Anti-PD-1/L1 Nanobodies As a Promising and Innovative Approach in Cancer Immunotherapy

Programmed cell death protein-1 (PD-1) and its ligands PD-L1 and PD-L2 form a central inhibitory axis that regulates immune tolerance and plays a key role in tumor immune evasion. Therapeutic blockade of this pathway with monoclonal antibodies has transformed cancer immunotherapy; however, clinical challenges such as limited tumor penetration, immune-related adverse events, high production costs, and resistance mechanisms highlight the need for next-generation PD-1/PD-L1 inhibitors. Nanobodies, single-domain antigen-binding fragments derived from camelid heavy-chain antibodies, have emerged as promising alternatives due to their small size, high stability, strong affinity, excellent tissue penetration, and ease of genetic engineering. Here, we review the structural and expression features of PD-1 and its ligands, as well as their molecular and biochemical roles in shaping tumor immunity. We then provide a comprehensive overview of current PD-1/PD-L1-targeting approaches, including antibodies, small molecules, aptamers, siRNAs, and peptides and compare their therapeutic potential. We highlight recent advances in the development and use of anti-PD-1/PD-L1 nanobodies for cancer diagnosis, modulation of the tumor microenvironment (TME), immune-cell reprogramming, targeted drug delivery, CAR-T cell engineering, and cancer vaccine design. Additionally, we discuss innovative bispecific nanobody constructs that simultaneously target immune checkpoints, remodel the TME, or act as T-cell engagers to boost antitumor immunity. Finally, we address current limitations and translational challenges in nanobody-based therapies and outline future directions to improve their clinical effectiveness. Overall, emerging evidence supports nanobody-mediated PD-1/PD-L1 blockade as a highly versatile and potent platform that could revolutionize next-generation cancer immunotherapy.