Background Ilixadencel (International Proprietary Name, INN) is an intratumoral immune primer, consisting of pro-inflammatory monocyte-derived dendritic cells (DCs) from allogeneic healthy donor material. Upon administration, these cells secrete high amounts of pro-inflammatory chemokines and cytokines and additionally trigger a rejection process due to their allogeneic nature (major histocompatibility complex mismatch). This promotes a local pro-inflammatory environment and triggers cross-presentation of cell-associated tumor antigens, including neoantigens, by recruited and activated endogenous DCs. Since ilixadencel expresses the immunosuppressive programmed cell death-ligand 1 (PD-L1), we studied whether the anti-PD-L1 antibody avelumab could further improve the ilixadencel immune priming potential in vitro.

Methods Ilixadencel DCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at a ratio 1:5 for 48 hours, +/- avelumab, atezolizumab (anti-PD-L1 antibody with a non-agonistic Fc-domain) or pembrolizumab targeting programmed cell death protein 1 (PD-1). The activation markers cluster of differentiation (CD)-69 and CD137 (4-1BB, tumor necrosis factor superfamily 9) on NK cells were analyzed by flow cytometry at the end of co-culture and culture supernatants were examined for chemokine and cytokine production by enzyme-linked immunosorbent assays. The supernatants collected after 48h were subsequently tested as maturation stimulus for ‘bystander’ immature monocyte-derived DCs and compared to standard maturation stimuli, including different toll-like receptor (TLR)-ligands, using flow cytometry.

Results Addition of avelumab, but not atezolizumab or pembrolizumab, to ilixadencel/allogeneic PBMC co-cultures was found to strongly increase the frequency of 4-1BB expressing NK cells and to substantially increase the production of C-C motif chemokine 5 (CCL5), interferon-gamma, tumor necrosis factor (TNF) alpha and interleukin 1 (IL-1) beta. Stimulation with supernatants from ilixadencel/allogeneic PBMC/avelumab co-cultures further induced the strongest phenotypical maturation of bystander DCs, including expression of C-C motif Chemokine Receptor 7 (CCR7), CD83 and CD86, when compared to the other supernatants. This combination also induced a stronger phenotypical maturation than direct stimulation with TLR-ligands alone, including polyinosinic:polycytidylic acid (poly I:C), lipoplysaccaride (LPS), resiquimod (R848) and cytidine-phosphate-guanosine (CpG).

Conclusions Taken together, the presented data indicate that intratumoral administration of ilixadencel in combination with systemic avelumab treatment may increase the pro-inflammatory tumor microenvironment through Fc/Fc-receptor interactions with Fc-receptor-expressing NK cells and potentially also Fc-receptor-expressing macrophages. This may promote increased intratumoral recruitment and activation/maturation of endogenous immune cells, including cross-presenting DCs, subsequently leading to increased priming and expansion of tumor-specific T cells. The data therefore support the exploration of a combination therapy comprising ilixadencel and avelumab for the treatment of solid tumors.