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increased survival if disease burden is low before adminis- tration of the cells,52 and that high disease burden53 is asso- ciated with an increased risk of side effects such as cytokine release syndrome. A potential advantage of using CAR T cells after HSCT is therefore the diminished leukemia burden due to the conditioning regimen and the transplant, which might translate into improved outcomes of the CAR T treatment and a lower incidence of cytokine release syndrome, but in the absence of informative ani- mal models this needs to be tested in a clinical trial.
An alternative haplo-HSCT approach using post-trans- plant cyclophosphamide for in-vivo T-cell depletion has shown promising results, although published work, to date, has focused primarily on adults.54,55 Although the use of aβ haplo-HSCT requires specific manufacturing expert- ise and upfront costs to establish the graft processing, we believe that the absence of post-HSCT pharmacological GvHD prophylaxis, the very low rate of severe GvHD and the low infection rate render this approach ideal for com- bination with post-HSCT adoptive immunotherapy. Eventually, it will need to be determined in prospective tri- als comparing aβ haplo-HSCT and post-transplant cyclophosphamide in children which alternative is the optimal treatment under what circumstances.
CAR T cells from healthy, allogeneic donors - which have preserved T-cell numbers and functionality and promise to overcome the manufacturing challenges and product variability of autologous CAR T cells29,56-59 – carry the potential to mediate GvHD if they still carry their endogenous TCR.60 Only a limited number of patients have been treated with allogeneic CAR T cells but the fre- quency of GvHD was surprisingly low when a co-stimu- latory domain derived from the CD28 molecule was used in the CAR construct,43 which raises the question of whether TCR deletion is necessary. Mechanistic studies suggest that the simultaneous activation of both the CD28-co-stimulated CAR and the TCR can lead to exhaustion and clonal deletion of alloreactive cells.44 The selective deletion of alloreactive T cells in this mode occurred, however, only at certain ratios between CAR T cells and target cells, and an excess of CAR T cells was able to induce GvHD.44 Furthermore, most CAR T-cell products are transfused without selection of the trans- duced cells and therefore contain untransduced cells not expressing a CAR which retain their alloreactive potential. Removal of the TCR from the cell surface, e.g., by genome editing approaches,37,38,46 is the best approach to reduce the risk of GvHD of allogeneic cells and additionally might prevent the induction of the T-cell dysfunction that can develop if the CAR and TCR are engaged on the same cell.61 CAR T cells with genome editing-based disruption of the TCR are currently being explored in clinical trials.62
Although CAR T cells with TCR knockout are often referred to as “universal” cells, they can still be rejected by the host immune system as it recovers from the immuno- depletion given prior to CAR T-cell infusion.63 Additional genetic engineering has been proposed to prevent recogni- tion by the host immune system, e.g., the use of genome editing to remove HLA class 1 expression64,65 and the expression of molecules that suppress natural killer cell activity.66 These strategies raise the issue that, if they suc- ceed, cells completely avoid recognition and clearance by the host immune system, but also escape immune surveil- lance in the case they become infected with viruses or turn malignant. Therefore, engineering an allogeneic CAR T-
cell graft that achieves bidirectional immune tolerance with a host immune system including satisfactory immune surveillance remains an unresolved challenge. The use of donor-derived T cells to create a TCR– CAR T- cell product that is administered after allogeneic HSCT enables HLA compatibility of the CAR T cells with the donor-derived host immune system after immune recon- stitution. A remaining limitation to full immune compati- bility is the nature or the CAR that we used, which is a synthetic protein with non-human parts and potential immunogenicity. Fully humanized CAR are currently in early stages of clinical trials.67 It is possible that in the post- transplant setting the development of an immune response to the CAR will not occur but that can only be tested in a human clinical trial.
It has previously been shown that transfusion of 104 T cells/kg can mediate rapid and protective immune recon- stitution,68 while among a cohort of 98 patients undergo- ing aβ haplo-HSCT who received a median of 4x104 TCRaβ+ cells, no patient developed high-grade acute GvHD and only one patient developed extensive chronic GvHD.8 With the TCRaβ+ depletion efficiency that we demonstrated, we estimate that therapeutically relevant doses of the cell product (theoretically up to 33x106 cells/kg) could be infused without administering more than 104 TCR+ cells/kg. It remains to be evaluated in a clin- ical trial whether the small number of aβ T cells that are transfused with the hematopoietic stem cell fraction, together with the residual TCR+ cells in the CAR T-cell product, substantially increase the GvHD risk.
In conclusion, we here establish a preclinical proof-of- concept for using the non-target fraction that is normally discarded during aβ+ T cell/CD19+ B-cell depletion to engineer a CD19-specific CAR T-cell product with a low risk of causing GvHD. aβ haplo-HSCT combined with graft-derived aβTCR-CD19 CAR T cells represents an appealing combination that: (i) allows a donor to be iden- tified for virtually every patient in need; (ii) overcomes the issues related to manufacturing autologous CAR T cells; (iii) abrogates the risk of GvHD through genome editing of the TRAC locus; and (iv) provides persistent targeted immune surveillance after HSCT. Furthermore, the use of adoptive post-HSCT immunotherapy can potentially translate in the future into desirable condition- ing regimens with lower toxicity and better preservation of fertility.
Disclosures
Patent submissions are planned. MHP serves on the scientific advisory boards of and has equity in CRISPR Tx and Allogene Therapeutics. Neither companies had any input into the design, execution, interpretation, or publication of this research. All the other authors declare that they have no competing financial con- flicts.
Contributions
Conception and design: VW, RB, AB and MHP; in vitro stud- ies: VW and PL; in vivo studies: VW and NM; off-target analy- sis: CML and GB; data collection: VW, CML; analysis and interpretation of data: VW, MHP and AB; supervision: AB, MHP and MGR; writing of the manuscript: VW and MHP. All authors reviewed the manuscript.
Acknowledgments
The authors thank Crystal Mackall, Rachel Lynn and Louai
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haematologica | 2021; 106(3)