Effective cancer immunotherapy requires physical contact between cytotoxic immune cells and malignant cells which is restricted by the tumour microenvironment (TME). The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 is able to enhance immune cell infiltration into 3D tumour-stroma spheroids.
We established 3D multicellular spheroids that mimic a solid tumour with a CXCL12-expressing TME. For this purpose, CXCL12-secreting murine stromal MS-5 cells were co-cultured with human cancer cell lines in ultra-low attachment plates. Peripheral blood mononuclear cells from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were dissociated for analysis of infiltrated immune cells by flow cytometry. In parallel, cellular distribution within the spheroids was assessed by immunohistochemistry. A reporter-based T cell activation assay was adapted to the 3D format in order to examine the combination of NOX-A12 with immune checkpoint blockade.
We found that CXCL12 inhibition with NOX-A12 enhanced infiltration of CD8+ T cells, CD4+ T cells and NK cells, and to a lesser extent of Treg and B cells into the homogeneous, CXCL12-expressing tumour-stroma spheroids. Monocyte infiltration was not increased by NOX-A12. By facilitating physical contact of T cells with matching tumour cells, NOX-A12 also enhanced activation of T cells and synergized with PD-1 checkpoint inhibition.
Mechanistically, NOX-A12 appears to generate CXCL12 gradients within the densely packed in vitro tumour structure and may thereby break the immune privilege of the TME in vivo. Furthermore, a lower monocyte-to-lymphocyte ratio, as found in NOX-A12 treated spheroids, has been recognized as an indicator for better prognosis in various cancer types. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T and NK cell-based therapies in cancer patients, such as CAR-T and CAR-NK.
Clinical trial identification
Legal entity responsible for the study
Noxxon Pharma AG
Noxxon Pharma AG
D. Zboralski, A. Kruschinski, D. Eulberg, A. Vater: Employee Noxxon Pharma AG