Invariant NKT cells as a platform for CAR immunotherapy and prevention of acute Graft-versus-Host Disease.

Pre-clinical and clinical evidence supports a critical role of donor iNKT cells in protection from aGVHD and their potential for its prevention.

iNKT cells provide optimal platform for chimeric antigen receptor-based immunotherapy of blood cancers.

iNKT cell-based, ‘off-the-shelf’ immunotherapy could be sourced from allogeneic, healthy donors without risk of aGVHD.

Introduction

Invariant NKT (iNKT)-cells are a rare (<0.1% of blood T-cells), evolutionarily conserved subset of TCRαβ T-cells sharing features of innate and adaptive immune responses., In humans they are characterised by an invariant TCRVα24Jα18 chain nearly always pairing with a diverse TCRVβ11 chain. iNKT-cells are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA class I-like molecule, and their development depends on CD1d-expressing double positive thymocytes.

iNKT-cells have a memory effector phenotype with pronounced ability to migrate and home to extra-lymphoid tissues. CD4+ and CD4- iNKT-cell subsets are functionally distinct (eg, Th1 vs Th1/Th2 profile, primarily cytotoxic vs immunoregulatory and differential migratory/homing profile respectively)., iNKT-cells modulate a variety of immune responses, that include enhancement (usually) of anti-tumour and anti-pathogen responses and protection from auto-immunity and allo-reactivity, in particular acute graft-versus-host disease (aGVHD)., As such, iNKT-cell-based immunotherapy can be sourced from 3rd party donors as ‘an-off-the shelf’ treatment.

Current state-of-the-art

iNKT cells for prevention of aGVHD

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative therapeutic approach for hematological malignancies. However, its wider applicability is prevented by aGVHD, the donor T-cell-mediated alloreactive process responsible for much of the morbidity and mortality associated with allo-SCT.,

Extensive pre-clinical and clinical observational studies show that donor iNKT cells can prevent aGVHD without increasing the risk of disease relapse. Adoptive transfer of donor CD4+ iNKT-cells (CD4- cells were not tested), either without manipulation,,, or following in vitro expansion in the presence of alpha-galactosylceramide (αGalCer), a glycolipid that selectively and powerfully activates iNKT-cells, prevents or alleviates established experimental aGVHD in a MHC mismatched setting. This protective effect is mediated through Th2 polarisation of alloreactive T-cells and expansion of donor regulatory T-cells (Tregs).,,, Indeed, adoptively transferred donor iNKT-cells are at least 10 times more potent than Tregs in protecting mice from lethal aGVHD without compromising the graft-versus-leukaemia effect.,,, In addition, adoptively transferred in vitro αGalCer -expanded human iNKT-cells ameliorate xenogeneic aGVHD and improve survival. This protective effect is mediated by CD4- but not CD4+ human iNKT-cells and involves depletion of murine dendritic cells (DC) in vivo, reduction of T-cell activation and induction of their Th2/Th17 polarisation in vivo. These findings are in line with previous work showing that human iNKT-cells can be activated by allogeneic DC in a CD1d- and activating KIR-dependent manner followed by lysis of the DC.

The protective impact of iNKT-cells in the context of allo-SCT was further highlighted in clinical observational studies. Upon multivariate analysis, amongst several clinical and biological parameters including various immune cell subsets, donor graft CD4- iNKT-cell dose (> 0.03 × 106/kg) was associated with a 4.27-fold reduction in the relative risk of clinically significant aGVHD in a T-cell replete allo-SCT setting. CD4+ iNKT-cell dose had a significant impact on univariate analysis and a trend towards significance on multivariate analysis, suggesting that as shown in murine pre-clinical models of aGVHD, there might still be a role for this subset in protection from clinical aGVHD. In line with these findings, early recovery of iNKT-cells post T-cell-depleted allo-SCT is associated with reduced risk of aGVHD while a higher CD4- iNKT-cell dose and CD4- iNKT/CD3 T-cell ratio in the graft were associated with protection from aGVHD.,

To-date, no clinical trials involving adoptive transfer of donor iNKT-cells to allo-SCT recipients have been reported and therefore adverse effects cannot be predicted. However, in a study involving infusion of up to 109 in vitro expanded autologous iNKT-cells in cancer patients only grade 2 toxicity was observed.

iNKT-cells as a platform for CAR immunotherapy

Chimeric antigen receptor (CAR)-T cell immunotherapy for B lineage malignancies has yielded very promising therapeutic results., Several aspects of CAR immunotherapy are under further optimization including the development of technologies which would allow sourcing of CAR-T cells from healthy donors for ‘off-the-shelf’ use. For this purpose and in order to prevent aGVHD mediated by allogeneic T-cells, the endogenous TCR is deleted by means of gene editing., Since allogeneic iNKT-cells do not cause aGVHD they would be an ideal platform for ‘off-the-shelf’ CAR immunotherapy without need for deletion of their TCR.

Metelitsa et al provided the first evidence that CAR engineering of iNKT-cells and their clinical scale expansion was feasible., Indeed anti-GD2 and -CD19 human CAR-iNKT-cells were reactive against experimental neuroblastoma and CD19+ lymphoma, respectively, without evidence of xenogeneic aGVHD. However, in both xenograft tumor models, in vivo anti-tumor activity of CAR-iNKT-cells required either repeated CAR-iNKT cell dosing or IL-2 administration.,

More recent work demonstrated that compared to CAR19-T-cells, a single dose of 2nd generation CAR19-iNKT-cells without in vivo cytokine support, exerts a more effective anti-lymphoma effect in vivo through dual targeting of CD19 by CAR19 and of CD1d, often expressed on B lymphoma cells, by the endogenous iTCR (Fig. 1). Notably, intravenously administered CAR19-iNKT but not CAR19-T-cells swiftly eradicated secondary brain lymphoma. This finding might be related to the higher expression by iNKT-cells of integrins and chemokine receptors required for crossing the blood- and choroidal plexus-brain barriers. Another salient feature was enhancement of the anti-lymphoma effect of CAR19-iNKT-cells by transcriptional upregulation of CD1d expression in lymphoma and CLL B-cells using all-trans retinoic acid (ATRA). Mechanistically, this involved de-repression by ATRA of CD1D transcription restrained by the co-operative interaction of RARα with EZH2, a component of the Polycomb repressive complex.

Figure 1

Potential advantages of CAR-iNKT cells over CAR19-T cells for the treatment of CD1d-expressing B lineage malignancies. The higher proliferative and cytotoxic potential of CAR19-iNKT cells against lymphoma cells, their ability to secrete higher levels of anti-tumour molecules and the prospect of enhancing CAR-iNKT cell-based immunotherapy with ATRA and αGalCer are highlighted.

Future perspectives

Taken together the pre-clinical and clinical evidence outlined above lay the foundations for clinical interventional studies that will explore the role of donor iNKT cells in the prevention of aGVHD.

While the pre-clinical potential of CAR-iNKT-cells in other CD1d-expressing malignancies such as multiple myeloma will be doubtless explored, clinical development of CAR-iNKT cells is already under way. Early phase clinical trials aiming to test safety and efficacy of autologous 2nd generation CARGD2-iNKT-cells co-expressing IL-15 for children with neuroblastoma (NCT03294954) and of allogeneic CAR19-iNKT-cells co-expressing IL-15 for B lineage malignancies including CLL and ALL (NCT03774654) have been registered. These and additional studies will define the future role of iNKT-cells as a potentially highly promising and versatile immunotherapy platform in the treatment of B lineage blood cancers and perhaps beyond.