Doctoral Thesis Hofstrom 2013

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Camilla HofströmCamilla radionuclidefor molecules Engineering m affibody of ISBN 978-91-7501-613-9 TRITA-BIO Report 2013:2 ISSN 1654-2312 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting

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Doctoral thesis in Biotechnology KtH 2013 stockholm, sweden 2013 www.kth.se

Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting Camilla Hofström Royal Institute of Technology School of Biotechnology Stockholm 2013

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

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

ISBN 978-91-7501-613-9 TRITA-BIO Report 2013:2 ISSN 1654-2312 III ______

Camilla Hofström (2013): Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting. School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden

Abstract Affibody molecules are small (-7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets.

In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock- down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE- tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6- tagged counterpart even when radiolabeling was at the C-terminus using alternative 99m 111 125 chemistries to attach Tc, In or I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR.

Keywords: Affibody molecules, EGFR, HER2, IGF-1R, H6-tag, radionuclide molecular imaging, tracer, intracellular targeting.

This thesis is based on the papers listed below, which will be referred to in the text by their Roman numerals (I-V). The papers are included in the Appendix.

I. Tolmachev V, Hofström C, Malmberg J, Ahlgren S, Hosseinimehr S J, Sandström M, Abrahmsén L, Orlova A, Gräslund T. (2010). HEHEHE-tagged Affibody molecule may be purified by IMAC, is conveniently labeled with [99mTc(CO)3]+, and shows improved biodistribution with reduced hepatic radioactivity accumulation. Bioconjug. Chem. 21(11):2013-22.

II. Hofström C, Orlova A, Altai M, Wangsell F, Gräslund T, Tolmachev V. (2011). Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of Affibody molecules site-specifically labeled with 99mTc, 111In, and 125I. J Med Chem. 54(11):3817-26.

III. Hofström C, Altai M, Honarvar H, Strand J, Malmberg J, Hosseinimehr S J, Orlova A, Gräslund T, Tolmachev V. (2012). HAHAHA, HEHEHE, HIHIHI or HKHKHK: influence of position and composition of histidine 99m + containing tags on biodistribution of [ Tc(CO)3] -labeled Affibody molecules. Manuscript.

IV. Orlova A, Hofström C, Strand J, Varasteh Z, Sandstrom M, Andersson K, 99m + Tolmachev V, Gräslund T. (2012). [ Tc(CO)3] -(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor 1 receptor expression in malignant tumors. Eur J Nucl Med Mol Imaging. Epub ahead of print, PMID: 23179942.

V. Hofström C, Stadler C, Vernet E, Gräslund T. (2012). Step-wise down- regulation of the epidermal growth factor receptor by affinity-based intracellular redirection. Manuscript.

All papers are reproduced with permission from the copyright holders

VI ______ Other publications

Larsson K, Hofström C, Lindskog C, Hansson M, Angelidou P, Hökfelt T, Uhlén M, Wernérus H, Gräslund T, Hober S. (2011). Novel antigen design for the generation of antibodies to G-protein-coupled receptors. J immunol. Methods. 70(1-2):14-23.

Lindberg H*, Hofström C*, Altai M, Honorvar H, Wållberg H, Orlova A, Ståhl S, Gräslund T, Tolmachev V. (2012). Evaluation of a HER-targeting Affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus. Tumor biol. 33(3):641-51.

Tolmachev V, Malmberg J, Hofström C, Abrahmsén L, Bergman T, Sjöberg A, Sandström M, Gräslund T, Orlova A. (2012). Imaging of insulin-like growth factor type 1 receptor in prostate cancer xenografts using the Affibody molecule 111In- DOTA-ZIGF1R:4551. J Nucl Med. 53(1):90-7.

* These authors contributed equally to this work VII ______

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IX ______Contents

Introduction ...... 1 1. Affinity protein classes ...... 3 1.2 Antibodies ...... 4 1.3 Antibody derivatives ...... 6 1.3 Alternative scaffolds ...... 8 1.3.1 Affibody molecules ...... 9 1.3.2 DAPRins ...... 10 1.3.3 Adnectins ...... 11 1.3.4 Anticalins ...... 12 1.3.5 Knottins ...... 12 2. In vivo molecular imaging of cancer ...... 13 2.1 Radiolabeling ...... 15 2.2 Biodistribution ...... 18 3. Intracellular targeting ...... 20 3.1 Intrabody formats ...... 20 3.2 Intrabody mode of action ...... 22 3.3 Intrabody for therapy ...... 22 3. Intrabody expression and gene delivery ...... 24 4. Present investigation ...... 26 4.1 Affibody molecules for radionuclide molecular imaging of HER2 and IGF-1R ... 27 4.1.2 A HEHEHE-tagged Affibody molecule may purified by IMAC, is 99m + conveniently labeled with [ Tc(CO)3] , and shows improved biodistribution with reduced hepatic radioactivity accumulation (paper I) ...... 30 4.1.3 Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of Affibody molecules site-specifically labeled with 99mTc, 111In, and 125I (paper II) ...... 33 4.1.4 HAHAHA, HEHEHE, HIHIHI or HKHKHK: influence of position and 99m + composition of histidine containing tags on biodistribution of [ Tc(CO)3] - labeled Affibody molecules (paper III) ...... 36 99m + 4.1.5 [ Tc(CO)3] -(HE)3-ZIGF1R:4551 , a new Affibody molecule conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumors (paper IV) ...... 39 4.2 Step-wise knock-down of the epidermal growth factor receptor by affinity protein- based intracellular retention (paper IV) ...... 41 4.3 Retargeting of viral particles using Affibody molecules for cell-specific gene delivery (work in progress) ...... 46

X ______

5. Concluding remarks and future perspectives ...... 49 Acknowledgments ...... 52 References ...... 54 XI ______Abbreviations

%IA/g Percent of injected activity per gram %ID/g Percent of injected dose per gram Ab Antibody CD Circular dichroism CD-MPR Cation-dependent mannose-6-phospahte receptor CDR Complementarity determining region E.coli Escherichia coli EGFR Epidermal growth factor receptor (ErbB1, HER1) Fab Fragment antigen binding Fc Fragment crystallizable FcRn Neonatal Fc receptor H6-tag Hexahistidine tag HER2 Human epidermal growth factor receptor 2 (ErbB2) iDab Intracellular single domain antibody Ig Immunoglobulin IGF-1R Insulin-like growth factor 1 receptor IMAC Immobilized metal affinity chromatography KD Equilibrium dissociation constant mAb Monoclonal antibody p.i. Post. Injection pAb Polyclonal antibody PET Positron emission tomography RP-HPLC Reversed phase high-performance liquid chromatography RNAi RNA interference scFv Single-chain variable fragment sdAb Single-domain antibody SPECT Single-photon emission computed tomography t 1/2 Half-life Tm Melting temperature VH Variable domain of an antibody heavy chain VHH Variable domain of a camelid heavy chain antibody VL Variable domain of an antibody light chain Camilla Hofström ______Introduction

The concept of selective interaction between particular biomolecules is essential for the function of living organisms and is involved in most cellular processes. Exemplified by e.g. hormones binding to receptors, transcription factors binding to genetic elements or enzymes recognizing substrates, proteins are commonly involved in such molecular recognition events since they posses an excellent capacity for selective interaction with other biomolecules. Through the establishment of methods for protein engineering, i.e. the ability to employ gene technology to make e.g. site- specific changes, truncations and extensions in a protein, it has become possible to engineer proteins with tailored interaction capacities for use in a vast number of life science applications, including basic research and environmental, diagnostic and medical applications.

Proteins utilized primarily for their capacity to bind to a desired target molecule, may be called affinity proteins. The most explored class of affinity proteins to date is the immunoglobulin class, more commonly known as antibodies, produced by the immune system and used by living organisms to recognize and combat foreign invaders. These naturally occurring affinity proteins were discovered over a century ago by von Behring and Kitasato (von Behring 1890) after they successfully implemented passive immunity to diphtheria and tetanus in animals, by transferring sera from an already immune animal. Since then, it was found that nature is able to create enormous repertoires of antibody molecules with sequence diversity located to the regions used for the molecular recognition. This allows for selective recognition and interaction with a wide range of molecular targets, so called antigens, of a wide range of compositions and sizes. The binding affinity between an antibody and its antigen is dependent on several non-covalent interactions between the antibody and the antigen.

Antibodies are currently the most widely used class of affinity proteins in many life science applications. However, the size, complex structure, means of production, and inherent effector functions of antibodies may not be optimal for all applications. This has attracted the attention to smaller and less complex antibody derivatives and also to alternative, non-immunoglobulin based “scaffold” proteins as powerful complementary affinity reagents.

1 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

In this thesis, a class of alternative scaffold proteins named Affibody molecules has been investigated for use in different applications. Small affinity proteins of this class have previously been generated towards a number of clinically relevant targets, e.g. oncogenic growth factor receptors and disease-related receptor ligands. In this thesis, Affibody molecules showing high-affinity binding to the oncogenic receptors EGFR, HER2 and IGF-1R, respectively, have been functionalized by introduction of various tags followed by evaluation of their performance as in vivo tumor imaging tracers (paper I-IV) and intracellular targeting reagents (paper V), which are applications where full-length antibodies may not be optimal reagents.

2 Camilla Hofström ______1. Affinity protein classes

Advances in many fields of recombinant DNA technology, including basic cloning techniques, de novo gene synthesis, gene amplification and site-directed mutagenesis, have enabled the construction of mutated and/or truncated versions of full-length antibodies, so-called antibody derivatives. These advances have also enabled the generation of novel affinity proteins derived from a non-immunoglobulin protein origin, so-called alternative scaffold proteins, that most commonly originates from a protein with a natural molecular recognition for another biomolecule.

To change the molecular recognition property of an affinity protein, several methods are available. Here, depending on whether the desired change refers to an alteration of the binding to the native partner or if efforts are made to obtain recognition to an entirely different molecule, different approaches can be used. Amino acids involved in a protein’s natural recognition may for example be rationally substituted one at the time for other amino acids to alter the affinity for its native target. By a different approach, several amino acids of the interaction interface may be varied randomly and simultaneously to generate a pool of variants, a so-called library. From these libraries, candidates with desired recognition properties may be isolated, including those with affinity for novel targets, for which the parental protein had no affinity.

There are several techniques available for the functional selection of novel affinity proteins from a library and among the most widely employed are phage display (Smith 1985), ribosome display (Mattheakis 1994; Hanes 1997), mRNA display (Nemoto 1997; Roberts 1997) and yeast display (Boder 1997) as well as several intracellular techniques (Fields 1989; Visintin 2002; Pelletier 1998; Löfdahl 2009; Pellis 2012). A common feature for these selection techniques is that a link is created between the protein member of the library and its encoding gene, i.e. a linkage between phenotype and genotype. This is essential since the function of the protein is used as criteria for selection, and it is impractical to analyze the composition of the protein library member itself. Analysis of the gene encoding a protein library member is less complex and may be performed by DNA sequencing.

For certain applications of affinity proteins, their ability to perform selective molecular recognition may be sufficient. However, for other applications the affinity protein may need to be functionalized through conjugation with an effector, which is the case for many diagnostic and therapeutic applications. This effector may for example be a

3 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______toxin for killing cells, a viral particle for therapeutic-gene delivery, an enzyme for pro- drug activation, or a radioisotope for in vivo imaging. Today, numerous antibody derivatives and alternative scaffold proteins with or without linked effector groups are being evaluated in pre-clinical and clinical applications for diagnosis and treatment of a large number of medical indications (Ståhl 2013; Löfblom 2011; Gebauer 2009).

1.2 Antibodies

Immunoglobulins (Igs), or antibodies (Abs), are in nature produced by B-cells as crucial components of the immune system that serve to protect, eliminate, and mediate immunity, against a diverse range of foreign substances. They accomplish this task through two distinct functional regions, the variable (V) region and the constant (C) region. The V region holds the molecular recognition capacity and allows for a bivalent target-interaction that may be of both high affinity and selectivity for a particular antigen. Once bound, the fragment crystallizable region (Fc region) located within the C region marks the antigen for elimination by interacting with effector cells and/or complement system mediators to induce ADCC (antibody-dependent cell- mediated cytotoxicity) or CDC (complement-dependent cytotoxicity), respectively (Chan 2010). The Fc region also provides antibodies with an extended serum half-life, typically several days, through endosomal recycling mediated by the neonatal Fc receptor, (FcRn (Roopenian 2007)).

A typical human antibody isotype, such as IgG, folds into a complex multi-domain Y- shaped structure, with each domain adapting the characteristic immunoglobulin fold comprised of two anti-parallel β-sheets forming a β-sandwich. The overall Y-shaped structure is made up of two identical pairs of heavy chains (H-chain, ∼50 kDa) and light chains (L-chain, ∼25 kDa) giving an overall molecular weight of ∼150 kDa, figure 1 and figure 3A. The structure is held together and stabilized by inter- and intra-molecular disulfide bonds and non-covalent interactions. Depending on the H- chain’s amino acid sequence in the C region, antibodies may be divided into five different main isotypes: IgA, IgD, IgE, IgG and IgM, with IgG being the most abundant in serum and the major isotype utilized within life science applications today.

Three loop-shaped regions, referred to as the complementarity determining regions

(CDRs), located within the VH and VL regions, respectively, comes together to form

4 Camilla Hofström ______the antigen-binding surface comprised of six CDRs in total located at each tip of the Y-shaped structure, figure 1. The enormous sequence diversity found within the CDRs is due to rearrangements and imprecise joining of gene elements encoding antibody variable domains in the B-cell germ line DNA upon maturation (Tonegawa 1983). Upon antigen stimuli, somatic hypermutations within the antibody’s gene further improve the encoded antibody’s affinity for its antigen, a process refereed to as affinity maturation (Tomlinson 1996).

In many instances, antibodies for life science applications are generated by animal immunization, followed by antibody harvest from their sera, often via affinity chromatography using either group-specific reagents such as the broadly immunoglobulin-binding protein A or protein G (Hober 2007; Grodzki 2010), but preferably via antigen-affinity capture (Ayyar 2012; Agaton 2004). These isolated pools of polyclonal antibodies, pAbs, recognize the injected antigen, but different members of the pool may recognize different sites, or epitopes, in the antigen (Rockberg 2008). This can be an advantage in certain applications, for example where the status of the antigen during analysis may differ. Immunization will evoke different immune responses in different species of animals and the response may also differ between individual animals of the same species, resulting in batch-to-batch variation of the pAbs (Hjelm 2012).

In 1975 however, the hybridoma technology was developed which offered the production of monoclonal antibodies, mAbs, where individual antibody producing B- cells from mice were immortalized by fusion with a myeloma cell to form so-called hybridoma (Kohler & Milstein 1975). Hybridomas may be grown in vitro and represent a means for a renewable source of a defined single mAb. The hybridoma technique was a major breakthrough for the use of antibodies for life science applications and enabled the production of well-defined and homogeneous reagents. Due to the non-human origin of mouse mAbs, different strategies have been developed to address immunogenicity issues to facilitate human therapeutic use, e.g. the engineering of mouse/human chimeric antibodies with ∼70% of the amino acids being of human origin (Boulianne 1984; Morrison 1984), and humanized antibodies with ∼95% of human origin (Jones 1986). Later, the ability to generate human antibodies from transgenic mice was described, such mice carrying human immunoglobulin genes and having their own immunoglobulin genes deactivated (Neuberger 1996; Tomizuka 2000). Today, human antibodies may also be isolated in vitro from human antibody repertoires created by various strategies (Bradbury 2011;

Camilla Hofström ______

Bivalent, as well as multivalent formations of scFvs and Fabs have been constructed to enhance the avidity to e.g. a cell with multiple copies of the antigen present, and hence increase the retention time of the scFv or Fabs on the cell. A successful approach to create bi- or multivalent scFvs has been to shorten the linker connecting VH and VL, which promotes inter-chain interactions between VH and VL from several scFv domains (Hudson 1999; Holliger 2006), figure 1. Depending on the length of the linker, from zero to five amino acids, bivalent diabodies (∼55 kDa), trivalent triabodies (∼80 kDa) and tetravalent tetrabodies (∼110 kDa) have been generated (Todorovska 2001; Holliger 2006).

During the 1990s it was discovered that a subset of the antibody repertoire in certain camelids (Hamers-Casterman 1993) and sharks (Greenberg 1996) were devoid of a light-chain, so-called heavy-chain antibodies, HcAbs. These HcAbs recognize and bind to antigens though one single variable domain denoted VHH from camelids and

VNAR from sharks, figure 1. The VHH domain, referred to as Nanobody, represents the smallest antibody-based derivative with a molecular weight of ∼11-15 kDa. Nanobodies employ only three CDRs for antigen binding, with the CDR3 loop commonly extended and typically stabilized by forming a disulfide bond with a cysteine residue present in CDR1 (Muyldermans 1994; Vu 1997). Hydrophilic amino acids have been found in the region of Nanobodies corresponding to the hydrophobic interface between VH and VL in human antibodies (Desmyter 1996; Muyldermans 1994) with the nanobodies CDR3 loop falling back over this region. This may explain how Nanobodies have evolved into soluble and stable single-domain antibodies (Riechmann 1999). Due to their extended CDR3 loop, it has been shown that Nanobodies may recognize “hidden” and “buried” epitopes such as active sites of enzymes (Desmyter 2002), which may be difficult for conventional antibodies to recognize. For clinical applications, humanization of VHH domains has been performed (Vincke 2009).

Initial attempts to generate single-domain antibodies, sdAbs, from human antibodies were associated with poor stability and solubility due to solvent exposure of the hydrophobic interface that normally connects the VH and VL domains. However, since the discovery of HcAbs, attempts to replace the amino acids in this interface with more hydrophilic amino acids have been performed. This engineering in combination with stringent in vitro selection conditions has yielded stable and soluble sdAbs (Jespers 2004).

7 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

Bispecific variants of antibodies and derivatives thereof have generated attention in the last couple of years, especially for therapeutical applications. So far one bispecific antibody, Catumaxomab, (Removab®), has been approved for clinical use that simultaneously targets CD3 on cytotoxic T-cells and the epithelial cell adhesion molecule (EpCAM) expressed on many tumor cells for treatment of patients with malignant ascites (Linke 2010). Another promising bi-specific T-cell engager, or so- called BiTE, is Blinatumomab that is currently being evaluated in several clinical trial for directing cytotoxic T-cell activity to B-cell derived malignancies (Nagorsen 2012). Other promising bispecific formations under development are e.g. aiming for simultaneous targeting of two cancer-associated growth factor receptors to inhibit their ligand-induced signaling (McDonagh 2012).

1.3 Alternative scaffolds

In nature, numerous proteins besides antibodies possess molecular recognition capacities and may serve as suitable frameworks for engineering of affinity proteins with desired molecular recognition properties. Alternative scaffold proteins are typically isolated by functional affinity selection from a combinatorial library of variants as a single candidate or a pool of candidates with desired properties. Further improvement to the binding affinity and specificity for their intended targets may be achieved by affinity maturation, either through rational design or by random approaches. This has allowed for isolation of affinity proteins with binding affinities in the low picomolar range (Orlova 2006a; Zahnd 2007).

The scaffold chosen for engineering of novel affinity proteins must be carefully considered with regard to e.g., molecular size, stability, complexity, immunogenicity, solubility and proper display for subsequent selection in a given system (Nygren 2004; Skerra 2007; Binz 2005). Also, scaffolds devoid of naturally occurring cysteine residues may be preferable if the affinity protein is to be later conjugated to a effector, or payload, since a unique thiol-reactive cysteine may then be site-specifically introduced and utilized as a labeling handle, which may yield well-defined homogenous reagents suitable for e.g. diagnostic or medical applications (Lundberg 2007). However, other chemistries for payload attachment are also possible, e.g. by utilizing a lysine residue, or by site-specific introduction of unique moieties during peptide synthesis (Perols 2012).

Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

Once isolated, Affibody molecules may be produced by either standard recombinant expression or, because of the small size, also through peptide synthesis, which can facilitate a site-specific introduction of particular functional groups, such as a site- specific chelator for radionuclide labeling (Tran 2009; Perols 2012). Also, smaller Affibody molecule derivates, comprised of essentially helix 1 and helix 2, have also been constructed (Webster 2009; Rosik 2012). Over the last decade, Affibody molecules have been isolated towards a large number of molecular targets, many of clinical importance such as different tyrosine kinase receptors and immune regulatory substances (Nygren 2008; Löfblom 2010). Three of these, recognizing EGFR, HER2 and IGF-1R, respectively, have been employed in this thesis, Chapter 4.

1.3.2 DARPins

In nature, certain proteins with molecular recognition capacity consist of, or contain stretches of, repetitive units, or domains (Kobe 2000). Designed ankyrin repeat proteins, or DAPRins, consist of a number of a consensus version of the cysteine free ankyrin repeat domain derived from humans, which is one of the most common protein-protein interaction motifs present in eukaryotes (Binz 2003). Each ankyrin repeat consists of 33 amino acids (3.5 kDa) that folds into a β-turn followed by two α- helixes. Typically 3-4 ankyrin repeats are connected in a head-to-tail configuration and the structure is stabilized through C- and N-terminal capping-units (Binz 2003; Interlandi 2008), figure 3B. By randomizing 6-7 surface-exposed amino acids located in the β-turn and one located in one of the α-helices in each ankyrin repeat, a diversified library may be created from which candidates with desired affinity for particular antigens can be isolated, typically by ribosomal display. For therapeutical applications, a VEGF-A-specific DARPin for treatment of neuvascular eye diseases has recently finished Phase II clinical trials (Stahl 2013; press release: Molecular partners). A HER2-specific DARPin has also been evaluated for imaging of HER2-expressing tumors in pre-clinical studies (Zahnd 2010), and has also been evaluated for targeting viral-mediated gene delivery to HER2 expressing cells (Münch 2011; Dreier 2011). Other repeat proteins that have served as stable scaffolds for engineering of novel affinity proteins include e.g., the armadillo (Varadamsetty 2012) and the leucine-rich (Stumpp 2003) repeat proteins.

Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

1.3.4 Anticalins

Anticalins are engineered from the protein family of lipocalins, which are found in a variety of organisms, from humans to bacteria, and function as transporters of small- molecule substances (Flower 1996). All lipocalins share a highly conserved β-barrel fold comprised of eight antiparallel β-strands that typically contain two disulfide bonds (Skerra 2001), figure 3F. The scaffold consists of 160-180 amino acid residues with a molecular size of ∼20 kDa. Four loop-shaped regions located on one side of the barrel forms a deep pocket-like binding site. These loop regions have shown to be highly diverse in regards to sequence and length, which resembles the CDRs found in antibodies and may reflect the variety of molecular targets they can bind (Skerra 2008). To avoid potential immunogenicity issues in humans, therapeutical Anticalins have been engineered from human-derived lipocalins, such as the Lnc2/NGAL protein, which has e.g. generated variants with affinity for the extracellular domain of CTLA-4 (Schönfeld 2009). Anticalins are being evaluated for medical applications (Skerra 2007) and a VEGF-specific anticalin was recently evaluated for treatment of patients with solid tumors in a phase I trial (Gebauer 2012; press release: Pieris).

1.3.5 Knottins

Cysteine-knot miniproteins, or knottins, are small polypeptides typically 28-37 amino acids in length with a molecular size of ∼4 kDa (Kolmar 2009). All members of the knottin family share a common fold comprised of three antiparallel β-strands connected by protruding loops of various lengths and sequences, figure 3E. The structure is held together by three disulfide bonds arranged in a way that gives the scaffold its characteristic knot-like fold, as well as its remarkable heat and chemical stability (Kolmar 2009). Many knottins have also shown high resistance to serum and intestinal proteases, which may potentially enable therapeutic knottins to be orally delivered (Werle 2006). Knottins have been shown to naturally function as protease inhibitors, ion-channel inhibitors, as well as antimicrobial agents. One such knottin, Ziconotide (Prialt®), derived from the venom of a marine snail has been approved by the FDA for clinical use for treatment of chronic pain (William 2008). Engineered knottins have shown great potential as tracers for in vivo tumor imaging due to their favorable pharmacokinetics related to their small molecular size. For example, an integrin-specific knottin, EETI-II, have shown promising biodistribution and tumor targeting capacity in pre-clinical studies (Kimura 2009; Miao 2009).

12 Camilla Hofström ______2. In vivo molecular imaging of cancer

Cancer is generally the consequence of multiple harmful mutations transforming a normal cell into a malignant cell that thereby acquires properties such as uncontrollable proliferation, unresponsiveness to growth-inhibiting signals and apoptosis inducing signals, as well as the ability to induce angiogenesis to support tumor growth (Hanahan 2000; 2011). As large malignant lesions start to form, malignant cells may detach and enter into the bloodstream to ultimately establish tumor growth elsewhere in the body, i.e. metastases, which is often an event associated with decreased patient survival.

To determine the abnormalities on either the protein or genetic level underlying a particular malignant state, biopsies are typically collected from the tumor. However, depending on the anatomical location of the tumor, sampling may be difficult or even impossible. Furthermore, the collection of biopsies may also be a contributing factor of tumor disseminations, i.e. cancer spreading (Robertson 2011), and may yield false- negative results due to the heterogeneous nature of many tumors (Marusyk 2012).

Anatomic visualization and molecular characterization of suspected tumors may be performed in the clinic by in vivo molecular imaging techniques (Massoud 2003; Weissleder 2006). Molecular imaging relies on an imaging tracer generating an informative signal in the form of e.g. visible light (in optical imaging), body- penetrating radiation (in radionuclide imaging) or magnetic resonance (in magnetic resonance imaging (MRI)) that can be detected outside of the body to spatially localize the source of the signal. In this thesis, radionuclide-based molecular imaging has been employed, paper I-IV (Chapter 4.1), which will be the focus of this chapter.

The most commonly used radionuclide-based imaging tracer for in vivo tumor visualization in the clinics today is fluorodeoxyglucose, 18F-FDG (Nabi 2002), which target tumors in a nonspecific manner. The use of 18F-FDG relies on the fact that most tumors have an increased glucose metabolism compared to surrounding tissue, to sustain their rapid growth. However, other tissues in the body may sometimes also metabolize high levels of glucose, such as the brain and inflamed tissues, which may obscure tumors or metastases at these locations (Abouzied 2005).

13 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

Tumors may also be selectively targeted by affinity protein-based tracers for visualization. These tracers recognize particular cancer-associated biomarkers, most commonly being oncogenic growth factor receptors that are overexpressed by malignant cells in comparison to normal cells. Preclinical evaluations of such engineered tracers are employed in this thesis, papers I-IV (Chapter 4.1). In addition to cell surface receptors, soluble ligands locally associated with tumor growth region have also been targeted for visualization (Nagengast 2007; Vosjan 2012). Besides revealing size and anatomic location of tumors, radionuclide molecular imaging tracers offer a non-invasive assessment of the current expression status of a particular biomarker. This valuable information may be of predictive and/or prognostic value, and also enable selection of patients who would most likely benefit from a particular treatment (Tolmachev 2010a; Weissleder 2006). Multiple imaging sessions are also feasible for monitoring the response to a particular drug and to follow the course of a treatment.

A protein or peptide-based radionuclide molecular imaging tracer typically consists of three functional parts: (i) a tumor targeting unit such as an affinity protein, (ii) a chelator or prosthetic group to enable conjugation of the radionuclide to the tumor- targeting unit and, (iii) a radioisotope for signal emission. The individual properties of all three components influence the imaging properties of a tracer and factors such as affinity for its target, molecular size, excretion pathway and radioactive decay properties will influence the quality of the obtained image (Tolmachev 2010b)

As seen in table 1, only a handful of tumor-targeting tracers are at this point used clinically, and their targeting unit is based on antibodies, Fab fragments or peptide hormone analogues. Over the last decade, imaging tracers engineered from smaller alternative scaffold proteins such as Affibody molecules, DAPRins, cysteine knots and Adnectins, and from antibody derivatives such as Diabodies and Nanobodies, are under pre-clinical and clinical development (Miao 2011; Romer 2011). Many pre- clinical studies have thus so far pointed to the fact that smaller tracers formats with high affinity for their targets are cleared rapidly from blood and non-targeted tissues and reach maximum tumor uptakes already within hours after injection, thus enabling high contrast images much faster than larger tracer formats. Their high affinities are crucial for high retention in the tumor (Schmidt 2009; Zahnd 2010), whereas larger tracer, with slower vascular permeability rates, will have more time to re-bind to their target (Rudnick 2009).

14 Camilla Hofström ______

Table 1: Tumor-targeting radionuclide molecular imaging tracers used in the clinics.

Tracer name Trade name Target Format Indication Ibritymomab Zevalin® CD20 Antibody Non-hodgkin’s tiuxetan lymphoma Capromab Prostascint® PSMA Antibody Prostate cancer

Satumomab OncoScint® TAG-72 Antibody Colorectal, pendetide ovarian cancer Nofetumomab Verlum® EpCAM Fab Small-cell lung cancer Arcitumomab CEA-scan® CEA Fab Colorectal cancer

Somatostatin OctreoScan® Somatostatin Peptide Neuroendocrine analouges receptors tumors

Small tracers may also penetrate solid tumorous tissues much faster, as they diffuse more rapidly through the intratumoral space (Thurber 2008). Interestingly, a model based on empirical data from the literature (Schmidt 2009) suggests the lowest tumor uptakes for intermediate-sized tracers, such as scFv (∼28 kDa), and with the highest tumor uptakes for either smaller or larger tracers, such as Affibody molecules (∼7 kDa) and antibodies (∼150 kDa), respectively. Even though antibodies may reach high accumulation in the tumor, it generally takes several days in comparison to only a few hours for e.g. Affibody molecules (Orlova 2009) and Nanobodies (Vaneycken 2011). This is an important factor since imaging on the same day as tracer injection is highly convenient, both for the patient and the clinicians.

2.1 Radiolabeling

Some factors to consider when designing novel imaging tracers are scaffold stability and the possibility for site-specific radiolabeling. The scaffold of choice must be stable enough to withstand the labeling procedure, which may require harsh chemical environments as well as high temperatures. Another desirable feature is the possibility for site-specific radiolabeling on a unique labeling handle such as a lysine, cysteine, tyrosine, or a short peptide sequence (Tolmachev 2010c), to ensure homogeneously labeled imaging tracers. Nonspecific radiolabeling may also be employed, but can result in an impaired target affinity of the tracer if the radiolabeling occurs on residues contributing to target recognition.

15 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

Depending on the radionuclide used, imaging modalities such as single-photon emission computed tomography, SPECT (Groch 2000) or positron emission tomography, PET (Turkington 2001), is employed that can generate quantitative anatomic images of high resolution. PET offers a higher spatial resolution and quantification accuracy than SPECT, but PET procedures are typically more expensive. Both modalities may be combined with standard CT (computed tomography) to provide the anatomical context of an image (Jaffer 2005).

Some commonly used radionuclides for SPECT or PET are presented in table 2. Matching the physical half-life of a chosen radionuclide with a tracer’s biological half- life is important to ensure proper signal detection and to avoid unnecessary radioactive exposure to the patient. Therefore, tracers such as antibodies that have a biological half-life of several days are typically labeled with radionuclides with a longer half-life in order to generate a detectable signal upon imaging, whereas tracers with shorter biological half-lives may be labeled with radionuclides with short half-lives (Wållberg 2012). However, strategies such as pretargeting of antibodies for both imaging and therapeutical purposes have been developed to reduce the radioactive exposure to patients and to enable conjugation to more short-lived radionuclides. Pretargeting strategies are based on bispecific antibodies with binding affinity for both its tumor associated target and its radiolabel. The unlabeled bispecific antibody is first injected and allowed to clear from blood and non-target tissues, after which the radiolabel is injected (Sharkey 2012).

Table 2: Commonly used SPECT and PET radionuclides for radionuclide molecular imaging. Radionuclide Half-life Detection

(t1/2) modality 99mTc 6 h SPECT 111In 2.8 days SPECT 123I 13.2 h SPECT 18F 110 min PET 124I 4.2 days PET 68Ga 68 min PET 11C 20.4 min PET 55Co 17.5 h PET

16 Camilla Hofström ______

Once a radiolabeled tracer is bound to its cancer-associated target, a cellular internalization (either active or through the target’s natural turnover rate) most commonly follows, with subsequent lysosomal degradation. Depending on the biophysical nature of the radiocatabolites generated, different events may occur. Radiometals most often generate hydrophilic, charged and bulky radiocatabolites with residualizing properties (Thorpe 1993) These radiocatabolites cannot diffuse freely through the lysosomal or plasma membrane resulting in radioactive entrapment within the tumorous cells that facilitates their visualization. Radiohalogens on the other hand, more commonly generate uncharged and hydrophobic radiocatabolites of non-residualizing nature, which may over time diffuse away from tumor sites and hence reduce the tumor-associated signal and increase background interference (Tolmachev 2003) High accumulation of radioactivity in tumors, but not in healthy non-targeted tissues and blood, is crucial for obtaining high contrast images.

The choice of chelator or prosthetic group depends on the radionuclide to be used. Radiometals are chelated by chemical groups or short peptide sequences through electrostatic interactions (Waibel 1999; Brechbiel 2008), whereas radiohalogens are covalently coupled to prosthetic groups or may also be directly conjugated to the tracer (Wilbur 1992; Tolmachev 2010c). The radionuclides must remain stably attached to the chelator-tracer conjugates until imaging is performed, to avoid the occurrence of free radioactivity in blood and non-target tissues. Furthermore, a stable chelation will also minimize the possibility for trans-chelation of certain radiometals to iron-binding proteins (Fe3+), such as transferrin (Welch 2005; Smith 2005). Technetium-99m, 99mTc, is the most utilized radionuclide for SPECT imaging due to its low cost, excellent availability, and favorable emitting energy that is almost ideal for SPECT detection, as well as conferring low dose burden to patients (Banerjee 2005).

99m Tc may be chelated by short peptide sequences, such as SN3 and N3S chelators consisting of a thiol-containing residue with three adjacent amino acids (Tran 2007; Ahlgren 2009), an approach utilized in paper II (Chapter 4.1.3). Furthermore, tri- 99m 99m + carbonyl Tc, [ Tc(CO)3] , has shown to be stably chelated by a hexahistidine-tag,

H6-tag, (Waibel 1999), which is an alternative labeling strategy employed in paper I, III, IV (Chapter 4.1.2; 4.1.4-5). Peptide-based chelators enable site-specific radionuclide labeling and may conveniently be introduced on a genetic level for subsequent production in fusion with a tumor-targeting affinity protein. Recently, a

17 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

99m + novel histidine-based [ Tc(CO)3] -chelator have been developed, which will be further discussed in Chapter 4.1, papers I-IV.

2.2 Biodistribution An optimal imaging tracer should preferably give high and selective tumor accumulation with rapid clearance from blood and non-target tissues. Such a tracer may generate high contrast images shortly after being injected into patient, preferably on the same day for clinical convenience. Accumulation of radioactivity in healthy tissues and blood may reduce the contrast of an image and/or obscure tumors and metastases associated with these tissues. Thus, an optimal biodistribution of a tracer must yield high tumor-to-nontumor ratios of measured radioactivity, which will translate into high contrast images. Furthermore, high tumor-to-nontumor ratios may allow for radionuclide-based therapy, by simply replacing the imaging radionuclide for a therapeutic one.

Kidney and liver are the two main excretion organs of the body and therefore the two most common sites associated with inappropriate radioactive uptake. Since liver metastases are far more common than kidney metastases (Disibio 2008), renal excretion of tracers is generally the preferred elimination route. As a tracer is internalized by cells in the kidneys or liver it may become metabolized, resulting in cellular entrapment of radioactivity if residualizing radiocatabolites are generated.

Inappropriate kidney uptake and tracer degradation occurs in the proximal tubule of the kidneys (Vegt 2010) after which residualizing radiocatabolites become entrapped and non-residualizing radiocatabolites either re-enter blood circulation or are excreted back to urine. Since smaller proteins are most commonly filtered through the kidneys, many tracer formats smaller than the kidney cut off (∼60 kDa) have been associated with high renal accumulation of radioactivity, including e.g. Affibody molecules (paper I-IV), DARPins (Zahnd 2010), Nanobodies (Vaneycken 2011) Adnectins (Hackle 2012a) and cysteine knots (Jiang 2010). Attempts to reduce the radioactive accumulation in kidneys by introducing minor amino acid substitutions in the tracers have successfully been reported where hydrophilic amino acids, both negatively and positively charged, have been suggested to contribute to an increased level of radioactivity (Hackel 2012b; Wållberg 2011). Inappropriate uptake of tracers may also occur in the liver. Here, tracers or radioactive catabolites there of that are not being retained may either re-enter into the blood 18 Camilla Hofström ______stream or be excreted into the bile duct and subsequently into the gastrointestinal tract (Funk 2008). Both hepatic uptake and excretion of radioactivity must be avoided so that potential tumors and metastases associated with the liver, as well as extrahepatic abdominal tissues, may not be obscured upon imaging. Inappropriate uptake of tracers in liver has been observed for a number of tracer formats (Orlova 2006b; Berndorff 2006; Hosseinimehr 2012), and successful attempts to reduce liver uptake and excretion of radioactivity have been reported when the hydrophobicity of tracers have been decreased by introducing hydrophilic chelators or amino acid residues into the tracer sequence (Hosseinimehr 2012, paper I). Furthermore, it has also been proposed that the presence of hydrophobic patches, rather then the overall hydrophobicity of a tracer, may be a factor promoting elevated liver uptake (Rusckowski 2001), which is also indicated in paper I-IV (Chapter 4.1).

In general, optimizing a tracer with regards to biodistribution by rational engineering is quite difficult, since the fate of a tracer is dependent on several parameters such as the pharmacokinetic properties of the tumor targeting affinity protein, as well as the components of the radiolabel. Minor amino acid substitution made in the tracer may have a profound impact on accumulation of radioactivity, and during its development phase into a properly behaving tracer for clinical usage, multiple variants with minor amino acid alterations radiolabeled through different chemistries are typically investigated in pre-clinical models.

19 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______3. Intracellular targeting

Proteins govern many cellular processes through interaction with other biomolecules, such as other proteins or nucleic acids. Protein-protein or protein-nucleic acid interactions are key determinants to enable e.g. DNA replication, cell signaling, transport of biomolecules, protein translation and catalysis of metabolic reactions. Dysfunctional or aberrant cellular expression of many proteins leads to apoptosis or to cellular phenotypes associated with disease. To increase the knowledge of proteins and their functions, as well as to aid in the understanding of mechanisms underlying a certain disease, overexpression or knocking down the function of the investigated protein are common approaches followed by analysis of the cellular consequences. Methods employed for knocking down the function of specific proteins are nowadays often based on RNA interference (RNAi) techniques (Brummelkamp 2002; Davidson 2011) or the utilization of so-called intrabodies. While RNAi-based techniques enable post-transcriptional silencing of an entire targeted gene, intrabody technology offers post-translational silencing of a targeted protein, or a region thereof.

Intrabodies are affinity proteins utilized intracellularly, usually with the purpose to inhibit a target-protein’s function by directly binding to its function-related interaction interface, thereby physically blocking the functional site. Other approaches have aimed to indirectly inhibit a protein’s function by retaining it in, or redirecting it to, a subcellular compartment where it cannot exert its function (Biocca 1995a; Manikandan 2007), two strategies investigated in paper V (Chapter 5). Compared to RNAi, which silences the function of the gene-encoded protein, intrabodies may interact with a particular part of a targeted protein to interfere with a particular property, e.g. by inhibiting the activity of a tyrosine kinase receptor by specifically blocking its phosphorylation (Paz 2005).

3.1 Intrabody formats

Antibodies were the first reagents explored for interfering with the function of intracellular proteins and the name “intrabody” is derived from “intracellular antibodies”. In this thesis, affinity proteins of non-immunoglobulin origin will also be defined as intrabodies when they are used for intracellular targeting. Due to an antibody’s complex structure, stabilized by several disulfide bonds that often cannot form properly in the reducing cellular environment, antibodies are typically difficult

20 Camilla Hofström ______to adjust for intracellular use. Their expression inside compartments with a reducing environment in cells most often results in nonfunctional intrabodies with low solubility and impaired or absent binding affinity for their intended target. There are however, subcellular compartments, such as the mitochondria and secretory compartments that hold a more favorable redox milieu for disulfide bond formation (Biocca 1995b). The majority of intrabodies described have been based on smaller, less structurally complex antibody derivatives such as single-chain fragment variables (scFv), single-domain antibodies (sdAb) or iDabs (intracellular single domain antibody fragment) and Nanobodies, with scFvs being the most common format. More recently, alternative scaffold proteins, such as Affibody molecules, (Vernet 2008; 2009; paper V) and DARPins (Kummer 2012) have also been explored for intracellular applications.

Initially, functional intrabodies were difficult and time-consuming to generate, and it is still considered a challenge. Cloning from hybridoma-derived mRNA or isolation by standard in vitro selection techniques typically yielded few functional candidates (Martineau 1998). This has however been addressed by different strategies, such as e.g. rational engineering of stable frameworks deprived of cysteine residues (Wörn 1998; Proba 1998). By a different approach, intracellular selection techniques such as yeast-two hybrid (Fields 1989), PCA (protein-fragment complementation assay (Pelletier 1998)), IACT (intracellular antibody capture technique (Visintin 2002; Tanaka 2010)), and bacterial two-hybrid (Pellis 2012) may be employed, which allows for screening and isolation of functional intrabodies binding their target inside cells under reducing conditions. Furthermore, by using pre-determined stable frameworks, in vitro selection of functional intrabodies from libraries has been developed (Desiderio 2001;Philibert 2007; Contreras-Martinez 2007).

Nanobodies (VHH) have, unlike human antibodies, evolved into highly stable single- domain antigen binders (Riechmann 1999), but due to the framework disulfide bond, the reducing cellular environment may be demanding for them as well. Thus a stable

VHH framework has been generated that allows grafting of CDR libraries for subsequent isolation of functional intracellular nanobodies by vitro selection (Saerens 2005). Alternative scaffold-based affinity proteins, such as Affibody molecules and DARPins have also been explored for intracellular applications. Neither Affibody molecules nor DARPins contain cysteine residues in their frameworks, which increases their chances of being fully functional intrabodies in the reducing intracellular environment.

21 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______3.2 Intrabody mode of action

Intrabodies may knock down the function of a target protein by directly blocking a functional site in the target protein, or by withholding the target protein from its normal subcellular location where it exert its function. For the later approach, multiple strategies have been developed that utilize various organelle-associated localization signals genetically fused to the intrabody of interest. Initially, intrabodies were retained in secretory compartments after their translation due to the favorable non-reducing milieu present, with the endoplasmic reticulum (ER) being the most targeted compartment. Equipped with an ER-retention signal (Munro 1987; Biocca 1995b), the intrabody was shown to recycle its bound target back to the ER from the cis-Golgi network, forcing accumulation of the targeted protein mainly to the secretory compartments. This approach has been successful for a number of intrabodies (Böldicke 2007). Additional compartments with favorable redox milieu, such as the Golgi apparatus (Zhou 1998) and mitochondria (Biocca 1995a), have also been targeted by utilizing organelle-specific localization signals. Redirecting intrabodies to the cytoplasm is quite easily achieved by impairing the signaling peptide on the antibody that normally directs secretion of an antibody from mature B-cells (Biocca 1990). A relocation of their target protein to the nucleus may also be achieved by utilizing a nuclear-localization signal coupled to the antibody (Biocca 1990; Lecerf 2001).

3.3 Intrabody for therapy

The potential to target intracellular disease-associated proteins has been explored in a number of studies over the last decade. Intrabodies, typically based on scFv, sdAb (iDabs) or Nanobodies, are currently under development for treating a wide range of human diseases, such as neurodegenerative disorders, viral infections and cancer (Kontermann 2004; Lo 2008; Pérez-Martínez 2010). The use of intrabodies as intracellularly expressed therapeutics relies on efficient and cell-specific gene-delivery systems, which is a major hurdle to overcome. Nevertheless, intrabody technology may have a future potential for treating medical indications to which no drug treatments are currently available. Moreover, intrabodies also hold potential as tools for drug discovery to aid in target validation and to facilitate the development of small-molecule drugs that interfere with particular disease-associated protein-protein interactions (Arkin 2004; Fry 2006).

22 Camilla Hofström ______

Many intrabodies are in development for potentially treating viral infections such as HIV-1 (human immunodeficiency virus 1), HPV (human papilloma virus (Griffin 2006)), HBV (hepatitis B virus (Serruys 2009)) and influenza A virus (Mukhtar 2009). Most of these intrabodies have been designed to target different stages of the viral life cycle by e.g. interfering with their regulatory proteins, viral assembly or viral entry into host-cells. For example, intrabodies targeting regulatory proteins such as Vif, Tat, and Rev of HIV-1 have been shown to inhibit viral replication and HIV infectivity in cells (Marasco 1999; Aires da Silva 2004; Vercruyess 2010). Other intrabodies have aimed to prevent HIV-1 virus from entering into host-cells by e.g. down-regulating a cell surface receptor required for host-cell entry (Mukhtar 2005). Intrabodies have also been explored for treating neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases by interfering with the formation of misfolded amyloid-aggregates, which is characteristic for all three disorders. (Cardinale 2008; Paganetti 2005; Lynch 2008; Southwell 2008). For treatment of cancer, intrabodies have been investigated for e.g. targeting of overexpressed oncogenic receptors such as EGFR, HER2 and VEGFR-2. A common approach has been to inhibit receptor signaling by preventing their transport to the cell surface (Wheeler 2003). This approach has also been successful when utilizing an EGFR-specific and a HER2-specific Affibody molecule, respectively, retained in the ER-compartment (Vernet 2008; 2009). An intrabody may also be designed to inhibit or restore the function of dysregulated cell-signaling transducers such as p53 and Ras. For example, an iDab binding specifically to Ras in its active GTP-bound state was shown to efficiently inhibit tumor growth in a mouse model (Tanaka 2007). Another example is an scFv with binding specificity to a mutated form of p53 restored its activity and induced tumor regression in a mice model (Orgad 2010). By different strategies, tumors may also be targeted by intrabodies designed to mediate an effector function, such as inducing apoptosis (Tse 2000) or to mediate selective degradation of a targeted protein by the ubiquitin/proteasome pathway (Melchionna 2007).

Intracellular DARPins have also been generated for e.g. inhibition of mitogen- activated protein kinases such as ERK2 and JNK. It was shown that an ERK2-specific DARPin, as well as JNK-specific DARPins, were able to inhibit ERK2 and JNK signaling in cells, respectively, as determined by the reduced phosphorylation levels of the kinases (Kummer 2012; Parizek 2012). Affibody molecules with potential for intracellular targeting of e.g. c-Jun (Lundberg 2009), Ras and Raf (Grimm 2010; 2012) have been generated and await in vitro evaluation.

23 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______3.4 Intrabody expression and gene delivery

Functional intrabodies may today be isolated by in vivo or in vitro selection techniques, which has greatly facilitated the generation of intracellular regents for potentially treating various medical indications. Even though the intrabody technology has shown future potentials, there are some major limitations that still need to be addressed before they may ever reach the clinics. The most important limitation is lack of sufficiently good delivery systems to obtain the desired therapeutic effect.

One strategy for transporting an intrabody across the cellular membrane is to attach a cell-penetrating peptide, CPP (Gupta 2005; Marschall 2011). These polycationic or amphipathic peptides may have the ability to deliver their cargo across the cellular membrane, either by directly penetrating the phospholipid bi-layer or through endocytosis. It has however, been questioned if CPPs can promote the delivery of sufficient quantities of intrabodies in the target cell to reach a therapeutic effect (Marschall 2011). An alternative approach is to use viral vectors derived from e.g. adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) to deliver the gene encoding the intrabody. This may allow for both a high and long-term expression of the intrabody.

A second challenge to undertake is to ensure cell-specific delivery, so that only cells meant to be treated will receive or express the therapeutic intrabody. A strategy investigated for gene delivery using viral vectors has been to fuse affinity proteins with viral envelope proteins to obtain cell-specific re-targeting of the viral particles (Waehler 2007; Schaffer 2008). For this strategy to be viable, the native viral proteins mediating host-cell infection must be inactivated. This may be accomplished by rational engineering of viral coat-proteins to inactivate their ability to attach and enter into the host-cell. The affinity protein used to redirect viral tropism, typically holds molecular recognition for a specific cell-surface protein, such as an oncogenic growth factor receptor, or a cell-type specific marker such as CD20. There are several viral envelopes and coat proteins that have shown to allow insertion of affinity proteins. For example, the measles viral envelope protein has been successfully modified to incorporate a HER2-specific DARPin (Münch 2011) or a CD20-specific scFv (Funke 2008), resulting in infectious viruses retargeted to HER2-expressing xenografts in mice or CD20-expressing B-cells in culture, respectively. Furthermore, DARPins with binding specificity for the fiber knob protein of adenovirus was fused to a HER2-

24 Camilla Hofström ______specific DAPRin. This bi-specific adapter was shown to mediate adenovirus infection by linking the virions to HER2-expressing cells (Dreier 2011). Similarly, a HER2- specific Affibody molecule was incorporated into the adenovirus knob protein, resulting in specific infection of HER2 expressing cells in vitro and in a mouse model (Magnusson 2007; 2012). Also, the envelope protein derived from Sindbis virus has allowed insertions of a dimeric Z domain (Ohno 1997), which has enabled antibodies, through their Fc-region to be bound with the virions and mediate recognition of specific antigens (Morizono 2005). In other studies, the dimeric Z domain was exchanged for a CCR5-specific scFv (Aires da Silva 2005) or an Affibody molecule, as will be described in Chapter 4.3.

25 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

4. Present Investigation

In this thesis, a class of alternative scaffold-based affinity proteins named Affibody molecules has been investigated. In all studies performed, various functional tags have been designed and engineered for genetic fusion to Affibody molecules with the main objective to improve and evaluate the tagged Affibody molecules performance in in vivo and in vitro applications concerning radionuclide molecular imaging (paper I-IV) and intracellular targeting (paper V).

Paper I-IV: Radionuclide molecular imaging Two Affibody molecules interacting with HER2 (paper I - III) or IGF-1R (paper IV) were used and different histidine-containing tags were introduced either at the C- or N-terminus for purification of recombinantly produced Affibody molecules by immobilized metal-ion affinity chromatography (IMAC) (paper I-IV) and chelation of 99m + Tc [(CO)3] (paper I, III and IV). In paper II, the Affibody molecule was radiolabeled with alternative methods. The influence of position and composition of the histidine-containing tags on the biochemical properties of the Affibody molecule tracers and their biodistribution was investigated.

Paper V: Intracellular targeting In this paper, an EGFR-specific Affibody molecule was tagged with various modified variants of a cellular transport protein, the cation-dependent mannose-6-phospate receptor (CD-MPR). The aim was to develop a system for intracellular EGFR- entrapment or degradation. Upon intracellular expression of the variants, the consequence on EGFR localization and function was investigated.

26 Camilla Hofström ______4.1 Affibody molecules for radionuclide molecular imaging of HER2 and IGF-1R

In the studies presented in paper I-IV, radiolabeled Affibody molecules were used as tumor-targeting tracers for visualization of the two oncogenic cell surface receptors, HER2 and IGF-1R, respectively, which are two cell surface receptors often found to be overexpressed in different types of cancers. The Affibody molecules were conjugated to different radionuclides that allowed for tumor visualization by SPECT.

The HER2 receptor (ErbB2), together with three closely related family members, EGFR (HER1, ErbB1), HER3 (ErbB3) and HER4 (ErbB4), constitute the human epidermal growth factor receptor family, or the EGFR-family. These tyrosine kinase receptors are involved in cell-signaling pathways meditating cellular events such as cell proliferation, motility, adhesion and survival (Yarden 2001). Aberrant signaling from these family members is often detected in many different types of cancers (Roskoski 2004). For example, an overexpression of HER2 has been observed in e.g. breast, lung, colon, and ovarian carcinomas, and is often associated with a more aggressive course of the disease. Therefore, detection, characterization, and quantification of HER2 expression levels are important for patient management.

Overexpression, resulting in an increased signaling by the tyrosine kinase receptor IGF1-R has been observed in different types of cancer, including prostate, breast and colorectal carcinomas (Hellawell 2002, Helle 2004; Shiratsuchi 2011). IGF-1R is ubiquitously expressed on normal tissues e.g. in the pancreas, colon, lungs, salivary gland and stomach, and its signaling may activate cell proliferation signaling pathways (Pollak 2004). An increased signaling generated by IGF-1R has been found to confer resistance to some therapeutic drugs and treatments, e.g. resistance to targeted trastuzumab therapy in breast cancer (Nahta 2005). Similar to HER2, the expression level of IGF-1R is commonly associated with a more aggressive course of the disease. In contrast to HER2, IGF-1R expression levels in tumors may be quite low, which in combination with its ubiquitous expression in normal tissues, makes radionuclide imaging of IGF-1R challenging.

Affibody molecules have shown great potential as tracers for radionuclide molecular imaging, since they are usually rapidly cleared from blood and healthy tissues and can be generated with high affinity and specificity for tumor-associated targets. Furthermore, it is also possible to utilize many different types of chemistries for site-

27 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______specific radiolabeling of Affibody molecules. Over the last several years, a great effort has been put into the development of an Affibody molecule tracer for HER2 visualization on tumors. Several pre-clinical studies have been performed where different chelators and prosthetic groups, radionuclides, and amino acid alterations in the Affibody molecule scaffold have been evaluated with respect to influence on e.g. biodistribution and tumor targeting capacity. Two pilot clinical studies have been performed (Baum 2010; Sandberg 2012) that clearly show the potential of Affibody molecules for visualizing HER2-expressing tumors in humans, and recently the Affibody molecule tracer ABY-025 entered into clinical trials for imaging of HER2- expressing tumors in breast cancer patients (press release: Affibody AB).

Affibody molecules can be conveniently produced recombinantly in Escherichia coli and may be efficiently recovered by IMAC purification when fitted with a hexahistidine tag (H6-tag). Most Affibody molecules also refold upon heat-denaturation, which is convenient since many radiolabeling methods require harsh conditions, including an elevated temperature. An elevated temperature is also a facile way to precipitate endogenous E. coli proteins during purification of the recombinantly produced Affibody molecule. The H6-tag, besides enabling IMAC purification, may 99m 99m + also be used for stable chelation of tri-carbonyl Tc ([ Tc(CO)3] ), (Waibel 1999). This elegant and straightforward site-specific radiolabeling approach is convenient both for Affibody molecules, as well as for other recombinantly produced tracers, especially during their development phase since multiple variants may be produced, purified and labeled relatively easy in parallel.

Previous pre-clinical studies made on a dimeric HER2-specific Affibody molecule, 99m + radiolabeled with [ Tc(CO)3] on a N-terminally placed H6-tag resulted in relatively high liver uptake of the tracer (Orlova 2006b). A possible explanation might be the 99m + relatively hydrophobic nature of the [ Tc(CO)3] -hexahistidine chelate (Ballinger 2004), since hydrophobic substances are naturally taken up by the liver (Hosseinimehr 2012). Liver uptake, as well as hepatobiliary excretion of radioactivity is undesirable, since the liver is a common site for metastases of many tumors and their presence may therefore be obscured. Further evaluation of a monomeric HER2-specific Affibody molecule with or without an N-terminal H6-tag and site-specifically labeled at the C- terminus with 111In or 99mTc, respectively, confirmed that the elevated hepatic uptake of radioactivity was associated with the presence of the H6-tag (Ahlgren 2008;2009), suggesting that the H6-tag by itself promotes liver uptake.

28 Camilla Hofström ______

In parallel studies, where the HER2-specific Affibody molecule was generated by peptide synthesis, it was found that the composition of the N-terminal sequence of the HER2-specific Affibody molecule could appreciably affect the biodistribution of the tracer. In these studies it was found that by introducing a negatively charged glutamic acid into a N-terminal mercaptoacetyl-containing peptide chelator for 99mTc, the overall liver accumulation of radioactivity could be significantly lowered (Tran 2007; Ekblad 2008), in comparison to if a positively charged lysine residue was introduced in the same position (Tran 2008). Taken together, these investigations indicate that an H6-tag located at the N-terminus of the HER2-specific Affibody molecule was associated with elevated uptake in the liver, and that liver uptake may be reduced by increasing the hydrophilicity, particularly by introducing an amino acid with negative charge in or near the N-terminus.

An IGF-1R-specific Affibody molecule that competes with the receptor’s natural ligand IGF-1 for receptor-binding was previously generated (Li 2010), followed by affinity maturation to enhance its affinity for IGF-1R (unpublished data, Gräslund). A variant isolated after affinity maturation, ZIGF1R:4551, was evaluated for its feasibility to image IGF-1R-expressing xenografts in mice (Tolmachev 2012). The H6-tagged

ZIGF1R:4551, site-specifically labeled with Indium-111, was able to visualize prostate cancer (DU-145) xenografted tumors but also accumulated in IGF-1R-expressing organs such as stomach, pancreas, lung and salivary gland. Nevertheless, this study showed that the IGF-1R-specific Affibody molecule has the potential to be used for radionuclide molecular imaging of IGF-1R-expressing tumors in vivo.

29 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

4.1.2 A HEHEHE-tagged Affibody molecule may be purified by IMAC, is conveniently labeled with [99mTc(CO)3]+, and shows improved biodistribution with reduced hepatic radioactivity accumulation (Paper I)

This was the initial study to investigate how engineering of the H6-tag and its position affected the biochemical properties of the HER2-specific Affibody molecule tracer and its biodistribution when evaluated in mouse models. More specifically, the aims of this study were to:

1) Investigate if a relocation of the H6-tag from the N-terminus to the C- terminus would influence the liver accumulation of the HER2-specific 99m + Affibody molecule ZHER2:342 when radiolabeled with [ Tc(CO)3] , since an

N-terminal placement of a H6-tag has previously resulted in high liver uptake.

2) Investigate if liver uptake would be reduced if every second amino acid in an

N-terminal H6-tag would be replaced with negatively charged and hydrophilic glutamic acids, resulting in the novel HEHEHE-tag. This

modification would possibly still allow for IMAC purification of ZHER2:342 99m + and enable chelation of [ Tc(CO)3] , since the theoretical motif needed for chelation is H-X-H, where X could essentially be any amino acid (Novak- Hofer 2004).

For these purposes, three ZHER2:342 constructs were included in this study; (HE)3-

ZHER2:342 and ZHER2:342-H6, and the previously evaluated H6-ZHER2:342 as a control.

After recombinant production of (HE)3-Z HER2:342 and Z HER2:342-H6 in E. coli, 2+ ZHER2:342-H6 was purified by IMAC on a column with immobilized Co . Cell lysate containing HEHEHE-tagged Z HER2:342 was heat treated at 60°C for 10 min to precipitate endogenous E. coli proteins and successfully purified on a column with 2+ immobilized Ni , despite the fact that every second histidine in the H6-tag had been replaced by glutamic acid. A Ni2+-column, rather than a Co2+-column, was used for purification of (HE)3-Z HER2:342 since histidine-containing proteins binds more strongly to this column. Analysis by RP-HPLC showed purities over 90% for all three constructs with no obvious difference in retention time, indicating a similar overall hydrophobicity. Temperature-induced unfolding by circular dichroism (CD) analysis

30 Camilla Hofström ______revealed melting temperatures (Tm) of 65°C for both (HE)3-ZHER2:342 and ZHER2:342-

H6, which was similar to the previously determined Tm of 63°C for H6-ZHER2:342

(Orlova 2006a). CD spectra of (HE)3-ZHER2:342 and ZHER2:342-H6 before and after heating to 90°C were similar and with a high α-helical content, indicating proper refolding after thermal unfolding. The affinity of (HE)3-ZHER2:342 and ZHER2:342-H6 for HER2 was determined by a biosensor analysis and was found to be similar to the previously determined KD of 22 pM for H6-ZHER2:342 (Orlova 2006a).

99m + Radiolabeling with [ Tc(CO)3] was somewhat more efficient for H6-ZHER2:342 compared to the other two constructs, but a radiochemical yield over 80% was obtained for all three constructs. After removal of unconjugated radionuclides by desalting, a final radiochemical purity of 95 % was obtained for each construct. The 99m + 99m + (HE)3-[ Tc(CO)3] chelate was found to be as stable as the H6-[ Tc(CO)3] chelates when incubated with a 5000-fold excess of free histidine for 1 h, indicating 99m + that the H-X-H-motif may very well be sufficient for efficient Tc [(CO)3] chelation, as previously suggested (Novak-Hofer 2004). The binding of all three constructs to HER2-expressing cells could be blocked by pre-incubation with non- labeled ZHER2:342, suggesting that the constructs did not interact with cells unspecifically but with the HER2 receptor.

The tracers were injected into mice and uptake in different organs was measured at 4 h and 24 h post injection (p.i.), respectively. At 4 h p.i all three constructs showed the general pattern typically observed for Affibody molecules with rapid clearance of radioactivity from blood, major organs and carcass, except from kidneys. As expected, all three constructs showed high uptake of radioactivity in kidneys, which is commonly seen for small imaging tracers. The most profound difference in biodistribution of the tracers was with regard to radioactive accumulation in the liver.

As seen in figure 4, at 4 h p.i. the HEHEHE-tagged ZHER2:342 showed a remarkable

10-fold lower liver accumulation in comparison to the N-terminal H6-tagged

ZHER2:342. Moreover, a relocation of the H6-tag from the N- to the C-terminus resulted in a 2-fold lower accumulation of radioactivity in liver, but did also give a slower clearance from blood. The HEHEHE-tag provided the best overall biodistribution, i.e. the lowest radioactive accumulation in most organs, at both 4 h and 24 h p.i.

Camilla Hofström ______

4.1.3 Use of a HEHEHE purification tag instead of a hexahistidine tag improves biodistribution of Affibody molecules site-specifically labeled with 99mTc, 111In, and 125I, (Paper II) The aim in this study was to further compare Affibody tracers with an N-terminally placed HEHEHE-tag and a H6-tag placed either at the N- or C-terminus. A site- specific radiolabeling was performed on a unique C-terminal cysteine in the HER2- specific Affibody molecule by alternative radiolabeling chemistries. For this purpose, the two constructs generated in paper I was re-cloned and fitted with a C-terminal cysteine, resulting in constructs (HE)3-ZHER2:342-C and ZHER2:342-H6-C , respectively.

As a control, the previously evaluated Affibody molecule H6-ZHER2:342-C was included (Ahlgren 2009). For radiolabeling, the residualizing radiometals 99mTc and 111In, and the non-residualizing radiohalogen 125I, were utilized.

(HE)3-ZHER2:342-C and ZHER2:342-H6-C were recombinantly procured in E. coli. Cell lysates were heat treated at 60°C for 10 min prior to IMAC purification. Purification of the HEHEHE-tagged Affibody molecule was attempted on a column with immobilized Co2+, which in comparison to a column with immobilized Ni2+ (both used in paper I) have a weaker interaction with histidines. It was found that the 2+ HEHEHE-tag also allowed purification of ZHER2:342 on this Co -conjugated column. The two constructs were further purified by RP-HPLC and SDS-PAGE analysis on samples retrieved after the different purification steps showed essentially pure samples after IMAC purification. The final samples, including the control construct H6-

ZHER2:342-C, were found to have a purity over 95% when analyzed by RP-HPLC. The constructs also had similar retention times on the RP-HPLC column, indicating similar overall hydrophobicities. Biochemical characterization of (HE)3-ZHER2:342-C and ZHER2:342-H6-C was performed essentially as in paper I and it was found that the secondary structure content, Tm and affinity for HER2 were not affected significantly by introduction of the cysteine.

All three constructs were site-specifically labeled with 99mTc, 111In and 125I, respectively, on the introduced C-terminal cysteine. 99mTc was chelated by the three

C-terminal amino acids present in each construct (VDC on (HE)3-ZHER2:342-C and 111 H6-ZHER2:342-C; HHC for ZHER2:342-H6-C). In was attached in a two-step reaction where the thiol-reactive maleimidomonoamide-DOTA chelator was first covalently attached, followed by 111In-chelation. 125I was also attached in a two-step reaction where the prostatic group HPEM was covalently attached to 125I, followed by

Camilla Hofström ______

After production in E. coli, all constructs could be successfully recovered by IMAC on 2+ the column with immobilized Co , except (HI)3-ZHER2:342, which was recovered on the column with immobilized Ni2+. After further purification by RP-HPLC, all variants had a purity exceeding 95%. Similar retention times on the RP-HPLC column were observed for all constructs, but with a somewhat longer retention time for the two HIHIHI-tagged ZHER2:342, indicating a slightly higher overall hydrophobicity in comparison to the other eight constructs, which was expected. Also in this study, biochemical characterization of the constructs was performed essentially as in paper I and II. It was found that the secondary structure content, Tm and affinity for HER2 were not affected significantly. The exception was the thermal unfolding analysis of (HI)3-ZHER2:342, where the protein precipitated around 80°C and a Tm could therefore not be determined.

99m + Radiolabeling of all constructs with [ Tc(CO)3] followed by desalting resulted in a final radiochemical purity between 97-99%. The N-terminally tagged ZHER2:342 constructs showed a higher stability of the radiolabel than the C-terminally tagged

ZHER2:342 constructs when incubated with a 5000-fold excess of free histidine for 4 h.

Unspecific binding to cells was observed for (HI)3-ZHER2:342, since pre-incubation with non-labeled ZHER2:342 did not significantly decrease its binding. The binding of all other constructs to cells could be efficiently blocked, indicating a HER2-specic interaction.

The constructs were injected into mice and biodistribution was determined 4 h and 24 h p.i. The measured uptake of radioactivity at 4 h p.i. clearly showed that the amino acid composition and position of the histidine-containing tag had a great influence on biodistribution. Most obvious was the influence on uptake in the liver and spleen, and the level of hepatobiliary excretion as indicated by accumulation of radioactivity in GI-tract. In these organs, as seen in figure 10, the composition of the N-terminally placed tags had a much more profound effect on biodistribution than the C-terminally placed tags. For C-terminal placement, the (HK)3-tagged ZHER2:342 had a significantly higher liver uptake of radioactivity than the other constructs, figure 10B. Elevated liver accumulations upon the introduction of a C-terminal lysine in a chelator sequence have been observed in other biodistribution studies made on a HER2-specific Affibody molecule (Wållberg 2011; Altai 2012). For N-terminal placement, the introduction of the highly hydrophobic isoleucine-containing histidine-tag (HIHIHI-tag) shifted the tracer’s uptake from kidneys to the liver, as well as eliciting high radioactive uptake in spleen, figure 10A. As expected, an N-

Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

Immunofluorescence (IF) confocal microscopy was performed to visulize the expression levels of the five constructs, as well as their subcellular distribution. It was found that the control construct (ZEGFR:1907)2 mainly accumulated to the ER, thus representing the normal distribution of the Affibody molecule processed by the cellular machinery for secretion, figure 17A. It is also likely that a portion of

(ZEGFR:1907)2 were secreted from the cells since no retention signal were introduced in this construct. Affibody-CD-MPR constructs with different localization signals were shown to accumulate in their expected organelles where (ZEGFR:1907)2-CD-MPR localized to Golgi and endosomes, which represents the normal trafficking route of

CD-MPR (Sun 2006). (ZEGFR:1907)2-CD-MPR-pm accumulated to the plasma membrane and (ZEGFR:1907)2-CD-MPR-Ala5 and (ZEGFR:1907)2-CD-MPR-LAMP1 were found to accumulated mainly in the lysosomes, figure 17A. The cellular distribution of all five constructs was also found to be overlapping the respective markers for ER, Goligi and lysosomes was detected.

Figure 17: Immunofluorescence confocal microscopy analysis of Affibody constructs and EGFR. A: staining for Affibody molecules (red); B: staining for EGFR (green); C: co-localization of the lysosomal protein Prosaposin (green) and (ZEGFR:1907)2-CD-LAMP1 (red). All cells are counter-stained with the nuclear probe DAPI (blue). Scale bar: 15 μm.

Camilla Hofström ______5. Concluding remarks and future perspectives The work presented in this thesis has been centered around Affibody molecules with the main objectives to design and engineer various functional tags and to further evaluate their influence on Affibody molecules for radionuclide molecular imaging and intracellular targeting applications. More specifically, for radionuclide molecular imaging applications the aim was to develop a tag that could enable IMAC purification, radiolabeling and improve biodistribution of Affibody molecule tracers when evaluated in vivo. For intracellular targeting, the aim was to develop a receptor- retention modeling system that would allow a step-wise reduction of a cell surface expressed oncogenic receptor in a controlled manner, to potentially be used for studying cellular effect upon different levels of receptor down-regulation. In a proof-of principle study, paper I, we developed a novel histidine-containing tag by replacing every second histidine in a hexahistidine-tag for the negatively charged amino acid glutamic acid, resulting in a HEHEHE-tag. This tag enabled IMAC 2+ purification of the HER2-specific Affibody molecule ZHER2:342 on a Ni -conjugated resin (paper I), as well as on a Co2+-conjugated resin (paper II), and allowed stable chelation of tri-carbonyl 99mTc. When evaluating its influence on biodistribution in vivo, the N-terminally HEHEHE-tagged ZHER2:342 provided a 10-fold lower uptake of radioactivity in liver in comparison to the N-terminally H6-tagged ZHER2:342 , and was equally efficient in targeting HER2 expressing xenografts.

In paper II, we further showed that even when ZHER2:342 is site-specifically labeled at the C-terminus by alternative radiolabeling chemistries, an N-terminally placed HEHEHE-tag still provides the lowest uptake of radioactivity in liver compared to its radiolabeled counterpart having a N-terminally placed H6-tag. The obtained levels of radioactivity in liver were also found to be similar or even lower in comparison to previous studies made with the same tracer without the presence of a H6-tag and radiolabeled by the same chemistry.

In paper III, we further investigated the influence on biodistribution of the non- chelating amino acids present in a histidine-containing tag for chelation of three- 99m carbonyl Tc. The H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2:342. As previously observed, the N-terminally HEHEHE-tagged tracer provided the lowest uptake of radioactivity in liver, whereas the introduction of the positively charged amino acid

49 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______lysine or more hydrophobic amino acids, especially isoleucine, provoked much higher levels of radioactive uptake in liver. It was also shown, as indicated from previous studies, that a C-terminal placement of a histidine-containing tag for three-carbonyl 99mTc chelation promotes higher levels of radioactivity in blood, which also correlated well with the observed reduced stability of the radiolabel.

In paper IV, we showed that the HEHEHE-tag can also enable IMAC purification of an IGF-1R-specific Affibody molecule tracer, ZIGF1R:4551, as well as improving its overall biodistribution in mouse models to provide higher tumor-to-organ ratios of measured radioactivity when compared to the initially evaluated H6-tagged ZIGF1R:4551.

In paper V, we developed an intracellular receptor-retention model system that enabled a step-wise down-regulation, 50 %, 60 %, 80 % and 96 %, respectively, of EGFR surface expression levels in an ovarian cancer cell line. The system was shown to be stable over a period of two months and for one of the Affibody molecules constructs relocating EGFR to the lysosomes, a potential lysosomal degradation of EGFR and the Affibody constructs were indicated. The cellular consequence of down regulating 80% or more of the EGFR surface levels resulted in a 15% lower proliferation rate of cells. In a related study concerning cell-specific delivery of transgenes for intracellular expression of proteins, Affibody molecules were shown to enable retargeting of viral particles for gene-delivery to HER2 expressing cell. More than a 4-fold higher infectivity was seen for viral particles retargeted by Affibody molecules in comparison to untargeted viral particles.

Taken together, we have shown that for radionuclide-based molecular imaging applications in vivo, the use of a novel HEHEHE-tag enables IMAC purification, three-carbonyl 99mTc radiolabeling, and improves the biodistribution of Affibody molecules ZHER2:342 and ZIGF1R:4551, respectively, when evaluated in preclinical models. In fact, we hope that this tag may be beneficial even for other Affibody molecules, as well as for other tracer formats, currently under development. We have further shown that Affibody molecules may be used for intracellular targeting of an oncogenic receptor in vitro by interfering with its secretion pathway to obtain a step-wise down- regulation of its cell surface expression levels mediated by a receptor-retention system. To further validate this system, additional cell lines and oncogenic receptors such as HER2 and IGF-1R, respectively, will be investigated in a near future. Lastly, we have shown that Affibody molecules may also be employed for retargeting of viral particles pseudotyped with the envelope derived from Sindbis virus for cell-specific transgene

50 Camilla Hofström ______delivery. To potentially increase the infectivity of the retargeted viruses, an affinity maturation to enhance the affinity of the anti-idiotypic Affibody molecule ZZTaq for

ZTaq, as well as employing alternative packing cell lines overexpressing the ZTaq- containing envelope protein, will be investigated.

51 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______Acknowledgments

Pust! Provtryck om några timmar och endast en sida kvar att skriva, men den är minst lika viktig då det finns så himla många jag vill tacka för all hjälp, trevligt sällskap på konferenser och alla glada stunder i labbet som förgyllt min vardag och gjort min tid på här på plan 3 till något jag alltid kommer att minnas!

Torbjörn! Tack för att du gav mig chansen att stanna kvar och doktorera efter mitt exjobb och för alla spännande projekt vi har gjort, jag har lärt mig så himla mycket under min tid i din grupp. Jag har även uppskattad din positiva och stöttande inställning till det mesta, speciellt de senaste veckorna!

Till Per-Åke, tack för ovärderlig input i diverse projekt, samt i denna avhandling. Track changes kan visst vara läskiga! Proteinklubbar och lit.sem skulle inte vara detsamma utan dig med dina frågor och kommentarer som får oss att tänka till!

Tack till Amelie för att du granskade denna avhandling, och tack till alla andra PI’s som bidrar till den trevliga och stimulerande arbetsmiljön vi har här på avdelningen. Ett stor tack till John för att du alltid tar dig tid att svara på mina frågor, och för värdefull input under avhandlingsskrivandet!

Till samarbetsparters Anna och Vladimir, thank you so much for teaching me all I know about imaging, I feel very fortunate to have had the opportunity to work with you both and with all of your PhD students! Thank you Mohammed and Anna for demonstrating how the animal experiments are performed, luckily I didn’t faint!

Jag vill även tacka värdens bästa och trevligaste företag Affibody AB för att jag fick möjligheten att utföra en selektion hos er. Extra stort tack till Elin, Susanne, Gunilla och Per för all hjälp och handledning. Håll tummarna för att de binder! Tack till fördetta plan3:aren Tove Alm på GE Healthcare för att jag fick komma och köra er fina BIAcore, och tack för all hjälp!

Till min exjobbshandledare Erik V, tack för att du lärde mig allt mellan himmel och jord under exjobbet och för att du inte avskräckte mig helt, bara lite, från att doktorera. Jag kan fortfarande höra din gälla stämma när jag står och ympar vid sterilbänken!

52 Camilla Hofström ______

Till ”snyggrummet”, som rätt ofta även blev ”snackisrummet” med Basia, Anna P, Helena, Johanna S och Feifan. Tack för alla sköna pauser, skvaller och för allt godis jag fick norppa!

Tack till mina exjobbare Magnus C och Johan S för era kanoninsatser i projekten. Kul att du stanna kvar i gruppen Johan, snart så är det din tur! Stort tack till Bahram för alla klockrena sekvenser, och tack till Emma Ö för att du håller ordning och reda här på plan 3, det är verkligen uppskattat! Och ett stort tack till proteinfabriken, molbio gruppen och immunotech för all hjälp under dessa år med diverse saker som antibiotika, IPTG, sekvenseringar, westernblot reagenser, och framförallt multipipetter!

Till mina rumskamrater, Daniel, Emilie, Tarek, Jesper, Sahar och Thiru, tack för att ni har stått ut med mig de senaste veckorna, jag har nog varit lite extra känslig! Och Danne, jag är förmodligen skyldig dig ett 24-pack coca cola nu! Och till före detta postdoken Peter J, bästa rumskamraten ever! Stort tack till Kristina W, räddaren i nöden som PyMOL expert!

Tack till tjejgänget i Boston på PEGS med Anna P, Anna K, Johanna S, Hanna, Lisa, Basia och Nina, shit vad kul vi hade! Forskning, shopping, amerikansk fotboll och clam chowder! Och tack Konrad för att du orkade släppa dit min poster! Till San Diego gänget på Antibody Engineering, Lisa, Filippa, Johan N, Feifan, John och Andreas! Tack för den trevliga födelsedagsmiddan, och tack Andreas för härliga sightseeing! Tack Anna P, Ulrika, Helena, Basia och Johanna för alla sköna middagar och vinkvällar, ribs it is! Och tack Anna för att du fick mig att börja sticka, en halv socka so far… Och tack för alla mysiga syjuntor med Anna P, Ulrika, Kristina W, Basia, Helena, Sara, Tove, Magdalena, Maja, Hanna… och många fler.

Till KTH:arna Sara, Charlotte, Linda och Darko! Kul att vi hållit kontakten efter utbildningen även om vi oftast befinner oss på olika platser i världen! Det är alltid lika nice att ses och snacka lite skit!

Till sist min familj, en stor kram till barndomsvännerna Lina, Julia, Sandra och Maryam, min extended family. Tack för att ni finns i mitt liv och för att ni alltid kommer att finnas där! Till min mamma Monica, tack mamma för att du och pappa alltid har uppmuntrat och trott på mig, oavsett vad det än gäller! Jag älskar er båda! Sist men inte minst, bästa storasyrran Annelie! Tack för att du alltid har stöttat mig och för att du alltid finns där när det behövs, mitt personliga skyddsnät. Oavsett min ålder så kommer jag alltid vara din ”kiddo”!

53 Engineering of Affibody molecules for Radionuclide Molecular Imaging and Intracellular Targeting ______

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