A nanobody-enzyme fusion protein targeting PD-L1 and sialic acid exerts anti-tumor effects by C-type lectin pathway-mediated tumor associated macrophages repolarizing

Aberrant sialylated glycosylation in the tumor microenvironment is a novel immune suppression pathway, which has garnered significant attention as a targetable glycoimmune checkpoint for cancer immunotherapy to address the dilemma of existing therapies. However, rational drug design and in-depth mechanistic studies are urgently required for tumor sialic acid to become valuable glycoimmune targets. In this study, we explored the positive correlation of PD-L1 and sialyltransferase expression in clinical colorectal cancer tissues and identified their mutual regulation effects in macrophages. Subsequently, we characterized a new sialidase with excellent properties from human oral symbiotic bacteria and then developed a novel nanobody-enzyme fusion protein, designated as Nb16-Sia, to concurrently target the PD-L1 and sialic acid. Results from syngeneic colon tumor models reveal superior efficacy of Nb16-Sia over monotherapy and combinations, which could remodel the tumor immune microenvironment. Mechanistically, Nb16-Sia, which could repolarize macrophages from the tumor-promoting M2 to anti-tumor M1 phenotype via the C-type lectin pathway, exerted its antitumor efficacy mainly by regulating tumor-associated macrophages. Our strategy of nanobody-enzyme fusion protein effectively enables the delivery of sialidase, allows the collaboration between anti-PD-L1 nanobody and sialidase in combating tumors, and holds considerable promise for further development.