Enhancing the therapeutic effect of biological drugs with protein engineering : Focusing on pre-clinical Alzheimer’s disease therapy

Aggregation of the amyloid-β peptide (Aβ) is one of the main pathological hallmarks in Alzheimer’s disease (AD). The soluble Aβ aggregates (oligomers and protofibrils) have shown to be the most harmful species. Hence, targeting these aggregates can be of therapeutic potential. Protein therapy is one of the fastest growing fields in drug development with more than 100 FDA approved protein-drugs in the last decade. Despite that, protein-drugs (mainly antibodies) targeting Aβ displayed limited beneficial effects in AD clinical trials. This might be attributed to the presence of the blood-brain barrier (BBB) that hinders the entry of big molecules such as proteins into the brain.  In paper I, we fused somatostatin peptide (SST) to the previously developed BBB transporter (scFv8D3). The new protein, SST-scFv8D3, exhibited a 120-times longer plasma half-life compared to SST, and reached the brain at high levels when intravenously administered. When tested in APPswe mouse model of AD, SST-scFv8D3 significantly enhanced neprilysin (NEP)-mediated degradation of hippocampal Aβ42 after only three injections. In paper II, treatment with SST-scFv8D3 displayed a wide-ranging effect on AD brain proteome. Mitochondrial and neuronal growth proteins were among the most altered protein-groups, where SST-scFv8D treatment shifted them towards wild-type levels. There is potential to increase the binding strength and selectivity of antibodies to small Aβ aggregates (oligomers), which are thought to be the most toxic Aβ species. In paper III, we developed a multivalent antibody format with additional binding sites having short distances between them. The new antibody format displayed a 40-fold reduction in the dissociation rate from Aβ protofibrils. Furthermore, the multivalent antibody could strongly bind small Aβ oligomers, which has been difficult to achieve with conventional IgG antibodies. In paper IV, we developed a bispecific version of the multivalent antibody capable of passing the BBB. A single intravenous injection of the new antibody format was enough to significantly clear soluble Aβ aggregates from the brain of tg-ArcSwe mice.  In paper V, we developed recombinant proteins with NEP linked to an Fc-fragment to provide long half-life and to the above-mentioned BBB transporter. When applied at therapeutic doses, these proteins significantly degraded plasma Aβ, but displayed limited effects on brain Aβ concentration, probably due to their short retention times in the brain. In conclusion, we developed new protein-drugs with improved binding properties to Aβ, ability to cross the BBB, and therapeutic potential in pre-clinical mouse models of AD.

Enhancing the therapeutic effect of biological drugs with protein engineering : Focusing on pre-clinical Alzheimer’s disease therapy

Aggregation of the amyloid-β peptide (Aβ) is one of the main pathological hallmarks in Alzheimer’s disease (AD). The soluble Aβ aggregates (oligomers and protofibrils) have shown to be the most harmful species. Hence, targeting these aggregates can be of therapeutic potential. Protein therapy is one of the fastest growing fields in drug development with more than 100 FDA approved protein-drugs in the last decade. Despite that, protein-drugs (mainly antibodies) targeting Aβ displayed limited beneficial effects in AD clinical trials. This might be attributed to the presence of the blood-brain barrier (BBB) that hinders the entry of big molecules such as proteins into the brain.  In paper I, we fused somatostatin peptide (SST) to the previously developed BBB transporter (scFv8D3). The new protein, SST-scFv8D3, exhibited a 120-times longer plasma half-life compared to SST, and reached the brain at high levels when intravenously administered. When tested in APPswe mouse model of AD, SST-scFv8D3 significantly enhanced neprilysin (NEP)-mediated degradation of hippocampal Aβ42 after only three injections. In paper II, treatment with SST-scFv8D3 displayed a wide-ranging effect on AD brain proteome. Mitochondrial and neuronal growth proteins were among the most altered protein-groups, where SST-scFv8D treatment shifted them towards wild-type levels. There is potential to increase the binding strength and selectivity of antibodies to small Aβ aggregates (oligomers), which are thought to be the most toxic Aβ species. In paper III, we developed a multivalent antibody format with additional binding sites having short distances between them. The new antibody format displayed a 40-fold reduction in the dissociation rate from Aβ protofibrils. Furthermore, the multivalent antibody could strongly bind small Aβ oligomers, which has been difficult to achieve with conventional IgG antibodies. In paper IV, we developed a bispecific version of the multivalent antibody capable of passing the BBB. A single intravenous injection of the new antibody format was enough to significantly clear soluble Aβ aggregates from the brain of tg-ArcSwe mice.  In paper V, we developed recombinant proteins with NEP linked to an Fc-fragment to provide long half-life and to the above-mentioned BBB transporter. When applied at therapeutic doses, these proteins significantly degraded plasma Aβ, but displayed limited effects on brain Aβ concentration, probably due to their short retention times in the brain. In conclusion, we developed new protein-drugs with improved binding properties to Aβ, ability to cross the BBB, and therapeutic potential in pre-clinical mouse models of AD.

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.De två första författarna delar förstaförfattarskapet</p

Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease

Background Aggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB). Methods To provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples. Results When tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain. Conclusion A one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.Fadi Rofo and Nicole G. Metzendorf shared first authorship</p

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

Amyloid-beta (A beta) oligomers and protofibrils are suggested to be the most neurotoxic A beta species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble A beta aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional A beta-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of A beta 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 A beta aggregates derived from brains of tg-ArcSwe and App(NL-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 A beta 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 A beta aggregates

Enhanced neprilysin-mediated degradation of hippocampal A beta 42 with a somatostatin peptide that enters the brain

Background: Aggregation of the amyloid-beta (A beta) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading A beta, with its activity enhanced by the neuropeptide somatostatin (SST). SST levels are decreased in the brains of AD patients. The poor delivery of SST over the blood-brain barrier (BBB) and its extremely short half-life of only 3 min limit its therapeutic significance. Methods: We recombinantly fused SST to a BBB transporter binding to the transferrin receptor. Using primary neuronal cultures and neuroblastoma cell lines, the ability of the formed fusion protein to activate neprilysin was studied. SST-scFv8D3 was administered to mice overexpressing the A beta-precursor protein (A beta PP) with the Swedish mutation (APPswe) as a single injection or as a course of three injections over a 72 h period. Levels of neprilysin and A beta were quantified using an Enzyme-linked immunosorbent assay (ELISA). Distribution of SST-scFv8D3 in the brain, blood and peripheral organs was studied by radiolabeling with iodine-125. Results: The construct, SST-scFv8D3, exhibited 120 times longer half-life than SST alone, reached the brain in high amounts when injected intravenously and significantly increased the brain concentration of neprilysin in APPswe mice. A significant decrease in the levels of membrane-bound A beta 42 was detected in the hippocampus and the adjacent cortical area after only three injections. Conclusion: With intravenous injections of our BBB permeable SST peptide, we were able to significantly increase the levels neprilysin, an effect that was followed by a significant and selective degradation of membrane-bound A beta 42 in the hippocampus. Being that membrane-bound A beta triggers neuronal toxicity and the hippocampus is the central brain area in the progression of AD, the study has illuminated a new potential treatment paradigm with a promising safety profile targeting only the disease affected areas

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

ABSTRACTSoluble 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 β 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

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

Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide

Alzheimer's disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-beta (A beta) peptide. A potential therapeutic intervention in Alzheimer's disease is to enhance A beta degradation by increasing the activity of A beta-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound A beta 42 degradation in the hippocampus of mice overexpressing the A beta-precursor protein with the Swedish mutation (APPswe). Using LC-MS, we further evaluated the anti-Alzheimer's disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC-MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated a (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer's disease therapies based on SST peptides