Blood-brain barrier transport, plasma pharmacokinetics, and

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Blood-brain barrier transport, plasma pharmacokinetics, and neuropathology following chronic treatment of the Rhesus monkey with a brain penetrating humanized monoclonal antibody against the human transferrin receptor William M. Pardridge, Ruben J. Boado, Daniel J. Patrick, Eric Ka-Wai Hui, and Jeff Zhiqiang Lu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00730 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018

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Molecular Pharmaceutics

Blood-Brain Barrier Transport, Plasma Pharmacokinetics, and Neuropathology Following Chronic Treatment of the Rhesus Monkey with a Brain Penetrating Humanized Monoclonal Antibody Against the Human Transferrin Receptor

William M. Pardridge†* Ruben J. Boado† Daniel J. Patrick‡ Eric Ka-Wai Hui† Jeff Zhiqiang Lu†

†ArmaGen, Inc. Calabasas, California 91302, United States ‡MPI Research, Mattawan, MI 49071, United States

*Address correspondence to: Dr. William M. Pardridge ArmaGen, Inc. 26679 Agoura Road, Suite 100 Calabasas, CA 91302 Ph: 818-252-8202 Fax: 818-252-8214 Email: [email protected]

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Table of contents graphic:

A transferrin receptor (TfR) monoclonal antibody (MAb) interacts with both the TfR type 1 (TfR1), via the variable region of the antibody, and with the Fc receptor (FcR), via the constant region of the antibody.


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Abstract A monoclonal antibody (MAb) against the blood-brain barrier (BBB) transferrin receptor (TfR) is a potential agent for delivery of biologic drugs to brain across the BBB. However, to date, no TfRMAb has been tested with chronic dosing in a primate model. A humanized TfRMAb against the human (h) TfR1, which cross reacts with the primate TfR, was genetically engineered with high affinity (ED50=0.18 ± 0.04 nM) for the human TfR type 1 (TfR1). For acute dosing, the hTfRMAb was tritiated and injected intravenously (IV) in the Rhesus monkey, which confirmed rapid delivery of the humanized hTfRMAb into both brain parenchyma, via transport across the BBB, and into cerebrospinal fluid (CSF), via transport across choroid plexus. For chronic dosing, a total of 8 adult Rhesus monkeys (4 males, 4 females) were treated twice weekly for 4 weeks with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb via a 60 min IV infusion for a total of 8 doses prior to euthanasia and microscopic examination of brain, and peripheral organs. A pharmacokinetics analysis showed the plasma clearance of the hTfRMAb in the primate was non-linear and plasma clearance was increased over 20-fold with chronic treatment of the low dose, 3 mg/kg, of the antibody. Chronic treatment of the primates with the 30 mg/kg dose caused anemia associated with suppressed blood reticulocytes. Immunohistochemistry of terminal brain tissue showed microglia activation, based on enhanced IBA1 immuno-staining, in conjunction with astrogliosis, based on increased GFAP immunostaining. Moderate axonal/myelin degeneration was observed in the sciatic nerve. Further studies need to be conducted to determine if this neuropathology is induced by the antibody effector function, or is an intrinsic property of targeting the TfR in brain. The results indicate that chronic treatment of Rhesus monkeys with a humanized hTfRMAb may have a narrow therapeutic index, with associated toxicity related to microglial activation and astrogliosis of the brain.


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Key words: Blood-brain barrier, transferrin receptor, monoclonal antibody, Rhesus monkey, astrogliosis, microglia, anemia, reticulocytes.


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Introduction Biologic drugs are large molecules that do not cross the blood-brain barrier (BBB). While it is suggested that therapeutic antibodies for the CNS penetrate the BBB to produce a brain level that is 0.1% of the plasma level,1,2 this presumption is actually based on antibody distribution into cerebrospinal fluid (CSF), not the brain.3 All proteins in blood enter into CSF via transport across the relatively leaky choroid plexus, which forms the blood-CSF barrier, and which filters molecules in plasma inversely related to molecular weight, such that the CSF/plasma ratio of IgG is 0.1-0.2%.3 However, CSF is not a surrogate for brain tissue, and when the antibody concentration is actually measured for brain tissue, the brain/plasma ratio is 98%. The [3H]-hTfRMAb was shipped overnight to MPI Research (Mattawan, MI) for next day intravenous (IV) injection in a 7.4 kg adult male Rhesus monkey. The injection dose (ID) was 1771 uCi in a volume of 4.3 mL, which is equivalent to an ID of 0.15 mg/kg. Plasma was taken for radioactivity measurements at 2, 5, 15, 30, 60, and 120 minutes after injection. At 120 minutes, the primate was euthanized, and brain and peripheral organs were removed for measurement of tissue radioactivity, per gram tissue. Additional samples of brain were taken for the capillary depletion method, as described previously.18 The plasma concentration curve was fit to a mono-exoponential function using the PAR subroutine of the BMDP Statistical Software (Statistical Solutions, Boston, MA), and plasma pharmacokinetics (PK) parameters were computed as described previously.7 The BBB permeability-surface area (PS) product, which is a quantitative measure of BBB transport, was computed from the ratio of the brain uptake, %ID/gram, at 2 hours, divided by the 2 hour plasma area under the concentration curve (AUC), %ID•min/mL, and expressed as uL/min/gram brain.10 Chronic treatment of Rhesus monkeys with the humanized hTfRMAb. Rhesus monkeys (Macaca mulatta) of mixed sex (4 males, 4 females) were studied at MPI Research, Inc. (Mattawan, MI), as described previously.19,20 All procedures were in compliance with the Animal Welfare Act Regulations, and were approved by the Institutional Animal Care and Use Committee. Each pair of monkeys (1 male, 1 female) were treated with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb administered as an intravenous infusion in the saphenous vein over a 60-min period in 50 mL of normal saline, and animals were dosed twice/week for 4 weeks for a total of 8 doses. Treatment was administered on days 1, 4, 8, 11, 15, 18, 22, and 25, followed by euthanasia on day 28, or 3 days after the last dose. For the 0 mg/kg dose, the animals were treated with the ABST formulation buffer diluted in normal saline.


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Adult experimentally naïve male and female Rhesus monkeys, 4-5 kg body weight, were studied. A complete physical exam, ophthalmoscopic exam, and electrocardiogram (ECG) were conducted on all animals pre-test and prior to euthanasia. Cageside observations were made twice per day, including a detailed clinical exam of each animal during the first 1 hr post-dose. Body weights were measured pre-test and twice a week during the study. Food consumption was measured daily during the study. Clinical chemistry tests were performed pretest and prior to euthanasia, and included urinalysis, coagulation tests (prothrombin, activated partial thromboplastin time, fibrinogen), hematologic profile (complete blood count, differential, reticulocytes, platelets), and chemistry panel with serum iron and total iron binding capacity. At necropsy, organ weights were measured and tissue sections of the brain and peripheral organs were collected for microscopic evaluation. The organs examined included the liver, lung, both kidneys, the heart (both ventricles, the myocardial septum, myocardial apex), and the left and right sciatic nerves. The formalin fixed tissues were embedded in paraffin and 5 micron thick sections were stained with hematoxylin and eosin (H&E). Brain (cerebrum, midbrain, cerebellum, medulla/pons, anterior cervical spinal cord) was also evaluated by GFAP and IBA1 immunohistochemistry, and by Fluoro Jade B fluorescence microscopy. After head-only perfusion fixation with 10% formalin, the brain was trimmed to evaluate 12 different regions and embedded in paraffin, as described by Garman.21 All tissue sections were evaluated by a boardcertified veterinary pathologist at MPI Research. The primary antibody used for the GFAP IHC was a 1:12,000 dilution of a rabbit polyclonal antibody (Agilent-DAKO, Santa Clara, CA), and the secondary antibody was an OmniMap horseradish peroxidase (HRP) conjugate of an antirabbit antibody (Roche Diagnostics, Rotkreuz, Switzerland). The primary antibody used for the IBA1 IHC was a 1:3,000 dilution of a goat polyclonal antibody directed against the carboxy


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terminus of human IBA1 (Abcam, Cambridge, MA), and the secondary antibody was an OmniMap anti-goat IgG-HRP conjugate (Roche Diagnostics). The IHC sections were counterstained with hematoxylin. Plasma pharmacokinetics. Blood was removed for PK analysis on Day 1 (start of study) and Day 25 (end of study). Blood (2 mL) was removed from the femoral vein and collected in tubes with K2-EDTA at 0, 2, 5, 30, 60 min, 2, 4, and 23 hrs after the IV infusion. The ‘0’ time point is pre-infusion, and all other time points start at the end of the 60 min IV infusion. Cerebrospinal fluid (CSF) was removed via the cisterna magna at 0, 4, and 23 hrs after the IV infusion on Day 1. Plasma and CSF hTfRMAb were measured by ELISA, as described above. Plasma concentrations, A(t), were fit to either a mono-exponential curve, A(t)=A1e-k1t, or to a biexponential curve, A(t)=A1e-k1t + A2e-k2t, where t=time after injection, and A=the Cmax, or maximal plasma concentration. The curve fit, e.g. mono- exponential or bi-exponential, which produced the lowest residual sum of squares was used. The PK parameters were derived from intercept (A) and slope (k) of each exponent, as described previously.7 Anti-drug antibodies. Terminal plasma was tested for the formation of anti-drug antibodies (ADA) by ELISA, as described previously.7 The capture agent is the humanized hTfRMAb. The detector reagent is a complex of the biotinylated humanized hTfRMAb and a streptavidin-peroxidase conjugate (Vector Labs, Burlingame, CA). The humanized hTfRMAb was biotinylated with sulfo-N-hydroxysuccinimide-LC-LC-biotin (Thermo-Fisher), and biotinylation was confirmed by Western blotting as described previously.7 Statistical differences were determined by ANOVA including a paired T-test using the 7D program of the BMDP Statistical software.


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Results The chimeric and humanized hTfRMAb, transiently expressed in COS cells, were purified to homogeneity based on reducing SDS-PAGE (Figure 1A), and non-reducing SDSPAGE (not shown), and the identity confirmed by human IgG Western blotting (Figure 1B). The LC and HC migrated with a MW of 28 kDa and 58 kDa, respectively. The SDS-PAGE and human IgG Western blotting of the CHO-derived antibody was identical to that shown in Figure 1. The MW of the heavy chain of the CHO-derived humanized hTfRMAb was reduced 3 kDa by N-glycanase treatment (Methods). The potency of the chimeric and humanized hTfRMAb was examined with a human TfR1 ELISA. The ED50 of binding of the COS-derived humanized hTfRMAb was actually lower, 0.36 ± 0.04 nM, than the ED50 of binding of the chimeric hTfRMAb, 0.75 ± 0.04 nM (Figure 2). No binding of the humanized hTfRMAb to the mouse TfR1 ECD was observed. The ED50 of binding to the human TfR1 of the CHO-derived humanized hTfRMAb was 27.5 ± 5.6 ng/mL (0.18 ± 0.04 nM). The plasma concentration of the [3H]-hTfRMAb decreased from 0.283 ± 0.003 %ID/mL, at 2 minutes after IV injection, to 0.136 ± 0.018 %ID/mL, at 120 minutes after injection, and the plasma TCA precipitability was >99% at all time points. The humanized hTfRMAb was removed from plasma in the Rhesus monkey with a clearance rate of 0.30 ± 0.04 mL/min/kg, a systemic volume of distribution, Vss, of 55 ± 3 mL/kg, and a plasma halftime, T1/2, of 129 ± 17 min (Table 1). Peripheral organ uptake at 120 minutes was 7.3 ± 0.9 %ID/100 gram for liver, 4.0 ± 0.8 %ID/100 gram for spleen, 2.1 ± 0.7 %ID/100 gram for kidney, 1.5 ± 0.1 %ID/100 gram for lung, 0.92 ± 0.08 %ID/100 gram for heart, 0.74 ± 0.05 %ID/100 gram for fat, and 0.16 ± 0.04 %ID/100 gram for skeletal muscle. The uptake of the humanized hTfRMAb by choroid plexus was high, 4.4 %ID/100 gram. The brain uptake at 2 hrs after injection of the hTfRMAb was 1.1


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±0.1 %ID/100 grams, and the BBB PS product was 0.51 ± 0.04 uL/min/gram in frontal cortical gray matter (Table 1). The brain uptake in frontal cortical white matter, in cerebellar gray matter, and in cerebellar white matter, was 0.77 ± 0.05 % ID/100 gram, 1.12 ± 0.05 % ID/100 gram, and 1.06 ± 0.15 % ID/100 gram, respectively. Capillary depletion analysis showed the brain volume of distribution (VD) of the humanized hTfRMAb in the post-vascular supernatant was greater than the VD in the vascular pellet (Table 2), indicating the majority of the vascular bound antibody had penetrated into the brain parenchyma by 2 hrs after administration. The distribution in brain of the humanized hTfRMAb was high compared to a non-specific IgG (Table 2). The VD values for the non-specific IgG represent distribution of an IgG confined to the vascular volume of brain. Adult male and female Rhesus monkeys were then treated 8 times over a 28 day period with 0, 3, 10, or 30 mg/kg of the humanized hTfRMAb by IV infusion. The plasma concentrations of the hTfRMAb at different times after infusion of the dose administered either on Day 1 (start of study) or Day 25 (end of study) are given in Tables 3-5 for the 3, 10, and 30 mg/kg doses, respectively. The plasma PK parameters for the Day 1 and Day 25 plasma clearance are summarized in Table 6. On Day 1 and Day 25, there is a dose-dependent increase in the plasma AUC, and a dose-dependent decrease in the plasma clearance, which is consistent with saturation of the peripheral TfR by the higher infusion doses (Table 6). The plasma mean residence time (MRT) is 7.7 ± 1.2 hrs, 12.9 ± 1.7 hrs, and 16.4 ± 1.8 hrs, at the 3 mg/kg, 10 mg/kg, and 30 mg/kg infusion doses, respectively, on Day 1 (Table 6). Although the rates of clearance of the hTfRMAb at Days 1 and 25 are comparable for the 30 mg/kg dose, there is differential clearance of the antibody at Days 1 and 25 at the 3 and 10 mg/kg doses. At the 3 mg/kg dose, the plasma clearance is accelerated 21-fold following chronic administration at Day


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25 as compared to Day 1 (Table 6). At the 10 mg/kg dose, the plasma clearance is increased 4fold following chronic administration at Day 25 as compared to Day 1 (Table 6). The penetration of the humanized hTfRMAb into the CSF of the Rhesus monkey is shown in Table 7. The mean CSF/plasma ratio is 4.8%, 1.2%, and 0.85%, at the 3 mg/kg, 10 mg/kg, and 30 mg/kg doses, respectively, indicating transport of the antibody across the choroid plexus, which forms the blood-CSF barrier, is saturated by the increased infusion dose. The CSF/plasma ratio for the hTfRMAb at all doses was higher than the typical IgG that leaks into CSF to produce a CSF/plasma ratio of 0.1-0.2%.3 The CSF/plasma ratio was higher in the female, as compared to the male, at each treatment dose (Table 7). The primates were monitored by a veterinarian and no clinical findings were observed and no FIRs were recorded in any animal. Analysis of terminal blood showed no changes in blood chemistry, coagulation, serum iron, serum total iron binding capacity; there were no changes in urinalysis, physical exam, electrocardiogram, or opthalmoscopic exam. There was no ADA formation as assessed by ELISA (Methods). However, there was a reduction in hematocrit (Hct) (Figure 3A), which was associated with suppressed reticulocytes (Figure 3B). The reduction in Hct and reticulocyte count at the 30 mg/kg dose was statistically different from the placebo control animals (Figure 3). After euthanasia, the brain, major peripheral organs (liver, lung, kidney, heart), and bilateral sciatic nerve were removed, fixed and examined. There were no gross morphologic changes for any organ, or changes in organ weights. There were no microscopic findings with the H&E stain in brain, liver, lung, kidney, or heart. There was no evidence of neural degeneration in brain based on H&E or Fluoro Jade B fluorescent microscopy. However, the GFAP immunohistochemistry (IHC) of brain showed a diffuse increase in the chromogenic


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labeling of astrocyte bodies within the cortical gray matter as well as thickened and/or increased branching of astrocytic processes, as shown in Figure 4A, for the monkey treated with 30 mg/kg of the antibody, as compared to Figure 4B for the monkey treated with 0 mg/kg of the hTfRMAb. Higher magnification of the GFAP IHC is shown in Figure 4C for the 30 mg/kg dose and in Figure 4D for the 0 mg/kg dose of the antibody. In addition, brain IBA1 IHC was characterized by a diffuse increased staining of microglial cells within the white and gray matter which was also accompanied by microglial cells that were enlarged, had fewer processes, and/or were clustered, as shown in Figure 4E for the monkey treated with 30 mg/kg of the hTfRMAb, as compared to Figure 4F for the monkey treated with 0 mg/kg of the antibody. The increased brain GFAP staining was observed both in males and females at ≥3 mg/kg/dose, and increased brain IBA1 staining was observed in females at ≥3 mg/kg/dose and in males at ≥10 mg/kg/dose. H&E microscopic examination of both left and right sciatic nerve sciatic nerve showed axonal/myelin degeneration in females at ≥3 mg/kg/dose and males at ≥10 mg/kg/dose, as shown in Figure 5A. The severity ranged from minimal to moderate in males and minimal to mild in females. The H&E stain of the sciatic nerve of the monkey treated with 0 mg/kg of the antibody is shown in Figure 5B.


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Discussion The results of the present study are consistent with the following conclusions. First, the humanized hTfRMAb binds to the human TfR1 with high affinity (Figure 2), and rapidly penetrates the BBB in the primate with a brain uptake of 1.1% ID/100 gram (Table 1). Second, the humanized hTfRMAb exhibits a high uptake by choroid plexus (Results), which is associated with dose-dependent penetration into the CSF (Table 7). Third, the pharmacokinetics of plasma clearance of the antibody is non-linear, which is consistent with saturation of antibody uptake by peripheral tissues as the ID increases from 3 to 30 mg/kg (Table 6); in addition, chronic administration of the hTfRMAb to monkeys causes a 21-fold increase in plasma clearance of the antibody at the ID of 3 mg/kg (Table 6). Fourth, chronic administration of the hTfRMAb to Rhesus monkeys causes a decrease in hematocrit, which is associated with a suppressed reticulocyte count at the high dose of 30 mg/kg (Figure 3). Fifth, chronic administration of the hTfRMAb to Rhesus monkeys induces microscopic changes suggestive of a direct or indirect astrocytic and microglial reaction in the CNS (Figure 4), and axonal degeneration in the peripheral nervous system (Figure 5). Biologics can be re-engineered for BBB delivery by genetic fusion with antibodies directed against exofacial epitopes on either the insulin or transferrin receptors. The safety of fusion proteins derived from the HIRMAb has been demonstrated in either acute 2-week or chronic 6-month GLP toxicology testing in Rhesus monkeys with repeat doses as high as 30 mg/kg.6,7,8 Human subjects have been treated with a HIRMAb fusion protein for at least 52 weeks with a low incidence, 2%, of reversible infusion related reactions or transient mild hypoglycemia.9 In contrast, safety issues have been raised with monovalent TfRMAbs, and 2 types of acute toxicity are observed: (a) first injection reactions (FIR) with severe clinical signs


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associated with acute cytokine release to blood, and (b) acute suppression of blood reticulocytes.11,12 In contrast, bivalent TfRMAb’s have been administered to mice in multiple models and no FIRs are observed.22-27 The FIR is dose dependent and more likely to be observed with low affinity monovalent TfRMAbs, which require higher IDs up to 50 mg/kg to achieve therapeutic effects in brain.11,12 Conversely, therapeutic effects in mouse models of neural disease are observed at low doses, 1 mg/kg, of high affinity bivalent TfRMAbs.22-26 The effector function effects of TfRMAbs in the mouse can be reduced or eliminated with mutations of certain amino acids in the Fc region of the IgG.11 A N297G mutation, which removes constant region N-linked glycosylation, eliminates both FIRs and suppressed reticulocytes after a single injection in the primate of a TfRMAb against the human TfR1.28 However, TfRMAbs have not been tested in primates subjected to chronic dosing with a TfRMAb, and the need for such a study provides the rationale for the current investigation. The humanized hTfRMAb engineered here has high affinity for the human TfR1 (Results), and shows significant penetration into the brain of the Rhesus monkey with a brain uptake of 1.1% ID per 100 grams of brain (Table 1). Brain uptake is expressed per 100 grams, because the weight of the brain in the Rhesus monkey is 100 grams.29 Capillary depletion analysis of brain radioactivity shows the majority of the hTfRMAb taken up by brain penetrated the brain parenchyma at 2 hours after IV administration (Table 2). All of the radioactivity in brain represents unmetabolized hTfRMAb, since the TCA precipitability of the plasma radioactivity was >99% for at least 2 hours after IV administration (Results). The results of the capillary depletion method indicate the hTfRMAb gains access to brain parenchyma via direct passage across the BBB or brain microvascular barrier. The capillary depletion method has been shown previously to correlate with morphologic studies confirming trans-vascular transport in


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brain including light microscopic emulsion autoradiography30 or electron microscopy of brain following perfusion of gold conjugated TfRMAb.31 The humanized hTfRMAb also shows high uptake by the choroid plexus of the primate (Results), and a high CSF/plasma ratio of 4.8% following the IV infusion of 3 mg/kg of the antibody (Table 7). The CSF/plasma ratio decreases as the ID is increased to 10 and 30 mg/kg (Table 7), which indicates the transport of this high affinity TfRMAb across the blood-CSF barrier at the choroid plexus is saturated. The delivery of the hTfRMAb into CSF is mediated by transport across the choroid plexus, whereas the delivery of the hTfRMAb into brain parenchyma is mediated by transport across the capillary endothelium of brain parenchyma, which forms the BBB.3 The pharmacokinetics (PK) of plasma clearance of the hTfRMAb in the Rhesus monkey was analyzed following the measurements of the immunoreactive hTfRMAb in monkey plasma after administration of 3, 10, or 30 mg/kg on either Day 1 or Day 25 of the study (Tables 3-5). The PK of plasma clearance of the hTfRMAb is non-linear as the clearance is decreased 66% as the ID is increased on Day 1 from 3 to 30 mg/kg (Table 6). Chronic dosing of the primate causes a 21-fold increase in the rate of plasma clearance, and a 14-fold increase in the systemic volume of distribution (Vss), at the low dose, 3 mg/kg, of the hTfRMAb at Day 25 of the study, as compared to Day 1 (Table 6). The rates of plasma clearance of the hTfRMAb are increased 3.7fold and 1.7-fold at Day 25 for the higher doses of the hTfRMAb, 10 and 30 mg/kg, respectively (Table 6). In contrast, the clearance of the HIRMAb in the primate is constant at 3, 10, and 30 mg/kg doses,19 and the plasma clearance in the Rhesus monkey of HIRMAb derived fusion proteins is unchanged after 6 months of chronic treatment.7 The accelerated clearance of the hTfRMAb at Day 25, as compared to Day 1, is not related to the formation of anti-drug


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antibodies, as the ADA titer was low as assessed by ELISA (Results). The increase in plasma clearance of the humanized hTfRMAb, particularly at the low dose of 3 mg/kg (Table 6), following chronic dosing in the primate was not observed in the mouse.27 Chronic dosing of mice with repeat intravenous injections of 2 mg/kg of a TfRMAb fusion protein directed against the mouse TfR caused no changes in the rate of systemic clearance from blood of the TfRMAb fusion protein.27 Chronic treatment over 4 weeks of the Rhesus monkey with the hTfRMAb causes a reduction in hematocrit, in association with a suppressed reticulocyte count (Figure 3). The blood halftime of erythrocytes in the Rhesus monkey is 17 days.32 Therefore, an acute suppression of reticulocytes at the high doses, 10-30 mg/kg of the hTfRMAb, within the first 24 hours of dosing would be expected to cause measureable decreases in hematocrit 28 days later. The reticulocyte suppression may be related to effector functions within the constant region of the TfRMAb.11 There must be some specific interaction between the FcR and the TfR1, because the constant region of the HIRMAb studied in GLP primate toxicology,6-8 and infused in humans over 52 weeks,9 is the same constant region, human IgG1κ, that was used to engineer the constant region of the humanized TfRMAb tested in this study. However, chronic administration of HIRMAb fusion proteins in either primates6-8 or humans9 is not associated with either first injection reactions, or reticulocyte suppression and anemia. No clinical findings or first-injection reactions of any kind were observed in any of the monkeys dosed with the humanized hTfRMAb in the present study (Results). The effect of the hTfRMAb on hematocrit and reticulocytes, in this sample of 8 primates, appears to be dose related, as there are minimal effects observed at the 3 mg/kg dose (Figure 3). A projected therapeutic dose for this high affinity, bivalent TfRMAb would be 3 mg/kg, which is the optimal therapeutic dose for high affinity, bivalent HIRMAb


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fusion proteins in humans.9 Nevertheless, a TfRMAb which has therapeutic dose of 3 mg/kg, but exhibits significant side effects at 10-30 mg/kg, would have a narrow therapeutic index. If a TfRMAb induces effector function effects in the periphery, a TfRMAb that penetrates the BBB, would be expected to also induce effector function effects in the CNS. The FcR is widely expressed in the CNS on multiple cells in brain, including microglia.33 Microglia are resident macrophages and comprise about 12% of the cells in the brain.34 Microglial function and astrocyte activation are closely linked in states of neuroinflammation.35,36 In the present study, astrocytic (Figure 4A, 4C), and microglial changes (Figure 4E), were observed in primates subjected to chronic dosing with the hTfRMAb. Fluoro Jade B fluorescent microscopy showed no evidence of neurodegeneration in the primate treated with the humanized hTfRMAb (Results). However, mild to moderate axonal degeneration was observed in a bilateral examination of the sciatic nerve (Figure 5). In contrast, weekly intravenous infusions of high doses, 30 mg/kg, of HIRMAb derived fusion proteins to Rhesus monkeys for 6 months caused no astrogliosis in brain or neural degeneration of the sciatic nerve.7,8 Treatment of subjects with MPSI and cognitive impairment for 52 weeks with a HIRMAb fusion protein stabilized the decline of mental function and the loss of gray matter volume in these subjects.9 Chronic treatment of the primate with the hTfRMAb leads to axonal and myelin degeneration in the sciatic nerve (Figure 5), and the mechanism of this effect is not known. Little information is available as to whether a TfRMAb traverses the blood-nerve barrier similar to antibody transport across the BBB, much less any species differences between rodents and primates. Transferrin is expressed in Schwann cells of myelinated fibers of the sciatic nerve in the rat,37 and the TfR1 is expressed on Schwann cells of the injured sciatic nerve in the rat.38 Further investigations are needed to determine if the effect of chronic treatment with a TfRMAb


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on the primate sciatic nerve is due to the effector function domain or the TfR binding domain of the antibody. In summary, the present study describes the chronic treatment of Rhesus monkeys with 330 mg/kg IV infusion doses of a humanized hTfRMAb. This antibody readily penetrated the parenchyma of brain, via transport across the brain capillary endothelium, which forms the BBB, and the CSF, via transport across the choroid plexus, which forms the blood-CSF barrier. The hTfRMAb exhibited a non-linear plasma PK profile, and accelerated clearance on chronic dosing. High doses, 30 mg/kg, of the hTfRMAb induced anemia associated with suppressed reticulocytes. This study describes microglial and astrocytic changes in brain of the primate subjected to 4 weeks of repeat dosing with a TfRMAb. The study highlights the potential for antibody-induced neuropathology following the chronic administration of a TfRMAb to primates, or humans, particularly at high doses.

Acknowledgements The authors are indebted to MPI Research, Inc. (Mattawan, MI) for execution of the primate study. Winnie Tai, Phuong Tran, and Yuen Yin Lee provided expert technical assistance.


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Table 1. Plasma pharmacokinetics and brain uptake of [3H]-humanized hTfRMAb in the Rhesus monkey following intravenous administration

parameter

units

value

Body weight

kg

7.4

T1/2

min

129 ± 17

Vss

mL/kg

55 ± 3

clearance

mL/min/kg

0.30 ± 0.04

AUC (120 min)

%ID•min/mL

21.7 ± 0.8

Brain uptake*

%ID/100 gram

1.11 ± 0.09

BBB PS product*

µL/min/gram

0.51 ± 0.04

*Gray matter in frontal cortex. The injection dose is 0.15 mg/kg (Methods); T1/2=plasma halftime; Vss=systemic volume of distribution, AUC=plasma area under the concentration curve; PS=permeability-surface area


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Table 2. Transvascular delivery of humanized hTfRMAb in the primate brain in vivo

Non-specific IgG

hTfRMAb

Homogenate VD

18 ± 4

69 ± 4

Post-vascular

16 ± 1

39 ± 7

1.1 ± 0.4

29 ± 2

supernant VD Vascular pellet VD

Volume of distribution (VD), uL/gram brain, of the [3H]-humanized hTfRMAb in the total brain homogenate, the post-vascular supernatant, and the vascular pellet of brain removed 2 hours after the IV administration of the [3H]-humanized hTfRMAb in the Rhesus monkey was measured with the capillary depletion method. The VD values for a non-specific IgG with no receptor specificity in the Rhesus monkey are reported previously.29


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Table 3. Plasma concentration of humanized hTfRMAb after IV infusion at 3 mg/kg on Day 1 and Day 25

minutes

Plasma concentration (ng/mL) Day 1

Day 25

0

0

22 ± 20

2

57,955 ± 10,138

5

48,998 ± 3,437

30

44,314 ± 9,456

3,716 ± 3,973

60

35,713 ± 4,361

2,915 ± 2,830

120

24,337 ± 3,506

1,275 ± 1,152

240 360

782 ± 658 14,758 ± 3,219

480 1380

343 ± 247 1,793 ± 370

54 ± 56

2880

16 ± 13

4320

Blood-brain barrier transport, plasma pharmacokinetics, and

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