Targeting EGFR and HER2 for Molecular Imaging of Cancer

The epidermal growth factor (EGF) receptor (EGFR, HER1, ErbB1) and human epidermal growth factor receptor type 2 (HER2, ErbB2) belong to the ErbB family of type I tyrosine kinases (TKs). This family of receptor TKs also includes another two closely related members HER3/ErbB3 and HER4/ErbB4. The general structure of these cell surface receptor proteins contains an extracellular ligand-binding domain, a hydrophobic transmembrane domain, an intracellular tyrosine kinase domain and a non-catalytic carboxyl terminal tail (Ferguson et al., 2000; Kari et al., 2003; Mitsudomi & Yatabe, 2010; Pines et al., 2010). Various ligands for EGFR, HER3 and HER4 have been identified, with EGF the most extensively characterized for its binding and activation of EGFR. These ErbB proteins are present in the plasma membrane as monomers. Upon ligand binding, the ErbB receptors can associate with each other to form different receptor dimers, which may be homodimers (e.g., EGFR-EGFR) or heterodimers (e.g., EGFR-HER2). The dimerization results in the activation of kinase activity and downstream signaling pathways, which often leads to cell proliferation and malignant tumor growth. HER2 is an exception, with no compatible ligand identified. However, it is the preferred heterodimerization partner for all other ErbB members (Mishani & Hagooly, 2009; Niu et al., 2008; Tzahar et al., 1996; Yarden & Sliwkowski, 2001).


Introduction
The epidermal growth factor (EGF) receptor (EGFR, HER1, ErbB1) and human epidermal growth factor receptor type 2 (HER2, ErbB2) belong to the ErbB family of type I tyrosine kinases (TKs). This family of receptor TKs also includes another two closely related members HER3/ErbB3 and HER4/ErbB4. The general structure of these cell surface receptor proteins contains an extracellular ligand-binding domain, a hydrophobic transmembrane domain, an intracellular tyrosine kinase domain and a non-catalytic carboxyl terminal tail (Ferguson et al., 2000;Kari et al., 2003;Mitsudomi & Yatabe, 2010;Pines et al., 2010). Various ligands for EGFR, HER3 and HER4 have been identified, with EGF the most extensively characterized for its binding and activation of EGFR. These ErbB proteins are present in the plasma membrane as monomers. Upon ligand binding, the ErbB receptors can associate with each other to form different receptor dimers, which may be homodimers (e.g., EGFR-EGFR) or heterodimers (e.g., EGFR-HER2). The dimerization results in the activation of kinase activity and downstream signaling pathways, which often leads to cell proliferation and malignant tumor growth. HER2 is an exception, with no compatible ligand identified. However, it is the preferred heterodimerization partner for all other ErbB members (Mishani & Hagooly, 2009;Tzahar et al., 1996;Yarden & Sliwkowski, 2001).
As EGFR and HER2 play important roles in many physiological and pathological processes, it is not surprising that deregulation of EGFR and HER2 is associated with various types of malignancies (Kari et al., 2003;Yarden & Sliwkowski, 2001). Both overexpression and mutation of EGFR have been identified in various types of cancers, while overexpression is a more commonly employed mechanism in the case of HER2 (Yarden & Sliwkowski, 2001). In these cancer cells, the TK activity is either elevated or constitutively activated. EGFR-and HER2-targeted anti-cancer medicines have been developed by either targeting the extracellular ligand-binding domain through antibody blocking, or by inhibiting the tyrosine kinase activity using low-molecular-weight molecules (Baselga, 2006;Pines et al., 2010;Speake et al., 2005). Cetuximab (a humanized mouse antibody) and panitumumab (a fully human antibody) have been approved for selected EGFR-targeted cancer treatment. Although these antibodies are designed to down-regulate EGFR signaling by inhibiting ligand-binding, preventing receptor dimerization and www.intechopen.com accelerating receptor degradation, antibody-dependent cell-mediated cytotoxicity is also considered as an important mechanism for IgG1 isotype antibody cetuximab Okamoto, 2010). Trastuzumab (a humanized mouse antibody) is a widely used treatment for breast cancer patients with HER2 overexpression . Because TKs depend on ATP to provide phosphate for the phosphorylation process, small molecules that bind to the ATP-binding pocket of the kinase domain can inhibit its enzymatic activity. These TK inhibitors (TKIs) may bind either reversibly or irreversibly. Reversible TKIs erlotinib and gefitinib are EGFR-specific, while lapatinib inhibits both EGFR and HER2 (Pines et al., 2010).
Molecular imaging agents with specific targets can provide information regarding biological processes at a cellular level before anatomical changes occur. Similar to the approaches used to develop EGFR and HER2 therapeutic agents, two strategies could be used for EGFR-and HER2-targeted tumor imaging. The first class of imaging agents targets extracellular domain of the receptor. Examples of these imaging agents include whole antibodies, antibody fragments, affibodies and nanobodies. The EGFR ligands, which bind to EGFR extracellular domain at high affinity, could also be used for this purpose. The second class of molecules includes TKIs and analogs that bind reversibly or irreversibly to the kinase domain of the receptor. Actually, some of the EGFR-and HER2-targeted therapeutic agents mentioned above have been used for tumor imaging after labeling with different functional groups, such as radionuclides for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging, and fluorescent dyes for optical imaging (Mishani et al., 2008;Mishani & Hagooly, 2009;.

Anti-EGFR antibodies
PET imaging with 64 Cu (t 1/2 = 12.7 h)-labeled cetuximab ( 64 Cu-DOTA-cetuximab) was employed to measure the EGFR expression in seven xenograft tumor models in vivo. The data was compared with EGFR levels in the tumors determined by Western blot. A reasonable linear correlation (R 2 = 0.80) between the tumor uptake of 64 Cu-DOTA-cetuximab (measured by PET) and the EGFR protein expression level (measured by Western blot) was discovered. It was also found that 64 Cu-DOTA-cetuximab had increasing tumor accumulation over time in EGFR-positive tumors but relatively low uptake in EGFRnegative tumors (Cai et al., 2007). In a following study, 64 Cu-DOTA-cetuximab was used to monitor the early response of EGFR degradation induced by 17-allyamino-17demethoxygeldanamycin (17-AAG) treatment . 17-AAG is an Hsp90 inhibitor, which induces the degradation of EGFR protein (Lang et al., 2007). In a PC-3 prostate tumor model, 17-AAG treatment induced significant reduction of 64 Cu-DOTAcetuximab uptake in the tumor. The reduction of 64 Cu-DOTA-cetuximab uptake correlated well with the lower EGFR protein level in the treated tumor as determined by immunofluorescence staining and Western blot .
Cetuximab has also been labeled with fluorescent dyes for optical imaging. Cy5.5 (Ex/Em: 675/695 nm)-labeled cetuximab (cetuximab-Cy5.5) was taken up specifically by head and neck squamous cell carcinoma (HNSCC) xenografts. The fluorescence from mouse tumors treated with cetuximab-Cy5.5 was significantly higher compared to a control probe IgG1-Cy5.5 (Rosenthal et al., 2007). To reduce the background fluorescence signal, a two-step activation strategy was designed. EGFR-overexpressing tumors (A431) were pre-targeted with biotinylated cetuximab, followed by administration of neutravidin conjugated with BODIPY-FL fluorescent dye (nAv-BDPfl). As the fluorescence of nAv-BDPfl increases dramatically (~ 10-fold) upon binding to biotin, the nAv-BDPfl signal will be specifically activated at the tumor site, where the biotinylated cetuximab was concentrated. Not surprisingly, this approach produced a higher tumor-to-background ratio . Another activatable probe employed the fluorescence quenching of ICG after conjugation with antibodies. The fluorescence could be restored by disrupting the molecular interaction between ICG and antibodies either in vitro (SDS plus 2-ME treatment) or in vivo (target binding and internalization). ICG-labled panitumumab (labeling ratio 1:5) exhibited a 58-fold signal increase upon SDS/2-ME treatment. EGFR-overexpressing tumors (A431 and MDA-MB-468) could be identified by this probe. Two other antibodies, trastuzumab (anti-HER2) and daclizumab (anti-CD25) were also labeled with ICG and used to image their respective targets in this study .
In an attempt to extend EGFR-targeted imaging with cetuximab to clinical scenario, cetuximab was labeled with 99m Tc (t 1/2 = 6 h). The resulting imaging probe 99m Tc-EC-C225 was evaluated by cell uptake assay, and in tumor-bearing mice and rats. SPECT imaging on a patient with squamous cell carcinoma visualized the tumor 2 h after 99m Tc-EC-C225 administration (Schechter et al., 2003). Assessment of the radiation dosimetry of this probe indicated that it has reasonable dosimetric properties for a diagnostic nuclear medicine agent (Schechter et al., 2004).
EGFR variant III (EGFRvIII) is a common in-frame deletion mutant that lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain (Ekstrand et al., 1992). EGFRvIII is constitutively activated, and associated with glioblastoma and some other tumors, such as prostate and breast cancer (Wikstrand et al., 1998). EGFRvIII could be recognized by ch806, a chimeric anti-EGFR antibody that selectively binds an EGFR epitope exposed only on mutant, overexpressed, or ligandactivated forms of the receptor (Panousis et al., 2005). 124 I (t 1/2 = 4.2d)-labeled ch806 ( 124 I -IMP-R4-ch806) was able to detect EGFRvIII expressing tumors (U87MG.EGFRvIII) at 24 h after probe injection, and prolonged retention was evident up to 168 h post-injection. There was a good correlation between in vivo tumor PET quantitation of 124 I -IMP-R4-ch806 and ex vivo measurement from dissected tissues. Remarkably, this probe was tumor-specific, and no significant binding to normal tissue was observed (Lee et al., 2010). This is in contrast to cetuximab, which accumulates in EGFR-expressing tissues such as liver .

EGFR-specific antibody fragments
Due to their large size (~150 kDa), the antibody-based imaging agents suffer from drawbacks such as long biodistribution time, poor penetrating capability, and slow clearance from the blood and normal tissues, which causes limited imaging contrast early post-injection, high background signal and non-uniform tumor penetration (Gong et al., 2010;Schier et al., 1996). Antibodies could be dissected into minimal monovalent binding fragments, such as Fab (~55 kDa) and scFv (~28 kDa), which retain the binding specificity (Holliger & Hudson, 2005). These antibody fragments have also been used for molecular imaging studies.

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The Fab fragment of a fully human antibody that recognizes the native extracellular domain of EGFR was labeled with 125 I (t 1/2 =59.4 d). The binding of 125 I-Fab to EGFR was confirmed by immunoprecipitation (IP) and fluorescence-activated cell sorting (FACS). In an animal imaging study, 125 I-Fab was able to distinguish the tumors with different levels of EGFR expression (Xu et al., 2009). An EGFR-specific scFv was isolated from a phage display library, and labeled with either quantum dot (QD) for fluorescence imaging, or magnetic iron oxide (IO) for magnetic resonance imaging (MRI). The resulting EGFR-targeted nanoparticles were discovered to accumulate in pancreatic xenograft tumors by tissue section fluorescence imaging and whole animal MRI (Yang et al., 2009). To achieve sitespecific labeling, an anti-EGFR scFv was fused to the SNAP-tag creating a fusion protein 425(scFv)SNAP (~48 kDa) (Kampmeier et al., 2010). SNAP-tag is a 20-kDa protein derived from the human DNA repair protein O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which reacts with the fluorescent dye conjugated benzylguanine (BG) substrate, leading to covalent labeling of the fluorescent dye on the fusion protein (Keppler et al., 2004). The fusion protein 425(scFv)SNAP labeled with the near-infrared (NIR) substrate BG-747 accumulated rapidly and specifically at the tumor site. Due to the efficient clearance, the tumor to background ratio (TBR) of this probe was significantly higher compared to the fulllength antibody cetuximab (Kampmeier et al., 2010).
Camelids contain a unique type of antibodies lacking the light chain (Hamers-Casterman et al., 1993). The single variable domain of heavy chain (VHH) has been isolated, and termed nanobody (~15 kDa). Several EGFR-specific nanobodies were labeled with 99m Tc for SPECT imaging. These 99m Tc-lableded nanobody molecules showed high specificity towards EGFRoverexpressing A431 tumors. The clearance from the blood was fast. The probe accumulation in the kidney was much higher than that in the liver, indicating a kidney clearance route (Gainkam et al., 2008;Huang et al., 2008). We labeled an EGFR-specific nanobody molecule (a kind gift from Dr. Paul van Bergen en Henegouwen at Utrecht University) with a NIR fluorescent dye IRDye® 800CW (Ex/Em: 774/789 nm) and used it for in vivo optical imaging. A431 xenograft tumors could be clearly identified at 1d postinjection with different dosages (0.4, 0.8 and 1.6 nmol per mouse) (Fig. 1). Consistent with SPECT results, the kidney accumulation of this fluorescent probe was high (Gong et al., unpublished data). To increase the binding affinity and optimize the pharmacokinetic properties, a bivalent form of nanobody molecule was created by fusing two monovalent Fig. 1. In vivo fluorescence imaging of nude mice bearing A431 tumors using an EGFRspecific nanobody labeled with IRDye 800CW. Images (dorsal view) were acquired at 1 d after injection of 1.6 nmol, 0.8 nmol or 0.4 nmol imaging agents, respectively. Arrows indicate A431 tumors. The nanobody was a gift from Dr. Paul van Bergen en Henegouwen, Utrecht University.
www.intechopen.com nanobody (EG 2 ) molecules to the human Fc fragment. The resulting molecule, EG 2 -hFc (80 kDa) was labeled with the fluorescent dye Cy5.5, and compared with Cy5.5-labeled monovalent EG 2 , and Cy5.5-labeled pentavalent derivative V2C-EG 2 (128 kDa). In vivo pharmacokinetic and biodistribution studies in mice revealed that the plasma half life of EG 2 -hFc-Cy5.5 was much prolonged compared to the other two probes. The retention of EG 2 -hFc-Cy5.5 in EGFR/EGFRvIII-expressing orthotopic brain tumors was also significantly higher, resulting in an improved tumor fluorescence signal (Iqbal et al., 2010).
Both Cy5.5-and IRDye 800CW-labeled EGF (named as EGF-Cy5.5 and EGF800 respectively) have been successfully used for fluorescence optical imaging of EGFR-expressing tumors. The specificity of both probes was verified by competition with excess cetuximab (Ke et al., 2003;Kovar et al., 2006). However, a direct comparison between these two probes revealed that EGF800 produced a significantly lower background and a higher tumor-to-background ratio, implying that IRDye 800CW is superior to Cy5.5 for this application (Adams et al., 2007). Analysis of the excised tumors from EGF800-treated mice demonstrated a good correlation between tumor wet weight and in vivo tumor fluorescence signal. Repeated administration of EGF800 probe allowed for the non-invasive tracking of orthotopic prostate tumor growth ( Fig. 2) (Kovar et al., 2006). Another study with EGF800 showed that probe accumulation in tumors reflected relative EGFR expression and EGFR occupancy by cetuximab (Manning et al., 2008).
QDs have also been used to label EGF for fluorescence optical imaging. An EGFR targeting nanoprobe was formed by coupling NIR QDs to EGF. The tumor-specific accumulation of this nanoprobe was demonstrated by both whole animal imaging and ex vivo tissue analysis (Diagaradjane et al., 2008).

EGFR-specific affibody
Affibody molecules are a class of affinity proteins composed of 58 amino acid residues that are derived from one of the IgG-binding domains of staphylococcal protein A. EGFRspecific affibody molecules (~7 kDa) have been selected by phage display technology (Friedman et al., 2007). A head-to-tail dimeric form (Z EGFR:955 ) 2 that has a higher binding affinity was labeled with 111 In and successfully used for in vivo imaging of A431 tumors www.intechopen.com (Nordberg et al., 2008). To further improve the binding affinity and specificity, the affibody was optimized by affinity maturation process (Friedman et al., 2008). The second generation of EGFR-specific affibody has been conjugated with various labels, and characterized in detail. Fig. 2. Tracking of orthotopic prostate tumor growth with EGF800. Male NOD/SCID mice were injected orthotopically with 22Rv1 tumor cells. Animals were injected with EGF800 at different time points and imaged. The left column shows the tumor progression of a representative mouse in color-coded fluorescence images superimposed on the white light images. In the right column, total fluorescence in an ROI encompassing the tumor region was quantified. Fluorescence intensity is color-coded to assist visualization. Adapted from (Kovar et al., 2006) with permission.
In vivo imaging with 64 Cu-or Cy5.5-labeled Z EGFR:1907 showed fast tumor (A431) targeting and good tumor-to-normal tissue contrast. Both agents accumulated at a high level in tumor, liver and kidney as revealed by biodistribution study . A dimeric form of EGFR-specific affibody (13.7 kDa) was labeled with IRDye 800CW (named as Eaff800), and used to image A431 xenograft tumors. The tumor could be visualized 1 h post-injection, and it became most prominent after 1 d (Fig. 3). The binding and uptake of Eaff800 was EGFR-specific because it only produced minimal signal when reacted with SK-OV-3 cells (HER2-overexpressing) in both cell-based assay and in vivo imaging study. It is notable that the liver uptake of Eaff800 was high, possibly due to the cross-reaction between Eaff800 and murine EGFR expressed in the liver (Gong et al., 2010). The monomer Z EGFR:1907 and dimer (Z EGFR:1907 ) 2 were labeled with 111 In and 125 I, respectively. The resulting four variants, 111 In-Bz-DTPA-Z EGFR:1907 , 111 In-Bz-DTPA-(Z EGFR:1907 ) 2 , 125 I-PIB-Z EGFR:1907 and 125 I-PIB-(Z EGFR:1907 ) 2 were compared in A431 tumor bearing nude mice. At 24 h after injection, the use of 111 In provided higher tumor radioactivity level than did 125 I for both the monomer and the dimer. It was also discovered that the tumor uptake of monomer was higher than that of dimer, despite a superior cellular retention of radioactivity for dimer in cell-based assays. The best probe, 111 In-Bz-DTPA-Z EGFR:1907 , produced a tumor-to-blood ratio of 100 at 24 h after injection . To investigate the effect of injected protein dose, an aliquot of labeled affibody 111 In-DOTA-Z EGFR:2377 was diluted in different amounts of unlabeled affibody before injection to A431 tumor bearing mice. A bell-shaped dose-responsive curve was observed. The initial increase in uptake was reasoned to be associated with saturation of EGFR in normal tissues, resulting in more 111 In-DOTA-Z EGFR:2377 available for tumor targeting. Further increase in the unlabeled affibody caused the competition of EGFR in the tumor site and reduced 111 In-DOTA-Z EGFR:2377 uptake .

TKI derivatives
TKIs targeting EGFR are small organic molecules developed based mainly on the anilinoquinazoline moiety. These molecules bind to ATP-binding site of the kinase domain either reversibly or irreversibly (Mishani & Hagooly, 2009). Reversible TKI agent gefitinib was labeled with 18 F (t 1/2 = 110 min) to image the EGFR status of different cancer cells. However it was discovered that [ 18 F]-gefitinib uptake did not correlate with EGFR expression levels due to high non-specific cellular uptake (Su et al., 2008). This finding is in agreement with other studies showing that most reversible TKIs were inadequate for in vivo imaging despite impressive in vitro profile, including high affinity and specificity toward EGFR. These radio-labeled reversible agents often exhibit low uptake in targeted tumors and high uptake in non-targeted tissues (Mishani & Hagooly, 2009). Nevertheless, a recent study with 11 C (t 1/2 = 20 min)-labeled erlotinib, another reversible TKI, showed that [ 11 C]erlotinib accumulated in HCC827 tumors that expresses high level of EGFR and is sensitive to erlotinib treatment. HCC827 xenograft tumors could be visualized by micro-PET scanning with [ 11 C]-erlotinib, whereas A549 and NCI358 xenografts (both express low level of EGFR) could not. However HCC827 cells also harbor an in-frame deletion mutation in exon 19 that contributes to the enhanced sensitivity to erlotinib. It is not clear whether this mutation causes the accumulation of [ 11 C]-erlotinib in the tumor (Memon et al., 2009).
To enhance binding efficacy, much effort has been invested in the development of irreversible TKIs as therapeutic drugs and imaging agents (Mishani et al., 2008;Mishani & Hagooly, 2009). An irreversible TKI-based radiotracer, morpholino-[ 124 I]-IPQA, has been developed and characterized. Morpholino-[ 124 I]-IPQA covalently binds to the ATP-binding site of the activated (phosphorylated) EGFR, but not the inactive EGFR. A431 tumors on immunocompromised rats and mice were successfully visualized by PET imaging with this probe (Pal et al., 2006). A newer version of this probe, [ 18 F]F-PEG6-IPQA, which has better water solubility, has also been developed. [ 18 F]F-PEG6-IPQA could detect NSCLC xenograft tumors harboring L858R activating EGFR mutations, and did not recognize NSCLC xenografts expressing either wild type or L858R/T790M dual mutant EGFR (Yeh et al., 2011). Another irreversible TKI analog ML04 exhibited excellent specificity to EGFR in vitro.

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However, in vivo biodistribution analysis of [ 18 F]ML04 showed that the probe uptake in EGFR-positive tumors was mainly non-specific, suggesting that further optimization of this probe is needed (Abourbeh et al., 2007;Mishani et al., 2008).
3 Imaging HER2-overexpressing tumors 3.1 Anti-HER2 antibodies HER2 amplification was initially observed in human breast cancer and was subsequently identified in ovarian cancer and other types of cancers Slamon et al., 1989;Zhang et al., 1989). HER2 expression is highly conserved between primary breast lesions and metastases in lymph node, bone marrow as well as distant locations (Gancberg et al., 2002;Lopez-Guerrero et al., 2006;Regitnig et al., 2004;Simmons et al., 2009;Simon et al., 2001;Tapia et al., 2007;Vincent-Salomon et al., 2007); and remains so throughout therapy Gong et al., 2005;Pectasides et al., 2006). Thus, accurate assessment of HER2 expression levels is of great interest for identifying breast cancer patients who will benefit from HER2-targeted therapy.
Trastuzumab (Herceptin; Genentech, CA), a recombinant humanized monoclonal antibody directed against the extracellular domain of the HER2 protein, was engineered by inserting the complementarity determining region of a murine antibody (clone 4D5) into the framework of a consensus human IgG1 (Carter et al., 1992). Tratuzumab has been shown to be effective in early-stage breast cancer that overexpresses HER2. Clinical trials indicate that combining tratuzumab treatment with standard chemotherapy for early-stage HER2 positive breast cancer reduces the risk of recurrence and death when compared to chemotherapy alone, thereby improving disease-free and overall survival rates in patients (Baselga et al., 2006;Romond et al., 2005).
Trastuzumab was radio-labeled with 111 In using DTPA as a chelator. 111 In-DTPAtrastuzumab selectively bound to t h e h u m a n H E R 2 r e c e p t o r i n in vitro analysis. Biodistribution and tumor targeting were studied in SK-OV-3 (HER2+) and GLC4 (HER2-) tumor-bearing athymic mice. The SK-OV-3 tumor showed substantial uptake of the labeled antibody after 5 h. The difference in uptake between SK-OV-3 and GLC4 tumors was even more pronounced 3 d after injection. At that time, the SK-OV-3 tumor was clearly visualized by radioimmunoscintigraphy (Lub-de Hooge et al., 2004). Research has also demonstrated that fluorescent dye-labeled trastuzumab enabled differentiation between breast cancer cells expressing high and low levels of HER2. Serial imaging before and during trastuzumab therapy revealed a significant reduction in probe uptake with treatment (Gee et al., 2008). Trastuzumab was also used to mediate the cellular internalization of pH-activatable fluorephores. These probes produced minimal fluorescence outside of the cell, and were activated by sensing the pH change in the lysosome after internalization (Urano et al., 2009). Other optical imaging based applications include imaging SK-BR-3 tumors with Cy5.5labeled trastuzumab (Hilger et al., 2004), and distinguishing between HER2+ pulmonary metastases and HER2-pulmonary metastases using rhodamine green-conjugated trastuzumab .
Another antibody against HER2 used for imaging applications is pertuzumab, a humanized antibody derived from the murine antibody 2C4. Unlike trastuzumab, pertuzumab sterically www.intechopen.com hinders HER2 hetero-dimerization with EGFR and HER3. Pertuzumab conjugated with 177 Lu showed significant tumor uptake in an in vivo study using gamma camera imaging (Persson et al., 2005).

HER2-specific antibody fragments
Trastuzumab-derived antibody fragments Fab and F(ab') 2 have also been employed for molecular imaging. 111 In-or 99m Tc-labeled trastuzumab Fab conjugates were used to detect HER2-expressing tumors using whole-body scintigraphy. Tumors were visualized 6 h postinjection . Trastuzumab and trastuzumab-derived Fab fragment (Fab4D5) labeled with 111 In were compared in a MMTV/HER2 mouse allograft model. Although Fab4D5 showed accumulation in the tumor as early as 2 h, rapid wash-out through the kidneys was observed by 6 h. On the contrary, trastuzumab was slow for tumor deposition and slow for clearance from the normal tissues. To improve the pharmacokinetic properties of the Fab fragment, an albumin binding sequence was introduced to generate a bifunctional molecule (AB.Fab4D5). Similar to Fab4D5 alone, AB.Fab4D5 visualized the tumor at 2 h post-injection, but its presence was sustained beyond 24 h, which resembles trastuzumab. Intravital microscopy revealed that tumor cell staining by AB.Fab4D5 was more uniform than for Fab4D5 or trastuzumab. The association of AB.Fab4D5 with albumin altered the clearance route of the probe, and minimal probe accumulation in the kidney was observed (Dennis et al., 2007). In another report, trastuzumab F(ab') 2 fragment labeled with 68 Ga was successfully used to quantify the loss and recovery of HER2 induced by HSP90 inhibitor 17-AAG in mice bearing BT-474 tumors (Smith-Jones et al., 2004). Table 1 summarizes trastuzumab-based agents for nuclear, optical and dual-modality imaging. This table includes only representative agents in each class, thus is in no way a complete list. The dual-labeled probes listed here will be discussed in detail later.

HER2-specific affibody
Phage display selection yielded affibody molecules that specifically bind to HER2 extracellular domain. One of these molecules, His 6 -Z HER2:4 (affibody Z HER2:4 linked to a histidine tag) targets the extracellular domain of HER2 with an affinity (K D ) at about 50 nM. The affibody does not interfere with trastuzumab binding to HER2, since the binding sites are different (Wikman et al., 2004). The specificity of the affibody was illustrated by the uptake of 125 I-His 6 -Z HER2:4 in HER2-overexpressing SK-BR-3 breast cancer cells Wikman et al., 2004). A bivalent version of the affibody (Z HER2:4 ) 2 , which has an increased binding affinity (K D = 3 nM) was later created (Steffen et al., 2005). When 125 I-labeled (Z HER2:4 ) 2 was investigated in an animal study, a tumor-to-blood ratio of about 10:1 was obtained 8 h after injection, and the tumor region could be visualized using gamma camera imaging (Steffen et al., 2006). However a comparison of 125 I-labeled  or 18 F-labeled (Cheng et al., 2008) second generation affibody molecules (see below) demonstrated that the monomeric form provided better tumor targeting than the dimeric form, which is the same as EGFR-specific affibody molecules ).
The second generation of HER2-specific affibody was generated through a single-library affinity maturation step. One of them, Z HER2:342 , showed a >2200-fold increase in affinity (22 pM for Z HER2:342 vs 50 nM for Z HER2:4 ). SK-OV-3 tumor xenografts were imaged 6 h after injection of 125 I-labeled Z HER2:342 . The tumor uptake at 4 h post-injection was improved by a factor of 4 compared to the parental molecule Z HER2:4 . Comparison of 124 I-labeled Z HER2:342 and trastuzumab demonstrated that better tumor-to-organ ratios were obtained with Z HER2:342 due to the more rapid clearance . By changing the radioisotope to 111 In, the labeled molecule 111 In-Benzyl-DTPA-Z HER2:342 maintain a K D of 21 pM. Detection of SK-OV-3 tumors was achieved at 4 h post-injection with a gamma-camera. Biodistribution analysis revealed a tumor-to-blood ratio of 100 . HER2-specific affibody molecules were also labeled with fluorescent dyes. The binding affinities and specificities of the conjugated molecules were unchanged or minimally affected by the modifications. In vivo NIR optical imaging revealed that affibody fused with an albumin-binding domain (ABD-(Z HER2:342 ) 2 ) exhibited a better performance compared to either monomer or dimer alone .
Site-specific radio-labeling of Z HER2:342 was achieved by synthetic chemistry. The resulting 111 In-DOTA-Z HER2:342-pep2 has a binding affinity of 65 pM. High contrast gamma camera images were obtained in the tumor region as early as 1 h after injection. Although pretreatment with trastuzumab did not compete with the binding of 111 In-DOTA-Z HER2:342-pep2 in tumors, degradation of the HER2 receptor using the heat-shock 90 inhibitor 17-AAG before probe administration reduced tumor uptake . These data taken together indicate that, similar to the first generation affibody Z HER2:4 , the binding site of Z HER2:342 is different from that of trastuzumab, but specific to the HER2 receptor. Site-specific labeling of 99m Tc was mediated by incorporation of either histidine tag (His 6 ), natural peptide sequences CCG, CGGG, or maGGG, maSSS, maSKS, maESE chelators into the Z HER2:342 sequence. All 99m Tc-labeled probes showed tumor-specific uptake at 6 h post-injection or earlier using gamma camera imaging Tran et al., 2008).
suggesting their potential to localize HER2-positive metastases not amenable to biopsy (Baum et al., 2010).
The HER2-specific affibody was further optimized by reengineering the nonbinding surface of Z HER2:342 to improve its properties such as storage stability, surface hydrophilicity, melting point and amenability for peptide synthesis. The new generation of affibody was created and characterized (Ahlgren et al., 2010;Feldwisch et al., 2010).
The fact that HER2-specific affibodies and trastuzumab target separate epitopes might be advantageous when developing these affibodies as imaging agents. These imaging agents will not interfere with trastuzumab binding when being used to monitor the therapeutic effect of trastuzumab (Baum et al., 2010;Orlova et al., 2007). One cause of concern of these affibody-base imaging agents is the high non-specific kidney accumulation. This is due to the small molecular weight of the affibody molecules which renders their clearance mainly through the kidney.

Dual-labeled trastuzumab
Probes dual-labeled with a radionuclide and a NIR fluorophore enhance the advantages and compensate the disadvantages of each modality, and could offer unique opportunities for the combination of non-invasive whole body imaging and subsequent intraoperative guidance during surgery. To investigate this possibility, trastuzumab was conjugated with either 111 In (for SPECT imaging) or 64 Cu (for PET imaging), and IRDye 800CW to image HER2 overexpression in mouse xenograft tumors (Sampath et al., 2010;Sampath et al., 2007).
( 111 In-DTPA) n -trastuzumab-(IRDye800) m was synthesized using a three step process wherein, trastuzumab was first conjugated to DTPA and then with IRDye 800CW. Prior to animal imaging, radio-labeling with 111 In was performed. The agent was validated for binding specificity in vitro using the fluorescence microscopy. High probe uptake in HER2overexpressing SKBr3-luc tumors was demonstrated using NIR fluorescence, SPECT and planar scintigraphy imaging. The tumor-to-muscle ratios were comparable between optical and nuclear imaging modalities, but NIR fluorescence imaging had a higher signal-to-noise ratio. Since NIR fluorescence imaging has improved sensitivity due to high photon count, it can be used to monitor the lymphatic uptake of the dual-labeled antibody after intradermal administration into the dorsal aspect of the foot pad. Accumulation into the lymph nodes was observed at picomole doses within 1 h after administration, while clearance was observed by 24 h (Sampath et al., 2007).
To test the ability of dual-labeled ( 64 Cu-DOTA) n -trastuzumab-(IRDye800) m to detect metastasis, Balb/c mice bearing 4T1.2/R (murine breast cancer cell, HER-) and 4T1.2neu/R (murine breast cancer cell, HER+) tumors were used. The diagnostic capability of this duallabeled probe was also compared with PET imaging agent 18 FDG. ( 64 Cu-DOTA) ntrastuzumab-(IRDye800) m showed significantly higher uptake in 4T1.2neu/R primary tumors compared to 4T1.2/R tumors, indicating in vivo specificity of the dual-labeled imaging agent. In contrast 18 FDG did not show any preferential uptake between the two tumor types. Lung metastases of HER2-overexpressing 4T1.2neu/R cells were successfully detected with whole body PET imaging after administration of ( 64 Cu-DOTA) n -trastuzumab-(IRDye800) m . Unfortunately, due to its limited penetration capability, whole body NIR fluorescence imaging could not detect lung metastases although it detected superficial skin metastases successfully. But agent uptake was evident with ex vivo NIR fluorescence imaging. Ex vivo NIR fluorescence imaging also visualized the trafficking of imaging agent from the primary tumor to lymph nodes, which was not possible for nuclear imaging with 64 Cu. When 18 FDG was investigated, it could not detect metastases under these conditions (Sampath et al., 2010).
In another study, it was found that trastuzumab labeled with both ICG and 111 In mimicked the cocktail of ICG-trastuzumab and 111 In-trastuzumab when injected into 3T3/HER2 tumor-bearing mice. As the fluorescence of ICG in this probe was only activated after binding to the HER2 receptor on the cell surface and being internalized, the HER2overexpressing tumor could be visualized with minimal background signal ).

Antibodies labeled with fluorescent dyes
Fluorescence optical imaging has the advantage of multiple channels, which can be employed to image two or more targets simultaneously. Cetuximab and trastuzumab were labeled with Cy5.5 and Cy7, respectively. The binding and internalization of cetuximab-Cy5.5 by A431, and trastuzumab-Cy7 by 3T3/HER2+ cells were confirmed by fluorescence microscopy. On the contrary, no binding of cetuximab-Cy5.5 to 3T3/HER2+, and trastuzumab-Cy7 to A431 was observed. When mice were injected with a cocktail of cetuximab-Cy5.5 and trastuzumab-Cy7, A431 and 3T3/HER2+ tumors could be detected distinctly based on the Cy5.5 and Cy7 spectral images (Barrett et al., 2007). In a subsequent study three antibodies (cetuximab, trastuzumab and daclizumab) were labeled with three different fluorophores (Cy5, Cy7 and AlexaFluor700), respectively. Spectrally resolved fluorescence imaging showed that these probes clearly distinguished their respective targeting tumors (A431, 3T3/HER2+ and SP2-Tac) based on their distinct optical spectra .

Affibody molecules labeled with fluorescent dyes
The EGFR-or HER2-specific Affibody molecules were labeled with IRDye 800CW and DY-682, respectively. The labeled probes, Eaff800 and Haff682, were taken up at high levels by EGFR-overexpressing A431 and HER2-overexpressing SK-OV-3 cells, respectively. Whole animal imaging showed that Eaff800 mainly accumulated in A431 tumor, while more Haff682 signal was found in SK-OV-3 tumor (Fig. 4A). Scanning tissue sections of dissected tumors confirmed the in vivo data. The liver uptake of Eaff800 was much higher than that of Haff682, suggesting a role of murine liver EGFR in the uptake of Eaff800. When the labeled fluorophores on the affibody molecules were exchanged (i.e. EGFR-specific affibody labeled with DY-682 and HER-specific affibody labeled with IRDye 800CW), the specificities of the affibody molecules were not affected (Fig. 4B). These results demonstrated that the tumor uptake of these imaging agents is receptor-mediated, and is independent of the fluorophore labeled on the affibody molecules (Gong et al., 2010).

Closing remarks
In vivo molecular imaging has become a valuable tool in biomedical research and drug development (Weissleder & Pittet, 2008;Willmann et al., 2008). The radionuclide-based imaging technology, such as PET and SPECT, has been used in clinical applications. In the research field, fluorescence optical imaging is becoming more and more popular due to its www.intechopen.com low cost, ease of use, longer time window for image capture and ability to track multiple probes simultaneously. Compared to fluorophores in the visible spectra, imaging with NIR fluorophores could reduce the autofluorescence, maximize tissue penetration, and are ideally suitable for non-invasive animal imaging applications (Kovar et al., 2007;Weissleder, 2001). However, even with NIR fluorophores, the direct clinical application of fluorescence imaging may be limited to superficial tissues such as breast and skin, or for visualization during endoscopy and image-guided-surgery procedures (Kampmeier et al., 2010).
EGFR-and HER2-targeted molecular imaging may aid in the selection of patients for individualized therapy by noninvasively assessing the expression level of EGFR and HER2 in tumors, and guide drug dosage and regime by measuring target-drug interaction and receptor occupancy. It can also be used to assess the responses to therapy in pre-clinical and clinical research, and provide valuable information for drug development and validation (Kampmeier et al., 2010).
EGFR and HER2 mediate a complex network of signaling pathways that interweaves with many other signaling networks. Interpretation of therapeutic output based solely on EGFRand HER2-targeted imaging results might be oversimplified. Analysis of downstream molecules such as p-Erk1/2 may complement EGFR-and HER2-targeted imaging and provide new insights for cancer treatment . For EGFR-targeted therapy, it is not uncommon that drug effects are independent of EGFR expression levels in cancer cells.
In these situations, targeting activated forms of the receptor instead of total receptor level may be a more reliable method to predict drug responsiveness ).