Application of in Vitro Transcytosis Models to Brain Targeted Biologics
Kangwen Deng, Yifeng Lu, Sjoerd J. Finnema, Kostika Vangjeli, Junwei Huang, Lili Huang, Andrew Goodearl
Abstract
The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation.
Introduction
Due to the global increase in age-related central nervous system (CNS) diseases, there is substantial demand for new CNS medicines. Biologics has become the new trend for CNS drug discovery. Despite the large number of candidates in the pipeline and clinical trials, the success rate of CNS biologics drug is extremely low [1]. Low blood–brain barrier (BBB) permeability has been one of the major causes of failure for new CNS drug candidates. The BBB is formed by specialized brain microvascular endothelial cells (BMECs) and other supporting cells of the neurovascular unit including pericytes, astrocytes, and neurons [2]. The low permeability is caused by decreased transcytotic activity and by tight junctions and adherents’ junctions that limit paracellular passage [3]. BBB plays a key role as a critical protecting barrier for the CNS against toxic and infectious agents while maintaining the ionic and volumetric environments. The barrier properties also create an obstacle for effective systemic drug delivery to the CNS from blood in which 0.01–0.4% percent of all large molecule drugs gets access [4]. Therefore, there has been a great interest in cell models which mimic BBB permeation properties for the purposes of drug screening and engineering [5, 6].
Materials and methods
Antibodies and bi-specific molecules
Sources of commercial anti-human TfR antibodies: RVS10 (Abcam ab25651), 29806 (R&D system MAB2474), OKT-9 (eBioscience 16-0719-81), MEM-189 (Novus Biologicals NB500-493), MEM-75 (Novus Biologicals NB100-77895), 3B8.2A1 (Thermo Scientific Pierce MA1-40198), ICO-92 (Thermo Scientific Pierce MA1-7657), CY1G4 (BioLegend 334102), L01.1 (BD Bioscience 347510), BGX.24 (Thermo Scientific Pierce MA1-12156), DF1513 (AbD Serotec MCA1148), Ber-T9 (Santa Cruz Biotech sc-19675). Anti-human TfR.B1 is an antibody that specifically recognizes human and monkey TfR. Anti-mouse TfR antibodies AB221, AB403, AB404, AB405 and DVD-Igs AB405-LS-RGMa and AB405-SL-RGMa are the same molecules as described before [9]. Anti-hTfR.B1 and anti-mTfR constructs were cloned into and expressed in HEK293 cells and purified according to established methods [32]. Production quantity was determined by absorbance measured with Nanodrop. Percentage of monomer was determined by size exclusion chromatography (SEC).
Results
hCMEC/D3 model fails to discriminate transcytosis of anti-TfR antibodies from control antibody
The hCMEC/D3 model [21] is widely used for transport assay on variety of BBB targeted drug, such as small molecules with different properties [34]. Its utility on BBB targeted antibody screening remains limited [35]. This is likely due to the low TEER value [36]. As a first step towards establishing our in vitro BBB models, we evaluated the feasibility of using hCMEC/D3 in in vitro transcytosis assay. Aligned with other publications, TEER value of hCMEC/D3 model remained low regardless of the presence of astrocytes plated to the opposite side of transwell (Fig 1A) and the addition of a cocktail with supplements including retinoic acid, RO-20-1724, apo-transferrin and putrescine, [37] that might help boosting TEER. In this article, all in vitro transcytosis assays, except the one using commercial kits, were performed in a mono-culture setup (Fig 1B). Human TfR expression in hCMEC/D3 cells was confirmed in FACS analysis (Fig 1C). Using mono-culture setup, transcytosis of two anti-human TfR antibodies, Ber-T9 and DF1513, and mouse IgG control were performed 7-day after seeding hCMEC/D3 cells to transwells. TEER value of hCMEC/D3 monolayer reached 25 ± 2 ohm.cm2 at the time of antibody treatment.
Discussion
In this study, we evaluated multiple in vitro transcytosis models for their potential application for CNS targeted biologics drug screening. By comparing transcytosis of anti-hTfR antibody Ber-T9, DF1513 and control IgG in hCMEC/D3 and Caco-2, we found out the importance of high TEER value in transcytosis assays. This phenomenon is also observed in the murine models by comparing the transcytosis of anti-mTfR molecules in bEnd3 and mIEC models. Models with TEER value below 100 ohm.cm2, such as hCMEC/D3 and bEnd3, show high level of paracellular transport of antibody which masks the transcytosis of anti-TfR through the RMT route. The monkey model with TEER value between 200 to 500 ohm.cm2 showed good differentiation between TfR-targeted antibody and control IgG indicating a model threshold of a few hundred ohm.cm2 tightness might be sufficient to block antibody diffusion. Consistent with this notion, it has been reported that a rat RBE model with 100–300 ohm.cm2 TEER value can differentiate the transcytosis of anti-IGF1R molecules [27].
Conclusions
Transcytosis of anti-TfR targeted antibodies were evaluated in multiple in vitro transcytosis models. High TEER value is a key parameter to differentiate RMT-mediated transcytosis from non-specific paracellular transport of antibodies. Our data showed good differentiation of transcytosis of a panel of anti-hTfR antibodies in Caco-2 and we confirmed good transcytosis of anti-hTfR.B1 in MBT model and monkey in vivo PET study. The validation using the mouse model strengthened our confidence on the predictive value of the human in vitro BBB model despite there is no human in vivo brain uptake data and limited NHP brain uptake data for biologics. Both mouse and human in vitro transcytosis models will serve as important screening assays for BBB targeted antibody / bispecific selection.
Acknowledgments
The authors are thankful to AbbVie employees John E. Harlan, Ji-Quan Wang, Kyle C. Wilcox, David Reuter, Martin J. Voorbach and Dustin Wooten for their expert contributions to the PET study and thank former AbbVie employee Farid Gizatullin for his help with MSD-ELISA assay. We are also thankful to AbbVie employee Axel Meyer and former AbbVie employee Denise Karaoglu Hanzatian for their support on hCMEC/D3 model.
Citation: Deng K, Lu Y, Finnema SJ, Vangjeli K, Huang J, Huang L, et al. (2023) Application of In vitro transcytosis models to brain targeted biologics. PLoS ONE 18(8): e0289970. https://doi.org/10.1371/journal.pone.0289970
Editor: Mária A. Deli, Eötvös Loránd Research Network Biological Research Centre, HUNGARY
Received: April 19, 2023; Accepted: July 31, 2023; Published: August 23, 2023
Copyright: © 2023 Deng 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.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: All authors (K.D., Y.L., S.J.F., K.V., J.H., L.H., and A.G.) are employees of AbbVie. This study was fully funded by AbbVie. AbbVie participated in design and conduct of experiments, interpretation of data, and review and approval of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
List of abbreviations: BBB, Blood-brain barriers; bEnd3, murine brain endothelial cell; Caco-2, Human Caucasian Colon Adenocarcinoma Epithelial Cells; CNS, central nervous system; DVD-Ig, Dual-variable-domain Immunoglobulin; hCMEC/D3, immortalized human capillary endothelial cell; LLOQ, lower limit of quantitation; MBT, monkey (Macaca irus) brain capillary endothelial cells, brain pericytes and astrocytes. BBB Kit; mIEC, murine intestinal epithelial cells (Cl-muADINTESTI); MSD, Electrochemiluminescence-Meso Scale Discovery; NHP, nonhuman primate; p-SCN-Bn-Deferoxamine, 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18.21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazheptaeicosine]thiourea; PET, Positron emission tomography; RGMa, Repulsive guidance molecule a; RMT, receptor-mediated transcytosis; SUV, standardized uptake value; TfR, transferrin receptor 1
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0289970#abstract0
