| 1. |
- Morrison, Jamie, et al.
(författare)
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Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways
- 2023
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 20:3, s. 1564-1576
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Stenler, Sofia, 1980-, et al.
(författare)
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Over the BBB and into the cell : Pursuing intracellular targets for immunotherapy of Parkinson’s disease
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The aim of our research is to modify therapeutic antibodies so that they can reach their dementia target inside cells located on the other side of the blood brain barrier. While the aggregates associated with Alzheimer’s are located extracellularly and thus readily available for antibodies that have crossed the BBB barrier, this is not the case for Parkinson’s disease. In this study, we focus on developing a peptide shuttle that can deliver antibodies not only over the BBB but also into neuronal cells where the Tau and a-synuclein aggregates can be found.For this purpose, we have investigated the use of a peptide which binds to a receptor that co- localizes with the aggregates. Our in-house experience suggests that the peptide is not an efficient BBB transporter despite the fact that some groups have used it as such, but that it might be more suitable as a transporter for intracellular delivery.We have successfully expressed recombinant antibodies with the peptide on the N-terminal of an antibody targeting the aggregates associated with Parkinson’s disease. Our initial studies indicate that the tyrosine on the N-terminal of the peptide needs to be free and unmodified to be able to enhance uptake into neuronal cells. This hinders the use of the normal labelling method which attaches radiolabelled iodine to tyrosines where the affinity for peptide target would be destroyed. We have been pursuing alternative methods, such as using click chemistry to attach the peptide which will leave the antibody free to be radiolabelled, as well as methods to detect unlabelled antibodies in vivo and in vitro.We have assessed the peptide-assisted increase in uptake in appropriate neuronal cell line models. Furthermore, we have studied uptake and retention in brain in mouse models for Parkinson’s disease.
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| 3. |
- Banka, Vinay, et al.
(författare)
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Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau
- 2023
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Ingår i: Frontiers in nuclear medicine. - : Frontiers Media S.A.. - 2673-8880. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging.METHODS: We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [18F]SFB as the prosthetic group, all four 18F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18F]SFB in acetonitrile/0.1 M borate buffer solution (final pH ~ 8.5) with an incubation of 20 min at room temperature, followed by purification on a PD MiniTrap G-25 size exclusion gravity column.RESULTS: Based on both in vitro and ex vivo evaluation, the bispecific antibodies displayed much higher brain concentrations than the unmodified antibody, supporting our subsequent F18-radiolabeling. [18F]SFB was produced in high yields in 60 min (decay-corrected radiochemical yield (RCY) 46.7 ± 5.4) with radiochemical purities of >95%, confirmed by analytical high performance liquid chromatography (HPLC) and radio-TLC. Conjugation of [18F]SFB and bispecific antibodies showed a 65%-83% conversion efficiency with radiochemical purities of 95%-99% by radio-TLC.CONCLUSIONS: We successfully labeled four novel and specifically engineered bispecific antibodies with [18F]SFB under mild conditions with a high RCY and purities. This study provides strategies to create brain-penetrable F-18 radiolabeled antibody probes for the selective detection of tau and Aβ aggregates in the brain of transgenic AD mice via in vivo PET imaging.
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| 4. |
- de la Rosa, Andrés
(författare)
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Design, expression, and analysis of antibody-based blood-brain barrier shuttles
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibody therapeutics, with their strong and highly selective target binding, are now used to treat various diseases. However, to enable their use to treat brain disorders, they must be delivered across the blood-brain barrier (BBB), as without active transport, only around 0.01% of intravenously injected doses reach the brain. Brain delivery can be done by BBB shuttles capable of binding receptors that naturally transport proteins, e.g., the Transferrin receptor (TfR). This thesis has studied strategies for designing TfR-binding shuttles and how to enhance the protein expression of antibody therapeutics. In Paper I, we shared our updated transient gene expression (TGE) protocol and developed a small-scale version to surmount the cost limitations of testing many conditions. Large variations of protein expression were observed for both protocols, prompting future studies investigating its cause(s). In paper II, we investigated if binding to the glycosaminoglycan heparan sulfate (HS) present at the BBB could improve brain delivery. Our results indicate that the BBB shuttle scFv8D3 is not dependent on the HS-binding sites identified, and adding new HS-binding sites did not enhance delivery. However, further studies are required due to HS's complexity and heterogeneity. Decreasing the TfR affinity of BBB shuttles has been shown to boost the delivery of therapeutic doses of high affinity anti-TfR antibodies, e.g., bivalent 8D3 antibodies. In Paper III, we applied the strategy to a monovalent single-chain fragment variable (scFv) of 8D3 (scFv8D3) based BBB shuttle. Our affinity mutants exhibited lowered TfR affinity, longer blood half-life, and higher brain concentration. Using our In-Cell BBB Trans assay, we concluded that the increased brain concentration is likely due to extended blood half-life. In paper IV, we fused the TfR ligand holo-transferrin to the TfR binding arms of the partly bivalent RmAb158-scFv8D3 antibody. Our results indicate that the TfR binding shifted from partly to fully bivalent, resulting in markedly decreased in vitro transcytosis. The potential transcytosis-promoting effect of the fused holoTf was absent and/or counteracted by the bivalent binding of the design. However, the strategy may still prove useful for monovalent TfR binders. In conclusion, monovalent and low-to-moderate affinity are likely beneficial binding properties for TfR-mediated brain delivery at therapeutic doses. However, whether it is possible to enhance brain delivery with HS-binding or holoTf-fusion requires further study.
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| 5. |
- de la Rosa, Andres, et al.
(författare)
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Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3
- 2022
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
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| 7. |
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| 8. |
- Fang, Xiaotian T., et al.
(författare)
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Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells
- 2017
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Ingår i: Biological Procedures Online. - : Springer Science and Business Media LLC. - 1480-9222. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).Results: The protocol has been used to generate multiple variants of tetra-and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (A beta) aggregates in vivo.Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.
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| 9. |
- Fang, Xiaotian T., et al.
(författare)
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High detection sensitivity with antibody-based PET radioligand for amyloid beta in brain
- 2019
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Ingår i: NeuroImage. - : Elsevier BV. - 1053-8119 .- 1095-9572. ; 184, s. 881-888
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Tidskriftsartikel (refereegranskat)abstract
- PET imaging of amyloid-beta (A beta) deposits in brain has become an important aid in Alzheimer's disease diagnosis, and an inclusion criterion for patient enrolment into clinical trials of new anti-A beta treatments. Available PET radioligands visualizing A beta bind to insoluble fibrils, i.e. A beta plaques. Levels of prefibrillar A beta forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of A beta leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary A beta , but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the A beta N-terminus and the transferrin receptor (TfR). Natively expressed at the blood-brain barrier (BBB), TfR could thus be used as a brain-blood shuttle. Di-scFv 3D6-8D3 bound to A beta 1-40 with high affinity and to TfR with moderate affinity. Di-scFv [I-124] 3D6-8D3 was injected in two transgenic mouse models overexpressing human A beta and wild-type control mice and PET scanned at 14, 24 or 72 h after injection. Di-scFv [I-124] 3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking A beta . This difference was observed from 24 h onwards, and at 72 h, 18 months old transgenic animals, with high load of A beta pathology, displayed SUVR of 2.2-3.5 in brain while wildtype showed ratios close to unity. A subset of the mice were also scanned with [C-11] PIB. Again wt mice displayed ratios of unity while transgenes showed slightly, non-significantly, elevated SUVR of 1.2, indicating improved sensitivity with novel di-scFv [I-124] 3D6-8D3 compared with [C-11] PIB. Brain concentrations of di-scFv [I-124] 3D6-8D3 correlated with soluble A beta (p < 0.0001) but not with total A beta, i.e. plaque load (p = 0.34). We have successfully created a small bispecific antibody-based radioligand capable of crossing the BBB, subsequently binding to and visualizing intrabrain A beta in vivo. The radioligand displayed better sensitivity compared with [C-11] PIB, and brain concentrations correlated with soluble neurotoxic A beta aggregates.
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| 10. |
- Gustafsson, Sofia, et al.
(författare)
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Blood-Brain Barrier Integrity in a Mouse Model of Alzheimer’s Disease With or Without Acute 3D6 Immunotherapy
- 2018
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Ingår i: Neuropharmacology. - : Elsevier BV. - 0028-3908 .- 1873-7064. ; 143, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- The blood-brain barrier (BBB) is suggested to be compromised in Alzheimer's disease (AD). The concomitant presence of vascular amyloid beta (AD) pathology, so called cerebral amyloid angiopathy (CAA), also predisposes impairment of vessel integrity. Additionally, immunotherapy against A beta may lead to further damage of the BBB. To what extent this affects the BBB passage of molecules is debated. The current study aimed to investigate BBB integrity to large molecules in transgenic mice displaying abundant A beta pathology and age matched wild type animals, with or without acute anti-A beta antibody treatment. Animals were administered a single i.v. injection of PBS or 3D6 (10 mg/kg), i.e. the murine version of the clinically investigated A beta antibody bapineuzumab, supplemented with [(125)]3D6. Three days post injections, a 4 kDa FITC and a 150 kDa Antonia Red dextran were administered i.v. to all animals. After termination, fluorescent detection in brain and serum was used for the calculation of dextran brain-to-blood concentration ratios. Further characterization of antibody fate and the presence of CAA were investigated using radioactivity measurements and Congo red staining. BBB passage of large molecules was equally low in wild type and transgenic mice, suggesting an intact BBB despite A beta pathology. Neither was the BBB integrity affected by acute 3D6 treatment. However, CAA was confirmed in the transgenes and local antibody accumulations were observed in the brain, indicating CAA-antibody interactions. The current study shows that independently of A beta pathology or acute 3D6 treatment, the BBB is intact, without extensive permeability to large molecules, including the 3D6 antibody.
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