Novel HER3 and IGF-1R Peptide Mimics and Synthetic Cancer Vaccines

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Megan Miller

Graduate Program in Microbiology

The Ohio State University

2014

Dissertation Committee:

Dr. Pravin Kaumaya, Advisor

Dr. Larry Schlesinger

Dr. Abhay Satoskar

Dr. Nicanor Moldovan

Megan Miller

2014

Abstract

Overexpression and constitutive activation of protein tyrosine kinases, including HER1 and HER2, are found in many human cancers and are critical factors in the development and malignancy of tumors. The downstream signaling networks of HER1 and HER2 have been extensively targeted by cancer therapeutics, and agents such as therapeutic monoclonal antibodies and small tyrosine kinase inhibitors (TKI) have been developed to block ligand binding, receptor dimerization, and intracellular tyrosine kinase activity.

Drugs approved by the FDA include TKIs such as gefitinib and erlotinib and therapeutic monoclonal antibodies such as cetuximab, pertuzumab and trastuzumab. HER3 (ErbB3) and IGF-1R are receptor tyrosine kinases that have only recently been recognized as important for the development and progression of cancer. These receptors are frequently upregulated in cancer and also may provide routes for resistance to agents that target

HER1 or HER2. Several recent studies have shown that HER3 and IGF-1R may be attractive targets against many types of cancer, including breast, ovarian, pancreatic, prostate, colon, head and neck, etc. Although there are no FDA approved therapies that target HER3 or IGF-1R, several monoclonal antibodies have been developed and are currently being evaluated in clinical trials. Our laboratory has proposed using peptides as candidate vaccines or therapeutic agents that target the HER family and IGF-1R. In particular, this study evaluates novel HER3 and IGF-1R peptide mimics expected to elicit an enduring immune response with protein reactive high affinity peptide antibodies. Our ii laboratory has identified several peptides of the HER3 and IGF-1R extracellular domain as potential B cell epitopes for active immunotherapy against HER3 and IGF-1R driven cancers. The peptide vaccines were immunogenic in outbred rabbits, and the vaccine antibodies and peptidomimetics induced anti-tumor responses, such as: inhibition of cancer cell proliferation, inhibition of receptor phosphorylation, induction of apoptosis and antibody dependent cellular cytotoxicity (ADCC). In addition, treatment with the peptidomimetics or vaccine antibodies significantly inhibited growth of xenografts originating from both pancreatic and breast cancers. In conclusion, we provide novel candidates for HER3 and IGF-1R targeted cancer therapy.

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Dedication

This document is dedicated to my husband.

Acknowledgments

I wish to thank The Ohio State University Microbiology Department for their willingness to support me as a Graduate Research/Teaching Associate. I would also like to thank my advisor, Dr. Pravin Kaumaya for the opportunity to work in his lab, as well as for his support, encouragement and constructive criticism.

I thank all of the members of the Kaumaya lab, especially Dr. Kevin Chu Foy and Jay

Overholser for their assistance, stimulating conversations and advice over the past 5+ years. I am also grateful for my committee members, Dr. Abhay Satoskar, Dr. Larry

Schlesinger and Dr. Nicanor Moldovan for their time and guidance.

Finally, I would like to thank God, my family and friends. I thank my parents, grandmother, mother-in-law and father-in-law for their love, advice, inspiration and encouragement to pursue my interests. I thank my brother, sister, sister-in-law, brother- in-laws and friends for being there for me and helping me maintain my sanity throughout this process. Lastly, I would like to thank my beloved husband for always believing in me and pushing me to be better in all areas of my life. Thank you, Luke, for being my rock!

Vita

June 2003 ...... Westland High School, Galloway, OH

June 2007 ...... B.S. Life Sciences, Otterbein University

2008 to present ...... Graduate Teaching and Research Associate,

Department of Microbiology, The Ohio

State University

Publications

Foy KC, Liu Z, Phillips G, Miller MJ, Kaumaya PT. Combination Treatment with HER-2 and VEGF Peptide Mimics Induces Potent Anti-tumor and Anti-angiogenic Responses in

Vitro and in Vivo. J Biol Chem 2011; 286, 13626-13637.

Foy KC, Miller MJ, Moldovan N, Carson III WE, Kaumaya PTP. Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo. OncoImmunology

2012; 1:1048–60

Foy KC, Wygle RM, Miller MJ, Overholser JP, Bekaii-Saab T, Kaumaya PT. Peptide vaccines and peptidomimetics of EGFR (HER-1) ligand binding domain inhibit cancer vi cell growth in vitro and in vivo. J Immunol. 2013; 191(1):217-27.

Miller MJ, Foy KC, Kaumaya PT. Cancer immunotherapy: present status, future perspective, and a new paradigm of peptide immunotherapeutics. Discov Med 2013;

15(82):166-76.

Fields of Study

Major Field: Microbiology

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

List of Tables ...... xi

List of Figures ...... xii

Abbreviations ...... xiv

Chapter 1 : Introduction to Peptide Immunotherapy and HER3/IGF-1R Signaling

Pathways in Cancer ...... 1

Targeted therapeutics in oncology ...... 1

Protein Tyrosine Kinases ...... 2

Monoclonal antibodies and tyrosine kinase inhibitors ...... 3

Peptide therapeutics in oncology...... 6

Human epidermal growth factor receptors and cancer...... 9

HER3 and Cancer ...... 11

HER3-targeted therapeutics in cancer ...... 13

The type I insulin-like growth factor receptor (IGF-1R) ...... 15

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Therapeutically targeting IGF-1R ...... 18

Drug resistance associated with HER-targeted therapeutics and receptor crosstalk ..... 19

HER3 and IGF-1R peptide mimics and vaccine antibodies ...... 20

Central Hypothesis and Overview of Chapters Two-Five ...... 21

Chapter 2 : Novel HER3 peptide mimics exhibit antitumor activity against HER3 positive cancers in vitro and in vivo ...... 23

Introduction ...... 23

Materials and Methods ...... 26

Results: ...... 30

Discussion ...... 35

Chapter 3 : Immunogenicity of HER3 peptide mimics and evaluation of cancer cell treatment with anti-HER3 peptide mimic antibodies in vitro and in vivo...... 49

Introduction: ...... 49

Materials and Methods: ...... 51

Results: ...... 56

Discussion ...... 59

Chapter 4 : IGF-1R peptide mimics, immunogenicity and anti-tumor activity ...... 70

Introduction: ...... 70

Materials and Methods: ...... 72

Results: ...... 77

Discussion: ...... 82

Chapter 5: Future directions and Conclusions ...... 101

References ...... 104

List of Tables

Table 2.1 The amino acid sequences of the synthetic HER3 peptide mimics and the chimeric peptides incorporating the MVF promiscuous T helper cell epitopes...... 38

Table 4.1 The amino acid sequences of all the IGF-1R peptides...... 86

List of Figures

Figure 2.1 Currently published monoclonal antibody: HER3 crystal structure complexes

...... 39

Figure 2.2 The crystal structure of the HER-3 extracellular domain (pdb 1M6B, modified)

...... 40

Figure 2.3 Western blot analysis of the expression of HER1, HER2, HER3 and IGF-1R in various cancer cell lines...... 41

Figure 2.4 HER3 peptide mimics inhibit proliferation of HER3 positive cancer cell lines.

...... 42

Figure 2.5 HER3 peptide mimics slightly inhibit phosphorylation of HER3 positive cancer cell lines...... 44

Figure 2.6 HER3 peptide mimics induce apoptosis...... 46

Figure 2.7 HER3 peptide mimics delay tumor growth in two transplantable mouse models ...... 47

Figure 3.1 Immunogenicity of MVF HER3 peptides (top) and ability of anti-HER3 vaccine antibodies to recognize HER3 (bottom) ...... 63

Figure 3.2 Direct binding of anti-peptide antibodies to native HER3 receptor. with goat anti-rabbit IgG-Alexa fluor 488 secondary antibody...... 65

Figure 3.3 HER3 vaccine antibodies induce apoptosis...... 66

Figure 3.4 MVF HER3 antibodies have the ability to elicit ADCC...... 67 xii

Figure 3.5 HER3 vaccine antibodies delay tumor growth in a transplantable mouse model...... 68

Figure 4.1 The crystal structure of the IGF-1R extracellular domain. pdb 1IGR, modified.

...... 87

Figure 4.2 IGF-1R peptide mimics inhibit proliferation of pancreatic and breast cancer cell lines ...... 88

Figure 4.3 Immunogenicity of IGF-1R peptides and ability of anti-IGF-1R vaccine antibodies to recognize recombinant IGF-1R ...... 90

Figure 4.4 Direct binding of anti-peptide antibodies to native IGF-1R receptor...... 92

Figure 4.5 IGF-1R peptide mimics and vaccine antibodies induce apoptosis ...... 93

Figure 4.6 IGF-1R peptide mimics and vaccine antibodies inhibit phosphorylation of breast and pancreatic cancer cell lines ...... 95

Figure 4.7 MVF IGF-1R antibodies have the ability to elicit ADCC...... 97

Figure 4.8 IGF-1R peptide mimics delay tumor growth in two transplantable mouse models...... 98

Figure 4.9 IGF-1R vaccine antibodies delay tumor growth in a transplantable mouse model...... 100

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Abbreviations

Akt protein kinase B

ErbB erythroblastosis viral oncogene homolog

Erk extracellular signal-related kinases

FDA Food and Drug Administration

Grb2 growth factor receptor bound protein 2

HER human epidermal growth factor receptor

HPLC high-pressure liquid chromatography

IGF-1 insulin-like growth factor 1

IGF-2 insulin-like growth factor 2

IGF-1R type I insulin-like growth factor receptor

IGF-2R type II insulin-like growth factor receptor

IR insulin receptor

IRS-1 insulin receptor substrate mAbs monoclonal antibodies

MALDI matrix-assisted laser desorption ionization mass spectroscopy

MAPK mitogen-activated protein

MEK mitogen activated protein kinase kinase xiv

PDGFR platelet-derived growth factor receptor (PDGFR)

PDK1 phosphoinositide-dependent kinase-1

PH pleckstrin homology domain

PI3K phosphoinositide 3 kinase

PIP3 phosphatidylinositol (3,4,5)-triphosphate

PTEN phosphatase and tensin homolog

PTKs protein tyrosine kinases

Ras rat sarcoma

SCFR/c-KIT mast/stem cell growth factor receptor

SH2 src homology 2 domain

Shc src homology domain containing transforming protein

SOS guanine nucleotide exchange factor son of sevenless

STATs signal transducers and activators of transcription

TKIs tyrosine kinase inhibitors

VEGFR vascular endothelial growth factor receptor

Chapter 1 : Introduction to Peptide Immunotherapy and HER3/IGF-1R Signaling

Pathways in Cancer

Targeted therapeutics in oncology

During the late 1800’s, bacteriologist Paul Ehrlich used histological cell staining techniques and hypothesized that “magic bullets” could be developed to selectively target human disease. This “magic bullet” concept referred to drugs that go straight to their intended target (pathogens or tumor cells) without eliciting harmful effects in normal cells and tissues. Erhlich’s achievements in the laboratory eventually expanded to the development of chemotherapy and have more recently inspired scientists to develop

“personalized and tailored” drugs to target cancer 1. In the past, chemotherapies were restricted to cytotoxic agents that target DNA synthesis and events that regulate cell division. These drugs are not specific for cancer and are notorious for causing a broad range of toxic side effects 2, 3. As a result, the prototype for cancer treatment has changed over the last few decades from cytotoxic drugs to more selective, targeted therapies 2.

Targeted therapeutics are designed to exploit genetic abnormalities and signal transduction pathways specific to cancer cells 4. One of the first rationally designed targeted anti-tumor drugs was tamoxifen, an anti-estrogen inhibitor that was introduced

1 into the clinical setting in the early 1970s. Since then, scientists have uncovered numerous other pathways that are deregulated in human cancers, and several novel targets for anticancer therapy have been identified. Targeted cancer therapies approved for use in specific cancers include drugs that interfere with cell signaling, inhibit tumor blood vessel development, promote apoptosis of cancer cells, stimulate the immune system to destroy cancer cells and deliver toxic agents to cancer cells.

Protein Tyrosine Kinases

Over the last few decades, molecular and genomic technologies have led to the discovery of new targets for cancer therapy. Among these targets, are protein tyrosine kinases

(PTKs), an important family of signaling proteins involved in many protein-protein interactions that form the basis of many cellular processes. Examples of PTKs include: members of the human epidermal growth factor receptor family (HER1, HER2, HER3 and HER4), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), insulin growth factor receptor (IGFR) and Mast/stem cell growth factor receptor (SCFR/c-KIT). PTKs are involved in cellular growth, proliferation, migration, differentiation and apoptosis 5. Most PTKs are transmembrane receptors that are activated upon ligand binding, while others are cytosolic proteins that become activated downstream of transmembrane receptors or other signaling proteins.

Activation of PTKs results in phosphorylation of intracellular signaling proteins that link

PTKs with multiple signaling pathways, such as phosphoinositide 3 kinase

(PI3K)/protein kinase B (Akt), Rat sarcoma/mitogen-activated protein kinase

(Ras/MAPK), signal transducers and activators of transcription (STATs), phospholipase

C (PLCγ), etc. 6-8. It is now well known that deregulation of kinase signaling pathways is extremely common in cancer, and this can occur via genetic mutations, gene amplification, overexpression and/or chromosomal translocation 9-11. The human genome encodes 90 genes with putative tyrosine kinase activity, and there are approximately 60 receptor tyrosine kinases that have been identified. Of these, 20 have already been implicated in cancer 12, 13. PTKs are now widely recognized as attractive targets for anticancer drugs, and there is an intense research effort to design inhibitors that target deregulated kinase signaling pathways. Today, several inhibitors of PTKs have been approved by the Food and Drug Administration (FDA) for treatment of specific types of cancer, and others are being studied in clinical trials and preclinical testing.

Monoclonal antibodies and tyrosine kinase inhibitors

At present, there are two main PTK targeted therapies available for use in clinical practice, and they include monoclonal antibodies (mAbs) and tyrosine kinase inhibitors

(TKIs). Over the last couple of decades, monoclonal antibody-based therapy has become established as a successful and important strategy for treating patients with various malignancies and solid tumors. Therapeutic mAbs are synthetically derived in the laboratory and are designed to mimic the antibodies naturally produced by the human body’s immune system to combat pathogens, vaccines and other invaders. Antibodies

3 have been selected as anti-cancer therapeutics due to their long serum half-lives and capacity to bind with high specificity and affinity to a wide variety of molecules 14. In addition, their ability to mediate target-specific inhibition and immune-mediated tumor suppression has enabled antibodies to demonstrate improved efficacy over standard chemotherapeutic drugs. MAbs exert an anti-tumor effect through several different mechanisms, including: induction of apoptosis, inhibition of tumor cell signaling and activation of complement dependent cytotoxicity or antibody dependent cellular cytotoxicity 15-20. The first therapeutic anti-cancer antibody to be approved by the FDA was rituximab, a humanized anti-cluster of differentiation (CD) 20 antibody used for treatment against B-cell lymphoma. Since then, the FDA has approved 12 antibodies for use in oncology 15, and these therapies have significantly improved treatment and clinical outcomes for many cancer patients around the world. Tyrosine kinase inhibitors (TKIs), on the other hand, represent a class of small molecule compounds that compete with adenosine triphosphate (ATP) binding and inhibit kinase activity. In 2000, imatinib was introduced into clinical settings, and this small molecule drug binds to the ATP pocket of the Abelson kinase (ABL), as well as c-KIT and PDGFR 21-23. Imatinib has demonstrated successful results in the clinic because breakpoint-cluster region-ABL

(BCR-ABL) fusion proteins are present in over 90% of human patients with chronic myeloid leukemia (CML), and approximately 15-30% of adult patients with acute lymphoblastic leukemia (ALL) 24-27. In general, TKIs are relatively inexpensive to synthesize, and they are usually orally available and easily administered 28. Their small size (usually <500 Da) also allows them to translocate through the plasma membrane and

4 interact with the cytoplasmic domain of cell-surface receptors and intracellular signaling molecules. To date, approximately 15 kinase inhibitors have received FDA approval as cancer treatments, and there are considerable efforts to develop selective small molecule inhibitors against other kinases implicated in cancer and other diseases 29 .

Although mAbs and TKIs have significantly improved treatment and clinical outcomes for millions of cancer patients around the world, the harsh reality is that the 40 year old

“War on Cancer” still prevails. Conventional chemotherapeutic drugs remain the backbone of current treatment, and major limitations, such as acquisition of drug resistance and patient relapse still exist with currently marketed mAbs and TKIs.

Monoclonal antibody limitations include: repeated treatments, high costs, inadequate pharmacokinetics and tissue accessibility, limited duration of action, undesired immunogenicity and toxicity 14. Antibodies are also large proteins (around 150 kDa) and are widely considered incapable of passing through the cellular membrane, therefore, these drugs can only act on molecules that are either expressed on the cell surface or secreted. Significant hurdles also exist for the development of tyosine kinase inhibitors.

In general, TKIs are thought to be less specific than mAbs, their half-lives are much shorter, and there is a high level of variation in toxic side effects. Developers of small molecule inhibitors are also limited to the number of potential drug candidates they can target, because they lack a cavity for the small organic inhibitors to bind and interact with protein partners through extensive and flat surfaces. New drugs targeting protein-protein interactions often require larger interaction sites than small molecules can offer, and

5 screening efforts have consequently been limited to a small fraction of the proteome (ion channels, nuclear receptors, GPCRs or enzymes) 30-32. In a recent study, an estimated

60% of small molecule drug discovery projects fail due to the biological target being‘”undruggable” 33, 34. Ultimately, the future success of targeted therapeutics relies on overcoming the obstacles associated with mAbs and TKIs.

Peptide therapeutics in oncology

A relatively newer class of targeted therapeutic drugs is emerging in cancer drug development, and these drugs are called peptide therapeutics. Peptides are short sequences of amino acids that can be produced biosynthetically, via natural or recombinant microbial fermentations, or chemically, through mechanisms such as solution–based or solid-phase peptide synthesis. The concept of using peptides as therapeutics has been historically disregarded, mainly due to peptide stability, susceptibility to degradation, size (>5000 Da) and consequent limitations in methods of delivery (low oral bioavailability). In recent years, however, the development of peptides as drugs is attracting increasing attention from the pharmaceutical industry; especially as the technologies for peptide development and manufacturing continue to mature. New synthetic strategies to improve productivity and reduce metabolism of peptides, along with alternative routes of administration have been developed in recent years, and a large number of peptide-based drugs are now being marketed 35. As of 2010, there were approximately 60 therapeutic peptides on the market, 150 in clinical phases and 400 in

6 advance pre-clinical stages. Peptide drugs are now being universally marketed, and more than 100 pharmaceutical and biotech companies are now active in the peptide field. Of the products now approved, about 30 peptide drugs are currently marketed in the U.S., and 17% are used in the cancer clinical setting 36. The peptide therapeutics foundation anticipates that pharmaceutical and biotechnology industries will continue to focus on these versatile molecules because of the increased acceptance of injected drugs on the market, the availability of new formulation and delivery technologies, and the relatively high approval success rates.

Development of peptide therapeutics has the potential of overcoming some of the obstacles observed with mAbs and TKIs. Currently, one of the greatest strengths of peptides as potential therapies lies in the powerful new approaches for discovering and screening new drug candidates. In theory, peptide-based drugs have given oncologists an opportunity to expand the repertoire of “druggable proteins.” As mentioned before, mAbs and TKIs are limited to the number of candidates they can effectively target; mAbs are too large and complex for entry into the cell, while small molecule inhibitors are limited to proteins with well-defined small binding pockets. Peptides are also small enough to penetrate the cell membrane, and they can be rationally designed to target almost any protein of interest 37. Peptides now have a broad range of potential clinical benefits against numerous protein targets, with applications in some of the most prevalent disease categories 38. These molecules are rationally designed to mimic the binding region between two or more proteins involved in various disease states. A perfect

7 peptide agent is designed rationally and has the ability to reach, bind and antagonize the function of a target protein with high selectivity and affinity 31. Their small size and specific binding properties make peptides ideal candidates for physiologically disrupting on-target functional protein-protein complexes. Peptide-therapeutics may consequently aide in the reduction of current therapeutic problems, such as off-target side effects and the requirement of high dosage treatment.

Other advantages of peptide therapeutics include: low cost, higher specificity and potency; due to their superior compatibility with targeted proteins, ability to penetrate the cell membrane, reduced immunogenicity and improved safety. In addition to their high specificity, peptides are now easily synthesized and very amenable to site-specific modification 39. Currently, chemical synthesis has enabled easy conjugation of small molecules and incorporation of non-natural amino acids by design, and peptides can now be easily modified to enhance their pharmacokinetic and pharmacodynamic profiles 40.

The most common modifications found in marketed peptides include: glycosylation, pegylation, lipid incorporation, antibody incorporation and radiolabeling. Modifications such as cyclisation, glycosylation, pegylation and incorporation of D-amino acids all help to extend the half-life of a peptide by rendering it unrecognizable to proteases 40-45.

Peptide modifications can also overcome current therapeutic limitations, such as inefficient drug delivery to the target site and adverse off-target effects. Today, peptides can be developed to act as homing devices for specific tissue types or organs, and cell- penetrating peptides provides a promising solution to drug delivery 46, 47.

Human epidermal growth factor receptors and cancer

The human epidermal growth factor receptor (HER or ErbB) family belongs to subclass I receptor tyrosine kinases (RTKs) and consists of four homologous members: HER1 (also known as EGFR), HER2, HER3 and HER4 (also known as ErbB2, ErbB3 and ErbB4, respectively). These receptors are expressed in tissues of epithelial, mesenchymal and neuronal origin and, under normal physiological conditions, they play fundamental roles in cellular growth and development 48, 49. All members have common structural features, including: an extracellular ligand-binding domain, a transmembrane region, and an intracellular domain containing a tyrosine kinase and carboxy-terminal region 50-57. The extracellular region consists of approximately 620 amino acids arranged into four domain structures with two L domains (I and III) and two cysteine-rich domains (II and IV) 58.

In response to a ligand , the receptor tyrosine kinases can form homodimers or heterodimers and initiate a complex network of cellular signaling that mediates many cellular processes, such as proliferation, differentiation, migration and survival 59, 60.

Specifically, ligands bind to domains I and III and cause a conformational change, in which domain II becomes exposed, and the receptor can then undergo dimerization 61.

Receptor dimerization is an essential requirement for HER function and for the signaling activity of these receptors 49.

HER family members are also involved in tumor genesis when their signaling functions are inefficiently regulated 50, 59, 60, 62-65. This role in cancer was first implicated in the early 1980’s, when the avian erythroblastosis tumor virus was found to encode an aberrant form of the human epidermal growth factor (EGF) receptor 66. Since then, intense research has focused on the role of these receptors in cancer development, and

ErbB receptors (particularly EGFR and HER2) have become widely known targets for cancer therapy. Deregulation of HER family signaling is relatively common in cancer and can occur via gain-of-function mutations, gene amplification, overexpression and/or chromosomal translocation 9, 67, 68. Over the last few decades, numerous approaches have been taken to target ErbB receptors, and two types of antagonists have been successful in the clinic: mAbs) and small molecule TKIs. Many of these therapies that target HER1 and/or HER2 are either in clinical use or in advanced clinical development.

TKIs that target HER1, such as gefitinib and erlotinib, have been approved for the treatment of non-small cell lung carcinomas (NSCLC) and head and neck squamous cell carcinomas (HNSCC). Other small molecule inhibitors, such as lapatinib, are being evaluated in advanced clinical trials. MAbs have also been developed against the extracellular domains of the HER family members, and they exert their anti-tumor effect via several different mechanisms, including: inhibition of ligand binding or dimerization, induction of apoptosis, inhibition of tumor cell signaling and activation of complement dependent cytotoxicity or antibody dependent cellular cytotoxicity 15-20. Cetuximab, a chimeric anti-HER1 antibody was approved for treatment of patients with advanced colorectal cancer in 2003, while trastuzumab, a humanized anti-HER2 antibody, has been

10 approved for treatment of HER2 positive breast carcinomas. Other monoclonal antibodies in clinical development include pertuzumab (a humanized anti-HER2 antibody), panitumumab (a humanized anti-HER1 antibody) and matuzumab (a humanized anti-HER1 antibody). Although development of HER-targeted therapeutics

(mAbs and TKIs) has improved cancer treatment significantly, these current treatments are not completely curative, and they have several drawbacks, including: the necessity of repeated administration, severe cardio toxicity, and high cost. Intrinsic or developed resistance to these targeted therapies is also a major problem frequently occurring in the clinic.

HER3 and Cancer

Despite their structural homology, the HER family members differ with respect to preferred ligands, affinity for ligands, tyrosine kinase activity, and rate of cellular down regulation 69. In particular, HER2 has no known ligand binding activity and exists in a constitutively active conformation. When HER1 or HER3 bind to their respective ligands, HER2 is frequently recruited to these ligand-receptor complexes, where it serves as a common co-receptor 70. In contrast to HER2, HER3 is the only member of the family lacking intrinsic tyrosine kinase activity, and it is entirely dependent on heterodimerization for signaling 71-74. HER3 also fails to transform cells when overexpressed or constitutively activated by continuous ligand stimulation, and there are no known mutational alterations known to confer oncogenic activities to HER3 75. As a

11 result, its role in cancer has long been underestimated, and efforts at targeting HER3 have lagged behind. In recent years, however, HER3 has emerged as a key player in the establishment of malignancy. An increasing amount of evidence has demonstrated that

EGFR and HER2 are the preferred dimerization partners for HER3, and that HER3 is frequently up-modulated in cancers with EGFR or HER2 over-expression 76-79. In addition, the heterodimer of HER2/HER3 constitutes the most potent receptor with respect to strength of interaction, ligand-induced tyrosine phosphorylation, and downstream signaling 49, 80. It forms a high affinity heregulin receptor with kinase activity and mediates the most mitogenic signal in vitro 74, 76, 77, 81, 82. The combination of HER2 and HER3 receptors may be critical in breast cancer growth and progression, and HER3 may be a necessary partner for the oncogenic activity of HER2 83-87. Recent studies have also demonstrated that HER3 is an obligate partner for HER2 mediated transformation 77 and that it may provide a route for resistance to agents that target

EGFR or HER2.

Most tumors require the PI3K/Akt (phosphoinositol 3-kinase/protein kinase B) signaling for their survival, and this is often achieved by inactivation of PTEN (phosphatase and tensin homolog), mutational activation of PI3K or upstream receptor tyrosine over- activity. When compared to other ErbB receptors, HER3 has the highest binding affinity for PI3K and serves as a crucial activator in downstream signaling of the kinase. HER3 phosphorylated tyrosine residues recruit PI3K, which activates membrane phosphoinositides and generates phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3

12 then recruits and activates the pleckstrin homology (PH) domain of phosphoinositide- dependent kinase-1 (PDK1) and protein kinase B (AKT/PKB). AKT phosphorylates and controls the activities of many downstream signaling molecules involved in cellular growth, proliferation, apoptosis and metabolism 88, 89. Induction of HER3 activation and subsequent PI3K/AKT signaling has been shown to 1) induce apoptosis resistance in a wide range of cancers 68, 90-98 and 2) provide a route for resistance to agents that target

EGFR and HER2 86, 99-101. An increasing amount of evidence suggests that HER3 contributes to escape from therapeutic suppression by several tyrosine kinase inhibitors in cancer 83, 86, 87. In fact, HER3 expression or signaling is associated with resistance to:

HER2 inhibitors in HER2-amplified breast cancers 86, 87 EGFR inhibitors in lung cancers

102, pertuzumab resistance in ovarian cancers 103, anti-estrogen therapies in ER-positive breast cancers 103-106, EGFR inhibitors in head and neck cancer , and hormone resistance in prostate cancers 107.

HER3-targeted therapeutics in cancer

Currently, there are no FDA approved therapies that target HER3, but several monoclonal antibodies that bind the HER3 extracellular domain are under investigation. Merrimack

Pharmaceuticals has developed MM-121, and this antibody was shown to effectively block ligand-dependent activation of HER3 and inhibit tumor growth in xenograft models of ovarian, prostate and kidney cancers. In 2008, MM-121 became the first selective

HER3 antagonist to enter into human clinical development, and it is currently being

13 evaluated in clinical trials 98, 108. Other antibodies developed include Amgen/U3-

Pharma’s AMG 888 (U3-1287) and Novartis/Sanofi Aventis’ LMJ718 98, 108-110. U3-1287 has been shown to bind to the extracellular domain and elicit anti-tumor effects in vitro and in vivo via receptor internalization and inhibition of ligand-induced receptor signaling 110. U3-1287 has also been evaluated in a phase I clinical trial, and it was shown to be well-tolerated and cause some disease stabilization. LJM716 does not compete with ligand binding to HER3, but it has been shown to potently inhibit ErbB3/

Akt phosphorylation and proliferation of HER3 dependent cell lines in vitro and in vivo.

111. These antibodies have also been shown to act synergistically with EGFR/HER2 inhibitors, suggesting that combination treatment may be essential to completely shut- down Erbb signaling 98, 112. In addition, dual-specific antibodies against HER2:HER3 or

EGFR: HER3 heterodimers are also being evaluated 113, 114. Genentech has developed

MEHD7945A (DL11), which binds to the extracellular domains of both HER1 and

HER3, and shuts down signaling of both receptors simultaneously 114. Merrimack

Pharmaceuticals has also developed an antibody, called MM-111, which recognizes both

HER2 and HER3. When compared to mono-specific therapeutics, both dual-specific antibodies were more broadly efficacious in multiple tumor models 115.

The type I insulin-like growth factor receptor (IGF-1R)

The insulin-like growth factor (IGF) signaling axis involves the coordinated function of three cell surface receptors (insulin receptor, type I insulin-like growth factor receptor, and type II insulin-like growth factor receptor ) the ligands IGF-1 and IGF-2, and a system of at least six binding proteins (IGF binding proteins 1-6) and attendant proteases. The type I insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase consisting of two extracellular (ligand binding) α chains and two membrane- spanning β chains that encompass an intracellular kinase domain devoted to the initiation of signaling cascades. The IGF-1R has the highest affinity for IGF-I and binds IGF-II and insulin with 5-10 and 100 fold lower affinities, respectively. Following ligand binding to the α chains, the intrinsic kinase activity of the β chains is activated, resulting in auto-phosphorylation of several tyrosine residues that provide docking sites for several substrates, including insulin receptor substrate (IRS-1) and Src homology domain containing transforming protein (Shc). Activated IRS-1 can initiate the phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) pathway and recruit the adaptor protein growth factor receptor bound protein 2 (Grb2), which can associate with son of sevenless (SOS) to activate the mitogen activated protein kinase (MAP) kinase kinase (MEK)/extracellular signal-related kinases (ERK) signaling pathway. These signaling pathways primarily result in cellular proliferation, transformation and inhibition of apoptosis.

The closest relative of IGF-1R is the insulin receptor (IR) with more than 70% sequence homology. Historically, IGF-1R was considered a redundant receptor used by cells only when signaling from the IR was absent or defective. In the past few years, however,

IGF-1R has emerged as a receptor with unique characteristics that differentiate it sharply from the IR. To simplify the differences between the two receptors, IGF-1R seems to play an important role in cancer biology, and it is the predominant receptor involved in mitogenesis, transformation and protection from apoptosis. Early evidence for this role in cancer was provided by experiments demonstrating that mouse embryonic fibroblasts lacking IGF-1R could not be transformed by the SV40 T antigen 116. Since then, several lines of evidence have linked the IGF-1: IGF-1R signaling pathway to the growth and development of many different types of malignancies 117 and resistance to anti-cancer drugs available in the clinic 118, 119. It is now well documented that IGF-1R plays a significant role in the initial steps of malignant transformation and also in the later stages of tumor progression 120, 121. Up-regulated levels of the receptor and its cognate ligands have been observed in a variety of solid human tumors 122-124. For example, it is now widely known that IGF-1R is present in all ovarian cancer cells 125 and its signaling causes decreased apoptosis, increased proliferation and increased cancer cell survival 126.

Small interfering RNAs targeting the insulin-like growth factor receptor-1 have been shown to decrease growth of OVCAR3 ovarian cancer cells in vitro and in vivo 127 and recent studies have also implicated IGF-IR as a novel therapeutic target in ovarian cancer

128. IGF-1R overexpression has also been shown to play a critical role in cancers of the lung 129, breast 130, pancreas 131, colon 132-135, prostate 136, 137, kidney 138 skin 139 and head

16 and neck 140. In addition, elevated levels of circulating IGF-1 have been associated with an increased risk of developing breast, prostate and colorectal cancers 141.

Signaling through the IGF-1 receptor appears to be important for most of the hallmarks of cancer, including evasion from apoptosis (survival), angiogenesis, self-sufficiency in growth signals and tissue invasion/metastasis. The role of IGF-1R in resistance to apoptosis was first described in the early 1990’s, when Baserga and colleagues demonstrated that downregulation of IGF-1R resulted in massive apoptosis of tumor cells growing in anchorage independent conditions 142. Since then, several reports have consistently shown that downregulation of IGF-1R causes apoptosis and growth inhibition of a variety of different cancer cells , including glioblastoma, melanoma, neuroblastoma, prostate cancer, colon cancer, rhabdomyosarcoma, lung cancer, Ewing’s sarcoma and medulloblastoma 143. It is now widely known that IGF-1R activation is a critical determinant of tumor cell resistance to apoptosis, and this occurs primarily through the PI3K signaling pathway 144-146. IGF-1R has also been shown to play a role in angiogenesis, or the growth of new blood vessels from pre-existing vasculature. The continued growth of solid tumors depends on an adequate blood supply for oxygen and nutrients, and angiogenesis is a necessary process for solid tumors to grow beyond a few millimeters. IGF-1R has been shown to regulate expression and activity of several critical mediators of angiogenesis. In particular, IGF-1 was shown to activate expression of VEGF-A 147, the major pro-angiogenic factor, in several cancer cell types, including

17 thyroid carcinoma 148, colon carcinoma 149, multiple myeloma 150 and pancreatic cancer

147.

Therapeutically targeting IGF-1R

While development of HER1 inhibitors began approximately 15 years ago, the IGF axis has only recently been recognized as a drug target, and there are no FDA approved therapies directly targeting this signaling pathway. However, various strategies have been used at the preclinical level to interfere with IGF-1R function, and recent estimates suggest that about 30 agents have been developed to target this receptor 151, 152.

Currently, there are at least 58 active clinical trials evaluating these agents alone or in various combinations 152, and the most clinically viable options include usage of antagonistic mAbs and small molecule TKIs. Unfortunately, design of IGF-1R TKIs has been complicated due to the fact that the kinase domain of IGF-1R and IR are 85% homologous, and the ATP binding cleft is 100% conserved 153. Some groups have attempted to exploit the subtle differences in sequences between the IGF-1R and IR kinase domain, and inhibitors such as nordihydroguaiaretic acid and cyclolignan picropodophyllin (PPP) have been developed. These inhibitors act as competitive inhibitors of substrate phosphorylation and have been shown to inhibit IGF signaling and growth of human breast and prostate cancer cells in vitro 154, 155. Antibodies targeting

IGF-1R are much more selective for the receptor, and reports have suggested that such antibodies can block ligand binding, decrease cell surface receptor expression, and block

18 intracellular signaling, particularly through the PI3K pathway 156-159. IMC-A12, a fully humanized mAb has been shown to produce significant growth inhibition of breast, pancreatic and renal xenografts. Other antibodies, such as EM164 (AVE-1642), CP-

751,871 (figitumumab) and MK-0646, have also been developed that show anti-tumor activity in a variety of different carcinomas 160-163. These antibodies are specific for IGF-

1R, and cross-reactivity with IR has not been observed. IGF-1R antibodies have also been shown to produce additive effects when combined with traditional chemotherapeutic agents, such as gemcitabine, doxorubicin and vinorelbine 161-163.

Drug resistance associated with HER-targeted therapeutics and receptor crosstalk

Although development of HER-targeted therapeutics (mAbs and TKIs) has improved cancer treatment significantly, the harsh reality is that intrinsic or developed resistance to these targeted therapies is a major problem frequently occurring in the clinic. Several molecular mechanisms of acquired resistance to these agents have now been documented, and extreme cross-talk exists between the HER family and other receptors, like IGF-1R and VEGFR 164. Compensatory HER3 signaling and sustained PI3K/AKT activation provide a route for resistance to agents that target EGFR and HER2 86, 99-101. In addition to HER3, recent data indicates that HER family member signaling and IGF-1R crosstalk is important for carcinoma growth and survival 165-168. Heterodimers between IGF-1R and HER1/HER2 have been identified as one mechanism of resistance of breast cancer cell lines to erlotinib and trastuzumab 169-174. In addition, increased expression of IGF-

1R causes induction of HER1 171, 172 and stimulates angiogenesis by upregulating VEGF expression 175. Promising and new alternative strategies taken to overcome drug resistance include combination therapy and development of multi-target inhibitors 11.

HER3 and IGF-1R peptide mimics and vaccine antibodies

In this study, we have evaluated the in vitro and in vivo efficacy of a relatively new type of targeted therapy that involves using peptide therapeutics to inhibit HER3 and IGF-1R.

The innovation of our approach lies in the fact that we incorporate chimeric peptide vaccines that stimulate both B and T cells to elicit high affinity antibodies and establish immunological memory. Previously, the design of structurally defined epitopes in our laboratory was dependent upon information obtained from computer-aided analysis and molecular modeling. However, the crystal structures of HER3 and IGF-1R have both been solved 52, 176, and we have gained significant insights into the ligand: receptor interactions. We have synthesized peptides based on the contact sites between IGF-I and

IGF-IR. In addition, HER3 crystal structures in complex with three therapeutic monoclonal antibodies (DL11, LMJ716 and RG7116) have been published over the last few years, and we have identified several HER3 peptides (based on antibody: receptor binding) as potential B cell epitopes for active immunotherapy against HER3 positive cancers. This research project stems from work conducted in our laboratory over the last several years. Our lab has developed effective vaccines against HER-2 and novel therapies based on blockade of receptor: ligand interactions, such as B7:CD28 63, 80, 177,

178. Two HER-2 B cell epitope vaccine candidates established their anti-tumor effects in preclinical studies, and we were able to translate our novel findings into a phase I clinical trial in which patients were vaccinated with a combination of the two vaccine epitopes

179-183. All patients had heavily treated advanced solid tumors and were unresponsive to all prior drug regimens. The vaccine was immunogenic in most of the patients, no serious effects of toxicity were reported and 25% showed clinical benefits 183. Clinical findings from this study validated our hypothesis that the antibodies to the vaccine were able to disrupt signaling pathways in cancer.

Central Hypothesis and Overview of Chapters Two-Five

In this study, we report on the anti-tumor effects of novel HER3 and IGF-1R peptide mimics and vaccine antibodies. The main objectives of this study were 1) to develop

HER3 and IGF-1R peptide mimics that could disrupt HER3/IGF-1R signaling pathways by preventing ligand binding or dimerization and 2) to identify epitopes of the IGF-1R and HER3 extracellular domains that could activate the immune system to produce highly specific antibodies that will target tumor cells. The driving motivation behind these studies rests upon the hypothesis that immunotherapy targeting HER3/IGF-1R will provide effective antitumor immunity, tumor regression and control of HER-3/IGF-1R overexpressing cancers. Chapter two focuses on the design and synthesis of novel HER-

3 peptide mimics and evaluation of the anti-tumor effects of the peptide mimics in vitro and in vivo. Chapter three describes the rational design of chimeric HER3 peptide

21 vaccines, immunogenicity and anti-tumor responses of the vaccine antibodies both in vitro and in vivo. Chapter 4 evaluates design, synthesis and treatment with novel IGF-1R peptide mimics and anti-IGF-1R peptide antibodies. In these chapters, we show that the

IGF-1R/HER3 peptide mimics and vaccine antibodies inhibited cancer cell proliferation, inhibited receptor phosphorylation, induced cancer cell apoptosis and significantly inhibited growth of xenografts originating from both pancreatic and breast cancers. In

Chapter 5, we hypothesize that targeting HER3 in combination with other HER family members or IGF-1R will produce synergistic anti-tumor effects with long-term control of many different types of cancer.

Chapter 2 : Novel HER3 peptide mimics exhibit antitumor activity against HER3 positive cancers in vitro and in vivo

Introduction

In 1989, HER3 became the third member of the human epidermal growth factor receptor family, and it maps to human chromosome 12q13 184. Since then, research dealing with the ErbB receptors has mainly focused on the dysregulated kinase activities of HER1 and

HER2. HER3 is the only member of the family lacking intrinsic tyrosine kinase activity, and its role in cancer has long been underestimated 71-73. Recently, however, an increasing amount of evidence has demonstrated that HER3 is an obligate partner for

HER receptor dimerization and a key mediator of primary and acquired resistance to

HER2 or HER1 targeted therapies. In addition, upregulation of HER3 is commonly observed in various malignancies, such as: breast cancer, colorectal carcinoma, head and neck carcinoma, melanoma, gastric cancer, ovarian cancer, prostate cancer and bladder cancer 76, 101, 185-187. The C terminal region of HER3 contains six consensus phosphotyrosine sites which bind the Src homology 2 (SH2) domain of PI3K, indicating a crucial role in the PI3K/Akt signaling pathway 97, 188, 189. Compensatory HER3 signaling and subsequent PI3K/Akt activation have been shown to play an important role

23 in resistance to HER-targeted therapeutics 49, 86, 190-192. Currently, there are no FDA approved therapies that target HER3, but several monoclonal antibodies that bind to the

HER3 extracellular domain are under investigation, including MM-121, MM-111,

MEHD7945A, RG7116, LMJ716 and AMG 888 98, 108, 111, 112, 114, 115. These monoclonal antibodies have been shown to inhibit tumor cell proliferation in vitro and in vivo, and some of them are being evaluated in clinical trials.

In this study, we evaluated the in vitro and in vivo efficacy of a relatively new type of targeted therapy that involves using peptide mimics to inhibit HER3. The main objective of this study was to develop HER3 peptide mimics that could disrupt HER3 signaling pathways by preventing ligand binding or dimerization. Peptide mimics are small protein-like chains of amino acids designed to antagonize a protein by mimicking one portion of the entire protein; usually a biologically active site or binding site of an enzyme 193, 194. These drugs offer the benefits of being water soluble, non-immunogenic, and having the ability to easily cross tissue barriers [83]. Since protein-protein interactions play many key roles in cancer, including cancer cell growth, drug resistance, metastasis and survival, peptide mimics can be developed to antagonize these interactions. Peptide mimics that block receptor-ligand interactions can be designed by screening combinatorial libraries of compounds, but the preferred approach is to use structural-based design. This research stems from work conducted in our laboratory over the last several years, in which we have developed effective peptide mimics against HER2 and novel peptide-therapies based on blockade of receptor: ligand interactions, such as B7:CD28 80, 177, 178.

Previously, the design of structurally defined epitopes in our laboratory was dependent upon information obtained from computer-aided analysis and molecular modeling.

However, the crystal structure of HER3 was solved in 2002, and since then, several therapeutic monoclonal antibodies have been generated against HER3. HER3 crystal structures in complex with three monoclonal antibodies, DL11, LMJ716 and RG7116, have been published over the last few years, and we have gained significant insights into the key HER3 amino acids involved in binding to the antibodies 111, 114, 195. Our laboratory identified several regions of HER3 that overlap the mAb binding sites, and we developed peptides encompassing residues 99-122, 140-162, 237-269 and 469-471 of

HER3 as potential peptide mimics for therapy against HER3 positive cancers. The basic hypothesis in the design of these HER3 peptide mimics is that many proteins exert their biological activity through relatively small regions of their folded surfaces, and the key residues of the binding epitope (receptor:ligand or receptor: receptor) may be transferred to a much smaller molecule while retaining binding capabilities [149]. Many biochemical processes require intermolecular interaction and recognition between complementary binding sites, and disruption of these interactions by small peptide antagonists has merit for rational drug design. The goal is to maintain the conformational space and orientation of the bioactive surface while retaining sufficient flexibility to bind cooperatively with a given receptor. The HER3 monoclonal antibodies discussed above have been shown to either inhibit ligand binding or receptor dimerization, and we hypothesize that our smaller molecule peptide mimics will act correspondingly. In this study, we report on the anti-tumor activity of the four HER3 peptide mimics in vitro and in vivo. 25

Materials and Methods

Design, Synthesis and Characterization of the HER3 peptide mimics. Peptide synthesis was carried out on a Milligen/Biosearch 9600 peptide solid phase synthesizer

(Bedford, MA) using Fmoc/t-butyl chemistry as previously described 196. CLEAR acid or amide resins were used for synthesis of the constructs. After synthesis, peptides were cleaved from the resin using cleavage reagent B (trifluoroacetic acid/phenol/water/TIS

90:4:4:2), and crude peptides were purified by semi-preparative reversed-phase HPLC

(high pressure liquid chromatography). After synthesis, cleavage and purification, the peptides were characterized by analytical HPLC and MALDI (matrix-assisted laser desorption ionization mass spectroscopy) at Chemical Instrumentation Center (The Ohio

State University, Columbus, OH). After confirming the correct molecular weight (Figure

2, 3), the peptides were lyophilized and dissolved prior to use in subsequent assays.

Protein Expression in cancer cell lines. Western blotting for total Erbb3, Erbb1, Erbb2 and IGF-1R was performed to determine total protein expression in cancer cell lines.

One million cells/well were plated in 6 well plates and incubated at 37⁰C until cells were

70-80% confluent. Culture media was then removed from the wells, and cells were washed with PBS. Cells were lysed with 1X RIPA Lysis Buffer (Santa Cruz) for 2.5 hours at 4⁰C. Cell lysates were spun at 13000xg and debris-free supernatants were transferred into clean tubes. Protein concentration of each sample was measured by

Coomassie plus protein assay reagent kit (Pierce). Lysates were frozen at -80⁰C. Protein expression was measured using western blotting with rabbit polyclonal antibodies for

HER1 (Cell Signaling #4405), HER2 (Cell Signaling #4290), HER3 (Santa Cruz sc-285) and IGF-1R (Cell Signaling #9750). A β-actin antibody (Abcam Ab8227) was used to control for loading. Detection was accomplished using goat anti-rabbit HRP secondary antibody (Bio-rad 170-5046) and Immun-StarTM HRP Chemiluminescent Kit (Bio-Rad).

All procedures were performed according to the manufacturer’s instructions.

Cell lines. BT474, MCF7, MDA-MB-453, MDA-MB-468, BxPC3, HT-29, SKOV-3,

T47D cancer cells were purchased from the ATCC. JIMT-1 cells were generously provided by Rita Nahta’s lab in Atlanta, Georgia. BxPC3 and T47D cells were cultured in RPMI1640 supplemented with 10% FBS, 1% pen-strep. MCF7, MDA-MB-453,

MDA-MB-468, JIMT-1 and BT474 cells were cultured in DMEM supplemented with

10% FBS, 1% pen-strep. All cells were grown at 37⁰C in 95% air, 5% CO2.

MTT Inhibition Assay. Cells were seeded in 96 well flat bottom plates at 1 x 104 cells/well in 100 ul growth media and allowed to adhere overnight at 37⁰C. Growth media was then replaced with low-sera (1% FBS) media, and cells were incubated overnight. Media was removed from the wells and replaced with peptides made up in 1% growth media. Plates were incubated for 1 hour at 37⁰C, and 50ng/mL HRG was added in 1% growth medium. Plates were incubated an additional 72 hours at 37⁰C before 25

27 uL of 5mg/mL MTT was added to each well. Plates were incubated for 2 hours at 37⁰C, then 100 uL extraction buffer (20% SDA,50% DMF, pH 4.7) was added. Plates were incubated overnight at 37⁰C and read on an ELISA reader at 570nm.

HER3 phosphorylation Assay. A Human phosphor-ErbB3 ELISA kit (R & D Systems) was used to measure the amount of phosphorylated HER3. One million cells/well were plated in 6 well plates and incubated at 37⁰C overnight. Culture media was removed from the wells, and cells were washed with PBS. Cells were treated with 150 µg HER3

237 peptide or anti-peptide antibodies in binding buffer (0.2& BSA, RPMI 1640, 10mM

HEPES (pH 7.2) for 1 hour at 37⁰C. 5nM Heregulin was added, and plates were incubated at RT for 10 min. After stimulation, cells were lysed with 1X RIPA Lysis

Buffer (R + D systems) for 2.5 hours at 4⁰C. Cell lysates were spun at 13000xg and debris-free supernatants were transferred into clean tubes. Protein concentration of each sample was measured by Coomassie plus protein assay reagent kit (Pierce). Lysates were frozen at -80⁰C. Phosphorylated HER3 was measured using the DuoSet IC for human phsopho-ErbB3 (R + D Systems, Minneapolis, MN).

Apoptosis Assay. The capsase glo 3/7 kit (Promega) was used to measure the ability of the peptides to induce apoptosis. 1x106 cells were seeded and incubated overnight at

37ºC. Cells were then treated with the peptides or vaccine antibodies for 1 hour prior to ligand stimulation. Cells were incubated for 8, 24 or 48 hours before addition of the

28 caspase glo detection reagent. Results only show data for 24 hour treatment. Apoptosis is directly related to the amount of luminescence (RLU).

Xenograft studies. Female BALB/c SCID mice 5-6 weeks old (Jackson Laboratories) were challenged subcutaneously with 5 x 106 human cancer cells (BxPC3 or JIMT-1).

Starting at day zero (day of tumor challenge), mice were treated intravenously with 200

µg of each peptide mimic weekly for 7-8 weeks. Tumor growth was monitored twice a week using Vernier calipers. Tumor volume was calculated by the formula (long measurement x short measurement2)/2.

Statistical analysis. Tumor sizes and weights were analyzed using Stata’s XTGEE cross- sectional generalized estimating equation, which fits general linear models that allow you to specify within animal correlation structure in data involving repeated measurements.

For other experiments, t-test was carried out to observe the statistical relevance in between different sets of experiments as well as the significant difference between treated and untreated cells.

Results:

HER3 crystal structure analysis and design, synthesis and characterization of HER3 peptide mimics. The three dimensional structure of a protein can be elucidated by crystallography analysis. When proteins are crystallized in complex with their binding partners and analyzed by X-ray diffraction, very detailed atomic information can be obtained. X-ray crystallography can show every atom in a protein and give details of ligands, inhibitors, ions, and other molecules that are incorporated into the crystal.

Peptide mimics can be designed with knowledge of the structural configuration of a protein. In 2002, the crystal structure of the unliganded HER3 extracellular domain was published by Cho et al. 52. A ribbon diagram showed that Domains I and III exhibit the expected B-helical structure, and domains II and IV are extended repeats of seven small disulfide-containing modules. Since then, several other crystal structures of the HER3 extracellular domain have been solved in complex with therapeutic monoclonal antibodies. In 2011, the crystal structure of the HER3 extracellular domain was published in complex with the Fab portion of a monoclonal antibody, MEDHD7945A

(DL11) 114. The HER3: DL11 complex revealed the residues of HER3 that were important for binding. The residues critical for DL11 binding are all found in Domain III of HER3, and these residues are also important for HRG binding (Figure 2.1). DL11 has the ability to block the HRG: HER3 interaction and inhibit downstream signaling of

HER3. Several residues are important for DL11 binding, and we have selected one peptide that includes these key residues. HER3 461-479 directly overlaps with the

DL11:HER3 binding site, and may play a role in signaling of the HER3 protein. The

HER3 461-479 epitope includes part of β-strand and α-helix in domain III of HER3

(Figure 2.2). In 2013, two other crystal structure complexes were solved for HER3 binding to a monoclonal antibody. The monoclonal antibodies described were RG7116 and LJM716 111, 195. The RG7116 monoclonal antibody: HER3 complex demonstrates that this antibody binds to domain I of the HER3 extracellular region (Figure 2.1).

RG7116 inhibits ligand binding and subsequent phosphorylation of HER3. As a result, our lab has synthesized two HER3 peptides overlapping this binding region: HER3 99-

122 and HER3 140-162. In the native HER3 protein, the HER3 99-122 residues fold into an alpha-helix followed by a random coil, while the HE3 140-162 peptide contains two anti-parallel β-sheets separated by a random coil. The complex of HER3 binding to

LJM716 demonstrated that the antibody binds to domain 2 and 4 of the HER3 extracellular region (Figure 2.1). LMJ716 was shown to lock HER3 into an inactive conformation and inhibit downstream signaling of the receptor. The residues encompassing our HER3 237-269 sequence overlap with the LJM716:HER3 binding site.

These residues encompassing HER3 237-269 fold into two anti-parallel β-sheets separated by a random coil in domain II of the HER3 extracellular region (Figure 2.1).

All HER3 peptide locations within the native protein are shown in (Figure 2.2). The four chosen epitopes were synthesized by solid-phase peptide chemistry either as the peptide mimic or as a chimeric peptide vaccine. After initial screening, two of our selected peptides (HER3 237-269 and HER3 461-479) were linked to a promiscuous T-helper cell epitope derived from the measles virus fusion protein (MVF residues 288-302). MVF

31 peptide constructs were subsequently used as peptide vaccines. (See chapter 3). Table

2.1 shows the peptide sequences and corresponding molecular weights.

Receptor expression in various cell lines. In order to determine HER3 and IGF-1R expression in different cancer cells and to compare expression levels with other HER family receptors, western blotting with commercial Erbb3, Erbb1, Erbb2 and IGFR primary antibodies were used (R & D Systems) (Figure 2.3). HER3 expression was detected in all cell lines except SKOV-3 cells. HER1 expression was only detected in

MDA-MB-468 cells and BxPC3 cells. High levels of HER2 were detected in BT474,

MDA-MB-453 and SKOV3 cells. Lower levels of HER2 were detected in Capan-2 and

HT-29 cells. IGF-1R expression was detected in all cell lines, but low amounts were found in MDA-MB-468, MDA-MB-453 and SKOV-3 cells. Overall, the results indicate that HER3 expression is predominant in most cancers and is always expressed together with other family receptors and/or IGF-1R.

MTT inhibition assay with peptide constructs: To test the ability of the peptide mimics to elicit anti-tumor effects, HER3 positive cells were treated with the inhibitors and examined in a MTT inhibition assay. The anti-proliferative effects of the peptides were tested against breast (JIMT-1, MCF7, MDA-MB-468) and pancreatic (BxPC3) cancer cells at various concentrations (Figure 2.4). Taxol, an inhibitor of mitosis, was used as a positive control (data not shown). Results from the various cell lines revealed that the

HER3 peptides inhibited proliferation of all cell lines in a dose-dependent manner. The most robust response was observed when all cell lines were treated with the HER3 99-

122 and the HER3 461-479 peptides, and the cell lines that were most responsive to treatment were the MDA-MB-468 and BxPC3 cells.

Phosphorylation assay with peptide constructs: HER3 activation is promoted by dimerization upon heregulin binding and triggers proliferation, migration and survival of cancer cells. To test the ability of the peptide mimics to block signaling of the HER3 protein, cells were treated with the inhibitors and phosphorylated HER3 was measured via ELISA and western blotting (Figure 2.5). Cancer cells were treated with the peptide mimics for 1 hour prior to ligand stimulation and cell lysis. Phosphorylated levels of

HER3 were measured with a human phospho-HER3 sandwich ELISA kit (R+D Systems) and by western blotting with a phospho-HER3 antibody (Cell Signaling). No inhibition was observed when cells were treated with the HER3 140-162 and HER3 237-269 constructs. The HER3 99-122 and HER3 461-479 peptides slightly inhibited phosphorylated HER3 (~20% and ~30%, respectively). Western blotting results also indicated a decrease in phosphorylation levels of HER3 with the HER3 99 and 461 peptides, which confirmed results already obtained with the ELISA. Both results indicate that these two epitopes were able to block receptor phosphorylation either by blocking

HER3 ligand binding or receptor heterodimerization.

HER3 peptide mimics elicit apoptosis: Targeting apoptotic regulatory pathways in cancer is a promising strategy for therapeutic agents, and it is now well known that most tumors require the PI3K/Akt (phosphoinositol 3-kinase/protein kinase B) signaling for their survival. HER3 serves as a crucial activator in downstream signaling of the PI3K kinase, and this pathway has been shown to induce apoptosis resistance in a wide range of cancers 68, 90-98. We hypothesized that blocking HER3 signaling and subsequent activation of PI3K with our peptide mimics would induce apoptosis in HER3 positive cancer cells. To test this hypothesis, apoptosis was measured using a Caspase 3/7 Glo kit

(Promega). MCF7, BxPC3 and JIMT-1 cells were treated with the inhibitors for 24 hours prior to measuring activity of caspases 3 and 7. The results obtained showed a significant increase in the amount of caspase 3 and 7activity when compared to the negative control

(IRR peptide), indicating that the HER3 peptide mimics can induce apoptosis (Figure

2.6). The highest amount of apoptosis was observed when cells were treated with the

HER3 461-479 and HER3 237-269 peptide mimics.

HER3 peptide mimics and two transplantable mouse models (BALB/c SCID mice and BXPC3 cells or JIMT-1 cells): To test the in vivo effects of the peptide mimics, we used two transplantable mouse models. Mice were challenged with either BXPC3 or

JIMT-1 cells and treated with the peptide mimics starting at day zero (day of tumor challenge) and weekly for a total of 8-9 treatments. Tumor growth was measured biweekly, and all mice were euthanized at the end of treatment; tumors were extracted and weighed, and the percentage of tumor weight was calculated. Both peptide

34 constructs had the ability to significantly delay tumor growth in mice challenged with

BXPC3 cells. As a result, only the HER3 461 peptide was used in JIMT-1 tumor model

(Figure 2.7). Overall, the HER3 461 peptide construct showed greater anti-tumor effects than the HER3 237 construct and significantly decreased the % tumor weight.

‘

Discussion

Protein tyrosine kinases trigger a wide variety of cellular pathways involved in cancer and represent a target class for drug development. Due to its lack of intrinsic kinase activity and inability to homodimerize, HER3 was not considered a target for cancer immunotherapy until recently. This view has changed because of mounting evidence suggesting that HER3 heterodimers (with HER1 or HER2) are the most active signaling dimers, and that HER3 offers a major mechanism by which HER-driven tumors escape from targeted therapy 86, 87, 99-101, 103-107, 197-200 . Currently, there are no FDA approved

HER3 targeted therapies available in the clinic today. As a result, development of novel

HER3 inhibitory therapies that help overcome resistance to other HER family therapies is of great priority. In this study, we have evaluated the in vitro and in vivo efficacy of a type of targeted therapy that involves using peptide therapeutics to inhibit HER3. Peptide therapeutics can be designed to inhibit protein-protein interactions, because the active or binding sites of proteins are typically confined to a small set of amino acids. Smaller sequences, like peptides, can be designed to mimic these regions and potentially act antagonistically against the target protein. Peptides therapeutics offer benefits such as:

35 low cost, high specificity and potency, ability to penetrate the cell membrane, low immunogenicity and safety 36, 201-205. Several published crystal structures of HER3 in complex with monoclonal antibodies (DL11, LMJ716 and RG7116) have provided our laboratory with significant insights into the key HER3 amino acids involved in binding to the antibodies 111, 114, 195. In pre-clinical studies, these monoclonal antibodies have been shown to 1) inhibit signaling of HER3 and 2) elicit anti-tumor effects both in vitro and in vivo. As a result, we developed four HER3 peptides that mimic the binding sites of these monoclonal antibodies (Figure 1.1, Table 1.1). We evaluated the anti-tumor effects of our peptide mimics on pancreatic and breast cancer cells that overexpress HER3. The peptide mimics were able to inhibit proliferation of HER3 positive cells in a dose dependent manner, suggesting that the peptides have the ability to block ligand-induced activation of HER3 (Figure 2.4). The HER3 99-122 and HER3 461-479 peptides elicited the strongest anti-proliferative effect, and these were the only two peptides that were able to slightly inhibit HER3 phosphorylation (Figure 2.5). The peptide mimics did not fully inhibit HER3 phosphorylation, suggesting that the peptides do not completely inhibit ligand binding or dimerization of the HER3 protein.

One major mechanism of cancer cell growth is to block apoptosis and anti-cancer agents should be able to induce apoptosis, resulting in programmed cancer cell death. Since phosphorylated HER3 activates PI3K, a crucial mediator of cancer cell survival, we hypothesized that blocking HER3 signaling could induce apoptosis of cancer cells. We then used the caspase assay to show that these peptide inhibitors were able to cause

36 release of caspase enzymes (Figure 2.6). These studies indicate that therapeutic treatment with the peptide mimics would be effective candidates for targeting HER3. After demonstrating the in vitro anti-tumor effects of these peptide inhibitors, we evaluated the in vivo effects using two transplantable mouse models, one of which is driven by high expression of the HER family of receptors (JIMT-1) and the other is highly dependent on

EGFR: HER3 expression (BxPC3). Two of our peptide mimics (HER3 237 and 461 constructs) were tested in these models, in which both elicited a significant delay in onset of tumor growth (Figure 2.7) Our results, however, suggest that the HER3 461 epitope has superior anti-tumor effects when compared to the HER3 237 peptide. This effect may be due to the inability of the HER3 237 peptide to inhibit phosphorylation of HER3 or via another unknown mechanism of action. Future analysis will involve 1) testing the remaining peptide mimics, HER3 99-122 and HER3 140-162, in vivo and 2) testing the mechanism of action of the HER3 peptide mimics via measuring peptide effect on downstream signaling molecules, such as p-AKT/p-MAPK or peptide induced receptor internalization.

Table 2.1 The amino acid sequences of the synthetic HER3 peptide mimics and the chimeric peptides incorporating the MVF promiscuous T helper cell epitopes. The table also includes corresponding molecular weights of all peptides after synthesis. MVF constructs were not synthesized for the HER3 99-122 and MVF HER3 140-162 peptides.

Figure 2.1 Currently published monoclonal antibody: HER3 crystal structure complexes.

Adapted from published papers 111, 114, 195. A) DL11: HER3 complex B) RG7116: HER3 complex C) LJM716: HER3 complex. Selected HER3 peptides shown at bottom of each complex.

Figure 2.2 The crystal structure of the HER-3 extracellular domain (pdb 1M6B, modified). Location of all synthesized HER3 peptides are shown in various colors.

Three monoclonal antibodies (DL11, RG7116 and LJM716) are listed with an arrow pointed towards their respective binding domains.

Figure 2.3 Western blot analysis of the expression of HER1, HER2, HER3 and IGF-1R in various cancer cell lines. Cells were grown in 6 well plates to 70-80% confluency prior to cell lysis. Cell lysates were solved in SDS-PAGE, transferred to PVDF membranes

(Thermo Scientific) and commercial rabbit antibodies for HER1 (Cell Signaling), HER2

(Cell Signaling), HER3 (Santa Cruz) and IGF-1R (Cell Signaling) were used to probe for expression of the different receptors. A goat anti-rabbit IgG HRP secondary antibody and ECL reagents (Bio-Rad) were used for detection.

Figure 2.4 HER3 peptide mimics inhibit proliferation of HER3 positive cancer cell lines.

Cancer cells (MDA-MB-468, MCF7, JIMT-1, BXPC3) were treated with peptide mimics at different concentrations (25-200 μg/mL) for 1 hour prior to ligand stimulation with

HRG (50 ng/mL) After 72 hours of incubation in the presence of the peptide mimics,

MTT was used to measure cell proliferation. Percent inhibition was calculated by taking absorbance (abs) readings at 570 nm and using the following equation: (abs. untreated- abs. treated)/abs. untreated *100. An irrelevant (IRR) peptide was used as a negative

42 control. Values are representative of three independent experiments in triplicate (n=3) with error bars indicating SEM. Statistical significance was determined by using student t-test (two-tail and paired), and p values of less than 0.05 were determined to be statistically significant. In all cases, p values were determined by comparing treatment groups (200 ug/mL) to cells treated with 200 ug/mL of the IRR peptide.

Figure 2.5 HER3 peptide mimics slightly inhibit phosphorylation of HER3 positive cancer cell lines. Cancer cells were treated for 1 hour prior to ligand stimulation with 10 ng/mL HRG for 15 minutes. After treatment, cells were lysed in 1X RIPA lysis buffer

(Santa Cruz) and phosphorylated HER3 was measured via a phosphor-HER3 ELISA kit from R+D Systems (Panel A). Percent inhibition was calculated by taking absorbance

(abs) readings at 450 nm and using the following equation: (abs. untreated-abs. treated cells)/abs. untreated x 100. Results displayed are representative of two independent experiments with duplicate samples. Error bars represent S.D. of the mean. To confirm

ELISA results, cell lysates were also subjected to western blotting. Lysates were solved in SDS-PAGE, transferred to PVDF membrane and probed for expression of pHER3 with a commercial phosphor-tyr-HER3 antibody from Cell Signaling (Panel B). Results displayed are representative of two independent experiments. In all experiments, an irrelevant peptide was used as a negative control. Statistical significance was determined

44 for ELISA results by using a student T-test (two-tail and paired), and p values of less than

0.05 were determined to be statistically significant. All p values were calculated by comparing individual treatment groups with IRR peptide.

Figure 2.6 HER3 peptide mimics induce apoptosis. HER3 positive cancer cells were plated in 96 well plates and treated with the peptide mimics for 24 hours prior to cell lysis. Apoptosis (directly proportional to amount of luminescence produced) was measured using the Caspase Glo 3/7 kit (Promega). After 24 hour treatment, caspase glo reagent was added, and plates were incubated for 3 hours before being read on a luminometer. An irrelevant (IRR) peptide was used as a negative control. Results are representative of two independent experiments with duplicate samples. Error bars represent S.D. of the mean. Statistical significance was determined by comparing treated cells with IRR peptide group with the student t-test (one tail, unpaired), and p values of less than 0.05 were determined to be statistically significant.

Figure 2.7 HER3 peptide mimics delay tumor growth in two transplantable mouse models. SCID mice (n=5) at the age of 5-6 weeks old were challenged with either

BXPC3 cells or JIMT-1 cells and treated weekly with peptide mimics intravenously (200

μg). Tumor growth was monitored over time. After euthanasia, tumors were extracted and weighed. Results displayed include tumor volume over time (top panel) and % weight tumors when compared to total mouse weight (bottom panel). Statistical significance was determined using random effects linear regression for tumor volume over time and via ANOVA for % tumor weight. In all cases, treatment groups were compared to the untreated mice, and p values of less than 0.05 were considered statistically significant. In BXPC3 mouse model, peptide treatment caused a significant 47 delay in tumor growth with both peptide constructs HER3 237-269 (p<0.02) and HER3

461-479 (p,0.001), and the effects were also observed in percentage tumor weight per body mass; HER3 237-269 (p<0.05) and HER3 461-479 (p<0.04). Only the HER3 461-

479 construct was used in JIMT-1 mouse model, and it caused both a significant delay in tumor growth and % tumor weight (p= 0.02 and p<0.05, respectively).

Chapter 3 : Immunogenicity of HER3 peptide mimics and evaluation of cancer cell treatment with anti-HER3 peptide mimic antibodies in vitro and in vivo.

Introduction:

Humoral immunity is mediated by proteins called antibodies, which are produced by B lymphocytes. Antibodies are large proteins (>150 KDa) that get secreted into the circulation and mucosal fluids, and they neutralize and eliminate pathogens/ microbial toxins. To date, passive immunotherapy with monoclonal antibodies is a well-established option in clinical oncology, and classic mAbs, such as: trastuzumab (Herceptin®), cetuximab (Erbitux®) and rituximab (Rituxan®) are being applied in cancer patients worldwide. These mAbs elicit anti-tumor effects through several different mechanisms, including: manipulation of tumor related signaling, induction of apoptosis, inhibition of tumor cell signaling and activation of complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). Despite the success, mAb therapy has limitations, so our lab has developed peptide vaccines that harness the patient’s own immune system to fight cancer. In general, anti-cancer vaccines are less advanced than mAbs, with the exception of prophylactic vaccines against oncogenic viral infections.

Since many proteins are over-expressed in cancer, tumor associated antigens (TAAs) are 49 self-proteins, and induction of tolerance remains a challenge when developing cancer vaccines 206-208.

Our lab has hypothesized that rationally designed peptide vaccines targeting specific B cell epitopes from the HER3 extracellular domain can overcome tolerance and result in the production of antibodies capable of inhibiting the growth of HER3 overexpressing cancer cells. Over the last several years, we have utilized chimeric peptides composed of both B and T cell epitopes and demonstrated that target peptides from over-expressed antigens can serve as ideal candidates for therapeutic and active immunotherapies against cancer. Our lab has developed effective vaccines against HER-2 and novel therapies based on blockade of receptor: ligand interactions, such as B7:CD28 63, 80, 177, 178. Two of our HER-2 B cell epitope vaccine candidates have established their anti-tumor effects in preclinical studies, and we were able to translate our novel findings into a phase I clinical trial in which patients were vaccinated with a combination of the two vaccine epitopes

179-183. Clinical findings from this study validated our hypothesis that the antibodies to the vaccine were able to disrupt signaling pathways in cancer. To enhance the immune response and overcome tolerance, a promiscuous T-helper cell peptide derived from the measles virus fusion protein (MVF), amino acid residues 288-302, was incorporated into our vaccines. MVF has been shown to bind to render our peptide constructs capable of overcoming MHC genetic polymorphisms by having the ability to bind several human

MHC class II molecules 179, 182, 209.

The main objective of this part of the study was to identify epitopes of the HER3 extracellular domain that could activate the immune system to produce highly specific antibodies that will target tumor cells. The innovation of our approach lies in the fact that we incorporate a chimeric peptide vaccine that stimulates both B and T cells to elicit high affinity antibodies and establish immunological memory. As previously described in Chapter two, HER3 crystal structures in complex with three therapeutic monoclonal antibodies (DL11, LMJ716 and RG7116) have allowed us to identify several HER3 peptides (based on antibody: receptor binding) as potential peptides for therapy against

HER3 positive cancers. Since these sequences are also potential B cell epitopes, or epitopes capable of being recognized by antibodies, we linked them to the MVF sequence and evaluated the peptide mimics’ abilities to generate antibodies that can specifically bind and inhibit HER3. We then evaluated the anti-tumor effects of our novel HER3 vaccine antibodies both in vitro and in vivo.

Materials and Methods:

Rabbits. Two female New Zealand white outbred rabbits (Harlan) were immunized intramuscularly with 1mg of MVF HER3 237 or MVF HER3 461 peptide dissolved in

500 uL PBS and emulsified in 500 uL of Montanide ISA720 vehicle with 100 ug muramyl dipeptide adjuvant, nor-MDP (N-acetylglucosamine-3yl-acetyl-L-alanyl-D- isoglutamine). Subsequent booster injections were given every three weeks after primary immunization. Sera of rabbits immunized with MVF HER3 237 or MVF HER3 51

461were collected weekly, and complement was inactivated by heating to 56⁰C for 30 min. The titer of anti-HER3 peptide antibody was quantified by ELISA. High-titered sera was purified on a protein A/G column (Pierce, Rockford, IL) and eluted antibodies were concentrated and exchanged in PBS using 100 kDa cutoff centrifuge filter units

(Millipore). Antibody concentrations were determined by Coomassie plus protein assay reagent kit (Pierce).

Cell lines. JIMT-1 cells were generously provided by Rita Nahta’s lab in Atlanta,

Georgia. All other cell lines were purchased from ATCC. BxPC3 cells were cultured in

RPMI1640 supplemented with 10% FBS, 1% pen-strep. MCF7, MDA-MB-453, MDA-

MB-468, JIMT-1 and BT474 cells were cultured in DMEM supplemented with 10%

FBS, 1% pen-strep. All cells were grown at 37⁰C in 95% air, 5% CO2.

Flow cytometry. Five x 105cells were washed twice in 1 mL of staining buffer (PBS,

1%BSA,0.02% sodium azide). After washing, cells were treated with 100 ug of anti-

HER3 peptide antibody in 100uL staining buffer for 30 min. Following incubation, cells were washed twice with 1mL staining buffer and treated for 30 min with 1:100 dilution of goat anti-rabbit IgG - Alexa Fluor 488 secondary antibody (Invitrogen) in 100 uL staining buffer. After washing, cells were fixed in 3.7% paraformaldehyde in PBS and analyzed on a BD FACSCalibur system (DHRLI Flow Cytometry Core Lab, OSU.

Normal rabbit IgG was used as the negative control, and a commercial anti-Her3 antibody was used as the positive control (Abgent).

rhHER3 ELISA. Ninety-six well plates were coated with 100 uL of rh HER3 at 2µg/ml in PBS overnight at 4⁰C. Nonspecific binding sites were blocked for 1 hour with 200 uL

PBS-1% BSA, and plates were washed with PBST. Anti-HER3 237 antibodies in PBT were added to antigen-coated plates in duplicate wells, serially diluted 1:2 in PBT, and incubated for 2 hours at room temperature. After washing the plates, 100 uL of 1:500 goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) were added to each well and incubated for 1 hour. After washing, the antibody was detected using 50 µL of

0.15% H2O2 in 24 mM citric acid and 5mM sodium phosphate buffer (pH 5.2) with

0.5mg/ml 2,2’-aminobis(3ethylbenzthiazole-6-sulfonic acid) as the chromophore. Color development proceeded for 10min, and the reaction was stopped with 25uL of 1% SDS.

Absorbance was read at 415nm using a BioRad Benchmark ELISA plate reader

(Hercules, CA).

MTT Inhibition Assay. Cells were seeded in 96 well flat bottom plates at 1 x 104 cells/well in 100 ul growth media and allowed to adhere overnight at 37⁰C. Growth media was then replaced with low-sera (1% FBS) media, and cells were incubated overnight. Media was removed from the wells and replaced with anti-peptide antibodies made up in 1% growth media. Plates were incubated for 1 hour at 37⁰C, and 50ng/mL

HRG was added in 1% growth medium. Plates were incubated an additional 72 hours at

37⁰C before 25 uL of 5mg/mL MTT was added to each well. Plates were incubated for 2 hours at 37⁰C, then 100 uL extraction buffer (20% SDA,50% DMF, pH 4.7) was added.

Plates were incubated overnight at 37⁰C and read on an ELISA reader at 570nm.

Apoptosis Assay. The capsase glo 3/7 kit (Promega) was used to measure the ability of the vaccine antibodies to induce apoptosis. 1x106 cells were seeded and incubated overnight at 37ºC. Cells were then treated with the peptides or vaccine antibodies for 1 hour prior to ligand stimulation. Cells were incubated for 8, 24 or 48 hours before addition of the caspase glo detection reagent. Results only show data for 24 hour treatment. Apoptosis is directly related to the amount of luminescence (RLU).

ADCC Assay. A biolouminescence cytotoxicity assay was used to detect the ability of anti-peptide antibodies to elicit ADCC (aCella-TOXTM). All procedures were done according to the manufacturer’s instructions. Non-radioactive reagents were used to measure the amount of GAPDH enzyme released by dead or dying cells. The effector cells were normal human PBMC’s from healthy donors (American Red Cross). Target cells used were MDA-MB-468 cells. Effector: target cell ratios used were 25:1, 12.5:1, and 6.25:1. Percent lysis was calculated by the following equation: (sample – E:T spontaneous lysis)/maximum lysis of target cells x 100).

Xenograft studies. Female BALB/c SCID mice 5-6 weeks old (Jackson Laboratories) were challenged subcutaneously with 5 x 106 human cancer cells (BxPC3 or JIMT-1).

Starting at day zero (day of tumor challenge), mice were treated intravenously with 200

µg of each peptide mimic or 500 ug/mL vaccine antibodies weekly for 7-8 weeks.

Tumor growth was monitored twice a week using Vernier calipers. Tumor volume was calculated by the formula (long measuremtn x short measurment2)/2.

For other experiments, t-test was carried out to observe the statistical relevance in between different sets of experiments as well as the significant difference between treated and untreated cells.

Results:

Synthesis and Characterization of Peptides. After synthesis of B cell epitopes, a fraction of each peptide (only HER3 237 and HER3 461) was cleaved from the resin, while the remaining peptide resin was linked to a promiscuous T-helper cell epitope derived from the measles virus fusion protein (MVF residues 288-302). Each peptide was synthesized collinearly with the MVF epitope and a flexible residue linker (GPSL) to allow independent folding of each epitope. After synthesis, cleavage and purification, the

MVF HER3 peptides were characterized by analytical HPLC and MALDI (matrix- assisted laser desorption ionization mass spectroscopy) at Chemical Instrumentation

Center (The Ohio State University, Columbus, OH). After confirming the correct molecular weight (Table 2.1), the peptides were lyophilized and dissolved prior to use in subsequent assays.

Immunogenicity of the MVF HER3 peptide constructs and cross-reactivity of vaccine antibodies to human HER3. We explored the ability of two of our MVF HER3 peptides to stimulate the production of antibodies that can specifically bind to the native

HER3 receptor. To determine the immunogenicity of the MVF peptides, we evaluated the immune responses elicited by two of our chimeric peptide vaccines (MVF HER3 237-

269 and MVF HER3 461-479) in pairs of outbred rabbits. The MVF HER3 237-269 and

MVF HER3 461-479 peptides were emulsified in ISA720 with nor-MDP as an adjuvant,

56 and rabbits were immunized every three weeks, for a total of three immunizations. Both peptide constructs were highly immunogenic by three weeks after the third immunization.

Antibody titers against MVF HER3 461 and MVF HER3 237 were ≥ 16,000 and

≥128,000, respectively (Figure 3.1, top panel). High antibody titers were also generated against B cell epitope constructs HER3 461-479 and HER3 237-269 with antibody titers

>16,000 and 128,000, respectively (data not shown). The ability of the peptide constructs to elicit high titered antibodies is not useful for anti-tumor effects if antibodies do not bind to the native protein. The polyclonal antisera to the peptides were purified on a protein A/G column, and total IgG was used to test the ability of HER3 anti-peptide antibodies to recognize and bind to recombinant human HER3 protein in an ELISA

(Figure 3.1, bottom panel). The MVF HER3 anti-peptide antibodies were able to specifically recognize their respective epitopes in a dose dependent manner when the rh

HER3 was used as an antigen in the ELISA assay (Figure 3.1, bottom panel). We also explored the ability of our vaccine antibodies to bind to the surface of HER3 positive cancer cells. Flow cytometric analysis was conducted with HER3 expressing BXPC3 and MCF7 cells (Figure 3.2). Pre-immune antibodies were used as the negative control.

The antibodies elicited by the MVF-HER3-237 and MVF-HER3-461 constructs bound to the HER3 protein expressed by the cell lines, and the most robust response occurred when BXPC3 cells were treated with the HER3 461 vaccine antibodies (Figure 3.2).

HER3 vaccine antibodies elicit apoptosis: Targeting apoptotic regulatory pathways in cancer is a promising strategy for therapeutic agents. Although monoclonal antibody- targeted cell killing is frequently associated with ADCC and CDC, they can also induce cell death by apoptotic processes. Antibody therapeutics can induce apoptosis of target cells by a number of mechanisms, including: antigen crosslinking, activation of death receptors, and blockade of ligand-receptor growth or survival pathways 210. The ability of our vaccine antibodies to induce apoptosis was tested using the Caspase 3/7 Glo kit

(normal rabbit IgG), indicating that the HER3 peptide mimics and vaccine antibodies can induce apoptosis (Figure 3.3). The highest amount of apoptosis was observed when cells were treated with the HER3 461-479 vaccine antibodies.

Vaccine antibodies mediate ADCC. Antibodies can exert their anti-tumor effects via

ADCC, because the Fc region can interact with peripheral blood mononuclear cells

(PBMCs) and attract them to specific targets. As a result, we examined the ability of our vaccine antibodies to induce ADCC against various cancer cell lines (Figure 3.4).

Target cells (MCF7, BxPC3 and JIMT-1) were treated with the vaccine antibodies and human PBMCs at different concentrations (effector cells). Cell lysis was measured using a bioluminescence cytotoxicity assay kit. A significant increase in cell lysis was

58 observed following treatment with the vaccine antibodies, and the effects were greater when an effector to target ratio of 100:1 was used. These results suggest that the vaccine antibodies are capable of stimulating PBMCs to cause cancer cell death (ADCC).

Two transplantable mouse models (BALB/c SCID mice and BXPC3 cells or JIMT-1 cells) and passive treatment with HER3 vaccine antibodies: We also used our

BALB/c SCID mice and JIMT-1 cell tumor models to explore the ability of our vaccine antibodies to delay tumor growth. Mice were challenged with tumor cells, and immediately after tumor challenge, mice were treated weekly with the vaccine antibodies for a total of 8-9 treatments. Tumor volume was monitored over time, and all mice were euthanized at the end of treatment; tumors were extracted and weighted, and the percentage of tumor weight was calculated. The results demonstrated in Figure 3.5 display tumor volume and % tumor weight. Only mice treated with the MVF HER3 461 vaccine antibodies resulted in a significant delay of tumor growth.

Discussion

Antibodies are important therapeutic agents for cancer. These large proteins have been selected due to their ability to mediate target-specific inhibition and immune-mediated tumor suppression via mechanisms such as ADCC and CDC. Passive immunotherapy with mAbs has established a relatively large role in modern medicine, and the FDA has approved 12 antibodies for use in oncology 15. These therapies have significantly 59 improved treatment and clinical outcomes for many cancer patients around the world.

However, there are a number of concerns that exist with the usage of mAbs, including their short duration of efficacy and necessity of repeated treatment. Repeated treatment also increases the chance for drug resistance and possible undesired immunogenicity, in addition to the associated costs. As a result of these shortcomings, mAbs are not completely curative, and an active immunity approach could offer an optimistic alternative. A therapeutic that induces the body’s own immune system to fight the tumor may give a sustained immune response due to immunological memory and lower toxicity.

Peptides can be used as antigens to generate high affinity antibodies specific for an entire protein. These peptides can be rationally designed based on conformational structural analysis and must include an antigenic determinant (B cell epitope) in which the residues are usually hydrophilic and exposed in the native protein. Our goal was to develop an effective peptide vaccine that elicits an enduring immune response by designing chimeric

HER3 peptide mimics consisting of both B and T cell epitopes. We linked HER3 peptide mimics to a promiscuous T helper cell epitope, MVF, and evaluated the immunogenicity of the peptides. Although we designed four HER3 peptide vaccines, we only analyzed two of the constructs, MVF HER3 237-269 and MVF HER3 461-479. These peptides were made prior to the other constructs, and our lab is currently evaluating the remaining peptides (MVF HER3 99-122 and MVF HER3 140-162). Our peptide vaccine constructs were highly immunogenic in outbred rabbits, and purified IgG antibodies raised against

60 each of the vaccines were able to bind rhHER3 protein in an ELISA assay (Figure 3,1).

In order for the vaccine antibodies to inhibit growth of HER3 expressing cancer cells, they must be able to recognize the native receptors on the surface of the cancer cells. As a result, we also tested the ability of the vaccine antibodies to bind HER3 expressing cells via flow cytometry (Figure 3.2). The binding effect was highest with the HER3 461 vaccine antibodies (Figure 3.2). The vaccine antibodies were not able to bind cells that do not express HER3 (SKOV-3) (results not shown). These results suggest that we were able to engineer a vaccine that is immunogenic with high binding affinity to the HER3 receptor. The inhibitory effects of the vaccine antibodies greatly rely on their ability to inhibit proliferation of cancer cells and induce apoptosis. One major mechanism of cancer cell growth is to block apoptosis and anti-cancer agents should be able to induce apoptosis, resulting in programmed cancer cell death. We then used the caspase assay to show that these peptide inhibitors or vaccine Abs were able to cause release of caspase enzymes (Figure 3.3). To further study the mechanism of action of these vaccines, we performed an assay to measure ADCC. ADCC is a key mechanism of action of most

Abs, and we showed that two of the peptide vaccine polyclonal Abs (237 and 461 constructs) were able to stimulate PBMCs to cause killing of breast and lung cancer cells

(Figure 3.4). These studies indicate that either vaccination with the chimeric epitopes or therapeutic treatment with the peptide mimics would be effective candidates for targeting

HER3. After demonstrating the in vitro anti-tumor effects of these peptide inhibitors, we evaluated the in vivo effects using two transplantable mouse models, one of which is driven by high expression of the HER family of receptors (JIMT-1) and the other is

61 highly dependent on EGFR: HER3 expression (BxPC3). Only passive treatment with the

MVF HER3 461 peptide vaccine antibodies caused a slightly significant delay in onset of tumor growth (Figure 3.5). Unfortunately, these mouse models were not ideal for studying active immunization with our peptide mimics. We were limited to using passive immunotherapy with xenografted nude mice. We anticipate that a better response would have been observed if mice were immunized with our peptide constructs.

Figure 3.1 Immunogenicity of MVF HER3 peptides (top) and ability of anti-HER3 vaccine antibodies to recognize HER3 (bottom). Two outbred New Zealand white rabbits were immunized with either MVF HER3 237-269 or MVF HER3 461-479 every three weeks for a total of three immunizations. Anti-peptide antibody titers were determined by ELISA with plates coated with the appropriate MVF HER3 peptide immunogen (top panel). Titer defined as the reciprocal of the highest dilution of sera that gave an absorbance reading above 0.2 after subtracting the background. Results are representative of three independent experiments plated in duplicate with error bars representing S.D. of the mean. Anti-HER3 peptide antibodies were purified from high tittered sera on a protein A/G column. The ability of the peptide specific antibodies to 63 recognize rh HER3 was tested in an ELISA (bottom panel). Results are averages of three independent experiments plated in duplicate with error bars representing S.D. of the mean. Pre-immune serum was used as a negative control.

Figure 3.2 Direct binding of anti-peptide antibodies to native HER3 receptor. Figure shows flow cytometric analysis of BXPC3 (left) cells and MCF7 (right) cells. Cells were treated with 50ug/mL anti-HER3 antibodies for 2 hours. HER3 binding was detected with goat anti-rabbit IgG-Alexa fluor 488 secondary antibody. Cells were analyzed on a

BD FACS Calibur system. Pre-immune serum was used as a negative control.

Figure 3.3 HER3 vaccine antibodies induce apoptosis. HER3 positive cancer cells were plated in 96 well plates and treated with the vaccine antibodies for 24 hours prior to cell lysis. Apoptosis (directly proportional to amount of luminescence produced) was measured using the Caspase Glo 3/7 kit (Promega). After 24 hour treatment, caspase glo reagent was added, and plates were incubated for 3 hours before being read on a luminometer. Pre-immune antibodies were used as a negative control. Results are representative of two independent experiments with duplicate samples. Error bars represent S.D. of the mean. Statistical significance was determined using the student t- test (one tail, unpaired), and p values of less than 0.05 were determined to be statistically significant. In all cases, p values were determined by comparing treatment groups with cells treated with pre-immune antibodies.

Figure 3.4 MVF HER3 antibodies have the ability to elicit ADCC. HER3 positive cancer cells (BxPC3 and MCF7 cells) were used as target cells. Target cells were seeded and incubated in the presence of human PBMCs at different effector: target cell ratios (100:1,

20:1, 4:1). Cells were then treated for one hour with MVF HER3 antibodies (100

μg/mL)prior to cell lysis. The Acella-tox kit (Cell Technology) was used to measure the relative amount of ADCC, and cell lysis was measured according to manufacturer’s directions. Results represent three different experiments (with each sample performed in triplicate) and display the % lysis of treatment groups when compared to 100% target cell lysis. Normal rabbit IgG (Pierce) was used as a negative control.

Figure 3.5 HER3 vaccine antibodies delay tumor growth in a transplantable mouse model. SCID mice at 4-6 weeks old were challenged subcutaneously with either JIMT-1 cells or BXPC3 cells and treated weekly with peptide vaccine antibodies (500 μg) via IP injection. Tumor growth was monitored over time. After euthanasia, tumors were extracted and weighed. Results displayed include average tumor volume over time (top) and % weight tumors when compared to total mouse weight (bottom). Error bars indicate standard deviation of the mean. Normal rabbit IgG (Pierce) was used as a negative control. Statistical significance was determined using random effects linear regression for tumor volume over time and via ANOVA for % tumor weight. In all cases, treatment groups were compared to the untreated mice, and p values less than 0.05 were considered 68 statistically significant. In both mouse models, only treatment with the MVF HER3 461 vaccine antibodies caused a significant delay in tumor growth (p<0.05 for both mouse models.

Chapter 4 : IGF-1R peptide mimics, immunogenicity and anti-tumor activity

Introduction:

A considerable amount of pre-clinical and clinical studies suggest that various parts of the IGF-1R signaling system have a significant impact on both the development and progression of cancer. Signaling through this receptor appears to be important for most of the hallmarks of cancer, including cancer cell growth, survival, angiogenesis and tissue invasion/metastasis. Increased levels of both the receptor and its cognate ligands are implicated in many different types of cancer. For instance, high plasma levels of IGF-1 are associated with an increased risk of breast, lung, head and neck, colorectal, pancreas and prostate cancers 129-132, 136, 140, 141, 211, while strong overexpression of the receptor contributes to an aggressive tumor phenotype, tumor progression and drug resistance in ovarian, prostate, endometrial, gastric, bladder, colorectal and breast cancers 123, 212, 213.

Over the past few years, development of IGF-1R inhibitors and therapeutic antibodies has become a promising alternative strategy, and the clinical potential of the IGF-1R as an anti-cancer treatment target is now being tested. Monoclonal antibodies, such as CP-

751,871 and AMG 479 have been evaluated in phase I clinical studies with modest activity as single agents, and these antibodies have also been well-tolerated 214-216.

In this study, we focused on IGF-1R and report on the anti-tumor effects of novel peptide vaccines that specifically target IGF-IR in cancer. The three-dimensional crystal structure of the L1/Cys-rich/L2 extracellular domain of IGF-1R was published in 1998

176, and this was quickly followed by the crystal structure of the IGF-1R kinase domain

217. Although these structures have provided high resolution templates for the development of selective small molecule TKIs, there are no other crystal structures available of the whole IGF-1R ectodomain in complex with a ligand or a therapeutic monoclonal antibody. As a result, we relied on experiments conducted by others, in which alanine scanning mutagenesis and binding studies with IGF-1R chimeras were used to determine potential IGF-1: IGF-1R binding sites. In the early 1990’s, binding studies with IR/IGF-1R chimeras were used to indicate determinants of specificity for insulin and IGF-1 binding to the two receptors. These results suggested that residues

131-315 of the IGF-1R extracellular region were responsible for IGF-1 binding 218, 219.

In addition, alanine scanning mutagenesis has been carried out on several different regions of the IGF-1R extracellular domain 220, 221. In between 1997 and 2001, twenty- nine residues from the L1 domain were mutated to alanine, and 10 mutants were shown to cause a significant impairment of IGF-1 binding. These residues included Asp8,

Asn11, Tyr28, His30, Leu33, Leu56, Phe58, Arg 69 and Phe90 of the IGF-1R L1 domain. In addition, another 25 residues from the Cys-rich region (predicted to be accessible to ligand on the basis of the IGF-1R fragment 3D structure) were also mutated, and 4 mutants, residues 240-242 and Phe251, also produced significant decreases in affinity for IGF-1R 220, 221. We hypothesized that targeting areas of IGF-1R that bind to

IGF-1 with our peptide mimics could inhibit receptor activation and subsequent signaling in cancer cells. Our lab synthesized four peptides based on the contact sites between

IGF-I and IGF-IR. The B-cell epitopes were synthesized alone (for peptide mimic therapy) or linked with the MVF T cell epitope to produce a chimeric peptide vaccine.

The main objective of this study was to develop IGF-1R peptide mimics that could disrupt the IGF-1R signaling axis and activate the immune system to produce highly specific antibodies that will target tumor cells. Here, we report on the anti-tumor effects of our novel IGF-1R peptide mimics and vaccine antibodies both in vitro and in vivo.

Materials and Methods:

Peptide Synthesis. Peptide synthesis was carried out on a Milligen/Biosearch 9600 peptide solid phase synthesizer (Bedford, MA) using Fmoc/t-butyl chemistry as previously described 181, 222. CLEAR amide resins were used for synthesis of the constructs. After synthesis, a fraction of the peptide was cleaved from the resin using cleavage reagent B (trifluoroacetic acid/phenol/water/TIS 90:4:4:2), and crude peptides were purified by semi-preparative reversed-phase HPLC. The remaining peptide resin was linked to a promiscuous T-helper cell epitope derived from the measles virus fusion protein (MVF residues 288-302). The peptide was synthesized collinearly with the MVF epitope and a flexible residue linker (GPSL) to allow independent folding of each epitope. After synthesis, cleavage and purification, the MVF HER3 peptide was characterized by analytical HPLC and MALDI (matrix-assisted laser desorption

72 ionization mass spectroscopy) at Chemical Instrumentation Center (The Ohio State

University, Columbus, OH). After confirming the correct molecular weight (Figure 2, 3), the peptide was then lyophilized and dissolved prior to use in subsequent assays.

Cell lines. MCF7 and BxPC3 cancer cells were purchased from the ATCC. JIMT-1 cells were generously provided by Rita Nahta’s lab in Atlanta, Georgia. BxPC3 cells were cultured in RPMI1640 supplemented with 10% FBS, 1% pen-strep. MCF7 and JIMT-1 cells were cultured in DMEM supplemented with 10% FBS, 1% pen-strep. All cells were grown at 37⁰C in 95% air, 5% CO2.

MTT Inhibition Assay. Cells were seeded in 96 well flat bottom plates at 1 x 104 cells/well in 100 μl growth medium and allowed to adhere overnight at 37⁰C. Growth medium was then replaced with various concentrations of peptides or vaccine antibodies dissolved in low-sera (1% FBS) medium. Plates were incubated for 1 hour at 37⁰C, and

50ng/mL HRG was added in 1% growth medium. Plates were incubated an additional 72 hours at 37⁰C before 25 uL of 5mg/mL MTT was added to each well. Plates were incubated for 2 hours at 37⁰C, then 100 uL extraction buffer (20% SDA,50% DMF, pH

4.7) was added. Plates were incubated overnight at 37⁰C and read on an ELISA reader at

570nm.

Phosphorylation Assay. A Human phosphor-IGF-1R ELISA kit (R & D Systems) was used to measure the amount of phosphorylated IGF-1R. One million cells/well were 73 plated in 6 well plates and incubated at 37⁰C overnight. Culture media was removed from the wells, and cells were washed with PBS. Cells were treated with 150 µg HER3

237 peptide or anti-peptide antibodies in binding buffer (0.2& BSA, RPMI 1640, 10mM

HEPES (pH 7.2) for 1 hour at 37⁰C. 5nM IGF-1 was added, and plates were incubated at

RT for 10 min. After stimulation, cells were lysed with 1X RIPA Lysis Buffer (R + D systems) for 2.5 hours at 4⁰C. Cell lysates were spun at 13000xg and debris-free supernatants were transferred into clean tubes. Protein concentration of each sample was measured by Coomassie plus protein assay reagent kit (Pierce). Lysates were frozen at -

80⁰C. Phosphorylated IGF-1R was measured using the DuoSet IC for human phsopho-

IGF-1R (R + D Systems, Minneapolis, MN).

Apoptosis Assay. The capsase glo 3/7 kit (Promega) was used to measure the ability of the peptides to induce apoptosis. 1x106 cells were seeded and incubated overnight at

37ºC. Cells were then treated with the peptides or vaccine antibodies for 1 hour prior to ligand stimulation. Cells were incubated for 8, 24 or 48 hours before addition of the caspase glo detection reagent. Results only show data for 24 hour treatment. Apoptosis is directly related to the amount of luminescence (RLU).

Rabbits. New Zealand white outbred rabbits (Harlan) were immunized intramuscularly with 1mg of MVF IGF-1R peptide dissolved in 500 uL PBS and emulsified in 500 uL of

Montanide ISA720 vehicle with 100 ug muramyl dipeptide adjuvant, nor-MDP (N- acetylglucosamine-3yl-acetyl-L-alanyl-D-isoglutamine). Subsequent booster injections

74 were given every three weeks after primary immunization. Sera of rabbits immunized with MVF HER3 237 were collected weekly, and complement was inactivated by heating to 56⁰C for 30 min. The titer of anti-IGF-1R peptide antibodies were quantified by

ELISA. High-titered sera was purified on a protein A/G column (Pierce, Rockford, IL) and eluted antibodies were concentrated and exchanged in PBS using 100 kDa cutoff centrifuge filter units (Millipore). Antibody concentrations were determined by

Coomassie plus protein assay reagent kit (Pierce).

Flow cytometry. Five x 105cells were washed twice in 1 mL of staining buffer (PBS,

1%BSA,0.02% sodium azide). After washing, cells were treated with 100 ug of anti-

IGF-1R peptide antibody in 100uL staining buffer for 30 min. Following incubation, cells were washed twice with 1mL staining buffer and treated for 30 min with 1:100 dilution of goat anti-rabbit IgG - Alexa Fluor 488 secondary antibody (Invitrogen) in 100 uL staining buffer. After washing, cells were fixed in 3.7% paraformaldehyde in PBS and analyzed on a BD FACSCalibur system (DHRLI Flow Cytometry Core Lab, OSU.

Normal rabbit IgG was used as the negative control.

rhIGF-1R ELISA. Ninety-six well plates were coated with 100 uL of rh IGF-1R at

2µg/ml in PBS overnight at 4⁰C. Nonspecific binding sites were blocked for 1 hour with

200 uL PBS-1% BSA, and plates were washed with PBST. Vaccine antibodies in PBST were added to antigen-coated plates in duplicate wells, serially diluted 1:2 in PBT, and

75 incubated for 2 hours at room temperature. After washing the plates, 100 uL of 1:500 goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) were added to each well and incubated for 1 hour. After washing, the antibody was detected using 50 µL of

0.15% H2O2 in 24 mM citric acid and 5mM sodium phosphate buffer (pH 5.2) with

0.5mg/ml 2,2’-aminobis(3ethylbenzthiazole-6-sulfonic acid) as the chromophore. Color development proceeded for 10min, and the reaction was stopped with 25uL of 1% SDS.

Absorbance was read at 415nm using a BioRad Benchmark ELISA plate reader

(Hercules, CA).

ADCC Assay. A biolouminescence cytotoxicity assay was used to detect the ability of vaccine antibodies to elicit ADCC (aCella-TOXTM). All procedures were done according to the manufacturer’s instructions. Non-radioactive reagents were used to measure the amount of GAPDH enzyme released by dead or dying cells. The effector cells were normal human PBMC’s from healthy donors (American Red Cross). Target cells used were IGF-1R positive cancer cells. Effector: target cell ratios used were 25:1, 12.5:1, and

6.25:1. Percent lysis was calculated by the following equation: (sample – E:T spontaneous lysis)/maximum lysis of target cells x 100).

Xenograft studies. Female BALB/c SCID mice 5-6 weeks old (Jackson Laboratories) were challenged subcutaneously with 5 x 106 human cancer cells (BxPC3 or JIMT-1).

Starting at day zero (day of tumor challenge), mice were treated intravenously with 200

µg of each peptide mimic or 500 ug/mL vaccine antibodies weekly for 7-8 weeks.

Tumor growth was monitored twice a week using Vernier calipers. Tumor volume was calculated by the formula (long measurement x short measurement2)/2.

For other experiments, t-test was carried out to observe the statistical relevance in between different sets of experiments as well as the significant difference between treated and untreated cells.

Results:

Peptide Selection, Design, Synthesis and Characterization. We have identified peptide mimics derived from the IGF-IR ligand binding domain for peptide therapy and active immunization against IGF-1R positive cancers. These B-cell epitopes/peptide mimics were designed based on the receptor: ligand binding site using crystallographic structures, mutagenesis studies and models of the complex between the receptor ligand interactions

176, 218-221, 223. All four chosen peptide sequences encompass key amino acids of the IGF-

1R extracellular domain involved in ligand binding, and these residues are IGF-1R 6-26,

IGF-1R 26-42, IGF-1R 56-81 and IGF-1R 233-252. All IGF-1R peptide locations

77 within the native protein are shown in (Figure 4.1), and these epitopes were synthesized by solid-phase peptide chemistry. After synthesis, a fraction of each peptide was linked to a promiscuous T-helper cell epitope derived from the measles virus fusion protein (MVF residues 288-302). MVF peptide constructs were subsequently used as peptide vaccines.

Table 1 shows the peptide sequences and their corresponding molecular weights.

IGF-IR Peptide mimics inhibit proliferation of breast and pancreatic cancer cells.

Ligand binding to IGF-1R results in receptor activation and downstream signaling that results in increased cellular proliferation. To test the ability of the peptide mimics to elicit anti-proliferative effects, HER3 positive pancreatic cancer cells (BXPC3) and breast cancer cells (MCF7 and JIMT-1) were treated with the peptide mimics and examined in a MTT inhibition assay (Figure 4.2). The cancer cells were treated with the inhibitors at different concentrations and incubated for three days before adding MTT.

Taxol, an inhibitor of mitosis, was used as a positive control (data not shown). Although all IGF-1R peptides inhibited cellular proliferation in a dose-dependent manner, the most robust response was observed when all cell lines were treated with the IGF-1R 56-81 and

IGF-1R 233-252. Based on these results, we decided to use the two best epitopes to construct our peptide vaccines by collinearly synthesizing IGF-1R 56-81 and IGF-1R

233-252 with our promiscuous T helper epitope as previously described 63.

Immunogenicity of IGF-IR peptide vaccines and cross-reactivity of vaccine antibodies with recombinant human IGF-IR. The two B-cell epitopes that showed the best inhibitory effects in the proliferation assay (IGF-1R 56-81 and IGF-1R 233-252) were collinearly synthesized with the MVF-T helper cell epitope to produce the chimeric vaccine constructs. We evaluated the immune response of each of the two chimeric peptide vaccine (MVF IGF-1R 56-81 and MVF IGF-1R 233-252) constructs in outbred rabbits. The MVF IGF-1R 233-252 construct was highly immunogenic by three weeks after the third immunization with antibody titers against this peptide that were ≥128,000

(Figure 4.3, top panel). The MVF IGF-1R 56-81 peptide construct was not as immunogenic, and antibody titers were no higher than 32,000. Similar antibody titers were also generated against B cell epitope constructs (Data not shown). We also explored the ability of two of our MVF IGF-1R peptides to stimulate the production of antibodies that can specifically bind to the native IGF-1 receptor. Total IgG was purified from high tittered sera, and the ability of the IGF-1R vaccine antibodies to recognize and bind to recombinant human IGF-1R was tested in an ELISA. Compared to pre-immune sera antibodies, the vaccine antibodies bound to rhIGF-IR in the ELISA assay, and the binding was considered specific since dilution of antibodies caused a gradual decrease in binding (Figure 4.3, bottom panel). The MVF IGF-1R 233 vaccine antibodies were able to bind more efficiently to their respective epitopes when compared to the MVF IGF-1R

56-81 vaccine antibodies (Figure 4.3, bottom panel). We also explored the ability of our vaccine antibodies to bind to the surface of IGF-1R expressing ovarian, pancreatic and colon cancer cells using flow cytometric analysis. The antibodies elicited by the MVF

IGF-1R 56-81 and MVF IGF-1R 233-252 constructs bound to the IGF-1R protein expressed by the cell lines (Figure 4.4).

Apoptosis determination by caspase activity assay. Targeting key apoptotic regulatory mechanisms in cancer is a promising strategy for the development of improved therapeutic agents, and reduced signaling of IGF-1R can result in induction of apoptosis of cancer cells. To test the ability of our peptide mimics and vaccine antibodies to inhibit

IGF-1R signaling and promote apoptosis, cancer cells were treated with the inhibitors and analyzed in a caspase activation assay (Promega). A significant increase in luminescence suggested that the IGF-1R peptides/antibodies stimulated higher levels of caspase 3 and 7

(Figure 4.5). Treatment caused more than a 10 fold increased in caspase release when compared to the negative controls (normal rabbit IgG or IRR peptide), and (Figure 4.5) this was indicative of increased apoptosis.

IGF-1R phosphorylation Assay. Signaling through IGF-1R results in activation of downstream proteins like MAPK, pAKT and also increased expression and phosphorylation of the receptor itself. We tested the ability of our peptide mimics and vaccine antibodies to downregulate expression of phosphorylated IGF-1R on the surface of cancer cells. We evaluated the effects of our peptide mimics and vaccine antibodies on IGF-1 signaling in MCF-7, JIMT-1 and BxPC-3 cells. After treatment, cell lysates were used to measure phosphorylated levels of the IGF-1 receptor using a human- phospho-IGF-IR ELISA kit (R&D diagnostics). Treatment significantly inhibited

80 receptor phosphorylation in all three cell lines, indicating that the peptide mimics and vaccine antibodies were able to prevent ligand binding and activation of the receptor

(Figure 4.6).

Vaccine antibodies and ADCC. We hypothesized that active immunization with our

MVF-IGFR peptides would induce a potent immune response against cancer cells that involves ADCC. We tested the ability of our antibodies to cause ADCC of cancer cells by inducing PBMCs to lyse target cancer cells in a bioluminescence assay. MCF-7,

JIMT-1 and BxPC-3 cells were treated with the antibodies, and cell lysis was measured with various ratios of effector: target cells. An increase in cell lysis was observed following treatment with the vaccine antibodies, and the effects were greater when an effector to target ratio of 100:1 was used (Figure 4.7). These results suggest that the vaccine antibodies are capable of stimulating PBMCs to cause cancer cell death via

ADCC.

Two transplantable mouse models (BALB/c SCID mice and BXPC3 cells or JIMT-1 cells) and treatment with peptide mimics or HER3 vaccine antibodies: To test the in vivo effects of the peptide mimics and vaccine antibodies, we used a transplantable breast and pancreatic mouse model where we injected BxPC-3 pancreatic and JIMT-1 breast cancer cells subcutaneously into the flanks of SCID mice. After tumor challenge, the mice were treated intravenously with the peptide mimics or vaccine antibodies starting at

81 day zero (day of tumor challenge) and weekly for a total of 7-9 weeks. Tumor growth was monitored twice weekly and at the end of treatment, all mice were euthanized and the tumors extracted and weighed and the percentage tumor weight calculated. Results display tumor volume and % tumor weight (Figure 4.8). Both peptide constructs demonstrated a significant delay in tumor growth and decreased the percentage tumor weight, but the IGF-1R 56-81 peptide construct showed greater anti-tumor effects and

(Figure 4.8). When mice were challenged with the vaccine antibodies, only the MVF

IGF-1R 233-252 vaccine antibodies were capable of significantly delaying tumor growth

(Figure 4.9).

Discussion:

Although development of HER family inhibitors began approximately 15 years ago, the

IGF axis has only recently been recognized as a drug target. As a result, previous research in our laboratory has focused on developing novel peptide vaccine strategies and

B cell peptide mimics that can specifically target the ErbB family of receptors. IGF-IR, however, is now an established target in various forms of cancer, including breast and pancreatic cancers, and signaling through this protein is important for cancer cell growth, survival, angiogenesis and tissue invasion/ metastasis. IGF-1R is also highly implicated in several mechanisms of drug resistance that develop with ErbB targeted therapies, such as trastuzumab and cetuximab 117, 224. In addition, there is currently no FDA approved

IGF-IR targeted therapy available in the clinic, and development of novel IGF-1R targeted therapies is of great priority. Novel targeted therapies that block IGF-IR protein- protein interactions (such as ligand: receptor binding or dimerization) have the potential of becoming clinically effective and non-toxic drugs against cancer. The use of peptides as therapeutic tools is becoming an increasingly effective and non-toxic option for cancer treatment, and peptides are highly advantageous when compared to other treatment modalities 202. In the present study, we extended our approach of targeting HER family receptors to IGF-IR using our peptide immunotherapeutic strategies. We targeted the extracellular domain of IGF-IR and synthesized four peptide mimics (IGF-1R 6-26, IGF-

1R 26-42, IGF-1R 56-81 and IGF-1R 233-252) based on receptor: ligand binding.

The inhibitory effects of our peptide mimics were tested on proliferation of breast and pancreatic cancer cells that overexpress IGF-1R. Since IGF-1R is highly implicated in the growth and metastasis of these cancer cells, we hypothesized that blocking the receptor with our peptide mimics would prevent proliferation of the cells. Two of our peptide mimics, IGF-1R 56-81 and IGF-1R 233-252, significantly inhibited proliferation of all cell lines in a dose dependent manner, suggesting that the peptides have the ability to block ligand-induced activation of IGF-1R (Figure 4.2). As a result, these two epitopes were chosen for further examination, and the peptides were collinearly synthesized with our promiscuous T helper cell epitope. The MVF IGF-1R peptide constructs were immunogenic in rabbits (Figure 4.3, top panel) and the vaccine

83 antibodies were able to specifically bind the rhIGF-IR (Figure 4.3, bottom panel). In order for the vaccine antibodies to inhibit growth of IGF-IR expressing cancer cells, they should be able to recognize the native receptors on the surface of cancer cells, and we evaluated this binding using flow cytometry.

The vaccine antibodies demonstrated binding to IGF-1R expressed on the surface of the cells (Figure 4.4), suggesting that we were able to engineer immunogenic vaccines eliciting antibodies with high binding affinity to the IGF-1R receptor.

The anti-tumor effects of our peptide mimics and vaccine antibodies greatly rely on their ability to inhibit proliferation of cancer cells and induce apoptosis. One major mechanism of cancer cell growth is to block apoptosis and anti-cancer agents should be able to induce apoptosis, resulting in programmed cancer cell death. We then used the caspase assay to show that these peptide inhibitors or vaccine Abs were able to cause release of caspase enzymes (Figure 4.5). After treatment, we observed an increase in caspase 3/7 levels, indicating that treatment induced apoptosis (Figure 4.5). The HER family receptors and IGF-IR are also known to form homo and heterodimerization after activation by their ligands. Dimerization leads to downstream signaling and intracellular phosphorylation of the tyrosine kinase domain. We hypothesized that our peptide mimics and vaccine antibodies would block IGF-1R signaling by preventing ligand binding or receptor: receptor interaction. To test the effects of peptide mimics and peptide vaccine antibodies on receptor signaling, we measured phosphorylated levels of the receptor after

84 treatment with the inhibitors. The results in Figure 4.6 demonstrated a significant inhibition of receptor phosphorylation with the vaccine antibodies and peptide mimics.

To further study the mechanism of action of these vaccines, we also performed an assay to measure ADCC (Figure 4.7). We showed that our antibodies were capable of stimulating PBMCs to cause killing of breast and pancreatic cancer cells (Figure 4.7).

These studies indicate that either vaccination with the chimeric epitopes or therapeutic treatment with the peptide mimics would be effective candidates for targeting IGF-1R.

After demonstrating the in vitro anti-tumor effects of these inhibitors, we evaluated the in vivo effects using two transplantable mouse models that rely on IGF-1R overexpression.

Both of our peptide mimics (IGF-1R 56-81 and IGF-1R 233-252) elicited a significant delay in the onset of tumor growth (Figure 4.8), but only the MVF IGF-1R 233-252 vaccine antibodies displayed statistically significant anti-tumor effects (Figure 4.9). To address this caveat, we should not limit ourselves to using passive immunotherapy with xenografted nude mice. We anticipate that a better response would have been observed if immuno-competent mice were immunized with our MVF peptide constructs.

Table 4.1 The amino acid sequences of all the IGF-1R peptides. Includes a list of both B- cell epitopes alone and corresponding MVF-peptides. The table also includes corresponding molecular weights of all peptides after synthesis.

Figure 4.1 The crystal structure of the IGF-1R extracellular domain. pdb 1IGR, modified.

Location of all synthesized IGF-1R peptides are shown in various colors.

Figure 4.2 IGF-1R peptide mimics inhibit proliferation of pancreatic and breast cancer cell lines. Cancer cells (MCF7, JIMT-1, BXPC3) were treated with peptide mimics at different concentrations (25-200 μg/mL) for 1 hour prior to ligand stimulation with IGF-1

(50 ng/mL) After 72 hours of incubation in the presence of the peptide mimics, MTT was used to measure cell proliferation. Percent inhibition was calculated by taking absorbance (abs) readings at 570 nm and using the following equation: (abs. untreated- abs. treated)/abs. untreated *100. An irrelevant (IRR) peptide was used as a negative control. Values are representative of three independent experiments in triplicate (n=3) with error bars indicating SEM. Statistical significance was determined using the student t-test (two tail, paired), and p values of less than 0.05 were determined to be statistically

88 significant. All p values were determined by comparing treatment groups to cells treated with pre-immune antibodies at highest concentration.

Figure 4.3 Immunogenicity of IGF-1R peptides and ability of anti-IGF-1R vaccine antibodies to recognize recombinant IGF-1R. Two outbred New Zealand white rabbits were immunized with either MVF IGFR 56-81 or MVF IGFR 233-252 peptide every three weeks for a total of three immunizations. Anti-peptide antibody titers were determined by ELISA with plates coated with the appropriate MVF IGFR peptide immunogen (top panel). Titer defined as the reciprocal of the highest dilution of sera that gave an absorbance reading above 0.2 after subtracting the background. Results are representative of three independent experiments plated in duplicate with error bars representing S.D. of the mean. Anti-HER3 peptide antibodies were purified from high tittered sera on a protein A/G column. The ability of the peptide specific antibodies to

90 recognize rh IGFR was tested in an ELISA (bottom panel). Results are averages of three independent experiments plated in duplicate with error bars representing S.D. of the mean. Pre-immune serum was used as a negative control.

Figure 4.4 Direct binding of anti-peptide antibodies to native IGF-1R receptor. Figure shows flow cytometric analysis of JIMT-1, BXPC3 and MCF7 cells. Cells were treated with 50ug/mL anti-IGFR antibodies for 2 hours. IGFR binding was detected with goat anti-rabbit IgG-Alexa fluor 488 secondary antibody. Cells were analyzed on a BD FACS

Calibur system. Pre-immune serum was used as a negative control.

Figure 4.5 IGF-1R peptide mimics and vaccine antibodies induce apoptosis. IGF-1R positive cancer cells were plated in 96 well plates and treated with the peptide mimics

(top panel) or vaccine antibodies (bottom panel) for 24 hours prior to cell lysis.

Apoptosis (directly proportional to amount of luminescence produced) was measured using the Caspase Glo 3/7 kit (Promega). After 24 hour treatment, caspase glo reagent was added, and plates were incubated for 3 hours before being read on a luminometer.

An irrelevant (IRR) peptide and normal rabbit IgG (Pierce) were used as negative controls. Results are representative of two independent experiments with duplicate samples. Error bars represent S.D. of the mean. Statistical significance was determined using the student t-test (one tail, unpaired), and p values of less than 0.05 were

93 determined to be statistically significant. All p values were determined by comparing treatment groups with cells treated with either IRR peptide or pre-immune antibodies.

Figure 4.6 IGF-1R peptide mimics and vaccine antibodies inhibit phosphorylation of breast and pancreatic cancer cell lines. Cancer cells were treated with peptide mimics

(top panel) or vaccine antibodies (bottom panel) for 1 hour prior to ligand stimulation with 50 ng/mL IGF-1 for 10 minutes. After treatment, cells were lysed in 1X RIPA lysis buffer (Santa Cruz) and phosphorylated IGF-1R was measured via a phosphor-IGF-1R

ELISA kit from R+D Systems. Percent inhibition was calculated by taking absorbance

(abs) readings at 450 nm and using the following equation: (abs. untreated-abs. treated cells)/abs. untreated x 100. Results displayed are representative of two independent

95 experiments with duplicate samples. Error bars represent S.D. of the mean. In all experiments, either an irrelevant peptide (IRR) or normal RIgG (Pierce) was used as a negative control. Statistical significance was determined using the student t-test (one tail, unpaired), and p values of less than 0.05 were determined to be statistically significant.

In all cases, p values were determined by comparing treatment groups with cells treated with pre-immune antibodies.

Figure 4.7 MVF IGF-1R antibodies have the ability to elicit ADCC. IGF-1R positive cancer cells (BxPC3 and MCF7 cells) were used as target cells. Target cells were seeded and incubated in the presence of human PBMCs at different effector: target cell ratios

(100:1, 20:1, 4:1). Cells were then treated for one hour with vaccine antibodies (100

μg/mL) prior to cell lysis. The Acella-tox kit (Cell Technology) was used to measure the relative amount of ADCC, and cell lysis was measured according to manufacturer’s directions. Results represent three different experiments (with each performed in triplicate) and display the % lysis of treatment groups when compared to 100% target cell lysis. Normal RIgG (Pierce) was used as a negative control.

Figure 4.8 IGF-1R peptide mimics delay tumor growth in two transplantable mouse models. SCID mice (n=5) at the age of 5-6 weeks old were challenged with either

BXPC3 cells or JIMT-1 cells and treated weekly with peptide mimics intravenously (200

μg). Tumor growth was monitored over time. After euthanasia, tumors were extracted and weighed. Results displayed include tumor volume over time (top panel) and % weight tumors when compared to total mouse weight (bottom panel). Statistical significance was determined using random effects linear regression for tumor volume over time and via ANOVA for % tumor weight. In all cases, treatment groups were compared to the untreated mice, and p values less than 0.05 were considered statistically significant. In BXPC3 mouse model, peptide treatment caused a significant delay in tumor growth with both peptide constructs IGFR 56-81 (p<0.001) and IGFR 233-252

(p,0.005), and the effects were also observed in percentage tumor weight per body mass;

IGFR 56-81 (p<0.04) and IGFR 233-252 (p<0.04). In the JIMT-1 mouse model, similar results were obtained. Both IGFR 56-81 and IGFR 233-252 caused a statistically significant delay in tumor growth (P<0.001).

Figure 4.9 IGF-1R vaccine antibodies delay tumor growth in a transplantable mouse model. SCID mice (n=5) at the age of 5-6 weeks old were challenged with JIMT-1 cells and treated weekly with vaccine antibodies via IP injection (500 μg). Tumor growth was monitored over time. After euthanasia, tumors were extracted and weighed. Results displayed include tumor volume over time (top panel) and % weight tumors when compared to total mouse weight (bottom panel). Statistical significance was determined using random effects linear regression for tumor volume over time and via ANOVA for

% tumor weight. In all cases, treatment groups were compared to the untreated mice, and p values less than 0.05 were considered statistically significant. Only treatment with the

MVF IGFR 233 vaccine antibodies caused a significant delay in tumor growth (p<0.004).

100

Chapter 5: Conclusions and Future Directions

The ultimate goal of cancer therapy is to effectively destroy tumor cells without eliciting harmful effects in normal cells or tissues. Fortunately, targeted therapies against cancer have been maturing over the last several decades, and they have demonstrated improved efficacy over conventional chemotherapeutics. In the pharmaceutical spotlight, mAbs and TKIs have improved cancer treatment significantly, and millions of cancer patients around the world have benefited from these drugs. Despite the improvements, however, these targeted therapeutics are not completely curative, and emergence of drug resistance is a problem frequently faced in the clinic. Several molecular mechanisms of acquired resistance to these agents have now been documented and suggest that most tumors have multiple molecular drivers. Known mechanisms of resistance to mAbs and TKIs include: acquisition of secondary mutations, amplification of the drug target itself, expression of transporters that alter drug efflux and activation of alternative or complementary signaling pathways, often via molecular feedback loops and cross talk 102, 225. The future success of targeted therapeutics will be dependent on overcoming these molecular mechanisms of drug resistance, and promising new alternative strategies taken to accomplish this include combination therapy and development of multi-target inhibitors.

Progressively, therapies are being designed to target multiple kinase pathways, and this can be achieved by using a single multi-target inhibitor or a combination of highly 101 selective agents. The advantages of simultaneously inhibiting multiple pathways include increased clinical efficacy and reduced drug resistance by common escape pathways 11.

An increasing amount of evidence indicates that crosstalk between the HER family members represents a major factor affecting clinical efficacy. Blocking the function of one HER receptor can be compensated by another HER family member, or alternative signaling pathway, via several molecular mechanisms that are not yet fully understood.

For instance, HER3, IGF-1R and EGFR have been identified as key contributors to acquired resistance against HER2 targeting agents, while HER3and IGF-1R have also been shown to be involved in regulating acquired resistance to EGFR inhibitors 99, 226, 227.

HER3 expression is associated with drug resistance to HER targeted therapeutics in breast cancers, lung cancers, ovarian cancers, head and neck cancers and prostate cancers.

Some investigators have combined HER3 monoclonal antibodies with HER1 or HER2 inhibitors and shown synergistic anti-tumor effects, while others have taken the approach of developing dual specific monoclonal antibodies that specifically bind to two of the

HER family receptors. In addition to HER3, recent studies have shown that resistance to

HER1/HER2 inhibitors (ie. erlotinib and trastuzumab) is mediated by increased signaling and crosstalk through IGF-IR and VEGF 106, 169, 228, 229. IGF-1R has been shown to form heterodimers with HER family members, and these associations contribute to drug resistance.

102

Recently, a bispecific antibody targeting both EGFR and IGF-1R was developed, and this antibody was able to prevent both EGFR and IGF-1R associated activation, trigger IGF-

1R receptor internalization and cause downstream activation 230. IGF-1R overexpression also appears to be associated with resistance to HER2 inhibitors in breast cancer and cetuximab resistance in colorectal cancer 224, 231, 232, and this indicates that a combined approach targeting the HER family receptor and IGF-IR may be a better therapeutic approach.

In our lab, we have proposed using peptide therapeutics and vaccines to target IGF-1R and HER3. Advances in peptide synthesis technology have allowed the application of peptides as antagonists in protein-protein interactions 194, 233. We hypothesize that targeting more than one receptor at the same time with our peptide therapeutics will prevent cross-talk and decrease the possibility of drug resistance due to activation of alternative signaling pathways. We intend to establish a multi-targeted approach that will avoid cross-talk between HER1, HER2, HER3, HER4, IGF-IR and VEGF.

103

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