Co-Stimulatory Bispecific Antibodies Induce Enhanced T Cell Activation and Tumor Cell Killing in Breast Cancer Models

Although T cell-recruiting CD3-binding bispecific antibodies (BiMAb) have been proven to be clinically effective for hematologic malignancies, the success of BiMAb targeting solid tumor-associated antigens (TAA) in carcinomas so far remains poor. We reasoned that provision of co-stimulatory BiMAb in combination with alphaTAA-alphaCD3 BiMAb would boost T cell activation and proliferative capacity, and thereby facilitate the targeting of weakly or heterogeneously expressed tumor antigens. Various alphaTAA-alphaCD3 and alphaTAA-alphaCD28 BiMAb in a tetravalent IgG1-Fc based format have been analyzed, targeting multiple breast cancer antigens including HER2, EGFR, CEA, and EpCAM. Moreover, bifunctional fusion proteins of alphaTAA-tumor necrosis factor ligand (TNFL) superfamily members including 4-1BBL, OX40L, CD70 and TL1A have been tested. The functional activity of BiMAb was assessed using co-cultures of tumor cell lines and purified T cells in monolayer and tumor spheroid models. Only in the presence of tumor cells, alphaTAA-alphaCD3 BiMAb activated T cells and induced cytotoxicity in vitro, indicating a strict dependence on cross-linking. Combination treatment of alphaTAA-alphaCD3 BiMAb and co-stimulatory alphaTAA-alphaCD28 or alphaTAA-TNFL fusion proteins drastically enhanced T cell activation in terms of proliferation, activation marker expression, cytokine secretion and tumor cytotoxicity. Furthermore, BiMAb providing co-stimulation were shown to reduce the minimally required dose to achieve T cell activation by at least tenfold. Immuno-suppressive effects of TGF-beta and IL-10 on T cell activation and memory cell formation could be overcome by co-stimulation. BiMAb-mediated co-stimulation was further augmented by immune checkpoint-inhibiting antibodies. Effective co-stimulation could be achieved by targeting a second breast cancer antigen, or by targeting fibroblast activation protein (FAP) expressed on another target cell. In tumor spheroids derived from pleural effusions of breast cancer patients, co-stimulatory BiMAb were essential for the activation tumor-infiltrating lymphocytes and cytotoxic anti-tumor responses against breast cancer cells. Taken together we showed that co-stimulation significantly potentiated the tumoricidal activity of T cell-activating BiMAb while preserving the dependence on TAA recognition. This approach could provide for a more localized activation of the immune system with higher efficacy and reduced peripheral toxicities.

• Warwas, K. M.
• Meyer, M.
• Goncalves, M.
• Moldenhauer, G.
• Bulbuc, N.
• Knabe, S.
• Luckner-Minden, C.
• Ziegelmeier, C.
• Heussel, C. P.
• Zornig, I.
• Jager, D.
• Momburg, F.