Development of an in-vivo platform to evaluate the PK-PD relationship of an anti-PD-L1 mAb in a syngeneic mouse model of melanoma

Abstract

Cancer immunotherapy represents one of the most recent research fields to develop new treatment strategies and attain the cure of this disease. The understanding of tumor immunology has highlighted the role of the immune system in controlling tumor proliferation and hence, its potential as therapeutic target. On that matter, immunotherapy has changed the landscape for the treatment of melanoma. Over the last 30 years, dacarbazine and interleukin-2 were the standard care treatments for this cancer, with low response rate and some life threatening adverse effects. Nowadays, there are eight new immunotherapy molecules approved by the FDA for melanoma treatment, including the checkpoint inhibitor monoclonal antibodies (mAb) targeted to the PD-1/PD-L1 axis (anti-PD-1.PD-L1). Immune checkpoint pathways are involved in mechanisms of tumor resistance due to their capability to down-regulate T cell activity and induce lymphocyte death. Hence, the development of checkpoint inhibitor agents is of particular interest. These therapeutic molecules are able to promote and reestablish innate and adaptive immune effector mechanisms in order to neutralize the tumor immune scape, leading to an enhanced anti-tumor immune response. Besides, demonstrated features such as durable efficacy associated with extended survival, and considerable low toxicity profile, have taken these new mAbs to breakthrough development and accelerated approval as first-line treatment for melanoma patients. In order to improve clinical outcomes for patients, the pharmacokinetics (PK) and pharmacodynamics (PD) characterization of anti-PD-1/PD-L1 mAbs, may help to establish adequate dose-regimens and to identify those biomarkers associated with PD to select as early as possible the patients who benefit from these therapies. In this regard, a pre-clinical platform was developed in the present work in order to explore, evaluate and characterize the PD and the PK-PD relationship of an anti-PD-L1 mAb, using a syngeneic mouse model of melanoma with B16-OVA cells. In-vitro results have demonstrated that anti-PD-L1 mAb was bound specifically to PD-L1. This was compatible with a mechanism of ligand blockage by the mAb at the cell surface, followed by the internalization of the ligand-mAb complex. Thus, anti-PD-L1 mAb exerted a specific effect over PD-L1 cellular availability, down-regulating the ligand turnover with dependence of the exposure time and mAb concentration. Additionally, anti-tumor capability and increased survival were observed on B16-OVA tumor bearing mice after anti-PD-L1 therapy, with independence of the initial tumor size. Besides, there was no clear dose dependence for the anti-tumor effect, although antibody tumor levels showed a linear dose-concentration relationship. The tumor immune response triggered by anti-PD-L1 mAb led to a rapid tumor lymphocytic infiltration characterized by an increment of tumor specific CD8+ (OVA-CD8+) lymphocytes, suggesting an enhanced intra-tumor immune response. In fact, the time profile of OVA-CD8+ response followed the same profiles as mAb tumor concentrations, and peripheral blood lymphocytes (PBLs). These results suggest that PBLs might be a possible biomarker of anti-PD-L1 mAb therapeutic activity. Finally, an overall integration of in-vitro and in-vivo findings allowed the development of a PK-PD model in order to describe the relationship between anti-PD-L1 mAb concentrations and drug effect. This model, based on Simeoni s tumor growth model, describes the drug effect throughout a delay compartment that may be associated with the ligand-mAb binding and the process of complex internalization. Moreover, this anti-tumor mechanism provided by the drug was combined with another based on the ability of the tumor itself to regulate the inhibition of tumor cells proliferation. Therefore, it can be concluded that anti-PD-L1 mAb was able to induce the anti-tumor effect by activating the immune response at the target tissue.