Optimizing treatment of metastatic colorectal cancer ...

Review

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Introduction

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EGFR and colorectal cancer: involvement in cell transformation and in the prediction of prognosis

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Univ Studi di Napoli on 01/03/13 For personal use only.

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Evolution of the antibodies raised against EGFR: cetuximab

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Mechanisms of primary resistance to EGFR-targeted mAbs: KRAS mutations

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New-generation antibodies raised against EGFR

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Conclusion

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Expert opinion

Optimizing treatment of metastatic colorectal cancer patients with anti-EGFR antibodies: overcoming the mechanisms of cancer cell resistance Teresa Troiani, Silvia Zappavigna, Erika Martinelli, Santolo R. Addeo, Paola Stiuso, Fortunato Ciardiello & Michele Caraglia† †

Second University of Naples, Department of Biochemistry, Biophysics and General Pathology, Naples, Italy

Introduction: A number of anti-EGFR monoclonal antibodies (mAbs) have been recently developed for the treatment of refractory metastatic colorectal cancer (mCRC). These mAbs, blocking ligand/receptor interactions, exert their biological activity via multiple mechanisms, including inhibition of cell cycle progression, potentiation of cell apoptosis, inhibition of angiogenesis, tumor cell invasion and metastasis and, potentially, induction of immunological effector mechanisms. Areas covered: Cetuximab is an anti-EGFR mAb currently used in mCRC treatment. Despite the evidence of efficacy of cetuximab in the treatment of mCRC patients, the observation of low response rates was the proof of concept of resistance to anti-EGFR mAbs treatment. An increasing number of molecular alterations have been more recently hypothesized to be involved in resistance to anti-EGFR mAbs in CRC: mutations in BRAF, NRAS and PIK3CA, loss of expression of PTEN and, now, activation of HER2 signaling through HER2 gene amplification and/or increased heregulin stimulation. Expert opinion: This review focuses on the development of new strategies such as combination with other agents blocking alternative escape pathways, cancer cell prioritization hyperactivating EGFR pathway, combination with immune system, development of nanotech devices to increase efficacy of antibody-based therapy and overcome the mechanisms of cancer cell resistance. Keywords: anti-EGFR monoclonal antibodies, BRAF, colorectal cancer, EGFR signaling, immunoresistance, KRAS, nanobodies Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

Cell growth is regulated by many kinds of growth factors. These growth factors act through different pathways: autocrine, paracrine, juxtacrine, intracrine, endocrine and other ways. The specific binding of a growth factor to its own receptor activates a variety of signal cascades, which finally leads to the biologic responses of the cells. The whole process results in the cell growth and differentiation. In physiological conditions, the growth of the cells depends on the balanced regulatory networks. When this balance is interrupted, pathologic changes of the cells will occur. Much evidence suggests that altered cell growth of malignant cells is due, at least in part, 10.1517/14712598.2012.756469 © 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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Deregulation of EGFR signaling is a hallmark of many cancers, including colorectal cancers (CRCs). Although EGFR is overexpressed in up to 82% of CRCs and is associated with tumor development and progression, its role is not entirely clear. Cetuximab, is an anti-EGFR mAb currently used in metastatic colorectal cancer (mCRC) treatment. New-generation antibodies and nanobodies raised against EGFR have been developed to overcome limitations of cetuximab. An increasing number of molecular alterations in EGFR-Ras-MAPK cascade have been more recently hypothesized to be involved in resistance to anti-EGFR drugs in CRC. In order to optimize treatment of mCRC patients with anti-EGFR drugs, new strategies such as cancer cell prioritization, combination with immune system and development of nanotechnologies are needed to overcome the mechanisms of cancer cell resistance.

This box summarizes key points contained in the article.

to the aberration of the regulatory networks. Growth factors that have stimulatory effect on cell proliferation and their receptors are often found to be overexpressed in tumors [1]. Molecular therapies targeting the abnormal regulatory molecules are of great interest in cancer therapy research. 2. EGFR and colorectal cancer: involvement in cell transformation and in the prediction of prognosis

EGFR is a cell membrane growth factor receptor characterized by tyrosine kinase activity that plays a crucial role in the control of key cellular transduction pathways in both normal and cancerous cells. EGFR is overexpressed in a variety of human tumors, including head and neck, breast, lung, colorectal, prostate, kidney, pancreas, ovary, brain and bladder cancer [2]. The 170 kDa protein function depends either on the formation of EGFR -- EGFR homodimers or heterodimers -- that comprise the three members of the EGFR (human epidermal receptor 1 (HER1)) family of growth factor receptors (HER2, HER3 and HER4) following binding of an EGFR-selective ligand. The activating ligands include the EGF, TGF-a, epiregulin, amphiregulin or neuregulin. The binding EGFR/ ligand results in conformational changes that allow the activation of EGFR tyrosine kinase and the phosphorylation of specific tyrosine residues within the EGFR intracellular carboxyl-terminal domain. Phosphorylated tyrosine residues serve as docking sites for several signaling proteins finally stimulating cell proliferation, loss of differentiation, invasion, angiogenesis and blocking of apoptosis. Within a few hours of activation, receptors are internalized into cytoplasm, where they are either degraded or recycled back to the membrane. The complex signaling network generated by triggering 2

EGFR includes the Ras- and mitogen-activated protein kinase (MAPK) pathway that leads to cell proliferation, the phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt) pathway driving cell cycle progression and cell survival [3,4]. There is also evidence that EGFR can translocate to the nucleus, where it acts as a transcription factor (Figure 1) [5-7]. Deregulation of EGFR signaling is a hallmark of many cancers, including colorectal cancers (CRC). In general, the EGFR pathway can be hyperactivated by overproduction of ligand, overproduction of receptor or constitutive activation of receptors [5]. Although EGFR is overexpressed in up to 82% of CRCs [6] and is associated with tumor development and progression, its role is not entirely clear. Roberts et al. elegantly demonstrated the involvement of EGFR signaling in a genetic mouse model of intestinal tumorigenesis. A role for EGFR signaling in establishment of initiated microadenomas was therefore put forward although EGFR function in normal intestinal development and crypt homeostasis still remains poorly understood [8]. In fact, EGFR expression progressively increases with malignant transformation from normal colon, through adenoma, to the poorly differentiated and metastatic cancer, suggesting its role in oncogenesis. EGFR overexpression is observed in tumors of more advanced stage, worse histologic grade and with lymph vascular invasion and it is associated with poor prognosis in the majority of studies [8].

Evolution of the antibodies raised against EGFR: cetuximab

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An anti-EGFR monoclonal antibody (anti-EGFR mAb), cetuximab, is currently used in metastatic colorectal cancer (mCRC) treatment [9,10]. Cetuximab (C225, Erbitux) is an immunoglobulin (Ig) G1 human--murine chimeric counterpart of the murine mAb M225 [11]. It binds to the EGFR with high affinity and promotes receptor internalization and subsequent degradation, resulting in receptor down-regulation. Cetuximab has been evaluated in both chemorefractory and untreated mCRC. In chemorefractory mCRC, cetuximab monotherapy was associated with response rates (RR) of 9 -- 12%. RR of approximately 20% were achieved when cetuximab was used in combination with irinotecan in patients who became refractory to irinotecan [12,13]. The impact of cetuximab plus irinotecan in second-line metastatic colorectal EGFR-expressing cancer patients was examined in a multinational Phase III trial known as the EPIC (Erbitux Plus Irinotecan for Metastatic Colorectal Cancer) study. The results revealed that treatment with cetuximab improved the progression-free survival (PFS), RR and health-related quality of life. However, no differences were seen in overall survival (OS), probably because almost half the patients crossed over to cetuximab treatment after the failure of irinotecan monotherapy [14]. These studies provided the bases for further development of cetuximab for its use in

Expert Opin. Biol. Ther. [Early Online]

Optimizing treatment of metastatic colorectal cancer patients with anti-EGFR drugs

Epidermal growth factor Epidermal growth factor receptor

PI3K SOS


K-ras GDP

PIP2 Grb2

K-ras GTP

PIP3 PTEN

P

P

P

P Akt

BRAF MEK

Cell survival and growth ERK

Cell proliferation

Figure 1. EGFR signaling pathway. The signaling cascade initiated by binding of the EGF and other ligands to the EGFR comprises two main axes. On one side, the KRAS--RAF--MAPK signaling pathway is thought to control cell growth, differentiation and apoptosis. After Grb2/SOS-mediated activation, guanosine triphosphate-bound KRAS recruits the serine protein BRAF, thus starting a cytoplasmic phosphorylation cascade leading to the activation of transcription factors. The other axis involves membrane localization of the lipid kinase PIK3CA, which promotes AKT activation, ensuring a parallel intracellular propagation of the signal.

first-line treatment of mCRC. In pivotal Phase III study, 1198 patients were randomized to receive cetuximab combined with first-line chemotherapy regimen of irinotecan, infusional fluorouracil and leucovorin (FOLFIRI) versus FOLFIRI alone. The primary objective of the so-called CRYSTAL study (Cetuximab combined with iRinotecan in first-line therapY for metaSTatic colorectAL cancer) was PFS. The study demonstrated that cetuximab improved the standard chemotherapy regimen, in particular significantly reduced the risk of progression (8.9 vs 8 months, hazard ratio (HR) 0.85; p = 0.048), enhanced tumor response (46.9 vs 38.7%, odds ratio 1.40; p = 0.004) and R0 (radical) resection of metastasis with curative intent (p = 0.002). OS analysis did not appear to be statistically significant different between treatments groups (19.9 vs 18.6; HR 0.93, p = 0.31). The lack of meaningful result regarding median OS could depend on the fact that the two-thirds of patients in each group received subsequent chemotherapy and in nearly 25% of cases cetuximab as well [15]. Cetuximab in combination with FOLFOX-4 (oxaliplatin, folinic acid and 5-fluorouracil) was investigated in Phase II randomized trial. The OPUS (Oxaliplatin and Cetuximab in First-line Treatment of mCRC) was a smaller study randomizing 337 patients. The primary objective was overall response rate (ORR) assessed

by an independent review committee according to modified WHO criteria. The results of this trial have demonstrated a higher ORR in cetuximab arm (46 vs 36%, p = 0.0064) that was not statistically significant in terms of PFS or OS. [16]. Subsequently, a Phase III trial has evaluated the efficacy of cetuximab in combination with an oxaliplatin-based first-line combination chemotherapy for the treatment of mCRC patients [17]. In this randomized controlled trial, 1630 patients were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B) or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomization. Unfortunately, the primary end point was not met. In fact, even if the addition of cetuximab to oxaliplatin--based chemotherapy increases RR, no evidence of benefit in terms of PFS or OS was achieved between arm A and B. Interestingly, subgroup analyses have showed that improved PFS with cetuximab was seen in patients treated with fluorouracil-based therapy (HR 0.72, 95% confidence interval (CI) 0.53 -- 0.98, p = 0.037), but not in those treated with capecitabine-based therapy (HR 1.02, 95% CI 0.82 -- 1.26, p = 0.88; p = 0.10 for interaction). Moreover, the addition of cetuximab (arm B) had an effect on


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time on treatment in the capecitabine-based therapy groups. In fact, the overall median duration of treatment was 29 weeks in atients receiving fluorouracil-based therapy, but 25 weeks in those given capecitabine-based therapy (p = 0.0028 adjusting for treatment group). The lack of benefit in patients treated with capecitabine could be accounted for at least in part by the increase in toxic effects recorded, and the resulting reduction in dose intensity of the chemotherapy administered or by other undetermined factors [17]. Consistent with the present COIN data, the NORDIC-VII study provides no evidence that cetuximab adds a significant benefit to oxaliplatin-based first-line treatment of mCRC. In fact, there was no significant trend toward a higher ORR (49 vs 41%) in the patients receiving FLOX plus cetuximab (arm B) as compared with FLOX alone (arm A), as well as no numerical or statistical difference in PFS or OS between the arms was found [18]. One major factor that could affect the benefit of the addition of cetuximab to chemotherapy is the precise nature of the agents used in combination. The only Phase III trial in first-line therapy showing an OS benefit to date used irinotecan and infusional fluorouracil as the chemotherapy backbone [15]. By comparison, the trials using oxaliplatin have not shown improved OS and this failure has raised the possibility of a negative interaction between oxaliplatin and cetuximab. It is hard to explain why cetuximab would have no effect when combined with oxaliplatin containing regimen in mCRC. At the moment, there are insufficient clinical data to answer this question. Preclinical mechanistic studies have shown both synergistic and antagonistic modulation by cetuximab of the effects of oxaliplatin in CRC cell lines in vitro [19-22].

Mechanisms of primary resistance to EGFR-targeted mAbs: KRAS mutations 4.

Despite the evidence of efficacy of cetuximab in the treatment of mCRC patients, the observation of low RR was the proof of concept of resistance to anti-EGFR mAbs treatment. In this setting, resistance to anti-EGFR therapies is likely attributable to the constitutive activation of signaling pathways acting downstream of EGFR [23]. Kirsten (K)RAS belongs to the gene family of oncogenes (KRAS, HRAS and NRAS) encoding guanosine di/triphosphate-binding proteins that act as an important, but not exclusive effector of EGFR [24,25]. When mutated, it results to be constitutively active leading the cells to become independent from the EGFR signaling activation. Somatic mutations of KRAS occur in 30 -- 40% of CRC and mostly occur in codon 12 (about 70 -- 80%) and codon 13 (about 15 -- 20%) of exon 2. The remaining mutations are mainly located on codons 61, 146 and 154 [24]. Initial retrospective analyses revealed that patients with CRC carrying activating KRAS gene mutations do not benefit from cetuximab therapy [25]. KRAS mutations have since emerged as the major negative predictor of efficacy in patients receiving cetuximab. 4

Studies in the first line and subsequent lines of treatment have shown that patients with tumors harboring a KRAS mutation neither respond to EGFR-targeted mAbs nor experience survival or quality-of-life benefit from such treatment [26-28]. Specifically, patients with wild-type (WT) KRAS generally have doubled PFS times compared with patients with mutant KRAS. The evidence that KRAS mutations were associated with the lack of response to cetuximab in chemorefractory mCRC patients, leading the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) to restrict the use of cetuximab monotherapy or in combination with chemotherapy, only to patients with KRAS WT tumors [29]. However, it is intriguingly now coming to light that perhaps not all KRAS mutations are created equal in their impact on mediating EGFR resistance. Various reports have described that patients with KRAS mutated tumors may occasionally respond to either cetuximab or panitumumab. Initially, these findings were considered as erroneous, and were thought to be related to either the inaccuracy of the mutational test, or, to response to chemotherapy that was administered in combination with anti-EGFR-targeted compounds [28]. In fact, recently it has been reported by De Roock et al. that chemo-refractory mCRC patients which carry a KRAS mutation in codon 13 may have longer PFS (median, 4.0 vs 1.9 months) and longer OS (median, 7.6 vs 5.7 months) compared with patients harboring other KRAS mutations when treated with cetuximab [30]. The evidence of some KRAS G13D not achieving a clinical benefit from anti-EGFR-targeted treatment indicates that these results have to be considered with caution and their impact should be carefully assessed in well-designed prospective trials. Nevertheless, it can be hypothesized that KRAS mutations might have different roles depending on the context where they develop and thus to which subgroup of CRC they belong. Moreover, the limited sensitivity of current sequencing methods in detecting point mutations may at least partially account for ‘apparent’ absence of mutations. Molecular markers predictive of primary resistance to mAbs: beyond KRAS mutations

4.1

The identification of additional genetic determinants of primary resistance to EGFR-targeted therapies in CRCs is important to prospectively identify patients who should not receive cetuximab, thus avoiding their exposure to ineffective and expensive therapy. Recent work has therefore been focused on the analysis of the molecules involved in downstream EGFR signaling. BRAF is a cytoplasmatic serine/threonine kinase directly interacting with RAS [31]. BRAF mutations are mainly located in the kinase domain, with a single substitution of glutamic acid for valine at codon 600 (V600E) accounting for 80% of all mutations; other, less frequent, activating mutations affect the same residue, including V600A, V600D, V600G, V600K, V600M and V600R [32]. The V600E amino acid




substitution is thought to be responsible for the oncogenic properties of BRAF by inserting a negatively charged amino acid in the activation segment, thus mimicking the phosphorylation of the kinase and causing it to be constitutively active [32]. The BRAF V600E mutation occurs in approximately 4 -- 15% of CRC and is known to be mutually exclusive with KRAS in CRC [33]. At least three studies have demonstrated that mutation in BRAF impair responsiveness to cetuximab in patients with mCRC [33,34]. BRAF mutation was also associated with a trend toward shorter PFS and with significantly shorter OS [35,18]. In line with these studies, a wide retrospective cohort analysis of chemorefractory patients from a European Consortium (n = 761) demonstrated that KRAS WT and BRAF mutant patients treated with cetuximab showed a significantly lower RR, shorter PFS and OS [35]. On the contrary, recent results from firstline randomized studies (OPUS and CRYSTAL) made the relationship between the BRAF mutation and the response to cetuximab not completely clear since they showed a trend in favor of cetuximab treatment for BRAF V600E patient. Based on these contradictory results, it cannot be yet concluded if the BRAF V600E mutation is a negative predictive marker of response to anti-EGFR mAbs but it can certainly be hypothesized that it might play a different role in heavily pretreated and chemonaı¨ve patients [36]. Since RAF is an important effector downstream of RAS, targeting this effector could be an effective strategy for treating KRAS or BRAF mutated tumors. Surprisingly, in a study evaluating 19 mCRC BRAF V600E patients treated with PLX4032 (vemurafenib), a selective RAF inhibitors, that showed pronounced activity in BRAF-mutant melanoma patients, only a modest clinical activity was observed suggesting that the biology of BRAF activation in patients with mCRC is clearly more heterogeneous than in melanoma [37]. Recently, it has been investigated the mechanisms underlying the limited therapeutic effect of vemurafenib in colon cancer patients harboring the BRAF V600E oncogenic lesion [38]. It has been shown that in multiple BRAF V600E mutant colon cancers cells, vemurafenib caused a rapid feedback activation of EGFR which supports continued proliferation in the presence of BRAF V600E inhibition. Moreover, the inhibition of EGFR by the antibody drug cetuximab or the smallmolecule drugs gefitinib and erlotinib was strongly synergistic with vemurafenib, both in vitro and in vivo. These data were also supported by the evidence that melanoma cells, harboring the same mutation and very sensitive to vemurafenib, have a very low levels of EGFR and are, therefore, not subjected to this feedback activation. Consistent with this, ectopic expression of EGFR in melanoma cells was sufficient to cause resistance to vemurafenib [38]. These data provide a strong rationale for a clinical trial combining BRAF and EGFR inhibitors in BRAF V600E mutant CRCs, which have a very poor clinical outcome and for which no targeted therapeutic strategies are effective after failure of standard therapeutic regimens. At first glance, it would seem

counterintuitive to consider treating a BRAF mutant colon cancer with an EGFR inhibitor, as multiple clinical studies in colon cancer have shown that EGFR inhibition is without clinical benefit when either KRAS or BRAF is mutated downstream of EGFR [25,26,31]. The strong synergistic interaction between inhibition of BRAF and EGFR described here is explained by an unexpected and powerful feedback activation of EGFR caused by BRAF inhibition, providing a rationale for the poor clinical response of BRAF V600E mutant colon cancer to vemurafenib monotherapy [38]. NRAS mutations might impair responsiveness to cetuximab treatment. Even if KRAS, NRAS and BRAF mutations are mutually exclusive in CRC, presumably because there is no advantage for a tumor cell to alter both genes since they act in the same linear signaling pathway, all of them can contribute to the selection of non-eligible patients for treatment with antiEGFR agents. By considering the 40% of KRAS and the 10% of BRAF and NRAS mutated tumors, the selection of non-responders can be improved up to 45 -- 55% [36]. One of the main pathways activated by EGFR is the PI3K/ PTEN/AKT signaling. It can be deregulated, either by the inactivation of the PTEN phosphatase or by activating mutations of the PIK3CA p110 subunit. Mutations in the PIK3CA gene mainly occur in exons 9 and 20 and are found in approximately 15% of mCRC tumors whereas loss of PTEN expression by immunohistochemistry (IHC) is reported in 19 -- 42% [39,40]. The impact of these mutations on response to cetuximab was investigated and conflicting results were reported. In fact in 110 mCRC patients, PIK3CA mutations and PTEN loss were statistically significantly associated with lack of response to cetuximab, lower PFS with poorer OS in tumor carrying PTEN mutation [41]. This negative association with PFS was also confirmed in a second study [42]. By contrast, another study was not supporting a significant association between PIK3CA mutations and lack of response to cetuximab. The role of PIK3CA mutations in resistance to EGFR treatment became more complicated due to the fact that these mutations did not co-occur with KRAS and BRAF mutations, probably identifying another CRC molecular subgroup different from the single mutation ones. The role of PTEN loss and its relationship with response to anti-EGFR-targeted agents is still under investigation. Initial analyses on the impact of PTEN status on response to cetuximab were conflicting [43]. In fact, even if in four studies it has been shown that PTEN-positive tumors have a better outcome than negative ones, two other ones failed to confirm this observation. A possible explanation of this equivocal result is that the alteration of PTEN is currently being evaluated by IHC with lack of standardization in the clinical setting. ErbB2 and IGF-R signaling as an alternative escape mechanism to EGFR

4.2

The identification of plausible mechanism of resistance to anti-EGFR therapies has enriched the population of patients


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Table 1. Markers of resistance to anti-EGFR mAbs. Type of resistance

Gene

Frequency (%)

Primary

KRAS BRAF NRAS PI3KA

30 -- 40 10 -- 15 3 -- 5 15

PTEN ErbB2

19 -- 42 36 -- 40

Secondary

Evidence

Robust Robust Robust Contrasting depend on mutation Contrasting Robust


mAbs: Monoclonal antibodies.

that will benefit from these treatments. When considering the cumulative incidence of KRAS mutations, as well as NRAS, BRAF and possibly PIK3CA, in all mCRC, more than 50% of tumors are expected to be resistant to anti-EGFR therapies (Table 1). Unfortunately, objective response to singleagent anti-EGFR treatment is still confirmed to 10 -- 20% of cases indicating that other mechanisms of resistance rather than genetic mutations are implicated. Therefore, both de novo and acquired resistance mechanisms significantly limit the efficacy of anti-EGFR mAbs in the medical management of mCRC patients. The molecular bases of secondary resistance to cetuximab in CRC are poorly understood. Recently, it was shown that molecular alterations (in most instances point mutations) of KRAS were causally associated with the onset of acquired resistance to anti-EGFR treatment in CRCs [44]. The simplest hypothesis to account for the development of resistance to EGFR blockade is that rare cells with KRAS mutations pre-exist at low levels in tumors with ostensibly WT KRAS genes. Although this hypothesis would seem readily testable, there is no evidence in preclinical models to support it, nor are there data from patients. To test this hypothesis, Diaz et al. [45] determined whether mutant KRAS DNA could be detected in the circulation of 28 patients receiving monotherapy with panitumumab, a therapeutic anti-EGFR antibody. They found that 9 out of 24 (38%) patients whose tumors were initially KRAS WT developed detectable mutations in KRAS in their sera. The appearance of these mutations was very consistent, generally occurring between 5 and 6months following treatment. Mathematical modeling indicated that the mutations were present in expanded subclones before the initiation of mAbs treatment [46]. These results suggest that the emergence of KRAS mutations is a mediator of acquired resistance to EGFR blockade and why solid tumors develop resistance to targeted therapies in a highly reproducible fashion [45]. In this scenario,Bertotti et al. identified HER2 gene amplification as a potential mechanism of resistance to cetuximab in mCRC that harbor normal, WT KRAS/NRAS/BRAF/ PIK3CA genes [47]. In fact, HER2 gene amplification was found to occur in approximately 2% of unselected mCRC, and in a significantly higher frequency in patients with 6

KRAS WT tumors who did not benefit from treatment with anti-EGFR mAbs. The molecular mechanisms that are involved in the primary, de novo resistance to antiEGFR drugs are likely to play a role also in the acquired resistance. In this regard, the findings by Bertotti et al. [47] are in agreement with a recent study showing that activation of HER2 signaling mediates acquired resistance to antiEGFR mAbs in different human cancer cell lines, including CRC [48]. By generating cetuximab-resistant cell lines, Yonesaka et al. [48] identified multiple clones with amplifications of HER2 and subsequent increases in total and phospho-HER2 levels. The depletion of HER2 in the resistant clones restored sensitivity to cetuximab, confirming the importance of HER2 in the resistant phenotype. In addition to HER2 amplification in a subset of cetuximab-resistant clones, the acquired resistance was mediated instead by increased levels of heregulin, a ligand that binds HER3 and HER4. These elevated levels of heregulin resulted in increased association between HER2 and HER3. Both these mechanisms caused acquired resistance to cetuximab treatment of human cancer cell lines by leading to persistent activation of ERK signaling. As a complement to these in vitro results, Yonesaka et al. [48]. were able to demonstrate that alterations in ErbB2 signaling correlate with acquired resistance to cetuximab in a clinical setting. Although the number of analyzed patient samples was limited, amplification of HER2 and increased heregulin levels were observed after patients became non-responsive to cetuximab therapy. Yonesaka et al. also presented more comprehensive clinical data indicating that HER2 amplification and elevated heregulin play a role in de novo resistance as well. In patients with CRC treated with cetuximab, levels of serum heregulin protein and tumor heregulin mRNA, though widely variable, were significantly higher. These higher levels of heregulin appeared to correlate with reduced PFS and OS [48]. The presence of HER2 amplification in a larger patient cohort also correlated with worse OS. Importantly, elevated heregulin or HER2 amplification resulted in worse survival among patients with WT KRAS when examined independently [49]. Finally, the finding that increased heregulin secretion can lead to HER2-mediated resistance suggesting that HER2 gene amplification is not essential and that increased HER2 signaling can induce resistance to anti-EGFR drugs in absence of HER2 gene amplification. Similarly, increased signaling via IGF-1R pathway was shown to be correlated with resistance to anti-EGFR therapy in several cancer types in vitro and in vivo. The role of IGF pathway in the pathogenesis of CRC has been demonstrated in different preclinical studies. The IGF-1R is widely expressed throughout the gastrointestinal tract with the highest expression detected in the proliferating cells at the bases of the colonic crypts, whereas the type II IGF receptor has an unknown physiological role. IGF-1 is a potent mitotic, anti-apoptotic peptide and, in addition, has been demonstrated to promote angiogenesis by increasing the production of VEGF. Significant cross-talk has been observed between



Table 2. mAbs raised against EGFR. Generic name

Characteristics

Cetuximab

Chimeric IgG1

Panitumumab

Fully human IgG2

Matuzumab Nimotuzumab Zalutumumab

Humanized IgG1 Humanized IgG1 Fully human IgG1

Status

Tumors

Approved Phase II -- III Approved Phase I -- III

CRC, SCCHN Pancreatic cancer, NSCLC, breast cancer and other solid tumors CRC NSCLC, rectal cancer, bladder cancer, breast cancer, gastric cancer and ovarian cancer Various solid tumors including NSCLC, cervical cancer and gastric cancer Various solid tumors with a focus in NSCLC SCCHN, NSCLC

Phase II Phase III Phase III


CRC: Colorectal cancer; mAbs: Monoclonal antibodies; NSCLC: Non-small cell lung cancer; SCCHN: Squamous cell carcinoma of the head and neck.

the IGF pathway and several other receptors. There is a crucial interaction between the IGF and EGF pathways. EGF is able to stimulate IGF-2 and vice versa. In addition, EGF can suppress the expression of IGFBP-3 and increase the ability of free IGFs. This interaction provides a rationale for combined therapy against these different pathways with the goal to improve antitumor activity [50]. Several mAbs and small inhibitors of the IGF-R have recently entered into clinical development. IMC-A12, a fully human mAb currently in clinical development, binds to IGF-1R with high affinity. In xenograft tumor models, IMC-A12 results in significant growth inhibitions of breast, pancreatic and colon tumors [50]. The safety and efficacy of IMC-A12 has been evaluated in a Phase II randomized open-label study with or without cetuximab in patients with metastatic refractory anti-EGFR mAbs, CRC [51]. In this trial, IMC-A12, alone or in combination with cetuximab, did not demonstrate meaningful antitumor activity and of 64 patients treated, only 1 patient did achieve a durable partial response (PR) to the combination treatment. The limited clinical efficacy of IMC-A12 in this study suggest that additional preclinical work will be required to identify predictors of IGF-1R dependence in CRCs. Another fully humanized mAb directed against IGF-1R, CP-751,871, is in advanced clinical development. In experimental models, it inhibits IGF-1 binding to cells and IGF-1-induced receptor phosphorylation, and results in down-regulation of IGF-1R expression at the plasma membrane through internalization of the receptor. Inhibition of tumor growth has been documented in multiple xenograft models, and its combination with standard chemotherapeutic agents enhances its antitumor efficacy. The combination of CP-751,871 with 5-fluorouracil (5-FU) in a Colo-205 xenograft model resulted in improved antitumor activity compared with either agent given alone [52]. Ganitumab (AMG 479) is a fully human mAb against the IGF-1R and a Phase II trial has been presented by Eng et al. comparing panitumumab with panitumumab plus ganitumab (AMG 479) and panitumumab plus rilotumumab (AMG 102), an antibody against hepatocyte growth factor (HGF). In preliminary data, ganitumab as single agent and in pretreated patients failed to generate any signal in this randomized trial [53]. In addition, a Phase II with FOLFIRI in

combination with ganitumab versus FOLFIRI in KRAS mutant mCRC is currently in progress. Amphiregulin/epiregulin expression as potential predictive markers

4.3

As mentioned above, patients affected by mCRC that carry the mutated active forms of KRAS, BRAF, NRAS and PIK3CA genes are associated with lack of response to antiEGFR mAbs. Although these markers are good negative predictive factors, still remain unable to selectively identify patients who will benefit from therapy. Thus, the identification of positive predictive factors of response to anti-EGFR therapy would be much more useful than negative predictors in the clinical practice [49]. Khambata-Ford et al. were the first to publish a gene signature obtained from snap-frozen liver metastasis of mCRC patients who were treated with cetuximab as monotherapy [54]. In this signature, two EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), were found among the top genes as predicting the cetuximab response. The predictive value of EREG and AREG expression to cetuximab sensitivity was confirmed by Jacobs et al. analyzing primary CRC formalin-fixed-paraffin embedded (FFPE) tumors from refractory metastatic patients treated with cetuximab-based therapy. In line with these findings, Tabernero et al. found that in mCRC patients receiving first-line cetuximab combined with an irinotecan-based regimen, AREG and EREG expression were elevated in tumors of patients without disease progression, either in the total population or in the KRAS WT tumor subgroup [55,56]. Even if AREG and EREG expression in mCRC WT for KRAS strongly correlated with sensitivity to cetuximab, there are also some KRAS WT tumors with low ligands expression that respond to it but show worse PFS. It is likely that tumors overexpressing these markers are more dependent on EGFR activation and therefore more likely to respond to anti-EGFR mAbs.

New-generation antibodies raised against EGFR

5.

Despite the evidence of efficacy of cetuximab in CRC therapy, in some cases cetuximab infusion can be associated


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with the risk of anaphylactic reactions. This has led to the development of new-generation anti-EGFR antibodies (Table 2). Panitumumab is a fully human anti-EGFR antibody, which binds to EGFR with a high affinity (~ 50 pmol/l) and is able to completely regress certain human xenografts in animal models as a single-agent therapy. It, like cetuximab, competitively inhibits EGFR ligand binding, promotes receptor internalization and prevents tyrosine kinase phosphorylation. Unlike cetuximab, however, panitumumab does not induce receptor degradation on internalization, suggesting that the EGFR may be recycled to the cell surface [57]. As an IgG2 subclass antibody, panitumumab does not mediate a significant level of antibody-dependent cellular cytotoxicity (ADCC) on EGFR-expressing tumor cells. In a multicenter, open-label, single-arm study, a total of 148 CRC patients were grouped into two cohorts: cohort A with higher EGFR staining intensity (2+ or 3+ in > 10% evaluated tumor cells (104 patients)) and cohort B with lower EGFR staining intensity (1+ or 2+ or 3+ in < 10% evaluated tumor cells (44 patients)). The antibody was well tolerated, with the major toxicity, skin rash (including 3% grade 3), occurring in 95% of patients. Interim analysis showed PR in 11% of cohort A patients and 9% of cohort B patients; the median OS time was 7.9 months [58]. As seen in the cetuximab trials, there seems to be a direct correlation between tumor response and skin rash, but not between tumor response and EGFR staining intensity in the tumors, in panitumumab-treated patients. Panitumumab (Amgen Co., CA, USA) was approved in 2006 for the treatment of patients with EGFR expressing metastatic CRC with disease progression on or following fluoropyrimidine-, oxaliplatin- and irinotecan-containing chemotherapy regimens. In the pivotal Phase III trial that led to its approval, panitumumab treatment significantly improved PFS and resulted in an 8% ORR [59]. In 2007, the Panitumumab Advanced Colorectal Cancer Evaluation (PACCE) trial, a Phase IIIb randomized, open-label clinical trial evaluating oxaliplatin- and irinotecan-based chemotherapy and bevacizumab with and without panitumumab in the first-line treatment of patients with mCRC had been discontinued. The trial enrolled 1054 patients (824 patients were randomized to receive oxaliplatin-based chemotherapy and 230 patients were randomized to receive irinotecan-based chemotherapy). A preplanned interim efficacy analysis scheduled after the first 231 events (death or disease progression) revealed a statistically significant difference in PFS in favor of the control arm (bevacizumab plus chemotherapy). An unplanned analysis of OS also demonstrated a statistically significant difference favoring the control arm. In addition, increased incidence of pulmonary embolism was observed in patients who received panitumumab compared with those who did not (4 and 2%, respectively) [60]. While the exact explanation for the negative interaction between EGFR and VEGF inhibitors is unknown, several hypotheses can be postulated. Although pharmacokinetic interactions between 8

antibodies or between antibodies and chemotherapy are uncommon, we cannot exclude this possibility. Potentially, a pharmacodynamic interaction induced by EGFR inhibition could have led to a blunting of the therapeutic effects of bevacizumab and/or chemotherapy. Possible mechanisms include EGFR-mediated alterations of downstream targets required for the activity of bevacizumab and/or chemotherapy or the induction of EGFR-mediated cell-cycle arrest leading to resistance to cytotoxics. Amado et al. first demonstrated that the response to panitumumab monotherapy and the improvement in PFS was limited only to patients with WT KRAS tumors. No patient harboring a KRAS mutation (46%) responded to panitumumab. Recently, analysis of three large randomized trials, the OPUS, the CRYSTAL and the PRIME (the Panitumumab Randomized Trial in Combination with Chemotherapy for Metastatic Colorectal Cancer to Determine the Efficacy), showed these data: when administered in first line, cetuximab and panitumumab, either in combination with an oxaliplatin-based or an irinotecan-based chemotherapy, result to be effective only in KRAS WT mCRC patients [28]. Nimotuzumab (YM Biosciences, Mississauga, ON, Canada, and Center of Molecular Immunology, Havana, Cuba) is a humanized IgG1 form of the murine IgG2a antibody R3 specific for EGFR. It binds to the EGFR extracellular domain with a moderate affinity (about 1 nmol/l), blocks EGF binding to its receptor- and ligand-dependent receptor autophosphorylation, and inhibits cell growth in EGFRexpressing cells [61]. Studies have shown that the antitumor effect of nimotuzumab may result from its combined effects on tumor cell proliferation, survival and angiogenesis [62]. Multiple clinical trials are currently being conducted to examine the therapeutic efficacy of the antibody in a number of cancers, including a Phase III trial in pediatric pontine glioma and Phase II studies in patients with carcinomas of the pancreas, esophagus and stomach, cervix and hormone-refractory prostate cancer [63]. The antibody was very well tolerated without grade 3/4 adverse events. None of the patients developed acneiform rash or allergic reactions. Nimotuzumab is also being tested in combination with radiotherapy. The combination therapy was well tolerated. Aside from infusion reactions, no skin or allergic toxicities were observed. OS was significantly increased after the use of the higher antibody doses [64]. Matuzumab is a humanized anti-EGFR antibody (Merck KGA, Germany) that has demonstrated antitumor activity in preclinical tumor models both as a single agent and in combination with chemotherapy and radiation [65]. Furthermore, as an IgG1 antibody, matuzumab induced potent ADCC against tumor cells in vivo, which distinguishes matuzumab from the recently approved panitumumab. To date, over 320 patients have been treated with the antibody and have tolerated it well, with the most commonly reported toxicities being skin rash, fever and headache. Clinical trials are also



Cetuximab; panitumumab

1) Target prioritization

3) Nanotechnologies EGF

Cytotoxic agents IFNα EGFR IFNR

Up-regulation Jak1

Tyk2 PI3K AKT


RAS RAF Stat

Jnk1

p38

PTEN

EGFR

MAPK

Growth inhibition apoptosis

MHC and peptide

EGFR NK cell

Anti-CTLA-4 HSP T cell

FC receptor

Complement

Dentritic cell 2) Combination with immune system

Figure 2. New strategies to overcome the mechanisms of cancer cell resistance. In order to overcome the mechanisms of cancer cell resistance, there is a need for the development of new strategies such as cancer cell prioritization (through upregulation and hyperactivation of EGF- and Ras-dependent MAPK cascade) (1), combination with immune system (monoclonal antibodies act directly on immune system inducing CDC, ADCC, T cells activation and antibodymediated cross-presentation of antigen to dendritic cells, inhibition of T-cell inhibitory receptors, such as cytotoxic T lymphocyte-associated antigen 4, CTLA4) (2), development of nanotech devices (3) to increase efficacy of antiEGFR antibodies.

being carried out to investigate the antitumor activity of matuzumab in combination with chemotherapy. Although results from several Phase II trials are expected, initial Phase I trials have shown matuzumab is well tolerated, with rash and diarrhea the most common toxicities. In Phase I trials, activity has been evaluated in colorectal, cervical and esophageal cancers and in squamous cell cancer of the head and neck. Current therapeutic targets in Phase II trials include cervical and gastric cancers, and non-small cell lung cancer (NSCLC) [66]. Zalutuzumab (Genmab A/S, Copenhagen, Denmark) is a human IgG1 anti-EGFR mAb, which blocks the binding of growth factors to tumor cells, inhibits phosphorylation of EGFR and cell proliferation and causes tumor cell killing by ADCC, and directly slows the rate of tumor growth [67]. In animal studies, complete eradication of tumors was observed

between 9 and 14 days after three injections of the antibody. A recent mechanistic study showed that the antibody locks the EGFR into an inactive configuration, preventing the growth factor from binding and the subsequent receptor dimerization and activation. In an open-label, randomized Phase III trial, zalutumumab did not increase OS but PFS was extended in patients with recurrent squamous cell carcinoma of the head and neck who had failed platinum-based chemotherapy [67]. The most common adverse events included rigors, fatigue, pyrexia, nausea, flushing and increased sweating. Nanotechnologies for EGFR targeting Nanoparticulate delivery systems equipped with anti-EGFR antibodies and loaded with therapeutic molecules have been widely used in literature for their biocompatibility and stability. 5.1


9


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Acharya et al. showed by fluorescence spectroscopy that nanoparticles (NP) formed from poly(lactide-co-glycolide) acid (PLGA) and equipped with mAb directed to the extracellular domain of EGFR, containing a fluorescence dye accumulated 13 times more compared with the non-targeted NP in MCF7 cell line [68]. Multiple cell lines with different EGFR expression levels were tested for gold nanoparticles (Au NP) accumulation. Cellular accumulation of the loaded particles was positively correlated to EGFR density. Moreover, in vivo effects of drug-loaded EGFR-targeted particles were superior in comparison with non-targeted particles. In addition to drug-loaded tumor-targeted nanoparticulate systems, immunoconjugates and immunotoxins have also been used as cellular-directed therapeutics in targeting ErbB receptor positive tumor cells. For instance, scFv (single-chain antibody fragment) antibody fragments directed to ErbB1 were fused with a fragment of a bacterial exotoxin A (ETA) to generate a scFv antibody--toxin construct. Michaelis et al. studied the effects of the scFv--ETA immunotoxin on neuroblastoma cell lines insensitive to ErbB receptor tyrosine kinase inhibitors or mAbs and showed that immunotoxins administered together with cisplatin decreased the viability of cells in comparison with single administration of each [69]. Nanobodies ErbB receptor targeting with nanobodies is still a developing area and not many studies have been conducted yet. Nanobody is the smallest antigen-binding fragment of heavy chain antibodies. Tijing et al., showed a positive correlation between nanobody uptake and ErbB receptor density in three model cell lines expressing high, moderate and low amounts of ErbB receptors (expression) [70]. This study also demonstrated that nanobodies are able to accumulate in solid tumors but experience a disadvantage in vivo since they are cleared rapidly from the blood circulation before reaching their target. A strategy to prevent the rapid clearance of heavy chain antibodies from the blood is based on the use of Llama heavychain antibodies consisting of three domains that have been recombinantly fused. The resulting proteins contain one albumin and two EGFR binding units (aEGFR--aEGFR--aAlb). Besides improved pharmacokinetics and tumor accumulation, the albumin-binding nanobody derivatives were also successful in delaying tumor growth [70]. 5.2

the nanobodies. In addition, chimeric antibodies -- bivalent nanobodies fused with the human Fc fragment -- have also been designed. Bell et al. [71] compared monovalent nanobodies (15 kDa), pentabodies (120 kDa) and chimeric antibodies (80 kDa) in terms of affinity, retention time in the body and targeting to tumor tissues. In this study, the half-life of the pentabody increased two- to four-fold compared with monovalent nanobody and the retention in the tumor was similar for both. Currently, there are two studies where anti-ErbB1 nanobodies are coupled to liposomes and polymeric micelles for tumor targeting and down-regulation of ErbB receptor pathways. Remarkably, nanobody-liposomes induced up to 90% ErbB1 internalization which subsequently led to receptor down-regulation. The ability of the liposomal construct to down-regulate the ErbB1 pathway is promising for further studies, because the drug carrier system has an intrinsic therapeutic activity [72]. 6.

Significant steps toward personalized treatment of patients with mCRC have been taken in the past few years. The demonstration that patients who carry mutations of the KRAS gene do not benefit of treatment with anti-EGFR agents represented an important innovation for medical oncology [24]. However, an increasing number of molecular alterations have been more recently hypothesized to be involved in resistance to anti-EGFR drugs in CRC: mutations in BRAF, NRAS and PIK3CA, loss of expression of PTEN and, now, activation of HER2 signaling through HER2 gene amplification and/or increased heregulin stimulation. These findings suggest that the resistance to anti-EGFR agents involves a complex network of molecular alterations that seems to be in most of the cases mutually exclusive, although it cannot be excluded that subclones of tumor cells with different molecular features might co-exist in the same tumor, and represent the ‘seed’ for the development of anti-EGFR treatment resistance. The complexity and the heterogeneity of the molecular alterations that are being identified as resistance mechanisms to anti-EGFR therapies suggest that a comprehensive molecular characterization of mCRC will likely be necessary in the next future in order to choose the most appropriate therapy for each individual patient. 7.

Bivalent antibodies, pentabodies and chimeric antibodies

Conclusion

Expert opinion

5.3

The terms bivalent and bispecific point out the existence of two domains of functional nanobodies in one construct. In the first construct, there are two of the same functional domains and in the latter there are two domains with different specificities. Similarly, pentabodies [71] and other multivalent nanobodies were engineered to increase the affinity and size of 10

The identification of ErbB2 as a mediator of de novo and acquired cetuximab resistance suggests that blockade of HER2 might prevent or revert resistance to anti-EGFR mAbs in selected patients. Indeed, combinations of different agents, which selectively target the EGFR (cetuximab or gefitinib) and HER2 (lapatinib or pertuzumab) were able to significantly inhibit the growth of cetuximab-resistant CRC cells in experimental models [47,48]. Whether a strategy of




combined EGFR and ErbB2 inhibition will be successful in malignancies amenable to EGFR inhibition remains to be seen. Another therapeutic strategy that could be used to treat CRC resistant to anti-EGFR agents might be represented by combinations of anti-EGFR drugs with inhibitors of intracellular signaling pathways. In this regard, the study by Yonesaka et al. indicated in ERK signaling a main pathway involved in HER2-induced acquired resistance to cetuximab [48]. In this respect, a potentially effective combination could be the use of anti-EGFR drugs and MEK inhibitors. Recently, it has been demonstrated that MEK inhibitor PD0325901 inhibits proliferation of BRAF V600E, KRAS/BRAF WT and KRAS mutant cancer cells. On the other hand, MEK inhibitors as single agents are unlikely to determine a significant antitumor effect mostly for the activation of compensatory signaling loops between the MEK/ERK and the PI3K/AKT pathways. A more effective therapeutic strategy could be the combined blockade of both the MEK/ERK and the PI3K/AKT pathways. Cancer cell prioritization A new strategy to overcome the mechanisms of cancer cell resistance could be cancer cell prioritization (Figure 2). In fact, it has been reported that tumor cells exposed to IFN-a undergo a prioritization of the target (through up-regulation of EGFR) which makes them more susceptible to inhibitors. IFN-a and other anti-proliferative agents, such as cytosine arabinoside, 5-aza-2¢-deoxycytidine and 8-chloro-cAMP (8ClcAMP), increase the expression and function of the EGFR at the surface of human cancer cells and hyperactivate EGF- and Ras-dependent MAPK pathway [73-75]. On the basis of these findings, it has been hypothesized that increased EGFR expression and function could be part of an inducible survival pathway, which is activated in the tumor cells by the exposure to IFN-a or other cytotoxic agents. Moreover, the up-regulation of growth factor receptors could be a common event in growth inhibited tumor cells and could represent a protective response toward the antiproliferative stimuli. It has been described that the selective targeting of these hyperactivated survival pathways by the combination of IFN-a with selective EGFR inhibitor (gefitinib) or the farnesyl-transferase inhibitor R115777 may enhance the antitumor activity of these drugs in cancer cells. In fact, tumor cells exposed to IFN-a become highly sensitive to specific signaling inhibitors (‘target prioritization’), avoiding the need of a wide inhibition of multiple survival signals. In other words, by treating the cells with IFN-a, we could mimic the effect of the kinase domain mutations of EGFR described in different types of cancer [75]. Mutated EGFR showed enhanced tyrosine kinase activity in response to EGF and increased sensitivity to inhibition by gefitinib or other EGFR inhibitors. 7.1

Combination with immune system Immune resistance, like drug and radio resistance, may depend on the degree of cancer cell heterogeneity and thus 7.2

on tumor burden. One possibility to overcome this type of resistance is to combine the immunotherapy with radiotherapy and/or chemotherapy and simultaneously affect the phenotype of tumor cells, thus making them more susceptible to the therapy (Figure 2). The authors and others have previously described the ability of cytotoxic drugs such as triazenes, 5-FU, VP-16, CPT-11, to sensitize tumor cells to the cytolytic activity of Ag-specific cytotoxic T lymphocytes (CTLs). Similarly, several heat shock proteins released or extracted by tumor cells can deliver multiple tumor cell Ag epitopes directly to DCs expressing the specific receptors [76-78]. In this contest, Basu et al. [79] have shown that the occurrence of necrosis is indispensable to obtain a sufficient release of heat shock proteins (including the HSP-90, HSP-96 and HSP-70), which in turn deliver a partial maturation signal into the DCs. Therefore, the results of the latter study could suggest that the immune adjuvant property of GOLF (Gemcitabine, Oxaliplatin, 5-Fluorouracil and Leucovorin) regimen is dependent on its ability to induce in the tumor cells either necrosis and apoptosis. This multiple modality of killing may, in fact, provide a stronger danger signal to either dendritic cells (DCs) or lymphocytes precursors giving rise to a more efficient CTL response. Recently, it has been shown that the antitumoral effects of mAb may be due to their ability to act directly on the immune system (Figure 2) [76,80]. In general terms, the use of mouse chimeric antibodies may elicit immune responses specifically for the mouse portion of the molecule leading to destruction of the antibody; in some cases, however, also leading to the destruction of targeted tumoral cells [80]. In addition, several reports have described, both in vitro and in vivo, how these antibodies are able to elicit ADCC, complement-mediated cytotoxicity (CMC) or both. These effector responses are due to the binding of the Fc portion of antibodies to the Fc receptors expressed on the surface of different cell types. This binding leads to a wide array of effects, from uptake to killing. It should be noted that macrophages, DCs, neutrophils, eosinophils, B cells, mast cells, natural killer (NK) cells, platelets and Langerhans cells express Fc receptors capable of discriminating different Ig classes. The effects of ligation of the Fc portion of the antibody with the Fc receptors on the cells depend on the specificity of the Fc receptors for a given Ig class and on the cell types. Finally, it should be remembered that the ability to interfere with the signaling initiated by EGFR affects many other receptor systems, for instance, chemokine and cytokine receptors, Toll-like receptors, that are critical for immune responses [80]. Furthermore, a better understanding of the interactions between the mAb used as therapy for solid tumors and the immune system could be critical in designing new approaches for immunotherapy in cancer. Nanotechnologies Researchers are investigating NPs to overcome the limitations of mAbs and immunoconjugates. Furthermore, in some cases, 7.3


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NPs can be designed in such a way as to protect the therapeutic agent from premature release and degradation. Additionally, since NPs can be functionalized with multiple therapeutic agents, they may be able to circumvent multidrug resistance in tumors (Figure 2). Finally, because solid tumors have an increased level of angiogenesis and leaky vasculature, mAb-targeted nanoconjugates may preferentially accumulate in tumors allowing for more effective ‘passive’ drug delivery [81]. Indeed, many mAb-targeted NP platforms have been investigated, including those derived from liposomes, micelles, polymers, metals and non-metals. Results from both in vitro and in vivo studies have generally been promising. However, several challenges still remain before mAbtargeted NPs can be effectively utilized as drug delivery platforms to treat cancer in humans. First, the molecular characterization of the multitude of different tumors must continue to be undertaken in order to effectively pinpoint cancer-specific targets for future mAbs. Also, new methods of conjugation of mAbs and chemotherapeutic agents to NPs must continue to be developed in order to facilitate coupling that maintains the potency of drug delivery Bibliography

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Acknowledgements FC and MC were supported by a grant from Italian Association for Cancer Research. MC received financial support from Italian Ministry of Education and University (PRIN 2009) and from Regione Campania for project Laboratori Pubblici ‘Hauteville’. T Troiani and S Zappavigna these two authors equally contributed to the manuscript.

Declaration of interest F Ciardiello and M Caraglia were supported by a grant from Italian Association for Cancer Research. M Caraglia received financial support from Italian Ministry of Education and University (PRIN 2009) and from Regione Campania for project Laboratori Pubblici “Hauteville”. The authors state no other conflict of interest and have received no payment in preparation of this manuscript.

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Affiliation Teresa Troiani1, Silvia Zappavigna2, Erika Martinelli1, Santolo R. Addeo2, Paola Stiuso2, Fortunato Ciardiello1 & Michele Caraglia†2 † Author for correspondence 1 Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale F. Magrassi e A. Lanzara, Seconda Universita` degli Studi di Napoli, Via S. Pansini 5, 80131 Napoli, Italia 2 Second University of Naples, Department of Biochemistry, Biophysics and General Pathology, Via Costantinopoli, 16 80138 Naples, Italy Tel: +00390815665871; Fax: +00390815665863; E-mail: [email protected]

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