Research ArticleAlzheimer’s Disease

Boosting Brain Uptake of a Therapeutic Antibody by Reducing Its Affinity for a Transcytosis Target

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Science Translational Medicine  25 May 2011:
Vol. 3, Issue 84, pp. 84ra44
DOI: 10.1126/scitranslmed.3002230


Monoclonal antibodies have therapeutic potential for treating diseases of the central nervous system, but their accumulation in the brain is limited by the blood-brain barrier (BBB). Here, we show that reducing the affinity of an antibody for the transferrin receptor (TfR) enhances receptor-mediated transcytosis of the anti-TfR antibody across the BBB into the mouse brain where it reaches therapeutically relevant concentrations. Anti-TfR antibodies that bind with high affinity to TfR remain associated with the BBB, whereas lower-affinity anti-TfR antibody variants are released from the BBB into the brain and show a broad distribution 24 hours after dosing. We designed a bispecific antibody that binds with low affinity to TfR and with high affinity to the enzyme β-secretase (BACE1), which processes amyloid precursor protein into amyloid-β (Aβ) peptides including those associated with Alzheimer’s disease. Compared to monospecific anti-BACE1 antibody, the bispecific antibody accumulated in the mouse brain and led to a greater reduction in brain Aβ after a single systemic dose. TfR-facilitated transcytosis of this bispecific antibody across the BBB may enhance its potency as an anti-BACE1 therapy for treating Alzheimer’s disease.


A promising approach to facilitate delivery of protein therapeutics across the blood-brain barrier (BBB) is to take advantage of receptor-mediated transcytosis, an endogenous endocytic process in which a ligand is transported across an endothelial cell barrier (14). Although antibodies directed against brain endothelial receptors have been reported, the extent of antibody uptake into the brain under therapeutic dosing conditions remains largely unexplored. Antibodies targeting the transferrin receptor (TfR), which is highly expressed by endothelial cells that make up the BBB, have been reported to cross the BBB (3, 57), but whether endocytosis of anti-TfR antibodies into brain vasculature can lead to antibody release and accumulation in brain parenchyma remains controversial (5, 810). These previous studies have typically used radiolabeled high-affinity antibodies for TfR administered in trace amounts. Although high affinity ensures binding to TfR and uptake into brain endothelium despite low concentrations in blood, it also likely reduces the probability of antibody being released from the central nervous system (CNS) vasculature, and thus potentially prevents accumulation of antibody in the brain parenchyma.

How can uptake of therapeutic antibodies by receptor-mediated transcytosis be improved? We propose that one possible solution is to use low-affinity antibodies to TfR to increase release of antibody from brain vascular endothelium and enhance uptake and distribution in the brain. In a therapeutic setting, high peripheral concentrations of the antibody would compensate for the low affinity and ensure binding to TfR on the vascular luminal surface, thus ensuring effective uptake from the periphery. Here, we provide evidence in support of this idea and demonstrate the feasibility of the approach using a bispecific therapeutic antibody with a low affinity for TfR and a high affinity for the enzyme β-secretase (BACE1), an Alzheimer’s disease drug target.


A high-affinity anti-TfR antibody that binds to brain vasculature has modest uptake

We first explored the potential of a high-affinity anti-TfR antibody to be taken up into mouse brain in both trace amounts and at therapeutic doses. We generated a human chimeric anti-murine TfR (muTfR) antibody (anti-TfRA) that does not compete with endogenous binding of transferrin to TfR and then compared its uptake in the mouse brain with a human control immunoglobulin G (IgG) through a double-labeled experiment. A single trace dose of [131I]anti-TfRA and [125I]control IgG was injected into wild-type mice intravenously, and brain uptake was measured at various time points. A significant increase in [131I]anti-TfRA uptake in the brain, measured as a percentage of injected dose per gram of brain, was observed at all time points (Fig. 1A, P = 0.0056). At its peak 1 hour after injection, there was a >11-fold difference in [131I]anti-TfRA brain accumulation compared to [125I]control IgG (n = 6; P < 0.0001). When unlabeled anti-TfRA (4 mg/kg of body weight) was coadministered, brain accumulation of [131I]anti-TfRA was reduced almost to the level of control IgG, indicating specific target-driven uptake. We next used more therapeutic relevant doses of either anti-TfRA or control IgG [20 mg/kg, intravenously (iv)], and determined human antibody concentrations in mouse cerebral cortex and serum at 1 and 24 hours. In these experiments, animals were thoroughly perfused with phosphate-buffered saline (PBS) to flush out the vasculature of any remaining antibody before harvesting brains to measure drug concentrations. Compared to control IgG, the concentration of anti-TfRA was significantly higher in the brain 24 hours after antibody administration (Fig. 1B, P = 0.0002, n = 10). Furthermore, the brain-to-serum ratio for anti-TfRA at 24 hours demonstrates improved brain uptake compared to control IgG (Fig. 1C, P = 0.003, n = 10).

Fig. 1

Systemically administered anti-TfRA antibody localizes to the brain vasculature. (A) Brain uptake after intravenous administration of trace doses of [131I]anti-TfRA and [125I]control IgG in mice was quantified as a mean percentage of injected dose per gram of brain tissue at 5 and 30 min, and then at 1, 4, 24, 48, and 72 hours after intravenous injection (n = 6 per group and time point). The enhanced uptake of [131I]anti-TfRA was reduced by co-injection with unlabeled anti-TfRA (4 mg/kg). (B) Mean antibody uptake in the brain 1 and 24 hours after an intravenous injection of anti-TfRA (20 mg/kg) showed increased uptake of anti-TfRA compared to control IgG (***P = 0.0002, n = 10 per group and time point). (C) Ratio of mean percent brain to serum concentrations of antibody (**P = 0.003, n = 10 per group and time point). (D and E) Immunohistochemical staining of brain sections after intravenous injection with anti-TfRA reveals colocalization of antibody (green) with collagen IV (red). Intravenous injection with control IgG exhibited vascular distribution in the brain at 1 hour but an absence of antibody staining after 24 hours. Images were taken from the mouse cortex for these and all subsequent images. Scale bar, 50 μm. Magnification, ×40.

These results show that systemically administered high-affinity anti-TfRA can accumulate in the brain when administered at both low and high doses, but does not address whether the antibody is released into the parenchyma. To determine antibody distribution after systemic delivery of anti-TfRA or control IgG (20 mg/kg), we sectioned and stained PBS-perfused brains with fluorescent anti-human secondary IgG. After 1 hour, anti-TfRA had a pronounced vascular distribution, as indicated by its colocalization with the basement membrane marker anti–collagen IV (Fig. 1D, left column). Although less pronounced, control IgG also localized to the vasculature (Fig. 1E, left column). At 24 hours, anti-TfRA exhibited modest parenchymal staining but remained largely vascular (Fig. 1D, right column), whereas control IgG was absent in the brain tissue (Fig. 1E, right column). Thus, our results confirm and extend previous studies using trace amounts of high-affinity antibody (9, 10) by showing that high-affinity anti-TfRA was predominantly confined to endothelial cells of the BBB and largely excluded from brain parenchyma when used in either trace amounts or at therapeutic doses.

Lower-affinity anti-TfR antibodies show increased brain uptake

We next explored whether reducing the affinity of the anti-TfR antibody for TfR would enable greater accumulation of the antibody in the brain parenchyma. Alanine mutations were introduced into the complementarity-determining regions of anti-TfRA to generate variants (anti-TfRB, anti-TfRC, and anti-TfRD) with a range of binding affinities for TfR [Fig. 2A; IC50 (median inhibitory concentration) values are 1.7 ± 0.1, 6.9 ± 0.4, 65 ± 12, and 111 ± 16 nM for anti-TfRA, anti-TfRB, anti-TfRC, and anti-TfRD, respectively]. These were evaluated in both a nonsaturating (trace dosing) and a saturating (therapeutic dosing) TfR paradigm. Trace amounts of 125I-labeled versions of the four antibodies were administered to mice intravenously, and brain uptake was measured. Anti-TfR antibodies delivered at trace doses showed a direct correlation between affinity and brain uptake, with lower-affinity antibodies showing the lowest uptake (Fig. 2B). In marked contrast to trace dosing, however, brain uptake of these same variants at therapeutic doses (20 mg/kg assessed at 1 and 24 hours) showed an inverse correlation between affinity and brain uptake at 24 hours (Fig. 2C; n = 10, P < 0.0001) and an increase in the brain-to-serum ratio of antibody (Fig. 2D; P = 0.0032). Furthermore, the lowest-affinity variant anti-TfRD showed a greater than fivefold increase in brain antibody concentration compared to control IgG 24 hours after injection (fig. S1).

Fig. 2

Lower-affinity anti-TfR antibodies at therapeutic doses show increased brain uptake. (A) Anti-TfR variant antibodies show distinct affinities for TfR by a competitive ELISA assay. IC50 values are 1.7 ± 0.1, 6.9 ± 0.4, 65 ± 12, and 111 ± 16 nM for anti-TfRA, anti-TfRB, anti-TfRC, and anti-TfRD, respectively. (B) Mean brain uptake after intravenous injection of trace doses of [125I]anti-TfR variants after 5 min and 1, 4, and 24 hours (n = 3 per group and time point) shows reduced brain uptake of lower-affinity anti-TfR variants. (C) Mean brain uptake after an intravenous injection of anti-TfR variants (20 mg/kg) at 1 and 24 hours (n = 10 per group and time point) shows increased brain uptake (P < 0.0001 at 24 hours). (D) Ratio of mean percent brain to serum concentrations of antibody (n = 10) at 1 hour (control IgG = 0.17, anti-TfRA = 0.30, anti-TfRB = 0.81, anti-TfRC = 0.55, anti-TfRD = 0.68) and at 24 hours (control IgG = 0.36, anti-TfRA = 0.91, anti-TfRB = 2.11, anti-TfRC = 1.43, anti-TfRD = 2.04).

On the basis of these results and our initial hypothesis, we propose the following model (Fig. 3). Compared to a high-affinity antibody that binds tightly to brain endothelial receptors, a low-affinity antibody is less likely to bind and remain bound to luminal TfR receptors in the vasculature when serum concentrations of antibody are low (that is, trace dosing). Thus, under nonsaturating concentrations, receptor-mediated transcytosis of the low-affinity antibody is reduced and brain uptake is low (Fig. 3, left panels). At a higher dose (that is, therapeutic dosing), luminal TfR would be saturated regardless of antibody affinity, resulting in similar endothelial uptake for both high- and low-affinity antibodies (Fig. 3, right panels); however, a low-affinity antibody could lead to greater total brain accumulation through an increased dissociation from TfR and release into the brain. Moreover, a lower-affinity antibody decreases the probability of receptor-mediated efflux out of the parenchyma because concentrations in the brain are likely to be no longer saturating.

Fig. 3

Model illustrating the inverse relationship between the antibody’s affinity for TfR and its uptake in brain. (Top, left panel) With trace dosing, a higher-affinity anti-TfR antibody will bind more receptors expressed on the luminal (blood) side of the endothelial cells that make up the BBB compared with trace dosing of lower-affinity antibodies (bottom, left panel), resulting in more high-affinity antibodies associated with brain endothelium. At therapeutic doses, saturating concentrations of antibody will result in antibody binding to receptors on the luminal side of the BBB epithelium regardless of antibody affinity (top and bottom right panels). The dissociation of antibody after receptor-mediated transcytosis will be more likely with a lower-affinity antibody, resulting in accumulation of lower-affinity antibody in the brain parenchyma (bottom right panel). Furthermore, the probability of efflux out of the brain parenchyma will be lower for a low-affinity TfR antibody because concentrations in the brain are likely to be no longer saturating (similar to left panels, but in the brain to blood direction).

Broad distribution of low-affinity anti-TfR antibody in mouse brain parenchyma

Although increased brain accumulation under therapeutic dosing conditions fit with our model (Fig. 3), we also wanted to examine parenchymal distribution of antibody at therapeutic doses. To address this, we compared antibody distribution in brain tissue for each affinity variant (Fig. 4, A to D). Mice were dosed at 20 mg/kg and PBS-perfused after 24 hours. Brain sections were stained with fluorescent anti-human secondary IgG to visualize antibody distribution. As described above, the high-affinity anti-TfRA antibody localized predominantly to the vasculature (Fig. 4A). In contrast, lower-affinity variants localized with the neuronal marker NeuN, suggesting their broad distribution in the brain parenchyma surrounding neurons (Fig. 4, B to D). In particular, anti-TfRD exhibited noticeable and pronounced parenchymal staining (Fig. 4D). A representative high-magnification image further illustrates neuronal localization of the anti-TfRD variant (Fig. 4E). This parenchymal distribution was especially pronounced when analyzing three-dimensional (3D) reconstructed confocal images that were converted into rotating movies comparing control IgG, anti-TfRA, and anti-TfRD (movies S1, S2, and S3, respectively). Together, these results indicate that lowering the antibody affinity for TfR can markedly enhance accumulation of antibody in brain parenchyma.

Fig. 4

Brain distribution of low-affinity anti-TfR antibody. (A to D) Comparison of immunohistochemical staining of brain sections 24 hours after intravenous injection of mice with one of four anti-TfR variants (anti-TfRA, anti-TfRB, anti-TfRC, or anti-TfRD) (20 mg/kg). Staining reveals differences in antibody distribution depending on whether the antibodies have high or low affinity for TfR. (B to D) Lower-affinity antibodies (green) show localization around NeuN-positive neuronal cell bodies (red) in the mouse brain, whereas the high-affinity anti-TfRA antibody (A) remains largely associated with the vasculature. Scale bar, 50 μm. (E) Representative high-magnification image showing localization of low-affinity anti-TfRD antibody (green) around neurons (NeuN, red). Scale bar, 20 μm.

Generation and characterization of an anti-TfR/BACE1 bispecific antibody

To further confirm transport of anti-TfR antibodies across the BBB and to test whether antibody can accumulate in physiologically relevant amounts, we generated a bispecific antibody (anti-TfR/BACE1) that binds to both TfR and BACE1 (Fig. 5A). BACE1 is expressed in the CNS and is considered to be the primary contributor of amyloid-β (Aβ) peptide formation through its ability to cleave amyloid precursor protein (APP) in the brain (11, 12). An antibody to BACE1 effectively inhibits BACE1 activity in vivo, resulting in reduced Aβ1–40 production in the periphery as well as in the brain, albeit at very high peripheral doses [see Atwal et al. (13), this issue]. When choosing an anti-TfR antibody from which to generate the anti-TfR arm of the bispecific anti-TfR/BACE1, we noted that there was considerable loss of affinity for TfR compared to the starting monospecific TfR antibodies because of loss of avidity and bivalent binding. Thus, when we generated the bispecific antibody using the higher-affinity anti-TfRA, the resulting bispecific antibody had an affinity for TfR that was comparable to native anti-TfRC and anti-TfRD (Fig. 5B; anti-TfRA: IC50 = 1.7 ± 0.1 nM; anti-TfR/BACE1: IC50 = 45 ± 3 nM). The anti-TfR/BACE1 bispecific antibody inhibited Aβ1–40 production in a stably transfected human embryonic kidney (HEK) 293 cell line expressing human APP with slightly reduced potency compared to monospecific anti-BACE1, indicating that bivalent binding is not required to inhibit BACE1 activity in cells (Fig. 5C).

Fig. 5

An anti-TfR/BACE1 bispecific antibody blocks Aβ production in vitro and accumulates in the brain in vivo. (A) Model of the bispecific antibody, which was engineered to bind to both TfR and the enzyme β-secretase (BACE1). (B) Affinities of the parental anti-TfRA (IC50 = 1.7 ± 0.1 nM) and anti-TfR/BACE1 (IC50 = 45 ± 3 nM) as measured by a competitive ELISA assay showed a significantly reduced affinity of the bispecific antibody for TfR. (C) Quantification of human Aβ1–40 in HEK293-hAPPWT cells after titration with anti-TfR/BACE1, anti-BACE1, and control IgG in a cell-based assay reveals slightly reduced potency of anti-TfR/BACE1 compared with anti-BACE1. (D) Mean brain uptake after trace doses of 125I-labeled antibody for 30 min and 6, 24, and 48 hours after intravenous injection in mice (n = 4 per group and time point) shows reduced uptake of anti-TfR/BACE1 compared with anti-TfR. (E and F) Mean antibody uptake (at 48 hours: anti-BACE1 = 0.12 ± 0.03%; anti-TfR/BACE1 = 0.57 ± 0.04%) (E) and average brain-to-serum ratio at 1, 12, 24, and 48 hours after an intravenous injection of an antibody (20 mg/kg) in mice (n = 10 per group and time point) (F), showing a robust uptake of anti-TfR/BACE1 with therapeutic dosing (at 48 hours: anti-BACE1 = 0.28 ± 0.08%; anti-TfR/BACE1 = 7.40 ± 0.58%). (G) Immunohistochemical staining of brain sections from mice 24 hours after an intravenous injection with either anti-TfR/BACE1 or anti-BACE1 (20 mg/kg). Images show broad staining in the brain parenchyma for the anti-TfR/BACE1 bispecific antibody compared with minimal staining for the monospecific anti-BACE1 antibody (see also fig. S2B).

Bispecific anti-TfR/BACE1 uptake and distribution in brain

When administered at trace doses, higher brain accumulation was observed with the [125I]anti-TfR/BACE1 bispecific antibody compared to [125I]anti-BACE1 antibody up to 48 hours after injection (Fig. 5D, n = 4, P = 0.05). Consistent with our model, brain uptake of [125I]anti-TfRA was much greater than that of the lower TfR affinity [125I]anti-TfR/BACE1 in these trace dosing experiments. At therapeutic doses, however, the anti-TfR/BACE1 bispecific antibody exhibited much higher brain uptake compared to either anti-BACE1 or anti-TfRA (Fig. 5E and fig. S2, also compare to Fig. 1B), again consistent with our model. Peak brain accumulation at 24 hours achieved concentrations of ~20 nM and remained elevated at 48 hours after injection (fig. S2A). Enhanced uptake of the bispecific antibody is especially apparent when comparing the average percent of antibody in the brain versus in the serum (Fig. 5F). Similar to low-affinity anti-TfR antibody localization, anti-TfR/BACE1 showed broad distribution throughout the brain parenchyma in addition to vascular staining (Fig. 5G and fig. S2B). The localization of anti-TfR/BACE1 to brain parenchyma was especially evident when analyzing 3D reconstructed confocal images [movie S4, also compare control IgG (movie S1) and anti-TfRA (movie S2)]. In contrast, mice injected with anti-BACE1 antibody showed very little brain vascular or parenchymal staining at 24 hours, similar to control IgG (compare Fig. 5G and fig. S2B with Fig. 1E).

Anti-TfR/BACE1 bispecific antibody reduces brain Aβ

On the basis of the impressive brain uptake and localization of the anti-TfR/BACE1 bispecific antibody after a single injection, we next examined whether BACE1 inhibition in vivo could be boosted using the bispecific antibody compared to the monospecific anti-BACE1 antibody, as assessed by a reduction in brain Aβ. Indeed, at 25 mg/kg, anti-TfR/BACE1 was able to significantly reduce brain Aβ1–40 concentrations compared to control IgG 24 hours after injection (control IgG, 1598.7 pg/g; anti-TfR/BACE1, 1031.5 pg/g; P = 0.001, n = 10) and 48 hours after injection (control IgG, 1791.3 pg/g; anti-TfR/BACE1, 1453.2 pg/g; P = 0.0003, n = 10). In contrast, the anti-BACE1 monospecific antibody had no effect on brain Aβ1–40 over this time period (Fig. 6A). At 50 mg/kg, anti-TfR/BACE1 had an even more marked effect in reducing brain Aβ1–40 at both time points compared to control IgG (Fig. 6B; at 24 hours: control IgG, 1903.0 pg/g; anti-TfR/BACE1, 1005.7 pg/g; at 48 hours: control IgG, 1733.7 pg/g; anti-TfR/BACE1, 867.5 pg/g; P < 0.0001, n = 10). Administration of anti-BACE1 at this dose also significantly reduced brain Aβ1–40 concentrations compared to control (Fig. 6B; 1480.9 pg/g at 24 hours and 1433.1 pg/g at 48 hours; P < 0.0001, n = 10 at 24 hours; P = 0.006, n = 10 at 48 hours), although to a lesser extent than did the bispecific anti-TfR/BACE1. The maximal effect of anti-TfR/BACE1 was achieved 48 hours after injection at 50 mg/kg, with a 50.0 ± 1.9% reduction in brain Aβ1–40 compared to control IgG (Fig. 6E). These reductions after a single dose are impressive when considering that mice lacking BACE1 show a maximal Aβ1–40 reduction of ~80% (13). A significant decrease in peripheral Aβ1–40 was also observed at both doses and time points for anti-TfR/BACE1, and at the 24-hour time point for anti-BACE1 (Fig. 6, C and D).

Fig. 6

Systemic dosing with anti-TfR/BACE1 reduces brain Aβ. (A to D) Quantification of Aβ1–40 concentrations in mouse brain (A and B) and plasma (C and D) after an intravenous injection of control IgG, anti-BACE1, or anti-TfR/BACE1 (25 or 50 mg/kg). Asterisks (*) represent significance compared to control IgG, whereas pound signs (#) represent significance compared to anti-BACE1. */#P < 0.05, **/##P < 0.01, ***/###P < 0.001; n = 10 per group and time point. (E) Mean Aβ1–40 reduction from data in (A) to (D) calculated as a percentage of Aβ1–40 concentrations relative to those in mice injected with control IgG. Anti-TfR/BACE1 reduces brain Aβ concentrations at both doses and time points when compared to either control IgG or anti-BACE1 antibody.

We next determined how brain concentrations of anti-TfR/BACE1 and anti-BACE1 relate to Aβ reduction. As expected, the reductions in Aβ1–40 concentrations in the brain correlated with much higher brain concentrations of anti-TfR/BACE1 compared to anti-BACE1 and control IgG (fig. S3). Neither anti-TfRA (the original arm used to generate the anti-TfR/BACE1 bispecific antibody) nor anti-TfRC (with a binding affinity for TfR similar to that of anti-TfR/BACE1) reduced Aβ1–40 appreciably (fig. S4), indicating that the anti-TfR arm of the anti-TfR/BACE1 bispecific antibody did not contribute to the marked reduction in Aβ1–40 after a single dose. Together, these results indicate that increasing the concentration of a BACE1-targeting antibody in the brain by exploiting TfR-mediated transcytosis can significantly enhance the potency of this potential therapeutic antibody.


Our results reveal an unexpected relationship between antibody affinity for TfR, a target of receptor-mediated transcytosis, and the extent of brain uptake of the antibody and its distribution in the brain parenchyma. The reduction in Aβ production by the anti-TfR/BACE1 bispecific antibody also demonstrates that the TfR pathway of receptor-mediated transcytosis is sufficiently robust to deliver a concentration of antibody across the BBB that is therapeutically relevant, at least in mice. This insight is particularly relevant in a clinical setting where the serum antibody concentration that is administered can typically be high, and where we predict that enhanced CNS delivery using TfR or another receptor-mediated transcytosis pathway can be achieved by reducing the affinity of the antibody for the receptor. To test this approach in primates, an anti-TfR antibody that binds to monkey and human TfR must be generated. Furthermore, although the anti-TfR antibody used in this study does not block binding of transferrin to TfR, the acute and chronic safety considerations of using antibodies raised against TfR to increase uptake in brain have not been explored. For example, investigation of iron transport and uptake (mediated by TfR) will be essential for determining the clinical feasibility of this approach. In addition, the broader consequences of increased exposure of brain tissue to antibody will require further investigation.

Although the TfR pathway appears to be a particularly robust route for CNS uptake of large molecules, it should be expected that, at some point, brain uptake will be limited by the receptor concentration and the rate of transcytosis. These limitations may vary for different receptor-mediated transcytosis targets, and so, the full potential of this approach requires investigation of these other targets. Furthermore, other receptor-mediated transcytosis targets may provide the added benefit of BBB-specific expression, in contrast to the broadly expressed TfR. Nevertheless, our findings demonstrate substantial promise for brain-penetrating bispecific therapeutic antibodies that exploit receptor-mediated transcytosis, and provide evidence that this approach may be useful in targeting a wide range of CNS diseases with antibody therapy.

Materials and Methods

Cloning and purification of muTfR

Amino acids 122 to 640 corresponding to the extracellular domain of muTfR were polymerase chain reaction (PCR)–amplified and inserted into the protein expression vector (Genentech) with the herpes simplex virus glycoprotein D signal sequence and an N-terminal 8× His tag. muTfR was expressed in HEK293 cells. Expression media containing muTfR were conditioned with 1 mM sodium azide, 0.5 mM phenylmethylsulfonyl fluoride, 0.3 M NaCl, and 15 mM imidazole and adjusted to pH 7.0. Precharged nickel resin (Ni-NTA superflow, Qiagen) was added to media for batch absorption at 4°C overnight and then packed into a column and washed with PBS and 0.3 M NaCl (pH 7.4), followed by 25 mM imidazole, 0.3 M NaCl, and 1 mM sodium azide (pH 8.0). The column was eluted with 250 mM imidazole, 0.3 M NaCl, and 1 mM sodium azide (pH 8.0). The eluent was concentrated [10,000 molecular weight cutoff (MWCO) Amicon Ultra, Millipore] and dialyzed (10,000 MWCO dialysis cassette, Pierce) into PBS and 0.3 M NaCl and furthered purified by size exclusion chromatography (Superdex 200, GE Healthcare) to remove aggregate and contaminants. The final pool was sterile-filtered and characterized by SDS–polyacrylamide gel electrophoresis, optical density at 280 nm, endotoxin assay, laser light scattering, and mass spectrometry.

Generation of anti-TfR and anti-TfR variant antibodies

Variants of anti-TfRA were generated by substituting alanine at key positions in the complementarity-determining regions. Affinities were determined with the xTfR competition enzyme-linked immunosorbent assay (ELISA) that measured the inhibition of biotinylated anti-TfRA binding to immobilized muTfR. Variants anti-TfRB, anti-TfRC, and anti-TfRD were selected as representative variants that covered a wide affinity range. All IgGs were expressed with human constant domains in Chinese hamster ovary cells and purified by conventional means. The anti-TfR/BACE1 bispecific antibody was generated using knobs into holes technology (14). Each heavy chain (anti-TfRA and anti-BACE1) was expressed with its corresponding light chain in separate Escherichia coli cultures. Individual half-antibodies were purified and combined, and the bispecific antibody was purified by conventional means.

Radiolabel studies

A modified Chizzonite radioiodination protocol was used to label antibodies with 125I or 131I (2, 15). Male CB-17 wild-type mice were randomized into two groups. All groups received control ([125I]control antibody, 5 mCi iv) plus either tracer ([131I]anti-TfR, 5 mCi iv) or tracer + cold ([131I]anti-TfRA, 5 mCi + 4 mg/kg iv). After injection, blood and brain tissues were collected at designated time points. Collected samples were analyzed for total radioactivity per gram of tissue. Brain tissue radioactivity was corrected for contribution of blood concentration to counts (n = 6 per group).

Antibody affinity measurements

The anti-TfR competition ELISA was performed in MaxiSorp plates (Nunc) coated with purified muTfR-His (2.5 μg/ml) in PBS at 4°C overnight. Plates were washed with PBS and 0.05% Tween 20 and blocked with Superblock blocking buffer in PBS (Thermo Scientific). A titration of anti-TfRA, anti-TfRB, anti-TfRC, or anti-TfRD (0.2 to 4000 nM, 1:3 serial dilution) was combined with 1 nM biotinylated anti-TfRA and added to the plate for 1 hour at room temperature. Plates were washed with PBS and 0.05% Tween 20, and horseradish peroxidase (HRP)–streptavidin (SouthernBiotech) was added to the plate and incubated for 1 hour at room temperature. Plates were washed with PBS and 0.05% Tween 20, and biotinylated anti-TfRA bound to the plate was detected with 3,3′,5,5′-tetramethyl benzidine (TMB) substrate (BioFX Laboratories).


To determine antibody distribution in the brain, we intravenously injected wild-type mice with an indicated antibody (20 mg/kg). After the indicated time, mice were perfused with Dulbecco’s PBS (D-PBS) (Invitrogen) at a rate of 2 ml/min for 8 min, and brains were fixed in 4% paraformaldehyde for ~18 hours, followed by 30% sucrose for 24 hours. Floating microtome sagittal sections of hemibrains were blocked in 5% bovine serum albumin (BSA) and 0.3% Triton X-100 before staining with primary antibodies overnight in 1% BSA and 0.1% Triton X-100 (rabbit anti–collagen IV, Cosmo Bio Co.; anti-mouse NeuN, Millipore). Anti-TfR and anti-TfR/BACE1 were visualized with Alexa488 anti-human secondary antibody, whereas collagen IV and NeuN were visualized with Alexa594 anti-rabbit and anti-mouse secondary antibodies, respectively (Molecular Probes, Invitrogen). Epifluorescent images of cortical regions (all images) were obtained with the Zeiss AxioCam 20×/0.50-mm and 40×/0.75-mm aperture objectives and the AxioVision software. Confocal z-stack images of cortical regions were obtained with the Zeiss LSM-710, and 3D reconstructions were generated with Imaris.

Measuring antibody concentrations in mouse brain and serum

The animals’ care was in accordance with institutional guidelines. Wild-type female C57B/6 mice ages 6 to 8 weeks were used for all studies. Mice were intravenously injected with anti-TfR variants, control IgG, or anti-TfR/BACE1 (20 mg/kg). Total injection volume did not exceed 250 μl, and antibodies were diluted in D-PBS when necessary (Invitrogen). After the indicated time, mice were perfused with D-PBS at a rate of 2 ml/min for 8 min. Brains were extracted, and the cortex and hippocampus were isolated and homogenized in 1% NP-40 (Calbiochem) in PBS containing Complete Mini EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics). Homogenized brain samples were rotated at 4°C for 1 hour before spinning at 14,000 rpm for 20 min. Supernatant was isolated for brain antibody measurement. Whole blood was collected before perfusion in serum separator microcontainer tubes (BD Diagnostics), allowed to clot for at least 30 min, and spun down at 5000g for 90 s. Supernatant was isolated for antibody measurement in serum.

Antibody concentrations in mouse serum and brain samples were measured with an ELISA. Nunc 384-well Maxisorp immunoplates were coated with F(ab′)2 fragment of donkey anti-human IgG, Fc fragment–specific polyclonal antibody (Jackson ImmunoResearch) overnight at 4°C. Plates were blocked with PBS and 0.5% BSA for 1 hour at 25°C. Each antibody (control IgG, anti-TfR variants, anti-BACE1, and anti-TfR/BACE1 bispecific) was used as an internal standard to quantify respective antibody concentrations. Plates were washed with PBS and 0.05% Tween 20 with a microplate washer (Bio-Tek Instruments Inc.), and standards and samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA, 0.05% Tween 20, and 15 parts per million (ppm) Proclin were added for 2 hours at 25°C. Bound antibody was detected with HRP-conjugated F(ab′)2 goat anti-human IgG, Fc-specific polyclonal antibody (Jackson ImmunoResearch) and developed with TMB (KPL Inc.), and absorbance was measured at 450 nm on a Multiskan Ascent reader (Thermo Scientific). Concentrations were determined from the standard curve with a four-parameter nonlinear regression program. The assay had a lower limit of quantification values of 3.12 ng/ml in serum and 15.6 ng/g in the brain. Statistical analysis of differences between experimental groups was performed with two-tailed unpaired t test.

Cellular Aβ1–40 assays

Inhibition of Aβ1–40 production in HEK293 cells stably expressing wild-type human APP was assessed with HEK293-APPWT cells and seeded overnight at a density of 3 × 104 cells per well in a 96-well plate. Fresh media [50 μl of Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS)] containing an anti-BACE1 antibody or a control IgG1 antibody were added for 24 hours at 37°C and then assayed for the presence of Aβ1–40 with a Aβ1–40 homogeneous time-resolved fluorescence (HTRF) assay (CisBio) according to the manufacturer’s instructions. Aβ1–40 values were normalized for cell viability, as determined with the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Experiments were performed at least three times, and each point in each experiment was repeated in duplicate.

Measuring mouse Aβ1–40 in mouse brain and plasma

Mice were intravenously injected with the indicated antibody (25 or 50 mg/kg) for either 24 or 48 hours. Antibody treatment and perfusions were performed in WT mice as stated above. For Aβ1–40 measurements, hemibrains were homogenized in 5 M guanidine hydrochloride buffer and samples were rotated for 3 hours at room temperature before diluting (1:10) in 0.25% casein and 5 mM EDTA (pH 8.0) in PBS containing freshly added aprotinin (20 mg/ml) and leupeptin (10 mg/ml). Diluted homogenates were spun at 14000 rpm for 20 min, and supernatants were isolated for Aβ1–40 measurement. For antibody concentration measurements, the corresponding hemibrain from each mouse was homogenized in 1% NP-40 as stated above. Whole blood was collected in EDTA microcontainer tubes (BD Diagnostics) before perfusion and spun at 5000g for 15 min, and supernatant was isolated for measuring plasma mouse Aβ1–40 and anti-TfR/BACE1 concentrations. The concentrations of total mouse Aβ1–40 in plasma and brain were determined with a sandwich ELISA following similar procedures described above. Rabbit polyclonal antibody specific for the C terminus of Aβ1–40 (Millipore) was coated onto plates, and biotinylated anti-mouse Aβ monoclonal antibody M3.2 (Covance) was used for detection. The assay had lower limit of quantification values of 1.96 pg/ml in plasma and 39.1 pg/g in the brain. Statistical analysis of differences between experimental groups was performed with two-tailed unpaired t test.

Supplementary Material

Fig. S1. Lower-affinity anti-TfR antibodies show increased concentrations in the brain after intravenous injection.

Fig. S2. Enhanced brain uptake and parenchymal distribution of the anti-TfR/BACE1 bispecific antibody.

Fig. S3. Increased brain antibody levels of anti-TfR/BACE1 compared to anti-BACE1 in a mouse efficacy study.

Fig. S4. Anti-TfR antibodies do not reduce Aβ levels in vivo.

Movies S1 to S4. 3D reconstructed confocal z-stack images showing distribution of injected antibody (green) with neuronal cell bodies (NeuN, red).


  • * These authors contributed equally to this work.

  • Citation: Y. J. Yu, Y. Zhang, M. Kenrick, K. Hoyte, W. Luk, Y. Lu, J. Atwal, J. M. Elliott, S. Prabhu, R. J. Watts, M. S. Dennis, Boosting Brain Uptake of a Therapeutic Antibody by Reducing Its Affinity for a Transcytosis Target. Sci. Transl. Med. 3, 84ra44 (2011).

References and Notes

  1. Acknowledgments: We thank A. Boswell and L. Khawli for design and preparation of radiolabeled antibodies. We thank J. Maloney for cloning; R. Tong, A. Koch, and J. M. Scheer for protein purification; and A. Bruce for the generations of illustrations. We also thank M. Sheng, J. Lewcock, M. Tessier-Lavigne, R. Port, F. Peale, and many others for their insight and collaborative assistance. Funding: Genentech Inc. Author contributions: R.J.W. and M.S.D. designed the project. Y.J.Y. and Y.Z. contributed to the study design and data collection. M.S.D. and Y.Z. designed and generated the antibodies and performed in vitro binding experiments and competitive ELISA experiments. M.K. and S.P. designed and performed all radiolabeled brain uptake studies. J.A. designed, performed, and analyzed cell-based assays. R.J.W. and Y.J.Y. designed, performed, oversaw, and analyzed in vivo experiments. K.H., W.L., and Y.L. ran pharmacokinetic and pharmacodynamic assays. J.M.E. produced the bispecific antibody. Y.J.Y., R.J.W., and M.S.D. wrote the manuscript, with valuable input from S.P., Y.Z., and M.K. Data and materials access: Data and materials are available from Genentech under a materials transfer agreement. Competing interests: All authors are paid employees of Genentech Inc. Genentech has filed a patent application related to this work on blood-brain barrier transport using transferrin receptor antibodies.
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