Chemical Synthesis of Affibody Molecules for Detection and Molecular Imaging

TORUN EKBLAD

Royal Institute of Technology School of Biotechnology Stockholm 2008 © Torun Ekblad Stockholm 2008

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 978-91-7415-152-7 TRITA BIO-Report 2008:22 ISSN 1654-2312 iii

Torun Ekblad (2008): Chemical Synthesis of Affibody Molecules for Protein Detection and Molecular Imaging. School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden. Abstract are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue -derived Z domain scaffold. In the first paper, solid phase synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.

In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl- glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.

Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications.

Keywords: Affibody, peptide synthesis, protein detection, molecular imaging

© Torun Ekblad 2008 iv

List of publications

This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-VII). The seven papers are found in the appendix.

I Engfeldt T., Renberg B., Brumer H., Nygren P-Å. and Eriksson Karlström A. (2005) Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein. ChemBioChem 6: 1043-50

II Renberg B., Shiroyama I., Engfeldt T., Nygren P-Å. and Eriksson Karlström A. (2005) Affibody protein capture microarrays: synthesis and evaluation of random and directed immobilization of affibody molecules. Anal Biochem 341:334-43

III Engfeldt T., Orlova A., Tran T., Bruskin A., Widström C., Eriksson Karlström A. and Tolmachev, V. (2007) Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence. Eur J Nucl Med Mol Imaging 34:722-33

IV Engfeldt T., Tran T., Orlova A., Widström C., Feldwisch J., Abrahmsén L., Wennborg A., Eriksson Karlström A. and Tolmachev V. (2007) 99mTc-chelator engineering to improve tumour targeting properties of a HER2-specific Affibody molecule. Eur J Nucl Med Mol Imaging 34:1843-53

V Tran T., Engfeldt T., Orlova A., Sandström M., Feldwisch J., Abrahmsén L., Wennborg A., Tolmachev V. and Eriksson Karlström A. (2007). 99mTc-maEEE-

ZHER2:342, an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors. Bioconjug Chem 18:1956-64

VI Ekblad T., Tran T., Orlova A., Widström C., Feldwisch J., Abrahmsén L., Wennborg A., Eriksson Karlström A. and Tolmachev V. (2008) Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake. Eur J Nucl Med Mol Imaging in press DOI 10.1007/s00259-008-0845-7 v

VII Ekblad T., Tolmachev V., Orlova A., Lendel C., Abrahmsén L. and Eriksson Karlström A. (2008) Synthesis and chemoselective intramolecular cross-linking of a HER2-binding Affibody. Submitted

Related publications not included in this thesis

Tran T., Engfeldt T., Orlova A., Widström C., Bruskin A., Tolmachev V. and Eriksson Karlström A. (2007) In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules. Bioconjug Chem 18:549-58

Orlova A., Tran T., Widström C., Engfeldt T., Eriksson Karlström A. and Tolmachev V. 111 (2007) Pre-clinical evaluation of [ In]-benzyl-DOTA-ZHER2:342, a potential agent for imaging of HER2 expression in malignant tumors. Int J Mol Med 20:397-404

Elfström N., Juhasz R., Sychugov I., Engfeldt, T., Eriksson Karlström, A. and Linnros J. (2007) Surface charge sensitivity of silicon nanowires: size dependence. Nano Lett 7:2608-2612

Tran A. T., Ekblad T., Orlova A., Sandström M., Feldwisch J., Wennborg A., Abrahmsén L., Tolmachev V. and Eriksson Karlström A. (2008) Effects of lysine-containing mercaptoacetyl- based chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules. Bioconjug Chem in press vi

Contents

INTRODUCTION ...... 1 1. Proteins...... 1 2. Affinity proteins...... 4 2.1 ...... 5 2.2 fragments...... 8 2.3 Alternative scaffolds...... 10 2.3.1 Single loops on a rigid framework...... 11 2.3.2 Several loops forming a contiguous surface...... 12 2.3.3 Engineered interfaces resting on a secondary structure ...... 13 2.3.4 Affibody molecules...... 14 3. Protein and peptide chemistry ...... 18 3.1 Peptide and protein production...... 18 3.2 Modifications of and proteins ...... 19 3.3 Chemical synthesis...... 22 3.3.1 History and development of Solid Phase Peptide Synthesis...... 22 3.3.2 Principles of Fmoc SPPS...... 25 3.3.3 Orthogonal protection strategies...... 27 3.3.4 Limitations of SPPS...... 28 3.3.5 Peptide ligation of synthetic fragments...... 29 4. Applications of antibodies and alternative binding proteins in bioassays ...... 33 4.1 Monitoring biomarkers ...... 33 4.2 Detection of proteins in vitro ...... 34 4.2.1 Fluorescent detection in protein microarrays ...... 36 4.3 Detection of proteins in vivo ...... 38 4.3.1 Molecular imaging...... 38 4.3.2 Tumor targeting ...... 39 4.3.3 Radionuclides for in vivo studies...... 40 4.3.4 Radionuclide imaging techniques ...... 42 4.3.5 Radiolabeling of targeting peptides and proteins ...... 43 4.3.6 Technetium labeling ...... 44 4.3.7 Biodistribution...... 46 vii

PRESENT INVESTIGATION...... 48 5. Chemical synthesis of Affibody molecules for in vitro protein detection assays ...49 5.1 Synthesis of triple-labeled Affibody molecules for specific protein detection (Paper I) ...... 49 5.2 Synthesis of Affibody molecules for protein microarrays (Paper II)...... 53 6. Chemical synthesis of Affibody molecules for molecular imaging in vivo...... 55 6.1 Synthesis of an Affibody molecule containing the 99mTc-chelator MAG3 for imaging of HER2-expressing tumors (Paper III)...... 55 6.2 Synthesis of Affibody molecules with engineered 99mTc-chelator sites for improved HER2-imaging (Papers IV, V and VI)...... 58 6.3 Synthesis and chemoselective intramolecular cross-linking of a HER2-binding Affibody molecule (Paper VII)...... 63 7. Conclusions and future perspectives ...... 65 8. Abbreviations ...... 67 9. Acknowledgements...... 68 10. References...... 71 viii TORUN EKBLAD

INTRODUCTION

1. Proteins

Proteins are polymeric biomolecules, assembled from a repertoire of 20 amino acids. An amino acid consists of an amino group, a carboxyl group and a distinctive side chain, all of which are bonded to a central ơ-carbon atom. Condensation reactions between the amino and carboxyl groups result in the formation of a peptide bond (Figure 1). Polymeric sequences of amino acids joined by peptide bonds are called peptides, or polypeptides. The peptide backbone is not very reactive chemically, but presents potent donors and acceptors for hydrogen bond formations. Hydrogen bonding between CO- and NH-groups in the peptide backbone makes polypeptides fold into secondary structures, such as alpha helices or beta sheets (Pauling, 1951a, Pauling, 1951b). Additional hydrogen bonds, as well as weak, reversible interactions, such as electrostatic and van der Waals interactions, with the amino acid side chains assist in the folding of proteins into three-dimensional structures. The spatial organization of a polypeptide is termed tertiary structure. Generally, the tertiary structure of proteins in aqueous solutions is characterized by a surface of polar groups whereas non-polar groups are buried in the interior. Polypeptides can vary enormously in terms of length and may be as long as 25 000 amino acids (Kiehn, 1970). In general, short polypeptides are referred to as peptides and longer polypeptides are termed proteins. However, the significant difference lies in the presence of a well-defined three-dimensional structure, which is not always correlating with the length of the polymer.

1 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

The amino acid building blocks are characterized by their different side chains, which all differ in size, shape, charge, hydrogen-bonding capacity and chemical reactivity. The order in which the amino acids are assembled is called the primary sequence and is of great importance since it dictates the structure and thereby also the function of the folded protein (Anfinsen, 1973). With few exceptions, all proteins in all species are constructed from the same set of 20 amino acids, and differ only in the number of amino acids, and the sequence in which the various amino acids occur. The average eukaryotic protein is linear and unbranched and consists of 280 amino acids (Creighton, 1997). With 20 amino acids to choose from, and the unrestricted freedom to organize them sequentially, a huge number of primary sequences can be generated. The diversity and versatility of the 20 amino acids allows for an overwhelming range of biological functions to be generated.

Figure 1. Formation of a peptide bond between two amino acids

Indeed, proteins have many different biological functions. Most chemical reactions in biological systems are catalyzed by proteins with enzymatic functions. Proteins play essential roles in the regulation of cell growth and differentiation. In particular protein hormones are important regulators. Proteins also serve important functions in transport and storage of small molecules and ions, such as myoglobin transportation of oxygen and ferritin for iron storage. Many proteins, such as keratin or , provide mechanical support. Antibodies is another important type of proteins that serve to protect its host from foreign substances.

In addition to the functionalities that are provided in the amino acid side chains, many proteins are subjected to various chemical modifications during or after assembly of the peptide chain. Proteolytic processing, alterations of the chain termini and coupling of functional groups to amino acid side chains are examples of modifications that are naturally introduced to tailor proteins for improved performance in certain functions. Proteins can be glycosylated to alter the specificity in their interactions with other biomolecules, to improve the solubility, or to lengthen the biological half-life. Lipids can be attached in order to tether proteins to membranes. Phosphorylation of serine, threonine, and tyrosine residues is an important 2 TORUN EKBLAD regulator of protein activity, and hydroxylation is an essential modification for folding and assembly of collagen.

Important classes of proteins are those that have the ability to interact specifically with other proteins. This thesis focuses on such affinity proteins, and methods to utilize molecular recognition for specific protein detection. In order to develop highly sensitive bioassays, the affinity proteins often need to be modified with functional groups that improve their performance. Chemical synthesis is a production route available for small proteins, and offers a means to introduce such functional probes site-specifically to modify the function of the protein.

3 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

2. Affinity proteins

A specific interaction between two molecules is termed molecular recognition. In nature, proteins and peptides are involved in various non-covalent interactions with other biomolecules, such as antigen-antibody, receptor-ligand, DNA-protein, sugar-lectin and RNA- ribosome interactions. Such natural examples of molecular recognition have been transferred to applications in biotechnology and medicine, including for example bioseparation using lectins as ligands for carbohydrate-recognition, and development of diagnostic assays such as skin tests to detect bacterial infections (Campos-Neto, 2001) or allergies. Natural protein interactions have also been used in clinical therapy for regulation of signaling pathways, as in the use of insulin receptor interactions to regulate glucose homeostasis, or for targeted delivery of therapeutic payloads, such as the use of growth hormones for delivery of toxins or radionuclides to epidermal growth factor receptors (Sundberg, 2003). In addition to protein interaction systems derived from natural interaction pairs, development of engineering strategies to generate novel affinity proteins for defined biomolecular recognition has been an intensive research field since the 1990s.

Depending on the intended application, different requirements are set for the affinity proteins. Most importantly, an affinity reagent has to bind with adequate affinity and selectivity to its target or group of targets. For applications where the bound protein should be recovered, it is essential to have a possibility to break the interaction by addition of suitable agents. The route by which the affinity protein can be produced is an important consideration. Ideally, it should be possible to obtain large quantities in a cost-efficient manner. For many applications it is also necessary to have a production route that allows for the introduction of chemical modifications. Stability to chemical, physical and enzymatical challenges are important issues, as are biophysical parameters such as size and polarity. For in vivo use, the species from which the affinity proteins originate has to be regarded for immunogenicity reasons.

The most widely used affinity proteins for biotechnological and medical applications are antibodies. Antibodies have played an essential role in the development of most applications that rely on molecular recognition. The importance of this class of affinity proteins can be illustrated by the number of Nobel Prizes earned in the field of antibody research, which is no less than seven (http://www.nobelprize.org). There are currently 32 antibodies and antibody- derived molecules approved by the US FDA to be used in the clinic for diagnostics and

4 TORUN EKBLAD therapy (Leader, 2008). However, certain shortcomings of antibodies have been identified, which have generated an interest in development of alternative reagents for molecular recognition. Using the conceptual architecture of antibodies and the principles by which variability can be created, strategies have evolved to develop new classes of affinity proteins, including fragments derived from antibodies and affinity proteins developed from alternative scaffolds. Such molecules have come forward as complement or alternatives to antibodies in various settings where characteristics different from those of antibodies are useful.

2.1 Antibodies

The existence and survival of an organism rely heavily on its capacity to protect itself from foreign invaders. The immune system of humans, and higher vertebrates, offers an ingenious source of diversification and means for selection of high affinity antibodies that recognize most imaginable alien substances. Antibodies have evolved to serve two functions, to specifically interact with foreign substances in the body, and to recruit effector functions of the immune system.

A human antibody consists of four polypeptide chains, two identical light chains (L) and two identical heavy chains (H), each of which is organized into multiple globular domains of hydrogen-bonded, antiparallel Ƣ-strands with connecting loops and stabilizing bonds. The domains all have a similar fold, called the immunoglobulin fold, but differ in terms of variability, and are divided into constant domains with strictly conserved sequences and variable domains with high sequence diversity. The antibody is Y-shaped, with constant domains of the heavy chains forming the stem, and each arm formed by two constant domains

(CH1 and CL) and two variable domains (VH and VL) at the very tip (Figure 2).

The sequence of the constant regions of the heavy chain can only vary according to certain patterns, which divides the antibodies into classes of different isotypes; IgG, IgM, IgA, IgD and IgE, and their subclasses. This part of the molecule is also responsible for a variety of biological effector functions, such as activating the complement system and directing cytotoxic activities.

5 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

Figure 2. Schematic representation of an IgG antibody

In the structures of immunoglobulins, high variability is observed in the loops that connect the Ƣ-strands, both in terms of length and amino acid sequence. Three loops each in the variable domains of the heavy and light chain respectively are hypervariable and called complementarity determining regions, (CDRs). The rest of the variable domains are known as framework regions and act as a scaffold that brings the six CDRs together to form the surface that is responsible for antigen binding. The great diversity in terms of composition and topology (flat, crevice-like or protruding) that the binding surface of antibodies can have allows for highly specific interactions with ligands of varying size and shape, from small haptens to large macromolecules. By different mechanisms occurring within the immunoglobulin-encoding genes in maturing B-cells (i.e. random gene segment shuffling, frame-shifting, nucleotide insertions, and somatic mutations), a vast diversity of binding specificity can be generated from a limited number of original genes. Individual members of this library of antibody variants is displayed by B-cells in the circulation, which upon encountering its cognate antigen, and after receiving stimulatory signals from helper T-cells, will start to differentiate into plasma cells, secreting large amounts of a specific antibody. The fine specificity and affinity of the antibody

6 TORUN EKBLAD can be further improved during the life of the B-cell lineage via somatic hypermutations in the CDR regions (Goldsby, 2003).

It has been suggested that basically any macromolecular structure can be immunogenic, if presented properly to the immune system (Benjamin, 1984). This fact has been employed in efforts to generate antibodies with desired specificities. By immunizing laboratory with an antigen of interest, an immune response can be raised, generating a pool of antibodies recognizing different sites (epitopes) of the antigen. This heterogeneous ensemble of molecules is termed polyclonal antibodies and has proved to be useful as affinity reagents in many biotechnological applications in vitro such as ELISA (Engvall, 1971), microarrays (Bertone, 2005), and affinity proteomics (Uhlén, 2005). However, immunizations may not generate an entirely reproducible response, which might be an issue in regulated manufacturing procedures. Also, for in vivo applications it is desirable to use reagents of more well-defined character.

The reproducible production of defined antibodies became possible with the development of the hybridoma-technology (Kohler, 1975). By fusion of mouse myeloma and mouse spleen cells (i.e. antibody-producing B-cells) from an immunized donor, individual in vitro-dividing cell-lines were generated that each produced a particular antibody variant, a . The development of the hybridoma technology came to revolutionize the use of antibodies and was later awarded the Nobel Prize. Some limitations of monoclonal antibody production by the hybridoma technology are that it is laborious, time-consuming and cannot be realized in a high-throughput fashion.

Important early steps towards a by-pass of both immunization and the hybridoma technology for obtaining antibodies were provided when recombinant production of antibodies was reported for bacterial cells. The first recombinant production of complete H- and L-chains in Escherichia coli was described in 1984 (Cabilly, 1984). However, the proteins were obtained as insoluble aggregates and refolding efforts resulted in poor recovery of activity. In 1988, two studies coincidently reported on recombinant production of functional antibody fragments (Skerra, 1988), (Better, 1988). Both reports agree that in order to achieve biologically active assembled heterodimers, the production route had to mimic the native assembly pathway in that the two chains had to be translated simultaneously, and secreted, either to the extracellular culture medium (Better, 1988), or to the periplasmic space (Skerra, 1988).

7 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

The successful cloning of antibody genes also allowed for , which could be used to address some of the issues regarding immunogenicity caused by the murine origin of monoclonal antibodies obtained by the hybridoma technology. By using genetic engineering, chimeric antibodies with the constant region of human origin and the variable domains carrying the antigenic specificity derived from mouse have been generated (Morrison, 1984). Another important progress was the approach to graft only the CDRs from the mouse clone onto a human antibody framework to generate humanized antibodies (Jones, 1986). Thereby, all constant domains are encoded by human genes, resulting in reduced immunogenicity and conservation of the biological effector functions, thus rendering the antibodies likely to trigger immune system reactions such as the complement activation or Fc receptor binding.

Cloning of antibody genes and the realization of recombinant production routes have been crucial to allow for different types of protein engineering of antibodies, such as site-directed mutagenesis or generation of fusion proteins. Furthermore, it inspired the development of genetically engineered antibody-derived fragments, which have been found to be better suited both for expression and in vitro selection. Small recombinant antibody fragments and engineered variants thereof have emerged as alternatives to full-sized antibodies in several applications where size and complexity become decisive issues.

2.2 Antibody fragments

For many applications, high-yield production and solubility of the affinity protein are critical factors. Furthermore, small size may prove essential to achieve a desired biodistribution in vivo, for applications such as molecular imaging. Size reduction can also be used as a strategy to reduce the immunogenicity of an affinity protein of non-human origin. Many attempts to reduce the size of antibodies while retaining its antigen-binding properties have been reported, some of which are described below.

The initial steps towards dissecting the antibody molecule were made by enzymatic digestion, revealing fragments with retained antigen-binding, namely Fab and F(ab)’2 (Porter, 1959). The Fab fragment has a size of 54 kDa and is made up of the complete light chain and the variable domain and the first constant domain from the heavy chain, which form the arm of the Y- shaped antibody structure (Figure 2). The difference between the fragments Fab and Fab’ is that the latter also includes the free cysteine residue that originally is included in the first hinge disulfide with the other heavy chain. F(ab)’2 is the disulfide-bonded dimer of two Fab’ 8 TORUN EKBLAD fragments. Fab fragments were the first functional antibody-derived fragments to be successfully produced in bacterial expression systems (Better, 1988), (Skerra, 1988).

The smaller Fv fragment consists of only the variable domains of the heavy and light chain (VH and VL). The domains associate in a non-covalent manner, which have been difficult to accomplish in recombinant expression settings. This has been by-passed with the development of single chain Fv fragments, scFv (27 kDa) (Bird, 1988), where a flexible peptide loop that links the variable domains allows for recombinant production of a single polypeptide chain and also brings a higher stability to the molecule. The scFv’s have found many applications as affinity reagents, such as in microarrays (Wingren, 2005). Dimers of scFv’s, called diabodies, have also been generated (Holliger, 1993). By using a linker that is too short to allow for interactions between domains on the same chain, dimeric molecules are forced to assemble by pairing with the complementary domain of another chain. The fact that diabodies have two functional antigen binding sites brings increased apparent affinity (avidity) compared to the monomer. Alternatively, this feature can be used to create molecules with dual binding affinities.

The variable domains VH and VL might be used individually as small (15 kDa) antigen binding units (Ward, 1989). However, a large hydrophobic surface, which originally makes up the interaction surface of the two domains, is exposed upon their separation, resulting in poor solubility of the individual fragments. Efforts to minimize the exposed hydrophobic interface by site-directed mutagenesis have resulted in successful production of such binders. In nature, a unique class of antibodies devoid of the light chain and with antigen-binding sites composed of a single variable domain has been identified in camelids (Hamers-Casterman, 1993) and cartilaginous fish (Greenberg, 1995). These represent the smallest available antigen-binding fragments (15 kDa) derived from antibodies.

The successful cloning and expression of antibody genes in bacterial cells and the progress made in the design of antibody fragments, was followed by the development of methods to generate and handle large complex libraries of antigen-binding fragments. In parallel, sophisticated in vitro selection systems were developed so that clones with distinct antigen- binding specificities could be isolated. Such systems mimic the native clonal expansion of B- cells and rely on a method for diversification or harvesting a natural diversity, followed by repeated cycles of selection and amplification of variants with desired properties. is the most prominent example of such systems, which employs the coat proteins on filamentous phages for display of peptides whose corresponding genes have been engineered

9 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING into the phage genome (Smith, 1985). Genes for complete antibody V-domains have been amplified by PCR and the corresponding proteins successfully displayed on phages so that clones with binding activities to a desired antigen could be selected using affinity chromatography (McCafferty, 1990). Cloning the repertoire of antigen-binding fragments from an immunized donor into a phage display vector and selection of antigen-specific clones has since become a routine method (Winter, 1994) (Hoogenboom, 1998). Other in vitro selection systems such as mRNA display, ribosome display, and cell surface display on different types of organisms, such as yeast, and gram-positive bacteria, are being used for the same purpose and are reviewed by Lin and Cornish (Lin, 2002).

2.3 Alternative scaffolds

One inconvenient feature related to antibodies is that they are difficult to express recombinantly as correctly folded and biologically active proteins at high yields in bacterial expression systems. With powerful in vitro selection systems at hand, the use of alternative scaffolds, characterized by a more easily folded framework than the immunoglobulin, could be considered for harboring novel binding sites. The use of such alternative scaffolds can bring advantages such as favorable expression routes in terms of yield, stability, and folding as well as attractive biophysical characteristics and might be a way to realize some applications where antibodies are not an option. Examples of such applications are interactions with immuno- evasive clefts and grooves in a target protein (Stijlemans, 2004), and co-crystallization (Högbom, 2003), (Binz, 2004).

Rational de novo design of peptides or proteins with specific binding capacity is a very difficult task. However, a completely different route to developing proteins with new functions was taken in 1990 when it was reported that novel specific ligands could be selected from combinatorial libraries of short peptides with randomly generated sequences without any prior knowledge about the nature of the interaction surface (Cwirla, 1990), (Devlin, 1990), (Scott, 1990). In efforts to overcome the poor binding affinities of the flexible peptides, conformationally constrained peptides were applied (O'Neil, 1992), (Koivunen, 1995), and the idea to use already folded proteins for development of novel affinity reagents evolved.

Scaffold engineering involves the introduction of a novel binding specificity to a structural framework made up of a folded protein. This can be accomplished either by grafting a peptide sequence with a known function onto the scaffold, or by site-specific mutations of surface- 10 TORUN EKBLAD exposed residues in the scaffold to replace a parental binding specificity or to create a novel binding site. More than 50 different scaffolds have been suggested as alternative affinity proteins, and the topic has been thoroughly reviewed by others (Nygren, 2004), (Binz, 2005), (Hey, 2005), (Hosse, 2006), (Skerra, 2007) The following sections serve to present some representative examples and highlight the Affibody scaffold, which has been the focus of the studies that are presented in this thesis. Affinity proteins based on alternative scaffolds differ widely in their structural architecture and the way in which they interact with their target molecules. A classification based on the properties of the interaction surface results in the following classes; single loops on a rigid framework (chapter 2.3.1), several loops forming a contiguous surface (chapter 2.3.2) and engineered interfaces resting on a secondary structure (chapter 2.3.3). Some examples of affinity proteins derived from alternative scaffolds are summarized in Table 1.

2.3.1 Single loops on a rigid framework

In their simplest form, engineered affinity proteins consist of a single loop peptide with randomized sequence grafted onto a stable protein framework. Kunitz domains are a class of serine inhibitors of such design. Some 60 amino acids make up a stable fold, presenting the protease binding site as an exposed loop with varying sequence. By randomizing residues in the loop, libraries have been generated from which protease inhibitors with novel specificities have been selected using phage display (Dennis, 1994, Nygren, 2004). The main application of Kunitz domains has so far been as protease inhibitors, such as the DX-88 inhibitor of plasma , which is now in clinical trials for treatment of hereditary (Williams, 2003).

Cystine-knot miniproteins, or knottins, represent another family of small (25-35 aa) proteins that has been used as alternative scaffolds for peptide loop grafting in combinatorial engineering. The cystine-knot structural motif is characterized by a well-defined organization of three intramolecular disulfide bonds where one disulfide bridge is interlocking the macrocycle formed by the other two (Kolmar, 2008, Le Nguyen, 1990, Smith, 1998). This framework has proved to bring remarkable stability to chemical and thermal denaturation as well as . Apart from the disulfide framework, little sequence similarity has been found among naturally occurring knottins, and the interspersed peptide loops are highly variable in both length and sequence (Christmann, 1999). A squash-type protease inhibitor with the cystine-knot motif has been used as a scaffold for rational peptide loop grafting and 11 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING was shown to be amenable to cell surface display on E.coli. Another member of the knottin family is the cellulose–binding domain, CBD, from fungal cellobiohydrolase, which has been subjected to combinatorial engineering to generate binders to alpha amylase (Lehtiö, 2000) and alkaline phosphatase (Smith, 1998). Furhtermore, knottins have been engineered to generate chimeric molecules that can accommodate multiple binding specificities on different faces of the scaffold (Le Nguyen, 1989). Another interesting feature of these small affinity proteins is that they can be produced by chemical synthesis, in addition to recombinant production. The head-to-tail cyclic cystine-knot protein Kalata BI has been synthesized and characterized in terms of folding pathways. It was discovered that fully deprotected peptides were able to fold independently with correct organization of the cystine-knot motif. However, cyclization of the peptide chain prior to oxidation of the thiol groups was found to increase the propensity for correct folding (Daly, 1999). As an alternative, a semisynthetic strategy has been applied for production of cyclic cystine-knot proteins, where recombinant production of a linear precursor peptide allowed for efficient folding and oxidation, and the head-to-tail cyclization was performed by chemical synthesis (Avrutina, 2008).

Another example of scaffolds that gain novel properties by variations of amino acids in a single continuous loop is thioredoxin. This small enzyme (108 aa) displays a disulfide- constrained solvent-exposed peptide loop in its active site that has been replaced by a 20 residue randomized sequence to generate molecules with novel binding capacities. Examples of such thioredoxin-derived peptide aptamers are those designed to interfere with the intracellular protein interactions of cyclin-dependent kinase (Cdk2) (Cohen, 1998) and the tyrosine kinase domain of ErbB2 (Kunz, 2006).

2.3.2 Several loops forming a contiguous surface

The description of this category of affinity proteins also fits the architecture of antibodies, in which pairs of variable Ig domains bring the six CDR loops together to form the antigen binding surface. The fibronectin type III domain (FN3) has a structure similar to that of an antibody VH domain but with only seven Ƣ-strands and no disulfide bonds. The FN3 framework provides several suitable properties such as being small (94 aa), monomeric, and with a ligand binding site formed by surface loops, and has been used as a scaffold to develop novel binding proteins (Koide, 1998) called or AdNectins. Targets to which FN3- variants have been selected include vascular endothelial growth factor receptor 2 (VEGFR2)

12 TORUN EKBLAD

(Getmanova, 2006), TNF-ơ (Xu, 2002), the SH3 domain of human c-Src (Karatan, 2004) and the estrogen receptor (Koide, 2002).

The lipocalins constitute another class of affinity proteins with a structurally conserved Ƣ– sandwich framework, which display loop regions with high variability in both length and sequence. The folding motif of the lipocalins is a Ƣ-barrel, made up of eight antiparallel Ƣ- sheets, with one end closed by densely packed hydrophobic residues and the other end forming an open ligand-binding pocket, flanked by four extended loops. Lipocalins with novel binding specificities, called (20 kDa), have been generated by random mutagenesis directed to sites in the loop regions. The scaffold allows for high affinity interactions with small hapten molecules as well as large proteins. The bilin-bindig protein BBP has been engineered to selectively interact with fluorescein (Beste, 1999) and digoxigenin (Schlehuber, 2000), whereas lipocalins of human origin have been utilized for development of anticalins for intended medical applications, such as an apolipoprotein D-derived hemoglobin-binder (Vogt, 2004) and a human neutrophil lipocalin engineered for specific interaction with CTLA-4 (Schlehuber, 2005).

2.3.3 Engineered interfaces resting on a secondary structure

This class of affinity proteins is derived from scaffolds that provide a secondary structure element with exposed side chains that have potential to be involved in molecular recognition. One such scaffold is represented by repeat proteins. In essence, repeat proteins are built up of structural units that are stacked into an elongated domain with a continuous binding surface. Repeat proteins are abundant in many species and are known to mediate protein-protein interactions (Kobe, 2000). Scaffolds have been designed based on consensus sequences of the repeated motifs in e.g. ankyrin repeats (Forrer, 2003), leucine-rich repeats (Stumpp, 2003), and armadillo repeats (Parmeggiani, 2008), and used to generate combinatorial libraries containing both conserved modular residues and variable surface-exposed residues. An ankyrin repeat is built up by an N-terminal capping domain, followed by a variable number (normally 2-6) of repeat domains and a C-terminal capping domain. The single structural motif of the repeat domain consists of a Ƣ-turn followed by two antiparallel ơ-helices and a loop that connects to the turn in the next unit. The binding surface is made up of variable residues in the Ƣ-turn and the first helix and has been subjected to combinatorial engineering. Binders with high affinity to targets such as maltose binding protein (Binz, 2004), MAP-kinase (Amstutz, 2006), and

13 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

HER2 (Zahnd, 2007) have been selected by ribosome display and are denoted designed ankyrin repeat proteins, .

Affibody molecules are small three-helix bundle proteins with a binding surface made up of residues spread in helices I and II. This class of affinity proteins has been used in all the studies in this thesis and I have therefore devoted a whole chapter (2.3.4.) to describe them in detail.

Table 1. Examples of affinity proteins derived from non-immunoglobulin scaffolds

Class of affinity Structure of Size (aa) Number of Chemical protein binding surface synthesis Kunitz domains Single loop 58 3 Not reported

Thioredoxin-derived Single loop 108 1 Not reported peptide aptamers

Cystine-knot Single loop 28-34 3 Le Nguyen, 1989 miniproteins Daly, 1999

Anticalins Loop rich 160-180 0-2 Not reported

AdNectins Loop rich 94 - Not reported

DARPins Secondary structure 166 - Not reported

Affibody molecules Secondary structure 58 - Nord, 2001 Engfeldt, 2005

2.3.4 Affibody molecules

The Affibody scaffold has been derived from staphylococcal protein A, (SPA), a receptin found in the cell wall of Staphylococcus aureus where it interacts with immunoglobulins (Langone, 1982). SPA consists of five homologous domains, E, D, A, B and C, all of which bind to the Fc region of human IgG (IgG1, 2 and 4, but not IgG3) and also to the Fab part of antibodies from the human VH3 subclass (Jansson, 1998). SPA also binds to immunoglobulin molecules from several other species (Lindmark, 1983). These interactions have been extensively used for affinity purification of antibodies and in different types of immunoassays. The B domain of SPA has been used to derive the Z domain, which is the scaffold that Affibody molecules originate from (Figure 3). The Z domain differs from the B domain in that it has a Gly to Ala substitution in position 29 for increased stability to hydroxylamine. Moreover, Ala1 has been 14 TORUN EKBLAD substituted for Val for subcloning reasons (Nilsson, 1987). Upon mutating residue 29, the Fab binding capacity was significantly reduced (Jansson, 1998). Z is made up of a single polypeptide chain of 58 amino acids with no cysteines, which folds rapidly (Arora, 2004) and independently into an anti-parallel three-helix bundle structure. The Fc-binding has been located to 11 surface-exposed residues distributed over helices I and II (Deisenhofer, 1981). The strategy from which Affibody molecules have spurred relies on the idea to introduce amino acid substitutions at specific sites in order to displace the native binding specificity but preserve the structural folding. 13 residues, including the 9 of the 11 Fc-binding residues, along with 4 additional residues located at the same face, were selected for genetic randomization to create a library of Affibody variants (Nord, 1995) (Figure 3). The first Affibody molecules with novel binding specificities were generated by incorporating a library of ~107 members into a phagemid vector allowing for phage display and selection against Taq DNA polymerase, human apolipoprotein A-1 variant, and human insulin (Nord, 1997). It was shown that the IgG interaction had been replaced with the novel binding specificities. Affibody molecules have since been selected to bind target molecules of various size, shape and origin in different applications, reviewed by Nygren (Nygren, 2008).

The binding affinity of Affibody molecules selected from the initial library was typically in the low micromolar range. Strategies to improve the affinity of those binders by affinity maturation have been successful in several cases, reaching equilibrium dissociation constants,

KD, in the nanomolar range (Gunneriusson, 1999) (Nord, 2001). However, by increasing the 9 library size to ~10 members, high affinity binders (nanomolar KD) could be selected directly from naive libraries. Examples of such Affibody molecules are the ones targeted to HER2 (Wikman, 2004), transferrin (Gronwall, 2007b), amyloid beta peptide (Gronwall, 2007a) and EGFR (Friedman, 2007). Directed evolution strategies have subsequently generated picomolar binders interacting with e.g. the HER-2 receptor (KD=22 pm) (Orlova, 2006), which are used in papers III-VII of this thesis. Also the EGFR-binder has been subjected to affinity maturation, resulting in a 30-fold increase in affinity (Friedman, 2008).

15 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

Figure 3. Schematic overview of the IgG binding domains of staphylococcal protein A, the Z domain, and the Affibody scaffold

The structure of Affibody molecules has been studied using NMR and X-ray crystallography. These studies have been accompanied by circular dichroism experiments and reveal that most selected Affibody variants fold into a compact three-helix bundle structure similar to the parental Z domain, despite the rigorous amino acid substitutions induced by the randomization, that is affecting more than 20 % of the residues. High-precision solution structures, solved by NMR, of the Affibody molecules ZTaq and anti-ZTaq, and their complex

(ZTaq:anti-ZTaq), reveal well-folded three-helix bundles, both in their free forms and in co- complex (Lendel, 2006). However, the Affibody molecule ZSPA-1, with lower target binding affinity, has a molten globule character in solution and only folds into the three-helix bundle structure upon binding to its target, Zwt (Lendel, 2002). A solution structure of an Affibody- dimer in complex with its target amyloid Ƣ-peptide, AƢ(1-40), have revealed an unexpected structural topology, in which the N-terminus of the Affibody molecules forms a beta strand, that can form hydrogen bonds with the AƢ-peptide (Hoyer, 2008).

16 TORUN EKBLAD

Affibody molecules have proven to have potential for use in several applications, ranging from bioseparation, diagnostics and viral targeting, to therapy. One important feature of the Affibody scaffold is that it is amenable to chemical synthesis protocols, described in chapter 3.3. Its small size (58 aa) allows for total synthesis with respectable yields, and the high solubility and independent folding mechanism allows for facile recovery of correctly folded proteins. Peptide synthesis brings measures for chemical modifications, such as incorporation of non-coded residues, site-specific introduction of functional probes or other features, that might be difficult to achieve by recombinant production routes.

The small size of Affibody molecules is an advantageous feature that has been employed also for in vivo applications based on molecular recognition. The rather large size and the presence of an Fc domain can be limiting factors for full-sized antibodies in applications such as in vivo molecular imaging due to long circulation times and poor tissue penetration (see chapter 4.3.7 for further details). Affibody molecules directed to tumor-associated antigens have been studied as alternative targeting agents (Nordberg, 2008, Steffen, 2006). Particularly, a HER2- binding Affibody has been extensively characterized in several preclinical and pilot clinical studies (Baum, 2006, Feldwisch, 2006, Orlova, 2007a). Paper III-VII of this thesis describes how Affibody molecules have been engineered to serve as targeting agents in in vivo imaging of HER2-expressing tumors.

17 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

3. Protein and peptide chemistry

3.1 Peptide and protein production

There are three principally different ways in which peptides and proteins can be obtained; either by extraction from natural sources, through recombinant production technologies, or by peptide synthesis.

Extraction of peptides and proteins from natural sources, such as plants, microorganisms, marine species or mammalian tissues has traditionally been a valuable method to obtain proteins. One important example of a therapeutic protein that was originally obtained from a native source is insulin, which is used for treatment of diabetes mellitus. Protein extraction from natural sources suffers from several drawbacks. The availability is limited, especially human organs are scarce, and the necessary methods are costly procedures. Furthermore, the risk of transmission of viral and prion diseases through the use of extracts from animals or humans limit the application of proteins derived from such sources.

The development of the recombinant DNA technology has provided a means for cheap, large scale recombinant production of proteins. Recombinant protein production has been of great value both to increase the understanding of the nature of proteins and in applied research. Two Nobel prize-rewarded achievements have been crucial for the development of recombinant DNA technologies, the discovery of restriction enzymes (Linn, 1968) and the invention of the polymerase chain reaction, PCR (Saiki, 1985). PCR is a powerful method for multiplication of DNA segments, whereas restriction enzymes and ligase allows for precise cutting and ligation of DNA fragments. The combination of the two technologies facilitates the introduction of protein-coding genes to host organisms for recombinant protein production.

In recombinant protein production strategies, the protein synthesis machinery of cells is employed to produce proteins from engineered DNA sequences. There are numerous recombinant DNA technologies that can be used for isolation and incorporation of a protein- coding gene into a host cell. The cells can originate from a wide range of organisms such as 18 TORUN EKBLAD bacteria, yeast, insects or mammals. A huge diversity of strategies for recombinant protein production has been reported, as well as down-stream processing protocols to obtain the pure protein (Gräslund, 2008). Although most proteins have the potential to be produced by recombinant technologies, certain types of proteins are not well suited for such routes. For instance, it is difficult to produce proteins that are toxic to the cell. Furthermore, membrane proteins and other poorly soluble proteins might be difficult to express successfully.

Another technology that has earned its inventor the Nobel Prize is solid phase peptide syntheis, SPPS (Merrifield, 1963). Peptide synthesis is an alternative to recombinant protein production routes, available to peptides and small proteins. In peptide synthesis, methods developed in the field of organic chemistry are applied to assemble polypeptides in an artificial manner. This strategy is discussed in further detail in chapter 3.3.

Depending on the production pathway, different options arise for modification of the peptides and proteins. The possibility to incorporate functional probes has been valuable to expand the applications of natural, recombinant and synthetic polypeptides.

3.2 Modifications of peptides and proteins

Peptides and proteins can be equipped with functional groups to make them better suited for a certain application. This has been an important strategy to expand the means in which polypeptides can be used, both as research tools but also in the clinic. Chemical modifications of different character can be realized through a diverse set of strategies commonly referred to as bioconjugation. In essence, bioconjugation is the process of coupling two biomolecules together via a covalent link.

Natural and recombinant proteins present several reactive groups that can be subjected to modifications by bioconjugation (Figure 4). The thiol function in the side chain of cysteine residues represents one such group, which can be addressed by different thiol-selective reagents. Cysteine modifications are typically accomplished via mixed disulfide formation, alkylation with ơ-halo carbonyl compounds, or addition to maleimide groups. However, cysteine is one of the least common amino acids in proteins. In addition, cysteines are often occupied in disulfide bonds and therefore not amenable to chemical modification without disruption of the protein structure. The N-terminal ơ-amino group and the ƥ-amino groups of lysine residues are other reactive functions present on the surface of proteins. These can be 19 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING modified by various reagents including active esters. Most often there are many amino groups in a protein, and it might be difficult to discriminate between them using amine-reactive reagents, which results in a heterogeneous mixture of modified species. Some of the reporter groups described below requires very precise positioning. For instance, certain probes respond to minor changes in the microenvironment and therefore require atomic accuracy in the conjugation method in order to realize its full potential. Such correctness might be difficult to accomplish by bioconjugation to amino groups, but can be addressed through chemical synthesis.

Figure 4. Schematic view of a protein surface with some representative functional groups

There is a plethora of different probes that can be used to modify the structure and function of peptides and proteins. One important class of reporter groups are those that can be used in protein detection assays, both for biotechnological and diagnostic applications. Affinity probes are widely used in biotechnology, as e.g. ligands in affinity chromatography to purify proteins, but also as immobilization handles to attach proteins onto solid supports. Biotin is a small hapten with a high-affinity interaction with streptavidin that has been extensively used as an affinity probe for many purposes. One important application has been to immobilize proteins onto surfaces, such as microarray slides. The labeling of proteins and peptides with fluorescent probes has been extensively used for various protein detection assays. The ability to fluoresce can be given a protein either through coupling of a fluorescent probe, or through fusion with a fluorescent protein such as the Nobel Prize-awarded green fluorescent protein, GFP (Chalfie, 1994) (Tsien, 1998). In particular, fluorescent probes have played a central role in the

20 TORUN EKBLAD development of protein microarrays. Fluorescence is further discussed in chapter 4.2.1. Another type of reporter groups that facilitate detection is molecular structures that are capable of coordinating radionuclides. Such chelators can be introduced to generate radiopeptides for various protein binding assays, including ELISA, but has also been of significant importance in the development of tracer molecules for diagnostic in vivo detection assays such as molecular imaging (molecular imaging is further described in chapter 4.3.). An example of probes that can be used to facilitate detection in biophysical studies of proteins is spin labels, that provides a means for studying peptide conformations in solution by electron spin resonance (ESR) (Marchetto, 1993). Another interesting type of chemical modification is polymer conjugation. Addition of polyethyleneglycol, PEG, is commonly used as a strategy to prolong the half-life of therapeutic peptides and proteins in vivo. PEGylation has been found to not only improve the circulation time, but bring additional benefits in terms of physical and thermal stability, resistance to proteolytic degradation, solubility and immunogenicity (Bailon, 2001).

Bioconjugation offers several valuable methods to modify proteins, however its scope of use is limited to the decoration of an already existing peptide backbone and cannot be applied for sequential integration of non-coded amino acids. A general strategy for introduction of non- natural amino acid derivatives at specific sites during recombinant production of proteins is offered by the nonsense suppression mutagenesis method (Noren, 1989). The method involves site-directed mutagenesis to introduce an amber stop codon at a site of interest, and in vitro aminoacylation of the corresponding tRNA. The non-natural amino acid is thereafter incorporated into the polypeptide chain by means of the ribosomal translation machinery. In a way, nonsense suppression mutagenesis can be regarded as a means to expand the genetic code beyond the use of the 20 natural amino acids. An important development in suppressor tRNA techniques has been the incorporation of amino acids with side chains carrying unique chemical groups for bioconjugation. Two examples are keto-carboxyl-containing amino acids for hydrazone or oxime bond forming modifications, and amino acids with azido functional groups for generation of triazole derivatives (Dieters, 2003). Many amino acid derivatives have been introduced into proteins using nonsense suppression, however the method is restricted by limits as to which side chains the ribosome can accommodate.

In addition to chemical modification of functional groups displayed on the protein surface, sophisticated gene fusion-based labeling methods can be used to modify the performance of recombinant proteins for certain applications. Such methods include the genetic fusion with immobilization tags, i.e. the hexahistidine tag or epitope tags that allow for detection with

21 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING specific antibodies. Gene fusion tags that allow for specific modifications inside cells have been reported, such as the Halotag, and the biotag. The Halo tag is based on a modified bacterial haloalkane dehalogenase and can be used for covalent bond formation with chloroalkane-fuctionalized probes (Los, 2008). The biotag consist of a biotin acceptor peptide fused to the gene for Escherichia coli biotin ligase (BirA) and can be used for producing biotinylated recombinant proteins in mammalian cells (Kulman, 2007).

Bioconjugation, genetic engineering and nonsense suppression offer valuable methods to modify functions of peptides and proteins, although they do not provide the operational freedom required for realization of modification patterns of complex or sensitive nature. As an alternative, organic synthesis is not limited to the use of natural or DNA-coded building blocks and offers means to introduce reporter groups and reactive probes in a site-specific manner, with the only assumption that they have to withstand the employed reaction conditions.

3.3 Chemical synthesis

3.3.1 History and development of Solid Phase Peptide Synthesis

The field of peptide chemistry begins with the synthesis of short amino acid oligomers by Emil Fischer in 1901 (Fischer, 1901) and Theodor Curtius in 1902 (Curtius, 1902). It was also then that the word peptide was first introduced, as Fischer named his product glycyl-glycine a dipeptide. A few years later, Fisher reported the synthesis of an octadecapeptide using acid chlorides for peptide bond formation (Fischer, 1907). Some 30 years later, Bergman and Zervas introduced the benzyloxycarbonyl (Cbz or Z) group as an amine protecting group that could be removed without cleavage of the peptide bonds, thus enabling controlled N-terminal chain elongation (Bergmann, 1932). The first biologically active synthesized peptide was oxytocin, reported by Du Vigneaud in 1953 (Du Vigneaud, 1953), for which he was later awarded the Nobel Prize in Chemistry. Although improved techniques and new reagents allowed for successful synthesis of an impressive number of small peptides, technical difficulties, such as poor solubility of protected peptides and slow reaction rates hampered the realization of synthesis of longer polypeptides. Also, purification of the product from by- products became a major issue as the number of residues increased.

22 TORUN EKBLAD

A new era for peptide synthesis began in 1963, when Bruce Merrifield introduced the concept of solid phase peptide synthesis, (SPPS). The Nobel Prize-awarded technology relies on the anchoring of the C-terminal amino acid to a solid polymer support, and the subsequent coupling and deprotection of N-protected amino acids in a stepwise manner and release from solid support upon completed synthesis (Figure 5). The first report of this technology was the synthesis of a tetrapeptide using chloromethylated polystyrene as solid support, carbobenzoxy- protected amino acids as building blocks and deprotection with strong acid (Merrifield, 1963). The major achievement was that reagents and impurities could be removed by washing and filtration, thus allowing for excess use of reagents to drive the coupling reactions to completion. When Merrifield later introduced the t-butyloxycarbonyl (Boc) group as an amine protecting group and used a benzyl ester as a linker to the solid phase, milder reaction conditions could be used (Merrifield, 1964), allowing for synthesis of longer sequences, such as insulin (Marglin, 1966), and the 124 aa bovine pancreatic ribonuclease A (Gutte, 1971).

The repetitive acid treatments during syntheis with the Boc/benzyl strategy, and the use of strong acid in the final deprotection step caused a number of undesired side reactions. It became evident, that for biologically active proteins to become readily attainable by synthetic means, even milder reaction conditions were required. The base-labile 9- fluorenylmethyloxycarbonyl (Fmoc) protection group was introduced by Carpino and Han in 1972 (Carpino, 1972) and realized for solid phase synthesis by Atherton and coworkers (Atherton, 1978a, Atherton, 1978b). By using Fmoc as a temporary amino protecting group in combination with acid labile tert-butyl derivatives for side chain protection, a complete differentiation in reaction conditions for the different deprotections was achieved, which was not possible when using acidic reagents of varying strength. This chemical differentiation is often referred to as orthogonality. In the context of synthetic chemistry, an orthogonal system is defined as a set of completely independent classes of protecting groups, where each class of groups can be removed in any order and in the presence of all other classes (Barany, 1977). Orthogonality is further discussed in chapter 3.3.3.

23 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

Figure 5. The principles of solid phase peptide synthesis

24 TORUN EKBLAD

3.3.2 Principles of Fmoc SPPS

The underlying principle of most peptide bond-forming coupling methods is the activation of the carboxyl group with an electron-withdrawing leaving group that renders the carbon atom susceptible for nucleophilic attack by the amino group. The activating group or reactions should be carefully chosen in order to achieve high reaction efficiency and at the same time avoid side reactions. There are several different coupling methods employed in solid phase synthesis. Traditionally, carbodiimides have been used as in situ activating reagents (Sheehan JACS 1955). Carbodiimides have also been used to generate pre-activated symmetrical anhydrides. The addition of triazolols, such as 1-hydroxy-benzotriazole (HOBt) or 1-hydroxy- 7-aza-benzotriazole (HOAt) in the pre-activation of Fmoc-amino acids with carbodiimides was introduced later and found to result in active esters with slightly reduced reactivity and reduced propensity for racemization. More recently, in situ activating reagents have emerged that totally omit the use of carbodiimides, such as the uronium or phosphonium-based salts HBTU, HATU and PyBOP. In situ activating coupling reagents are the most widely used procedure today, being suitable for use in automated synthesis and giving fast reactions also for sterically hindered amino acids.

Using any of the activation methods above, amino acids are coupled sequentially in a stepwise manner to amino groups presented on the resin. In order to allow for unambiguous peptide bond-formation, all reactive sites but the carboxyl group of the amino acid has to be masked. Not all amino acids require side chain protection groups, but certain reactive groups need special attention. The alcohol functions of serine and threonine, the phenol of tyrosine, the guanidine of arginine and the thiol of cysteine can all undergo acylation and alkylation and need protection during the synthesis to prevent the formation of branched peptides. Histidine can undergo both acylation and racemization if not protected properly. The carboxyl groups in the side chains of aspartic and glutamic acids must be protected in order to achieve unambiguous activation. Asparagine and glutamine might be used without protection of the primary amide functionalities, however dehydration during activation can be avoided by use of protecting groups.

In the Fmoc/tBu strategy, fluorenylmethoxycarbonyl- is used as a temporary Nơ-protection group and tert-butyl-derivatives (tBu and tBoc) are used for permanent protection of side chains (Figure 6). Since this original setting was invented, the repertoire of side chain protection groups has been extended beyond the use of tBu and numerous alternative groups are currently in use (Figure 6), all designed to serve particular needs. The trityl group (Trt) is commonly used on Asn, Gln, His, and Cys, and the Pbf or Pmc groups are used for guanidine 25 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING protection on Arg. For routine Fmoc synthesis, all side chain protection groups are chosen to be TFA-labile. A special class of protecting groups is those used for orthogonal protection discussed in chapter 3.3.3.

Figure 6. Protection groups used in Fmoc chemistry

Upon completed coupling of an amino acid to the resin, the Nơ-protection group Fmoc is removed by base treatment (typically 20 % piperidine) to expose a new amino group for the next amino acid to react with. Chain elongation continues in a repeated cycle of amino acid activation, coupling and deprotection, until the sequence is completed. The polymer support to which the peptide is anchored is insoluble and stable to the chemical reactions carried out in the synthesis. The solid support facilitates washing and filtration so that excess of reagents can be removed, but it also promotes solvation of the poorly soluble protected peptide. Merrifield used cross-linked poly(styrene-divinylbenzene) in his initial settings. This matrix is still in use but has been accompanied by more polar polymers with increased mechanical stability and improved solvation behavior, such as cross-linked poly(dimethylacrylamide) (Atherton, 1975), co-polymerized ethylene oxide and polystyrene (Bayer, 1991) and grafted PEG-polystyrene (Sherrington, 1998, Zalipsky, 1994). The term resin is often used when referring to the polymer support equipped with a linker. The linker serves to attach the first (C-terminal) amino acid to the polymer support and form a bond that is stable throughout the synthesis. The linker is cleaved only when the peptide has been completed. This final deprotection step often also includes the simultaneous deprotection of the permanent side chain protecting groups (often with strong acid, such as 95 % TFA). C-terminal peptide amides are typically synthesized by anchoring the peptide via an amide bond to Rink amide, Pal, or Sieber resins,

26 TORUN EKBLAD whereas ester bonds to Wang, HMPB, Sasrin, or tritylchloride resins are used to generate C- terminal peptide acids. By using other linkers, the peptide can be designed with various alternative C-terminal end groups (Alsina, 2003).

An attractive feature of solid phase peptide synthesis is that it is not restricted do natural or DNA-encoded amino acids, but allows for incorporation of many different types of building blocks, including polymers and fatty acids. The ability to introduce non-natural residues that alter the structure or function of polypeptides has been applied to improve the pharmacokinetics of proteinaceous drugs. For example, D-amino acids have been incorporated in synthetic polypeptides as a strategy to improve the stability to enzymatic challenges (Dooley, 1994). Other unnatural amino acid derivatives, such as ơ-aminoisobutyric acid, have been valuable in peptide ligand design as a way to introduce conformational constraints to improve binding affinity (Karle, 1990).

3.3.3 Orthogonal protection strategies

As discussed above, the Fmoc/tBu chemistry is in itself orthogonal, with Fmoc being liberated by piperidine through a Ƣ-elimination reaction and tBu by acidolysis with TFA. Additional dimensions of orthogonality can be added to the peptide by using side chain protecting groups with unique deprotection mechanisms (Figure 7). Such groups can be selectively removed in the presence of other side chain protection groups to liberate a single functional site for subsequent modification and have been reviewed by (Albericio, 2000). Orthogonal protecting groups can be divided into classes depending on their deprotection mechanism.

Certain protection groups have been developed for selective unmasking with acid treatment that is weak enough to leave other acid labile side chain protecting groups unaffected. The methyl trityl group (Mtt) is one such group that can be used for selective protection of ƥ-amino groups of lysine residues and of residues with side chain hydroxyl or thiol functions. Mtt is liberated with 1 % TFA in DCM. Even more acid labile derivatives have been reported, such as the monomethoxytrityl (Mmt) and dimethoxytrityl (Dmt) groups (Matysiak, 1998). A more strict orthogonal deprotection mechanism is Pd(0)-catalyzed allyl transfer, which can be used to displace allyl-based protecting groups (Stevens, 1950). The allyl and allyloxycarbonyl (Alloc) groups can be used to protect a carboxylic acid, hydroxyl, or amino group and are stable to both acid and base treatments. Thiolysis is a mild procedure that can be employed for the liberation of dithiasuccinylamino groups (Dts) on Nơ-amines (Barany, 1977). Another strategy 27 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING is the use of nucleophiles, such as hydrazine, as selective deprotection agents for Dde and ivDde groups (Bycroft, 1993). As an additional alternative, photosensitive protecting groups are removed through exposure to light (Patchornik, 1970). Also photolabile linkers have been reported (Hammer, 1990) and photoorthogonality can be accomplished by using groups with sensitivity to distinct wavelengths.

Figure 7. Examples of orthogonal protecting groups

3.3.4 Limitations of SPPS

Despite ingenious design of numerous protection groups and activation protocols, some side reactions are associated with the synthesis of peptides. The activation of the carboxyl group generates a planar carbon, which is susceptible to racemization in the presence of organic bases. Amino acid racemization is a serious side reaction since enantiomeric peptides are known to have very different biological activity. Another observed side reactions in peptide synthesis is the formation of aspartimide by the sequence-dependent cyclization of aspartic acid (Yang, 1994).

In addition to specific side reactions, certain sequences are known as “difficult”, being resistant to efficient assembly by solid phase peptide synthesis (Chan, 2004). The difficulty is thought to be due to self-association by hydrogen bond formation leading to aggregation of the resin-

28 TORUN EKBLAD bound peptide, resulting in poor solvation and, consequently, incomplete deprotection and coupling (Tam, 1995), (Tickler, 2004). This phenomenon is length and sequence dependent, and is mainly a problem for peptides rich in hydrophobic and Ƣ-branched residues. The resin type and loading has been identified as critical parameters to prevent aggregation. By using a lower loading the probability of intermolecular interactions leading to aggregation is reduced. Polymer supports with polar character have been found to reduce the propensity of hydrophobic peptides to undergo self-aggregation. Different organic solvents vary in their capacity to solvate peptides with difficult sequences and in addition, chaotropic salts have been used to break aggregates. Structural changes in the backbone can be introduced by employing pseudoproline derivatives to prevent aggregation (Mutter, 1995).

Due to the iterative nature of the solid phase peptide synthesis method, every step has to be performed with high efficiency to allow for production of longer polypeptide chains with high yield. Generally, peptide sequences longer than 100 amino acids have been difficult to assemble with a reasonable yield. Already in Merrifield’s pioneering paper (Merrifield, 1963) he identified several pitfalls that still engage peptide chemists. Incomplete deprotection results in truncated peptides. However, the effect might also be temporary, and followed by complete deprotection in a later cycle, resulting in a deletion peptide. Truncation and deletion peptides also arise when the coupling reaction is incomplete, however formation of deletion peptides might be overcome by introducing a capping step that terminates further elongation of such peptides. The formation of truncated side products decrease the overall yield and are potentially difficult to separate from the desired full-length product.

3.3.5 Peptide ligation of synthetic fragments

Already in 1902, Emil Fischer predicted that peptide synthesis should facilitate the preparation of synthetic enzymes. Although large peptides, up to the size of proteins, with biological activity have indeed been generated, the realization of larger proteins relies on condensation strategies where the sequence is split into smaller fragments that can be efficiently synthesized individually and thereafter linked together using a variety of peptide ligation chemistries. Peptide ligation was initially developed as a strategy to overcome the size-limit of peptide synthesis and realize the synthesis of larger polypeptides and proteins (Schnolzer, 1992), but has since found useful for generating branched peptides, peptide dendrimers, and production of peptid e and protein mimetics.

29 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

In one type of peptide ligation, very acid labile linkers are used to enable release of peptide fragments from the resin in a protected form. Protected peptides can be coupled using standard carboxy-activation chemistry. However, free protected peptides are poorly soluble in most solvents, which result in low yields. In addition, care has to be taken in order to prevent racemization at the ligation site. A more efficient chemical ligation is selective linking of unprotected peptide fragments by means of mutually reactive groups that are unreactive to all other functional species in the conditions used. An important advantage of using unprotected peptides is that the ligation can be performed in aqueous solution at neutral pH. Several peptide ligation chemistries have been developed for unprotected peptide fragments, and the approaches can be divided into two groups depending on the character of the resulting bond; i) ligation reactions that generate native peptide bonds and ii) non-amide ligation strategies that result in alternative linkages (Table 2).

Native chemical ligation has been reported by Kent and coworkers as a strategy where a peptide bond is formed between two unprotected peptide fragments in a two-step reaction (Dawson, 1994). The first reaction step is a chemoselective capture reaction between the first segment, carrying a C-terminal thioester, and the second segment, with an N-terminal cysteine residue, resulting in a thioester-linked intermediate. Due to the favorable geometric arrangement of this intermediate, a proximity-driven intramolecular rearrangement occurs spontaneously to yield a native peptide bond. This peptide bond-forming reaction discriminates one specific ơ-amine from other ơ- and ƥ-amines without the use of protecting groups and consequently fits in the definition of orthogonal systems, described above. Tam and coworkers refers to this type of peptide ligation reaction as orthogonal ligation, and have reported on various alternative electrophiles and nucleophiles to accompany the ơ-acyl- and ơ- amine- moieties in similar entropic activation strategies for peptide bond formation (Liu, 1994a) (Chan, 2004). Amide ligation approaches only allow for head-to-tail ligation of peptide fragments. In order to accomplish other rearrangements, non-amide ligation approaches can be employed.

Similar to orthogonal ligation approaches, non-amide ligation utilize chemoselective reactions between a pair of mutually reactive groups on two segments. Such selectivity can be provided by using the reaction of aldehydes with hydroxylamines, hydrazines or aminothiols, resulting in the formation of oxime (Shao, 1995), (Rose, 1994), hydrazone (Fisch, 1992) or thiazolidine linkages (Liu, 1994b). Peptide aldehydes can be generated by oxidation of a precursor peptide carrying a 1,2-diol or a 1,2-aminoalcohol group, or from resins that yield C-terminal aldehydes. Thiols and haloacetyl groups are another example of pairs of functionalities that can be

30 TORUN EKBLAD employed for chemoselective ligation (Lu, 1991). Both alkyl and acyl thiols can be used, and haloacetyl moieties can be site-specifically incorporated to the side chains of lysine residues. The ligation method is called thioalkylation and the resulting bond is a thioether. This strategy has been used in paper VII and is discussed further in chapter 6.3.

Table 2. Examples of peptide ligation chemistries

Non-amide ligation chemistries provide more flexibility in the joining of peptide segments compared to orthogonal methods. Various efforts have generated ligated peptide products with amino end-to-amino end, carboxyl end-to-amino end or end-to-side chain structures. By incorporating the reactive functions into the same sequence, side chain-to-side chain cyclized peptides can be produced (Chan, 2004).

One successful example where peptide ligation strategies have been used is the synthesis of the 166- amino acid erythropoiesis protein, Epo. Kochendoerfer et al. were able to assemble a biologically active protein with precisely defined covalent structure (Kochendoerfer, 2003). The protein sequence was divided into four peptide fragments that were synthesized individually. Oxime ligation was used for site-specific introduction of polymers to mimic a post-translational glycosylation pattern, and native chemical ligation was used for assembly of

31 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING the peptide fragments. The synthetic variant had comparable specific activity in vitro, but superior duration of action in vivo, relative to human erythropoietin.

Peptide ligation strategies provide means for precise atom-by-atom control of the covalent structure, and can be used in combination with orthogonal protection strategies and site specific modifications as a valuable tool to systematically correlate protein structure with function. Furthermore, ligation of peptide fragments is an attractive strategy to assemble large proteins, which cannot be obtained from one synthesis. Ligation chemistries have also been used in semisynthetic preparation of large proteins to assemble fragments from recombinant and synthetic productions. Semisynthesis is an attractive approach for the preparation of membrane proteins. The soluble intra- and extra-cellular domains can be produced by recombinant technologies whereas the less soluble transmembrane parts can be produced by chemical synthesis, followed by ligation using chemoselective strategies.

Protein splicing is a post-translational modification process in which protein precursors undergo self-catalyzed intramolecular rearrangements, resulting in the linking of an N-terminal and C-terminal extein fragment by the removal of a separating intein fragment. This process has been exploited in biotechnology to ligate peptide fragments of interest by fusion to inteins. Expressed protein ligation and protein trans-splicing are examples of intein-based semisynthetic approaches to protein assembly (Muralidharan, 2006). have also been used for ligation of peptide fragments, and by genetic engineering it has been possible to generate mutant proteases, favoring the ligation reaction over hydrolysis of the peptide bond. This strategy is exemplified by subtiligase, which is a mutant of the serine protease subtilisin BPN' that can be used for assembly of peptide fragments (Abrahmsén, 1991), (Chang, 1994). Another semisynthetic approach is cyanogen bromide cleavage at methionine residues in recombinant proteins to generate a homoserine lactone (Villa, 1989) that can be used to form a hydrazone linkage to peptides or proteins with aldehyde or keto groups.

32 TORUN EKBLAD

4. Applications of antibodies and alternative binding proteins in bioassays

4.1 Monitoring biomarkers

The levels of proteins in cells and extracellular fluids can vary as a consequence of medical conditions, due to increased or reduced endogenous protein expression related to the disease, leakage of cellular proteins from damaged tissue, or to the introduction of exogenous proteins from viruses or bacteria. Cancer, inflammation, viral infections and various genetic disorders are examples of conditions that can generate altered protein levels. A protein with a disease- regulated presence and a defined “normal’ concentration level can be used as a biomarker. There are different types of biomarkers, and three important categories are prognostic, predictive and pharmacodynamic biomarkers (Sawyers, 2008). Prognostic biomarkers provide information regarding the natural course of a disease and can be used to distinguish good outcome from poor. Predictive biomarkers can be used to assess the probability that a patient will benefit from a particular treatment and the pharmacodynamic biomarkers are used to guide dose selection.

Molecular biomarkers in blood plasma that could classify complex and severe diseases, and identify presymptomatic individuals would be particularly useful and yield substantial therapeutic and health-economic benefits. Single biomarkers have been identified that can be used individually to diagnose a disease, such as the prostate specific antigen, PSA, that is routinely detected in the clinic as a diagnostic biomarker for prostate cancer (Catalona, 1991). However, not all diseases can be identified by the regulation of a single protein, and it is more likely that multiple biomarkers have to be studied in parallel in order to identify unique molecular fingerprints associated with certain conditions. One example of such efforts is a study of plasma samples from individuals with Alzheimer's disease, where a regulated pattern of 18 predictors could be identified from studies of 120 known signaling proteins (Ray, 2007).

Powerful protein detection methods are needed to identify biomarkers. The focus of several large-scale proteomics efforts is to identify biomarkers that can aid an early detection of

33 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING disease and allow for individualized treatment. Traditionally, expression proteomics has been performed by using two-dimensional gel electrophoresis to separate proteins from complex mixtures (Gorg, 2004). Differences in isoelectric point and size result in a unique pattern of separated protein spots. Biomarkers can be identified by comparing such patterns, generated from different samples. Differentially expressed proteins can be identified by mass spectrometric analysis of samples excised from the gel. Alternatively, a strategy named shotgun proteomics is based on enzymatic digestion of whole samples, separation of the digested peptide fragments by liquid chromatography, and the subsequent identification of the fragments by mass spectrometry (MacCoss, 2002). A completely different approach has been taken in the Swedish Human Proteome Atlas project, where the aim is to generate monospecific antibodies to all human proteins and provide an atlas of the detailed localization profile of each protein in 48 normal human tissues and 20 different cancers (Uhlén, 2005).

Well-characterized biomarkers are valuable tools that can bring an increased understanding of many complex diseases and disorders. In particular, biomarkers can be used in diagnostics to study the progress of a disease or to identify individuals with an increased risk of being affected by a certain disease. In order to achieve such information, assays that allows for highly sensitive detection of several relevant protein biomarkers in parallel from complex clinical samples are needed. Several in vitro detection methods exist for monitoring proteins. In relation to certain diseases such as cancer, it is also desirable to study protein expression and distribution within the body using in vivo protein detection assays.

4.2 Detection of proteins in vitro

Proteins possess many biophysical properties that can be used for specific identification, including size, charge and mass that can be determined by electrophoresis, isoelectric focusing, and mass spectrometry, respectively. In addition, hydrophobicity can be evaluated by reversed phase chromatography and the amino acid composition can be studied by UV absorbance. Generally, these are all methods for biochemical analysis of single proteins in samples with relatively simple composition, although their applicability can be expanded to separation and identification of specific target proteins from complex mixtures. Typically, this is achieved by combining several techniques, such as in two-dimensional gel electrophoresis or strategies based on sophisticated settings of liquid chromatography and mass spectrometry. A different type of detection assays is those that are based on molecular recognition. Affinity-based detection methods rely on affinity reagents for specific binding to target proteins and a 34 TORUN EKBLAD reporter system that convert the binding event to a detectable signal, such as fluorescence, luminescence or radioactivity. Assuming that specific affinity reagents can be developed for the target protein of interest, such methods should allow for protein capture and identification from samples of very diverse composition.

The first affinity-based detection method was the radioimmunoassay (RIA), developed in the late 1950’s by Yalow and Berson (Yalow, 1959) (Yalow, 1960). In its original setting, RIA was a competitive assay format where radiolabeled bovine insulin competed with unlabeled human insulin for binding to polyclonal antibodies. Separation and detection of ligand-bound antibodies were performed by paper chromatography. The concept of immobilizing the affinity reagent on a solid phase to facilitate removal of unbound ligands was first introduced by Porath and coworkers (Wide, 1966) and later used in the development of the enzyme-linked immunosorbent assay, ELISA (Engvall, 1971). Also ELISA was originally described as a competitive assay, with alkaline phosphatase-linked IgG competing with native IgG for binding to anti-IgG antibodies on a cellulose matrix. Later, non-competitive sandwich assays were developed, using one antibody for ligand capture and a secondary enzyme-linked antibody, directed to a different site on the ligand, enabeling detection.

The need to simultaneously study expression levels of panels of proteins has encouraged the development of multiplexed microarray technologies for protein analysis. Ekins and coworkers were the first to describe a multi-microspot immunoassay in which antibodies with different binding specificities were immobilized in a pattern of rows and columns to allow simultaneous, parallel detection of many different proteins (Ekins, 1990, Ekins, 1989). However, it was not until the early 2000’s, largely inspired by the successful development of DNA microarrays for expression profiling, that protein microarray technologies were developed and reported as useful research tools. Since the initial reports on protein arrays, several technological advancements have been accomplished, and protein microarray technology has now been successfully applied for the identification, quantification and functional analysis of proteins in basic and applied proteome research (MacBeath, 2002) (Templin, 2003).

The array format has been amenable to miniaturization so that very small amounts of many, typically hundreds to thousands, different molecules can be arrayed on a small surface. A miniaturized assay format requires minute sample volumes, which is particularly attractive for clinical diagnostics of precious patient samples. A critical parameter in the development of protein microarrays is the availability of highly specific and high-affinity capture molecules. High affinity is essential to achieve a sensitive detection assay. Polyclonal and monoclonal

35 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING antibodies, antibody-derived fragments, affinity proteins from alternative scaffolds, peptides and nucleic acid aptamers are examples of biomolecules that have been used as capture molecules on protein microarrays (Templin, 2003).

Immobilization of a protein onto a solid support with retained biological activity is not a trivial task. Proteins have a three-dimensional structural fold that is intimately associated to its biological function, and the immobilization has to be performed in ways that do not interfere with the folding. Most commonly, glass microscope slides are used as the array surface. In order to provide a biocompatible structure for protein attachment, different polymeric matrices have been investigated for derivatization of the glass surface (Zhu, 2003). A variety of strategies can be applied to couple the affinity protein, including passive adsorbtion, covalent cross-linking and affinity tag interactions. As an alternative to planar microarrays, bead-based systems have been used for multiplexed analysis of a limited number of analytes (Joos, 2002). In such systems, color- or size-coded beads are used as solid support on which capture molecules are immobilized and flow cytometry systems enable the simultaneous discrimination of bead types and the quantification of an analyte with suitable reporter dyes.

The use of high affinity ligands and signal amplification methods are strategies that have been used to improve the detection limit and assess low abundant proteins. By using a sandwich- based detection strategy, a higher specificity can be achieved. However, not all antigens are accompanied by two corresponding high affinity binding molecules. Most protein array settings use fluorescence as the detection method, both because of its high sensitivity, but also because fluorescence microarray scanners early were developed and made commercially available for use in DNA microarray experiments.

4.2.1 Fluorescent detection in protein microarrays

Fluorescence is a photophysical process in which light is emitted as a consequence of electronic excitation of certain molecules (Figure 8). Fluorescent molecules are called fluorophores. Typically, fluorophores consist of conjugated systems of unsaturated bonds. Electrons in such systems can be excited from their ground state to a higher electronic singlet state by the absorption of a photon. The excited electron falls back to the lowest vibrational level and loses some of its energy through internal conversion before it can relax to the ground state by emitting the remaining excess energy in the form of a new photon. The energy difference between the exciting and the emitted photons is termed the Stoke’s shift and allows for excitation of a fluorophore at one specific wavelength and fluorescent detection at another.

36 TORUN EKBLAD

Figure 8. A. The principle of fluorescence. Photons of energy hƭex result in the excitation of an electron from the ground state, S0, to the excited state, S1’, from which the electron relaxes to S1 and falls back to the ground state by emission of a photon of energy hƭem. B. Structures of the commonly used fluorophores Cy3 and Cy5. C. The principle of FRET. The emission from an exited donor fluorophore results in the excitation of a proximal acceptor fluorophore due to a spectral overlap

Proteins can be labeled by using a number of reactive derivatives of fluorescent dyes, such as N-hydroxysuccinimide esters or isothiocyanates for amino group modification or maleimides or haloacetyls for conjugation to thiols. In protein microarray experiments, fluorescence can be used in several conceptually different strategies. The whole sample can be labeled before it is allowed to bind on the array, and the captured proteins can be detected through their fluorescence after removal of unbound proteins (Wingren, 2005). Alternatively, captured proteins from unlabeled samples can be detected by using a fluorescently labeled secondary affinity reagent in a sandwich assay (Gao, 2005). Protein expression from different samples can be compared on one single microarray in a two-color expression analysis, similar to the strategy developed for expression profiling on DNA microarrays. In such strategies, one sample can be compared to a reference sample by labeling with different fluorescent probes, typically the organic dyes Cy3 and Cy5 (Figure 8). The samples are pooled and incubated on

37 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING the array and the intensities of the fluorescent signals can be compared. Differences in protein expression are identified by increased representation of the particular dye (Sreekumar, 2001).

Although regular fluorescence is the most common detection method for protein microarrays, several attempts have also been made to utilize other fluorescence-based technologies for detection on protein microarrays. Various types of nanoparticles with high fluorescence intensity, photostability, and biocompatibility have been developed and used for detection in protein microarrays (Wu, 2008), and carbon nanotubes have been used as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format (Chen, 2008). Different aspects of environmentally sensitive probes have been investigated for microarray applications (Nagl, 2008) and fluorescence resonance energy transfer has been used as a detection method in protein arrays (Usui, 2004). Fluorescence resonance energy transfer (sometimes also referred to as Förster resonance energy transfer after its discoverer (Förster, 1948)), FRET, is a phenomenon where energy is transferred from one fluorophore to another through non-radiative dipole-dipole interactions. For FRET to occur, a spectral overlap of the donor emission wavelength and the acceptor absorption spectrum is required (Figure 8). Furthermore, the donor and acceptor dipole orientations must be approximately parallel, and the molecules must be positioned in close proximity, typically less than 100 Å, for efficient energy transfer.

4.3 Detection of proteins in vivo

4.3.1 Molecular imaging

Molecular imaging is a methodology to study disease-related molecular alterations in vivo. Oncology is one field where imaging techniques have been valuable for detection of tumors, and to characterize their molecular phenotype. The number of patients diagnosed with cancer have been increasing in recent years, with the most common types of cancers world wide being lung, breast, prostate and colon cancer (Parkin, 2005). Most often, it is not the primary tumor, but its distant metastases that are the main cause of death. Screening programs, such as mammography are used in many countries to enable early detection of tumors and improve the chance of successful treatments. Molecular imaging has emerged as an accompanying method with the ability to assess also the molecular status of the tumor. This is accomplished by using labeled agents that accumulate at the tumor site, either due to specific molecular

38 TORUN EKBLAD recognition of tumor markers (Mankoff, 2008), or as an effect of an elevated biological activity at the tumor site, such as increased rate of glucose metabolism (Plathow, 2008), or DNA synthesis (Bading, 2008). In targeted molecular imaging, a patient is given a labeled substance that binds to a target molecule of interest and accumulates at sites where that particular molecule is abundant. The label is detected from outside the body and reflects the biodistribution of the tracer. Molecular imaging has potential to allow for a precise localization as well as molecular classification of primary tumors and metastatic lesions. The method is non-invasive, as compared to alternative methods such as endoscopy or methods, which rely on in vitro examination of excised biopsy samples. Different detection methods can be used, such as ultrasound (Dayton, 2002), optical fluorescence (Contag, 2002) or magnetic resonance imaging (Hogemann, 2002), however the far most widely adopted strategy is the use of radioisotopic labeling.

4.3.2 Tumor targeting

The perfect tumor target should be exclusively associated with the cancer cells and absent in normal tissue. Although cancer cells are unique in many aspects, such targets have been difficult to identify. However, molecular structures that are overexpressed in tumors and expressed only to a limited extent in normal cells have been identified and proved useful as targets. Examples of such molecules are cell-surface proteins, both on solid tumors and on circulating malignant cells, and antigens associated with the tumor-surrounding vasculature or extracellular matrix. Cell surface structures are well suited as targets since they can be expected to be easily accessible to agents circulating the blood stream. Several such molecules have been identified as markers for various cancers, including members of the epidermal growth factor receptor (EGFR) family (Yarden, 2001). An increased understanding of tumor biology has lead to the development of drugs that target particular molecular structures or associated signalling pathways in cancer. Detection of expression of such targets is important for selection of patients who would benefit form particular types of therapy. A few imaging agents are approved for in vivo diagnosis of cancer, including ProstaScint (anti-PSA mAb for detection of prostate, colon and ovarian cancers), OncoScint (mAb specific for tumor-associated glycoprotein in colon and ovarian cancer) and CEA-scan (anti CEA-antibody for colon and breast cancer detection) (Leader, 2008).

Some critical parameters that have to be considered when selecting a targeting agent for molecular imaging are affinity, selectivity, biological half-life and catabolism pathways. These 39 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING factors all affect the pharmacokinetic properties of the imaging agent and are further discussed in chapter 4.3.7. In addition to analogs of natural peptide receptor ligands, antibodies and antibody fragments have been used for targeted molecular imaging. The humanized monoclonal antibody trastuzumab (Herceptin) targets the human epidermal growth factor receptor 2 (HER2) and has been approved for treatment of metastatic breast cancer since 1998 (Walsh, 2003). Radiolabelled trastuzumab has also been used for imaging of HER2 expression in tumors (Behr, 2001). However, smaller (Fab)’2 fragments of trastuzmab have been found to be favorable as imaging agents due to a more rapid blood clearance (Smith-Jones, 2004). Other examples of antibody fragments that have been used as HER2-imaging reagents in mice with superior tumor diffusion capacity compared to full-sized antibodies are scFv’s and diabodies

(Adams, 2004). A camelid single VHH domain antibody (nanobody) was recently reported to yield high tumor-to-organ ratios when used to target EGFR in mice (Huang, 2008). In addition, several of the affinity proteins described in chapter 2.3 have potential as tumor targeting agents. A HER2-binder has been generated using the DARPin scaffold and evaluated for tumor interaction ability (Zahnd, 2007). Furthermore, Affibody molecules have been investigated as targeting agents directed to HER2 (Wikman, 2004) (Orlova, 2006) and EGFR (Friedman, 2008). Papers III-VII in this thesis describe efforts to tailor Affibody molecules for in vivo diagnostic imaging of HER2.

4.3.3 Radionuclides for in vivo studies

Radionuclides are atoms with unstable nuclei that decay to more stable states through emission of ionizing radiation. The emission can take the form of alpha particles, beta particles, or gamma radiation, depending on the nature of the decay. For clinical imaging, it is important that the radiation can penetrate the human body and be detected by an external detector. For this reason, two classes of nuclides are used for medical imaging; positron-emitting nuclides and nuclides that decay by electron capture or isomeric transition and emit gamma-quanta with energy of 80-300 keV.

Besides the type of decay, other aspects of the radionuclide that has to be considered in the context of diagnostic imaging are half-life, chemical properties, cost and availability. Ideally, the nuclide should be chosen so that its physical half-life matches the biological half-life of the targeting molecule. Radionuclides with too short half-life will lose a lot of its activity through decay before the targeting agent reaches the tumor site, whereas the use of too long-lived nuclides requires an increased concentration of the radiolabeled substance in order to generate 40 TORUN EKBLAD enough decays before excretion. The methods by which radionuclides are produced vary widely, with some requiring cyclotrons whereas others are produced using generators. A generator consists of a column with an immobilized mother radionuclide that decays to a more short-lived daughter nuclide that can be eluted periodically. This offers an inexpensive source of diagnostic nuclides. Cyclotron products can only be generated at sites with particle accelerators, which increase the cost and also limit the availability of such nuclides. Some of the nuclides that are currently in use for diagnostic imaging are listed in Table 3.

Table 3. Radionuclides for diagnostic imaging

Radionuclide Emission T1/2 Application Production

99mTc Ȗ 6 h SPECT 99Mo/99mTc-generator

123I Ȗ 13.3 h SPECT Cyclotron

111In Ȗ 2.8 days SPECT Cyclotron

67Ga Ȗ 3.3 days SPECT Cyclotron

11C ȕ+ 20 min PET Cyclotron

68Ga ȕ+, Ȗ 1.1 h PET 68Ge/68Ga-generator

18F ȕ+ 1.8 h PET Cyclotron

64Cu ȕ+, Ȗ 12.7 h PET Cyclotron

76Br ȕ+, Ȗ 16.2 h PET Cyclotron

124I ȕ+, Ȗ 4.18 days PET Cyclotron

Traditionally, radioiodination with 123I or 124I has been the labeling method of choice for generation of tracer substances (Hnatowich, 1990), mainly due to its straightforward labeling chemistry. 123I is a widely used nuclide in the clinics, however it is expensive, which limits its application. 18F has been widely used for tumor imaging with the glucose analog 2-deoxy-2- fluoro-D-glucose (18F-FDG). Although few examples of efficient radiofluorination of peptides exist, an oxime-based labeling method was recently suggested, in which 18F-labeled aldehydes or ketones were used for radiofluorination of aminooxy-functionalized peptides (Poethko, 2004). This method has been applied to label several proteins, including Affibody molecules (Namavari, 2008). Other biogenic elements such as 11C, 13N and 15O, allows for radioactive labeling without altering the native set of atoms in a tracer. However, their short-lived nature 41 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

(half-lives of 20 min, 10 min and 2 min respectively) can not match the kinetics of peptides or proteins and their use is limited to labeling of small molecules such as e.g. amino acids and hormones. 111In is a radiometal that offers good imaging properties with emission of ƣ- photons of 173 and 247 keV that are well-suited for ƣ-scintigraphy. An attractive alternative to costly cyclotron-generated nuclides, such as 111In, are those that can be produced from generators. The positron emitter 68Ga is one such example that can be produced from 68Ge 99 99m (T1/2 271 days) (Maecke, 2005). A Mo/ Tc generator system has been successfully used during decades for inexpensive production of 99mTc. The majority of all radionuclide diagnostic applications use 99mTc as a label. Its gamma energy of 140 keV is ideal for gamma camera imaging and the half-life of 6 h corresponds well to the in vivo kinetics of peptide-based targeting agents. Technetium labeling is further discussed in chapter 4.3.6.

4.3.4 Radionuclide imaging techniques

Gamma radiation consists of photons of discrete energies that are emitted from excited nuclei as a means to gain a lower energy state without altering the number of protons or neutrons. Targeting agents carrying radionuclides that emit gamma radiation can be detected using single photon imaging methods such as planar gamma camera imaging or single photon emission computerized tomography (SPECT). The detection of single photons in a gamma camera occurs in large planar NaI(TI)-crystals that scintillate in response to incident gamma radiation (Anger, 1958). A lead collimator is used to select only photons that are emitted perpendicular to the crystal. The resulting scintillation events are detected in photomultiplier tubes and summed to generate a two-dimensional projection of the distribution of the tracer element. The range of gamma energy that can be used for single photon imaging is restricted by the design of the collimator grid and the thickness of the detector crystal. Typically, energies in the range 80-300 keV are suitable. One limitation of planar scintigraphy is that it generates a depth-integrated two dimensional illustration of a distribution that in reality covers three dimensions. In SPECT, gamma camera projections are collected from multiple different angles to generate a three dimensional image of the biodistribution of the tracer. A typical SPECT investigation of a patient can take up to 30 min, and therefore a steady state distribution of the radionuclide is preferable.

In positron emission tomography, (PET), positron-emitting nuclides deposited into tissue decay with emission of positrons that travel a short distance before they are stopped and undergo annihilation. The energy is released as pairs of anti-parallel 511 keV photons. The 42 TORUN EKBLAD positron camera is made up of rings of small detecting units, and when two detectors are hit simultaneously it can be assumed that the photons originate from the same decay. Thus, decay events can be detected in all directions and the three-dimensional radionuclide distribution can be quantified in absolute values (Lundqvist, 1998). PET imaging is a more rapid detection method compared to SPECT and does not require a steady state radioactivity distribution, which allows for monitoring of distributional changes of the tracer. Although PET offers several advantages such as higher spatial resolution and sensitivity compared to single photon imaging (Ott, 1996), it is also associated with some limitations. The short half-lives of some of the most common PET nuclides put a requirement on geographical co-localization of a cyclotron for nuclide production, radiochemical laboratory for labeling of targeting agents, and a PET scanner for detection. Gamma cameras are more widely available than PET scanners and generally, the radionuclides for single photon imaging are less expensive.

4.3.5 Radiolabeling of targeting peptides and proteins

The radiolabeling method has to be chosen to be efficient, with tolerable effect on the targeting agent, and result in a conjugate that is stable in vivo. Each radionuclide requires its own considerations.

Radiohalogenation can be performed using direct or indirect labeling methods in which stable covalent bonds are formed between halogen and carbon atoms. Positively charged halogen ions can undergo electrophilic substitution in the phenyl ring of tyrosine side chains. Generation of such ions require strong oxidizing agents such as chloramine T, with potential to oxidize essential groups in the targeting protein. Indirect labeling methods make use of bifunctional linkers with one site for radionuclide attachment and one for conjugation to the targeting molecule. Thereby, the harsh oxidation reaction can be performed separately prior to conjugation and sensitive groups on the targeting protein are thereby spared. The conjugation function can be designed to direct the linker to any of the reactive groups displayed on the targeting agent. Labeling at the side chains of amino acids might alter the binding properties of the targeting molecule if residues involved in the molecular recognition are affected. In particular, tyrosine residues are overrepresented in the CDR loops of antibodies (Nikula, 1995) and a direct labeling approach might disturb the antigen interaction.

Metals do not posses the same capacity as halogens to form stable bonds with the atoms available in biomolecules, and even though direct radiometal labeling has been applied to 43 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING antibodies (Sundrehagen, 1982), such interactions are generally too unstable for in vivo applications. Instead, most radiometal labeling approaches rely on the use of coordination complexes, which offer sites for columbic interactions and electron donors for dative bonds. Depending on the ionic radii, different metals require different chelators. Nơ-diethylene- triaminopentaacetic acid, DTPA (Krejcarek, 1977), and its conformationally restricted analogs are strong chelating groups that often are used for labeling with 111In. Indium can also be complexed with the macrocyclic chelator 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’- tetraacetic acid, DOTA (Fichna, 2003). In addition, DOTA forms stable complexes with other trivalent nuclides such as 66Ga, 68Ga, and 86Y, as well as with some divalent nuclides including 64Cu. However, 1,4,8,11-tetraazocyclotetradecane-1,4,8,11 tetraacetic acid, TETA, is often preferred as a copper chelating agent.

4.3.6 Technetium labeling

More than 80 % of all radiopharmaceuticals currently in clinical use are labeled with 99mTc. Technetium offers several advantageous features such as low cost and high availability as well as very favorable dosimetric properties. The half-life (6 h) is long enough to allow time for both peptide radiolabeling and collection of imaging data, but still short enough to permit administration of adequate amounts of radioactivity without risking increased doses to normal tissues in the patient.

99mTc has a complex redox chemistry represented by nine oxidation states (from –I to +VII). 99mTc is eluted with saline from 99Mo/99mTc-generators in the form of sodium pertechnate, 99m Na TcO4. Currently, there is no method for stable attachment of pertechnetate ions to peptides, and most methods rely on the reduction of pertechnetate to lower oxidation states, from which stable complexes can be formed. The reduction reaction is difficult to control, being dependent both on the nature of the reducing agent and the chelator, as well as on the reaction conditions (pH, temperature etc). Several technetium cores have been used for radiolabeling of biomolecules, as reviewed by Liu and Edwards (Liu, 1999). Technetium labeling of proteins can be divided into direct and indirect methods, where the former uses endogenous groups in the protein and the latter is achieved through conjugation of bifunctional chelating agents. 99mTc can be chelated by nitrogen-, sulfur-, and phosphorus- based systems. Depending on the oxidation state of the radiometal, different organizations of the donor atoms can be applied. By framing the coordination sphere with surrounding molecular groups, the metal centre can be protected from transchelation and re-oxidation. 44 TORUN EKBLAD

In the case of large proteins, such as antibodies, a direct labeling strategy can be applied in which the technetium is attached to reduced disulfide bonds (Griffiths, 1992). This strategy is generally not applicable to smaller proteins or peptides, since reduction of disulfide bonds often disturbs the folding. As an alternative, certain arrangements of amino acid side chains have been found to provide sites for Tc attachment. Particularly stable complexes have been achieved with histidine residues and the tricarbonyl core, in which technetium is in its +1 oxidation state. This strategy has been applied to labeling of a recombinant scFv fragment with a C-terminal His-tag (Waibel, 1999). Another example on genetic incorporation of 99mTc- chelation sites in recombinant proteins is the C-terminal Gly4Cys-tag, fused to a scFv fragment (George, 1995).

As an alternative, a chelator can be used for attachment of technetium in proteins. Chelators have traditionally been pre-organized macrocyclic molecules, such as DOTA, or other more or less rigid organic structures, with a functional linker, such as a NHS-ester, maleimide or hydrazide for conjugation to proteins using the chemistries described in chapter 3.2. The radiolabeling can be accomplished in two ways, either the chelator is covalently attached to the protein and the whole conjugate is labeled, or the chelator can be radiolabeled prior to coupling to the protein. The multidentate MAG3 chelator (mercaptoacetylglycylglycylglycine) was originally developed to coordinate 99mTc as a small non-specific imaging agent for renal function studies (Fritzberg, 1986). Due to the stable complex formation, MAG3 has since been further explored and used as a bifunctional agent for conjugation to various targeting molecules. Hydrazino nicotinamide, HYNIC, (Abrams, 1990) is a 99mTc-chelating structure that can occupy only some of the coordination sites and therefore requires the use of accompanying co-ligands to complete the coordination sphere. The nature of the co-ligands can be varied and used to modify the properties of the radiolabeled conjugate. In addition to conjugation of chelating moieties to proteins via functional linkers, peptides can be synthesized with the metal-chelating sequence incorporated in the molecule. Genetic incorporation of chelating sites is restricted to the use of natural amino acids, however, by using peptide synthesis, other classes of building blocks can be used in addition to amino acid derivatives (Lister-James, 1996). Such strategies fall between the definitions of direct and indirect labeling methods, but they have nonetheless proved very efficient for technetium labeling of targeting agents (Blok, 2004).

Several studies indicate that the biophysical nature of the chelating moiety has an impact on the biodistribution of radiometal-labeled targeting molecules (Zhang, 2000) (Decristoforo,

45 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

1999). Stability of the chelate is essential since released technetium may form colloids that accumulate in non-targeted organs and cause increased background. Furthermore, the serum stability of 99mTc-labels is challenged by transchelation to sulfhydryl groups on plasma proteins such as transferrin. Radiometal chelators can be engineered for altered biodistribution by modifying biophysical parameters, such as charge or polarity. Such strategies are further discussed in chapter 6. Paper III-VI describes chemical synthesis of Affibody molecules with built-in 99mTc-chelating sites and engineering efforts to optimize their performance as tumor targeting agents in diagnostic imaging of HER2-expressing tumors

4.3.7 Biodistribution

The efficacy of a targeting agent depends on its ability to reach and retain at its target site in adequate quantities. The pharmacokinetics of a radiolabeled targeting agent is to a large extent governed by the biophysical characteristics of the biomolecule, but also properties inherited from the radiolabel are of importance.

The residence time in the circulation is dependent on the size of the targeting agent (Batra, 2002), with large molecules having a longer circulation time compared to small. The serum half-life of intact antibodies is long, typically weeks, and regulated by interaction with Fc receptors (Ober, 2001). Smaller molecules, such as antibody fragments or scaffold-derived affinity proteins where the Fc binding site is omitted, have a shorter half life. In diagnostic imaging, slow blood clearance is unfavorable due to interfering background radioactivity from the blood pool, but also from organs that are in equilibrium with blood. Such background signals reduce the concentration ratio between tumor and normal tissues. A more rapid clearance allows for shorter clinical protocols. The molecular size also influences the rate of diffusion from the vasculature into tumors as well as the ability to penetrate into tumor mass.

Affinity is another important parameter for successful tumor localization and retention of a targeting agent. High affinity is related with good tumor targeting, and ensures that the tracer is durably retained on its target. High selectivity is also essential since cross-reactivity results in poor discrimination between tumor and non-tumor areas. Typically, an affinity higher than 10-7 M is required to significantly retain the targeting agent at the tumor antigen (Fujimori, 1990), however, higher binding strengths have been reported to increase the tumor uptake (Adams, 1998) and are preferable for molecular imaging applications. Strategies to improve the functional affinity include increasing the valency by multimerization of the targeting molecule, 46 TORUN EKBLAD such as in the formation of diabodies described in chapter 2.2, or by using self-associating peptides (Willuda, 2001). However, reports have also suggested that too high affinity might limit the tumor localization and intratumoral diffusion (Adams, 2001).

Small proteins and peptides (with a molecular weight < 60 kDa) are cleared from the blood by glomerular filtration in the kidneys. Large proteins are unable to cross the basement membrane of the glomerus and are withheld, whereas smaller molecules are excreted. After excretion, some peptides might be re-absorbed by proximal tubular cells via an electrostatic attraction. This results in high accumulation of radioactivity in the kidneys in molecular imaging studies. The renal uptake of proteins and, in particular, the reabsorbtion in proximal tubuli, can be modified by altering the surface charge of the protein (Pavlinkova, 1999) (Kim, 1999). In addition, the lipophilicity of the tracer affects the biodistribution. Targeting peptides with high lipophilic character might be excreted via bile despite a small size, resulting in accumulation of radioactivity in chime and faeces, which complicates imaging of abdominal targets.

Targeting molecules labeled with radiohalogens and radiometals differ in the fates that their resulting catabolites face. Binding to cell surface antigens most often results in internalization and eventually degradation of the tracer molecule. Depending on the lipophilicity of the radiocatabolite, they are subjected to different excretion pathways. Residualizing labels, i.e. radiometals and their chelate complexes, are charged or highly polar and therefore trapped intracellularly until exocytosis occurs. Radiohalogens on the other hand are lipophilic enough to penetrate cellular membranes and display shorter cellular retention (Tolmachev, 2008).

47 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

PRESENT INVESTIGATION

The main objective of the work in this thesis has been to explore peptide synthesis as a means to prepare Affibody molecules with designed features for specific protein detection.

In the first paper of this thesis, solid phase peptide synthesis of Affibody molecules was described. A chemical protocol was developed and optimized to avoid undesired side reactions and allow for efficient synthesis and characterization of Affibody molecules. It was confirmed that the synthetic Affibody molecules were very similar to the corresponding recombinant proteins in terms of biophysical properties. Furthermore, it was established that the Affibody scaffold can accommodate various chemical modifications, whose introduction is enabled by the synthetic production route. These important findings have been the basis for the studies undertaken in all the following papers.

Solid phase peptide synthesis was used to generate Affibody molecules with reporter groups and immobilization handles to function as capture agents for monitoring of protein levels in a solution assay (paper I) and a microarray format (II). In the following papers (III-VII), Affibody molecules were evaluated as radiolabeled targeting agents for molecular imaging.

48 TORUN EKBLAD

5. Chemical synthesis of Affibody molecules for in vitro protein detection assays

5.1 Synthesis of triple-labeled Affibody molecules for specific protein detection (Paper I)

In this paper, peptide synthesis was investigated as a means for preparing Affibody molecules to be used as fluorescence-based biosensors for protein detection. Total chemical synthesis was expected to enable production of full-sized Affibody molecules, and also allow for introduction of several functional probes. Fmoc/tBu chemistry was chosen since it is compatible with the use of a wide range of orthogonal protecting groups that enable site- specific chemical modifications. Furthermore, Fmoc/tBu chemistry has often been the preferred choice for synthesis of longer peptides and small proteins since milder reaction conditions are applied. When setting up a new lab, the Fmoc chemistry has an additional advantage in that it does not require access to the specialized HF-resistant equipment normally used in Boc chemistry.

Zwt was synthesized and analyzed in an initial pilot experiment. When analyzing the crude synthetic product by reversed phase HPLC, the full-length peptide was represented by the main peak, accompanied by multiple smaller fragments. Mass spectrometric analyses were performed to identify those fragments. It was discovered that one of the major side products had a -18 Da mass difference. Upon fragmentation and MS/MS analysis it could be confirmed that this side product was caused by an aspartimide-forming reaction at the Asp2-Asn3 sequence. In the subsequent syntheses, an AspĺGlu substitution was introduced at this site to circumvent the side reaction.

The first synthesis of the Affibody molecule ZIgA resulted in low yield of full-length product. MS analysis of sampled fractions from a reversed phase HPLC analysis of the synthetic 49 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING product revealed multiple sites in the sequence were insufficient coupling had resulted in truncated peptides. In order to optimize the protocol, the coupling reactions at these specific sites were performed twice to increase the yield in later synthesis. These efforts generated a synthetic protocol that resulted in 20-30 % yield of full-length peptides. A representative chromatogram of a synthesis of ZIgA using the optimized protocol is shown in Figure 9.

Figure 9. HPLC profile of crude synthetic product obtained from the optimized synthesis of ZIgA

Previously, Karlström and Nygren had explored Affibody molecules as affinity reagents in a FRET-based detection assay that allowed for detection of unlabeled target proteins (Karlström, 2001). The detection method utilizes energy transfer from an excited donor fluorophore (EDANS) to an acceptor fluorophore (NBDX), which becomes excited by the emitted light from the donor. The hypothesis was that by positioning the pair of fluorophores so that they flank the binding site, interactions with the target protein will induce structural changes that alter the efficiency of the energy transfer. This phenomenon can be detected as a shift in fluorescence towards wavelengths characteristic of the donor fluorophore, and used to quantify binding of the target protein. Precise positioning of the FRET donor/acceptor pair is essential for the assay. In order to be able to relate fluorescent events in the detection assay to a specific protein interaction, it is also important to have a labeling method that generates a homogeneously labeled product with reproducible incorporation of the fluorophores in defined positions. In the initial study, a recombinant Z domain was used and selective fluorescent labeling of the N-terminal relied on the difference in pKa of the N-terminal ơ- amino group and the ƥ-amino group of the lysine residues in the protein. When applied to other Affibody molecules, this strategy resulted in a heterogeneous mix of labeled species and poor yields of correctly dual labeled molecules. The successful chemical synthesis of Affibody molecules offers an alternative way of introducing the fluorescent probes. In this paper, an orthogonal protection scheme was designed to allow for site-specific introduction of the

50 TORUN EKBLAD functional probes into synthetic Affibody molecules (Figure 10). Piperidine deprotection was used to liberate the Nơ-protecting Fmoc group prior to NBDX coupling. The ƥ-amino group of the C-terminal lysine residue (Lys58) was protected with an alloc group that could be selectively deprotected with Pd(0)-catalyzed allyl transfer, allowing for site-specific coupling of biotin. The third functional probe, EDANS, was coupled via a succinyl linker to the side chain of Lys23, which had been protected with an acid-labile Mtt group.

Figure 10. The orthogonal protection strategy

Two model proteins, the Z domain and the Affibody ZIgA, were produced by solid phase peptide synthesis and as recombinant His6-tag fusions in E.coli for reference. The synthesized proteins were compared to their corresponding unmodified recombinant proteins in terms of biophysical properties. It was discovered that the synthetic Affibody molecules similarly to the recombinant proteins had a secondary structure with a high helical content, characteristic of the three-helix bundle that characterizes the Z domain. Furthermore, the production route and the introduction of chemical modifications did not have a negative influence on the target binding affinity.

The two model protein interactions were evaluated in the FRET-based binding assay, Zwt binding to IgG and the Affibody molecule ZIgA binding to IgA. The doubly labeled binding proteins were investigated in the binding assay with titrated target protein, and the target protein detection was found to be specific in both cases. The relatively low sensitivity of the assay was largely an effect of the relatively high target molecule concentrations needed to generate a measurable fluorescent signal.

51 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

A third functional probe, biotin, was introduced in addition to the fluorophores to function as an immobilization handle for attachment to a solid phase, such as a microarray slide. The immobilized proteins displayed a retained target binding specificity.

Conclusions and Future Perspectives: The main conclusions from this paper are that Affibody molecules can be produced by solid phase pe ptide synthesis and that multiple orthogonal protecting groups can be used to introduce different reporter groups. Importantly, the synthetically assembled polypeptide chain had the ability to fold into a functional protein. It was shown that synthetic Affibody molecules have a secondary structure and target protein binding affinity that is similar to its recombinant analogue. Furthermore, a variety of functional probes can be introduced through orthogonal protecting group strategies.

The fact that synthetic Affibody molecules, modified with several functional groups, could be immobilized onto a solid support with retained binding specificity encouraged further investigation to apply the FRET-based solution phase assay to a solid phase format on microarr ay glass slides. Unfortunately, the high energy photons needed for excitation of the donor fluorophore used (EDANS) also generated high background fluorescence from the glass slide, which hampered detection of FRET-specific fluorescence. It would be interesting to investigate alternative FRET-pairs that operate at higher wavelengths and that are compatible with conventional array scanners. Derivatives of fluorescein and rhodamine might be an alternative, or other fluorophores that are compatible with the conditions of SPPS.

When designing the assay, it was hypothesized that binding of a target protein would induce a change in the efficiency of FRET. However, it is possible that the observed effect is due to other events, induced by changes in the local environment surrounding the fluorescent probes, as the performance of both NBDX and EDANS is known to be sensitive to the microenvironment (Shi, 2005), (Pletneva, 2005). Other aspects of probes that are sensitive to changes the microenvironment are interesting to explore further as alterative methods for detecting binding of unlabeled proteins. In a separate study, we have investigated the possibility to introduce such probes at particular sites in the Affibody molecule and preliminary results show that binding of target protein can induce singlet-triplet electronic state transitions that can be monitored with fluorescence correlation spectroscopy (Sandén et al., unpublished).

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5.2 Synthesis of Affibody molecules for protein microarrays (Paper II)

It has previously been reported that the orientation in which antibodies and antibody fragments are immobilized onto protein array surfaces has an influence on their performance as capturing agents. Immobilization of capturing agents can be performed in a random fashion, or in a directed manner with a specified orientation of the binding site. Peptide synthesis of small affinity proteins allows for introduction of immobilization handles at specific sites and offers a way to perform the immobilization in a highly controlled fashion. The aim of this study was to compare the performance of Affibody molecules that had been immobilized on surfaces in a random or directed orientation.

Following completed peptide synthesis, an orthogonal protecting group was specifically removed from the ƥ-amine in the side chain of the C-terminal lysine residue to allow for introduction of a unique cysteine residue or a biotin moiety. These functions were used for directed immobilization of the Affibody molecules. Additional constructs were synthesized in which the affinity handles were distanced from the peptide by an aminohexanoic acid linker. Directed immobilization of Affibody molecules to chip surfaces by thiol-coupling or biotin- streptavidin interaction was compared to random coupling through any of the amino groups on the protein. The performance of the immobilized Affibody molecules was evaluated using surface plasmon resonance (SPR) or fluorescence for detection of bound target proteins.

In the SPR-experiments, it was found that directed coupling through thiol chemistry resulted in the highest specific activities of the immobilized proteins (Figure 11). It was also found that the use of a spacer to increase the distance between the surface and the Affibody molecule generated higher target binding signals, both for the cysteine and biotin immobilization chemistry in both detection methods, suggesting that the protein is more accessible to binding when equipped with a spacer. When the immobilization chemistries were evaluated using SPR, the streptavidin strategy was only more efficient than random coupling when the spacer was used.

In the microarray format, directed thiol-coupling and random immobilization were comparable, with high fluorescence signals and low background. Directed immobilization onto streptavidin-coated slides through biotin resulted in the least efficient performance with high

53 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING and uneven background fluorescence. The background might be partly due to the fact that the streptavidin-coated slides were lacking the dextran layer present on the other chip surfaces.

Figure 11. Specific activity of the Affibody molecule ZTaq when immobilized via amino groups, biotin or a unique cysteine residue

Conclusion and Future Perspectives: The use of small affinity proteins such as Affibody molecules offers an advantage compared to larger capture agents in that they can be produced by peptide synthesis. This study illustrates the value of chemical methods that allow for introduction of affinity probes at selected sites in a peptide sequence. Many aspects have to be considered for development of highly sensitive protein microarray-based assays, including surface chemistry and printing protocols as well as choice of detection method and capture agent. The use of solid phase peptide synthesis for affinity protein production can provide a means to address some of these issues by allowing for site-specific chemical modifications to introduce linkers or other functional probes such as polymers or reporter groups.

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6. Chemical synthesis of Affibody molecules for molecular imaging in vivo

6.1 Synthesis of an Affibody molecule containing the 99mTc-chelator MAG3 for imaging of HER2-expressing tumors (Paper III)

The human epidermal growth factor receptor type 2, HER2, is an important biomarker in relation to breast cancer. The monoclonal antibody trastuzumab, directed to HER2, has been successfully used for treatment of breast cancer, however, the treatment is only efficient in patients that have an overexpression of HER2. Molecular imaging has been used as a method to monitor HER2 expression and thereby select patients who are likely to respond to trastuzumab therapy. In general, small affinity proteins, including antibody-derived fragments and affinity proteins derived from alternative scaffolds, are attractive alternatives to full-sized antibodies in molecular imaging experiments due to better tissue penetration and a more rapid clearance from blood, allowing for high contrast imaging and short clinical protocols. The fact that Affibody molecules are amenable to solid phase peptide synthesis and chemical modifications for reporter group introduction, make them attractive for medical imaging applications.

The selection and affinity maturation of an HER2-binding Affibody molecule has previously been described (Wikman, 2004) (Orlova, 2006). Variants of this Affibody molecule have been labeled with a number of radioisotopes, including 125I (Orlova, 2007a), 111In (Orlova, 2007b), (Tolmachev, 2006), 90Y (Fortin, 2007), 68Ga, 177Lu (Tolmachev, 2007) and 18F (Kramer-Marek, 2008), (Namavari, 2008) and evaluated for tumor targeting properties. In this paper, the aim was to develop a strategy for efficient 99mTc-labeling of Affibody molecules. Such a protocol would offer a cheap method to generate targeting agents for gamma camera imaging, as 99mTc is an inexpensive radionuclide that is readily available through 99Mo/99mTc generators and has a photon energy that is very well suited for gamma camera scintigraphy.

55 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

The mercaptoacetyl-glycyl-glycyl-glycine sequence (“MAG3”) is a well-documented 99mTc chelator that has been used for many applications. For example, 99mTc-labeled MAG3 has been widely used for investigation of renal function. MAG3 has also been coupled to proteins, peptides and oligonuclides to enable labeling with 99mTc and 186/188Re. In this paper, solid phase peptide synthesis was used to produce HER2-binding Affibody molecules with the chelating sequence added as an N-terminal extension of the peptide. By assembling the chelating moiety one residue at the time on the peptide terminus, a homogeneous product with well-defined modification is assured.

The peptide sequence was assembled using standard Fmoc-protected building blocks and S- trityl-protected mercaptoacetic acid. The pure full-length product, including the chelating sequence, was obtained by reversed phase HPLC and the correct mass was confirmed by mass spectrometry. The secondary structure of the modified protein was observed in a circular dichroism experiment and found to be similar to the unmodified Affibody molecule with characteristic high helix content. The thermal stability was not impeded by the introduction of the chelator moiety, which is important for the protein to withstand the harsh radiolabeling conditions. The HER2 binding affinity was somewhat lower for Affibody molecules with the chelator, however still high enough to allow for efficient tumor targeting.

The pharmacokinetics and tumor targeting performance were evaluated by injecting 99mTc- labeled Affibody molecules into mice and excising individual organs for radioactivity measurements. Biodistribution data are summarized in Figure 12. Xenografts of HER2- expressing LS174T cells had been established in the mice and were used as a tumor model.

Important results of this study were that MAG3 could be used to label an Affibody molecule with 99mTc, and that the chelate was stable. Technetium chemistry is complex, and the efficiency of the labeling is often influenced by the molecular environment at the site in the protein where the label should be introduced (Qu, 2001). Therefore, labeling protocols generally have to be optimized for each individual protein. Furthermore, it was found that the Affibody molecules indeed accumulated at the HER2-expressing xenograft, with an uptake of 99m 7 % IA/g at 4 h p.i. The biodistribution of Tc-MAG3-ZHER2:342 was characterized by a low uptake and rapid clearance in most organs, with the exception of kidneys, where high radioactivity was detected. This study also indicated that 99mTc-labeled Affibody molecules provided similar or better tumor-to-organ ratios compared to radioiodination. Furthermore, it was discovered that almost 30 % of the injected activity was accumulated in the intestines in normal mice, indicating a high degree of hepatobiliary excretion. The elimination of

56 TORUN EKBLAD biomolecules from the body occurs primarily by two major routes of excretion, gastrointestinal (hepatobiliary) and renal (glomerular filtration and tubular secretion). Most proteins are cleared through a mix of those routes. The gastrointestinal route is slow, and radiolabeled tracers that linger in the intestines cause high background signals, which might obstruct efficient detection of metastases in the abdominal area. From an economical perspective, and with the best patient comfort in mind, it is ideal if the imaging can be performed at the day of injection. However, with imaging agents that have a high degree of hepatobiliary excretion, the clearance is slow and the detection may need to be performed at a later time point.

Figure 12. Biodistibution of MAG3-ZHER2:342 in tumor-bearing mice at 2, 4, and 6 h p.i.

Conclusions and Future Perspectives: Chemical synthesis of targeting agents for molecular imaging provides a means to introduce radiometal chelators in a well-defined manner to generate uniformly labeled radioconjugates. A homogeneous population of tracer molecules is essential when evaluating in vivo behavior of proteins in relation to their structure. In this study, MAG3 was found to provide a stable 99mTc-chelation site at the N-terminal of a HER2-targeting Affibody molecule. The tumor uptake was receptor-specific and high enough to allow for imaging with reasonable contrast to healthy tissue. Several attractive features of the 99mTc-labeled Affibody molecules encouraged further studies to address the issue of hepatobiliary excretion and reduce the gastrointestinal uptake.

57 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

6.2 Synthesis of Affibody molecules with engineered 99mTc-chelator sites for improved HER2-imaging (Papers IV, V and VI)

The studies that are reported in papers IV-VI were all part of an effort to optimize the performance of the Affibody molecule ZHER2:342 as a SPECT imaging agent. The strategy for doing so was based on introduction of amino acid substitutions in the previously evaluated 99mTc-chelating sequence mercaptoacetyl-glycyl-glycyl-glycyl (“MAG3” or MaGGG). Increased hydrophilic character has previously been reported to result in an increased renal excretion of proteins. Therefore, amino acids with hydrophilic and charged side chains were considered as alternatives to glycyl residues in the MAG3 chelator to redirect the excretion pathway of 99mTc- labeled Affibody molecules to a renal route rather than through the intestines.

A panel of Affibody molecules carrying different chelating sequences (Figure 13) were generated by solid phase peptide synthesis and subjected to thorough biophysical characterization prior to evaluation of tumor targeting performance and in vivo biodistribution pattern. Independent of the chelating sequence, all variants were found to have a secondary structure and helical content typical of Affibody molecules, and to bind to HER2 with high affinity.

The biodistribution of the Affibody molecules carrying the different chelator sequences were studied in normal mice (Figure 14). The renal uptake of Tc-labeled Affibody molecules was increased when glycine residues in the chelating sequence were substituted with serine or glutamic acid. At the same time, a decreased uptake in the intestines was observed. The engineered chelator sequences were also found to form more stable 99mTc-complexes, as indicated by the reduced radioactivity concentration in blood and stomach. The results indicate that the excretion pathway was shifted from hepatobiliary towards renal by substitutions with hydrophilic amino acids, and the effect was more pronounced when all glycine residues in the chelator were altered. In particular, a dramatic effect with a 10-fold decrease was observed when all three glycine residues were substituted with glutamic acid. However, there was a very high reabsorbtion in the kidneys associated with the triple-substituted conjugate. As an alternative, chelators with a mixed seryl and glutamyl composition were expected to combine the low intestinal uptake with a reasonably high kidney accumulation.

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Figure 13. Structures of the 99mTc-chelators

Figure 14. Biodistribution in NMRI mice 4 h p.i. of Affibody molecules carrying the 99mTc- chelator sequence MaGGG, MaGSG, and MaSSS (left) or MaGGG, MaGEG and MaEEE (right)

When evaluating the tumor targeting properties of the mixed seryl/glutamyl chelator conjugates, it was found that all investigated Affibody molecules had a specific accumulation to HER2-expressing xenografts, with tumor uptake in the range of 7-12 % IA/g (Table 4). Interestingly, the mercaptoacetyl-diglutamyl-seryl variants differed significantly in the kidney uptake despite having identical amino acid composition. This effect might be explained by

59 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING different renal retention of the corresponding radiocatabolites. The high tumor-to-blood ratios are results of quick blood clearance and high stability to transchelation to plasma proteins. The results from these studies illustrate how minor changes, such as single amino acid substitutions, but also the order in which the chelating amino acids are arranged, can have dramatic effects on the pharmacokinetic properties of Affibody molecules.

Table 4. Biodistribution data from mice injected with 99mTc-labeled Affibody molecules obtained at 4 h p.i. Animals were BALB/c nu/nu mice with SKOV-3 xenografts, (*) BALB/c nu/nu mice with LS174T xenografts, or (#) non-tumor bearing BALB/c nu/nu mice

Chelator Tumor uptake Kidney uptake Intestinal uptake Tumor-to- (% IA/g) (% IA/g) (% IA/whole sample) blood ratio MaGGG* 7 6 30 # 24

MaSSS 11 34 11 76

MaEEE 8 102 4 31

MaEES 12 68 4 55

MaSEE 10 71 2 68

MaESE 10 33 2 58

From the panel of potential 99mTc-chelating sequences that were evaluated in these studies, the mercaptoacetyl-glutamyl-seryl-glutamyl- sequence (MaESE) was identified as the most promising candidate for clinical imaging of HER2-expressing tumors and metastases. Although other variants indicated equally good or higher tumor uptake and tumor-to-blood ratio, the MaESE chelator offered an attractive compromise of low hepatobiliary excretion and tolerable kidney uptake. Affibody molecules with the MaESE chelator were used to achieve high contrast imaging of HER2-expressing xenografts in mice in a gamma camera imaging experiment (Figure 15).

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Figure 15. Gamma camera image of mice with HER2-expressing SKOV-3 xenografts 99m obtained 5 h p.i. with Tc-MaESE-ZHER2:342. The on the right-hand side was pre- injected with excess of non-labelled Affibody molecules

Conclusions and Future Perspectives: Molecular imaging is an important tool to detect expression of molecular targets in metastases, to tailor treatment protocols to individual patients, and to monitor the response to therapy. For such efforts to be accurate, it is essential that the tumor targeting agents do not generate background signals that obstruct high contrast imaging. Amino acid substitutions directed to the 99mTc-chelating sequence were found to be an efficient way to alter the pharmacokinetics of Affibody molecules. In addition, the different amino acid side chains were found to bring increased stability to the 99m Tc-chelator complex, which decreased the background in normal tissue further.

Other amino acids with hydrophilic or charged side chains could be considered, such as lysine or aspartic acid. Lysine has indeed been investigated as an alternative to bring increased hydrophilicity to the chelator sequence (Tran et al., in press). The positively charged side chain of lysine did not generate improvements in terms of tumor targeting performance, but rather resulted in increased liver uptake. Aspartic acid could offer an interesting alternative but was omitted from the study due to its susceptibility to aspartimide formation under the conditions in peptide synthesis.

In theory, the chelator could be positioned anywhere in the sequence. In these studies the N- terminal was chosen as it represents a flexible region of the Affibody molecule that was expected to allow for efficient radionuclide labeling with little influence on the target binding surface. An alternative strategy in which the chelator is positioned as far away from the binding surface as possible has also been considered. Since the microenvironment has proved to have a

61 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING high impact on the behavior of the Affibody molecules, both in terms of label stability and biodistribution, such a strategy might offer a general labeling protocol for Affibody molecules with different binding surfaces. To investigate this further, branched Affibody molecules, with the 99mTc-chelating sequence introduced in the middle of helix III have been synthesized (Ekblad et al. unpublished). A complete characterization of the in vivo behavior of these conjugates is still ongoing, however the biophysical characterization suggests a similar structure and binding capacity compared to the earlier evaluated variants.

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6.3 Synthesis and chemoselective intramolecular cross-linking of a HER2- binding Affibody molecule (Paper VII)

An important parameter for molecular imaging agents is resistance to elevated temperatures, as such conditions might be necessary in radiolabeling reactions. For tumor targeting agents with therapeutic potential, stability to proteolytic digestion is an important issue to assure increased circulation time. In this study, a strategy to bring increased stability to a HER2-binding Affibody molecule by intramolecular cross-linking was evaluated.

Chemoselective ligation can be used to modify the backbone of synthetic peptides and substitute native peptide bonds with analogs with altered stability. The chemistry can also be used to introduce intramolecular cross-links by ligation of amino acid side chains. In this paper, thioalkylation between chloroacetyl and thiol groups was used to form a thioether bond between the side chain of a C-terminal lysine residue and a cysteine residue in the loop region connecting helices I and II. The tethered Affibody molecule was found to have a HER2- binding affinity comparable to a linear control. However, the thermal stability differed significantly, with the cross-linked protein having a melting temperature ten degrees higher than the linear control (Figure 16).

Figure 16. Thermal stability study of cross-linked and linear Affibody molecules

63 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

The cross-linked and linear control Affibody molecules were labeled with 111In at an N- terminally introduced DOTA chelator, and their tumor targeting performances were evaluated in tumor-bearing mice. The tumor uptake was found to be better for the Affibody molecule with the intramolecular cross-link. Unfortunately this improvement was accompanied by elevated levels of radioactivity in the blood pool.

Conclusions and Future Perspectives: The primary rationale for introducing an intramolecular cross-link in the Affibody molecule was to bring increased thermal stability. Furthermore it was speculated that such strategy might be used to improve the binding affinity and allow for higher tumor uptake. Traditionally, disulfide bridges have been used to increase the structural stability of proteins, however such bonds were not considered sufficiently stable for use in imaging agents that are to be subjected to the reducing milieu in vivo.

In addition to thermal stability, tethered Affibody molecules might also have increased proteolytic stability, resulting in improved in vivo properties. Alternative strategies to the one applied in this paper could be to generate head-to-tail cyclic molecules. Such proteins would lack both the N- and C-termini and might have a higher stability to exo-proteases. Furthermore, proteins with multiple intramolecular cross-links can be designed, in order to generate an even more compact structure.

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7. Conclusions and future perspectives

The work presented in this thesis is focused on chemical synthesis of Affibody molecules and methods to introduce functional probes that facilitate specific protein detection.

Chemical synthesis is an alternative production route available for peptides and small proteins that offer enormous potential for engineering of various molecular features such as biophysical parameters, structural properties and biological functions that can not be realized using recombinant expression systems. Not only does solid phase peptide synthesis permit the use of building blocks beyond the repertoire of the 20 DNA-encoded amino acids, but it also offers the possibility to introduce chemical modifications in a site-specific manner, which would be next to impossible by bioconjugation strategies, and to engineer the protein backbone. In the work described, chemical synthesis has been explored as a method for generating Affibody molecules as affinity reagents in various protein detection assays.

The ability to introduce chemical modifications is essential for the development of affinity reagents for both in vitro and in vivo applications. Depending on the context in which the affinity protein is being used, different characteristics might be desirable. In addition to introduction of reporter groups, chemical modifications can also be used to bring increased solubility, affinity or thermal stability, which are all interesting parameters when developing affinity reagents for in vitro protein detection assays such as protein microarrays. Controlled engineering of such biophysical parameters is also important when developing affinity reagents for in vivo applications. In such contexts, chemical modifications are valuable tools for altering cellular uptake, reducing immunogenicity or affecting the clearance.

From the work presented in this thesis, it is apparent that protein modifications have a fundamental impact on the way Affibody molecules behave in vivo when used as tumor targeting agents in molecular imaging. Chemical synthesis was found to be a powerful method to tailor imaging reagents with desired characteristics. Chemical synthesis allows for fine-tuning of the molecular properties of a protein with atomic level accuracy. This is of particular advantage when designing affinity proteins with probes that respond to fluctuations in the immediate molecular environment. High-precision modifications

65 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING and the possibility to purify the functionalized protein to homogeneity is essential for developing protein detection assays based on such attributes, for example the FRET-based assay described in this thesis.

All studies in this thesis have been centered on Affibody molecules. However, the chemical tools that have been used are general in the sense that they can also be applied to other small proteins, such as different scaffold proteins, or to alternative .

A lot of efforts have been, and will continue to be put into large scale proteomics and other - omics analyses to unravel the functions of proteins. Development of novel sensitive assays will be important to cover the gaps were existing technologies fall short. Such assays can hopefully result in identification of new biomarkers for diseases. Ideally, with biomarkers for all severe diseases in hand, it will be possible to diagnose patients and suggest suitable, personalized treatment before the disease has progressed to a serious state. That type of diagnosis relies also on sensitive, yet facile in vitro and in vivo detection methods. New labeling methods will emerge, and probably will physical measures not yet thought of play important roles in the development of protein detection assays in the future. However, I do believe that affinity proteins for specific molecular recognition will remain important.

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8. Abbreviations

aa Amino acid CDR Complementarity determining region

CH Constant domain of the antibody heavy chain

CL Constant domain of the antibody light chain DNA Deoxyribonucleic acid ELISA Enzyme-linked immunosorbent assay Fab Fragment, antigen binding (Antibody) Fc Fragment, crystallizable (Antibody) FDA Food and drug administration FRET Fluorescence resonance energy transfer HER2 Epidermal growth factor receptor-2 (HER2/neu, ErbB-2) HSA Human serum albumin IgG

KD Equilibrium dissociation constant kDa Kilodalton mAb Monoclonal antibody MAG3 Mercaptoacetyl glycylglycylglycine PET Positron emission tomography p.i. Post injection scFv Single chain variable fragment SPA Staphylococcal protein A SPECT Single photon emission computerized tomography SPPS Solid phase peptide synthesis

VH Variable domain of the antibody heavy chain

VL Variable domain of the antibody light chain

67 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

9. Acknowledgements

Först och främst vill jag tacka min handledare Amelie Eriksson Karlström. Tack för att du delar med dig av din kunskap och för att du alltid tagit dig tid och engagerat dig helhjärtat i mina projekt. Du har ett fantastiskt ordningssinne och en förmåga att alltid kunna sätta fingret på vad som är viktigt. Det har räddat mig i många kaotiska situationer, inte minst under författandet av denna avhandling.

Ett varmt tack vill jag också ge till Per-Åke Nygren. Tack för alla gånger jag har fått sitta ner (eller stå, i brist på plats) i ditt kaoskontor och prata forskningsproblem. Jag har alltid gått därifrån med uträtade frågetecken och ökad förståelse.

”Plan 3” har varit mitt andra hem under ett antal (ganska många) år nu. Det finns många att tacka för den trevliga atmosfären på labbet. Mathias Uhlén är en stor inspiratör som med ett glatt flin får allt att verka enkelt. Tack till Stefan Ståhl för din härliga attityd till det mesta, den är absolut något jag skall komma ihåg från den här tiden. Stort tack också till alla övriga PI’s för ert gedigna forskningsintresse och för att ni är så generösa med er kunskap. Tillsammans sätter ni en unik prägel på avdelningen som jag har uppskattat otroligt mycket under min doktorandtid och som jag tror är väldigt värdefull, och värd att värna om, i en tid när projekten växer och tempot ökar.

Ett stort tack vill jag rikta till Björn, som fyllde mina första doktorandår med sarkasm, cynism och colaburkar. Tack för sällskap i synteslabbet, guidning i microarrayvärlden, underhållning i Mellanstadiet, koncentrationssvårigheter i skrivrummet, och för att jag blev speciellt utvald att följa med på löparrundorna på Gordon-konferensen.

I would also like to thank Vladimir Tolmachev for the enthusiasm you have shown throughout the project on technetium-labeled Affibody molecules. Thank you for sharing your expertise so generously and for always taking the time to answer my questions. I am also very grateful to Anna Orlova, for a very fruitful collaboration, and for performing the animal experiments with great skills. Tack också till Thuy Tran för ett himla bra samarbete.

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Tack till Lars Abrahmsén och Joachim Feldwich på Affibody AB för värdefull kunskap kring allt som har med Affibody-molekyler att göra. Tack också till Malin Lindborg för hjälp med att designa HER2-kinetikstudierna.

Harry Brumer has helped me a lot with ESI-MS analyses. Även Gustav Sundqvist har varit fantastisk och hjälpt mig med massor av massspec-prover.

Jag har haft fantastiska kollegor, och det finns många att tacka för alla goa garv, uppmuntrande ord, hjälpande händer, roliga fester, trevliga luncher, udda musikkvällar och svettiga träningspass. Jag vill särskilt tacka: Alla i ”Mellanstadiet”, för en väldigt speciell labatmosfär och för att jag fick vara ”also starring”. Mina rumskamrater genom åren; Malin, Max, Maria, Erik, Björn, John, Emelie, Jojje och PerÅke, för fruktstunder, fruktflugor och fruktbara diskussioner. Hela tjejligan, för allt kul vi hittat på! Emelie, Emma, Cilla, Sara, Carro, Cissi, mfl, tack för fredagsöl, förfester, teater, tennis, pyssel och fikapauser som har förgyllt livet på labbet. Tack till Per, My och Anna för att ni är så sköna typer! Speciellt tack till Tove för din aldrig sinande omtänksamhet, Esther för att du är en sådan färgsprakande vän, och Malin för vänskap, och alla förtroliga snack om forskning, livet och hur man skall få ihop allt. Ett jättetack också till Mikaela, för all hjälp med korrekturläsning och färdigställandet av avhandlingen. Du är en fantastisk tjej med en härligt rak attityd och det har varit kanon att jobba med dig.

Ylva, tack för ovärderlig hjälp att granska språk och innehåll i avhandlingen, du är en klippa och en fantastisk vän.

Stort tack till Bo och Ingrid för er otroliga gästvänlighet som har gjort det möjligt att färdigställa det här arbetet trots utlandsflytt. Tack också för alla härliga dagar på Ornö. Om jag själv får en svärdotter någon gång så skall jag försöka komma ihåg hur coola ni var när jag kraschade er nya bil...

Christina och Berndt, tack för att ni ställer upp i alla lägen! Ni har alltid funnits till hands, oavsett om jag behövt låna en bil, se en fotbollsmatch eller skrapas ihop efter ett marathonlopp. Hycklinge har varit en oas där jag har kunnat härja fritt i skogarna, vila ut efter tuffa perioder och samla ihop mig inför nya projekt. Christina; dina insatser under hösten som gått har varit ovärderliga! Det värmer att se din glädje över att pendla med okristligt tidiga flyg till Horw för att steka pannkakor, storstäda och ta hand om en sjuk Frida.

69 CHEMICAL SYNTHESIS OF AFFIBODY MOLECULES FOR PROTEIN DETECTION AND MOLECULAR IMAGING

Tack till Mamma och Pappa för all kärlek och omtanke. Ni har gett mig mod och självförtroende att göra det jag gör och vara den jag är.

Uno, en stolt storebror är bland det bästa som finns. Tack för stöd och uppmuntran genom åren. Tack också för galenskap och roliga upptåg, från Kosterfjorden i styv kuling till ”Puerto Rican Day” i New York.

Frida, tack för mysigt sällskap vid oändliga proteinreningar på HPLC’n. Du ger mig perspektiv på tillvaron och är en ständig källa till glädje.

David, du har varit ett fantastiskt stöd under hela min doktorandtid. Du får mig att skratta och ger mig kraft. Du får svårigheter att verka överkomliga och påminner mig om mina framsteg. Du har ett hjärta av guld och jag älskar dig.

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10. References

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