Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Colorado State University on 08/21/14 For personal use only.

Review

Blood--brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody

1.

Introduction

2.

Transferrin receptors

3.

TfR monoclonal antibodies

William M Pardridge

4.

Iron delivery to brain via

ArmaGen Technologies, Inc., Calabasas, CA, USA

retro-endocytosis of transferrin at the BBB 5.

Transcytosis of transferrin and the TfRMAb at the BBB

6.

Brain uptake of the TfRMAb

7.

TfRMAb fusion proteins for targeted BBB delivery of biologic drugs

8.

TfRMAb--avidin fusion proteins for targeted delivery of biotinylated agents

9.

BBB delivery with a low-affinity TfRMAb Trojan horse

10.

Safety pharmacology of TfRMAb fusion proteins

11.

Expert opinion

Introduction: Biologic drugs are large molecules that do not cross the blood-brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. Areas covered: This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG--decoy receptors, IgG--lysosomal enzyme therapeutics and IgG--avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson’s disease, stroke, Alzheimer’s disease and lysosomal storage disorders. The review covers the engineering of TfRMAb--avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. Expert opinion: The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials. Keywords: biologic drugs, blood--brain barrier, fusion proteins, monoclonal antibody, transferrin receptor Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Biologic drugs include recombinant proteins, therapeutic antibodies, decoy receptors, neurotrophins, enzyme therapeutics, peptides and nucleic acid therapeutics. Development of these pharmaceuticals for brain diseases is difficult, as all of these large molecule drugs do not cross the blood--brain barrier (BBB) [1]. No biologic drug is currently FDA approved for treatment of a neural disease, wherein drug action requires transport across the BBB. Biologic drugs have entered into Phase III clinical trials over the last 20 years, but this drug development work proceeded in the absence of any BBB drug delivery strategy. Biopharmaceuticals, such as neurotrophins or therapeutic antibodies,

10.1517/17425247.2014.952627 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

1

W. M. Pardridge

Article highlights. .

. .


.

.

The blood--brain barrier (BBB) transferrin receptor (TfR) is a transcytosis system that mediates the delivery from blood to brain of transferrin and certain TfR monoclonal antibodies (MAb). A high-affinity TfRMAb penetrates the brain at a rate comparable to lipid-soluble small molecules. A BBB-penetrating TfRMAb can be used as a molecular Trojan horse to ferry into the brain biologic drugs that alone do not cross the BBB. BBB delivery of neurotrophins, decoy receptors, lysosomal enzymes and therapeutic antibodies is enabled following the re-engineering of the drug as a TfRMAb fusion protein. The TfRMAb fusion proteins have high therapeutic indices, as the chronic administration results in therapeutic effects in neural disease, without tissue toxicity or downregulation of TfR activity in vivo.

This box summarizes key points contained in the article.

were administered by systemic injection (subcutaneous [SQ], intravenous), and the clinical trials failed because the biologic drug does not penetrate the BBB from blood. In other trials, the BBB was bypassed, and the neurotrophin was injected directly into the brain or cerebrospinal fluid (CSF). Diffusion limits drug distribution to brain following injection into either brain or CSF [1], and clinical trials with transcranial drug delivery to the brain also failed. None of these prior biologic drug development programs for the brain employed BBB drug targeting technology. The penetration of the BBB by biologic drugs requires the re-engineering of the agent with a BBB drug delivery system. This review is limited to one platform technology, which develops BBB molecular Trojan horses to deliver a biologic drug across the BBB. A BBB molecular Trojan horse is an endogenous peptide, or a peptidomimetic MAb, that crosses the BBB via transport on an endogenous receptor-mediated transport (RMT) system [2]. This review focuses on the use of a MAb against the transferrin receptor (TfR), designated the TfRMAb, as a model molecular Trojan horse for biologic drug delivery across the BBB. 2.

Transferrin receptors

There are two TfRs, designated TfR1 and TfR2 [3,4]. The TfR1 is encoded by a 5 -- 6-kb mRNA, whereas TfR2 is encoded by a 2.8-kb mRNA [4]. These TfRs have tissuespecific gene expression. Polymerase chain reaction (PCR) studies show expression of TfR2 is 40-fold greater than TfR1 in liver, whereas expression of TfR1 is sevenfold greater than TfR2 in spleen in the mouse [5]. The amino acid identity between TfR1 and TfR2 is 39% in the mouse [6], so it is possible that a TfRMAb against TfR1 could cross-react with TfR2. The TfR1 comprises a small amino-terminal cytoplasmic domain, a transmembrane domain and a large 2

extracellular domain (ECD) [7]. The functional binding unit is a tetrameric structure, formed by a TfR1 disulfide-linked dimer that binds two Tf molecules. In the mouse, the ECD is formed by amino acids 89 -- 763 (NP_035768), and in the rat, the TfR1 ECD is formed by amino acids 140 -- 761 (NP_073203). The existence of a high affinity, low nM KD, binding site for Tf at the BBB was identified with isolated human brain capillaries and a radio-receptor assay [8]. Subsequent BBB genomics studies identified the BBB TfR as TfR1 [9]. 3.

TfR monoclonal antibodies

The type of TfRMAb that proves to be an effective molecular Trojan horse is an antibody that binds an epitope within the ECD of TfR1, because only epitopes within the ECD domain of the receptor are exposed to an antibody in the bloodstream. The TfRMAb should target a sequence within the ECD that allows for endocytosis of the TfRMAb/TfR1 complex into the cell. The TfRMAb should bind a sequence on the TfR1 ECD that is spatially removed from the endogenous Tf binding site so that there is no interference between binding of Tf and binding of the TfRMAb to TfR1. TfRMAbs that act as molecular Trojan horses at the BBB are almost always species-specific. The OX26 antibody is a mouse MAb against the rat TfR [10], which is transported across the BBB in the rat [11]. The RI7-217 antibody [12], or the 8D3 antibody [13], is a rat antibody against the mouse TfR, and both antibodies cross the BBB in the mouse [14], whereas the OX26 MAb against the rat TfR does not cross the BBB in the mouse [14]. The 128.1 antibody is a mouse MAb against the human TfR [15], and this antibody crosses the BBB in Old World primates such as the cynomolgus monkey [16]. The activity of a given TfRMAb as a BBB molecular Trojan horse is a function of the transcytosis property of the antibody. If the TfRMAb was only endocytosed into the endothelial compartment of brain, the antibody would not be an effective molecular Trojan horse. Transcytosis studies have been performed for both Tf and BBB-penetrating TfRMAbs, as reviewed below. The transcytosis of the TfRMAb through the BBB parallels the transcytosis of Tf through the brain capillary endothelial barrier, and the function of Tf transport across the BBB is the delivery of iron from blood to brain.

Iron delivery to brain via retro-endocytosis of transferrin at the BBB

4.

Iron in brain is derived from iron in blood, and Tf is the principal transport protein for blood-borne iron. In the retro-endocytosis model, it is posited that holo-Tf undergoes receptor-mediated endocytosis at the blood side of the brain capillary endothelium, followed by dissociation of Tf and iron within the endothelium, followed by retro-endocytosis of apo-Tf from the endothelium back to blood [17,18]. The iron then undergoes exocytosis from the endothelium

Expert Opin. Drug Deliv. (2014) ()


A.

C.

B.

D.


E.

Brain

Brain

H.

Brain

Blood

F.

Blood

G.

Figure 1. (A, B) Light microscopic silver staining of rat brain following a 10-min internal carotid artery infusion of a conjugate of 5 nm gold and the OX26 MAb against the rat TfR. Prior to perfusion fixation of rat brain with 2% glutaraldehyde, the brain vasculature was cleared with a saline infusion. (C, D, E) Electron microscopic examination of rat brain shown in panels A and B; the OX26 MAb--gold conjugate is observed in the intra-endothelia compartment within 100 nm endosomes (arrows in panels C and D). The conjugate is observed exocytosed into the brain interstitium in panel E (arrow). (F) Dark field light microscopy of rat brain following a 5-min internal carotid artery infusion of [125I]-rat holo transferrin. Prior to removal of the brain for freezing and frozen sectioning, the brain vasculature was cleared with a saline infusion. The autoradiography shows rapid distribution of the holo-transferrin into the entire postvascular compartment of brain. (G, H) Confocal microscopy of freshly isolated rat brain capillaries shows labeling of the BBB TfR by the OX26 MAb against the rat TfR. In panel G, the binding of the OX26 MAb was detected with a fluorescein conjugated secondary antibody; the TfRMAb is observed binding to the TfR on the abluminal membrane of the capillaries, as well as sequestered within intra-endothelial endosomes. In panel H, the binding of the TfRMAb to the TfR on both the luminal and abluminal membranes of the brain capillary is demonstrated. A,B: With permission from [26]. C -- E: With permission from [26]. F: With permission from [24]. G,H: With permission from [22]. BBB: Blood--brain barrier; TfR: Transferrin receptor.

to the brain interstitial fluid by an unknown mechanism, followed by endocytosis of the iron into brain cells via a mechanism that is also unknown. The retro-endocytosis model of Tf transport at the BBB is a theory that is based on problematic experimental design with respect to both the radio-tracer experiments and the morphologic experiments that support the model. In the radio-tracer model, a complex of 59Fe/125I-Tf is injected, and brain 59Fe radioactivity is shown to be enriched over time relative to brain 125I radioactivity. However, this observation is also consistent with the

transcytosis of holo-Tf across the BBB. The 59Fe becomes enriched in brain with time, relative to the 125I-Tf, because the Tf domain exits the brain in parallel with retention by brain of the iron. Following transcytosis of holo-Tf through the BBB, the holo-Tf is endocytosed by brain cells, iron is sequestered in brain cells, followed by reverse transcytosis of apo-Tf from brain back to blood. The reverse transcytosis of apo-Tf from brain to blood was shown with Brain Efflux Index method [19]. The efflux of apo-Tf from brain back to blood is rapid, and apo-Tf in brain undergoes exodus from brain with a T1/2 of 39 min [19]. Moreover, the 125I radioactivity escapes from brain via a second pathway, as 125I-labeled drugs that enter brain from blood are subject to deiodination in brain, followed by rapid efflux of the 125I radioactivity via BBB active efflux transport systems for the iodide anion [20]. In the morphologic experiments supporting the retroendocytosis model, immunoreactive TfR was shown to exist only on the luminal membrane of the BBB using a pre-embedding labeling method with fixed whole brain [18]. However, abluminal BBB receptors are not exposed to the antibody reagents using fragments of whole brain, and abluminal BBB receptors cannot be detected with pre-embedding labeling methods [21]. The presence of the TfR on both luminal and abluminal membranes of the brain capillary endothelium was demonstrated by confocal microscopy of freshly isolated brain capillaries [22], as shown in Figure 1H.

Transcytosis of transferrin and the TfRMAb at the BBB

5.

The transcytosis of 125I-holo-Tf across the BBB in the rat has been demonstrated by arterial perfusion methods [23,24]. Using methods originally developed to show transcytosis of insulin through the BBB in vivo [25], the carotid arterial perfusion method was combined with emulsion autoradiography of brain to demonstrate rapid movement of labeled Tf through the BBB in vivo [24], as shown in Figure 1F. This study confirms that 125I-holo-Tf rapidly moves through the BBB and fills the entire brain parenchyma during carotid arterial perfusions of duration as short as 5 min [24]. The radioactivity in brain was reduced > 90% by competition of transport of the 125 I-holo-Tf with the high concentrations of Tf in plasma [24]. The internal carotid arterial perfusion method was also combined with the capillary depletion method to show that transcytosis of either Tf or a TfRMAb through the BBB was rapid and occurred at comparable rates in vivo [24]. In contrast to the autoradiographic methods, it is difficult to demonstrate movement of the TfRMAb into brain parenchyma with morphologic methods. A conjugate of 5 nm gold (Au) and the TfRMAb was infused in the carotid artery of a rat for 10 min, followed by saline clearance of the brain compartment and perfusion fixation with glutaraldehyde [26]. The distribution of the Au--TfRMAb in brain at the light microscopic level was demonstrated by immunogold-silver staining [26], as shown in Figures 1A and 1B. The Au--TfRMAb is detected


3

W. M. Pardridge

Table 1. Brain uptake of TfRMAb versus small molecules in the rat. Drug

Brain uptake (%ID/g)


Diazepam OX26 TfRMAb Morphine Sucrose

0.81 ± 0.03 0.44 ± 0.07 0.080 ± 0.007 0.0090 ± 0.0010

Brain uptake (%ID/brain)* 1.6 0.88 0.16 0.02

*Based on brain weight in rat of 2 grams. Data from [28]. ID: Injected dose; TfR: Transferrin receptor

only within the intra-endothelial compartment of brain, and not within the brain parenchyma. This observation could be interpreted within the context of a model that the TfRMAb is sequestered within the capillary endothelium without movement out of the endothelium into the extravascular volume of brain. However, such a theory does not account for the volumetrics of brain. The intra-endothelial volume in brain is very small, 0.8 µl/g [27], whereas the extravascular volume of brain is nearly a 1000-fold larger, 700 µl/g. Therefore, the Au--TfRMAb undergoes a nearly 1000-fold dilution as the antibody exits the endothelial compartment and enters the extravascular volume of brain, and this 1000-fold dilution prevents the detection of the antibody in brain parenchyma with light microscopy. The distribution of the Au--TfRMAb in brain was also examined with electron microscopy [26]. The Au--TfRMAb complex is found on the luminal membrane of the brain capillary endothelium, as well as within intracellular endosomes, which have a diameter of about 100 nm (Figure 1C). Higher magnification at the electron microscopy level shows movement of the endosomes carrying the Au--TfRMAb toward the abluminal endothelial membrane (Figure 1D), followed by exocytosis into the brain interstitium (Figure 1E). The distribution of the TfRMAb into the endothelial endosomal compartment is also shown with confocal microscopy of freshly isolated rat brain capillaries (Figure 1G). In summary, the body of experimental data supports the theory that both Tf and the TfRMAb undergo RMT through the brain endothelial barrier followed by entry into brain parenchyma. 6.

Brain uptake of the TfRMAb

The pharmacokinetic rule The brain uptake of a TfRMAb is expressed as a percentage of injected dose (ID)/gram brain, and is a function of the plasma AUC and the BBB permeability-surface (PS) area product [28]: (1) 6.1

%ID /gram = (plasma AUC) × (BBB PS product )

It is not possible to compare the %ID/gram brain parameter among different species of different body weight (BW). This is because the plasma AUC is inversely related to the blood 4

volume and BW. Since the %ID/gram parameter is a direct function of AUC, the %ID/gram is also inversely related to BW. Therefore, the %ID/gram parameter should be converted to the %ID/brain parameter, based on the brain weight of the given species. The brain weight in the mouse, rat, Rhesus monkey and human is 0.5, 2.0, 100 and 1200 g, respectively. Brain uptake of a TfRMAb versus small molecules The brain uptake of the TfRMAb in the rat is 0.44% ID/g, or 0.88% ID/brain (Table 1), since the brain weight in the rat is 2 g. Before concluding whether this level of brain uptake of the TfRMAb is either high or low, the result should be placed in the context of brain uptake of small molecules. The brain uptake of diazepam in the anesthetized rat is 0.81% ID/g (Table 1). Diazepam is a lipid-soluble small molecule that is nearly 100% extracted by brain on a single pass. Diazepam sets the upper limit of brain uptake of drugs. The brain uptake of morphine, another small-molecule drug of less lipid solubility as compared to diazepam, is 0.08% ID/g in the rat (Table 1). The brain uptake of sucrose, which is a watersoluble small molecule with minimal penetration of the BBB, is ninefold lower than that of morphine (Table 1). Therefore, the brain uptake in the rat of the TfRMAb is within the boundaries of brain uptake of substances that are pharmacologically active in the brain as defined by levels of brain uptake of morphine and diazepam (Table 1). This analysis indicates the brain uptake of the TfRMAb is high and in the range that is expected to cause CNS pharmacological effects following systemic administration. 6.2

Brain uptake of TfRMAb relative to related molecules

6.3

Lactoferrin (Lf) is an iron-binding protein found in milk, and Lf is said to cross the BBB, based on observations with an in vitro model of the BBB [29]. However, studies with in vitro models of BBB transport are not predictive of BBB transport in vivo, owing to the leaky nature of the in vitro BBB, and the downregulation in cell culture of BBB transporters [30]. With respect to Lf transport across the BBB in vivo, this is very low, if not nil. The brain uptake of Lf in the rat is 0.02% ID/g [31], which is comparable to the brain uptake of sucrose (Table 1), a molecule used to define the lowest limit of BBB transport. In another example of the poor predictive properties of in vitro BBB model systems, the BBB transport of p97, also called melanotransferrin (MTf), an iron-binding protein that is expressed in malignant melanoma, was said to be high based on transfer across an endothelial monolayer in culture [32]. However, subsequent in vivo work could not demonstrate measureable transport of MTf across the BBB [33]. Although Tf can deliver 59Fe across the BBB in vivo [17], no brain uptake of a complex of 59 Fe and MTf is observed [34]. The use of Tf as a BBB delivery agent is not advisable, even though Tf crosses the BBB. This is



because the plasma concentration of Tf, 25 uM, is > 1000fold larger, than the KD of Tf binding to the BBB TfR [8]. Therefore, exogenous Tf must compete for binding to the TfR with the large pool of endogenous Tf [24]. The advantage of a TfRMAb as a BBB delivery agent is that the TfRMAb binding site on the TfR is spatially separated from the binding site for Tf, and there is no competition for antibody binding to the TfR.

TfRMAb fusion proteins for targeted BBB delivery of biologic drugs


7.

In an early study, a model neurotrophin, nerve growth factor (NGF), was re-engineered as a TfRMAb fusion protein [35]. NGF is produced within the cell as a 241 amino acid prepropeptide, and the preproNGF is cleaved at the junction of amino acids 122 and 123 to produce the 119 amino acid mature NGF from the carboxyl-terminal half of the precursor protein. The biology of NGF processing presents the conundrum of how to engineer a TfRMAb--NGF fusion protein such that the new molecule retains both high-affinity binding to the TfR and high-affinity binding to the NGF receptor, trkA. If the NGF is fused to the carboxyl terminus of the IgG chain, for example, the heavy chain, then only the mature NGF, not the preproNGF, can be fused. Fusion of the preproNGF to the carboxyl terminus of the antibody chain would result in cleavage and release of the mature NGF from the fusion protein. If the mature NGF is fused to the carboxyl terminus of the antibody chain, then the antibody domain of the fusion protein will preferentially fold into a three-dimensional protein co-translationally prior to folding of the fused NGF, and this could lead to improper folding of NGF, and to a loss of biological activity of the NGF domain of the fusion protein. When mature NGF was fused to the carboxyl terminus of the TfRMAb chain, there was a loss of biological activity of the neurotrophin domain of the fusion protein [35]. Conversely, when the preproNGF was fused to the amino terminus of the TfRMAb chain, there was retention of biological activity of the neurotrophin domain of the fusion protein [35]. However, the problem with fusion of a biologic to the amino terminus of the antibody chain is that this causes steric hindrance of antibody binding to the target receptor, for example, the TfR. The steric hindrance caused by amino-terminal fusions occurs because the complementarity-determining regions (CDR), which determine IgG binding to the target antigen, are near the amino terminus of the IgG heavy chain or light chain. Fusion of a lysosomal enzyme, b-glucuronidase (GUSB), to the amino terminus of a MAb against the human insulin receptor (HIR) allowed for retention of high GUSB enzyme activity, but resulted in a > 95% decrease in HIRMAbbinding activity at the HIR [36]. Subsequent work has shown that biologic drugs can be fused to the carboxyl terminus of the IgG heavy chain, with retention of activity of the biologic domain of the IgG fusion protein [2].

Properties of IgG fusion proteins for BBB delivery Models of IgG fusion proteins for BBB delivery are given in Figure 2. In the case of a neurotrophin, decoy receptor or lysosomal enzyme drug, the mature biologic agent, without the signal peptide or propeptide, is fused to the carboxyl terminus of each heavy chain of the IgG (Figure 2A). Model neurotrophins, such as glial-derived neurotrophic factor (GDNF) or erythropoietin (EPO), have been fused to the carboxyl terminus of the heavy chain of a chimeric MAb against the mouse TfR, designated the cTfRMAb [37,38]. A model decoy receptor, the ECD of the type II TNF-receptor (TNFR), was fused to the carboxyl terminus of the heavy chain of the cTfRMAb [39]. Model lysosomal enzymes, such as iduronidase (IDUA) or iduronate 2-sulfatase (IDS), have been fused to the carboxyl terminus of the heavy chain of the cTfRMAb [40,41]. In all cases, there was retention of biologic activity of the neurotrophin, decoy receptor or enzyme, as well as retention of high-affinity binding of the cTfRMAb to the murine TfR [37-41]. If the biologic agent is a therapeutic antibody, the problem is the engineering of a bispecific antibody (BSA). In this event, a singlechain antibody, such as a single-chain Fv (ScFv) antibody, is fused to the carboxyl terminus of each antibody heavy chain (Figure 2B). The model therapeutic antibody was a MAb against the Ab-amyloid peptide of Alzheimer’s disease (AD), and a ScFv form of the anti-amyloid antibody (AAA) was fused to the carboxyl terminus of the heavy chain of the cTfRMAb [42]. The BSA retained high-affinity binding to both the mouse TfR and to the Ab peptide of AD [42]. The desired properties of TfRMAb fusion proteins are listed in Table 2. Following the engineering of the TfRMAb-biologic drug fusion protein, it is necessary to demonstrate the bifunctionality of the fusion protein. Both the biologic activity of the drug domain, and the high-affinity binding to the TfR of the antibody domain, must be retained (Table 2). The highaffinity binding to the TfR by the TfRMAb domain of the fusion protein will insure a high level of brain uptake of the fusion protein, for example, > 1 -- 2% ID/g in the mouse (Table 2). This level of brain uptake is generally required to induce the desired pharmacologic effect in brain of the fusion protein at an acceptable ID, for example, 1 mg/kg [2]. Finally, it should be shown that administration of therapeutic doses of the fusion protein in an animal model of neural disease produces the desired pharmacologic effect in brain without causing systemic toxicity [2]. 7.1

Pharmacologic effects of TfRMAb fusion proteins in Parkinson’s disease

7.2

A mouse model of Parkinson’s disease (PD) was produced by the intracerebral injection of the neurotoxin, 6-hydroxydopamine. The toxin is injected on only one side of brain, which causes a unilateral motor deficit. The motor deficits in the mouse model of PD are quantified by observations of rotation behavior induced by either apomorphine, which causes 360 rotations in the direction contralateral to the lesion, or


5

W. M. Pardridge

A.

B.

VH VL

VH

CH1

VL CL CH2

CH1

TfR MAb

CL

Hinge

CH2

CH3

FcRn CH3


Protein drug

Anti-Aβ ScFv VH VL

Figure 2. (A) Structure of IgG fusion protein is shown, and is formed by fusion of a protein drug to the carboxyl terminus of each heavy chain of a MAb against an endogenous BBB receptor, such as the TfR. The heavy chain is composed of the following domains: variable region (VH), CH1, hinge, CH2, CH3, and protein drug. The light chain is composed of the following domains: variable region (VL) and constant region (CL). The protein drug alone is not transportable across the BBB, and may be a therapeutic enzyme, neurotrophin or decoy receptor. (B) Tetravalent, bispecific antibody formed by fusion of a ScFv antibody to the carboxyl terminus of each heavy chain of a TfRMAb. In this model, the therapeutic antibody is the ScFv domain, and the BBB delivery antibody is the TfRMAb domain. The model therapeutic antibody is an antibody against the Ab peptide of AD. With permission from [2]. AD: Alzheimer’s disease; BBB: Blood--brain barrier; ScFv; Single chain Fv; TfR: Transferrin receptor.

Table 2. Properties of TfRMAb molecular Trojan horses for the BBB. Property Trojan horse targets a BBB receptor that is a transcytosis, not an endocytosis, system Trojan horse binding to BBB receptor is high affinity (low nM binding KD) High-affinity binding of Trojan horse to BBB receptor (low nM binding KD) is retained following fusion or conjugation of drug to Trojan horse High-affinity binding or high activity of drug or enzyme is retained following fusion or conjugation of the drug to the Trojan horse High brain uptake of Trojan horse-drug molecule by brain, for example, > 1 -- 2% ID/g brain in the mouse In vivo CNS pharmacologic effects are observed following IV administration of Trojan horse-drug molecule With permission from [2]. BBB: Blood--brain barrier; ID: Injected dose; TfR: Transferrin receptor.

amphetamine, which causes 360 rotations in the direction ipsilateral to the lesion. The biochemical correlate of the lesion is quantified by measurements of either tyrosine hydroxylase (TH) enzyme activity in the striatum, or immunoreactive TH in the striatum by immunocytochemistry. Chronic treatment of mice with PD with thrice-weekly IV injections of 1 mg/kg of the cTfRMAb--GDNF or the cTfRMAb--EPO fusion protein caused a reversal of aberrant motor activity and caused an increase in striatal TH enzyme activity [43,44]. Of note is the observation that chronic treatment of mice with the cTfRMAb--EPO fusion protein induced only minor 6

changes in hematocrit [44]. The weak erythropoietic effect of the cTfRMAb--EPO fusion protein is due to the rapid clearance (CL) of the fusion protein from blood. The erythropoietic effect of EPO is proportional to the plasma AUC of EPO [45], and the plasma AUC of the cTfRMAb--EPO fusion protein is > 10-fold lower than the plasma AUC of EPO [38,44]. Mice with PD were treated with chronic IV doses of 1 mg/kg of the cTfRMAb--TNFR decoy receptor fusion protein [46]. In this study, the mice were also treated with equal doses of the TNFR--Fc fusion protein (etanercept). The ECD of the type II TNFR decoy receptor is fused to the carboxyl terminus of the heavy chain of the cTfRMAb as shown in Figure 3A. In contrast, etanercept is formed by fusion of the TNFR ECD to the amino terminus of the human IgG1 Fc region (Figure 3B). Despite the opposite orientation of the TNFR in the cTfRMAb--TNFR fusion protein, as compared to etanercept, the affinity of binding of the cTfRMAb--TNFR fusion protein to TNF-a is equal to the binding affinity of etanercept for TNF-a (Figure 3C). Chronic dosing of mice with PD with etanercept had no effect on either the aberrant motor activity or on striatal TH enzyme activity [46], which is consistent with other observations that etanercept does not cross the BBB [47]. In contrast, the cTfRMAb--TNFR fusion protein does cross the BBB in the mouse [39], and chronic dosing of PD mice with the cTfRMAb--TNFR fusion protein reversed the aberrant motor activity, and increased both striatal TH enzyme activity and immunoreactive TH in the striatum [46]. These findings show that the re-engineering of biologic TNF inhibitors (TNFI), such as the TNFR decoy receptor, as a BBB-penetrating IgG--TNFI fusion protein enables drug



A.

B. Binds BBB TfR Binds TNF-α

VH VL CH1

TNFR-II CL CH2 CH2 CH3

TNFR-II Binds TNF-α

C.

0.4

[125I]-TNF-a bound


CH3

0.3

cTfRMAb-TNFR KD = 347 ± 77 pM

0.2

Etanercept KD = 280 ± 80 pM

0.1

0 0

2000

4000 TNF-a (pM)

6000

Figure 3. (A) The cTfRMAb--TNFR fusion protein is formed by fusion of the ECD of the type II human TNFR to the carboxyl terminus of each heavy chain of the TfRMAb. (B) The etanercept fusion protein is formed by fusion of the same ECD of the type II human TNFR to the amino terminus of the Fc fragment of human IgG1 heavy chain. (C) Radio-receptor assay shows saturation of binding of human TNF-a to either the cTfRMAb--TNFR fusion protein or to etanercept. The binding dissociation constant, KD, was determined by nonlinear regression analysis. The horizontal line at 1.5% binding represents the nonspecific binding observed when either human IgG1 or mouse IgG1 was plated in lieu of the fusion protein. With permission from [46]. ECD: Extracellular domain; TfR: Transferrin receptor.

development of TNFIs for brain disease. TNFIs that are re-engineered for BBB penetration can be just as potent therapeutics for neural disease as the conventional TNFIs are for chronic inflammation in peripheral disease. Pharmacologic effects of TfRMAb fusion proteins in stroke

7.3

Neuroprotective agents have failed in clinical trials of acute ischemic stroke, because: i) the neuroprotective agents do not cross the BBB; and ii) the BBB is intact in the early hours after stroke when neuroprotection is still possible [2]. Therefore, if

neuroprotective agents are to be developed as new treatments for stroke, these biologic drugs should be first re-engineered for BBB penetration. The therapeutic effects of BBB penetrating TfRMAb fusion proteins in experimental stroke in the mouse were demonstrated for a decoy receptor, TNFR, and a neurotrophin, GDNF [48,49]. An acute ischemic stroke in the mouse was induced by the transient (60 min) middle cerebral artery occlusion (MCAO) followed by release of the occlusion and reflow of blood to the affected region of brain. Neuroprotection was quantified by staining coronal sections of mouse brain with 2,3,5-triphenyltetrazolium chloride, which


7

W. M. Pardridge

Blood-brain barrier BBB

Aβ ScFv TfRMAb


Aβ Aβ ScFv TfRMAb

Blood

Aβ amyloid plaque TfR

Aβ ScFv TfRMAb

FcRn

Interstitium

Figure 4. The cTfRMAb--ScFv fusion protein clears amyloid from brain in AD via three sequential steps, and each of these three sequential steps uses separate parts of the fusion antibody molecule. Step 1 is the influx of the fusion antibody from blood to brain across the BBB, which is mediated by binding of the fusion antibody to the BBB TfR. Step 2 is binding of the fusion antibody to the amyloid plaque in AD, which promotes disaggregation of the amyloid plaque, and this binding to the plaque is mediated by the anti-Ab ScFv part of the fusion antibody. Step 3 is the efflux of the fusion antibody from brain to blood across the BBB, which is mediated by binding of the fusion antibody to the BBB FcRn at the CH2--CH3 interface of the Fc region of the fusion antibody. Reprinted with permission from [52]. Copyright 2007 American Chemical Society. AD: Alzheimer’s disease; BBB: Blood--brain barrier; FcRn: Fc receptor; ScFv: Single-chain Fv; TfR: Transferrin receptor.

is oxidized to a red chromogen, triphenylformazan, in the presence of healthy brain tissue. This dye staining of brain sections allows for the determination of the infarct volumes in the brain ipsilateral to the MCAO lesion. Treatment with either IV GDNF alone or IV etanercept alone had no neuroprotective effect, because neither agent crosses the BBB [48,49]. However, treatment of the mice with experimental stroke with an IV dose of 1 mg/kg of either the cTfRMAb--TNFR fusion protein or the cTfRMAb--GDNF fusion protein was neuroprotective and the fusion protein treatment lowered stroke volume and improved neural deficit [48,49]. GDNF and the TNFR induce neuroprotection in brain via different molecular mechanisms, and the maximal therapeutic effect in the stroke model was observed with combination fusion protein therapy, wherein both the cTfRMAb--GDNF and cTfRMAb--TNFR fusion proteins are co-injected following the acute stroke [49]. Pharmacologic effects of TfRMAb fusion proteins in Alzheimer’s disease

7.4

The dementia of AD correlates with the deposition in brain of amyloid plaque formed by the 40 -- 43 amino acid Ab-amyloid peptide [50]. AAAs are potential new treatments 8

of AD, because AAAs that bind the Ab-amyloid peptide can either reduce the formation of amyloid plaque or cause disaggregation of pre-existing amyloid plaque. However, a pivotal Phase III clinical trial failed following chronic IV dosing of AD patients with an AAA [51]. This negative result of the clinical trial is consistent with the following considerations: i) the AAAs do not cross the BBB [52]; ii) the amyloid plaque in brain resides behind the BBB; iii) the BBB is intact in human AD [53]; and iv) the AAA must come into physical contact with the amyloid plaque in order to reduce the amyloid burden in brain [54]. Given these findings, it would seem apparent that an AAA therapeutic for AD must be first re-engineered for penetration through a nondisrupted BBB. A model AAA was converted to a ScFv form of the therapeutic antibody and fused to the carboxyl terminus of the heavy chain of the cTfRMAb [42], as depicted in Figure 2B. Double transgenic AD mice, aged 12 -- 14 months, were treated with daily SQ 5 mg/kg doses of the cTfRMAb--ScFv fusion protein for 12 weeks [55]. At the end of the 3 months of treatment, brain amyloid plaque was quantified by confocal microscopy following staining of brain sections with either thioflavin-S or the 6E10 MAb against the Ab-amyloid fibrils [55]. Fusion protein treatment caused a 60% decrease in amyloid plaques in the cortex and hippocampus of the AD mice. In conventional AAA passive immune therapy in murine models of AD, the AAA treatment causes cerebral microhemorrhage, which correlates with 1000-fold increase in plasma Ab-amyloid peptide concentration [56]. The cerebral microhemorrhage observed with conventional AAAs may be caused by the large increase in plasma Ab peptide, which is associated with BBB disruption [57,58]. The large increase in plasma Ab-amyloid peptide is caused by the long residence time in plasma of the AAA, which is cleared slowly from blood with a half-time of 3 weeks [59]. In contrast, chronic treatment of AD mice with the cTfRMAb--ScFv fusion protein causes neither an elevation in plasma Ab-amyloid peptide concentration nor any cerebral microhemorrhage [55]. Chronic administration of the cTfRMAb--ScFv fusion protein does not increase plasma Abamyloid peptide, because this fusion protein is rapidly cleared from blood with a mean residence time in mice of 3 h [42], which is > 100-fold faster than the CL from plasma of a conventional AAA with no receptor specificity [59]. The cTfRMAb--ScFv fusion protein is a trifunctional molecule that incorporates the three properties required to clear Ab-amyloid from brain (Figure 4). First, the cTfRMAb domain enables influx of the fusion protein from blood to brain across the BBB. Second, the ScFv domain of the fusion protein enables binding of Ab-amyloid plaque and free Abamyloid peptide. Third, the CH2--CH3 domain of the constant region of the antibody heavy chain enables binding to the BBB neonatal Fc receptor (FcRn), which allows for efflux of the fusion protein/Ab complex from brain to blood across the BBB [52]. The BBB expresses the FcRn [60], and IgG molecules in brain undergo reverse transcytosis from brain to blood across the BBB via an FcR-dependent mechanism [61].



Table 3. Plasma clearance, AUC, BBB PS product, and brain uptake in the mouse for TfRMAb fusion proteins. Fusion protein [reference]

Plasma CL (ml/min/kg)

Plasma AUC (%ID.min/ml)

BBB PS product (ml/min/g)

Brain uptake (%ID/g)

cTfRMAb--ScFv [42] cTfRMAb--GDNF [37] cTfRMAb--TNFR [39] cTfRMAb--EPO [38] cTfRMAb--IDS [41]

0.47 ± 0.13 0.65 ± 0.08 2.1 ± 0.1 2.3 ± 0.2 5.9 ± 0. 3

1752 ± 32 1031 ± 19 778 ± 13 533 ± 25 390 ± 25

2.0 3.0 3.6 3.7 3.3

3.5 3.1 2.8 2.0 1.3

± ± ± ± ±

0.2 0.2 0.6 0.2 0.2

± ± ± ± ±

0.5 0.1 0.5 0.1 0.1


BBB: Blood--brain barrier; CL: Clearance; EPO: Erythropoietin; GDNF: Glial-derived neurotrophic factor; ID: Injected dose; IDS: Iduronate 2-sulfatase; PS: Permeability-surface area; ScFv; Single-chain Fv; TfR: Transferrin receptor.

The BBB transport of IgG molecules mediated by the FcR is asymmetric and only enables IgG transport in the brain-toblood direction, not the blood-to-brain direction [61]. Pharmacologic effects of TfRMAb fusion proteins in lysosomal storage disease

7.5

There are > 50 lysosomal storage disorders and about 75% affect the brain [62]. Lysosomal storage diseases are treated with weekly enzyme replacement therapy (ERT) and IV infusions of the recombinant lysosomal enzyme. However, lysosomal enzymes are large molecule drugs that do not cross the BBB [63], and ERT does not treat the neural manifestations of the lysosomal storage disease [64]. Therefore, lysosomal enzymes must be re-engineered for BBB delivery if the serious CNS effects of lysosomal storage disorders are to be treated. Mucopolysaccharidosis (MPS) Type I, also called MPSI, or Hurler syndrome, is caused by mutations in the gene encoding the IDUA lysosomal enzyme [65], which is responsible for the hydrolysis of unsulfated a-L-iduronosidic linkages in heparan and dermatan sulfate. The murine form of IDUA was cloned by the PCR method and fused to the carboxyl terminus of the heavy chain of the cTfRMAb to form the cTfRMAb--IDUA fusion protein [40]. The cTfRMAb--IDUA fusion protein bound to the murine TfR with high affinity and exhibited an IDUA enzyme activity comparable to recombinant IDUA [40]. Aged 6-month old MPSI mice were treated with twice-weekly IV injections of 1 mg/kg of the cTfRMAb--IDUA fusion protein for 8 weeks. Treatment caused reduction in glycosoaminoglycans in peripheral organs, and caused a 73% reduction in the lysosomal inclusion bodies in brain cells [40]. MPS Type II is caused by mutations in the gene encoding the lysosomal enzyme, IDS [66], which catalyzes the hydrolysis of sulfate groups of glycosoaminoglycans such as heparan or dermatan sulfate. The cTfRMAb--IDS fusion protein has been engineered and this fusion protein retains high-affinity binding to the murine TfR, and exhibits an IDS enzyme activity comparable to recombinant IDS [41]. Pharmacokinetics and brain uptake of TfRMAb fusion proteins in the mouse

7.6

A series of cTfRMAb fusion proteins have been engineered and tested in mouse models (Table 3). A pharmacokinetics

(PK) analysis was performed following the IV injection of the radiolabeled fusion protein, which enabled the determination of the rate of plasma CL and plasma AUC for each fusion protein (Table 3). The brain uptake, %ID/gram and the BBB PS product were also determined for each fusion protein (Table 3). With the exception of the cTfRMAb--ScFv fusion protein, the BBB PS product for neurotrophin, decoy receptor and lysosomal enzyme fusion proteins was comparable, and in the range 3.0 -- 3.7 µl/min/g (Table 3). The BBB PS product for the BSA was reduced about one-third to 2.0 µl/min/g (Table 3). The brain uptake of the fusion proteins in the mouse varied over a range of nearly threefold from 1.3% ID/g for the cTfRMAb--IDS fusion protein to 3.5% ID/g for the cTfRMAb--ScFv fusion protein (Table 3). This variation in brain uptake is due to differences in the rate of plasma CL of the fusion protein. As described above in discussion of the Pharmacokinetic Rule, the brain uptake is directly proportional to the plasma AUC, which is inversely related to the rate of plasma CL of the fusion protein (Table 3). The plasma CL rates of the fusion proteins in the mouse differ by > 10-fold and range from 0.47 ml/min/kg for the cTfRMAb--ScFv fusion protein, to 5.9 ml/min/kg for the cTfRMAb--IDS fusion protein (Table 3). The differential plasma CL of the fusion proteins is due to different rates of CL of the fusion protein by peripheral tissues that is triggered by the therapeutic domain of the fusion protein. For example, lysosomal enzymes such as IDS are rapidly cleared from blood via the mannose 6-phosphate receptor in liver and spleen [63].

TfRMAb--avidin fusion proteins for targeted delivery of biotinylated agents 8.

Avidin (AV) or streptavidin binds biotin with extremely high affinity with a KD of 10-15 M, and a dissociation T1/2 of 3 months [67]. A TfRMAb--AV delivery system was initially developed by chemical cross-linking the AV to the TfRMAb [68], for BBB delivery of biotinylated agents such as peptide radiopharmaceuticals [69] or nucleic acid drugs [70]. Subsequently, a TfRMAb--AV fusion protein was engineered and expressed in myeloma cells, and tested in rats [71]. However, the problem in the expression of IgG--AV fusion proteins in eukaryotic systems in the presence of very high concentrations


9

W. M. Pardridge

A. TfRMAb

Avidin

H

H

S

HN

O

NH

Aβ (1 – 40) peptide

Biotin

B.

Brain uptake (% ID/g)


2.5 2 1.5

1 0.5 0

Figure 5. (A) Conjugate of a TfRMAb--avidin fusion protein and a mono-biotinylated Ab (1 -- 40) amyloid peptide radiopharmaceutical is shown. The biotin group on the peptide binds to the avidin domain of the fusion protein to form a high-affinity linkage between the TfRMAb molecular Trojan horse and the peptide radiopharmaceutical. (B) Brain uptake of [125I]-biotinyl Ab (1 -- 40) amyloid peptide following injection of labeled peptide either alone (open bars) or conjugated to the TfRMAb--AV fusion protein (closed bars). Data are mean ± SE (n = 3 mice per point). A: With permission from [1]. B: Reprinted with permission from [73]. Copyright 2011 American Chemical Society. AV: Avidin; TfR: Transferrin receptor.

of biotin in the cell culture medium. The IgG--AV fusion protein expressed in such systems is fully loaded with biotin, and owing to the very high affinity of AV binding of biotin, it is very difficult to dissociate the biotin from the fusion protein [72]. An IgG--AV fusion protein fully bound by biotin will have limited utility since > 90% of the biotin binding sites are saturated. Therefore, IgG--AV fusion proteins were expressed in stably transfected Chinese hamster ovary (CHO) cells, and these CHO cells were exposed to a biotindepletion program in culture for production of IgG--AV fusion proteins not saturated by biotin [72]. A fusion protein of AV and the cTfRMAb was also engineered and expressed in biotin-depleted CHO cells [73]. The BBB transport properties of this fusion protein in the mouse were investigated using mono-biotinylated Ab (1 -- 40) as the model pharmaceutical agent. The Ab-amyloid peptide of AD avidly binds the 10

amyloid plaque of AD, and is a potential peptide radiopharmaceutical for imaging of the amyloid burden in brain in AD [69]. The cTfRMAb--AV fusion protein was formulated in one vial, and the mono-biotinylated, radio-iodinated Ab (1 -- 40)-amyloid peptide was formulated in a second vial, as depicted in Figure 5A. The two vials were mixed prior to IV injection in the mouse [73]. Owing to the very high affinity of AV binding of biotin, the AV--biotin linkage connects the Ab peptide radiopharmaceutical to the cTfRMAb molecular Trojan horse. The Ab peptide alone does not cross the BBB in the mouse, as the brain uptake following IV injection is background, 0.1% ID/g (Figure 5B). However, the brain uptake of the Ab peptide radiopharmaceutical is high, 2.1% ID/g, following conjugation of the peptide to the cTfRMAb--AV BBB delivery system (Figure 5B). This level of brain uptake of the Ab peptide radiopharmaceutical is comparable to the brain uptake in the mouse of lipid-soluble small molecule Ab-amyloid imaging agents [73,74]. Therefore, the imaging of the amyloid burden in brain in AD need not be limited to lipid-soluble small molecules. BBB-penetrating peptide radiopharmaceuticals can be developed with the combined use of the TfRMAb molecular Trojan horse and AV--biotin technology. Such an approach could also be used to deliver nucleic acid drugs across the BBB [70].

BBB delivery with a low-affinity TfRMAb Trojan horse

9.

Engineering low-affinity bivalent TfRMAb The mouse TfR ECD was produced and used to isolate variable regions of an antibody against the mouse TfR [75]. These variable regions were fused to constant regions of a human IgG of unknown isotype to produce a TfRMAb designated anti-TfRA [75]. This antibody exhibited a high affinity for the mouse TfR, KD of 1.7 nM and a high brain uptake in the mouse of 3% ID/g. The genes encoding the antiTfRA and an antibody against b-secretase-1 (BACE1), a potential AD therapeutic, were then used to express an antiTfRA/anti-BACE1 BSA using knob-in-hole technology [75]. Knob-in-hole BSAs are monovalent for each antibody, and this monovalency causes low affinity of each antibody domain for each target antigen, including the TfR on the BBB. The KD of binding of the anti-TfRA/anti-BACE1 BSA for the mouse TfR was reduced 25-fold to 45 nM [75]. So as to test the value of a low-affinity TfRMAb Trojan horse, alanine mutagenesis of the CDRs of the anti-TfRA antibody was performed to produce a low-affinity antibody designated anti-TfRD, which exhibited a > 60-fold reduction in affinity for the mouse TfR, and a binding KD of 111 nM [75]. The anti-TfRD antibody was proposed as an improved Trojan horse over the anti-TfRA antibody based on measurements of brain uptake 24 h after IV injection of high doses, 20 mg/kg, of the high and low-affinity TfRMAb antibodies. Although the low-affinity TfRMAb did not show an improved brain uptake at 1 h after injection, the brain uptake 9.1




at 24 h was higher for the low-affinity TfRMAb, as compared to the high-affinity TfRMAb [75]. However, this is the expected result, given the very different binding properties of the low- and high-affinity TfRMAb. As discussed above in the context of the Pharmacokinetic Rule, the brain uptake is directly proportional to both the plasma AUC and the BBB PS product. An ID of 20 mg/kg will produce a plasma concentration of the TfRMAb that is many-fold greater than the KD of the high-affinity TfRMAb, and this will lead to saturation of the TfR by the high-affinity TfRMAb, but not for the low-affinity TfRMAb. The selective saturation of the TfR at the BBB by the high-affinity TfRMAb leads predictability to a reduction in PS product for the high-affinity TfRMAb that is not observed for the low-affinity TfRMAb. With respect to the plasma AUC component of the pharmacokinetic rule, the low-affinity TfRMAb will be cleared from plasma so slowly that the plasma AUC at 24 h for the low-affinity TfRMAb will be much higher than for the high-affinity TfRMAb. In summary, the injection of such a high dose of antibody, 20 mg/kg, selectively masks the BBB transport properties of the high-affinity TfRMAb, relative to the low-affinity TfRMAb. The use of a low-affinity TfRMAb commits the drug developer to the requirement for systemic ID that are at least a log order higher than IDs used for a high-affinity TfRMAb. As discussed below, such large IDs can induce off-target effects in peripheral tissues and reduce the therapeutic index of the TfRMAb. Engineering monovalent TfRMAb Gantenerumab is an AAA being developed as a treatment for the amyloid plaques of AD [76]. So as to enable BBB transport, this antibody was re-engineered as a BSA, where the other domain was composed of a monovalent single-chain TfRMAb [77]. The monovalent TfRMAb was low affinity, but was chosen over the high-affinity bivalent TfRMAb based on the hypothesis that the high-affinity TfRMAb causes downregulation of cell surface TfR [77]. While downregulation of the TfR may occur in cell culture [78], such downregulation is not observed in vivo following chronic treatment of mice with the TfRMAb (see next section on safety pharmacology). The problem with the monovalent, low-affinity TfRMAb is the very low brain uptake of this construct. The brain concentration of the monovalent TfRMAb is only 2 nM after the IV injection of 13.3 mg/kg [77], which indicates the brain uptake is < 0.1% ID/g in the mouse. This is a background level of brain uptake observed for TfRMAbs that have minimal penetration of the BBB in the mouse [14]. Future work may continue to explore the value of development of a TfRMAb that is either a low-affinity bivalent TfRMAb [75] or a low-affinity monovalent TfRMAb [77]. The use of a low-affinity TfRMAb as a BBB delivery system results in a reduced level of brain uptake (%ID/gram). The reduced brain uptake can be offset by increases in the ID of the fusion protein. For example, BSAs engineered with the low-affinity TfRMAb are administered to mice at systemic 9.2

doses of 25 -- 100 mg/kg [79], which are up to 100-fold higher than the 1 mg/kg therapeutic doses that are effective in mouse models of neural disease observed with a high-affinity TfRMAb [40,43,44,46,48,49]. The requirement to administer such large doses of a low-affinity TfRMAb fusion protein can induce off-target toxicity in peripheral organs and lower the therapeutic index of the fusion protein. For example, if a BSA was engineered from a low-affinity TfRMAb and adalimumab, which is a MAb against TNF-a, the dose of the adalimumab domain of the BSA administered for treatment of the brain would be so high as to exceed the dose of adalimumab that is currently used to treat chronic inflammation in peripheral tissues. The therapeutic dose of adalimumab is 0.5 -- 1 mg/kg for peripheral indications. Doses of a BBBpenetrating BSA that was > 10-fold higher, for example, > 10 mg/kg, might cause immunosuppression and other toxic effects caused by administration of such a large dose of adalimumab. Conversely, therapeutic effects with TfRMAb fusion proteins derived from a high-affinity TfRMAb are observed following the IV administration of the fusion protein at a dose of 1 mg/kg, without systemic toxicity [80].

Safety pharmacology of TfRMAb fusion proteins

10.

The single administration of the low-affinity TfRMAb, designated anti-TfRD, to mice causes severe lethargy within 5 min of administration, in association with hemolytic anemia, and elevated reticulocyte count [81]. The symptoms are not observed in the complement knockout mouse, indicating the acute toxicity of the anti-TfRD antibody is due to complement fixation [81], perhaps related to amino acid sequences within the human constant region used to engineer the antibody [75]. The acute toxicity caused by administration of the genetically engineered low-affinity TfRMAb [81] is not a general property of TfRMAbs. The safety of acute or chronic administration of cTfRMAb fusion proteins in the mouse was demonstrated by a study that examined potential toxicity following 12 weeks of twice-weekly IV injections of 2 mg/ kg/injection of the cTfRMAb--GDNF fusion protein [80]. The findings of the study are summarized in Table 4. No acute or chronic toxicity was observed in the mice, and no change in the serum iron level or total iron-binding capacity was observed [80]. The extent to which the TfR might be downregulated in vivo following 12 weeks of chronic treatment with the TfRMAb fusion protein was examined with an end-ofstudy PK and brain uptake evaluation [80]. If the TfR was downregulated in vivo, then the rate of plasma CL, and the rate of brain uptake, of the fusion protein would be suppressed. However, the plasma CL rate and the level of brain uptake of the TfRMAb fusion protein were unchanged after 12 weeks of chronic treatment [80]. In another study, the cTfRMAb--ScFv fusion protein was administered daily to mice by SQ injection for 12 consecutive weeks with no acute or chronic toxicity [55]. The extent to which anti-drug


11

W. M. Pardridge

Table 4. cTfRMAb--GDNF safety pharmacology: findings after 12 weeks of chronic treatment. Parameter

Result

Organ histology

No pathologic changes in brain, cerebellum, pancreas, kidney, liver, spleen or heart No change No change in 23 serum clinical chemistry parameters, including no change in serum iron or total iron-binding capacity Low (< 1 OD/µl) No change in plasma clearance or volume of distribution No change in brain uptake of cTfRMAb--GDNF fusion protein


Body weight Clinical chemistry

ADA titer Plasma pharmacokinetics Brain uptake

Data from [80]. ADA: Anti-drug antibodies; GDNF: Glial-derived neurotrophic factor; TfR: Transferrin receptor.

antibodies (ADA) form following chronic treatment of mice with TfRMAb fusion proteins has been examined with sandwich ELISAs designed to measure ADAs against the specific fusion protein [40,44,46,55,80]. The ADA titers are typically low, for example, < 1 OD/µl, and are not associated with any clinical signs of immune response following chronic treatment. In the case of a TfRMAb--GDNF fusion protein, the drug was administered IV twice-weekly for 12 weeks [80]. At the end of the study, the plasma ADA titer against the TfRMAb--GDNF fusion protein was low, and was not associated with any change in the rate of CL of the fusion protein from plasma, or the rate of fusion protein transport across the BBB [80]. In another study, a BSA, composed of a TfRMAb and a second antibody against the Ab-amyloid peptide of AD, was administered by daily SQ injection for 12 weeks, and this treatment resulted in only a low ADA titer, which was not associated with any clinical signs of immune response [55]. 11.

Expert opinion

There has been recent progress in the re-engineering of biologic drugs for brain penetration using monoclonal antibodies directed against endogenous receptors on the BBB such as the TfR or the insulin receptor [1]. The key finding in recent years is that almost any type of recombinant therapeutic protein, for example, a neurotrophin, a lysosomal enzyme, a therapeutic antibody, or a decoy receptor, can be re-engineered as a bifunctional BBB-penetrating IgG fusion protein [2]. By bifunctional, it is meant that: i) the affinity of the IgG fusion protein for the target BBB receptor is comparable to the affinity of the antibody prior to genetic fusion to the therapeutic protein; and ii) the pharmacologic activity of the IgG fusion protein is comparable to the pharmacologic activity of the

12

therapeutic protein prior to genetic fusion to the antibody. Some therapeutics such as peptides or nucleic acid drugs, for example, antisense agents or short-interfering RNA, cannot be re-engineered as an IgG fusion protein. However, peptides and antisense agents can still be delivered across the BBB with the combined use of BBB molecular Trojan horses and AV--biotin technology. In this approach, the peptide or antisense agent is mono-biotinylated, in parallel with the production of a Trojan horse--AV fusion protein. The development of a BBB penetrating antibody-based molecular Trojan horse enables the re-engineering of almost any class of recombinant protein as a BBB-penetrating IgG/ therapeutic drug fusion protein. Thus, it is not enough to discover a novel biologic drug that has promise to treat the brain. Following drug discovery, the therapeutic protein must be reengineered as a BBB-penetrating Trojan horse fusion protein. As the IgG fusion proteins enter clinical trials, future challenges will include the safety pharmacology and potential immunogenicity of the fusion protein in humans. Clinical trials will start soon in the treatment of lysosomal storage disorders with IgG--lysosomal enzyme fusion proteins. These fusion proteins have undergone extensive safety pharmacology studies in monkeys and have been shown to have acceptable safety profiles [82,83]. No adverse events were observed in monkeys at any dose of an IgG--enzyme fusion protein during the course of 6 months of treatment with weekly IV infusions [83]. The initial clinical trials will use a MAb against the HIR as the BBB Trojan horse [82,83]. However, biologic drugs could also be delivered to the human brain using a MAb against the human TfR. This is because the abundance of the HIR or the human TfR at the human BBB is comparable, based on radio-receptor assays using isolated human brain capillaries [8,84]. Recent BBB proteomics work using liquid chromatography/mass spectrometry shows the level of expression of the HIR or human TfR in isolated human brain capillaries is comparable [85]. Initial clinical trials will administer the IgG fusion protein via weekly IV infusions. However, for future treatment of neurodegenerative disease, it may be necessary to increase the frequency of drug administration to every other day or every day dosing. This is possible with SQ routes of administration of IgG fusion proteins formulated at high concentration, for example, 50 mg/ml. Other future challenges include the isolation of host cell lines of ever increasing levels of IgG fusion protein productivity, so that market demand can be met for diseases such as Alzheimer’s disease or Parkinson’s disease that afflict millions of patients worldwide.

Declaration of interest The author is consultant and shareholder in ArmaGen Technologies, Inc.



Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.


2.

Pardridge WM. Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab 2012;32(11):1959-72 Pardridge WM, Boado RJ. Reengineering biopharmaceuticals for targeted delivery across the blood-brain barrier. Methods Enzymol 2012;503:269-92

3.

Cheng Y, Zak O, Aisen P, et al. Structure of the human transferrin receptor-transferrin complex. Cell 2004;116(4):565-76

4.

Kawabata H, Yang R, Hirama T, et al. Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family. J Biol Chem 1999;274(30):20826-32

5.

Wallace DF, Summerville L, Lusby PE, Subramaniam VN. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin. Gut 2005;54(7):980-6

6.

Fleming RE, Migas MC, Holden CC, et al. Transferrin receptor 2: continued expression in mouse liver in the face of iron overload and in hereditary hemochromatosis. Proc Natl Acad Sci USA 2000;97(5):2214-19

7.

8.

.

9.

10.

.

Lai A, Sisodia SS, Trowbridge IS. Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain. J Biol Chem 1995;270(8):3565-73 Pardridge WM, Eisenberg J, Yang J. Human blood-brain barrier transferrin receptor. Metabolism 1987;36(9):892-5 Demonstration of high affinity transferrin receptor (TfR) on human brain capillary, in context of model of receptor-mediated transport of transferrin through the blood--brain barrier (BBB). Li JY, Boado RJ, Pardridge WM. Blood-brain barrier genomics. J Cereb Blood Flow Metab 2001;21(1):61-8 Jefferies WA, Brandon MR, Hunt SV, et al. Transferrin receptor on endothelium of brain capillaries. Nature 1984;312(5990):162-3 Demonstration of TfR on vasculature of rat brain with OX26 mouse MAb against rat TfR.

11.

12.

13.

.

14.

15.

16.

.

17.

.

18.

.

19.

Pardridge WM, Buciak JL, Friden PM. Selective transport of an anti-transferrin receptor antibody through the bloodbrain barrier in vivo. J Pharmacol Exp Ther 1991;259(1):66-70 Lesley J, Hyman R, Schulte R, Trotter J. Expression of transferrin receptor on murine hematopoietic progenitors. Cell Immunol 1984;83(1):14-25 Kissel K, Hamm S, Schulz M, et al. Immunohistochemical localization of the murine transferrin receptor (TfR) on blood-tissue barriers using a novel antiTfR monoclonal antibody. Histochem Cell Biol 1998;110(1):63-72 Demonstration of TfR on vasculature in mouse brain with 8D3 rat MAb against mouse TfR. Lee HJ, Engelhardt B, Lesley J, et al. Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse. J Pharmacol Exp Ther 2000;292(3):1048-52 White S, Taetle R, Seligman PA, et al. Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects. Cancer Res 1990;50(19):6295-301 Friden PM, Olson TS, Obar R, et al. Characterization, receptor mapping and blood-brain barrier transcytosis of antibodies to the human transferrin receptor. J Pharmacol Exp Ther 1996;278(3):1491-8 Delivery of MAb against human TfR to brain of cynomolgus monkey. Taylor EM, Crowe A, Morgan EH. Transferrin and iron uptake by the brain: effects of altered iron status. J Neurochem 1991;57(5):1584-92 Evidence for retro-endocytosis model of transferrin uptake by brain endothelium. Roberts RL, Fine RE, Sandra A. Receptor-mediated endocytosis of transferrin at the blood-brain barrier. J Cell Sci 1993;104(Pt 2):521-32 Evidence for retro-endocytosis model of transferrin uptake by brain endothelium. Zhang Y, Pardridge WM. Rapid transferrin efflux from brain to blood


.

across the blood-brain barrier. J Neurochem 2001;76(5):1597-600 Evidence for rapid efflux from brain to blood of apo-transferrin, which supports the transcytosis model of BBB transport of transferrin.

20.

Okamura T, Igarashi J, Kikuchi T, et al. A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain. Life Sci 2009;84(23-24):791-5

21.

Vorbrodt AW. Ultracytochemical characterization of anionic sites in the wall of brain capillaries. J Neurocytol 1989;18(3):359-68 Study shows abluminal receptors on the brain capillary endothelium cannot be detected with pre-embedding immune labeling of brain tissue.

.

22.

.

23.

.

24.

.

Huwyler J, Pardridge WM. Examination of blood-brain barrier transferrin receptor by confocal fluorescent microscopy of unfixed isolated rat brain capillaries. J Neurochem 1998;70(2):883-6 Demonstration of TfR on both luminal and abluminal plasma membranes of brain capillary endothelium. Fishman JB, Rubin JB, Handrahan JV, et al. Receptor-mediated transcytosis of transferrin across the blood-brain barrier. J Neurosci Res 1987;18(2):299-304 Arterial perfusion method provides evidence for transferrin transcytosis through the BBB. Skarlatos S, Yoshikawa T, Pardridge WM. Transport of [125I] transferrin through the rat blood-brain barrier. Brain Res 1995;683(2):164-71 Arterial perfusion method, combined with emulsion autoradiography and capillary depletion methods, provides evidence for transcytosis of either transferrin or TfRMAb through the BBB in vivo.

25.

Duffy KR, Pardridge WM. Blood-brain barrier transcytosis of insulin in developing rabbits. Brain Res 1987;420(1):32-8

26.

Bickel U, Kang YS, Yoshikawa T, Pardridge WM. In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary

13

W. M. Pardridge

.


27.

endothelium. J Histochem Cytochem 1994;42(11):1493-7 Electron microscopy of BBB transcytosis of TfRMAb. Gjedde A, Christensen O. Estimates of Michaelis-Menten constants for the two membranes of the brain endothelium. J Cereb Blood Flow Metab 1984;4(2):241-9

28.

Pardridge WM. Brain drug targeting. CUP, Cambridge; 2001

29.

Fillebeen C, Descamps L, Dehouck MP, et al. Receptor-mediated transcytosis of lactoferrin through the blood-brain barrier. J Biol Chem 1999;274(11):7011-17

30.

31.

37.

14

.

Boado RJ, Hui EK, Lu JZ, et al. Reversal of lysosomal storage in brain of adult MPS-I mice with intravenous trojan horse-iduronidase fusion protein. Mol Pharm 2011;8(4):1342-50 Demonstration of reduction in lysosomal inclusion bodies in brain following systemic injection of lysosomal enzyme re-engineered for BBB transport with TfRMAb.

Ji B, Maeda J, Higuchi M, et al. Pharmacokinetics and brain uptake of lactoferrin in rats. Life Sci 2006;78(8):851-5

42.

Boado RJ, Zhou QH, Lu JZ, et al. Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor. Mol Pharm 2010;7(1):237-44

43.

Fu A, Zhou QH, Hui EK, et al. Intravenous treatment of experimental Parkinson’s disease in the mouse with an IgG-GDNF fusion protein that penetrates the blood-brain barrier. Brain Res 2010;1352:208-13

Pan W, Kastin AJ, Zankel TC, et al. Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier. J Cell Sci 2004;117(Pt 21):5071-8

36.

40.

Zhou QH, Boado RJ, Hui EK, et al. Brain-penetrating tumor necrosis factor decoy receptor in the mouse. Drug Metab Dispos 2011;39(1):71-6

Zhou QH, Boado RJ, Lu JZ, et al. Brain-penetrating IgG-iduronate 2sulfatase fusion protein for the mouse. Drug Metab Dispos 2012;40(2):329-35

33.

.

39.

Zhou QH, Boado RJ, Lu JZ, et al. Re-engineering erythropoietin as an IgG fusion protein that penetrates the bloodbrain barrier in the mouse. Mol Pharm 2010;7(6):2148-55

41.

Demeule M, Poirier J, Jodoin J, et al. High transcytosis of melanotransferrin (p97) across the blood-brain barrier. J Neurochem 2002;83(4):924-33

35.

38.

Pardridge WM, Triguero D, Yang J, Cancilla PA. Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier. J Pharmacol Exp Ther 1990;253(2):884-91

32.

34.

mouse. Drug Metab Dispos 2010;38(4):566-72

Richardson DR, Morgan EH. The transferrin homologue, melanotransferrin (p97), is rapidly catabolized by the liver of the rat and does not effectively donate iron to the brain. Biochim Biophys Acta 2004;1690(2):124-33 McGrath JP, Cao X, Schutz A, et al. Bifunctional fusion between nerve growth factor and a transferrin receptor antibody. J Neurosci Res 1997;47(2):123-33 Genetic engineering and testing of fusion protein of TfRMAb and nerve growth factor. Boado RJ, Pardridge WM. Genetic engineering of IgG-glucuronidase fusion proteins. J Drug Target 2010;18(3):205-11 Zhou QH, Boado RJ, Lu JZ, et al. Monoclonal antibody-glial-derived neurotrophic factor fusion protein penetrates the blood-brain barrier in the

44.

Zhou QH, Hui EK, Lu JZ, et al. Brain penetrating IgG-erythropoietin fusion protein is neuroprotective following intravenous treatment in Parkinson’s disease in the mouse. Brain Res 2011;1382:315-20

45.

Elliott S, Pham E, Macdougall IC. Erythropoietins: a common mechanism of action. Exp Hematol 2008;36(12):1573-84

46.

Zhou QH, Sumbria R, Hui EK, et al. Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor. J Pharmacol Exp Ther 2011;339(2):618-23 Demonstration of neuroprotection in Parkinson model with BBB penetrating biologic tumor necrosis factor inhibitor.

.

47.

Boado RJ, Hui EK, Lu JZ, et al. Selective targeting of a TNFR decoy


receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein. J Biotechnol 2010;146(1-2):84-91 48.

Sumbria RK, Boado RJ, Pardridge WM. Brain protection from stroke with intravenous TNF-alpha decoy receptortrojan horse fusion protein. J Cereb Blood Flow Metab 2012;32(10):1933-8

49.

Sumbria RK, Boado RJ, Pardridge WM. Combination stroke therapy in the mouse with blood-brain barrier penetrating IgG-GDNF and IgG-TNF decoy receptor fusion proteins. Brain Res 2013;1507:91-6

50.

Naslund J, Haroutunian V, Mohs R, et al. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA 2000;283(12):1571-7

51.

Salloway S, Sperling R, Fox NC, et al. Two phase 3 trials of bapineuzumab in mild-to-moderate Alzheimer’s disease. N Engl J Med 2014;370(4):322-33

52.

Boado RJ, Zhang Y, Zhang Y, et al. Fusion antibody for Alzheimer’s disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation. Bioconjug Chem 2007;18(2):447-55 Tetravalent bispecific antibody formed by fusion of a transporter antibody and a therapeutic antibody.

.

53.

.

Schlageter NL, Carson RE, Rapoport SI. Examination of blood-brain barrier permeability in dementia of the Alzheimer type with [68Ga]EDTA and positron emission tomography. J Cereb Blood Flow Metab 1987;7(1):1-8 The BBB is intact in Alzheimers disease.

54.

Seubert P, Barbour R, Khan K, et al. Antibody capture of soluble Abeta does not reduce cortical abeta amyloidosis in the PDAPP mouse. Neurodegener Dis 2008;5(2):65-71

55.

Sumbria RK, Hui EK, Lu JZ, et al. Disaggregation of amyloid plaque in brain of Alzheimer’s disease transgenic mice with daily subcutaneous administration of a tetravalent bispecific antibody that targets the transferrin receptor and the Abeta amyloid peptide. Mol Pharm 2013;10(9):3507-13 Daily administration of TfRMAb fusion protein to mice for 12 weeks has no toxicity, and produces desired

.


Proc Natl Acad Sci USA 1991;88(21):9695-9

therapeutic effect in mouse model of Alzheimers disease. Wilcock DM, Alamed J, Gottschall PE, et al. Deglycosylated anti-amyloid-beta antibodies eliminate cognitive deficits and reduce parenchymal amyloid with minimal vascular consequences in aged amyloid precursor protein transgenic mice. J Neurosci 2006;26(20):5340-6

66.

57.

Jancso G, Domoki F, Santha P, et al. Beta-amyloid (1-42) peptide impairs blood-brain barrier function after intracarotid infusion in rats. Neurosci Lett 1998;253(2):139-41

68.

58.

Su GC, Arendash GW, Kalaria RN, et al. Intravascular infusions of soluble beta-amyloid compromise the bloodbrain barrier, activate CNS glial cells and induce peripheral hemorrhage. Brain Res 1999;818(1):105-17

Yoshikawa T, Pardridge WM. Biotin delivery to brain with a covalent conjugate of avidin and a monoclonal antibody to the transferrin receptor. J Pharmacol Exp Ther 1992;263(2):897-903

69.

Saito Y, Buciak J, Yang J, Pardridge WM. Vector-mediated delivery of 125I-labeled beta-amyloid peptide Abeta1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the Abeta1-40/vector complex. Proc Natl Acad Sci USA 1995;92(22):10227-31


56.

59.

60.

.

61.

.

62.

63.

64.

65.

Black RS, Sperling RA, Safirstein B, et al. A single ascending dose study of bapineuzumab in patients with Alzheimer disease. Alzheimer Dis Assoc Disord 2010;24(2):198-203 Schlachetzki F, Zhu C, Pardridge WM. Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier. J Neurochem 2002;81(1):203-6 Presence of Fc receptor (FcRn) at the BBB. Zhang Y, Pardridge WM. Mediated efflux of IgG molecules from brain to blood across the blood-brain barrier. J Neuroimmunol 2001;114(1-2):168-72 IgG molecules are rapidly effluxed across the BBB from brain to blood via a FcR. Cheng SH, Smith AE. Gene therapy progress and prospects: gene therapy of lysosomal storage disorders. Gene Ther 2003;10(16):1275-81 Boado RJ, Hui EK, Lu JZ, et al. Blood-brain barrier molecular Trojan horse enables imaging of brain uptake of radioiodinated recombinant protein in the rhesus monkey. Bioconjug Chem 2013;24(10):1741-9 Wraith JE, Scarpa M, Beck M, et al. Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur J Pediatr 2008;167(3):267-77 Scott HS, Anson DS, Orsborn AM, et al. Human alpha-L-iduronidase: cDNA isolation and expression.

67.

Wilson PJ, Morris CP, Anson DS, et al. Hunter syndrome: isolation of an iduronate-2-sulfatase cDNA clone and analysis of patient DNA. Proc Natl Acad Sci USA 1990;87(21):8531-5 Green NM. Avidin. Adv Protein Chem 1975;29:85-133

70.

Pardridge WM, Boado RJ, Kang YS. Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo. Proc Natl Acad Sci USA 1995;92(12):5592-6

71.

Shin SU, Wu D, Ramanathan R, et al. Functional and pharmacokinetic properties of antibody-avidin fusion proteins. J Immunol 1997;158(10):4797-804

72.

Boado RJ, Zhang Y, Zhang Y, et al. Genetic engineering, expression, and activity of a chimeric monoclonal antibody-avidin fusion protein for receptor-mediated delivery of biotinylated drugs in humans. Bioconjug Chem 2008;19(3):731-9

73.

.

74.

Zhou QH, Lu JZ, Hui EK, et al. Delivery of a peptide radiopharmaceutical to brain with an IgG-avidin fusion protein. Bioconjug Chem 2011;22(8):1611-18 Brain uptake of peptide radiopharmaceuticals, mediated by TfRMAb Trojan horse, is comparable to brain uptake of lipid soluble small molecule radiopharmaceuticals. Ono M, Cheng Y, Kimura H, et al. Novel 18F-labeled benzofuran derivatives with improved properties for positron emission tomography (PET) imaging of beta-amyloid plaques in Alzheimer’s brains. J Med Chem 2011;54(8):2971-9


75.

.

Yu YJ, Zhang Y, Kenrick M, et al. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med 2011;3(84):84ra44 Production of low affinity, bivalent TfRMAb and knob-in-hole bispecific antibody (BSA).

76.

Bohrmann B, Baumann K, Benz J, et al. Gantenerumab: a novel human anti-abeta antibody demonstrates sustained cerebral amyloid-beta binding and elicits cellmediated removal of human amyloid-beta. J Alzheimers Dis 2012;28(1):49-69

77.

Niewoehner J, Bohrmann B, Collin L, et al. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron 2014;81(1):49-60 Production of low affinity, monovalent single chain TfRMAb and BSA.

.

78.

Lesley J, Schulte R, Woods J. Modulation of transferrin receptor expression and function by antitransferrin receptor antibodies and antibody fragments. Exp Cell Res 1989;182(1):215-33

79.

Atwal JK, Chen Y, Chiu C, et al. A therapeutic antibody targeting BACE1 inhibits amyloid-beta production in vivo. Sci Transl Med 2011;3(84):84ra43

80.

Zhou QH, Boado RJ, Hui EK, et al. Chronic dosing of mice with a transferrin receptor monoclonal antibody-glial-derived neurotrophic factor fusion protein. Drug Metab Dispos 2011;39(7):1149-54 Chronic administration of TfRMAb fusion protein in mice has no toxicity; end-of-study brain uptake analysis shows no down-regulation of BBB TfR in vivo.

.

81.

.

82.

Couch JA, Yu YJ, Zhang Y, et al. Addressing safety liabilities of TfR bispecific antibodies that cross the bloodbrain barrier. Sci Transl Med 2013;5(183):183ra157; 181-112 Certain TfRMAbs that activate complement cause acute toxicity in mice. Boado RJ, Hui EK, Lu JZ, Pardridge WM. IgG-enzyme fusion protein: pharmacokinetics and anti-drug antibody response in rhesus monkeys. Bioconjug Chem 2013;24(1):97-104

15

W. M. Pardridge

83.

Pardridge WM, Eisenberg J, Yang J. Human blood-brain barrier insulin

receptor. J Neurochem 1985;44(6):1771-8 85.

.

Uchida Y, Ohtsuki S, Katsukura Y, et al. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J Neurochem 2011;117(2):333-45 Mass spectrometry and isolated brain capillaries used to quantitate insulin receptor and TfR at human BBB.


84.

Boado RJ, Hui EK, Lu JZ, Pardridge WM. Insulin receptor antibody-iduronate 2-sulfatase fusion protein: pharmacokinetics, anti-drug antibody, and safety pharmacology in rhesus monkeys. Biotechnol Bioeng 2014. [Epub ahead of print]

16


Affiliation William M Pardridge ArmaGen Technologies, Inc., 26679 Agoura Road, Calabasas, CA 91302, USA Tel: +1 818 252 8202; Fax: +1 818 252 8214; E-mail: [email protected]

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain.

14C1, an antigen associated with human ovarian cancer, defined using a human IgG monoclonal antibody.

Pulmonary monoclonal antibody delivery via a portable microfluidic nebulization platform.

HSA nanocapsules functionalized with monoclonal antibodies for targeted drug delivery.

Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.

Transient monoclonal proteins in drug hypersensitivity reactions.

A new human fusion partner, HK-128, for making human-human hybridomas producing monoclonal IgG antibodies.

Therapeutic evaluation of monoclonal antibody-maytansinoid conjugate as a model of RON-targeted drug delivery for pancreatic cancer treatment.

Mucus as a barrier to drug delivery – understanding and mimicking the barrier properties.

Insulin receptor antibody-sulfamidase fusion protein penetrates the primate blood-brain barrier and reduces glycosoaminoglycans in Sanfilippo type A cells.

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.

A monoclonal anti-peptide antibody recognizes the adrenocorticotropic receptor.

Monoclonal antibody to GABA binding protein, a possible GABAB receptor.

The effects of transferrin receptor antibody, transferrin receptor antibody bound to Pseudomonas exotoxin and transforming growth factor-alpha bound to Pseudomonas exotoxin on human tenon's capsule fibroblast proliferation.

Production and characterization of a monoclonal antibody to dopamine D2 receptor: comparison with a polyclonal antibody to a different epitope.

Preferential generation of monoclonal IgG-producing hybridomas by use of vesicular stomatitis virus-mediated cell fusion.

Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants.

FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

Improving the systemic drug delivery efficacy of nanoparticles using a transferrin variant for targeting.

Evaluation of antibody-chemokine fusion proteins for tumor-targeting applications.

Insulin receptor antibody-iduronate 2-sulfatase fusion protein: pharmacokinetics, anti-drug antibody, and safety pharmacology in Rhesus monkeys.

Immobilized transferrin Fe3O4@SiO2 nanoparticle with high doxorubicin loading for dual-targeted tumor drug delivery.