CD137 is a potent costimulatory immunoreceptor and a member of the TNF-receptor (TNFR) superfamily. The receptor, also known as 4-1BB, is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells. While multiple lines of evidence show that CD137 is a highly promising therapeutic target, current approaches using monospecific antibodies may display a limited therapeutic window due to peripheral T cell and NK cell activation, leading to unwanted toxicity. To overcome this limitation, we have generated a bispecific protein therapeutic designed to achieve a tumor-target driven activation of immune cells via binding to CD137 and to a differentially expressed tumor target, HER2. Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins. Using phage display technology a CD137-specific Anticalin was identified. The Anticalin was recombinantly fused to a trastuzumab variant at either the C or N terminus of the antibody's heavy or light chain, yielding four different constructs covering a range of distances between the binding sites of the T cell-target and the tumor cell target. To minimize Fcγ-receptor interaction of the resulting bispecific and concomitant potential toxicity towards CD137-positive cells, the backbone of trastuzumab was switched from IgG1 to an engineered IgG4 isotype. Using ELISA or cell-based assays it was shown that all bispecific constructs bound their targets CD137 and HER2 with similar affinity compared to the parental building blocks, and both targets could be simultaneously bound. Binding to human receptors FcγRI and FcγRIII was significantly reduced in the bispecific constructs compared to non-engineered trastuzumab, while binding to the neonatal Fc receptor (FcRn) was retained. All constructs were shown to have excellent drug-like properties including thermal stability and plasma stability. HER2-dependent agonistic engagement of CD137 was demonstrated in ex-vivo T-cell activation assays utilizing HER2-positive human cell lines. The functional activity of the bispecific constructs was found to be dependent on their geometry. In conclusion, we report the first bispecific therapeutic protein that targets the potent costimulatory immunoreceptor CD137 in a tumor-target dependent manner, utilizing HER2 as the tumor target. Compared to currently existing CD137-targeting antibodies, this approach has the potential to provide a more controlled activation of the immune system in the tumor microenvironment with reduced peripheral toxicity. Bispecific T-cell engagers based on CD137 and HER2 have potential utility in HER2-positive cancers where there is a significant unmet medical need.
Hinner, M. J., Aiba, R.-S. B., Wiedenmann, A., Schlosser, C., Allersdorfer, A., Matschiner, G., … Olwill, S. A. (2015). Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein. Journal for ImmunoTherapy of Cancer, 3(S2). https://doi.org/10.1186/2051-1426-3-s2-p187