Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn

brain research 1534 (2013) 13–21

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn Philip R. Coopera,n,1, Gary J. Ciambroneb,1, Connie M. Kliwinskic, Eva Mazeb, Lowell Johnsonb, Qianqiu Lid, Yiqing Fenga, Pamela J. Hornbya a

Antibody Drug Discovery, USA Drug Product Development, USA c Biologics Pharmacology and Toxicology, USA d Bio-Stats PA, USA b

art i cle i nfo

ab st rac t

Article history:

Monoclonal antibody (mAb) engineering that optimizes binding to receptors present on

Accepted 17 August 2013

brain vascular endothelial cells has enabled them to cross through the blood–brain barrier

Available online 23 August 2013

(BBB) and access the brain parenchyma to treat neurological diseases. However, once in the

Keywords: Blood–brain-barrier IgG FcRn Efflux Intracranial Intranasal

brain the extent to which receptor-mediated reverse transcytosis clears mAb from the brain is unknown. The aim of this study was to determine the contribution of the neonatal Fc-receptor (FcRn) in rat brain efflux employing two different in vivo drug delivery models. Two mAb variants with substantially different affinities to FcRn, and no known neuronal targets, (IgG1 N434A and H435A) were administered to rats via intranasal-to-central nervous system (CNS) and intra-cranial dosing techniques. Levels of full-length IgG were quantified in serum and brain hemispheres by a sensitive enzyme-linked immunosorbent assay (ELISA). Following intra-nasal delivery, low cerebral hemisphere levels of variants were obtained at 20 min, with a trend towards faster clearance of the high FcRn binder (N434A); however, the relatively higher serum levels confounded analysis of brain FcRn contribution to efflux. Using stereotaxic coordinates, we optimized the timing and dosing regimen for injection of mAb into the cortex. Levels of N434A, but not H435A, decreased in the cerebral hemispheres following bilateral injection into the rat cortex and higher levels of N434A were detected in serum compared to H435A after 24 h. Immunohistochemical staining of human IgG1 in sections of cortex was consistent with these results, illustrating relatively less intense immunostaining in N434A than H435A dosed animals. Using two in vivo methods with direct cranial administration, we conclude that FcRn plays an important role in efflux of IgG from the rat brain. & 2013 Elsevier B.V. All rights reserved.

Abbreviations: FcRn, neonatal Fc receptor; IgG, immunoglobulin G; WT, wild type n Corresponding author at: Biologics Research, Janssen R&D, Pharmaceutical Companies of Johnson & Johnson, Welsh & McKean Roads, PO Box 776, Spring House, PA 19477, USA. E-mail address: [email protected] (P.R. Cooper). 1 Philip R. Cooper and Gary J. Ciambrone are joint-first authors. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.08.035

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1.

brain research 1534 (2013) 13–21

Introduction

The blood brain barrier (BBB) is the protective layer separating the systemic circulation from the brain extracellular fluid. It is made up of tight junctions between endothelial cells on cranial capillaries, a thick basement membrane and astrocytic end-feet. The BBB serves to restrict bacteria and other large hydrophilic molecules from entering the brain parenchyma, while allowing small hydrophobic molecules and nutrients to enter. Pharmacologic treatment of neurologic diseases has relied on brain penetration of small lipophilic molecules. However, where high selectivity and potency is desirable, an alternative therapeutic approach could be the use of monoclonal antibodies (mAbs). Immunoglobulin-Gs (IgGs), along with other plasma proteins, are large hydrophilic molecules that are unable to pass through the BBB in sufficient quantity to be efficacious when systemically administered (Poduslo et al., 1994). Researchers are currently experimenting with receptorbased brain endothelial transcytosis, such as using the transferrin receptor (Bickel et al., 1994; Pardridge et al., 1991; Yu et al., 2011) or insulin receptor (Boado et al., 2007; Pardridge et al., 1995) for IgGs to enter the brain parenchyma. However, once mAbs enter the brain, the extent to which they are cleared by receptor-mediated reverse transcytosis is not wellknown. Evidence of the involvement of an Fc-receptor in the clearance of IgG from the central nervous system (CNS) has been shown by a shorter half-life of IgG, compared to IgM (antibody that lacks Fc region), in both rat and monkey cerebrospinal fluid (Bergman et al., 1998). Moreover, efflux of IgG though the BBB is competitively inhibited by the addition of Fc fragments (Boado et al., 2007; Zhang and Pardridge, 2001). Indeed, the Fc-receptor mediated Aβ-IgG efflux mechanism has been shown to facilitate the clearance of IgG complexes from brains (Deane et al., 2005). There are data to both support and refute the role of the neonatal Fc-receptor (FcRn) in IgG efflux from the brain. Using non-compartment mathematical modeling in mice which lack FcRn functionally, there was no apparent difference in efflux compared to wild-type mice based on labeled IgG and residual blood volume (Abuqayyas and Balthasar, 2013; Garg and Balthasar, 2009). FcRn is visualized by confocal microscopy in brain microvasculature endothelial cells (Schlachetzki et al., 2002), but whether the receptor is involved in efflux in addition to its role in recycling IgG is unknown. In vascular endothelial cells, IgG is taken up from the circulation by non-specific fluidphase pinocytosis where it binds to FcRn in the acidic endosome. It is recycled to the capillary lumen where it has a long half-life (Roopenian and Akilesh, 2007). It is therefore postulated that expression of FcRn located in brain endothelial cells (Schlachetzki et al., 2002) may be involved in the efflux of IgGs from the brain. The aim of the current study was to define the role of FcRn in IgG efflux from the rat brain using two variants of a recombinant human IgG1 mAb that either had increased FcRn binding (IgG1 N434A) or decreased FcRn binding (IgG1 H435A) compared to wild-type by incorporating mutations at the 434 and 435 amino acid positions (EU numbering) (Firan

et al., 2001; Yeung et al., 2009). The monoclonal antibodies were generated to target respiratory syncytial virus (RSV) and would not be expected to bind to targets in the brain. A human mAb was used to avoid potentially faster clearance of mouse mAb dosed to rats, and enable detection of the human Fc in rat tissues. Studies were 24 h or less to avoid differences in serum levels due to the relationship of FcRn binding affinity and circulating half-life. The two variants have been shown to have rat FcRn binding affinities, of 77 nM for N434A and 41000 nM for H435A at pH 6.0 (Kliwinski et al., 2013).

2.

Results

2.1. Physicochemical characteristics of the FcRn binding variants Both variants had identical pI values of 7.2. The circular dichroism (CD) spectra for both the near and far ultra-violet ranges showed very similar secondary and tertiary protein structure for both of the variants. They had the same Size Exclusion Chromatography (SEC) profiles with no covalent aggregates, and were stable at 25 1C for 4 d. There was no interaction with mucins, which would confound their delivery by intranasal route (data not shown).

2.2. Pharmacokinetics of FcRn binding variants after intranasal-to-CNS administration FcRn binding variants (H435A and N434A) were administered intranasally into each nostril of rats (40 nmol/rat) and plasma was collected after 20, 40, and 90 min post-dose. The levels of the FcRn binding variant increased to levels that reached
200 ng/mL in the circulation at a greater rate than the nonFcRn binding variant (Fig. 1A). Rat brain hemispheres were collected after brain perfusion, at 20, 40, and 90 min postdose from different rats. FcRn binding variants delivered into the brain (ng/g) were detected by an ELISA-based MSD assay that detects full-length mAb (Fig. 1B). N434A entered the brain at a faster rate than H435A and peaked at a higher level at 20 min. Despite the greater degree of uptake of N434A, levels of this variant dropped to very low levels within the same 90 min timeframe as H435A. Statistical comparison of the AUC values generated for each variant showed a statistically significant difference (N434A AUC 1637 ng min/g vs. H435A AUC 827 ng min/g, Po0.05), representing an approximately two-fold faster rate of efflux for N434A compared to H435A. To monitor that test article was correctly deposited with the tube insertion technique; olfactory epithelia from both nostrils were collected at 20, 40, and 90 min post-dose and analyzed for FcRn binding variants. The PK profiles of each are shown in Fig. 1C and D. In both epithelia, the N434A variant was cleared at a much faster rate than the H435A, and the AUC values for each were significantly different (left AUC H435A 2.2  107 ng min/g vs. left AUC N434A 1.4  107 ng min/g, P¼ 0.01; right AUC H435A 2.6  107 ng min/g vs. right AUC N434A 1.6  107 ng min/g, Po0.01). These data are indicative of an active FcRn mediated efflux mechanism removing the FcRnbinding N434A variant from the olfactory epithelium.

15

60

300 N434A High FcRn Binder 250

Hemisphere Levels B21M Variants (ng/g)

Plasma Levels B21M Variants (ng/ml)

brain research 1534 (2013) 13–21

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Left Olfactory Epithelium IgG conc (ng/g)

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90

450,000

P<0.05

400,000 350,000 300,000 250,000 200,000 150,000 100,000 50,000 20

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Time (min)

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Time (min)

Fig. 1 – PK profiles of variants after intranasal-to-CNS administration: H435A or N434A were administered intranasally to rats. A total of 50 ll, of a 60 mg/ml solution of either variant, was applied to each nostril sequentially over 12 min (4  25 ll, right and left, 4 min interval). (A) Serum was collected at 20, 40, and 90 min post-dose and full-length IgG quantified. Animals were euthanized at 20, 40, and 90 min post-dose and (B) hemispheres (n ¼ 12) and (C) left and (D) right olfactory epithelia (n¼ 6) analyzed for N434A (■) or H435A (●). Values are mean 7 SEM.

2.3.1. Serum levels of the FcRn binding IgG variant, N435A, increase with time The contribution of FcRn in IgG brain efflux was suggestive of FcRn-mediated efflux but not conclusive after intranasal administration due to the relatively low brain levels and differences in serum levels of the variants. Therefore, we complimented these studies by direct intracranial stereotaxic administration. Preliminary experiments were performed to determine a dose that, when administered into the brain via stereotaxic coordinates to the parietal cortex, would result in detectable serum levels. To do this, rats were maintained under anesthesia for 4 h after unilateral administration of the FcRn binding variant (N434A; 2.0 mg/mL; 1.2 mL) into the right anterior SiFl region of the somatosensory cortex. Serum levels of intact IgG were measured at 5, 30, 60, 120, 180, and 240 min. Following intra-cranial administration of the antibody, low but detectable levels of full-length IgG in serum were detected by 30 min. Serum levels continued to increase up to the termination of the experiment at 4 h. The rate of efflux was fairly stable from 0 to 180min with an average efflux rate of 0.4 ng/mL/h. The rate increased to 0.9 ng/mL/h between 180 and 240 min, with serum levels of 2.170.5 ng/mL at the final time point (Fig. 2).

Serum levels of IgG1 N434A (ng/mL)

3

2.3. Pharmacokinetics of FcRn binding variants after intra-cranial administration

2

1

LLOQ: 0.5 ng/mL 0 0

1

2

3

4

Time (hr) Fig. 2 – Time-course of serum levels of IgG1 N434A: serum levels (ng/mL) were measured from rats (n ¼15) at 5 and 3 min, and 1, 2, and 4 h following intra-cranial administration of IgG1 N434A (2.4 lg; 2 mg/ml). Intact IgG1 was quantified using the MSD assay.

2.3.2.

Unilateral intra-cranial dosing of IgG variants

Having established that intact IgG serum levels following intra-cranial administration increased over time, but had not reached maximal levels after 4 h, serum levels of FcRn binding variants (N434A, with the FcRn low binding control IgG, H435A) were measured up to 24 h. A 2.4 mg dose (2.0 mg/mL) of either

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brain research 1534 (2013) 13–21

Serum levels of B21M IgG1 variants (ng/mL)

2.3.3. 40

B21M IgG1 N434A B21M IgG1 H435A

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B21M IgG1 N434A B21M IgG1 H435A

140 120 100 80 60 40 20 0 5 mins

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5 mins

4

24

Time (hr) Fig. 3 – Unilateral intra-cranial dosing: anesthetized Sprague Dawley rats (n ¼6/group) were intra-cranially (anterior right hemisphere) administered (2.4 lg; 2 mg/ml) of either IgG1 N434A or IgG1 H435A. Intact IgG1 was quantified using the MSD assay. (A) Serum levels (ng/mL) were measured at 5 min, and 4 and 24 h following intra-cranial administration of IgG1 N434A (■) or IgG1 H435A (●). (B) After the final blood draw, brains were removed, homogenized and intact IgG quantified IgG1 N434A (solid) or IgG1 H435A (stripes).

N434A or H435A was administered into the right anterior SiFl region of the cortex of anesthetized rats. The animals in this study were anesthetized until after the 4 h blood draw then allowed to recover. Consistent with the preliminary study, levels of full-length IgG in the serum at 5 min were below the LLOQ for all rats dosed, thus confirming that no surgical damage was performed that would lead to systemic contamination. Levels of N434A and H435A were similar 4 h after administration (4.471.9 and 3.471.9 ng/mL, respectively), but after 24 h there tended to be higher levels of the N434A FcRnbinding variant (20.675.8 and 11.973.1 ng/mL, respectively) which did not attain a level of statistical significance (Fig. 3A). In brain tissue at the earliest time point of 5 min, levels of N434A (FcRn binding variant) were 17167354 ng/g of tissue and similar to that expected based on dose administered (average mass of a hemisphere was 1.0 g). Levels decreased by approximately 40% after 24 h whereas levels of the nonbinding variant H435A in the brain hemispheres were unchanged over time up to 24 h (Fig. 3B). Levels in the cerebellum, brainstem, and lymph nodes were low and no difference was detected between the variants (data not shown).

Bilateral intra-cranial dosing of IgG variants

A high concentration of mAb administered unilaterally may saturate the FcRn receptor in the vicinity of the injection, thus limiting the contribution of receptor-mediated efflux. This could explain the lack of a clear difference between the two variants following unilateral administration; therefore, the variants were dosed bilaterally. The total brain dose of 2.4 mg was the same as in the previous study, but a 1.2 mg (0.6 mL) dose of either N434A or H435A was administered bilaterally. Serum levels were measured at 5 min, 4, and 24 h and rats were allowed to recover from anesthesia immediately after surgery. After 24 h, N434A serum levels reached 25.975.2 ng/mL which was significantly greater (Po0.001) than the serum levels of the low FcRn binding variant, H435A, at 6.670.6 ng/mL. The rate of efflux of N434A was calculated to be three times greater than H435A (Fig. 4A). In a separate group of animals, brain hemispheres were excised 5 min after either N434A or H435A antibody administration. Levels of N434A detected at 5 min were 2.270.5 mg/g of tissue (average mass of the hemispheres was 1.0 g) which was the expected range after administration. After 24 h, levels of N434A in the hemispheres decreased by 48%. Levels of the non-FcRn binding mAb, H435A, did not change over the same time period (Fig. 4B). In order to visualize the presence of human IgG immunohistochemically in the rat brain, three animals were dosed either N434A or H435A bilaterally into the SiFl region of the cortex similar to the previous experiment. After 24 h rats were perfused and brains prepared for immunohistochemistry using an anti-human Fc rabbit polyclonal in thin (5 mm) brain sections. Brains were processed for routine histology (hematoxylin and eosin and Luxol fast blue), as well as human IgG and anti-rabbit IgG negative controls. At 24 h after dosing both N434A and H435A mAb variants were visualized in brain tissue (Fig. 5). There was a range of staining intensity visualized in sections at an equivalent coronal plane in the region of the somatosensory cortex in pyramidal and supporting cells that was localized to the cortex or spread into the corpus callosum region. The staining intensity of N434A (Fig. 5A) in the cortex was consistently less than brains injected with H435A (Fig. 5B). There was no specific staining detected in PBS microinjected brains (Fig. 5C) or mAb injected brains incubated in serum (Fig. 5D). Semi-quantitative immunostaining scores supported the impression that less of the FcRn binding variant was visualized in brain after 24 h (HA¼7.071.4 vs. NA¼ 4.471.7 of a total of 12.0 maximum staining; P¼0.3 by unpaired t-test), which is consistent with greater efflux of the FcRn binding mAb variant.

3.

Discussion

This study used two in vivo drug delivery models in rats, intranasal-to-CNS and direct intra-cranial dosing, to determine the contribution of FcRn to IgG receptor-dependent reversetranscytosis (efflux) from the brain parenchyma to the circulation. Following administration into the brain by these two methods, greater amounts of the FcRn binding variant N434A were detected in the circulation compared to the low-binding FcRn variant control, H435A. Following intra-cranial administration, levels of H435A in the brain hemispheres did not

Serum levels of B21M IgG1 variants (ng/mL)

brain research 1534 (2013) 13–21

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P < 0.001 B21M IgG1 N434A B21M IgG1 H435A

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Time (hr)

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P < 0.01

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Fig. 4 – Bilateral intra-cranial dosing: anesthetized Sprague Dawley rats (n ¼ 10/group) were intra-cranially (anterior hemisphere: right and left) administered (1.2 lg each site; 2 mg/ml) either IgG1 N434A or IgG1 H435A. Intact IgG1 was quantified using the MSD assay. (A) Serum levels (ng/mL) were measured at 5 min, and 4 and 24 h following intracranial administration of IgG1 N434A (■) or IgG1 H435A (●). (B) After the final blood draw, hemispheres were removed, homogenized, and intact IgG quantified (ng/g brain tissue) in total hemispheres. Key: 5 min (solid), 24 h (squares).

change over a 24 h period while the levels of N434A significantly decreased over time. Intranasal-to-CNS delivery was used initially because it is a non-invasive technique. For maximal delivery to the brain hemispheres, the test article had to be applied directly to the olfactory epithelium of the nose (Thorne et al., 2004). Test article then moves in a paracellular fashion, driven by diffusion, past the olfactory epithelium, and into the nasal lamina propria beneath (Dhuria et al., 2010). This space is contiguous with channels through the cribriform plate, which contain the axons of the olfactory neurons. Earlier studies have shown that a certain percentage of cerebrospinal fluid (CSF) exits the brain through the cribriform plate and enters nasal lymphatics which drain into the cervical lymph nodes (Bradbury et al., 1981; Bradbury and Westrop, 1983; Cserr et al., 1992). This process could represent a hindrance to molecular flow into the brain. However, subsequent ultra-structural studies have demonstrated the existence of neuronal channels that traverse the subarachnoid space (Field et al., 2003; Li et al., 2005) thus preventing direct interaction with CSF. These channels are believed to provide direct access into the parenchyma of the brain hemispheres (Dhuria et al., 2010; Lochhead and

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Thorne, 2012). Functional studies using both small and large molecules have shown delivery to the hemispheres via this mechanism despite this potential hindrance. The main driving force for uptake is the concentration gradient of the test article (Barakat et al., 2006; Evseev et al., 2010; Furrer et al., 2009; Gorbatov et al., 2010; Hoekman and Ho, 2011; Romanova et al., 2010; Sipos et al., 2010). However, there are two main factors that do impinge on the efficiency of CNS uptake of a molecule using this technique: those related to formulation, and those related to physicochemical characteristics. The formulation considerations include the type, pH, and tonicity of the buffer, and/or the presence of excipients representing transport enhancers, stabilizers, and muco-adhesives (Pujara et al., 1995; Washington et al., 2000). The important physicochemical characteristics include molecular size, hydrophobicity/hydrophilicity, surface charge, and mucus compatibility (Vyas et al., 2006). The test articles used in these studies are similar in terms of their physicochemical characteristics. Both H435A and N434A have pI values of 7.2, differ by just one amino acid, and have virtually identical secondary and tertiary structures as measured by circular dichroism. Functionally, they only differ in their affinity for the FcRn receptor, have no known targets in the brain and therefore are uniquely suited to use address the role of FcRn in IgG efflux from the brain. Following intra-nasal administration, the data suggested that FcRn contributed to reverse-transcytosis of IgGs from the rat brain, but were inconclusive, in part because of different levels of mAb remaining in the olfactory epithelium. With lower than expected brain levels after dosing intranasally at 40 nmol/ rat, and unexpected 100-fold higher serum levels (compared to intra-cranial administration), it is unclear whether variants entered the brain hemispheres prior to their entering the vascular compartment and confounded the interpretation of brain FcRn contribution to levels in the serum. However, AUC calculated data of the variant levels that did enter the hemispheres did provide a statistical significant difference in the rate of efflux of the two variants that did enter the brain. These data suggest a role of FcRn in the reverse-transcytosis of IgGs from the rat brain. Following unilateral stereotaxic administration, there was a tendency for greater amounts of N434A in the serum after 24 h compared to H435A. Importantly, potential damage to the tissue vasculature during the surgery could have resulted in systemic contamination; however, the serum levels at 5 min after administration were below the lower level of quantification in all animals suggesting the absence of such damage. Sample size (n ¼6) may have been the reason for the lack of statistical significance, as well as potential saturation of FcRn due to high local concentrations of the compound over the duration of the study (r 24 h). To avoid this problem, the study was repeated with the mAbs dosed bilaterally at the same co-ordinates for both right and left brain hemispheres. The total brain dose was identical to the unilateral dosing, but the local concentration at either brain hemisphere was halved. A significant increase in serum levels at 24 h and a decrease in total brain hemisphere levels were noted for the higher affinity FcRn binding variant compared to the low FcRn binding variant. Together these data confirm the trends shown in the intranasal and unilateral intra-cranial administration studies; and are in agreement with the previous

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N434A; anti-human IgG

PBS; anti-human IgG

H435A; anti-human IgG

H435A; rabbit serum

Fig. 5 – Greater levels of IgG1 H435A than IgG1 N434A in brain: representative examples of IgG-immunoreactivity in rat brains 24 h after intra-cranial administration of (A) IgG1 N434A; anti-hIgG1; (B) IgG1 H435A; anti-hIgG1; (C) PBS, anti-hIgG1, and (D) IgG1 H435A, rabbit serum.

studies that propose FcRn mediated efflux (Deane et al., 2005; Deane et al., 2009; Zhang and Pardridge, 2001). The opposite conclusion with regard to the role of FcRn in IgG brain efflux was reached by two studies using two different experimental methods. β-2-microglobulin knockout mice, which lack functional FcRn, and wild-type controls showed no difference in brain-to-plasma AUC ratios after intravenous administration of an I125 labeled mAb, leading the investigators to conclude that FcRn does not significantly contribute the low exposure of mAb in brain (Abuqayyas and Balthasar, 2013; Garg and Balthasar, 2009). In contrast another study reported that brain clearance after systemically administered mAb was reduced in FcRn/mice (Deane et al., 2005; Deane et al., 2009). The most significant difference between the protocol used in the studies with opposing conclusions (Abuqayyas and Balthasar, 2013; Garg and Balthasar, 2009), and the protocol used in the current study, and others (Zhang and Pardridge, 2001), was that IgG was administered directly into the brains of rats and serum and brain levels were measured. The other studies (Abuqayyas and Balthasar, 2013; Garg and Balthasar, 2009) involved administration of IgG into the circulation of wild-type and FcRn knock-out mice and relied on the ability of IgG to cross into the brain to measure AUC differences between brain content and serum levels. There was no direct

evidence that the IgG crossed the BBB into the brain parenchyma as the group did not measure IgG levels in the brain directly, but instead measured levels in residual blood. It is also unclear what the affinity of their IgG antibody was to murine FcRn. Therefore, there is limited evidence that FcRn had the ability to play a role in the efflux within that previously published study. Another disadvantage of this protocol was their use of FcRn knock-out mice. With no FcRn, the recycling and salvation of IgGs would not be present in these mice so IgG half-life would be substantially decreased. Although the study involving these mice was shortened to 4 d to compensate for this, there would be significantly less IgG in the circulation after 4 d (95% less). This adds differences in AUC of mAb in WT and knock-out mice confounding brain exposure. Indeed, clearance was eight-fold faster in FcRn knock-out mice compared to the other strains, as would be expected (Abuqayyas and Balthasar, 2013). In addition, the observed brain to plasma AUC ratio was greater in mice in the second study and the data was adjusted for differences in hematocrit (Abuqayyas and Balthasar, 2013; Garg and Balthasar, 2009). The emphasis on mathematical modeling may account for the differences in their conclusions compared to the observations in FcRn knock-out mice where brain clearance of a systemically administered mAb was lower than wild type controls (Deane et al., 2005; Deane et al., 2009).

brain research 1534 (2013) 13–21

In summary, this study demonstrates that FcRn plays an important role in the efflux of IgGs. These results need to be taken into account in future studies evaluating therapeutic IgGs containing an Fc portion when targets in the brain are investigated. As the variants in the present study did not have a neuronal target, future studies should consider the impact of target receptor occupancy for the therapeutic target to determine the maintenance of IgG brain levels or when investigating the relevance of FcRn-dependent efflux.

4.

Experimental procedure

4.1.

Animals

Male Sprague Dawley rats, 7–10 weeks old (200–300 g) (Charles River, Wilmington, MA, USA) were kept in plastic filter-topped cages and allowed free access to food and water. All animal studies were performed in accordance with the Federal Animal Welfare Act and methods approved by the Institutional Animal Care and Use Committee at Janssen R&D.

4.2.

Monoclonal antibody preparation

The mAbs used in this study were variants of a recombinant chimeric human IgG1 monoclonal antibody specific for human respiratory syncytial virus (RSV). For intranasal dosing the FcRn binding mutants, IgG1 H435A and IgG1 N434A, underwent buffer exchange from phosphate-buffered saline (PBS). The buffer used for exchange was 10 mM histidine/5.5% sucrose (pH 5.3), 150 mM NaCl. After three rounds of exchange, the mutants were concentrated to
66.67 mg/mL and propylene glycol was added to a final concentration of 10%, making the final concentration of the mutants 60 mg/mL. These preparations were used for intranasal dosing.

4.3. Physicochemical characteristics of the FcRn binding variants The physiochemical characteristics of the two variants were assessed and compared as this is a factor which can contribute to a difference in intra-nasal uptake. The predicted isoelectric point (pI) values of the variants were derived using Vector NTI sequence analysis software (Invitrogen). Circular dichroism (CD) spectroscopy to analyze structure was performed on the variants (0.25 mg/mL in PBS) and compared to PBS alone. Spectral acquisition was measured at 6 spectra from 190–260 nm at 1 nm path length and 1 nm intervals with a 2 s signal at 20 1C (Circular Dichroism Spectropolarimeter, Model 400, Aviv). The CD spectra were averaged and the net spectrum of the variants obtained by subtracting the average PBS scores. Spectra were fitted for species content using an MWR of 106 g/mol (150 kDa).

4.4.

Intranasal-to-CNS administration

Pre-dose plasma samples were collected from the animals via tail vein a day prior to dosing. On the day of dosing rats were anesthetized with sevoflurane (5.0–6.0% sevoflurane, 3.0 L/min O2; Abbott Labs., Princeton, NJ, USA) while placed in a supine

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position on an acrylic support with their heads positioned at a 451 angle to the horizon. A microcannula (BioTime, Berkeley, CA, USA) was inserted to a depth of 1.5 cm into the right nostril and either H435A or N434A (1.5 mg in 25 mL at a rate of 50 mL/ min) was infused by syringe pump (Harvard Apparatus, Cambridge, MA, USA). After 4 min, the same variant was applied into the left nostril and the alternating procedure repeated for a total application of 50 mL/nostril, therefore 400 mmol/L. After the final dose, the microcannula was removed and inhalant anesthetic was continued for 8 min with the animal supine and the head angle maintained at a 451 angle. At 20 min after the start of the first dose, animals were euthanized and tissues collected. For longer time points, anesthesia was maintained for 20 min and then removed and animals were allowed to awaken.

4.5.

Intra-cranial administration

The animals were placed in a chamber for induction of anesthesia with isoflurane (initially 2–4%) and then removed and placed in a stereotaxic device (Knopf) on a surgical pad maintained at 37 1C with a nose cone for maintenance of anesthesia (2% isoflurane) (Cetin et al., 2006). Buprenorphine (0.01–0.05 mg/kg) analgesia was administered sub-cutaneous. A midline incision was made to expose the bregma and was used to locate the ipsilateral primary somatosensory forelimb (SiFl) (þ0.2 mm anterior and 4.0 mm lateral3.0 mm deep). For unilateral administration, only the right hemisphere was dosed. A drill with a small burr attachment was used to introduce a 1 mm hole into the skull to allow entry for a Hamilton syringe (5 mL) to inject the test article, either IgG1 N434A or IgG1 H435A into the brain (antibodies were dosed in pH-neutral buffers (pH 7.4), unless otherwise stated). For unilateral administration a 1.2 mL bolus of test article (2.0 mg/mL) was administered at a rate of 0.4 mL/min (total dose¼2.4 mg, or 14 mmol/L). For bilateral administration the volume administered to each side was halved (0.6 mL); therefore, the total dose administered was equal to the unilateral administration. Care was taken to perform the surgical procedure following aseptic techniques and a surgical plane of anesthesia was maintained throughout the entire procedure. The incisions of animals were closed and the animals were allowed to recover from anesthesia following the 5 min baseline blood draw.

4.6.

Blood and brain tissue collection and homogenization

Animals were given isoflurane to facilitate retro-orbital blood draws of approx 100 mL. All blood samples were allowed to stand for at least 30 min, but no longer than 1 h, centrifuged at 3500 rpm for 15 min and the serum separated and stored at 80 1C. Following the final blood collection a full body perfusion was performed to remove residual blood from the brain via cardiac puncture using 60 mL (15 mL/min, syringe pump) of a PBS solution containing protease inhibitors (1 tablet/ 10 mL, Complete Mini, Roche, Indianapolis, IN, USA) and 5 mM EDTA. Following perfusion, the anterior cervical lymph nodes were removed and placed in FastPrep tubes. The head was then decapitated and the brain removed and halved into two hemispheres (excluding the olfactory bulbs), brainstem (hypothalamus to level of cistern magna of hindbrain), and cerebellum. The olfactory epitheliums were collected following

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intranasal-to-CNS administration only. Brain tissues were placed into pre-weighed lysis Matrix D tubes (MP Biomedical, Cat# 6913-500) or IKA tubes (IKA Cat# 3703100) then reweighed and quickly frozen on dry ice and stored at 80 1C until homogenization. Brain hemispheres were homogenized in 1:10 homogenization buffer (assume 1 g of tissue is 1 mL volume) (TBS 25 mM Tris [pH 7.8], 150 mM NaCl, 1% Triton X-100, 5 mM EDTA and cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets [1 tablet/10 mL]). The brain hemispheres were thawed and homogenized in buffer solution using an IKA instrument (Ultra Turrax, IKA Cat# 3645001) (setting# 6, 20 s). The left and right olfactory epithelia were diluted in the same homogenization buffer, and homogenized using the Bio101 FastPrep instrument (6.5 m/s; 40 s). Full length IgG in the brain and tissue samples was quantified within 24 h of homogenization.

4.7.

Full-length human IgG detection

Full-length IgG antibodies were quantified using the Meso Scale Discovery (MSD) electrochemiluminescent assay. The mAbs used in this study were a recombinant chimeric human IgG1 monoclonal antibody specific for human respiratory syncytial virus (RSV). An anti-idiotypic antibody specific for the anti-RSV IgGs was labeled with sulfo-NHS-LC-biotin and used as a capture antibody. A pan anti-human IgG1/IgG2 antibody labeled with MSD Sulfo-TAG NHS Ester was used as the detection antibody. The electrochemiluminescent labels added emit light when electrochemically stimulated. Electrodes bound to the sample-sandwich initiate the detection process. A standard curve for the anti-RSV IgGs in neat rat serum was observed over the range of 50,000–50 pg/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL using 25 mL of neat rat serum.

4.8.

Immunohistochemistry

At 24 h after intracranial dosing (3 dosed N434A, 3 dosed H435A, and one control dosed PBS) and whole body blood perfusion (10% neutrally buffered formalin, NBF), the brain hemispheres were removed and a 1 mm section proximal to the site of dosing was placed between two biopsy sponges and fixed in 10% NBF. The rats used in this study were separate from the previous efflux study. Following dehydration, the tissues were exposed to the primary antibody, human IgG1 (1 mg/mL; 1 h; Epitomics), then stained and counter-stained and dehydrated further. The above procedures were completely automated using the TechMate 500 (BioTek Solutions). Positive staining was indicated by the presence of a brown chromogen (DABHRP) reaction product. Representative images were obtained with an Olympus Microfire digital camera (M/N S97809) attached to an Olympus BX60 microscope. Samples were individually scored on a semi-quantitative basis for human IgG immunostaining. Each sample received an intensity human IgG score per region of staining from each hemisphere. These regions were Pyramidal Cells, Other Neural & Support Cells, and Corpus Callosum Area. Intensity scores were graded on a 5 point scale; 0, 1þ, 2þ, 3þ, or 4þ staining intensity (where 0¼no staining and 4þ ¼ completely saturated staining). The scores

were then added together to obtain a sample total score (maximum¼12).

4.9.

Statistical analyses

4.9.1.

Intra-cranial administration

Data were plotted as mean 7 standard error of the mean (SEM). Statistical tests used were either a one- or two-way ANOVA with post-test analysis performed using Bonferroni's multiple comparison test. P-values less than 0.05 were considered significant.

4.9.2.

Intranasal-to-CNS

A fixed effects model, including group, time, and group by time interaction as fixed effects, was fit to the FcRn variant concentration data (Wolfsegger and Jaki, 2005; Wolfsegger, 2007). For each FcRn variant group, with application of the trapezoidal rule, area under the curve (AUC) measurements: AUC (0-Last) (AUC of mean concentration from 0 min to 90 min) were calculated based on the mean concentrations across three time points and under the assumption of zero concentration at time 0. Group mean concentrations by time were estimated and 95% confidence intervals were constructed. FcRn variant effects were examined using group AUCs as linear functions of the mean concentrations at three times. The Delta method was applied for the inference of between-group AUC differences. All the 95% confidence intervals were two-sided t-type intervals and all P-values were from two-sided t-tests. For all tests, P-values less than 0.05 were considered significant (Wolfsegger and Jaki, 2005; Wolfsegger, 2007).

Acknowledgments We are grateful to Steve Jarantow, Deidra Bethea and Bethany Swencki-Underwood for their assistance in the physiochemical characterizing of the antibodies, and Bernie Scallon for helpful discussion.

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