Antibody-targeted nanoparticles for cancer therapy

381

Review

Immunotherapy (2011) 3(3), 381–394 ISSN 1750-743X10.2217/IMT.11.5 © 2011 Future Medicine Ltd

Antibody-targeted nanoparticles for cancer therapy

Despite the advances in our understanding of the molecular and cellular basis of cancer, con-ventional front line cancer treatment remains centered on systemic chemotherapy and radio-therapy. These regimes are plagued by broad distribution, frequently resulting in suboptimal dosing of the tumor and/or induction of toxic side effects in normal tissues [1–3]. These issues have also inhibited the development of new drugs, where experimental agents have demon-strated exciting efficacies in vitro, but have failed in their ability to alleviate the disease in vivo. This can be due to inadequate dosing of the target area and drug accumulation in normal tissue [4–6].

The improved formulation and delivery of chemotherapies has the potential to address these current hurdles in the effective drug-ging of the diseased tissue. The application of nanoparticle (NP) delivery systems has been extensively studied for a range of drug molecules, with various perimeters such as size, entrap-ment efficiencies and release profiles examined in depth. Indeed, nanoparticulate formula-tions of daunorubicin (DaunoXome®; NeXstar Pharmaceuticals, CO, USA) and doxorubicin (Doxil®/Caelyx®, Janssen, UK and Myocet®, Sopherion Therapeutics, Inc., NJ, USA) are used clinically to treat breast cancer and Kaposi’s sarcoma [7].

Although the use of NP formulations has improved drug bioavailability, targeting of these entities has the potential to further improve both the therapeutic effectiveness of drug compounds

at the disease site whilst also reducing off-target effects. This targeting can be achieved through the coating of a broad range of molecules on the surface of the NP to achieve targeting and local-ization of the drug conjugate. In the following sections, the main classes of NP are briefly dis-cussed before a more in-depth review of the cur-rent progress with targeting strategies, focusing in particular on antibodies, are examined.

Nanoparticle fabrication diversity � Gold nanoparticles

The therapeutic application of gold-based NPs has been documented in the treatment of rheuma toid arthritis since the 1930s and has been extensively investigated in diagnostic imag-ing and therapeutic applications. Gold NP-based diagnostic strategies have been developed, exploiting gold interaction with near infrared radiations, based on surface plasmon scatter-ing, or surface plasmon resonance, which can be used to detect NPs at concentrations as low as 10–16 M [8]. For therapeutic purposes, gold NPs have been used as photosensitizers, emitting heat to bring about cell death when excited by minimal near infrared radiation [9].

Research into the use of gold-based NPs has examined different compositions and shapes, such as the conventional nanospheres, gold nanoshells (composed of a silica core enclosed in a thin layer of gold) [10] and gold nano-rods [11,12]. Each of these particles exploits the physiochemical proper ties of elemental gold [13]. Gold is resistant to oxidative corrosion and

In recent years, nanoparticulate-mediated drug delivery research has examined a full spectrum of nanoparticles that can be used in diagnostic and therapeutic cancer applications. A key aspect of this technology is in the potential to specifically target the nanoparticles to diseased cells using a range of molecules, in particular antibodies. Antibody–nanoparticle conjugates have the potential to elicit effective targeting and release of therapeutic targets at the disease site, while minimizing off-target side effects caused by dosing of normal tissues. This article provides an overview of various antibody-conjugated nanoparticle strategies, focusing on the rationale of cell-surface receptors targeted and their potential clinical application.

KEYWORDS: anticancer � drug delivery � immunotargeting � liposome � nanoparticle � quantum dot � receptor-mediated endocytosis

Francois Fay1 & Christopher J Scott†1

1School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK†Author for correspondence:Tel.: +44 289 097 2350 [email protected]

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is therefore considered stable to the various environments encountered within the human body [13]. However, although some studies have shown that gold would appear safe in vivo [14], others have demonstrated a significant reduction in cell viability when exposed to high concen-trations of gold-based NPs [15–18]. Nonetheless, gold-based NPs have now entered clinical examination. For example, polyethylene glycol (PEG)-coated NPs, conjugated to recombinant TNF-a, have been examined in a Phase I clinical study. In this study, patients with non responsive advanced and/or metastatic solid organ cancers were treated with single or several injections of various doses (from 50 to 600 mg/m2) of the NPs, without encountering dose limiting toxicity or immunogenicity [19].

� Single-wall carbon nanotubesSingle-wall carbon nanotubes consist of single-atom thick cylindrical structures [20]. Carbon nanotubes have been shown to absorb light of near infrared light wavelengths, making them useful for imaging [21], biosensing [22–24] and therapeutic strategies. Single-wall carbon nano-tube surfaces can be functionalized by poly-mers or biomolecules, and various therapeutic applications have been developed, including anti microbial [25,26] and photothermal tumor therapy [27,28]. In these strategies, similar to gold NPs, the carbon nanotubes are used as photosensitizers to kill targeted cells.

� Quantum dotsQuantum dots (QDs) are semiconductor crystal nanostructures. QDs have fluorescent properties that can be modified as a result of changing the diameter or composition of the particle [29]. These properties make them ideal for biological applications, as in the case of bio-imaging [30–32]. Furthermore, the conjugation of the QD to other molecules, such as targeting proteins, does not affect their spectral properties. However, as the core of the QD contains toxic metal, leakage from this has raised some toxicity concerns [33–35].

� LiposomesLiposomes are spherical vesicles composed of one or more aqueous cores, which are enclosed in a single or multilamellar lipid bilayer (Figure 1). The development of liposomal vesicular carri-ers has been examined, attempting to mimic natural lipid bilayers since the first description of an endosome/lysosome in the early 1960s. Liposomal drug formulations can provide longer

drug half lives as well as tailored drug-release pro-files, reducing high peak plasma concentrations. Moreover, it has been shown that liposomes can accumulate within the tumor tissue as a result of the enhanced permeability and retention (EPR) effect; a consequence of leaky vasculature asso-ciated with the developing cancer [36,37]. A fore-most example of the application of liposomes in drug delivery is in the application of doxorubicin. Doxorubicin can induce off-target myocardial toxicity, which is significantly reduced when the drug is encapsulated within a liposomal prepa-ration [38,39]. Consequently, liposomal prepara-tions of doxorubicin (Doxil/Caelyx and Myocet) and daunorubicin (DaunoXome), have been clinically approved to date [40–43].

Liposomal lipid bilayers are usually composed of naturally derived phospholipids such as egg phosphatidylethanolamine, hydrogenated soy phosphatidylcholine and cholesterol. Cationic surfactants, such as diol-eolyl-phosphatidyl-etha-nol-amine, or ionizable cationic lipid, such as dio-leoyl-trimethyl ammonium propane (DODAP), have also been used to produce positively charged structures that promote interaction with nega-tively charged molecules such as DNA and gene-silencing RNA (siRNA) [44–49]. Liposomes may also have potential in the co encapsulation of different types of hydrophilic biologics, such as siRNA in combination with imatinib, to treat chronic myeloid leukemia [50].

� NiosomesNiosomes, or nonionic surfactant vesicles, have a similar structure to liposomes but their membranes are only composed by nonionic surfactants, such as polyglyceryl-alkyl ethers or N-palmitoylglucosamine [51]. Niosomes comprise a wide spectrum of vesicles, from small, unila-mellar to large, multilamellar vectors and con-stitute an alternative to liposomes with improved chemical stability in vivo [52]. Various studies have examined niosome formulations for oral and sys-temic delivery of molecules, such as doxorubi-cin, camptothecin and insulin, demonstrating an increase in drug bioavailability [53].

� DendrimersDendrimers essentially encompass a range of branched polymer complexes. Generally, they consist of an initiator core, surrounded by a fur-ther layer of selected polymer, which is grafted to this core, forming a tree-like branched macro-molecular complex. Dendrimer synthesis is per-formed through controlled repetitive chemical polymerization steps. These repetitions permit

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Antibody-targeted nanoparticles for cancer therapy Review

tight regulation and control of features, such as shape, size and surface functionality [54]. Dendrimers, produced using polymers such as poly(amidoamine) or poly(l-lysine), have excel-lent water-solubility, biocompatibility and are considered sufficiently nonimmunogenic [55]. Dendrimers have been used in various therapeu-tic and diagnostic applications for the delivery of a broad range of molecules including DNA [56,57], RNA [58,59], bioimaging contrast agents [54] and chemotherapies [60].

� Polymeric micellesPolymeric micelles are composed by aggregates of amphiphilic polymers constituted into hydropho-bic cores, surrounded by a corona of hydrophilic polymeric chains exposed to the aqueous envi-ronment [61]. Polymers used in recent studies are mainly heterobifunctional copolymers, composed of a hydrophilic block of PEG, poly(vinyl pyrrol-idone) and hydrophobic poly(l-lactide) or poly(l-lysine) that will form the particle core [62,63]. Encapsulation of anti cancer drugs, such as doxo-rubicin [64,65] and camptothecin [66,67] has been developed through conjugation to the hydropho-bic core. Furthermore, as with liposomes, formu-lations of cationic micelles have been developed to carry DNA or RNA molecules [61,68].

� Polymeric nanoparticlesThese nanoparticulate structures encom-pass both nanospheres and nanocapsules. Nanospheres are amongst the simplest NP for-mulations, consisting simply of a solid matrix of polymer, whist the nanocapsule contains an aqueous core (Figure 1). The formulation used is essentially dependent on the solubility of the drug substance; poorly water-soluble drugs are more readily encapsulated within the non-aqueous environment in nanospheres, while water-soluble and labile drug substances, such as DNA and proteins/peptides, are more easily encapsulated within nanocapsules.

Various polymers have been exam-ined for use in these formulations, includ-ing poly (acr y lamides ) , poly (e ster s ) , poly(alkylcyanoacrylates), poly(lactic acids), poly(glycolic acids), poly(lactic-coglycolic acid) (PLGA), chitosan (copolymer of d-glucosamine and N-acetyl-glucosamine) and alginates (copolymer of guluronic acid and mannuronic acid) [69–74]. PLGA is considered by some to be particularly attractive owing to its proven biocompatibility. Indeed, various PLGA-based clinical products such as bone fixations [75], con-trolled-release drug implants or micro particles including Nutropin™ Depot (Genentech, CA,

Nanospheres Nanocapsules Liposomes Dendrimers

Polymeric micelles

Polymeric matrix

Lipid core

Hydrophilic drug

Hydrophobic drug

Phospholipid

Polymer

Amphiphilic polymer

Polar head group

Hydrophobic tail

Figure 1. Diversity of nanoparticles used in drug delivery strategies. Nanoparticulate formulations can be tailored based on the required drug to be encapsulated/carried. While hydrophobic molecules can be incorporated inside the core of the nanoparticles, hydrophilic molecules can be carried more readily within an aqueous core protected by a polymeric or lipidic shell.


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USA), Trelsta™ Depot (Pfizer, CT, USA), Sandostatin™ LAR Depot (Novartis, MO, USA) are already available on the market.

Nanoparticle drug delivery: targeting strategiesAlmost as intense an area of research as NP formulation is NP targeting. The targeting of nanoparticulate formulations focuses on both the development of new diagnostic tools and improving the efficacies of therapeutic agents. Targeting approaches can be broadly classified into two areas; passive and active targeting.

� Passive targetingPassive targeting exploits the normal biodistri-bution that unadorned NPs will take within the body. Upon intravenous delivery, plain NPs are rapidly removed from circulation by opsoniza-tion and macrophage engulfment [76] or accumu-late in the liver and spleen [77]. Therefore, this clearance can be exploited to treat hepatic disorders such as leishmaniasis, a parasitic dis-ease [78], or for the targeting of accumulated macrophages in atherosclerosis [79].

Despite these opportunities to exploit opson-ization, the treatment of most diseases, in par-ticular tumors, requires the circumvention of this clearance mechanism. To avoid opsoniza-tion and subsequent phagocytosis, NPs coated with PEG have been widely investigated and shown to promote significant improvements in bioavailability of the particles [80,81]. By pre-senting a corona of hydrophilic polymer, the absorption of protein on the NP surface (which triggers opsonization) is prevented [82]. The form ation of a PEG shield can be achieved by using PEG during particle formulation [83–86] or by adsorption of PEG polymer onto preformed particles [87–89].

It has also been established that nontargeted NPs can accumulate in developed tumors. Studies have suggested that particles up to 400 nm can be passively targeted to tumors [90]. This is due to the ability of these particles to leach into the diseased tissue through the leaky vasculature network that is commonly associ-ated with tumorigenesis; a phenomenon called the EPR effect [91]. The increased permeability of the vasculature in tumors is due to incom-plete or disordered endothelial cell junctions that are frequently found in rapidly grow-ing tumors. Consequently, tumor vessels are more permeable to nanoparticulate formula-tions than the well-defined vasculature found in normal differentiated tissue. Furthermore,

tumors tend to have poor lymphatic drainage, leading to further accumulation of the NP at the diseased site. Indeed, the clinical use-fulness of Doxil has been attributed, at least in part, to the EPR effect with a significant decrease of doxorubicin side effects, such as cardiomyo pathy and myelo suppression; a result of decreased exposure of these normal tissues and cells to the drug [92].

� Active targetingActive targeting involves the modification of the NP with a targeting moiety. This modi-fication is usually on the corona of the par-ticle, introducing a ligand, which facilitates the homing, binding and internalization of the formulation to the targeted cells (Table 1). Although this approach has been used to target normal cells in vaccine formulations (e.g., tar-geting of gastro intestinal tract epithelial cells in oral delivery strategies [84,93]), most research has focused on the specific targeting of cells expressing disease-associated biomarkers, as in the case of cancer. Various moieties have been examined as targeting agents, including vita-mins [87,94], carbo hydrates [95], aptamers [85,96], peptides (e.g., Arg-Gly-Asp, allatostatin, trans- activating transcriptional activator) [84,97–99] and proteins (e.g., lectins, and transfer-rin) [98,100–103]. However, the majority of research to date has focused on antibodies, which will now be discussed in more depth.

Antibody-targeted nanoparticlesAntibodies are considered by many as ideal anticancer therapeutic agents and have been an area of intense research since customized monoclonal antibody production was reported in the mid-1970s. Indeed, antibody produc-tion display and screening innovations, such as phage display, mean that antibodies can be derived or engineered to bind with exceptional specificity to a wide range of target antigens. Importantly however, although antibodies are used very successfully as therapeutic agents in their own right, they also have the ability to be exploited as targeting agents [104,105].

The application of antibodies to deliver con-jugated agents to disease sites can be utilized for imaging or diagnostic purposes as in the example of fluorophores [106] or radioisotopes for lymphomas [10]. Alternatively, they can be used for the delivery of active therapeutics such as cytokines [107], prodrug activation enzymes (e.g., b-lactamase and carboxypepti-dase) [108,109] and chemotherapy toxins [110–112].



Clinically, immunoconjugates have been used in cancer treatment. For example, gem-tuzumab (Mylotarg®; Wyeth, CT, USA) con-sists of a CD-33 specific monoclonal antibody conjugated to a calicheamicin chemotherapy, and was used for the treatment of acute myeloid leukemia [113]. Furthermore, conjugation of radioisotopes with targeting antibodies has been developed for both imaging (immuno-scintigraphy) and radioimmunotherapy strat-egies. For example, anti-CD20 ibritumomab-tiuxetan (Zevalin®; Spectrum Pharmaceuticals, CA, USA) has been applied incorporating either 90Y metal isotope for use as a clinical therapy [114,115] or, alternatively, g-emitter 111In for in vivo imaging strategies in hematological malignan-cies [10]. Antibody–NP conjugates have poten-tial benefits over these current approaches as they have the ability to circumvent some of the issues associated with direct conjugates, such as the possible inactivation of the drug entity and the necessary release of the drug once internal-ized into endosomal/lysosomal vesicles through pH labile or reducible linkers. Furthermore, whereas stoichiometric ratios of drug to anti-body are only possible with direct conjugates, the potential of much higher drug to antibody ratios are possible with antibody–NP com-plexes; thereby maximizing the concentration of drug that can be targeted to the disease site.

This current background has led to focused interest in the development of antibody-coated nanoparticulates; both for lipid-based [116,117] and non-lipid-based NPs [118]. The majority of nanoparticulate antibody-targeting research has focused on antitumor strategies, using the antibody to target cell-surface markers of disease that are frequently upregulated or expressed specifically on either the tumor or tumor-associated cells (Table 2). In the following sections, the main targets examined for anti-body–NP conjugates to date are discussed in more depth.

� EGFR/HER1EGF receptor (EGFR/HER1) is a member of the human epidermal receptor family. This recep-tor binds various, closely related ligands, such as EGF or TGF-a [119]. When activated by ligand binding, it has been shown to trigger down-stream signaling to promote proliferation, migra-tion and angiogenesis [119]. This receptor is over-expressed or dysregulated in many solid tumors, such as breast or esophageal cancers [120–122] and its expression levels have been linked with poor prognosis [122,123]. Consequently, numerous antibody-based strategies have been developed to block EGFR activation and are used clini-cally, as in the example of cetuximab (colorec-tal and head/neck carcinomas) [124,125], panitu-mumab (colo rectal cancer) [125] or nimotuzumab (head/neck cancer) [126].

This proven effectiveness of antibody thera-pies towards EGFR, has led to much interest in EGFR as a target for immunoconjugates includ-ing antibody conjugated liposomes [64,92,127] and NPs [128–131]. EGFR-targeted antibody–NP tar-geting strategies have also been developed for diagnostic purposes, using gold NPs as the con-trast agent in optoacoustic tomography. Using this approach, it has been possible to differenti-ate between cells preincubated with either EGFR antibody-labeled gold NPs, or nonspecific gold NPs [130]. More recently, it has been shown that cetuximab-conjugated gold NPs could be used in conjugation with radiotherapy to specifically target and kill cells expressing high levels of EGFR in mixed cultures [132].

The effectiveness of targeting chemotherapy-loaded NPs with EGFR antibodies has also been demonstrated. This successful targeting was exploited to deliver rapamycin (a poorly water soluble, antiproliferative drug), inducing a significant increase of cytotoxicity compared with native rapamycin and nontargeted loaded rapamycin NPs towards breast adenocarcinoma MCF7 cells [120].

Table 1. Main nanoparticle intracellular uptake mechanisms.

Endocytosis pathway

Particles size limit

Proteins involved Stimuli Ref.

Phagocytosis Up to 20 µm

Actin, dynamin Antibody Fc region, complement, vitronectin, phosphatidylserine

[175,176]

Macropinocitosis ~1 µm Actin Growth factor, antigen binding [177,178]

Caveolae-mediated endocytosis

~80 nm Caveolae, actin, dynamin

Cholera toxin, tetanus toxin, folic acid

[179,180]

Clathrin-mediated endocytosis

~200 nm Clathrin, actin, dynamin

Transferrin, low-density lipoprotein, EGF

[181–183]

Endocytosis pathways can be subdivided into four main categories: phagocytosis, macropinocitosis, caveolae-mediated and clathrin-mediated. These pathways are principally initiated through receptor activation on the cell membrane by various molecules.


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In addition to these in vitro investigations, EGFR targeting strategies have also now been examined in vivo. Administration of anti-EGFR monoclonal antibody-labeled dendrim-ers conjugated with nonradioactive boron-10 to treat F98 EGFR glioma-bearing rats, fol-lowed by boron neutron capture therapy, demonstrated an increase of lifespan of 107% when compared with untreated controls [129]. This work highlights that the successful tar-geting observed in vitro can be preserved and exploited in vivo, paving the way for clinical applications in the future.

� Her2/neuHuman EGF receptor 2 (Her2/neu, ErbB-2, CD340) is a membrane tyrosine kinase recep-tor from the same epidermal growth factor receptor family as EGFR. This receptor has been linked with the signal transduction of cell growth and differentiation pathways [133,134]. Moreover, it is a prominent cancer target, overexpressed in a signif icant number of breast cancer cases (20–30%) and linked with poorer prognosis and higher risk of disease recurrence [135–137].

Tra s tu zumab (Hercept in®; Roche Pharmaceuticals, CA, USA), is a humanized, monoclonal antibody raised against the extra-cellular domain of HER2/neu that is currently used clinically to treat HER2 positive breast cancer [138]. However, its precise in vitro and in vivo antitumor mechanisms remain con-troversial. Using experimental models, diverse molecular and cellular mechanisms have been proposed including inhibition of HER2 extra-cellular cleavage, inhibition of intra cellular signaling pathways and in vivo antibody-dependent, cell-mediated cytotoxicity [139,140]. Nonetheless, trastuzumab has been granted by various national agencies to treat early-stage HER2-positive breast cancer in combination with chemotherapy [141,142].

In addition to its clinical application as a direct therapeutic target, HER2 has also been examined as a target for drug delivery. Cirstoiu-Hapca and coworkers observed that anti-Her2 labeled NPs were able to bind to, and inter-nalize into, SKOV-3 (HER2+) cells [143]. In agreement with these findings, another study observed similar internalization effects using NP conjugated to trastuzumab. This study also revealed that upon NP uptake, a 40% decrease in surface receptor density was observed. Furthermore, through incubation of a range of silver and gold NP formulations with SK-BR-3 cells overexpressing HER2, this study demon-strated that while nude particles had little effect, trastuzumab-conjugated NPs induced caspase-9 and caspase-3 activation, leading to a twofold enhancement in cytotoxicity compared with trastuzumab treatment alone [144]. This indi-cated that the density of the antibody paratopes on the surface of the NP could cross-link and activate the receptor. Moreover, by modifying the diameter, they were also able to demonstrate that internalization was dependent on NP size, with the most efficient uptake occurring within the 25–50-nm diameter range. Interestingly, although larger particles were less readily taken up, as was anticipated, so too were particles smaller than this optimum range. The authors argue that although these small particles can target and bind to the receptors on the surface, once the particles get to a critical size, the dis-played antibodies induce multivalent recep-tor crosslinking; stimulating ligand–complex internalization. This research suggests that the most efficient particle size is likely to be a com-promise between being large enough to induce receptor cross-linking, while small enough to facilitate efficient membrane wrapping. This multivalent receptor cross-linking has also been observed with anti-Her2 antibody-conjugated liposomes [145], as well as for receptors such as IgE [146], transferrin [147] and Fas [148].

Table 2. Main tumor-associated antigens used as targets for nanoparticle-based tumor therapy.

Antigen Antigen function Cell types targeted Ref.

Her2/neu EGF receptor Breast, colorectal and ovary cancers [55,104,140,143,144,184,185]

EGFR EGF receptor Brain tumor glioma, oral and pharyngeal, liver, head and neck cancers

[64,92,120,127–129, 129–132]

VEGF VEGF receptor Lymphoma breast, colorectal and lung cancers

[151–154]

PSMA Prostate-specific membrane antigen

Prostate cancers [164,165]

Tfr Transferrin receptor Brain tumor glioma, pancreatic, colorectal, lung and breast cancers

[102]



The application of anti-HER2-conjugated NPs has also been examined in vivo. Established control or HER2-expressing xenograft tumors, treated with anti-HER2 grafted dendrimers, revealed significant NP localization and inter-nalization into the HER2-expressing tumors in comparison with control tumors after 15 h; thus highlighting that antibody target-ing could be maintained in vivo [55]. Indeed, another study demonstrated that the efficacy of paclitaxel-loaded PLGA NPs could be improved in SKOV-3 xenografts with trastu-zumab targeting [104]. The authors argue that the effectiveness of the conjugates is a combi-nation of both passive targeting by EPR and receptor-mediated internalization.

� Vascular endothelial growth factor receptorsThe VEGF receptors (VEGFRs) are tyrosine kinase receptors, which, when activated by their ligand, promote angiogenesis and vasculogen-esis [149]. VEGF and VEGFR are upregulated in angiogenic tumors [150] and, as such, are of interest in both the development of diagnostics and therapeutics – particularly in development of antagonistic antibodies to block activation of VEGFR. Soman and Giorgio developed anti-VEGF labeled QDs as an early diagnos-tic strategy and demonstrated the possibility to measure the concentration of VEGF in solu-tion within a sensitivity limit of 50 × 10–15 M by flow cyto metry [151]. In another diagnostic approach, superparamagnetic iron oxide NPs, conjugated with anti-VEGF-R2 antibody, were used as probes to detect VEGF-R2 positive glioma- bearing mice to monitor progression of the disease using MRI [152].

The application of anti-VEGFR conjugated gold NPs has been examined as an ex vivo therapeutic approach for the treatment of B-cell chronic lymphocytic leukemia. It was demonstrated that the diseased cells extracted from patients, were able to take up the parti-cles regardless of the presence of antibody into early/late endosomes and in multivesicular bod-ies. However, the anti-VEGFR NPs were able to significantly increase apoptosis compared with naked, gold NPs and antibodies alone [153].

The potential of using VEGFR antibody conjugates to deliver radiotherapy has also been pursued. VEGFR-targeted dextran magnetic NPs containing the radioisotope iodine-131 were applied towards human liver HepG2 xeno-grafts, in combination with a magnetic field applied to the tumor to target the particles to

this site. Analysis of the particle biodistribution revealed that the majority of the radioactivity was localized within the tumors; demonstrating the potential of combinative antibody-based and magnetic targeting to induce tumoral localiza-tion of the targeted NP. This improved localiza-tion of the double-targeted iodine-131, encapsu-lated in VEGFR-targeted dextran magnetic NP, was evidenced by the improved tumor inhibi-tion rate (89%) compare with free iodine-131 (27%), or iodine-131 tartgeted only by anti-VEGFR antibody (77%) or dextran magnetic NPs (81%) [154]. This highlights the possible future application of this ‘double-targeting’ approach in clinical radioimmunotherapy.

� Prostate-specific membrane antigen Prostate-specific membrane antigen (PSMA) is a nonsecreted type II transmembrane glyco-protein expressed mainly in prostatic epithe-lium. However, it is only expressed in prostate cancer cells and its expression level increases dur-ing the progression of malignancy [112]. PSMA has also recently been detected in kidney and bladder carcinoma [155]. A radioimmunoscinti-graphic diagnostic method, using anti-PSMA radioactive indium conjugates (ProstaScint®; Cytogen Corporation, NJ, USA) is already widely used for diagnosing prostate cancer in patients [156]. PSMA has also been examined as a target in various therapeutic approaches for prostate cancer including cancer vaccine deliv-ery [157,158], antibody–drug conjugates [159–161] and radioimmunotherapy [162,163].

To investigate the application of anti-PSMA (clone J591) conjugated dendrimers as an effective targeted dual drug/imaging delivery system, in vitro studies were carried out using human prostate cancer LNCaP cells (PSMA+) incubated with nontargeted or J591-conjugated dendrimer. The presence of the J591 antibody on the particles led to preferential cellular adherence and internalization over controls. Moreover, competition assays using free J591 blocked this inter action, demonstrating the specificity of the binding of the conjugates to the surface PMSA [164]. Using the same antibody (J591) to conjugate PEGylated QD (QD-PSMA), Gao and coworkers performed an in vivo study with nude mice bearing PSMA+ human prostate cancer cells (C4–2) in sub-cutaneous xenografts in order to investigate the biodistribution of QD-PSMA compared with QDs with free carboxylic acid moieties (QD-COOH) or coated with PEG (QD-PEG). Upon tail vein administration, only targeted


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QD-PSMA induced a strong tumor signal, enabling the visualization of the tumor using a whole-body macro illumination system [165]. However, these studies also revealed a high sig-nal in the livers of treated animals, demonstrat-ing that further optimization of the QD bio-availability is required before clinical evaluation can begin.

� Transferrin receptorThe transferrin receptor (Tfr, CD71) is a mem-brane carrier protein that is involved in the reg-ulation of intracellular iron levels as it internal-izes the complexes formed by the blood plasma protein, transferrin, and iron ions [166]. As this receptor has been shown to be overexpressed in proliferating cells in comparison with resting cells [167], it has been examined as a target in tumor strategies. The main ligand developed to target the Tfr has been transferrin itself [168–170]; however, anti-transferrin antibody-conjugated NPs have also been developed and investigated. Using Tfr overexpressing human B-cell lym-phoma (Ramos) cells incubated with fluores-cent 200 nm size PEG-based NPs, Wang and coworkers demonstrated that 80% of the cells were labeled with the anti-Tfr NP after only 4 h, in comparison with 10% with control anti-body conjugates [102]. Interestingly, both anti-Tfr and even transferrin-coated NPs exhibited a cytotoxic effect, with 70% cell death observed, while control NPs did not show any toxicity, even though no drug was encapsulated. The cytotoxic effect of the anti-Tfr NP was fully suppressed by addition of ferric ammonium sulfate, suggesting that the toxicity was primar-ily caused by iron deprivation. However, these studies also revealed that the Tfr NP uptake correlated with receptor density and that the size of the particles was also crucial; incubation of anti-Tfr antibody coated with larger 5-µm microparticles resulted in no internalization and only negligible toxicity. This work clearly highlights that the size and composition of the particulate is also a key consideration in active targeting strategies.

As the Tfr is also constitutively expressed in the blood–brain barrier, Ulbrich and cowork-ers developed an analgesic (loperamide)-loaded NP, conjugated to either transferrin or anti-Tfr antibodies [103]. The analgesic effect of tail vein injection of various loperamide-loaded particle solutions was subsequently tested by monitor-ing mice responses to heat. While free analge-sic or loperamide-loaded NPs did not achieve any analgesic effect in vivo, both transferrin or

anti-Tfr antibody conjugated, drug-loaded NPs elicited significant effects, highlighting their future therapeutic utility.

� Dendritic cell receptorsDendritic cells (DCs) are key regulators in antigen-specific immunity, owing to their abil-ity to internalize, process and present antigens to T cells. Therefore, in order to increase T-cell-mediated immune responses in vivo, targeted delivery of antigens to DCs using antibodies has been examined. The main receptors targeted are endocytic transmembrane receptors such as C-type lectins (i.e., DC-SIGN and CD205), integrins (i.e., MACI and CD11c–CD18) or FcR (i.e., FcgRI and FcgRIII) families [171]. The in vivo application of antigen-loaded particles or liposomes conjugated with DC-targeted anti-CD205 has demonstrated the enhanced efficacy of DC-targeted carriers as a tumor immuno-therapy in animal studies [172]. In this work, mice were vaccinated against ovalbumin by tail injections of free ovalbumin or ovalbumin-loaded conjugated NPs conjugated to either anti-CD205 antibodies or isotype control. After 7 days, enhanced ovalbumin epitope-specific cytotoxic T-lymphocyte activity was measured in spleen cells isolated from mice vaccinated with the anti-CD205 monoclonal antibody conjugated ovalbumin-loaded NPs compared with free ovalbumin and ovalbumin-loaded NPs (conjugated with isotype control antibody). Similarly, targeting of DCs through application of a humanized DC-SIGN particles conjugate has been examined. Interestingly, analysis of the uptake and antigen presentation in the tar-geted DCs revealed that although DC-SIGN targeting increased intracellular accumula-tion and antigen presentation, the targeting of the larger macro particles did not significantly enhance either internalization or antigen pre-sentation. This again highlights that particle size is a critical parameter in these targeting strategies [173].

Conclusion & future perspectiveTo date, developments in the field of antibody-conjugated NPs have demonstrated attractive results both in vitro and in vivo. The cell- specific receptor-mediated endocytosis of the NP at the site of disease provides an attractive feature for a variety of diagnostic and therapeutic strategies. Furthermore, an additional feature of receptor targeting is the possibility of inducing multi-valent receptor activation to trigger receptor-activated signaling, leading to downstream



Executive summary

� Antibody and antibody fragments can be efficiently conjugated to the surface of most types of nanoparticles by facile cross-linking approaches.

� Antibody-conjugated nanoparticles can target cells and be exploited for a range of diagnostic and therapeutic approaches, particularly in diseases such as cancer.

� For specific cell-surface antigens, the high density of antibody moieties on the nanoparticle surface can induce multivalent receptor activation, which can be exploited to trigger receptor-mediated internalization and other downstream signal transduction.

effects such as cell degranulation [146], apop-totic signaling [144,146,148] or iron depletion [147]. In addition, some of these studies have shown that NP size and surface ligand density are critical key features [144,146,147]. Therefore, optimizations of these parameters, as well as PEGylation and antibody density, are required to ensure that the functionality of each modi-fication is not compromised. As many antibod-ies are currently being developed towards novel antitumor targets or biomarkers, the ability to further exploit these molecules for targeting NPs is expected to be a rapidly expanding area. However, some key questions remain before targeted NPs are to be used clinically such as biodistribution, retention, metabolism and long-term toxicity of some particle materials [174]. In addition, the production of these particles to

a current good manufacturing practice stan-dard in a cost effective manner will also need to be addressed. Nonetheless, taking together the exciting preclinical efficacies that these NP conjugates have demonstrated, with the limited clinical evaluation that has been undertaken to date, highlights exciting opportunities for their commercialization and clinical application.

Financial & competing interests disclosureChristopher Scott owns shares in Fusion Antibodies Ltd, Belfast, UK. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Antibody-targeted nanoparticles for cancer therapy