Antibody-based delivery of cytokine payloads to carbonic anhydrase IX leads to cancer cures in immunocompetent tumor-bearing mice

Antibody-cytokine fusion proteins can have the potential to increase the density and activity of subsets of leukocytes within the tumor mass. Here were describe the design, production and characterization of four novel antibody-cytokine fusion proteins directed against human carbonic anhydrase IX, a highly validated marker of hypoxia which is over-expressed in clear cell renal cell carcinoma and in other malignancies. As immunomodulatory payloads we used tumor necrosis factor, interleukin-2, interferon-alpha2 (corresponding to products which are in clinical use), and interleukin-12 (as this cytokine potently activates T cells and NK cells). Therapy experiments were performed in BALB/c mice, bearing CT26 tumors transfected with human carbonic anhydrase IX, in order to assess the performance of the fusion proteins in an immunocompetent setting. The biopharmaceuticals featuring tumor necrosis factor, interleukin-2 or interleukin-12 as payloads cured all mice in their therapy groups, whereas only a subset of mice was cured by the antibody-based delivery of interferon-alpha2. While the antibody fusion with tumor necrosis factor mediated a rapid hemorrhagic necrosis of the tumor mass, a slower regression of the neoplastic lesions (which continued after the last injection) was observed with the other fusion proteins and treated mice acquired protective anticancer immunity. A high proportion of tumor-infiltrating CD8+ T cells was specific to the retroviral antigen AH1, however the LGPGREYRAL peptide derived from human carbonic anhydrase IX was also present on tumor cells. The results described herein provide a rationale for the clinical use of fully human antibody-cytokine fusions specific to carbonic anhydrase IX.