Affinity Protein Based Inhibition of Cancer Related Signaling Pathways

Affinity protein based inhibition of cancer related signaling pathways

Erik Vernet

Royal Institute of Technology School of Biotechnology Stockholm 2009

Royal Institute of Technology School of Biotechnology AlbaNova University Center SE-106 91 Stockholm Sweden

Printed by Universitetsservice US-AB Drottning Kristinas väg 53B SE-100 44 Stockholm Sweden

ISBN 978-91-7415-232-6 TRITA BIO-Report 2009:1 ISSN 1654-2312 iii

Erik Vernet (2009): Affinity protein based inhibition of cancer related signaling pathways. School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.

Abstract Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.

In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.

Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy.

Keywords: Affibody, Cancer, Endoplasmic reticulum retention, EGFR, HER2, IGF-1R, Raf, Ras.

List of publications This thesis is based on the following articles, referred to in the text by their Roman numerals (I-IV). The articles are reprinted with permission from the copyright holders.

I. Vernet E, Konrad A, Lundberg E, Nygren P-Å and Gräslund T. (2008). Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule. J. Immunol. Methods 338(1-2) 1-6.

II. Vernet E, Lundberg E, Friedman M, Rigamonti N, Klausing S, Nygren P-Å and Gräslund T. (2009). Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain. New Biotechnology. Accepted for publication 2009-02-03.

III. Li J, Lundberg E, Vernet E, Höidén-Guthenberg I and Gräslund T. (2009). Selection of affibody molecules blocking hormone-binding to the insulin-like growth factor 1 receptor. Manuscript.

IV. Lundberg E*, Grimm S*, Shibasaki S, Vernet E, Skogs M, Nygren P-Å and Gräslund T. (2009). Selection and characterization of affibody molecules interfering with the interaction between Ras and Raf. Manuscript.

* Authors contributed equally.

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Contents

Introduction ...... 1 1 Protein trafficking ...... 3 1.1 Exocytosis ...... 3 1.2 Endocytosis ...... 4 1.3 Cellular localization signals ...... 5 1.4 Protein degradation ...... 6

2 Growth‐related signaling pathways in cancer ...... 7 2.1 Self sufficiency in growth signals ...... 7 2.2 Signaling pathways ...... 8 2.3 Cancer related proteins ...... 11

3 Generation of affinity reagents from protein based libraries ...... 15 3.1 Antibodies ...... 15 3.2 Library‐based methods for generation of affinity reagents ...... 17 3.3 Phage display selection ...... 18 3.4 Other selection technologies ...... 18 3.5 Affibody molecules ...... 19

4 Regulating protein expression and activity ...... 21 4.1 Protein interference ...... 21 4.2 Small molecule interference ...... 25 4.3 RNA interference ...... 26

5 Delivery systems for genes and proteins ...... 29 5.1 Viral vectors ...... 30 5.2 Non‐viral vectors...... 33

Present Investigation ...... 35 6 Affibody molecule mediated ER retention (Paper I, Paper II) ...... 37 6.1 Affibody molecule mediated ER retention of HER2 (Paper I) ...... 37 6.2 Affibody molecule mediated ER retention of EGFR (Paper II) ...... 40

7 Novel affibody molecules for cancer related targets (Paper III, Paper IV) ...... 42 viii

7.1 Selection of IGF‐1R specific affibody molecules (Paper III) ...... 43 7.2 Selection of Raf specific and Ras specific affibody molecules (Paper IV) ...... 46

8 Concluding remarks ...... 49 9 Acknowledgements ...... 51 10 References ...... 53

List of abbreviations ADCC Antibody-dependent cell-mediated cytotoxicity ADCP Antibody-dependent cell-mediated phagocytosis CPP Cell penetrating peptide EGF Epidermal growth factor EGFR Epidermal growth factor receptor ER Endoplasmic reticulum ERK Extracellular signal regulated protein kinase Fab Fragment antigen binding Fc Fragment crystallizable Ig Immunoglobulin IGF-1 Insulin-like growth factor 1 IGF-1R Insulin-like growth factor 1 receptor mAb Monoclonal antibody MAPK Mitogen-activated protein kinase MEK Mitogen-activated or extracellular signal-regulated protein kinase PI3K Phosphoinositide 3-kinase

PIP3 Phosphatidylinositol-3,4,5-triphosphate PKC Protein kinase C PLC Phospholipase C PPI Protein-protein interaction PTM Posttranslational modification RNAi RNA interference RTK Receptor tyrosine kinase scFv Single-chain variable fragment siRNA Small interfering RNA TGN Trans-Golgi network TKI Tyrosine kinase inhibitor TF Transcription factor

"Concern for man and his fate must always form the chief interest of all technical endeavors… Never forget this in the midst of your diagrams and equations." Albert Einstein xii

ERIK VERNET 1

Introduction

This thesis is focused on proteins, their functions in cancer related cellular processes and the inhibition thereof. From a chemical point of view, proteins are polymers of amino acids connected by covalent amide bonds. Proteins are built from a repertoire of 20 different amino acids, each with distinct chemical and physical properties, and even a very small protein can thus be endowed with unique properties depending on the amino acid composition and order. The amino acid sequence determining the structure and the function of the protein is in turn determined by the deoxyribonucleic acid (DNA) sequence coding for the specific protein. The transfer of information from DNA (the blueprint of life) to protein is mediated by ribonucleic acid (RNA), the third player in the central dogma of molecular biology (1).

Proteins are crucial components of all living cells, and it is therefore not very surprising that dysregulation of protein function can cause major problems for the affected cell and organism. Given the fact that there are approximately 20 500 unique protein coding genes in the human genome (2), it is obvious that a complex system, maintaining the delicate spatial and temporal control of protein synthesis, activation, localization and degradation, is needed. With recent advances in genomics, transcriptomics and proteomics, scientists today have a better possibility than ever before to understand protein function, protein- protein interactions and the signaling pathways.

Chapter 1 and 2 of this thesis will discuss parts of the mammalian cellular machinery controlling protein function, including a brief overview of signaling pathways related to cancer. In Chapter 3 through 5, natural and engineered molecular tools for inhibition of these harmful processes will then be described, together with a number of current viral and non-viral delivery systems that can be used to deliver protein or nucleic acid based entities for inhibition of protein function. The contribution from the work in this thesis will then be summarized in Chapter 6 and 7, followed by some concluding remarks.

It has been said that “Some books are to be tasted, others to be swallowed, and some few to be chewed and digested” (3). I hope you will enjoy this protein rich diet.

2 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

ERIK VERNET 3

1 Protein trafficking

In cells, proteins are synthesized by ribosomes, large cytosolic complexes consisting of proteins and ribonucleic acids that translate the protein-coding information of mRNAs. The nascent polypeptide chains are folded into functional proteins in the cytosol, the endoplasmic reticulum (ER) and in mitochondria, each organelle containing a specialized set of chaperones and folding enzymes to facilitate the folding process, which in some cases is initiated as the protein is still being synthesized. In addition, proteolytic systems degrade unfolded proteins to avoid malfunction and toxic aggregation (4).

The correct function of a protein is in many cases dependent on its localization. In order to control protein function and localization, the cytoplasm contains numerous vesicular compartments and accessory proteins for correct sorting and processing of secreted or internalized molecules, both endogenous and exogenous. A majority of the protein sorting occurs in the trans-Golgi network (TGN), at the plasma membrane and in endosomes (see below) (5). Compartments in these protein trafficking pathways contain domains with distinct lipid and protein compositions, which enable them to carry out different functions. These include transmembrane proteins, displaying motifs in the cytoplasmic domains that interact with sorting proteins for correct transporting, for example by kinesin-mediated transportation on microtubules (6, 7).

1.1 Exocytosis In mammalian cells, ribosomes are located in the cytosol and become membrane associated when synthesizing proteins destined for transport through the secretory compartments (8). These proteins contain signal sequences recognized by a signal recognition particle (SRP) in the cytosol (9) and are then transported through pores in the ER membrane known as the Sec61 translocon (10). The ER is a subcellular compartment with extracellular properties in terms of ionic strength and redox potential. Here, nascent proteins interact with chaperones and other proteins to be correctly folded and undergo necessary covalent posttranslational modifications (PTMs), including signal peptide removal, disulfide bond formation and N-glycosylation (4). This processing is intimately linked to a multistep process called ER quality control (11), where proteins that fail to fold undergo a retrograde transport called ER-associated degradation to the cytosol where they are degraded by the ubiquitin-proteasome system (Chapter 1.4). Unless tagged to be retained in the ER (Chapter 1.3.1), proteins that pass the quality control steps are transported onward to the Golgi complex, and then on to the final destination at e.g. the cell surface or the extracellular space. Transport to the Golgi complex from the ER occurs in the lumen of transport vesicles coated with the coat protein complex-II (COPII) (12), a 4 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

process mediated by specific ER export processes that depends on a interplay between cargo and adaptor proteins not fully understood (13).

The Golgi complex consists of several stacks, known as cisternae, where additional covalent PTMs such as glycosylation and phosphorylation occur. The Golgi complex also plays a major role in protein sorting, which mainly takes place in the outer Golgi vesicles, also known as the trans-Golgi network (TGN). These tubular domains show a dynamic behavior, forming on arrival of proteins and being consumed as export carriers (14). For example, proteins to be transported to the lysosomes, the plasma membrane and other compartments can be glycosylated with mannose-6-phosphate (M6P) and transported from the TGN by the membrane bound M6P receptors CD-MPR and CI-MPR (15). In summary, these elaborate systems for protein compartmentalization and modification enable a multitude of processes to take place simultaneously.

1.2 Endocytosis Endocytosis is the process of energy-dependent vesicle formation at the plasma membrane, with the purpose of engulfing external soluble molecules (such as nutrients) or membrane bound receptors (such as growth signal receptors). The mechanism of vesicle formation can be actin-driven, as in the case of phagocytosis (“cell-eating”) or a special form of pinocytosis (“cell-drinking”) called macropinocytosis, where cell membrane protrusions “zipper up” around the particle or collapse back onto the cell surface, respectively (16). Other types of pinocytosis, all formed by invaginations in the plasma membrane, include clathrin-mediated endocytosis (CME) (17), caveolin-mediated endocytosis (18) and clathrin- and caveolin-independent endocytosis (19). After formation, the vesicle is commonly transported to the early endosomes, from which the internalized molecules are distributed to different parts of the cell (20). Internalized soluble molecules, including most nutrients, are often degraded through the lysosome pathway, going from early to late endosomes and finally ending up in lysosomes (Chapter 1.4). Internalized molecules (e.g. membrane bound receptors) can also be recycled to the plasma membrane, either directly from the early endosomes or through recycling endosomes, a distinct subpopulation of endosomes that contain internalized receptors but lack the dissociated ligands (21), located near the microtubule-organizing center (MTOC).

Endocytosis plays an important role in regulating signaling pathways. Internalization of receptors is a common way for cells to control receptor activity by degradation in lysosomes or to separate different signaling pathways, as signaling from ligand-bound receptors is now known to occur not only from the plasma membrane but also in early endosomes (22). For example, binding of the epidermal growth factor (EGF) to its receptor EGFR initiates several different signaling pathways (Chapter 2), with the phospholipase C- ERIK VERNET 5

protein kinase C (PLC-PKC) pathway and the phosphatidylinositol 3-kinase Akt pathway (PI3K-Akt) pathway being activated by surface bound receptor while Ras-mediated signaling through the MAP-kinase pathway is initiated in endosomes, after receptor-ligand co-internalization (23, 24). EGFR activity can also be modulated by other ligands, including transforming growth factor α (TGF-α), and the actual signals transmitted is dependent on the ligand bound, which is related to the stability of receptor-ligand complexes in endosomes (25). In addition, the route preference for different surface-bound receptors is not completely determined, but many receptors can enter the cell through at least two different mechanisms. For example, a strong mitotic signal mediator, transforming growth factor β (TGF-β), internalizes through clathrin-coated pits as well as caveolae (26). The route is important, as clathrin-coated pit endocytosis leads to signaling from endosomes while caveolin-mediated endocytosis leads to rapid ubiquitylation and degradation (27).

1.3 Cellular localization signals It is now obvious that localization plays a major role in protein function. In 1999, Günter Blobel was awarded the Nobel Prize in Physiology or Medicine for the discovery that “proteins have intrinsic signals that govern their transport and localization in the cell”. This work was initiated in the 1970’s when he formulated the “signal hypothesis” (28). Extensive work carried out in this field over the last thirty years has provided a list of localization signals, a few of which are found in Table 1.

Signal Sequence Notes References Nuclear PKKKRKV Sequence from SV40 large T (29) localization antigen signal (NLS) ER retention KDEL Variants ([KRHQSA]-[DENQ]- (34) signal E-L) exist in humans and other species (30-33) Mitochondrial MSVLTPLLLRGLT N-terminal presequence of (35) targeting signal GSARRLPVPRAK cytochrome c oxidase (MTS) Table 1. Examples of subcellular localization tags. One-letter abbreviations are used for amino acids with alternative amino acids in brackets. Sequences are written from N- to C- terminal.

Interestingly, many localization signals can be expressed as fusions to recombinant proteins to direct protein transport to desired compartments within the cell, as will be described later in this thesis and which has also been described in literature (36, 37). 6 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

1.3.1 ER retention Not all soluble proteins that enter the ER are meant for onward export. An ER retention signal, composed of the C-terminal amino acids KDEL (lysine-aspartic acid-glutamic acid- leucine) was identified some twenty years ago in a number of proteins that were all retained in the ER but secreted when this tag was removed (34). The authors could also show that addition of the KDEL sequence to a protein that was normally secreted, led to its retention inside the cell. It was further observed that KDEL-tagged proteins obtained Golgi-specific carbohydrate modifications, which suggested that a retrograde transport from the cis-Golgi to the ER took place. This model was later supported by experimental data when the ERD receptor was found to bind to KDEL-tagged proteins and mediated the retrograde transport (38, 39). ERD binding is dependent on phosphorylation of the cytosolic domains of the receptor, allowing binding of the KDEL sequence in the cis-Golgi and transport it back to the ER in coat protein complex-I (COPI) coated vesicles (40). This mechanism has also been shown to be functional in plants (41).

1.4 Protein degradation Protein degradation is an important process in order to control the activity of both endogenous and exogenous (including pathogenic) proteins. In cells, proteins are actively degraded by proteases that catalyze the breakage of the covalent peptide bonds. As it would be detrimental for a cell to have active, non-discriminating proteases in the cytosol, these enzymes are therefore restricted to certain compartments and/or substrate specificities.

Macromolecules contained in vesicles, including proteins taken up by endocytosis, are usually degraded in the lysosomes. Lysosomes are low pH vesicular compartments in the cytoplasm containing various enzymes, including proteases, effectively degrading most macromolecules. Larger subcellular compartments, such as mitochondria, can also be engulfed by lysosomes, a process referred to as macroautophagy (42). Even cytosolic proteins can be transported into lysosomes, a process referred to as microautophagy (43).

In addition to the lysosomal protein degradation pathway, a cytosol located machinery referred to as the ubiquitin-proteasome system exists. This system involves several classes of enzymes that catalyze the formation of covalent bonds between the small protein ubiquitin and the target protein, which directs transport of the proteins to the proteasome for degradation (43). The ubiquitin-proteasome system has been utilized as a research tool, where the cellular concentration of a protein of interest can be regulated by fusing it to a domain that is degraded in the absence of a cell-penetrating stabilizing ligand, creating an inducible system for protein degradation (44).

ERIK VERNET 7

2 Growth-related signaling pathways in cancer

In general terms, a signaling pathway can be described as a number of units (e.g. proteins) that transmit a signal from one place (e.g. the extracellular space) to another (e.g. the nucleus). In cell biology, this can either be done by a single molecule, for example by cell surface receptors that translocate directly to the nucleus (45), or by a signaling relay where information is transmitted from one protein to another. These relays are often complicated, as there are numerous examples of proteins being involved in several pathways, effectively building up very complex signaling networks. The signals transmitted may be controlled by protein localization and concentration but are also controlled by activation and inactivation of the protein. The active and inactive states often differ by subtle conformational changes, such as the binding of a cofactor (e.g. a metal ion or a small organic molecule) or minor covalent modifications. Phosphorylation is one of the most common covalent protein modifications, and recent investigations of protein expression throughout the cell cycle has indicated that temporal control of phosphorylation plays a fundamental role, perhaps even more important that the temporal control of protein expression (46, 47).

The uncontrolled division and invasive spreading of abnormal cells, commonly known as cancer, is one of the major causes of human death and suffering in developed countries. In spite of numerous ambitious research programs aiming at reducing the number of deaths, such as the National Cancer Act of 1971 (also known as Nixon’s “War on cancer”), cancer still prevails today. However, in the last forty years, astonishing advancements have been made in many disciplines and today the scientific community have more knowledge than ever before about the origin, development, life, and death of cancer cells. This work has lead to the wide-spread opinion that the origin of cancer is a stepwise progressive process where a series of genetic events must occur in a single cell for tumor initiation and progression. This is in contrast with diseases like cystic fibrosis or sickle-cell anemia that are caused by single mutations. The idea that at least two mutations are needed for tumor development (the “two-hit hypothesis”) was communicated in the 1970s after studies of retinoblastoma patients (48). Today, cancer is regarded as a group of diseases rather than a single disease and current theories rely on the existence of a number of traits or hallmarks believed to be shared by most or all cancer types (49). “Self sufficiency in growth signals” is one of them, and this aspect of cancer will be further discussed in the following section.

2.1 Self sufficiency in growth signals The life cycle of a cell is to a large extent controlled by external growth signals, but it has long been known that cancer cells have a lower requirement for exogenous growth factors (50). Oncogenes, often encoding proteins which are parts of transcription activating 8 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

pathways, can be over expressed or mutated, leading to protein variants which are constitutively active, thus equipping the cell with its own proliferation activation signals. For example, the activity of cell surface receptors is often dysregulated in tumor cells. This can be caused by mutations that may lead to altered substrate specificities (for example for kinases) or expression levels. The latter can also be caused by gene duplications, which may lead to altered signaling patterns due to the formation of additional ligand-receptor complexes. This may result in increased signaling, but may also lead to a decrease in receptor internalization and signal attenuation (25). The activity can also be affected by increased ligand concentration, which is especially dramatic when activation of receptor leads to new ligand expression through positive autocrine feedback loops.

2.2 Signaling pathways Pathways involved in relaying growth signals include the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, the phospholipase C (PLC) protein kinase C (PKC) pathway and the phosphatidylinositol 3-kinase (PI3K) Akt pathway. These signaling cascades are commonly activated by transmembrane proteins such as receptor tyrosine kinases (RTKs) and relay different messages to the nucleus, resulting in transcription factor activation and altered gene expression (Figure 1). The RTK super family consists of 58 different kinases. In most cases, upon ligand binding, these receptors form dimers which leads to autophosphorylation and initiation of downstream signaling pathways (51).

Figure 1. Examples of signaling pathways involved in gene expression regulation. Arrows indicate signal transmission, flat arrows indicate inhibition. ERIK VERNET 9

2.2.1 The ERK-MAPK pathway The mitogen-activated protein kinase (MAPK) pathways are central for cell proliferation, differentiation and survival (52). A number of pathways have been identified; including the extracellular signal-related protein kinase (ERK) cascade, the c-Jun amino-terminal and stress-related protein kinase (JNK/SPK) cascade and the p38 MAPK cascade. These complex signaling networks are responsible for signal transduction from a large number of external stimuli to evoke an intracellular response, in many cases induction or repression of transcription. Signaling through the MAPK pathways is often induced by receptor dimerization and subsequent phosphorylation of intracellular tyrosine residues, a dynamic process that depends on the precise timing and spacing of the constituents (53). The onward signaling to the nucleus is mediated by a number of kinases, where MAPK is phosphorylated by a MAPK kinase (MAPKK), in turn phosphorylated by a MAPKK kinase (MAPKKK) (Figure 2).

Figure 2. Schematic drawing summarizing the RTK activated ERK-MAPK pathway. Zigzag lines indicate protein-protein interactions; TF, transcription factor; P, phosphorylation. Modified from (54).

The division into separate pathways is by no means strict, as they have been shown to be interconnected in several ways and share a number of common constituents. The nomenclature is yet more confused by the fact that the ERK1/2 pathway, which is associated with receptor tyrosine kinases (RTKs) such as EGFR or HER2 and is a 10 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

subfamily of the MAPK cascades, is sometimes referred to as “the MAPK cascade” in the literature. This Raf-MEK-ERK cascade is of particular interest in cancer research since several of the proteins have been shown to be mutated or over expressed in different cancers, and it is also central to the work in this thesis (Figure 2). This signaling pathway can be activated by RTKs such as ErbB receptors (55) and IGF-1R (56), described in the following sections. It should further be noted that some proteins in the cascade, including Ras and Raf, exist in multiple isoforms, performing specialized, sometimes tissue specific, functions (57).

2.2.2 The PI3K-Akt pathway The phosphoinositide 3-kinase (PI3K)-Akt pathway plays an important role in insulin- dependent metabolism (58). Similar to the Raf-MEK-ERK cascade, it is a complex pathway functioning downstream of different RTKs and its members are also frequently mutated in cancer (59). The most thoroughly investigated PI3Ks are the subclass 1A PI3Ks, which consist of a p85 regulatory subunit and a p110 catalytic subunit that are assembled into heterodimers upon binding to phosphorylated residues in the cytoplasmic domains of RTKs. PI3Ks catalyze the phosphorylation of phosphoinositides, including the formation of the important second messenger phosphatidylinositol-3,4,5-triphosphate

(PIP3). Due to its involvement in cancer related processes, kinase inhibitors occupying the ATP-binding pocket are being developed (59).

The membrane-bound phospholipid PIP3 enables activation of Akt kinases (also referred to as protein kinase B, PKB). The Akt gene codes for three isoforms of the Akt protein which is involved in various cellular processes, including cell survival and proliferation. Akt1 is ubiquitously expressed, while Akt2 and Akt3 are tissue specific (60). Over expression of Akt has been reported in pancreatic, ovarian and head and neck cancers (61). In addition, a mutation (E17K) in the PIP3 binding domain of Akt leads to cell transformation in vitro (62). This pathway also plays a role in MAPK signaling, as Akt suppress Raf activity upon external IGF-1 stimulus (63).

2.2.3 The PLC-PKC pathway The PLCs constitute a family of lipases that cleave phospholipids, including the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), the latter activating PKC. This example shows that also glycerides, in addition to phosphorylated proteins or phosphorylated lipids, can mediate signals, further underscoring the diversity in intracellular signaling pathways. Members of the PLCγ subfamily can bind to phosphorylated tyrosine residues on activated ErbB proteins. This serves two purposes, both activating PLCγ by phosphorylation and tethering it to the plasma membrane, thus placing it in close proximity to its lipid substrates (64). ERIK VERNET 11

PKCs are typically lipid-dependent serine/threonine kinases. Similar to many other intracellular kinases involved in signal transduction, different PKC isozymes have individual substrate specificities and are expressed in different tissues and different subcellular compartments, including endosomes and the nucleus (65). Activation of PKC leads to both short term effects (e.g. metabolic) and long term effects (e.g. cell differentiation and proliferation). For example, PKC is known to activate MAPKs such as ERK1/2 by interactions with Ras-Raf complexes (66), and total PKC expression levels has been shown to be elevated in breast tumor tissue (67, 68).

2.3 Cancer related proteins

2.3.1 The ErbB family The ErbB family is a subclass of the RTK super family and consists of four homologous transmembrane proteins with different ligand specificities and intracellular domains. Numerous studies have described the intricate signaling system that these receptors constitute, and it is therefore productive to study them simultaneously in order to understand their biology and relevance in cancer signaling (69). Two of these proteins are of particular interest to the work presented in this thesis and will therefore be further described here.

The first ErbB member, ErbB1, also known as HER1 or epidermal growth factor receptor (EGFR), is important in both development and adult cell function. It interacts with a number of soluble ligands, including the epidermal growth factor (EGF) and transforming growth factor α (TGF-α). Upon ligand binding, the conformation changes from the tethered, or closed, state to the ligand-activated, or open, state, thereby exposing the “dimerization arm” (70). Homo- or heterodimerization (for example with HER2) of EGFR leads to phosphorylation of the cytoplasmic tyrosine kinase domain and onward signaling through several pathways, including the MAPK pathways (71). EGFR is rapidly internalized upon EGF ligand binding, which leads to both receptor down regulation (by lysosomal degradation) or signal diversification (by signaling from endosomal compartments) (24). EGFR was the first tyrosine kinase receptor found to be involved in cancer (72) and it is often over expressed and/or mutated in several different tumor types, such as head and neck, bladder, lung and breast cancer (73, 74). Mutated EGFR is found to be more sensitive to treatment with gefitinib (Table 2) compared to wild-type EGFR, but unfortunately drug resistance often evolves, probably by mutations in the ATP binding domain (75) or through Ras mutations (76).

The second member, ErbB2, also known as HER2, has no known ligand and is believed to be constitutively in an active “open” conformation with an accessible domain for homo- or 12 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

heterodimerization with other receptors in the family (77, 78), thereby activating the intracellular tyrosine kinase domain for downstream signaling (79). In contrast to EGFR, HER2 is not rapidly internalized upon homodimer formation, leading to prolonged signaling from surface located complexes (69). Amplification of the HER2 gene and subsequent receptor over expression is oncogenic (80) and found in various cancers, including breast cancer, where over expression of HER2 is correlated with poorer patient prognosis (81). In addition to over expression, mutations in the tyrosine kinase domain have been reported, for example in lung cancers (4-10 % frequencies) (82).

Due to their involvement in cancer, the members of the ErbB family are interesting targets for therapy and diagnostics. HER2 for example is the target of Trastuzumab (83), the first antibody to be approved for breast cancer therapy (84). Table 2 summarizes some current ErbB targeted therapeutics in use.

Compound Type Target Status Trastuzumab Humanized HER2 Approved for the treatment of HER2 (Herceptin) mAb over expressing breast cancer Cetuximab Chimeric mAb EGFR Approved for the treatment of (Erbitux) colorectal cancer Gefitinib TKI EGFR Approved for the treatment of non- (Iressa) small colony lung cancer after failure on other available treatments Erlotinib TKI EGFR Approved for the treatment of non- (Tarceva) small colony lung cancer after failure on other available treatments Table 2. A selection of ErbB targeted therapeutic compounds (commercial names in brackets). mAb, monoclonal antibody; TKI, tyrosine kinase inhibitor. Modified from (85).

2.3.2 The Ras and Raf proteins Ras proteins (H-Ras, N-Ras, K-Ras4A and K-Rab4B) are highly homologous GTP- binding proteins involved in the MAPK pathways (Figure 2), interacting with over 20 downstream proteins (86). They can be anchored to the inner face of the plasma membrane by a farnesyl-anchor (87). Ras cycles between the active GTP-bound state and the inactive GDP-bound state. The binding of GTP is facilitated by guanine nucleotide-exchange factors (GEFs) such as SOS, while inactivation is catalyzed by GTPase-activating proteins (GAPs) such as neurofibromin (NF1). The four Ras isoforms share several domains, including the GTP-binding P-loop, and two regions (switch I and switch II) regulating protein binding (88). Ras is mutated in about 30 % of all human cancers (89) although the frequency varies substantially between different cancers, with about 90 % occurrence in pancreatic solid tumors (90). The most frequent amino acid changes are found at residues Gly12 and Gln61. These mutations prevent GTP hydrolysis, resulting in constitutively ERIK VERNET 13

active Ras which lead to cell proliferation and inhibition of apoptosis (91). It is noteworthy that germline Ras mutations are associated with non-cancer diseases, indicating that Ras mutations alone are insufficient to initiate tumorigenesis (88). Ras is believed to play a central role in most cancers, and has been called “the holy grail” in drug development (92). Accordingly, numerous Ras inhibitors have been investigated for therapeutic applications (52). For example, an anti-Ras scFv antibody fragment has been shown to inhibit oncogenic transformation when expressed with a plasma membrane targeting signal (93).

The Raf proteins (A-Raf, B-Raf and Raf-1; the latter also being known as C-Raf) have recently received increasing attention owing to their new-found direct involvement in human disease, being mutated in cancer and other diseases (94, 95). The Raf proteins are kinases in the ERK-MAPK pathway catalyzing the phosphorylation of mitogen-activated or extracellular signal-regulated protein kinases (MEKs; Figure 2) (96). The Raf proteins also have individual functions (97) and have been shown to play additional roles in apoptosis (98) and NFκB activation (99), indicating that the traditional, linear kinase- dependent MAPK signaling is only one part of a complicated network. On the molecular level, Raf proteins share three conserved regions: CR1, is a cysteine-rich domain that contains part of the Ras-binding domain (RBD) and also interacts with membrane lipids; CR2 which is a serine/threonine rich region; and CR3, the kinase domain, which can bind the substrate MEK. A small molecule inhibitor of B-Raf and Raf-1, Sorafenib (brand name Nexavar) is approved for the treatment of advanced kidney cancer or unresectable hepatocellular carcinoma (100, 101), with several other small molecule inhibitors of Raf currently under investigation (102). Since Ras-Raf interactions are central in the pro- proliferative pathway, it is an interesting protein-protein interaction to interfere with for cancer therapy. A set of small molecules inhibiting Ras-dependent activation of Raf have been selected using two-hybrid selection (Chapter 3.4.2) (103).

2.3.3 The IGF-1 receptor The insulin-like growth factor 1 receptor (IGF-1R) is a tetrameric RTK consisting of two α-chains (involved in ligand binding) and two β-chains (containing intracellular tyrosine kinase domains) (104). IGF-1R is widely expressed on normal tissues, but elevated expression is found in various cancers, including common cancers such as breast and prostate (105, 106). Ligand binding to the extracellular part of IGF-1R leads to phosphorylation of the tyrosine kinase domains which in turn activates the PI3K-Akt pathway and the ERK-MAPK pathway (107). IGF-1R has several soluble ligands, including the insulin-like growth factors 1 and 2 (IGF-1 and IGF-2). In contrast to other RTK-activating peptide ligands, such as EGF, the IGFs not only stimulate the producer cell and cells in the vicinity (so called autocrine and paracrine signaling, respectively) but IGF levels in the circulation also affect the entire organism (endocrine signaling) significantly complicating the understanding of the role of IGF-1R in human disease (108). 14 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

Both in vitro and in vivo studies show that IGF-1 levels influence tumor growth, however the frequent gene amplification and ligand-independent activation events described for HER2 are not common for IGF-1R (108). As a consequence of its importance in human aging and disease, drugs targeting IGF-1 and IGF-1R are under development. These include anti-ligand, anti-receptor, and kinase inhibitor strategies using both affinity proteins and small molecules (108, 109).

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3 Generation of affinity reagents from protein based libraries

Generation of affinity reagents to novel targets (so called antigens) are traditionally accomplished by in vivo immunization of laboratory animals, in which the immune system of the animal recognizes the antigen as foreign and develops antibodies against it. These antibodies can subsequently be isolated directly from the serum of the animal (polyclonal antibodies) and have proven extremely useful in many applications. However they suffer from lack of reproducibility as different immunizations will give different clone compositions. In addition, some target proteins (including self-antigens) are not immunogenic and will not initiate an immune response in the host. Another strategy to isolate affinity reagents from the immunized animal was by hybridoma technology (110). In this case, a specific antibody producing cell from the animal was fused to a tumor cell, creating an immortal antibody producing cell that could be expanded into a large population. This is however a challenging and time consuming method, since a multitude of clones have to be screened in order to find one that produces high levels of the desired antibody.

While it is still common to generate antibodies from immunized animals, this route has been challenged in recent years by so called protein library techniques, by which immunoglobulin-based reagents can be generated without the use of laboratory animals. In contrast to using animals for affinity reagent generation, these techniques are not limited to reagents based on molecules of the immune system. As we shall see in the following sections affinity reagents based on non-immunoglobulin scaffold proteins can as easily be generated using these techniques

3.1 Antibodies Among the most important constituents of the immune system of vertebrates are the antibodies or immunoglobulins (Ig), proteins naturally found on certain B lymphocytes and in serum. Through an elaborate procedure, involving genetic recombination with positive and negative selection, new antibody variants are constantly generated to specifically bind to essentially all molecules (called antigens) that are deemed foreign to the host organism. Antibodies have also found much use in medical and technical applications. Antibodies produced by immunizing animals with an immunogen (an antigen with the ability to evoke an immune response) are polyclonal since they consist of a mixture of antibodies produced from different cell clones and thus have different amino acid compositions that may bind different surfaces (called epitopes) on the antigen. In contrast, monoclonal antibodies (mAbs) are identical in terms of amino acid sequence and thus bind to the same epitopes. 16 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

In structure, antibodies are large glycosylated proteins consisting of two light chains and two heavy chains interconnected by disulphide bonds (Figure 3). There are five antibody isotypes (subclasses) in humans (A, D, E, G and M) with IgG being is the most abundant isotype in human serum and the most common for technical applications. For that reason, mainly IgG molecules will be considered in this text. All chains of an IgG molecule consist of domains of the immunoglobulin fold, which is a conserved 25 kDa domain with intramolecular disulfide bridges. Full-size antibodies (150 kDa) have two antigen binding (Fab) parts and one crystallizable (Fc) part. The Fab fragments each contain a variable part

(Fv), consisting of the variable domains of the heavy and light chains (VH and VL) and constant (non-antigen binding) domains (CH1 and CL). The variable domains interact with the antigen, primarily through their hyper-variable loops, referred to as the CDRs (complementarity-determining regions) (111). The Fc part on the other hand contains the other constant domains of the heavy chain (CH2 and CH3) which determines the isotype of the antibody. The Fc part can interact with molecules such as components of the complement system, the FcγIII receptor and the neonatal Fc receptor (FcRn) for lysis of bacterial cells, activation of antibody-dependent cell-mediated cytotoxicity (ADCC) and for prolonged antibody half-life in serum (112), respectively.

Figure 3. Schematic drawing of an IgG antibody and a single chain variable fragment (scFv). The antigen binding parts (Fab) are in gray, the hinge region in black and the crystallizable (Fc) part is in white. S indicates sulfur atoms, forming covalent disulfide bonds under oxidizing conditions.

To overcome some of the limitations of antibodies for technical applications (Chapter 4), antibody fragments have been developed using recombinant DNA technology. These fragments include the non-covalently associated variable fragment (Fv) (113), the disulfide bond associated fragments (dsFv) (114) or the covalent single-chain variable fragment (scFv) (115). Of these, the scFv is the most studied (Figure 3). By removing everything but the variable antigen binding part, the molecular weight is reduced from 150 kDa to approximately 25 kDa. The need for intermolecular disulfide bonds (to link the VH and VL ERIK VERNET 17

domains) is in this case eliminated by inserting a covalent linker (most commonly a

(Gly4Ser)3 linker) between the two chains.

3.2 Library-based methods for generation of affinity reagents Advances in recombinant DNA technology have in recent years enabled cloning of antibody genes, e.g. for recombinant expression in CHO cells (116). In addition, repertoires of antibody genes can also be constructed, which is called an antibody gene library. These libraries can originate from non-immunized animal in which case a naïve library is created, or from animals immunized with a particular antigen in which case the library is enriched for genes encoding antibodies reactive to the antigen, a so called immune library. It is also possible to create a fully synthetic library by synthesizing oligonucleotides coding for the library members. With the creation of antibody-based libraries, it was soon realized that analogous libraries could be created using other proteins frameworks (117) such as the 58 amino acid Z domain derived from protein A (Chapter 3.2). Regardless of the scaffold protein utilized, the library can then be used for selections, where the different protein variants compete with each other, and the molecules exhibiting the sought-for properties are enriched for. Instead of selecting the sought-for “winner” by competition, the variants of a library can be examined individually by screening, where a number of potentially useful variants are examined individually. This process is generally more time-consuming but can be useful in finding properties that cannot be selected for.

A key feature of an efficient affinity protein is the ability to bind the intended target with high selectivity and affinity. In directed evolution strategies, these proteins (the “books”) are isolated by the use of a selection system. A physical linkage between the genotype and the phenotype is necessary to allow for determination of the DNA (or RNA) sequence of the variant which in turn allows for determination of the protein sequence. This linkage is a general property of all selection systems. A large library size is desirable as the greater the number of variants in the library, the greater the chance of it containing the sought-for variant. In cell-based systems, library size is often limited by the transformation efficiencies. Finally, a low background is desirable. Characterization of potential binders is a time consuming process which often includes sub-cloning and various in vitro assays. A high number of false positives is therefore a problem.

In general, the process of directed evolution can be regarded as a four-step-process consisting of diversification, protein expression, selection/screening and gene amplification. Over the years, a number of different selection systems have been developed, some of which will be covered here. 18 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

3.3 Phage display selection Phage display (118) is the most commonly used artificial system for selection of both antibody fragments and alternative scaffold proteins interacting with various target antigens. Systems derived from filamentous phages such as M13, which assemble in the bacterial periplasm, are most commonly used. The genomic library is contained inside the phage particles, while the corresponding gene product is expressed on the phage surface as a fusion protein with one of the phage coat proteins (cp), most commonly cp3 or cp8. The number of recombinant protein copies per phage varies from a few (for cp3 display) to several thousand (for cp8 display). Due to steric hindrance, expression of large fusion proteins is more limited on cp8 than on cp3. The cp3 protein, expressed on the tip of the phage, is therefore used for protein or peptide library expression while the cp8 is used for peptide libraries. Since all coat proteins are displayed in several copies, the library protein will also be present in multiple copies when fused to them. This may be undesirable and could lead to higher apparent affinity of the library members to the antigen as a consequence of display number rather than true affinity. One common approach to circumvent this problem is to create mosaic phages, where one or zero copies of the library member is expressed on each phage particle. This can be achieved by the use of a 3+3 or 8+8 phagemid system (119, 120). In this system, a phagemid (phage plasmid) contains the library-cp3 gene construct with a phage packaging signal, while a helper phage supplies the rest of the phage genome (including wild type cp3), but has a disabled packaging signal, which means that the cp3 fusion protein gene from the phagemid will be favored during phage assembly. Selection is traditionally done by bio-panning, where the phage library is incubated with the target antigen (118). Eluted phages are then allowed to infect a host organism.

3.4 Other selection technologies

3.4.1 Cell display selection A protein library can also be expressed on the surface of cells, including bacteria (121), yeast (122) and mammalian cells (123). One advantage of using these larger entities for display is the possibility to directly sort the selected cells in a cell sorter (e.g. a flow cytometer), where the affinity for the target molecule can also be determined directly on the cells, abolishing the need for time-consuming sub-cloning before KD determination (124).

3.4.2 Two-hybrid systems As noted, phage (and cell) display technologies have been used extensively for extracellular selections. However, if a selected molecule is to be used in a complex environment, such as the cytosol, a protocol involving a step where intracellular ERIK VERNET 19

interactions with the intended target are selected for is desirable. The two-hybrid system was first described in yeast (125), but has now been expanded to bacteria (126) and mammalian cells (127). For intracellular proteins lambda phages have also been used, for example in a phage two-hybrid system (128). Two-hybrid systems are generally based on transcription factor-controlled activation of a reporter gene. Here, the transcription factor is split into two parts, so that the DNA-binding domain is genetically fused to one of the proteins under investigation (the “bait”). The activating domain of the transcription factor is then genetically fused to the other protein (the “prey”). The downstream reporter gene will then be transcribed only if the two parts of the transcription factors are brought into close proximity by bait-prey interactions.

As with most other cell-dependent systems, a major limitation is the transformation efficiency. This means that most yeast libraries are about a hundredfold smaller than a standard phage library. In bacteria the libraries can be made larger, but since the cellular machinery is less sophisticated (e.g. with regard to PTMs) it might be less suited for investigation of for example human proteins. The two-hybrid systems are sometimes also hampered by high background, since there is a chance of transcription factor proximity without the bait-prey interaction.

3.4.3 Intracellular antibody capture systems Development of antibodies or antibody fragments with intracellular activity has been a challenge (Chapter 4.1). Intracellular antibody capture (IAC), a combination of phage display and the yeast two-hybrid system, was developed to create stable antibody scaffolds for intracellular applications, into which the antigen binding sites of an in vitro selected antibody can be inserted (129). After a number of rounds of phage display based selection, a pool of DNA from antigen binding phages can be sub-cloned to a yeast expression system for further selection. This technology has been used for development of single chain antibody domains with affinity for Ras (93, 130). Intracellular expression of antibody fragments has also been used for selection of variants that not only bind to an antigen but also neutralize it, hence selection for function in addition to binding (131).

3.5 Affibody molecules For certain technical applications the intrinsic physical and chemical properties of antibodies are limiting the success of the application (117). It is therefore desirable to develop affinity proteins based on other protein scaffolds. One of these, the affibody scaffold, is central to the work presented here (Figure 4).

20 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

Figure 4. Schematic drawing of the IgG binding domains of SPA, the engineered Z domain and affibody scaffold, with the 13 positions used for randomization.

Affibody molecules are derived from one of the IgG binding domains of the staphylococcal Protein A (SPA). This domain (the B domain), a 58 amino acid three-helix bundle of about 6 kDa, was then engineered to create the Z domain, by Gly29Ala and Ala1Val mutations for hydroxylamine resistance and facilitated cloning, respectively (132). This molecule was initially used as an affinity tag for purification and detection of antibodies and other proteins (133, 134). After constructing a library by randomization of 13 amino acid positions in two of the helices, it has been possible to generate small, stable, soluble affinity proteins (so called affibody molecules) towards different targets (135). These are in many cases readily expressed in E.coli, highly soluble, and can be purified using denaturing conditions as they can be refolded. To date phage display (Chapter 3.4) has been the selection method of choice, although other selection methods, including cell display and ribosome display, are under investigation. Affibody molecules can also be synthesized by chemical methods, such as solid phase peptide synthesis (136).

ERIK VERNET 21

4 Regulating protein expression and activity

As seen in Chapter 2, dysregulation of protein function can have grave consequences for the fate of a cell. Today, natural and engineered systems are available for regulation of protein function, including RNA interference (RNAi), protein interference and small molecule interference. The two latter are based on the concept of affinity, a measure of the rates of association and dissociation between molecules. The association and dissociation reactions of protein A and its ligand B can in its simplest form be set up as the equilibrium

Here the rate of protein-ligand associ ation is given by the differential equation

where k1 and k-1 are the rate constants for the association and dissociation, respectively. When equilibrium is reached, the rate of association and dissociation will be equal, and by rearrangement, the dissociation constant KD can be defined as

Where ΔG° is the change in Gibbs free energy, R is the gas constant and T is the temperature. Using SI units, the unit for KD will be molar (M). Under in vitro conditions, dissociation constants for many protein-ligand interactions are in the nanomolar range. There are some exceptions, including the tetrameric protein avidin which binds to vitamin

B7 (biotin) with a KD of around 1 femtomolar (137). Molecules that have a low KD value (typically below 10 nM) for the interaction with a specific target molecule while binding poorly to other molecules (KD typically above 10 μM) are referred to as high affinity ligands and are of great interest in both in vitro and in vivo applications.

4.1 Protein interference Affinity proteins, such as antibodies or alternative scaffold molecules, can be used to interfere with unwanted protein-protein interactions in vivo. Protein interference may in some cases be used to interfere with a subpopulation of the protein, for example depending 22 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

on posttranslational modifications. The affinity protein’s interactions with the target molecules can have different effects, some of which are listed here.

1. Antagonistic binding. By binding, the ligand inhibits one or more of the native functions of the protein. This is commonly achieved by binding to an epitope overlapping the epitope of a natural activating (agonistic) ligand. This is a common mode of action for both protein inhibitors, such as antibodies (138), and small molecule inhibitors (139).

2. Agonistic binding. In this case, affinity ligand binding activates the target protein. This could lead to a number of different effects, including downstream signaling or receptor internalization (140). Different agnostic binders can thus mediate different cellular responses.

3. Target protein relocalization. When combined with localization signals (Chapter 1.3), affinity proteins can also be used to specifically retarget its cellular target to a specific part of the cell. This can be done in order to achieve a more selective effect but also to sequester or degrade (141) a potentially harmful molecule.

4.1.1 Protein affinity and stability It is crucial that the affinity proteins can function in the relevant intracellular or extracellular context; however this is by no means guaranteed by using in vitro selection unless this is a property specifically selected for. The milieu can significantly affect the affinity and specificity, especially if the protein contains structural elements that will not fold correctly in the relevant cell compartment. Another crucial factor is the stability of the protein, and it has been argued that stability is more important than affinity in intracellular applications (142-144), implying that the half-life of the protein cannot be too short for successful therapy. Attempts have been made to account for these factors by incorporating heat (145), acid (146) or protease (147) stability as a criteria in the affinity protein generation.

4.1.2 Antibodies against extracellular targets Owing to their versatility and accessibility, antibodies are used as affinity proteins in a wide range of in vivo applications, including therapeutic applications (148, 149). As most monoclonal antibodies are of rodent origin, they are usually engineered to avoid immune response. These modifications include chimerization (where the Fc part is replaced with a human ortholog) (150) or humanization (where the CDRs are grafted into a human antibody) (151). Antibodies have long half-lives in sera, which is beneficial for some applications. This is partly due to their large size which prevents renal clearance but also ERIK VERNET 23

due to the mechanism of the neonatal Fc receptor (FcRn) (Chapter 3.1). The large size and long half-lives can however be a problem, for example for manufacturing (117, 152), in vivo imaging and tumor penetration (153) or intracellular applications (see below). For example, anti-tumor antibodies have shown to be more effective against circulating malignant cells than against solid tumors, possibly due to size dependent accessibility issues (154, 155).

Antibodies can also be used to target another functional entity to a specific location and thus mediate secondary effects. These entities may be constituents of the immune system, includes antibody-dependent cell-mediated cytotoxicity (ADCC). For example, the HER2 binding antibody Trastuzumab (Chapter 2.3.1, (83)) has been shown to (partially) function by antibody-dependent cell-mediated phagocytosis (ADCP), while an IgE isotype of the same antigen binding domains instead mediated ADCC (156). The secondary effect may also be performed by foreign molecules such as cytotoxic drugs (154, 157), or radioisotopes for cancer therapy or tumor imaging.

4.1.3 Antibodies against intracellular targets Inspired by the broad success in extracellular applications, antibodies have also been investigated for use in intracellular applications. One early example was the microinjection of full-size antibodies for neutralization of SV40 tumor antigen (158). However, antibodies are not suitable for intracellular applications since they are large and in many cases do not fold correctly. This is largely due to the lack of intra- and intermolecular disulfide bridges, important for correct folding and stability, which are not able to sustain the reducing environments (35, 159).

4.1.4 Antibody fragments against extracellular targets A large number of antibody fragments are currently in clinical trials for tumor imaging and therapy. Typically, by fusing different molecules to the antibody fragment, different mode of actions can be achieved in addition to antagonistic binding. Especially scFvs have been explored for targeted delivery of toxic moieties, for example the fibronectin-specific scFv L19 used for delivery of IL-2 (160) or IFNγ (161) for inhibition of new blood vessel formation (anti-angiogenesis). Bispecific antibody fragments, including bispecific scFv fragments targeting related cell surface receptors such as members of the ErbB family (162) or fragments directing the immune system towards HER2 expressing cells (163), is a rapidly growing field in cancer therapy. In addition, several Fab fragments are today approved for therapeutic or diagnostic use (153). 24 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

4.1.5 Antibody fragments against intracellular targets Antibody fragments have also been investigated for intracellular applications. The first expression of an intracellular antibody fragment – or intrabody as they are sometimes referred to (164) – was the use of a yeast alcohol dehydrogenase I-specific antibody fragment in Saccharomyces cerevisiae (165). Some authors mark the difference between antibody fragments in cytosolic or nuclear milieu and those residing in the extracytoplasmic space (such as the endoplasmic reticulum) (166). Although antibody fragments are promising tools for intracellular applications, some limitations still exist including short half-lives (167) and the inability to form intramolecular disulfide bridges. These problems may potentially be circumvented by the use of alternative intracellular selection strategies, such as intracellular antibody capture (IAC) (93, 129) or protein fragment complementation assay (PCA) (168) to select for affinity proteins with correct function in reducing environments. The stability issue can also be addressed by Fc fusions (169), possibly influenced by the formation of intrabody dimers (170).

A special field in intracellular antibody based research is the targeting of subcellular compartments by fusing the intrabody to a localization signal (Chapter 1.3) and a number of scFvs towards different proteins has now been expressed in the ER of mammalian cells. Important examples with relevance to the work presented in this thesis are anti-HER2 scFvs, as expression of these has shown to inhibit soft agar cell growth (171) as well as in vivo tumor cell growth (172). A few examples of intrabodies are found in Table 3.

Target Localization Delivery References protein signal HER2 KDEL Viral vector (171) Integrin α4 KDEL Proprietary transfection reagent (173) (Lipofectamine), electroporation P53 NLS Calcium phosphate transfection (174) CCR5 KDEL Viral vector (175) EGFR None (cytosolic) Proprietary transfection reagent (176) (FuGene6) VEGFR-2 KDEL Proprietary transfection reagent (177) (Lipofectamine), viral vector Tau (τ) None (cytosolic) Proprietary transfection reagent (141) (Lipofectamine) Table 3. Examples of intracellular scFv fragments.

4.1.6 Affibody molecules against extracellular targets As described elsewhere (117, 178), affibody molecules have several interesting features for use in diagnostics and their potential use in tumor imaging is now well established (179, 180). For extracellular protein therapy, some limitations of native affibody molecules ERIK VERNET 25

are short in vivo half-life and lack of recruitment of immune system effectors. These issues are potentially solved by the use of albumin-binding domains (181) or antibody-domains (182), respectively, as fusion partners. Since the affibody scaffold is naturally devoid of cysteine residues, it is possible to introduce a terminal cysteine for specific labeling. In addition, since affibody molecules can be synthesized by chemical synthesis, it is possible to introduce non-natural amino acids that can alter the characteristics, for example by creating cross-linked proteins (183). A number of affibody molecules with significance in cancer research have been selected and characterized (Table 4). In this thesis, novel affibody molecules binding to the extracellular domain of IGF-1R (Chapter 2.3.3, Paper III) are selected and characterized.

Target protein Binder affinity References CD25 130 nM (184) EGFR 5 nM (185) HER2 22 pM (186) Table 4. Examples of affibody molecules with affinity for cancer related proteins. Binder affinity was determined by Surface Plasmon Resonance (SPR).

4.1.7 Affibody molecules against intracellular targets As noted earlier, for an intracellular protein binder to be effective it must not only show high specificity and selectivity for its target but also have a high stability. Being small, stable and devoid of disulfide bridges, the affibody scaffold has all the necessary theoretical prerequisites for use in intracellular applications. The first truly intracellular target for an affibody molecule was the transcription factor c-jun (187), and several new binders towards the cancer related proteins Ras and Raf (Chapter 2.3.2) are developed in Paper IV. In addition, in Paper I and Paper II, two affibody molecules, preciously selected to bind to the extracellular domains of HER2 and EGFR (Chapter 2.3.1), respectively, are utilized for ER retention.

4.2 Small molecule interference Small molecules, often organic, can be natural or engineered ligands for many proteins. Typically, these molecules function by activating signaling (agonists) or by blocking the target’s interactions with other cellular components (antagonists). Owing to their small size and hydrophobicity, small organic molecules can be distributed orally and even traverse the plasma membrane for targeting of intracellular proteins. Amongst the most investigated small molecule inhibitors are enzyme inhibitors. Small molecules have proven to be particularly useful for these applications, as enzyme active sites are small, often contained within a deep cleft devoid of water molecules that may hinder substrate (or inhibitor) docking (188). One example with relevance to cancer is the tyrosine kinase inhibitors (TKIs), inhibiting phosphorylation of tyrosine residues in signaling pathways 26 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

(139), including those described in Chapter 2. Interestingly, inhibition of tyrosine kinase activity can often be tolerated in normal cells (189), indicating that a low level of kinase activity is sufficient for patient survival. The success of these molecules as pharmaceuticals has led to intensive drug development research of small molecule inhibitors of protein-protein interactions (PPIs) as well. In contrast to protein based inhibitors of PPIs, whose large binding surfaces (typically around 2000 Å2 (190)) have a high probability of inhibiting the sought protein-protein interactions (188), protein-small molecule interfaces are generally small (around 500 Å2 (191)) and spatially separated from the protein-protein interaction meaning that there are rarely good starting ligands for further engineering of ligands intended to interfere with PPIs. However, even though PPIs are large, there are often specific important “hot-spots” in the target protein that account for a majority of the Gibbs free energy of binding. Small molecule inhibitors of protein- protein interactions with KD values similar to the native protein ligands are now available against numerous cancer related targets, including the p53-HDM2, GRB2-SH2 and Ras- Raf interactions (188, 191).

4.3 RNA interference RNA interference (RNAi) refers to post-transcriptional, sequence-specific downregulation of gene expression by small (around 25 nucleotides) RNAs. It has been extensively investigated in nematodes, where it was discovered that sense RNA could regulate gene expression as effectively as antisense RNA (192) and that long double stranded RNAs were even more potent inhibitors of gene expression (193). This work led to a new model of how RNA levels are regulated and was awarded with the Nobel Prize in Physiology or Medicine in 2006. RNAi is suggested to play a role on the pre-transcriptional level, inducing DNA methylation (194) and chromatin remodeling (195), but the most studied effects are post-transcriptional where the interfering RNA can inhibit protein translation by catalytic degradation of mRNA (196) or by blocking the translation initiation (197). These post-transcriptional actions are performed by miRNA a complex called RISC (RNA induced silencing complex).

This endogenous system can be harnessed using small interfering RNAs (siRNAs), 21-23 bases long RNA molecules (198) with near-perfect base pairing with its target sequence. SiRNAs can be translocated into cells, either directly as siRNA or in a long dsRNA format that can be processed by the protein Dicer before RISC loading. This leads to a transient effect, but siRNAs can also be expressed in the cell, commonly by using a RNA polymerase III-dependent short hairpin RNA (shRNA) expression form (199, 200). This allows for long term expression of siRNAs and is a promising technology with an increasing number of applications, including HIV therapy (201).

ERIK VERNET 27

RNAi has been proven to be a highly efficient mechanism for control of gene expression. However, there are a number of limitations to consider. One is saturation of the endogenous RNAi machinery, as high expression of shRNAs has proven to be lethal (202). This problem may be circumvented by the simultaneous use of different promoters utilizing different nuclear export pathways (203). There is also a possibility that systemic delivery of RNAs may induce an immune response involving Toll-like receptors and interferons. This risk is clearly correlated to RNA length as dsRNA longer than 27 bp are generally highly immunogenic while for shorter molecules, the response seems to be sequence-dependent (204). It is also not clear how successfully shRNA expressing cells will proliferate in vivo, and the expression in vitro is reported to decrease over time, possibly linked to non-acute cytotoxicity (205). One additional advantage of protein based interference over RNAi is the ability to inhibit only a specific function of a protein instead of down regulating the protein expression altogether (206).

28 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

ERIK VERNET 29

5 Delivery systems for genes and proteins

As described in the previous chapter, both nature and man have developed several distinct systems for inhibition of protein function. However, no molecule is better than the system used to deliver it to the relevant localization, and there is accordingly a dire need for efficient delivery systems. Traditionally, small molecules (Chapter 4.2) have been used for in vivo applications of protein defunctionalization, and many of these molecules can be distributed in the human body after oral delivery with uptake through the gastrointestinal wall, which is convenient for the patients. Proteins and other biological macromolecules, on the other hand, cannot normally be distributed orally since they are degraded in the gut. Therefore alternative methods, such as subcutaneous or intravenous injections, must be used. Molecules that cannot be directly replicated (such as proteins and small molecules) are degraded over time and thus give a transient effect which is desirable for certain applications.

An alternative to the direct delivery of the functional protein or RNA is gene delivery. The fact that genes, nowadays defined as genomic sequences directly encoding functional product molecules (207), can be deleted, modified or even interchanged between species opens up the possibility for recombinant or gene modified organisms. The term “gene therapy” usually refers to the introduction of a supplementing functional gene (and possibly the genetic replacement of a defective mutated allele); however, the introduction of novel genes is also a possibility. Regardless of the aim, genes can be introduced using a number of different techniques, some of which are described here. Both in vivo and ex vivo strategies are possible. In vivo therapy is generally considered to be more cost-efficient, especially if surgery is required to retrieve the cells of interest (which may be altogether impossible in some cases) for ex vivo therapy. In vivo therapy is however limited by the amount of treatable cells, their accessibility and there is also a risk of evoking an immune response against the delivery vector, especially if repeated treatments are necessary. In ex vivo therapy the target cells are collected from the patient and can be treated with the gene therapy vector, cultivated and sorted before reintroduction. However it is desirable to reduce the ex vivo time as much as possible due to the risk of cell differentiation.

A great deal of optimism about gene therapy was noted around year 2000, especially concerning viral delivery as a number of patients with severe immunodeficiencies were apparently cured (208, 209). However, several research programs were thereafter discontinued after a number of cases of vector-induced leukemia in patients (See Chapter 5.1.3). Today gene therapy for primary immunodeficiencies is showing promising results, although vector-related side effects remains a major concern (210). Research in the area is intense (Table 5): At the end of 2008, there were over 1400 gene therapy clinical trials in progress (211). 30 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

Regardless of the macromolecule to be delivered, there are several steps that have to be accomplished for successful intracellular therapy, including cell surface interactions, internalization, and transport to the subcellular compartment of interest. For these reasons, delivery systems (also known as vectors) are used.

Therapeutic entity Gene therapy vector Stage of development IgG1 Fc and TNF-α AAV Phase 2 in rheumatoid arthritis receptor Herpes simplex Retrovirus (ex vivo) Phase 3 in graft-versus-host disease thymidine kinase TNF-α Adenovirus (inducible Phase 3 in pancreatic cancer promoter) HLA-B7 and β2 micro DNA plasmid with non- Phase 3 in chemotherapy-naïve globulin complex viral vector patients with metastatic melanoma Table 5. A selection of recent gene therapy clinical trials. Modified from (212).

5.1 Viral vectors The ability of viruses to infect cells has been known for a long time. Viruses are small (nanometer range) entities containing single- or double stranded genetic material (DNA or RNA) surrounded by a protein coat called a capsid and, sometimes, an envelope. The envelope can contain host cell components (lipids and proteins) as well as viral proteins and is used to increase the efficiency of infection by fusing with the target cell membrane. Some viruses, such as retroviruses (Chapter 5.1.3), have two distinct life cycles. They can either go through the lysogenic pathway, where integration of the virus genome into the host genome typically leads to low production of viral proteins and maintained cell integrity. The alternative is the lytic pathway, where massive production of viral proteins leads to cell lysis and release of virions. Viruses are normally not considered “living” since they do not have a metabolism of their own and most of them are not able to replicate by themselves. Accordingly they are not classified in the same way as living organisms, but into seven groups (denoted I-VII) according to how their genetic material is stored. It should however be noted that viruses sometimes are regarded as living and have thus been given a domain (Acytota; meaning “without cell”) and full taxonomy. The term virus-like particle (VLP) is sometimes used to clarify the use of a non-complete virus. As of 2007, 70 % of gene therapy clinical trials use viral vectors (213). The viral vector is usually assembled in a cell line. The budded viral particles are then collected and can be concentrated and purified if needed before being used to infect the cells of choice.

5.1.1 Adenovirus-based vectors Adenoviruses are non-enveloped, double-stranded DNA viruses. Being a non-integrating virus, adenovirus-based gene therapy leads to a transient transfection of the target cells ERIK VERNET 31

which might be an advantage or a disadvantage depending on the application. Another potential problem with the vectors is preexisting immunity towards the most common adenovirus serotypes, as most people have had a number of infections during childhood. One advantage with these vectors is the broad tropism, including many dividing and non- dividing cell types. Other advantages are that adenovirus-based vectors can be produced to very high titers and high purity, which is important if they are to be used in the clinic. Adenovirus-based vectors have also been engineered to restrict its tropism. For example an anti-HER2 affibody molecule was inserted into the virus surface protein to restrict infection only to cells expressing HER2 (214). Adenovirus-based vectors have also been used in combination with tissue-specific promoters, to specifically lyse a certain cell type (215). In many instances the vectors are made non-replicating by excluding several viral genes from the DNA that gets packed into the particles. After infection, the adenoviral vector can thus express the transgenes but not express all necessary self-genes or produce progeny virions.

5.1.2 Adeno-associated virus-based vectors Adeno-associated viruses (AAVs) are non-enveloped single-stranded DNA viruses originally identified as a contaminant of adenovirus preparations (216). As the name suggests, these viruses are dependent on a helper virus (e.g. adenovirus, herpes simplex or vaccinia virus) infection for replication. The AAVs are non-pathogenic in humans and infection normally leads to a weak immune response. There are a number of different AAV serotypes, with different surface proteins and tissue specificity. In the absence of the helper virus, the AAV enters the lysogenic pathway and the genome integrates at a specific locus (denoted AAVS1) on chromosome 19 (217). If a cell is infected by the helper virus, the AAV can enter the lytic lifecycle (218) leading to formation and release of infectious virions.

The low immunogenicity, non-pathogenicity, tissue targeting ability and specific integration mechanism make AAV-based gene delivery vectors very interesting alternatives for use in gene therapy. However, recent clinical trials have found stronger immune responses than expected, possibly from previous AAV infections (219). One study also detected vector sequences in germ line cells although this was a transient effect (220). Additional drawbacks of the system include difficulties of scaling up the virus production together with complicated and expensive purification protocols.

5.1.3 Retrovirus-based vectors Retroviruses store their genetic information in the form of RNA and use reverse transcriptase and integrase enzymes to convert this to DNA and permanently integrate into the host cell’s genome. This is done by recombination, introducing the viral genome 32 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

flanked by the 5’ and 3’ long terminal repeats (LTRs). In fact, a substantial part of the human genome (up to 1 %) is considered to be non-functional remains of previous retroviral infections (221). After integration, the provirus can use the cell’s machinery (including ribosomes for protein production and RNA polymerase II for RNA synthesis) to produce proteins and RNA molecules for progeny viral particles. The 5’LTR can act as a RNA polymerase II promoter, and the 3’LTR terminate transcription and promote polyadenylation of the transcripts. Only RNA sequences containing the specific Ψ packaging signal will be included in the produced virions. In contrast to other common viral infections, which are often lethal to the cell, retroviral infections are chronic and the viral particles are instead budded from the cell, retaining some of the cell’s membrane in its envelope. Up to date, four human retroviruses are known to cause disease: Human T- lymphotropic virus (HTLV) I and II and Human immunodeficiency virus (HIV) I and II (although the two latter are sub classified as lentivirus, Chapter 5.1.4).

Today, retroviral vectors (RVs) based on the murine leukemia virus (MLV or MuLV) are commonly used. These systems are based on a simple enveloped gammaretrovirus with single stranded RNA containing the gag, pol and env genes encoding the viral capsid proteins, viral enzymes and envelope proteins, respectively (222) (Figure 5). The viruses are frequently pseudotyped with Vesicular Stomatitis Virus G glycoprotein (VSV-G) as envelope protein since it allows high titers and is promiscuous in mammalian cell infections (223). However promiscuity is a drawback if the target cells are found among other cell types not wanted for infection, which is often the case in systematic distribution. Targeting of pseudotyped retroviruses to specific cell types is therefore desirable and has been investigated using Fc-binding proteins such as the Z domain (Chapter 3.5) (224, 225).

Figure 5. Schematic drawing of the genomic components of (A) a retrovirus and (B) a replication-deficient retroviral delivery system. P: Promoter; Ψ: packaging signal. Modified from (226).

For most applications a replication-deficient retrovirus (RDR), capable of only one round of infection, is desirable. In fact, a major concern using viral delivery is the generation of replication-competent retroviruses (RCR). By inserting the gene of interest and omitting the Ψ packaging signal from all sequences except the transgene to be delivered, a RDR ERIK VERNET 33

delivery system can be constructed. To further improve the safely of the system, the genes are divided between several (typically three or four) different plasmids before the virus particles are assembled in a packaging cell line. The drawback of this is decreased efficiency and increased inconvenience. Other current concerns and limitations include decreasing transgene expression over time and the possibility of insertional mutagenesis as the vector is randomly inserted into the host genome (227). Gene therapy trials for X- linked severe combined immunodeficiency (SCID-X1) treatment around year 2000 tragically visualized these issues as retroviral genome insertions in proximity of the LIM domain only 2 (LMO2) gene led to the development of leukemia (228).

5.1.4 Lentivirus-based vectors Lentiviruses constitute a subclass of the retroviral family and have been increasingly used in the last few years to overcome the limitations of ordinary retroviral delivery systems, such as the incapability to integrate in non-dividing cells. The most well known lentivirus is doubtless the human immunodeficiency viruses HIV I and II, and the lentiviral delivery systems are based on these. Although the basic genome organization and lifecycle is the same as for the retroviruses described in Chapter 5.1.3 (sometimes referred to as oncoretrovirus or gammaretrovirus), the lentivirus have additional genes and a more complex mechanism of replication. For example, owing to the expression of the rev protein the lentiviral RNA genome is able to travel to the nucleus of both non-dividing and terminally differentiated cells. These viruses are therefore more useful for gene therapy treatment of primary cells and have been shown to give transgenic expression in vivo (229). To improve safety, a minimum of viral genes are included: for example, the nef gene is frequently deleted and the vpu gene is defective due to an internal deletion (230).

5.2 Non-viral vectors Non-viral vectors are explored as an alternative to viral vectors in order to overcome limitations such as including limited insert lengths, the risk of insertional mutagenesis and large scale production and purification issues. Non-viral delivery systems are based on physical properties of a molecule co-distributed with the drug, either covalently bound (for example as a fusion protein) or in a mixture with the delivery molecules. The complexes are in many cases coated with polymers such as polyethylene glycol (PEGylation), aiming at reducing the immunogenicity and increasing the in vivo half-life. The efficiencies of the non-viral systems are ever increasing, but are in most cases still considerably lower than for viral vectors, possibly linked to toxicity (231) or transcription (232) issues. A few different groups of molecules with distinct molecular mechanisms are described here. 34 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

5.2.1 Naked DNA The simplest vector is, obviously, no vector. Indeed, some tissue types have shown to take up injected naked DNA with efficiencies comparable to those achieved with transfection reagents in cell cultures (233). The exact uptake mechanism is not understood, it is possibly different from the endocytic routes outlined in Chapter 1.2 (234). Using improvements such as a gene gun or electroporation, high expression levels can be achieved (235) although these methods are limited to superficial tissues such as the skin.

5.2.2 Cell-penetrating peptides Cell-penetrating peptides (CPPs) are peptides that increase the translocation efficiencies of macromolecules over the plasma membrane. CPPs are either natural (protein-derived) such as the commonly used TAT peptide (from HIV-1 tat protein) (236) or penetratin (from a transcription factor in D. melanogaster) (237), or rationally designed such as the polyarginines (238). CPPs received a great deal of attention some ten years ago, as early reports showed that they could mediate intracellular delivery of large cargo proteins to cells in vivo, even crossing the blood-brain barrier (239). Initially uptake was seemingly energy independent with fast translocation to the nucleus, but experimental artifacts were later shown to account for these phenomena (240, 241) and today endocytosis is the most commonly proposed uptake mechanism. Some success has been noted with tumor- targeting (242) CPPs, although other studies report on the non-specific uptake in vivo (243). Taken together, in spite of the early enthusiasm, CPPs have largely failed to show usability for in vivo applications, and uptake efficiencies are typically around 0.1 % (244) with endosomal release being a major hurdle.

5.2.3 Fusogenic proteins As noted earlier, endosomal release is a major hurdle for the introduction of genes and proteins in cells. Many endocytosis dependent viruses therefore express so called fusogenic proteins that facilitate this process. These proteins can be used to pseudotype other viruses (245) or to construct non-viral vector, for example by creating a fusion protein with a CPP (246). The most commonly used protein is HA2, derived from haemagglutinin naturally expressed on influenza virus particles. At low pH, HA2 folds into an alpha helix secondary structure with destabilizing effect on cellular membranes (247, 248), significantly increasing the translocation efficiency of macromolecules. Other examples of peptides with similar effects are the E1 proteins from alpha viruses such as the Semliki Forest Virus (SFV) (249).

ERIK VERNET 35

Present Investigation

The work in this thesis is focused on the inhibition of cancer related proteins, by developing and evaluating affinity ligands that interact with these proteins. This is a field with potential for the treatment of cancer, as the affinity proteins may inhibit the growth related signaling pathways that these receptors participate in, for example those presented in Chapter 2. One alternative to achieve defunctionalization is by relocalizing the target protein to an environment where it cannot perform its biological function. In Paper I and Paper II, affibody molecules targeting the cell surface receptors EGFR and HER2 (Chapter 2.3.1) are utilized for receptor capturing and retention in the secretory compartments. In both cases, expression of specific affibody molecules equipped with ER retention signals led to significant downregulation of the cell surface expression of the endogenous receptors. The downregulation also correlated with a decrease in cell proliferation rate, underscoring the importance of these receptors for malignant cell growth.

An alternative strategy for protein interference is to use of affinity proteins that interfere with the binding of an endogenous activating (agonistic) ligand to a protein in a signaling pathway. One example of this is presented in Paper III, where novel affibody molecules interacting with IGF-1R (Chapter 2.3.3) are developed to compete with IGF-1 binding to the receptor. One of these had stronger affinity for IGF-1R than IGF-1 itself, and this affibody was shown to have significant impact of cell growth. In addition to interference with receptor-ligand interactions, affinity proteins have also been developed for interference with intracellular protein-protein interactions. In Paper IV, an affibody molecule interacting with the cancer related kinase Raf (2.3.2) was developed. This affibody molecule also interfered with Ras-Raf interaction in vitro. In this paper, several affibody molecules targeting Ras were also generated and characterized.

36 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

ERIK VERNET 37

6 Affibody molecule mediated ER retention (Paper I, Paper II)

Cell surface receptors are transported through the secretory compartments on their route towards the plasma membrane where they can become activated by several different mechanisms. One strategy to hinder their signaling is to block their transport towards the plasma membrane. To investigate such a strategy using a novel class of affinity proteins, affibody molecules specific for their respective target were expressed as fusion proteins with an N-terminal export signal and a C-terminal KDEL tag interacting with ERD for ER retention (Chapter 1.3.1). This construct targeted the affibody molecule into the ER but retained it in the ER/cis-Golgi compartments. A schematic drawing of the principle is shown in Figure 6. As depicted here, successful ER retention relies on the interactions between three proteins: Binding of a KDEL-tagged affibody molecule to the normally membrane bound target receptor and simultaneous binding of this complex to the KDEL receptor.

Figure 6. Schematic drawing of the ER retention principle using an affibody molecule to retain a protein destined for the plasma membrane.

6.1 Affibody molecule mediated ER retention of HER2 (Paper I) HER2 is a cell surface bound RTK, constitutively in “open” conformation and is therefore a strong activator of MAPK signaling (Chapter 2.3.1). It is over expressed in 25-30 % of breast and ovarian cancers and over expression is correlated with a higher rate of relapse 38 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

and a poorer prognosis of affected patients (81, 250). To inhibit its signaling, the HER2 binding affibody molecule ZHER2:00477, with 32 pM affinity for HER2 (186), equipped with an export signal peptide and a KDEL tag (ZHER2:00477-KDEL) was expressed in SKOV3 cells. This cell line is a widely used model for ovarian cancer with a very high surface expression level of HER2. The affibody encoding gene was delivered using a MLV retrovirus-based vector (Chapter 5.1.3). A number of control constructs were also expressed, including ZTaq-KDEL, containing the signal peptide and the ER retention signal but with an affibody molecule binding to the irrelevant target DNA polymerase from

Thermus aquaticus (251) and ZHER2:00477, containing the signal peptide but lacking the ER retention signal. Expression and subcellular localization of the KDEL-tagged constructs was verified using immunoblotting, showing expression of full-length fusion proteins with no detectable degradation. Cells were also stained for affibody expression and endogenous HER2 and examined by confocal fluorescence microscopy (Figure 7).

Figure 7. Localization of expressed affibody molecules and HER2 in SKOV3 cells. A) Cell expressing ZHER2:00477-KDEL; B) Cell expressing ZTaq-KDEL. Left panels: HER2 (green); middle panels: affibody molecule (red); right panels: merge.

When the ZTaq-KDEL affibody fusion protein was expressed a strong intracellular staining can be seen for the affibody molecule along with a clearly separated staining for HER2 in the cellular membrane (Figure 7B). By contrast, when the HER2 binding ZHER2:00477-KDEL affibody fusion protein was expressed, the strong staining for HER2 receptors is shifted from the cellular membrane to the interior of the cell, resulting in colocalization with the affibody molecule, suggesting a specific capability of the HER2 binding ZHER2:00477-KDEL affibody fusion protein to act in an inhibitory manner on the export of HER2 to the plasma membrane (Figure 7A). Using flow cytometry for the quantification of surface density of

HER2, it was shown that expression of ZHER2:00477-KDEL reduced the surface expression of HER2 by 60 %, as measured five days after gene delivery, while expression of any of the control constructs did not affect the surface expression of HER2 significantly (Figure 8). ERIK VERNET 39

In addition, this decrease in HER2 levels correlated with a significantly increased population doubling time, compared to SKOV3 cells not expressing affibody proteins or cells expressing the control construct ZTaq-KDEL.

Earlier studies on ER retention of HER2 using scFvs (171) have shown upon a greater relative down regulation of HER2 than was seen in the present study, and we therefore wanted to determine the limiting factor in our setup. The affibody ZHER2:00477-KDEL was expressed in three additional cell lines having different basal levels of HER2 expression. Interestingly, about the same level of downregulation of HER2 (~60 %) was observed in these cell lines (LS174T, RT4 and SKBR3) as in SKOV3. This indicates that the expression levels of the affibody construct is high enough for efficient retention, as the down regulation in HER2 rich cell lines were much greater in absolute numbers. It may rather be a question of too high expression levels (blocking the crucial simultaneous affibody-ERD and affibody-HER2 interactions), insufficient affinity between the components (affibody molecule interacting with ERD and HER2) or residual HER2 on the plasma membrane, secreted prior to affibody expression.

Figure 8. Quantification of the surface HER2 expression level in SKOV3 cells. The means for the populations expressing the different constructs were calculated and compared to cells expressing ZTaq-KDEL with HER2 level arbitrary set to 1.

In summary, this project shows that an affibody molecule selected by phage display to bind to the extracellular domain of HER2 can be expressed in a human cell and recognize native HER2 in the secretory compartment. To our knowledge, this is the first example of a non-immunoglobulin based affinity protein used for capturing a cell surface receptor in the secretory compartments. 40 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

6.2 Affibody molecule mediated ER retention of EGFR (Paper II) Paper I provided proof-of-principle for the use of affibody molecules in ER retention applications. However, as discussed, the success of this principle depends on a number of factors, including the affinity of the affibody molecule for the target protein and the accessibility of the binding epitope during the exocytic process. To investigate the possibility to use this strategy for other receptors, a head-to-tail dimer of the affinity- maturated EGFR binding affibody ZEGFR:01907 (185) fused to an export signal peptide and an ER retention tag ((ZEGFR:01907)2-KDEL) was expressed in A431 cells, a cell line with high EGFR expression. A MLV retrovirus-based expression system was used to deliver the genes encoding the affibody constructs. EGFR is an RTK over expressed and/or mutated in several different cancers but in contrast to HER2, EGFR has soluble ligands (e.g. EGF) and EGF binding leads to conformational changes that expose a dimerization arm in one of the extracellular domain, enabling receptor dimerization and autophosphorylation of the C-terminal kinase domain (Chapter 2.3.1). Similar to the work in Paper I, a number of control constructs were also expressed, including ZTaq-KDEL, containing the signal peptide and the ER retention signal but with an affibody molecule binding to the irrelevant target DNA polymerase from Thermus aquaticus (251) and

(ZEGFR:01907)2, containing the signal peptide but lacking the ER retention signal. Expression of the KDEL-tagged constructs was verified using immunoblotting, showing expression of full-length fusion proteins with no detectable degradation. The subcellular localization of the affibody fusion proteins were visualized by confocal fluorescence imaging and showed a strong intracellular colocalization of (ZEGFR:01907)2-KDEL and EGFR, while the staining for the control affibody molecule ZTaq-KDEL and EGFR was non-overlapping (Figure 9).

Figure 9. Localization of expressed affibody molecules and EGFR in A431 cells. A) Cells expressing (ZEGFR:1907)2-KDEL. B) Cells expressing ZTaq-KDEL. Top left panels: DAPI nucleic acid staining (blue); top right panels: affibody molecule (red); bottom left panels: EGFR (green); bottom right panels: merge. ERIK VERNET 41

Colocalization of (ZEGFR:01907)2-KDEL and EGFR indicated a specific interaction which was verified by a co-capture experiment where EGFR from a cell lysate of affibody- expressing A431 cells was captured by antibody bound beads. Bound proteins were separated by SDS-PAGE and transferred to a blotting membrane where co-captured affibody molecules were visualized. In the same experiment, no interaction was detected between EGFR and the control affibody ZTaq-KDEL. Flow cytometry analysis showed a strong (~80 %) down regulation of surface located EGFR on A431 cells expressing

(ZEGFR:1907)2-KDEL relative to cells expressing ZTaq-KDEL (Figure 10A). The surface density of HER2 was also investigated and shown to be unaffected by expression of (ZEGFR:1907)2-KDEL, verifying that EGFR capture is specific without cross-reactivity towards the homologous HER2 protein (Figure 10B), consistent with results from the affibody selection where cross-reactivity towards the extracellular domain of HER2 was analyzed in vitro (185). These results also indicate that no significant interactions exist between EGFR and HER2 in the secretory compartments, supporting the current view that receptor dimerization occurs after binding of soluble EGFR ligand, which occurs at the cell surface (78).

Figure 10. Quantification of the surface levels of EGFR and HER2 in affibody expressing A431 cells. The means for the populations expressing the different constructs were calculated and compared to cells expressing ZTaq-KDEL with EGFR/HER2 levels arbitrary set to 1. A) Relative surface level of EGFR. B) Relative surface level of HER2.

In accordance with the results from Paper I, the growth rate was significantly lower for cells expressing the specific receptor binding affibody molecules compared to cells expressing the control affibody molecules or un-infected A431 cells. As EGFR signaling is initiated by ligand binding and subsequent phosphorylation of the intracellular kinase domain, EGFR capture in the secretory compartments might inhibit EGF-mediated activation of EGFR signaling. Cells were therefore stimulated with EGF and EGFR phosphorylation was detected by immunoblotting, showing that capture of the receptor in 42 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

the secretory compartments could be correlated with a significantly lower degree of phosphorylation of tyrosine 992 in the kinase domain of EGFR (Figure 11). Phosphorylated Tyr992 is a known binding site for one of the SH2-domains of PLCγ, which leads to the activation of the PLC-PKC and ERK-MAPK pathways (Chapter 2.2). These results further show the feasibility of intracellular affibody expression to retain cell surface proteins in the secretory compartments. Furthermore, the inhibition of phosphorylation of the intracellular tyrosine kinase domain of EGFR proveides a molecular explanation for the growth inhibitory effect of affibody mediated ER retention of EGFR.

Figure 11. Phosphorylation of EGFR and rate of proliferation in affibody expressing cells.

A) Lysates from EGF-stimulated cells expressing (ZEGFR:1907)2-KDEL or ZTaq-KDEL were separated by SDS-PAGE and immunoblotted using an antibody recognizing EGFR phosphorylated at Tyr992. B) Determination of the proliferation of affibody expressing cells by an MTT-assay. The proliferation rate of cells expressing ZTaq-KDEL was arbitrarily set to 1. Error bars represent one standard deviation.

7 Novel affibody molecules for cancer related targets (Paper III, Paper IV)

Affinity proteins based on alternative scaffolds, including affibody molecules (Chapter 3.3), have proven useful for various in vitro and in vivo applications. In an effort to expand the arsenal of affibody molecules with affinity for cancer related proteins, we performed selections from a large combinatorial library using phage display technology towards three cancer related proteins. ERIK VERNET 43

7.1 Selection of IGF-1R specific affibody molecules (Paper III) IGF-1R is a RTK over expressed in various cancers, including breast, ovarian, pancreatic and prostatic (252) and as signaling through IGF-1R is associated with cell proliferation and survival (Chapter 2.3.3), it is therefore of great interest to develop affinity ligands that can inhibit its function (109). In addition, information on IGF-1R status can have prognostic value, for example for breast cancer patients (253) and robust affinity reagents that can be used for qualitative and quantitative analysis of receptor status is also of great importance. IGF-1R undergoes conformational changes upon IGF-1 or IGF-2 binding, leading to phosphorylations in the intracellular kinase domains that in turn lead to the activation of the PI3K-Akt and MAPK cascades. In order to select for antagonistic affinity proteins it is preferable to be able to enrich for variants binding to an epitope that is crucial for target protein function, and in this selection we therefore intended to enrich for affibody molecules interacting with an epitope over-lapping the IGF-1 site of interaction on IGF-1R by eluting bound phages from the target protein using IGF-1.

Phage display based selections against a hexahistidine tagged extracellular domain of IGF- 1R was performed by bio-panning in four rounds from a previously constructed library. During the first two rounds of selection, receptor/phagemid complexes were isolated by a Co2+/NTA functionalized solid support specifically interacting with the hexahistidine tag present in the receptor. To minimize co-isolation of phagemid particles that bound unspecifically to the Co2+/NTA functionalized solid support, a streptavidin coated solid support was instead used in cycles three and four, in which cases the target receptor used was in vitro biotinylated to allow for capture. An increase in the number of eluted phagemids was observed as the rounds progressed, indicating enrichment for phagemids specifically interacting with the receptor and/or unspecifically with other components in the system. After the fourth round of selection, 55 clones were isolated and affibody genes were expressed. Clones were investigated for binding to IGF-1R in an ELISA format and the DNA sequences of interacting affibody molecules were determined, resulting in three different sequences (Z4:26, Z4:40 and Z4:52).

In addition, three control selections were performed using varying concentrations of target protein and detergent. Also during these selections, a Co2+/NTA functionalized solid support interacting with the hexahistidine tag in the receptor (cycle one and two) or a streptavidin coated solid support (cycle three and four) was used to capture the receptor/phagemid complexes. Elution from the solid support during cycles one and two was carried out by addition of imidazole to break the interaction between the receptor and the solid support. During cycles three and four, elution was instead performed by lowering the pH to break the interaction between the phagemid and the receptor. Also during these 44 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

selections, an increase in the number of eluted phagemids was observed as the rounds progressed. After the fourth round of selection 42 randomly picked colonies from each of the control experiments (total of 126) were cultivated and their harbored affibody gene was expressed and tested for binding to the receptor in an ELISA experiment. Positive clones were detected in all three control experiments (total of 54) and their DNA sequences were determined. The similarity of all sequences isolated during selection with IGF-1 competition during elution and the control selections was compared (Figure 12). The two most abundant variants from the selection with IGF-1 competition during the elution (Z4:26 and Z4:40) cluster with each other but show no similarities to any of the other sequences. The third variant from the selection with elution by competition with IGF-1

(Z4:52) show more similarity to the variants isolated in the control selections rather than to

Z4:40 or Z4:26.

Figure 12. Clustering of sequences obtained from four different selections.

In order to evaluate the ability of the selected variants to bind to the native full-length receptor, a total of eleven affibody variants were sub-cloned, produced recombinantly and used to stain MCF-7 cells, a cell line expressing about 20 000 copies of IGF-1R per cell (254). Some of the positive binders are shown in Figure 13, together with the positive control IGF-1.

ERIK VERNET 45

Figure 13. MCF-7 cells were stained with biotinylated affibody molecules or IGF-1 followed by neutravidin-Alexa 488 and analyzed in a flow cytometer. Thin lines correspond to non- stained cells, thick lines corresponds to stained cells.

The Z4:40 affibody was chosen for further characterization as it showed strong staining that could be blocked by adding an excess of non-biotinylated affibody, demonstrating its specificity. For this variant, the KD for IGF-1R was determined to be 1.2±0.32 nM, which is 2-fold stronger than the IGF-1/IGF-1R interaction. Z4:40 also proved to compete with IGF-1 for binding to IGF-1R, mapping the epitope of this affibody molecule to the binding site of IGF-1 as expected. The affibody Z4:40 was also used to visualize MCF-7 cells by confocal fluorescence imaging, giving results comparable to those obtained using a commercial antibody (Figure 14).

Figure 14. Immunofluorescence images of MCF-7 cells stained for IGF-1R using A) affibody molecule Z4:40 or B) a commercial antibody.

This affibody variant was also used for pull-out of IGF-1R from a MCF-7 cell lysate. In addition, when Z4:40 was added to the growth medium of MCF-7 cells a significant and dose-dependent decrease in proliferation rate was observed compared to cells treated with the control affibody Zwt, shown to interact with MCF-7 cells. These results demonstrate the usability of Z4:40 for tissue staining and in vitro applications, and motivate the further investigation of in vivo therapeutic applications. Since IGF-1R is a strong activator of ErbB downstream signaling (through the PI3K-Akt pathway), combinations of anti-ErbB and anti-IGF-1R treatment has been suggested (255). One interesting strategy would be to take advantage of the ER retention system investigated in Paper I and Paper II using one of the affibody molecules investigated in Paper III, possibly in a heterodimer fusion protein combination with affibody molecules interacting with EGFR or HER2.

46 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

The high-affinity affibody molecules selected here provide examples of how affibody molecules binding to a certain epitope can be selected using competitive elution strategies.

Mapping experiments confirmed that Z4:40 and Z4:52 both interacted with a site on the receptor that over-lapped the binding site of IGF-1. From the control selections, several additional affibody molecules binding to IGF-1R could be isolated. Interestingly, the site of interaction of one of these variants (Z2:38) with the receptor was also mapped and found to be over-lapping that of IGF-1, and as Z4:52 had homologous sequences found in the control selections, we concluded that several of the variants isolated during the control selections interacted with the IGF-1 binding site of the receptor, indicating that this was a

“hot-spot” for affibody interaction. However, neither of the high-affinity binders Z4:40 or

Z4:26 were isolated during the control selections, where instead variants with lower affinity such as Z2:38 were enriched. An explanation could be that affibody molecules were enriched during the control selections for reasons other than affinity, such as higher production levels or better display on the phages. Z4:40 and Z2:26 could also have been outcompeted by the frequently isolated variant Z3:24 which contain a cysteine residue that could interact unspecifically with the selection system to give it an advantage during the selection. The results of these four selections emphasize the important influence the elution conditions have on the final result of a panning procedure.

7.2 Selection of Raf specific and Ras specific affibody molecules (Paper IV) The MAPK pathway is frequently over-active in transformed cells, for example due to aberrant tyrosine kinase signalling or by activating mutations or over-expression of proteins that relay the signals. Two protein families in this pathway are the Ras and Raf proteins and recent progress in the understanding of their biology suggests that interfering with their interaction could prove be a viable strategy in cancer therapy. We therefore investigated the possibility of developing affibody molecules using Ras and Raf proteins as targets.

Selections against Ras performed using truncated H-Ras as the target. This variant (amino acid 1-170) contains all necessary parts of the protein for its normal function except for the C-terminal part of the protein that becomes farnesylated and interacts with the plasma membrane. Selection against Raf was performed using truncated Raf-1 (ΔRaf-1) as a target. This variant (amino acid 5-143) contains the Ras binding domain and was used instead of full-length Raf-1 to increase the likelihood of selecting binders to the Ras binding domain that could interfere with the Ras-Raf interaction. Both target proteins were expressed in E. coli as fusion proteins with hexahistidine (his6) tags for purification and albumin binding protein (ABP) tags for purification and increased solubility. The two ERIK VERNET 47

recombinant proteins were analyzed using real-time biospecific interaction analysis, showing that his6-ABP-ΔRaf-1 bound specifically to immobilized his6-ABP-HRas.

Three selections against H-Ras were carried out where parameters such as target protein concentration, types of solid support, number of washing steps and number of cycles performed were varied. In all three cases affibody ligands were obtained with specific binding to the Ras protein (Figure 15A-C). During the selection against ΔRaf-1, one predominant variant (ZRaf322) was isolated (Figure 15D) with a moderate affinity of 2.5 µM. The specificity of the selected affibody molecules was analysed and found to be very high using dot blot against 18 human proteins. In contrast to the H-Ras binders, the affibody variant ZRaf322 was able to interfere with the Ras/Raf interaction in a biacore experiment (Figure 16).

Figure 15. Biosensor binding analysis of the selected affibody ligands. Sensorgrams obtained after injection of (A) ZRas122, (B) ZRas220 (C) ZRas521, (D) ZRaf322 over a his6-ABP-

HRas surface (for A, B, C) and his6-ABP-∆Raf-1 surface for (D) shown in a solid black line, and the control proteins his6-ABP (solid gray line) and human IgG (dotted black line).

This emphasizes the importance of using a selection procedure that will enrich for ligands with binding to the preferred site of interaction in the target protein, in this case by using only the part of the target protein that is responsible for Ras interaction. The affinity proteins selected here were all specific for their targets, which opens up the possibility for cellular experiments where the binder against Raf-1 could be directly expressed to inhibit Ras/Raf interaction or either of the variants against H-Ras or Raf-1 could be expressed with a fusion partner that will relocalize the affibody construct together with the target protein to e.g. the proteasome for degradation (141). 48 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

Figure 16. Competition studies of Z(Raf322)2. (A) Sensorgrams documenting the binding of

Raf-1-RBD (dashed black line), a mixture of Raf-1-RBD and Z(Taq)2 (solid black line) or a mixture of Raf-1-RBD and Z(Raf322)2 (dotted black line) to captured ABP-HRas, respectively. The solid grey documents the dissociation of captured ABP-HRas.

All affibody variants isolated during the H-Ras selections (ZRas122, ZRas220 and ZRas521) contained at least one cysteine residue. Similar results have been obtained during other selections e.g. against the transcription factor c-Jun (187). Since a lower affinity for the respective target was observed both against Ras in the current study and in the previous study against c-Jun, a possible explanation is that these variants can gain an advantage during the selection by forming multimers that gives a higher apparent affinity. However, all three variants failed to inhibit the Ras/Raf interaction when analyzed in a biacore experiment. Even though the three variants were selected using different conditions, they were all found to bind to overlapping epitopes on Ras as found in an ELISA competition assay. This suggests that there is a preferred affibody interaction site on Ras that is separated from the Ras/Raf interaction. During future selections against Ras it could be possible to find binders to other sites by e.g. blocking Ras with one of the binders described in this study or by employing a competitive elution strategy using e.g. the Ras binding domain of Raf-1 rather than low pH for elution.

ERIK VERNET 49

8 Concluding remarks

Inhibition of protein activity, for example by using protein-based affinity reagents, can be used to investigate protein function and for therapeutic applications. In this thesis, several protein-based affinity reagents (so called affibody molecules) have been generated and evaluated for use in cancer research.

In Paper I and Paper II, affibody molecules with high affinity for the oncogenic cell surface receptors HER2 or EGFR were expressed in human cell lines as fusions to a signal peptide and an ER retention signal. The affibody molecules were resident in the secretory compartments and hindered a significant portion of newly synthesized targeted receptor from reaching the cell surface, which also led to a decrease in proliferation. These results demonstrate that affinity proteins based on alternative scaffolds can be used for protein relocalization in mammalian cells.

The concept of ER-retention-mediated knock-down of receptor function is not used in the clinic today. One reason is the lack of sufficiently effective and specific gene delivery vehicles. However, the clinical use of EGFR inhibitors has proven efficacious and there is a correlation between decreased phosphorylation of EGFR and downregulation of MAPK signaling constituents, such as ERK (256). As a significantly decreased phosphorylation of EGFR is seen also by ER retention (Paper II and (78)), it is possible that ER-retention- mediated knock-down of EGFR could become a viable strategy for cancer therapy when efficient delivery systems have been developed. Interestingly, the current poor efficacy problems with anti-HER2 antibody therapy seem to be at least partly due to the inability of Trastuzumab to prevent HER2 from forming heterodimers with other ErB receptors (257). ER retention is therefore an appealing approach, as it hinders the receptor from reaching the membrane altogether. For both HER2 and EGFR, the relative ER retention efficiency was similar in all cell lines investigated, which indicates that a higher affibody expression does not necessarily give a higher degree of retention. This phenomenon has been described previously and may be due to insufficient levels of the KDEL receptor (ERD) to quantitatively transport the proteins (258). One possible improvement of this system would therefore be to express the affibody molecule as a fusion protein with ERD. The relocalization strategy might also be feasible using other membrane bound transport proteins in the secretory compartments, including the cat-ion dependent mannose phosphate receptor CD-MPR (Chapter 1.1), that circulates between the TGN, early endosomes and late endosomes.

In Paper III and Paper IV, novel affibody molecules towards cancer related proteins have been generated and characterized. In Paper III, several affibody molecules targeting the hormone binding site of IGF-1R were developed. One of these, Z4:40, was shown to bind 50 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

with 1.2 nM affinity (KD) to MCF-7 cells and could interact with IGF-1R in receptor pull- down experiments as well as in fluorescent imaging of fixed cells expressing IGF-1R. It also decreased the proliferation rate of MCF-7 cells and may thus prove useful for (prognostic and diagnostic) tissue staining, for investigations of the IGF-1R related proliferative pathways and for therapeutic applications.

In Paper IV, one affibody molecule against Raf-1 and three affibody molecules against H- Ras were developed. All four affibody molecules were specific to their target and in addition, the Raf-1 binding affibody molecule completely disrupted the Ras-Raf interaction in biosensor analysis. As the complex signaling network involving different Raf proteins is far from fully understood, affinity proteins interfering with Ras or Raf are valuable tools for further understanding of signaling in both normal and malignant cells. For example, B-Raf has been shown to activate C-Raf/Raf-1 independently of Ras (259).

In conclusion, affinity proteins based on alternative scaffolds such as the affibody molecules presented in this thesis can be generated to interact with a number of cancer relevant target proteins. I show that it is possible to utilize them to inhibit protein function by different strategies, either by hindering receptor tyrosine kinases from reaching their proper site for signaling (the plasma membrane) or by inhibiting important protein-protein interactions directly such as IGF-1/IGF-1R interactions and Ras/Raf interactions. The use of the affinity proteins and strategies described here may be valuable tools in the war on cancer, initially for the further investigation of cancer related signaling pathways in order to expand our current understating of cancer but eventually also for the development of reagents suitable for cancer therapy.

ERIK VERNET 51

9 Acknowledgements

Jag vill naturligtvis tacka alla som gjort den här texten möjlig: Torbjörn, för din entusiasm och optimism i alla lägen, inte minst alla gånger vi diskuterat FACS resultat över en dålig telefonförbindelse med skrikande barn i bakgrunden. Har lärt mig så oerhört mycket (säkert du också!) under de här åren. Uppskattar din vilja att alltid göra häftig vetenskap även om jag nog aldrig förstått storheten i varken bridge eller Slayer. Per-Åke, tack för att du alltid tagit dig tid att diskutera vetenskap och alltid haft nya förslag på experiment. Plan 3 skulle inte vara samma sak utan dina frågor på alla seminarier, de har jag verkligen uppskattat. Tack Emma, först och främst för alla fina cellbilder till artiklarna och för att du visade mig allt som Tobbe inte hann med när jag började på labbet … Var kalas att labba bredvid dig innan du drog vidare, vi hann med en hel del skvaller jag aldrig skulle hört annars. Tack till all mina andra samarbetspartners: Jingjing and Sebastian for collaborations with the IGF-1R and Ras/Raf binders, Anna K och Mikaela för era insatser med ER retention systemet. Janne och Lukas på KS för samarbete med S100A6 projektet, jag lärde mig mycket biologi. Hjalmar för många timmar framför mikroskopet med CPPs, QDs och andra spännande idéer. My thesis students, I just realized that you all are PhD students now – glad I didn’t scare you off. Nicolò, my first student, we had a great time (when the cloning finally worked). Sandra, my second thesis student, for great work with the lentiviral system although I never asked about your weekend. Camilla, tredje exjobbare och gruppens nya stjärna. Lycka till med allt och lova att inte jobba på lördag. Anna S och Marie för att ni tagit så väl hand om AC-labbet, jag kommer sakna virushooden... Mina nuvarande och tidigare rumskamrater Johan, Johanna, Jorge, Karin, Malin, Marcus, Sebastian, Sverker, Ulrika och Valtteri för hård arbetsdisciplin kombinerat med sköna pauser i arbetet och intressanta diskussioner om allt möjligt. Alla andra som jobbar på plan 3 för att ni gör det till ett härligt ställe att arbeta på med mycket skratt, practical jokes och kakor. Kalle Hult för intressant syntesjobb sommaren 2004 och exjobbet som fick mig att vilja forska. KTH för finansiering.

Sen vill jag tacka alla andra vänner som förgyllt mina lediga stunder under dessa år: “Da gang” (Andy, Pat, Paw, Hawk, Ciz, Tez, Sara, Cilla och es.yarps) för fester, luncher, resor, spelkvällar etc. Anna, Daniel, David, Erik, Henny, Holger, Karin, Maja, Martin, Mats, Mårten, Rasmus och Sara för middagar, segling, skidåkning, spelkvällar, resor och annat skoj. Kemisektionen för fem fina år, extra tack till Anna, Daniel, Henny, Mimmi och Peter för att ni drog ner mig i sektionsträsket och till pH-04 för att ni är så gulliga och skäggiga. Jonas, Mattias och Leffe för sköna pauser från forskandet med spel, surfing och en och annan öl. CD, vi har ju känt varandra ett bra tag nu, tack för alla upptåg genom åren med resor, fester och konstiga TVM-helger. Cat, för alla glöggfester och fikastunder i Helsingborg och våra diskussioner om allt möjligt. Ett stort tack till medlemmarna i Teatersällskapet Paddan, för kulturarvet vi lämnat efter oss med musikdokumentärer, ostar 52 AFFINITY PROTEIN BASED INHIBITION OF CANCER RELATED SIGNALING PATHWAYS

och andra produktioner. Vill också tacka boysen i Logement 1 på SkyddS för återträffar, taktikpärmar och andra minnen med och utan kryckor. Martin W för att du hjälpte mig komma tillrätta på Stanford.

Till slut vill jag tacka min fantastiska släkt med alla mina mostrar, morbröder, fastrar och farbröder, kusiner och andra, vi har alltid roligt ihop. Särskilt tack till farmor och farfar för alla middagar, lånade saker och hjälp när något måste sys… tack morfar för att du lärde mig kilamonkan, du är min idol. Tack till Kristin för att du är rolig att ha med på våra resor och för att du står ut med familjens dåliga humor. Rickard och Martin för den där särskilda broderskärleken som man bara kan uppnå genom slagsmål. Tack mor och far för att ni alltid uppmuntrat och aldrig ifrågasatt.

ERIK VERNET 53

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