Accepted Manuscript Title: Adaptive Responses to Antibody Based Therapy Author: Tamara Rodems Mari Iida Toni M. Brand Hannah Pearson Rachel Orbuch Bailey Flanigan Deric L. Wheeler PII: DOI: Reference:

S1084-9521(16)30001-5 http://dx.doi.org/doi:10.1016/j.semcdb.2016.01.001 YSCDB 1912

To appear in:

Seminars in Cell & Developmental Biology

Received date: Revised date: Accepted date:

12-11-2015 5-1-2016 5-1-2016

Please cite this article as: Rodems Tamara, Iida Mari, Brand Toni M, Pearson Hannah, Orbuch Rachel, Flanigan Bailey, Wheeler Deric L.Adaptive Responses to Antibody Based Therapy.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Adaptive Responses to Antibody Based Therapy Tamara Rodems1, Mari Iida1, Toni M. Brand1, Hannah Pearson1, Rachel Orbuch1, Bailey Flanigan1and Deric L. Wheeler1,2 1

Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, USA 2

Corresponding author: Deric L Wheeler PhD, Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, 1111 Highland Avenue, WIMR 3159, Madison, Wisconsin 53705. Phone: (608) 262-7837; fax: (608) 263-9947; e-mail: [email protected]. Running Title: Adaptive responses to antibodies

AUTHORS INFORMATION Tamara Rodems Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email: [email protected] Mari Iida Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email : [email protected] Toni M. Brand Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email : [email protected] Hannah Pearson

1

Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email: [email protected] Rachel Orbuch Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email : [email protected] Bailey Flanigan Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3136 Madison, Wisconsin 53705 Phone: (608) 265-5446 Email : [email protected] Deric L. Wheeler Department of Human Oncology University of Wisconsin Comprehensive Cancer Center 1111 Highland Avenue WIMR 3159 Madison, Wisconsin 53705. Phone: (608) 262-7837; fax: (608) 263-9947 Email: [email protected]

2

ABSTRACT Receptor tyrosine kinases (RTKs) represent a large class of protein kinases that span the cellular membrane. There are 58 human RTKs identified which are grouped into 20 distinct families based upon their ligand binding, sequence homology and structure. They are controlled by ligand binding which activates intrinsic tyrosine-kinase activity. This activity leads to the phosphorylation of distinct tyrosines on the cytoplasmic tail, leading to the activation of cell signaling cascades. These signaling cascades ultimately regulate cellular proliferation, apopotosis, migration, survival and homeostasis of the cell. The vast majority of RTKs have been directly tied to the etiology and progression of cancer. Thus, using antibodies to target RTKs as a cancer therapeutic strategy has been intensely pursued. Although antibodies against the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) have shown promise in the clinical arena, the development of both intrinsic and acquired resistance to antibody-based therapies is now well appreciated. In this Review we provide an overview of the RTK family, the biology of EGFR and HER2, as well as an in-depth review of the adaptive responses undertaken by cells in response to antibody based therapies directed against these receptors. A greater understanding of these mechanisms and their relevance in human models will lead to molecular insights in overcoming and circumventing resistance to antibody based therapy.

Abbreviations: CBL, casitas B-lineage lymphoma; CtxR, cetuximab-resistant; CtxS, cetuximabsensitive; CRC, colorectal cancer; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EMT, epithelial to mesenchymal transition; EPR, epiregulin; FDA, food and drug administration; HB-EGF, heparin-binding epidermal growth factor; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; HNSCC, head and neck squamous cell carcinoma; IGF-1R, insulin-like growth factor receptor 1; IgG, immunoglobulin G, ILK, integrin linked kinase; mAb, monoclonal antibody; MAPK, mitogenactivated protein kinase; MDGI, mammary-derived growth inhibitor; NSCLC, non-small cell lung cancer; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homologue; RALT, receptor-associated late transducer; RTK, receptor tyrosine kinase; SFKs, Src-family kinases; STAT, signal transducer and activator of transcription; TGF-, transforming growth factor receptor alpha; TGF-, transforming growth factor receptor beta; TKD, tyrosine kinase domain; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Key Words: EGFR, MET, HER2, antibodies, therapeutic resistance, receptor tyrosine kinases.

3

Introduction The therapeutic use of antibodies dates back to the late 1800s when patients were injected with the sera of immunized animals for the treatment of diphtheria and other infectious diseases1. Over the last 40 years, significant strides have been taken to improve antibody therapy, beginning with the identification of tumor-specific targets and development of monoclonal antibodies (mAbs) from hybridomas2. In early development, murine mAbs produced from hybridoma technology were developed for clinical use, with OKT3 being the first murine mAbs approved by the food and drug administration (FDA) in 1986 to reduce organ transplant rejection3. In an effort to abrogate the negative side effects of treating humans with murine mAbs, chimeric antibodies were generated, which contained the murine antigen-specific variable domain fused to a human constant domain4. Taking this concept one step further led to humanized antibodies, which contain only a small portion of the murine variable domain – the complementarity-determining region – fused to a human antibody scaffold5. Humanization provided a significant step forward in antibody-based therapy due to the diminished immunogenicity and increased effector function of human antibodies. Advances such as phage display technology have brought antibody therapy development to the forefront, allowing antibodies to be screened and chosen from libraries and giving researchers the power to select, design, and optimize antibodies in a similar fashion to traditional small molecule drug discovery6-10. These technologies have been utilized in the clinic to treat a variety of conditions including cardiovascular disease, tissue transplant rejection, arthritis, and cancer. In the last few decades, antibody therapeutics has been focused on the treatment of both solid and hematological malignancies. Several antibodies have been granted FDA approval with many more in early preclinical development (Table 1). The advantages of therapeutic antibodies

4

over traditional small molecules or chemotherapeutics include increased specificity, lower toxicity, and the ability to deliver antibody-conjugated drugs. Another advantage of antibody therapy in cancer is the range of mechanisms by which an antibody can elicit a therapeutic response. Mechanisms of action include receptor blockade, agonist function, signal abrogation, immune response regulation, and modulation of the tumor microenvironment. Despite these advantages, however, a constant barrier in the progression of targeted therapies has been the rapid development of therapeutic resistance by tumor cells in response to antibody treatment. Tumor cells have demonstrated the ability to escape the effect of antibody therapies, regardless of the mechanism of action of the antibody. Known escape mechanisms include target antigen mutation, downregulation, alternative signaling pathway activation, immune suppression, and changes in tumor vascularity. Particular emphasis in recent years has been placed on developing strategies to overcome resistance to antibody therapies as well as understanding the biological significance of the escape mechanisms employed by resistant tumor cells. In this review, we focus on adaptive mechanisms of resistance to therapeutic antibodies against one of the most targeted class of proteins, the RTKs. We discuss the role of EGFR and HER2 in cell biology and cancer as well as the development of therapeutic antibodies against these receptors. We also provide an in-depth discussion of the adaptive responses of cells that lead to resistance to these antibody therapies.

Receptor Tyrosine Kinases as Targets in Cancer In 1962, Stanley Cohen characterized a salivary gland protein that induced eyelidopening and stimulated the proliferation of epithelial cells. This protein was thus named the epidermal growth factor (EGF)11,

12

. In subsequent years, Graham Carpenter identified the

5

presence of specific binding receptors for EGF on target cells.

In 1975, Carpenter and

colleagues identified EGFR as a 170Kda membrane protein that increased

32

P incorporation in

response to EGF treatment of A431 epidermoid carcinoma cells13,14. In 1984, a group of collaborators isolated, cloned and characterized the sequence of the human EGFR from normal placental cells and A431 tumor cells15. Around this time it was also discovered that modification of proteins by phosphorylation on tyrosine residues might be a critical step in tumorigenesis16, 17. Shortly after these discoveries, EGFR was recognized as a receptor protein tyrosine kinase. This two-decade effort led to the identification of the prototypical RTK and its ligand.

The

identification of the EGFR as an RTK contributed to pivotal studies advancing our understanding of RTK activation18, 19. Following these early discoveries, several additional RTKs have been identified and characterized. Upon completion of the human genome project, a broader appreciation of RTKs began to emerge. Early classification of the family had identified 58 human RTKs that fall into 20 subfamilies based on the ligands they bind, their sequence homology, and their structures. The architecture of all RTKs is highly conserved from the nematode Caenorhabditis elegans, with an extracellular ligand-binding domain, a single transmembrane α-helix, an intracellular tyrosine kinase domain, and a tyrosine-rich C-terminal tail. RTKs are activated upon the binding of ligand, which lead to receptor homo- or hetero-dimerization, kinase domain activation, and subsequent phosphorylation of tyrosine residues located within the cytoplasmic tail. Phosphorylated tyrosines serve as docking sites for a variety of intracellular adaptors and effector enzymes that transmit signals to the cytoplasm and nucleus, resulting in changes in cell function or fate. The importance of many RTKs in mammalian development has been displayed through the study of mice with genetically altered RTKs, resulting in either severe

6

developmental abnormalities or embryonic lethality. Collectively, RTK activation of signaling networks provide an essential mechanism by which cells communicate to regulate a multitude of different cellular responses. RTKs regulate both developmental and regulatory cellular processes, and thus, abnormalities in RTK structure or activity can result in human disease. Several diseases result from RTK point mutations, partial deletions, constitutive activation and/or overexpression (often due to gene amplification) as well as autocrine ligand-receptor stimulations20. Some examples of these diseases include diabetes, inflammation, arteriosclerosis, angiogenesis, autoimmune disorders, skeletal diseases, and cancer. Due to this causal relationship, therapeutic targeting of specific RTKs is clinically approved for treatment of several human cancers. Despite the clinical success these therapies, intrinsic and acquired resistance to antibody-based therapies targeting RTKs has been observed in many cellular systems of human cancer. Perhaps the most well studied aspect of RTKs has been their signaling pathways. RTKs and their pathways represent some of the most prevalent mechanisms of tumor survival, proliferation, and progression. As such, they are largely agreed to be a promising platform for targeted cancer therapy despite the issue of therapeutic resistance. The combined effort to understand RTK targeting and subsequent therapeutic resistance may prove beneficial for cancer patients. Ongoing intensive research is being performed to determine methods of attenuating resistance to allow full therapeutic response of RTK targeted antibodies in cancer patients. EGFR Biology. The human epidermal growth factor receptor (HER) family is comprised of four transmembrane type I growth factor receptors: EGFR (HER1, ErbB1), HER2 (HER2/neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR, often labeled as the prototypical RTK, is the

7

most studied and best-understood HER family member because it was the first RTK discovered. These receptors contain an extracellular ligand-binding domain (domains I, II, III, IV), a single membrane-spanning region, a juxtamembrane nuclear localization signal (NLS), a cytoplasmic tyrosine kinase domain (TKD) and a C-terminal tail which houses several tyrosine residues for propagating downstream signaling21, 22. Currently, seven ligands are known to bind and activate the EGFR including epidermal growth factor (EGF), transforming growth factor alpha (TGF), heparin-binding EGFR-like growth factor (HB-EGF), amphiregulin, betacellulin, epigen and epiregulin23. Ligand binding to leucine-rich repeats in domains I and III of the EGFR extracellular domain triggers a conformational change in the receptor that exposes the dimerization loop (domain II) to other receptors on the cell surface24. Exposure of domain II allows for homo- or hetero-dimerization with other HER family members, activating EGFR kinase function. Receptor dimerization then allows several C-terminal tyrosine residues to be phosphorylated, resulting in the activation of a variety of downstream signaling cascades25. The major signaling pathways activated by EGFR include the RAS/RAF/MAPK, PI3K/AKT, and JAK/STAT pathways22. EGFR signaling is regulated mainly by receptor internalization and subsequent degradation or recycling in order to maintain normal cellular growth, usually carried out by the E3-ubiquitin ligase CBL26, 27.

EGFR and its role in cancer. The signaling cascades activated by EGFR typically result in increased survival, proliferation, and differentiation and are therefore important in development as well as normal cellular growth. However, overexpression or hyper-activation of the receptor and its subsequent pathways is frequently linked to cancer. EGFR is found to be upregulated in nearly all types of human tumors and associated with more aggressive tumor phenotypes21.

8

EGFR protein levels and gene amplification are found to be elevated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and nearly half of all head and neck squamous cell carcinoma (HNSCC). Furthermore, breast, ovarian, cervical, bladder, pancreatic, gastric, and brain cancer have been associated with dysregulated EGFR expression28-30. In many of these cancers, EGFR overexpression is associated with decreased overall survival rates31. Due to the prominent role of EGFR in the tumor biology of many cancers, several independent approaches have been taken to develop therapeutic antibodies targeting the EGFR.

Therapeutic antibodies targeting the EGFR. There are currently two FDA approved antibody therapies targeting the EGFR and many others under review or in clinical trials. The first, and perhaps the hallmark of EGFR-targeted therapy, is cetuximab (Erbitux), a chimeric IgG1 antibody targeted to the EGFR and currently approved for use in metastatic CRC and HNSCC. Cetuximab functions by binding competitively with EGFR preventing ligand stimulation and ultimately downstream signal activation32, 33. Along with the shutdown of downstream signaling, cetuximab causes increased receptor internalization and downregulation32. With reduced receptor availability, signals directing the cell to senesce or go through apoptosis dominate and tumor shrinkage is observed. The second EGFR-targeted antibody approved by the FDA is panitumumab. Panitumumab is a fully human IgG2 antibody approved for the treatment of wildtype K-RAS metastatic CRC. Panitumumab works in a similar fashion to cetuximab in that it binds EGFR causing reduced proliferation and increased apoptosis34. Also necitumumab, a fully human IgG1 EGFR targeted antibody for the treatment of NSCLC is currently in review for FDA approval35. Several other EGFR targeted antibodies are in clinical trials for the treatment of HNSCC, CRC, gastric cancers, glioblastoma, and triple negative breast cancer (TNBC).

9

Adaptive Responses of antibody-based targeting of the EGFR. Despite positive clinical evidence, resistance to EGFR-targeted antibodies has consistently been observed in vitro, in vivo, as well as in the clinic and spans a wide range of mechanisms. Studies on cetuximab and panitumumab resistance have led to new combinations of therapies in the clinic as well as insight into novel pathways and mechanisms in EGFR biology36.

Angiogenesis. In 2001, Viloria-Petit et al. showed that increased vascular endothelial growth factor (VEGF) expression was present in variants of an epidermoid carcinoma cell line, A431, which demonstrated resistance to cetuximab treatment in vivo. These resistant variants showed increased angiogenic potential due to increased VEGF production and faster in vitro growth rate compared to the parent A431 cetuximab-sensitive (CtxS) cell line37. Additionally, resistant variants showed enlarged blood vessel formation in tumors in vivo, which is characteristic of neoangiogenesis37. While this study showed an increase in VEGF production in CtxR tumors, the connection between VEGF and treatment resistance was not immediately clear. Further studies have elucidated possible mechanisms by which EGFR inhibition and subsequent acquired resistance promotes increased angiogenic potential. Ciardiello et al. saw increased VEGF production in CtxR variants of human GEO colon cancer xenografts. In addition to increased VEGF, COX-2 and phospho-MAPK expression also increased compared to parental lines38. COX-2 has been shown to support neovascularization and neoangiogenesis39, 40 and both COX-2 and MAPK have been shown to cause upregulation of VEGF39, 41. EGFR activation of MAPK and the subsequent promotion of transcription and stabilization of COX-2

10

mRNA by active MAPK provides a possible connection between acquired resistance to antiEGFR therapy and increased VEGF production42. Further evidence for the possible involvement of angiogenic factors in cetuximab resistance comes from the reported success of targeting EGFR along with vascular endothelial growth factor receptor (VEGFR). Jung et al. showed in 2002 that the combination of cetuximab and DC101, a VEGFR-2-targeted antibody, was more effective in reducing the growth of gastric tumors than either treatment alone and was able to overcome cetuximab resistance in vivo

43

.

Similarly, Ciardiello et al. showed that GEO colon cancer xenografts treated with ZD6474, an inhibitor of VEGFR that also has anti-EGFR activity, do not show any acquired resistance unlike those treated with cetuximab38. Overall, these findings support a role for angiogenesis in resistance to EGFR blockade.

Altered Degradation and Internalization of the EGFR. EGFR degradation and internalization typically occurs following receptor activation and is mediated in part by the E3-ubquitin ligase CBL. Dysregulation of this process has contributed to resistance to EGFR-targeted therapies, particularly in resistance to cetuximab. Friedman et al reported in 2005 that combinations of antiEGFR mAbs down-regulate EGFR better than each mAb alone in human KB cells. They also observed that down-regulation of EGFR by the combination of the antibodies was not only more extensive than each Ab alone, but also evolved more rapidly44. In 2007, Lu et al. found that a colon cancer cell line, DiFi, and its CtxR counterpart, DiFi5, had disparate levels of EGFR and CBL association. When the receptors were co-immunoprecipitated, the resistant DiFi5 cells showed increased association between EGFR and CBL compared to DiFi cells. The resistant cells also showed increased ubiquitination and decreased expression of the EGFR protein. This

11

suggests a role for downregulation of EGFR by increased internalization and degradation in cetuximab resistance45. Alternatively, Wheeler et al. found that CtxR HNSCC and NSCLC cell lines did not internalize EGFR upon EGF stimulation, and instead saw an increase in EGFR protein expression in resistant cell lines. This was visualized by immunofluorescent staining where resistant cells showed an accumulation of EGFR at the plasma membrane with no endocytic EGFR, while parental cells showed a lower amount of surface EGFR and an accumulation of endocytic EGFR. This study also revealed through co-immunoprecipitation that EGFR in the resistant cell lines did not recruit CBL46. In 2009, Nevo et al. demonstrated another mechanism of dysfunctional EGFR internalization in breast cancer cells. MDA-MB-231 cells were made to express GFP only or GFP plus mammary derived growth inhibitor (MDGI). MDGI was not present in cultured cells, but had been found in primary cancer cultures and patient tumors. MDGI-expressing cells are resistant to cetuximab and showed increased intracellular EGFR and decreased surface EGFR compared to GFP only cells. The internalized EGFR accumulated, but was not degraded and could still be phosphorylated and signal as if it were at the surface, leading to cetuximab resistance47. Together, these findings indicate a possible role for dysregulated degradation and internalization in resistance to cetuximab.

Oncogenic Shift. Oncogenic shift, or the activation of other RTKs when an RTK that the cancer cell relies upon is therapeutically targeted, has been observed in cancer cells resistant to cetuximab. In 2008, Wheeler et al. performed a high-throughput screen on CtxR cell lines and their respective parent lines and found an upregulation of HER2, HER3, and MET in several

12

resistant clones. Immunoprecipitation and subsequent western blotting revealed that the resistant clones demonstrated increased EGFR hetero-dimerization with these three RTKs versus the parental lines. These RTKs were activated upon the repeated cetuximab exposure used to create resistant clones, suggesting that their activation plays a role in conferring resistance 46. In 2014, Iida et al. highlighted the role of HER3 in cetuximab resistance by demonstrating that dual targeting of EGFR and HER3 was able to overcome cetuximab resistance. Three CtxR clones derived from one parental lung cancer cell line were treated with U3-1287, a HER3 specific antibody, and cetuximab. Neither antibody showed a significant effect on proliferation in the resistant clones when used alone, but a robust decrease in proliferation was seen when cells were treated with both antibodies48. Sohn et al. similarly confirmed the role of MET in cetuximab resistance by simultaneously targeting MET and EGFR. TNBC cell lines that demonstrated intrinsic resistance to cetuximab and the MET tyrosine kinase inhibitor EMD1214063 showed a decrease in proliferation and in downstream signaling activation when treated with both drugs at once49. In 2015, Mancini et al reported that a combination of three antibodies against the EGFR (mAb565 or cetuximab), HER2 (mAB12 or trastuzumab) and HER3 (mAb33) exhibited superior ability to overcome resistance. Furthermore, they also demonstrated that combination treatment with novel TKIs and a triple mAb mixture held promise showing additive or synergistic efficacy in drug resistance50. An emerging role for the receptor tyrosine kinase AXL in multiple cancers has come to light over the last decade51-54. In 2014, Brand et al. found that AXL played a prominent role in cetuximab resistance. In this study, they utilized both NSCLC and HNSCC models of acquired resistance to cetuximab, where AXL was overexpressed, activated, and highly associated with the EGFR in CtxR clones. Using siRNA approaches and novel anti-AXL targeting agents, AXL

13

was found to regulate CtxR cell proliferation, EGFR activation, and MAPK signaling. Interestingly, EGFR regulated AXL mRNA expression directly through MAPK and the transcription factor c-Jun in CtxR clones, which created a positive feedback loop that maintained AXL-induced EGFR activation. Furthermore, CtxS parental cells acquired resistance to cetuximab upon stable overexpression of AXL or stimulation with EGFR ligands. This led to increased AXL activity and AXL-EGFR association. This relationship was further investigated in cell line xenografts, which showed that with prolonged cetuximab treatment and subsequent development of cetuximab resistance, AXL was hyperactivated and associated with EGFR. Finally, examination of patient derived xenografts established from surgically resected HNSCCs indicated AXL was robustly overexpressed and activated in tumors that were intrinsically resistant to cetuximab55. The authors expanded on this work with a second paper indicating the prominent role of AXL in therapeutic resistance in HNSCC56. Collectively, this study identified AXL as a key player in cetuximab resistance and provided a rationale for the clinical evaluation of anti-AXL therapeutics for treatment of CtxR cancers.

Nuclear localization of the EGFR. In 2009, Liao and Carpenter showed that EGFR moves to the nucleus in response to cetuximab treatment, but the implications of this translocation for cetuximab resistance were not known57. Reports have now linked nuclear trafficking to cetuximab resistance. Li et al. showed that an increased amount of EGF and src family kinases (SFKs) in CtxR clones as compared with the parental CtxS line led to nuclear translocation of EGFR. The role of nuclear EGFR (nEGFR) in cetuximab resistance was confirmed by adding a nuclear localization sequence to EGFR in the CtxS parental line, which led to increased nEGFR and subsequent resistance to cetuximab58. Blockade of SFKs with dasatinib was able to block

14

nEGFR translocation and re-sensitize the resistant clones to cetuximab. These findings shed light on the role of EGFR nuclear translocation in cetuximab resistance and support the targeting of SFKs as a potential route to overcoming this resistance. Brand et al. further demonstrated a role for SFK-mediated nEGFR in cetuximab resistance TNBC cell lines and human TNBC patients. Using a battery of TNBC cell lines and human tumors, the authors showed through EGFR-knockdown that TNBC cell lines retained dependence on EGFR despite their resistance to cetuximab. This result was explained by the reported expression of nEGFR. Furthermore, SFK were shown to influence nEGFR translocation in TNBC cell lines and in in vivo tumor models where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab59. Collectively, these data further strengthen the role of nEGFR leading to resistance to cetuximab therapy. This work highlighted that targeting both the nEGFR signaling pathway through the inhibition of its nuclear transport and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of TNBC patients.

Epithelial to Mesenchymal Transition (EMT). EMT is a cellular process in which a cell transitions from an epithelial phenotype into a mesenchymal phenotype by changes in expression of proteins such as vimentin, e-cadherin, and other adhesion molecules60. EMT is a hallmark of progressive cancers and is typically necessary for solid tumors to spread and metastasize60. Several studies implicated EMT as a mechanism of resistance to EGFR inhibitors. In 2008, Fuchs et al. found that based on protein and gene expression analysis human hepatoma cell lines, which are classified as having more mesenchymal than epithelial phenotype were also resistant

15

to various EGFR inhibitors including cetuximab. These cells also showed increased expression of downstream molecules such as AKT, STAT3, and integrin linked kinase (ILK). Inhibition of ILK was sufficient to reduce AKT expression, promote the transition back to an epithelial phenotype, as well as re-sensitize the cells to cetuximab treatment61. Basu et al. reported a similar finding in squamous cell carcinoma cell lines. Subsets of mesenchymal-like cells in two predominantly epithelial cell lines were isolated. The mesenchymal subpopulations showing relatively low e-cadherin expression were intrinsically resistant to cetuximab, while those with higher e-cadherin expression showed more sensitivity62. These small subsets of cells with resistance may provide an explanation for tumor recurrence and resistance development post-cetuximab treatment. Holz et al. also noted a decreased sensitivity to cetuximab in HNSCC cell lines that were more mesenchymal in nature and had high expression of the transcription factor SNAIL1. They found, however, that these cell lines were very sensitive to a combined treatment of radiation and cetuximab, and thus they suggested SNAIL1 as a possible biomarker for determining patients that would benefit from combined treatment63. These studies indicate a role for EMT in both acquired and intrinsic resistance to antibody therapy and suggest EMT blockade as a method to overcome resistance.

Activation of Effector Molecules. Several reports have implicated various downstream effector molecules as factors in in resistance to EGFR-targeted treatment. In 2006, Lievre et al. found a correlation between a lack of cetuximab response in CRC patients and KRAS mutation. They suggested that mutated KRAS could still activate other molecules in the MAPK pathway even in the presence of EGFR targeted therapies, including cetuximab. The RAS/MAPK pathway is

16

important for proliferation and therefore the tumors were able to escape cetuximab treatment through continued activation of this pathway. In light of this, KRAS mutation status could be used as a biomarker for CRC patients likely to be unresponsive to cetuximab64. Di Fiore et al. further confirmed this finding in 2007. CRC patient tumors were tested for KRAS mutations and a strong correlation was found between mutation status and resistance to therapy65. In 2009, Wheeler et al. found increased SFK activation in NSCLC cells that were resistant to cetuximab. These cells were also more sensitive to the SFK inhibitor dasatinib than the CtxS parental line, suggesting dependence of resistant cells on SFK activation of survival pathways66. In 2011, Dunn et al. found that dasatinib was able to re-sensitize CRC cells harboring KRAS mutations to cetuximab. This effect was also observed in vivo67. Another group found that HIF-1α was overexpressed in CtxR CRC cells with KRAS mutations, which led to a positive feedback loop between increased HIF-1α production and KRAS activation. Inhibition of HIF-1α in these cells resulted in re-sensitization to cetuximab68. In 2014, HNSCC tumors with KRAS and PIK3CA mutations were found to be unresponsive to cetuximab. Upon further investigation, these tumors were found to retain activation of mTOR, a molecule downstream of EGFR leading to proliferation. Targeting mTOR along with EGFR produced robust antiproliferative effects in the HNSCC xenografts with mutated KRAS and PIK3CA, supporting the role of constitutive activation of mTOR in cetuximab resistance69. Kim et al reported that constitutive activation of AKT was responsible for acquired cetuximab resistance in NSCLC cells. They demonstrated that AKT activation was mediated by an increased instability of phosphatase and tensin homologue (PTEN), a tumor suppressor that causes deactivation of the PI3K/AKT pathway70. Rebucci et al. also implicated AKT activation in cetuximab resistance in HNSCC cell lines. Targeting the PI3K/AKT pathway in these cells led

17

to cetuximab re-sensitization, confirming a role for this pathway in resistance71. AKT was also implicated in CRC cetuximab resistance by Kawakami et al in 2014. In this model, the HER3 ligand heregulin was found to be upregulated in resistant clones, leading to HER3 activation and ultimately AKT activation, even in the face of cetuximab treatment. Treatment of these cells with patritumab, the HER3 targeted antibody, re-sensitized the CRC cells to cetuximab treatment72. Another ligand implicated in cetuximab resistance is the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Hatakeyama et al. showed in 2010 that, in HNSCC, the overexpression of HB-EGF along with lowered expression of miR-212, an HB-EGF-regulating micro RNA, was associated with CtxR. With blockage of HB-EGF in resistant clones, sensitivity to cetuximab was recovered. This study also suggested that HB-EGF may have a direct role in cetuximab resistance and EMT potentially by activation of fibroblast growth factor receptor in the setting of prolonged cetuximab exposure73. In addition, recent evidence has implicated another EGFR ligand, hepatocyte growth factor (HGF), in CtxR in CRC cells. HGF is a ligand for MET and Yonesaka et al. showed that high HGF levels were correlated with poor response to cetuximab in patients. This finding suggests that the circulating level of HGF can be used as a biomarker for patients that will respond to cetuximab74. This growing body of evidence for the activation of effector molecules as contributors to therapeutic resistance provides ample avenues for further study into overcoming resistance to EGFR-targeted therapies.

EGFR Mutation. In 2015, Braig et al. isolated CtxR CRC clones and noted that, contrary to previously reported data, these clones showed no overexpression of other RTKs or effector molecules. Furthermore, when treated with other EGFR inhibitors such as panitumumab or small molecule inhibitors, CtxR clones showed sensitivity. Based on this evidence, Braig et al.

18

hypothesized that cetuximab targets a specific EGFR site, and that there was a mutation specifically in their EGFR epitope. Through next-generation sequencing, they confirmed that an EGFR S492R mutation was present in resistant clones, and thus identified this mutation related to cetuximab resistance75. Another EGFR mutation implicated in resistance to both cetuximab and panitumumab is G465R. This mutation was found in 2 patients with gastrointestinal cancer and was confirmed to exist in in vitro models as a promoter of resistance to EGFR-targeted antibody therapy75. A 2015 study on 37 CRC patient samples showing cetuximab resistance confirmed the presence of the G465R mutation and furthermore notified 4 other resistancerelated EGFR mutations: R451C, K467T, S464L, and I491M. The authors reported that these mutations prevented cetuximab from binding to EGFR, allowing the cells to become resistant to its effects76. Assaying for these mutations prior to treatment may help determine if a patient will benefit from treatment.

HER2 Biology. HER2 is similar to EGFR in structure, containing an extracellular domain, transmembrane domain, and intracellular kinase domain. HER2, however, unlike the prototypical EGFR, has no known ligand. Without a ligand to activate it, HER2 is thought to be in an open conformation, but its signaling ability depends on its heterodimerization with other members of the HER family, preferentially with HER324, 27, 77. The propagation of signals by HER3 similarly depends on dimerization due to its lack of inherent kinase activity. In 1996, Pinkas-Kramarski et al firstly demonstrated that HER3 formed active heterodimers with HER278. It has also been demonstrated that HER2 and HER3 dimers result in the most robust signaling within the HER family27,

77

. Similar to EGFR, HER2 activates many of the same signaling pathways and is

19

important in normal cellular function and development of many tissues including cardiac, neuronal, and mammary tissues27.

HER2 and its role in cancer. HER2 overexpression has been identified in approximately 25-30% of primary breast cancers and is a significant prognostic factor in terms of nodal status, tumor grade, overall survival rate and probability of relapse in breast cancer patients 79-81. Therefore, it was postulated that HER2 could have a role in the pathogenesis of breast cancer. Preclinical experiments demonstrated that overexpression of HER2 in mouse fibroblast cells resulted in malignant cellular transformation and tumorigenesis 82, 83. In addition to its well-established role in breast cancer, HER2 overexpression and amplification has also been identified in numerous other human cancers, including gastric 84, esophageal 88

85

, oropharyngeal

86

, endometrial 87, lung

, and bladder 89, and in these cancers it is associated with poorer prognosis.

Therapeutic antibodies targeting HER2. The monoclonal antibody trastuzumab (Herceptin) gained FDA approval in 1998 as a HER2 targeted antibody available for use in humans. Trastuzumab is now approved for the treatment of HER2-positive breast cancer and gastric cancer. Trastuzumab is a humanized IgG1 antibody that binds HER2 and works to diminish the effects of overexpressed HER2 by causing the removal of the receptor from the cell surface90. The actions of this antibody limits HER2 availability for dimerization with other HER family members, shutting down the powerful proliferative signals that would otherwise be transmitted specifically signals through the MAPK and PI3K/AKT pathways90. Several other anti-HER2 antibodies remain in clinical trials for the treatment of breast and gastric cancers. In 2013, TDM1 - an antibody drug conjugate linked to the cytotoxic drug DM1 and containing trastuzumab

20

was approved by the FDA91. While initial results in HER2-positive breast cancer patients treated with T-DM1 seemed favorable, recent reports have shown no difference in response in patients treated with T-DM1 versus patients treated with trastuzumab plus standard chemotherapy92. Thus, there remains a strong need for continued therapy development.

Adaptive Responses of antibody-based targeting of HER2. Another motivation for continued research on anti-HER2 antibody therapy is to overcome the development of resistance to trastuzumab seen in vivo, in vitro, and in the clinic after FDA approval. Numerous studies have discussed the various mechanisms cancer cells use to escape the effects of trastuzumab and the implications of those mechanisms for future HER2 targeted therapies.

Epitope Masking. The presence of a truncated, intracellular form of HER2 known as p95HER2 has been reported to give HER2-positive cancer cell lines a growth advantage as well lead to trastuzumab resistance93,

94

. p95HER2 does not contain the extracellular domain to which

trastuzumab binds but is still active in its kinase domain. Therefore blocking the effects of trastuzumab due to loss of the epitope while still allowing HER2 signaling to take place94, 95. Reports have shown that the cleaved HER2 extracellular domain is detectable in the serum of patients and correlates with more severe disease and increased trastuzumab resistance96. Screening for this form of HER2 prior to treatment may help determine which patients will benefit more from trastuzumab therapy. A second type of epitope masking that occurs in trastuzumab-resistant cells can be attributed to molecules other than HER2 itself. This variety of resistance is seen in cancers with overexpression of CD44 and its ligand hyaluronan, or in the glycoprotein complex MUC4. Palyi-

21

Krekk et al. showed in 2007 that CD44 associates with HER2 in trastuzumab-resistant cells. This association causes an inability of trastuzumab to bind to its epitope on HER2 if the relatively large hyaluronan is bound to CD44 97. In 2002, melanoma and breast adenocarcinoma cells were found to be resistant to trastuzumab upon expression of MUC4. The authors conclude that MUC4 blocks trastuzumab’s ability to bind HER2 by steric hindrance at the cell surface. The specificity of MUC4 for blocking trastuzumab binding was demonstrated MUC4 was unable to significantly block other antibodies from binding to their targets98. Nagy et al. also implicated MUC4 in trastuzumab resistance. JIMT-1, a trastuzumab-resistant breast cancer line, showed increased MUC4 expression at the cell surface compared to trastuzumab sensitive cell lines. Furthermore, genetic ablation of MUC4 allowed increased trastuzumab binding in JIMT-1 cells. Treatment of JIMT-1 cells with APMA, which activates mellanoproteases that cleave membrane proteins including MUC4, also allowed increased trastuzumab binding. The authors suggest that due to this intrinsically occurring escape mechanism, HER2 status alone cannot be used as a marker for trastuzumab benefit99.

Oncogenic Shift. It has been reported that trastuzumab is unable to inhibit HER2 signaling when HER2 is heterodimerized100. In 2001, Lu et al. found that insulin-like growth factor receptor (IGF-1R) signaling causes resistance to trastuzumab in HER2-positive breast cancer. They demonstrated that overexpression of IGF-1R led to resistance and abrogation of IGF-1R signaling, which restored sensitivity101. In 2005, Nahta et al. reported that resistance due to IGF1R could be attributed to its heterodimerization with HER2. They reported that the HER2/IGF1R heterodimer occurs only in resistant cells and disruption of dimerization results in sensitivity. Furthermore, another HER2 targeted antibody, pertuzumab, disrupted heterodimerization,

22

implying that HER2 is still targetable even in cases of trastuzumab resistance102. Further demonstrating the oncogenic shift occurring with the targeting of HER2, Narayan et al. showed that HER3 and EGFR were upregulated in trastuzumab-resistant cell lines after continual treatment with trastuzumab. HER3 was upregulated in all five cell lines tested, suggesting that shifting dependence to a different set of receptors is a common escape mechanism underlying resistance103. Due to the preference of HER2 to dimerize with HER3, it is possible that this form of heterodimerization is responsible for resistance as well100. Ritter et al. showed higher levels of HER2/EGFR heterodimers in trastuzumab resistant BT-474 breast cancer cells. They induced apoptosis by treating the cells with a dual EGFR/HER2 drug, lapatinib, and inhibited growth with EGFR tyrosine kinase inhibitors. This suggests an oncogenic shift from HER2 to EGFR dependence in trastuzumab resistant populations104. Involvement of signaling through other HER family members is also implicated by the observation by Anastasi et al. that loss of signaling by receptor-associated late transducer (RALT), a HER family inhibitor, results in trastuzumab resistance. SKBR3 and BT-474 breast cancer cell lines treated with HER family ligands were insensitive to trastuzumab and were more proliferative when RALT signaling was abrogated. When RALT signaling was induced in cells, trastuzumab successfully reduced growth and mitogenic signaling105. Another RTK implicated in trastuzumab resistance is MET, which has been shown to be upregulated in trastuzumab-resistant, HER2-positive breast cancer tumors106. Shattuck et al. reported that MET was upregulated in trastuzumab resistant breast cancer cell lines upon trastuzumab treatment. Additionally, knock down of MET with siRNA or blockade with a tyrosine kinase inhibitor in two breast cancer lines resulted in an increased response to trastuzumab, further implicating MET in trastuzumab resistance. Analysis of downstream

23

signaling pathways post-trastuzumab treatment revealed that cells expressing active MET were able to retain activation of survival pathways normally shut down by trastuzumab106. These data suggest that targeting HER2 and other interacting RTKs in combination is another possible strategy to overcome trastuzumab resistance.

Activation of Effector Molecules. Several HER2 effector molecules have been implicated in trastuzumab resistance. In 2004, Nahta et al. found that p27kip1 was downregulated in trastuzumab-resistant breast cancer clones compared to the sensitive parental line. They linked this molecule to resistance by treating resistant cells with exogenous p27kip1 or a proteasome inhibitor to induce p27kip1 expression, both of which increased sensitivity to trastuzumab107. PTEN has also been shown to be responsible for trastuzumab resistance. Nagata et al. reported that PTEN deficiency resulted in resistance to trastuzumab in vivo and in patient samples. A PTEN antisense oligonucleotide was used to knockdown PTEN in breast cancer cells injected into mice. Mice with this treatment were less sensitive to trastuzumab than their control treated counterparts. HER2-positive breast cancer patient samples were then examined for PTEN deficiency and a correlation was observed between lower PTEN levels and poor response rates to trastuzumab therapy. The authors also demonstrated that resistant cells could be re-sensitized with PI3K inhibitors, due to the role of PI3K in PTEN inactivation108. Berns et al. confirmed that PTEN was solely identified in an shRNA screen as an effector of trastuzumab resistance. Furthermore, constitutively active PI3K transfected into trastuzumab sensitive cells was able to confer almost complete resistance to those cells109. Naturally occurring gain of function mutations in PI3K have also been found, resulting in cellular dependency on PI3K signaling, as well as resistance to trastuzumab treatment. PI3K inhibition successfully inhibited growth in this

24

setting as well110. Trastuzumab in combination with PI3K inhibition has also been shown to be effective regardless of resistance status, suggesting that this combination could be widely therapeutic in patients111. In addition, PI3 Kinase activity was shown to be upregulated by TGFβ expression through HER3 activation by increasing HER3 ligand production. Expression of TGF-β in trastuzumab-sensitive cells resulted in resistance, suggesting TGF-β production as another escape mechanism through PI3K112. Another player in the PTEN/PI3K mechanism of resistance is SRC. As PTEN is known to deactivate SRC, deficiency of PTEN through PI3K signaling in breast cancer cells has been reported to result in more phosphorylated SRC and increased resistance113. Zhang et al. showed that SRC inhibition was successful in re-sensitizing resistant cell lines to trastuzumab, providing another way to attack this powerful signaling node113.

Altered Internalization. Valabrega et al. demonstrated an inability of trastuzumab to internalize and downregulate HER2 in breast cancer cells treated with TGF-α. TGF-α functionally delayed the internalization and lysosomal sorting in SKBR3 cells, a process that is partially responsible for the effectiveness of trastuzumab114. Dokmanovic et al. found disrupted internalization in a SKBR3-resistant clone, which overexpress the GTPase RAC1. Treatment with RAC1 inhibitor, NSC23766, restored the ability of trastuzumab to internalize HER2 and inhibit growth. Additionally, a constitutively active mutant of RAC1 successfully conferred resistance to parental SKBR3 cells and inhibited the internalization of HER2 by trastuzumab115. These studies establish a role for dysregulated internalization of HER2 in trastuzumab resistance and suggest possible targets for overcoming resistance in the clinic.

Future directions

25

De novo and acquired resistance to HER family-targeting antibodies plague patients with tumors that overexpress EGFR and/or HER2 and may otherwise benefit from these therapies, which carry little of the toxicity associated with traditional chemotherapy and/or radiation. It is clearly evident that the treatment of a tumor with a single molecularly targeted antibody can lead to rapid resistance through several mechanisms. It is now becoming clear that improved antibodies against the EGFR that targeting multiple epitopes, leading to its degradation116-120 or antibodies that targeting an entire family of receptors rather than a single receptor are much more desirable and are showing promising results in the pre-clinical arena121-124. As we advance the field of RTK targeting at the cell surface, there is little doubt that the greatest benefit will be achieved by targeting RTK receptors and effector molecules such as SFKs and PI3K/AKT simultaneously. This is an exciting time in the field of antibody-based therapies against HER family members, and work in the next decade will only advance our understanding of resistance. Increased understanding of the mechanisms that underlie this resistance will help clinicians predict which patients will become resistant and more importantly, what we can do to circumvent this resistance.

Reference 1. 2. 3. 4.

5.

Llewelyn, M.B., Hawkins, R.E. & Russell, S.J. Discovery of antibodies. BMJ 305, 126972 (1992). Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-7 (1975). Smith, S.L. Ten years of Orthoclone OKT3 (muromonab-CD3): a review. J Transpl Coord 6, 109-19; quiz 120-1 (1996). Morrison, S.L., Johnson, M.J., Herzenberg, L.A. & Oi, V.T. Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A 81, 6851-5 (1984). Jones, P.T., Dear, P.H., Foote, J., Neuberger, M.S. & Winter, G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321, 522-5 (1986).

26

6. 7. 8. 9. 10.

11. 12. 13.

14. 15.

16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26.

Addepalli, B., Rao, S. & Hunt, A.G. Phage display library screening for identification of interacting protein partners. Methods Mol Biol 1255, 147-58 (2015). Deantonio, C., Cotella, D., Macor, P., Santoro, C. & Sblattero, D. Phage display technology for human monoclonal antibodies. Methods Mol Biol 1060, 277-95 (2014). Fujiwara, D. & Fujii, I. Phage selection of peptide "microantibodies". Curr Protoc Chem Biol 5, 171-94 (2013). Nelson, A.L., Dhimolea, E. & Reichert, J.M. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9, 767-74 (2010). Zhao, A., Tohidkia, M.R., Siegel, D.L., Coukos, G. & Omidi, Y. Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol, 1-14 (2014). Cohen, S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem 237, 1555-62 (1962). Cohen, S. The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biol 12, 394-407 (1965). Carpenter, G., Lembach, K.J., Morrison, M.M. & Cohen, S. Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. J Biol Chem 250, 4297-304 (1975). Carpenter, G., King, L., Jr. & Cohen, S. Epidermal growth factor stimulates phosphorylation in membrane preparations in vitro. Nature 276, 409-10 (1978). Ullrich, A. et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 41825 (1984). Eckhart, W., Hutchinson, M.A. & Hunter, T. An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell 18, 925-33 (1979). Hunter, T. & Sefton, B.M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A 77, 1311-5 (1980). Riedel, H., Dull, T.J., Schlessinger, J. & Ullrich, A. A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor. Nature 324, 6870 (1986). Schlessinger, J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci 13, 443-7 (1988). Hynes, N.E. & MacDonald, G. ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 21, 177-84 (2009). Herbst, R.S. Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 59, 21-6 (2004). Wieduwilt, M.J. & Moasser, M.M. The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci 65, 1566-84 (2008). Singh, A.B. & Harris, R.C. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal 17, 1183-93 (2005). Baselga, J. & Swain, S.M. Novel anticancer targets: revisiting ERBB2 and discovering ERBB3. Nat Rev Cancer 9, 463-75 (2009). Yarden, Y. & Sliwkowski, M.X. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2, 127-37 (2001). Burke, P., Schooler, K. & Wiley, H.S. Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking. Mol Biol Cell 12, 1897-910 (2001).

27

27. 28.

29. 30. 31. 32. 33. 34. 35.

36. 37.

38.

39. 40.

41. 42.

43.

44.

Citri, A., Skaria, K.B. & Yarden, Y. The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res 284, 54-65 (2003). Hirsch, F.R., Varella-Garcia, M. & Cappuzzo, F. Predictive value of EGFR and HER2 overexpression in advanced non-small-cell lung cancer. Oncogene 28 Suppl 1, S32-7 (2009). Spano, J.P. et al. Impact of EGFR expression on colorectal cancer patient prognosis and survival. Ann Oncol 16, 102-8 (2005). Kalyankrishna, S. & Grandis, J.R. Epidermal growth factor receptor biology in head and neck cancer. J Clin Oncol 24, 2666-72 (2006). Nicholson, R.I., Gee, J.M.W. & Harper, M.E. EGFR and cancer prognosis. European Journal of Cancer 37, S9-S15 (2001). Harding, J. & Burtness, B. Cetuximab: an epidermal growth factor receptor chemeric human-murine monoclonal antibody. Drugs Today (Barc) 41, 107-27 (2005). Baselga, J. The EGFR as a target for anticancer therapy--focus on cetuximab. Eur J Cancer 37 Suppl 4, S16-22 (2001). Weber, J. & McCormack, P.L. Panitumumab: in metastatic colorectal cancer with wildtype KRAS. BioDrugs 22, 403-11 (2008). Dienstmann, R. & Tabernero, J. Necitumumab, a fully human IgG1 mAb directed against the EGFR for the potential treatment of cancer. Curr Opin Investig Drugs 11, 1434-41 (2010). Bardelli, A. & Siena, S. Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer. J Clin Oncol 28, 1254-61 (2010). Viloria-Petit, A. et al. Acquired resistance to the antitumor effect of epidermal growth factor receptor-blocking antibodies in vivo: a role for altered tumor angiogenesis. Cancer Res 61, 5090-101 (2001). Ciardiello, F. et al. Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clin Cancer Res 10, 784-93 (2004). Wu, G. et al. Involvement of COX-2 in VEGF-induced angiogenesis via P38 and JNK pathways in vascular endothelial cells. Cardiovasc Res 69, 512-9 (2006). Chien, M.H. et al. Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway. Carcinogenesis 30, 2005-13 (2009). Leung, W.K. et al. Cyclooxygenase-2 upregulates vascular endothelial growth factor expression and angiogenesis in human gastric carcinoma. Int J Oncol 23, 1317-22 (2003). Xu, K. & Shu, H.K. EGFR activation results in enhanced cyclooxygenase-2 expression through p38 mitogen-activated protein kinase-dependent activation of the Sp1/Sp3 transcription factors in human gliomas. Cancer Res 67, 6121-9 (2007). Jung, Y.D. et al. Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model. Eur J Cancer 38, 1133-40 (2002). Friedman, L.M. et al. Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: implications for cancer immunotherapy. Proc Natl Acad Sci U S A 102, 1915-20 (2005).

28

45.

46. 47. 48. 49. 50. 51.

52.

53.

54. 55. 56. 57. 58. 59. 60. 61.

62.

63.

Lu, Y. et al. Epidermal growth factor receptor (EGFR) ubiquitination as a mechanism of acquired resistance escaping treatment by the anti-EGFR monoclonal antibody cetuximab. Cancer Res 67, 8240-7 (2007). Wheeler, D.L. et al. Mechanisms of acquired resistance to cetuximab: role of HER (ErbB) family members. Oncogene 27, 3944-56 (2008). Nevo, J. et al. Mammary-derived growth inhibitor alters traffic of EGFR and induces a novel form of cetuximab resistance. Clin Cancer Res 15, 6570-81 (2009). Iida, M. et al. Overcoming acquired resistance to cetuximab by dual targeting HER family receptors with antibody-based therapy. Mol Cancer 13, 242 (2014). Sohn, J. et al. cMET Activation and EGFR-Directed Therapy Resistance in TripleNegative Breast Cancer. J Cancer 5, 745-53 (2014). Mancini, M. et al. Combining three antibodies nullifies feedback-mediated resistance to erlotinib in lung cancer. Sci Signal 8, ra53 (2015). Graham, D.K., DeRyckere, D., Davies, K.D. & Earp, H.S. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer. Nat Rev Cancer 14, 769-85 (2014). Linger, R.M., Keating, A.K., Earp, H.S. & Graham, D.K. Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors. Expert Opin Ther Targets 14, 1073-90 (2010). Linger, R.M., Keating, A.K., Earp, H.S. & Graham, D.K. TAM receptor tyrosine kinases: biologic functions, signaling, and potential therapeutic targeting in human cancer. Adv Cancer Res 100, 35-83 (2008). Verma, A., Warner, S.L., Vankayalapati, H., Bearss, D.J. & Sharma, S. Targeting Axl and Mer kinases in cancer. Mol Cancer Ther 10, 1763-73 (2011). Brand, T.M. et al. AXL mediates resistance to cetuximab therapy. Cancer Res 74, 515264 (2014). Brand, T.M. et al. AXL Is a Logical Molecular Target in Head and Neck Squamous Cell Carcinoma. Clin Cancer Res (2015). Liao, H.J. & Carpenter, G. Cetuximab/C225-induced intracellular trafficking of epidermal growth factor receptor. Cancer Res 69, 6179-83 (2009). Li, C., Iida, M., Dunn, E.F., Ghia, A.J. & Wheeler, D.L. Nuclear EGFR contributes to acquired resistance to cetuximab. Oncogene 28, 3801-13 (2009). Brand, T.M. et al. Nuclear epidermal growth factor receptor is a functional molecular target in triple-negative breast cancer. Mol Cancer Ther 13, 1356-68 (2014). Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 64674 (2011). Fuchs, B.C. et al. Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. Cancer Res 68, 2391-9 (2008). Basu, D. et al. Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity. Oncogene 29, 4170-82 (2010). Holz, C. et al. Epithelial-mesenchymal-transition induced by EGFR activation interferes with cell migration and response to irradiation and cetuximab in head and neck cancer cells. Radiother Oncol 101, 158-64 (2011).

29

64. 65. 66. 67. 68.

69. 70.

71. 72. 73.

74. 75. 76. 77. 78.

79. 80. 81.

82. 83.

Lievre, A. et al. KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res 66, 3992-5 (2006). Di Fiore, F. et al. Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy. Br J Cancer 96, 1166-9 (2007). Wheeler, D.L. et al. Epidermal growth factor receptor cooperates with Src family kinases in acquired resistance to cetuximab. Cancer Biol Ther 8, 696-703 (2009). Dunn, E.F. et al. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab. Oncogene 30, 561-74 (2011). Wang, Y., Lei, F., Rong, W., Zeng, Q. & Sun, W. Positive feedback between oncogenic KRAS and HIF-1alpha confers drug resistance in colorectal cancer. Onco Targets Ther 8, 1229-37 (2015). Wang, Z. et al. mTOR co-targeting in cetuximab resistance in head and neck cancers harboring PIK3CA and RAS mutations. J Natl Cancer Inst 106 (2014). Kim, S.M. et al. Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells. Cancer Lett 296, 150-9 (2010). Rebucci, M. et al. Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance. Int J Oncol 38, 189-200 (2011). Kawakami, H. et al. The anti-HER3 antibody patritumab abrogates cetuximab resistance mediated by heregulin in colorectal cancer cells. Oncotarget 5, 11847-56 (2014). Hatakeyama, H. et al. Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma. PLoS One 5, e12702 (2010). Yonesaka, K. et al. Circulating hepatocyte growth factor is correlated with resistance to cetuximab in metastatic colorectal cancer. Anticancer Res 35, 1683-9 (2015). Braig, F. et al. Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer. Oncotarget 6, 12035-47 (2015). Arena, S. et al. Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in Colorectal Cancer. Clin Cancer Res 21, 2157-66 (2015). Moasser, M.M. The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 26, 6469-87 (2007). Pinkas-Kramarski, R. et al. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J 15, 2452-67 (1996). Slamon, D.J. et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235, 177-82 (1987). Slamon, D.J. et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244, 707-12 (1989). Berger, M.S. et al. Correlation of c-erbB-2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading. Cancer Res 48, 1238-43 (1988). Di Fiore, P.P. et al. erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science 237, 178-82 (1987). Hudziak, R.M., Schlessinger, J. & Ullrich, A. Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci U S A 84, 7159-63 (1987).

30

84.

85. 86.

87.

88. 89. 90. 91. 92. 93.

94. 95. 96. 97.

98.

99.

100. 101.

102.

Yano, T. et al. Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer. Oncol Rep 15, 65-71 (2006). Mimura, K. et al. Frequencies of HER-2/neu expression and gene amplification in patients with oesophageal squamous cell carcinoma. Br J Cancer 92, 1253-60 (2005). Khan, A.J. et al. Characterization of the HER-2/neu oncogene by immunohistochemical and fluorescence in situ hybridization analysis in oral and oropharyngeal squamous cell carcinoma. Clin Cancer Res 8, 540-8 (2002). Morrison, C. et al. HER-2 is an independent prognostic factor in endometrial cancer: association with outcome in a large cohort of surgically staged patients. J Clin Oncol 24, 2376-85 (2006). Hirsch, F.R. et al. Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas. Br J Cancer 86, 1449-56 (2002). Latif, Z. et al. HER2/neu overexpression in the development of muscle-invasive transitional cell carcinoma of the bladder. Br J Cancer 89, 1305-9 (2003). Vu, T. & Claret, F.X. Trastuzumab: updated mechanisms of action and resistance in breast cancer. Front Oncol 2, 62 (2012). Hayashi, T. et al. Targeting HER2 with T-DM1, an Antibody Cytotoxic Drug Conjugate, Is Effective in HER2 Over-Expressing Bladder Cancer. J Urol (2015). Ellis, P.A. et al. in ASCO Annual Meeting (2015). Scott, G.K. et al. A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 13, 2247-57 (1993). Scaltriti, M. et al. Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst 99, 628-38 (2007). Pedersen, K. et al. A naturally occurring HER2 carboxy-terminal fragment promotes mammary tumor growth and metastasis. Mol Cell Biol 29, 3319-31 (2009). Ali, S.M. et al. Serum HER-2/neu and relative resistance to trastuzumab-based therapy in patients with metastatic breast cancer. Cancer 113, 1294-301 (2008). Palyi-Krekk, Z. et al. Hyaluronan-induced masking of ErbB2 and CD44-enhanced trastuzumab internalisation in trastuzumab resistant breast cancer. Eur J Cancer 43, 2423-33 (2007). Price-Schiavi, S.A. et al. Rat Muc4 (sialomucin complex) reduces binding of anti-ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance. Int J Cancer 99, 783-91 (2002). Nagy, P. et al. Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res 65, 473-82 (2005). Baselga, J. A new anti-ErbB2 strategy in the treatment of cancer: prevention of liganddependent ErbB2 receptor heterodimerization. Cancer Cell 2, 93-5 (2002). Lu, Y., Zi, X., Zhao, Y., Mascarenhas, D. & Pollak, M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 93, 1852-7 (2001). Nahta, R., Yuan, L.X., Zhang, B., Kobayashi, R. & Esteva, F.J. Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization

31

103. 104.

105.

106.

107.

108. 109. 110.

111.

112.

113. 114. 115.

116.

117. 118.

119.

contributes to trastuzumab resistance of breast cancer cells. Cancer Res 65, 11118-28 (2005). Narayan, M. et al. Trastuzumab-induced HER reprogramming in "resistant" breast carcinoma cells. Cancer Res 69, 2191-4 (2009). Ritter, C.A. et al. Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network. Clin Cancer Res 13, 4909-19 (2007). Anastasi, S. et al. Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin. Oncogene 24, 4540-8 (2005). Shattuck, D.L., Miller, J.K., Carraway, K.L., 3rd & Sweeney, C. Met receptor contributes to trastuzumab resistance of Her2-overexpressing breast cancer cells. Cancer Res 68, 1471-7 (2008). Nahta, R., Takahashi, T., Ueno, N.T., Hung, M.C. & Esteva, F.J. P27(kip1) downregulation is associated with trastuzumab resistance in breast cancer cells. Cancer Res 64, 3981-6 (2004). Nagata, Y. et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6, 117-27 (2004). Berns, K. et al. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12, 395-402 (2007). Kataoka, Y. et al. Association between gain-of-function mutations in PIK3CA and resistance to HER2-targeted agents in HER2-amplified breast cancer cell lines. Ann Oncol 21, 255-62 (2010). Junttila, T.T. et al. Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 15, 429-40 (2009). Wang, S.E. et al. Transforming growth factor beta engages TACE and ErbB3 to activate phosphatidylinositol-3 kinase/Akt in ErbB2-overexpressing breast cancer and desensitizes cells to trastuzumab. Mol Cell Biol 28, 5605-20 (2008). Zhang, S. et al. Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways. Nat Med 17, 461-9 (2011). Valabrega, G. et al. TGFalpha expression impairs Trastuzumab-induced HER2 downregulation. Oncogene 24, 3002-10 (2005). Dokmanovic, M., Hirsch, D.S., Shen, Y. & Wu, W.J. Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer. Mol Cancer Ther 8, 1557-69 (2009). Dienstmann, R. et al. Safety and Activity of the First-in-Class Sym004 Anti-EGFR Antibody Mixture in Patients with Refractory Colorectal Cancer. Cancer Discov 5, 598609 (2015). Iida, M. et al. Sym004, a novel EGFR antibody mixture, can overcome acquired resistance to cetuximab. Neoplasia 15, 1196-206 (2013). Skartved, N.J. et al. Preclinical pharmacokinetics and safety of Sym004: a synergistic antibody mixture directed against epidermal growth factor receptor. Clin Cancer Res 17, 5962-72 (2011). Koefoed, K. et al. Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor. MAbs 3, 584-95 (2011).

32

120. 121. 122.

123. 124.

Pedersen, M.W. et al. Sym004: a novel synergistic anti-epidermal growth factor receptor antibody mixture with superior anticancer efficacy. Cancer Res 70, 588-97 (2010). Nielsen, C.H. et al. In vivo imaging of therapy response to a novel Pan-HER antibody mixture using FDG and FLT positron emission tomography. Oncotarget (2015). Jacobsen, H.J. et al. Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity. Clin Cancer Res 21, 4110-22 (2015). Francis, D.M. et al. Pan-HER receptor inhibition augments radiation response in human lung and head and neck cancer models. Clin Cancer Res (2015). Hu, S. et al. Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk. Cancer Res 75, 159-70 (2015).

33

FDA approved

Name:Antibody

Target

Antibody Type

1998 (US)

Herceptin:Trastuzumab

HER2

Humanized, IgG1

2004 (US)

Erbitux:Cetuximab

EGFR

2004 (US)

Avastin:Bevacizumab

VEGF

2006 (US)

Vectibix:Panitumumab

EGFR

HER2+ Breast, Gastric Chimeric, IgG1 CRC, HNSCC CRC, NSCLC, Breast, RCC, Humanized, IgG1 Glioblastoma, Cervical, Ovarian Human, IgG2 CRC, NSCLC

2011(US)

Yervoy:Ipilimumab

CTLA-4

Human IgG1

2012(US)

Perjeta:Pertuzumab

HER2

2013(US)

Kadcyla:Ado-trastuzumab emtansine (T-DM1)

HER2

2014(US)

Cyramza:Ramucirumab

VEGFR2

2015(US)

Necitumumab

EGFR

Humanized, IgG1 Breast Humanized, IgG1, DrugCancer conjugate Humanized, IgG1 Gastric Non-small cell Human IgG1 lung cancer

Phase III

TheraCIM h– R3R:Nimotuzumab

EGFR

Humanized, IgG1 HNSCC

Oncoscience, Biotech Pharma, YM Biosciences, Biocon, CIMAB SA

Phase III

Zalutumumab (2F8) (HuMax–EGFrR)

EGFR

Human IgG1

Genmab A/S, Medarex

Cancer

Melanoma

HNSCC

Company Genentech/Roche Imclone/Lilly Genentech/Roche Amgen Medarex/Bristol-Myers Squibb Genentech/Roche Genentech/Roche ImClone/Lilly Lilly

34

Phase II/III

Phase II/III

MetMAb:onartuzumab

AMG 102:Rilotumumab

c-MET

c-MET

Phase II/III

Onartuzumab

c-MET

Phase II

Sym004

EGFR

Phase II

Patritumab

HER3

Phase II

IMC-A12: cixutumumab

IGF1R

Phase II

AMG479 :ganitumab

IGF1R

Phase II

MM-121, SAR256212

HER3

humanized monovalent monoclonal antibody fully human, anti-HGF/SF neutralizing antibody a monoclonal monovalent (one-armed) antibody a mixture of two synergistic fulllength anti-EGFR antibodies human monoclonal antibody fully human IgG1 monoclonal antibody fully human IgG1 monoclonal antibody fully human monoclonal

NSCLC, Gastroesophageal Genentech, Roche Cancer, TNBC, CRC Gastric, KRAS wild-type mCRC, Glioblastoma

Amgen

Colon, Non-small cell lung cancer, Glioblastome, Gastric, Breast

Genentech

KRAS wild-type mCRC, HNSCC

Symphogen/EMD Serono

Breast, NSCLC

Daiichi-Sankyo

mCRC, mHNSCC KRAS wild-type mCRC

Amgen

Breast, Ovarian,

Merrimack/Sanofi 35

antibody

Phase II

Phase II Phase II

Phase II

Phase II

Receptor activator of fully human XGEVA:Denosumab nuclear factor monoclonal kappa-B antibody ligand humanised IgG4 LY2875358:Emibetuzumab c-MET antibody LY3012207, IMCPDGFRα human IgG1 3G3:Olaratumab Hepatocyte growth factor Ficlatuzumab:AV-299 HGF/c-MET (HGF) inhibitory antibody

KB004

EphA3

Phase II

Margetuximab

HER2

Phase II

MEDI-573

IGFI, IGFII

Phase I/II

ABT-414

EGFR

Monoclonal antibody targeting the EphA3 receptor Fc-optimized monoclonal antibody monoclonal antibody Antibody-Drug

NSCLC

Amgen

Gastric, NSCLC

Lilly

Prostate, Ovary, NSCLC, brain

Lilly

NSCLC

AVEO

Acute myelogenous leukemia, KaloBios Pharmaceuticals myelodysplastic syndrome, myelofibrosis Breast, gastroesophageal MacroGenics cancer Breast

AstraZeneca/MedImmune

Glioblastoma,

Abbvie 36

Phase I/II

EGFRvIII CAR

EGFR

Phase I

Sym013

Pan-HER

Phase I

MM-151

EGFR

Phase I

MM-141

IGF1R and HER3

Phase I

MM-302

HER2

Phase I

AMG595

anti-EGFRvIII

Conjugate EGFRvIII-specific murine Chimeric antigen receptor transduced T cells a mixture of six humanized full length monoclonal antibodies targeting EGFR, HER2 and HER3 mixture of three fully human monocolonal antibodies (oligoclonal ) bispecific monoclonal antibody an antibody drug conjugated liposomal doxorubicin Antibody-Drug Conjugate

SCC EGFRvIII expressed Glioblastoma

Kite Pharma

Pancreatic

Symphogen

CRC, Non-small cell lung cancer, TNBC

Merrimack

Hepatocellular Carcinoma

Merrimack

HER2+ Breast

Merrimack

Glioblastoma

Amgen 37

Phase I

AMG780

Tie2, Ang1, Ang2

Phase I

FS102

HER2

Phase I

SAR307746:nesvacumab

Ang2

Phase I

ABT-700

c-MET

RG7221:Vanucizumab

Ang2 and VEGF

Phase I

RG7597, MEHD7945A: Duligotuzumab

HER3 and EGFR

Phase I

RG7116:Lumretuzumab

HER3

Phase I

RG7155:Emactuzumab

Exploratory LJM716

colony stimulating factor-1 receptor HER3

Monoclonal Antibody Monospecific antibody fragments Angiopoietin-2 monoclonal antibody

Advanced Solid Tumors HER2-Positive Breast and Gastric Cancer

Amgen BMS

Solid Tumors

Sanofi Oncology

Advanced Solid Tumors

Abbvie

Colon

Roche/Pharma Research and Early Development

Kras mutation tumors

Genentech Research & Early Development

Breast

Roche Pharma Research & Early Development

humanized monoclonal antibody

Solid tumor

Roche Pharma Research & Early Development

Human IgG1

HNSCC, Gastric,

Novartis

Humanized, IgG1 bi-specific monoclonal antibody dual-action, phage-derived human IgG1 monoclonal antibody glyco-engineered humanised monoclonal antibody

38

pre-clinical

Sym015

c-MET

a mixture of two antibodies directed at the MET receptor

Breast NSCLC, breast, melanoma, prostate, gastric, colorectal

Symphogen

39