Invariant NKT Are a More Effective and Versatile Platform Than T Cells for CAR Immunotherapy of CD1d-Expressing B Lineage Malignancies: Cellular and Molecular Mechanisms

Anti-CD19 chimeric antigen receptor T cell (CAR19T) immunotherapy has shown promising clinical potential in relapsed/refractory mature B cell malignancies. However, only about half of patients benefit, highlighting the need for more effective CAR-based strategies.

iNKT cells are rare but powerful immunoregulatory and cytotoxic T lymphocytes, playing a pivotal anti-tumor role. iNKT cells are restricted by CD1d, a non-polymorphic, glycolipid-presenting HLA I-like molecule. We previously showed that CD1d, as well as on normal B cells, is also expressed on malignant CD19+ B cells in mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL) cells, while in up to 50% of patients with chronic lymphocytic leukemia (CLL) CD1d expression is very low or negative.

We tested the hypotheses that a) bi-specific CAR iNKT 19 cells, targeting simultaneously CD19 and CD1d via the CD19-specific CAR and their natural invariant TCR respectively, would be more effective than CART19 cells against CD19+CD1d+ B cell malignancies and b) transcriptional enhancement of CD1d expression would increase the CAR19iNKT cytotoxic effect.

We optimized a novel protocol for manufacturing CAR19iNKT cells. Their in vitro reactivity was assessed in cytotoxicity (flow cytometry), cytokine and cytotoxic granule intracellular staining and cytokine release assays (flow cytometry and Luminex technology) as well as real-time Incucyte proliferation assays. In vivo reactivity was assessed in systemic NSG xenograft assays with growth of the C1RCD1d B cells monitored by bioluminescence and magnetic resonance imaging. Transcription factor binding and histone mark enrichment at the promoter of CD1d was assessed by ChiP- and ChIP-re-ChIP-qPCR assays.

We manufactured clinical scale 2^nd and 3^rd generation CAR19iNKT cells using a highly optimized protocol (>75% transduction efficiency, n=8). In vitro validation, using singly- or dual-positive CD1d and CD19 targets, demonstrated that CAR19iNKT cells are CD19-specific, retain their natural CD1d-restricted reactivity and exert additive dual-specific cytotoxicity against CD1d+CD19+ targets. Compared to same donor CAR19T, CAR19iNKT cells display a significantly higher (P<0.0001) expandability and proliferative potential and are equally or more effective in killing CD19+CD1d+ lymphoid cell lines (C1RCD1d and Farage) and consistently more effective against primary MCL, MZL and CLL cells.

Since CD1d expression is low/negative in most cases of CLL, we sought to restore CD1d expression on leukemic cells for the purpose of CAR19iNKT cell immunotherapy. We found that surface CD1d expression can be restored by clinically relevant concentrations of ATRA in a time-dependent manner. Mechanistic dissection of CD1d transcriptional regulation in the CD1d-negative B lineage cell line U266 demonstrated the co-existence of bivalent chromatin marks at the promoter of CD1d commensurate with enriched binding and association of EZH2 with RARα. Notably, an EZH2 inhibitor exerted a synergistic effect along with ATRA in restoring CD1d transcription and surface expression and, in line with our hypothesis, 2^nd and 3^rd generation CAR19iNKT but not CAR19T cells displayed higher cytotoxic activity against ATRA-treated CLL cells.

Finally, in an NSG xenograft model of lymphoma, while survival of T and iNKT cell-treated animals was the same as that of untreated animals (n=7, P>0.05), both CAR19T and CAR19iNKT cell-treated animals (n=19) had significantly and comparably improved overall survival (OS; P<0.0001). However, compared to CAR19T, CAR19iNKT cell immunotherapy led to a significantly improved OS (P=0.01) and also better disease control including of CNS lymphoma, with earlier, more profound and sustained complete responses resulting in a significantly improved tumor-free survival (P=0.03).

We conclude thatCARiNKT19 are more effective than CART19 cells against CD19+CD1d+ B cell malignancies in vitro and in vivo and ATRA-mediated restoration of CD1d expression enhances the anti-lymphoma effect of CAR19iNKT immunotherapy in vitro . These properties together with the previously demonstrated ability of donor iNKT cells to protect from aGVHD raise the prospect of developing effective 'off-the-shelf' CAR19iNKT immunotherapy of lymphomas, which can mitigate against CD19-negative immune escape upon CAR treatment and can be further enhanced by transcriptional and epigenetic treatment.