Chemical Engineering of Small Affinity Proteins

Joel Lindgren

KTH Royal Institute of Technology School of Biotechnology Stockholm 2014

KTH 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-106 91 Stockholm Sweden

ISBN 978-91-7595-004-4 TRITA-BIO Report 2014:3 ISSN 1654-2312

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Joel Lindgren (2014): Chemical Engineering of Small Affinity Proteins. School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

Abstract Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS).

In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I).

In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III).

In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge- mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V).

Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.

Keywords: Affibody molecules, albumin binding domain, ligation, protein synthesis, solid phase peptide synthesis

List of publications

This thesis is based upon the following five papers, which are referred to in the text by their Roman numerals (I-V) The papers are included in the Appendix of this thesis and are reproduced with permission from the copyright holders.

I. Lindgren J., Ekblad C., Abrahmsén L. and Eriksson Karlström A. (2012). A native chemical ligation approach for combinatorial assembly of Affibody molecules. ChemBioChem 13(7): 1024-1031.

II. Lindgren J., Wahlström A., Danielsson J., Markova N., Ekblad C., Gräslund A., Abrahmsén L., Eriksson Karlström A. and Wärmländer S. K. (2010). N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity. Protein Sci 19(12): 2319-2329.

III. Lindgren J., Segerfeldt P., Sholts S. B., Gräslund A., Eriksson Karlström A. and Wärmländer S. K. (2013). Engineered non- fluorescent Affibody molecules facilitate studies of the amyloid-beta (Abeta) peptide in monomeric form: low pH was found to reduce Abeta/Cu(II) binding affinity. J Inorg Biochem 120: 18-23.

IV. Lindgren J., Refai E., Zaitsev S., Abrahmsén L., Berggren P-O. and Eriksson Karlström A. (2014). A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life. Submitted

V. Lindgren J. and Eriksson Karlström A. (2014). Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability. Submitted

Contents

Introduction

1 Proteins ...... 2 1.1 Protein therapeutics ...... 6 1.2 Protein engineering ...... 7 1.2.1 Protein engineering by rational design ...... 7 1.2.2 Protein library technology ...... 8 1.3 Affinity proteins ...... 9 1.3.1 Affibody molecules ...... 11 1.3.2 Albumin binding domain (ABD) ...... 14 2 Protein synthesis ...... 17 2.1 The pre-solid phase peptide synthesis era of chemical protein synthesis ...... 18 2.2 Solid phase peptide synthesis (SPPS) ...... 19 2.3 Peptide ligation strategies...... 24 2.3.1 Native chemical ligation (NCL) ...... 24 2.3.2 Traceless Staudinger ligation ...... 26 2.3.3 Expressed protein ligation (EPL) ...... 26 2.3.4 Enzyme-catalyzed ligation ...... 27 2.3.5 Non-native ligation methods ...... 27

Present investigation

3 Aim of the thesis ...... 30 4 Paper I – A native chemical ligation approach for combinatorial assembly of Affibody molecules ...... 32 5 Paper II – N-terminal engineering of amyloid-beta-binding Affibody molecules yields improved chemical synthesis and higher binding affinity ...... 37

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6 Paper III – Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (Aβ) peptide in monomeric form: low pH was found to reduce Aβ/Cu(II) binding affinity ...... 42 7 Paper IV – A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life ...... 45 8 Paper V – Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability ...... 50 9 Concluding remarks ...... 56 Populärvetenskaplig sammanfattning ...... 59 Acknowledgments ...... 63 References ...... 65

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Introduction Chemical Engineering of Small Affinity Proteins

1 Proteins

Every living organism, including bacteria, fungi, plants and animals, is dependent on proteins and they perform a vast amount of distinct functions both inside and outside of cells. Many proteins are enzymes, which catalyze chemical reactions. One example is the conversion of carbon dioxide and water to carbohydrates in plants, algae, and cyanobacteria using the energy from sunlight. This reaction is catalyzed by a series of enzymes and all life on earth is dependent on this process. Other proteins act as molecular motors (e.g. myosin in muscles), which make it possible for us to move by converting stored energy to motion. Antibodies are another important protein class which is in charge of recognizing invaders in our body, and without antibodies an ordinary cold could be lethal. A fourth example is the protein hemoglobin, further discussed below, which is responsible for transporting oxygen from the lungs to the rest of our body. This very small fraction of mentioned functions gives a clue of the importance of proteins. Indeed, the word Protein is derived from the Greek word proteios meaning “of the first rank”, proposed for the first time by the Swedish scientist Jöns Jacob Berzelius in 1838. (Vickery, 1950) Proteins truly are very important for every living organism, and also highly abundant, comprising approximately 60% of the dry mass of a mammalian cell. (Alberts, 2002)

Proteins are typically built up from an ensemble of 20 naturally occurring amino acids joined together by peptide bonds to form polypeptides (see figure 1), and polypeptides with a defined secondary structure are commonly referred to as proteins. Each amino acid has a carboxyl group, an amino group and a side chain specific for the amino acid, which gives each of the 20 amino acids its specific properties. Even though the number of different residues (amino acids) is relatively low, they can be combined in a very large number of different ways. Polypeptides and proteins vary greatly in size, from very large proteins (e.g. the muscle protein titin with more than 34,000 residues (Li, 2012), to very small peptides consisting of only a few amino acids, e.g. the 2

Joel Lindgren

O R2 H N OH 2 N H R1 O

O O Leucine (Leu, L) H3C Alanine (Ala, A) H3C OH OH

NH2 CH3 NH2

NH2 O O

Arginine (Arg, R) H2N Lysine (Lys, K) HN NH OH OH

NH2 NH2 O O S H2N Asparagine (Asn, N) Methionine (Met, M) OH H3C OH NH O NH2 2 O O HO Phenylalanine (Phe, F) OH Aspartic acid (Asp, D) OH NH O NH2 2 O O H HS OH Cysteine (Cys, C) N Proline (Pro, P) OH

NH2 O O O

HO OH Glutamic acid (Glu, E) HO OH Serine (Ser, S)

NH2 NH2

NH2 O CH3 O

O OH Glutamine (Gln, Q) HO OH Valine (Val, V)

NH2 NH2 O O

OH Glycine (Gly, G) OH Tryptophan (Trp, W)

NH2 NH2 NH O O

N Tyrosine (Tyr, Y) OH Histidine (His, H) OH NH N 2 NH2 HO H CH3 O CH3 O Valine (Val, V) H3C Isoleucine (Ile, I) OH H3C OH NH NH2 2 Figure 1. The chemical structure of a dipeptide (top) and the 20 natural amino acids (bottom) are shown. In the dipeptide, the peptide bond is marked with a dashed square and R1 and R2 represent the two amino acid side chains. For the 20 amino acids the amino group, carboxyl group, and alpha carbon, common for all different amino acids, are shown in grey while the unique side chains are shown in black. Note that glycine has no side chain other than a hydrogen atom and that the side chain of proline is connected to the amine nitrogen.

Chemical Engineering of Small Affinity Proteins

bioactive polypeptide angiotensin-(1-7) built up by only seven residues. (Santos, 2003) These are extremes, while a protein in nature typically consists of approximately 300 amino acids. (Brocchieri, 2005) In humans there are approximately 20,000-25,000 different protein-encoding genes and the proteins are often further processed by post-translational modifications increasing the estimated number of unique protein molecules in humans to 50,000-500,000. (Uhlén, 2005)

The primary structure of proteins, i.e. the amino acid sequence, is determined by the gene sequence stored in the DNA of every cell, together with possible post-translational modifications, including enzymatic cleavage of the protein. The usually long string of amino acids often forms secondary structure segments where hydrogen bonds between the C=O and the N-H groups of different peptide bonds play an important role. Two major forms of secondary structure are alpha-helices (Pauling, 1951) and beta-sheets (Pauling, 1951) often connected by more flexible loops or turns. In most proteins the formed secondary structures subsequently fold in what is called tertiary structures of which several can assemble into quaternary structures, exemplified by hemoglobin in figure 2.

While hydrogen bonds within the protein backbone are most important for the secondary structure formation, the side chains are most important for forming the tertiary and quaternary structure of a protein. Also in these higher levels of protein structure hydrogen bonds are involved, but other interactions based on hydrophobic effects (exclusion of water from hydrophobic parts of the protein), electrostatic effects (ionic bonds), and van der Waals interactions are often more important.

Many proteins are involved in interactions with other molecules, e.g. other proteins or small molecules. Such proteins can be responsible for sensing the environment and make the cells, and finally the whole organism, respond to changes. One example are the proteins acting as receptors for binding the hormone melatonin, a hormone secreted in higher levels when it is dark,

Joel Lindgren which induces a number of reactions which together make us feel more sleepy. (Witt-Enderby, 2003) The action of melatonin receptors can be used to treat people with sleeping problems or to more quickly get rid of jet-lag, by simply administering melatonin to activate the receptors and induce sleepiness.

VHLTPEEKSAVTALWGK

Figure 2. The different levels of protein structure illustrated using hemoglobin. Top left: The primary structure (the amino acid sequence) of a part of the protein is shown. Top right: The secondary structure of a part of one domain is shown, here as an alpha-helix. Bottom left: The tertiary structure, where the different folded secondary structure segments are packed against each other to create a defined 3D-structured domain, is illustrated. Bottom right: Here the complete structure of hemoglobin is shown, including the quaternary structure built up by two pairs of identical domains, i.e. four domains in total. The four heme groups, responsible for the oxygen binding, are shown in cyan, green, orange and red.

Other proteins are responsible for us feeling pain and developing an inflammatory response, and one well known example is cyclooxygenase (COX), an enzyme which induces an inflammatory response when the immune system has recognized an intruder and is involved in pain signaling.

Chemical Engineering of Small Affinity Proteins

Even though the pain and inflammatory response help the body to fight the intruder, these effects sometimes need to be reduced. This can be done by blocking the activity of COX, by for example taking the drug aspirin (acetyl salicylic acid), which irreversibly inhibits the action of COX and thereby exhibits an anti-inflammatory effect and reduces the experienced pain. (Vane, 2003)

The two examples illustrate how proteins can be activated or blocked to induce a pharmacological effect. As a matter of fact, the vast majority of all drugs that are used today target proteins, where G-protein coupled receptors (GPCRs) is the most common class of target proteins. (Rask-Andersen, 2011)

1.1 Protein therapeutics

Proteins themselves can also be used as drugs, either as replacement therapy when an endogenous protein is missing or present at reduced levels, or by administration of other proteins having a pharmacological effect. Two classical examples of replacement therapy are insulin, used by diabetic patients, and human growth hormone, used by patients with growth hormone deficiency. Protein drugs, or protein therapeutics, specifically generated for targeting other types of diseases, have during recent years gained interest and taken some focus from the classical low molecular weight drugs. Protein therapeutics often show excellent properties such as high potency and selectivity, and thus often show a lower risk of severe side effects compared to low molecular weight drugs. Moreover, protein-based drugs tend to have a faster clinical development time, which is a very costly part of the drug development process. (Leader, 2008;Carter, 2011;Dimitrov, 2012)

However, protein therapeutics are typically not compatible with oral delivery, and are instead generally administered though injection or infusion. An additional problem with oral delivery of proteins is the often large size; they often have too large hydrodynamic radii to be efficiently absorbed into the blood stream from the intestine, but by the aid of additives and different formulations uptake of small- to medium-sized proteins such as the 51 amino

Joel Lindgren acid long protein insulin have been reported. (Yun, 2013; Park, 2011; Swaminathan, 2012; Renukuntla, 2013)

The importance of understanding protein function and the growing importance of proteins as therapeutics have resulted in a massive amount of ongoing work in the protein research field.

1.2 Protein engineering

Nature has been modifying and improving proteins for their functions for billions of years through what we often refer to as evolution. By naturally occurring changes in the protein-encoding sequence, such as point mutations, insertions and deletions, together with natural selection, proteins have been able to evolve and contribute to the host´s ability to adapt to changes in the environment. This process has been going on in nature for billions of years and is responsible for that life has progressed on earth. In that time perspective, we as humans have only very recently learnt how to modify proteins to better fit our needs by what we refer to as protein engineering. Such modifications often involve amino acid substitutions, deletions or insertions, but also the creation of fusion proteins and the introduction of various protein tags, e.g. His-tags for affinity purification.

1.2.1 Protein engineering by rational design

Rational protein engineering often relies on available detailed structural information about a protein and defined amino acids are typically targeted for mutations. Rational protein engineering is also referred to as knowledge-based engineering, because already known information about the protein is used to decide what part of a protein, or even which specific amino acid, to target and mutate or modify.

In 1951 the amino acid sequence of a protein (insulin) was determined for the first time, which was an important milestone in protein engineering. (Sanger, 1951;Sanger, 1951) Seven years later, in 1958, the first high resolution 3-D

Chemical Engineering of Small Affinity Proteins structure of a protein (myoglobin) was determined using X-ray crystallography. (Kendrew, 1958) The exact amino acid sequence together with a high resolution 3-D structure of the protein of interest can make it possible to identify important parts and key residues in that particular protein. However, the toolbox to manipulate and modify specific residues in a controlled and systematic manner became available in the late 1970s, when general recombinant DNA technology together with techniques for site- directed mutagenesis and the polymerase chain reaction (PCR) were introduced, making it possible to mutate codons for single amino acids in a relatively straightforward manner. (Hutchison, 1978;Mullis, 1986) Since the 1980s much research has been performed using the rational approach. One early example is the work done on subtilisin, a protein commonly used in washing detergents. This protein has been extensively studied and engineered for increased catalytic efficiency and enhanced stability to high temperature, high and low pH, and oxidative inactivation. (Wells, 1988)

Even though rational protein engineering can be very useful, it is often hard to predict the effect certain mutations will have on function, structure and stability of the protein. Typically, many amino acids are involved in interacting with other molecules, and these are commonly dependent on the overall structure of the protein for correct positioning. One single amino acid substitution can have a huge effect on the structure and function of a protein. In an engineered domain from the streptococcal protein G (SPG), a single mutation was shown to promote the protein to adopt a completely different fold, with both the secondary and tertiary structures of large parts of the protein are changed. (He, 2012) It can be argued that this is a non-natural protein domain and perhaps not very representative for most proteins, but it still indicates how fragile proteins can be.

1.2.2 Protein library technology

Protein library technology relies on the creation of large repertoires of protein variants (libraries) from which particular members can be identified via

Joel Lindgren screening or selection based on desired properties, e.g. improved catalysis, increased stability, or high affinity to a target protein.

This approach can be seen as a mimic of the natural selection process in nature, but at a much shorter time span. Typically there has to be a physical link between the phenotype, which is the protein in this case, and the genotype, which is the DNA or RNA sequence encoding for that protein. Moreover, an efficient screening or selection system has to be used to be able to fish out and identify proteins with the desired properties. There are many different types of selection systems available today. Cell-based systems, such as phage display, bacterial display and yeast display, all rely on living cells to generate the link between phenotype and genotype (Smith, 1985;Gai, 2007;Löfblom, 2011), while the cell-free systems, such as ribosome display and mRNA display, are truly in vitro systems with no need of living host cells. (Lipovsek, 2004)

Even when a library with many billion different proteins is used, typically only a fraction of the theoretical diversity is covered. If for example 13 positions are randomized, and all 20 amino acids are allowed in each position, there are theoretically 1320 different possible protein variants. Practically, the library size is often limited to ~109 members. (Reetz, 2008) To circumvent this potential problem one can introduce additional diversity during the selection process, which even further mimics evolution, e.g. by using error prone PCR. (Cadwell, 1992) Today libraries are often constructed based on prior information about protein function, structure and sequence, in a semi-rational approach. Constraints on which amino acid positions that are varied and which amino acids that are allowed in those positions may reduce the theoretical library size dramatically and often yield libraries with a higher degree of functional proteins. (Lutz, 2010)

1.3 Affinity proteins

As already mentioned, many proteins recognize and interact with other molecules. Some proteins are specialized in recognizing and binding very

Chemical Engineering of Small Affinity Proteins tightly to only one, or a group of, other target molecule. It is said that this type of proteins has high affinity and selectivity for that target; thus such proteins are often referred to as affinity proteins. The most well-known class is antibodies, or immunoglobulins (Ig), which is the golden standard when it comes to affinity proteins. Antibodies are naturally occurring and vital for the immune system of humans and higher vertebrates, but they have also been used for a long time in man-made applications as research reagents and later also for in vivo diagnostics and therapy of cancer. (Carter, 2001) Antibodies, and variants of antibodies, are still the most widely used and generally most successful affinity protein, but there are today a number of non-antibody based classes of affinity proteins, or alternative scaffold proteins as they are often called. Some selected alternative scaffold proteins are listed in table 1. (Friedman, 2009;Löfblom, 2011;Ruigrok, 2011) Another interesting approach is the combination of designed polypeptide scaffolds to which small molecules can be attached to create selective high affinity binders. (Tegler, 2011)

Common for most alternative scaffolds is that they are relatively small and stable proteins that fold independently and have high solubility in water. The scaffold should have a high tolerance towards mutations of certain amino acids, without compromising the structure of the protein, which is a prerequisite to be able to create large libraries of well-folded proteins. Two affinity proteins central for this thesis are the Affibody molecule and the albumin binding domain (ABD), which I will introduce in section 1.3.1 and 1.3.2, respectively.

Joel Lindgren

Table 1. Examples of alternative scaffold proteins.

Number of Number of Name MW (kDa) Binding motif Selected references amino acids disulfide bonds

Adnectin 94 10 - LoopsLipovsek, 2011

Affibody 58 7 - Helices Löfblom, 2010

Anticalin ≈170 ≈20 0 - 2 Loops Schonfeld, 2009

DARPin 67 + n x 33 14 + n x 3 - Helices + loops Binz, 2004

Knottin ≈ 30 3 3 - 4 Loop Schmidtko, 2010

Kunitz domain 58 7 3 Loop Williams, 2003

Peptide aptamer ~100 ~20 1 Loop Kunz, 2006

1.3.1 Affibody molecules

One important class of non-antibody derived affinity proteins is the Affibody molecule, derived from one of the immunoglobulin-binding domains of staphylococcal protein A (SPA). SPA has five homologous domains that are individually folded and all separately capable of binding immunoglobulins. One of the domains, the 58 residue B-domain, had earlier been optimized for use as affinity fusion partner through amino acid substitutions to yield the very stable Z-domain. (Nilsson, 1987) This Z-domain folds in three anti- parallel α-helices that form a compact three-helix bundle with a hydrophobic core (illustrated in figure 4). (Wahlberg, 2003;Lendel, 2006;Lindborg, 2013)

Chemical Engineering of Small Affinity Proteins

Side view Top view Side view Top view

Side view Top view Figure 3. The structure of an Affibody molecule is shown, here illustrated by the original Z domain (pdb: 1H0T). The side chains of the thirteen amino acid positions in helices 1 and 2, which are randomized to create large Affibody molecule libraries, are shown in green.

To construct unbiased Affibody molecule libraries, thirteen surface-exposed amino acids in helices one and two are typically randomized. (Nord, 1995;Nord, 2001;Wikman, 2004;Grönwall, 2007;Grimm, 2011;Kronqvist, 2011;Lindborg, 2011) Nine of the thirteen residues are amino acids known to be important in binding to IgG, thereby the affinity to IgG has been removed for the absolute majority of the different library members. (Deisenhofer, 1981;Cedergren, 1993) A further optimized scaffold has been created by substituting additional nine residues not involved in the binding interface. (Feldwisch, 2010) From libraries based on either the original or the second generation scaffold, a large number of Affibody molecules with specificity to different targets have been selected and used in a large variety of applications, some of which are shown in figure 4. (Löfblom, 2010)

Affibody molecules typically fold quickly and are highly stable, and are small relative to most other affinity proteins, including IgG, which has more than

Joel Lindgren

20 times higher molecular weight (7 kDa for Affibody molecules compared to 150 kDa for IgG). The small size makes it possible to synthesize Affibody molecules using solid phase peptide synthesis (SPPS), an advantage discussed further in the following chapters. The high stability and quick folding and re- folding, together with the small size and relatively simple production, make the Affibody molecules highly successful in biotechnological applications, including affinity separation of antibodies (in e.g. MabSelect SuRe sold by GE Healthcare). The small size gives Affibody molecules fast biodistribution and tissue penetration, together with a rapid clearance of unbound molecules, which are ideal properties for use as imaging agents in vivo, for e.g. cancer diagnostics. (Tolmachev, 2008)

SPIO SPECT Enzyme fusion

Conjugated Fc fusion fluorescence PET Bacterial toxin

Viral MRI Fusion proteins Nuclide Nanoparticle Fused fluorescent protein Optical Particles Liposome

Receptor Enzyme Blocking Blocking Cell-Cell Detection interaction

Conjugations Enzyme fusion Separation Enzyme

IP MabSelect SuRe Protein [GeHealthcare] ligand

Chemotoxin Radiotherapy Microarray, FRET

Figure 4. Examples of applications of Affibody molecules (figure adopted from Löfblom et al. 2010 with permission of the copyright holder).

Chemical Engineering of Small Affinity Proteins

1.3.2 Albumin binding domain (ABD)

The second affinity protein central for this thesis is the albumin binding domain (ABD). Similarly to the Affibody molecule, ABD is also derived from a bacterial protein, namely streptococcal protein G (SPG) from strain G148. SPG contains three homologous immunoglobulin-binding domains and three homologous albumin-binding domains. (Olsson, 1987) The third albumin- binding domain of protein G from the streptococcal strain G148 (abbreviated G148-GA3), hereafter simply referred to as ABD, has been extensively studied. The solution structure of ABD was solved using NMR, which showed that also this protein folds as a three-helix bundle (figure 5). (Johansson, 2002)

Side view Top view Figure 5. The structure of the albumin binding domain (ABD) studied in this thesis is shown, here illustrated by the structure of the last 46 amino acids in of G148-GA3 (pdb: 1GJT) The residues important for albumin binding are highlighted in red (based on mutational analysis (Linhult, 2002))

The binding strength of proteins is often illustrated and compared by their equilibrium dissociation constants (KD) to their target proteins. ABD has a KD of approximately 5 nM to human serum albumin (HSA), which is a rather high affinity, comparable to the affinity many biotechnologically used

Joel Lindgren monoclonal antibodies (mAbs) have to their targets. (Linhult, 2002) By introducing point mutations in ABD and comparing the affinity to HSA, important amino acids were identified and it was determined that the binding to HSA is predominantly restricted to the second helix and the loops flanking it. (Linhult, 2002) The affinity to HSA has been improved significantly by creating libraries and using phage display to select high affinity variants of

ABD, resulting in a variant denoted ABD035 with a KD of approximately 120 fM to HSA. (Jonsson, 2008) Furthermore, other variants based on ABD035 have also been created and used for half-life extension, discussed below. (Frejd,

2012)

1.3.2.1 ABD for half-life extension of biotherapeutics

Both IgG and HSA have remarkably long in vivo circulation half-lives, with a half-life of approximately three weeks. Because of their large size, neither IgG (150 kDa), nor HSA (67 kDa) are renally cleared. Moreover, the neonatal Fc receptor (FcRn) binds endocytosed IgG and albumin and transports them to the outside of the cell, where they are released back into the blood circulation. The two proteins are hereby rescued from degradation in lysosomes and hence have very long in vivo circulation half-lives. (Anderson, 2006)

It has been shown that it is possible to take advantage of the long half-life of albumin by genetically fusing a protein of interest to albumin and thereby getting a longer circulation half-life of the partner protein. One example where this approach is used is in Albiglutide, a fusion protein of HSA and a dimer of the 30 amino acid peptide GLP-1. (Bush, 2009)

An alternative approach to direct fusion is to use a high affinity binder to albumin as gene fusion or conjugate partner to the protein of interest. Albumin, being the most abundant protein in blood with a concentration of ~40 g/L (0.6 mM), will then likely form a non-covalent, but tightly bound complex, with the protein conjugate in vivo and thereby potentially increase the half-life of the protein of choice (illustrated in figure 6). One example of this approach was the genetic fusion of an Affibody molecule dimer with an

Chemical Engineering of Small Affinity Proteins engineered variant of ABD. The ABD–Affibody molecule fusion protein showed similar in vivo half-life as the albumin control, thereby indicating that the ABD fusion protein is rescued from degradation by FcRn as a complex together with albumin. (Andersen, 2011)

The possibility to control the circulation time for a protein used in vivo can be very useful. For some applications a fast in vivo clearance is desired (e.g. in cancer diagnostics using molecular imaging) while for other applications a long circulation time is desired (e.g. for many protein therapeutics).

(1) Bloodstream pH 7.4

(5) (2) Inside of cell

(3) (4) Recycling Albumin Sorting endosome endosome ABD pH5-6 (6) Fusion partner Lysosome Other serum protein FcRn

Figure 6. Schematic illustration of FcRn-mediated rescue of an ABD-fusion protein. ABD with its fusion partner bind the highly abundant albumin (~40 mg/ml) in the bloodstream (1). Protein are taken up by hematopoietic or endothelial cells by endocytosis (2). In the sorting endosome the pH is lowered and albumin together with associated proteins bind to membrane-bound FcRn (3). Recycling endosomes then transport the complex back to the cell surface (4), and upon exposure to physiological pH albumin, and proteins bound to albumin, are released from the FcRn and recycled back to the bloodstream (5). Proteins not bound to FcRn in the sorting endosome will be degraded in the lysosome (6).

Joel Lindgren

2 Protein synthesis

Proteins can essentially be produced by two different methods, through recombinant production or by chemical synthesis. In recombinant production the protein producing machinery of host cells is used, where cells from bacteria, yeast, insects or mammals are grown under controlled conditions. The gene encoding for the protein of interest is inserted into the host cells, and the protein of interest can often be produced in high yield at a relatively low cost. After some time the cells are typically harvested and the protein of interest purified from the complex mixture of host cell proteins. Recombinantly produced proteins can then be modified using bioconjugation chemistry, e.g. by coupling to cysteine residues, using maleimide chemistry (Monji, 1978), or to primary amines (lysine residues and the N-terminus), using succinimidyl esters.

The alternative production route is to use chemical synthesis, which has many advantages compared to recombinant production in host cells. Using chemical synthesis expands the possible building blocks beyond the twenty DNA- encoded amino acids, enabling the use of non-natural amino acids to change the properties of the protein or to introduce new functionalities. It also makes it possible to build branched proteins and introduce non-amino acid building blocks, such as polyethylene glycol (PEG), carbohydrates, and lipids. Using chemical protein synthesis it is possible to site-specifically label a protein with e.g. fluorescent probes, chelators for radiolabeling, or spin labels for electron paramagnetic resonance (EPR) (Altenbach, 1990) still with an atom-to-atom precision, which is an advantage for many applications, but particularly important when using high resolution spectroscopic methods. Chemical synthesis makes it possible to engineer proteins to perform better in a particular application; as an example it is possible to extend the in vivo half-life of a protein by increasing the hydrodynamic radius in a controlled manner by site-specific PEGylation. (Jevsevar, 2010) Furthermore, recombinant production of proteins in gram-negative bacteria, including E. coli, often yields a product with contaminants such as lipopolysaccharides (LPS, often referred

Chemical Engineering of Small Affinity Proteins to as endotoxins). These can be toxic if injected into the bloodstream of a patient, even in very small amounts, and LPS can often be challenging to remove in downstream processing of protein therapeutics. (Bito, 1977;Liu, 1997)

Chemical protein synthesis indisputably opens doors closed in classical molecular biology, and the importance of the research field is demonstrated by the several Nobel laureates exploring different aspects of protein synthesis, some mentioned below.

2.1 The pre-solid phase peptide synthesis era of chemical protein synthesis

The quest to be able to synthesize proteins chemically was initiated by Emil Fischer, when he in 1901 was able to link two amino acids together with a peptide bond. He used the simplest amino acid glycine to make glycyl-glycine, and named it a dipeptide; thus the word peptide was introduced. (Fischer, 1901) Interestingly, Fischer was awarded the Nobel Prize in chemistry only one year later, not for his work on peptides, but instead for previous work on sugar and purine synthesis.

In the reaction when a new peptide bond is formed, the primary amine of one amino acid attacks the carbonyl carbon of another amino acid, forming a new covalent bond between the nitrogen and the carbonyl carbon (see figure 7). Fischer made a homodipeptide and therefore did not need to control which amino acid that was supposed to act as a nucleophile and which to act as an electrophile. The coupling of two different amino acids would be more problematic since both amino acids have a primary amine, acting as a nucleophile, and a carboxyl group to be attacked. This problem was solved in 1932 when Bergmann and Zervas introduced the Nα-protecting group benzyloxycarbonyl (abbreviated Cbz or Z), thereby making it possible to protect the primary amine on one amino acid to prevent that residue to act as a nucleophile. (Bergmann, 1932) To drive the reaction in the desired direction

Joel Lindgren various activation strategies of the carboxyl group have been used, making the carbonyl carbon more electrophilic and thus more reactive. In 1965 Sheehan and co-workers reported the use of carbodiimides for activation of the carboxyl group, which still today is used for some applications. (Sheehan, 1965) Many of the twenty amino acids have functional groups on the side chains that can participate in chemical reactions and lead to unwanted side reactions during the synthesis. Protecting groups for the different amino acid side chains were early introduced, but have undergone further development to function better under different conditions. (Isidro-Llobet, 2009)

O O O R2 H N H N 2 + 2 H2N OH OH OH N + H2O H R R 1 2 R1 O

O O O R2 PG NH OH PG NH H2N + H AG AG + OH N H R1 R2 R1 O

Figure 7. Top scheme: The reaction scheme for the coupling of two amino acids is shown. In nature this reaction is catalyzed by the ribosome. Bottom scheme: The use of an amino protecting group (PG) and a carboxyl activating group (AG) is illustrated.

After the early attempts, continuing research in the field further developed peptide synthesis, and one major breakthrough was in 1953, when du Vigneaud and his colleagues synthesized the first biologically active peptide. (duVigneaud, 1953) The peptide was the nine amino acid long peptide hormone oxytocin, and du Vigneaud was two years later awarded the Nobel Prize in chemistry for the achievement.

2.2 Solid phase peptide synthesis (SPPS)

Up to the early 1960s, all peptide synthesis was performed using classical organic chemistry in solution, which is time-consuming and very laborious, and associated with severe solubility problems of the fully protected peptides.

Chemical Engineering of Small Affinity Proteins

In 1963 Bruce Merrifield published a paper that would truly revolutionize the world of peptide synthesis. In that paper Merrifield founded the concept of solid phase peptide synthesis (SPPS) that gave him the Nobel Prize in chemistry in 1984 (the basic concepts of SPPS are illustrated in figure 7). (Merrifield, 1963)

The key feature of SPPS is that the growing peptide being synthesized is anchored to a solid support (resin) via the carboxyl group of the C-terminal amino acid, thus permitting the use of excess reagents in each coupling step, which then can be removed by simple filtration. The alpha-amine of each subsequent amino acid is reversibly protected when it is added to the peptide immobilized on the resin. Merrifield initially used the Cbz protecting group, but the harsh conditions needed for deprotection were not ideal. Merrifield quickly developed another protecting group, the tert-butyloxycarbonyl (t- Boc), which could be cleaved under milder conditions, thus increasing the synthesis yield. (Merrifield, 1964) The next amino acid to be incorporated is added in excess, with an Nα-protecting group and an activated with an electron-withdrawing group, making the carbonyl more reactive to nucleophilic attack by the amino group of the previously coupled residue. After coupling of the amino acid, any unreacted reagents can easily be filtered away and the product bound to the solid support can be carefully washed. To avoid polypeptides that have deletions, meaning that they miss one or several amino acids in the middle of the sequence, irreversible capping of potential unreacted primary amines is usually performed after the coupling step. This can be done using acetic anhydride, which efficiently caps all free amines and hinders them to react with other activated amino acids later in the synthesis. This procedure yields truncated peptides instead of polypeptides with deletions, which typically are harder to separate from the desired, full-length product.

Next, the N-terminal protecting group can be removed and another amino acid coupled, and this is repeated until the peptide chain is finished. Finally, the polypeptide can be released from the solid support and the side chain

Joel Lindgren

H O T-PG N AG HO Linker Resin Rn PG

Attachment of first amino acid to the solid support

H O T-PG N O Linker Resin Rn PG

Repeat for each amino acid Deprotection of the temporary amino protecting group

H2N O Linker Resin Rn PG H O T-PG N AG Coupling of next amino acid Rn-1 PG

Rn-1 H O N T-PG N O Linker Resin H O Rn PG

Final deprotection and removal of side chain protecting groups Cleavage from the solid support

AG Carboxyl activating group

O R H O T-PG Temporary amino protecting group H N N 2 N OH H PG Side chain protecting group R1 O Rn n-2 Figure 8. The basic principles of solid phase peptide synthesis are illustrated.

Chemical Engineering of Small Affinity Proteins protecting groups removed; usually this is done in a one-pot reaction, yielding the fully unprotected product in solution.

However, using Cbz or t-Boc as N-terminal protection requires repetitive acid treatments and the use of acids with varying strengths to be able to selectively deprotect the N-terminus or the side chains, or release the polypeptide from the solid support. Typically one has to use extremely strong and toxic acids, e.g. hydrofluoric acid (HF), to cleave the peptide from the solid support, which is not only dangerous to handle, but can also lead to undesired side reactions. Another approach, introduced by Carpino and Han in 1972, makes it possible to use much milder reaction conditions. They developed the base- labile 9-fluorenylmethyloxycarbonyl (Fmoc) group that can be used for protection of the alpha-amino group of amino acids. (Carpino, 1972) This method, often referred to as Fmoc chemistry, is the most commonly used approach in SPPS today. The Fmoc protecting group is removed by base treatment (typically 20% piperidine), while the removal of the side chain protecting groups and the cleavage from the solid support are achieved by treatment with 95% triflouroacetic acid (TFA). Furthermore, today there is a number of orthogonal protecting groups available, meaning that they can be selectively deprotected, and thereby exposing only selected functional site(s) for further modification. (Albericio, 2000;Isidro-Llobet, 2009)

Another important factor for successful protein synthesis is the activation of the carboxyl group with an electron-withdrawing group, making the carbonyl carbon more electrophilic and more susceptible to a nucleophilic attack by the amino group of the previously coupled amino acid. The already mentioned carbodiimides were first developed, where the C-terminal carboxylic acid is replaced with a highly reactive O-acylisourea. (Sheehan, 1965) These coupling reagents are very efficient, but can cause racemization of the chiral alpha carbon, which potentially gives the polypeptide undesired properties. Relatively soon the combination of carbodiimides and additives in the form of triazoles such as 1-hydroxybenzotriazole (HOBt) was introduced, yielding an active ester, which gives a reduced risk of racemization. (Künig, 1970) Some

Joel Lindgren years later new coupling reagents were developed which made it possible to activate the carboxyl group without the use of carbodiimides, e.g. 2-(1H- benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). (Fields, 1991) There is today a large number of other commonly used activators for amino acid coupling and SPPS. (Albericio, 2004;Pattabiraman, 2011)

The relatively simple and repetitive chemistry used in SPPS makes it well suited for automation, and there are a number of different commercial peptide synthesizers, for small scale parallel synthesis to larger scale synthesis. By using Fmoc chemistry there is no need for special lab equipment other than materials commonly found in biotechnological laboratories.

Even though SPPS is a very attractive method for the generation of proteins, it does have some limitations, with the most severe one being the limitation in protein length possible to synthesize, as mentioned previously. Due to the iterative process and many coupling and deprotection steps in SPPS, the synthesis yield of each reaction has to be very high to give a reasonable over all synthesis yield. The theoretical total yield for a relatively short protein of 50 amino acids would only be 60% even if the yield is 99% in each step, while it would drop to below 8% with a 95% yield in each step. In practice, the coupling yield is not constant but instead differs between different coupling steps and regions in the sequence, e.g. some amino acids are harder to couple due to steric hindrance, and some regions of a sequence can be difficult due to self-association and aggregation of the resin-bound peptide. Even though methods to overcome these problems have been developed, such as low loading resins and pseudo-prolines (Mutter, 1995;Valente, 2005), peptide sequences of over 100 amino acids have been hard to synthesize with reasonable yields. (Albericio, 2004;Kent, 2009) Given that the average protein is approximately 300 amino acids long, other approaches have to be considered to be able to produce a synthetic full-length protein.

Chemical Engineering of Small Affinity Proteins

2.3 Peptide ligation strategies

One approach to circumvent the size-limitation of SPPS can be to fuse smaller peptide fragments to create a full-length protein. An ideal ligation method should be selective and possible to use with unprotected side chains without any side reactions, it should be possible to perform at mild reaction conditions, and it should yield a native peptide bond at the site of ligation. Some selected ligation methods are described in this section.

2.3.1 Native chemical ligation (NCL)

A significant contribution to the field of peptide ligation has been made by the group of Stephen Kent. Interestingly, Kent did his post-doctoral work in the lab of the founder of SPPS, the legendary Bruce Merrifield. The first ligation approach suggested from the Kent group relied on the formation of a non- amide bond between two peptide fragments. (Schnolzer, 1992) However, already two years later the same group reported a ligation method that would yield a native peptide bond at the site of ligation, which they named “Native Chemical Ligation” (NCL). (Dawson, 1994) The principle of NCL is illustrated in figure 9, and described here in short. NCL relies of the reaction between two polypeptides, one carrying a C-terminal thioester (Fragment 1), and the other having an N-terminal cysteine (Fragment 2). The first step of the ligation is a nucleophilic attack from the side chain of the N-terminal cysteine residue in fragment 2 to the C-terminal carbonyl of fragment 1, yielding a thioester linkage between the fragments. This orients the N- terminal amine of fragment 2 in a favorable position for another nucleophilic attack on the same carbonyl of fragment 1, resulting in the second, irreversible, step of the ligation, the S-to-N acyl shift, which yields a native peptide bond at the site of ligation. NCL can be performed in aqueous solution at neutral pH with fully unprotected peptides, and it is even possible to have other cysteine residues in the peptides without them interfering with the ligation reaction. The requirement of a C-terminal thioester was initially a limitation of NCL when using Fmoc chemistry since the thioester is not stable

Joel Lindgren during the repetitive base treatments used used for Fmoc deprotection. However, today many methods for forming the thioester after completed synthesis have been developed, where the use of a diaminobenzoyl linker is especially straightforward. (Blanco-Canosa, 2008)

The high selectivity and efficient ligation reaction of NCL has made it very popular and widely used. The limitation of the demand of a cysteine, which are relatively rare in natural proteins, at the site of ligation has been circumvented by the possibility to convert the cysteine to the more common alanine by desulfurization after the ligation reaction. (Pentelute, 2007)

One interesting example where NCL has proved very successful is for the synthesis of the 166 amino acid erythropoietin (EPO) glycosylated at all of the four native glycosylation sites, resulting in a fully synthetic wild type EPO. (Wang, 2013)

O O O

R H2N H2N Fragment 1 S NH Fragment 2 OH

Transthioesterification O (reversible)

H2N Fragment 1 S O

NH Fragment 2 OH H2N O

SNacylshift (irreversible)

O SH O

NH Fragment 2 OH H2N Fragment 1 NH

O Native peptide bond

Figure 9. The principle of the native chemical ligation (NCL) reaction is shown.

Chemical Engineering of Small Affinity Proteins

2.3.2 Traceless Staudinger ligation

Similarly as NCL, also the traceless Staudinger reaction yields a native peptide bond at the site of ligation without any residual atoms. (Nilsson, 2000;Saxon, 2000) An advantage of the traceless Staudinger ligation is that it is not restricted to any special amino acid residues. The method relies on the ligation of one fragment having a C-terminal phosphinothioester and another fragment with an N-terminal azide.

The traceless Staudinger ligation has not yet been used as much as NCL and the practical significance of the method needs to be further verified. However, there are some examples where the method has been fruitful. The ligation method has for example been used to synthesize a number of different glycoproteins (Liu, 2006) and to prepare small cyclic peptides. (Kleineweischede, 2008) The method is well suited to be used together with NCL since the chemistries are orthogonal, as exemplified by the synthesis of the 124 amino acid ribonuclease A (RNase A) with an isotope-labeled proline in position 114 as a probe for NMR studies of the protein. (Nilsson, 2003) In this example the traceless Staudinger ligation was performed with the peptide still attached to the solid phase.

2.3.3 Expressed protein ligation (EPL)

Expressed Protein Ligation (EPL) combines classical molecular biotechnology with protein synthesis in an ingenious way. The method makes it possible to combine a recombinantly produced protein with a synthetic peptide yielding a native peptide bond at the site of ligation. (Muir, 1998) The method takes advantage of the naturally occurring protein splicing, which is a post- translational modification that cleaves out a protein fragment (an intein). The desired protein gene is simply cloned into a special vector containing the sequence of an intein. After expression and purification of the fusion protein, the ligation reaction is performed by the addition of the peptide of interest, containing an N-terminal cysteine, and a thiol catalyst (e.g. thiophenol), resulting in the formation of a C-terminal thioester, which rapidly undergoes

Joel Lindgren

NCL. EPL has been used to chemically modify proteins in a large number of ways, including introduction of fluorescent probes, post-translational modifications, stable isotopes, and unnatural amino acids. (Muir, 2003)

2.3.4 Enzyme-catalyzed ligation

Another method to combine recombinant production with SPPS is by protein ligation catalyzed by enzymes. Two examples of well-studied enzymes are Subtiligase and Sortase A, which can ligate two proteins, or peptides, in a clean reaction resulting in a native peptide bond. (Abrahamsen, 1991;Mao, 2004) For Sortase A-mediated ligation, the C-terminal protein fragment needs to have an N-terminal glycine tag (two or three glycine residues), while the N- terminal fragment has to include a C-terminal LPXTGG-motif (X is an arbitrary amino acid). Sortase-mediated ligation can thus also be used with two synthetic peptides or two recombinantly produced proteins since no chemical modifications are needed. However, the product will have an - LPXTGGG- sequence between the ligated fragments. Sortase A-mediated ligation can also be used to prepare backbone-cyclized proteins. (Proft, 2010)

2.3.5 Non-native ligation methods

There are a number of other ligation methods available that do not form a native peptide bond at the site of ligation, e.g. oxime-, hydrazone-, and thiazolidine ligation that all rely on the reaction of an aldehyde with a hydroxylamine, a hydrazine or an aminothiol, respectively. (Shao, 1995) Cycloaddition reactions (“Click chemistry”) are other popular methods used by protein chemists to ligate two peptide fragments. (Kolb, 2001)

The selective reaction between thiols and haloacetyl groups is a popular and efficient method used for peptide ligation. (Lu, 1991) The resulting bond is a thioether which is stable and insensitive to reducing conditions, as compared to disulfides which can be reduced and undergo thiol/disulfide exchange. Using SPPS, haloacetyl groups can straightforwardly be site-specifically introduced in polypeptides, e.g. on the side chain of lysine, and be used for

Chemical Engineering of Small Affinity Proteins conjugation to thiol-bearing proteins, peptides or other biomolecules. Thioalkylation can also be used for intramolecular crosslinking, by the reaction of an introduced haloacetyl group with the side chain of cysteine. (Ekblad, 2009)

Present investigation Chemical Engineering of Small Affinity Proteins

3 Aim of the thesis

The overall aim of this thesis was to investigate if rational design and chemical protein engineering could be used to improve the properties of the affinity proteins ABD and Affibody molecules.

Paper I The aim of this study was to investigate if native chemical ligation could be used to synthesize Affibody molecules, and to develop a synthetic scheme enabling a combinatorial assembly of Affibody molecules.

Paper II The aim of this study was to investigate if the dimeric Affibody molecule targeting the amyloid-beta peptide, relevant for the development of Alzheimer’s disease, could be prepared by SPPS and if the synthetic yield could be increased by N-terminal engineering without reducing the affinity to the amyloid-beta peptide.

Paper III The aim of this study was to investigate if an Affibody molecule devoid of intrinsic fluorescence targeting amyloid-beta peptide could be developed for use as a research tool to study the copper binding of the amyloid-beta peptide by fluorescence spectroscopy.

Paper IV The aim of this study was to investigate the feasibility of covalently linking ABD to GLP-1, a bioactive peptide proven to be very beneficial for diabetic patients, for potentially increased in vivo half-life and less frequent administration.

Joel Lindgren

Paper V The aim of this study was to investigate if intra molecular cross-linking of ABD could enhance the protease stability of the protein, as a step towards orally available ABD fusion proteins.

Chemical Engineering of Small Affinity Proteins

4 Paper I – A native chemical ligation approach for combinatorial assembly of Affibody molecules

Depending on the application of the affinity protein, various modifications or functional groups often need to be introduced. A fluorescent probe can be introduced for in vitro visualization and if the protein should be used for in vivo diagnostics, labeling with a radionuclide can be performed by introduction of a chelator. If the affinity molecule instead is intended for use in therapy, it may be conjugated to an effector molecule such as a cytotoxic group. By using SPPS for the production of the affinity protein, site-specific introduction of such reporter groups or effector molecules may be facilitated. Moreover, several modifications can site-specifically be introduced in the same molecule, which is generally hard to achieve with a recombinantly produced protein.

As previously discussed in the introduction of this thesis, proteins are often larger than 50-60 amino acids and thus challenging to produce with reasonable yields using standard SPPS. Nevertheless, the 58-residue large Affibody molecules have previously been successfully synthesized using direct SPPS. (Renberg, 2005;Engfeldt, 2007;Ekblad, 2009;Heskamp, 2012) In these examples the Affibody molecules were also site-specifically labeled with reporter groups such as biotin moieties, fluorophores, and radionuclide chelators for use in both in vitro and in vivo applications. However, the synthesis yield of the Affibody molecules is highly sequence-dependent and the synthesis often needs to be optimized for each Affibody molecule variant, and the use of expensive pseudo-prolines is sometimes required to get acceptable yields. A classical method to increase the synthesis yield in organic chemistry is to use parallel synthesis of smaller sub-fragments and later combine them to give the desired molecule. The same approach is vital also for protein synthesis, where the shorter fragments are synthesized with high yield followed by an efficient condensation reaction where the fragments are joined together to form the full-length protein.

Joel Lindgren

Figure 10. Schematic illustration of the combinatorial assembly approach using NCL for preparation of Affibody molecules. The target selectivity of Affibody molecules is determined by the N-terminal amino acid sequence found in helices 1 and 2 (I). The C-terminal part contains helix 3 (II), and is constant in all Affibody molecules. Various modifications can readily be introduced in helix 3 at this stage. After the peptides are cleaved from the solid support (III), NCL is used to yield the full-length Affibody molecules (IV).

In this study we wanted to investigate if the NCL method (Dawson, 1994) could be used to produce Affibody molecules using a fragment condensation approach. Affibody molecules consist of one variable part (helices 1 and 2), determining the target binding specificity, and one constant part (helix 3), needed for protein stability, making a fragment condensation approach very appealing. It would enable combinatorial assembly of the protein, where the same, or different, constant part could be ligated to different variable parts, thus creating full-length proteins with different specificities and overall properties. Thus, if different batches of constant parts are modified with reporter groups or effector molecules, a panel of Affibody molecules with

Chemical Engineering of Small Affinity Proteins different specificities and various modifications can readily be prepared, as illustrated by the schematic figure above (figure 10).

The flexible loop between helix 2 and helix 3 was chosen as NCL site; more specifically the peptide bond between glutamine 40 and serine 41 of the original Affibody scaffold. To enable for the NCL two substitutions were introduced; in position 40 a glutamine to glycine substitution for increased flexibility and minimal steric hindrance, and in position 41 a serine to cysteine substitution for the introduction of the required 1,2-aminothiol.

The generation of the constant C-terminal fragment with the N-terminal cysteine was straightforward and the synthetic yield of this fragment was high with few side products. The last amino acid, lysine 58 in the original Affibody molecule scaffold, was introduced with a 4-methyltrityl (Mtt) protecting group that could be selectively deprotected to give a free amine used for site- specific conjugation to reporter groups.

The N-terminal fragments containing the C-terminal thioester were more challenging to prepare. Since the thioester is not stabile to the repeated Fmoc deprotections, the thioester has to be formed at the end of the synthesis. Initially three different methods for thioester formation were evaluated; the approaches of using a “safety catch” sulfamylbutyryl linker (Quaderer, 2001) or an aryl hydrazine support (Camarero, 2004) were not successful in our hands, while the use of a diaminobenzoyl linker (Blanco-Canosa, 2008) worked very well. Using this linker, a peptide with a C-terminal N-acyl- benzimidazolinone (Nbz) group was prepared and cleaved from the resin. The peptide-Nbz is stable at low pH and under the conditions normally used during workup and purification of peptides. However, in aqueous solution at neutral pH the Nbz group is quickly cleaved by thiolysis and replaced by a thioester, which can be used in NCL. The formation of the thioester was done in situ during the NCL by the addition of the thiol catalyst 4- mercaptophenylacetic acid (MPAA).

Joel Lindgren

The NCL reaction proved to be very selective and with few side reactions other than the competing hydrolysis of the thioester. The reaction was complete after approximately 3 hours incubation, illustrated by figure 11, and after overnight incubation typically no adducts or by-products could be detected, thus providing flexibility in the practical work procedure. The very clean reaction also makes it possible to use unpurified (crude) starting materials in the ligation reaction, making the preparation time significantly shorter and less laborious.

MPAA Z (1-40)-Nbz A HER2 400 Z(41-58) 300 200 100 0

0 1020304050 U

A MPAA m

/ B A 400 Z(41-58) 300 ZHER2(1-58) 200 100 0 0 1020304050 t /min

Figure 11. A typical ligation reaction between purified starting materials ZHER2(1–40)- Nbz and Z(41–58) analyzed using RP-HPLC is shown. The chromatograms show the absorbance at 220 nm for the ligation mixture at A) t=0, and B) t=3 h. MPAA (4- mercaptophenylacetic acid) is the thiol catalyst used in the reaction.

Three different Affibody molecules were prepared using the NCL approach,

ZIgG(1-58) targeting IgG, ZInsulin(1-58) targeting insulin, and ZHER2(1-58) targeting human epidermal growth factor receptor 2 (HER2). The three Affibody molecules were analyzed using surface plasmon resonance (SPR), where the binding to the respective target proteins was verified and the binding affinity (KD) to each respective target correlated well with previously reported values.

Chemical Engineering of Small Affinity Proteins

The constant C-terminal fragment was synthesized in very high yield, providing a perfect starting point for further modifications. This was exemplified by the site-specific introduction of the fluorescent probe (5-(6)- carboxyfluorescein (CF)) on the side chain of lysine in position 58. This labeled fragment was ligated to the HER2-specific helices 1 and 2 to yield the full-length labeled Affibody molecule ZHER2(1-58)-CF. The function of this labeled Affibody molecule was verified using fluorescence microscopy, which clearly showed the expected staining of a HER2-overexpressing cell line and not the negative control cell line.

To conclude, in this paper we describe a method to use NCL for the synthesis of Affibody molecules, enabling a combinatorial assembly approach that makes it straightforward to prepare a panel of site-specifically modified Affibody molecules in relatively high yield.

Joel Lindgren

5 Paper II – N-terminal engineering of amyloid-beta- binding Affibody molecules yields improved chemical synthesis and higher binding affinity

Still today more than one hundred years after Alzheimer’s disease (AD) was first described, the exact cause of the disease remains unknown. However, at present, the short and aggregation-prone amyloid-beta (Aβ) peptides are believed to be the most important factor for the onset and development of AD. (Gilbert, 2013) Aβ peptides vary in length between 39 and 43 residues, where Aβ (1-40) is the most abundant while Aβ (1-42) is more aggregation- prone and generally considered to be the most toxic peptide. The Aβ peptides were identified in the insoluble tangles (fibrils) in the post mortem brains of patients diagnosed with AD for the first time almost thirty years ago. (Masters, 1985) It was long believed that the fibrils caused the disease, while today it is generally believed that soluble oligomers of Aβ peptides are the most toxic species. (Gilbert, 2013) Nevertheless, the exact mechanism and cause of the disease is still unknown, and therefore more knowledge about the Aβ peptides is needed.

Previously an Aβ peptide binding Affibody molecule, denoted ZAβ3, was selected using phage display. (Grönwall, 2007) ZAβ3 has been shown to bind to Aβ peptides with high affinity and was demonstrated to have the capacity to both inhibit aggregation of Aβ peptides and dissolve previously formed fibrils.

(Hoyer, 2008;Luheshi, 2010) Furthermore, ZAβ3 has in fruit fly models shown potential to attenuate the neurotoxic effect of Aβ peptides. (Luheshi, 2010)

The three-dimensional structure of the Aβ (1-40)/ZAβ3 complex was solved using NMR and it was seen that the Affibody molecule binds the peptide as a disulfide-linked homodimer. (Hoyer, 2008) It was also seen that the normal three-helix bundle structure of the Affibody molecule was not retained for this protein, but instead that the major part of the first helix is unstructured, while the rest of it forms a beta strand when binding to Aβ peptides. In complex with ZAβ3 the Aβ (1-40) peptide forms an anti-parallel β-hairpin flanked by a

Chemical Engineering of Small Affinity Proteins

β-strand of each Affibody molecule monomer, together forming a four- stranded anti-parallel β-sheet (see figure 12). Interestingly, the Aβ peptide is locked in a β-hairpin conformation, which is very similar to the conformation found in fibrils. Moreover, when the Aβ peptide is locked by the Affibody molecule it cannot aggregate and form oligomers or fibrils.

In this study we wanted to investigate if homodimeric Affibody molecules targeting the Aβ peptide could be prepared using SPPS, which would facilitate site-specific modifications of the Affibody molecule for possible future applications. We also wanted to examine if the unstructured N-terminal part of the Affibody molecule ZAβ3 could be omitted with retained binding affinity to the Aβ peptide, and if this N-terminal truncation would result in a higher synthesis yield.

Based on the three-dimensional solution structure of the Aβ (1-40)/ZAβ3 complex, six N-terminally truncated variants of ZAβ3 were designed and synthesized using standard SPPS, according to figure 12. The shortest variant was truncated in the region that forms a beta strand when binding to the Aβ peptide, and was thus not expected to be able to bind the peptide.

Figure 12. To the left is the 3D-structure of Aβ (1-40) peptide (in red) in complex with the Affibody molecule dimer ZAβ3 (in dark and light blue) (based on pdb-file: 2OTK). To the right are the sequences of the ZAβ3(1-58) Affibody molecule. The arrows indicate the sites of truncation giving rise to the six different variants.

The synthesis yield was considerably higher for the shorter variants, where

ZAβ3(12-58), ZAβ3(15-58) and ZAβ3(18-58) had a synthetic yield of approximately 30%, compared to 8% for the full-length ZAβ3(1-58). The

Joel Lindgren

different ZAβ3 variants were then dimerized and the corresponding homodimers were isolated (hereinafter all ZAβ3 Affibody molecules referred to in the text are understood to be disulfide-linked dimers).

Surface plasmon resonance (SPR) was used to investigate the Aβ (1-40) peptide binding affinity of the ZAβ3 variants (see figure 13). These experiments showed that the shortest variant, ZAβ3(18-58), as expected had lost its affinity to Aβ peptide, while both ZAβ3(15-58) and ZAβ3(12-58) showed high binding affinities with KD values of 0.5 nM and 0.7 nM, respectively. This is actually more than ten times higher affinities than the affinity determined for the full- length ZAβ3(1-58) variant, which showed a KD of 9.5 nM. The higher binding affinity for the shorter variants is related to an approximately ten-fold faster on-rate compared to the full-length variant, together with essentially unaffected off-rate kinetics.

60 50 40 ZA3(15-58) 30 ZA3(12-58) 20

10 ZA3(1-58)

ZA3(18-58)

Relative response (RU) 0 0 200 400 600 800 1000 1200 1400 1600 Time (s)

Figure 13. SPR sensorgram showing the responses when different Affibody homodimers were injected over a chip surface harboring Aβ (1–40) peptides. Injected concentrations:

ZAβ3(18–58) 200 nM; Z Aβ3 (15–58) 20 nM; Z Aβ33(12–58) 20 nM; Z Aβ33(1–58) 20 nM.

Circular dichroism spectroscopy was also used to study changes in the secondary structure content upon titration with Aβ (1-40) peptide. These experiments showed that there is a uniform decrease in signal when titrating

Aβ peptide to the full length ZAβ3(1-58), indicating a lower helical content in the ZAβ3(1-58)/Aβ peptide complex than the Affibody molecule alone. This is not as pronounced for the shorter variant ZAβ3(12-58) and especially not for

Chemical Engineering of Small Affinity Proteins

ZAβ3(15-58). These results indicate that the N-terminal part of the full-length

ZAβ3(1-58) is, at least partially, folded as an α-helix in the free state, but unfolds upon binding to the Aβ peptides. In the shorter variants, ZAβ3(12-58), and ZAβ3(15-58), the N-terminal part is deleted and thus no unfolding is required for Aβ peptide binding, which is a plausible explanation for the faster on-rate kinetics of the truncated variants. Again no difference was seen in the signal from ZAβ3(18-58), alone or when Aβ peptide has been added to the sample, again verifying the SPR results where ZAβ3(18-58) did not show any binding to the peptide.

Isotope-labeled Aβ (1-40) peptide and 2D-NMR spectroscopy (1H15N- Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC-NMR) was used to compare the three-dimensional structure of Aβ peptide alone and together with ZAβ3(12-58), ZAβ3(15-58) or ZAβ3(18-58).

Once again no difference was seen when ZAβ3(18-58) was added to the Aβ peptide, indicating that no interaction takes place between the peptide and

ZAβ3(18-58). On the contrary, clear shifts of the signals were seen when

ZAβ3(12-58) or ZAβ3(15-58) was added to the Aβ peptide, indicating that the Affibody molecules bind the peptide and induce a structural change in the peptide, which alter the environment for certain atoms and thereby inducing a chemical shift that is recorded using NMR. The spectra from ZAβ3(12-58) and

ZAβ3(15-58) in complex with the Aβ peptide are similar, but not identical.

Furthermore, the spectrum from ZAβ3(12-58) is consistent with the previously reported spectrum for the Aβ peptide in complex with the original ZAβ3 Affibody dimer, indicating a very similar structure of the Aβ peptide in complex with ZAβ3(12-58) as in the previously determined structure in complex with ZAβ3. (Hoyer, 2008)

In conclusion, by omitting the 11 or the 14 N-terminal amino acids of ZAβ3 the synthesis yield was increased almost four times and the binding affinity to

Aβ peptide was enhanced 10-20 times compared to the full-length ZAβ3(1-58).

The ZAβ3(12-58) binds the Aβ peptide similarly as the previously reported full-

Joel Lindgren

length ZAβ3 and locks the Aβ peptide in a biologically relevant β-hairpin conformation, previously shown to efficiently prevent peptide aggregation.

Chemical Engineering of Small Affinity Proteins

6 Paper III – Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (Aβ) peptide in monomeric form: low pH was found to reduce Aβ/Cu(II) binding affinity

The aggregation of Aβ peptides is believed to be one important factor for the onset and development of AD, described in more detail in the summary of paper II. The aggregation of Aβ peptides is strongly pH-dependent, where a low pH increases the aggregation rate. Inflammatory conditions, which lead to physiological acidosis (low pH), are suggested to be involved in AD progression. (Fuster-Matanzo, 2013) Therefore it is important to study the behavior of the Aβ peptide with varying pH.

Furthermore, metal ions, such as copper, zinc and iron, are known to accelerate Aβ peptide aggregation, and deposits of copper and zinc have been found to co-localize with amyloid plaques in human AD brain tissue; consequently metal ions are believed to be involved in AD progression. (Gaggelli, 2006;Eskici, 2012) Previous copper binding studies using NMR have shown that the predominant binding site is located to the N-terminal part of the Aβ peptide, where three histidine residues (H6, H13 and H14) together with the N-terminus bind the copper ion. (Danielsson, 2007)

Another method to study the Cu(II) binding of Aβ peptide is to use fluorescence spectroscopy and measure the fluorescence from the single fluorescent amino acid tyrosine 10 in the Aβ peptide. Cu(II) binding in the proximity of the Tyr10 residue will quench the fluorescence, which makes it possible to determine the binding affinity between Cu(II) and Aβ peptide by titrating Cu(II) to the peptide.

In this study we wanted to investigate if an engineered variant of the Affibody molecule ZAβ3 could be produced and functionally used to lock the Aβ (1-40) peptide in the biologically relevant β-hairpin conformation without interfering with the fluorescence spectroscopy measurements. For this experiment it is an

Joel Lindgren advantage if the Affibody molecule is devoid of any fluorescent amino acids, which otherwise would interfere with the fluorescence spectroscopy measurements, and the original ZAβ3 contains one fluorescent amino acid, a tyrosine residue in position 18.

Previous studies of the Aβ peptide in complex with ZAβ3 have shown that the flexible N-terminus of the peptide is not involved in binding and can be omitted with retained binding affinity. (Hoyer, 2008; paper II) Therefore we used ZAβ3(12-58), the previously N-terminally engineered variant of ZAβ3, as a starting point when designing three non-fluorescent Affibody molecules targeting the Aβ peptide. Based on the results from the original phage display selection of Affibody variants towards the Aβ peptide (Grönwall, 2007) we chose to synthesize the homodimers of ZAβ3(12-58)Y18L and ZAβ3(12-

58)Y18R and the heterodimer ZAβ3(12-58)Y18L/R, since variants with leucine or arginine in position 18 also were found to bind the Aβ peptide.

Using SPR it could be concluded that all three non-fluorescent variants of

ZAβ3(12-58) bind the Aβ peptide, both at physiological pH (pH 7.4) and acidic pH (pH 5.0). By studying the off-rate and comparing the dissociation phase of the sensorgrams of the different variants it was seen that ZAβ3(12-

58)Y18R/L showed the slowest off-rate at pH 7.4, while ZAβ3(12-58)Y18L and

ZAβ3(12-58)Y18R showed a slightly faster off-rate indicating a potentially weaker binding. At pH 5.0 on the other hand, ZAβ3(12-58)Y18L showed the slowest off-rate, indicating a higher affinity. Based on these results and the more straightforward production route for the homodimer form, ZAβ3(12- 58)Y18L was the variant chosen for further studies. The dissociation constant

(KD) for ZAβ3(12-58)Y18L binding Aβ peptide was determined to 5.5 nM at pH 7.4 and 0.55 nM at pH 5.0.

Isotope-labeled Aβ peptide was used to investigate the binding region of

Cu(II) to the peptide in complex with ZAβ3(12-58)Y18L using 2D-NMR (1H15N-HSQC NMR). The data showed that all N-terminal cross-peaks assigned in the spectrum without Cu(II) disappeared after addition of Cu(II). On the other hand, a number of the assigned cross-peaks coming from the

Chemical Engineering of Small Affinity Proteins region in the Aβ peptide that is known to bind the Affibody molecule were unaffected by the addition of addition of Cu(II) and the assigned C-terminal cross-peaks displayed reduced intensity, but were still visible in the presence of Cu(II). Taken together, these results strongly indicate that Cu(II) binds the N-terminal part of the Aβ peptide when the peptide is in complex with

ZAβ3(12-58)Y18L, similarly as previously shown for the free Aβ peptide.

Next we determined the binding constant between Cu(II) and free Aβ peptide and Aβ peptide in complex with ZAβ3(12-58)Y18L, both at physiological (7.4) and low (5.0) pH. This was done by titrating Cu(II) to the peptide, measuring the fluorescence intensity, and calculating the KD from the titration curves.

For the free peptide the KD was determined to 0.86 μM at pH 7.4 and to 51 μM at pH 5.0. Thus, a more than 50-fold decreased affinity was observed at pH 5.0 compared to at pH 7.4. This can possibly be related to protonation of the histidine residues, with a theoretical pKa of 6, which are involved in copper binding. When instead the Aβ peptide is in complex with ZAβ3(12-

58)Y18L, the KD for Cu(II) was determined to 5.4 μM at pH 7.4 and 198 μM at pH 5.0. Again, there is approximately a 50-fold difference in affinity between pH 7.4 and 5.0 consistent with the results for free peptide. There is a reduced affinity, approximately five-fold increased KD, between Cu(II) and Aβ peptide when the peptide is bound by ZAβ3(12-58)Y18L compared to free peptide.

In conclusion, this paper showed that it is possible to use a fluorescently silent variant of ZAβ3(12-58) to lock the Aβ peptide in a monomeric and biologically relevant β-hairpin conformation to facilitate the use of fluorescence spectroscopy to study copper binding of the Aβ peptide.

Joel Lindgren

7 Paper IV – A GLP-1 receptor agonist conjugated to an albumin-binding domain for extended half-life

After we have eaten, insulin is released to the blood stream from the beta-cells, which makes the liver, muscles and fat tissue take up glucose from the blood for immediate use or storage as glycogen for future use. This process is absolutely necessary for us to be able to live a normal life. Diabetic persons either lack the ability to produce insulin (diabetes mellitus type 1) or have developed insulin resistance (diabetes mellitus type 2). The classical treatment for diabetes mellitus type 1 is by subcutaneous injection of insulin or insulin derivatives, while diabetes mellitus type 2 sometimes can be improved by increased exercise and dietary modification. However, diabetes mellitus type 2 often also demands medication by injected insulin. Even though insulin injections have a long history, there are risks associated with this treatment, if insulin is taken in too large dose there is a risk of hypoglycaemia, or too low blood sugar, which can lead to unconsciousness. Since the frequency of type 2 diabetes has dramatically increased during the last couple of decades (estimated 30 million in 1985 and estimated 217 million in 2005 (Smyth, 2006)), the need for safer and more convenient medications is desired.

Glucagon-like peptide 1 (GLP-1) is a peptide released from the gut in response to food intake and it is involved in blood glucose regulation. GLP-1 promotes secretion of insulin from the beta cells and suppresses the pancreatic secretion of glucagon, a peptide hormone that triggers the liver to convert its glycogen to glucose. Moreover, the action of GLP-1 is glucose-dependent, resulting in little risk of hypoglycaemia. (Baggio, 2007;Nauck, 2009) GLP-1 exists in two equipotent variants, the 30 amino acid GLP-1(7-37) and the 29 amino acid GLP-1(7-36)-NH2 with a C-terminal amide. However, the in vivo half-life of these two peptides is less than two minutes, making it impractical to use the endogenous peptides for therapy.

A number of different analogues of GLP-1 with enhanced in vivo circulation half-lives have been developed and many show promising results. GLP-1

Chemical Engineering of Small Affinity Proteins analogues have shown to be beneficial for patients suffering from type 2 diabetes since the peptide has shown potential to increase insulin levels, decrease glucose levels, and restore glucose sensitivity of the beta cells. (Meier, 2012;Jones, 2013) The most successful GLP-1-based drug so far is Liraglutide developed by Novo Nordisk (Denmark), which was approved for clinical use in 2009 and sold for approximately $1.7 billion in 2012 alone. Liraglutide binds non-covalently to albumin via a fatty acid introduced in the side chain of lysine 34, thereby showing a dramatic 300-fold prolonged in vivo half-life (13 hours) allowing for administration once daily. (Agerso, 2002;Elbrond, 2002;Juhl, 2002)

The aim of this study was to investigate the feasibility of covalently linking ABD (introduced in section 1.3.2) to GLP-1, as a potential alternative strategy to extend the in vivo half-life of GLP-1. By binding very strongly to HSA, which has an in vivo half-life of approximately 3 weeks, the ABD-GLP-1 conjugate could potentially be suited for administration once a week rather than once daily. A prerequisite for this approach to be successful is that the ABD-GLP-1 conjugate shows a retained strong binding to HSA and an agonistic effect on the GLP-1 receptor (GLP-1R).

An engineered variant of ABD (Jonsson, 2008;Frejd, 2012) was prepared by standard SPPS with a lysine to cysteine mutation in position 14 to be used for conjugation to GLP-1. The position was chosen based on the determined three-dimensional X-ray crystal structure of a homologue ABD in complex with HSA (Lejon, 2004) and previous studies where that position was mutated and still showing retained HSA binding. (Alm, 2010)

GLP-1(7-37), with an alanine to glycine mutation in position 8 and a lysine to arginine mutation in position 34 similar to Liraglutide, was synthesized using SPPS. Lysine in position 26 was introduced with a 4-methyltrityl (Mtt) protecting group, enabling site-specific introduction of a polyethylene glycol (PEG)-based linker to be used as a handle in the conjugation to ABD (see figure 14). PEG-based linkers with three different lengths were introduced, corresponding to two, four, and six PEG units, to avoid potential steric

Joel Lindgren hindrance and interference with either HSA binding or GLP-1R activation. The linker was functionalized with a haloacetyl group, as a chemical handle for the reaction with a thiol.

The ABD and functionalized GLP-1-PEG were cross-linked by the formation of a thioether bond between cysteine 14 in ABD and the functionalized lysine

26 in GLP-1. The ABD-PEGn-GLP-1 conjugates were readily prepared and the ligation reaction proved to be clean without the formation of by-products (schematic illustration shown in figure 14).

AcCl PEG based spacer Lys26

Cys

ABD GLP-1

GLP-1-(PEG)2-ABD

GLP-1-(PEG)4-ABD

GLP-1-(PEG)6-ABD

Figure 14. Schematic figure showing the ligation reaction and the three GLP-1-ABD conjugates with varying linker lengths.

SPR was used to confirm retained HSA binding of the ABD-PEGn-GLP-1 conjugates. The conjugates were first ranked and compared to free ABD reference protein by comparing the dissociation phases after injecting the different ABD proteins over a surface harboring HSA. The experiments showed that the off-rate kinetics of the three conjugates were similar to each other and to the reference ABD. When determining the overall affinities to

Chemical Engineering of Small Affinity Proteins

HSA via both on-rate and off-rate kinetic measurements it became evident that the conjugates displayed similar dissociation rate constants (kd), but that the association rate constants (ka) were slightly lower for the ABD-PEGn-GLP- 1 conjugates than for free ABD. Nevertheless, the three conjugates still showed a very high affinity, with overall KD values of approximately 200 pM to HSA.

To investigate if the ABD-PEGn-GLP-1 conjugates were functional and could activate the GLP-1 receptor, insulin secretion from mouse pancreatic islets was measured after addition of the conjugates. GLP-1 was used as a positive control and the experiments were done in triplicates at low glucose concentration (3 mM) and high glucose concentration (11 mM). No significant increased insulin secretion was seen at low glucose concentration, which was expected since the action of GLP-1 is glucose-dependent. At high glucose concentration, all conjugates as well as free GLP-1 showed increased insulin secretion compared to the control, although at different levels. The increase was most significant for GLP-1 and the ABD-PEG2-GLP-1 variant.

) 3 mMGlucose 11 mM Glucose % 600 600

P = 0.037 P = 0.044

400 400 P = 0.092 P = 0.120

200 200 alized Insulin secretion (

m 0 0 Normalized Insulin secretion (%) Nor l ol D D D o -1 D D D tr B B B tr P B B B n LP-1 -A -A -A n L -A -A -A o G 2 4 6 o G 2 4 6 C G G C G G G EG E E E E -P -P -P -P -P -1 -1-PE 1 -1 -1 -1 P- P P P LP LP L L L L G G G G G G

Figure 15. Normalized insulin secretion from mouse islets at low (3 mM) glucose concentration, and high (11 mM) glucose concentration. The mean values ± SEM are shown.

Joel Lindgren

In conclusion, in this paper it was demonstrated possible to link a biologically active peptide to “the backside” of ABD via a thioether-anchored PEG linker and yield a conjugate with retained very high affinity to HSA and retained biological activity of the conjugated peptide.

Chemical Engineering of Small Affinity Proteins

8 Paper V – Intramolecular thioether cross-linking of therapeutic proteins to increase proteolytic stability

Compared to classical low molecular weight (LMW) drugs, protein therapeutics potentially have a number of advantages, including higher selectivity and potency, and have therefore gained interest and importance during recent years. (Carter, 2011;Dimitrov, 2012) Protein-based therapeutics have historically shown a lower risk of severe side effects and typically a faster clinical development and approval time compared to classical LMW drugs. (Leader, 2008;Reichert, 2003)

On the other hand, there are some drawbacks related to protein therapeutics, such as an often high production cost and the risk of inducing an immune response. (Jiskoot, 2012) Another significant disadvantage of protein drugs is the incompatibility of most proteins with oral delivery of the therapeutic. Proteins that we eat are degraded by the digestive system in the gastrointestinal (GI) tract, where the low pH in the stomach and the proteases pepsin, trypsin and chymotrypsin are responsible for most of the degradation, and the body does not distinguish proteins intended as therapeutics from proteins from e.g. a steak. Therefore protein therapeutics generally need to be administered by injection or infusion, which can be inconvenient for the patient.

Even though the vast majority of natural proteins do not withstand the conditions of the GI tract, there are exceptions. Two very stable protein classes found in nature are cyclotides and cysteine-knot mini proteins, which both have been carefully studied and engineered for various applications and have proved to be possible to administer orally. (Werle, 2006;Craik, 2011) Common for these protein classes is that they are stabilized by intramolecular cross-linking by several disulfide bonds and cyclotides have additionally a head-to-tail cyclic backbone. Intramolecular cross-linking have also shown promising results for stabilizing peptides naturally devoid of intramolecular disulfides by the introduction of thioether bridges. (Kluskens, 2009;Rink,

Joel Lindgren

2010) The thioether bond is potentially even more stable than the disulfide since it is resistant to reducing conditions and thiol-disulfide exchange.

The aim of this study was to investigate if the proteolytic stability of small therapeutic proteins can be enhanced by the introduction of intramolecular thioether bridges. As a model we used the 46-residue ABD, described in the introduction of this thesis, which binds HSA with high affinity. (Jonsson, 2008;Frejd, 2012)

A ABD: LAEAK GVS AKT ALP

ABD_CL1: LAEAK GCS AKT AKP

ABD_CL2: LAEAK GVS CKT AKP

ABD_CL3: CAEAK GVS AKT AKP

ABD_CL12: LAEAK GCS CKT AKP

ABD_CL13: CAEAK GCSAKT AKP

3 B O C SH Cl ABD NH 2 3: Cys1 –Lys29 ABD 2: Lys5 –Cys28

1: Cys17 –Lys45 O or ABD NH 1: Cys17 –Cys45 S

ABD 1 Figure 16. A) Schematic illustration of the design of the thioether cross-linked proteins, with the cross-linking positions indicated by yellow, red, and blue. B) The thioalkylation reaction between the side chain of cysteine and the chloroacetylated side chain of lysine. C) Schematic representation of the three-helical protein, with the three cross-linking sites indicated by colored circles connected by solid lines.

Five different ABD variants were designed to contain one or two intramolecular cross-links according to figure 16. The cross-linking positions were chosen based on the structure of ABD G148-GA3 (pdb file: 1GJT)

Chemical Engineering of Small Affinity Proteins

(Johansson, 2002) and designed to link the beginning of helix 1 to the loop region between helices 2 and 3 (in ABD_CL2 and ABD_CL3) or the end of helix 3 to the loop region between helices 1 and 2 (in ABD_CL1), or a combination of the two (in ABD_CL12 and ABD_CL13).

The five ABD variants were synthesized by SPPS and prepared for intramolecular cross-linking by the introduction of a cysteine and a lysine residue that are in close proximity if the proteins fold as the expected three- helical bundle. The lysine residues intended to be used for cross-linking were introduced with a 4-methyltrityl (Mtt) protecting group that selectively was deprotected and functionalized with a chloroacetyl group. The cross-linking reaction was initiated by dissolving the functionalized protein in buffer, where the thiol group of the cysteine performs a nucleophilic attack on the α-carbon of the chloroacetyl group and thus forming a thioether. The five cross-linked ABD variants were all successfully prepared; the cross-linking reaction proved to be were selective with few side reactions, especially for the three variants containing one cross-link, consequently it was not necessary to purify the linear proteins before the reaction.

The HSA binding of the five cross-linked proteins was analyzed using SPR and compared to a reference ABD. The experiment showed that ABD_CL2 and ABD_CL12 had lost the ability to bind HSA, while ABD_CL13 showed a considerably affected low affinity binding to HSA. On the other hand, both ABD_CL3 and ABD_CL1 still showed high affinity binding to HSA. The binding kinetics to HSA for ABD_CL1 and the ABD reference protein were determined, and both displayed affinities in the low pM range, approximately 20 pM.

The secondary structure content of the cross-linked ABD variants was studied using circular dichroism (CD) and compared to the ABD reference protein. These experiments showed that ABD_CL1 and the reference protein both are predominantly α-helical, showing the typical CD spectra with distinct local minima at 208 and 221 nm. The four other cross-linked proteins do not show

Joel Lindgren the characteristic CD spectra, indicating a partly or completely lost alpha- helical secondary structure content for these variants.

The solution three-dimensional structure of ABD G148-GA3 shows that the N-terminus of the protein is part of the first α-helix. This can possibly explain the radical loss of helicity for all variants having a cross-link to that part of the protein (i.e. all variants except ABD_CL1). Additionally, the last three amino acids of the C-terminus seem unstructured, which potentially makes this part of the protein less sensitive to modifications.

The melting temperatures for ABD_CL1 and the reference protein were determined using CD measurements, where the Tm for ABD_CL1 (63°C) was found to be 5°C higher than for the ABD reference protein (58°C), indicating an enhanced thermal stability of ABD_CL1 compared to the reference protein. None of the other cross-linked proteins showed a clear transition and therefore no Tm could be calculated from the variable temperature measurements.

Next the proteolytic stability of the cross-linked ABD variants was studied and compared to the reference protein. Initial screening experiments showed a considerably increased stability toward both pepsin and the combination of trypsin and chymotrypsin for ABD_CL1 compared to the reference ABD, while none of the other variants showed an improved stability under the conditions used in these experiments.

The protease stability of ABD_CL1 and the reference protein was studied more thoroughly by following the degradation over time when challenged with pepsin or trypsin and chymotrypsin. The results from these experiments clearly showed that the proteolytic stability of ABD_CL1 was substantially enhanced, particularly against pepsin, visualized by figure 17. When comparing the area under curve (AUC) of ABD_CL1 to the ABD reference protein, it is evident that the total amount of intact protein present during the 60 minutes challenged with pepsin is at least 17 times higher for ABD_CL1,

Chemical Engineering of Small Affinity Proteins and almost 5 times higher for ABD_CL1 when challenged with the combination of trypsin and chymotrypsin.

ABD_CL1 100 100 ABD_CL1 ABD ABD

50 50

Residual intact protein(%) 0 Residual intact protein(%) 0 0 20 40 60 0 20 40 60 Time (min) Time (min)

Figure 17. Proteolytic degradation of ABD_CL1 and the ABD reference protein by pepsin at pH 2.6, 37°C to the left, and by trypsin and chymotrypsin at pH 7.4, 37°C to the right.

A variant of ABD_CL1 compatible with recombinant production of the ABD protein was also synthesized, having another cysteine in position 45 instead of the lysine residue. By using a dibromide linker the two cysteine residues form thioether bridges with the linker, thus cross-linking the ABD in the same positions as ABD_CL1. This variant unfortunately showed lower proteolytic stability than ABD_CL1, and only displayed slightly improved proteolytic stability compared to the ABD reference protein. The Tm of the variant was also decreased (Tm ~55°C) and found to be lower than the Tm of the ABD reference protein. A possible explanation for the dissimilarity between this variant and ABD_CL1, even though cross-linked in the same positions, can be related to the structural difference of the linkers. Even though the linkers are of approximately the same length, the investigated haloacetyl linkers are more bulky and possible interfere with the protein folding, making the three-helical bundle less compact and thus more susceptible for proteolytic degradation and less thermostable.

Mass spectrometry (MS) was used to investigate the major degradation products of the ABD reference protein. The peptide bond between leucine 42 and alanine 43 was rapidly cleaved when challenged with pepsin, and the corresponding protein fragment is the dominating product after 3 minutes. The fragment is then relatively rapidly further cleaved, especially between

Joel Lindgren phenylalanine 20 and tyrosine 21. When the ABD reference protein instead is challenged with the combination of trypsin and chymotrypsin, the most susceptible peptide bond is between tyrosine 21 and lysine 22, which proved to be the predominant cleavage site. Taken together, these results show that the region Phe20-Tyr21-Lys22-Arg23 in the ABD reference protein is a region especially sensitive to proteolysis by the three investigated enzymes. The cross-linking in ABD_CL1 between Cys17 and Lys45 evidently suppresses the proteolysis in this region, as well as between position 42 and 43, potentially by sterically interfering with the access of the proteases to these susceptible regions. In previous work, the amino acids in position 20-24 were found to be important for albumin binding. (Linhult, 2002) This indicates that traditional mutation of susceptible amino acids may not be feasible to use for this protein. Moreover, the concept of using an intramolecular cross-linking possibly also increases the stability towards a wider range of proteases compared to single amino acid substitutions.

To conclude, in this study it was shown that it is possible to substantially increase the proteolytic stability by the introduction an intramolecular cross- link. In this paper a tethered variant of ABD was presented, cross-linked by a thioether bridge, resulting in increased thermostability and enhanced stability towards pepsin, trypsin and chymotrypsin.

Chemical Engineering of Small Affinity Proteins

9 Concluding remarks

The five papers that this thesis is based upon all involve chemical synthesis of the affinity proteins ABD or Affibody molecules. Both protein classes are small and can therefore relatively easily be synthesized, which makes it possible to chemically modify the proteins with acceptable total yield. Furthermore, the small size and the high stability of the proteins make them highly competitive to other affinity protein classes, including antibodies.

In the first paper, an alternative production route for Affibody molecules using a fragment condensation approach was described. The method makes it possible to prepare a large set of full-length Affibody molecules with different C-terminal modifications from a set of pre-produced sub-fragments, which can be very convenient if several variants need to be screened. In the second and third papers, Affibody molecules targeting the Aβ peptide involved in AD were studied. Firstly, the synthesis yield was considerably improved by omitting the first 11 residues, but it somewhat surprisingly also increased the binding affinity to the Aβ peptide at least ten times for ZAβ3(12-58). These results were recently further explored in affinity maturation studies of the ZAβ3 Affibody molecule using staphylococcal display, where N-terminally truncated variants were shown to have both increased surface expression level and affinity to the Aβ peptide compared to full-length variants. (Lindberg, 2013)

In the third paper, the improved variant ZAβ3(12-58) was further modified, and this study was facilitated by the increased synthesis yield. Moreover, the increased affinity of ZAβ3(12-58) was highly beneficial as it could compensate for the reduced (approximately ten-fold) affinity caused by the substitution of the fluorescent tyrosine residue to create the fluorescently silent ZAβ3(12- 58)Y18L. In paper III it was also shown that it is possible to lock the Aβ peptide in a biologically relevant β-hairpin conformation and thereby study the peptide without any risk of aggregation, thus making it easier to study the peptide in conditions that normally would be problematic due to oligomerization and fibril formation. The possibility to synthesize site- specifically modified Aβ-binding Affibody molecules in high yield gives

Joel Lindgren improved opportunities to develop new methods for studying the Aβ peptide, e.g. fluorimetric assays based on fluorescent-labeled Aβ-binding Affibody molecules.

ABD was studied in the fourth and fifth papers. In paper IV we linked ABD to the biologically active peptide GLP-1 via the linkage of two side chains. Position 14 in ABD was chosen for conjugation since it is positioned on the opposite side as the albumin-binding site, potentially resulting in low risk of interference with albumin-binding. An advantage of using chemical synthesis is that any position in ABD can be selected for conjugation, and as predicted, conjugation to GLP-1 via this position did not reduce the albumin-binding properties of the conjugate. The conjugation of biologically active proteins, peptides, or even small molecules to ABD for increased circulation half-life seems very promising. It would be very interesting to evaluate the ABD-GLP- 1 conjugates in vivo, to investigate both the biological effect, and the effect of ABD conjugation on the in vivo circulation half-lives, in diabetic mouse models. I believe that in the near future this, or similar, approach will be much more widely spread. The approach has the potential to expand the portfolio of methods used to tailor molecules as drugs that in themselves are impossible to use due to poor in vivo properties, as for example GLP-1.

In paper V a further example of the use of chemical synthesis was illustrated by the introduction of thioether bridges to improve the properties of ABD. This resulted in a variant, ABD_CL1, with considerably enhanced proteolytic stability. With the increasing interest of protein-based therapeutics, there is a need for stabilizing proteins to withstand the harsh conditions related to oral administration. ABD_CL1 shows substantially increased stability towards the three most important proteases in the GI tract and the low pH in the stomach, compared to the non-crosslinked ABD reference protein. Such an improved stability can result in significantly higher bioavailability of a protein after oral delivery, and the thioether cross-linking approach is possibly general and can also be used for other protein-based therapeutics. With the use of alkylating agents, such as dihalides or trihalides, intramolecular cross-linking (investigated in paper V) of larger proteins, not possible to produce with

Chemical Engineering of Small Affinity Proteins standard SPPS, becomes available. It also makes it possible to use selection systems such as phage display to screen libraries of peptides or proteins constrained by thioether bridges. (Angelini, 2012;Chen, 2013)

Taken together the work presented in this thesis demonstrates the potential of using solid phase peptide synthesis and engineering by rational design to modify small proteins.

Since protein therapeutics show such great potential, there is a need to develop efficient production routes for such drugs. I believe that synthetic, and especially semi-synthetic, production routes will be further developed to meet the new needs. The possibility to improve the properties of proteins and peptides by the introduction of unnatural amino acids, building blocks such as PEG or lipids, and the introduction of stabilizing links such as intramolecular thioether bonds, makes synthetic or semi-synthetic production of the protein drug highly competitive compared to recombinant production.

Joel Lindgren

Populärvetenskaplig sammanfattning

Proteiner beskrivs ibland som cellers arbetare. De sköter de flesta funktionerna i cellen, men är även ansvariga för mycket annat utanför själva cellerna. Hos oss människor är det till exempel proteiner som plockar upp syre från lungorna och transporterar ut det till resten av kroppen. En annan typ av välkända proteiner är antikroppar tillverkade av vårt immunsystem och med uppgift att känna igen ovälkomna gäster i våra kroppar, såsom t.ex. bakterier. Antikropparna binder ofta hårt till den främmande molekylen och gör så att vår kropp kan känna igen och oskadliggöra inkräktaren. Proteiner som binder andra molekyler hårt, med hög s.k. affinitet, kallas för affinitetsproteiner och kan vara väldigt användbara för många tillämpningar. Olika antikroppar, riktade mot olika målmolekyler, används t. ex. världen över som verktyg i laboratorier samt för diagnostik och behandling av t.ex. bröstcancer.

Idag finns det även flera typer av affinitetsproteiner som kan designas för att binda till olika typer av molekyler; jag har jobbat med s.k. Affibody-molekyler som kan ses som en typ av konstgjorda antikroppar. Genom olika metoder kan Affibody-molekyler som binder med hög affinitet till ett speciellt protein fiskas fram. T. ex. finns det Affibody-molekyler som binder till proteinet HER2, som är överuttryckt på vissa cancerceller och som kan användas för att diagnostisera cancerpatienter. En annan framtagen Affibody-molekyl binder till peptiden amyloid-beta, som tros vara inblandad i utvecklingen av Alzheimers sjukdom. Affibody-molekyler är mindre och mer stabila än antikroppar vilket ger dem en mängd fördelar; de tränger t.ex. in bättre i tumörer och kan ge snabbare svar om de används för cancerdiagnostik. För många tillämpningar behöver proteinet märkas in med reportergrupper, t. ex. färgämnen eller radioaktiva ämnen, som gör det möjligt att se var proteinet har ansamlats i kroppen och på så vis använda Affibody-molekyler för cancerdiagnostik.

Ett annat protein som jag har arbetat med är ett litet albuminbindande protein, förkortat ABD, som binder stenhårt till albumin som är det mest

Chemical Engineering of Small Affinity Proteins förekommande proteinet i blodet och som har en väldigt lång cirkulationstid i kroppen. Tack vare att ABD binder till albumin åker också ABD runt i blodet längre tid, och genom att sätta ihop ett läkemedel med ABD kan effekten av läkemedlet förlängas. På så vis kan ABD användas för s.k. halveringstidsförlängning av i stort sett vilka läkemedel som helst.

Både Affibody-molekyler och ABD är små proteiner, bestående av 58 respektive 46 byggstenar (s.k. aminosyror) vilket gör det möjligt att tillverka dem genom kemisk syntes. Det underlättar och är i vissa fall det enda sättet för att modifiera proteinerna på ett kontrollerat sätt och det möjliggör också användandet av andra byggstenar än de 20 naturligt förekommande aminosyrorna, vilket kan ge proteinet nya fördelaktiga egenskaper. Det är också lättare att få proteinet helt rent om det produceras genom kemisk syntes, jämfört med produktion i bakterier som är det vanligaste andra sättet. Det är självklart en stor fördel om proteinet ska användas som läkemedel att det inte finns några rester från bakterier kvar. Det är dock fortfarande inte helt lätt att tillverka proteiner genom kemisk syntes på grund av de många syntesstegen, men bättre och effektivare syntesmetoder utvecklas hela tiden. Att undersöka möjligheten att genom kemisk syntes tillverka och förbättra Affibody- molekyler och ABD är en central del i denna avhandling.

I projekt I undersökte vi om det går producera Affibody-molekyler genom att tillverka två separata fragment som sedan sätts ihop. De aminosyror som avgör vad Affibody-molekylen binder till sitter i den första delen av proteinet, medan den sista delen är konstant i alla Affibody-molekyler. Därför vore det behändigt om man separat kunde göra en konstant del, med eventuella reportergrupper, som kan sättas ihop med önskad första del av proteinet och således bilda ett funktionellt protein. Resultaten visar att det är möjligt och vi beskriver en ny metod för att effektivt producera olika Affibody-molekyler på ett smidigt sätt genom kemisk syntes.

I projekt II och III har vi jobbat med Affibody-molekyler som binder amyloid- beta-peptiden, en peptid som kan bilda långa trådar (fibriller) som ofta hittas i hjärnan hos avlidna personer som har haft Alzheimers sjukdom. I projekt II

Joel Lindgren undersökte vi om det går göra just den Affibody-molekylen mindre så att den blir enklare att tillverka genom kemisk syntes. Resultaten från denna studie visade att det går göra proteinet mindre utan att förstöra funktionen, vilket gav ökat syntetiskt utbyte. Affiniteten till amyloid-beta-peptiden visade sig inte försämras, utan i stället har vår förkortade variant, som vi kallar ZAβ3(12-58), minst tio gånger starkare bindning till amyloid-beta-peptiden. I projekt III abetade vi vidare med den förbättrade Affibody-molekylen för att undersöka om den går använda som ett verktyg för att låsa amyloid-beta-peptiden i en speciell struktur och studera peptidens bindning till kopparjoner. Förhöjda halter av koppar har hittats hos personer med Alzheimers sjukdom och det spekuleras i om dessa påverkar amyloid-beta-peptiden och på så vis har inverkan på sjukdomsförloppet. Resultaten från denna studie visar att den modifierade Affibody-molekylen lämpar sig bra för att användas i studier av amyloid-beta-peptiden och kan vara ett hjälpmedel i framtida forskning för att undersöka hur Alzheimers sjukdom uppstår, något man idag inte vet helt säkert.

I de sista två projekten har jag arbetat med proteinet ABD istället för Affibody-molekyler. I projekt IV undersökte vi om det går sätta ihop ABD med en peptid, GLP-1, som har visat sig ge positiv effekt på personer som har typ 2-diabetes. GLP-1-peptiden bryts ned på mindre än två minuter i kroppen och kan därför inte själv användas som läkemedel. Det finns ett liknande läkemedel baserat på GLP-1 (Liraglutid) på marknaden som tas genom en spruta en gång om dagen. Detta läkemedel sålde för över 11 miljarder kronor under 2012, vilket visar vilken enorm marknad det finns. Vår variant skulle potentiellt kunna reducera antalet injektioner från en om dagen till en per vecka, något som självklart skulle vara fördelaktigt för användarna. Resultaten från denna studie visar att ABD-GLP-1-molekylen fortfarande binder hårt till albumin, och borde därför ha en lång halveringtid i kroppen. I våra försök visar molekylen jämförbar verkan som GLP-1, vilket typer på att den fortfarande är aktiv och borde ha samma biologiska effekt i kroppen som GLP-1 eller läkemedlet Liraglutid.

Chemical Engineering of Small Affinity Proteins

Gemensamt för alla proteinläkemedel är att de inte kan tas som ett piller utan behöver i stället injiceras (som t. ex. insulin). Det beror på att vår kropp inte skiljer på proteiner från maten vi äter och proteiner som ätits för att fungera som läkemedel, utan båda bryts effektivt ned i magen och tunntarmen och tas upp som små byggstenar för att tillverka nya proteiner i kroppens celler. Det forskas en hel del på att få fram proteinbaserade läkemedel som kan tas som piller istället för att injiceras. Ett exempel på detta är att det danska läkemedelsbolaget Novo Nordisk (samma bolag som tagit fram Liraglutid) nyligen gick ut med att de satsar 23 miljarder kronor på att utveckla läkemedel mot diabetes som kan tas i tablettform istället för med spruta. I projekt V studerade vi möjligheten att göra ABD mer stabilt mot proteaser. Detta gjordes genom att modifiera vissa aminosyror i ABD och låta dem bilda nya bindningar till varandra. Resultaten från detta projekt visade att vår bästa variant, som vi kallar för ABD_CL1, har avsevärt högre stabilitet mot de tre viktigaste proteaserna i mag-tarmkanalen. De övertygande resultaten från denna studie indikerar att sådan korslänkning kan användas för att stabilisera proteiner och är ett steg mot ett proteinläkemedel som kan tas som ett piller.

I majoriteten av alla läkemedel som används idag, som t. ex. alvedon och magnecyl, är den aktiva substansen inte proteiner utan s.k. små-molekyler. Idag har dock mycket forskningsfokus skiftat från små molekyler till större proteinbaserade läkemedel. Proteinläkemedel har ofta färre sidoeffekter och är därför säkrare än små molekyler, de har historiskt sett också snabbare tagit sig genom de väldigt kostsamma kliniska prövningar som görs innan ett läkemedel blir godkänt, vilket självklart är en fördel för läkemedelsbolagen. Det är dock fortfarande många problem som måste lösas innan den fulla potentialen hos proteiner som läkemedel kan utnyttjas och proteinläkemedel kan ta en större del av marknaden, där det faktum att de måste injiceras är ett stort problem.

Sammantaget visar arbetet som behandlas i denna avhandling att det genom kemisk syntes går modifiera små proteiner och optimera deras egenskaper för att få dem att fungera bättre i de avsedda tillämpningarna.

Joel Lindgren

Acknowledgments

Då var avhandlingen snart färdig för tryck, det känns skönt. Det är många jag vill tacka som gjort det möjligt för mig att att skriva denna bok och om allt går vägen hämta ut min doktorstitel om någon månad.

Först och främst Amelie, tack för att du har varit en helt fantastisk handledare! Du har från dag ett alltid tagit dig tid för frågor och diskussioner, även om du många gånger egentligen haft alldeles för mycket att göra. Tack för en väldigt trevlig och lärorik tid!

Min bihandledare Per-Åke, vi har inte direkt pratat varje vecka under de här åren, men du har ändå alltid haft full koll på vad jag jobbat med och de inlägg och förslag du har är alltid högst relevanta. Tack också för noggrann genomläsning av boken!

Torbjörn, tack för att du kvalitetsgranskade avhandlingen!

Tack alla PIs på plan 3 för att ni, även om forskarvärlden generellt är ganska pressande och stressig, möjliggör en skön och avslappnad atmosfär på jobbet!

Sen vill jag passa på att tacka VINNOVA och Vetenskapsrådet för finansiering.

Projektledarna som jag samarbetat med från Affibody AB: Lars, Caroline och Fredrik. Det är alltid lika roligt att komma till Affibody för möten med er. Ni är alltid väl insatta och har input i världsklass till våra projekt. Tack!

Alla övriga som jag kommit i kontakt med på Affibody AB: Först Tove, du var verkligen den bästa handledare jag kan tänka mig som exjobbare. Sen blev jag kvar i proteinreningsgruppen med Pelle som chef, det var en trevlig tid, även om det inte blev så länge. Tack Elin för att du tipsade mig om att Amelie sökte en doktorand. Sen vill jag också tacka alla andra på Affibody AB för en trevlig tid hos er, det är fortfarande alltid trevligt att komma och hälsa på och köra CD.

Chemical Engineering of Small Affinity Proteins

Jag vill också tacka medförfattarna i amyloid-beta projekten, särskilt Sebastian W, det har varit väldigt roligt att samarbeta med dig.

I would also like to thank the group of P-O Berggren at KI for a very interesting collaboration within the ABD-GLP-1 project.

Vidare vill jag tacka alla alla ni som har jobbat på plan 3 under min doktorandtid. Alla behövs för att skapa den härliga helhet som vi har där på AlbaNova. Speciellt vill jag tacka de jag jobbat närmast, alla i syntesgruppen: Andreas, Anna K, Anna P, Alexis, Danne, Cissi, Emily, Kristina, Melina, Nima, Patrik, Peter, Sara och Viktor, för att ni har gjort arbetet extra roligt.

Sen vill jag också tacka alla andra vänner som gjort att jag kommit ihåg att det finns en värld utanför forskningen på KTH.

Min familj med Mamma, Pappa, Malin och Erik som gett mig en trygg bra uppväxt, utan den skulle jag aldrig skrivit den här boken. Tack!

Tack också Furbergs för att ni tagit emot mig med öppna armar, och för hjälp med barnpassning då och då, det har underlättat arbetet.

Till sist och viktigast av alla, min nya lilla växande familj. Sara, tänk att det redan gått drygt 9 år sedan vi var på bio tillsammans för första gången, det har hänt en hel del sedan dess. Tack för att du är du! Viktor min son, du kom som en härlig vitaminkick mitt i doktorandtiden. Tack för att du är helt underbar och gör det väldigt enkelt att sätta saker och ting i perspektiv och förstå vad som verkligen är viktigt. Sen till vårt ofödda lilla knyte, tack för att du satte lite tidspress på mig, utan dig hade det säkert tagit lite längre tid att bli färdig. Jag älskar er tre över allt annat!

Joel Lindgren

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