraction of naïve and CM cells.
Antileukemic efficacy in vivo
We determined the activity of the aβTCR-CD19 CAR
T-cell product in vivo using a standard Nalm6 xenograft model.47 We transplanted 5x105 CD19+ Nalm6 cells i.v. into NSG mice to create the CD19+ leukemia model. Four days later, we i.v. infused 1x106 or 5x106 aβTCR-CD19 CAR T or control cells and followed leukemia burden by bioluminescence imaging. The higher dose of CAR T cells led to rapid and complete eradication of leukemia which lasted for at least 3 months, while the lower dose led to a transient decrease in leukemia burden and improved sur- vival although the mice eventually relapsed (Figure 4A). While control mice became moribund and died from dis- ease within 4 weeks, life was significantly extended at both doses of CAR T cells (Figure 4B) (log-rank test: P<0.01 for 5x106 cells, P<0.05 for 1x106 cells). No xeno- geneic GvHD was observed in any of the mice, demon- strating the low GvHD potential of the T-cell products. We repeated the experiment at the CAR T-cell dose level of 5x106 cells per mouse using two different control groups, either mock-treated T cells (expressing their endogenous TCR) or RNP-treated T cells (TCR knock- out), which confirmed comparable CAR T-cell efficacy and showed no difference between those control groups (Online Supplementary Figure S3A, B).
Off-target evaluation
While the sgRNA specificity has previously been evalu- ated in an integration-deficient lentivirus capture assay,40 we extended the specificity analysis to measure the off-tar- get activity of the TRAC-targeting RNP using targeted next-generation sequencing. We created a list of predicted off-target sites determined by the COSMID online tool (Figure 5A) and performed targeted deep sequencing of the sites in T cells, from six different donors, electroporated with the RNP (or mock electroporated to determine back- ground). Sequencing confirmed the specificity of the endonuclease with high activity at the on-target site, but no detectable insertions/deletions at off-target sites above the detection limit of 0.1% in any of the samples (Figure 5B).
Discussion
In the case of persistent minimal residual disease after HSCT, relapse risk is high, but treatment options are lim- ited during the time of engraftment and immune recov- ery. Many immune-based therapies are futile in this peri-
od as the immune system is only slowly developing, and transplant protocols typically include immune suppres- sion which would inhibit any adoptive cell-based therapy suchs as CAR T cells. aβ haplo-HSCT represents an excellent platform for adoptive immunotherapy not only because it helps to overcome the limited availability of HLA-matched donors, but also because post-HSCT immunosuppression is not required. It has shown robust clinical results in pediatric patients,8 but some patients still relapse.
We here hypothesize that a donor-derived CAR T-cell product with TCR knockout after haplo-HSCT has the potential to dissociate the beneficial anti-leukemic activi- ty from the harmful GvHD, two phenomena that are inherently connected to each other when infusing unma- nipulated donor-derived lymphocytes (donor lymphocyte infusion). This would take advantage of both the graft- versus-leukemia effect of allogeneic HSCT and the antileukemic activity of CAR T cells. It will also supple- ment the polyclonal, HLA-dependent immune response that the transplanted immune system elicits after HSCT with the antigen-specific, HLA-independent cytotoxicity of CAR T cells, in order to address relapses after allogene- ic HSCT that occur due to downregulation of HLA mole- cules.48-50 Moreover, manufacturing CAR T cells from the donor would maintain immune tolerance between the CAR T-cell product and the donor immune system (which are HLA identical) while taking advantage of the beneficial features of healthy donor T cells.
Our innovative approach avoids the risk of manufactur- ing failures that comes with the use of autologous T cells, but is distinct from allogeneic, “off-the-shelf” CAR T cells, as it creates a personalized CAR T-cell product for every patient from the respective haploidentical donor. It will therefore not benefit from the same cost-effective- ness that “off-the-shelf” CAR T cells promise, which aim to reduce prices by manufacturing doses for multiple patients during a single run. On the other hand, creating aβTCR-CD19 CAR T cells from the left-over cell fraction and administering them after HSCT will be more eco- nomic than the common practice of following the admin- istration of autologous CAR T cells with allogeneic HSCT,51 which carries the high price tag of current CAR T-cell products but then results in their eradication by the donor immune system. Our proposed protocol, in con- trast, allows for increased CAR T-cell persistence, since the cells are HLA-identical to the immune system after HSCT. This could create prolonged antileukemic surveil- lance from a single cell dose, or alternatively enable administration of multiple CAR T-cell doses for the same patient created from one manufacturing run. Moreover, the cells would benefit from the lymphopenia after HSCT, enabling their engraftment and prolonged activity without additional lymphodepleting therapy. Importantly, our approach allows for the creation of both the product containing the hematopoietic stem cells and the gene edited CAR T cells from a single apheresis, as the CAR T cells are made from the otherwise discarded cell fraction. This avoids an additional procedure and thereby leads to cost reduction and mitigates the risks and discomfort for the donor. This will be of particular importance when very young persons are the HSCT donors, e.g., younger siblings of pediatric patients, or the children of adult patients.
Clinical trials have shown that CAR T cells lead to
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