Enhanced VLA4-dependent endothelial cell adhesion underpins the higher efficacy of CAR-iNKT over CAR-T cells in limiting leptomeningeal acute leukemia Free

Invariant natural killer T (iNKT) cells are rare innate lymphocytes that bridge innate with adaptive immunity. Their invariant T cell receptor (iTCR) recognises lipid antigens presented by CD1d, a nonpolymorphic MHC class I-like glycoprotein. iNKT cells have a protective effect against acute-graft-versus-host-disease (aGVHD) and they can be deployed as a powerful off-the-shelf CAR immunotherapy platform.

Emerging pre-clinical evidence shows that CAR-iNKT cells outperform CAR-T against blood cancers, including B-cell acute lymphoblastic leukemia (B-ALL). Our previous transcriptome analysis showed higher expression of ITGA4 (alpha-4) and ITGB1 (beta-1), the constituent chains of VLA-4 by iNKT, compared to same donor T cells. VLA-4 mediates immune cell adhesion to endothelial cells via binding to vascular cell adhesion molecule 1 (VCAM-1), a pre-requisite for immune cell trafficking across the blood brain barrier (BBB), migration across the choroid plexus epithelium and entry into the central nervous system via the cerebrospinal fluid. Since B-ALL is associated with high incidence of leptomeningeal leukemia, we hypothesised that CAR-iNKT would be more effective in trafficking into the leptomeningeal space and limiting leptomeningeal leukemia than CAR-T cells.

To address this, we investigated the expression of VLA-4 in resting and CD3/CD28 antibody-stimulated healthy donor T and iNKT cells. We observed that while 86.99% of resting iNKT cells highly co-expressed the two subunits of VLA-4, only 29.85% of T cells had the same level of co-expression (n=10, p < 0.0001). iNKT cells were also found to bind significantly more soluble VCAM-1 than T cells, assessed by flow cytometry, (n=5, p < 0.01) suggesting higher conformationally active VLA-4 expression in iNKT cells.

Next, we evaluated VLA-4 expression and activity on CAR-iNKT and CAR-T cells using CARs targeting B-ALL (CD19-CD133 bispecific CAR) or multiple myeloma (BCMA CAR). First, we compared the cytotoxic activity of same donor CAR-T, CAR-iNKT cells and same donor non-transduced (NT) T and iNKT cells against CD19/CD133 (SEM) and BCMA (MM1.S)-expressing cell lines. CAR-iNKT showed significantly higher cytotoxicity of targets than same donor CAR-T cells, with very limited non-specific killing observed by NT cells. CAR-T and CAR-iNKT cells had the same levels of beta-1 expression, however only 30% of CAR-T cells exhibited similar level of alpha-4 expression to CAR-INKT cells (n=5, p < 0.01).

To assess the functional role of VLA-4 in CAR-T and CAR-iNKT cells, we measured their adhesion capacity on immobilized soluble VCAM-1 and VCAM-1-expressing HUVEC and BBB-derived hCMEC/D3 endothelial cells, under static and shear stress conditions, either in the presence of natalizumab, an alpha-4 blocking antibody, or its IgG4 isotype. Both CAR- and NT-iNKT cells showed significantly higher static adhesion than their T cells counterparts, with CAR-iNKT cells exhibiting the highest overall adhesion capacity under flow (n=5, p < 0.001), followed by NT-iNKT cells (n=5, p < 0.01); these differences were sustained at the highest shear stress level. Natalizumab blocking significantly decreased cell adhesion, less so in iNKT cells, consistent with the higher expression levels of conformationally active VLA-4 compared to T cells. These findings highlight iNKT cells' inherent ability for firmer cell adhesion than T cells, in a VLA-4-dependent manner.

To test our hypothesis in vivo, we employed a luciferase-expressing SEM B-ALL xenograft model in NSG mice. Treatment on day 17, when leukemia cells are readily identified in the meningeal space, showed that 10⁷CD19/CD133 CAR-iNKT cells were more effective, compared to same dose CAR T cells, in reducing leukemia burden in the bone marrow, spleen and in near elimination of leukaemia cells in the meninges at 24 hours and even more profoundly at 48 hours post treatment.

We conclude that CAR-iNKT cells are inherently better poised than CAR-T cells for treating high-burden pre-clinical meningeal leukemia, in part due to their higher, VLA-4-mediated ability to adhere to endothelial cells of the BBB and increased rentention in the leptomeningeal space. These findings have wider implications for cellular immunotherapy of blood and solid tumor cancers affecting the brain and the meninges.