Anticalin Protein Research Articles

Abstract B023: Costimulatory T-cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein based on Anticalin® technology

Abstract Background: 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 are not designed to achieve a tumor-target driven activation, which may reduce the available therapeutic window via peripheral T cell activation and toxicity. To overcome this limitation, we applied Anticalin® technology to generate a bispecific protein therapeutic binding to CD137 and a differentially expressed tumor target, HER2. Methods: Anticalin® proteins are 18 kD protein therapeutics derived from human lipocalins which enable straight-forward multimeric drug targeting across several formats. We utilized phage display to generate an Anticalin protein binding to CD137 with high affinity and specificity. The CD137-specific Anticalin protein was genetically fused to a Trastuzumab variant, 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 Fc-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. Results: All four bispecific constructs bound the targets CD137 and HER2 with a nearly identical affinity compared to the parental building blocks, and both targets could be simultaneously bound. Compared to non-engineered Trastuzumab, binding to human receptors FcγRI and FcγRIII was significantly reduced, while binding to the neonatal Fc receptor (FcRn) was retained. Functional activity was demonstrated in human T cell activation assays, and shown to be tumor target (HER2) dependent. 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 localized activation of the immune system with reduced peripheral toxicity. Bispecific T cell engagers based on CD137 and HER2 may have utility in HER2-positive cancers where there is a significant unmet medical need, such as bladder, ovarian and gastric cancer. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Alexander Wiedenmann, Corinna Schlosser, Andrea Allersdorfer, Gabriele Matschiner, Christine Rothe, Ulrich Moebius, Shane A. Olwill. Costimulatory T-cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein based on Anticalin® technology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B023.

Engineered Human Lipocalin as an Antibody Mimetic: Application to Analysis of the Small Peptide Hormone Hepcidin

To the Editor: Anticalin® proteins are human lipocalins that are engineered to bind relevant targets with high affinity and specificity (1). By applying a mutation and selection process, it is possible to select Anticalin proteins that can specifically bind to very diverse protein targets in a monovalent fashion. Anticalin proteins are also suited for engaging small and compact ligands owing to their cuplike binding pocket. Compared with monoclonal antibodies, Anticalin proteins are about 8 times smaller and can be recombinantly produced in bacterial cells in large amounts. The hepatic peptide hormone hepcidin is a highly conserved molecule of only 25 amino acids with 4 disulfide bridges that plays a central role in body iron metabolism (2). Therefore, it could become a useful biomarker. Numerous hepcidin assays, by use of either mass spectrometry (MS)-based1 techniques or traditional immunochemical assays, have been described to quantify hepcidin in biological fluids (2). However, both MS and immunochemical approaches have disadvantages, including costly and sophisticated instrumentation, low throughput, need for highly specialized personnel, and difficulties in obtaining high-quality antibodies. The latter is ascribed to the small size of hepcidin, its folded nature, and its conservation throughout evolution, which complicate the generation of hepcidin antibodies for immunochemical assays such as ELISA. Thus, there is a need and opportunity for alternative approaches. We developed …