Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [⁶⁸Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining ⁶⁸Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.

Dendritic cell [DC] vaccines can induce durable clinical responses, at least in a fraction of previously treated, late stage cancer patients. Several preclinical studies suggest that shielding programmed death-ligand 1 [PD-L1] on the DC surface may be an attractive strategy to extend such clinical benefits to a larger patient population. In this study, we evaluated the use of single domain antibody [sdAb] K2, a high affinity, antagonistic, PD-L1 specific sdAb, for its ability to enhance DC mediated T-cell activation and benchmarked it against the use of the monoclonal antibodies [mAbs], MIH1, 29E.2A3 and avelumab. Similar to mAbs, sdAb K2 enhanced antigen-specific T-cell receptor signaling in PD-1 positive (PD-1^pos) reporter cells activated by DCs. We further showed that the activation and function of antigen-specific CD8 positive (CD8^pos) T cells, activated by DCs, was enhanced by inclusion of sdAb K2, but not mAbs. The failure of mAbs to enhance T-cell activation might be explained by their low efficacy to bind PD-L1 on DCs when compared to binding of PD-L1 on non-immune cells, whereas sdAb K2 shows high binding to PD-L1 on immune as well as non-immune cells. These data provide a rationale for the inclusion of sdAb K2 in DC-based immunotherapy strategies.

Recently, a paradigm shift has been established for oncolytic viruses (OVs) as it was shown that the immune system plays an important role in the specific killing of tumor cells by OVs. OVs have the intrinsic capacity to provide the right signals to trigger anti-tumor immune responses, on the one hand by delivering virus-derived innate signals and on the other hand by inducing immunogenic cell death (ICD), which is accompanied by the release of various damage-associated molecules from infected tumor cells. Here, we determined the ICD-inducing capacity of Talimogene laherparepvec (T-VEC), a herpes simplex virus type 1 based OV, and benchmarked this to other previously described ICD (e.g., doxorubicin) and non-ICD inducing agents (cisplatin). Furthermore, we studied the capability of T-VEC to induce the maturation of human BDCA-1⁺ myeloid dendritic cells (myDCs). We found that T-VEC treatment exerts direct and indirect anti-tumor effects as it induces tumor cell death that coincides with the release of hallmark mediators of ICD, while simultaneously contributing to the maturation of BDCA-1⁺ myDCs. These results unequivocally cement OVs in the category of cancer immunotherapy.