A PD1-CD137L Fusion Protein More Potently Enhances Anti-Tumour Immune Responses Than Targeting PD-L1 and CD137 Separately.

Tumour immune evasion frequently involves PD-L1-mediated inhibition of T cell activity, limiting the efficacy of PD1/PD-L1 blockade, particularly in PD-L1-low or immunologically 'cold' tumours. To overcome this limitation, we developed a dual targeting human PD1-CD137L (hPD1-CD137L) fusion protein that blocks PD-L1-PD1 inhibitory signalling and simultaneously delivers CD137-mediated T cell costimulation. The fusion protein was produced with high purity and demonstrated specific binding to PD-L1-expressing tumour cells and CD137-positive T cells. The hPD1-CD137L retained a stable multimeric structure under acidic conditions and during prolonged storage, supporting its suitability for systemic administration and activity within the tumour microenvironment. Functionally, hPD1-CD137L enhanced T cell activation, as evidenced by increased NF-κB signalling and activation marker expression, and promoted robust tumour cell cytolysis in both 2D and 3D co-culture systems. Anti-tumour activity was observed across multiple tumour models with varying PD-L1 expression, including nasopharyngeal carcinoma (NPC), rhabdomyosarcoma, lung carcinoma and patient-derived colorectal cancer organoids. Notably, hPD1-CD137L enhanced cytolysis of C666 NPC cells by patient-derived tumour-infiltrating lymphocytes at low effector-to-target ratios, and was more potent than a combination of an anti-PD-1 antibody and a CD137 agonist. In vivo, PBMC-humanised NSG mice tolerated hPD1-CD137L without significant weight loss, systemic inflammation or survival impact. Subsequent efficacy study in PBMC-humanised, Rd18 rhabdomyosarcoma-engrafted or C666 nasopharyngeal carcinoma-engrafted NSG mice showed tumour growth suppression without overt toxicity. This study validates a human PD1-CD137L fusion protein as a potent drug candidate for cancer immunotherapy.