Targeting IgV-like Domain Immune Checkpoint Receptors with Novel Nucleic Acid and Protein-based Therapeutics

摘要

The immune system is heavily regulated by negative checkpoint pathways; a network of cell signaling events governed by immune inhibitory ligand-receptor interactions. Physiologically, these negative checkpoint pathways are crucial for maintenance of self-tolerance in peripheral tissues. These pathways are often usurped by tumors as a mechanism to dampen anti-tumor immune responses. Clinically, the targeted blockade of PD-1:PD-L1 and CTLA-4:CD80/86 checkpoint ligand-receptor interactions using monoclonal antibodies has proved to be a viable means to provoke effective and durable anti-tumor responses. Conversely, agonists that stimulate these natural immunoinhibitory signaling pathways could potentially serve as immune-suppressants for treating patients with inflammatory and autoimmune disorders. The central theme of this thesis was to derive novel biotherapeutics that agonize or antagonize clinically-relevant checkpoint receptors. Specifically, the thesis focuses largely on targeting checkpoint receptors with short, single-stranded oligonucleotides, termed DNA aptamers, which bind molecular targets with affinity and specificity rivalling that of monoclonal antibodies. As a proof-of-principle, I first describe the derivation of DNA aptamers which bind to murine PD-1 and antagonize the PD-1:PD-L1 pathway to release anti-tumor immune responses and suppress the growth of a murine colon carcinoma tumor implant in-vivo. Second, I report the development of aptamers that bind to CD200R1 and agonize this inhibitory receptor. Strikingly, these anti-CD200R1 agonists were found to act as strong immunosuppressant's in mouse models of transplant rejection and allergy. Lastly, I described the engineering of a novel multimeric fusion protein, consisting of the extracellular IgV-like domain of the checkpoint ligand VISTA, fused to a short helical pentamerization domain derived from the cartilage oligomeric matrix protein. This soluble agonist readily inhibited T-cell activation in-vitro, and suppressed inflammation in models of transplant rejection and autoimmune hepatitis. Notably, this construct represents the first agonist targeting the as-of-yet discovered VISTA-receptor to stimulate immunoinhibitory signaling in-vivo.