1. Volume 89, Issue 1, Pages 70-82 (January 2016)
Discovery of Novel Blood-Brain Barrier Targets to Enhance Brain Uptake of Therapeutic Antibodies 
Y. Joy Yu Zuchero, Xiaocheng Chen, Nga Bien-Ly, Daniela Bumbaca, Raymond K. Tong, Xiaoying Gao, Shuo Zhang, Kwame Hoyte, Wilman Luk, Melanie A. Huntley, Lilian Phu, Christine Tan, Dara Kallop, Robby M. Weimer, Yanmei Lu, Donald S. Kirkpatrick, James A. Ernst, Ben Chih, Mark S. Dennis, Ryan J. Watts 
Neuron 
Volume 89, Issue 1, Pages (January 2016)
DOI: /j.neuron
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2. Figure 1 Lack of Brain Uptake of Antibodies Targeting Lrp1 and InsR
(A) Screening cascade used to determine success of potential receptor-mediated transport (RMT) targets.
(B) Phage-derived anti-Lrp1 and anti-InsR antibodies bind to their corresponding murine receptors transfected in HEK293 cells by flow cytometry. See also Figure S1 for FACS profiles showing a lack of nonspecific binding in untransfected HEK293 cells.
(C) Immunohistochemical staining with fluorescent anti-human secondary IgG of antibody localization in mouse cortical tissue 1 hr after a 5 mg/kg intravenous injection of the indicated antibody. Scale bar, 50 μm.
(D) Brain uptake of trace doses of I125-labeled antibodies at various time points postdose after intravenous administration in wild-type mice, quantified as mean ± SEM percent injected dose per gram of brain tissue (n = 3 per group and time point).
(E) Antibody concentrations in brain 1 and 24 hr after a 20 mg/kg dose of the indicated antibody. Bar graphs represent mean ± SEM (n = 6 per group and time point; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ compared to control IgG at the same time point).
Neuron  , 70-82DOI: ( /j.neuron )
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3. Figure 2
mRNA Enrichment at the BBB Is Not Sufficient to Identify an RMT Target
(A) Microarray expression profiling of FACS-purified brain and liver/lung endothelial cells from wild-type mice identifies genes enriched at the BBB (Tam et al., 2012).
(B) FACS analysis of anti-Ldlrad3 and anti-CD320 antibodies shows binding to their corresponding murine antigens expressed in HEK293 cells.
(C) Immunohistochemical staining of antibody localization in mouse cortical tissue 1 hr after a 5 mg/kg intravenous injection of the indicated antibody. Scale bar, 50 μm.
(D) Brain uptake of trace doses of I125-labeled antibodies at various time points postdose after intravenous administration in wild-type mice, quantified as mean ± SEM percent injected dose per gram of brain tissue (n = 3 per group and time point).
(E and F) Antibody concentrations in brain 1 and 24 hr after a 20 mg/kg dose of the indicated antibody. Data in (E) were obtained concurrently with data in Figure 1E as part of a single experiment; thus the same control IgG and anti-TfRA are shown. Bar graphs represent mean ± SEM (n = 6 per group and time point; ∗p ≤ 0.05, ∗∗∗∗p ≤ compared to control IgG at the same time point).
Neuron  , 70-82DOI: ( /j.neuron )
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4. Figure 3
Proteomic Identification of Highly Expressed Transmembrane Proteins at the BBB
(A) Primary wild-type mouse brain endothelial cells (CD31+/CD45−) were isolated by FACS as previously described (Tam et al., 2012) and analyzed by mass spectrometry (see Experimental Procedures for details).
(B) AUC represents the average of two technical replicates for the integrated intensity of the top three most abundant peptide counts as determined by mass spectrometry for endothelial cell proteins (Glut1, Pg-p, ZO-1, Esam, Cldn5) and other brain cell-specific proteins (Fasn, Aldoc, Glul, Plp1).
(C) AUC integrated intensity for the top three most abundant peptide counts as determined by mass spectrometry for previously evaluated (i.e., TfR, Lrp1, InsR, Ldlrad3, CD320) and newly identified (i.e., Glut1, basigin, CD98hc) RMT candidates.
(D) Summary table of potential RMT targets identified by microarray, RNA-seq (Zhang et al., 2014), and mass spectrometry.
Neuron  , 70-82DOI: ( /j.neuron )
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5. Figure 4
Evaluation of Antibodies against High-Expressing BBB Proteins for RMT
(A) FACS analysis of RMT candidate antibodies binding to HEK293 cells transfected with murine antigen.
(B) Immunohistochemical staining of antibody localization in mouse cortical tissue 1 hr after a 5 mg/kg intravenous injection of the indicated antibody. Scale bar, 50 μm.
(C–E) Brain uptake of trace doses of I125-labeled antibodies at various time points post-dose after intravenous administration in wild-type mice, quantified as mean ± SEM percent injected dose per gram of brain tissue (n = 3 per group and time point).
(F–H) Antibody levels in brain 1 and 24 hr after a 20 mg/kg dose of the indicated antibody. Data in (F) and (G) were obtained concurrently as part of a single experiment with Figure 2F; thus the same control IgG and anti-TfRA are shown. Bar graphs represent mean ± SEM (n = 6 per group and time point; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001,∗∗∗∗p ≤ compared to control IgG at the same time point). See also related Figure S2.
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6. Figure 5 CD98hc as a Novel BBB Transport Target
(A) Competitive ELISA comparison of bivalent anti-CD98hc (solid) versus bispecific anti-CD98hc/BACE1 antibodies (dashed) binding to murine CD98hc (IC50: anti-CD98hcA- 1.5 nM, anti-CD98hcA/BACE1- 4.0 nM anti-CD98hcB- 4.6 nM, anti-CD98hcB/BACE  nM)
(B) Brain uptake of trace doses of I125-labeled antibodies at various time points postdose after intravenous administration in wild-type mice, quantified as mean ± SEM percent injected dose per gram of brain tissue (n = 3 per group and time point).
(C–F) Antibody concentrations in plasma (C) and brain (D), and Aβx-40 concentrations in brain (E) and percent Aβx-40 reduction compared to control IgG (F) at each time point after a single 50 mg/kg intravenous administration of antibody. See also related Figure S3.
Neuron  , 70-82DOI: ( /j.neuron )
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7. Figure 6
CD98hc Expression, Stability, and Function Are Unperturbed by Antibody Treatment
(A) Wild-type IMCD3 cells were treated with the indicated antibodies and concentrations for 24 hr. Lysates were probed for endogenous CD98hc and actin as the loading control.
(B) Quantification of the western blots averaged from three independent experiments each performed in triplicate. Bars represent mean ± SEM (n = 3).
(C) IMCD3 cells stably overexpressing mouse CD98hc were treated with 1 μM of the indicated antibodies for 1 hr at 37°C, fixed, and stained for human IgG, mouse CD98hc, and lysosomal marker, Lamp1. Representative images of cellular uptake of Control IgG, anti-CD98hcA/BACE1, and anti-CD98hcB/BACE1 costained with Lamp1.
(D) CD98hc puncta were analyzed and quantified for colocalization with Lamp1. Bars represent mean ± SEM (n = 5). Scale bar, 5 μm
(E–H) Western blot analysis of CD98hc expression in brain lysates after a single 50 mg/kg dose of the indicated antibodies at various days postdose (n = 5 per group and time point).
(I) Quantification of CD98hc levels in the western blots. All graphs represent mean ± SEM (n = 5 per group and time point).
(J) IMCD3 cells stably overexpressing mouse CD98hc cells were treated with 1 μM of indicated antibodies for 24 hr, and amino acid uptake activity was assessed by the amount total internalized HPG, a methionine analog. BCH (2-amino-2-norbornane-carboxylic acid), an inhibitor of a system L amino acid transporter, was used as a positive control. Methionine uptake was expressed as a percentage of Control IgG and plotted against each data point. Bars represent mean ± SEM (n = 12). See also related Figure S4.
Neuron  , 70-82DOI: ( /j.neuron )
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