The impact of glycosylation on immune checkpoint inhibitors

Immune checkpoints are regulators of the immune system. They are crucial to prevent the immune system from attacking cells indiscriminately. Immune checkpoints engage when proteins on the surface of immune cells called T cells recognize and bind to partner proteins on other cells. When the checkpoint and partner proteins bind together, they send an off signal to the T cells. PD-1 is a receptor expressed on the surface of several circulating immune cells that acts as a off switch preventing T-cells from attacking other cells. It does this when it attaches to the PD-L1 ligand expressed on a variety of cells. When PD-1 binds to PD-L1 it transmits inhibitory signals that suppress T-cell function and thus limits autoimmunity. Tumor cells can take advantage of these natural protective inhibitory mechanisms by displaying PD-L1 that binds to PD-1, preventing T cells from killing tumor cells in the body. Blocking the binding of tumor PD-L1 to PD-1 with an inhibitory molecule (e.g monoclonal antibodies) prevents the off signal from being sent and allows the T cells to kill tumor cells. Several anti-PD-1/PD-L1 antibodies are being used in cancer immunotherapy but so far their success is limited. Alternative strategies propose the use of small compounds and peptides to block PD-1/PD-L1 interaction. For example, soluble recombinant PD-1 is able to bind and neutralize PD-L1, acting as ligand trap (decoy). PD-L1 and PD-1 are proteins decorated with sugars (glycosylation). These sugars (glycans) mediate the interaction of PD-L1/PD-1 but an in-depth analysis of the role of glycosylation in PD- L1/PD-1 interactions is not yet available. The type of glycans on recombinant proteins depend on the host glycosylation machinery. Plants have entered the fight against cancer because they constitute low-cost and efficient hosts to produce proteins in a timeframe of weeks. In particular, Nicotiana benthamiana is well suited for the transient expression of recombinant proteins without the need for genetic transformation. Importantly, plants have a small glycosylation repertoire and lack many mammalian glycosyltransferases. This can be used as an advantage to facilitate a flexible stepwise overexpression of glycosyltransferases required for a specific glycan modification. The project aims to take advantage of the tolerance of plants for glyco-engineering to synthetize proteins with homogenous tailored glycans and identify which type of glycans can improve the affinity PD-L1 to PD-1. This will enable us to design decoy molecules with optimized glycans that will bind to tumor PD-L1 preventing it to bind to PD-1 to switch off T-cells. Studies on the functional activity of glyco-optimized proteins are likely to provide major insights into glycan-dependent interactions that will help guide therapeutic applications in cancer treatment.

The main goal of this project was to better understand and manipulate a key mechanism that cancer cells use to evade the immune system. This mechanism relies on two proteins, PD-1 and PD-L1, which normally help regulate immune responses. Cancer cells can exploit this system by producing PD-L1, effectively "turning off" immune cells and avoiding attack. While antibodies targeting PD-1 or PD-L1 have transformed cancer therapy, their effectiveness varies, and side effects or treatment resistance remain challenges. Our project aimed to develop new tools to improve understanding of this pathway and explore alternative therapeutic strategies. Using plants as tiny biofactories, we produced human proteins involved in this immune checkpoint system. Plants are cost-effective, fast, and safe hosts for producing complex human proteins, allowing us to create different versions of PD-1, PD-L1, and antibodies with specific sugar modifications. These sugars, or glycans, play a critical role in the proteins' function and interactions, but their influence is often poorly understood. By controlling the sugar patterns on these proteins, we could study how glycosylation affects their ability to block or engage with their targets. Our team successfully generated plant-derived PD-1 fusion proteins and Durvalumab (an anti-PD-L1 antibody) variants with carefully designed sugar profiles. We showed that these proteins can efficiently block the PD-1/PD-L1 interaction in laboratory tests, with some forms showing improved stability, longer circulation times, and reduced binding to inhibitory receptors-features that are therapeutically advantageous. Notably, the plant-derived PD-1 proteins recognized PD-L1 regardless of its sugar modifications, suggesting they could serve as robust diagnostic tools to help identify patients most likely to benefit from immune checkpoint therapies. The project demonstrated that plant molecular farming is a powerful and flexible platform for producing therapeutic proteins and testing new cancer immunotherapy strategies. The results provide important insights into how sugar modifications affect immune checkpoint proteins and open new avenues for cost-effective production of biologics for both therapeutic and diagnostic purposes. Beyond cancer, this work could help advance treatments for infectious and other immune-related diseases, offering potential societal, medical, and economic benefits by making innovative therapies more accessible.