Generation and in vivo validation of multi-functional antibody-fusion proteins for cancer therapy

Cancer remains a leading cause of mortality worldwide, necessitating the development of innovative therapeutic approaches. Immunotherapy has revolutionized cancer treatment, introducing novel strategies such as checkpoint inhibitors, antibody-drug conjugates, and CAR-T cell therapies. Recombinant cytokines have been explored for decades in cancer treatment, but only some patients benefit from their administration. The major drawback of recombinant cytokines is the development of systemic side effects. Immunocytokines, which combine antibodies against a tumor-associated antigen with the immunomodulatory properties of cytokines, represent a promising strategy to overcome these limitations. This approach aims to concentrate their biological activity at the site of disease, while limiting systemic exposure and sparing healthy organs. To date, several immunocytokines have demonstrated efficacy in clinical settings. Our group has developed multiple immunocytokines based on the L19 and F8 antibodies, which target EDB and EDA spliced variants of fibronectin, which are highly expressed in most malignancies. Immunocytokines such as L19-IL2, L19-TNF and L19-IL12 are showing promising results in the clinic for the treatment of progressive solid tumors, including sarcoma, melanoma, renal cell carcinoma and glioblastoma. In this work, I contributed to the generation and preclinical characterization of three antibody-based fusion proteins for cancer therapy. The first project focused on Tripokin, a multi-specific immunocytokine that combines interleukin 2 (IL2) and tumor necrosis factor (TNF) with the L19 antibody. Tripokin demonstrated enhanced tumor targeting, macroscopic tumor necrosis and robust immune response activation in preclinical models. It exhibited promising pharmacokinetics and potent therapeutic efficacy across different tumor models. It also proved synergistic effects when combined with existing therapies, making it a promising candidate for clinical applications. The second project described in this thesis involved the development and characterization of a dual-cytokine antibody-fusion protein designed to target Fibroblast Activation Protein (FAP). This immunocytokine, composed of IL2 and TNF, was validated in vitro and evaluated for its in vivo biodistribution and its therapeutic efficacy. The fusion protein demonstrated preferential localization to the tumor site and induced partial tumor retardation in immunocompromised mouse models. The third project involved the generation of a tumor-targeted checkpoint inhibitor combining anti-PD1 with the L19 antibody. The tumor-targeted immune checkpoint inhibitor has been characterized in vitro and preliminary results in vivo indicated corrected localization of the fusion protein to the neoplastic lesion, sparing healthy organs. However, the therapeutic efficacy of the targeted anti-PD1 was compared to the anti-PD1 monoclonal antibody and did not achieve an improved therapeutic effect. Our fusion protein served as a building block for the generation of a more complex immunocytokine that includes IL2 as an additional immunostimulatory payload.