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Journal of Controlled Releas

Binding specificity and internalization properties of an antibody–avidinfusion protein targeting the human transferrin receptor

José A. Rodríguez a, Gustavo Helguera a, Tracy R. Daniels a, Isabel I. Neacato a,Héctor E. López-Valdés b, Andrew C. Charles b, Manuel L. Penichet a,c,d,⁎

a Division of Surgical Oncology, Department of Surgery; University of California, Los Angeles (UCLA), United Statesb Department of Neurology, University of California, Los Angeles (UCLA), United States

c Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles (UCLA), United Statesd Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles (UCLA), United States

Received 15 June 2007; accepted 17 August 2007Available online 25 August 2007

Abstract

The human transferrin receptor (hTfR1) is a membrane-bound protein involved in transferrin (Tf)-mediated iron uptake and is highly expressedon malignant cells. A second version of the receptor (hTfR2) also mediates Tf-dependent iron import. We previously developed a proteincomposed of avidin fused to a mouse/human chimeric IgG3 specific for hTfR (anti-hTfR IgG3-Av) that was originally designed to deliverbiotinylated drugs into cancer cells. We have now found that anti-hTfR IgG3-Av does not cross-react with hTfR2 and binds hTfR1 expressed onthe surface of cells, attached to a solid surface, and in solution. We also found that the hemochromatosis protein (HFE), another ligand of the TfR,does not inhibit the binding of anti-hTfR IgG3-Av to the receptor. In addition, using live cell laser scanning confocal microscopy (LCLSCM) wedemonstrated that anti-hTfR IgG3-Av and anti-hTfR IgG3 are internalized into cells expressing hTfR1 at a similar rate. Furthermore, ourproliferation and morphological studies demonstrated the effective cytotoxicity of a biotinylated toxin delivered by anti-hTfR IgG3-Av only intocells expressing hTfR1. Our results better define the properties of anti-hTfR IgG3-Av and pave the way for the rational design of future in vitroand in vivo studies for the treatment of human malignancies.© 2007 Elsevier B.V. All rights reserved.

Keywords: Drug delivery; Antibody; Avidin; Transferrin receptor; Transferrin

1. Introduction

Iron is essential for a number of cellular functions includingDNA synthesis and electron transport. The human transferrinreceptor (hTfR) is a type II transmembrane homodimericprotein responsible for transferrin (Tf) mediated iron uptake intocells [1]. The hTfR family has two members, hTfR1 (alsoknown as CD71) and hTfR2, as well as two ligands, Tf and thehemochromatosis protein (HFE). The hTfR1 is ubiquitouslyexpressed at low levels, while hTfR2 is expressed mostprominently in the liver tissue [2]. Importantly, hTfR1 is highly

⁎ Corresponding author. Division of Surgical Oncology, Department ofSurgery, UCLA 10833 Le Conte Avenue CHS 54-140, Los Angeles, CA 90095-1782, United States. Tel.: +1 310 825 1304; fax: +1 310 825 7575.

E-mail address: penichet@microbio.ucla.edu (M.L. Penichet).

0168-3659/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2007.08.020

expressed in rapidly dividing cells including a variety of humancancers, in many cases several orders of magnitude higher thanin normal tissue [1]. In addition, studies have also suggestedthat hTfR may play a role in cellular signaling and proliferationstimuli [3–5]. The high levels of expression of TfR in cancercells, its extracellular accessibility, its ability to internalize, andits central role in the pathology of human cancer make thisreceptor an attractive target that can be exploited as a “TrojanHorse” for the delivery of cytotoxic agents into tumor cells ashas also been done with antigens such as folate receptor [6,7].

A number of ligand and antibody-based therapies have beendeveloped for the delivery of cytotoxic agents into cells by TfRmediated targeting and endocytosis [6,8–13]. In fact, Tfconjugated with a mutant form of the diphtheria toxin(CRM107) has been successfully used in local administration topatients with brain malignancies in both Phase I and Phase II

36 J.A. Rodríguez et al. / Journal of Controlled Release 124 (2007) 35–42

clinical trials [10], leading to a Phase III trial for treatment ofglioblastoma multiforme (NCI, NCT00087230). Additionally, avariety of antibody-based therapies have been used for toxindelivery via hTfR targeting including 454A12-RTA, a murineanti-hTfR IgG1 carrying an enzymatically active ricin A chain.This antibody conjugate was tested in a phase I clinical trial for theintraventricular treatment of leptomeningeal neoplasia, achievingconcentrations that would be tumoricidal in vitro but showed noacute or chronic drug toxicity in vivo [14]. These studies suggestcytotoxic conjugates targeting the hTfR can have minimal sideeffects when administered locally or systemically [9,15].

We have developed antibody–avidin fusion proteins todeliver biotinylated anti-cancer agents into cells by receptor-mediated endocytosis. These antibody fusion proteins consist ofeither the variable region of the mouse monoclonal antibodyOX-26 or 128.1 specific for the rat and human TfR respectively,and a human IgG3 constant region genetically fused to chickenavidin [16] (Fig. 1). Deduced from fast protein liquidchromatography (FPLC) analysis under non-denaturing condi-tions, the proposed quaternary structure of theses proteins insolution is that of a non-covalent dimeric (tetravalent) protein,due to the natural non-covalent association of avidin [16,17]. Auniversal delivery system such as the proposed anti-TfRantibody–avidin fusion protein eliminates the need to make aspecific construct for each individual application, or a chemicalconjugation with each agent delivered.

We have demonstrated in previous studies that IgG3-Av fusionproteins targeting both rat and human TfR (rTfR and hTfR) arecapable of delivering biotinylated FITC into rat and humanmalignant cells respectively [16]. However, we unexpectedly

Fig. 1. Schematic representation of anti-hTfR IgG3-Av and its proposed dimericstructure. Panel A shows anti-hTfR IgG3-Av, a mouse/human chimeric IgG3specific for human TfR genetically fused to chicken avidin at the carboxy-terminusof the CH3 domain. Panel B illustrates the extended hinge region of human IgG3,which provides spacing and flexibility to the molecule thereby facilitatingsimultaneous binding of antigen and biotinylated compounds. The non-covalenttetrameric interaction between its four avidin moieties results in a homodimerictetravalent molecule in solution, capable of binding up to four biotinylated agentsfor delivery.

found that both fusion proteins possess a strong intrinsic anti-proliferative/pro-apoptotic activity against malignant hematopoi-etic cell lines [16,18]. This activity seems to be, at least in part, aresult of iron deprivation induced by TfR degradation duringrecycling [18]. Therefore, we have already demonstrated that it ispossible to transform an antibody specific for the TfR, with lowcytotoxic activity, into a novel drug with significant intrinsic anti-proliferative/pro-apoptotic activity against certain malignant cellsby fusing it with avidin. Importantly, this cytotoxic activity maybe potentially enhanced through its delivery of biotinylatedcompounds into cells. The present study offers critical insights onthe use of antibody–avidin fusion proteins as novel therapeuticagents capable of a two-pronged attack onmalignant cells throughtoxin delivery and direct induction of apoptosis.

2. Materials and methods

2.1. Cell lines and reagents

The soluble form of human TfR1 (sTfR1) used in the ELISAstudies was a gift from Dr. Chi-Hong Betty Chen, UCLADepartment of Chemistry and Biochemistry, and Dr. PamelaBjörkman at the California Institute of Technology in Pasadena,CA, who also provided us with the human HFE protein. Themurine anti-human TfR2 antibody (9F81C11) used as a positivecontrol for cell-surface TfR2 binding was a kind gift from Dr.Caroline Enns at the OregonHealth Sciences University, PortlandOR.Derived fromCHO-TRVb cells (a cell linewithout detectablecell surface TfR levels) [19], the CHO-TRVb-hTfR1 (expressingonly human TfR1), CHO-TRVb-hTfR2 (expressing only humanTfR2), and CHO-TRVb-neo cells (transfected with the emptyneomycin vector) [2] were a gift of Dr. Phillip Koeffler at CedarsSinai Medical Center, Los Angeles CA. All CHO-TRVb cellswere cultured in F-12 (HAM)medium (Invitrogen Inc., Carlsbad,CA) supplemented with 10% fetal bovine serum (AtlantaBiologicals Inc., Lawrenceville, GA) and 1 mg/ml G418 as aselectable marker (Invitrogen Inc.).

2.2. Antibody production and purification

Anti-hTfR IgG3 and anti-hTfR IgG3-Av as well as theirisotype-matched negative controls targeting the dansyl (DNS)hapten 5-dimethylamino naphthalene-1-sulfonyl chloride, anti-DNS IgG3 and anti-DNS IgG3-Av, were expressed in murinemyeloma cells and purified from tissue culture supernatants usingprotein G or A immobilized on Sepharose 4B fast flow beads(Sigma-Aldrich Chemical Co., St. Louis, MO) as previouslydescribed [16,20]. Purity was assessed by Coomassie Bluestaining of SDS-PAGE gels, and all protein concentrations weredetermined by the bicinchoninic acid based protein assay (BCAProtein Assay, Pierce Biotechnology Inc., Rockford, IL) andquantitative ELISA [16,18,20].

2.3. Protein ELISA

The binding specificity of anti-hTfR IgG3-Av and anti-hTfRIgG3 to sTfR was tested by ELISA. Immulon 2H-B 96-well

37J.A. Rodríguez et al. / Journal of Controlled Release 124 (2007) 35–42

ELISA plates (Thermo Labsystem, Franklin, MA) coated withsTfR (0.5 μg/ml) were incubated overnight at 4 °C with serialdilutions of either anti-hTfR IgG3 or anti-hTfR IgG3-Av alongwith their appropriate isotype-matched negative control, anti-DNS IgG3 and anti-DNS IgG3-Av respectively. The plates werewashed and bound anti-hTfR IgG3 or anti-hTfR IgG3-Av weredetected by incubating the plates with alkaline phosphatase(AP) labeled anti-humanκ (Sigma Chemical, St. Louis, MO) for60 min at 37 °C. After washing, p-nitrophenyl phosphatedisodium dissolved in diethanolamine buffer (Sigma-AldrichCo.) was added; the plates were incubated for 30 min at roomtemperature and absorbance at 410 nm detected. To confirm theability of anti-hTfR IgG3 or anti-hTfR IgG3-Av to bind sTfR insolution, a competition assay was performed by adding aconstant amount (5.0 μg/ml) of sTfR to the dilution of theantibodies or antibody fusion proteins.

2.4. Cell-ELISA

For detection of binding to cells on a solid surface, CHO-TRVb-hTfR1, CHO-TRVb-hTfR2, and CHO-TRVb-neo cellswere plated into tissue culture 96-well plates at 20,000 cells/welland incubated in F-12 (HAM) medium with 10% fetal bovineserum and 1mg/ml G418 at 37 °C for 24 h. Cells were fixed with100 μl/well of 4% paraformaldehyde, blocked with 100 μl of 3%BSA in PBS, and stored at 4 °C overnight. The detection ofantibody binding to the hTfR was performed following theprotein ELISA protocol described above and modified to matchthat of Suter et al. [21]. Briefly, wells were incubated overnightwith a serial dilution of primary antibodies along with theirisotype-matched controls as previously described. Plates werecoated with secondary antibodies (AP conjugated anti-mouseIgG or anti-human IgG, from Sigma-Aldrich Co.), followed byincubation with AP substrate dissolved in diethanolamine buffer.Absorbance levels were determined after 60 min of incubationwith the AP substrate. Inhibition of anti-hTfR IgG3-Av bindingto TfR1 in the presence of HFE was also tested by cell-ELISA,performed according to the cell ELISA studies described abovewith an additional constant (5.0 μg/ml) of HFE co-incubated inthe primary antibody dilutions.

2.5. Live cell laser scanning confocal microscopy

Live cell confocal microscopy was used to image anti-hTfRIgG3-Av uptake into CHO-TRVb-hTfR1 cells. CHO-TRVb-hTfR1 cells were seeded at near confluency on poly-L-lysinecoated Fisherbrand microscope cover glasses (Fisher ScientificCo., Fairlawn, NJ) in 12-well plates (Corning ScientificInstruments, Med-field, MA) and incubated overnight in F-12(HAM)mediumwith 10% fetal bovine serum and 1mg/ml G418(Invitrogen Inc.). The cover glass was used as the bottom of anopen perfusion chamber mounted on an inverted microscope(Diaphot T200; Nikon) with a chamber volume of 200 μl. Themicroscope design is a variant of that by Sanderson and Parker[22] with a 475 nm diode laser using a 60× (Numerical Aperture1.4) oil immersion objective. Emitted fluorescence was filteredat 510 nm and detected using a photomultiplier tube (Hama-

matsu Photonics Inc., Bridgewater, NJ), while two oscillatingmirrors scanned excitation and emission (GSI Lumonics Inc.,Moorpark, CA). Images were captured with a Raven board (BitFlow Inc., Woburn, MA) using Video Savant software at amaximum image resolution of 980×730 pix at 4 frames per min.Cells were incubated in media for 1 min at 25 °C with 20 μg/mlTf-Alexa 488 (Invitrogen Inc.), or 8 μg/ml (40 nM) of anti-DNSIgG3, anti-hTfR IgG3, anti-DNS IgG3-Av, or anti-hTfR IgG3-Av labeled with Zenon Human IgG labeling kit containing Alexa488 (Invitrogen Inc.). After a 1 min incubation, cover glasseswere continuously washed with medium containing 10% FBS ata flow rate of 2–3 ml/min at 25 °C. Intracellular and membraneintensity measurements over time were taken from 15 cells usinga custom made macro for the NIH ImageJ software availableonline (http://rsb.info.nih.gov/ij/). Briefly, cells were outlinedand the total cell intensity measured. A cytoplasmic region wasthen outlined for each cell and its total intensity also measured.The ratio of total cytoplasmic intensity to membrane intensitywas determined as the average of 15 individual cell measure-ments and plotted as a function of time.

2.6. Toxin conjugation, delivery, and cytotoxicity

The custom mono-biotinylated saporin toxin, a ribosomeand protein synthesis inhibitor derived from Saporina officinalis(b-SO6) was purchased from Advanced Targeting Systems (SanDiego,CA). Conjugation of b-SO6with the avidin fusion proteinswas carried out at 4 °C for 30 min in a 1:1 molar ratio before theaddition of cell culturemedium to prevent the formation of proteinaggregates. CHO-TRVb cells expressing hTfR1, hTfR2, or noTfR were treated with b-SO6 alone or conjugated to either anti-DNS IgG3-Av or anti-hTfR IgG3-Av, as well as with anti-hTfRIgG3-Av, anti-hTfR IgG3, anti-DNS IgG3-Av alone, or buffer ascontrols. The effect of the various treatments on cell proliferationwas determined by the [3H]-thymidine incorporation assay aspreviously described [18]. Briefly, 2000 cells per well (96-wellplates) were seeded on the day prior to treatment and incubatedovernight in F-12 (HAM) medium with 10% fetal bovine serumand 1 mg/ml G418 (Invitrogen Inc.). Cells were then treated with0.1 nM of anti-hTfR IgG3-Av/b-SO6, anti-DNS IgG3-Av/b-SO6,b-SO6 alone, anti-hTfR IgG3-Av, anti-DNS IgG3-Av, anti-hTfRIgG3, or anti-DNS IgG3 for 72 h. [3H]-thymidine (MPBiomedicals, Solon, OH) was added at a concentration of0.5 μCi per well for the final 16 h of the treatment period. Cellswere then harvested onto glass fiber filters (Printed Filtermat A,PerkinElmer Life and Analytical Science Inc., Waltham, MA) byusing a 11050 Micro Cell Harvester (Skatron, Lier, Norway).Betaplate Scint (PerkinElmer Life and Analytical Science Inc.)scintillation fluid was added to a sample bag (PerkinElmer Lifeand Analytical Science Inc.) containing the fiber filter andradioactivity was counted in a 1205 Betaplate Liquid ScintillationCounter (PerkinElmer Life and Analytical Science Inc.). The rateof proliferation of treated cells is reported as a percent of [3H]-thymidine incorporated into buffer treated cells, with statisticalsignificance determined using a Student's t-test. To assess cellmorphology, differential interference contrast (DIC) images of thetreated cells were obtained using a Zeiss Axiovert 40 CFL (Carl

Fig. 2. Binding of anti-hTfR IgG3-Av and anti-hTfR IgG3 to CHO cellsexpressing hTfR1, hTfR2, or no TfR, and to sTfR1 immobilized on a solidsurface or in solution. Panel A shows CHO-TRVb cells expressing hTfR1,hTfR2, or no hTfR were coated on ELISA plates and incubated with anti-DNSIgG3, anti-DNS IgG3-Av, anti-hTfR IgG3, anti-hTfR IgG3-Av, or the 9F81C11mouse anti-TfR2 monoclonal antibody. Results are shown as the average oftriplicate samples and error bars indicate the standard deviations. Panel B (left)shows the binding to ELISA plates coated with sTfR1 and incubatedwith seriallydiluted anti-hTfR IgG3 alone or in the presence of 5.0 μg/ml sTfR1 in solution asa competitor along with anti-DNS IgG3 alone or in presence of sTfR1. Alsoshown in panel B (right) is the binding of anti-hTfR IgG3-Av alone or in thepresence of sTfR1 as a competitor. Anti-DNS IgG3-Av alone or in presence ofsTfR1 was included as a control. Bars indicate the standard deviation of triplicatedata points for experiments that were repeated twice with similar results.

Fig. 3. Live cell scanning confocal microscopy of CHO-TRVb-hTfR1 cellsincubatedwith fluorescently labeled anti-hTfR IgG3, anti-hTfR IgG3-Av, and anti-DNS IgG3-Av. Panel A shows confocal microscopy images of 2 to 18 min timelapsed uptake of anti-hTfR IgG3, anti-hTfR IgG3-Av, and anti-DNS IgG3-AvAlexa 488 labeled antibodies into CHO-TRVb-hTfR1 cells. The cells were pulsedfor 1 min with the ligands, washed 1 min and incubated at 25 °C under continuousperfusion. Images are representative samples taken from a single field of view heldconstant throughout the duration of the experiments. Panel B shows a plot of theinternalization profiles of the fluorescently labeled antibodies. To determine the rateof internalization of labeled antibody into CHO-TRVb-hTfR1 cells as a function oftime, confocal microscopy images were used to measure the average fluorescenceintensity of a fixed area within the cytoplasm divided by that of a fixed area on themembrane giving dimensions of (Intensitycyto / Intensitymem·min), or min−1.Images were sampled at a rate of four frames per minute for 15 cells chosen atrandom within a constant field of view and are representative of the whole field,data points indicate the minute average of these measurements and error barsdenote standard error. This experiment was performed twice with similar results.

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Zeiss Inc., Thornwood, NY) microscope mounted with a 20×objective and a digital camera.

3. Results

3.1. Anti-hTfR IgG3-Av specifically binds hTfR1

Using ELISA we examined whether the anti-hTfR IgG3-Avcan specifically bind hTfR1 expressed on the cell surface, on asolid surface, or in solution. We found that both anti-hTfR IgG3-Av and anti-hTfR IgG3 bound CHO-TRVb-hTfR1 cells(Fig. 2A), and that isotype-matched controls targeting DNS didnot show binding to the antigen. As expected, neither anti-hTfRIgG3-Av, anti-hTfR IgG3, nor their respective isotype-matchedcontrols bound to CHO-TRVb-hTfR2 or CHO-TRVb-neo cells(Fig. 2A). 9F81C11 (an antibody specific for hTfR2) was used asa positive indicator of cell surface hTfR2 expression. Moreover,co-incubation of anti-hTfR IgG3-Avwith a 10-fold excess ofHFEdid not preclude its binding to CHO-TRVb-hTfR1 cells (data notshown), which is consistent with previous results showing Tfbinding to TfR without inhibition by anti-hTfR IgG3-Av [18].

Additionally, binding of anti-hTfR IgG3-Av and anti-hTfR IgG3to sTfR1 immobilized on a solid surface was dose-dependent andcould be prevented by co-incubation with a 10-fold excess ofsTfR1 in solution (Fig. 2B). Thus, both anti-hTfR IgG3 and anti-hTfR IgG3-Av are capable of binding the ectodomain of humanTfR1 immobilized on a solid surface and in solution.

3.2. Internalization of anti-hTfR IgG3-Av into cells expressinghTfR1

We used LCLSCM to investigate the rate of internalization ofanti-hTfR IgG3-Av into CHO-TRVb-hTfR1 cells. CHO-TRVb-

39J.A. Rodríguez et al. / Journal of Controlled Release 124 (2007) 35–42

hTfR1 cells were pulsed for 1 min in the presence of fluorescentlylabeled anti-hTfR IgG3-Av, anti-hTfR IgG3, anti-DNS IgG3-Av,or Tf. Fig. 3A shows a montage of images taken at 2, 7, 13, and18 min after the addition of the fluorescently labeled antibodies.We observed time dependent internalization of fluorescent anti-hTfR IgG3-Av and anti-hTfR IgG3, but not of the isotype controlanti-DNS IgG3-Av. Uptake of fluorescently labeled Tf was alsoused as a control for functional TfR1 endocytosis (SupplementaryFig. 1). In the case of anti-DNS IgG3-Av, traces of antibody

remained on the cell surface after 2 min, but no increase in thefluorescence intensity within the cells was detectable over time(Fig. 3A). A pulse with the negative control anti-DNS IgG3 didnot exhibit any detectable signal (data not shown). Fig. 3B showschanges in the ratio of cytoplasmic to membrane fluorescenceintensity as a function of time for CHO-TRVb-hTfR1 cells treatedwith anti-hTfR IgG3-Av and anti-hTfR IgG3. Supplementary Fig.2 (Videos A and B) shows a time lapsed video of anti-hTfR IgG3-Av and anti-hTfR IgG3 internalization into CHO-TRVb-TfR1cells for comparison. We found that anti-hTfR IgG3-Av wasinternalized into CHO-TRVb-hTfR1 cells at a rate comparableand not significantly different from anti-hTfR IgG3 under theconditions tested.

3.3. The cytotoxic effect of anti-hTfR IgG3-Av complexed tob-SO6 is specific to cells expressing hTfR1

The capacity of anti-hTfR IgG3-Av to deliver a biotinylatedtoxin specifically to hTfR1-expressing cells was evaluated usingthe [3H]-thymidine incorporation assay. We tested the ability ofanti-hTfR IgG3-Av to deliver the b-SO6 protein, a potent inhibitorof protein synthesis, intoCHOcell lines expressing hTfR1, hTfR2or not expressing hTfR by monitoring the proliferation of treatedcells. SO6 was used in a mono-biotinylated form in order to avoidthe generation of multimer complexes when conjugated with theavidin-fusion proteins. We observed that the controls, anti-hTfRIgG3, b-SO6 alone, as well as anti-DNS IgG3-Av alone orcombined to b-SO6 exhibit no inhibition of proliferation in thethree cell lines tested (Fig. 4A). However, anti-hTfR IgG3-Avcomplexed to b-SO6 showed potent anti-proliferative effectsagainst CHO-TRVb-hTfR1 cells, with more than 95% inhibitionof proliferation at 0.1 nM (Student's t-test pb0.02) (Fig. 4A, Toppanel). As expected, the anti-hTfR IgG3-Av/b-SO6 complex didnot have an effect on CHO-TRVb-hTfR2 or CHO-TRVb-neocells (Fig. 4A, middle and bottom panels respectively). Themorphology of CHO-TRVb-hTfR1, CHO-TRVb-hTfR2, andCHO-TRVb-neo cells treated with anti-hTfR IgG3-Av/b-SO6was assessed using DIC images. These data demonstrate that thecomplex not only blocks cell proliferation but, given the decreasein total number of cells and their changed morphology, also

Fig. 4. Cytotoxic effects elicited by anti-hTfR IgG3-Av conjugated to b-SO6 inCHO cells expressing hTfR1. Panel A shows CHO-TRVb-hTfR1, CHO-TRVb-hTfR2, and CHO-TRVb-neo cells treated with 0.1 nM anti-DNS IgG3-Av, anti-hTfR IgG3, anti-hTfR IgG3-Av, b-SO6 alone, anti-DNS IgG3-Av/b-SO6, oranti-hTfR IgG3-Av/b-sSO6 for 72 h. The anti-proliferative effects of the toxinconjugate were monitored using the [3H]-thymidine incorporation assay. Data ispresented as the percent of the mean of triplicate samples of [3H]-thymidineincorporated into negative control cells (100%mark is indicated by a punctuatedline). Data shown is representative of three independent experiments and errorbars indicate the standard deviation. Asterisks indicate significant differencecompared to negative control (Student's t-test pb0.02). Panel B shows themorphology of the cytotoxic effects induced by the toxin conjugate anti-hTfRIgG3-Av/b-SO6 on CHO-TRVb-hTfR1. CHO cells expressing hTfR1, hTfR2,or no TfR were treated with 0.1 nM anti-DNS Av/b-SO6, or anti-hTfR Av/b-SO6 for 48 h. Representative differential interference contrast (DIC) images areshown. Toxicity is only observed in cells expressing hTfR1 treated with the anti-hTfR IgG3-Av/b-SO6 complex, not hTfR2 or the negative control. Images arerepresentative of 3 independent experiments.

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induces cell death (Fig. 4B). Our studies demonstrate that anti-hTfR IgG3-Av can selectively deliver b-SO6 into cells expressinghTfR1 such as CHO-TRVb-hTfR1 cells.

4. Discussion

Several strategies for specific drug targeting and deliveryinto malignant cells are under development [6] with the goal oflimiting toxic side effects against healthy tissue and improvingthe therapeutic index of the drug. Antibody-based targetedtherapies have been useful for the delivery of toxic compoundsbecause they can selectively target tumor associated antigensand can be applied systemically [23]. Recent advancements ingenetic engineering have facilitated the development and FDAapproval of anti-cancer therapeutics such as gemtuzumab (anti-CD33mAb-calcheamicin), ibritumomab (anti-CD20mAb-90Y),and tositumomab (anti-CD20 mAb-131I), antibodies armed withdifferent anti-cancer agents that have shown effectivenessagainst various malignancies [23]. Anti-hTfR IgG3-Av wasdesigned as a universal vector for the delivery of biotinylatedagents including a variety of toxins, DNA/RNA, nanoparticles,and radioactive compounds into malignant cells expressing highlevels of hTfR.

Delivery of agents via anti-hTfR IgG3-Av eliminates the needto make specific conjugate constructs for each individualapplication by taking advantage of the high affinity avidin–biotin interaction. However, before anti-hTfR IgG3-Av can beused to deliver a cytotoxic payload it must exhibit selectivetargeting of cells that express high levels of hTfR1. The variableregion of anti-hTfR IgG3-Av belongs to themonoclonal antibody128.1 that was developed prior to the discovery of hTfR2, aprotein with 66% similarity to the extracellular domain of TfR1[2]. Therefore, since the binding of anti-hTfR IgG3-Av to cell-surface hTfR2 would have a strong effect on its pharmacokineticproperties and its capacity to deliver biotinylated payloads intohTfR1-expressing cells, the potential cross-reactivity of anti-hTfR IgG3-Av against hTfR2 was investigated. Our studiesindicate that anti-hTfR IgG3-Av specifically binds cell-surfacehTfR1 expressed on CHO cells with no detectable binding toCHO-TRVb-hTfR2 or CHO-TRVb-neo cells, which reducespotential hepatotoxic side-effects given the high level ofexpression of TfR2 in the liver [2]. Moreover, HFE does notpreclude the binding of anti-hTfR IgG3-Av to TfR consistentwith what has been previously observed using Tf [18], whichwould potentially diminish its capacity for delivery in vivo. Thisis a significant advantage over Tf-based delivery systems, whichare inhibited by both of the endogenous ligands of the receptor[24].

In addition to its specific targeting of CHO-TRVb-hTfR1 cells,anti-hTfR1 IgG3-Av also binds sTfR1, a truncated version of thereceptor that exists in serum as a result of receptor proteolysis[25]. Importantly, the binding of anti-hTfR IgG3-Av to sTfR1immobilized on a solid surface was prevented by an excess ofsTfR1 in solution, an interaction which may preclude its bindingto cell-surface hTfR1 under physiological conditions. However,trastuzumab (Herceptin®,Genentech Inc., San Francisco, CA), anantibody therapeutic known to bind the soluble formof theHER2/

neu receptor (ECDHER2) that has been FDA approved for use inpatients with breast cancer over-expressing HER2/neu, has beeneffective in clinical trials despite the fact that patients havedetectable ECDHER2 levels present in serum [26]. It is alsoimportant to note that the normal concentration of sTfR in serumis approximately 19.6 nM on average [27]; a threefold lowerconcentration than that used in our inhibition assays, but nodifferent than that of cancer patients [28]. Additionally, a phase Iclinical trial using the murine monoclonal IgA 42/6 targetinghTfR has shown that sTfR levels decrease in a dose-dependentmanner after infusion with the antibody [28], which would favorselective targeting. Moreover, despite the presence of a humananti-mouse response, these studies achieved antibody levels inblood ranging from 1 to 36 μg/ml without significant toxicity[28]. A human anti-mouse reaction would be less likely againstanti-hTfR IgG3-Av since it contains a fully human IgG3 Fcregion. However, its avidin moiety could potentially beimmunogenic and preclude multiple sequential administrationsin vivo. Nevertheless, this immunogenicity may be reduced orabsent in humans because of exposure to chicken avidin by eatingeggs, which may render avidin tolerogenic [29]. In fact, avidinwas chosen instead of the bacterial protein streptavidin since it isexpected to have reduced immunogenicity [30]. Additionally, thepossible development of a human immune response mountedagainst anti-hTfR IgG3-Av can be avoided by using it in com-bination with commercially available immune suppressive agentssuch as rituximab (Rituxan®, Genentech Inc., San Francisco, CAand IDEC Pharmaceutical Corp., San Diego, CA), an antibodytherapy used in the depletion of B cells and humoral immunity[31]. Moreover, it is possible that immunogenicity against theavidin moiety may be lower in patients with hematologicalmalignancies that have an impaired immune response [31].

For antibody-based targeted therapies, the valency of anantibody could affect its ability to deliver a cytotoxic payload. Theproposed tetravalent structure of anti-hTfR IgG3-Av (Fig. 1),deduced from FPLC data indicating that anti-hTfR IgG3-Av hasan approximate mass of 400 kDa corresponds to two monomericantibody fusion proteins bound non-covalently [16], whichwouldbe expected due to the tetrameric structure of avidin [17]. Thetetravalency of the antibody as well as its four avidin moietieswould result in an increased capacity to target and to internalize itspayload, carrying with it up to four biotinylated compounds. Ourgroup previously demonstrated that anti-hTfR IgG3-Av is capableof delivering the fluorochrome FITC conjugated to biotin intocancer cells [16]. In the present studies we have shown, in realtime, using LCLSCM that fluorescently labeled anti-hTfR IgG3-Av can bind and be internalized by CHO-TRVb-hTfR1 cells.Interestingly, the rate of internalization of anti-hTfR IgG3-Avwascomparable to that of anti-hTfR IgG3, presumably since theyshare the same variable region. Importantly, the profiles of anti-hTfR IgG3-Av and anti-hTfR IgG3 internalization into CHO-TRVb-hTfR1 may be due in part to the high level of hTfR1expression on these cells, suggesting that similar behavior mayoccur in human malignant cells, which also express high levels ofthe receptor. In fact, we have also conducted LCLSCM studiesusing a human U87 astrocytoma cell line endogenouslyexpressing hTfR, in which similar results to those obtained

41J.A. Rodríguez et al. / Journal of Controlled Release 124 (2007) 35–42

using the CHO-TRVb-hTfR1 cells were observed (Rodriguezet al., unpublished results).

Several types of protein toxins have been used for targeteddelivery of hTfR on cancer cells [6]. Tf-based SO6 conjugateswere cytotoxic to the human erythroleukemia K562 cell line aswell as the human hepatoma cell line HepG2 [32], and alsoagainst primary glioma cells [24]. However, Tf-SO6 cytotoxicitywas heavily influenced by the variable affinity of the Tf conjugate,such that Tf-SO6 was effectively out-competed even in thepresence of low concentrations of endogenous Tf [24]. In otherstudies, SO6 was conjugated to the murine monoclonal anti-human TfR IgA 42/6 with similar efficacy to that of Tf-SO6, butwithout the disadvantages of an iron saturation requirement orcompetition with the endogenous ligand. However, the chemicalconjugation process required to synthesize these immunotoxinscan be problematic due to the heterogeneity of the final conjugatepopulation and the yield of the conjugation reaction [33]. Incontrast, the avidin–biotin system employed by anti-hTfR IgG3-Av offers a broad range of biotinylated conjugate options, withgreater homogeneity. Moreover, our in vitro studies show that theinhibition of proliferation induced by 0.1 nM anti-hTfR IgG3-Avcomplexedwith b-SO6 ismore than 95% for CHO-TRVb-hTfR1,in contrast to the undetectable effect of the toxin alone, theantibody alone, or their isotype-matched controls. In fact, weexpected that anti-hTfR IgG3-Av alone to have no effect since theCHO cells are artificially transfected with the TfR and do notrequire it for normal ironmetabolism. Furthermore, the anti-hTfRIgG3-Av/b-SO6 complex did not affect the proliferation of CHO-TRVb-hTfR2 or CHO-TRVb-neo cells, comparable to the effectof the antibody and toxin controls. Similar effectiveness indelivery and selectivity would be expected against malignant cellsexpressing high levels of cell-surface hTfR1, since a therapeuticwindow exists between these cells and their normal tissuecounterparts [1]. Toxin delivery can overcome resistance totreatment with anti-hTfR IgG3-Av alone, since recent studiesusing human malignant hematopoietic cells with various degreesof sensitivity to anti-hTfR IgG3-Av alone were rendered highlysensitive to the antibodywhen complexed to b-SO6 (Daniels et al.submitted).

The present studies demonstrate that anti-hTfR IgG3-Av canspecifically target hTfR1 and that it can be internalized specificallyby CHO-TRVb-hTfR1 cells. In addition, our data support the useof anti-hTfR IgG3-Av as a potential universal vector for thedelivery of cytotoxic agents, including b-SO6, intomalignant cellsexpressing high levels of hTfR1. Three key features of anti-hTfRIgG3-Av establish its superiority over ligand-based targetingapproaches; these are its specificity for hTfR1, its lack ofcompetition with endogenous TfR ligands (Tf and HFE), and itsstable binding to cell-surface receptor followed by rapidendocytosis. Moreover, the use of this therapeutic is not limitedto targeting of myeloma cells systemically; anti-hTfR IgG3-Avcan also be used for ex vivo approaches such as the efficientpurging of myeloma cells in autologous transplantation protocolsfor multiple myeloma patients. Finally, this universal vectordelivery strategy is not necessarily limited to targeting hTfR1.Since the expression plasmid used to produce this fusion proteinhas amodular design, it is possible to change the variable region to

target other relevant antigens such as HER2/neu or folate receptorand expand its applicability beyond the targeting of hTfR1 relatedmalignancies [7,20,34,35].

Acknowledgments

We would like to thank Dr. Sherie L. Morrison (UCLADepartment of Microbiology, Immunology and MolecularGenetics) for her support to this project, Dr. Caroline Enns(Oregon Health Sciences University, Portland OR) for hergenerous donation of the 9F81C11 monoclonal antibody, andDr. H. Phillip Koeffler (Cedars SinaiMedical Center, Los AngelesCA) for providing us with the CHO-TRVb cells. The sTfR1protein was a kind gift from Dr. Chi-Hong Betty Chen (UCLADepartment of Chemistry and Biochemistry) and Dr. PamelaBjorkman (California Institute of Technology, Pasadena CA) whoalso provided uswithHFE. Sincere appreciation is extended to theMARC-NIH, CARE, and SPUR programs at UCLA for theirgenerous support and funding. This work was supported in part bygrants CA86915, CA107023, and supplement CA107023-02S1from theNational Institutes ofHealth (NIH) and by the 2004BrianD. Novis InternationalMyeloma Foundation Senior Grant Award.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.jconrel.2007.08.020.

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