Multivalent design of the monoclonal SynO2 antibody improves binding strength to soluble α-Synuclein aggregates.

Soluble aggregates are reported to be the most neurotoxic species of α-Synuclein (αSyn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of αSyn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble αSyn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular αSyn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the αSyn oligomer-specific antibody SynO2, to increase avidity binding to soluble αSyn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to αSyn aggregates, while binding to αSyn monomers and unspecific binding to amyloid ß protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble αSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble αSyn aggregates was increased to ~ 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extracellular αSyn aggregates. The ability of the multivalent antibodies to bind a wider range of αSyn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.

Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways.

The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.

A single-chain fragment constant design enables easy production of a monovalent blood-brain barrier transporter and provides an improved brain uptake at elevated doses.

The interest for developing antibody-driven therapeutic interventions has exponentially grown over the last few decades. Even though there have been promising leaps in the development of efficacious antibody therapies, problems revolving around production and site-directed delivery of these large macromolecules persist. This is especially pertinent when it comes to designing and producing antibodies to penetrate the blood-brain barrier (BBB) to tackle neurodegenerative diseases. One of the most effective approaches to alleviating this problem is to employ a "Trojan Horse" approach, using receptor-mediated transcytosis, such as those governed by the transferrin receptor (TfR)-mediated pathways, to deliver large protein payloads into the brain. Even though this method is effective, ideal limiting factors, related to how the antibody binds to the TfR, need to be elucidated to improve BBB penetrance. With this said, we have designed and produced a single-chain Fc antibody, conjugated to an scFv8D3 TfR binding motif, creating a single-chain monovalent BBB transporter (scFc-scFv8D3). This recombinant protein is easy to produce and purify, demonstrates monovalent binding to the TfR and is structurally stable at physiologically relevant temperatures. Using an in vitro BBB model system, we show a positive correlation between the concentration of administered antibody and transcytosis efficacy, with scFc-scFv8D3 demonstrating significantly higher transcytosis levels compared with scFv8D3-conjugated bivalent antibodies at elevated administered concentrations. Furthermore, in vivo studies recapitulate the in vitro results, with the scFc-scFv8D3 demonstrating an elevated brain uptake at higher therapeutic doses in wild-type mice, comparable with that of the scFv8D3-conjugated bivalent antibody control. In addition, the half-life of the single-chain monovalent BBB transporter is comparable with that of standard IgG antibodies, indicating that the scFc format does not exacerbate physiological degradation. Our results lead us to the conclusion that valency and affinity are important variables to consider when discerning optimal transport across the BBB using TfR-mediated transcytosis pathways. In addition, we believe the single-chain Fc antibody we have described, which can easily be manipulated to accommodate a bispecific target tactic, provides a simple and efficacious approach for delivering therapeutic payloads to the brain milieu.

Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3.

The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.

A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice.

Amyloid-ß (Aß) oligomers and protofibrils are suggested to be the most neurotoxic Aß species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble Aß aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional Aß-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of Aß protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble Aß aggregates derived from brains of tg-ArcSwe and AppNL-G-F mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble Aß aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble Aß aggregates.