Pre-clinical characterization of avelumab (anti-human PD-L1) reveals an enhanced anti-tumor efficacy in hIgG1 isotype

Background: Antibodies blocking PD1/PDL1 axis have been designed as either hlgG4 or as an engineered hlgG1 isotypes which has low or no binding to the FcγR (referred as hlgG1 silent). Avelumab is a fully human antibody of the immunoglobulin (IgG) 1 isotype that specifically binds to programmed death ligand 1 (PD-L1). Because PD-L1 can be expressed on activated T cells, in-depth understanding of PDLVs expression pattern within the tumor microenvironment is required for optimal isotype selection. Dahan and colleagues recently reported that FcγRs have different impact on anti-PD1/PDL1 antibodies' activity by using mouse surrogate antibodies and FcγR deficient mouse models. Since avelumab cross-reacts to mouse PD-L1, and has been investigated in multiple clinical trials, we engineered avelumab into a hlgG1 silent isotype, to investigate their mechanism of action (MOA) in preclinical models. Material and Methods: We generated avelumab hlgG1 silent (hlgG1 D265A/N297A mutant) and compared its efficacy to avelumab hlgG1 in pre-clinical models. CD4, CD8, and NK in vivo depletion studies and tumor infiltrating lymphocyte (TIL) profiling by FACS were conducted to distinguish the MOA between avelumab hlgG1 and hlgG1 silent in vivo. We also conjugated avelumab with fluorophore and used it for immunophenotyping PD-L1 on TILs isolated from cancer patients. Results: Avelumab hlgG1 and hlgG1 silent have identical antigen Dinding affinity based on biosensor analysis. Human lgG1 silent has been confirmed to have minimal detectable binding affinity towards all FcvR subfamilies. In two independent murine syngeneic models, enhanced antitumor efficacy was observed with avelumab hlgG1 compared to the hlgG1 silent version. Avelumab hlgG1 treatment did not decrease CD4 or CD8 T cell numbers in either tumor or spleen. We also examined the frequency of different myeloid subpopulations within the tumor, and compared the PD-L1 expression levels on each of the populations. Consistent with a previous report, avelumab hlgG1 decreased the most abundant, PD-L1 + myeloid suppressor population within the tumor. CD8 and NK in vivo depletion study results confirmed that engagement of NK-mediated ADCC and CD8 effector T cells are critical for anti-PD-L1 antibody's efficacy. Interestingly, CD4 in vivo depletion study indicate that removal of inhibitory signal between CD4 T cells and myeloid suppressor cells might be another critical contributor towards avelumab hlgGVs activity comparing with its