Brain Over Level 55

[Achill Baldwin]

Thesebrain scans highlight dopamine receptors, with areas of highest density shown in red. The meth abuser has severely reduced receptor levels. Other drugs, including alcohol, cocaine, and heroin, have the same effect. Images courtesy Dr. Nora Volkow, Brookhaven National Laboratory.

Effective delivery of protein therapeutics into the brain remains challenging because of difficulties associated with crossing the blood-brain barrier (BBB). To overcome this problem, many researchers have focused on antibodies binding the transferrin receptor (TfR), which is expressed in endothelial cells, including those of the BBB, and is involved in receptor-mediated transcytosis (RMT). RMT and anti-TfR antibodies provide a useful means of delivering therapeutics into the brain, but the anti-TfR antibody has a short half-life in blood because of its broad expression throughout the body. As a result, anti-TfR antibodies are only maintained at high concentrations in the brain for a short time. To overcome this problem, we developed a different approach which slows down the export of therapeutic antibodies from the brain by binding them to a brain-specific antigen. Here we report a new technology, named AccumuBrain, that achieves both high antibody concentration in the brain and a long half-life in blood by binding to myelin oligodendrocyte glycoprotein (MOG), which is specifically expressed in oligodendrocytes. We report that, using our technology, anti-MOG antibody levels in the brains of mice (Mus musculus) and rats (Rattus norvegicus) were increased several tens of times for a period of one month. The mechanism of this technology is different from that of RMT technologies like TfR and would constitute a breakthrough for central nervous system disease therapeutics.

Copyright: 2019 Nakano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Competing interests: We have the following interests Ryosuke Nakano, Sayaka Takagi-Maeda, Tomoko Osato, Kaori Noguchi, Kana Kurihara-Suda and Nobuaki Takahashi are employed by Kyowa Hakko Kirin Co., LTD.. The technology Accumubrain described in our manuscript was patented and published in June 2018 (Patent number WO2018123979). There are no further patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Therapeutic enzymes are effective against, and have been approved for, lysosomal storage diseases such as Fabry disease and Gaucher disease, but they are not effective against central nervous system conditions because their molecular weight is too high to allow for passage across the BBB [6,7].

To increase the concentration of therapeutic proteins in the brain, direct intrathecal and intracerebral injection has been tested, but the procedure is highly invasive [8]. Furthermore, it is reported that IgG are rapidly discharged from the brain into circulating blood by neonatal Fc receptor (FcRn) [9,10].

To overcome these problems, extensive research has been undertaken into technologies for delivering drugs into the brain. Most popular technologies use a receptor-mediated transcytosis (RMT)-based mechanism, and there are many reports of antibodies which bind to the transferrin receptor (TfR), which is expressed in endothelial cells, including those of the BBB, and allows for transport across the BBB by RMT [11,12]. Additionally, other receptors, such as the insulin receptor, are also reportedly transported across the BBB by means of RMT [13].

For clinical use, anti-TfR antibody or anti-insulin receptor antibody are fused with therapeutic antibodies or enzymes. One example is the anti-BACE1 anti-TfR bispecific antibody, which is transported across the BBB by RMT of TfR and has been found to be present in the brain at levels four times higher than that in control mice [14]. Other examples include anti-insulin receptor antibodies fused with glial cell-derived neurotrophic factor (GDNF) or iduronidase (IDUA). Anti-insulin receptor antibody GDNF fusion protein concentration is reported to display a 10-fold increase in the brains of Rhesus monkeys (Macaca mulatta) [13].

Anti-TfR antibody and anti-insulin receptor antibody are clearly useful in applications requiring transport of large molecules across the BBB, and for increasing antibody concentrations in the brain. However, TfR and the insulin receptor are expressed not only in the vascular endothelial cells of the brain, but also in those of other organs, as well as in other cells types in, for instance, the liver [15]. These technologies therefore deliver drugs to tissues other than the brain. Consequently, anti-TfR antibody has a short half-life in the blood [11].

For this reason, the effectiveness of therapeutic antibodies that natively have a long half-life in the blood is significantly reduced if they bind to TfR. In order to increase the half-life of anti-TfR antibodies in the blood, some studies have attempted to reduce their binding strength or valence [14,16]. However, there remains a trade-off relationship for anti-TfR antibodies between high delivery ability into the brain and long half-life in the blood.

Therefore, we adopted a different approach that did not make use of the RMT concept. RMT enhances antibody transport across the BBB, but antibodies are also distributed in the body wherever antigens like TfR are expressed. We focused on slowing down antibody export from the brain, by binding the antibodies to a brain-specific antigen.

Here, we report the use of an anti-MOG antibody to increase antibody and enzyme concentrations in the brain. We named this technology AccumuBrain. This new technology is expected to increase the levels of therapeutic proteins like antibodies or enzymes in the brain, and to maintain these levels for longer periods than technologies which use anti-TfR antibodies.

Male ICR mice (Mus musculus) were purchased from Japan SLC (Japan) at 4 weeks of age. Sprague Dawley (SD) rats (Rattus norvegicus) were purchased from Charles River Japan (Japan) at 6 weeks of age. Mice and rats were housed in individually ventilated cages (IVC) with Paper-Clean chips (Japan SLC) and maintained in a specific pathogen-free (SPF) environment with a 12-hour light/dark cycle, controlled temperature and humidity, and free access to water and solid diet throughout the duration of the study.

After a quarantine/acclimation period of 1 week, mice were used at 5 weeks of age and rats were used at 7 weeks of age. During the quarantine/acclimation period, no animals were found to show abnormal clinical signs.

Animals were assigned to groups in an alternating manner, in order of decreasing body weights (heaviest to lightest) so that the variance between the groups was minimal. SD rats or ICR mice were intravenously injected with each antibody (see below). After the indicated time, rats or mice were perfused with 1% heparin-PBS (150 mL for rats and 6 mL/min for 8 min for mice) from the left ventricle, and their brains were extracted under anesthesia with pentobarbital. Blood was collected before perfusion. Brain weights were measured, after which brains were homogenized in citric buffer (pH 3.5). After centrifugation, the supernatant was isolated as a brain sample, and the volume was measured. Pellets were homogenized and eluted in citric buffer two more times (pH 3.5 and pH 2.5). Eluted samples were neutralized with 1 M tris-HCl (pH 9.0). Antibody concentrations were measured using an AlphaLISA kit (PerkinElmer). For each time point, three samples of antibody were eluted at pH values of 3.5, 3.5, and 2.5; the antibody concentration of each sample was then calculated (per gram brain), and the three antibody concentrations were added to determine the final concentration.

Monoclonal anti-avermectin antibody was used as a negative control and was isolated by means of the standard hybridoma method [23]. Briefly, 4-week old SD rats were immunized four times with bovine serum albumin-avermectin (BSA-AVM). Aluminum hydroxide and Bordetella pertussis were added as adjuvants for the first immunization. The spleen was removed 3 days after the final immunization, and 1 108 splenocytes were fused with 1 107 P3-U1 cells in the presence of polyethylene glycol 1000 (Junsei, Tokyo, Japan). The screening of cultured hybridoma cells was performed using binding enzyme-linked immunosorbent assay (ELISA). From ELISA-positive hybridoma, cDNA was prepared, sequenced, and identified as anti-AVM antibody.

For recombinant MOG01 and MOG14 production, DNA encoding the VH and VL of MOG01 or MOG14 and constant region of lambda light chain were cloned into N5KG4PE in which the human heavy chain IgG1 constant region was replaced with IgG4 with S228P and S235E mutations in N5KG1 [24]. MOG01 scFv and the hinge-CH2-CH3 region of human IgG4PE were amplified with PCR and cloned into N5KG4PE to create MOG01 scFv-Fc. As a negative control, VH and HV of rat anti-avermectin antibody were cloned into N5KG4PE. VH and VL of the anti-rat transferrin receptor antibody OX-26 [25] were cloned into N5KG4PE.

For symmetric bispecific antibody expression vector construction, the constant region of human IgG and MOG01scFv were amplified and assembled with PCR to construct CH1-Hinge-CH2-CH3-MOG01scFv, and then cloned into a pCI vector (Promega). Light chain and VH of the anti-AVM antibody were amplified with PCR and cloned into the MOG01 scFv expression vector. As a negative control, the constant regions of human IgG and anti-AVM scFv were amplified and assembled to construct CH1-Hinge-CH2-CH3-AVMscFv, with an inserted NheI-BamHI site in the anti-AVM antibody, and cloned into the MOG01 scFv expression vector.

For membrane-bound native MOG expression, full length DNA of human, rat, mouse, and cynomolgus monkey (Macaca fascicularis) MOG were cloned into pEF6/V5-His (Thermo Fisher Scientific). To construct the Fc-fusion protein, the extracellular domain of rat, mouse, and human MOG and FLAG tag-fused human Fc were cloned into INPEP4 (IDEC). To construct the GST-fusion protein, the extracellular domain of rat, mouse, and human MOG and GST were cloned into N5K (IDEC).

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