Authors:
Estelle Adam, Emma Jenkins, Floriane Laurent, Jennifer Hillman, Maureen West, Emma Stanley, David Sanford, Min Fang, Agnieszka Kownacka, Celine Mothes, Alexandra Oudot, Vincent
Matthew, David Gilfoyle, Barry Jones, William Bachovchin, Amrik Basran, Cyril Berthet
Abstract:
Programmed Cell Death-Ligand 1 (PD-L1) is part of the immune checkpoint system involved in preventing autoimmunity. PD-L1 is upregulated on tumor cells and binds to its receptors, PD-1, expressed by immune cells in the tumor microenvironement. Anti-PD-L1 immunotherapies have shown to be effective treatments both as monotherapies and in combinations with chemotherapies and radiotherapies, providing long term responses in a subset of patients. We have developed a PD-L1 Affimer antagonist that could differentiate itself from the current clinically approved mAbs due to its smaller size, improved tissue penetration and alternative routes of delivery such as a subcutaneous injection. It also opens up the possibility of generating additional PD-L1 antagonist therapeutics such as bispecifics and cytokine fusions.
The Affimer biotherapeutic scaffold is a 14kDa monomeric protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. We have identified a range of high-affinity competitive binders to human PD-L1 confirmed by SPR using Fc formatted antigen, competition ELISA and blockade of PD-L1/PD-1 axis using various cell-based assays.We demonstrated efficacy in a humanized PD-L1 MC38 tumor growth inhibition model and compared it to approved PD-L1 antibody therapies. Pharmacokinetics (PK) and biodistribution are key parameters that influence the efficacy and safety of therapeutic proteins. Half-life extension of lead Affimer proteins was achieved by fusing to a human Fc region of an IgG (designated AVA004).
Pharmacokinetic analysis of AVA004 Fc molecules were performed in C57Bl/6, humanized FcRn/HSA mice and cynomolgus after single intravenous administration.
Tissue distribution following intravenous administration of AVA004 Fc to humanized NOG mice bearing orthotopic MDA-MB-231 tumor cells was evaluated using radiolabeled 125I- AVA004 Fc. Biodistribution imaging studies were conducted in mice bearing A375 tumor cells using IRDye800 conjugated to AVA004 Fc followed by ex vivo imaging. A more quantitative biodistribution study was conducted in the same model using SPECT imaging DOTAGA bioconjugated AVA004 Fc molecules radiolabeled with 111Indium. The imaging biodistribution allowed us to select the most favorable molecule based on its tumor/plasma and tumor/liver ratios.