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REVIEW

The transferrin receptor part I: Biology and targetingwith cytotoxic antibodies for the treatment of cancer

Tracy R. Daniels a, Tracie Delgado a, Jose A. Rodriguez a,Gustavo Helguera a, Manuel L. Penichet a,b,c,*

a Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USAb Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine,UCLA, Los Angeles, CA 90095, USAc Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA

Received 7 June 2006; accepted with revision 16 June 2006Available online 10 August 2006

1521-6616/$ — see front matter D 200doi:10.1016/j.clim.2006.06.010

* Corresponding author. Division of SuAngeles, CA 90095-1782, USA. Fax: +1

E-mail address: penichet@microbio

KEYWORDSTransferrin receptor;Iron;Receptor-mediatedendocytosis;Monoclonal antibodies;Recombinantantibodies;Immunotherapy;Cancer

Abstract The transferrin receptor (TfR) is a cell membrane-associated glycoprotein involvedin the cellular uptake of iron and in the regulation of cell growth. Iron uptake occurs via theinternalization of iron-loaded transferrin (Tf) mediated by the interaction with the TfR. Inaddition, the TfR may also contain other growth regulatory properties in certain normal andmalignant cells. The elevated levels of TfR in malignancies, its relevance in cancer, and theextracellular accessibility of this molecule make it an excellent antigen for the treatment ofcancer using antibodies. The TfR can be targeted by monoclonal antibodies specific for theextracellular domain of the receptor. In this review, we summarize advancements in the basicphysiology of the TfR including structure, function, and expression. We also discuss the efficacyof targeting the TfR using cytotoxic antibodies that inhibit cell growth and/or induce apoptosisin targeted malignant cells.D 2006 Elsevier Inc. All rights reserved.

Contents

The transferrin receptor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Transferrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145Transferrin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Clinical Immunology (2006) 121, 144—158

6 Elsevier Inc. All rights reserved.

rgical Oncology, Department of Surgery, UCLA 10833 Le Conte Avenue CHS 54-140 Mail code 178218 Los310 825 7575..ucla.edu (M.L. Penichet).

Transferrin receptor 145

Transferrin receptor expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Iron uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Structural interaction between Tf and TfR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Other functions of the TfR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Transferrin receptor 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149TfR2 expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Targeting the TfR with monoclonal antibodies that directly inhibit proliferation and/or induce apoptosis . . . . . 150Rat anti-mouse TfR antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Murine anti-human TfR antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Chimeric anti-TfR antibody fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

The transferrin receptor system

Transferrin

Iron is a required cofactor of heme and nonheme proteinsinvolved in a variety of cellular processes including metab-olism, respiration, and DNA synthesis [1,2]. Dietary iron isabsorbed by the intestine [3]. Iron in the ferric state (Fe3+) isreduced to the ferrous state (Fe2+) in the intestinal lumenand Fe2+ is transported across the duodenal epithelium bythe divalent metal transporter 1. Ferroportin then exportsFe2+ into the portal circulation where it is oxidized to Fe3+.At physiological conditions, free Fe3+ can play a key role inthe production of free radicals or it can be hydrolyzed toform insoluble polymers, both of which are toxic to cells. Tfis part of a family of proteins that includes serum Tf,ovotransferrin, and lactoferrin [4], which binds circulatingFe3+ and prevents it from traveling throughout the body inits toxic form. Tf transports and delivers iron into cells viainteractions with its receptor [5]. For the purpose of thisreview, only serum Tf and its therapeutic potential todeliver molecules into tumor cells will be discussed. TheTf monomer (80 kDa) is a glycoprotein that consists of twosubunits (40 kDa each) known as the N-lobe and the C-lobe,which are separated by a short spacer sequence. Each lobe iscapable of binding one iron atom and therefore each Tfmolecule (apo-Tf) can transport one (monoferric Tf) or two(diferric Tf) iron atoms. The association constant for diferricTf and the TfR is 30-fold higher than that of monoferric Tf,and 500-fold higher than apo-Tf [1,6]. Differic Tf representsapproximately 10—30% of circulating Tf, leaving Tf to bindwith other metal ions [1,4]. Synthesis of Tf occurs primarilyin hepatocytes, but small amounts are also synthesized inSertoli, ependymal, and oligodendroglial cells [5].

Transferrin receptor

The TfR (CD71) is an essential protein involved in ironuptake and the regulation of cell growth [7]. TfR knockoutembryos die before embryonic day 12.5 due to defects inerythropoiesis and neurological development [8]. Deliveryand uptake of iron from Tf into cells occur through theinternalization of iron-loaded Tf and are mediated by theTfR [1,2,4,9—11]. The TfR is a type II transmembraneglycoprotein found primarily as a homodimer (180 kDa)consisting of identical monomers joined by two disulfide

bonds [12] (Fig. 1). Each monomer (760 amino acids,molecular weight 90—95 kDa) contains a large extracellularC-terminal domain (671 amino acids) known as the ectodo-main that contains the Tf-binding site, a single-passtransmembrane domain (28 amino acids), and a shortintracellular N-terminal domain (61 amino acids). Theectodomain contains 3 N-linked glycosylation sites and oneO-linked glycosylation site. Glycosylation at these sites isrequired for adequate function of the receptor. Phosphory-lation of the TfR on serine 24 in the intracellular domain hasbeen previously demonstrated but is not required forinternalization or recycling of the receptor.

Regulation of TfR expression has been studied in greatdetail and was reviewed in Pantopoulos et al. [3] (Fig. 2).TfR expression is primarily regulated at the post-transcrip-tional level in response to intracellular iron levels. The 3Vuntranslated region (UTR) of the TfR transcript is large andplays an important role in the regulation of mRNA stability.This region contains 5 iron response elements (IRE) thatconsist of about 30 nucleotides each that form loopstructures and are involved in the post-transcriptionalregulation of TfR expression. These IRE are recognized bytwo RNA-binding iron regulatory proteins (IRP). IRP-1 isubiquitously expressed in all cells and knockout micedemonstrate no change in phenotype. IRP-1 is homologousto the mitochondrial aconitase, an iron-sulfur cluster (4Fe-4S) containing enzyme of the citric acid cycle. Like themitochondrial aconitase, IRP-1 contains a 4Fe-4S clusterthat regulates its function. In high iron conditions, the 4Fe-4S cluster of IRP-1 is assembled and the protein acts as anaconitase. Thus, IRP-1 does not bind to the IRE regions ofthe TfR under these conditions and the TfR mRNA isdegraded. High iron also impedes the binding of IRP-1 tothe mRNA of the iron storage protein ferritin allowing itstranslation. The net result in iron replete conditions is theinhibition of further iron uptake and promotion of ironstorage by the cell. In iron-deficient conditions the 4Fe-4Sdisassembles and the aconitase activity of IRP-1 is lost,allowing it to bind and stabilize the IREs of the TfR mRNA.The mRNA is translated and the levels of TfR on the surfaceof the cell increase. IRP-1 also binds ferritin mRNA, but inthis case inhibits the translation of ferritin mRNA. Theoverall result in iron-deficient cells is to increase ironuptake via the Tf-TfR system and block iron storage byferritin.

The second IRP protein, IRP-2, also regulates theexpression of TfR. It is 61% identical to IRP-1 at the

Figure 1 Schematic representation of the TfR. This receptoris a type II receptor found on the cell surface as a homodimerconsisting of two monomers linked by disulfide bridges atcysteines 89 and 98 (n). The TfR contains an intracellulardomain, a transmembrane domain, and a large extracellulardomain. There is an O-linked glycosylation site at threonine 104(E) and three N-linked glycosylation sites on arginine residues251, 317 and 727 (.). The extracellular domain of the TfRconsists of three subdomains: apical (A), helical (H) andprotease-like domain (P).

T.R. Daniels et al.146

amino acid level. Similar to IRP-1, binding of IRP-2 to theIREs in the TfR mRNA increases TfR expression. UnlikeIRP-1, IRP-2 contains only mRNA-binding activity. It lacksaconitase activity and is not regulated by the labile iron-sulfur cluster. IRP-2 is regulated instead by iron-inducedproteolysis in response to iron and oxygen levels. In irondepleted or hypoxic cells IRP-2 is stable and increases TfRexpression, while in iron replete or normoxic cells, IRP-2undergoes proteosomal degradation. IRP-2 knockout miceshow aberrant iron homeostasis and accumulation of ironin the intestinal mucosa and the central nervous system[3].

Transferrin receptor expression

The TfR is ubiquitously expressed at low levels on normalcells and is expressed at greater levels on cells with a highproliferation rate such as cells of the basal epidermis andintestinal epithelium [13—15]. Activated peripheral bloodmononuclear cells express high levels of the TfR [16—18].

The TfR is also expressed on cells that require large amountsof iron such as placental trophoblasts, the cells responsiblefor the delivery of iron to the fetus, and maturing erythroidcells that require iron for heme synthesis [18]. However,mature erythroid cells do not express the TfR. TfR expres-sion has also been observed on nonproliferating cellsincluding those of the vascular endothelium of braincapillaries, endocrine pancreas, seminiferous tubules ofthe testes, cells of the pituitary, luminal membranes ofthe breast, hepatocytes, Kupffer cells of the liver, andtubules of the kidney [19—21]. Little or no TfR expressionhas been detected on pluripotent hematopoietic stem cells(from the mouse, rat, or human) [22,23], while late cyclingmurine and human erythroid progenitor cells do express thereceptor [20,24—26].

Various studies have also shown elevated levels ofexpression of the TfR on cancer cells when compared totheir normal counterparts [13—15,19,20,24,27—45]. Thiscould be attributed to the increased need for iron as acofactor of the ribonucleotide reductase enzyme involved inDNA synthesis of rapidly dividing cells. When benign andmalignant breast epithelium were compared in the samesection, TfR expression could be up to 4- to 5-fold higher inmalignant breast cells when compared to nonneoplasticbreast cells [33, 34]. Bladder transitional cell carcinomas,breast cancer, gliomas, lung adenocarcinoma, chroniclymphocytic leukemia and non-Hodgkin’s lymphoma alsoshowed increased TfR expression that correlated with tumorgrade and stage or prognosis [30,32,36,46—48]. Primarybladder malignancies with high expression of TfR demon-strated a higher rate of recurrence than those with low TfRexpression [49]. Increased TfR expression was also detectedon peripheral blood mononuclear cells from lymphoma,myeloma, or leukemic tumor patients compared to thosetaken from normal patients [38]. These data suggest thatTfR expression is increased on circulating tumor cells, tumorprecursor cells, or cells that have been activated duringtumorigenesis.

Iron uptake

The pathway by which iron-bound transferrin is internalizedwith the TfR has been studied extensively [1—5,9—11,18,50]. Diferric Tf binds the receptor and both areinternalized in clathrin-coated pits through receptor-medi-ated endocytosis (Fig. 3). The decrease in pH in theendosome facilitates a conformational change in Tf [51]and its subsequent release of iron, while increasing theaffinity of apo-Tf for the receptor. Tryptophan641 andphenylalanine760 residues of TfR and the histidine349 residueof Tf interact to trigger the conformational change thatresults in the release of iron from the C-lobe of Tf [52]. Theapo-Tf/TfR complex returns to the cell surface where apo-Tf is released. Diferric Tf has the highest affinity for the TfRand is 10- to 100-fold greater than that of apo-Tf atphysiological pH [2]. The TfR is constitutively internalizedindependently of ligand binding [24,53,54]. This is incontrast to other receptors, such as the epidermal growthfactor receptor, that are internalized only after interactionwith its ligand (ligand-mediated internalization). Approxi-mately 10% of the recycling TfR undergoes a slowerrecycling pathway through the Golgi complex [1].

Figure 2 Regulation of TfR and ferritin expression in response to cellular iron levels. (A) Expression of both proteins is post-transcriptionally regulated by the binding of iron regulatory proteins (IRP) to iron response elements (IRE) in untranslated regions(UTR) of the mRNA. (B) Low iron conditions cause the dissociation of 4Fe-4S clusters from IRP-1 as well as the de novo synthesis ofIRP-2. This allows binding of both IRP-1 and IRP-2 to the single IRE in the 5V UTR of the ferritin mRNA as well as multiple IREs in the 3VUTR of the TfR mRNA. Binding of IRPs to IRE sites in the 3V UTR of TfR stabilizes its mRNA, while binding of IRPs to the IRE of ferritinmRNA inhibits its translation. (C) High iron conditions allow the association of 4Fe-4S clusters to cysteine residues within the IRE-binding site of IRP-1 as well as the ubiquitination and proteosomal degradation of IRP-2. High iron conditions eliminate IRP binding toboth ferritin and TfR mRNA allowing the translation of ferritin mRNA and triggering the degradation of TfR mRNA throughendonucleosis of its 3V UTR.

Transferrin receptor 147

Structural interaction between Tf and TfR

The crystal structures of Tf and the TfR have beenindividually characterized [55]. Recent studies have char-acterized the structural interaction between Tf and itsreceptor and have further developed our understanding ofthe process of iron uptake by the cell [52,56—59]. Cryo-electron microscopy (cEM) was used to develop a densitymap of the diferric Tf-TfR complex [56]. An atomic modelof the complex was constructed by fitting the crystalstructures of the N-lobe of iron-bound Tf [51], the C-lobeof rabbit Tf [60] and the TfR ectodomain [61] into thedensity map. The resulting model shows that the C-lobe ofdiferric Tf interacts with the lateral surface of the TfR

and the N-lobe binds the stalk region the receptor. The N-lobe thus fits in the space between the cell membraneand the ectodomain of the TfR. The two stalks of the TfRare connected by two disulfide bonds [12] and arepredicted to rest between the N-lobes of the two Tfmolecules bound to the TfR homodimer. The C-lobe of Tfbinds the receptor at a single site, while the N-lobe bindsat two distinct sites. Mutational analysis [52], X-rayhydroxyl radical footprinting [57] and radiolytic footprint-ing followed by mass spectrometry [58] have all confirmedthe suggested binding residues predicted by the cEMmodel. Interestingly, this implies that the apical regionof the receptor does not directly bind Tf, and thus itsfunction remains unknown.

Figure 3 Cellular uptake of iron through the Tf system via receptor-mediated endocytosis. (A) The TfR consists of a dimer on thesurface of the cell. Each receptor monomer binds one Tf molecule that consists of two lobes (the N and C lobes). Each lobe of Tf bindsone iron molecule and thus two diferric Tf molecules bind to the receptor with high affinity. (B) Endocytosis of the diferric Tf/TfRcomplex occurs via clathrin-coated pits and the complex is delivered into endosomes. Protons are pumped into the endosomeresulting in a decrease in pH that stimulates a conformational change in Tf and its subsequent release of iron. The iron is thentransported out of the endosome into the cytosol by the divalent metal transporter 1 (DMT1). Apotransferrin remains bound to theTfR while in the endosome and is only released once the complex reaches the cell surface.

T.R. Daniels et al.148

In addition to Tf, the hereditary hemochomatosis protein(HFE) is also involved in the regulation of iron homeostasisthrough its interaction with the TfR [62]. HFE is a homolog tothe major histocompatibility complex molecules and mustassemble with b2 microglobulin [63]. Hereditary hemochro-matosis (HH) is a genetic disorder characterized by excessabsorption of dietary iron and progressive iron deposition invarious tissues, especially the liver. The most commonmutation in patients with HH occurs in the HFE gene andprevents the proper folding and interaction of HFE with b2microglobulin. Mutations in other iron-related proteins havebeen identified in patients with HH [64]. HFE remains bound

to the TfR in endosomes and this interaction may target theTfR to lysosomal degradation [65]. While Tf and HFE bindoverlapping sites on the TfR and compete for receptorbinding, HFE does not interfere with the release of ironfrom Tf in the endosome [52,66,67]. The model proposed byCheng et al. [56] demonstrates that the TfR homodimer canbind HFE on one monomer and diferric Tf on the other,providing evidence that HFE directly competes with Tf forreceptor binding. Thus, HFE not only competes with Tf forbinding to the TfR, but it decreases the level of cellularuptake of iron by disrupting the recycling pathway of TfR andleads to degradation of the receptor.

Transferrin receptor 149

Other functions of the TfR

Several studies suggest that the TfR may play additional rolesin cell growth and proliferation besides its function in ironuptake. It has recently been suggested that the TfR may alsocontain immunoregulatory properties. For example, incontrast to FcaRI receptor that binds both IgA1 and 2, ithas been reported the TfR selectively binds monomeric IgA1antibodies [68]. TfR expression is upregulated on culturedmesangial cells and glomerular mesangial cells in patientswith IgA nephropathy suggesting that the TfR may play animportant role in the pathogenesis of this disease [68].

The TfR may also play an immunoregulatory role in T cellactivation. The TfR can provide the second stimulus requiredfor the activation of T cells and may also be involved inantigen-independent pathways of T cell activation [69,70].J64 is a monoclonal anti-human TfR IgG1 antibody that doesnot interferewith Tf binding. J64 in combinationwith phorbolmyristate acetate (PMA), antibodies to the T3/antigenreceptor complex (that serve has agonists in T cell activa-tion), or calcium ionophores induced IL-2 secretion by HUT78T lymphoma cells [69]. J64 showed other PMA-like activitiessuch as increased IL-2 receptor expression and inhibition ofcell growth [69]. Another murine monoclonal anti-human TfRIgG1 antibody (FG 1/6) also induced Tcell proliferation in thepresence of submitogenic doses of phorbol esters that areknown to activate protein kinase C [70]. The rat monoclonalanti-murine TfR IgG antibody C2F2 selectively inhibited IL-1-dependent T cell activation, but did not block IL-2 inducedactivation [71]. Taken together, these studies suggest thatthe TfR plays selective roles in Tcell activation that have notyet been fully elucidated.

In addition, the TfR directly associates with the T cellreceptor (TCR) zeta chain and the zeta-binding ZAP70tyrosine kinase [72]. Stimulation of the TfR in the humanJurkat T leukemia cell line by treatment with the FG 1/6antibody resulted in the increase in tyrosine phosphoryla-tion of the TCR zeta chain. Conversely, stimulation of theTCR using an the F(abV)2 fragment of the OKT3 murineanti-TCR IgG2a antibody resulted in tyrosine phosphoryla-tion of the TfR. It was proposed that the TfR-zeta complexis responsible for transducing the TfR-induced signals thatserve to amplify those delivered by antigen binding to theTCR. Although the TfR is typically absent from lipid rafts atsteady state, stimulation with a mitogenic mixture of anti-CD3 antibodies and human Jurkat T cells leads to a rapidcompartmentalization of the TfR into lipid rafts accompa-nying that of CD3 epsilon and Lck protein tyrosine kinase[73]. The FG 1/6 antibody blocked the formation of theselipid rafts and the immunological synapse. These studiessuggest that the TfR is involved in the formation of theimmunological synapse and may also play a role in signaltransduction during T cell activation.

It has also been suggested that the TfR is required for thenormal maturation of thymocytes. TfR knockout mice die byembryonic day 12.5 due to severe anemia [8]. TfR�/�

chimeric mice were generated by introducing TfR�/� stemcells into wild-type blastocysts [74]. Mice were then analyzedto determine which tissues require TfR for development, orthose derived exclusively from the wild-type stem cells.TfR�/� embryonic stem cells from this chimeric mouse gaverise to most tissues and organs but not to hematopoietic

tissues on a wild-type C57BL/6J background. This suggeststhat both adult erythropoiesis and lymphopoiesis require TfRexpression. Chimeric mice were also generated by introduc-ing TfR�/�mouse embryonic stem cells into immunodeficientRAG2�/� blastocysts [74]. In these mice, all mature lympho-cytes are derived from the TfR�/� stem cells. In this modelsystem, TfR�/� stem cells support B cell development at leastto the IgM+ stage. Tcells, however, are arrested very early intheir development at the CD4�CD8�CD3� stage. This suggeststhat the TfR is required for Tcell development and to a muchlesser extent in B cell development, a claim furthersupported by a study that used hypotransferrinaemic(TfRhpx/hpx) mice to investigate the possible involvement ofTf in T lymphocyte differentiation in vivo [75]. TfRhpx/hpx

mice carry a spontaneous point mutation in a splice donor siteof the Tf gene that results in severe deficiency in serum Tf,less than 1% of the levels found in normal mice. TfRhpx/+ micecarry half the normal levels of circulating Tf and do not differfrom wild-type mice. TfRhpx/hpx mice are profoundly anemicand are not viable unless given Tf during the first 4 weeksafter birth. These mice also show iron overload in parenchy-mal tissues as a result of increased iron absorption andincreased clearance of non-Tf-bound plasma iron. The overallnumber of thymocytes was reduced in TfRhpx/hpxmice and thisreduction was not attributed to increased apoptosis. In thethymus immature T cells, termed TN thymocytes (CD4�,CD8�, and CD3�), are divided into four subpopulations: TN1(CD44+, CD25�), TN2 (CD44+, CD25+), TN3 (CD44�, CD25+),and TN4 (CD44�, CD25�). The TN4 cells are direct precursorsto double positive (CD4+, CD8+) thymocytes. This studyindicated that TfRhpx/hpx mice show a significant blockagein Tcell development between the TN3 and TN4 stages as wellas no reduction in the rate of thymocyte proliferation,suggesting that the thymus has alternative Tf-independentmechanisms of iron uptake. This study also suggests thateither Tf or the interaction of Tf with its receptor is requiredfor early T cell differentiation in vivo.

Transferrin receptor 2

A second transferrin receptor (TfR2) was identified by twoindependent groups and has been previously reviewed [76].Glockner et al. [77] identified the TfR2 gene while cloningthe entire region of the human chromosome 7q22. Kawabataet al. [78] also identified this protein serendipitously whensearching for new transcription factors associated with thedevelopment of cancer. This latter group subsequentlyidentified TfR2 in mice [79] and also further characterizedhuman TfR2 [80, 81]. There are two transcripts of the humanTfR2 gene, a and b, that are produced by alternative splicing[78]. TfR2 a shows similar domain structure to TfR1. TheTfR2 a extracellular domain is 45% identical and 66% similarto the TfR1 ectodomain. However, the N-terminal cytoplas-mic regions of TfR1 and TfR2 a show no similarity. TfR2 btranscript lacks exons 1—3 of TfR2, thus it does not contain atransmembrane or cytoplasmic domain. TfR2, like TfR1, isglycosylated, however, it has a 25-fold lower affinity for Tfthan TfR1 [81]. TfR1 and TfR2 are able to form hetero-dimers, but preferentially form homodimers [82].

Unlike TfR1, the expression of TfR2 is not regulated inresponse to intracellular iron levels [80,81]. Interestingly,the TfR2 mRNA does not contain IRE sequences. In the

T.R. Daniels et al.150

human erythroleukemic cell line K562, treatment withdesferrioxamine (DFO) did not result in the upregulation ofthe TfR2 mRNA, while TfR1 levels increased [81]. Instead ofbeing regulated by intracellular iron levels, TfR2 expressionmay be regulated by the cell cycle [81]. In the MG-63 humanosteosarcoma cell line, high levels of the protein wereobserved in the late G1 phase of the cell cycle andexpression was lost in G0/G1. This was in contrast to theexpression of TfR1 where expression peaked in both late G1and G2/M. TfR2 expression is also regulated by diferric Tf[83,84]. In fact, TfR2 protein levels increased in a time- anddose-dependent manner after diferric Tf treatment but notafter apo-Tf or free iron treatment [83,84]. Taken together,these studies suggest that the regulation of TfR1 and TfR2expression are considerably different.

TfR2 expression

The pattern of TfR2 expression appears to be different fromthat of TfR1. In contrast to TfR1, TfR2 expression appears tobe limited to hepatocytes and enterocytes of the smallintestine [80,85]. Surface expression of TfR2 was detectedin a wide variety of human cell lines derived from solidtumors and selected B and myeloid cell lines [85]. Highlevels of TfR2 mRNA have also been detected in HepG2(human hepatoma), K562, and HEL-R (both human erythro-leukemic) cell lines [78,80]. The human lymphoid cell linesRaji and MOLT-16 and the human myeloid cell lines U937,NB4, HL-60, KCL22, and KG-1 expressed low or undetectablelevels of TfR2 mRNA [80]. In contrast, TfR1 mRNA expressionwas detected in all the lymphoid, myeloid, and erythroidcell lines tested, the highest observed in OCI-M1 erythroidcells. TfR2 mRNA was also detected in human CD34+

erythroid precursors isolated from normal peripheral blood.An analysis of TfR2 expression in patients showed thatmyeloid and nonerythroid leukemia cases show significantlyelevated TfR2 levels compared to nonmalignant bonemarrow cells and that this upregulation contributed to abetter prognosis [80,86]. Thus, TfR2 may be a novelprognostic factor for patients with acute myelogenousleukemia [80]. The proposed intracellular b transcript ismore widely distributed but at much lower levels and itsfunction remains unclear [78].

In addition to the above evidence that TfR2 is expressedin erythroid precursors, both human and murine TfR2promoters contain 2 typical GATA-1 consensus sequencesand a doublet of the CAAT sequence [79]. GATA-1 is anerythroid-specific transcription factor that is essential forthe early stages of erythrocytic differentiation. GATA-1 wasshown to enhance TfR2 promoter activity, which is consis-tent with expression analysis and suggests that TfR2transcription is regulated by erythroid-specific machinery.Despite the fact that the above studies suggest that TfR2 isexpressed in erythroid precursors, this remains a controver-sial issue. Calzolari et al. [87] found that TfR2 was notexpressed at the protein level in normal erythroid cells atany stage of differentiation. These authors point out thatsome of the previous studies only tested TfR2 mRNA levels.They suggest that although there may be some detectablemRNA (as confirmed by their own mRNA studies), the proteinis not expressed in normal erythroid cells. These authorsalso point out that the lack of TfR2 expression in erythroid

cells is supported by their studies on an inactivatingmutation of the TfR2 gene where they demonstrated thatthis inactivation does not affect erythroid cell homeostasis.

Targeting the TfR with monoclonal antibodiesthat directly inhibit proliferation and/or induceapoptosis

Rat anti-mouse TfR antibodies

Various studies have demonstrated the effectiveness of anti-TfR antibodies to inhibit the growth of many cell types(Table 1). The rat anti-mouse TfR IgM antibody R17 208blocks iron uptake in S194/5.XXO.BU.1 murine myelomacells and mouse L cells [40]. R17 208 also inhibits the growthof S194/5.XXO.BU.1 by arresting them in the G2/M phase ofthe cell cycle. Surprisingly, R17 208 demonstrated no growthinhibitory effects on mouse L cells. This could be due todifferential iron requirements or alternative iron uptakemechanisms between the two cell types [40]. To determineif antibody class plays a role in the cytotoxic effects of anti-TfR antibodies, Lesley et al. [88] investigated the cytotoxiceffects of five rat monoclonal anti-murine TfR antibodies ofdifferent classes. IgM antibodies, R17 208 and REM17,prevented cell growth of the AKR1 mouse T lymphoma cellline while the IgG antibodies, R17 217 (IgG2a), RL34-14(IgG2a), and RR24 (IgG2b), did not significantly inhibit cellgrowth in these cells. Neither IgM nor the IgG antibodiesblocked Tf binding to its receptor. The IgM antibodies thatextensively cross-linked the TfR blocked internalization ofTfR and iron uptake [88,89]. Cells treated with IgGantibodies demonstrated downregulation of the surfaceTfR level and increased TfR degradation without significantgrowth inhibitory effects [88]. However, monovalent (Fab)antibody fragments of these IgG antibodies did not down-regulate TfR surface expression or demonstrate degradationof the TfR [89]. The addition of a secondary anti-IgGantibody to the IgG anti-TfR-treated cells mimicked thegrowth-inhibiting effects of IgM antibodies. Taken together,these studies suggest that cross-linking of cell surfacereceptors is vital for the cytotoxic effects of the anti-TfRantibodies.

The in vivo effects of the R17 208 and REM17 IgMantibodies have also been examined in mice challengedwith syngeneic AKR/J SL-2 leukemic cells [90]. Even thoughthe in vitro studies showed that the two antibodies hadsimilar cytotoxic effects, R17 208 prolonged the survival ofmice-bearing AKR/J SL-2 leukemic cells in a dose-dependentmanner, whereas, REM17 did not affect the survival rate ofthe mice. Although acute toxicity was minimal, R17 208treatment decreased bone marrow stem cell activity andincreased the number of erythroid and cellular colonyforming units in the spleens of these mice.

Additional in vivo studies with two rat monoclonal anti-murine TfR IgG2a (C2 and RL34) antibodies demonstratedthat the combination treatments could block 38C13 mouse Bcell lymphoma growth in C3H/HeN mice immunocompetentmice [91]. Combination treatment of the C2 antibody withthe iron chelator deferoxamine (DFO) blocked lymphomagrowth in this model, however, no anti-tumor activityagainst established tumors was observed. Neither DFO nor

Table 1 Antibodies to the TfR that inhibit proliferation/cell growth or induce apoptosis

Antibody Isotype Comments References

Rat anti-mouse TfRR17 208 IgM Blocks internalization of the Tf-TfR complex [40,88,90]

Does not interfere with Tf bindingCells arrest in G2/M phase of the cell cycleInhibits cell growth of hematopoietic tumor cells in vitro and in vivo

REM17 IgM Blocks Tf function [88,90]Inhibits cell growth of hematopoietic tumor cells in vitro and in vivo

R17 217 IgG2a Does not inhibit cell growth of hematopoietic tumor cells [88,89]Does not block Tf-TfR internalizationDownregulates surface TfR expression

RL34-14 IgG2a Does not inhibit cell growth of hematopoietic tumorcells unless cross-linked or used in combination

[88,91]

RR24 IgG2b Does not inhibit cell growth of hematopoietic tumorcells unless cross-linked

[88]

C2F2 IgG2a Inhibits IL-1 dependent T cell activation and proliferation [71]C2 IgG2a Does not inhibit cell growth of hematopoietic tumor

cells unless cross-linked or used in combination[91]

Murine anti-human TfRJ64 IgG1 Does not block Tf binding [69]

Promoted IL-2 secretion in HUT78 T lymphoma cells(as a second stimulus)

FG (1/6) IgG1 Second stimulus for T cell proliferation [70]B3/25 IgG1 Does not block Tf binding [95]

Cytotoxic to hematopoietic tumor cells43/31 IgG* Inhibits binding of Tf to TfR [94,96,97]

Cytotoxic to normal granulocyte/macrophage progenitor cellsNot cytotoxic to mitogen stimulated mononuclear or CCRF-CEM cells

42/6 IgA Generally cytotoxic to hematopoietic tumor cells [16,92,94,97,98,104—107]

Generally more potent than the IgG antibodiesSolid tumors show minimal cytotoxic effects)Inhibits binding of Tf to TfR (steric hindrance) and decreases iron uptakeDownregulates surface TfRCells accumulate in the S-phase of the cell cyclePhase I Clinical Trial

E2.3 IgG1 Cytotoxic to IL-6 dependent hematopoietic tumor cells [101]D65.30 IgG1 Inhibited growth of CCRF-CEM cells [96]A27.15 IgG1 Cytotoxic to IL-6 dependent hematopoietic tumor cells [100]A24 IgG2b Competes with Tf for TfR binding and blocks iron uptake [68,102]

Reduces TfR expression and impairs TfR recyclingInduces apoptosis of activated T cells

RBC4 IgM Inhibits cell growth of hematopoietic tumor cells in vitro [93]

Chimeric anti-human TfR fusion proteinsAnti-hTfR IgG3-Av IgG3 128.1 Fv region, human Fc region [112]

Does not block Tf bindingInduces apoptosis in K562 cells

Anti-rTfR IgG3-Av IgG3 Fv from OX26 antibody, human Fc region [112]Does not block Tf bindingInduces apoptosis in Y3-Ag1.2.3 and C58(rat T cell lymphoma cell line) but not in BC47bladder carcinoma or 9L gliosarcoma

* IgG isotype not described.

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C2 alone exhibited consistent anti-tumor activity whengiven as a single therapy. The DFO and C2 combinationtreatment was accompanied by an increase in the risk of

bacterial infection. The two anti-TfR antibodies C2 and RL34used in combination (without DFO) also resulted in tumorregression, but were associated with sporadic mortality.

T.R. Daniels et al.152

Murine anti-human TfR antibodies

In general, human hematopoietic cells and cell lines aremore sensitive to the growth inhibitory effects of monoclo-nal antibodies than nonhematopoietic cell types [18,92].Like antibodies to the murine TfR, monoclonal IgG anti-bodies specific for the human TfR do not inhibit TfRinternalization and generally have less of an effect on cellgrowth when compared to multivalent isotypes such as IgM,IgA, or IgG cross-linked with secondary antibodies (Table 3).The murine monoclonal anti-human TfR IgM antibody RBC4inhibits the proliferation of a panel of human T, B andmyeloid cell lines as well as activated peripheral bloodlymphocytes [93], but does not affect unstimulated lym-phocytes. The murine monoclonal anti-human TfR IgGantibodies B3/25 (does not block Tf-binding TfR) and 43/31 (blocks Tf binding) inhibit the growth of granulocyte/macrophage progenitors (CFU-GM) isolated from normalvolunteers and patients with chronic myelogenous leukemiaand the human myeloid leukemia cell lines KG-1 and HL60[94—96]. CFU-GM from leukemia patients and the cell lineswere generally more sensitive than normal CFU-GM. How-ever, B3/25 and 43/31 did not inhibit growth in mitogenstimulated mononuclear or CCRF-CEM human Tcell leukemiacell line [97,98]. The murine anti-human TfR IgG1 antibody128.1 did not inhibit the growth of CCRF-CEM or K562 cells[97].

Like IgG antibodies to the mouse TfR, treatment ofeither murine anti-human TfR IgG antibody OKT9 or B3/25downregulates the surface expression of TfR and results inthe increased degradation of the receptor [88,99]. Howev-er, in most cases IgG antibodies are not growth inhibitoryand are only cytotoxic when used in combination [97,100]or when cross-linked by a secondary anti-murine antibody[98]. However, there are a few exceptions. The murinemonoclonal anti-TfR IgG1 antibody D65.30 was found tohave a cytotoxic effect against the CCRF-CEM humanleukemic cell line, an activity that was maximized whencombined with various other murine IgG antibodies target-ing the transferrin receptor [97]. Also the murine mono-clonal anti-human TfR IgG1 antibodies E2.3 and A27.15inhibited the growth of the IL-6-dependent OCI-My4 humanmyeloma cell line and the human acute myelogenousleukemia cell line UCSD/AML1 [101]. However, cell linesthat are not dependent on IL-6 for growth (human myelomacell lines 8226 and U266) were resistant to the anti-growtheffects of E2.3 and A27.15. Another murine monoclonalanti-human TFR IgG2b A24 antibody demonstrates anti-tumor effects on T lymphocytes isolated from patients withacute T cell leukemia [102]. A24 blocks Tf binding to thereceptor and iron uptake in activated T cells. A24 alsoinhibits T cell proliferation through the induction apoptosisof adult T cell leukemia cells ex vivo. Cell death was shownto occur as a result of reduced expression and impairedrecycling of TfR. The combination therapy of A24 pluschemotherapies such as VP16 (plant alkaloid, topoisome-rase II inhibitor) leads to synergistic enhancement of celldeath.

The murine monoclonal anti-human TfR IgA antibody 42/6 has demonstrated the most potent cytotoxic effectsagainst human malignancies [92,94,97,103]. This antibodyinhibits the binding of Tf to its receptor by noncompetitive

inhibition and blocks growth due to iron deprivation [98].Treatment with 42/6 also leads to the downregulation ofTfR on the surface of the cells and inhibits the proliferationof mitogen activated T cells, the human T leukemic cell lineCCRF-CEM, and the human myeloid leukemic cell lines KG-1and HL60 [94,98,104,105]. In addition, the cytotoxic effectof 42/6 was enhanced in HL-60 and KG-1 leukemic cellsgrown in serum-free conditions [106]. In contrast to mousecells treated with R17 208 that arrest in the G2/M phase[40] discussed above, CCRF-CEM cells treated with 42/6accumulate in S-phase of the cell cycle and the addition offree iron did not overcome these cytotoxic effects [105].42/6 also inhibits proliferation that eventually leads to celldeath of human lymphocytes stimulated by phytohemag-glutinin [103]. Removal of 42/6 from the culture mediumwithin 48 h of treatment initiation reversed the anti-proliferative effect of the antibody on stimulated lympho-cytes. 42/6 was also shown to cause dose-dependentgrowth inhibition of (CFU-GM) from normal and chronicmyelogenous leukemia patients [94]. CFU-GM isolated fromchronic mylogenous leukemia patients were generally moresensitive to the cytotoxic effects of 42/6 when compared toCFU-GM isolated from normal volunteers.

Due to the in vitro success of 42/6 in hematopoietictumor cell killing, a Phase I clinical trial was conducted in 27patients with advanced refractory cancer of differentorigins (epithelial, mesenchymal, and hematopoietic)[107]. These patients received 24-h intravenous infusionsof 42/6 at doses ranging from 2.5 to 300 mg/m2. This wasthe first clinical trial of a mouse IgA antibody, the first touse an anti-TfR antibody, and one of the few trials using anantibody that reacts with a broad range of normal tissues.The treatment was well tolerated and three patientsdemonstrated a mixed anti-tumor response. Interestingly,these three patients were all affected with hematopoieticcancer: follicular lymphoma, Hodgkin’s disease, and chroniclymphocytic leukemia. The lack of more effective thera-peutic results was explained at least in part by theextremely rapid clearance of IgA in circulation comparedto IgG antibodies and by the presence of human anti-mouseIgA antibodies that were detected in several patients.Treatment with 42/6 induced an initial decrease in marrowerythroid progenitor (BFU-e) growth, however, this decreasewas not significant. This trial demonstrated that 42/6infusions could inhibit malignant cell growth with minimalnonspecific effects.

Chimeric anti-TfR antibody fusion proteins

The use of monoclonal antibodies in the clinical treat-ment of cancer started in the early 1980s. However, thereare major setbacks in using murine monoclonal antibodiesfor therapeutic purposes in humans [108]. Due to theshort half-life of the antibodies in the blood, murinemonoclonal antibodies must be administered frequently inorder to maintain therapeutic levels. In addition, patientstreated with these murine antibodies often producehuman anti-mouse antibodies (HAMA) that limit thetreatment capacity of the murine antibody and potential-ly lead to severe allergic reactions. Effector functionssuch as antibody-dependent cellular cytotoxicity (ADCC)and complement-dependent cytotoxicity (CDC) are an

Figure 4 Y3-Ag 1.2.3 cells internalize biotinylated proteinscomplexed with the anti-rat TfR IgG3—avidin fusion protein. (A)Confocal microscopy of cells incubated with the complexes onice and at 378C (scale bar, 2 Am). (B) Internalization ofbiotinylated h-galactosidase complexed with anti-rat TfRIgG3-Av. Cells were incubated at 378C, for 3 h with 0.1 Ag ofanti-dansyl IgG3-Av (narrow line) or anti-rat TfR IgG3-Av (boldline) bound to biotinylated h-galactosidase (1:1 molar ratio).After washes, intracellular h-galactosidase activity wasdetected using a membrane permeable, fluorogenic h-galacto-sidase substrate, and flow cytometry. Reprinted from Ng et al.,2002. Proc. Natl. Acad. Sci, USA. 99(16) 10706:10711. Copyright2002 with kind permission of The National Academy of Sciences,USA.

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important aspect of antibody therapy. The human effectorsystem does not effectively interact with murine constantregions diminishing the full therapeutic potential of theantibody. These problems have been circumvented by thedevelopment of chimeric, humanized, and totally humanantibodies [108—110]. To date, the FDA has approvednumerous monoclonal and recombinant antibodies withhuman constant regions for the treatment of cancer thatare used alone or in combination with chemotherapeuticdrugs [109,111]. There is much optimism in this field forthe further development of these antibodies that bindnovel targets and improve targeting for the treatment ofcancer.

A study published by our group also provides evidencethat recombinant anti-TfR antibodies have tremendoustherapeutic potential [112]. Two chimeric antibodies werederived from either the variable region of the murinemonoclonal anti-rat TfR IgG2a antibody OX26 or thevariable region of the murine monoclonal anti-human TfRIgG1 antibody 128.1. The CH3 regions of both of thesechimeric antibodies were genetically fused to chickenavidin to generate antibody fusion proteins (anti-rTfRIgG3-Av and anti-hTfR IgG3-Av) that could serve as universalvectors to deliver biotinylated therapeutic agents (bacte-rial or plant toxins, chemotherapy drugs, etc.) to malignantcells. These antibodies are the first to contain IgG3 humanFc regions and target the TfR. Avidin was chosen due to itsdecreased immunogenicity when compared to the highlyimmunogenic bacterial protein streptavidin. Since oralantigens are known to induce tolerization, avidin is lessimmunogenic due to the consumption of chicken eggs[113]. These antibody fusion proteins do not inhibit thebinding of Tf to its receptor [20,97]. Human IgG3 waschosen due to its extended hinge region to facilitateantigen binding by the separation of the antigen-bindingsite and the Fc region that carries that therapeuticcompound. In addition, the human IgG3 Fc region willmaximize the effector functions of the antibody fusionproteins since it is the most effective of the IgG isotypes incomplement activation [114] and like IgG1 binds all threeFcc receptors [115].

The biological activities of the anti-rTfR IgG3-Av andanti-hTfR IgG3-Av fusion proteins have been previouslyreported [112]. Anti-rTfR IgG3-Av efficiently deliveredbiotinylated-FITC (Fig. 4A) and biotinylated b-galactosidase(Fig. 4B) into Y3-Ag1.2.3 rat myeloma cells throughreceptor-mediated endocytosis. Surprisingly, anti-rTfRIgG3-Av treatment alone inhibited proliferation (Fig. 5A)and directly induced apoptosis (Fig. 5B) in the Y3-Ag1.2.3and the C58 (NT) D.1G.OVAR.1 rat T cell lymphoma celllines. Inhibition of proliferation and the induction ofapoptosis were not observed in two rat cell lines not ofhematopoietic lineage, bladder carcinoma BC47 and glio-sarcoma 9L (Fig. 5B). The anti-hTfR IgG3-Av fusion proteinalso demonstrated dose-dependent anti-proliferative activ-ity in the K562 human erythroleukemic cell line, whichcorrelated with the rate of apoptosis in this cell line. Anontargeting fusion protein (anti-dansyl IgG3-Av) and theparental anti-hTfR 128.1 antibody did not demonstrate anycytotoxic effects.

The rat fusion protein was shown to be a dimer, probablydue to the tetrameric structure of avidin that would lead to

the interaction of two anti-rTfR IgG3-Av molecules (Fig. 6).The dimeric structure of anti-rTfR IgG3-Av suggests its toxicactivity was due to cross-linking of surface TfR, which issupported by the fact that anti-rat TfR IgG3 (not conjugat-ed to avidin) cross-linked with secondary antibody alsoexhibited anti-growth activity comparable to that of thefusion protein. However, cytotoxicity may also be due toother factors such as the positively charged avidin moietythat is known to bind heparin [116], the IgG3 extendedhinge region [117], or binding of the Fc region to Fccreceptors. Effector functions, including antibody-depen-dent cellular cytotoxicity (ADCC) and complement-depen-dent cytotoxicity (CDC), may also further enhance the anti-tumor effects of the fusion protein in vivo. Importantly, theintrinsic cytotoxic effects of anti-rTfR IgG3-Av or anti-hTfRIgG3-Av may be enhanced by the chemical conjugation tobiotinylated agents. In this way, the anti-hTfR IgG3-Av

Figure 5 The anti-rat TfR IgG3-Av fusion protein inhibits the proliferation and induces apoptosis of rat hematopoietic tumor cell lines.(A) (top panel) Y3-Ag1.2.3 were treated with anti-rat TfR IgG3-Av (n), anti-dansyl IgG3-Av (5), anti-rat TfR IgG2a (D), anti-rat TfR IgG3(.), or anti-dansyl IgG3 (o) at the indicated concentrations for 24 h. (bottom panel) Y3-Ag1.2.3 (n), BC47 (.), and 9L (D) cells weretreated with various concentrations of the anti-rat TfR IgG3-Av fusion protein for 24 h. Proliferation was determined by [3H]-thymidineincorporation and expressed as percent of control cells. Assays were performed in quadruplicate. (B) Y3-AG1.2.3, C58 (NT) D.1G.OVAR.1,BC47, and 9L were incubated with buffer alone (left panels) or anti-rat TfR IgG3-Av (right panels) for 48 h. Apoptosis was determined byannexin/propidium iodide staining and flow cytometry. Percentage of cells in each quadrant is shown in the corner. Reprinted from Ng etal., 2002. Proc. Natl. Acad. Sci, USA. 99(16) 10706:10711. Copyright 2002with kind permission of The National Academy of Sciences, USA.

T.R. Daniels et al.154

fusion protein may be used as a universal vector system todeliver a wide variety of therapeutic agents (chemothera-peutic drugs, toxic proteins, and high molecular weight

Figure 6 FPLC analysis of the anti-rat TfR IgG3—avidin fusionprotein shows a dimeric structure. The anti-rat TfR IgG3monomer is shown in the top panel, the anti-rat TfR IgG3—avidin fusion protein in the middle panel, and the standardprofile of dimeric IgA and monomeric IgG in the bottom panel.Fraction size is 1 ml. Reprinted from Ng et al., 2002. Proc. Natl.Acad. Sci, USA. 99(16) 10706:10711. Copyright 2002 with kindpermission of The National Academy of Sciences, USA).

compounds) (reviewed in [118]). Therefore, anti-hTfR IgG3-Av is advantageous because of its dual functionality,including its intrinsic cytotoxic effects as well as its abilityto deliver therapeutic agents to cancer cells. Ongoingstudies in our laboratory are focused on determining theefficacy of anti-hTfR IgG3-Av as a dual function therapeuticfor the treatment of hematopoietic malignancies.

Conclusion

Recent studies on the structural interaction between Tfand the TfR have refined our knowledge on how these twomolecules interact and mediate the uptake of iron. Otherstudies have demonstrated that the TfR not only binds Tfbut may interact with other proteins as well and thus mayhave additional functions besides its role in iron homeo-stasis. The TfR is an attractive targeting molecule thatcould potentially be used to treat a variety of malignan-cies. Targeting the TfR through the use of certainmonoclonal or recombinant antibodies specific for TfRcauses cytotoxic effects including growth inhibition andinduction of apoptosis in malignant cells. The chimericanti-TfR IgG3-Av antibody fusion protein is a uniquemolecule that can be used as a universal vector systemthat exhibits intrinsic cytotoxic activity in tumor cells andthat can also be used to deliver biotinylated cytotoxicmolecules to enhance its intrinsic tumor cytotoxicity.Further studies are warranted to refine the effects oftargeting the TfR with antibodies to maximize theirtherapeutic potential.

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Acknowledgments

This work was supported in part by grants K01 CA86915 andR01 CA107023 from NCI/NIH, the 2004 Brian D. NovisInternational Myeloma Foundation Senior Grant Award, andthe 2003 Jonsson Cancer Center Foundation InterdisciplinaryGrant ‘‘Targeted Therapy of Multiple Myeloma.’’

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