Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

Malaria Biomimetic for Tumor Targeted Drug Delivery.

Malaria infected erythrocytes utilize the parasite protein VAR2CSA to bind to a unique presentation of chondroitin sulfate (CS) for their placenta specific tropism. Interestingly, many cancers express a similar form of CS, thereby termed oncofetal CS (ofCS). The distinctive tropism of malaria infected erythrocytes and the identification of oncofetal CS, therefore, represent potentially potent tools for cancer targeting. Here we describe an intriguing drug delivery platform that effectively mimics infected erythrocytes and their specificity for ofCS. We used a lipid catcher-tag conjugation system for the functionalization of erythrocyte membrane-coated drug carriers with recombinant VAR2CSA (rVAR2). We show that these malaria mimicking erythrocyte nanoparticles (MMENPs) loaded with docetaxel (DTX) specifically target and kill melanoma cells in vitro. We further demonstrate effective targeting and therapeutic efficacy in a xenografted melanoma model. These data thus provide a proof of concept for the use of a malaria biomimetic for tumor targeted drug delivery. Given the broad presentation of ofCS found across various types of malignancies, this biomimetic may therefore show potential as a broadly targeted cancer therapy against multiple tumor indications.

Long-term effects of immunotherapy with a brain penetrating Aß antibody in a mouse model of Alzheimer's disease.

BACKGROUND: Brain-directed immunotherapy is a promising strategy to target amyloid-ß (Aß) deposits in Alzheimer's disease (AD). In the present study, we compared the therapeutic efficacy of the Aß protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis. METHODS: AppNL-G-F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL-G-F mice, with evaluation after 3 days. Second, to assess the antibodies' ability to halt the progression of Aß pathology, 3 months old AppNL-G-F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL-G-F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aß aggregates and total Aß42 were quantified with ELISA and immunostaining. RESULTS: Neither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aß protofibrils or insoluble Aß1-42 after a single injection treatment. After three successive injections, Aß1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aß aggregates, but a reduction in total Aß42 was seen in the cortex of mice treated with both antibodies. CONCLUSIONS: Both RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aß immunotherapy.

Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice.

BACKGROUND: The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3Hu). V-aCD3Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system. METHODS: We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically "cold" 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically "hot" CT26 colon carcinoma model. RESULTS: V-aCD3Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model. CONCLUSIONS: Our findings suggest that V-aCD3Mu combined with an immune checkpoint inhibitor renders immunologically "cold" tumors "hot" and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.

First-in-human use of a modular capsid virus-like vaccine platform: an open-label, non-randomised, phase 1 clinical trial of the SARS-CoV-2 vaccine ABNCoV2.

BACKGROUND: Capsid virus-like particles (cVLP) have proven safe and immunogenic and can be a versatile platform to counter pandemics. We aimed to clinically test a modular cVLP COVID-19 vaccine in individuals who were naive to SARS-CoV-2. METHODS: In this phase 1, single-centre, dose-escalation, adjuvant-selection, open-label clinical trial, we recruited participants at the Radboud University Medical Center in Nijmegen, Netherlands, and sequentially assigned them to seven groups. Eligible participants were healthy, aged 18-55 years, and tested negative for SARS-CoV-2 and anti-SARS-CoV-2 antibodies. Participants were vaccinated intramuscularly on days 0 and 28 with 6 µg, 12 µg, 25 µg, 50 µg, or 70 µg of the cVLP-based COVID-19 vaccine (ABNCoV2). A subgroup received MF59-adjuvanted ABNCoV2. Follow-up was for 24 weeks after second vaccination. The primary objectives were to assess the safety and tolerability of ABNCoV2 and to identify a dose that optimises the tolerability-immunogenicity ratio 14 days after the first vaccination. The primary safety endpoint was the number of related grade 3 adverse events and serious adverse events in the intention-to-treat population. The primary immunogenicity endpoint was the concentration of ABNCoV2-specific antibodies. The trial is registered with ClinicalTrials.gov, NCT04839146. FINDINGS: 45 participants (six to nine per group) were enrolled between March 15 and July 15, 2021. Participants had a total of 249 at least possibly related solicited adverse events (185 grade 1, 63 grade 2, and one grade 3) within a week after vaccination. Two serious adverse events occurred; one was classified as a possible adverse reaction. Antibody titres were dose-dependent with levels plateauing at a vaccination dose of 25-70 µg ABNCoV2. After second vaccination, live virus neutralisation activity against major SARS-CoV-2 variants was high but was lower with an omicron (BA.1) variant. Vaccine-specific IFNγ+ CD4+ T cells were induced. INTERPRETATION: Immunisation with ABNCoV2 was well tolerated, safe, and resulted in a functional immune response. The data support the need for additional clinical development of ABNCoV2 as a second-generation SARS-CoV-2 vaccine. The modular cVLP platform will accelerate vaccine development, beyond SARS-CoV-2. FUNDING: EU, Carlsberg Foundation, and the Novo Nordisk Foundation.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Functionalization of Radiolabeled Antibodies to Enhance Peripheral Clearance for High Contrast Brain Imaging.

Small molecule imaging agents such as [11C]PiB, which bind to the core of insoluble amyloid-ß (Aß) fibrils, are useful tools in Alzheimer's disease (AD) research, diagnostics, and drug development. However, the [11C]PiB PET signal saturates early in the disease progression and does not detect soluble or diffuse Aß pathology which are believed to play important roles in the disease progression. Antibodies, modified into a bispecific format to enter the brain via receptor-mediated transcytosis, could be a suitable alternative because of their diversity and high specificity for their target. However, the circulation time of these antibodies is long, resulting in an extended exposure to radiation and low imaging contrast. Here, we explore two alternative strategies to enhance imaging contrast by increasing clearance of the antibody ligand from blood. The bispecific Aß targeting antibody RmAb158-scFv8D3 and the monospecific RmAb158 were radiolabeled and functionalized with either α-d-mannopyranosylphenyl isothiocyanate (mannose) or with trans-cyclooctene (TCO). While mannose can directly mediate antibody clearance via the liver, TCO-modified antibody clearance was induced by injection of a tetrazine-functionalized, liver-targeting clearing agent (CA). In vivo experiments in wild type and AD transgenic mice demonstrated the ability of both strategies to drastically shorten the circulation time of RmAb158, while they had limited effect on the bispecific variant RmAb158-8D3. Furthermore, single photon emission computed tomography imaging with TCO-[125I]I-RmAb158 in AD mice showed higher contrast 1 day after injection of the tetrazine-functionalized CA. In conclusion, strategies to enhance the clearance of antibody-based imaging ligands could allow imaging at earlier time points and thereby open the possibility to combine antibodies with short-lived radionuclides such as fluorine-18.

Reformation of the chondroitin sulfate glycocalyx enables progression of AR-independent prostate cancer.

Lineage plasticity of prostate cancer is associated with resistance to androgen receptor (AR) pathway inhibition (ARPI) and supported by a reactive tumor microenvironment. Here we show that changes in chondroitin sulfate (CS), a major glycosaminoglycan component of the tumor cell glycocalyx and extracellular matrix, is AR-regulated and promotes the adaptive progression of castration-resistant prostate cancer (CRPC) after ARPI. AR directly represses transcription of the 4-O-sulfotransferase gene CHST11 under basal androgen conditions, maintaining steady-state CS in prostate adenocarcinomas. When AR signaling is inhibited by ARPI or lost during progression to non-AR-driven CRPC as a consequence of lineage plasticity, CHST11 expression is unleashed, leading to elevated 4-O-sulfated chondroitin levels. Inhibition of the tumor cell CS glycocalyx delays CRPC progression, and impairs growth and motility of prostate cancer after ARPI. Thus, a reactive CS glycocalyx supports adaptive survival and treatment resistance after ARPI, representing a therapeutic opportunity in patients with advanced prostate cancer.

PET Imaging in Preclinical Anti-Aß Drug Development.

Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer's disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab, an antibody directed towards amyloid-beta (Aß) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aß pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aß drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aß, and its present and potential future role in the development of drugs aimed at reducing brain Aß levels as a therapeutic strategy to halt disease progression in AD.

Internalization and trafficking of CSPG-bound recombinant VAR2CSA lectins in cancer cells.

Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.

Detection of VAR2CSA-Captured Colorectal Cancer Cells from Blood Samples by Real-Time Reverse Transcription PCR.

Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility.

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.

Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta.

BACKGROUND: Amyloid-ß (Aß) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer's disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of Aß, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest. METHODS: We designed and recombinantly produced a hexavalent antibody based on mAb158, an Aß protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to Aß protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to Aß aggregates of different sizes. Finally, the ability of the antibodies to protect cells from Aß-induced effects was evaluated by MTT assay. RESULTS: Using real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble Aß aggregates. CONCLUSION: We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of Aß aggregates. This approach should be general and work for any aggregated protein or repetitive target.

Oncofetal Chondroitin Sulfate Is a Highly Expressed Therapeutic Target in Non-Small Cell Lung Cancer.

Broad-spectrum therapeutics in non-small cell lung cancer (NSCLC) are in demand. Most human solid tumors express proteoglycans modified with distinct oncofetal chondroitin sulfate (CS) chains that can be detected and targeted with recombinant VAR2CSA (rVAR2) proteins and rVAR2-derived therapeutics. Here, we investigated expression and targetability of oncofetal CS expression in human NSCLC. High oncofetal CS expression is associated with shorter disease-free survival and poor overall survival of clinically annotated stage I and II NSCLC patients (n = 493). Oncofetal CS qualifies as an independent prognosticator of NSCLC in males and smokers, and high oncofetal CS levels are more prevalent in EGFR/KRAS wild-type cases, as compared to mutation cases. NSCLC cell lines express oncofetal CS-modified proteoglycans that can be specifically detected and targeted by rVAR2 proteins in a CSA-dependent manner. Importantly, a novel VAR2-drug conjugate (VDC-MMAE) efficiently eliminates NSCLC cells in vitro and in vivo. In summary, oncofetal CS is a prognostic biomarker and an actionable glycosaminoglycan target in NSCLC.

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding.

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity.

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.

Enhanced neprilysin-mediated degradation of hippocampal Aß42 with a somatostatin peptide that enters the brain.

Background: Aggregation of the amyloid-beta (Aß) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading Aß, 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 Aß-precursor protein (AßPP) 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 Aß 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 Aß42 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 Aß42 in the hippocampus. Being that membrane-bound Aß 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.

SPECT imaging of distribution and retention of a brain-penetrating bispecific amyloid-ß antibody in a mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) immunotherapy with antibodies targeting amyloid-ß (Aß) has been extensively explored in clinical trials. The aim of this study was to study the long-term brain distribution of two radiolabeled monoclonal Aß antibody variants - RmAb158, the recombinant murine version of BAN2401, which has recently demonstrated amyloid removal and reduced cognitive decline in AD patients, and the bispecific RmAb158-scFv8D3, which has been engineered for enhanced brain uptake via transferrin receptor-mediated transcytosis. METHODS: A single intravenous injection of iodine-125 (125I)-labeled RmAb158-scFv8D3 or RmAb158 was administered to AD transgenic mice (tg-ArcSwe). In vivo single-photon emission computed tomography was used to investigate brain retention and intrabrain distribution of the antibodies over a period of 4 weeks. Activity in blood and brain tissue was measured ex vivo and autoradiography was performed in combination with Aß and CD31 immunostaining to investigate the intrabrain distribution of the antibodies and their interactions with Aß. RESULTS: Despite faster blood clearance, [125I]RmAb158-scFv8D3 displayed higher brain exposure than [125I]RmAb158 throughout the study. The brain distribution of [125I]RmAb158-scFv8D3 was more uniform and coincided with parenchymal Aß pathology, while [125I]RmAb158 displayed a more scattered distribution pattern and accumulated in central parts of the brain at later times. Ex vivo autoradiography indicated greater vascular escape and parenchymal Aß interactions for [125I]RmAb158-scFv8D3, whereas [125I]RmAb158 displayed retention and Aß interactions in lateral ventricles. CONCLUSIONS: The high brain uptake and uniform intrabrain distribution of RmAb158-scFv8D3 highlight the benefits of receptor-mediated transcytosis for antibody-based brain imaging. Moreover, it suggests that the alternative transport route of the bispecific antibody contributes to improved efficacy of brain-directed immunotherapy.

A simple method for detecting oncofetal chondroitin sulfate glycosaminoglycans in bladder cancer urine.

Proteoglycans in bladder tumors are modified with a distinct oncofetal chondroitin sulfate (ofCS) glycosaminoglycan that is normally restricted to placental trophoblast cells. This ofCS-modification can be detected in bladder tumors by the malarial VAR2CSA protein, which in malaria pathogenesis mediates adherence of parasite-infected erythrocytes within the placenta. In bladder cancer, proteoglycans are constantly shed into the urine, and therefore have the potential to be used for detection of disease. In this study we investigated whether recombinant VAR2CSA (rVAR2) protein could be used to detect ofCS-modified proteoglycans (ofCSPGs) in the urine of bladder cancer patients as an indication of disease presence. We show that ofCSPGs in bladder cancer urine can be immobilized on cationic nitrocellulose membranes and subsequently probed for ofCS content by rVAR2 protein in a custom-made dot-blot assay. Patients with high-grade bladder tumors displayed a marked increase in urinary ofCSPGs as compared to healthy individuals. Urine ofCSPGs decreased significantly after complete tumor resection compared to matched urine collected preoperatively from patients with bladder cancer. Moreover, ofCSPGs in urine correlated with tumor size of bladder cancer patients. These findings demonstrate that rVAR2 can be utilized in a simple biochemical assay to detect cancer-specific ofCS-modifications in the urine of bladder cancer patients, which may be further developed as a noninvasive approach to detect and monitor the disease.