Canine Immunoglobulins Against The Tumor Antigen Egfr Canine Immunoglobulins Against The Tumor Antigen Egfr Biology Essay

Masterarbeit

Zur Erlangung des akademischen Grades

Master of Science

der Fachhochschule FH Campus Wien

Vorgelegt von:

Judit Fazekas

Personenkennzeichen

1010544009

Betreuer/in:

Univ.Prof. Dr. Erika Jensen-Jarolim

Co-supervisor:

Dr. Josef Singer

Begutachter/in:

Univ.Doz. Dr. Ines Swoboda

Eingereicht am:

Abstract: English

Cancer is a global burden affecting both humans and pet dogs (Canis lupus familiaris) with similar incidence rates. Standard therapies in human and veterinarian oncology include surgery, radiation and chemotherapy. However, passive immunotherapy with monoclonal antibodies, like anti-EGFR (ErbB-1) IgG1 antibody cetuximab (Erbitux®, Merck) are today available for human patients only, although the molecular expression patterns of cancer antigens are remarkably similar in both species.

We previously demonstrated that the human and canine EGF receptors are highly homologous, with merely a 4 amino acid difference in the cetuximab binding epitope. Moreover, besides sequential and structural homology, also targeting of the canine homologue of EGFR with cetuximab led to similar biological effects in the canine cancer cells, i.e. silencing of growth signals and tumor growth inhibiton. Since overexpression of EGFR is frequently observed in canine mammary carcinomas, we proposed it as a promising anti-tumor immunotherapy target in dog patients.

Along this line this master’s study aims to introduce passive immunotherapy in veterinarian oncology, specifically for dog cancer patients. We thus employed the cetuximab variable regions and generated a canine-mouse chimeric IgG antibody (can225IgG) targeted against EGFR.

Upon transfection of CHO-DHFR- cells with plasmids encoding the heavy and light chain gene sequences, clones were screened to select those with highest productivity for subsequent large-scale production. Following purification with Streptococcal Protein G, the biochemical integrity and folding of the antibody was assessed using immunoblots and CD-spectroscopy. Specificity of the can225IgG towards the naturally expressed canine EGF receptor could be confirmed by flow cytometry performed with canine mammary carcinoma cell lines. Proliferation and viability assays were carried out to verify in vitro functionality; in these assays significant tumor cell growth inhibition could be observed upon can225IgG treatment.

We report in this study for the first time the generation of a recombinant canine anti-tumor IgG for subsequent clinical studies in dog cancer patients. This study emphasizes the need of comparative medicine to reveal conserved pathological processes between different species. Thereby, the development of novel therapeutic approaches could be speeded-up in human and veterinarian oncology.

Abstract: Deutsch

Krebs ist eines der weltweit größten und meistbeachteten Gesundheitsprobleme sowohl in der Human- wie auch der Veterinärmedizin, das Menschen wie auch Hunde (Canis lupus familiaris) mit ähnlichen Inzidenzraten betrifft. Standardtherapien der human- und veterinärmedizinischen Onkologie umfassen Chirurgie, Strahlentherapie sowie Chemotherapie. Passive Immuntherapie mit monoklonalen Antikörpern, wie z.B. anti-EGFR (ErbB-1) IgG1-Antikörper Cetuximab (Erbitux®, Merck), stehen jedoch nur menschlichen Patienten zur Verfügung, obwohl die molekularen Expressionsmuster von Krebsantigenen in beiden Spezies sehr ähnlich sind.

Bereits in einer früheren Studie konnten wir zeigen, dass die EGF-Rezeptoren von Menschen und Hunden äußerst homolog sind, mit lediglich 4 unterschiedlichen Aminosäuren im Cetuximab-Bindungsepitop. Neben der sequentiellen und strukturellen Homologie führt zielgerichtetes Attackieren des homologen kaninen EGFR durch Cetuximab auch zu ähnlichen biologischen Wirkungen, wie z.B. Stilllegen der Wachstums-Signale und Inhibition des Tumorwachstums in Hundekrebszellen. Da die Überexpression von EGFR ein häufiges Phänomen bei Hunde-Mammakarzinomen ist, würde unserer Meinung nach dieses Molekül ein vielversprechendes Zielprotein für eine Anti-Tumor-Immuntherapie in Hunde-Patienten darstellen.

Dieser Strategie folgend hat diese Masterarbeit das Ziel, passive Immuntherapie in der veterinären Onkologie einzuführen, insbesondere für Hunde-Krebspatienten. Aus diesem Grund haben wir mithilfe der variablen Regionen von Cetuximab einen Hund-Maus-chimären IgG-Antikörper (can225IgG) gegen EGFR erzeugt.

Nach Transfektion von CHO-DHFR- Zellen mit den entsprechenden Plasmiden wurden die Klone mit der höchsten Produktivität ausgewählt und weiterkultiviert. Nach Aufreinigung des Antikörpers mit Protein G wurde die biochemische Integrität und die Faltung des Proteins mittels Immunoblots und CD-Spektroskopie überprüft. Die Spezifität gegen das natürlich vorkommende kanine Antigen auf der Oberfläche von Krebszellen wurde durch Durchflusszytometrie bestätigt. Proliferations- und Viabilitätsstudien lieferten überzeugende Beweise über die Funktionalität des Antikörpers. In beiden Versuchsreihen konnte eine signifikante Wachstumsinhibition von EGFR überexprimierenden Tumorzellen nachgewiesen werden.

In dieser Studie berichten wir zum ersten Mal die Herstellung eines rekombinanten kaninen anti-Tumor-IgGs für nachfolgende klinische Studien in Hunde Krebspatienten. Die Ergebnisse unterstreichten die Notwendigkeit der vergleichenden Medizin um zwischen verschiedenen Spezies konservierte pathologische Prozesse zu enthüllen. Damit könnte die Entwicklung neuartiger therapeutischer Ansätze für die humane- und veterinärmedizinische Onkologie deutlich beschleunigt werden.

Table of contents

Abstract: English 1

Abstract: Deutsch 2

Table of contents 3

List of abbreviations 4

Introduction 5

Supplementary results 23

Discussion 29

Acknowledgement 33

Eidesstattliche Erklärung 35

Annex 36

List of abbreviations

Abbreviation

Full name

ADCC

Antibody-Dependent Cell-Mediated Cytotoxicity

ADCP

Antibody-Dependent Cellular Phagocytosis

BSA

Bovine Serum Albumin

CD

Circular Dichroism

CDC

Complement-Dependent Cytotoxicity

CDR

Complementarity Determining Region

EGF

Epidermal Growth Factor

EGFR

Epidermal Growth Factor Receptor; ErbB-1

ELISA

Enzyme-Linked Immunosorbent Assay

FT

Flow-Through

HB-EGF

Heparin Binding EGF

HER-2

Human Epidermal Growth Factor Receptor 2, ErbB-2

NK cells

Natural Killer Cells

PAGE

Poly-Acrylamide Gel Electrophoresis

TGF

Tumor Growth Factor

VEGF

Vascular Endothelial Growth Factor

Introduction

Comparative medicine

Human and veterinarian medicine are two separate, but closely related branches of the art to heal patients. There is no explicit reason to separate humans completely from the rest of the mammals, as most pathological processes are common and highly comparable in both [1].

Although the ancient Greeks were already aware of the similarities of human and animal anatomy and disease progression, the first chair of comparative medicine was founded merely 150 years ago in Lyon, France [2]. Since then, the discovery of the homology of vertebrate genomes [3-9] and the revealing of several highly conserved molecular pathways gave a considerable push towards new studies in this field. Nowadays, this discipline gets more and more public attention, thanks to the better communication of scientific findings to lay people.

The term 'comparative medicine' can be defined as the study of animals, including humans, in order to gain new insights into both human and veterinarian medicine. In comparative medicine, experimental tools are used to study and analyze naturally occurring diseases and conditions, to define the highly relevant experimental models including animal models and to evaluate the achieved information in comparison to similar conditions in other species. For instance, pet animals share similar environmental and dietary risk factors as their owners, and thus are often affected by related diseases. An important aim of comparative medicine is to promote the development and accessibility of new drugs and treatments not just for use in human, but also in veterinarian medicine.

Comparative oncology

Comparative oncology refers to a specific branch of comparative medicine, which aims at investigating the development and progression of spontaneously emerging tumors in animals and comparing it to malignant diseases occurring at the same site in human patients. Common cancer types in pets are lymphomas, melanomas, osteosarcomas and mammary carcinomas [10]. The high prevalence, good comparability and rapid progression of the disease in most animal patients makes them very apt for comparative cancer studies [11]. Like in comparative medicine, the goal of comparative oncology is to support the development of new anti-tumor therapeutic drugs and to provide pet animals with novel drugs available for cancer treatment [10].

A well-studied example for comparative oncology is lymphoma in dogs [12]. Like other companion pets, dogs (Canis lupus familiaris) also tend to spontaneously develop several different types of cancer, especially at an advanced age. For instance, the anatomy, high incidence rate and involved signaling pathways of canine mammary carcinoma are remarkably similar to human breast cancer; therefore it represents a more suitable and comparable model for breast cancer research, than the usually applied rodent systems [11, 13-16]

Breast cancer in humans and canines

Human breast cancer is a malignant disease showing by far the highest incidence rate (22.9%) as well as the highest mortality (13.7 %) among women according to global world statistics. However, the disease is more prone in developed countries compared to developing ones [17]. This phenomenon could be explained by the considerably different environmental factors, dietary habits and overall lifestyle. Approximately 1,384,000 new cases are diagnosed every year and 450,000 deaths can be accounted on breast cancer, which makes it the fifth cause of death in patients suffering from cancer [17].

In aged dogs, cancer is the most common cause of death. Similar to the situation in women, mammary carcinoma is the most frequent type of cancer in unspayed female dogs [18, 19]. About 50% of all canine mammary carcinoma cases are metastatic, resulting in a poor prognosis [15, 20]. A further complication is that diagnosis in pets is usually made at a later stage of disease, when the pet owner gets aware of the symptoms.

EGFR and the ErbB growth factor receptor family

Cancer research aims to target and interrupt proliferation and migration of cancer cells only, while as much as possible sparing healthy cells. Thus targeted therapies are needed to avoid the often harsh side effects of conventional unspecific therapies, such as chemotherapy. Promising targets for new therapy approaches are for instance growth factor receptors [21].

The Epidermal Growth Factor Receptor (EGFR or ErbB-1) is a receptor tyrosine kinase and belongs to the ErbB growth factor receptor family. Other members of this molecule family include ErbB-2 (HER-2), ErbB-3 and ErbB-4 [21]. EGFR is a 170 kDa glycoprotein and consists of an extracellular receptor domain responsible for ligand binding, a transmembrane domain for anchoring in the plasma membrane and an intracellular tyrosine kinase domain for the phosphorylation of various downstream molecules [21].

15 ligands of this receptor family have been identified so far (EGF, TGF alpha, HB-EGF, amphiregulin, etc); they bind to several members of the ErbB family, however, each of them have different specificity and affinity to the 4 different ErbB receptors [22]. Also different functions can be assigned to the various ligands, for instance, EGF induces rapid internalization and degradation of the activated ErbB-1 receptor, whereas TGF-α is more prone to activate the MAPK pathway and therefore stimulate invasion and metastasis [23].

Although EGFR is as well expressed by healthy ectoderm-originated cells, such as epidermal cells of the skin, the overexpression of this receptor is a clear and frequently observed feature of malignant cells of several tissues [21].

Activation of the ErbB-1 receptor and downstream signaling

Ligand binding to EGFR causes conformational changes of the molecule, resulting in dimerization (homo-, or heterodimers with other ErbB family members) of the receptor, leading to its internalization and autophosphorylation [21, 24, 25]. Upon activation, the tyrosine kinase domain of the intracellular domain of the receptor phosphorylates downstream molecules. The most important pathways involved are the Ras-Raf mitogen activated protein kinase (MAPK), the JNK and the phosphatidyl inositol 3’ kinase (AKT) pathway. Activation of these various pathways depends on the ligand and on the dimerization partner of ErbB-1 [25]. EGFR activation can therefore lead to a variety of cellular responses, such as enhanced proliferation, survival and invasion, depending on the ligand and the context of the stimuli [21, 23].

Crosstalk between EGFR and other signaling pathways can also be observed. For instance, EGFR activation leads to VEGF stimulation, which then promotes angiogenesis, which again supports tumor growth [21, 26].

EGFR and breast cancer

EGFR is known to play an important role in the development of epithelial tissues [23], e.g. the development of the mammary gland in mice [27]. On the other hand, it is often overexpressed in cancers from epithelial origin, like head and neck cancers, breast cancer or colon cancer [26]. Increased EGFR expression is generally a strong marker for overall poor prognosis [21, 23].

EGFR expression in non-malignant cells ranges from 40.000 – 100.000 receptors per single cell in humans, however, human breast cancer cell lines can express up to 2 million receptors per cell. This leads to enhanced signal intensity and increased activation of signaling pathways (MAPK; AKT), resulting in enhanced proliferation and malignancy. EGFR overexpression occurs in about 57% of all breast cancers in women [28]. This upregulation is particularly often observed in women with a remarkably aggressive and invasive, so called triple negative breast cancer (HER-2-, ER-, PR-) and has been also linked to inflammatory breast cancer [23].

Similarly to humans, canines also often overexpress EGFR in mammary carcinomas. According to previous studies, about 42.2% of all malignant mammary carcinomas in dogs are EGFR positive [29]. It has also been shown, that the human and canine EGFR are not just sequential and structural homologues, but also harbor remarkably similar biological functions [30]. Although the receptor density on the canine cell lines was about 100 fold lower compared to human cell lines [30], the receptor density should not play a role in the efficacy of Cetuximab treatment [31].

EGFR inhibitors

Conventional therapies like radiation or chemotherapy can effectively suppress tumor growth, however, in case of highly aggressive EGFR-overexpressing tumors; these options often turn out to be insufficient.

Previous studies could also show that radiation therapy in these particular tumors is not always beneficial, as it leads to activation of EGFR signaling and enhanced proliferation, therefore radiation-resistant (i.e. DNA damage resistant) subclones can emerge. In such cases, EGFR inhibition parallel to radiation appeared to be beneficial [21].

Inhibitory agents of EGFR are monoclonal antibodies targeted against the extracellular domain and small molecule tyrosine kinase inhibitors, which block the autophosphorylation of the tyrosine kinase domain of the receptor [21]. Tyrosine kinase receptors act on a broad range of molecules, by contrast, anti-EGFR antibodies act much more specific. Downsides of such monoclonal antibodies include ineffectiveness against mutated EGFR VIII receptor or immune responses to the antibody. Moreover, the intravenous administration can be both tiresome and cost-intensive.

Cetuximab 225 (Erbitux®, Merck) is a chimeric human-mouse anti-EGFR monoclonal antibody, which effectively blocks the binding of ligands to EGFR and the dimerization of the molecule, resulting in inhibition of downstream signaling of the receptor [32]. ‘225’ refers to the original murine clone, of which the clinically used human antibody was engineered. It was reported, that the affinity of the chimeric variant is highly increased in contrast to the original murine clone[32].

Cetuximab 225 has proven anti-proliferative effects, and has been used in clinical oncology for instance in the treatment of EGFR-overexpressing head and neck cancers or non-small-cell lung cancers [33-36]. Mild to moderate side effects, like folliculitis or skin dryness can occur upon Cetuximab treatment [37]. Although these symptoms can be unpleasant to the patients, they are often regarded as a sign for positive response to the treatment [38].

Singer et al. showed that due to the high homology between the receptor molecules, Cetuximab specifically binds to canine EGFR as well, and exhibits in vitro tumor-inhibitory functions in canine mammary carcinoma cell lines to a significant extent [30]. Because of the frequent overexpression of EGFR in malignant mammary tumors of dogs and due to the excellent binding and inhibitory function of Cetuximab towards the homologous canine receptor, the hypothesis of this master’s thesis was, that a recombinant caninized antibody with the exact specificity of Cetuximab 225 could provide a promising approach for the treatment of this disease.

Immunoglobulins

Immunoglobulins or antibodies are antigen targeting molecules, secreted by plasma cells in order to defend the host from pathogens at the site of invasion by binding to it and recruiting effector cells and complement activatory molecules to eliminate the threat [39]. At the same time, membrane-bound antibodies function as antigen receptors on the pre-form of plasma cells: B-cells. Immunoglobulins in general have specificity towards exogenous antigens, like bacteria and virus associated antigens or in itself harmless proteins, e.g. allergens.

Evolution of immunoglobulins

It was already known at the beginning of the 20th century, that lower vertebrates like turtles or alligators produced antitoxins (antibodies), hereby dating the evolution and first appearance of antibodies to about 220 million years back [40, 41]. Recent studies suggest however, that the first antibody-like proteins could have appeared much earlier in evolution, already in the common ancestors of mammals and chondrichthyes (cartilaginous fishes) about 500 million years ago, as not only all vertebrates, but also sharks are able to produce antibodies. A further evidence for the orthology of these genes is that the light chain sequence of the sandbar shark is homologous to the typical λ-chain of vertebrates with lungs. Nevertheless the nurse shark predominantly expresses light chains with a homology with the κ-light chain of mammals. This fact supports the hypothesis, that also the different light chain variants have been separated at a very early time point in the evolution, probably simultaneously with the appearance of the first functional antibodies [40].

The first antibody class that evolved was IgM. Already sharks express (besides their own unique IgW immunoglobulin) antibodies resembling the common vertebrate IgM molecule. By moving up the evolutionary tree towards higher vertebrates, also the diversity of antibody isotypes increases remarkably. For instance, amphibians and reptiles have besides IgM and their own distinct immunoglobulins IgX or IgN also a common IgY antibody, which has a slightly larger heavy chain than IgM. Birds however express the IgA isotype as well, responsible for mucosal immune-defense [40, 42]. Finally, about 160 million years ago with the appearance of the first Eutherians, also the immunoglobulin subset became complete with the well-studied IgM, IgG, IgA, IgD and IgE isotypes expressed in humans and higher mammals [40, 43-45].

Structure of IgG antibodies

In human serum, with about 75% of all antibodies, IgG is the most common isotype [46]. IgG is also in dogs the most abundant immunoglobulin class, representing about 86% of the total serum antibody concentration [47].

Human IgG antibodies are large, ~150 kDa glycoproteins, consisting of four individual protein chains, which are pair-wise identical. Two identical heavy chains of the size of ~50kDa are assembled with two identical light chains (~25 kDa) by distinct intermolecular disulfide bonds to form a symmetric, Y-shaped molecule (shown in Figure 1). The canine IgG antibody is slightly larger, than the human, but the structure is the same. IgG antibodies have two major glycosylation sites on the Fc portion, however, glycosylation can also occur in the Fab region [48]. The small region at the disulphide bridges between the two heavy chains is called hinge region and has a different degree of flexibility among the immunoglobulin classes.

IgGs can have one of the two light chain variants, either the λ-light chain, or the κ-light chain, but never both in the same antibody. The ratio of the κ- and λ-chain differs in humans and dogs. Humans have two-times more κ- chain than λ, whereas dogs show a 9:1 ratio of λ to κ-chain [39, 49]. Of which of the light chain variants the antibody is constructed, plays no role regarding its functionality [39].

On the other hand, the heavy chain defines the characteristic function and distribution of an antibody in an organism due to interaction with specific Fc-receptors on diverse cells, and is responsible for complement activation or effector cell activation [39].

The variable region of the molecule contains the so-called complementarity-determining regions (CDRs), which are responsible for the highly specific binding of the antibody to a unique epitope structure of an antigen [39].

Figure 1. Schematic structure of an IgG antibody. Blue color highlights the two heavy chains of the molecule, green color stands for the two light chains. Regions with the prefix C stand for constant regions, V stands for the epitope-specific variable regions of the antibody. The Fc part of the immunoglobulin consists of the CH2 and CH3 regions.

IgG subclasses in humans and canines

Dogs, as well as humans express 4 different subtypes of IgG [39, 47, 50-52]. The human subclasses are IgG1, IgG2, IgG3 and IgG4 [39, 51]. In contrast, the canine immunoglobulin G subclasses are termed as IgG-A, IgG-B, IgG-C and IgG-D [39]. The human and canine subclasses have not been matched to each other yet, and the exact biological functions of the dog IgG subclasses are still unknown. Subclasses of the gamma immunoglobulins generally differ in the amino acid sequence and length in the hinge-region of the antibody. Moreover, they differ in the number of cysteine residues determining the disulphide bridges between both heavy chains.

The biological role of the different classes is well studied in humans. Among the different subtypes, IgG1 is the most common, whereas IgG4 is the rarest. IgG1 and especially IgG3 are powerful complement-activators and are the most potent triggering antibodies for antibody-dependent cell mediated cytotoxicity [39, 51]. Immunity against viruses is mediated by IgG1 and IgG3 antibodies. On the other hand, antigens mainly consisting of carbohydrates usually trigger IgG2 production [51]. In contrast to the other 3 subclasses, the human IgG4 subclass has no complement-activating capacity, and thus acts anti-inflammatory. It is therefore very beneficial in allergic diseases, as it is able to neutralize allergen molecules by simply binding to them, thereby blocking the binding of in this case deleterious IgE antibodies [39, 53].

The existence of the four canine IgG subclasses is known for more than 40 years and also the cDNA sequences encoding them have been identified and sequenced about 12 years ago [47, 51]. Tang et al. could show, that the various subclasses share around 78% overall homology, however, similarly to humans, the hinge regions show high variability. Furthermore it was shown, that the number of cysteine residues in this region also vary, with IgG-A having 3, IgG-B 2, IgG-C 7 and IgG-D again 3 cysteins [51]. Data about the expression levels of the different antibodies however, are contradictory. Tang claims IgG-A and IgG-B to be the most common subclasses with 48,1% and 30,4%, and IgG-C and D the rarest, with a percentage of only 12% and 7% of all IgG encoding cDNA samples, respectively [51]. In contrast, Reynolds et al. measured the serum concentration of the different subclasses and found that IgG-D alone made up 39% of all serum IgG, IgG-A and IgG-B -measured together- accounted for 53% and IgG-C for only 8% of the total IgG found in the circulation of mixed-breed dogs [47].

About the functionalities of the different canine subclasses however, there is very few information available, probably also due to the limited number of available detection tools.

Cancer immunotherapy

Cancer cells arise from endogenous cells and are additionally inducing an immune-suppressive environment, therefore frequently repressing cytotoxic T-cells or the generation of antibodies targeting self antigens, so called autoantibodies [54, 55]. Nevertheless, passive immunotherapy can overcome this problem and provide a powerful tool against malignant tumors by specifically targeting tumor associated antigens and recruiting cytotoxic or phagocytic cells to the tumor site [56, 57].

In passive anti-cancer immunotherapy, therapeutical doses of cancer antigen-targeting IgG antibodies are intravenously administered to the patient [56, 57]. The first clinical trials of early therapeutic murine monoclonal antibody began in 1975. However, it soon became clear, that such immunoglobulins, just like other exogenous proteins are recognized as foreign by the human immune system and they trigger serum sickness, or even anaphylaxis upon the second administration. In addition to these side effects, non-human antibodies are also ineffective, as they are unable to bind to Fc receptors of xenogenic effector cells. Furthermore because of their foreign origin, they are rapidly degraded and cleared from the circulation. In order to create antibodies which are able to explicit cellular immune response in the injected patient, the ratio of human protein sequence had to be increased. The first clinical trials with human-mouse chimeric antibody, i.e. human constant and murine variable regions, started in 1987 and only a mere year afterwards, the first "humanized" monoclonal antibody with only the complementarity-determining regions retained from the original mouse monoclonal antibody also entered a clinical trial [58]. It is already known for about 20 years, that of different chimeric human-mouse anti-tumor monoclonal IgG isotypes, IgG1 was shown to be the most effective [59]. The first monoclonal IgG1 antibody used in passive anti-cancer therapy, Rituximab (MabThera®, Roche or Rituxan®, Genetech), was FDA approved in 1997. Rituximab is an anti CD20 antibody used for the treatment of various lymphomas [60]. Since then, numerous additional monoclonal antibodies have been approved for clinical use, like the above mentioned anti-EGFR IgG1 antibody Cetuximab, or the anti-HER-2 (ErbB-2) antibody Trastuzumab (Herceptin®, Roche), which has been widely used in the standard clinical care for breast cancer patients within recent years [33-36]. Cetuximab is currently applied in head and neck carcinomas and colon cancers [61, 62]. Moreover, there is also a completed phase II trial of its effectiveness as a therapeutic agent in combination with carboplatin in metastatic triple-negative breast cancer [63].

Mechanisms of anti-cancer Immunotherapy

The main mechanisms of immunoglobulin-mediated anti-cancer immunotherapy are the abilities to trigger antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) reactions. Some antibodies however, like Cetuximab, can also act through their epitope-specificity, via receptor blocking capacity, by inhibiting the activation of them and their downstream signaling pathways. A relatively new approach to increase the tumoricidic effects of antibodies is to generate toxin-, cytotoxic drug- or radioactive isotope - coupled antibodies to concentrate these tumoricidic drugs at the site of malignancy [39, 64, 65].

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC is triggered by the presence of antigen bound antibodies on the cell-surface. Tumor cells coated with a tumor-antigen specific monoclonal antibody are recognized by the Fcγ receptors of natural killer (NK) cells or neutrophils [39, 64]. NK-cells are derived from the lymphoid lineage; however, they lack highly specific antigen-binding molecules and belong therefore to the innate immune system [39]. NK cells bind to the antibody coated cancer-cells via their FcγRIII molecules, which are specifically recognizing IgG1 and IgG3 molecules. Their cytoplasm contains characteristic granules, loaded with membrane-disruptive perforin and special serine proteases, called granzymes [39, 64]. The release of the content of such granules results in rapid cell death, due to the fact, that perforin induces H2O permeable pores and leads to target cell lysis. In addition, the proteases co-released into the cytoplasm are responsible for apoptosis-induction [39].

Antibody-dependent cell-mediated phagocytosis (ADCP)

It was previously described, that solid tumors are frequently infiltrated by macrophages and that several cancer cells are secreting various chemotactic agents to attract macrophages [66-68]. However, these macrophages usually belong to the immunosuppressant M2 subset and are associated with poor prognosis due to their secretion of angiogenic and proliferative factors [67, 69-71]. In contrast, the M1 subset is rather pro-inflammatory and could promote tumor-cell elimination [71]. Ong et al. could show that co-culture of M1 macrophages with colorectal cancer cell lines is connected to release of Th1 cytokines like IL-8 and IFN-γ and results in growth-inhibition of the tumor cells [72]. The production of IFN-γ by tumor-associated macrophages is especially important, as it has been shown that this cytokine is able to convert M2 monocytes to the tumorsuppressive M1 subtype [73]. Furthermore, it was shown that tumor-associated M1 macrophages secrete chemotactic Th1-specific cytokines, for instance CCL2, 3 and 4 [72]. Upon recruitment of T-cells, these macrophages have been shown to express T-cell co-stimulatory receptors, like CD40, CD80 or CD54 [72]. In addition to the cytokines mentioned above, activated macrophages also release reactive nitrogen oxide species in order to damage the integrity of the cell membrane of tumor cells [39, 74]. It has been reported, that tumor cells, opsonized by anti-tumor antibodies (for instance Trastuzumab), are subject to remarkably high phagocytic activity by monocytes and that this binding occurs via the low affinity Fcγ receptors of monocytes and macrophages [66, 75]. Damaged tumor cells undergo endocytosis and subsequent elimination of the malignant cells occurs with the production of reactive oxygen compounds in the phagolysosomes of the macrophages [39, 74, 76, 77]. Induction of ADCP has many advantages in anti-tumor therapy. Phagocytosis and degradation of tumor cells results in antigen presentation to the formerly attracted Th cells. The activation of T-cells, furthermore the additional release of pro-inflammatory cytokines promotes the induction of an active immune response upon a passive anti-cancer antibody treatment [78, 79].

Aims of the study

The overall aim of this project was to generate and characterize a caninized α-EGFR antibody for immunotherapeutic studies in canine mammary carcinoma patients, using the following methodological approach:

Cloning of the 225 variable & canine gamma heavy chain fusion genes into the SV40 expression vectors

Amplification of vectors in E. coli

Transfection of CHO DHFR- cells

Selection of clone with highest

productivity & specificity

Large-scale production of the antibody

Purification of the can225IgG

Quality control: integrity, assembly and folding

Characterization of specificity and in vitro functionality

Major goals:

Production and purification of the can225IgG antibody

Quality assessment and characterization of can225IgG regarding protein assembly and correct folding, specificity and in vitro tumor-growth inhibitory functions.

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Supplementary results

Plasmid isolation

pIRES_dhfr_SV40_chim_gamma and pIRES_NEO_SV40_chim_kappa_225 plasmids were transfected into KCM competent E. coli DH5α. After overnight incubation, plasmid DNA was isolated, yielding:

pIRES_NEO_SV40_chim_kappa_225:

1491,7 µg/ml

pIRES_NEO_SV40_chim_gamma:

974,9 µg/ml

To confirm the identity of the isolated plasmids, pIRES_NEO_SV40_chim_

kappa_225 was digested by KpnI and pIRES_dhfr_SV40_chim_gamma was digested by SacI. Following digestion, 1% agarose gel electrophoresis showed that the isolated DNA were indeed the chimeric kappa_225 and gamma chain plasmids.

1

a)

b)

Figure 1: Restriction digest of the isolated plasmids. a) KpNI digest of the pIRES_NEO_chim_kappa_225 vector. Two bands were visible, at 1917bp and 3780bp, indicating a correct size of the expression vector. b) SacI digestion of pIRES_dhfr_SV40_chim gamma. A small band is visible at 312bp along with a large band at 5830bp as expected, again confirming the correct size of the plasmid.

Protein G bead purification – pilot experiment

In contrast to previously published data, Staphylococcal Protein A for canine IgG purification yielded unexpectedly negative results. Therefore Streptococcal Protein G purification was established using different buffer and eluent conditions (Table 1.). In order to assess the binding of recombinant can225 antibody and to determine optimal buffer conditions, small-scale purifications were performed using Protein G Sepharose beads.

Binding buffer

Washing buffer

Elution buffer

Result

Protein A column

20mM sodium phosphate; pH 8.0

20mM sodium phosphate; pH 8.0

0.1M citric acid; pH 3.0

negative

20 mM sodium phosphate; pH 7.3

20 mM sodium phosphate; pH 7.3

0.1M citric acid; pH 3.0

negative

0.1M Glycine, 0.1M NaCl; pH 7.5

0.1M Glycine, 0.1M NaCl; pH 7.5

0.1M Glycine; pH 3.0

negative

Protein G column

100mM Tris; pH 8.0

10mM Tris; pH 8.0

0.1M Glycine; pH 2.5

negative

20 mM sodium phosphate; pH 7.4

20 mM sodium phosphate; pH 7.4

0.1M Glycine; pH 2.5

unsatisfying

20 mM sodium phosphate; pH 7.2

20 mM sodium phosphate; pH 7.2

0.1M Glycine; pH 2.5

unsatisfying

20 mM sodium phosphate; pH 7.0

20 mM sodium phosphate; pH 7.0

0.1M Glycine; pH 2.5

positive

20 mM sodium phosphate; pH 6.9

20 mM sodium phosphate; pH 6.9

0.1M Glycine; pH 2.5

positive

20 mM sodium phosphate; pH 7.0

20 mM sodium phosphate; pH 7.0

0.1M Glycine; pH 2.7

optimal

Table 1. Buffer system attempts for the establishment of the can225IgG purification.

To control the outcome of the purification trials, samples of the respective fractions were analyzed in non-reducing 10% SDS-PAGE and immunoblot (Figure 2).

Optimal binding and elution was observed with 20mM sodium phosphate (pH 7.0) binding/washing buffer and 0.1M glycine (pH 2.7) elution buffer (Tab. 1). Elution fractions were directly neutralized by 1M Tris, pH 9.0. Upon purification, an immediate buffer exchange was necessary to maintain protein integrity on the long run. The tris-glycine buffer (pH ~7.0) containing the antibody was exchanged for phosphate buffered saline (pH 7.0) in order to enable further in vitro studies. The pre-experiments with the Protein G beads suggested that the large-scale purification was feasible using a Protein G affinity column in combination with the ÄKTA liquid chromatography system.

Figure 2. Representative non-reducing 10% SDS-PAGEs showing the fractions of a successful purification with Protein G Sepharose beads.

2

a)

b)a) Coomassie stained gel b) Western blot, detected by rabbit anti dog IgG HRP (Jackson ImmunoResearch). Sample volumes: 15µl for each fraction. Standard: 1µg/slot for Coomassie gel and 500ng/slot for Western blot were loaded.

2

a)

b)As Figure 2. shows, protein concentration of the can225 cell culture supernatant could be retained after dialysis. The dialysed supernatant was concentrated via centrifugation with Amicon 40kDa-cutoff columns prior to Protein G purification. The concentrated sample showed in SDS-PAGE a mild smear of other, smaller-sized proteins; however these contaminations are only visible because of the very high protein concentration. Analysis of the Amicon flow-through fraction (Lane Amicon FT in Fig. 2) confirmed that the column membrane was intact and no antibodies were lost during the concentration process. After 18h of gentle stirring, there was still some unbound antibody detectable in the Protein G flow-through (Lane Prot. G FT in Fig. 2). The first two elution fractions E1 and E2 contained most of the antibodies; while the 3rd fraction E3 contained almost no protein at all. The canine IgG standard confirms the correct height and specificity of the signals, and the negative control BSA was not detected on the immunoblot.

Specificity ELISA and Immunoblots

Due to the fact that the buffer conditions needed for efficient elution were relatively harsh, the specificity of the can225IgG had to be re-confirmed after the purification and buffer exchange procedures. Thus, ELISA (Fig. 3a) and immunoblot assays (Fig. 3b) were carried out using recombinant human EGFR extracellular domain protein.

3

a)

b)

Figure 3. EGFR/HER-2 specificity of the can225IgG. a) ELISA plates were coated with 1µg/ml recombinant EGFR or HER-2 extracellular domain and incubated with the corresponding concentration of can225IgG indicated on the X-axis. Bound can225 was detected by rabbit anti dog IgG HRP (Jackson ImmunoResearch) and plates were subsequently developed by ECL BD OptEIA™ substrate and read OD450nm. Mean values of x are shown from 2 determinations, +/- standard deviation. b) Western blot with 1µg of 1) EGFR and 2) HER-2 loaded. Blots were incubated with can225IgG and detected by rabbit anti dog IgG HRP (Jackson ImmunoResearch). Signal was developed with ECL substrate.

The result of both assays demonstrated clearly, that the can225IgG retained its specificity despite the low pH during elution and bound specifically to the rec. human EGFR, but not to the highly homologous HER-2 receptor. Furthermore, we could show, that the signal intensity in the EGFR-coated wells gradually decreased at lower can225IgG concentrations.

Proliferation assays

In addition to MTT-based cell viability assays (see manuscript; p. 55 (Fig.5) ), pilot-experiments were carried out to measure the direct impact of the can225IgG on the proliferation of cancer cells (see Annex - Supplementary methods; p. 72).

Figure 4. Cetuximab and can225IgG were used to stimulate cells and their effect on proliferation was detected by BrdU assay. The upper panels show the effects on EGFR positive human cell lines, the lower panels show ErbB-1 positive canine mammary carcinoma cell lines. Significant tumor-growth inhibition could be observed with all of the shown cell lines upon can225IgG application. (Two-tailed t-test, * p<0,05; ** p<0,005; *** p<0,001)

The results shown in Figure 4. further approve the results of the tetrazolium-based EZ4U® viability assays discussed in the manuscript. We could demonstrate the anti-proliferative effect of the can225IgG on four additional cell lines, two human colon carcinoma cell lines (Caco-2 TC-7, HT29) and two canine mammary carcinoma cell lines (P114, Sh1b). Thus we can conclude, that the can225IgG is not only specifically binding to the natural canine ErbB-1 receptor (demonstrated by flow cytometric analyses), but also exhibits tumor-growth inhibitory functions, comparable to those observed with the original Cetuximab in classical human EGFR reference-cell lines.

Discussion

Although canines tend to frequently develop spontaneous cancers [1], no passive anti-cancer immunotherapy has been approved yet for veterinarian use. The current, regularly applied tumor therapies include surgery, radiation therapy and chemotherapy [2], which are often not sufficient to provide a significantly higher survival. In human oncology however, several monoclonal anti-cancer IgG1 antibodies are available in clinical use, for instance Cetuximab®, a chimeric anti-EGFR (ErbB-1) antibody [3-5]. Humans and dogs share a high structural and functional homology between their ErbB-1 molecules and Cetuximab can specifically bind to and inhibit the receptor of both species [6]. Since it has been described, that canine mammary carcinomas (just like human breast cancer cells) also regularly overexpress ErbB-1 [7, 8], this tumor-associated antigen represents a very promising target for cancer immunotherapy in canine patients.

For the generation of this recombinant chimeric mouse-canine antibody we used the variable region of the above mentioned Cetuximab 225 due to its distinguished binding affinity towards the canine antigen. A further aspect was that the Cetuximab 225 binds only to extracellular subunit III of EGFR, and therefore blocks the ligand binding site, stabilizes the receptor in its inactive conformation and prevents dimerization of the receptor by sterical blockade [9, 10].Since antibodies have been reported to be able to either inhibit or activate receptor signaling [11], the precise site of binding was crucial in this case, as we aim to block this receptor and thereby its downstream pathways.

Unexpectedly we found out, that in spite of several research articles claiming binding of canine IgG to Protein A [12, 13], we were unable to effectively purify our recombinant antibody using this method. Thus we established the purification via Streptococcal Protein G, wherein we assessed various buffer compositions and pH-values. 20mM sodium phosphate (pH 7.0) ensured excellent binding conditions, however, only when the concentration of contaminants by other compounds of the cell culture supernatant were removed by adequate dialysis. To elute bound can225IgG, 0.1M Glycine (pH 2.7) followed by an immediate buffer exchange was proven to be optimal. The precise determination of the pH-range of the elution buffer was a critical key point, as high pH could have resulted in incomplete dissociation and therefore a low yield of the can225IgG. Too low pH however, could have caused damaged antibodies or loss of mono-specificity towards ErbB-1 [14-16].

For the evaluation of in vitro functionality of can225IgG, we decided to perform both viability and proliferation assays, as they investigate various cellular events at different time points of the cell cycle and are therefore able to provide a better overview of the involved processes. BrdU assays are commonly used for direct study of proliferation, as it is based on the incorporation of the labeled compound 5-bromo-2'-deoxyuridine into the DNA of proliferating cells. Thus it provides information about cells being directly in the S-phase during the labeling period. The tetrazolium-based EZ4U® viability assay, however, measures proliferation indirectly by the mitochondrial metabolic conversion of a transparent substrate into a red-color substance. In this case the division of cells is observed after completing the M-phase, when the resulting two metabolically active cells give a more intense signal, than only one, non-dividing cell. Such metabolism-based assays are however, not always reliable, as an enhanced signal intensity can also be a result of upregulated metabolic activity, not only of the increased cell numbers. The reproducibility of the results of the viability assay with BrdU indicate that the can225IgG has indeed tumor-growth suppressing functions and indicates that the significantly decreased live-cell number observed in the EZ4U assay was not a bias caused by downregulated metabolism.

In conclusion, we were able to generate the first recombinant canine anti-tumor IgG and effectively purify it using Protein G affinity chromatography. The results of the immunoblots and CD-spectroscopy demonstrated that our antibody is correctly assembled and folded. The precise specificity towards the recombinant human antigen and the naturally expressed human and canine EGF receptors was proven by a variety of assays including flow cytometry and immunohistochemistry. The favorable results of the proliferation and viability assays indicated a tumoricidic property of can225IgG similarly to that observed with the original human-mouse Cetuximab 225 [6, 17].

Therefore we propose this newly generated antibody a highly interesting tool for future comparative oncological trials. Upon application of this caninized anti-cancer antibody in a veterinary clinical immunotherapy setting, we will be able to gain important insights into the ErbB biology as well as canine immunology, with possible benefits for the development of novel immunotherapies for human patients.

List of references - Discussion

1. Dobson, J.M., et al., Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. Journal of Small Animal Practice, 2002. 43(6): p. 240-246.

2. Lana, S.E., et al., Tumors of the respiratory system—nasal tumors, in Small Animal Clinical Oncology. 2001, Saunders: Philadelphia. p. 370-377.

3. Bouché, O., et al., The role of anti-epidermal growth factor receptor monoclonal antibody monotherapy in the treatment of metastatic colorectal cancer. Cancer Treatment Reviews. 36(0): p. S1-S10.

4. Shirao K, et al., A phase I escalating single-dose and weekly Wxed-dose study of cetuximab pharmacokinetics in Japanese patients with solid tumors. Cancer Chemother Pharmacol, 2009. 64: p. 557-564.

5. Carey, L.A., et al., TBCRC 001: Randomized Phase II Study of Cetuximab in Combination With Carboplatin in Stage IV Triple-Negative Breast Cancer. Journal of Clinical Oncology. 30(21): p. 2615-2623.

6. Singer, J., et al., Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting. Molecular Immunology. 50(4): p. 200-209.

7. Gama, A., et al., Immunohistochemical expression of Epidermal Growth Factor Receptor (EGFR) in canine mammary tissues. Research in Veterinary Science, 2009. 87(3): p. 432-437.

8. Bo, A.-h., et al., Over-expression of EGFR in breast cancer. Chinese Journal of Cancer Research, 2008. 20(1): p. 69-72.

9. Li, S., et al., Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell, 2005. 7(4): p. 301-311.

10. Peipp, M., et al., Effector mechanisms of therapeutic antibodies against ErbB receptors. Current Opinion in Immunology, 2008. 20(4): p. 436-443.

11. Bradbury, A.R.M., et al., Beyond natural antibodies: the power of in vitro display technologies. Nat Biotech. 29(3): p. 245-254.

12. Scott, M.A., et al., Staphylococcal protein A binding to canine IgG and IgM. Veterinary Immunology and Immunopathology, 1997. 59(3-4): p. 205-212.

13. Warr, G.W., et al., Binding of canine IgM and IgG to protein A of Staphylococcus aureus: a simple method for the isolation of canine immunoglobulins from serum and the lymphocyte surface. American journal of veterinary research, 1979. 40(7): p. 922-926.

14. McMahon, M.J., et al., Polyreactivity as an acquired artefact, rather than a physiologic property, of antibodies: evidence that monoreactive antibodies may gain the ability to bind to multiple antigens after exposure to low pH. Journal of Immunological Methods, 2000. 241(12): p. 1-10.

15. Djoumerska, I., et al., The Autoreactivity of Therapeutic Intravenous Immunoglobulin (IVIg) Preparations Depends on the Fractionation Methods Used. Scandinavian Journal of Immunology, 2005. 61(4): p. 357-363.

16. Djoumerska-Alexieva, I.K., et al., Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis. FEBS Journal. 277(14): p. 3039-3050.

17. Wild, R., et al., Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels. Molecular Cancer Therapeutics, 2006. 5(1): p. 104-113.

Acknowledgement

First and foremost, I would like to thank to my supervisor Prof. Dr. Erika Jensen-Jarolim for her exemplary guidance, supervision, and continuous encouragement throughout this project. Her demand on high quality research was extraordinarily inspiring and motivating. I am especially grateful for her trust and promoting me to actively participate in several meetings and symposia, which helped me a lot to improve my communication skills and my self-confidence. Without her persistent help and providing of excellent ideas, this master’s thesis would not have been possible.

I wish to express my gratitude to Dr. Josef Singer, Dr. Marlene Weichselbaumer and Mag. Miroslawa Matz for supporting me and teaching me every important method. Your endless patience, advice and aid were of immense help and contributed greatly to the success of this work.

I am also thankful for the successful cooperation and valuable advice of Univ.Prof.DI Dr. Renate Kunert, DI Dr. Alexander Mader and Willibald Steinfellner from the Department of Biotechnology, VIBT – University of Natural Resources and Life Sciences, Vienna.

Furthermore, I would like to thank the entire division for comparative medicine and laboratory for comparative immunology and oncology for helping me and answering my questions at any time. I enjoyed spending time with you both in- and outside the lab. You are more than just colleagues to me. Thank you Anna Willensdorfer, Cristina Gómez-Casado, Franziska Roth-Walter, Anna Lukschal, Krisztina Szalai, Cornelia Schultz, Caroline Stremnitzer, Denise Heiden, Beatrix Pfanzangl, Irfete Fetahu, Doris Hummel, Abhishek Aggarwal, Julia Höbaus, Samawansha Tennakoon, Charlotte Gröschel, Enikő Kállay, Teresa Manhardt, Erika Bajna, Lucia Panakova, Sarah Meitz, Carmen Fisher, Kumiko Oida, Philipp Starkl, Silke Schrom, Diana Mechtcheriakova, Kathrin Thell, Anastasia Meshcheryakova, Martin Svoboda and everyone else!

Finally, I would like to express my special gratitude and thanks to my parents for their love and patience and for supporting and encouraging me throughout my entire studies. My thanks also go to my brother and my friends for cheering me up in difficult times. Without you, I could never have done all this.

This study was supported by the Austrian Science Fund project P23398-B11.

Eidesstattliche Erklärung

Erklärung:

Ich erkläre, dass die vorliegende Diplomarbeit/Masterarbeit von mir selbst verfasst wurde und ich keine anderen als die angeführten Behelfe verwendet bzw. mich auch sonst keiner unerlaubter Hilfe bedient habe.

Ich versichere, dass ich diese Diplomarbeit/Masterarbeit bisher weder im In- noch im Ausland (einer Beurteilerin/einem Beurteiler zur Begutachtung) in irgendeiner Form als Prüfungsarbeit vorgelegt habe.

Weiters versichere ich, dass die von mir eingereichten Exemplare (ausgedruckt und elektronisch) identisch sind.

Datum: Unterschrift:

Annex

Supplementary methods

BrdU proliferation assays

To directly measure DNA replication, BrdU Proliferation assays (Roche, Hertfordshire, United Kingdom) were performed. BrdU (5-bromo-2'-deoxyuridine), is a nonradioactive compound, which is incorporated into the genomic DNA of proliferating cells during DNA synthesis. EGFR expressing cell lines HT29, Caco-2 TC7, P114 and Sh1b were seeded onto 96-well cell culture plates at a density of 3x103 cells/well. After 18h of growing in full medium, cells were incubated with various concentrations of can225 IgG, Cetuximab or isotype controls for 24h prior to labeling with BrdU. After a labeling period of 4h, the incorporated BrdU was detected with an HRP-conjugated anti-BrdU antibody. Bound antibodies were detected with TMB and color reaction was measured at 450nm in an ELISA reader (Infinite M200 PRO, Tecan Group AG, Maennedorf, Switzerland).