1. ABSTRACT The blood-brain barrier (BBB) restricts the influx of biomolecules from the vasculature to the brain parenchyma. This attenuates exposure levels of the brain to systemically administered drugs, especially large- size molecules such as antibodies. This issue also makes systemic treatment of glioblastoma (GBM), the most devastating brain cancer, ineffective in most cases. Recent clinical studies have demonstrated that a measurable number of GBM cells, in particular cells near the growing edge of the infiltrative tumor area, exist behind an intact BBB. Collectively, the features of the BBB create a special challenge for effective treatment of central nervous system (CNS) diseases, including brain cancer, using drugs that have proven efficacy in other diseases. Antibody-drug conjugates (ADCs) are an emerging drug class with prominent target specificity, durable therapeutic efficacy, and high translatability in drug development. While promising, clinical benefits of ADCs in the treatment of brain diseases, in particular GBM, remain unconfirmed. Unfortunately, recent interim analysis in a Phase 3 study using the anti-EGFRvIII ADC Depatux-M (formerly called ABT-414) revealed no survival benefit for patients with newly diagnosed GBM receiving this ADC. Thus, improvement in BBB penetrability for ADCs is critically needed to advance this promising molecular format toward truly effective and safe systemic therapy for CNS diseases. We have developed novel ADC linker technologies, including: 1) branched linkers for site-specific and simultaneous installation of two distinct molecules onto a single antibody and 2) enzymatically cleavable linkers with exceptional circulation stability. Using these technologies, we have successfully constructed homogeneous conjugates appended with peptides that facilitate traversing the BBB through receptor-mediated transcytosis. One of the homogeneous peptide conjugates, as compared to a conventional heterogeneous variant, showed greater accumulation into the brain parenchyma in healthy mice (2.7-fold) and orthotopic GBM tumors in a xenograft mouse model (3.6-fold). Based on these findings, we hypothesize that homogeneous conjugation of properly designed BBB-penetrating peptides with ADCs will be a promising approach for systemic drug delivery to the brain. In this project, we will prepare a variety of BBB-penetrating peptides and construct antibody conjugates with various conjugation modalities (linker attachment site, linker structure, and stoichiometry of the peptides and payloads). All conjugates will be evaluated in vitro and in vivo for plasma stability, receptor-mediated transcytosis efficiency, pharmacokinetics, biodistribution, tolerability, and immunogenicity profiles. We will then evaluate a panel of BBB-permeable ADCs for tumor targeting efficiency as well as therapeutic efficacy in cell line-based and patient-derived xenograft mouse models of orthotopic GBM. We will also perform intravital fluorescen...