Anti-transferrin antibody-drug barrierantibody-drug conjugates cross the BBB. MATERIALSANDMETHODS Preparation ofAntibodyandAntibody Conjugates. OX-26 ... the anti-transferrin receptor

Proc. Natl. Acad. Sci. USAVol. 88, pp. 4771-4775, June 1991Medical Sciences

Anti-transferrin receptor antibody and antibody-drug conjugatescross the blood-brain barrier

(drug delivery/methotrexate)

PHILLIP M. FRIDEN*, LEE R. WALUS, GARY F. Musso,AND RUTH M. STARZYKAlkermes, Inc., 26 Landsdowne Street, Cambridge, MA 02139

Communicated by Hilary Koprowski, February 19, 1991

ABSTRACT Delivery of nonlipophilic drugs to the brain ishindered by the tightly apposed capillary endothelial cells thatmake up the blood-brain barrier. We have examined the abilityof a monoclonal antibody (OX-26), which recognizes the rattransferrin receptor, to function as a carrier for the delivery ofdrugs across the blood-brain barrier. This antibody, whichwas previously shown to bind preferentially to capillary endo-thelial cells in the brain after intravenous administration(Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams,A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London)312, 162-163), labels the entire cerebrovascular bed in adose-dependent manner. The initially uniform labeling ofbraincapillaries becomes extremely punctate =4 hr after injection,suggesting a time-dependent sequestering of the antibody.Capillary-depletion experiments, in which the brain is sepa-rated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capil-laries into the brain parenchyma, which is consistent with thetranscytosis of compounds across the blood-brain barrier.Antibody-methotrexate conjugates were tested in vivo to assessthe carrier ability of this antibody. Immunohistochemicalstaining for either component of an OX-26-methotrexate con-jugate revealed patterns of cerebrovascular labeling identicalto those observed with the unaltered antibody. Accumulation ofradiolabelod methotrexate in the brain parenchyma is greatlyenhanced when the drug is conjugated to OX-26.

The levels of various substances in the blood, such ashormones, amino acids, and ions, undergo frequent smallfluctuations that can be brought about by activities such aseating and exercise (1). If the brain were not protected fromthese variations in serum composition, the result could beuncontrolled neural activity. The blood-brain barrier (BBB)functions to ensure that the homeostasis of the brain ismaintained. Specialized characteristics of the endothelialcells that form brain capillaries are responsible for this barrier(1, 2). Brain capillary endothelial cells are joined together bytight intercellular junctions that form a continuous wallagainst the passive movement of substances from the bloodto the brain (3, 4). These cells lack continuous gaps orchannels connecting the luminal and abluminal membranes,which, in other endothelial cells, allow relatively unrestrictedpassage of blood-borne molecules into tissue.The isolation of the brain from the bloodstream is not

complete; were this the case, the brain would be unable tofunction properly due to a lack ofnutrients and because oftheneed to exchange hormones and other compounds with therest of the body. The presence of specific transport systemswithin the capillary endothelial cells, such as those for aminoacids, transferrin, glucose, and insulin (2, 5-8), assures that

MARJORIE A. TAYLOR, BERNARD MALFROY,

the brain receives, in a controlled manner, all of the com-pounds required for normal growth and function. For thetransferrin receptor, it has been reported (9) that brainendothelial cells are selectively labeled by anti-receptorantibodies in the rat after in vivo administration, presumablydue to a high density of transferrin receptors on the surfaceof brain capillary endothelial cells.A problem posed by the BBB is that, in the process of

protecting the brain, it also excludes many potentially usefultherapeutic agents. Currently, only substances that are suf-ficiently lipophilic or are recognized by an existing transportsystem can readily penetrate the BBB (1, 2). The lipidizationof drugs to enhance brain uptake is very nonspecific, in thatit will increase the ability of the particular compound to crossall cellular membranes, and is not possible for many com-pounds. A carrier system that has some degree of organselectivity and that could be used for a wide range ofcompounds would have a significant advantage over currentmethods for circumventing the BBB. Such a carrier systemcould be developed by exploiting a transport system knownto deliver compounds across the BBB, such as that fortransferrin.We report experimental results that suggest that anti-

transferrin receptor antibodies may have utility as drugdelivery carriers for the brain. This delivery system takesadvantage ofthe high density oftransferrin receptors on brainendothelial cells as well as the ability of these receptors toshuttle molecules across the BBB. Both qualitative andquantitative experiments show that, in the rat, the anti-transferrin receptor antibody OX-26 and antibody-meth-otrexate (MTX) conjugates bind to capillary endothelial cellsin the brain in a dose- and time-dependent manner. Inaddition, we present data that indicate that this antibody andantibody-drug conjugates cross the BBB.

MATERIALS AND METHODSPreparation of Antibody and Antibody Conjugates. OX-26

antibodies were purified from supernatants harvested fromcultures of the OX-26 hybridoma cell line [provided by AlanF. Williams, Medical Research Council, Oxford, U.K. (10)]by using protein A-Sepharose column chromatography. Thecontrol IgG2a antibody (UPC10) was purchased from Sigma.A hydrazone-linked conjugate between MTX and OX-26

was synthesized by incubating MTX t-hydrazide with anti-body that had been oxidized with sodium periodate to formaldehydes from the carbohydrate groups located on the Fcportion of the protein (G.F.M., S. Abelliera, and A. Morrow,unpublished data and ref. 11). The conjugate, which con-sisted of approximately six MTX molecules per antibody,was analyzed as described (12). The same procedure was

Abbreviations: BBB, blood-brain barrier; MTX, methotrexate.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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followed for synthesizing [3H]MTX-OX-26 conjugates, using[3',5',7-3H]MTX (Amersham).Immunohistochemistry. Female Sprague-Dawley rats

(100-125 g) were anesthetized with halothane for tail veininjections. At sacrifice, the animals were perfused withphosphate-buffered saline (PBS) to remove blood from thevasculature. The brains were frozen in liquid nitrogen forcryostat sectioning. Sections (-30 Im) were fixed in acetoneat room temperature and stored at -20TC. Immunolocaliza-tion of injected OX-26 in the brain sections was performedusing a horse anti-mouse IgG Vectastain ABC kit fromVector Laboratories by following the manufacturer's proto-col. MTX was detected using a rabbit anti-MTX antisera fromWestern Chemical Research and a biotinylated goat anti-rabbit IgG antisera from Vector Laboratories. All sectionswere counterstained with methyl green (0.05% in PBS).

Capillary Depletion. Antibodies were labeled with either[14C]acetic anhydride or [3H]succinimidyl propionate (Am-ersham) essentially as described (13, 14). For 14C, the numberof acetyl groups per antibody was in the range of 5 to 10; for3H, less than one propionyl group was added per antibody.Average specific activities were -10 Ci/mmol and 280 mCi/mmol (1 Ci = 37 GBq) for 3H and 14C, respectively. Rou-tinely, 1 x 106 dpm of3H and 5 x 105 dpm of 14C were injectedper animal. For all experiments, the radiolabeled compoundswere injected as a 400-Al bolus into the tail vein of femaleSprague-Dawley rats (100-125 g) under halothane anesthesiaand the animals were sacrificed at the appropriate time afterinjection using a lethal dose of anesthetic. A radiolabeledIgG2a control antibody (14C for 3H-labeled OX-26, or viceversa) was co-injected with the labeled OX-26 to serve as acontrol for nonspecific radioactivity in the brain due toresidual blood.

Capillary depletion experiments were performed essen-tially as described by Triguero et al. (15). This methodremoves >90% of the vasculature from the brain homoge-nate. The entire capillary pellet and samples of the homog-enate and supernatant were solubilized overnight with 2 ml ofSoluene 350 (Packard) prior to liquid scintillation counting.All data were collected as dpm by using a Beckman modelTD5000 liquid scintillation counter. Data are expressed aspercent of the injected dose per brain of the antibody in eitherthe parenchyma or capillary fractions. Blood samples werecentrifuged to pellet erythrocytes (which did not displaysignificant binding of radiolabeled materials) and the radio-activity in a sample of serum was determined using liquidscintillation counting.

RESULTSDose- and Time-Dependent Localization of an Anti-

Transferrin Receptor Antibody to Brain Capillaries in Vivo.Immunohistochemistry was used to localize the anti-transferrin receptor antibody OX-26 in the rat brain vascu-lature after intravenous injection by the tail vein as shown byJefferies et al. (9). One hour after injection of 0.5 mg ofpurified antibody, uniform intense staining of the capillarieswas observed throughout the brain (Fig. LA). Larger bloodvessels as well as capillaries showed the presence of boundantibody. This staining of the brain vasculature was clearlydetected at doses as low as 50,g per rat and appeared tosaturate at a dose of -0.5 mg per rat (data not shown). Asreported by Jefferies et al. (9), specific staining of capillarieswas not observed in any of the other organs that wereexamined (data not shown).

FIG. 1. Immunohistochemical detection of OX-26 in brain sections. Sections were cut from brains taken from rats sacrificed 1 hr (A) and4 hr (B) after injection of 0.5 mg of OX-26 into the tail vein. The tissue sections were stained with horse anti-mouse IgG. The brown reactionproduct outlines the cerebral vasculature. The punctate staining pattern is clearly visible 4 hr after injection (B). A methyl green counterstainwas used to label cell nuclei. (A, xlO00; B. x400.)

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To determine how early after injection the anti-transferrinreceptor antibody could be detected in the brain capillaries aswell as how long it persisted, a time course experiment wasperformed. Rats were injected with 0.5 mg of OX-26 peranimal and sacrificed at various times after the injection.Antibody was detected immunohistochemically within thebrain capillaries as early as 5 min after injection (data notshown). The staining observed at this time was uniformlydistributed along the capillary endothelium. By 1 hr afterinjection, the observed staining for antibody along the braincapillaries had become more intense (Fig. LA) and, as for the5-min time point, was quite uniform. By =4 hr after injection,the staining pattern of OX-26 in the brain vasculaturechanged dramatically and became very punctate (Fig. 1B),suggesting sequestration of the antibody in some manner.The pattern of localization was still punctate 8 hr afterinjection but returned to a uniform, fainter staining by 24 hrafter injection (data not shown).To ensure that the observed staining was unique to the

OX-26 antibody, a control antibody (UPC10) of the samesubclass (IgG2a) was injected into rats for immunohisto-chemical localization. No staining of brain vasculature wasobserved with the control IgG2a at doses and times compa-rable to those used with OX-26 (data not shown).

Distribution ofOX-26 in Brain Parenchyma and Capillaries.The above results show qualitatively that the anti-transferrinreceptor antibody OX-26 is present in the brain vasculatureafter i.v. administration. To demonstrate quantitatively thatthe anti-transferrin receptor antibody accumulates in thebrain parenchyma, homogenates of brains taken from ani-mals injected with radiolabeled OX-26 were depleted ofcapillaries by centrifugation through dextran to yield a braintissue supernatant and a capillary pellet. A comparison of therelative amounts of radioactivity in the different brain frac-tions as a function oftime should indicate whether the labeledmonoclonal antibody has crossed the BBB. Because thevasculature remains intact during the capillary depletionprocedure, the antibody that reaches the parenchyma hastraversed the basement membrane and perivascular cellsassociated with the capillaries. As a control for nonspecificassociation with the brain due to residual blood contamina-tion, a 14C-labeled IgG2a control antibody was co-injectedwith the 3H-labeled OX-26.The amounts of OX-26 in the brain parenchyma fraction

and in the brain capillary fraction plotted as a function oftimeafter injection are shown in Fig. 2. Initially, the amount ofOX-26 associated with the brain capillaries rises sharply,reaching a level of -0.6% of the injected dose by 1 hr afterinjection. This level then decreases over time, dropping to0.13% of the injected dose by 24 hr after injection. As theamount of antibody associated with the capillaries decreases,the amount associated with the brain parenchyma increases,reaching a value of 0.44% of the injected dose by 24 hr afterinjection. This redistribution of the radiolabeled OX-26 fromthe capillary fraction to the parenchyma fraction is consistentwith the time-dependent migration of the anti-transferrinreceptor antibody across the BBB.To address the question of stability of the radiolabeled

carrier in the circulation, total IgG was extracted from serumfollowed by polyacrylamide gel electrophoresis and autora-diography. This analysis did not reveal detectable levels ofOX-26 degradation as late as 48 hr after injection (data notshown).

Localization of OX-26-MTX Conjugates in the Brain Vas-culature. Ifan anti-transferrin receptor antibody is to functionas a drug carrier, it must retain its ability to bind to and beinternalized by brain capillary endothelial cells after conju-gation with drug. To test for targeting and binding to braincapillaries in vivo, an OX-26-MTX conjugate (-6 MTX per 1antibody) was prepared using a hydrazone linkage. One hour

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FIG. 2. Time-dependent changes in the disposition of 3H-labeledOX-26 between brain parenchyma and vasculature. Capillary deple-tion was performed on homogenates prepared from brains taken fromanimals 1, 4, and 24 hr after injection of radiolabeled OX-26. Thepercent injected dose of antibody per brain in the parenchymafraction (o) and vascular pellet (o) are shown. Similar results wereobtained using 14C-labeled OX-26 (data not shown). The valuesshown are mean ± SEM (n = 3 rats per time point). These results arerepresentative of other studies that have been done.

after its injection into the rat tail vein, the brain was removedand sectioned for immunohistochemistry as for the studiesdescribed above. Both the antibody carrier and the MTX"passenger" were visualized in the brain using either anti-mouse IgG or anti-MTX antisera.A staining pattern similar to that seen with OX-26 alone

was revealed when sections from the conjugate-injectedanimals were stained for the carrier antibody (Fig. 3A). Asimilar pattern was seen when these sections were stainedwith anti-MTX antisera (Fig. 3B), indicating colocalization ofOX-26 and MTX in the brain vasculature.When equivalent amounts of free MTX were injected into

rats, no staining of the brain vasculature was observed usingthe anti-MTX antisera, indicating that localization ofthe drugto the brain capillaries is dependent on OX-26 (data notshown). Also, no staining was observed when the anti-MTXantiserum was used on sections containing only OX-26.

Distribution of the OX-26-MTX Coajugate in Brain Paren-chyma and Capillaries. Capillary-depletion studies identicalto those described above for OX-26 were performed with anOX-26-MTX conjugate in which the MTX moiety was labeledwith 3H at the 3', 5', and 7 positions. As with unconjugatedantibody, the amount of label in the capillary fraction at anearly time after injection (30 min for these experiments) wasgreater than that in the parenchyma fraction (Fig. 4). Thisdistribution changed over time such that by 24 hr afterinjection, -5-fold more of the labeled MTX was in the brainparenchyma than was in the capillaries. These results areconsistent with those obtained with unconjugated antibodyand suggest that the OX-26-MTX conjugate crosses the BBB.To ensure that these results were not due to contaminating

amounts of free [3H]MTX or [3H]MTX that had been cleavedfrom the conjugate after injection, a mixture of labeled drugand antibody was injected into rats and a capillary-depletionexperiment was performed. The amount of [3H]MTX in thedifferent brain fractions was significantly lower for the mix-ture as compared to the conjugate (as much as 47-fold in thecapillary fraction 30 min after injection; Fig. 4). The

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A -~~~~~~~~~~~~~~~~~~~~A

iAjf 4.'~ ;'~'' i A

FIG. 3. Immunohistochemical detection of an OX-26-MTX conjugate in brain sections. Horse anti-mouse IgG (A) and rabbit anti-MTX (B)antisera were used to detect the carrier antibody and the passenger drug, respectively, in the brain vasculature after i.v. administration of 0.2mg of OX-26-MTX conjugate. Immunohistochemistry was as described in Fig. 1. (x100.)

[3H]MTX in the mixture also did not show the change indistribution of the label between the various brain fractionsover time as was seen with the antibody-MTX conjugate orantibody alone (Figs. 2 and 4).

DISCUSSIONThe results presented herein indicate that an antibody whichrecognizes an extracellular epitope ofthe transferrin receptorcrosses the BBB and can be used to deliver the chemother-apeutic drug MTX to the brain after intravenous administra-tion. Initially, immunohistochemistry was used to corrobo-rate and elaborate on earlier studies by Jefferies et al. (9)demonstrating that anti-transferrin receptor antibodies bindpreferentially to vascular endothelial cells within the brain.This phenomenon is most likely attributable to a higherdensity of these receptors on the endothelial cells that makeup the BBB as compared to those in other capillary beds.Physiologically, this is consistent with the fact that theprimary, if not only, pathway for iron to enter the brain is bythe transcytosis ofiron-loaded transferrin across the BBB (2,7, 8).Immunohistochemical staining of brain sections taken

from animals sacrificed at various times after injection showsthat the localization of OX-26 in the brain vasculaturechanges with time. A very punctate pattern of staining isobserved from o2 to 4 hr to -8 to 16 hr after injection ascompared to the more uniform pattern observed before andafter this period. This most likely represents endocytosis andconcentration ofthe antibody in an intracellular compartmentfollowed by eventual exocytosis. One hour after injection bythe carotid artery, ferrotransferrin-horseradish peroxidaseconjugates have been observed to label perivascular cleftsand cells, apparently as a result of crossing the endothelial

cell layer (16). The time frame in which the OX-26 punctatestaining occurs is consistent with uptake by perivascularcells, indicating BBB penetration.Although suggestive, the above results do not demonstrate

that the antibody crosses the BBB as opposed to simply

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FIG. 4. Enhanced delivery ofMTX across the BBB using OX-26as a drug carrier. Capillary depletion experiments were used tocompare the distribution of an OX-26-MTX conjugate to that of amixture of antibody and drug. The data are expressed as percent ofthe injected dose of 3H-labeled MTX per brain in unfractionatedhomogenate, the parenchyma fraction, and the capillary fraction.The values shown are mean + SEM (n = 3 rats per time point). Theseresults are representative of other studies that have been done.

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binding to the luminal surface of the capillary endothelialcells. To address these concerns as well as to obtain morequantitative data, OX-26 was radioactively labeled and itsdistribution within the brain was determined using the tech-nique of capillary depletion as developed by Triguero et al.(15). The time-dependent changes that were observed in thedistribution of radiolabeled OX-26 within the vascular andparenchymal fractions ofthe brain are consistent with modelsfor the transcytosis of blood-borne proteins across the BBB(2, 17). This pathway consists of (i) binding of the blood-borne compound to a receptor on the luminal side of thecapillary endothelial cell, (it) engulfment of occupied recep-tors into endocytic vesicles and transport of the vesicularcontents to the abluminal surface of the cell, and (iOh) exo-cytosis ofthe internalized materials on the abluminal surface.There is precedence for the binding of anti-transferrin recep-tor antibodies to the receptor on the cell surface with sub-sequent internalization of the antibody-receptor complex(18), suggesting that steps i and ii of the transcytosis pathwaydescribed above can occur. With regard to the release of theantibody on the abluminal surface of endothelial cells, it hasbeen demonstrated that certain antibodies to the low densitylipoprotein receptor-related protein undergo internalization,escape degradation in the lysosome, and recycle to the cellsurface where they dissociate from the receptor in an acid-precipitable form (19). A similar pathway may be followed bythe anti-transferrin receptor antibodies in traversing the braincapillary endothelial cells.The use of radiolabeled antibody in the capillary depletion

experiments also allows the quantitation of the amount ofcarrier in the different brain fractions at specific times.However, because the brain is a dynamic system with uptakefrom the blood and clearance to the cerebrospinal fluid andthe blood occurring concurrently, these calculations mostlikely underestimate the total amount of antibody thatreaches the brain parenchyma from the circulation.

Conjugates of OX-26 and MTX were synthesized andtested in vivo to examine the ability of this antibody to deliverdrugs to the brain. The results presented suggest that thetargeting of OX-26 to brain capillary endothelial cells is notmarkedly affected by the attachment of "passenger" drugmolecules. However, although we have clearly demonstratedenhanced delivery ofMTX to the brain by using OX-26 as acarrier, the amount that reaches the brain parenchyma islower than that of antibody alone (0.27 and 0.44% of theinjected dose, respectively, 24 hr after injection). It appearsthat this difference may be a reflection of the physical-chemical properties of the passenger compound, as we havefound that some conjugates of OX-26 exhibit enhanceduptake into the brain parenchyma relative to antibody alone(L.R.W. and P.M.F., unpublished data). The use of radio-labeled MTX in the capillary depletion experiments clearly

demonstrates that it is the drug which is delivered to the brainparenchyma.The results presented demonstrate the feasibility of deliv-

ering drugs across the BBB by using an anti-transferrinreceptor antibody as a carrier. Although the present studyestablishes delivery of a small drug molecule, neuroactivepeptides and neurotrophic factors whose effective brainconcentrations are in the nanomolar range (or lower) are alsopotential candidates for delivery using this system. OX-26has been shown to deliver proteins as large as 40 kDa to thebrain and to achieve concentrations in the brain estimated tobe in the 10-100 nM range (L.R.W., P.M.F. and M.A.T.,unpublished data). Thus, therapeutic levels of neuroactivecompounds may be achieved in the brain using an anti-transferrin receptor antibody.

We thank Biba Tehrani, Joseph Eckman, Susan Abelleira, andAnne Morrow for their excellent technical support. We also thankFloyd Bloom, Timothy Curran, John Kozarich, Paul Schimmel, andMichael Wall for their support and insightful comments. We appre-ciate the many helpful comments made by our colleagues at Alker-mes, Inc.

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