PEPTIDE-BASED B-CELL EPITOPE
VACCINES TARGETING HER-2/NEU
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy in the Graduate School of
The Ohio State University
By
Joan T. Garrett, B.S./B.A.
*****
The Ohio State University 2007
Dissertation Committee: Approved by
Professor Pravin Kaumaya, Advisor ______Advisor Professor Dehua Pei, Advisor
Professor Ross Dalbey ______Advisor Professor Thomas Magliery
Graduate Program in Chemistry ABSTRACT
HER-2/neu (ErbB2), a member of the epidermal growth factor family of receptors
(EGFR) is overexpressed in a significant fraction of breast cancers. It is an attractive target for receptor-directed antitumor therapy using monoclonal antibodies. Trastuzumab and pertuzumab are growth-inhibitory humanized antibodies targeting the oncogenic protein HER-2/neu. Although passive immunotherapy with trastuzumab is approved for treatment of breast cancer, a number of concerns exist with passive immunotherapy.
Treatment is expensive, and has a limited duration of action, necessitating repeated
administrations of the monoclonal antibody. Active immunotherapy with conformational
B-cell epitopes affords the possibility of generating an enduring immune response,
eliciting protein-reactive high-affinity anti-peptide antibodies.
The three-dimensional structure of human HER-2 in complex with trastuzumab
reveals that the antigen binding region of HER-2 spans residues 563-626 that comprises
an extensive disulfide bonding pattern. In order to minimally dissect the interacting
binding region of HER-2, we have designed four synthetic peptides with different levels
of conformational flexibility. Chimeric peptides incorporating the measles virus fusion
‘promiscuous’ T cell epitope via a four-residue linker sequence were synthesized,
purified, and characterized. All conformationally restricted peptides were recognized by
trastuzumab and prevented the function of trastuzumab inhibiting tumor cell
ii proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/n mice with antibodies against 597-626 and 613-626 recognizing
HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting antibodies which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their antibody- dependent cell-mediated cytotoxicity (ADCC). Moreover, immunization with the 597-
626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice.
Based on the three-dimensional structure of the HER-2: pertuzumab Fab fragment complex, we have designed three conformational peptide constructs to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo anti- tumor efficacy. All the constructs elicited high affinity anti-peptide antibodies and all the anti-peptide antibodies showed ADCC to varying degrees with the 266-296 constructs being equally effective as compared to trastuzumab. The 266-296 peptide vaccine statistically reduced tumor onset in both transplantable tumor models (FVB/n and
BALB/c) and significant reduction in tumor development in a transgenic mouse tumor model (Balb-neuT).
Finally, we report on a phase I clinical trial using the first generation peptide vaccines MVF 316-339 and MVF 628-647 with nor-MDP as adjuvant. The goals of the trial were to determine the safety and toxicity of the vaccine as well as the maximum tolerable dose. The vaccine was well-tolerated and the maximum tolerable dose was
iii identified as the highest dose level, 1.5 mg of each peptide. Additionally, patients
produced antibodies of the IgG isotype against the vaccine, and patients receiving the highest dose level had a statistically significant increase in the IgG antibody response compared to patients receiving the lowest dose level.
iv
Dedicated to my family particularly my husband, Steve Garrett and my parents, Bob and Diane Steele
v ACKNOWLEDGMENTS
I wish to thank my advisor, Dr. Pravin Kaumaya, for his support and
encouragement. His enthusiasm and interest have been essential for the success of this
project. I am also grateful to my committee members, Drs. Ross Dalbey, Thomas
Magliery, Dehua Pei, as well as Ming-Daw Tsai and Robert Coleman for their time and
guidance.
My sincere acknowledgements to Dr. Stephanie Allen, who worked with me on
several of the studies described in Chapter 3. I thank Dr. Sharad Rawale for his assistance with the synthesis of many of the peptides used in this project. I acknowledge
Drs. John Morris, Guido Forni, and Todd Reilly for providing cell lines and transgenic mice breeding pairs as well as critical reading of manuscripts. I am grateful to those who assisted in the clinical trial studies including Jacquie Lieblein, Danielle Carbin, Abby
Short, and Stephen Vincent. Additionally, I thank the clinical trial coordinators I have
worked with over the years including Tammy Lamb and Yahaira Kane. I am grateful to
all members of the Kaumaya lab past and present, especially Daniele Vicari, Marcus
Lynch, and Naveen Dakappagari for advice, problem-solving, and a helping hand.
Finally, I would like to thank my family and friends. I thank my parents for their
unconditional love and support. I am grateful to my sister, Marie, for her support given through many hours of phone conversations. I thank my husband, Steve, for all the love
vi and encouragement he has given me. He has helped me to maintain my sanity throughout the completion of this thesis as well as in all areas of life.
vii VITA
March 13, 1980...... Born – Cincinnati, Ohio
2002…………………………………………B.A./B.S. Chemistry/Computer Science, Ohio University
2002 – present………………………………Graduate Teaching and Research Associate, The Ohio State University
PUBLICATIONS
Research Publication
1. Allen, S.D., Garrett, J.T.*, Rawale, S., Jones, A.L., Phillips, G., Forni, G., Morris, J.C., Oshima, R.G., and Kaumaya, P.T. Peptide Vaccines of the HER-2/neu Dimerization Loop are Effective in Inhibiting Mammary Tumor Growth in vivo. J Immunol, 179: 472-482, 2007. (* joint first author)
2. Garrett, J.T., Rawale, S., Allen, S.D., Phillips, G., Forni, G., Morris, J.C., and Kaumaya, P.T. Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu. J Immunol, 178: 7120-7131, 2007.
3. Steele, J.T., Rawale, S., Kaumaya, P.T.P. Cancer Immunotherapy with Rationally Designed Synthetic Peptides. In: Kastin, A. (ed.), Handbook of Biologically Active Peptides. Elsevier. 511-518, 2006.
4. Dakappagari, N.K., Lute, K.D., Rawale, S., Steele, J.T., Allen, S.D., Phillips, G., Reilly, R.T., and Kaumaya, P.T. Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities. J Biol Chem, 280: 54-63, 2005.
FIELDS OF STUDY
Major Field: Chemistry
viii TABLE OF CONTENTS
Page
Abstract………………………………………...………………………………………….ii
Dedication……………………………………………………………………………...….v
Acknowledgments…………………………………………………………………..……vi
Vita……………………………………………………………………………………...viii
List of Tables…………………………………………………………………………….xi
List of Figures……………………………………………………………………………xii
Abbreviations……………………………………………………………………………xv
Chapters:
1. Introduction…………………………………………………………………………….1
1.1 Breast Cancer Epidemiology and AvailableTreatments…………………...….1
1.2 HER-2…………..……………………………………………………………..2
1.3 Cancer Immunotherapy………………………………………………………15
1.4 Hypothesis and Overview of Chapters 2-4.………………………………….30
1.5 Tables and Figures……….…………………………………………………..32
2. Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In
Vivo Antitumor Properties against HER-2/neu………………….………..………40
2.1 Introduction…………………………………………………………………..40
ix 2.2 Materials and Methods……………………………………………………….44
2.3 Results………………………………………………………………………..52
2.4 Discussion………………..…………………………………………………..61
2.5 Tables and Figures……….…………………………………………………..67
3. Peptide Vaccine Strategies Targeting the HER-2 Dimerization Loop…………….…86
3.1 Introduction…………………………………………………………………..86
3.2 Materials and Methods……………………………………………………….89
3.3 Results………………………………………………………………………..94
3.4 Discussion………………..…………………………………………………100
3.5 Tables and Figures……….…………………………………………………107
4. Effect of Dose on Humoral Immune Response in Patients Vaccinated with Multi-
Epitope Peptide-Based Vaccines Targeting HER-2…………………….…...…119
4.1 Introduction…………………………………………………………………119
4.2 Patients and Methods………..………………………………...……………121
4.3 Results………………………………………………………………………127
4.4 Discussion………………..…………………………………………………132
4.5 Tables and Figures……….…………………………………………………137
5. Summary and Future Perspectives…………………………………...……………...148
Bibliography…………………………………………………………………………....152
x LIST OF TABLES
Table Page
2.1 Amino Acid Sequence of HER-2 Trastuzumab-Binding Epitopes…..………………67
3.1 Amino Acid Sequence of HER-2 Pertuzumab-Binding Epitopes…..….…………..107
4.1 Dose Escalating Trial Design for Multi-Epitope HER-2 Peptide Vaccine………....137
4.2 Patient Characteristics………………………………………………………………138
4.3 Partial Response and Stable Disease Patients……………………………...……….139
4.4 Maximum Mean IgG Response to HER-2 Peptide Vaccines per Dose Level…...…140
xi LIST OF FIGURES
Figure Page
1.1 Proposed mechanisms of action of trastuzumab. …..……………………..…………32
1.2 Comparison of soluble EGFR monomer, soluble EGF-EGFR monomer complex, soluble 2:2 EGF-EGFR complex, and soluble HER-2 monomer…………………….….33
1.3 The structure of soluble HER-2 and Trastuzumab Fab complex…………………….35
1.4 The HER-2 heterodimerization and pertuzumab interface………………………..…37
1.5 The relationship of T cells, antigen-presenting cells, and B cells in the immune response…………………………………………………………………………………..38
2.1 Synthetic strategy for generating three disulfide pairings in peptide……………….68
2.2 Binding of trastuzumab to peptides…………………………………………………69
2.3 Cell proliferation by MTT assay……………………………………………………..70
2.4 Immunogenicity of HER-2 peptide constructs in FVB/n mice………………………71
2.5 Cross-reactivity of peptide antibodies to breast cancer cell lines……………………72
2.6 Effect of vaccination with 597-626 epitope in outbred rabbits………………………74
2.7 ELISA data demonstrating anti-597-626 Abs bind HER-2 and Trastuzumab inhibits anti-597-626 Abs binding to HER-2……………………………………………………..75
2.8 Anti-peptide Abs decrease cell viability and inhibit tumor cell proliferation in vitro.77
2.9 Anti-peptide Abs are capable of mediating ADCC………………………………….78
2.10 Cross-reactivity of HER-2 peptide antibodies with the rat neu receptor…………...79
xii 2.11 Immunogenicity and immunoprotective effects of HER-2 peptide epitope on autochthonous tumor development in BALB-neuT transgenic mice……………………80
2.12 Immunohistochemical images of infiltration by CD3+ T lymphocytes in solid
mammary tumors………………………………………………………………………...82
2.13 Immunohistochemical images of infiltration by macrophages in solid mammary
tumors…………………………………………………………………………………....84
3.1 Antibody responses elicited by peptide vaccines in outbred rabbits……………….108
3.2 Cross-reactivity of the antipeptide antibodies to native HER-2/neu……………….110
3.3 Anti-peptide antibodies mediate antibody-dependent cellular cytotoxicity in vitro..112
3.4 Antibody responses elicited by peptide vaccines in inbred FVB/n mice…………..113
3.5 NT2.5 tumor challenge in vaccinated FVB/n mice………………………………...114
3.6 Isotype distribution of HER-2 peptide constructs in FVB/N mice………………...115
3.7 Cross-reactivity of the MVF266 antipeptide antibodies to rat neu…………………116
3.8 In vivo suppression of transplantable tumor growth by active immunization with
MVF266 peptide epitopes………………………………………………………………117
3.9 In vivo suppression of autochthonous tumor growth by active immunization with
MVF266 peptide epitopes………………………………………………………………118
4.1 Vaccination and Phlebotomy Schedule…………………………………………….141
4.2 Dose Level 1 IgG Response to HER-2 Peptide Vaccines…………………………..142
4.3 Dose Level 2 IgG Response to HER-2 Peptide Vaccines…………………………..143
xiii 4.4 Dose Level 3 IgG Response to HER-2 Peptide Vaccines…………………………..144
4.5 Dose Level 4 IgG Response to HER-2 Peptide Vaccines…………………………..145
4.6 Cross-reactivity of patient antibodies to breast cancer cells over-expressing
HER-2…………………………………………………………………………………..146
4.7 Antibodies from vaccinated patients mediate antibody-dependent cell-mediated cytotoxicity in vitro……………………………………………………………………..147
5.1 The First and Second Generation HER-2 B-cell Epitope Vaccines………………..151
xiv ABBREVIATIONS
Ab antibody
ABTS 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
ADCC antibody-dependent cell-mediated cytotoxicity
Ag antigen
APC Antigen-presenting cell
ATP adenosine triphosphate
But tert-Butyl
BSA bovine serum albumin
CD circular dichroism
CDC complement-dependent cytotoxicity
CDR complementarity determining region
CTL cytotoxic T lymphocyte
CYC cyclized
DTT dithiothreitol
ECD extracellular domain
EGF epidermal growth factor
EGFR epidermal growth factor receptor
ELISA enzyme-linked immunosorbent assay
xv ER estrogen receptor
ESI-MS electrospray ionization mass spectrometry
Fab Antigen-binding fragment
FISH fluorescent in situ hybridization
HER human epidermal growth factor receptor
HLA human histocompatibility leukocyte Ag
IFN interferon (e.g., IFN-γ)
IGF-1R insulin-like growth factor receptor-1
IHC immunohistochemistry
IL interleukin (e.g., IL-2)
KLH keyhole limpet hemocyanin mAb monoclonal Ab
MALDI matrix-assisted laser desorption/ionization
MAP multiple antigenic peptide
MAPK mitogen-activated protein kinase
MHC major histocompatibility complex
MMTV mouse mammary tumor virus
MTT C,N-diphenyl-N'-4,5-dimethyl thiazol-2-yl tetrazolium bromide
MVF measles virus fusion protein amino acids 288-302
NC non-cyclized
xvi NK natural killer
NSCLC Non-small cell lung cancer
PBMC peripheral blood mononuclear cell
PBS phosphate-buffered saline
PI3K phosphatidylinositol 3-kinase
TAA tumor-associated ag
TCR T cell receptor for Ag
TGF transforming growth factor
Th cell T helper cell
TK tyrosine kinase
TKI tyrosine kinase inhibitor
TM transmembrane
TNF tumor necrosis factor
VEGF vascular endothelial growth factor
xvii CHAPTER 1
INTRODUCTION
1.1 Breast Cancer Epidemiology and Available Treatments
According to the American Cancer Society (http://www.cancer.org) breast cancer is the most frequently diagnosed cancer in women; it is the second leading cause of cancer
death in women, after lung cancer. There are slightly over 2 million women living in the
United States who have been treated for breast cancer in 2007. The probability of a
woman having invasive breast cancer some time during her life is about 1 in 8; the
chance of dying from breast cancer is about 1 in 33. More than 200,000 women in the
U.S. will be found to have invasive breast cancer in 2007 (1). About 40,910 people will
die from the disease in 2007, making this disease a high priority for cancer researchers.
Current treatments for breast cancer include local/regional treatment: lumpectomy
(surgical removal of tumor), mastectomy (surgical removal of the breast), with removal of axillary underam lymph nodes to determine the severity of disease (if cancer has spread to lymph nodes; node-positive or node-negative status), and irradiation. It can also entail adjuvant systemic therapy including DNA-targeted chemotherapy
(doxorubicin, cyclophosphamide, and paclitaxel). Additionally, if an individual’s tumor
1 specimen is estrogen receptor (ER) positive, hormone therapy (tamoxifen, aromatase
inhibitors) is a possible treatment. Monoclonal antibody therapy with trastuzumab is also
a possible treatment if a patient’s tumor specimen is HER-2 positive.
1.2 HER-2
Normal Function of HER-2. HER-2 (human epidermal growth factor receptor; also
known as erbB2) is a 185-kDa protein and a member of the HER family of receptor
tyrosine kinases that includes EGFR (epidermal growth factor receptor, erbB1), HER-3
(erbB3), and HER-4 (erbB4). The rat homolog of HER-2, neu, was originally identified
by its activation in ethylnitrosourea-induced rat neuroblastomas (2). HER-2 was subsequently identified using the avian erythroblastosis virus transforming gene, v-erbB as a screening probe (3). The gene for the HER-2 protein is localized to q21 of chromosome 17. An essential role for HER-2 in mid-gestation development was indicated by embryonic lethality of HER-2 deficient mice at embryonic day 11, indicating HER-2 is necessary for cardiac and neural development. Embryos showed dysfunctions associated with a lack of cardiac trabeculae. In addition development of cranial neural crest-derived sensory ganglia was markedly affected, as was development of motor nerves (4). Cre-Lox technology, a conditional mutagenesis approach relying on the Cre DNA recombinase-mediated deletion of a DNA segment flanked on both sides by two loxP sequences, has circumvented the mid-gestation cardiac defect in HER-2 knockout mice. When the recombinase is expressed under an appropriate promoter, Cre-
Lox technology allows the introduction of a mutation in a tissue-specific and inducible manner (5). Development of severe dilated cardiomyopathy resulted from conditional
2 mutagenesis of the HER-2 gene in murine ventricular cardiomyocytes (6). Other studies
have revealed the importance of HER-2 in different tissues (7). Mice lacking HER-2 in
skeletal muscles are viable, but lack muscle spindles and display defects in muscle
generation (8). HER-2 is essential for muscle spindles development and regulates the
formation of effective neuromuscular synapses (9).
Signaling through HER Receptors. All four HER receptors (EGFR, HER-2, HER-3, and
HER-4) share extracellular domains (composed of four subdomains, two of which are disulfide-rich) with high structural homology, a single transmembrane spanning helix, and a cytoplasmic portion that contains a conserved but not equally functional tyrosine kinase domain flanked by a carboxy-terminal tail with autophosphorylation sites. Only
EGFR and HER-4 are completely functional in terms of ligand binding and kinase activity. HER-2 appears to lack a high-affinity ligand (10) and HER-3 has impaired kinase activity (11), relying on the kinase activation of its heterodimerization partners for activation.
All HER ligands have in common an EGF-like domain of approximately 60
amino acids including six cysteine residues that interact covalently to form three loops
(12). HER ligands can be divided into three groups (5). The first group specifically binds EGFR, including EGF, amphiregulin (AR), and TGF-α. The second group exhibits dual specificity for EGFR and HER-4 including betacellulin, heparin-binding EGF, and epiregulin (13). The third group is composed of the neuregulins (NRG, also called heregulins, HRG), and include two subgroups based on their ability to bind HER-3 and
HER-4 or only HER-4.
3 HER-2 plays a major coordinating role in this network, since each receptor with a
specific ligand seems to prefer HER-2 as its heterodimeric partner (14, 15). HER-2
containing heterodimers potently amplify signaling because HER-2 reduces the rate of
ligand dissociation, allowing strong and prolonged activation of downstream signaling
pathways (16, 17). The most significant intracellular pathways activated by the HER
receptors are those involving the phosphatidylinositol-3-kinase (PI-3K) and mitogen-
activated protein kinase (MAPK) (18). In normal cells activation of the HER family
initiates a rich network of signaling pathways that control normal cell growth, differentiation, motility, and adhesion in several cell lineages (19-21). The formation of heterodimers and the resulting activation are temporally and spatially controlled in normal cells and tissues (22).
HER-2 and Cancer. Increased wild-type HER-2 expression levels causes dysregulation of the HER network in tumor cells, providing both growth and survival advantages for the tumor (23, 24). Early studies demonstrated that overexpression of HER-2 in normal fibroblasts (NIH 3T3 cells) resulted in transformation and tumorigenesis and resistance to the cytotoxic effects of TNFα (25-27). Slamon et al demonstrated that HER-2 amplification is a significant predictor of both overall survivor and time to relapse in breast cancer patients and over-expression of HER-2 occurs in 20-30% of invasive breast cancers (28, 29).
Initial conflicting reports regarding the prognostic factor of HER-2 were resolved with improved uniform methodologies (30), confirming the findings of Slamon et al.
Breast cancers can have up to 25-50 copies of the HER-2 gene and up to 100-fold
4 increase in HER-2 protein expression resulting in up to 2 million receptors expressed at
the tumor cell surface (31-33). FDA-approved tests for determining HER-2 status include
immunohistochemistry (IHC) for protein overexpression using the Herceptest KIT
(Dako) or gene amplification by fluorescent in situ hybridization (FISH). HER-2 over-
expression has been defined by IHC as being highest (reported as 3+) when receptor
levels near 2 million, or medium intensity (2+) when receptor levels are around 500,000.
Normal levels of membrane-bound HER-2 are approximately 20,000-50,000 per cell (34)
Although the HER-2 gene has been found to be mutated in tumors in a few cases (35), the
most common alteration in tumors is amplification of the gene.
Breast cancers over-expressing HER-2 have biological characteristics that
differentiate them from other types of breast cancers including resistance to certain
hormonal agents, increased sensitivity to certain cytotoxic chemotherapeutic agents, and
increased propensity to metastasize to the brain (36). Additionally, HER-2
overexpression and amplification is seen in subsets of gastric, esophageal, endometrial,
uterine, ovarian, and lung cancers (37-42).
HER-2 is an attractive therapeutic target in cancers for several reasons. The
amount of HER-2 expressed on cancer cells is much higher than in normal adult tissues
(43), potentially reducing the toxicity of HER-2 targeting drugs. Tumors with a high expression of HER-2 often show homogenous, intense IHC staining (44), signifying that
HER-2 targeted therapy would target most cancer cells in a given patient. Additionally,
HER-2 overexpression is found in both the primary and metastatic sites (45), suggesting that HER-2 targeted therapy may be effective in all disease sites.
5 Development of Trastuzumab. Soon after the discovery that the HER-2 oncogene was
amplified in many breast cancers, efforts began to develop inhibitors against HER-2.
Monoclonal antibody technology, first developed by Milstein and Kohler in 1975 (46),
was available at this time and since HER-2 is a membrane bound receptor, exposed to the
extracellular matrix, it was a highly rationale hypothesis that a mAb could bind the ECD
of HER-2 and interfere with signal transduction and thus inhibit tumorigenic HER-2
function (47). Over 100 mAbs directed against the ECD of HER-2 have been generated.
Dependent upon epitope specificity, the interaction of HER-2 and anti-HER-2 antibodies
(Abs) resulted in no effect, inhibition, or stimulation of tumor growth (48). One of these anti-HER-2 mAb was developed for clinical testing, initially identified as mouse mAb
4D5. 4D5 was created by the fusion of a mouse myeloma cell line with splenocytes from
BALB/c mice immunized NIH 3T3 cells stably transfected with HER-2 (49, 50). 4D5 was selected from a panel of mouse anti-HER-2 Abs at Genentech Inc. because it inhibited the growth of breast tumor cell lines over-expressing HER-2 in both anchorage- dependent and independent assays in vitro and inhibited tumor growth in mouse models
(49, 51).
To avoid human anti-mouse antibody responses, mouse mAb 4D5 was humanized
using recombinant engineering such that the human 4D5 contained only the antigen
binding loops (complementary determining regions; CDRs) from mouse 4D5 with human
variable region framework residues and a human IgG1 constant domain (52). The IgG1
isotype is favored due to its ability to support effector functions such as ADCC and CDC
(53). Eight humanized variants were constructed to probe the importance of several
framework residues. Some variants lost in vitro anti-tumor activity despite high affinity
6 binding to HER-2, but others retained activity and the most potent was selected for further clinical development (generic name: trastuzumab; trade name: Herceptin).
Trastuzumab proved to be more efficient than its murine counterpart 4D5 in mediating
ADCC (52, 54, 55). Additional preclinical studies demonstrated trastuzumab’s ability to reduce cell culture antiproliferative activity and antitumor efficacy in mouse xenograft studies (55-57).
Initial clinical trials of trastuzumab tested it as a single agent in HER-2
overexpressing metastatic breast cancer and demonstrated response rates of 12 to 34% for
a median duration of 9 months (58-60). The most beneficial clinical use of trastuzumab has been in combination with a variety of cytotoxic chemotherapies (47). Trastuzumab added to multiple chemotherapy treatments significantly increases their antitumor efficacy (61, 62). Trastuzumab’s largest impact has been in the treatment of patients with
potentially curable early-stage breast cancer (47). The addition of trastuzumab to
chemotherapy regimens to early-stage HER-2 over-expressing breast cancer patients who
receive chemotherapy after surgical resection, significantly prolongs disease-free survival
and reduces the chance of recurrence (63, 64). As a result of these studies, trastuzumab
has recently been approved in combination with doxorubicin, cyclophosphamide and
paclitaxel in adjuvant treatment for early stage breast cancer after primary therapy (65).
The clinical activity of trastuzumab is limited to HER-2 overexpressing tumors and
trastuzumab has no significant activity against breast cancers without HER-2
overexpression (60, 66).
Despite the impressive clinical results with trastuzumab, there are side effects
associated with this mAb including cardiotoxocity. Initial clinical trials revealed
7 trastuzumab had significant cardiotoxicity, with the incidence of cardiac dysfunction
ranging from 4% to 7% (67). Subsequent randomized clinical trials demonstrated
significant trastuzumab-associated cardiotoxicity, with 5-17% increase in left ventricular
ejection fraction, a measure of the pumping ability of the heart, and 1-3% increase in
congestive heart failure (68). The mechanism of trastuzumab-induced cardiac
dysfunction is not fully understood (69). HER-2 may aid in the repair of cardiac myocyte
injury. Thus, the use of trastuzumab and the loss of HER-2-dependent cardiac myocyte
survival pathways could make patients more vulnerable to cardiac damage (70).
Possible Mechanisms of Action and Resistance to Trastuzumab. Many studies have
attempted to decipher the molecular mechanisms underlying the clinical activity of
trastuzumab. Early studies supported trastuzumab mediated endocytosis and degradation
of the HER-2 receptor (71-73), with consequent inhibition of the downstream PI3K and
MAPK signaling pathways. However, other studies have shown trastuzumab does not
downregulate HER-2 (74, 75). Austin et al, using immunofluorescence and quantitative
immunoelectron microscopy, demonstrated that trastuzumab binds and internalizes with
surface HER-2 but reappears with HER-2 at the cell surface, only accompanying HER-2
passively along its normal endocytic recycling route (74). Trastuzumab binding to HER-
2 inhibits the proteolytic cleavage and shedding of HER-2 by matrix metalloproteases
(76, 77). This could be a part of the anti-tumor properties of trastuzumab since truncated
HER-2 is associated with increased kinase activity and increased transforming efficiency
(78, 79), although many HER-2 over-expressing breast cancer do not have significant
8 truncation of HER-2 (47). Trastuzumab is unable to inhibit HER-2 from dimerizing with other members of the HER family including EGFR and HER-3 (80, 81).
While the effect of trastuzumab on the function of its direct target HER-2 remains
to be identified, numerous studies have described the effects of trastuzuamb on
downstream signaling pathways. Cells treated with trastuzumab undergo arrest during
the G1 phase of the cell cycle modulated by the induction of p27, with a concurrent
reduction in proliferation (82-84). Overexpression of HER-2 in tumors is associated with
increased angiogenesis and expression of vascular endothelial growth factor (VEGF)
(85). Trastuzumab exhibits certain anti-angiogenic properties. Trastuzumab treatment on
HER-2 over-expressing breast cancers reduces tumor volume and decreases microvessel
density in vivo (86, 87) (Fig. 1.1). Trastuzumab may block PI3K signaling via disrupting
the interaction between HER-2 and Src tyrosine kinase, leading to the inactivation of Src
with subsequent activation of the PI3K inhibitor PTEN (88).
In light of recent data indicating that trastuzumab does not down-regulate cell
surface HER-2, there is a renewed interest in another possible mechanism of action of
trastuzumab, its ability to induce an immune response via its Fc domain. Trastuzumab is
highly efficient at activating antibody-dependent cell-mediated cytotoxicity (ADCC) in
vitro (52, 89). Natural killer (NK) cells, a major immune cell type involved in ADCC,
express the Fc-γ receptor III, to which the Fc domain of trastuzumab binds, activating
NK-mediated cell lysis (Fig. 1.1). Xenograft studies with mice bearing HER-2 over-
expressing tumors displayed a tumor regression rate of 96% when treated with
trastuzumab. Mice lacking the Fc receptor showed only 29% tumor growth inhibition
when treated with trastuzumab (57). This model provides convincing evidence that the
9 antitumor activity of trastuzumab is mediated through immunological targeting mechanisms (47).
The majority of metastatic breast cancer patients who initially respond to trastuzumab exhibit disease progression within one year (61, 90). Additionally, 15% of early-stage HER-2 over-expressing breast cancer patients who receive trastuzumab and chemotherapy after surgical resection (63, 64) eventually develop metastatic disease.
Preclinical studies examining trastuzumab resistance revealed the stable overexpression of insulin-like growth factor-I receptor (IGF-IR) reduced trastuzumab mediated inhibition of SKBR3 breast cancer cells (91). Additionally, it has been shown that IGF-IR physically interacts with and induces phosphorylation of HER-2 in trastuzumab-resistant cells, but not in trastuzumab-sensitive parental cells (92). Another potential mechanism of resistance is the masking of HER-2 with membrane-associated glycoprotein mucin-4
(MUC4), preventing it from binding to trastuzumab (93, 94). MUC4 is a member of the mucin family, which are highly glycosylated proteins that form protective barriers on epithelial cells and has been suggested to contribute to cancer progression by its ability to inhibit immune recognition of cancer cells (34).
Structural Studies with HER-2 and Trastuzumab. Recently, there have been numerous x- ray crystallographic structural studies revealing how the HER family initiates signal transduction. Crystal structures of ligand-less EGFR (95) and HER-3 (96) reveals that a finger-like β-hairpin loop from domain II make intramolecular contacts with domain IV
(Fig. 1.2A); this structure is considered the “closed” state due to this interaction between domains II and IV.
10 Structures of EGFR complexed with either EGF or TGF-α reveal ligand binds to domains
I and III causing dramatic and global changes in the conformation, exposing a long projection from domain II that is involved in dimerization (Fig. 1.2B, EGF-EGFR monomer complex) (97, 98). The 2:2 EGF-EGFR complex reveals that dimerization between two molecules of EGFR occurs through interaction within each molecules domain II (Fig. 1.2C). This data evokes a model for receptor activation in which ligand- less receptors exist in a closed form that upon ligand binding results in extensive domain rearrangement. The rearrangement brings domains I and III together for binding ligand, splitting the domain II-IV interaction and freeing the domain II extended projection to dimerize with other HER receptors and initiate signal transduction. Two independent groups published crystal structures of the extracellular region of HER-2 (99, 100). HER-
2 adopts an open conformation that resembles the ligand-activated state of EGFR (Fig.
1.2D). The contact between domains II and IV, observed in ligand-less EGFR and HER-
3 is absent in HER-2, instead an interaction between domains I and III is seen. This structure of HER-2 is similar to a ligand-activated state; showing HER-2 poised to dimerize with other HER receptors in the absence of direct ligand binding and explains the inability of HER-2 to bind any known ligands.
The structure of soluble HER-2-trastuzumab Fab complex reveals that trastuzumab binds on the C-terminal side of domain IV of the HER-2 ECD (Fig. 1.3A).
The trastuzumab-HER-2 interaction buries 1,350 Å2 of the HER-2 surface (Fig. 1.3B).
Three loops are observed to make contact with trastuzumab (Fig. 1.3C). Loop 1 contains residues 579-583 and loop 3 contains residues 615-625 and these loops are composed of primarily electrostatic interactions with trastuzumab. Loop 2 comprises residues 592-595
11 and this loop makes primarily hydrophobic contacts with trastuzumab. The specific details of the contact region between HER-2 and trastuzumab may allow for the design of possible small peptides or peptide analogues.
Pertuzumab: HER-2 Dimerization Inhibitor. From the panel of anti-HER-2 mAbs developed at Genentech Inc. the mAb 2C4 was also selected for further characterization.
Compared with mAb 4D5, 2C4 has diminished anti-proliferative activity in vitro with the
HER-2 over-expressing cell line SKBR3 (49). Agus et al showed that 2C4 but not
trastuzumab is able to block the association of HER-2 and HER-3 in breast cancer cell
lines (80), indicating 2C4 has properties distinct from trastuzumab. Additionally it was
reported that 2C4 inhibited heregulin mediated HER-2 phosphorylation and MAPK and
Akt activation in breast cancer cells not over-expressing HER-2 (80). In vivo pre-clinical
xenograft studies have demonstrated 2C4 has activity on breast, ovarian, and prostate
tumors including tumors without HER-2 over-expression (80, 101). 2C4 has been
humanized to be used for human clinical usage (102) and the resulting humanized mAb is
called pertuzumab (Omnitarg). Limited data has been presented on 2C4 or pertuzumab’s
ability to inhibit cell signaling pathways since these mAbs are not yet accessible to the
wider scientific community (47).
The crystal structure of the HER-2 ECD bound to pertuzumab reveals this mAb
binds a different epitope than trastuzumab, found at the center of domain II, near the
junction of domains I, II, and II (103). The contact buries 1210 Å2 on the HER-2 side of the interface. The interface between HER-2 and pertuzumab is mostly polar, with few hydrophobic or charged residues in the interface. Domain II in HER-2 and EGFR is
12 highly conserved and this is the same region involved in EGFR dimerization. This region overlaps with pertuzumab binding, indicating that pertuzumab binds at the HER-2 dimerization interface. This structure provides a model in which pertuzumab sterically interferes with HER-2 dimerizing with other members of the HER family (Fig. 1.4A, B) and thereby interrupts the growth signals mediated by HER-2 ligand activated heterodimers.
Phase I studies of pertuzumab revealed the agent was well-tolerated with side- effects of fatigue, nausea, and vomiting (104). A number of phase II trials have been conducted in breast , non-small cell lung cancer (NSCLC) (105), ovarian (106), and prostate (107) cancers. Clinical responses were seen in a Phase II trial of ovarian cancer patients, with an overall response rate of 4.3% (106). Pertuzumab has shown synergy with trastuzumab in vitro (108), and a phase II trial is investigating the effect of a combination of pertuzumab and trastuzumab in HER-2 over-expressing breast cancer
(109). The effects of pertuzumab on HER-2 expressing cancers await additional preclinical and clinical studies.
HER Kinase Inhibitors. Another therapeutic approach is the use of small molecule tyrosine kinase inhibitors (TKI) that inhibit the specific catalytic activity of HER kinases by interfering with the binding of ATP. This area of study was revolutionized by the discovery of modified quinazoline compounds as highly specific and potent inhibitors of
EGFR (110, 111). The clinical success of the ABL-kinase inhibitor imatinib mesylate
(Gleevec; Novartis) in the treatment of ABL-driven leukemia (112) raised hopes that
13 drugs targeting HER kinases implicated in other types of cancers would be similarly
efficacious. However, TKIs lack the singular target specificity of antibodies.
Various strategies involving TKIs have been developed to target EGFR and/or its
family members, and these are in a range of stages for clinical testing. Nearly all of these
agents are ATP analogs and inhibit kinase activity by binding the ATP pocket of the
catalytic domain (47). Gefitinib (Iressa; AstraZeneca) and erlotinib (Tarceva; OSI
Pharmaceuticals, Genentech), which bind to the ATP binding site of the intrinsic tyrosine kinase domain of EGFR, received fast-track approval by the US FDA in 2003 and 2004,
respectively, for patients with advanced NSCLC who had failed to respond to
conventional chemotherapy. In addition, lapatinib (Tykerb; GlaxoSmithKline), a dual
inhibitor of EGFR and HER-2 kinases, is being investigated in phase II and III trials in
combination with chemotherapy and/or hormonotherapy. In cell-based assays these
orally bioavailable ErbB family TKIs are effective at inhibiting both EGFR and HER-2
as well as EGFR and HER-2 phosphorylation. Despite this success, TKIs have shown
very limited clinical activity, marked by modest delays in tumor progression. Although
gefitinib received fast-track approval in 2003, the conditions of the approval were
restricted in 2004 because of negative results of the phase III Iressa Survival Evaluation
in Lung Cancer (ISEL) trial (113). Gefitinib is not presently clinically available in US.
Sergina et al has recently shown that TKIs failure to completely inhibit the kinase
activity of HER-2 allows oncogenic signaling to continue through kinase inactive HER-3
(114). This study points out that until compounds with the needed potency and
specificity can be identified, combinations of agents with the intention of undermining
14 the robustness of the HER signaling family may represent the most promising therapeutic approach.
1.3 Cancer Immunotherapy
Cancer immunotherapy involves exploiting the impressive powers of the immune system
for the treatment of cancer. The concept of active immunotherapy, that the immune
system can recognize and respond to tumors, was first popularized by Coley in the 19th century who noted the spontaneous regression of tumors was often preceded by infectious episodes (115). Within the past twenty years there have been significant advances in the development of a prophylactic vaccine against cancer. Despite the progress made, the treatment and/or prevention of cancer remain a considerable challenge because of cancer cells ability to evade the immune system. This is a consequence of tumor-associated antigens (TAAs) in many cases being expressed in normal tissues. As a result many of the lymphocytes reactive with epitopes derived from these TAAs may have been eliminated through mechanisms involved in immune tolerance.
There are several potential advantages associated with cancer immunotherapy.
The current conventional strategies to treat cancer include chemotherapy, radiotherapy, and surgical excision which target rapidly dividing cells. However, chemotherapy and radiotherapy also target other rapidly dividing cells including hair follicle cells, gastrointestinal epithelium and leukocytes resulting in adverse side effects.
Immunotherapy specifically targets only tumor cells expressing the antigen of interest with relatively few side effects compared to chemotherapy and radiotherapy. In addition
15 active immunotherapy against a TAA offers the advantage of generating immunological
memory leading to a sustained and long-lasting response against the TAA.
Overview of the Immune System. Innate immunity consists of defense mechanisms in
place prior to infection and poised to respond rapidly. The main components of innate immunity include phagocytic cells (neutrophils and macrophages) and NK cells.
Adaptive immunity, which develops in response to an infection, comprises two arms- humoral immunity and cell-mediated immunity. Humoral immunity is mediated by antibodies produced by cells called B lymphocytes. Cell-mediated immunity or cellular immunity is mediated by T lymphocytes or T cells. T cells can further be divided into helper T cells (Th cells) and cytolytic, or cytotoxic, T lymphocytes (CTLs).
CTLs (also referred to as CD8+cells) eliminate infected cells or tumor cells
through direct cytotoxic action on these targets. CTLs detect infected or malignant cells
through the recognition of major histocompatibility complex (MHC) class I molecules
that are complexed with peptides derived from proteins expressed within the cell. MHC molecules are the most polymorphic in the genome, and are the ones recognized by T lymphocytes during transplant rejection. They encode for a family of cellular antigens that aids the immune system from differentiating self from non-self. Both the humoral and cell-mediated arms of the adaptive immune system are dependent on Th cells. Th cells, also known as CD4+ T cells, recognize MHC class II molecules that are complexed to peptides derived from predominately exogenous proteins. All nucleated cells can present peptides derived from intracellular proteins on their surface bound to MHC I, whereas peptides derived from extracellular proteins are only presented by MHC II on
16 professional antigen presenting cells (APCs), such as dendritic cells, B cells, and
macrophages. The T-cell receptor (TCR) on the surface of the CTL or Th-cell forms a
complex with the MHC I/peptide-epitope complex or the MHC II/peptide-epitope
complex, respectively on APCs. These interactions are aided by the CD8 co-receptor on
CTLs and the CD4 co-receptor on Th cells.
The complex interplay of these recognition processes results in the initiation and
propagation of immune responses that control infection and cancer in humans (Fig. 1.5).
The aim of vaccination is to induce immunity towards these states by selectively
stimulating antigen-specific B cells or CTLs and Th cells (116). An effective vaccine must contain two antigenic epitopes: a Th-epitope and an epitope that will either induce specific B-cell or CTL responses.
Passive Versus Active Immunotherapy. Passive immunotherapy involves enhancing or
stimulating the immune system using exogenous cytokines, antibodies, immune cells, or
growth factors. Passive immunotherapy is highly specific; it leads to tumor cell death.
However there is a limited duration associated with mAbs; one study found at a dosage of
4mg/kg body weight the mean half-life of trastuzumab, a mAb used against the TAA
HER-2/neu, to be 5.8 days. Thus, there is a need for subsequent treatments. In addition
there is possible toxicity and immunogenicity associated with passive immunotherapy.
Another limitation is that mAb treatment is expensive. Many aspects contribute to the
high cost of mAb treatment including the high cost of manufacturing and the large total
doses that are frequently required (53).
17 Active immunotherapy involves a specific TAA eliciting an endogenous immune
or anti-tumor response. Weak but detectable levels of HER-2 specific Abs and T cells
have been found in early stage breast cancer patients that over-express HER-2/neu (117).
Thus, active immunotherapy could augment an established immunogenic response. In addition, immunologic memory is elicited with an active approach; the longevity of the
immune response could prevent the re-emergence of the tumor. Hence active
immunotherapy is an attractive target. Examples of passive and active immunization
strategies are detailed here followed by specific aspects of peptide vaccine formulations
with an emphasis on the HER-2 tumor antigen.
Passive Immunotherapy with Monoclonal Antibodies. The effectiveness of mAbs as a
treatment for cancer and other diseases was recognized soon after the development of
hybridoma technology in 1975. Antibodies have a high affinity for antigen and
discriminating specificity, thus mAbs have the ability to bind small quantities of antigen
and neutralize their function. In addition abs bound to antigen can trigger effector
functions via the Fc region. The Fc region can recruit lymphocytes or the complement
system that subsequently results in cell death of the antibody-bound cell (ADCC or
CDC). Nearly all mAbs used clinically today are engineered to be chimeric or fully
humanized because the rapid induction of human antimouse antibody (HAMA)
responses.
Ideal targets for passive immunotherapy with mAbs are lymphomas; this type of
cancer has the advantage over solid tumors that the tumors cells are accessible and more
likely to be penetrated by antibodies. A mAb targeting CD20 (IDEC-C2B8,
18 Rituximab/Rituxan) is approved for use in B cell non-Hodgkin’s lymphoma and is among
the most effective monoclonal antibody therapies. CD20 is an antigen present on mature
B cells and 90% of B cell lymphomas (118). Myelotarg (Gemtuzumab Ozogamicin) is a
humanized anti-CD33 antibody that is conjugated to calicheamicin. This mAb is approved for patients with CD33-positive acute myeloid leukemia. This is an example of an engineered antibody derivative in which calicheamicin, a cytotoxic antitumor antibiotic, has been coupled to the mAb to increase its effector function of killing the antibody-bound cell.
There have been a number of mAbs developed that target solid tumors. These targets include cell-surface proteins and antigens involved in tumor-associated vasculature that support tumor growth. Cetuximab (Erbitux) and panitumumab
(Vectibix) both target EGFR and are approved for treatment by the FDA to treat colorectal cancer. Both these anti-EGFR mAbs block ligand-receptor interactions.
VEGF (vascular endothelial growth factor) and its receptors (Flt-1 and KDR) have been implicated in promoting solid tumor growth and metastasis by stimulating tumor- associated angiogenesis. Bevacizumab (Avastin) is a recombinant humanized IgG1 mAb that is approved for treatment of lung and colorectal cancers. Bevacizumab binds VEGF
and prevents the interaction of VEGF with its receptors on the surface of endothelial
cells.
Peptide-Based Vaccines for Cancer. Vaccination has been one of the most successful
clinical interventions for the prevention of human diseases. Vaccines have contributed to
the eradication of smallpox, an extremely contagious and deadly disease. The occurrence
19 of other diseases such as polio, measles, mumps, rubella, chickenpox, and typhoid has
decreased significantly as a result of vaccination. Despite a large body of work, there is
still no effective cancer vaccine. This, in large part, is due to the immune system’s weak
and ineffective response to cancer, whereas the immune system responds efficiently to
bacteria, viruses, and other agents.
A number of formulations of cancer vaccines have been used in both pre-clinical
and clinical studies including peptide, DNA, whole-cell, dendritic cell, and adoptive
therapy. HER-2 vaccines have been designed that use whole cell expressing tumor
antigens (119-121), proteins (122), DNA (123-125), and B-cell epitope peptides (126-
129) as sources of antigen. Reilly et al. found that mice depleted of CD4+ T cells produced no neu-specific IgG after a whole cell vaccination with 3T3-neu/GM cells which express neu (130). The mice were unable to reject a subsequent tumor challenge, suggesting that both the cellular and humoral immune responses are needed for eradication of tumors. Another study used cytokine-engineered allogeneic HER-2/neu positive cells as a vaccine (131). IL-12 engineered cell vaccines had the most powerful immunoprotective activity equal to that of administration of recombinant IL-12 in
combination with HER-2/neu cell vaccine. The use of dendritic cells expressing neu has
produced promising results in a tumor mouse model (132). Chang et al. found that the
co-expression of IL-18 or GM-CSF with a HER-2/neu DNA plasmid increased antitumor
activity (133). BALB-neuT mice that received DNA plasmids at 10-week intervals had
total protection from invasive carcinomas by one-year of age (134).
It has long been thought because of tolerance to self, TAAs are not antigenic because TAAs are self-antigens. This is an argument against active immunotherapy.
20 However, it has been proposed that this issue can be circumvented using subdominant
epitopes with a synthetic peptide vaccine (135). The study of peptide vaccines is a
constantly evolving field, with advances in immunogenicity and the extension of peptide
half-life continuously improving existing vaccines. Peptide vaccines can be broadly
characterized into two groups: cellular (T cell) and humoral (B cell) vaccines. Most cancer studies are focused on T cell epitopes due to the fact that when presented in the context of MHC Class I, the resulting activated cytotoxic T cells can become activated and lyse transformed tumor or infected cells. This response is thought to be the most prominent mechanism utilized by the immune system to control viral replication and kill transformed tumor cells.
B cell epitope vaccines are designed to induce a protective humoral response.
This response includes creation of antibody-producing plasma cells as well as
immunologic memory. B cell epitopes have been used in a variety of vaccines. Oomen
et. al. designed a peptide mimic of the immunodominant β-turn of PorA from Neisseria meningitidis to protect against meningococcal meningitis (136). A malaria vaccine
against Plasmodium falciparum is made up of multiple tandem repeats of NANP, which
is the immunodominant B cell epitope of the circumsporozoite protein (137). United
Biochemical, Inc. has developed a B cell epitope vaccine for HIV targeting residues 39-
66 on the CD4 T cell molecule. Resulting antibodies are expected to cause steric
hindrance, preventing viral infection of CD4+ T cells (138).
Identification of B-cell epitopes. A fundamental step in the development of peptide
vaccines for treating cancer is the identification of epitopes that are recognized by B cells
21 to stimulate a neutralizing humoral response and T cells to induce a cellular response. In
the past ten years there has been a wealth of publications demonstrating the effectiveness
of epitope enhancement in the design of CTL peptide vaccines (139-141). This strategy
involves altering the anchor residues of class I epitopes to increase the affinity of peptide binding to MHC molecule or increase the affinity of the peptide-MHC complex for the
TCR (142). Identification of T cell epitopes has been reviewed previously (143).
B cell epitopes can be classified as either continuous or discontinuous, depending
on whether or not the residues involved in the epitope are contiguous in the polypeptide chain. Discontinuous epitopes are made up of residues that are not continuous in sequence but are assembled at the surface of the protein either by constraints imposed by the three-dimensional structure of the protein or by the folding of the polypeptide chain.
Identification of continuous epitopes can be determined from the primary structure of the protein, while determination of conformational epitopes usually entails analysis of the x- ray crystal structure of the protein. Crystallographic studies allow detailed information about antigen-antibody information. However crystallographic studies are not amenable to widespread use, given the time necessary to acquire and interpret such data and the necessity that the protein antigen must be isolated in pure crystalline form. Thus other methods have been developed to identify B-cell epitopes.
Various theoretical and experimental methods have been developed to determine
potential antigenic sequences of a protein. Antibody: antigen interfaces have been
assumed to generally be hydrophilic and transiently accessible to solvent surrounding the
antigen. This is the basis for the prediction of antigenic sites in the seminal work by
Hopp and Woods (144) in which hydrophilic stretches in a protein’s linear sequence are
22 identified. In addition one can predict a protein’s hydrophilicity, hydrophobicity,
antigenic index, and surface probability based on the primary sequence of the protein.
Kaumaya et al provides detailed analysis of computational methods available to identify
B-cell epitopes (145). Due to limited correlation between predicted and actual epitopes,
further testing on individual epitopes identified from computational methods need to be
performed to confirm that the sequence is immunodominant, immunogenic, and
possesses biological relevance.
Epitope mapping, or determination of the antigen binding site to an antibody, is a
useful tool in identifying B-cell epitopes of a protein. This procedure necessitates having
a panel of monoclonal or polyclonal antibodies available against the protein. One
experimental method to map protein epitopes involves the synthesis of peptides which
represent overlapping segments of the antigen sequence (146). However this procedure
can be costly and time prohibitive, even when multiple peptides are screened
simultaneously with a series of antibodies using the PEPSCAN approach. Another
approach involves cleavage of the protein by enzymes. The protein fragments are then separated, localized, and analyzed by gel electrophoresis, Western blotting, and microsequencing (147). Several procedures have been developed which use mass spectrometry to identify B-cell epitopes (148). MALDI mass spectrometry is used to identify non-binding peptides through a direct comparison of the spectra of a mixture of proteolytic peptide fragments before and after reaction with a mAb. Peptides that are part of the epitope are determined based on the absence of their ions in the spectrum of the antibody reaction mixture versus unreacted control spectrum.
23 The term mimotope is defined as a peptide capable of binding to the paratope of an antibody (the antigen-binding site on the antibody) but unrelated in sequence to the protein antigen used to elicit the antibody (149). These peptides are usually identified through synthetic peptide libraries or recombinant peptide libraries (mostly phage display libraries) (150). To be a true mimotope, the peptide should be able to elicit antibodies that recognize the epitope being mimicked, in addition to binding the antibody. This phenomenon is explained by the fact that dissimilar amino acid residues in the two cross- reactive peptides nevertheless contain similar atomic groups that are able to interact with atoms of the CDR regions of the antibody (151). Several groups have identified mimotopes that bind to trastuzumab using phage libraries (152, 153). Antibodies raised against one of these peptides recognized HER-2/neu and caused internalization of the receptor from the cell surface in a similar manner as trastuzumab (152). More recently these trastuzumab mimotopes were able to induce IgE antibodies in BALB/c mice immunized via the oral route. These IgE antibodies were able to mediate ADCC on a
HER-2 over-expressing cell line (154). A phage/algorithm approach has been described in which mAbs specific to HIV-1 are used to select specific phages from a combinatorial phage-display library that is then used as an epitope-defining database. This database can then be applied via a computer algorithm to analyze the crystalline structure of the original antigen (155), and epitopes on the protein that bind to the mAbs can be predicted.
Rational Design of Peptide Vaccines. Peptides have the potential to be safe, non-
infective, well-defined and stable vaccines. However many barriers remain in the
24 rational design of peptide-based vaccine in spite of a mounting knowledge of the
molecular recognition and stimulation of the immune system. Several factors need to be
considered in formulating an effective peptide vaccine; these factors include inclusion of a universal T helper epitope and the necessity to mimic the structure of the parent antigen to generate high-affinity Abs.
In the design of a synthetic peptide vaccine, it is necessary that the B-cell epitope be presented along with an appropriate helper T-cell epitope. The interaction between B cells and T cells recognizing the same antigen is essential for B cell proliferation and differentiation and the generation of high-affinity antibodies that are of the IgG isotype
(Fig. 1.5). For sequences shorter than 15 residues, peptides are traditionally coupled to a larger carrier protein such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). This approach can be problematic for several reasons including epitope suppression by the carrier protein and poorly defined constructs due to lack of control during the chemical coupling reaction. Several alternatives have been described to produce conformationally stable peptides.
One approach involves the use of branched oligo-lysines as a template for the
attachment of antigenic peptides known as multiple antigenic peptides (MAP) (156).
Since these compounds are of high molecular weight, adjuvants are not necessary for
antibody production. This approach has been found effective in a number of cases;
however, it’s possible that due to the density of the peptides in the construct, peptides
assume a conformation that in some cases does not mimic the structure of the peptide
monomer or the parent protein (157).
25 Another alternative involves B and T cell epitopes constructed in a co-linear
fashion. This has limited usage when the T helper epitope is recognized by one or a few
MHC class II alleles. Thus the identification of ‘universal’ or ‘promiscuous’ T helper epitopes that bind multiple MHC haplotypes is necessary in the design of an optimum vaccine. Universal T helper epitopes from tetanus toxoid (TT) and measles virus fusion protein (MVF) have been identified (158, 159). Eight predicted B-cell epitopes derived from the HER-2/neu oncoprotein were synthesized collinearly with a.a. 288-302 from the
MVF protein. All eight of the peptides investigated generated high-titered antibodies in outbred rabbits and importantly produced undetectable levels of antibodies against the
MVF TH epitope (126, 128), thus proving the utility of a chimeric vaccine construct
incorporating B and TH epitopes in a collinear fashion.
The introduction of functional humoral immunity with unstructured peptide vaccines may be difficult. Even if the protein contains a continuous epitope, peptides containing this sequence may induce antibodies that lack cross-reactivity with the parent
protein. To induce functional cross-reactive immune responses, it appears essential that
peptide design take into account conformational correctness (160). One approach to
achieve molecular mimicry to the parent protein is through constricting the peptide by
cyclization. Linear peptides are very flexible and can adopt an assortment of
conformations in solution, but only a few of these conformations are responsible for their
immunoreactivity. Cyclic peptides can cause favored spatial arrangements that reproduce
the bioactive conformation resulting in superior binding and immunological properties.
There are several extensive reviews on the cyclization of peptides (161, 162).
26 Disulfide bridges are important evolutionarily conserved structural motifs found
in many biologically important peptides and proteins including hormones, growth factors,
and immunogloblins. Artificial introduction of disulfide bonds into designed peptide
vaccines is performed with the goal to improve the cross-reactivity of the elicited
antibodies to the native protein and thus enhanced activity of the vaccine. Regioselective
pairing of disulfides can occur by controlled chemistries involving orthogonally
removable Cys protecting groups (see Chapter 2). There a number of methods used to
promote the oxidation of thiols to disulfides including air oxidation, dimethyl sulfoxide-
mediated oxidation, and iodine oxidation. This area of study is extensively reviewed by
Annis et al (163).
Constraining peptides by cyclization has been investigated as a diagnostic tool in
identifying the human papillomavirus type 16 (HPV-16) oncoprotein in cervical cancer
patients. Using a hydrazone link approach to mimic the helix secondary structure of
HPV-16, it was found that a conformationally restricted α-helix peptide showed strong
positive reaction with sera from women having invasive cervical carcinoma whereas the
linear version showed little reaction to the sera (164). The utility of peptide cyclization with the incorporation of the two native disulfide bonds of the 626-649 sequence of HER-
2/neu has been shown (127). Antibodies raised against the disulfide-paired peptide showed enhanced cross-reactivity to tumor cells over-expressing HER-2/neu and
demonstrated superior antitumor responses in the context of ADCC and IFN-γ induction.
In addition mice vaccinated with the disulfide-bonded epitope showed a significant
reduction in the development of exogenously administered tumors in vivo compared with
27 mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF)
control peptide.
Anti-Tumor Immunity Using Peptide Vaccines. To date most peptide cancer vaccine
strategies seek to induce a cellular antigen-specific T-cell response and there are
numerous reviews on this subject (165-167). However, the humoral arm could play a
critical role in the generation of an antitumor response. The successful clinical usage of
passively infused mAbs points to the effectiveness of the humoral arm of the immune
system. A recent study found that the induction of anti-HER-2/neu antibodies are both
necessary and sufficient in protecting mice transgenic for the HER-2/neu oncogene and
that have been depleted of CD4+ and CD8+ cells from developing tumors (168). Thus, an effective peptide vaccine should have components that activate both the cellular and humoral arms of the immune system. The vaccine should be composed of appropriate tumor antigen B-cell epitopes, CTL epitopes, and a universally immunogenic T-helper epitope. In addition there have been several recent advances in peptide vaccine formulations; the utility of adjuvants, cytokines and the use of multiple epitopes in vaccine are discussed below.
Adjuvants are usually defined as compounds that increase and/or moderate the
intrinsic immunogenicity of an antigen (169). Aluminum salts are the most widely used
adjuvants in humans and their mechanism of action is poorly understood. It’s thought
that aluminum salts serve as depot for antigen, that is the adjuvant serves as a depot for
the antigen at site of vaccination, from which antigen can be released, increasing the half-
life of the antigen. Oil-in-water emulsion adjuvants are thought to be linked to the depot
28 effect as well. Tiny drops of antigen are surrounded by oil, trapping the antigen. As a result the antigen is released slowly over a prolonged period and destruction of the antigen is delayed and an individual’s exposure to the antigen is lengthened. Montanide
ISA 720 is one such emulsion approved for human usage (170) and is used in the Phase I
Clinical Trial described in Chapter 4.
Different types of receptors on antigen-presenting cells (APCs) can be targeted by adjuvants. The cytosolic NOD-like receptors (NLRs) in APCs can sense signals from intracellular bacteria. Muramyl dipeptides are a ubiquitous constituent of bacterial cell walls and are recognized by APCs such as macrophages by NLRs. The muramyl dipeptide synthetic compound N-acetyl-glucosamine-3 yl-acetyl L-alanyl-D-isoglutamine can be used as an adjuvant to activate APCs and has been used extensively in our laboratory with success. Bacterial DNA containing CpG motifs can trigger APCs such as dendritic cells via Toll-like receptor-9 (TLR-9) found on APCs. CpG is an effective adjuvant in tumor vaccinations (171).
The use of recombinant cytokines has been exploited as an adjuvant for tumor vaccines. Cytokines lead to either a Th1 or Th2 response (172). The production of interleukin-2 (IL-2), IL-12, and interferon-gamma (IFN-γ) allows the selective enhancement of a Th1-type cellular response, while production of IL-4, IL-5, or IL-10 directs a Th2-type humoral immunity (173). IL-12 is a promising cytokine with the possibility of clinical usage as a vaccine adjuvant. It has strong inflammatory properties, causes degranulation of neutrophils, and recombinant IL-12 has a relatively long half-life in the body compared to other cytokines (174). A multiepitope HER-2/neu vaccine in combination with IL-12 caused a significant reduction in the number of pulmonary
29 metastases induced by challenge with syngeneic tumor cells overexpressing HER-2/neu
(126). Granulocyte-macrophage colony stimulating factor (GM-CSF) has been studied
extensively as a vaccine adjuvant because of its ability to recruit antigen presenting cells
at the site of vaccination. There have been a number of studies demonstrating the
adjuvant effects of GM-CSF using a range of cancer vaccine methods (175).
Antibodies against TAAs can mediate diverse effects. There are antibodies that
result in stimulation of cells overexpressing HER-2/neu (176). Therefore it is crucial to identify epitopes on TAAs that result either in stimulation or inhibition of tumor cell growth to develop an effective peptide vaccine that creates an antibody response for therapeutic benefit. The enhanced efficacy of a combination vaccine containing two
HER-2/neu B-cell epitopes as compared to a single epitope vaccine has been shown; a phase 1 human clinical trial with the multiepitope HER-2/neu vaccine was conducted based on the results (see Chapter 4). Ideally an optimum cancer vaccine should include not only a combination of epitopes from a single TAA, but epitopes from various TAAs
(166). This is a fundamental requirement to overcome the problems imposed by the antigenic heterogeneity of each tumor type and prevent the reemergence of tumors.
1.4 Hypothesis and Overview of Chapters 2-5
The structures of the HER-2-trastuzumab complex and the HER-2-pertuzumab complex have led to new insights into the mechanistic and biological activities of HER-2 antibodies as well as the process of ligand-induced receptor dimerization which in turn has empowered us to rationally design more effective HER-2 conformational epitopes with potentially increased efficacy for preventing and inhibiting tumor growth. To date
30 no conformational peptides have been designed and tested based on the crystal structure of the HER-2 ECD in complex with the trastuzumab or the pertuzumab Fab fragments, hence these studies are novel. We hypothesize that active immunization with these peptides will duplicate the effect mAbs trastuzumab and pertuzumab have in vivo but will not necessitate repeated weekly treatments that are needed for mAb treatment.
Chapter 2 of this thesis presents studies on the trastuzumab epitopes. One epitope, 597-626, was pursued for additional studies based on its ability to be recognized by trastuzumab and the ability of antibodies elicited against the peptide to bind HER-2 at the same site as trastuzumab. Antibodies against the 597-626 construct were able to mediate both direct and indirect mechanisms of anti-tumor activity against HER-2 in vitro. Additionally, the 597-626 peptide vaccine was able to impede the autochthonous tumor formation in BALB-neuT transgenic mice. Chapter 3 details the pertuzumab epitope studies. From these studies, we have identified the 266-296 construct as the most promising vaccine candidate based on the cross-reactivity of antibodies against this peptide to bind HER-2 and mediate anti-tumor properties in vitro. The 266-296 peptide vaccine was able to additionally reduce tumor burden in vivo using two transplantable tumor models and a transgenic mouse model. The results of a phase I clinical trial using two HER-2 epitopes identified with computer-aided analysis are detailed in Chapter 4.
The results from the clinical trial indicate that patients are able to mount strong antibody responses against the multi-epitope vaccine. Additionally the vaccine was well-tolerated with few side effects.
31 1.5 Figures
Figure 1.1 Proposed mechanisms of action of trastuzumab. (adapted from ref. (177)).
The mechanisms shown in the schematic is described in detail in the text.
32 Figure 1.2 Comparison of soluble EGFR monomer, soluble EGF-EGFR monomer complex, soluble 2:2 EGF-EGFR complex, and soluble HER-2 monomer. Proteins are shown in rainbow coloring (N terminus blue, C terminus red). (A) Ligand free soluble EGFR monomer. (B) soluble EGF-EGFR monomer complex; EGF is shown in
33 black. (C) 2:2 EGF-EGFR complex; both EGF are shown in black, the black. right EGFR is shown in purple. (D) soluble HER-2 monomer
.
34
Figure 1.3 The structure of soluble HER-2 and Trastuzumab Fab complex. (A)
Soluble HER-2 shown in rainbow color (N-terminus: blue, C-terminus: red). The 35 trastuzumab heavy chain (Trast. HC) shown in purple and the trastuzumab light chain
(Trast. LC) shown in cyan. Boxed area is expanded in (B), the HER-2-trastuzumab
interface. (C). The three loops of HER-2 that make contact with trastuzumab are indicated. Loop 1 incorporates residues 579-583. Residues 592-595 make up loop 2 and loop 3 includes residues 615-625.
36
Figure 1.4 The HER-2 heterodimerization and pertuzumab interface. [Adapted from
Johnson et al (178).] (A) Ligand (shown in black) binds to ligand binding site of one of the partners of HER-2 (EGFR, HER-3, or HER-4; shown in purple). HER-2 is shown at right, cyan color. The dimerization interface between the two molecules is circled. (B)
The pertuzumab Fab (green, heavy chain; blue, light chain) binds to HER-2 at its dimerization interface and prevents HER-2 from dimerizing with other HER members.
37 CD4+ T-helper cell B cell Activated A B CD4+ T-helper cell Signal- Activating signals activating molecules
T-cell receptor peptide Co-stimulatory Internalization MHC class II molecules of immunogen by BCRs APC Plasma cell Immunogen MHC class I and class II molecules with bound peptide
Memory B cell
Epitope-specific antibodies
Activating signals D C
+ Activated APC Naïve CD8+ CTL Activated CD8 CTL
Figure 1.5 The relationship of T cells, antigen-presenting cells, and B cells in the immune response. [Adapted from ref. (116).] (A). Antigen presenting cells (APCs) uptake an immunogen and the immunogen undergoes proteolysis to form peptides, some of which are bound by major histocompatibility complex (MHC) class II molecules that are then transported to the APC surface. T helper (Th) cells with T-cell receptors (TCRs) which are capable of interacting with the peptide/MHC II complexes can then interact with the APC. Additionally, co-stimulatory molecules on the APC and Th cell interact, resulting in the transmission of activation signals to the Th cell. (B) The activated Th cell is now able to respond to B cells that display the same peptide/MHC II complexes on the B cell surface, obtained as the result of internalization of the immunogen through the
38 B-cell antigen receptor (BCR). This interaction between the Th cell and B cell results in the B cell differentiating to memory B cells or plasma cells, which are capable of secreting antibodies of the same specificity as that of the immunoglobulin receptor. (C)
The Th cells interaction with APCs can also bring these APCs to a state capable of
stimulating naïve CTLs. The activated APC presents the appropriate peptide epitopes in
the context of MHC class I to a naïve CTL. (D) This results in the generation of CTLs
that are able to recognize and kill target cells that display a viral or tumor peptide in the
context of MHC class I molecules. Cytokines, soluble proteins produced by each of these
cell types, can strongly influence the type of immune response that is elicited.
39 CHAPTER 2
NOVEL ENGINEERED TRASTUZUMAB CONFORMATIONAL EPITOPES
DEMONSTRATE IN VITRO AND IN VIVO ANTITUMOR PROPERTIES
AGAINST HER-2/NEU
2.1 Introduction
The tumor antigen HER-2 (ErbB2), a member of the epidermal growth factor receptor family, consists of a cysteine rich extracellular domain (ECD) that has several glycosylation sites, a transmembrane (TM) domain, and an intracellular conserved tyrosine kinase (TK) domain (3). Lacking a high affinity ligand, HER-2 functions as a preferential heterodimerization signaling partner with other members of the EGFR family
(EGFR, HER-3, and HER-4) (10, 179) leading to cellular proliferation and differentiation. HER-2 is weakly detectable in epithelial cells of normal tissues (43), but is frequently overexpressed in cancers of the breast, ovary, uterus, lung, and gastrointestinal tract (40-42, 180, 181). HER-2 overexpression in breast cancer patients is associated with a poor prognosis (28). These findings make HER-2 an ideal target for cancer immunotherapy.
Numerous antibodies directed against the ECD of HER-2 have been generated by immunizing mice with cells expressing HER-2. Dependent upon epitope specificity, the
40 interaction of HER-2 and anti-HER-2 antibodies (Abs) resulted in no effect, inhibition, or
stimulation of tumor growth (48). Trastuzumab (Herceptin™), a humanized monoclonal
antibody (mAb) directed against HER-2, has been shown to cause phenotypic changes in
tumor cells including downregulation of the HER-2 receptor, inhibition of tumor cell
growth, and reduced vascular endothelial growth factor production (82). In addition, the
interaction of trastuzumab with the human immune system via its human IgG1 Fc domain may promote its antitumor properties. In vitro and in vivo studies prove that trastuzumab is very effective in mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against HER2-overexpressing tumor cell lines (176). Trastuzumab is FDA approved for passive immunotherapy in patients with metastatic HER-2 overexpressing breast cancer.
In addition recent studies indicate that 12 months of trastuzumab treatment along with chemotherapy significantly reduced disease recurrence in patients with early-stage breast cancer (63, 64). Trastuzumab has recently been approved in combination with doxorubicin, cyclophosphamide and paclitaxel in adjuvant treatment for early stage breast cancer after primary therapy (65).
There are a number of concerns despite the impressive clinical effects of passive
trastuzumab application; these include limited duration of action that necessitates
repeated treatments at considerable cost. Trastuzumab treatment has been linked with
side effects, including cardiac dysfunction and congestive heart failure (63, 64, 182, 183).
The induction of humoral immune responses against HER-2 utilizing active
immunotherapy generating a polyclonal, long lasting immune response has become a
desirable objective. To this end our laboratory has studied a number of epitopes from the
HER-2 ECD identified from computer-aided analysis; we reported the anti-tumor
41 properties of chimeric B-cell epitope sequences 628-647 and 316-339 that incorporate a
promiscuous T-cell epitope (MVF) (126, 128). These studies are the basis for the Phase I
clinical trial currently being conducted at the Ohio State University James Cancer
Hospital.
The crystal structure of the trastuzumab Fab fragment bound to HER-2 reveals
that it binds at the C-terminal portion of subdomain IV of the HER-2 ECD (99) which
should facilitate the design of new therapeutics and vaccines. Trastuzumab binds domain
IV of the extracellular region of HER-2 at amino acids 579 to 625. Binding of trastuzumab blocks activation of HER-2 by promoting receptor endocytosis as well as blocking proteolytic cleavage of the ECD. Thus, the selection, design and syntheses of selected regions allow us by active immunization to generate sequence specific anti- peptide antibodies. By examining the mechanistic effects of these antibodies such as induction of apoptosis, decreased cell proliferation, Her-2 down- regulation, dephosphorylation, inhibition of signal pathways, inhibition of homo/hetero-dimerization,
ADCC, and CDC, we are then able to select these readouts as guide for pursuing in vivo studies as well as for translating these vaccines to the clinic.
In particular, antibodies raised against a peptide that could closely mimic the native structure of the pocket-like trastuzumab-binding region of HER-2 are likely to provide more effective functional cross-reactive immune responses with potent antitumor
properties. We have successfully used several different strategies to mimic the
conformation of epitopes in native proteins (127, 184-186). We also demonstrated the
utility of peptide cyclization by incorporating native disulfide bonds in the 626-649
42 sequence of HER-2/neu which resulted in improved in vitro and in vivo activity compared with the linear non-cyclized peptide (127).
In this study we have designed several HER-2 epitopes at the HER-2/trastuzumab
interface requiring an elaborate scheme for the successful synthesis, purification, and
characterization of these complex epitopes. We show that with varying degrees of
reactivity all conformationally restricted peptides were recognized by trastuzumab and
blocked the function of trastuzumab inhibiting tumor cell proliferation. All the cyclic and
linear epitopes were highly immunogenic in inbred mice eliciting high tittered antibodies.
The 597-626 and 613-626 epitopes elicited strong native-like anti-peptide antibodies as
evidenced by their reactivity to BT474 and SK-BR-3 breast cancer cell lines using flow
cytometric analysis. The 597-626 epitope was immunogenic in outbred rabbits eliciting
antibodies that recognized HER-2 at the HER-2/trastuzumab interface, inhibited cancer cell growth in vitro and caused antibody-dependent cell-mediated cytotoxicity. To
investigate the efficacy of vaccination in a clinically relevant model, we examined the
ability of the conformationally-restricted 597-626 epitope to inhibit the development of
mammary tumorigenesis in a transgenic model of HER-2/neu in which the HER-2/neu
oncogene is expressed in a tissue-specific manner. We demonstrated that immunization
with both the cyclized and non-cyclized 597-626 epitope significantly reduced tumor
burden in this aggressive tumor model. We conclude the HER-2 597-626 sequence is a
potential vaccine candidate that could be translated to the clinic.
43 2.2 Materials and Methods
Synthesis of linear and disulfide-constrained peptides. HER-2 B-cell epitopes 563-598,
585-598, 597-626, and 613-626 were synthesized co-linearly with a promiscuous Th epitope derived from the measles virus fusion protein (a.a. 288-302). Peptide synthesis was performed on a Milligen/Biosearch 9600 peptide solid-phase synthesizer (Bedford,
MA) using Fmoc/t-But chemistry on preloaded CLEAR Acid resin (Peptides
International, Louisville, KY) and cleaved using reagent B (TFA:Phenol:Water:TIS,
90:4:4:2). Three disulfide bonds were introduced in the epitope HER-2 563-598 to more closely mimic the three-dimensional structure of the HER-2 protein. Chemoselective protectecting groups Cys(Trt), Cys(Acm), Cys(But) were used for desired disulfide paring. The protecting group from Cys(Trt) comes off in the global cleavage reaction as confirmed by electrospray ionization mass spectroscopy (ESI-MS) analysis. Pure fractions were analyzed using analytical Waters™ HPLC, pooled together and lyophilized in 1% acetic acid solution. Cleavage of the peptide, RP-HPLC purification and lyophilization in acidic medium and prevention of oxidation of free sulfhydryl groups of Cys residues as confirmed by ESI-MS analysis was performed.
Animals. Female New Zealand White outbred rabbits and FVB/n inbred mice were purchased from Harlan (Indianapolis, IN). Virgin female BALB-neuT mice (187),
BALB/c mice transgenic for the rat transforming neu oncogene expressed under the control of mouse mammary tumor virus promoter, were bred in our animal facility.
Animal care and use was in accordance with institutional guidelines.
44 Cell lines and antibodies. All cell culture media, fetal calf serum (FCS), and
supplements were purchased from Invitrogen Life Technologies (Carlsbad, CA). The
human breast tumor cell lines BT-474, SK-BR-3, and MDA-468 were purchased from
American Type Culture Collection (Manassas, VA) and maintained according to the
supplier’s guidelines. TUBO cells are a cloned cell line established in vitro from a
lobular carcinoma that arose spontaneously in a BALB-neuT mouse (188). TS/A cells
are derived from a spontaneous breast cancer of a wild-type BALB/c mouse. HER-2
mAb Ab-2 (clone 9G6) was purchased from Neomarkers (Fremont, CA). Rat neu mAb
Ab-4 was purchased from Calbiochem. Humanized mouse mAb Herceptin™
(trastuzumab) was generously provided by Genentech, Inc (South San Francisco, CA).
Immunoassays. To determine trastuzumab’s ability to bind various peptides, a
trastuzumab specificity ELISA was performed. Ninety-six -well plates were coated with
100 µl of peptide at 2 µg/ml in PBS overnight at 4°C. Nonspecific binding sites were
blocked for 1 h with 200 µl of PBS-1% BSA, and plates were washed with PBT.
Trastuzumab (2 mg/ml) was added to antigen-coated plates in duplicate wells, serially
diluted 1:2 in PBT, and incubated for 2 h at room temperature. After washing the plates,
100 µl of 1:500 goat antihuman IgG conjugated to horseradish peroxidase (Pierce,
Rockford, IL ) were added to each well and incubated for 1 h. After washing, the bound
antibody was detected using 50 µl of 0.15% H2O2 in 24 mM citric acid and 5 mM sodium phosphate buffer (pH 5.2) with 0.5 mg/ml of ABTS as the chromophore. Color development was allowed to proceed for 10 min, and the reaction was stopped with 25 µl
45 of 1% SDS. Absorbance was determined at 410 nm using a Benchmark Microplate
Reader (Bio-Rad Laboratories, Hercules, CA).
Determining the anti-peptide response was performed as previously described
(128). Ab titers were defined as the reciprocal of the highest serum dilution with an
absorbance of 0.2 or greater after subtracting the background. All data represent the
average of duplicate samples.
Mouse serum was isotyped using Mouse Typer Sub-Isotyping Kit (Bio-Rad,
Hercules, CA) which was used per the manufacturer’s instructions. Rabbit serum was
isotyped using goat anti-rabbit IgG, IgM, and IgA conjugated to horseradish peroxidase
(Novus Biologicals, Littleton, CO).
To determine the anti-HER-2 response, a sandwich ELISA was performed. Plates
were coated overnight at 4°C with 100µL of 10µg/mL of Ab-2, washed four times with
0.1% Tween/PBS, and blocked with of 100µL of PBS-1% BSA for 4 hours on a rocker.
Plates were washed four times with 0.1% Tween/PBS. Wells were then coated overnight
at 4°C with 50µL of either PBS-1% BSA or SK-BR-3 cell lysate (1 x 108 cells in 20 mL lysis buffer). Lysis buffer was composed of 1% Triton X-100, 10% glycerol, 150 mM
NaCl, 50 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 10 mM pyrophosphate, 100 mM
NaF, 0.2 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF. Plates were washed four times with 0.1% Tween/PBS and serial dilutions of rabbit sera (starting at 1:100) were added and the plates were incubated for 2 hours on a rocker. Ab binding was detected as described above.
The anti-HER-2 response was measured using an additional competitive inhibition assay: plates were coated overnight at 4°C with 100µL of 500ng/mL of
46 recombinant human HER-2 ECD/Fc chimera (R&D Systems Minneapolis, MN) and
plates blocked with 2%BSA for 1 hour at 25°C. A constant amount (1:2000 dilution) of
rabbit anti-597-626 CYC abs or anti-597-626 NC abs was added to the plates and at the
same time various amounts of inhibitor (trastuzumab or isotype control human IgG) was
added. Bound anti-peptide rabbit antibodies was detected with HRP-conjugated anti-
rabbit IgG antibody (minimized for binding human IgG). The inhibition rate was
calculated according to the following formula: (ODanti-peptide-Ab - ODanti-peptide-Ab + inhibitor)/
(ODanti-peptide-Ab) x 100.
Peptide immunization and antibody purification. Mice and rabbits were immunized s.c.
at multiple sites with a total of 1 mg (rabbits) or 100 µg (mice) of peptide dissolved in
H2O with 100µg of a muramyl dipeptide adjuvant, nor MDP (N–acetyl-glucosamine-3 yl–acetyl L–alanyl–D–isoglutamine). Peptides were emulsified (50:50) in Seppic
Montanide ISA 720 vehicle. The same dose of booster injections was administered twice at three and six weeks. Sera were collected, and complement was inactivated by heating to 56ºC for 30 min. High-titered sera were purified on a protein A/G agarose column
(Pierce) and eluted antibodies were concentrated and exchanged in PBS using 100 kDa cut-off centrifuge filter units (Millipore, Billerica, MA). The concentration of antibodies was determined by Coomassie plus protein assay reagent (Pierce).
BALB-neuT mice were immunized in the same manner described, commencing at
5-6 weeks of age. After the second boost, the transgenic mice received two subsequent
boosters at monthly intervals. Tumor size (length and width) in each of ten mammary
glands was measured twice weekly with Venier calipers beginning at 18 weeks of age.
47 Individual tumors were calculated by the formula (length x width2)/2. All mice were euthanized at 25 weeks of age.
Immunohistochemistry. Tumors excised from BALB-neuT mice were fixed in formalin
and embedded in paraffin. Paraffin embedded tissue was cut at 4 microns and placed on
positively charged slides. Slides with specimens were then placed in a 60 °C oven for 1
hour, cooled, and deparaffinized and rehydrated through xylenes and graded ethanol
solutions to water. All slides were quenched for 5 minutes in a 3% hydrogen peroxide
solution in water to block for endogenous peroxidase.
Prior to addition of primary antibody, slides were blocked with 10% normal rabbit
serum for 30 minutes. An enzymatic digestion was performed with proteinase K for 10
minutes at 37°C. The primary antibodies used included rat anti-mouse F4/80, rat anti-
mouse neutrophils, and rabbit anti-mouse CD3. The primary antibody was added at a
dilution ranging from 1:5 to 1:2500 and incubated for 1 hour at room temperature. The
secondary antibody used was Vector rabbit anti-rat, mouse adsorbed or goat anti-rabbit at
a dilution of 1:200 and incubated for 30 minutes. The detection system used was
Vectastain Elite for 30 minutes. The substrate chromogen used was DAB+. The slides
were then counterstained with Richard Allen hematoxylin, dehydrated through graded
ethanol solutions and coverslipped.
Images were viewed at all ranges and acquired at x40 or x20 original
magnification with a Nikon Eclipse E400 microscope and Image-Pro Plus v.5.0 software.
To determine significant differences between CD3-positive staining, three random fields
at x40 of each treatment groups were counted. Statistical analysis was performed using 48 the Student’s t test. The difference was considered statistically significant when the P- value was less than 0.05.
Flow cytometry. 1 x 106 BT474, SK-BR-3, MDA468, TUBO, or TS/A cells were
incubated with 1, 10, or 100 µg of mouse or rabbit antipeptide antibodies. HER-2-
specific mouse monoclonal antibody Ab-2 (Lab Vision Corp, Fremont, CA) and rat neu-
specific monoclonal antibody Ab-4 (Calbiochem, San Diego, CA) were used as positive
controls, and isotypic IgG was used as a negative control. Cells were incubated for 2 h at
4°C in 100 µl of PBS/2% FCS/.1% NaN3. The cells were washed twice in cold PBS and
incubated with FITC-labeled secondary antibody (1:50 dilution) for 30 min at 4°C in 100
µl of PBS/1% FCS/.1% NaN3. The cells were washed twice, fixed in 1% formaldehyde, and analyzed by a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 cells were counted for each sample, and final processing was performed.
Debris, cell clusters, and dead cells were gated out by light scattered assessment before
single-parameter histograms were drawn and smoothened.
Cell survival assay by trypan blue exclusion. This assay was adapted from Nahta et al.
(92). BT474 cells were seeded at a density of 1 x 105 cells per well in 12-well plates.
Twenty-four hours later cells were treated in triplicate with 20 µg/mL of anti-peptide
Abs, trastuzumab, or normal rabbit IgG for an additional 72 hours. Cells were trypsinized and stained with trypan blue dye and viable cells were counted under a microscope. Cell growth inhibition is expressed as the percentage of viable cells compared with untreated cells.
49
MTT cell growth inhibition assay. For peptides preventing Trastuzumab from inhibiting
tumor cell growth assay, BT474 cells were plated in 96-well microtiter plates at 2 x 104 cells/well and incubated overnight at 37ºC. PBS containing trastuzumab or normal human IgG (100µg/ml) with or without dilutions of peptide was added to the wells. The plates were incubated for three days at 37ºC. The number of viable cells was measured with MTT by reading OD570. The percentage of inhibition was calculated using the
formula (ODnormal human IgG - ODtrastuzumab+peptide)/ODnormal human IgG x 100. All experiments were performed in triplicate.
For anti-peptide antibodies inhibiting tumor cell growth, the assay was performed in a similar fashion with several modifications. BT474 cells were plated at 1 x 104
cells/well and the following day were treated with 0.1 µg/mL of rabbit anti-peptide Abs,
trastuzumab, or normal rabbit IgG in triplicate for an additional 72 hours. The percentage
of inhibition was calculated using the formula (ODnormal rabbit IgG - ODpeptide Ab)/ODnormal
rabbit IgG x 100.
Antibody-dependent cell mediated cytotoxicity assay (ADCC). Effector PBMCs from
normal human donors obtained by density gradient centrifugation in Ficoll-Hypaque
(Pharmacia Biotech, Piscataway, NJ) were washed twice in RPMI 1640-5%FCS and then
serially diluted in 96-well plates to give effector to target ratios of 25:1, 12.5:1, and
6.25:1. The following day 1 x 106 target cells received 50 µg of Protein A/G purified
anti-peptide rabbit Abs or trastuzumab. BT-474 and MDA-468 target cells (HER-2high
low 6 51 and HER-2 , respectively) were labeled with 100µCi/1×10 cells of Na CrO4 (Perkin
50 Elmer, Boston, MA) and incubated for one hour at 37°C. After three washings 5×103 target cells were delivered to each well so as to give a final volume of 0.2ml/well. The cells were incubated for four hours at 37°C, after which time 75 µl of cell free supernatants were harvested and radioactivity determined using a gamma counter. To assess nonspecific lysis, effector and target cells were co-incubated in the presence of normal rabbit antibodies. Cytotoxicity was calculated by the formula (%) lysis = (A-
B)/(C-B) x 100, where A represents 51Cr (cpm) from test supernatants, B represents 51Cr
(cpm) from target alone in culture (spontaneous release) and C represents maximum 51Cr release from target cells lysed with 5% Triton-X100. Results represent the average of triplicate samples.
Statistical Analysis. Differences in MTT cell proliferation assay were evaluated with the
Student’s t test. Tumor growth over time was analyzed using Stata’s® XTGEE (cross sectional generalized estimating equations) model which fits general linear models that allow you to specify with-in animal correlation structure in data involving repeated measures. The model includes terms for treatment group, time, and the interaction of treatment by time. This interaction term is used to calculate the differences in the slopes of each group. The XTGEE model assumes that the data are normally distributed and that volume is a continuous linear variable. Log transformation of the volume addresses both of these issues. The slopes by treatment of the log transformed tumor volumes were calculated and compared to determine if there was a statistically significant difference between treatments. The significance level was set at α = 0.01 to control for the overall
51 type I error rate when doing multiple comparisons. The results of the above regression
are transformed back into their original units.
2.3 Results
Design and synthesis of conformational and linear peptides. The crystal structure of the
extracellular region of HER-2 bound to the trastuzumab Fab fragment reveals that
trastuzumab binds subdomain IV of the HER-2 ECD (99). The antigen-antibody
interaction is mediated by three regions of HER-2. These regions are composed of loops
which consist of residues 579-583 (formed by two disulfide bonds; C563-C576 and
C567-C584), 592-595 (cysteine disulfide pairing between C587-C596) and 615-625
(cysteine disulfide bond between C600-C623). We have selected this 64 residue
cysteine-rich region of the HER-2 ECD for the design of several peptides to minimally
mimic the binding epitope. Four constructs (Table 2.1) encompassing residues 563-626
were designed to contain as least one region of the three binding sequences that make
contact with trastuzumab. In addition, we have incorporated the native disulfide bonds in
each of these epitopes.
Epitopes containing one or two intramolecular disulfide bond were cyclized using iodine oxidation and characterized as reported (127). In the case of epitope MVF 563-
598, which contains three intramolecular disulfide bonds, the first and second disulfide bond formation was performed with in situ reaction using I2/H2O, the first disulfide bond formation occurring in the first hour. The addition of water boosts the removal of Acm groups and concurrent formation of second disulfide bond. This was confirmed by PEO- maleimide reaction. Biotinylation agent PEO-maleimide, which attacks free sulfhydryl
52 groups to form addition product, can be used to determine the completion of disulfide
pairing and was confirmed by electrospray ionization mass spectroscopy (ESI-MS) (127).
Two Cys (But) groups remained intact during the cyclization procedure. The third
disulfide bond was formed by silyl-chloride-sulfoxide method and the completion of third
disulfide bond was confirmed by PEO-maleimide reaction; no PEO-Maleimide addition
was observed, which was confirmed by ESI-MS characterization (Fig. 2.1). Linear
peptide was generated by DTT reduction. These spectroscopic results indicate that we
have successfully synthesized conformational peptides incorporating three disulfide
bonds.
The HER-2 B-cell epitopes were synthesized co-linearly with a promiscuous Th
epitope derived from the measles virus fusion protein (MVF) (amino acids 288-302). A
four-residue linker sequence, GPSL, connects the Th and B cell epitopes; this linker
sequence forms a hairpin loop. The small, flexible nature of glycine and proline’s ability
to readily form the cis-conformer make these amino acids amenable to tight turns. Serine favors hydrogen bonds with the free amide of the backbone and interacts favorably with solvent and leucine is buried in the hydrophobic core. Independent folding of the Th and
B cell epitopes is achieved using the flexible linker sequence (145, 189).
Specificity of peptides binding to trastuzumab. To examine the ability of trastuzumab to bind the conformational cyclized synthetic peptides, a trastuzumab specificity ELISA was developed. Polystyrene plates were coated overnight with various peptides and the following day probed with trastuzumab. Trastuzumab binds to all four of the peptides
MVF563-598, 585-598, 597-626, and 613-626 but not the irrelevant control peptide
53 MVF316-339 (Fig. 2.2A). The 563-598 peptide exhibited the highest reactivity with the mAb. Moreover, trastuzumab binds the synthetic peptide MVF 563-598 that incorporates the natural disulfide pairings of HER-2 in a dose-dependent manner and to a lesser extent the linear non-cyclized peptide (Fig. 2.2B). These results suggest that the conformation induced by cyclization mimics that native sequence better than the flexible peptide.
Conformational peptides prevent trastuzumab inhibiting tumor cell growth.
Trastuzumab’s ability to bind the ECD of HER-2 and inhibit the downstream signaling of
HER-2, resulting in growth inhibition of HER-2 over-expressing cell lines has been demonstrated extensively (176). Since we have shown that trastuzumab recognizes all four of the peptides, we investigated whether theses peptide prevent trastuzumab from inhibiting tumor cell growth. By pre-incubating trastuzumab with peptide, the response rate of the breast cancer cell-line BT474 to trastuzumab was blocked by all peptides
(MVF563-598CYC, MVF585-598CYC, MVF597-626CYC, and MVF613-626) in a dose-dependent manner, while an equivalent concentration of control peptide had no effect (Fig. 2.3). At a concentration of 60 µg/mL only the 563-598 and 597-626 peptides had a statistically significant decrease (p < 0.05) in inhibition compared to nonspecific peptide (MVF127-144), indicating that both MVF563-598CYC and MVF597-626CYC peptides bind to trastuzumab and prevent the mAb from inhibiting tumor cell growth.
Immune response of peptide constructs in FVB/n mice. We investigated the immune response of each of these constructs in FVB/n mice (n=5-10). Each of the constructs elicited high-titered antibodies in mice, as evidenced by antibody titers over 100,000
54 (Fig. 2.4A). Notably both the cyclized (CYC) and linear (NC) forms of the 563-598
epitope were most immunogenic and induced titers greater than 100,000 in each mouse
two weeks after the second booster injection. In addition, mice receiving the cyclized
versions of 563-598 and 597-626 showed a greater immune response compared to mice
receiving the linear versions of the peptides. IgG1, IgG2a, and IgG2b were the major
isotypes in the mouse sera elicited by the various peptide constructs (Fig. 2.4B).
Interestingly, sera against peptide constructs MVF585-598CYC, MVF597-626CYC, and
MVF597-626NC generated the largest proportion of IgG2a (29-32% of total Ig), an isotype associated with an effective anti-tumor response (168, 188, 190).
Cross-reactivity of the peptide Abs with native HER-2. It is essential for antibodies raised against a synthetic peptide to recognize the native protein in order to be considered a potential vaccine candidate. We tested the binding of FVB/n purified antibodies to the
HER-2 overexpressing human breast cancer lines BT474 and SK-BR-3 by immunofluorescence staining of a single cell suspension (Fig. 2.5, left and middle panel).
Antibodies generated against 597-626 bound well within 1 log of HER-2 specific mouse monoclonal antibody Ab-2 in both BT474 and SK-BR-3 (Fig. 2.5C) cell lines and showed the largest shift of the anti-peptide antibodies. Antibodies against the 613-626 epitope were capable of recognizing the native protein (Fig. 2.5D). However, antibodies to 563-598 and 585-598 showed weak binding to both cell lines (Fig. 2.5A-B). Anti- peptide antibodies did not demonstrate binding to MDA468 (Fig. 2.5, right panel), a non-
HER-2 over-expressing breast cancer cell line.
55 The 563-598 sequence harbors a putative N-linked glycosylation site at residue
571 (Table 2.1, boxed residues). The published crystal structure of HER-2 bound to
trastuzumab was enzymatically de-glycosylated, whereas the crystal structure of HER-2
bound to the pertuzumab Fab fragment reveals a sugar moiety at position 571 (103).
Antibodies against 563-598 peptides did not cross-react with SK-BR-3 cells treated with
tunicamycin, which prevents addition of N-linked oligosaccharides to proteins (data not
shown). The lack of recognition of antibodies elicited to 563-598 may be due to
conformational differences when HER-2 is glycosylated. The 585-598 epitope is most
likely too short (containing only 14 amino acids) to elicit cross-reactive antibodies
specific to HER-2.
Effects of 597-626 peptide constructs in outbred rabbits. Based on the ability of
antibodies induced by the synthetic peptide vaccine 597-626 to bind the native receptor with high specificity and to be recognized by trastuzumab, this epitope was considered to be the most promising vaccine candidate. We therefore evaluated the immunogenicity of the 597-626 construct in outbred New Zealand White rabbits in order to generate a large quantity of Abs for in vitro studies. Both the cyclized and linear peptides elicited high- titered antibodies (Fig. 2.6A). IgG was the predominant isotype generated in rabbits.
Antibodies elicited by 597-626CYC construct contained 95.8% IgG, 3.7% IgM, and 0.5%
IgA, whereas antibodies elicited against 597-626NC had 94.4% IgG, 5.0% IgM, and
0.6% IgA (data not shown).
We examined the cross-reactivity of the rabbit Abs to the native protein utilizing
flow cytometry. Abs raised against both 597-626CYC and NC recognized both HER-2
56 over-expressing cell lines BT474 (Fig. 2.6B) and SK-BR-3 (Fig. 2.6C) but not MDA468
cells (Fig. 2.6D) which do not over-express HER-2. This data suggests that the
antibodies induced by the vaccine constructs were specific for the HER-2 protein.
In addition, the binding of anti-peptide antibodies to HER-2 was measured
utilizing two ELISA assays. First, we performed a sandwich ELISA in which SK-BR-3
cell lysate was used as a source of HER-2. Fig 2.7A reveals that anti-597-626 antibodies
(1:100 dilution) recognize HER-2 in a similar manner as trastuzumab (20µg/ml). Next,
the ability of 597-626 anti-peptide antibodies to bind recombinant human HER-2/Fc
chimera was examined. Both 597-626CYC and 597-626NC antibodies had a titer of
8000 (Fig. 2.7B) against the native protein. To test whether the 597-626 anti-peptide
antibodies bound to the same epitope as trastuzumab, the mAb or isotype control human
IgG were used as competitor for antigen binding in ELISA experiments. Anti-peptide
antibodies were allowed to bind to immobilized HER-2 in the presence and absence of
various concentrations of trastuzumab or human IgG. At a concentration of 1000ng/ml,
trastuzumab was able to inhibit the binding of anti-597-626CYC antibodies (Fig. 2.7C)
and anti-597-626NC antibodies (Fig. 2.7D) to HER-2 by 75.0% and 70.9%, respectively.
The results demonstrate that both anti-597-626CYC and anti-597-626NC antibodies
recognize the same or a similar determinant as that of trastuzumab.
Effect on breast cancer cell viability and proliferation. We next examined the effect of
597-626 peptide antibodies on tumor cell survival in vitro. BT474 cells were plated
overnight; the next day cells were treated with either 20 µg/mL of Abs elicited from
597CYC, 597NC, trastuzumab, normal rabbit IgG, or media alone. Cell viability was
57 measured after 72 hours by trypan blue exclusion. Cells treated with antibodies elicited
from 597-626CYC and 597-626NC had 61% and 46% viability, respectively compared
with untreated cells (Fig.2.8A) whereas trastuzumab treated cells had 21% viability,
indicating that antibodies against both conformational and linear form of 597-626 are
able to decrease BT474 cell viability. The trypan blue test only indicates if a cell is alive
and does not indicate the growth rate of cells as does the MTT assay in which cells with functional mitochondria are needed to convert the tetrazolium dye into its reduced form.
We therefore examined the ability of anti-peptide antibodies to effect in vitro
tumor cell proliferation using the MTT assay. As shown in Figure 2.8B antibodies
elicited by peptide epitopes 597CYC and 597NC had a similar effect on the proliferation
of BT474 cells (19 and 18% inhibition, respectively), whereas trastuzumab demonstrated
a 59% inhibition on tumor cell proliferation. These findings demonstrate that antibodies
against both conformational and linear forms of 597-626 are able to diminish cell
viability as well as have anti-proliferative effects on BT474 cells in vitro.
Ability of anti-peptide Abs to mediate ADCC. It has been well documented that in vivo
the Fc portions of Abs can be of foremost importance for efficacy against tumor targets
(57). When Fc binding is reduced or completely removed, trastuzumab loses virtually all
of its antitumor activity in vivo (191). Consequently, Fc-dependent ADCC is critical for
in vivo efficacy. Therefore, we measured the ability of the anti-peptide Abs to mediate
ADCC in vitro. Peptide antibodies elicited in rabbits against both the cyclized and linear
peptide invoked lysis of the breast cancer cell line BT474 in the presence of human
PBMCs, analogous to trastuzumab (Fig. 2.9). These results suggest that antibodies raised
58 against both conformational and linear form of 597-626 are able to mediate ADCC in a similar manner as trastuzumab.
Effects of peptide constructs in BALB-neuT mice. We used the BALB-neuT transgenic
mouse mammary cancer model as a measure of the ability of the peptide constructs to
reduce tumor progression. BALB/c inbred mice transgenic for the transforming activated
rat HER-2/neu oncogene under the control of a mammary-specific promoter is likely the
most aggressive model of HER-2/neu carcinogenesis (187). A point mutation that
replaces the valine residue at position 664 in the TM domain with glutamic acid favors
HER-2/neu homo- and heterodimerization and renders the neu gene product
constitutively active (192). Animals rapidly develop tumors; in preliminary studies utilizing untreated mice, all animals developed tumors by 25 weeks of age.
There is 88% sequence homology between human HER-2 and rat neu; the human
597-626 sequence has 93% homology with the rat neu sequence, with two disparate
amino acids (Fig. 2.10A). We examined whether Abs raised against 597-626 were
capable of recognizing the rat neu receptor using the TUBO cell line, a cell line
established in vitro from a lobular carcinoma that arose spontaneously in a BALB-neuT
mouse. As depicted in Fig. 2.10B, Abs against both cyclized and linear forms of 597-626
were shifted relative to normal rabbit IgG and were comparable to Ab-4, a mouse mAb
that binds rat neu. Flow cytometric analysis of the non-neu expressing TS/A cell line
demonstrated that no antibodies bound this cell line (Fig. 2.10C).
Based on these results, the conformationally restricted MVF597-626 was chosen
for study in female transgenic BALB-neuT mice. Previous studies have shown the age at
59 which mice are immunized is critical (168). Transgenic mice that began receiving dendritic cells transduced with adenovirus expressing the neu oncoprotein past 6 weeks old had larger tumor burden compared to mice receiving the vaccine at 5-6 weeks of age
(132). Transgenic mice were immunized with 597-626CYC beginning at 5-6 weeks of age. Mice received two booster immunization at three week intervals and subsequently two additional immunizations at four week intervals. Impressively, 597CYC elicited high-titer anti-neu antibody responses in all mice three weeks after the third immunization (Fig. 2.11A). At 25 weeks of age untreated mice and mice immunized with irrelevant peptide had an average tumor burden of 3486 mm3 (±1166) and 2720 mm3
(±1163) respectively (P=0.6441). Mice immunized with 597-626CYC had a significant reduction in tumor burden (P<0.0001), with an average tumor burden of 378 mm3
(±228.0) (Fig. 2.11B).
We wished to investigate the type of tumor infiltrating leukocytes present in solid tumors of BALB-neuT mice. Neutrophils are the first line of defense against infections and are known to infiltrate tumors (193). In addition, a majority of tumors appear to be infiltrated by macrophages, which are capable of killing tumor cells but are also able to stimulate growth by producing angiogenic factors (194). We performed immunohistochemistry to examine whether neutrophils, macrophages, and T cells had infiltrated the solid tumors of BALB-neuT mice. We also wished to examine differences in tumor infiltrating leukocytes between vaccinated and control mice. There was evidence of some CD3+ T cell infiltration in tumors, although there was no statistical difference in the number of infiltrating CD3+ T cells between untreated, MVF597CYC treated, and MVF treated (Fig. 2.12 A-C, left panel). This indicates that the vaccine did
60 not increase the number of T cells within the tumor microenvironment. Little or no
neutrophils were observed in any of the tumors (data not shown). F4/80 staining revealed
the presence of macrophages in all tumors. Interestingly, the staining pattern of F4/80
varied between MVF597CYC treated and control mice. Control mice (both untreated
and MVF treated) tumors had staining at the periphery of tumor, whereas a MVF597CYC
tumor showed F4/80 staining within the tumor proper (Fig. 2.13 A-C, left panel).
Macrophages found within the tumor of MVF597CYC treated mice are capable of
mediating ADCC, a possible mechanism of action of the vaccine.
The in vivo anti-tumor activity observed here correlates with the in vitro studies indicating the 597-626 construct is capable of inhibiting tumor cell growth as well as mediate ADCC in a similar manner as trastuzumab. These results indicate that the conformationally-restricted 597-626 epitope is capable of significantly inhibiting tumor growth in a mouse model that parallels several characteristics of the stepwise mammary carcinogenesis in women.
2.4 Discussion
HER-2 vaccines have been designed that use whole cells expressing tumor antigens (119-
121), proteins (122), as well as DNA expression plasmids (123-125). Most of the immunotherapies targeting the HER-2 oncoprotein have focused on T-cell epitopes, and several studies have produced notable results indicating that vaccinated patients can develop immunity to HER-2 peptides and native protein (135, 195, 196). Recent clinical trial results using a HER-2 CTL epitope showed an increase in disease-free survival in
61 vaccinated HER-2/neu-expressing breast cancer patients (85.7%) compared to the control group (59.8%) (197). However, the effectiveness of a CTL vaccine for clinical use is limited to patients who express the appropriate HLA haplotype. To date mAbs, based on
B-cell immune responses, and not vaccines to activate the T-cell immune responses, have been successful in clinical trials and approved for usage (198). In particular the clinical efficacy of trastuzumab suggests that the generation of a robust and focused humoral immune response may be biologically significant for tumor defense.
Prior to the publications of the three-dimensional structure of HER-2 (99, 100), the identification of HER-2 B-cell epitopes was achieved through computer-aided analysis (126, 128, 129). In addition several HER-2 B-cell mimotopes have been identified through phage display (48, 152, 153). Riemer et al. (152) utilized a constrained
10-mer random peptide phage display library to identify peptide mimotopes to trastuzumab; antibodies raised against one of these peptides recognized HER-2/neu and caused internalization of the receptor from the cell surface in a similar manner as trastuzumab. Although this peptide sequence bears no sequence homology to HER-2, it was matched to the third loop of HER-2 at the HER-2/trastuzumab interface using computational methods (199). Jiang et al. (153) identified another mimotope that matched to an epitope between loops 1 and 2 of HER-2 at the HER-2/trastuzumab interface. These studies indicate that all three loops are important for trastuzumab binding HER-2.
We have designed four peptide constructs that each contains at least one of the three loops of HER-2 involved in binding trastuzumab. The utility of synthetic peptides to represent protein domains is restricted by conformational issues. The protein fragment
62 of interest is stabilized by secondary and tertiary interactions in the native protein, but the matching peptide in solution will typically have a random coil structure. Lacking structural restraints, the flexibility of peptides can lead to varied conformations presented to the immune system, most of which are non-native (200). Linear peptides are highly flexible and can adopt a variety of conformations in solution. However, only a few of these conformations are responsible for their immunoreactivity (201). One approach to achieve molecular mimicry to the parent protein is through constricting the peptide by cyclization if the natural sequence bears cysteine residues that are paired to provide loop sequences with enhanced stability. Cyclic peptides can cause preferred spatial arrangements that duplicate the bioactive conformation, resulting in improved binding and immunological properties.
In our previous studies we demonstrated that conformational cyclic epitopes
HER-2 sequence 626-649 (127) had the desired secondary structural characteristics as determined by circular dicroism (CD) measurements. Antibodies against the cyclized epitope bound the HER-2 protein with a higher affinity than the non-cyclized epitope and were twice as effective in ADCC assay and in reducing tumor growth in transgenic mice.
However, in the present study, we were unable to differentiate between the conformations of the epitope region spanning residues 597-626. Similarly the antibodies that were generated were also very similar in reactivity and biological efficacy.
We have designed peptide constructs to include the native disulfide bonds of the
epitopes to more closely mimic the native structure. Each of the peptide constructs were
recognized by trastuzumab, with 563-598 showing the greatest recognition. In addition
all peptides were able to prevent trastuzumab from inhibiting tumor cell growth.
63 However, antibodies raised against the 563-598 epitope do not, to an appreciable extent, recognize HER-2 as measured by flow cytometry.
This may be due to the asparagine linked glycosylation site (571-NGS) found within this sequence. It has been reported previously that trastuzumab’s mouse counterpart mAb, 4D5 recognized glycosylated HER-2 and not un-glycosylated HER-2,
indicating that either the mAb recognizes a conformation of the protein attained only
when it is glycosylated, or, conversely, the epitope recognized by 4D5 comprises partly
of carbohydrate (49). Antibodies against the 563-598 peptide inability to bind native
HER-2 may due to a local conformational change when HER-2 is glycosylated, or,
alternatively, dominant epitopes in the 563-598 peptide are not surface exposed on the
native protein. However, antibodies raised against the third loop of HER-2 (597-626) did recognize HER-2. The goal of a B-cell vaccine is to have antibodies against the vaccine recognize the native antigen, not necessarily that the vaccine construct is able to bind a monoclonal antibody that recognizes the antigen. To this end we pursued the 597-626 peptide for further investigation. The 597-626 epitope was immunogenic in outbred rabbits; these polyclonal antibodies recognized HER-2. In addition, competition experiments revealed that trastuzumab was able to inhibit the binding of anti-597 Abs to
HER-2, indicating the anti-peptide Abs bound the same epitope as trastuzumab.
Trastuzumab is known to affect tumor growth by both direct and indirect mechanisms. The direct mechanisms involve binding to HER-2 and altering the receptor’s signaling properties that can result in tumor growth cell inhibition (176). Anti-
597 Abs were able to diminsh cell viability as well as inhibit tumor cell growth of the
BT474 cell line in a similar manner as trastuzumab. The indirect mechanisms involve the
64 classical pathways in which trastuzumab kills tumor cells by mediating ADCC and CDC.
We show here that anti-597 Abs were able to mediate ADCC in a manner similar to
trastuzumab.
In order to demonstrate in vivo efficacy of the 597-626 vaccine, we utilized the
BALB-neuT transgenic mouse model. Although a more desirable model would be mice transgenic for human HER-2, only recently has a model been described in which animals form tumors (202). Antibodies against the 597-626 epitope were cross-reactive with rat neu protein, thus we utilized this animal model. It has been shown that the induction of
anti-HER-2/neu antibodies are both necessary and sufficient for protection of BALB-
neuT mice from developing tumors as shown by depletion of CD4+ and CD8+ cells (168,
190). Thus, this animal model is advantageous to studies that identify B-cell epitopes necessary in the protection of BALB-neuT transgenic mice from developing tumors.
By 25 weeks of age, mice immunized with the 597CYC construct had a
statistically significant reduction in tumor burden compared to both naïve and MVF
immunized mice. Immunohistochemical analysis of tumors reveals no difference in the
number of CD3+ tumor infiltrating cells between 597CYC vaccinated and control mice,
discounting the possibility that a T cell response to the vaccine may have played a role in
efficacy. We are not aware of any studies that have reported the existence of MHC class
I-restricted T cell responses to the 563-626 sequence of HER-2 (203). A study has
identified HER-2 sequence 605-619 as a possible T helper epitope; this sequence was
predicted to bind numerous histocompatibility alleles using computer algorithms (204).
However, T helper cells specific for HER-2 605-619 were unable to proliferate in
response to processed HER-2 when either PBMCs or DCs were used as APCs. Based on
65 this information as well as studies demonstrating an inability to generate a T cell response in vaccinated BALB-neuT mice and immunohistochemical data we present here, we
attribute the anti-tumor response to the generation of anti-peptide antibodies. Since we
have demonstrated that antibodies against the 597-626 epitope bind to HER-2 at the trastuzumab interface, the mechanism of action of the endogenous tumor protection in
BALB-neuT mice most likely include down modulation of the HER-2/neu receptor as well as interaction with the immune system via the Fc domain of endogenous Abs against the 597-626 epitope.
In summary, we report here an epitope that mimics the HER-2/trastuzumab interface capable of inducing Abs with anti-tumor properties that significantly reduces tumor burden in vivo in transgenic mice. There are inherent limitations of passive immunotherapy with trastuzumab including unequal tissue distribution, limited half-life,
prolonged administration, possible immunogenicity with high dosages, and
cardiotoxicity. Immunotherapy with peptide vaccines that produces endogenous Abs
may be more valuable than repeated administration of an exogenous mAb. Peptide
vaccines are easy to produce, amenable to quality-control, and cost-effective. The active
generation of Abs with similar characteristics as trastuzumab has the potential to suppress
the development of HER-2 over-expressing breast cancers.
66 2.4 Tables and Figures
Table 2.1 Amino Acid Sequence of HER-2 Trastuzumab-Binding Epitopes
a Peptides containing disulfide bonds (CYC) are shown; linear versions (NC) were also synthesized (not shown in table)
b Residues involved in binding trastuzumab are shown in bold; a possible N-linked
glycosylation site in the 563-598 epitope is boxed. Underlined amino acids were mutated
from Cys to Leu so as not to interfere with natural disulfide formation.
67
Figure 2.1 Synthetic strategy for generating three disulfide pairings in peptide.
MVFGPSL-563-598 from the trastuzumab-binding region of HER-2. Disulfide bonds are selectively introduced in the epitope; the peptide was synthesized with chemoselective protected cysteine residues. Each cysteine pair of a disulfide bond was identically protected to achieve desired intramolecular cyclization. The PEO-Maleimide reaction and subsequent ESI-MS characterization confirmed the intramolecular disulfide pairing.
68
Figure 2.2 Binding of trastuzumab to peptides. Microtiter wells were coated overnight with 2 µg/ml of various peptides and then blocked with 1% BSA for one hour.
Trastuzumab was then added to plates at a concentration of 2000 µg/ml and serially diluted 1:2 with PBT. Bound trastuzumab was detected with HRP-conjugated anti- human IgG and then with substrate. (A) The OD415 value for peptides from Table I and an irrelevant control peptide (MVF316-339) using 2000 µg/ml of trastuzumab. Values shown are the mean of duplicate samples. SEM are indicated by error bars. (B) Titration of trastuzumab with the disulfide-bound (CYC) and linear (NC) forms of MVF563-598 along with irrelevant control peptide (MVF316-339). 69
Figure 2.3 Cell proliferation by MTT assay. BT474 cells were plated in 96-well
microtiter plates at 2 x 104 cells/well and incubated overnight at 37ºC. PBS containing trastuzumab or normal human IgG (100µg/ml) with or without peptide at the indicated concentrations was added to the wells. MVF127-144 is an irrelevant control peptide.
The plates were incubated for three days at 37ºC. The number of viable cells was measured with MTT by reading OD570. The percentage of inhibition was calculated using the formula (ODnormal human IgG - ODtrastuzumab+peptide)/ODnormal human IgG x 100. Values shown are the mean of triplicate samples. SEM are indicated by error bars.
70
Figure 2.4 Immunogenicity of HER-2 peptide constructs in FVB/n mice. A. Indirect
ELISAs were performed; results of individual mice are shown (n=5-10). Ab titers were
defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or
greater after subtracting the background. 1Y+3 indicates the titer of blood drawn three
weeks after the first immunization. Pre-immune sera was used as a negative control (data
not shown). B. Two weeks after the final immunization (3Y+2), the level of Ig subtypes
were measured using a mouse isotyping kit. The concentrations of IgG3, IgA, and IgM
were <10% (data not shown).
71
Figure 2.5 Cross-reactivity of peptide antibodies to breast cancer cell lines. The reactivity of purified antibodies from immunized FVB/n mouse sera was tested with
BT474 (left panel), SK-BR-3 (middle panel), which are breast cancer cell lines that over-
express HER-2, and MDA468 (right panel), a non-HER-2 over-expressing cell line,
using flow cytometric analysis. Antibodies shown were raised against peptides (A) 563-
598, (B) 585-598, (C) 597-626, and (D) 613-626. Ab binding was detected with goat-anti
mouse FITC-conjugated secondary abs. Histograms indicate linear peptide Abs (light
72 gray shading), cyclized peptide antibodies (dark gray shading), normal mouse IgG
(negative control, light gray line histogram), and Ab-2 (positive control, black shading).
73
Figure 2.6 Effect of vaccination with 597-626 epitope in outbred rabbits. (A),
Indirect ELISAs were performed to determine the immunogenicity of the cyclized and linear constructs in pairs of outbred rabbits. Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. 1y+3w indicates the titer of blood drawn three weeks after the first immunization. Pre-immune sera was used as a negative control (data not shown). (B)
BT474, (C) SK-BR-3, and (D) MDA468 cells were incubated with peptide vaccine
induced Abs; the extent of tumor binding was assessed by flow cytometry. Histograms
indicate 597NC Abs (light gray shading), 597CYC (dark gray shading), normal rabbit
IgG (negative control, dotted line histogram), and trastuzumab (positive control, black
shading).
74
Figure 2.7 ELISA data demonstrating anti-597-626 Abs bind HER-2 and
Trastuzumab inhibits anti-597-626 Abs binding to HER-2. (A), A sandwich ELISA was carried out to evaluate Abs specific to HER-2. SK-BR-3 cell lysate was added to plates containing capture Ab Ab-2. Sera was analyzed at 1:100 along with 20 µg/mL of trastuzumab (positive control). (B-D) Plates were coated overnight with 500 ng/ml of recombinant human HER-2/Fc chimera. (B) Serial dilution of rabbit anti-597 abs and
anti-CH354 abs (irrelevant control) starting at a 1:1000 dilution were added to plates. X-
axis represents dilution of rabbit sera. (C and D) A constant amount (1:2000 dilution) of
rabbit anti-597-626 CYC abs (C) or anti-597-626 NC abs (D) was added to the plates and
at the same time various amounts of inhibitor (trastuzumab or isotype control human
IgG) was added. Bound anti-peptide rabbit antibodies were detected with HRP-
conjugated anti-rabbit IgG antibody and then substrate. X-axis represents concentration 75 of inhibitor trastuzumab() or human IgG (). The inhibition rate was calculated according to the following formula: (ODanti-peptide-Ab - ODanti-peptide-Ab + inhibitor)/ (ODanti-
peptide-Ab)x 100. Values shown are the mean of duplicate samples. SEM are indicated by
error bars.
76
Figure 2.8 Anti-peptide Abs decrease cell viability and inhibit tumor cell proliferation in vitro. A Trypan blue exclusion assay showing the effects of purified antipeptide Abs on the viability of BT474 cells. 1 x 105 cells were incubated with media alone, 597CYC Abs, 597NC Abs, trastuzumab, and normal rabbit IgG (20 µg). The number of viable cells remaining after three days was determined by the trypan blue exclusion assay. Cell viability is given as a percentage of untreated cells. Data points represent the mean of three independent experiments; bars represent SEM. B. Effects of purified antipeptide Abs on the proliferation of BT474 cells. 1 x 104 cells were incubated
with 597CYC Abs, 597NC Abs, trastuzumab, and normal rabbit IgG (0.1 µg).
Bioconversion of MTT was used to estimate the number of viable tumor cells remaining
after 3 days. The proliferation inhibition rate was calculated using the formula (ODnormal
rabbit IgG - OD peptide Ab)/ODnormal rabbit IgG x 100. Error bars represent SEM. 77
Figure 2.9 Anti-peptide Abs are capable of mediating ADCC. 51Cr-labeled mammary
tumor target cell lines BT-474 was assayed in the presence of human PBMCs. The
percentage of cytotoxicity was calculated as described in "Materials and Methods." Bars
represent SEM of triplicate wells.
78
Figure 2.10 Cross-reactivity of HER-2 peptide antibodies with the rat neu receptor.
(A), The amino acid sequences of rat neu (top) and human HER-2 (bottom, light gray
shading) were aligned between human HER-2 sequence 597-626. Asterisks indicate
disparate residues. Flow cytometric analysis was performed on TUBO (B) and TS/A (C)
cell lines. TUBO are derived from a spontaneous breast cancer of a BALB-neuT
transgenic mouse and over-express rat neu; TS/A is a spontaneous breast cancer from a
wild-type BALB/c mouse. Histograms indicate 597NC Abs (light gray shading),
597CYC (dark gray shading), normal rabbit IgG (negative control, dotted line histogram),
and Ab-4 (anti-neu Ab, black shading). 79
Figure 2.11 Immunogenicity and immunoprotective effects of HER-2 peptide epitope on autochthonous tumor development in BALB-neuT transgenic mice.
BALB-neuT mice (n = 5-7) were immunized with MVF, MVF597CYC or left untreated.
Beginning at 5-6 weeks of age, mice were treated five times at 3- to 4-wk intervals. (A),
ELISAs were performed to determine the immunogenicity of the 597CYC construct in
BALB-neuT mice. Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. 1y+3w indicates
80 the titer of blood drawn three weeks after the first immunization. (B), Tumor
measurements were performed twice a week on each of ten mammary glands. Tumor
volumes were calculated by the formula (long measurement x short measurement2)/2.
The data are presented as the average tumor size per group and are reported as mm3. The group receiving MVF597CYC showed significant prevention of tumor growth compared with the naive group or the group receiving MVF (*p < 0.0001). Error bars represent
SEM.
81
Figure 2.12 Immunohistochemical images of infiltration by CD3+ T lymphocytes in solid mammary tumors. Sections of formalin fixed mammary tumor tissues were obtained from 25 week old BALB-neuT mice untreated (A), immunized with
MVF597CYC (B), and immunized with MVF alone (C). The left panel represents
82 addition of primary rabbit anti- mouse CD3 polyclonal Abs, whereas the right panel is the
rabbit isotype control IgG. Scattered CD3- positive T lymphocytes are present in solid
mammary tumors. No significant difference in the number of CD3- positive cells was found between each group (the mean of 3 random fields using the student’s t test; p <
0.05 considered significant). Images were acquired at x40 original magnification with a
Nikon Eclipse E400 microscope and Image-Pro Plus v.5.0 software.
83
Figure 2.13 Immunohistochemical images of infiltration by macrophages in solid mammary tumors. Immunohistochemical analysis for F4/80, a cell surface antigen expressed by cells of monocyte-macrophage derivation in the mouse, was performed on
BALB-neuT mice untreated (A), immunized with MVF597CYC (B), and immunized
84 with MVF alone (C). Images were acquired at x20 original magnification. The left panel represents addition of primary rat anti- mouse F4/80 mAb, whereas the right panel is the rat isotype control IgG. Note that F4/80 staining for untreated and to some extent MVF appears in the interstitial space, whereas F4/80 staining from MVF597CYC appears in the tumor proper.
85 CHAPTER 3
PEPTIDE VACCINE STRATEGIES TARGETING THE HER-2 DIMERIZATION
LOOP
3.1 Introduction
Members of the human epidermal growth factor receptor (HER) of receptor tyrosine kinases include EGFR (HER1, ErbB1), HER-2 (ErbB2), HER-3 (ErbB3), and
HER-4 (ErbB4). The receptors are positioned at the cell membrane and have an extracellular ligand-binding region, a transmembrane region and a cytoplasmic tyrosine kinase domain. Ligand binding to the receptors results in receptor hetero- and homo- dimers, activation of the kinase domain, and subsequent phosphorylation of specific tyrosine residues within the cytoplasmic tail (13, 18). Proteins are recruited to these phosphorylated residues, leading to signal transduction pathways that promote cell growth, proliferation, differentiation, and migration. Lacking a high-affinity ligand,
HER-2 is the preferred heterodimeric partner. HER-2 has been found to be overexpressed in cancers of the breast, ovary, uterus, lung, and gastrointestinal tract (40-
42, 180, 181). HER-2 protein overexpression or gene amplification, which occurs in approximately 30% of breast cancer, indicates poor clinical outcome (28, 29, 205). Thus,
HER-2 is a validated target in cancer. Trastuzumab (Herceptin), a humanized anti-HER-
86 2 mAb, was the first HER-2 targeted therapy approved by the U.S. FDA for the treatment of HER-2 over-expressing metastatic breast cancer. Recently, trastuzumab has shown
significant benefit when administered as a first-line therapy on women with HER-2 over-
expressing metastatic breast cancer (206).
Another mAb targeting HER-2, pertuzumab (Omnitarg) represents a new class of
targeted therapeutics known as HER-2 ‘dimerization inhibitors’ that block homo- and
hetero- dimerization of HER-2 (102). Pertuzumab is a humanized IgG1 mAb that binds to HER-2 at domain II of the ECD, sterically blocking heterodimerization of HER-2 with
EGFR and HER-3, thus inhibiting intracellular signaling (80, 101, 207). Pertuzumab, unlike trastuzumab which is only effective against HER-2 over-expressing tumors, is effective in tumor cells that express lower HER-2 levels (80). Phase II trials have been conducted with pertuzumab involving patients with prostate (104, 107), ovarian (106), breast and NSCLC (105). These trials indicate that pertuzumab is well-tolerated and clinically active.
Despite impressive clinical results with anti-HER-2 mAb therapy and other antibody therapeutics, there are several drawbacks including severe side effects such as cardiocytotoxicity that have been observed in some patients (61). Given that antibodies represent a growing class of human therapeutics, their use is as yet, rarely, if ever completely curative. Thus, there is considerable interest in developing both a prophylactic and therapeutic vaccine that could mediate anti-tumor activity, have sustained immune responses, and exhibit little toxicity with lower associated costs.
Indeed, patients have shown the ability to mount weak humoral and cellular immune responses against HER-2/neu (117, 208). Both HER-2/neu-specific cytotoxic T
87 lymphocytes (CTLs) and IgG antibodies directed against HER-2/neu have been detected in 30-50% of breast cancer patients (203, 209), indicating that it is possible to break tolerance and mount an immune response against the HER-2/neu receptor (135, 195). Our laboratory proposed using B cell peptide epitopes as candidate HER-2/neu vaccines, and developed several epitopes by mapping regions in the extracellular domain using computer-aided analysis (126, 128). Two HER-2/neu epitopes, 628-647 and 316-339, were identified as viable candidates for active immunotherapy and these are presently being studied in a Phase I trial.
The structure determination of the HER-2/pertuzumab Fab fragment complex showing the complexity of the binding site (residues 266-333) provides considerable insights for the development of new vaccine candidates (103). Pertuzumab binds to
HER-2 near the center of domain II; binding is predicted to sterically block the region necessary for HER-2 dimerization with other members of the HER family and thus prevent signal transduction independent of HER-2 overexpression (210). Corroborating the structural data, pertuzumab is unable to block signaling to HER-3 when tested with
HER-2 mutants in residues predicted to be important for this interaction. As a strategy to minimally dissect the conformational preference of this region, we selected and designed several peptides spanning sequences 266-296, 298-333 and 315-333 that overlap the pertuzumab binding site on the dimerization loop.
In this study, we report on the activity of several constructs containing complex, differential disulfide pairings (Table 3.1) as well as their linear counterparts in order to determine the best mimic of the pertuzumab-binding conformational region which can result in an effective vaccine that could be translated to the clinic. The immunogenicity
88 of each cyclized (CYC) and noncyclized (NC) constructs was determined in both mice
and rabbits, eliciting a high affinity, high titer antibody response. All the antibodies raised against the peptide constructs recognized the native HER-2/neu receptor with varying degrees of reactivity. The ability of the 266-296 peptide constructs to elicit the highest titer antibodies; highest recognition of native HER-2 by showing a greater shift by FACS analysis comparable to trastuzumab indicates that this sequence was the most cross-reactive to the native protein. Additionally, three of the six putative conformational
constructs, 266-296CYC, 266-296NC, and 315-333CYC were able to mediate ADCC.
Our results show that the 266-296 pertuzumab-like epitope constructs had statistically
reduced tumor onset in both transplantable tumor models (FVB/n and Balb/c) and
significant reduction in tumor development in a transgenic mouse tumor model (Balb-
neuT). These studies demonstrate that both the linear (NC) and conformational (CYC)
peptide vaccines corresponding to residues 266-296 of the dimerization region of HER-
2/neu are able to elicit an immune response with antitumor capabilities, resulting in a
peptide vaccine that will be able to mimic the effects of pertuzumab in vivo without the
harmful side effects associated with mAb therapy.
3.2 Materials and Methods
Cell Lines and Antibodies. All culture media, fetal calf serum (FCS), and supplements
were purchased from Invitrogen (Carlsbed, CA). The human breast cancer cell lines
BT474, SKBR-3 and MDA468 were purchased from American Type Culture Collection
(Manassas, VA) and maintained according to the supplier’s guidelines. Mouse breast
tumor cell lines NT2.5 and TUBO were kind gifts from Drs. R. Todd Reilly and John C.
89 Morris. Ab-1, a rabbit polyclonal antibody that binds the kinase domain of HER-2 and
Ab-4, a mouse monoclonal antibody that binds the extracellular domain of neu were
purchased from Calbiochem (La Jolla, CA).
Animals. Female New Zealand White rabbits, FVB/n and Balb/c mice were purchased
from Harlan (Indianapolis, IN). Virgin female Balb-neuT (187) mice were generated by
breeding wt Balb/c females with heterozygous Balb-neuT males. Animal care and use
was in accordance with institutional guidelines.
Synthesis and Characterization of Conformational and Linear Peptides. HER-2/neu B
cell epitopes 266-296, 298-333, and 315-333 were colinearly synthesized with a
promiscuous TH cell epitope derived from the measles virus fusion protein (MVF,
residues 288-302) using a four residue linker (GPSL). Peptide synthesis was performed
on a Milligen/Biosearch 9600 peptide solid-phase synthesizer (Bedford, MA) using
Fmoc/t-butyl chemistry as previously described (127). Trt was used as side chain
sulfhydryl protection for the 266 and 315 epitopes, and double protection for 298
included Trt on Cys 315 and 331 and Acm on Cys 299 and 311. Peptides were cleaved
from the resin using cleavage reagent B (trifluoroacetic acid:phenol:water:TIS, 90:4:4:2),
and crude peptides purified by semi-preparative RP-HPLC and characterized by
electrospray ionization mass spectroscopy (ESI-MS) (186). Intramolecular disulfide
bonds were formed using iodine oxidation as described (211) and disulfide bridge
formation was further confirmed by Maleimide-PEO2-biotin reaction and subsequent analysis using ESI-MS.
90
Immunization of Rabbits and Mice. NZW rabbits were immunized with 1 mg peptide
dissolved in ddH2O emulsified (1:1) in Montanide ISA720 vehicle with 100 µg of nor-
MDP (N-acetylglucosamine-3yl-acetyl-L-alanyl-D-isoglutamine). BALB/c and FVB/n mice, 6-8 wks old, were immunized with 0.1 mg peptide emulsified in ISA720 with 100
µg nor-MDP. Rabbits and mice were boosted with the respective doses at 3 week intervals. Blood was collected via the central auricular artery in rabbits and retro-orbital sinus in mice and sera tested for antibody titers. Balb-neuT mice (5-6 weeks old) were
immunized as described above. After the third vaccination, the Balb-neuT mice received
boosters at monthly intervals. Mice were euthanized at 25 weeks of age.
Antibody Purification. High-titered sera were purified on a protein A/G-agarose column
(Pierce, Rockford, IL) and eluted antibodies were concentrated and exchanged in phosphate-buffered saline using 100-kDa cut-off centrifuge filter units (Millipore,
Bedford, MA). The concentration of antibodies was determined by Coomassie plus
protein assay reagent kit (Pierce).
ELISAs. Antibody titers were determined as previously described (128), and is defined as
the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after
subtracting background.
Mouse Isotyping. Antibodies raised in mice were typed using a Mouse Typer Isotyping
Kit (Bio-Rad, Hercules, CA). The assay was performed according to the manufacturer’s
91 instructions, except that a 1:1000 dilution of goat anti-rabbit IgG horseradish peroxidase conjugate was used.
HER-2 ELISA. Plates were coated overnight at 4°C with 100 µL of 10 µg/mL of
trastuzumab (Herceptin™, Genetech, San Francisco, CA), washed four times with 0.1%
Tween/PBS, and blocked with of 100 µL of PBS-1% BSA 4 h. Plates were washed four
times with 0.1% Tween/PBS. Wells were coated overnight at 4°C with 50 µL of either
PBS-1% BSA or SK-BR-3 cell lysate (1×108 cells in 20 mL lysis buffer (1% Triton X-
100, 10% glycerol, 150 mM NaCl, 50 mM HEPES, 1.5 mM MgCl2, 1mM EDTA, 10 mM pyrophosphate, 100 mM NaF, 0.2 mM Na3VO4, 10 µg/mL aprotinin, 10 µg/mL
leupeptin, 1 mM PMSF)). Plates were washed four times with 0.1% Tween/PBS, a 1:100
dilution of rabbit sera added, and incubated 2 h on a rocker. Antibody binding was
detected using goat-anti rabbit IgG HRP.
Flow Cytometry. BT474 and MDA468 (1×106) cells were incubated with anti-peptide
antibodies in 100 µL of 2% FCS in PBS for 2 h at 4°C. Normal rabbit IgG was used as a
negative control and humanized trastuzumab used as a positive control. Unbound
antibodies were removed with PBS, and the cells incubated with fluorescein
isothiocyanate-conjugated (FITC) anti-rabbit antibodies for 30 min at 4°C in 100 µL of
2% FCS in PBS. Cells were washed in PBS and fixed in 1% formaldehyde before being
analyzed by Coulter ELITE flow cytometer (Coulter, Hialeah, FL). A total of 10,000
cells were counted for each sample. Debris, cell clusters, and dead cells were gated out
by light scatter assessment before single parameter histograms were drawn and smoothed. 92
ADCC. As described previously (127). Briefly, 1×106 BT474 cells were incubated with
51 50 µg of anti-peptide antibody and 100 µCi of Na CrO4 for 1 h at 37ºC. Unbound antibody and excess chromium was removed and the cells added to human PBMCs and incubated 4 h at 37ºC. Cell-free supernatant was collected and the release of chromium by lysed cells measured using a scintillation counter. % Lysis = 100 × (Experimental -
Spontaneous)/(Maximum - Spontaneous).
Tumor Challenge. NT2.5: Ten days after the third immunization, FVB/n mice were challenged with 3×106 NT2.5 cells. Mice were monitored twice weekly for the presence
of palpable tumors for a total of 24 days.
TUBO: Fourteen days after the third immunization, Balb/c mice were challenged with
1×105 TUBO cells. Mice were monitored twice weekly for the presence of palpable
tumors for a total of 39 days.
Tumor Measurements. Tumor Challenge: Palpable tumors were measured in a blinded
fashion with Vernier calipers and tumor volume calculated by the formula
(length×width2)/2.
Balb-neuT Tumors: Tumors in each of ten mammary glands were measured for tumor
volume as described above. Results are reported as total mean tumor volume per group.
Statistical Analysis. Tumor growth over time was analyzed using Stata’s® XTGEE (cross sectional generalized estimating equations) model which fits general linear models that 93 allow you to specify with-in animal correlation structure in data involving repeated measures. The model includes terms for treatment group, time, and the interaction of treatment by time. This interaction term is used to calculate the differences in the slopes of each group. The XTGEE model assumes that the data are normally distributed and that volume is a continuous linear variable. Log transformation of the volume addresses both of these issues. The slopes by treatment of the log transformed tumor volumes were calculated and compared to determine if there was a statistically significant difference between treatments. The significance level was set at α = 0.01 to control for the overall type I error rate when doing multiple comparisons. The results of the above regression are transformed back into their original units (212).
3.3 Results
Selection, Design, and Characterization of Peptides. The crystal structure of the Fab fragment of pertuzumab bound to the ECD of HER-2/neu (103) reveals that pertuzumab binds to HER-2/neu in subdomain II of the HER-2 ECD. Subdomain II contains an extensive disulfide-bonding network with seven disulfide-bonded modules. Within the central disulfide-bonded module is a β-hairpin (residues 269-288) that extends beyond the rest of the protein. Each EGFR monomer contains this same β-hairpin extension, where it forms one side of the dimerization interface (97, 98). The 266-333 region of
HER-2 was selected for the design of peptides with the objective of eliciting antibodies against the peptides capable of inhibiting dimerization of HER-2 with other members of the EGFR family. We examined three different sequences, 266-296, 298-333, and 315-
333 to determine the best minimal conformational epitope for effective B cell
94 immunization (Table 3.1). The 266-296 sequence contains the β-hairpin loop as well as
four residues that contain atoms which directly contact pertuzumab atoms (as indicated
by bold residues in Table 3.1). The 298-333 sequence contains 7 amino acids which
contact pertuzumab, whereas the 315-333 sequence has 4 residues in contact with
pertuzumab. Although the 266-296 contains the β-hairpin loop that protrudes from the protein and is involved in heterodimerization, residues in 298-333 and 315-333 (Ser 310,
Leu 317, and His 318) are essential for pertuzumab binding HER-2 based on mutagenesis studies (103). The correct disulfide pairings were achieved by selective side chain protection. For the 266-296 and 315-333 peptides, the side chain trityl was removed, and
+ the disulfide bridge formed by I2 oxidation in acetic acid (266-296, M+H Cal/Obs
5759/5760; 315-333, M+H+ Cal/Obs 4493/4495). The two disulfide bonds in the 298-333 peptide were achieved (M+H+ Cal/Obs 6278/6280) as previously described (127).
Immunogenicity of constructs in outbred rabbits. Extremely high antibody titers
(250,000-500,000) were obtained in rabbits immunized with both MVF266CYC and
MVF266NC (Fig. 3.1A). Rabbits immunized with MVF298CYC and MVF298NC also
elicited high titers (60,000-130,000) (Fig. 3.1B). Rabbits immunized with the
MVF315CYC or MVF315NC construct elicited slightly lower titers (40,000-60,000),
most probably due to the smaller size of the construct (Fig. 3.1C). The predominant
isotype generated from all peptides was IgG (greater than 95%), whereas IgM and IgA
antibodies were less than 5% of total Ig (Fig. 3.1D). The ability of the MVF266 peptide
constructs to elicit the highest titer antibodies indicates that this sequence was the most
immunogenic.
95
Cross-reactivity of peptide antibodies with the HER-2 receptor. Binding of the intact
HER-2/neu receptor was determined by immunofluorescence staining of a single cell suspension of BT474 cells, a HER-2 over-expressing cell line. Differential binding was observed for each construct at 5µg. Antibodies elicited by both MVF266CYC and
MVF266NC bound the receptor similar to the control HER-2-specific mAb trastuzumab
(Fig. 3.2A, left panel). Antibodies to the MVF298CYC construct bound just within one log of trastuzumab, while the MVF298NC antibodies did not exhibit any HER-2 protein binding (Fig. 3.2B, left panel). MVF315CYC antibodies bound within one log of trastuzumab, and MVF315NC induced antibodies did not bind (Figure 3.2C, left panel).
No binding was observed with the MDA468 cell line, a non-HER-2 overexpressing breast cancer cell line (Fig. 3.2A-C, right panel). An indirect ELISA using SKBR-3 cell lysate as a source of HER-2 was also used to determine the anti-peptide antibodies’ ability to bind the native receptor in comparison to the positive control rabbit polyclonal antibody, Ab-1 (Figure 3.2D). MVF266CYC and MVF266NC antibody binding confirmed the flow cytometry data, exhibiting the highest absorbance with the cyclic peptide binding strongly, suggesting that the cyclized epitope may better mimic the corresponding site on the native HER-2/neu protein. On the other hand, both the
MVF298CYC and MVF298NC antibodies bound with the next highest absorbance, while
MVF315CYC and MVF315NC antibodies bound with the lowest absorbance. The results of these studies show that of the six constructs, the MVF266 peptide antibodies exhibited the strongest binding to the native receptor.
96 Antitumor activity of peptide antibodies. We next tested the ability of the peptide
antibodies to mediate ADCC using human PBMCs as effector cells (213, 214). Both
MVF266CYC and MVF266NC (50µg) induced antibodies showed high levels of dose- dependent cell lysis with Effector:Target of 20:1 showing between 60-65% lysis as compared to the positive control trastuzumab with 75% lysis. MVF315CYC antibodies induced 46% cell lysis, while MVF315NC, MVF298CYC and MVF298NC antibodies did not exhibit lysis levels over background (Fig. 3.3). The ADCC assay results correlate with the ability of the antibodies against MVF266CYC, MVF266NC, and MVF315CYC to bind the native receptor in vitro indicating that these antibodies not only bind the
native receptor, but are able to effectively mediate anti-tumor activity.
Immunogenicity and tumor challenge in FVB/n mice. To investigate the in vivo efficacy of the peptide constructs, we initially studied the immunogenicity of each construct in
FVB/n mice that could then be challenged with the cell line NT2.5, derived from a spontaneous mammary tumor isolated from FVB/n202 transgenic mice (originally described by Guy et al. (215)). As a consequence of neu overexpression these mice
develop spontaneous mammary adenocarcinomas in a manner similar to that observed in human breast cancer patients (119), and are therefore a suitable model for human breast cancer studies. Fig. 3.4 demonstrates that the 266-296 constructs were extremely immunogenic compared to the 298-333 and 315-333 constructs. One week after the third immunization, twelve of sixteen mice immunized with the 266-296 constructs had antibody titers greater or equal to 64,000 (Fig. 3.4A). No mice immunized with the 298-
333 constructs had a titer greater or equal to 64,000 (Fig. 3.4B) and four of sixteen mice
97 immunized with the 315-333 constructs had antibody titers greater or equal to 64,000
(Fig. 3.4C).
To determine whether there was an immunoprotective effect conferred upon
FVB/n mice immunized with the constructs, ten days following the third immunization, mice were challenged with the NT2.5 cell line. The mean tumor volume in each treatment group is shown in Fig. 3.5. There was a significant difference between the tumor burden of mice immunized with MVF266CYC or MVF266NC versus MVF- immunized controls (p=<0.001 and p=0.002, respectively) that indicates both peptide vaccine constructs were protective (Fig. 3.5A). However, there was no significant difference between MVF control and mice immunized with the 298 and 315 constructs
(Fig. 3.5B,C). This data correlates with the ability of anti-peptide Abs against the 266 constructs binding HER-2 and mediating ADCC. Additionally, the lack of tumor protection in FVB/n mice immunized with the 298 and 315 constructs supports the poor
Ab responses elicited against the constructs in these mice.
We wished to examine the Ab isotype distribution elicited by each construct. The antibody isotype distribution is similar for protective peptides (MVF266CYC and NC)
and non-protective peptides (MVF298CYC and NC, MVF315CYC and NC) (Fig. 3.6).
IgG2a accounted for 32-45% of total Ig, an isotype associated with an effective anti-
tumor response (168, 188, 190). The same relative isotype distribution between
protective and non-protective peptides indicates that efficacy of the 266-296 vaccine was
due to elicitation of a strong humoral response and the affinity of the anti-peptide Abs for
the HER-2 dimerization interface.
98 In vivo studies with the BALB-neuT transgenic model. Based on the ability of the 266
construct to bind HER-2, mediate ADCC, and inhibit tumor cell proliferation and HER-2
phosphorylation (data not shown, see Allen et al. (216)), we pursued the 266 construct in
the BALB-neuT mouse model. This model is likely the most aggressive and consistent
model of rat HER-2 mammary carcinogenesis. Between the 10th and 20th week, in situ carcinomas around the nipple become invasive and metastasize to the bone marrow and lungs (217).
There is 97% sequence homology between human HER-2 and rat neu within the human HER-2 266-296 sequence with only one disparate amino acid (Fig. 3.7A). We performed flow cytometry to determine if MVF266 anti-peptide antibodies were cross- reactive with rat neu using NT2.5 cells. Antibodies raised against both MVF266CYC and MVF266NC (5µg) were shifted relative to isotype control IgG and bound the receptor similar to the control neu-specific antibody Ab-4 (Fig. 3.7B).
We initially investigated the in vivo efficacy using a transplantable model, challenging Balb/c mice with TUBO cells derived from a tumor of a BALB-neuT transgenic mouse (188). Groups of Balb/c mice (n=10) were immunized with
MVF266CYC, MVF266NC or MVF and antibody titers monitored on a weekly basis.
Lower titers were observed in the Balb/c mice as compared to the FVB/n mice (Fig.
3.4A), and no MVF-specific antibodies were detectable in immunized control mice
(Figure 3.8A). The mean tumor volume over time for each of the three treatment groups are shown in Fig. 3.8B. Statistical significance was found between mice immunized with
MVF versus mice immunized with MVF266CYC or MVF266NC (p=0.0002 and
99 p=0.0007, respectively), which confirms that immunization of mice with either
MVF266CYC or MVF266NC reduces tumor burden.
We then investigated the efficacy of the 266-296 epitope in transgenic BALB-
neuT mice. Animals rapidly develop tumors; in preliminary studies, 100% of untreated
mice developed tumors by 25 weeks of age. The MVF266 epitope elicited high-titer antibody responses three weeks after the third immunization (Fig. 3.9A). Mice immunized with MVF266 had a significant reduction in tumor volume as compared to mice immunized with MVF (p=0.0068) or left untreated (p=0.0067) (Fig. 3.9B). We conclude that the MVF266 peptide constructs are effective in eliciting protective immune responses by generating high titer antibodies that bind the native HER-2/neu receptor and
inhibit the growth and differentiation of cancerous cells.
3.3 Discussion
The HER-2/neu protein is an appealing target for cancer therapy as it is overexpressed in
a significant fraction of breast cancers. Patients with HER-2/neu overexpressing tumors
have shown the ability to mount weak immune responses to this antigen, indicating that
HER-2/neu is immunogenic. In addition, the HER-2/neu receptor is exposed to the
extracellular matrix making it available for direct antibody binding. Trastuzumab, a
humanized mAb, was one of the first target-specific molecules to be successfully
exploited for clinical use (218). Recent studies have demonstrated significant
improvements in disease-free survival of women with early stage HER-2-positive breast
cancer when trastuzumab was combined with adjuvant chemotherapy (63, 64).
Pertuzumab, another humanized mAb, is also a HER-2/neu inhibitor that has a different
100 mechanism of action from trastuzumab. Pertuzumab binds to the dimerization loop in the
ECD of HER-2/neu, preventing HER-2/neu from interacting with other members of the
ErbB family, thus blocking the activation of tyrosine kinase domains and inhibition of phosphorylation of tyrosine residues on the intracellular portion of the receptors and further signal transduction.
There are a number of issues with the use of passive cancer immunotherapy including the requirement for repeated dosing and its high cost, the development of resistance through loss of immunodominant epitopes, and undesired immunogenicity of humanized and chimerized antibodies. A vaccine would trigger the body to produce its own antibodies with the advantage of a sustained immune response due to long term immunologic memory.
Cardiocytotoxicity is a known side effect in patients receiving trastuzumab (61).
Active immunotherapy offers the possibility of avoiding this side effect. FVB/n,
BALB/c, and BALB-neuT mice immunized with the MVF266-296 peptide vaccine did not demonstrate any external heart impairment. In addition Jasinska et al. did not find signs of inflammation or toxicity in the heart, lung, liver, or kidney of mice immunized with HER-2 peptide vaccines (129). Patients that receive trastuzumab are infused with a large amount of the mAb every one to three weeks over a long period of time. We argue that the amount of mAb (i.e. trastuzumab or pertuzumab) passively infused in patients is much greater than the amount of HER-2 reactive antibodies in an individual receiving three vaccines at 3 week intervals and thus the cardiotoxicity is reduced or eliminated.
There have been a number of studies with peptide cancer vaccines, the majority of which target the cellular arm of the immune system by activating CD8+ cytolytic T cells
101 (CTL). To date, most of the HER-2/neu-specific peptide vaccines have been restricted to
T cell epitopes. The most studied HER-2 derived peptide is HER-2(p369-377), originally identified by Fisk et al. (219) as a CTL HLA-A2 binding epitope. The HER-2(p369-377)
(also referred to as E75) used in a clinical trial with breast cancer patients was able to induce intra- and interantigenic spreading (220). The Disis group (196, 221) vaccinated patients with HER-2/neu-overexpressing tumors with a mixture of potential Th epitopes
HER-2(p369-384) (containing the p369-377 CTL epitope), HER-2(p688-703), and HER-
2(p971-984) admixed with GM-CSF. Ninety-two percent of the patients developed a T cell response to the peptides, with a preferential response directed against the HER-
2(p369-384) epitope, and 38% of the patients continued to show immunity one year later
(196, 221). One of the major drawbacks of T cell based vaccines is the restricted applicability due to MHC haplotype specificity.
Our laboratory has focused on the humoral arm of the immune system by creating vaccines that combine a molecularly defined B cell epitope with a promiscuous Th activating epitope. Our work has focused on the identification, characterization and evaluation of the B cell epitopes of the ECD of the HER-2/neu oncoprotein. Recently, we examined the effect of conformationally constraining an epitope from near the trastuzumab-binding region of HER-2/neu and found that the cyclized, conformational epitope enhanced antitumor activity (127). Our ongoing efforts to enhance antibody affinity and cross-reactivity have led us to investigate whether constraining the dimerization loop peptide with the native disulfide bonds would enhance the affinity of the antibodies and consequently its biological in vivo efficacy.
102 Pertuzumab has shown activity in reducing cellular proliferation, signal transduction and tumor growth in xenograft models (80, 101, 207, 222). We identified three peptide epitopes (266-296, 298-333, 315-333) that mimic the dimerization loop of the HER-2/neu receptor and used them for vaccines to induce endogenous antibodies to this region. The immunogenicity of the disulfide-bonded (CYC) and linear (NC) peptides were evaluated in rabbits. High antibody titers were elicited with MVF266-
296CYC, MVF266-296NC, MVF298-315CYC, and MVF298-315NC constructs. Lower titers were seen in rabbits immunized with MVF315-333CYC and MVF315-333NC, presumably due to the small size of the epitope. The antibodies generated were tested by flow cytometry and we found that both the MVF266-296 constructs bound to HER-2 expressing cells with the cyclized form binding slightly better than the noncyclized form.
Antibodies against the MVF315-333CYC construct showed strong binding to the cognate receptor, while antibodies against the MVF298-333CYC, MVF298-333NC, and
MVF315-333NC constructs showed weak or no binding. These findings were further supported by the results of testing the antitumor effects of the antibodies by ADCC.
MVF266-296CYC, MVF266-296NC, and MVF315-333CYC all showed HER-2 specific cell lysis, while the other three constructs did not produce tumor lysis above background levels. Interestingly, when the antibodies were tested for binding to the native receptor in cell lysates, both the MVF298-333 constructs showed stronger binding than both of the
MVF315-333 constructs did. This could be attributed to the native protein not being tested in intact cells, but in cellular lysates which could have either modified the conformation of the receptor or exposed additional binding sites in the intracellular and transmembrane regions.
103 We initially performed in vivo studies on FVB/n mice, challenging mice with the
NT2.5 cell line as done previously in our laboratory (127). Both the 298-333 and 315-
333 constructs were weakly immunogenic in FVB/n mice, whereas the 266-296 produced a strong Ab response. In corroboration with the immunogenicity studies, only mice immunized with the 266-296 epitopes had a statistical reduction in tumor burden.
We further investigated the efficacy of the 266-296 construct using the BALB-
neuT mouse model. We used a tumor challenge model in which BALB/c mice were
immunized before being challenged with syngeneic TUBO tumor cells. Immunization
with MVF266CYC and MVF266NC significantly inhibited tumor growth. In addition,
we tested the vaccine constructs in a transgenic autochthonous tumor model of breast
cancer, which is more clinically relevant than challenge models as they actually go
through the process of tissue transformation and tumor development without external
manipulations. Active immunization of Balb-neuT mice with the MVF266 peptide
construct resulted in a reduction of tumor burden in treated mice as compared to control
mice. The results from these models echo the results found by other groups testing the
ability of dimerization blockers to reduce tumor growth in vivo.
There was a reduction in tumor burden but not complete protection of animals
vaccinated with the MVF266 peptide constructs using both syngeneic tumor transplants
and transgenic mice. We do not believe that residual tumor growth is due to a lack of
affinity of the antibodies to HER-2/neu based on binding studies of anti-266 Abs to HER-
2 (Fig. 3.2). Tumors present many barriers for endogenous antibodies to gain access
including heterogenous vascularization and high interstitial fluid pressure, opposing
convection and diffusion. Thus residual tumor growth could be due to the anti-peptide
104 Abs inability to access all tumor cells. Alternatively, the residual tumor growth could be
due to resistance of cells to HER-2/neu targeted therapy. Trastuzumab-resistance has been attributed to increased signaling by the PI3K/Akt pathway as well as loss of function of the tumor suppressor PTEN gene, the negative regulator of Akt (34). These mechanisms of resistance could explain the residual tumor growth. Additionally, of importance is that the Balb-neuT model is an extremely aggressive model of rat HER-
2/neu carcinogenesis. This transgenic model uses the mouse mammary tumor virus
(MMTV) promoter, an extremely potent promoter targeting the transgenes for mammary
glands. Residual tumor growth can also be attributed to the particularly aggressive nature
of this model.
Our studies indicate the 266-296 peptide epitope holds the most promise as a
prophylactic vaccine against HER-2/neu-expressing breast cancer. Results show that
while conformational restraints do not necessarily lead to enhanced antitumor effects for
this specific construct, the ability of MVF315CYC to better bind the native protein and
mediate ADCC activity as well as previous results (127) indicate that conformation is
important for some epitopes. It is possible that the MVF266NC construct folds in a
similar conformation to the MVF266CYC peptide even without the disulfide bridge,
while other constructs require the conformational constraint to more closely mimic the native protein. Given the mode of action of pertuzumab in that it sterically interferes with HER-2 dimerization and signaling pathways, it is expected that the 266-296 construct could be effective in both HER-2 overexpressing cancers as well as normal
HER-2 expressing cancers such as lung, ovarian and colon. Pertuzumab is being investigated in clinical trials in patients whose tumors do not contain the amplified ErbB2
105 gene since pertuzumab inhibits the dimerization of HER-2 with EGRR and HER-3.
Thus, our vaccine targeting the dimerization arm of HER-2 could be efficacious in patients who do not over-express HER-2 but have normal expression. Although the mechanisms by which antibodies exert their therapeutic effects are still being debated, the putative mechanisms are either direct (i.e., block signaling functions, internalization of receptors, reduce proteolytic cleavage of receptors) or indirect action mediated by the immune system (complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytoxicity (ADCC). For future studies we plan to investigate in detail the mechanism of action of the anti-peptide antibodies including the effect of downstream proteins of HER-
2 including Akt and MAPK. Additionally, such studies will also include combination vaccine therapy in which 266-296 will be added to epitopes from the trastuzumab binding site to determine if there is an additive/synergistic effect on antitumor capabilities.
106 3.4 Tables and Figures
Peptide Sequenceb Designationa
266-296 peptide MVF 266- H2N-KLLSLIKGVIVHRLEGVE-GPSL- with one disulfide 296CYC LHCPA LVTYNTDTFESMPNPEGRYTFGASCV –COOH bond
298-333 peptide MVF298- H2N-KLLSLIKGVIVHRLEGVE-GPSL- with two disulfide 333CYC ACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEK-COOH bonds
315-333 peptide MVF315- H2N-KLLSLIKGVIVHRLEGVE-GPSL- with one disulfide 333CYC CPLHNQEVTAEDGTQRCEK-COOH bond
Table 3.1 Amino Acid Sequence of HER-2 Pertuzumab-Binding Epitopes
a Peptides containing disulfide bonds (CYC) are shown; NC peptides are free peptides without disulfide bonds (not shown in table).
b MVF sequence is italicized. Cysteine residues are underlined to indicate the locations
of the disulfide bonds. Residues in bold indicate individual amino acids which directly
contact pertuzumab (<3.2 Å distance).
107
Figure 3.1 Antibody responses elicited by peptide vaccines in outbred rabbits. Two rabbits per group were immunized with one vaccine construct for a total of four
108 injections. Blood was drawn weekly, and sera surveyed for peptide-specific antibodies by ELISA. Each bar represents one rabbit. Titers are defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. 2y+3w indicates the antibody titer in blood drawn three weeks (3w) after the second immunization (2y). White bars indicate cyclized peptide immunization; gray bars indicate noncyclized peptide immunization. (A) Rabbits immunized with the
MVF266 constructs. (B) Rabbits immunized with the MVF298 constructs. (C) Rabbits immunized with the MVF315 constructs. (D) The levels of IgG, IgM, and IgA were determined for pooled sera obtained two weeks after the third immunization for each peptide construct. The y-axis represents the percentage of total Ig.
109
Figure 3.2 Cross-reactivity of the antipeptide antibodies to native HER-2/neu. Flow cytometry was used to assess the binding capabilities of antipeptide antibodies to the native receptor. Purified antibodies (5µg) from immunized rabbit sera were tested against 110 BT474 (left panel), a breast cancer cell line that over-expresses HER-2, and MDA468
(right panel), a non-HER-2 over-expressing cell line. Histograms contain overlays of
rabbit preimmunization IgG (negative control, dotted line), peptide antibodies (cyclized
(CYC), dark gray shading; non-cyclized (NC), light gray shading), and trastuzumab (5µg, positive control, black shading). Antibody binding was detected by goat-anti-rabbit
FITC-conjugated secondary antibodies. The x-axis represents fluorescent intensity, and the y-axis represents relative cell number. (A) Binding of MVF266CYC and
MVF266NC antibodies to BT474 (left panel) and MDA468 (right panel). (B) Binding of MVF298CYC and MVF298NC antibodies to BT474 (left panel) and MDA468 (right panel). (C) Binding of MVF315CYC and MVF315NC antibodies to BT474 (left panel) and MDA468 (right panel). (D) Binding of antipeptide antibodies to cellular lysate of
SKBR-3 cells as measured by indirect ELISA. Reported as averages ±SEM.
111
Figure 3.3 Anti-peptide antibodies mediate antibody-dependent cellular cytotoxicity in vitro. Target cell line BT474 was coated with 50µg of purified antipeptide antibodies from rabbits, normal rabbit IgG, normal human IgG (negative controls) or trastuzumab
(positive control), then cultured in the presence of human PBMC effector cells to give an effector:target ratio (E:T) of 100:1, 20:1, and 4:1 in triplicates. Results are representative results of three experiments, ±SEM.
112 .
Figure 3.4 Antibody responses elicited by peptide vaccines in inbred FVB/n mice.
Indirect ELISAs were performed to determine the immunogenicity of the cyclized and linear constructs in FVB/n mice (inbred). Each treatment group consisted of 8 mice and the results of individual mice are shown. Ab titers were defined as the reciprocal of the highest serum dilution with an absorbance of 0.2 or greater after subtracting the background. (A) Mice immunized with the MVF266 constructs. (B) Mice immunized with the MVF298 constructs. (C) Mice immunized with the MVF315 constructs.
1y+3w indicates the titer of blood drawn 3 weeks after the first immunization.
Preimmune and Th cell epitope (MVF) sera were used as negative controls (data not shown).
113
Figure 3.5 NT2.5 tumor challenge in vaccinated FVB/n mice. FVB/n mice were immunized with MVF266-296, MVF298-333, MVF315-333, or MVF (n=8) three times and immunogenicity determined (see Fig. 3.4). Ten days after the third immunization, mice were challenged with 3×106 NT2.5 cells subcutaneously. Tumor size was monitored twice weekly for a total of 24 days. (A) Mice immunized with the MVF266 constructs. (B) Mice immunized with the MVF298 constructs. (C) Mice immunized with the MVF315 constructs. For clarity three separate graphs are shown, although the control MVF is identical in each graph. Results are reported as average tumor size
(mm3)+SEM. *p=<0.001; **p=0.002.
114 60
IgG1 50 IgG2A IgG2B 40 IgG3 Ig l a t
o 30 t f % o 20
10
0
C C C C C C Y N Y Y N 6 5 6C 6 98N 5C 1 6 2 98C 2 1 3 2 2 F 3 F VF F V VF M V M VF MV M M M
Figure 3.6 Isotype distribution of HER-2 peptide constructs in FVB/N mice. Two weeks after the final immunization (3Y+2), the level of Ig subtypes were measured using a mouse isotyping kit. The concentrations of IgA, and IgM were <5% (data not shown).
115
Figure 3.7 Cross-reactivity of the MVF266 antipeptide antibodies to rat neu. (A)
The amino acid sequences of rat neu (top) and human HER-2 (bottom, light gray shading) were aligned between human HER-2 sequence 266-296. Neu disparate residues are underlined. (B) Flow cytometric analysis was performed on the NT2.5 cell line using 5 µg of Abs. Histograms indicate MVF266NC antibodies (light gray shading),
MVF266CYC (dark gray shading), normal rabbit IgG (negative control, dotted line histogram), and Ab-4 (anti-neu Ab, black shading).
116
Figure 3.8 In vivo suppression of transplantable tumor growth by active immunization with MVF266 peptide epitopes. (A) Immunogenicity of peptide vaccines determined in inbred Balb/c mice (n=10). Fourteen days after the third immunization, mice were challenged with 1×105 TUBO cells subcutaneously. (B)
Tumor size was monitored twice weekly for a total of 39 days. Results are reported as average tumor size (mm3)+SEM. *p=0.0007; **p=0.0002.
117
Figure 3.8 In vivo suppression of autochthonous tumor growth by active immunization with MVF266 peptide epitopes. Balb-neuT mice (n=5-8) were
immunized with MVF, MVF266NC or left untreated. Beginning at 5-6 weeks of age,
mice were treated five times at 3- to 4-wk intervals. (A) Immunogenicity of the
MVF266NC construct in Balb-neuT mice. (B) Tumor measurements were performed
twice a week on each of ten mammary glands. The data are presented as the average
tumor size (mm3)+SEM. *p=0.0067; **p= 0.0068
118 CHAPTER 4
EFFECT OF DOSE ON HUMORAL IMMUNE RESPONSE IN PATIENTS
VACCINATED WITH MULTI-EPITOPE PEPTIDE-BASED VACCINES
TARGETING HER-2
4.1 Introduction
The HER-2 oncogenic protein is a tumor antigen that is highly expressed in many
epithelial-derived cancers (29). Tumor over-expression of HER-2 in breast cancer is
associated with a poor prognosis and a high risk of cancer relapse (28). Trastuzumab
(herceptin), a humanized IgG1 mAb against the ECD of HER-2, strongly inhibits the
growth of HER-2 over-expressing cell lines and xenografts in preclinical models (49, 52).
In metastatic HER-2 over-expressing breast cancer patients, administration of trastuzumab alone or in combination with paclitaxel produced response rates between 20 and 50% (60, 90). More recent studies with early-stage HER-2 over-expressing breast cancer patients receiving trastuzumab in combination with chemotherapy regimens after surgical resection demonstrate the addition of trastuzumab significantly prolongs disease- free survival and reduces the chance of recurrence (63, 64). As a result of these studies, trastuzumab has recently been approved in combination with doxorubicin,
119 cyclophosphamide and paclitaxel in adjuvant treatment for early stage breast cancer after
primary therapy.
Despite clinical success with trastuzumab, there are limitations with trastuzumab
mAb therapy stemming from the short half-life of IgG, necessitating repeated treatments, resulting in high costs for treatment. Vaccines targeting HER-2 could avoid the limitations of trastuzumab via elicitation of a long-lasting endogenous immune response.
Detectable antibody and T-cell immune responses have been identified in cancer patients whose tumors over-express HER-2 (208, 223, 224). An advantageous method of immunizing cancer patients against weakly immunogenic self-proteins expressed in their tumors is via peptide vaccination. Peptide vaccines are made of fragments of the tumor antigen specifically designed to elicit a B-cell or HLA class I (CTL) and/or HLA class II
(Th) antigen-specific T-cell responses. There have been a number of clinical trials using
HER-2 peptides recognized by CTLs and Th cells (195, 197, 221). However, there is a paucity of data using B-cell epitope based vaccines targeting HER-2. B-cell epitopes do not have specific HLA restrictions as do CTL or Th cell epitope-based peptide vaccines and are thus appropriate in an outbred population. Additionally, no T-cell epitope cancer vaccine has been approved for usage in humans, whereas trastuzumab represents the proof-of-concept of the efficacy of antibody treatment.
Prior to the publication of the three-dimensional structure of HER-2 alone and in complex with two mAbs, we have used a computer based approach to determine prospective B-cell epitopes, reviewed by Kaumaya et al (145). Algorithms used in this approach will locate regions that are surface exposed based on analyzing a protein’s hydrophilicity, flexibility, mobility, amphiphilicity, solvent exposure, and protrusion.
120 One epitope sequence, 628-647, elicited antibodies which were cross-reactive to the native HER-2 receptor and were able to inhibit the proliferation of HER-2 over- expressing breast cancer cells and cause their antibody-dependent cell-mediated cytotoxicity in vitro. Additionally, immunization with the 628-647 peptide prevented the spontaneous development of HER-2 over-expressing mammary tumors in 83% of transgenic mice (128). Furthermore, abs induced by a combination of two vaccines, 316-
339 and 628-647, down-modulated HER-2 receptor expression and activated IFN-γ release better than individual vaccines. Moreover, this multi-epitope vaccine in combination with IL-12 caused a significant reduction in pulmonary metastases induced by challenge with syngeneic tumor cells over-expressing HER-2 (126). These preclinical data suggested that a multi-epitope construct consisting of 316-339 and 628-647 could be a clinically relevant vaccine in HER-2-expressing cancers.
The study described here is a phase I active immunotherapy clinical trial using
MVF-316-339 and MVF-628-647 in combination with nor-MDP adjuvant. The goal of this study was to determine the safety of this approach, assess the optimal dosing of this vaccine, and measure the immunogenicity of the vaccine. The results show the vaccine was well-tolerated and a subset of patients produced a humoral immune response against the vaccine.
4.2 Patients and Methods
Patient Characteristics and Clinical Protocols. The Human Institutional Review Board at the Ohio State University Medical Center approved the clinical protocol. The clinical trial was conducted under an investigational new drug application (IND-9803) approved
121 by the Food and Drug Administration. Patients with histologically confirmed metastatic
and/or recurrent solid tumors were eligible for enrollment. Additionally, in order to be
eligible for enrollment in the trial, patients had received standard therapy and were no
longer responding. Patients were not required to have HER-2 over-expression prior to
study entry. Study patients were required to be at least 18 years of age and meet the following eligibility criteria: patients with a prior history of treated brain metastases must
have stable disease for at least 3 months; must be ambulatory with an ECOG/Zubrod
performance status of 0, 1, or 2; normal organ function; must be at least 4 weeks past any
prior surgery, cytotoxic chemotherapy, other immunotherapy, hormonal therapy, or
radiation therapy; and be capable of giving written informed consent in accordance with federal and institutional guidelines. Patients were excluded from participation if they were pregnant; or had a significant concurrent illness such as an underlying immunological disease, uncontrolled or severe cardiac disease, or had active viral hepatitis, HIV, or other infectious agents. Additionally, patients were skin tested with
MVF-628-647 and MVF-316-339 for immediate hypersensitivity; those with a positive
skin test (>20 mm) were not eligible for the trial.
Vaccine. The combination vaccine was comprised of a mixture of MVF-HER-2 (628-
647) and MVF-HER-2 (316-339), two synthetic peptides consisting of B-cell epitopes of
HER-2 corresponding to amino acids 628 to 647 and 316-339. Individual immunogens were synthesized co-linearly via a 4-residue linker sequence (G-P-S-L) to the measles virus fusion protein Th epitope corresponding to amino acids 288 to 302. The peptides were produced in good manufacturing practices grade by the Synthetic Peptide
122 Application Lab, University of Pittsburg (Pittsburg, PA). The purity was guaranteed to
be greater than 98% by HPLC, amino acid analysis and mass spectrometry. The peptide
was also tested by amino acid analysis and mass spectrometry to ensure correct amino
acid composition. In addition, the peptide was tested for residual organic solvents.
Peptide content, counterions and water were quantified to ensure correct mass balance.
The remaining component of the vaccine was a synthetic adjuvant muramyl dipeptide,
nor-MDP (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine) purchased from
Pennisula Laboratories, Inc. (San Carlos, CA) and a saline-oil phase vehicle consisting of
4 parts squalene to 1 part mannide monooleate (Montanide ISA 720, SEPPIC, Paris) as an emulsifying agent. MVF-HER-2 (628-647), MVF-HER-2 (316-339), and nor-MDP dissolved in water were emulsified 50:50 in Montanide ISA 720 vehicle. The immunogen-adjuvant-vehicle was administered intramuscularly (1.0 mL volume) into gluteus maximus muscle and subsequent injections were administered in alternating gluteal muscles unless precluded by previous local reaction.
Vaccination Series. The study was performed as a dose-escalating safety trial (Table
4.1). Six patients for each dose level received three inoculations at three week intervals:
0.25 mg of MVF-316-339 and 0.25 mg of MVF-628-647 (dose level 1), 0.5 mg of MVF-
316-339 and 0.5 mg of MVF-628-647 (dose level 2), 1.0 mg of MVF-316-339 and 1.0 mg of MVF-628-647 (dose level 3), and 1.5 mg of MVF-316-339 and 1.5 mg of MVF-
628-647 (dose level 4). 4 weeks after the third inoculation (day 71) patients were restaged with appropriate imaging modalities. Patients with a clinical response or stable disease at day 71 were eligible for a 6 month booster vaccine.
123 Starting at dose level 1, 3 patients received each dose level and were observed for a minimum of 4 weeks. If 0-1 of these patients experienced dose-limiting toxicity (DLT, defined below), an additional 3 patients were entered at that dose level and observed for a minimum of 4 weeks. If 2-3 of the initial 3 patients at the dose level experienced DLT, additional enrollment at the dose level would have been terminated and the previous dose level identified as the maximum tolerable dose (MTD). In the absence of dose-limiting
toxicity, successive cohorts of patients were entered into the protocol at increasing doses of peptide vaccine. Thus, MTD was defined as dose level 4, since 0-1 patients experienced DLT for each dose level.
Toxicity and Response Assessment. Toxicity was assessed using the NCI Common
Toxicity Criteria 2.0. Patients who experienced any grade 3 non-hematological or hematological toxicity including grade 3 flu-like symptoms or any grade 3 injection site reaction were considered to have experienced a DLT. In the absence of DLT, successive cohorts of patients were entered onto the protocol at increasing dose level. Responses were evaluated using the NCI Response Evaluation Criteria in Solid Tumors (RECIST).
Procurement of Patient Serum and Peripheral Blood Mononuclear Cells. Blood draw for plasma and peripheral blood mononuclear cells (PBMCs) from patients was done before receiving each inoculation and at 4 hours post-vaccination (Fig. 4.1). Approximately forty mL of peripheral blood was drawn into heparinized tubes. Tubes were centrifuged for the isolation of plasma and PBMCs. Plasma samples were frozen at -70°C. PBMCs were separated by density gradient centrifugation with Ficoll-Paque Plus (Amersham)
124 and subsequently washed 3 times with RPMI-1640 media. PBMCs were cryoperserved
and stored at -134°C in liquid nitrogen. In addition, weeks in which patient did not
receive vaccine, approximately 8 mL of blood was drawn and plasma samples isolated
(Fig. 4.1). If at day 71 a patient had an antibody response against the vaccine, blood was
drawn monthly thereafter until no antibody response was detected.
Enzyme-linked Immunosorbent Assay. The presence of antibodies specific for peptide
vaccine constructs MVF-316-339 and MVF-628-647 in patient’s serum was directly
assessed using enzyme-linked immunosorbent assays (ELISA). 96-well plates were
coated with 100 µl of antigen at 2 µg/ml in PBS overnight at 4°C. Patient plasma samples were thawed at 25°C and centrifuged to remove particulate matter. Nonspecific binding sites were blocked for 1 h with 200 µl of PBS-1% BSA, and plates were washed with PBT. Patient’s serum, both pre-serum and a specific time point, (1:4 dilution) in
PBT was added to an antigen-coated plate in duplicate wells, serially diluted 1:2 in PBT, and incubated for 2 h at room temperature. After washing the plates, 100 µl of 1:500 goat
anti-human IgG conjugated to horseradish peroxidase (Pierce) were added to each well and incubated for 1 h. After washing, the bound antibody was detected using 50 µl of
0.15% H2O2 in 24 mM citric acid and 5 mM sodium phosphate buffer (pH 5.2) with 0.5 mg/ml 2,2'-aminobis(3-ethylbenzthiazoline-6-sulfonic acid) as the chromophore. Color
development was allowed to proceed for 10 min, and the reaction was stopped with 25 µl
of 1% SDS. Absorbance was determined at 410 nm using a Dynatech MR700 ELISA reader (Chantilly, VA). Pre-serum was subtracted from all samples.
125 Additionally, isotype ELISAs were performed as described above, starting with
1:16 dilution of patient serum. Secondary antibodies used were goat-anti human total Ig,
IgG, and IgM.
Flow Cytometry. The presence of patient antibodies that bound to HER-2 was assessed
using the HER-2 over-expressing cell line BT474. 1 x 106 BT474 cells were incubated with various dilutions of patient’s serum (ranging from 1:4 to 1:64). HER-2-specific humanized mAb trastuzumab (Genentech) was used as positive control, and patient’s pre- serum was used as negative control. Cells were incubated for 2 h at 4°C in 100 µl of
PBS/2% FCS/.1% NaN3. The cells were washed twice in cold PBS and incubated with
FITC-labeled secondary antibody, goat-anti human IgG (1:50 dilution) for 30 min at 4°C
in 100 µl of PBS/1% FCS/.1% NaN3. The cells were washed twice, fixed in 1% formaldehyde, and analyzed by a BD FACS Calibur flow cytometer (BD Biosciences,
San Jose, CA). A total of 10,000 cells were counted for each sample, and final processing was performed. Debris, cell clusters, and dead cells were gated out by light scattered assessment before single-parameter histograms were drawn and smoothened.
Antibody-Dependent Cell-mediated Cytotoxicity. Effector PBMCs from normal human
donors (American Red Cross) were obtained by density gradient centrifugation in Ficoll-
Paque Plus (Amersham) and washed twice in RPMI 1640-5%FCS and then serially
diluted in 96-well plates to give various effector to target ratios with or without 15ng/ml
of IL-2 to activate PBMCs. The following day 1 x 106 BT474 target cells received 1:10 or 1:40 dilution patient pre-serum or day 71 serum or 50 µg of trastuzumab or normal
126 6 51 human IgG. BT-474 target cells were labeled with 100µCi/1×10 cells of Na CrO4
(Perkin Elmer, Boston, MA) and incubated for one hour at 37°C. After three washings
5×103 target cells were delivered to each well so as to give a final volume of 0.2ml/well.
The cells were incubated for four hours at 37°C, after which time 75 µl of cell free supernatants were harvested and radioactivity determined using a gamma counter.
Cytotoxicity was calculated by the formula (%) lysis = (A-B)/(C-B) x 100, where A represents 51Cr (cpm) from test supernatants, B represents 51Cr (cpm) from target alone in
culture (spontaneous release) and C represents maximum 51Cr release from target cells
lysed with 5% Triton-X100. Results represent the average of triplicate samples.
Statistical Analysis. To estimate the significance of differences in maximum IgG values, the data was compared using a two-tailed Student’s t test, with a level of significance set at 0.05 (Microsoft Office Excel 2003).
4.3 Results
Patients. Twenty-four patients received 3 inoculations of the vaccine and the characteristics of the patients in the study are listed in Table 4.2. The mean age of these participants at enrollment was 62. Patients had a variety of malignancies, including breast (5), ovarian (5), colorectal (4), endometrial (2), cervical (1), pancreas (1), adrenal
(1), GIST (1), leiomyosarcoma (1), and unspecified squamous cell cancer (1). Among the patients, there were 19 women and 5 men. Twenty-one patients were white, and three black. Nine patient’s tumor biopsies tested positive for HER-2 over-expression, measured by gene amplification using FISH or protein over-expression using IHC. 15
127 patient’s tumor samples did not over-express HER-2. All of these patients received 3 vaccine doses at the intended dose level
Vaccine and Vaccination Series. The MVF-316-339 and MVF628-647 peptides were mixed with 0.025 mg of nor-MDP and injected intramuscularly in alternating gluteus maximus muscles. Table 4.1 provides the dose-escalation scheme. Patients were inoculated three times at three week intervals. If a patient failed to receive two inoculations, a replacement patient was given the same dose until each group was complete. Blood specimens were obtained weekly from patients in order to measure humoral response to the vaccines. Figure 4.1 summarizes the vaccine and phlebotomy schedule for the trial.
Toxicity. Patients were evaluated for toxicities at each visit (typically every 7 days).
Five patients (21%) experienced serious adverse events (SAE). SAE is defined as adverse experience that results in any of the following outcomes: death, a life-threatening experience, inpatient hospitalization or prolongation of existing hospitalization.
Additionally, any grade 3 (severe) toxicity from the NCI Common Toxicity Criteria 2.0 list is considered a SAE. Two were hospitalized for reasons felt to be unrelated to vaccine therapy. One patient developed grade 3 diarrhea. One patient had grade 3 pain, with grade 2 pain at baseline. One patient expired on day 118 but had been removed from the study on day 63 due to progressive disease. This same patient had grade 3 pain and hyperglycemia, with grade 1 pain and hyperglycemia at baseline.
128 Clinical Response. 24 patients receiving three immunizations of the HER-2 peptide
vaccines were assessable for disease response. Six (25%) patients were classified as having a partial response or stable disease, 4 patients with stable disease and 2 with a partial response (Table 4.3). Three patients from dose level 1, one patient from dose level 2 and two patients from dose level 4 had clinical responses. Per study guidelines these patients were eligible for a 4th inoculation six months after receiving the 1st inoculation and currently three of the five patients have received a 4th inoculation. The two patients from dose level 4 with clinical responses may receive an additional inoculation pending the results of future scans. Additionally, patient 1D received a 5th vaccination 2 years and 10 months from the patient’s first vaccination, due to this patient’s prolonged disease stabilization.
Antibody Response to Peptide Vaccine. ELISA assays were performed to determine patient’s IgG antibody response against each of the vaccine constructs. Patient 1F had high levels of antibodies against MVF 316-339 (OD values at 1:16 dilution of 0.6-1.2), and intermediate levels against MVF 628-647 (OD values at 1:16 dilution of 0.2-0.6).
All other patients at this dose level had low antibody levels (OD values at 1:16 dilution of less than 0.2) (Fig. 4.2). Interestingly, the 3 patients (1A, 1B, and 1D) who showed
clinical responses from dose level 1 produced low antibody responses against the peptide
vaccine constructs.
Patient 2A produced high antibody levels against both vaccine constructs (Fig.
4.3). This patient showed stable disease after radiologic reassessment and consequently
received a fourth inoculation. 37 weeks after the fourth inoculation antibody levels
129 remained high against the MVF 628-647 construct and intermediate against the MVF
316-339 construct. There were three additional intermediate responses against MVF 316-
339 (patients 2B, 2C, 2D), and an additional high and intermediate response against MVF
628-647 (patients 2C and 2D, respectively).
Within dose level 3, 2 patients (3D and 3E) produced high antibody levels against both MVF 316-339 and MVF 628-647 (Fig. 4.4). Patient 3F produced intermediate antibody levels against MVF 316-339 and MVF 628-647 and an additional patient (3B) had intermediate antibodies against MVF 628-647. No patients from this dose level showed a clinical response.
Currently, 5 of the 6 patient’s IgG response have been measured from dose level
4. Encouragingly, 4 of the 5 patients had high antibody levels against both MVF 316-339 and MVF 628-647 (Fig. 4.5). Patient 4C had an intermediate antibody response against
MVF 628-647. Patients 4D and 4E had clinical responses after radiologic reassessment four weeks after the third immunization and also produced robust IgG antibody responses against both MVF 316-339 and MVF 628-647.
Table 4.4 summarizes the mean maximum absorbance value for each dose level
against MVF 316-339 and MVF 628-647. With increasing dose level, there is an increase in the mean maximum value for both MVF 316-339 and MVF 628-647.
However, the only statistical difference in mean maximum response was between dose level 1 and 4 for both MVF 316-339 (p = 0.019) and MVF 628-647 (p = 0.0023). This data indicates that a dosage of 3 mgs total peptides (dose level 4) is able to elicit a larger antibody response against the vaccine compared to a dosage of 1 mg total peptides (dose level 1).
130 We wished to investigate whether patients who did not produce IgG antibodies
against the peptide vaccine were able to produce IgM antibodies against the peptide vaccines since IgM antibodies are a Th-cell independent isotype whereas IgG antibodies require Th cell activation. 6 patients elicited IgM antibodies ranging in absorbance from
0.2-0.48 at a 1:16 dilution of serum (data not shown). 4 of these 6 patents elicited a similar or greater IgG antibody response (1F, 2B, 2C, and 2D). Patient 1F produced an intermediate IgM response to both MVF 316-339 and MVF 628-647, whereas the patient’s IgG response was high and intermediate against MVF 316-339 and MVF 628-
647, respectively. 2B, 2C, and 2D had both intermediate IgM and IgG antibodies against the MVF 316-339 construct. 2 patients who did not produce IgG antibodies produced intermediate levels of IgM antibodies against the MVF 316-339 construct (1C and 3C).
This data indicates that IgG and not IgM is the primary antibody isotype elicited from the peptide vaccines.
Antibody Response to HER-2. We next investigated whether antibodies elicited in vaccinated patients could bind to HER-2. Serum from patients 1D and 2A were examined because both these patients showed clinical responses on the trial and additionally patient 2A mounted a strong antibody response against the vaccine (Fig.
4.3). In addition we tested patients’ 3D and 3E serum due to the strong antibody responses elicited in these patients against the vaccine (Fig. 4.4). Binding to HER-2 was assessed using flow cytometric analysis with the BT474 breast carcinoma cell line that over-expresses HER-2 and revealed little difference in binding between pre-serum and serum obtained one week after the third immunization (Fig. 4.6). There were slight shifts
131 in patients 1D, 3D, and particularly 3E. These findings using flow cytometric analysis indicate that these patients were not able to produce a large quantity of antibodies against the vaccine that could bind to HER-2.
Measurement of Patient’s Antibodies to Mediate Antibody-Dependent Cell-mediated
Cytotoxicity. Since it is postulated that one of the main of mechanisms of action of HER-
2 mAbs is ADCC (47), we wished to investigate whether patient’s antibodies elicited from the peptide vaccine constructs could mediate ADCC. We investigated patients 1D and 3E serum from day 1 and 4 weeks after the third vaccine. Both 1D and 3E were chosen due to the slight binding seen in these patients serum to HER-2 as measured by flow cytometric analysis (Fig. 4.6). Antibodies from patient 1D’s serum showed 15% cytotoxicity compared to trastuzumab’s 78% cytoxicity after subtracting background. In a separate assay, patient’s 3E serum demonstrated 11% cytotoxicity compared to trastuzumab’s 40% after subtracting background. This data indicates that antibodies elicited from the peptide vaccine constructs are able to some extent mediate ADCC.
4.4 Discussion
The clinical trial described here is one of the first reports evaluating HER-2 B-cell epitope peptide vaccines in patients. The vaccine strategy consists of two B-cell epitope peptides, MVF 316-339 and MVF 628-647, in combination with nor-MDP adjuvant in an oil-in-water vehicle. Results demonstrate that this vaccine strategy is well tolerated in patients with advanced malignancy. We have identified dose level 4 as the maximum tolerable dose based on the lack of dose-limiting toxicity seen in any dose level and the
132 robust antibody response generated in patients in dose level 4. The data presented here
demonstrate that patients receiving dose level 4 had a statistically significant increase in
IgG antibody response compared to patients in dose level 1 and that the major antibody
isotype elicited was IgG and not IgM.
The current trial was not designed to assess efficacy, however our results reveal a
clinical benefit rate (complete response, partial response, or stable disease) of 25% (6 out of 24 patients). One of these five patients was HER-2 positive as determined by FISH, the remaining five did not over-express HER-2. The three patients from dose level 1 demonstrating clinical benefit all had low IgG antibody responses against the vaccine, although antibodies from patient 1D at day 70 are to some extent able to mediate ADCC in vitro using a HER-2 over-expressing target cell line. The remaining three patients with clinical benefit, 2A, 4D, and 4E, developed a strong antibody response against both MVF
316-339 and MVF 628-647.
The majority of peptide vaccines targeting the HER-2 oncogene have targeted T-
cell immunity utilizing either CTL or Th cell peptide epitopes. The HER-2 CTL epitope
E75 (HER-2 sequence 369-377) was originally identified by Fisk et al. (219) as an
immunodominant HLA-A2 binding epitope recognized by tumor-associated lymphocytes
of ovarian cancer. Most published HER-2 peptide vaccine clinical trials have used E75.
One trial reported all patients receiving the E75 peptide in combination with GM-CSF
showed clonal expansion of E75-specific CD8+ T cells and there was an increase in disease-free survival in the E75 vaccinated group versus patients not treated with the vaccine (197). The Disis group has vaccinated patients with HER-2 over-expressing breast, NSCLC, and ovarian cancers with peptide derived from potential Th epitopes of
133 the HER-2 protein in combination with GM-CSF (196, 221). 92% of patients developed
T-cell immunity to the HER-2 peptides. Peptide vaccines targeting HER-2 to date have
proven to be safe and immunogenic. However, peptide based vaccines specifically
designed to elicit cellular immunity are limited to specific HLA haplotype restrictions to
predict efficacy. Studies have demonstrated that the induction of anti-HER-2/neu
antibodies are both necessary and sufficient for protection of BALB-neuT mice from
developing tumors as shown by depletion of CD4+ and CD8+ cells (168, 190). DNA and
adenovirus-based HER-2 vaccines have shown efficacy in these mice but no protection is
evident in immunoglobulin µ chain gene knockout BALB-neuT mice (134, 190, 225),
underscoring the importance of eliciting a humoral immune response against HER-2.
The HER-2 316-339 and 628-647 epitopes were identified from computer-aided
analysis as putative B-cell epitopes and have demonstrated both in vitro and in vivo anti- tumor properties (126, 128). A clinical trial using HER-2 peptide 328-345 induced antibodies in patients that suppressed the phosphorylation of HER-2 on tyrosine 1248, a residue important for HER-2 signaling (226). This peptide sequence overlaps the 316-
339 HER-2 sequence described in this study, suggesting this could be an important region for targeting HER-2 humoral immunity. Moreover, the crystal structure of the HER-2
ECD reveals this region is at interface of dimerization between HER-2 and other members of the ErbB family (103), suggesting endogenous antibodies raised against these epitopes sterically hinder HER-2 from dimerizing with other members of the ErbB and results in the inhibition of signal transduction.
We demonstrate here that the peptide vaccines elicited IgG antibodies and not
IgM antibodies, indirectly showing the efficacy of the vaccine to elicit Th cells since IgG
134 is a T helper-dependent isotype. IgG antibodies were elicited in patients at all dose
levels, indicating peptide-specific helper T-cell activation. In future studies we will
address this question directly, investigating the extent to which patient PBMCs proliferate
in response to the peptide vaccine. No studies have reported the existence of MHC class
I or class II-restricted T cell responses to the 316-339 or 628-647 sequence of HER-2
(203). Thus, the MVF Th epitope co-linearly synthesized to the B-cell epitopes is most
likely responsible for helper T-cell activation. This T-cell epitope has been identified as
broadly reactive in multiple MHC haplotypes (158).
The dose of a peptide-based vaccine can impact the development of the immune
response. We have shown here a statistically significant increase in the IgG antibody response of patients receiving dose level 4 versus patients receiving dose level 1. Within all dose levels seven patients each had high levels of IgG antibodies against MVF 316-
339 and MVF 628-647 and additionally four and five patients produced intermediate levels of IgG to MVF 316-339 and MVF 628-647, respectively. Although a robust antibody response against the vaccine was elicited, antibodies raised in patients against the peptide vaccine showed little binding to HER-2. HER-2 is a normal gene product that is over-expressed in a subset of human tumors. Mechanisms responsible for self tolerance can dampen the immune response against HER-2. B-cell elimination through tolerance could explain the paucity of antibodies elicited from patients that bind to HER-
2. However, the subset of B cells producing antibodies which bound the synthetic peptides but not HER-2 could have not been eliminated, thus explaining the strong antibody response against the vaccine elicited in some patients.
135 In conclusion, this study demonstrates the HER-2 multi-epitope peptide vaccine is safe and effective at eliciting IgG antibodies in a population of patients with metatstatic disease that had been heavily pre-treated. Additionally patients’ antibodies elicited after vaccination were able to mediate ADCC with HER-2 over-expressing breast cancer cells in vitro. The clinical benefit of this vaccine remains to be investigated in future Phase II clinical trials.
136 4.5 Tables and Figures
Table 4.1 Dose Escalating Trial Design for Multi-Epitope HER-2 Peptide Vaccine.
Each dose level consisted of 6 patients.
137
Patient ID Age Malignancy Gender HER-2 status (years) 1A 73 colon F - 1B 75 SCC F FISH + 1C 65 ovarian F FISH + 1D 78 endometrial F - 1E 74 breast F IHC 3+ 1F 37 breast F IHC 3+ 2A 57 adrenal M - 2B 55 pancreas M - 2C 67 ovarian F - 2D 59 rectal M - 2E 53 leiomyosarcoma F - 2F 75 endometrial F FISH + 3A 66 breast F IHC 3+ 3B 41 breast F IHC 3+ 3C 46 rectal F - 3D 58 colon M - 3E 61 colorectal M - 3F 66 breast F IHC 2+ 4A 65 ovarian F IHC 2+ 4B 35 GIST F - 4C 70 cervical F - 4D 74 ovarian F - 4E 71 ovarian F - 4F 49 NSCLC F
Table 4.2 Patient Characteristics. Abbreviations used: SCC, squamous cell carcinoma;
GIST, gastrointestinal stromal tumor; FISH, fluorescent in situ hybridization; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer.
138
Patient ID Characteristic 1A 1B 1D 2A 4D 4E
Malignancy colon SCC endometrial Adrenal ovarian ovarian
lungs, Sites of L abdomen, lung, L thigh liver, abdomen Progression retroperitoneum pelvis neck acites, Periaortic perigastric
Dose level 1 1 1 2 4 4
4 3 5 4 3* 3* Doses Received
Response SD SD PR SD PR SD
Table 4.3 Partial Response and Stable Disease Patients. Abbreviations used: SCC, squamous cell carcinoma; L, left; SD, stable disease; PR, partial response. * pending further doses.
139
Dose Level MVF 316-339 MVF 628-647
1 (n=6) 0.20 ± 0.12 0.11 ± 0.05
2 (n=6) 0.38 ± 0.17 0.57 ± 0.27
3 (n=6) 0.51 ± 0.22 0.62 ± 0.26
4 (n=5) 1.09 ± 0.32 1.26 ± 0.30
Table 4.4 Maximum Mean IgG Response to HER-2 Peptide Vaccines per Dose
Level. The maximum absorbance value for each patient’s serum at a 1:16 dilution after subtracting pre-serum value was determined against MVF 316-339 and MVF 628-647.
The numbers below MVF 316-339 and MVF 628-647 correspond to the mean value for each dose level ± SEM.
140 Day 1 8 15 22 29 36 43 50 57 64 71
▲ ○ ○ ▲ ○ ○ ▲ ○ ○ ○ ○
▲immunization with peptide vaccines, blood draw for plasma and PMBCs prior to immunization, blood draw for plasma and PMBCs 4 hours after immunization
○ blood draw for plasma
Figure 4.1 Vaccination and Phlebotomy Schedule. Patients on all dose levels received peptide vaccines at days 1, 22, and 43.
141
Figure 4.2 Dose Level 1 IgG Response to HER-2 Peptide Vaccines. Plates were coated overnight with either MVF316-339 or MVF628-647. The following day after blocking plates, various dilutions of patient’s sera were added to plates. Binding was detected using goat anti-human IgG conjugated to HRP. Results of each patient in dose level 1 (1A-1F) are shown, using a 1:16 dilution of patient sera. 1y+3w indicates blood drawn three weeks after the first immunization. The y-axis represents the absorbance after subtracting patient’s pre-sera (day 1) value.
142
Figure 4.3 Dose Level 2 IgG Response to HER-2 Peptide Vaccines. Plates were coated overnight with either MVF316-339 or MVF628-647. The following day after blocking plates, various dilutions of patient’s sera were added to plates. Binding was detected using goat anti-human IgG conjugated to HRP. Results of each patient in dose level 2 (2A-2F) are shown, using a 1:16 dilution of patient sera. 1y+3w indicates blood drawn three weeks after the first immunization. The y-axis represents the absorbance
after subtracting patient’s pre-sera (day 1) value.
143
Figure 4.4 Dose Level 3 IgG Response to HER-2 Peptide Vaccines. Plates were coated overnight with either MVF316-339 or MVF628-647. The following day after blocking plates, various dilutions of patient’s sera were added to plates. Binding was detected using goat anti-human IgG conjugated to HRP. Results of each patient in dose level 3
(3A-3F) are shown, using a 1:16 dilution of patient sera. 1y+3w indicates blood drawn three weeks after the first immunization. The y-axis represents the absorbance after subtracting patient’s pre-sera (day 1) value.
144
Figure 4.5 Dose Level 4 IgG Response to HER-2 Peptide Vaccines. Plates were coated overnight with either MVF316-339 or MVF628-647. The following day after blocking plates, various dilutions of patient’s sera were added to plates. Binding was detected using goat anti-human IgG conjugated to HRP. Results of each patient in dose level 4
(4A-4E) are shown, using a 1:16 dilution of patient sera. 1y+3w indicates blood drawn three weeks after the first immunization. The y-axis represents the absorbance after
subtracting patient’s pre-sera (day 1) value.
145
Figure 4.6 Cross-reactivity of patient antibodies to breast cancer cells over- expressing HER-2. The reactivity of serum from immunized patients was tested with
BT474, a breast cancer cell line that over-expresses HER-2, using flow cytometric analysis. Serum form patients 1D (top left panel), 2A (top right panel), 3D (bottom left panel), and 3E (bottom right panel) were used at a 1:50 dilution. Ab binding was detected with goat-anti human IgG FITC-conjugated secondary abs. Histograms indicate individual patient’s pre-serum (dashed line), serum taken 1 week after the third immunization (gray shading) and 10 µg of trastuzumab (positive control, black shading).
146
Figure 4.7 Antibodies from vaccinated patients mediate antibody-dependent cell- mediated cytotoxicity in vitro. Target cell line BT-474 (HER-2high) was coated with
1:10 dilution of patient 1D (A) or 3E (B) pre-serum or serum obtained 4 week after the third immunization, 50 µg of normal human IgG, or trastuzumab and cultured with normal donor human PBMC effector cells in the presence of interleukin-2 to give an effector:target (E:T) ratio of 20:1, and 4:1. The results represent the mean (+SEM) of triplicate samples after subtracting pre serum or nonspecific normal human IgG lysis.
147 CHAPTER 5
SUMMARY AND FUTURE PERSPECTIVES
The goal of the research presented in this dissertation was to continue the pursuit of an efficacious HER-2 B-cell epitope vaccine using a peptide vaccine strategy.
Chapters 2 and 3 described the identification and characterization of novel HER-2 B-cell epitopes taking advantage of the crystal structure of the HER-2 ECD bound to the trastuzumab and pertuzumab Fab fragments. Chapter 4 focused on clinical studies with the first generation HER-2 B-cell epitopes evaluating the safety and toxicity and determining the maximum tolerable dosage of the vaccine.
The first generation HER-2 peptide vaccines were identified prior to the publications of the crystal structure of the HER-2 ECD (99, 100, 103) using computer- aided analysis. The second generation HER-2 peptide vaccines were designed with the consideration of the structure of HER-2 bound to trastuzumab and pertuzumab, two mAbs with proven inhibitory properties against HER-2 expressing cancer cells.
Interestingly, the epitopes identified from the first and second generation are in close proximity to each other (Fig. 5.1), indicating that the first and second generation vaccines could work in a very similar manner.
148 However, the second generation of peptides is advanced from the first generation,
stemming from how they were identified. The previous generation of peptides was
identified from computer algorithms that predict portions of the protein that may be
surface-exposed. The second-generation peptides are advanced because they are based
on the three-dimensional structure of HER-2 in complex with monoclonal antibodies that
already have proven efficacy. The mAb trastuzumab was the first targeted treatment for
cancer approved in 1998. Trastuzumab has already been shown to be an effective
treatment of breast cancer patients who have a high level of HER-2 overexpression.
Thus, we believe the 597-626 epitope is likely to be superior over the previous generation
(i.e. 628-647) because this epitope is found at the interface between HER-2 and
trastuzumab, whereas 628-647 is not involved in binding trastuzumab.
The advantage of active immunotherapy over passive immunotherapy with mAbs
such as trastuzumab and pertuzumab is a fundamental aspect of this work. The half-life of IgG administered intravenously can range from 5-21 days. Thus, repeated treatments with trastuzumab are necessary; patients typically receive the mAb every week to three weeks. The repeated treatment with trastuzumab raises the cost of passive immunotherapy with this mAb to approximately $50,000 USD a year. Additionally, patients are constantly infused with a large quantity of trastuzumab, which contributes to the cardiotoxicity associated with trastuzumab treatment. Active immunotherapy with the second generation peptide vaccines affords the possibility of generating a long-lasting endogenous antibody response through the elicitation of memory B-cells. Active immunotherapy offers the possibility of being cost-effective and loss toxic compared to mAb treatment. Data from the clinical trial reveals that a patient receiving 4 inoculations
149 of the first generation vaccines still produced a robust antibody response against the vaccine nearly one year and two months after receiving the first inoculation, demonstrating the proof-of-concept that only a few inoculations are necessary to produce a long-lasting endogenous antibody response.
We have identified two HER-2 B-cell epitopes at the interface of trastuzumab and pertuzumab binding. Additionally, we have shown that the first generation vaccine was well-tolerated in patients and patients were able to produce high-affinity antibodies against the vaccine. The next step in this work should be pre-clinical studies investigating whether a combination vaccine with the trastuzumab-binding epitope (597-
626) and the pertuzumab-binding epitope (266-296) show additive and/or synergistic properties. In support of the proposed work, in vitro studies have demonstrated that a combination of pertuzumab and trastuzumab synergistically inhibit the survival of breast cancer cells (108). If a combination of these vaccines demonstrates additive or synergistic properties, the combination could be translated into the clinic to investigate the efficacy of the trastuzumab and pertuzumab-binding epitopes. Additionally, future studies should investigate possible mechanisms of resistance to these vaccines. Finally, the scope of this work may be broadened by the use a combination vaccine that incorporates T-cell and B-cell epitopes from other tumor-associated antigens such as
MUC-1 and p53 that may stimulate all arms of the immune system and potentially afford much greater clinical benefit.
150 266-296, pertuzumab binding site
316-339, clinical trial peptide
597-626, trastuzumab binding site
628-647, ? clinical trial peptide
Figure 5.1 The First and Second Generation HER-2 B-cell Epitope Vaccines.
Ribbon diagram of the HER-2 ECD is shown in cyan. The first generation peptides 316-
339 and 628-647 are shown in green and the second generation peptides 266-296 and
597-626 are shown in magenta as indicated in figure. The crystal structure of the HER-2
ECD was cut off at amino acid 627; the figure represents a putative structure for the 628-
647 fragment.
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