title: Improved Drug Delivery of next-generation Antibody-Drug Conjugates by utilizing tumor-associated proteases
Creators: name: Roßkopf, Janis Mario
Publisher: name: UNSPECIFIED
Year: 2019

description: Targeted therapy with tumor-specific antibodies established in clinical oncology over the past decades with a variety of approved drugs. However, antibodies targeting cell surface antigens also suffer from limitations. Therapeutic agents must reach all tumor cells in cancer therapy, as untreated regions can lead to tumor recurrence. Poor penetration and drug delivery of conventional antibody-based drugs remain a major challenge for effective treatment of solid tumors. In the context of antibody-drug conjugates (ADCs), many research efforts focused on new linker chemistry, optimization of cytotoxic drugs and site-specific conjugation technologies. Less attention was drawn to the underexplored targeting scaffold of the antibody portion or antibody alternatives for optimizing drug delivery. Especially, the molecular size of the ADC pharmacodelivery vehicle plays a key role to deliver the cytotoxic payload to tumor cells. In the presented study, the effect of molecular weight and valency on tumor penetration and efficacy was investigated, introducing a novel ADC design. In this novel design an additional protease cleavage site was engineered into the IgG1 hinge region. This protease site is sensitive to enzymes commonly active in the tumor microenvironment, resulting in the release of 2 Fab-drug conjugates from the original ADC. The hinge cleavable ADC was evaluated for the tumor penetration ability and efficacy of Fab-drug conjugates (FDCs) that are generated locally by extracellular tumor proteases while retaining the half-life of the full-length antibody in systemic circulation. Homogeneous ADCs and FDCs with cytotoxic payloads MMAE and MMAF were generated by site-specific conjugation to the light chains of antibodies or antibody fragments using sortase A. Proteolytic cleavage reactions and enzyme kinetics displayed a fast and efficient release of FDCs from the full-length ADC by tumor-associated proteases urokinase-type plasminogen activator (uPA) and matriptase (MT-SP1). The designed anti-HER2 Trastuzumab ADCs and FDCs retained antigen binding properties, were stable in mouse serum and demonstrated high in vitro potency and cancer cell killing ability in HER2-overexpressing cell lines. For assessment of a better tumor localization, preparation as antibody-fluorophore conjugates for imaging analysis was achieved with Alexa Fluor 488. Enzymatically generated Fab-fluorophore fragments were able to penetrate and distribute more evenly within tumor spheroids compared to full-length IgG antibodies. A correlation was observed between reduced molecular size of the pharmacodelivery vehicle and tumor penetration and distribution ability. In vitro imaging analysis of tumor spheroids demonstrated increased molecular size and increased cellular binding resulted in decreased tumor penetration. Hence, the presented work showed promising results for the in situ generation of FDCs from IgG-ADCs that might have a potential benefit against solid tumors in terms of tumor penetration and localization. Moreover, an improved tumor to blood ratio with FDCs can be expected that could also result in reduced adverse effects. Further in vivo evaluation is necessary to optimize the balance between optimum tumor penetration and accumulation, molecular size of the targeting scaffold and pharmacokinetic properties.