αHSA-proIL-12: A Tumor-Conditional IL-12 Prodrug with Reduced Systemic Toxicity and Potent Antitumor Activity.

Interleukin-12 (IL-12) potently promotes the recruitment and activation of immune cells within the tumor microenvironment (TME), highlighting its immense potential for cancer therapy. However, its dose-limiting systemic toxicity and short half-life severely hinder its clinical translation. Here, we design a tumor-conditionally activated IL-12 prodrug termed αHSA-proIL-12. This engineered molecule integrates a cleavable albumin-binding nanobody (αHSA) domain to extend the half-life and enhance tumor targeting alongside a masking domain that occludes the IL-12 receptor-binding site to prevent nonspecific activation. This construct undergoes selective cleavage by matrix metalloproteinases (MMPs) that are highly expressed in the TME to release highly penetrant, free, low-molecular-weight IL-12 locally. This tumor-restricted activation process directly facilitates the selective recruitment and activation of tumor-infiltrating lymphocytes (TILs). The intravenous administration of an extremely low dose of 108 pmol per dose of αHSA-proIL-12 completely cleared MC38 tumors without inducing systemic toxicity. The maximum tolerated dose of this molecule exceeded 2 nmol, yielding a remarkably wide therapeutic index >18.5. Mechanistically, αHSA-proIL-12 effectively reprogrammed the immunosuppressive TME by expanding the numbers of effector CD8