Antibody-Based Imaging of HER-2: Moving into the Clinic

Abstract

Human epidermal growth factor receptor-2 (HER-2) mediates a number of important cellular activities, and is up-regulated in a diverse set of cancer cell lines, especially breast cancer. Accordingly, HER-2 has been regarded as a common drug target in cancer therapy. Antibodies can serve as ideal candidates for targeted tumor imaging and drug delivery, due to their inherent affinity and specificity. Advanced by the development of a wide variety of imaging techniques, antibody-based imaging of HER-2 can allow for early detection and localization of tumors, as well as monitoring of drug delivery and tissue’s response to drug treatment. In this review article, antibody-based imaging of HER-2 are summarized and discussed, with an emphasis on the involved imaging methods.

INTRODUCTION

The epidermal growth factor (EGF) and EGF receptor (EGFR) are one of the first discovered pairs of growth factor ligand-receptor, with EGFR later recognized as a member of a receptor tyrosine kinase family, the human epidermal growth factor receptor (HER) family [1]. The HER family comprises four structurally related members: EGFR (HER-1 or erythroblastic leukemia viral oncogene homolog 1 [ErbB1]), HER-2 (ErbB2), HER-3 (ErbB3), and HER-4 (ErbB4) [2, 3]. The structure of every HER family member consists of an N-terminal extracellular domain (ECD), a single membrane-spanning region and a C-terminal cytoplasmic domain, among which the ECD acts as a receptor to bind with ligands [1, 4]. To date, a number of these ligands have been reported, such as EGF, β-cellulin, amphiregulin, and transforming growth factor-alpha (TNF-α) [2, 3]. Upon binding, the ligand induces the dimerization of a HER-kinase that either homodimerizes with itself, or heterodimerizes with other HER family members [1]. As a result of the dimerization, the HER-kinases undergo conformational changes, which promote the activation of the cytoplasmic domain with a subsequent phosphorylation leading to various downstream signaling pathways and cellular events [1].

Among the HER family, one key member, HER-2, behaves in a unique way since it has no identified natural ligand yet. HER-2 is thereby activated only through its heterodimerization with other HER family members that have been pre-activated by their own ligands. For other HER family members, HER-2 is actually the preferred partner to form heterodimers, and the HER-2 involved heterodimerization is the most potent signal transduction pathway among all dimerizations of the HER family [5]. Hence, by mediating complex signaling pathways, HER-2 plays pivotal roles in cell growth, migration, differentiation and survival [4, 6–8]. From a genetic perspective, the HER-2 gene was derived from a potent oncogenic mutant in neuroglioblastomas developed in carcinogen exposed rats, and was originally termed NEU [9–11]. The gene amplification and protein over-expression of HER-2 can increase and prolong signals that trigger the transformation of cells, leading to tumorigenesis and metastasis [4]. The up-regulation of HER-2 is thereby commonly observed in human cancers, such as breast, ovarian, gastric, and prostate cancer [4, 12–14]. To date, both HER-2 and its involved signaling pathways have been extensively investigated as targets in cancer therapy, during which a direct inhibition of these proteins/pathways can block tumor proliferation. Moreover, the ECD of HER-2 is regarded as an ideal target for tumor-directed drug delivery [4].

One major strategy for HER-2 targeting is to use monoclonal antibodies (mAbs), among which trastuzumab was the first Food and Drug Administration (FDA) approved anti-HER-2 antibody [11, 15]. Trastuzumab, also known as Herceptin, binds to the ECD of HER-2 and induces apoptosis in HER-2 over-expressing cancer cells [4, 11]. Another humanized anti-HER-2 mAb is pertuzumab, which binds to a different subdomain of HER-2’s ECD and thereby sterically inhibits the dimerization of HER-2 with itself and other HER family members [16]. Clinically, pertuzumab displayed promising therapeutic activities and tolerable toxicities [17]. A synergy of trastuzumab and pertuzumab also resulted in an enhanced antitumor effect in vivo [18, 19]. Besides antibodies, another strategy is to use tyrosine kinase inhibitors to target the intracellular domain of HER-2, among which lapatinib has been well characterized, and is currently in clinical trials [20]. Alternatively, some other protein inhibitors [21, 22], as well as agents to knock out HER-2 mRNA [23, 24], have also been reported.

Whereas an intensive attention has been paid to HER-2 targeted therapy, increasing interests also arise in HER-2 based molecular imaging. The effectiveness of HER-2 targeted therapy highly depends on accurate evaluation of its expression level in tumors. It is recommended by the American Society of Clinical Oncology (ASCO) to characterize HER-2 levels on primary breast cancer at the time of either diagnosis or recurrence [25]. By using contrast agents or probes, noninvasive molecular imaging techniques allow for direct visualization of the targeting process including bio-distribution and pharmacokinetics, as well as spatiotemporal monitoring of cellular activities/responses in the targeted region. As mentioned above, antibodies against HER-2 are the mainstream of the developed therapeutics for treating HER-2 over-expressing cancer. For HER-2 detection and imaging, they are also the most commonly used agents [11]. Antibody-based imaging not only renders more insights into the HER-2 targeting process, but also identifies and localizes the HER-2 expressing tumors, which facilitates the subsequent drug delivery and the follow-up evaluation of therapeutic results. Herein, we systematically summarize and discuss the applications of antibodies for diagnostic imaging of HER-2, using a variety of imaging techniques. Several practical examples including clinical applications are also highlighted for each imaging approach.

ANTIBODIES AS TARGETING AGENTS

Antibodies, also known as immunoglobulins, have become desirable molecules for use as research tools, diagnostic agents, and therapeutic drugs. There are five isotypes of human antibodies (i.e. IgA, IgD, IgE, IgG, and IgM), among which IgG is the most abundant and versatile type that constitutes 75 percent of serum immunoglobulins [26, 27]. IgG is a Y-shaped, multi-domain protein composed of two Fab arms and one Fc stem [28]. The antigen-binding sites are located on the tips of the Fab arms, while the Fc domain recruits effector functions and maintains long serum half-lives of the antibody, by interacting with a diverse set of receptors [29]. The introduction of the hybridoma technology by Kohlerand Milstein in 1975 [30] set the starting point of the antibody industry. Subsequently, other technical invention to generate humanized monoclonal antibody (mAb) and chimeric mAb further resolved the issues in immunogenicity and low efficacy [31, 32]. In many applications of antibodies such as imaging, the Fc-mediated effects are not necessary and may even compromise certain function/properties of the antibody [29]. Advanced by recombinant DNA technology and protein engineering, the Fc domain and associated effects can be removed to render monovalent and bivalent mAb fragments and engineered variants, such as Fab, F(ab′), single chain Fv (scFv), minibodies, diabodies, affibodies, nanobodies and anticalins [28, 33–35]. Among these engineered fragments, affibody is the most commonly used in imaging, which is a small 58-amino acid Z-domain scaffold derived from the IgG-binding domain of staphylococcal protein A [36]. The binding surface of affibody has a randomized sequence of 13 amino acids, which can be screened to generate a specific sequence recognizing the desired target [34, 37]. For HER-2 targeting, the affibody molecule His₆-Z_HER-2/neu:4 was shown to bind the ECD of HER-2 with nanomolar affinity [38].

To date, development of antibodies for diagnostic and therapeutic applications has become the fastest growing area in biopharmaceutical research, with over 100 mAbs currently in clinical trials [31] and more than 20 already approved for clinical use [39]. Besides being therapeutic agents, antibodies also play pivotal roles as targeting ligands to direct the delivery of chemotherapeutics and imaging contrast agents to the tissue of interest. Compared to traditional drugs, antibody-drug conjugates (ADCs) control the bio-distribution of the drugs that are cytotoxic in most cases, thereby sparing their contacts from normal tissues and providing a high intratumoral drug concentration [40]. Antibody specificity is crucial to the success of antibody directed drug delivery, where the targeted antigen should be substantially up-regulated on tumor cells but not on normal tissue cells [40]. This specificity allows the use of highly potent drugs that would otherwise be too toxic for healthy cells. Additionally, the stability of the linker (which ensures drug release only after targeting) and the conjugation method (which affects the drug loading stoichiometry and homogeneity) are both important factors to be considered [40]. Currently, there are three common methods for conjugation of mAbs: alkylation of reduced inter-chain disulfides, alkylation of genetically engineered cysteine, and acylation of lysine, among which the cysteine conjugation provides a greater degree of uniformity by allowing site-specific introduction of cysteines into the mAb and subsequent attachment of drugs at the defined stoichiometry [41–43].

There are several challenges in antibody-based imaging of HER-2. An efficacious delivery and high contrast imaging is often accompanied by antibodies’ high avidity to the target and their relatively long retention time in the target region. In reality, however, a large fraction of antibodies rapidly detach from their binding sites, especially under the high shear stress of physiological relevance. To generate antibodies with the desired high affinity, phage display technique was adopted for customized engineering of humanized antibodies [44]. Another issue besides the affinity is the formidable barriers imposed by solid tumors to resist the penetration of drugs or delivery agents, which largely diminishes the values of in vitro experiments [45]. It was also shown that once nanoparticles or microbubbles were conjugated to antibodies, the combined sizes of the resulting conjugates could slow down the delivery speed [45]. Despite these drawbacks, antibody-based imaging of HER-2 represents one of the most promising strategies for clinical management of breast cancer, which has recently attracted intensive research interests.

RADIOIMMUNOSCINTIGRAPHY (RIS)

Radiolabeling of antibodies was initially used to circumvent several problems encountered in the therapeutic development of antibody conjugated chemotherapeutics or toxins. To be efficacious, it is required that every malignant cell expresses the target antigen, which is usually not the case in practice [27]. However, drugs may be susceptible to degradation in endocytosis during drug delivery, and toxins can easily induce immunogenicity. Furthermore, a variable percentage of tumor cells can develop multidrug resistance. Compared to these traditional approaches, radioisotopes bear certain advantages in which tumor cells cannot develop resistance against irradiation and the particles emitted by radionuclides can kill adjacent tumor cells [27]. Whereas the volume of drugs to be conjugated may affect the antibodies’ transportation and delivery, an alternative conjugation with small sized radioisotope can achieve a high specificity with about 20: 1 tumor-to-normal-tissue ratio [27]. Therefore, radioimmunotheraphy (RIT) has become a common approach to complement surgery and chemotherapy, although its efficacy is still limited in certain cancer types such as medullary thyroid carcinoma (MTC) [29].

Another application of isotopes is radioimmunoscintigraphy (RIS), which can determine the extent of disease, monitor the effect of therapy, and detect tumor recurrence [46]. In a typical procedure, a radiolabeled antibody is injected into patients intravenously, and the gamma ray emission from the radiolabeled antibody can be captured by gamma cameras to generate two-dimensional images. RIS detects antigens at a cellular level and has certain advantages in sensitivity over other existing imaging techniques, such as magnetic resonance imaging (MRI), computerized tomography (CT), and ultrasound (US) [46, 47], which primarily detect general morphological changes and are thereby fundamentally different from RIS in the underlying mechanism [48]. RIS is also different from other imaging techniques such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and X-ray. In the case of diagnostic X-ray, an external radiation was employed to pass through the body for image acquisition. For PET and SPECT, they generate three-dimensional images.

The common radionuclides used in RIT and RIS are ⁶⁷Cu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹²⁵I, ¹¹¹In, and ^99mTc [27, 49]. In a pilot study in mouse xenograft models, ¹²⁵I-labeled 741F8-2 (scFv′)₂ homodimer displayed higher specificity to human SKOV-3 tumors than other antibody fragments, resulting in an enhanced tumor contrast in sagittal section autoradiography [50]. Subsequently, trastuzumab Fab was radiolabeled with ¹¹¹In, which specifically localized HER-2-positive BT-474 human breast cancer xenograft in athymic mice [51]. Around 24 hours after administration of the radiolabeled antibody, tumors as small as 3 ~ 5 mm in diameter could be detected by imaging. A much improved HER-2 imaging has been achieved with affibodies, such as ¹²⁵I-labeled (Z_HER-2:4)₂ [52] , ¹¹¹In-labeled Z_{HER-2:342-pep2} [53] or ABY-025 [54], and ^99mTc-labeled Z_HER-2:342 [55] (Fig. (1)), which all afforded high-contrast gamma camera images. Following the success in animal models, ¹¹¹In-labeled trastuzumab was administered to patients with HER-2 expressing breast cancer [56, 57]. Clinical imaging demonstrated an accurate detection of HER-2-positive tumors. It is noteworthy that this trastuzumab-based tracer did not induce undesired immune response in the body, and exhibited a satisfactory biodistribution in tumors and normal organs.

Fig. (1).

Radioimmunoscintigraphy (RIS) of HER-2-positive SKOV-3 tumor-bearing mice with ^99mTc-labeled Z_HER-2:342 affibody. (A) At 1 h and 2 h post-injection (pi), tumors were visualized on the right hind legs. (B) A better image contrast was obtained at 5 h pi. As a negative control, a pre-injected dose of excess unlabeled affibody blocked tracer uptake in the tumor. Adapted from [55].

POSITRON EMISSION TOMOGRAPHY (PET)

PET has been widely used in medical diagnostics and biochemical research including oncology, neurology, and cardiology [58–63]. The radiolabeled agents, using positron emitters, are intravenously injected to the body, circulated, and collectively retained in tissues according to their biological properties [64]. The positron-electron annihilation generates a pair of 511 keV gamma photons in opposite directions, which can escape from the body and be detected and reconstructed to generate three-dimensional images. The signal intensity of every voxel is proportional to the amount of radioactive agents, thereby allowing PET to quantitatively map the spatial dispersion of radioactive agents all over the body. When serial PET images over a time course are collected, the time-dependent spatial distribution of radiotracers can be obtained, thereby allowing the monitoring and characterization of biological phenomena of interest [64].

Two common approaches are adopted for immunoPET imaging [65]. In the first approach, PET probes are constructed based on fast kinetic antibodies, which can be applied for fast imaging at low radiation levels, to confirm the target presence and scout the proper person for following-up therapies [65, 66]. Smaller antibody fragments (scFv, diabodies, minibodies, etc) are routinely used in this approach, which retain the binding specificity of the parent antibodies, but have different pharmacokinetic characteristics such as quicker clearance from the body, and relatively low tumor uptake [67]. In the second approach, radiolabeled antibodies are administered for imaging in a pre- or early course of therapies, to gain a sight of the targeting efficiency, the in vivo behavior of antibodies, as well as the therapeutic responses [65]. In this regard, intact antibodies are well suited materials in terms of their long residence in circulation (1–3 weeks), which allows for a specific and prominent accumulation of radiotracers in tumor cells, as well as long intervals between administrations [67]. For both approaches, the decay half-life of the used radionuclides should match the circulation half-life of the antibodies to be conjugated to. Hence, radioisotopes ¹¹C, ⁶⁸Ga, ¹⁸F, ⁶⁴Cu, ⁸⁶Y, and ⁷⁶Br are commonly used for the first approach, while the long-lived positron emitters such as ⁸⁹Zr or ¹²⁴I are preferentially employed for the second approach [66–69].

In an early study, a minibody derived from trastuzumab was radiolabeled and shown by quantitative microPET to preferentially localize in the kidneys, thereby not ideal as an imaging agent [70]. By manipulating the size and format, a slightly larger antibody fragment, the 105 kDa scFv-Fc was designed and demonstrated to have an increased tumor uptake and a reduced kidney uptake. Compared to the whole antibody molecule, this scFv-Fc fragment was still tailored in structure to possess a fast clearance in blood and normal tissues [70]. Subsequently, several trastuzumab-based antibodies or affibodies were labeled with radioisotopes, especially those long-lived ones, for PET imaging of HER-2 expressing human tumor xenografts [71–74]. Among these reports, affibody targeting provided better imaging contrast than that of a radiolabeled intact antibody, due to its better radioactivity retention and tumor specificity [74]. It is also notable that with PET imaging of ⁸⁹Zr-trastuzumab, HER-2 down-regulation can be monitored during the tumor treatment by a heat shock protein 90 (HSP90) inhibitor [75] in a mouse model. Hence, immunoPET imaging of HER-2 can monitor the therapeutic responses of HER-2 positive tumors [73] (Fig. (2A,B)).

Fig. (2).

PET imaging of HER-2 expression with ⁸⁹Zr-trastuzumab. Serial transverse and coronal PET images of a mouse administered with ⁸⁹Zr-trastuzumab before (A) and after (B) three dosages of anticancer drug (HSP90 inhibitor) NVP-AUY922 are shown here. Arrows indicate the HER-2 positive tumors. (C) ⁸⁹Zr-trastuzumab PET scans of a patient already on trastuzumab treatment at different time points post-injection revealed an increase over time of the tumor-to-nontumor ratio of tracer uptake. Arrow indicates ⁸⁹Zr_trastuzumab uptake in the only lesion. (D) ⁸⁹Zr-trastuzumab PET of a patient with liver and bone metastases at 5 days post-injection. A number of lesions are indicated by arrows. Adapted from [73, 80].

Using other radiolabeled HER-2 targeting antibodies, one can independently measure the HER-2 levels affected by trastuzumab treatment. For example, the C6.5 diabody can selectively target ECD of HER-2, in a region that is distinct from that bound by trastuzumab [76]. The ¹²⁴I-labeled C6.5 diabody was thus shown to be specifically taken up by several HER-2-positive tumor cell lines, and was thereby employed to monitor HER-2 status and to evaluate the results of trastuzumab treatment [77, 78].

Clinically, ⁸⁹Zr-trastuzumab was stable for up to seven days in human serum, and had a minimal dose of 100 μg for optimal PET imaging [79]. ⁸⁹Zr-trastuzumab based PET images were demonstrated to result in an excellent spatial resolution and a good signal-to-noise ratio, with tumor uptake observed in HER-2-positive lesions such as metastatic liver, lung, bone and brain (Fig. (2C,D)) [80]. The radiotracer uptake can also be quantified via PET scanning [79, 80], which may help the definition of optimal dosage for patient-specific trastuzumab therapy regimens [81]. On the other hand, probes at low doses will be subjected to rapid hepatic clearance, presumably due to high levels of circulating HER-2 ECD in plasma [80].

SINGLE-PHOTON EMISSION COMPUTED TOMOGRAPHY (SPECT)

SPECT differs from PET in the method used to define the angle of incidence, in which the gamma rays are emitted as single photons and only those in the prescribed direction can be received by the detector [82, 83]. A variety of radioisotopes such as ^99mTc, ¹²⁵I, and ¹³¹I can be used for SPECT [82, 84–88], which does not require complicated facilities such as cyclotron for PET imaging. Since most of the incident photons are discarded by detectors, SPECT has a much lower sensitivity (by 1 to 2 orders of magnitude) than PET.

Several ^99mTc- or ¹¹¹In-labeled murine mAbs have been approved by the FDA for use in SPECT imaging [48]. For instance, capromab pendetide labeled by ¹¹¹In is a radioactive mAb against prostate-specific membrane antigen. Coupled with improved techniques in anatomic localization, SPECT via this radiolabeled mAb accurately localized soft tissue metastasis in prostate cancer patients [89]. Although there is few clinical trials for SPECT imaging using HER-2 targeting antibodies, related studies using SPECT/CT have been reported in mouse xenografts [90, 91]. After screening a set of nanobodies, 2Rs15d was selected, which is stable, specific, and does not compete with trastuzumab or pertuzumab in HER-2 targeting. As a result, ^99mTc-labeled 2Rs15d exhibited high uptake in HER-2 positive mouse tumor models, with fast blood clearance, high tumor-to-blood/muscle ratios, and low background in normal tissues [90] (Fig. (3)). Similar studies were carried out with an ¹¹¹In-labeled albumin-binding Fab, AB.Fab4D5, which was derived from trastuzumab and exhibited rapid targeting, excellent tumor accumulation and retention [91]. With respect to individual SPECT or CT images, the fused images were advantageous in assessing potential bone metastases and differentiating malignant from benign bone lesions [92].

Fig. (3).

Fused SPECT/CT images (transverse, coronal and sagittal views) of mice with HER-2 positive SKOV3 tumor. Images were obtained at 1 h after injection of ^99mTc-labeled HER-2 targeting nanobody (2Rs15d) or a control nanobody. Adapted from [90].

MAGNETIC RESONANCE IMAGING (MRI)

MRI detects nuclei of atoms inside the body based on their nuclear magnetic resonance. The signal of MRI depends on the longitudinal (T₁) and transverse (T₂) relaxation times of protons mostly from tissue water, whose relaxation differences result in different MRI contrast [93]. The relaxation time of tissue water is affected by its physiological environment, which only happens at a late stage of physiological process, and is not specific [93]. To achieve an early and specific detection, contrast agents that decrease the longitudinal and transverse relaxation time can be intravenously injected to enhance the appearance of blood vessels and tumors [93]. The relaxivity of these agents, r₁ or r₂, is defined based on their ability to decrease T₁ or T₂ relaxation time, respectively. MRI contrast agents are classified into two categories based on their r₂/r₁ ratios. The ones with a high r₂/r₁ ratio, such as iron oxide particles, can result in a negative contrast or dark spots in T₂-weighted images; while the ones with a low r₂/r₁ ratio, such as complexes of Gd³⁺, or Mn²⁺, generate a positive contrast or bright spot in T₁-weighted images [93].

Although MRI has a high spatial resolution [94], it has a low sensitivity that needs to be improved by contrast agents of a high relaxivity [93]. Gadolinium chelates were used in early studies where they were conjugated to antibodies as exogenous contrast agents [95] (Fig. (4)). However, Gd³⁺ suffers from low relaxivity and biocompatibility, which makes the alternative superparamagnetic iron oxide (SPIO) particles more attractive in MRI applications [94]. Other than the advantage in relaxivity, SPIO also improves the sensitivity due to its inherent composition of a great number of iron atoms [94]. These irons are biocompatible and can be metabolized in vivo. In addition, SPIO particles possess easily modifiable surface coating, size-dependent magnetic property for derivatization, and optical/electronic active spectra for facile detection [94, 96]. SPIO nanoparticles are commonly synthesized via precipitation-based methods, either by co-precipitation or by the reverse micelle technique [97, 98]. To be suitable for passage through capillary walls and micro-distribution within the tumor tissue, the size of these particles should be below 50 nm [99]. After synthesis, the particles can be further coated with a PEG-modified, phospholipid micelle shell [98] or an amphiphilic lipid bilayer [93], both of which can improve water solubility, stability, and circulation retention time of the particle, and provide a versatile platform for conjugation of various functional moieties including antibodies [93, 98, 99]. Based on the applications and probe design, MRI contrast agents can be broadly divided into four classes: non-specific contrast agents, targeted contrast agents incorporating ligands for specific molecular targeting, smart contrast agents only activated by certain events such as enzymatic cleavage, and cell labeling contrast agents comprising ligands for cellular targeting [93]. In this review article, we will focus on antibody-based imaging which mainly involves targeted contrast agents and cell labeling contrast agents.

Fig. (4).

T₂-weighted MRI images (Top: grayscale; Bottom: color-map) of tumor-bearing mice before and at 1 h after injection of trastuzumab-conjugated nanoparticles. The mice were implanted with both HER-2 positive SKBR-3 and HER-2 negative KB tumors. Adapted from [105].

Using antibodies as the targeting ligands, SPIO-based noninvasive MRI imaging has been widely studied in oncology [100]. In several early reports, anti-HER-2 antibodies were conjugated to iron oxide containing nanoparticles and showed effective in vitro imaging of breast cancer cells via T₂-weighted MRI [101–103]. Through a process that involved systematic evaluation of the magnetic spin, size and type of spinel metal ferrites, artificially engineered magnetic nanoprobes (Mn-doped magnetism-engineered iron oxide; MnMEIO) linked to trastuzumab were shown to be able to visualize small tumors implanted in a mouse while trastuzumab conjugated cross-linked iron oxide (CLIO) does not [104]. Recently, trastuzumab-conjugated iron oxide nanoparticles were successfully utilized in mice bearing breast cancer tumor, where the tumor site was detected by T₂-weighted MRI (Fig. (4)) [105].

Besides intact antibodies, anti-HER-2 affibody was also labeled with SPIO, and demonstrated to be a feasible, specific MRI contrast agent which accumulated to human ovarian SKOV-3 tumor lesions in an in vivo model, and can be detected by MRI at around 30 min after probe injection [106]. Recently, a protein was designed to contain a Gd³⁺ binding site, which exhibited a significantly improved T₁ relaxivity than other traditional MRI contrast agents [107]. Once conjugated with an anti-HER-2 affibody, the probe demonstrated a significant improvement in detecting HER-2 positive human cell lines both in vitro and in mouse xenograft models [107].

Although many clinical trials in patients with HER-2 expressing breast cancer involve MRI as the imaging/diagnostic tool [108, 109], there is no reported clinical use of a MRI contrast agent that is comprised of an anti-HER-2 antibody. Nonetheless, with the rapid development of antibody-based contrast agents, the clinical application is feasible and expected for antibody-based MRI of HER-2.

NEAR-INFRARED FLUORESCENCE (NIRF) IMAGING

Optical imaging has been a vibrant research area over the last decade, which is suitable for preclinical research in small animal models and a variety of clinical scenarios [110–115]. Photon penetration through living tissues is subjected to attenuation by tissue absorption and scattering. Given that near-infrared (NIR, 700–900 nm) light is lowly absorbed in most tissue chromophores such as hemoglobins, NIR light can penetrate several centimeters of tissue and can thereby be clinically useful [116, 117]. In addition, significant auto-fluorescence of tissue severely compromises the signal to background ratio of optical imaging using visible light, which is, however, dramatically reduced in the NIR region [117].

Much progress has been made in recent years to use noninvasive NIRF imaging in diagnostics and image-guided drug delivery, even though the detection of deep and small lesions remains a challenge [116]. Commonly used contrast agents are organic small molecules such as indocyanine green (ICG, a NIR dye) which has been FDA-approved, IRDye78 (a tetra-sulfonated heptamethine indocyanine fluorophore), cyanine dyes (e.g. Cy5, Cy5.5, Cy7, etc), among others [116–118]. Antibodies can be readily conjugated to these organic fluorophores for tumor imaging. For example, Cy7-conjugated trastuzumab was mixed with other fluorescently labeled mAbs and injected to tumor-bearing mice [119]. With multi-filter spectrally resolved fluorescence imaging at 24 h after injection of the antibody cocktail, different tumor types in mice can be distinguished by separate fluorescence from their bound antibodies, including the HER-2 positive NIH3T3 tumors. Several other fluorophores, such as Cy5.5 [120, 121], AF750 [121], RhodG dye [122], ICG dye [123], IRDye 800CW [124] (Fig. (5)), and rhodamine green [125] have all been conjugated to trastuzumab for NIRF imaging of HER-2 in tumor-bearing mice. Among these, several studies further demonstrated NIRF imaging as a useful tool for monitoring the effects of trastuzumab therapy [19, 121], or to guide the surgical removal of tumor tissues [125]. Recently, with the use of mathematical modeling, Alexa Fluor^®750 dye labeled Z_HER-2:342 based affibody was demonstrated to be useful for quantitative assessment of HER-2 expression in vivo [126].

Fig. (5).

Dualmodality imaging of HER-2 in subcutaneous SKBR-3 tumor-bearing mice. (A) NIRF imaging with IRDye800CW-labeled trastuzumab (sagittal view). (B) PET imaging with ⁸⁹Zr-trastuzumab (coronal view). (C) Images of mouse bearing human gastric cancer KATO-III intraperitoneal tumor captured by intraoperative camera. IRDye800CW-labeled trastuzumab was administered to mice on a diet that minimizes autofluorescence in the peritoneum. NIRF imaging was taken on day 4 after injection. White arrows indicate the tumors, which were also shown in green in the overlay of color picture and a NIRF image. Adapted from [124].

Conventional organic fluorophores suffer from certain limitations, such as their untunable excitation and emission wavelengths, low quantum yields in aqueous solutions, photobleaching, and limited number of sites for conjugation of targeting ligands, all of which undermine the feasibility and sensitivity of NIRF imaging with fluorescent dyes [116]. Alternatively, nanomaterials such as quantum dot (QD), carbon nanotubes, and gold nanoshells, have been investigated as inorganic fluorophores [127–129]. Among them, QD is the most intensively studied due to its unique optical properties including broadband absorption, high quantum yield, and low photobleaching [117, 130]. In addition to the advantages in optics, as a nanoparticle, QD also possesses multi-valency for drug loading as well as the enhanced permeation and retention (EPR) effect that can potentially facilitate its use for drug delivery. A biocompatibility study of QDs and trastuzumab-conjugated QDs in rats revealed that antibody conjugation can significantly control the toxicity of QDs and make them suitable for breast cancer imaging [129]. To date, advanced in vivo experiments using QDs are still lacking [131], with most of the reported HER-2 imaging conducted at the cellular level. Among a number of reports using anti-HER-2 antibody labeled QDs [132–135], one study clearly demonstrated that QD-based immunofluorescence imaging was more sensitive and gave stronger signals than traditional techniques [133]. Therefore, anti-HER-2 antibody modified QDs are considered to be promising agents for delineating the heterogeneity of HER-2-positive tumors.

PHOTOACOUSTIC TOMOGRAPHY (PAT)

Optical imaging techniques face challenges in imaging deep tissue, since biological tissues are always optically scattering, where most photons are unable to penetrate deeper than the optical transport mean free path (~ 1 mm) [136]. The spatial resolution of these imaging techniques is thereby largely impeded by the depth [137]. In addition to the aforementioned NIRF imaging, many other imaging approaches with kindles beyond optics are currently under development. Given that ultrasound scattering in soft tissues is two to three orders of magnitude weaker, it is believed that a better resolution can be achieved with ultrasound-based imaging, even at a depth greater than the optical transport mean free path [137]. Ultrasonography is an ultrasound-based medical imaging technique that relies on the acquisition, analysis, and display of acoustic signals generated by reflection or backscatter of sound at a frequency higher than the audible range of humans [138]. Ultrasound imaging without contrast agents suffers from weak contrast in its detection of mechanical properties of tissues, thereby much less sensitive than other imaging approaches [137]. Therefore, contrast agents such as microbubbles and polymeric particles have to be used in any cases for ultrasonography [137]. Recently, PAT was developed which overcame these limits by combining the strengths of optical imaging and ultrasound to achieve a good optical contrast and a high ultrasonic resolution in one single system for high-resolution imaging in vivo [136].

PAT is based on the photoacoustic effect, in which the tissue or organ generates acoustic waves after the absorption of electromagnetic energy such as radio-frequency or optical waves [136, 137]. The tissue is usually irradiated by a short pulsed electromagnetic wave/light/laser beam, upon the absorption of which acoustic transient pressure is raised as the initial source of acoustic waves, together with thermoelastic expansion. The photoacoustic waves reach the tissue surface with different time delays, and are recorded by ultrasonic transducers to map the spatial distribution of acoustic sources. The tunable ultrasonic bandwidth in PAT determines the imaging depth and the spatial resolution in the tissue, with a 10 MHz bandwidth for a 0.1 mm resolution [136, 137]. Since many diseases do not possess enough endogenous photoacoustic contrast, exogenous contrast agents are required to target the specific diseased regions for precise and enhanced PAT images.

To date, a number of nanoparticles have been described as contrast agents for PAT, such as iron oxide and gold-couple core-shell nanoparticles [139], gold nanorods [140, 141], and single-walled carbon nanotubes (SWNT) [142]. With antibody-conjugated nanoparticles, the functional and molecular activity of tumor cells and diseased tissues can be visualized by PAT. Gold nanoparticles conjugated with trastuzumab were demonstrated to selectively target human SKBR-3 breast cancer cells in a gelatin phantom resembling the breast tissue, where 1×10⁹ nanoparticles were detected at a depth of 6 cm [143]. Anti-HER-2 antibodies were also conjugated to gold nanorods [144], nanoparticles made by biodegradable polyactic acid [145], or embedded with ICG dye [146] to investigate their capability to target breast cancer and prostate cancer cells in vitro with a high contrast and a high efficiency [146]. HER-2-targeted PAT imaging using gold nanorods was also conducted in the OECM1 tumor-bearing mouse, where the photoacoustic intensity within the tumor region raised to 3 dB higher than the pre-injection level, at around 30 min after injection (Fig. (6)) [147].

Fig. (6).

(A) In vivo photoacoustic images of OECM1 tumor before and after injection of trastuzumab-conjugated gold nanorods. Eclipsed dashed circles indicate the tumor regions. (B) The time-dependent image signal intensity of the tumor region. Adapted from [147].

MULTIMODALITY IMAGING

Each imaging modality discussed above possesses both advantages and disadvantages, in terms of the physical characteristics and the detection of the signals. Multimodality imaging can potentially combine the advantages of these modalities to overcome their respective disadvantages. For instance, optical imaging provides a direct, fast, low-cost, and real-time screening for surface lesions. However, it suffers from a limited sensitivity in deep tissue and is not apt for quantitative and tomographic imaging. On the other hand, nuclear imaging is superior in deep tissue sensitivity and quantitative imaging, yet it can be at a higher cost and requires a more restricted target-specificity due to the always-on characteristics of ionizing radiation [148, 149].

Radiolabeled anti-HER-2 antibodies or antibody fragments have been employed in nuclear imaging, and the resulting images were fused with X-ray CT [1, 11]. The C6.5 diabody, after being labeled with radioiodine, exhibited a time-dependent HER-2-positive tumor targeting [77], and was used effectively with PET/CT to evaluate HER-2 levels in response to HER-2-directed therapeutic treatments [78]. Another example of dual-modality imaging is with combined SPECT/CT, which has been described previously. The fused SPECT/CT affords a clearer visualization and localization of radiotracers in mouse models, thereby facilitating the evaluation of their bio-distributions and specificity of tumor uptake [90, 91, 150, 151]. Besides CT, other optical imaging approaches have been conducted simultaneously with PET imaging, such as NIRF imaging. In one report, PET was used to verify the detection results of NIRF imaging, in which two antibody-based contrast agents for PET and NIRF imaging purposes were prepared accordingly [124]. Other than optical imaging, anti-HER-2 antibody-based PET was sometimes co-performed with MRI. The results of these two imaging approaches were cross-validated with each other, to confirm the position of radiolabeled antibody in vivo [80, 152]. Clinically, fusion images from PET/MRI were reported to facilitate the identification of previously undetected lesions and metastasis in patients [80].

Compared to contrast agents separately prepared for more than one modality, the development of a single contrast agent for multimodality imaging can allow for a better synchronized scan, and can minimize the errors/differences caused by different agents. Given the inherent multi-valency, nanoparticles are ideal platforms for multimodality imaging. The anti-HER-2 antibody coated water-soluble FePt nanoparticles were demonstrated to be biocompatible in mouse models and were applicable to CT/MRI imaging [153]. Other iron oxide nanoparticles were either labeled with anti-HER-2 antibodies pre-conjugated by fluorophores [154], or coated simultaneously with both fluorophores and HER-2 antibodies (Fig. (7)) [155], for both NIRF and MRI imaging in tumor-bearing mice. In addition, a streptavidin nanoparticle was reported to be modified simultaneously by biotinylated trastuzumab, Cy5.5 fluorophore, and a ¹¹¹In labeled chelator, which collectively allowed for fluorescence and SPECT imaging in mice bearing HER-2-positive tumors [149]. Other than nanoparticles, anti-HER-2 antibodies can also be utilized to carry both radionuclides and fluorescent dyes for combined RIS/NIRF imaging [148, 156], or combined PET/NIRF imaging [157].

Fig. (7).

Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo with both NIRF imaging and MRI. Adapted from [155].

CONCLUSION

We have summarized several essential imaging techniques in antibody-based imaging of HER-2, among which the most sensitive techniques appear to be radionuclide-based. One concern of radionuclide-based imaging is its potential radio-hazard that may cause adverse effects. Therefore, alternative imaging techniques are emerging, such as PAT that can combine the advantages of ultrasound and optical fluorescence. Other than these single modality imaging approaches, multimodality imaging has also attracted intensive research interests which combine the advantages and circumvent the disadvantages of each single approach. Due to the limited endogenous contrast, most of these imaging techniques rely on exogenous contrast agents to achieve the desired spatial resolution and contrast. Therefore, the development of antibody-based contrast agents remains a promising direction in image-guided diagnostics and therapy.

Antibody-based imaging of HER-2, especially quantitative imaging, is highly valuable at several stages of pharmaceutical development and therapeutic intervention. Preclinical imaging studies in animal models can reveal the efficiency of antibody drugs in tumor targeting and therapy. Clinical imaging in patients can be employed to determine the optimal antibody dosing for tumor targeting, to assess the toxicity based on nonspecific uptake in normal organs, and to reveal inter-patient variations in pharmacokinetics and pharmacodynamics. Importantly, biopsy of tumors is traditionally used to confirm antigen expression level and to select patients for HER-2 targeted therapy, which may not represent an overall in vivo HER-2 expression statussince tumors are highly heterogeneous and in many cases hardly accessible. Antibody-based imaging, in this regard, may have unique values for patient selection due to its noninvasive, quantitative, time-sensitive, and whole-body nature for the assessment of both tumors and normal tissues.

Currently, many clinical trials are ongoing for antibody-based imaging of HER-2, most of which make use of the FDA-approved trastuzumab (Table 1). It is notable that some clinical trials are already starting to use HER-2 imaging to evaluate the therapeutic effects of small molecule or antibody-based anticancer drugs. Among all available imaging techniques, PET appears to be the most useful/used in clinical trials for HER-2 imaging, presumably due to its excellent sensitivity and good imaging quality. Moreover, this diverse set of imaging techniques may be employed in future clinical studies, in the form of multimodality imaging, to complement with each other for validation purposes, and also for improved imaging sensitivity and resolution. Taken together, antibody-based HER-2 imaging has moved into the clinic, which is expected to make a significant impact in clinical patient management and personalized medicine in the near future.

Table 1.

Current clinical studies of antibody-based imaging of HER-2 (Information based on www.clinicaltrials.gov).

Clinical Trial Number	Radiolabeled antibody	Imaging technique	Treatment	Status
NCT01445054	111In-Trastuzumab	RIS		Phase I
NCT00605397	64Cu-Trastuzumab	PET		Pilot Trial
NCT00613847	68Ga-F(ab′)2-Trastuzumab	PET
NCT01081600	89Zr-Trastuzumab	PET	AUY922	Phase I/II
NCT01420146	89Zr-Trastuzumab	PET/CT		Phase I
NCT01565200	89Zr-Trastuzumab	PET/CT	T-DM1	Phase II
NCT01216033	111In-ABY-025	SPECT		Phase I/II
NCT00474578	111In-Trastuzumab	RIS/SPECT		Phase I