Abstract 1473: CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade
Authors: Zboralski Dirk , Bauer Lisa , Eulberg Dirk , Vater Axel 
Published: July 2016

Abstract
Immune checkpoint inhibition promotes T cell-mediated killing of cancer cells and can induce striking responses, but objective control of tumor growth is observed in only 10-30% of patients with cancer types that generally respond to this treatment (Fearon 2014, Cancer Immunol Res 2:187). A possible cause for this limitation of checkpoint inhibition may be an immune-privileged tumor microenvironment (TME) which excludes the cytotoxic T cells from the vicinity of cancer cells. 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 (olaptesed pegol) is able to enhance T cell infiltration in 3D tumor-stroma spheroids, thereby facilitating effective immunotherapy.
We established 3D multicellular microtissues that mimic a solid tumor with a CXCL12-abundant TME. For this purpose, CXCL12-expressing murine stromal MS-5 cells were co-cultured with solid human cancer cell lines in ultra-low attachment plates for three days. Primary human T cells isolated from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were washed and dissociated for T cell quantification by flow cytometry. T cell localization in the 3D microtissues was assessed by immunohistochemistry (IHC). In order to examine T cell activation in the spheroids, a bioluminescent reporter-based PD-1/PD-L1 blockade bioassay (Promega) was adapted to the 3D format: Jurkat-PD-1/Luc T cells were incubated with anti-PD-1 and added to NOX-A12-treated spheroids (CHO-PD-L1 + MS-5).
We found that NOX-A12 increases the amount of T cells in tumor-stroma spheroids in a dose-dependent manner; flow cytometry analyses revealed a 2-3 fold increase in spheroid T cell infiltration at 10 nM NOX-A12 in all examined 3D co-culture types. Enhanced T cell infiltration in the presence of NOX-A12 was corroborated by IHC. In line with this, we found increased infiltration and activation of Jurkat-PD-1/luc T cells in the MS-5/CHO-PD-L1 spheroids treated with NOX-A12. Importantly, NOX-A12 synergized with anti-PD1-induced T cell activation.
Taken together, in heterotypic 3D models that mimic the complexity of the TME, the CXCL12 antagonist NOX-A12 improved T cell-based tumor immunotherapy by increasing T cell infiltration. By modulating the CXCL12 gradients within the complex 3D structure, NOX-A12 appears to break the immune-privilege of the TME, thereby paving the way for T cell migration into the tumor. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T cell-based therapies in patients with solid cancer.
Citation Format: Dirk Zboralski, Lisa Bauer, Dirk Eulberg, Axel Vater. CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1473.