Targeting Strategies in Cancer Immunotherapy: Comparative Evaluation of Stromal and Cellular Antigens for Bispecific Antibodies and Immunocytokines

Cancer remains one of the leading causes of mortality worldwide, resulting in millions of deaths annually, despite significant advances in detection and treatment. Conventional therapies such as surgery, radiation, and chemotherapy have improved survival rates for many malignancies, but their efficacy is often limited by systemic toxicity, resistance, and lack of specificity. These challenges underscore an urgent need for novel therapeutic strategies that can selectively target tumors and minimize side effects to healthy tissues. The targeted delivery of immunotherapies, whether directed toward stromal components of the tumor microenvironment or cellular antigens expressed on the tumor cell surface, has a substantial influence on efficacy and safety. The optimal approach is highly dependent on the therapeutic modality. Bispecific antibodies and antibody–cytokine fusion proteins (immunocytokines) are two distinct classes of immunomodulatory agents with fundamentally different mechanisms of action. Bispecific antibodies require direct engagement between effector immune cells and tumor cells to mediate cytotoxicity, whereas immunocytokines act by delivering cytokines to the tumor site to broadly stimulate immune responses within the microenvironment. The aim of this thesis is to explore how different targeting strategies affect the performance of these modalities through two complementary projects: (i) a comparative evaluation of stromal versus cellular targeting for a T cell engaging bispecific antibody, and (ii) a comparable assessment for the immunocytokine L19- IL2. In the first project, we developed and characterized a novel murine T cell-engaging bispecific antibody, 2C11xL19, which binds CD3 on T cells (via the 2C11 clone) and the extra domain B (EDB) of fibronectin (via the L19 antibody). To directly compare stromal and cellular targeting using the same antigen, we engineered murine tumor cell lines (F9 and WEHI-164) to express a membrane-anchored form of EDB, while wild-type cell lines expressed EDB only in the stroma. Biodistribution studies revealed substantially higher tumor accumulation of 2C11xL19 in cellular EDB- expressing tumors compared to stromal EDB tumors. Therapeutic experiments confirmed significantly higher antitumor activity in the cellular models, while stromal targeting showed limited efficacy despite high antigen abundance. These findings highlight the critical importance of antigen localization and accessibility for T cell- engaging bispecific antibody-mediated tumor killing. The second project extended this targeting comparison to an immunocytokine format. L19-IL2 (Darleukin) consists of human IL-2 fused to the L19 scFv targeting EDB. We assessed its biodistribution and therapeutic activity in the same F9 and WEHI-164 models, comparing wild-type (stromal EDB) and membrane-anchored EDB expression. Unlike the previous findings with bispecific antibodies, radiolabeled biodistribution studies showed no significant differences in tumor accumulation between stromal and cellular EDB models. Therapeutic studies revealed comparable tumor growth inhibition in both groups. These results suggest that, for L19-IL2, stromal targeting is sufficient to achieve effective cytokine delivery and antitumor activity, and that membrane localization of the target does not result in additional benefit. This discrepancy from the previous results reflects the different mechanisms of action between the two modalities: bispecific antibodies require direct tumor cell engagement for activity, whereas immunocytokines can function via signaling within the tumor microenvironment.