site-specific labeling of affinity molecules for in vitro and in vivo studies

Site-specific labeling of affinity molecules

for in vitro and in vivo studies

ANNA PEROLS

Royal Institute of Technology

School of Biotechnology

Stockholm 2014

© Anna Perols

Stockholm 2014

KTH Royal Institute of Technology

School of Biotechnology

AlbaNova University Center

SE-106 91 Stockholm

Printed by Universitetsservice US-AB

Drottning Kristinas väg 53B

SE-100 44 Stockholm

Sweden

TRITA-BIO Report 2014:14

ISSN 1654-2312

ISBN 978-91-7595-252-9

Anna Perols (2014): Site-specific labeling of affinity molecules for in vitro and in vivo

studies. School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.

Abstract

The thesis is focused on site-specific labeling of affinity molecules for different applications

where two types of binding proteins, Affibody molecules and antibodies, have been used. For the

purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-

functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study,

two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide

chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395).

In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-

MMA-ZHER2:2395 counterpart, resulting in improved tumor-to-organ ratios. In a second study the

in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in

tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-

ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in

higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant.

In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1,

was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide

bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-

NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-

ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone

ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the

DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of

helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation

resulted in the highest contrast.

Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-

binding regions might influence the affinity. A method for site-specific labeling of antibodies

using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody

by photoconjugation was optimized. By investigation of positions most suitable for incorporation

of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses

important for both diagnostic and therapeutic applications. In addition, optimized variants were

used in combination with an incorporated click-reactive handle for selective labeling of the

antibody with a detection molecule.

Key words: Affibody molecules, molecular imaging, site-specific labeling, solid phase peptide

synthesis, IgG-binding domains, photoconjugation.

Sammanfattning

Avhandlingen är baserad på arbeten som varit inriktade på inmärkning av två olika typer av

affinitetsmolekyler, dels affibodymolekyler och dels antikroppar. Inmärkningen har skett på ett

kontrollerat sätt som för affibodymolekyler lett till förbättrad tumördiagnostik, och för

antikroppar bibehållen affinitet efter inmärkning.

I första delen av avhandlingen har egenskaper hos Affibodymolekyler som binder den tumör-

associerade biomarkören HER2 med hög affinitet studerats i syfte att förbättra visualiseringen av

HER2-uttryckande tumörer i djurmodeller. För att kunna urskilja en HER2-uttryckande tumör

från övriga organ krävs att den målsökande spårmolekylen ansamlas i tumörvävnaden, och

samtidigt har lågt upptag i omgivande vävnad, dvs. ger en bra kontrast. I dessa studier har olika

typer av kelatkomplex, DOTA, NOTA och NODAGA, testats för att binda den radioaktiva

isotopen 111In till affibodymolekylen. Utvärdering i djurmodeller med tumörer implanterade

visade att både val av kelatkomplex samt var kelatkomplexet positionerades i affibodymolekylen

resulterade i skillnader i kontrasten. Då metastaser vanligen förekommer i skelett för

prostatacancer och i lever för bröstcancer, är det viktigt att dessa organ har ett lågt, icke-specifikt

upptag av affibodymolekyler för att kunna urskilja möjliga metastasbildningar. Studierna

konkluderade att NOTA och NODAGA är lovande kelat-strukturer för visualisering av

prostatacancertumörer vid konjugering C-terminalt med maleimide/tiol-kemi. Uppföljande

studier visade vidare att konjugering via en amidbindning N-terminalt gav förbättrad kontrast

för DOTA för visualisering av metastaser i lever och skelett i jämförelse mot NOTA och

NODAGA, samt att en C-terminal amid-konjugering av DOTA är den mest lovande.

I andra delen av avhandlingen har en metod för inmärkning av antikroppar använts, där den

antikroppsbindande domänen Z har syntetiserats med en fotoreaktiv grupp för att kovalent

kunna korslänkas till en antikropp med UV-ljus, utan att påverka dess bindning. I studien

förbättrades konjugeringseffektiviteten genom att optimera placeringen av den fotoreaktiva

molekylen i domän Z. En optimerad variant användes i en efterföljande studie i kombination

med en funktionell grupp för klick-kemi, för att selektivt kunna koppla en reportergrupp till

antikroppen. Vanligen resulterar antikroppsinmärkning i en heterogen produkt vilket kan

påverka bindningen, men ovanstående studier presenterar ett lovade alternativ för selektiv

inmärkning av antikroppar med bibehållen affinitet.

Sammanfattningsvis visar studierna i denna avhandling att en kontrollerad inmärkning av

affinitetsproteiner är viktigt, dels för användning av Affibodymolekyler i tumördiagnostiskt syfte,

och dels i en metod för selektiv inmärkning av antikroppar utan förlust av dess specificitet.

List of publications

This thesis is based on the following articles or manuscript, which are referred to in the text by

their Roman numerals (I-VI). The articles can be found in the appendix.

I Tolmachev V, Altai M, Sandström M, Perols A, Karlström AE, Boschetti F, and Orlova A.

(2011) Evaluation of a maleimido derivative of NOTA for site-specific labeling of Affibody

molecules. Bioconjug Chem. 22:894-902

II Altai M, Perols A, Karlström AE, Sandström M, Boschetti F, Orlova A, and Tolmachev V.

(2012) Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA. Nucl Med Biol. 39:518-529

III Malmberg J*, Perols A*, Varasteh Z, Altai M, Braun A, Sandström M, Garske U,

Tolmachev V, Orlova A, and Karlström AE. (2012) Comparative evaluation of synthetic

anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA,

NOTA and NODAGA chelators in mice bearing prostate cancer xenografts. Eur J Nucl Med

Mol Imaging. 39:481-492.

IV Perols A*, Honarvar H*, Strand J, Selvaraju RK, Orlova A, Karlström AE, and Tolmachev

V. (2012) Influence of DOTA chelator position on biodistribution and targeting properties

of 111In-labeled synthetic anti-HER2 Affibody molecules. Bioconjug Chem. 23:1661-1670

V Perols A, and Karlström AE. (2014) Site-specific photoconjugation of antibodies using

chemically synthesized IgG-binding domains. Bioconjug Chem. 25:481-488.

VI Perols A, Famme MA, and Karlström AE. Site-specific antibody labeling by covalent

photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions.

Manuscript

* These authors contributed equally to this work.

All papers are reproduced with permission from the copyright holders.

Contributions to the papers

Paper I

Designed, performed and evaluated the protein characterization analyses prior to the

radiolabeling and in vitro and in vivo evaluation. Contributed to the writing and editing of the

paper.

Paper II

Designed, performed and evaluated the protein characterization analyses prior to the

radiolabeling and in vitro and in vivo evaluation. Contributed to the writing and editing of the

paper

Paper III

Synthesized the variants, designed, performed and evaluated the experiments for protein

characterization prior to the radiolabeling and in vitro and in vivo evaluation. Contributed to the

writing and editing of the paper

Paper IV

Synthesized and purified the variants used in the study. Performed all parts of protein

characterization experiments prior to the radiolabeling and in vitro and in vivo evaluation.

Wrote the paper together with co-authors.

Paper V

Synthesized the IgG-binding variants used in the study. Designed, performed the conjugation

optimization studies and analyzed the results. Created the images for the paper and wrote the

paper together with co-author.

Paper VI

Synthesized the IgG-binding variants used in the study. Design, performed and analyzed the

results for the conjugations optimization studies. Created the images for the paper and wrote the

paper together with co-authors.

Related publications

Strand J, Honarvar H, Perols A, Orlova A, Selvaraju RK, Karlström AE, and Tolmachev V.

(2013) Influence of macrocyclic chelators on the targeting properties of 68Ga-labeled synthetic

Affibody molecules: comparison with 111In-labeled counterparts. PLoS One 8: e70028

Rönnlund D, Xu L, Perols A, Gad AK, Karlström AE, Auer G, and Widengren J. (2014)

Multicolor fluorescence nanoscopy by photobleaching: concept, verification, and its application

to resolve selective storage of proteins in platelets. ACS Nano 8: 4358-65

Honarvar H, Strand J, Perols A, Orlova A, Selvaraju RK, Karlstrom AE, and Tolmachev V.

Position for site-specific attachment of a DOTA chelator to synthetic Affibody molecules has a

different influence on the targeting properties of 68Ga- compared to 111In-labeled conjugates.

Molecular Imaging, in press.

Contents

Introduction

Chapter 1 - Proteins ..................................................................................................................... 1

Protein chemistry ........................................................................................................................................... 1

Affinity proteins ............................................................................................................................................ 4

Antibody structure and function ........................................................................................................... 4

Antibody fragments ............................................................................................................................... 7

Antibody generation .............................................................................................................................. 8

Antibody therapy ................................................................................................................................... 8

Alternative scaffolds .............................................................................................................................. 9

Affibody molecules .............................................................................................................................. 10

Chapter 2 - Chemical protein synthesis ............................................................................... 14

From solution to solid phase ........................................................................................................................ 14

Solid phase peptide synthesis (SPPS) .......................................................................................................... 15

Fmoc/tBu strategy ................................................................................................................................ 15

Side chain protecting groups........................................................................................................................ 17

Aggregation ................................................................................................................................................. 20

Microwave synthesis ............................................................................................................................ 20

Limitations in solid phase peptide chemistry ............................................................................................. 22

Chapter 3 - Protein labeling.....................................................................................................23

Chemical approaches .................................................................................................................................. 23

Amino groups ...................................................................................................................................... 23

Thiol groups ......................................................................................................................................... 24

Bioorthogonal reactions for labeling proteins ............................................................................................ 24

Cu(I) catalyzed [3+2] alkyne-azide cycloaddition ............................................................................... 25

Strain-promoted [3+2] alkyne-azide cycloaddition ............................................................................ 27

Alternative ligation strategies .............................................................................................................. 28

Introduction of chemoselective handles in recombinantly produced proteins ................................... 28

Radiolabeling for molecular imaging .......................................................................................................... 29

PET and SPECT ................................................................................................................................... 29

Radiometals and chelators for imaging ............................................................................................... 30

Application-specific considerations ..................................................................................................... 32

Present investigation

Aim of the thesis ........................................................................................................................ 35

Chapter 4 - Functionalization of Affibody Molecules for Detection of HER2 using

Molecular Imaging .................................................................................................................... 36

HER2 as a molecular target......................................................................................................................... 36

Papers I, II and III - The influence of macrocyclic chelators and different conjugation chemistries

for radiolabeling of Affibody molecules in molecular imaging. .................................................................. 37

Papers I and II...................................................................................................................................... 37

Paper III ............................................................................................................................................... 40

Conclusions and future outlook ........................................................................................................... 42

Paper IV – The influence on biodistribution from the positioning of the DOTA chelator in

the Affibody molecule .................................................................................................................................. 43

Conclusions and future outlook .......................................................................................................................... 45

Chapter 5 - Site specific labeling of antibodies using IgG-binding domains ............. 47

Paper V – Site-specific photoconjugation of antibodies using chemically synthesized

IgG-binding domains .................................................................................................................................. 48

Conclusions and future outlook ............................................................................................................51

Paper VI – Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for

alkyne-azide cycloaddition reactions .......................................................................................................... 52

Conclusions and future outlook ........................................................................................................... 55

Concluding remarks and future outlook.............................................................................. 57

Acknowledgements ................................................................................................................... 60

References ................................................................................................................................... 62

List of acronyms

%ID/g Percent injected dosage per gram

ADC Antibody-drug conjugates

ADCC Antibody-dependent cell-mediated cytotoxicity

DAR Drug-antibody-ratio

DNA Deoxyribonucleic acid

DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

DTPA Diethylenetriaminepentaacetic acid

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

EGFR Human epidermal growth factor receptor

Fab Fragment, antigen binding

Fc Fragment crystalizable

FcRn Neonatal Fc receptor

FDA The Food and Drug Administration

FISH Fluorescence in situ hybridization

HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronoium hexa fluorophosphate

HC Heavy chain

HER2 Human epidermal growth factor receptor 2

HF Hydrofluoric acid

HOBt 1-hydroxybenzotriazole

kDa kilo Dalton

LC Light chain

MMA Maleimidoethylmonoamide

NODAGA 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane

NOTA 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid

p.i. post injection

PEG Polyethylene glycol

PET Positron emission tomography

SPECT Single-photon emission computed tomography

SPPS Solid phase peptide synthesis

TETA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid

TFA Trifluoroacetic acid

Imagination is more important than knowledge.

Knowledge is limited. Imagination encircles the

world.

- Albert Einstein

INTRODUCTION

Anna Perols

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~ Chapter 1 ~

Proteins

The molecular recognition by proteins is important for many processes in nature. The proteins

capable of selectively binding their target molecules are referred to as affinity proteins, and have

shown importance in many biotechnological and therapeutic applications. The main focus of this

thesis is the labeling of these affinity proteins for both in vitro and in vivo applications. Different

types of affinity proteins will be further discussed later in this chapter, however in order to

understand protein binding one has to understand proteins.

Protein chemistry

Protein research might have started already in 1839 when the term protein was coined by

Mulder, based on the greek word proteos with the meaning “of first importance”. Mulder also

stated the importance of proteins for all living organisms as “unquestionably the most important

of all known substances in the organic kingdom” (Tui 1952). The genetic information is however

stored in DNA, and transferred via RNA to form the proteins that are performing most of the

cellular functions. These fundamental biomolecules are responsible for cell structure, function

and regulation, such as the mechanical support of keratin in skin and collagen in for example

muscles, skin and bones. They are also responsible for all chemical processes facilitated by

enzymes, and the involvement in cell signaling by hormones and in transportation, such as the

transport of the oxygen in the blood by hemoglobin. Furthermore, proteins also have the ability

to selectively bind other proteins, DNA or vitamins.

Even though proteins show a vast variety of functions and sizes, they are built from the 20 amino

acids encoded in the DNA, where diversity is introduced by varying the order of amino acids in

the polypeptide chain. Each amino acid has a chiral carbon, the α-carbon, to which both an

amine and a carboxylic acid is attached, hence the name amino acid. To this α-carbon, a specific

functional side chain is also attached, making each amino acid only one of its kind (Figure 1).

Site-specific labeling of affinity molecules for in vitro and in vivo studies

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Two amino acids are linked together via the α-carboxyl group of the first amino acid and the

α-amino group from the second amino acid in a condensation reaction with elimination of a

water molecule forming a peptide bond, which together with the α-carbons make up the peptide

or protein backbone. The different amino acids can be divided into three groups depending on

the chemical characteristics of the functional side chain. The amino acids can be classified as

hydrophobic, i.e. the side chains have weak propensity to interact with water, or they could be

charged or polar which often give an energetically favorable contact with water. Glycine is a

special amino acid since it does not have a side chain, and is therefore difficult to assign to any of

the above mentioned classes, however, it can often be found on the surface of proteins, in loops,

giving flexibility. Another amino acid that can be distinguished from the other amino acids is

proline, in which the aliphatic side chain is linked to the backbone amide resulting in a cyclic side

chain, and it is in contrast to glycine a very rigid amino acid providing stability to the protein.

The linear sequence of amino acids, the polypeptide chain, is referred to as the primary

structure of a protein and is determined by the gene encoding the protein. The primary

structure will after synthesis form different motifs, such as α-helices where the backbone is

coiled around its own axis forming a spiral with the amino acids pointing outwards or as β-sheets

where the backbones are in a parallel or anti-parallel direction. The polypeptide chain can fold

into these α-helices and β-sheets, called the secondary structure of the protein. These

secondary structure elements will together fold into the final three-dimensional shape of the

protein, giving the tertiary structure. Certain proteins are composed of several polypeptide

chains and the overall organization and the interactions between the different chains create the

quaternary structure.

The intramolecular interactions that hold the secondary structure together are mainly hydrogen

bonds within the backbone, whereas for the tertiary structure the protein fold is mainly

determined by the amino acid sequence. The different interactions between the side chains in a

protein include salt bridges, hydrogen bonding, and van der Waals’ interactions among the non-

covalent interactions and disulfide bonds as a covalent bond. In most proteins hydrophobic

amino acids are buried in the interior of the protein and the hydrophilic amino acids are

accessible on the surface of the protein, although there are protein structures that have charged

or polar amino acids in the interior of the protein. These amino acids need to be paired to

compensate for their charges, thus forming salt bridges or ionic interactions.

Anna Perols

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Figure 1: Structure and name of the 20 natural amino acids.


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Affinity proteins

Proteins with the ability to bind to a desired target molecule are referred to as affinity proteins. A

specific interaction between an affinity protein and its target can for example be seen for the

proteins involved in host defense, such as between an antibody produced by the immune system

and its antigen. Also alternative affinity proteins have been developed, as the protein engineering

technology has emerged, with smaller and less complex structure. Different types of affinity

proteins, both naturally produced proteins and alternative binding proteins will be further

discussed below.

Antibody structure and function To survive the everyday life where we constantly are challenged by bacteria and other pathogens,

the immune system in most vertebrates has evolved to become the defense system that we have

today and this is branched into the early innate immune system and the specific adaptive

immune system. The innate immune system is the first-in-line defense comprised by a set of

recognition proteins and cells that bind to classes of proteins on bacteria and viruses, and are

built up from ancient history and encoded by germline DNA. The adaptive immune system both

relies on recombination of the germline encoded genes and somatic mutations creating diversity

to produce the antigen specific receptors and binders, the immunoglobulins (Igs). It also holds a

memory resulting in a fast onset of defense mechanisms upon the second encounter (Janeway

2002; Li 2004).

Immunoglobulins are key immune system proteins that specifically recognize foreign substances

or antigens in the body such as viruses or bacteria, and aid in their destruction. In humans, five

different isotypes of immunoglobulins have been identified, all with similar overall structure

comprised of two heavy and two light chains linked by disulfide bridges, and formed as Y-shaped

proteins. The different isotypes are classified based on the different heavy chains α, δ, ε, γ and μ,

and could either remain bound to the membrane of a B lymphocyte acting as a receptor (mIgM,

mIgD) or be secreted (IgA, IgD, IgE, IgG and IgM). In the 1960’s, IgG was found to have four

subclasses (Grey 1964; Terry 1964) designated IgG1, IgG2, IgG3 and IgG4 based on decreasing

abundance in human sera. The subclasses have later been found to have differences in the heavy

chain amino acids sequence, mainly in the hinge region where the number of disulfide bonds and

hinge flexibility differ (Figure 2). IgA also has been further divided into the subclasses IgA1 and

IgA2, whereas IgD, IgE and IgM have no subclasses. For the light chains, there are two types of

light chains, the kappa (κ) and the lambda (λ) chain, resulting in either IgG(κ) or IgG(λ) in a ratio

ranging from 3:1 to 1.5:1 in human serum (Jefferis 1980).

Anna Perols

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Figure 2: Schematic representation of the four subclasses of human IgG. Constant domains of both heavy and light chain are shown in dark grey and the variable domains are shown in light grey. The chains are held together by disulfide bonds, represented by black lines.

IgG is the most abundant Ig class in human serum, making up about 75% of total serum Igs

(Jefferis 2007). With its high abundance, IgG has become the golden standard in research and

diagnostics and the name is often used interchangeably with the definition antibody, which will

hereafter be used synonymously with IgG throughout this thesis unless stated otherwise. An

antibody consists of two identical heavy chains (HC), each with approx. 440 amino acids, and

two light chains (LC) with approx. 220 amino acids each, and forms a symmetrical protein with a

molecular weight of approx. 150 kDa. The heavy chain has four domains; one variable domain

(VH), followed by three domains (CH1, CH2, CH3) with a conserved amino acid sequence resulting

in a constant (CH) part. The light chain folds into two domains, one variable (VL) and one

constant (CL) domain. The hinge region in the middle of the heavy chain, located between CH1

and CH2 divides the antibody into two separate structures; the two arms responsible for antigen

binding (Fab-fragments) and the bottom part of the heavy chains, (Fc-fragment) responsible for

effector functions (Figure 3A). The light and the heavy chain as well as the two heavy chains are

held together by both noncovalent interactions as well as covalent disulfide bonds. Besides

connecting the two heavy chains together, the unstructured hinge region also provides flexibility

for efficient antigen binding by the two arms (Edelman 1973).

The Fab-fragment consists of parts from both the heavy and light chain, connected by the

disulfide bond, and at the tip of each arm the variable domains from the heavy and light chain

form the antigen binding site. Each domain is individually folded into a β-sandwich of two

antiparallel β-sheets. The amino acid sequence variation within these variable domains is not

evenly distributed but instead clustered into three regions separated from each other, by forming

the complementarity determining regions (CDR1, CDR2 and CDR3) (Wu 1970). The VH domain

is formed as a stabile framework with the three CDRs located in the connecting loops, and in the


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tertiary structure of the Fab-fragment these six loops are positioned in close proximity making

up the antigen binding site responsible for selective antigen binding (Figure 3A).

Figure 3: Schematic structure of an antibody and antibody derived fragments. A) An antibody with the Fc-fragment and Fab-fragments indicated, with the CDRs at the tip of each arm, forming the antigen binding site. B) Antibody fragments, F(ab’)2 and Fab-fragment, with retained antigen binding capacity but reduced molecular weight. C) Fragments composed of variable domains, either monovalent as scFv or bivalent as diabodies. Picture modified from (Holliger 2005)

With the Fab-fragments responsible for antigen binding, the Fc-fragment is important for the

antibody function in terms of activating the complement system, recruiting immune cells and for

the neonatal Fc-receptor recycling. The complement system is activated by binding of the first

complement protein to the Fc-region, followed by a cascade of binding events leading to lysis of

the microbe. In antibody dependent cell-mediated cytotoxicity (ADCC), effector cells such as

natural killer cells, macrophages and neutrophiles expressing Fcγ-receptors will bind the Fc-

region and be activated, and the target cell will be lysed. Similarly, in the process of opsonization

where a pathogen has been covered with antibodies, the Fc-binding will facilitate the

phagocytosis of the pathogen. These Fcγ-receptors expressed on the immune cells are not the

only type of Fc-receptor available, also the neonatal Fc-receptor (FcRn) responsible for the

prolonged circulation time is of great importance (Brambell 1969). The antibodies are recycled in

a process facilitated by the FcRn, where the receptor is actively rescuing the antibody from

endosomal degradation by retaining it to the receptor after endocytosis and instead recycling the

antibody to the surface of the cell. This results in an extended half life of approx. 21 days for IgGs

Anna Perols

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(with the exception of IgG3, which has a half-life of approx. 7 days) in serum, a large increase in

comparison to other classes of immunoglobulins which have a half-life of 2-8 days (Rogentine

1966; Chames 2009).

Antibody fragments The modular composition of IgGs, built up by distinctive individually folded domains, makes it

possible to split IgG proteins into smaller fragments, with retained structure. With the use of

smaller antibody fragments, there is a possibility to optimize the pharmacological properties for

different applications, such as half-life, tissue penetration and avidity (Nelson 2010a). The

implications of the molecular size in different applications will be discussed later in thesis.

Antibody fragmentation was traditionally performed by enzymatic digestion but is today most

often generated by recombinant protein engineering. Cleavage below the hinge region generates

the bivalent F(ab’)2 fragment with both arms available for antigen binding (Figure 3B). The

removal of the Fc-portion will eliminate the interactions with previously mentioned Fc-receptors

and the molecular weight will be reduced from 150 kDa to 110 kDa. More commonly used is the

monovalent Fab-fragments, comprised of CH1, CL, VH and VL, with a molecular weight of 50 kDa.

The two chains are stabilized by a disulfide bond and could be used as an alternative to the intact

antibody for applications where a smaller agent is wanted, such as the humanized Fab

ranibizumab (Lucentis®) which is an alternative to the full-length antibody bevacizumab

(Avastin®) targeting the endothelial growth factor (VEGF) (Klettner 2008).

An even smaller domain from an antibody with retained monovalent binding capacity binding is

the Fv-portion consisting of only VH and VL. This fragment cannot readily be obtained from an

intact antibody by proteolysis, and is instead typically recombinantly produced as a single chain

with a peptide linker holding the domains together, resulting in a single chain Fv (scFv) with a

molecular weight of 25 kDa (Bird 1988; Huston 1988). By changing the linker lengths between

the two domains, it has been shown that complexes of different size will form, such as dimers

(diabodies) and trimers (Hudson 1998). Single variable domains (VH or VL) could show poor

solubility and are in general prone to aggregate when unpaired with the corresponding variable

domain, however, engineered monomeric variants have been selected for certain targets, such as

HER2 (Baral 2012). In camelids and cartilaginous fish, a portion of the antibodies are expressed

as heavy-chain antibodies, with no light chains, and the heavy-chain single-domain can be

isolated, such as the variable domain VHH from camelids and V-NAR from sharks. The single-

chain antibody fragments from these antibody types differ from the traditional ones, especially

the CDRH3 region, which is in general longer and could improve binding into small cavities

where standard antibodies or antibody fragments would not bind (Holliger 2005); (Holt 2003).


Page | 8

Antibody generation Antibodies can naturally be generated by immunization with an antigen, resulting in an

activation of the immune system and the production of antibodies binding the antigen with high

specificity and affinity. The pool of antibodies produced, is generated against different parts of

the antigen, i.e. it recognizes different epitopes, and is referred to as polyclonal antibodies

(pAbs). For diagnostic and therapeutic applications a single type of antibody, a monoclonal

antibody (mAb), is desired. Thanks to the introduction of the hybridoma technology for

production of monoclonal antibodies by Köhler and Milstein, a renewable source for production

of identical antibodies of mouse origin toward almost any antigen was obtained (Köhler 1975). By

fusing an antibody-producing mouse B-cell with an immortalized myeloma cell line, the

combined properties resulted in an immortal cell line secreting monoclonal antibodies. However,

these antibodies were not suitable for in vivo applications such as human therapeutics or in vivo

imaging due to the non-human origin, and methods were developed for making the mouse

antibodies humanized, such as grafting antigen binding regions into a human antibody

framework (Nelson 2010b).

Human antibodies are also generated without using the immune system; instead the human

antibody gene libraries could be displayed by in vitro selection technologies such as phage and

ribosomal display. One commonly used display method is the display on filamentous phages

(Smith 1985). Expression and correct folding of the complex structure of the antibody are

difficult in most microorganism host expression systems, but was overcome by the use of smaller

fragments of the IgG-molecule such as Fab-fragments or scFvs lacking the Fc-region

(Schirrmann 2008). A combined system for display of full length antibodies has also been

designed, in which the antibodies produced were excreted to the bacterial periplasm and

captured for display on the inner membrane (Sidhu 2007).

Antibody therapy Antibodies can in themselves exhibit therapeutic effect either by antigen binding, usually by

blocking receptor activation or binding to the receptor ligands, or by activation of the ADCC or

the complement system by the Fc-region as previously described. A variety of monoclonal

antibodies are currently used in the clinics today for treatment of malignant diseases such as

cancer but also for autoimmune diseases, such as asthma and arthritis (Nelson 2010a). For

treatment of solid malignancies, the selective targeting properties could also be exploited for

efficient delivery of toxic molecules to a specific area rather than using systemic treatments, and

by conjugation of a cytotoxic drug, forming antibody-drug conjugates (ADC), the efficacy of the

antibody might be improved (Alley 2010). In order to reduce off-target toxicity, the ADC needs to

be stabile in the circulation and preferably have a similar biodistribution pattern as the naked

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antibody (Ducry 2009). Protein labeling often results in heterogenous labeled product, (further

discussed in Chapter 3), and differences in conjugation chemistry and conjugation site could

greatly affect the performance of the antibody-drug-conjugates (Webb 2013). Standard amine

coupling is one of the most common conjugation methods, and due to the several surface-

exposed lysine residues available for antibody labeling it will most likely result in a pool of

antibodies with different drug-to-antibody ratios (DAR). The different number of drugs on each

antibody might lead to differences in the pharmacokinetic properties, and could decrease the

therapeutic window (Jeger 2010). Two ADCs that have been FDA approved are the HER2-

targeting trastuzumab-emtansine T-DM1 (Kadcyla®) used for treatment of breast cancer and the

CD30-targeting brentuximan-vedodin (Adcetris®) that is used for treatment of Hodgkin’s

lymphoma. T-DM1 has a maytansinoid derivate conjugated, whereas brentuximan-vedodin is

conjugated with an auristatin derivate, where both cytotoxins have antimitotic mechanisms that

results in apoptosis of the targeted cancer cells (Sawant 2014).

In addition to conjugation with cytotoxic drugs for therapy, antibodies can also be labeled with

radionuclides, where the labeled antibody delivers a toxic level of radiation to the targeted site.

As the radiolabeled antibody has a long circulation time, pretargeting is a promising strategy as it

reduces the radioactive exposure in healthy tissues. By separating the injection of the targeting

tracer, the antibody, from the delivery of the radioactive moiety, often a small molecule with fast

clearance, the toxicity can be lowered. There are several strategies of pretargeting relying on the

high selectivity between streptavidin/avidin-biotin, complementary DNA strands, or bispecific

antibody-hapten systems, to mention the most common (Frampas 2013). A biotinylated antibody

targeting tenascin was reported to be in phase I/II clinical trials, where the biotin-avidin

selectivity was used together with the radionuclide Yttrium-90 for treatment of glioma, non-

Hodgkin’s lymphoma patients (Steiner 2011).

Alternative scaffolds Antibodies have traditionally been extensively used in applications as recognition molecules,

together with the smaller antibody fragments. However, with the improved protein engineering

techniques, also non-IgG like proteins could be modified using combinatorial engineering to

introduce novel binding properties, resulting in new affinity proteins (Skerra 2007). These

proteins typically provide advantages such as high thermal and proteolytic stability and are

efficiently produced in recombinant expression systems (Gebauer 2009).

Based on the human 10th fibronectin type III domain (10Fn3) with a structural similarity to the

IgG-domain, the alternative scaffold Adnectins was developed. This 94-residue protein shares

its structural fold, a stable β-sheet structure, with the IgG-domain but has in contrast to the IgG-


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domains no disulfide bridges for stabilization (Koide 1998). Scaffold proteins could also be based

on protein with no structure similarity with IgGs, such as the Anticalins, an alternative affinity

protein based on the protein family of lipocalins. The anticalins are larger than the adnectins,

with approx 160-180 residues in eight anti-parallel β-strands, forming a conical barrel with an

α-helix in the C-terminal part if the protein (Flower 2000; Skerra 2008). For both these affinity

proteins, the loops are subjected to randomization for generation of binders in selections systems

(Schlehuber 2005; Tolcher 2011).

Repeat proteins, such ankyrin or leucine-rich repeat proteins, with modules or motifs stacked

next to each other, are abundant in nature (Andrade 2001). The ankyrin repeat protein was used

as a framework for the alternative protein scaffold, Designed Ankyrin Repeat Proteins

(DARPins). Each module consists of 33 amino acids and usually 2-4 modules in a single

polypeptide chain are stacked to improve thermal and chemical stability, as well as increase the

binding surface for antigen binding, resulting in protein with a molecular weight of 14-21 kDa

(Stumpp 2008; Boersma 2011).

Cysteines are usually rare in proteins and often avoided in scaffolds, however, there is a class of

proteins that takes advantage of the unique properties of the thiol-containing side chain, the

cysteine knot proteins. The proteins share a similar structure in which the disulfide bonds

lead to the formation of a very rigid core and one of the three disulfide bonds creates a knot in

the cyclic peptide. The loops between the conserved cysteine positions are highly diverse, and

thus amenable for randomization or display of functional binding epitopes (Christmann 1999;

Kolmar 2008). One example of these small proteins is knottins, a small scaffold protein

comprised of only about 30 residues but with three intramolecular disulfide bonds (Moore 2012).

This constrained and conserved structure of engineered cysteine knot proteins results in

extraordinary proteolytic and thermal stability, especially advantageous for in vivo applications

such as oral delivery or for harsh in vitro preparation and can readily be prepared using solid

phase peptide synthesis.

Affibody molecules Affibody molecules are derived from one of the five homologous Ig-binding domains of

protein A, expressed on the surface on Staphylococcus aureus. All five domains, E, D, A, B and C,

individually bind the Fc-region of most antibodies and to the Fab fragment of VH3 IgG subtypes

(Jansson 1998), directing the neutralizing antibodies in the opposite direction compared to

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Figure 4: Generation of Affibody molecule. A schematic picture showing the origin of Affibody molecules from protein A. Based on domain B, domain Z (left) was generated, and further on also Affibody molecules (right), highlighting randomized residues generating specificity for e.g. HER2.


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a normal immune response and thereby evading neutrophil destruction (Gomez 2006). The

B-domain has been further engineered by an alanine to glycine substitution in position 29 to

remove a hydroxylamine cleavage site and to increase the alkaline stability, and additionally an

alanine to valine substitution in position 1 was introduced. The substitutions yielded domain Z

with retained affinity for the Fc-fragment, however the substitution in position 29 additionally

deleted the Fab-binding (Jansson 1998). The resulting Z-domain is an obligate Fc-binding

protein comprised of 58 amino acids formed as a triple helix bundle protein, with high thermal

and chemical stability. The Z-domain is rapidly folded and the abscence of cysteines and

disulfide bonds allow for folding in a reducing environment. The small size (approx. 7 kDa)

makes it amenable to efficient production using chemical synthesis (Engfeldt 2005) in addition

to the recombinant expression route in bacterial hosts. Even though the Z-domain is significantly

smaller than an antibody, the two types of affinity proteins have binding surfaces of similar size,

around 800 Å2, which is typical for protein-protein interactions (Nygren 2008).

The Z domain was the starting point for creating a stable non-immunoglobulin like affinity

protein. The natural binding surface of the protein formed by helices 1 and 2 with nine residues

involved in the Fc-binding together with four additional residues were chosen for randomization

to create a library from which binders could be generated using phage display (Nord 1995; Nord

1997) (Figure 4). Since then, Affibody molecules have been generated to a large number of

different targets including EGFR/HER1 (Friedman 2007), HER2 (Wikman 2004), and Aβ42

(Grönwall 2007). Besides the mentioned Affibody molecules, additional binders have been raised

for different applications such as viral targeting, bioseparation, diagnostics, and in vivo

molecular imaging (Löfblom 2010).

The Affibody molecule targeting HER2 is the Affibody used throughout this thesis. The first

variant selected (Wikman 2004) was subjected to a maturation selection using phage display

where the affinity was increased from low nM to 22 pM (Orlova 2006). The high affinity

targeting protein ZHER2:342 has been extensively used in molecular imaging and has shown great

value as a diagnostic tool in xenograft models. In order to site-specifically label the protein, a

unique Cys was introduced in the C-terminus resulting in ZHER2:2395, in which the high affinity for

HER2 was retained (KD = 27 pM) (Ahlgren 2007). The Affibody molecule scaffold was further

improved by substituting 11 residues in non-binding regions, which resulted in a scaffold with

higher thermal stability and increased hydrophilicity, but also increased the synthesis yield

(Feldwisch 2010). Based on the new HER2-binder, ZHER2:2891, changes in the N-terminus of

ZHER2:342 were introduced resulting in ZHER2:S1, a variant used for papers III-IV (Figure 5).

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Figure 5: Sequence alignment of the Z domain and the HER2-targeting Affibody molecules used in this thesis. Randomized positions indicated with (•) and amino acids substitutions in the scaffold (red).

The small size of Affibody molecules makes them amenable for production using chemical

synthesis, further reviewed in Chapter 3, and the use of the high affinity HER2-targeting

Affibody molecule is extensively discussed in papers I-IV.

Affinity proteins in molecular imaging and therapeutics

Alternative scaffold proteins have shown a great potential for diagnostic applications, such as in

molecular imaging (Stern 2013). Affinity proteins will have an improved tissue penetration in

solid tumors in comparison to large-sized antibodies, and smaller proteins will also be excreted

more quickly by glomerular filtration resulting in a shorter circulation half-life in comparison to

larger proteins (Schmidt 2009). The are several aspects influencing the renal filtration, such as

molecular size, shape and charge, but low molecular weight proteins usually pass the filter

whereas protein with a molecular weight of 40-50 kDa usually are retained (Kontermann 2011).

For applications where affinity proteins need to stay in circulation for a longer time, such as in

therapy, half-life extension could be obtained by increasing the hydrodynamic volume of the

proteins by PEGylation, to a molecular size above the threshold and thereby avoiding kidney

clearance (Harris 2003), or by fusion to for example human serum albumin (HSA) which is in

addition to IgGs recycled by the neonatal Fc-receptor (Dennis 2002). Molecular imaging in

general will be further discussed in Chapter 3, and the use of Affibody molecules in molecular

imaging are extensively discussed in papers I-IV.


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~ Chapter 2 ~

Chemical protein synthesis

Recombinant protein production is the most common way of producing proteins in large

amounts, and especially the E. coli expression system continues to be the preferred system in

protein engineering (Chen 2012). Bacterial expression systems are favored because of the fast

growth rate of bacteria which often results in high yields of the recombinant proteins. By

co-expression the protein with a purification tag, the protein can be purified and obtained in

large quantities to a fairly low cost. Larger proteins with complex structures can sometimes be

hard to produce in bacteria, and post-translational modifications might not be performed,

resulting in a non-functional protein. For production of such proteins, mammalian expression

systems might be better suited resulting in functional proteins with post-translational

modifications, but the yield is often lower than for bacterial expression. Other limitations of

using recombinant expression systems could be low yield when producing toxic proteins or the

presence of pyrogenes, endotoxins or lipopolysaccarides after lysis of the bacteria that could

impair the in vivo use. (Yin 2007)

Chemical protein synthesis has a number of advantages in comparison to recombinant

production of proteins, as the proteins can be assembled from building blocks exceeding the 20

amino acids encoded by DNA, incorporate D-amino acids or attach polymers to the protein. In

addition to non-natural amino acids that could be incorporated, also branched proteins and

peptides could be synthesized. This also allows for the possibility to introduce site-specific

modifications or chemical handles for bioorthogonal reactions, changes that could add new

functionalities to the protein. Examples of these bioorthogonal reactions are further discussed in

Chapter 3. Furthermore, many of these advantages of chemical synthesis were utilized in the

production of both the Affibody molecule used in papers III-IV and the IgG-binding domain in

papers V-VI.

From solution to solid phase

Many milestones have passed in order to synthesize proteins. The history of protein synthesis

started in the early years of the twentieth century when Emil Fischer described the first solution

synthesis of a dipeptide. His aim was to prepare protein fragments, “albumoses”, by total

chemical synthesis (Kent 1988). With the introduction of the first reversible protecting group by

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Bergmann, synthesis of longer peptides became feasible, but still required large amount of man

hours and resources (Mitchell 2008). In 1959, Bruce Merrifield, revolutionized the field of

peptide synthesis by attaching the C-terminus of the growing peptide chain to a solid support.

Merrifield was inspired by ribosomal protein synthesis but in contrast to biosynthesis, which

occurs in the opposite direction, the peptide was elongated at the N-terminus. (Birr 1978). Four

years later he had successfully synthesized the tetrapeptide Leu-Ala-Gly-Val on a solid matrix

(Merrifield 1963) and even though he had overcome the scientific hurdles, he was met by severe

resistance from other colleagues within the field. Despite the obstacles, the work was continued

and in 1969 Gutte and Merrifield published the solid phase synthesis of a Ribonuclease A (Rnase

A) with full activity, in shorter time and with higher yield compared to the solution condensation

strategy (Gutte 1969). In 1984 Merrifield was awarded with the Nobel Prize for his achievements.

Solid phase peptide synthesis (SPPS)

Since the introduction of synthesis on solid phase by Merrifield, two general strategies in

peptides synthesis have been widely used; the tert-butyloxycarbonyl (Boc)/benzyl (Bzl)

chemistry and the 9-fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl (tBu) chemistry. The first

solid phase chemical synthesis strategy utilized was based on Nα-Boc and benzyl-based side chain

protecting groups, which relies on differences in acid lability. Temporary N-terminal protecting

groups are cleaved off using trifluoroacetic acid (TFA). The final cleavage of side-chain protecting

groups and peptide release from the resin requires treatment with hydrofluoric acid (HF), an

even stronger acid than TFA. The Fmoc/tBu strategy utilizes an orthogonal deprotection strategy,

i.e. distinctly different reaction conditions for removing side-chain protecting groups and Nα-

protecting groups. In this strategy the Nα-protecting group is cleaved off by a secondary amine, a

base (Carpino 1970), whereas the side chain protecting groups are still removed by acidic

deprotection, and for cleavage from the solid support TFA is used instead of the hazardous acid

HF (Kent 1988). Fmoc/tBu is the standard chemistry used throughout the work of this thesis,

and will be the strategy of focus hereafter.

Fmoc/tBu strategy Peptide elongation is performed in a stepwise manner adding one amino acid at the time to the

growing chain. The C-terminal amino acid is anchored to a solid support (the resin) with an acid-

labile linker (Figure 6). During each coupling step, the newly introduced amino acid will have its

Nα-group temporarily protected to avoid unwanted side reactions.


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Figure 6: Schematic illustration of the repetitive addition of amino acids in SPPS using Fmoc/tBu strategy. The amino acid is activated (exemplified here by HOBt ester) before coupling to the solid support, in this case a Rink amide resin. The temporary protecting group, Fmoc, is cleaved off from the N-terminus and the next amino acid can be coupled. This is repeated until the last amino acid of the sequence. The side chain protecting groups are cleaved off simultaneously with cleavage of the peptide chain from the solid support.

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The carboxylic acid group is activated, typically by a coupling reagent to give an active ester,

e.g. by HBTU/HOBt to make a better leaving group. In order to avoid accumulation of deletion

peptides, due to incomplete acylation at the coupling step, a capping step is often performed

after each coupling ensuring that only the correct peptide chain is elongated. After each amino

acid that is coupled, a deprotection step is added where the temporary amino protecting group

is cleaved off before next coupling can be performed. When the synthesis has reached its end, the

temporary base-labile protecting group needs to be cleaved off before the peptide is released

from the solid support. Since both the linker between the C-terminus and the resin, as well as the

standard side chain protecting groups, are sensitive to acid, cleavage is usually performed as a

single step treatment using concentrated TFA.

Side chain protecting groups

Regardless of which of the two general synthesis strategies is applied, Boc/Bzl or Fmoc/tBu,

additional side chain protecting groups are needed. The concept orthogonal deprotection

was first described in 1977 by Baranay and defined as two or more protecting groups that are

removed by distinct mechanisms (Barany 1977). This could be exemplified by the removal of the

temporary protecting group Fmoc using the base piperidine and removal of the side chain

protecting group tBu using the acid TFA (Figure 7).

The guanidine group in arginine is often protected during synthesis to avoid side reactions,

where the most commonly used protecting group at present is 2,2,4,6,7-pentamethyl-

dihydrobenzofuran-5-sulfonyl (Pbf) which is removed with 80-95% TFA (Carpino 1993). For

histidine the two imidazole nitrogens are susceptible for reactions with electrophiles, however

trityl (Trt)-protection will be sufficient for most cases. Lysine is commonly used for

postsynthetic modifications thanks to the ε-amino group and its ability to form amide bonds. It is

therefore important to have efficient protecting groups for it during synthesis to avoid peptide

branching. The standard protection of Lys is Boc, which is simultaneously cleaved off with the

TFA cleavage from the resin. For on-resin modifications, side chain protecting groups with an

alternative deprotection strategy or weaker acid lability is used. The acid sensitive protection

groups Trt, generally cleaved off with 20% TFA, could be further sensitized by the introduction of

electron-donating substituents, resulting in the acid labile protecting groups 4-methyltrityl (Mtt)

and 4-methoxytrityl (Mmt). Mtt is removed by repetitive treatment of 1% TFA/DCM, and Mmt

can be cleaved off in even milder conditions using AcOH/2,2,2-trifluoroethanol (TFE)/DCM

(1:2:7) (Aletras 1995)


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Figure 7: Commonly used protecting groups in Fmoc/t-Bu strategy. Summarized from (Isidro-Llobet 2009).

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For occasions where a non-acid labile protecting group is preferred, introduction of the

allyloxycarbonyl (Alloc) protecting group, cleaved by palladium (Pd0) catalysis in the presence of

nucleophiles is an alternative. As an additional alternative for protecting the ε-amino group of

lysine, the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) group was developed.

Dde is considered quasi-orthogonal since it is semi-stable at conditions used for Fmoc removal

but the 5% hydrazine treatment used to remove the Dde group removes the Fmoc group as well

(Bycroft 1994). Improvement of the base stability was performed for the 1-(4,4-dimethyl-2,6-

dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), which also showed less propensity for lysine

migration (Chhabra 1998).

For aspartic acid and glutamic acid, the protection of the carboxylic acid side chains is

mandatory to avoid amide bond formation resulting in a branched peptide. Even though

protected, most widely with OtBu, Asp and in some cases Glu can undergo acid/base-catalyzed

backbone cyclization, forming cyclic imides, as aspartimide for Asp. The side reaction is sequence

dependent where Asp-Gly/Asn/Ala/Gln are especially susceptible and is commonly prevented

using backbone amide protecting groups (Quibell 1994; Yang 1994). In the case of asparagine

and glutamine, incorporation during peptide synthesis could be performed without protecting

groups, but introduction of the protecting group Trt will increase the low solubility of the side

chains, as well as block the amide group from involvement in secondary structure formation that

could potentially result in incomplete deprotection and reduced reaction rates. Also the

sulfhydryl group in the cysteine side chain needs to be protected during synthesis to avoid

alkylation. There are several protecting groups that can be used for selective deprotection, which

are especially useful in directed disulfide formation, however the acid-labile Trt group is most

commonly used for cysteine protection.

Side reactions for serine and threonine are not as common as for amine- and carboxylic acid

containing side chains, but these amino acids are commonly protected with tBu, which is cleaved

by a strong acid. Selective deprotection of Ser and Thr for postsynthesis modifications such as

glycosylation and phosphorylation is possible when using Trt, that due to the mild steric

hindrance of the hydroxyl group can be cleaved off by 1% TFA in DCM (Barlos 1998). The side

chain of tyrosine is prone to side reactions with residual intermediates from the Fmoc-removal,

and is therefore commonly incorporated as Tyr(tBu) (Isidro-Llobet 2009; Carganico 2010).

Lastly, tryptophan is stable during peptide synthesis but could be alkylated during TFA-

cleavage. By using Trp(Boc), the two-step mechanism of Boc removal will protect the indole ring

during TFA-cleavage leaving a carbamide acid derivate that will decompose during the work up

leaving a deprotected side chain.


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Aggregation

Efforts have been made in finding stable protecting groups, but the protected peptide chain could

meet other problems during the synthesis such as poor solubility, which could lead to self-

association or aggregation and consequently incomplete deprotection and reduced amino-

acylation reaction rate. These undesired problems are mainly due to secondary structure

formation, primarily where backbone hydrogen bonds form β-sheets. In order to avoid this

secondary structure formation, external factors such as elevated temperature and change of

solvents have been investigated in addition to lowering the peptide resin load and choosing

protecting groups with higher solubility.

Aggregation could also be reduced by using the backbone protecting group 2-hydroxy-4-

methoxybenzyl (Hmb), which blocks the hydrogen bond interaction of the amide, and the

protected amide also prevents aspartimide formation at Asp-residues as previously mentioned

(Packman 1995). Also the improved backbone protecting group 2,4-dimethoxybenyl (Dmb) could

be used (Figure 8A). Another way to avoid secondary structure formation is to incorporate

pseudoprolines into the peptide, a dipeptide derivate with structural similarity to prolines

(Figure 8B), which introduce a change in the backbone conformation. These derivatives are

formed by backbone cyclization of either Ser/Thr or Cys and are opened up during standard

cleavage from solid support, resulting in a reversible protecting group for both the backbone

amide as well as the side chain functionality (Wöhr 1996).

Microwave synthesis In the 1980s microwave technology was introduced in the field of organic chemistry to heat

reactions in a controlled manner to increase the reaction rate, and later it was also introduced to

the field of solid phase peptide synthesis in order to enhance the coupling efficiencies (Yu 1992).

Some material has the ability to be efficiently heated by microwave radiation by so called

“microwave dielectric heating”, where the waves are transformed into heat due to dipolar

depolarization or ionic conduction. Upon irradiation with oscillating electric field, the dipole

alignment will cause molecular friction and the energy loss will turn into heat, resulting in an

more uniform heating pattern in comparison to the more traditionally used oil or water baths

(Kappe 2004).

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Figure 8: Examples of amino acid derivatives reducing aggregation during synthesis. A) The backbone protecting group Dmb. B) A pseudoproline, where the side chain of serine is forming a proline-like structure.

With the introduction of microwave-assisted heating, it was proposed that the growing peptide

chain on the resin moves with the oscillating electrical field and thereby reduces aggregation and

increases yield. However, thermal heating itself would increase the final yield and in order to

show any additional non-thermal effect on the final purity comparisons between conventionally

heated SPPS and microwave heated SPPS do need to be performed at the same elevated

temperature, which is not trivial (Bacsa 2008). It could however be concluded, that the use of

microwave assisted heating in SPPS results in faster coupling (acylation) and deprotection

reactions, and for some sequences also leads to a higher final purity (Palasek 2007; Pedersen

2012).


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Limitations in solid phase peptide chemistry

Peptides and proteins up to 50 amino acids can routinely be synthesized in high yield, however

certain sequences are more difficult than other, and the linear assembly might not result in

sufficient yield of the protein. In order to assemble larger proteins (>50 amino acids), incomplete

acylation and deprotection reactions would accumulate by-products and the final protein yield

might be too low. If a 50-mer peptide is synthesized with 99% coupling efficiency, the final yield

is 60%, but if the coupling efficiency instead is lowered to 95% the final yield is only 7%. Since

the average length of a protein is approximately 250 amino acids (Berman 1994), new strategies

for protein synthesis have been developed in order to overcome the size limitations of SPPS.

Chemical ligation is a convergent strategy where two peptide fragments are coupled together,

often in an aqueous solution (Chandrudu 2013). Most successful among the strategies available

are native chemical ligation (NCL) (Dawson 1994), in which a segment with an N-terminal

cysteine is condensed with a fragment containing a C-terminal thioester, followed by

intramolecular rearrangement forming a native peptide bond. Ligation could also be performed

for recombinantly expressed fragments such as in the semisynthesic strategy, expressed protein

ligation (EPL), where a peptide is instead added to a recombinantly expressed protein in a

chemoselective reaction (Muir 1998). In protein semisynthesis, natural modifications such as

glycosylation and phosphorylation introduced during recombinant production can be added

together with the site specific modifications or unnatural amino acids derivates that can be

introduced during SPPS.

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~ Chapter 3 ~

Protein labeling

For many applications the proteins produced recombinantly or by chemical synthesis (as

described in Chapter 2) are labeled with a detection probe in order to follow the distribution or

localization of the protein, or to study its function. One example of a diagnostic application is

where a radionuclide is attached to a tumor-targeting protein to follow its distribution and

localize the tumor. Larger molecules, such as fluorescent dyes, and other detection molecules or

biophysical probes, could also be introduced for biotechnological applications such as protein

detection, analysis and quantification (Means 1990). One application of relevance for this thesis

is molecular imaging. Special considerations associated with protein labeling for molecular

imaging, will be further discussed in the end of this chapter.

Chemical approaches

Non-natural chemical modifications are usually introduced to a certain protein for the purpose of

studying biological processes where the protein is involved. Strategies have been developed to

efficiently label different biomolecules, with a fine balance between fast efficient reactions and

selectivity. Similarly to naturally occurring posttranslational modifications, chemical

modifications of proteins are useful to improve the understanding of the molecular mechanisms.

Even though most types of modification have a common purpose, i.e. to detect or monitor a

specific protein or process, the way of labeling could differ.

Amino groups A commonly used conjugation strategy is to use the ε-amino group in the side chain of lysine,

which is a reactive nucleophile when unprotonated. However, with a pKa of around 9 the

availability of the more reactive form at physiological pH is rather low. By using activated esters

such as N-hydroxysuccimide (NHS) esters, labeling is efficient in slightly alkaline aqueous

solvents (pH >8), but could also be performed in physiological conditions more suitable for

proteins (Bradburne 1993). Reactions using NHS-esters are irreversible resulting in an amide

bond with NHS as a leaving group (Figure 9A), and are an efficient in vitro labeling strategy, but

are rarely used for in vivo labeling (Baslé 2010). Since most proteins have several lysines at the


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surface of the protein, this labeling strategy often results in a heterogeneous mixture which could

be a disadvantage for certain applications. The NHS-esters could also react with sulfhydryl

groups and hydroxyl groups, but the resulting thioesters and esters are not stable in aqueous

solutions and the reaction products are quickly hydrolyzed. (Sinz 2003)

Thiol groups Cysteine on the other hand, is a residue with low abundance on protein surfaces and could be

introduced as a chemical handle for site-specific conjugation, often without loss of function.

A fraction of the thiol groups of cysteine will form thiolate anion near physiologial pH, a reactive

soft nucleophile (Baslé 2010). Naturally unpaired cysteines are rare in proteins since they are

often occupied in stabilizing disulfide bonds that need to be reduced before conjugation can take

place, for example by dithiothreitol (DTT) or tris[2-carboxyethyl]phosphine (TCEP). Thiols can

successfully be labeled using haloacetyls, disulfides and maleimides, preferably at physiological

pH, and the maleimide is by far the most commonly used group thanks to its fast and efficient

reaction (Kim 2008). The reaction is a Michael addition of the thiol to the maleimide, resulting

in a succinimidyl thioether and the conjugation reaction is considered a stable irreversible

reaction (Figure 9B) (Gregory 1955), even though maleimide exchange have been seen for in vivo

studies (Baldwin 2011; Shen 2012). The nucleophilic reactivity of maleimides towards sulfhydryl

groups has resulted in the generation of numerous N-functionalized maleimide reagents that are

commercially available. However, maleimides could undergo spontaneous hydrolysis, a reaction

mediated by a water attack on either of the two carbons in the imido group, resulting in a ring

opening and the formation of either of the two isoforms of succinamic acid thioether (Kalia 2007;

Lin 2008). The maleimide conjugation could also be reversible by substituting the maleimide

with bromide, which is cleaved with molar excess of thiols (Ryan 2011).

Bioorthogonal reactions for labeling proteins

In addition to previously mentioned amino acids available for modification, many additional

reactions have been added to the toolbox of protein labeling. A selective reaction between two

different functional groups in a biological environment is often referred to as a bioorthogonal

reaction. The reactants that are involved in a bioorthogonal reaction should be unreactive and

not toxic to the living system, and the reaction should yield a covalent linkage without toxic

by-products, preferably performed in physiological conditions (Lang 2014). Many different

reactions have been developed and the area has been extensively reviewed (Hackenberger 2008;

Sletten 2009). Only the strategies important for the work of this thesis will be discussed here.

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Figure 9: Reaction mechanisms of the two most commonly used bioconjugation strategies. A) Acylation reaction between the ε-amino group of Lys and a NHS-ester, and B) Michael addition reaction between the thiol group of Cys and a maleimide.

Cu(I) catalyzed [3+2] alkyne-azide cycloaddition In 1967, Huisgen first described the reaction between a terminal alkyne and an azide, a dipolar

cycloaddition generating a 5-membered ring, a 1,2,3-triazole. The low reactivity of the starting

material required elevated temperature and pressure in order to gain the product, however, a

mixture of both 1,4- and 1,5-regioisomers was often generated unless highly electron-deficient

alkynes were used (Figure 10A) (Bock 2006). In 2002, the groups of both Meldal and Sharpless,

independently, found that alkyne-azide coupling could be efficiently catalyzed using Cu(I)

(Rostovtsev 2002a; Tornøe 2002) and the improvement of the reaction resulted in an increased

reaction rate up to 107 times. The Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction

also improved the regioselectivity, resulting in that only the 1,4-regioisomer was produced

(Figure 10B).

Kolb and Sharpless coined the term “click chemistry” as a set of reactions where the starting

material should be readily available, the reaction should be carried out in high yields with only

benign reaction conditions and have a simple workup procedure, but could produce a wide

diversity through its reactive building blocks (Kolb 2001; Kolb 2003). Since then, many different

sets of reactions have been categorized as click-reactions, however the Cu(I)-catalyzed alkyne-

azide cycloaddition is the one that has gained most popularity and has almost become

synonymous with the term click reaction.


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Figure 10: Examples of alkyne-azide cycloaddition reactions. The thermal Huisgen alkyne-azide cycloaddition results in both 1,4 and 1,5 regioisomers, whereas the Cu(I)-catalyzed alkyne-azide cycloaddition yields only the 1,4-regioisomer.

The weak acid-base properties of both azides and alkynes make them almost inert to most

biomolecules, and the absence of azides in almost all natural compounds makes it possible to

introduce them into proteins and peptides as selective handles. In the Cu(I)-catalyzed click

reaction generating only the 1,4-regioisomer, the 1,2,3-triazole formed is very stable. It can

withstand most chemical conditions including oxidation, reduction and hydrolysis (Wu 2007)

but is also biologically interesting since the rigid ring structure mimics a peptide bond without

the susceptibility to hydrolysis. However, the distance between the two adjacent R1 and R2

groups is increased with 1.1 Å for the larger ring structure in comparison to the planar peptide

bond (Bock 2006). The reaction tolerates substitution of most organic groups, which makes it

suitable for conjugation of fluorophores, radionuclides, peptides and proteins. The application of

Cu(I)-catalyzed click chemistry for selective labeling of antibodies is further discussed in paper

VI.

Since Cu(I) is not stabile in aqueous solutions, another source of Cu(I) is needed. Most reactions

are performed using Cu(II) in combination with reducing agent such as CuSO4×5H2O/Na-

ascorbate (Rostovtsev 2002b). Also ligands, such as tris(benzyltriazolylmethyl)amine (TBTA),

can be added to stabilize Cu(I), protecting it from oxidation and disproportionation, and by

retaining the catalytic activity of Cu(I), TBTA can also increase the reaction rate (Chan 2004;

Moses 2007).

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Strain-promoted [3+2] alkyne-azide cycloaddition The Cu(I)-catalyzed click reaction has gained popularity, as the reaction is compatible with

biological conditions, such as ambient temperature and aqueous media. However, even trace

amounts of the catalytic copper could be toxic for living organisms, limiting the use of the

reaction in vivo (Link 2003), and residual copper could also have a detrimental effect on

radiolabeling yields for macrocyclic chelators in molecular imaging, leading to the need for

developing a Cu-free alkyne-azide cycloaddition reaction.

Bertozzi et al. reported in 2004 a metal-free reaction with a strain-promoted alkyne and an azide

in a [3+2] cycloaddition without additional reactants, based on the work by Wittig and Krebs in

the 1960s (Agard 2004). The reaction was performed with small azide-containing molecules

resulting in high yield conjugation and was further on validated for selectivity by biotinylation of

azide-modified living cells without any noticeable toxicity, however, the reaction rates were

somewhat slower than for the Cu(I)-catalyzed click reaction (Agard 2006). Further improvement

of the concept resulted in a more water soluble compound, dimethoxyazacyclooctyne, and

improvement of reaction rates was achieved by fluorination to give monofluorinated and

difluorinated cyclooctynes (Figure 11A) (Chang 2010).

Figure 11: Strain-promoted systems for Cu(I)-free click reactions. A) Cyclooctyne-based strain-promoted alkynes and B) dibenzocyclooctyne-based strain-promoted alkynes.


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Dibenzocyclooctyne compounds, where benzene rings are attached to the cyclooctyne to increase

the ring strain and expand the conjugated system and thereby increase the azide reactivity, were

also developed for labeling living cells (Figure 11B). Dibenzocyclooctyne is stable to other

nucleophiles but reactive towards azides, resulting in three orders of magnitude faster reaction

rates than for cyclooctynes (Ning 2008). A more water soluble compound with high reactivity

towards azides, aza-dibenzocyclooctyne, was synthesized, and the nitrogen introduced could also

serve as a conjugation handle for protein labeling, however it also introduced acid sensitivity

(Debets 2010).

Alternative ligation strategies The Staudinger ligation instead uses the reactivity of azides towards phosphines forming an ylide

in a modified Staudinger reaction, which can be used for bioconjugation. The nitrogen in the aza-

ylide intermediate is highly reactive, and the covalent adduct is not stable in water. By

introducing an electrophilic group within the structure, such as an ester, the nucleophilic aza-

yilde will rather by an intramolecular cyclization rearrangement form an amide bond. (Saxon

2000b) Further improvement led to the development of the traceless Staudinger ligation, where

an amide bond is formed directly between the two molecules and the phosphine linker is absent

from the conjugate. However, this reaction is slower and phosphonamide and amide by-products

are accumulated (Saxon 2000a).

Oxime ligation is another bioorthogonal reaction, which is a chemoselective reaction between an

aldehyde and a aminooxy group, resulting in a stabile oxime linkage. The aldehyde-aminooxy

condensation reaction is highly selective and efficient, and has proved useful for a variety of

conjugations to proteins and peptides (Thumshirn 2003). The reaction efficiency has been

greatly enhanced by aniline catalysis, when performed at low concentrations in ambient

temperatures (Dirksen 2008).

Introduction of chemoselective handles in recombinantly produced proteins In 2003, Schultz and co-workers showed that the genetic code could be expanded beyond the 20

naturally occurring amino acids. By using an amber nonsense codon less often used by the

expression host and introducing an orthogonal tRNA/aminoacyl-tRNA synthetase pair,

unnatural amino acids or other functional groups could be incorporated in the protein during the

translation (Deiters 2003; Wang 2005b). There is also a variety of enzymatic methods for site-

specific labeling of proteins, reviewed by (Rabuka 2010), and only a few examples will be

presented here. The formylglycine generating enzyme (FGE) is used in another strategy

compatible with recombinant production, in which a cysteine residue is enzymatically oxidized

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to formylglycine using a 6-mer consensus sequence recognized by the formylglycine-generating

enzyme (Carrico 2007). Co-expression of the formylglycine-generating enzyme with the 6-mer

tagged protein directly yields the aldehyde-containing protein. The aldehyde could later be

targeted in an oxime ligation using an aminooxy group or a hydrazine group. Another enzymatic

approach for modification, is using biotin ligase (BirA), which adds biotin to a specific lysine in a

15-mer recognition peptide tag (Chen 2005), where the introduced biotin later could be used for

probing the protein both in vitro and in vivo.

Radiolabeling for molecular imaging

A type of protein labeling of particular focus for this thesis is radiolabeling of affinity proteins for

molecular imaging, an emerging technique in which specific targets or biological processes can

be used for diagnosing and monitoring the disease progress. The biomarker detection for a

certain disease can provide important molecular information regarding prognosis, response to

therapy, and aid in the targeted therapy selection. Molecular imaging using radioactive

modalities is a noninvasive technique that often can be performed in combination with ongoing

treatments, and since it can be used in a repetitive manner disease progression or pre-and post

treatment changes can be followed (Knowles 2012). Two commonly used nuclear imaging

modalities are Positron Emission Tomography (PET) and Single Photon Emission

Computed Tomography (SPECT), which both have the sensitivity needed for detecting

interactions between physiological targets and their ligands.

PET and SPECT PET and SPECT are imaging techniques that detect gamma rays emitted from the radionuclide

used as tracer. For SPECT, the gamma rays emitted by the radionuclide (Figure 12A) are directly

measured by rotating the collimator detectors around the area of interest, giving a 3D image. In

addition to the 3D acquisition from the rotating gamma camera, SPECT is often used in

combination with computed tomography (CT) for co-registration of images that could provide

the anatomical information not given by the SPECT in a multimodality molecular imaging. For

PET imaging the radionuclide instead decays by emitting a positron. This positron is generated

with high energy from a beta-decay of an unstable isotope, and the kinetic energy allows the

positron to travel a short distance before it collides with its anti-particle, an electron, and

annihilates (Figure 12B). At the positron-electron annihilation two 511 keV photons are

generated, directed 180 degrees apart and the gamma rays can be detected by the ring of

scintillation detectors. These coincidence events can be seen as projections from the patient, and

after reconstruction will give a 3D image with higher resolution compared to SPECT.


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Figure 12: Schematic representation of radioactive decay. A) A gamma photon release for SPECT imaging. B) A positron is emitted and produces two 511 keV annihilation photons upon interaction with a neighbouring antiparticle, an electron.

As for SPECT, also PET can be used in combination with CT, allowing the localization of tumors

in regard to the anatomical structure. (Gambhir 2002; Massoud 2003; Vallabhajosula 2009)

PET has some advantages over SPECT as a greater sensitivity and a potential quantitative

possibility (Cai 2006), however, PET imaging is more expensive. The molecular information

achieved from both modalities regarding the global receptor expression on tumor cells could help

to prevent the false-negative results that might arise due to the heterogeneity of tumors, which

cannot be fully represented by a biopsy sampling (Kramer-Marek 2008). This makes molecular

imaging a promising tool in the ongoing development of personalized medicine.

Radiometals and chelators for imaging Nuclear imaging modalities are dependent on the radioactivity emitted by the radionuclide used.

There are many different radionuclides that can be used in PET and the half-lives vary largely

between them, such as 11C (20.3 min), 68Ga (68 min), and 64Cu (12.7 h). The most commonly used

PET radionuclide in the clinic today is 18F (109.8 min), but also more long-lived radionuclides are

available, such as 89Zr (78.4 h) (Ametamey 2008). The radionuclides used in SPECT in general

have a longer half-life in comparison to PET, which make them amenable for transport and thus

no production on site is necessary, as well as allowing for a longer monitoring window of

biological processes in vivo. 99mTc is currently the most commonly used radionuclide in SPECT

imaging thanks to it advantageous nuclear properties (T1/2= 6.0 h and 140 keV γ photon), but

also 111In (67.3 h) and 67Ga (78 h) are commonly used for labeling SPECT-tracers (Pimlott 2011).

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There are different ways to produce radionuclides, but in general the nuclei of stable atoms are

bombarded with subnuclear particles to create unstable nucleus. This could either be performed

with a primary source as in a reactor production, such as accelerators, or with a secondary source

using generators, in which a long-lived parent isotope is allowed to decay into its short-lived

daughter isotope that can be chemically separated and eluted (Owunwanne 1995).

Radiometals are in general hard nucleophiles, and peptides and proteins do not have the

functional groups needed for efficient labeling that could provide the stability to ensure intact

delivery of the radiometal to the tumor site due to competition with serum proteins and anions

(Maecke 2005). In order to use radiometals with biological tracers, the metals need to be

anchored to remain bound to the tracer to gain a high contrast image and reduce unwanted

exposure to healthy organs and unnecessary background noise. By donating a lone pair of

electrons present in atoms or molecules, such as N, O or S, a bond between the metal and the

ligand can be form, and by combining a number of ligands into a chelating agent or chelator,

these coordinate or dipolar bonds will bind the central metal to the structure (Vallabhajosula

2009). There are several chelating agents available, both based on polyamino carboxycylic acids,

such as EDTA and DTPA that are flexible, open chains and more rigid structures such as triaza-

or tetraaza-containing macrocyclic structures, such as NOTA and DOTA (Figure 13).

Figure 13: Structures of commonly used chelating agents.


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DOTA is the most widely used macrocyclic chelator for radiometal labeling, constituting of a

twelve-membered ring, with four carboxylic acid groups acting as arms keeping the radiometal in

place. However, different chelators have been shown to influence the target affinity, the cellular

processing and the biodistribution in different ways (Tolmachev 2010b), indicating that the

different chelator-radiometal combinations need to be evaluated. The NOTA chelator is a nine-

membered triaza ligand that has successfully been labeled with the radiometals 68Ga and 111In for

targeted molecular imaging (Eisenwiener 2002), where the gallium complex with NOTA was

shown to be more stable than the corresponding DOTA complex (Velikyan 2008). In NOTA, one

of the carboxylic acid groups is used for anchoring to the targeting molecule, and the

coordination properties could become compromised for radiometals preferring an octahedral

coordination. To preserve the coordination pattern of NOTA, while providing a covalent

conjugation site to a targeting vector, a derivate of NOTA with an additional carboxylic arm,

NODAGA, was designed with the preserved high labeling efficiency and in vivo stability

properties of NOTA (André 1998; Bartholomä 2012).

Application-specific considerations The tracers used in molecular imaging are often a combination of a molecular targeting part and

a radioactive-labeled part, in which the part responsible for molecular targeting brings the

radionuclide to the area of interest, as a solid tumor. An optimal tracer should hence bind its

target and have low uptake in non-target tissues for an optimal contrast and in order to retain

the contrast, also the in vivo stability of the complex need to be high. The radionuclide needs to

be retained to its chelator and the complex must in turn be conjugated to the protein with a

linker that is stabile under in vivo conditions. Furthermore, the tracer should have a fast

biodistribution with efficient tissue penetration in order to have a high uptake in targeted area,

and a fast clearance to reduce the background. As the radionuclides used in PET in general have

short half-lives, a fast labeling chemistry to produce the PET-tracers is desirable. The use of

Affibody molecules as targeting agents is further discussed in papers I-IV.

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PRESENT INVESTIGATION


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Aim of the thesis

The aim of this thesis was to explore site-specific labeling of affinity proteins for different in vitro

and in vivo applications, utilizing the advantages of solid phase peptide synthesis for protein

synthesis. The thesis is based on investigation of labeling of two different types of affinity

proteins, where the first part (papers I-IV) is focusing on site-specific labeling of chemically

synthesized Affibody molecules for molecular imaging, and the second part (papers V-VI) is

focusing on exploring a method for site specific labeling of antibodies.

In the first two papers, two novel bifunctional chelators, maleimide-NOTA and maleimide-

NODAGA, were evaluated, in order to improve the Affibody molecule as an imaging agent in

SPECT using the gamma-emitting radionuclide 111In. In the third paper, we looked further into

the differences between the chelators and the effect the choice of chelator and labeling chemistry

had on the molecular imaging properties. This time a synthetic Affibody targeting HER2 was

produced with the chelators conjugated to the N-terminus via an amide bond. The different

chelators were evaluated in a tumor model with moderate HER2-expression, increasing the

demands on the imaging agents with respect to tumor uptake and background, i.e. the contrast.

In the fourth paper, the effect of the positioning of the chelator was instead investigated in the

search for a better imaging agent.

In the second part, starting with the fifth paper, a photochemical method for site-specific labeling

of antibodies was explored. In a continuation of earlier efforts in the field, antibodies of more

subclasses and species were introduced, with the aim to develop a more general and versatile

labeling method, especially suitable for subclasses important for therapeutics or common in vitro

diagnostic assays. By improving the IgG-binding molecule the conjugation efficiency was

increased, and new variants were developed. In the sixth paper, one of the new variants

developed for efficient antibody labeling was used for labeling an antibody commonly used in

cancer treatment by click chemistry.


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~ Chapter 4 ~

Functionalization of Affibody molecules for detection of HER2 using molecular imaging

As mentioned in Chapter 1, small affinity proteins have shown great potential as targeting agents

in molecular imaging. The importance of molecular imaging as a diagnostic tool to monitor

diseases was discussed in Chapter 3, and in order to find an indication of tumor growth at the

earliest time point, the tracers used need to be improved to attain the best resolution or highest

contrast.

HER2 as a molecular target

In the present work, the development of radiolabeled Affibody-based tracers for molecular

imaging of tumors was evaluated using HER2-expressing tumors as a model. HER2 belongs to a

group of receptor tyrosine kinases, which are transmembrane proteins that by signaling affect a

wide range of cellular processes such as growth, differentiation, cell motility and survival (Mass

2004). The epidermal growth factor receptor family (EGFR family) has four members,

EGFR/HER1, HER2/neu, HER3 and HER4, where all members share a common structure

consisting of an extra-cellular ligand-binding domain, followed by a hydrophobic

transmembrane region and an intracellular tyrosine kinase domain (excluded in HER3) (Yarden

2001). HER2 is a more potent oncoprotein in comparison to the other members, and is the only

family member not having a known high affinity ligand. Instead HER2 is constitutively adopting

an activated-like structure ready for dimerization. The active structure of HER2 makes it the

preferred dimerization partner for the other EGFR family members (heterodimerization), but the

receptor can also undergo dimerization with other HER2 receptors (homodimerization), more

common at high expression levels of HER2 (Graus-Porta 1997).

HER2 is overexpressed in about 20% of all breast cancers, and is linked to other types of cancer,

including prostate cancer (Craft 1999), ovarian cancer (Verri 2005) and colorectal cancer

(Carlsson 2012). The amplification of the HER2 signaling in breast cancer has been associated

with increased cell proliferation and suppression of apoptosis, resulting in a malignant behavior

of the cancer, which is often associated with an aggressive tumor growth and poor prognosis

(Slamon 2001). Molecular profiling of the cancer would aid in the selection of therapy, today the

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patient stratification is mainly performed by methods such as immunohistochemistry and

fluorescence in situ hybridization (FISH) on tumor biopsies (Pauletti 2000).

Upregulated expression of HER2 has also been validated as a biomarker for androgen-

independent prostate cancer, where it can activate androgen-dependent pathways even in the

absence of androgen itself (Signoretti 2000). Tumor progression is often aggressive and less

responsive to standard therapy, than in other types of cancer with high HER2 expression.

However, HER2 expression is generally not as high as in breast cancer, resulting in higher

demands on the detection agents for correct diagnostics (Minner 2010).

Papers I, II and III - The influence of macrocyclic chelators and different conjugation chemistries for radiolabeling of Affibody molecules in molecular imaging.

Efficient patient stratification of patients with moderate HER2-expression using molecular

imaging requires a molecular tracer with efficient tumor targeting while non-target uptake

remains low, in order to achieve a good contrast. An increased imaging sensitivity, i.e. the

contrast, is especially important for targets with moderate expression levels, such as HER2 in

prostate cancer, and thus an important precondition for efficient patient stratification using

molecular imaging based methods are the high sensitivity and contrast, which is essential for

detection of small lesions.

Comparative studies of Affibody molecules have shown that small changes in the labeling

chemistry could have profound effects on biodistribution and tumor targeting, resulting in large

differences in tumor-to-organ ratios. By increasing the hydrophilicity of a peptide-based chelator

conjugated to the N-terminus, the hepatobiliary excretion pathway was reduced and redirected

towards renal excretion (Engfeldt 2007; Ekblad 2008). This suggests that the use of different

macrocyclic chelators may show different pharmacokinetic effects, and led to a head-to-head

study of a HER2-targeting affibody molecule conjugated with NOTA, NODAGA and DOTA for

the evaluation of the 111In-conjugates both in vitro and in vivo.

Papers I and II For this purpose the chelators NOTA and NODAGA were site-specifically conjugated to a unique

cysteine introduced in the C-terminal part of an HER2-targeting Affibody molecule (ZHER2:2395)

using maleimide chemistry (papers I and II).


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Figure 14: Schematic illustration of the Affibody molecules used in papers I and II. ZHER2:2395 with the different bifunctional chelators maleimide-DOTA, maleimide-NOTA and maleimide-NODAGA conjugated to the introduced Cys in the C-terminus of the protein.

The biophysical properties of all variants were carefully characterized and circular dichroism

(CD) measurements verified that all variants had a retained triple-helical structure and the

melting point determination showed no destabilizing effects, independent of chelator.

Importantly, to verify that the conjugation of the chelators did not influence the affinity for

HER2, all variants were analyzed for binding to HER2 using surface plasmon resonance (SPR)

for determination of the KD, with no apparent decrease in affinity (Table 1).

In the biodistribution studies performed in non-tumor bearing mice in papers I and II, both

NOTA and NODAGA-conjugated ZHER2:2395 in comparison to DOTA-MMA- ZHER2:2395 showed

biodistribution patterns typical for Affibody molecules, with high uptake in the kidneys due to

renal excretion. In paper I, biodistribution studies using NMRI mice with xenografts from a

prostate carcinoma cell line (DU-145) with moderate HER2-expression implanted on the flank,

showed an overall similar distribution pattern for NOTA- and DOTA-conjugated ZHER2:2395, with

somewhat higher tumor uptake for 111In-NOTA-MMA-ZHER2:2395 (8.3 %ID/g ) compared to 111In-

DOTA-MMA-ZHER2:2395 (7.3 %ID/g). 111In-NOTA-MMA-ZHER2:2395 also showed somewhat faster

clearance from blood and lower uptake in bone, resulting in higher ratios for both tumor-to-

blood and tumor-to-bone (Figure 15A). In the biodistribution studies using NMRI mice with

DU-145 xenografts in paper II, DOTA-ZHER2:2395 was instead compared to NODAGA-ZHER2:2395,

where the latter variant showed lower uptake in most organs, including the tumor. Even though

the tumor uptake was lower, the faster clearance from blood resulted in higher tumor-to-blood

and tumor-to-organ ratios, i.e. a better contrast (Figure 15B).

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Figure 15: In vivo evaluation of 111In-labeled conjugates in mice with DU-145 xenografts. A) Comparison of radioactivity uptake and tumor-to organ ratios for 111In-MMA-NOTA-ZHER2:2395 and 111In-MMA-DOTA-ZHER2:2395 and B) Comparison of radioactivity uptake and tumor-to-organ ratios for 111In-MMA-NODAGA-ZHER2:2395 and 111In-MMA-DOTA-ZHER2:2395. Data are presented as an average from four animals %ID/g± SD

In the two studies, papers I and II, two novel chelators were introduced to an Affibody molecule

and could successfully be labeled with 111In in high yield, where the resulting radiolabeled

conjugates showed high in vitro stability. Both conjugates showed rapid clearance from non-

target organs resulting in higher tumor-to organ ratios in comparison to the well studied DOTA

chelator. Taken together, both MMA-NOTA and MMA-NODAGA are promising bifunctional

chelators that can be used for site-specific labeling of cysteine-containing targeting agents.


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Paper III After showing that both NOTA and NODAGA conjugated to an Affibody molecule could

efficiently be labeled with 111In, all the chelators were included in a larger study (paper III). For

this study, a variant of the HER2-targeting Affibody was produced using chemical synthesis,

where the first 6 amino acids were substituted in order to gain a higher synthetic yield (ZHER2:S1).

The chelators were incorporated after completed chemical synthesis, prior to cleavage from the

solid support to the N-terminus of the protein, forming a stabile amide bond.

Figure 16: Schematic illustration of the Affibody molecules used in paper III. ZHER2:S1 with the different chelators DOTA, NOTA and NODAGA conjugated to the N-terminus of the protein.

As for the previous studies, the three variants were analyzed to verify that the conjugation did not

affect the affinity for HER2. However, the measured affinity for HER2 was lower for the variants

with N-terminally conjugated chelators than for the variants with chelators conjugated by

maleimide chemistry to the C-terminal Cys (Table 1). This decrease in affinity could be due to a

sterical hindrance by the large chelator in the interaction with HER2. Since only helices 1 and 2

take part in the interaction with HER2 (Eigenbrot 2010), conjugation to the N-terminal part of

the protein could be expected to have a larger influence on the affinity as modifications are

performed closer to the binding area in comparison to conjugation to the C-terminus. Taken

together this suggests that larger modifications should preferably be performed in the C-terminal

to avoid interference with target binding.

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Table 1: Data from SPR-measurements of the interaction with HER2. Each variant was analyzed in six concentrations in duplicates, where the KD was determined from the ka and kd.

ZHER2:2395 ZHER2:S1

Chelator ka

(M-1·s-1) kd

(s-1) KD

ka (M-1·s-1)

kd (s-1)

KD

NOTA 8.2 × 106 6.0 × 10-4 72 pM 5.7 × 106 8.0 × 10-4 140 pM

NODAGA 1.0 × 107 6.7 × 10-4 67 pM 6.6 × 106 5.9 × 10-4 90 pM

DOTA 5.3 × 106 3.9 × 10-4 74 pM 4.1 × 106 6.4 × 10-4 130 pM

Biodistribution studies in non-tumor bearing mice showed a similar biodistribution pattern as

for the variants in papers I and II, with fast excretion by the renal pathway and low uptake in

most organs, except the kidneys. However, an unfavorable increased uptake in liver, colon and

spleen was seen for 111In-NOTA-ZHER2:S1, which resulted in a discontinuation of the conjugate

from further in vivo studies (Table 2). It can be speculated that this elevated uptake is due to a

net positive charge of the conjugate, as NOTA only has two carboxylic arms in comparison to the

three arms available for DOTA and NODAGA, directing it towards hepatobiliary excretion.

Table 2: Biodistribution data from non-tumor bearing mice injected with 111In-labeled Affibody molecules. Data presented as mean±SD %ID/g from four mice.

Variant Blood Liver Spleen Colon Kidney

NOTA-ZHER2:S1 1.2±0.2 8.0±0.6 3.2±1.6 1.6±0.4 217±33

NODAGA-ZHER2:S1 0.8±0.1 2.5±0.4 0.7±0.05 0.85±0.02 206±22

DOTA-ZHER2:S1 1.8±0.1 2.9±0.1 1.2±0.2 0.6±0.1 170±17

In the biodistribution study where the tumor targeting of 111In-DOTA-ZHER2:S1 and 111In-NODAGA-

ZHER2:S1 was evaluated in HER2-expressing DU-145 xenografts, differences in the biodistribution

pattern was seen. 111In-NODAGA-ZHER2:S1 showed faster clearance from blood in comparison to

DOTA, as well as a lower tumor uptake implying that the small difference of the chelators results

in large differences in the biodistribution. 111In-NODAGA-ZHER2:S1 also showed an unfavorable

distribution with elevated uptake in both liver and bone, in comparison to 111In-DOTA-ZHER2:S1

(Figure 17).


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Figure 17: In vivo evaluation of 111In-NODAGA-ZHER2:S1 and 111In-DOTA-ZHER2:S1. A) Radioactivity uptake for tumor and normal organs and B) tumor-to-organ-ratios. Data are from 4 h p.i. and an average of four mice ±SD.

Conclusions and future outlook The different Affibody molecules produced using chemical synthesis has the advantage of site-

specific incorporation of chelators via a stabile amide bond. Introducing a unique cysteine at the

C-terminus opens up for efficient site-specific labeling of recombinantly produced proteins, both

as a conjugation site and for chelation of certain radiometals with thiophilicity, i.e. the possibility

to be coordinated by peptide chelators containing a cysteine (Maecke 2005). In papers I and II

we showed that a HER2-targeting Affibody molecule could be conjugated with both NOTA and

NODAGA and efficiently be labeled with 111In. Biodistribution studies showed better

characteristics of both NOTA and NODAGA in comparison to DOTA, such as higher tumor-to-

organ ratio and lower uptake in bone, a common metastatic site in prostate cancer (Bubendorf

2000). The maleimido-derivatives of both NOTA and NODAGA are thus promising bifunctional

chelators, also for other radionuclides (Strand 2013), for site-specific labeling of Affibody

molecules for in vivo molecular imaging.

In paper III, the biodistribution patterns for the different chelators showed greater diversity. The

elevated uptake of NOTA in the liver for the synthetic variant excluded it from further

biodistribution studies in tumor-bearing mice, and no comparison between NOTA and NODAGA

could be done in terms of tumor-to-organ ratios. Elevated hepatic uptake has been noted in

earlier studies for conjugates with a positive charge at the N-terminus (Tran 2008), which was

further supported by this study. The faster clearance of the NODAGA conjugate in comparison to

DOTA resulted in lower uptake in tumor, but it also showed an unfavorable elevated uptake in

bone. Despite the two-fold higher tumor-to-blood ratio for the NODAGA-conjugate, detection of

the metastatic sites commonly found in bone would be problematic, and as the tumor-to-bone

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ratio is higher for 111In-DOTA-ZHER2:S1, our data suggests that this would be the best imaging agent

for detecting HER2 in prostate cancer patients.

Based on papers I-III it could be suggested that the influence on the biodistribution from the

small changes in the N-terminal part of the HER2-targeting Affibody molecule is more

pronounced than for the changes in the C-terminal. This may be due to the modifications being

closer to the binding site (helices 1 and 2), potentially having a larger impact on the binding of

the conjugate to HER2, as shown in paper III.

As the conjugation site is of importance, as seen in the three first studies, this finding led to the

next study (paper IV) where the positioning of DOTA was more thoroughly investigated.

Paper IV – The influence on biodistribution from the positioning of the DOTA chelator in the Affibody molecule

As seen in papers I-III, not only different chelators can influence the biodistribution pattern but

also the site of conjugation to the protein can have a considerable influence. The different

Affibody variants used in papers I-III had the chelators at either the C- or N-terminus, however,

they differed in production route and sequence (at 5 residues) making head-to-head comparisons

difficult. However, the results indicated that placement of the chelator may influence the

phamacokinetics of the Affibody molecule, suggesting that this aspect of Affibody radiolabeling

should be further evaluated. Based on the best tracer from paper III, 111In-DOTA-ZHER2:S1, DOTA

was conjugated to three different positions on the HER2-targeting Affibody molecule ZHER2:S1; to

the amino group at the N-terminus, to the C-terminal amino acid lysine (K58) and as far away

from the binding surface as possible, in the middle of helix 3, also using a lysine residue (K50).


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Figure 18: Conjugation sites for DOTA chelator on Affibody molecule. The chelator was attached either to the N-terminus, to Lys50 in the middle of helix 3 or to the C-terminal residue Lys58.

All variants were successfully conjugated with DOTA, without affecting secondary structure

content and refolding properties. The melting point was decreased with 5°C to 57°C when the

chelator was introduced in the middle of helix 3 instead of to the C-terminus, indicating a

decrease in stability, probably due to distortions in the helix (Table 3). A small decrease in the

affinity for HER2 was seen for both the destabilized variant DOTA-K50-ZHER2:S1 and for the N-

terminally conjugated DOTA-A1-ZHER2:S1 in comparison to C-terminally conjugated DOTA-K58-

ZHER2:S1.

Table 3: Biophysical characteristics of the Affibody conjugates.

Variant Purity ka

(M-1·s-1)

kd

(s-1)

KD

(pM)

Tm

(°C)

DOTA-A1-ZHER2:S1

98 % 4.8 × 106 6.4 × 10-4 133 64

DOTA-K50-ZHER2:S1

97 % 7.2 × 106 7.7 × 10-4 107 57

DOTA-K58-ZHER2:S1

97 % 7.9 × 106 7.4 × 10-4 94 62

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Biodistribution studies were performed in tumor-bearing mice with colorectal carcinoma

(LS174T) xenografts with moderate HER2 expression (4×104 HER2-receptors/cell). All 111In-

conjugates showed a distribution pattern typical for Affibody molecules with a fast clearance

from blood and with renal filtration as the excretion pathway, with reabsorption in the kidneys

by the proximal tubule, resulting in high uptake in the kidneys. The tumor uptake was similar for 111In-DOTA-A1-ZHER2:S1 and 111In-DOTA-K58-ZHER2:S1, however, for 111In-DOTA-K50-ZHER2:S1, the

variant with DOTA conjugated in helix 3, the uptake was significantly lower resulting in low

tumor-to-organ ratios (Figure 19). Additionally, for Affibody molecules with DOTA conjugated to

the C-terminus, an elevated uptake was seen in lung, liver and spleen; however, it also provided

the fastest blood clearance. This resulted in significantly higher tumor-to-blood ratio for 111In-

DOTA-K58-ZHER2:S1 and a higher tumor-to bone ratio, however it also provided the lowest tumor-

to liver ratio even though the differences were smaller for the tumor-to-liver ratios. Taken

together, this suggested that 111In-DOTA-K58-ZHER2:S1 was the best tracer evaluated in this study.

Figure 19: Tumor uptake (left) and tumor-to-organ ratios (right) for 111In-DOTA-A1-ZHER2:S1, 111In-DOTA-K50-ZHER2:S1 and 111In-DOTA-K58-ZHER2:S1 at 4 h p.i. *Significant difference (p>0.05)

Conclusions and future outlook A number of studies have shown that even subtle differences between different conjugates may

lead to large differences in the image sensitivity. Even though some general trends have been

described such as positive charge (Tran 2008), lipophilicity and conjugate orientation

(Tolmachev 2010a) it is difficult to draw any final conclusions how a certain change might

influence the contrast.


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The tumor-to-organ ratios clearly showed that conjugating the chelator to helix 3 reduced the

overall contrast, mainly due to the low tumor uptake. As all variants had similar blood

concentrations, the low uptake of DOTA-K50-ZHER2:S1 could be a result of the in vivo decrease in

binding ability due to destabilization. Despite that conjugation to the N-terminus (Tran 2007)

has been shown to have larger impact on the biodistribution than corresponding changes in the

C-terminus (Tran 2009), 111In-DOTA-K58-ZHER2:S1 showed elevated uptake in the non-target

organs including lung, liver and spleen in comparison to the N-terminal conjugate.

Taken together, in this study it was shown that the positioning of the DOTA chelator influences

both the biodistribution and the targeting properties, suggesting that further studies might

enhance the sensitivity of molecular imaging, which is particular important for detection of small

lesions.

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~ Chapter 5 ~

Site-specific labeling of antibodies using IgG-binding domains

The use of antibodies for therapy has increased over the years, and today a number of

monoclonal antibodies are available for treatment of malignant diseases. Current antibodies used

in the clinics are mainly targeting receptors expressed on cells and modulate the cell function or

acting as neutralizing agents for soluble targets in the circulation (Golay 2012). Even though the

antibodies have a therapeutic effect by themselves, either by blocking receptor dimerization and

further signaling pathways or by recruiting the immune system in antibody dependent cell-

mediated cytoxicity, they can also be used as targeting agents delivering cytotoxic payloads to the

site of interest, or used for diagnostic applications, such as radiolabeling for detection of

metastases in molecular imaging (Scott 2012). Regardless of the applications, it is important that

the binding ability is not altered by the conjugation of the payload, which can be avoided by

directing the conjugation site away from the binding site (Strop 2013). The antibody-drug-

conjugate ado-trastuzumab emstansine, T-DM1 (Kadcyla®), has the cytotoxic drug DM1, a

maytansinoid derivate conjugated via a non-reducible thioether linkage (Junttila 2011). The

labeling results in a heterogeneous mixture with an average of 3.5 DM1 molecules per each

antibody, where a twofold heterogeneity was observed. Firstly, the antibodies have different

numbers of drugs conjugated, and secondly, antibodies with the same number of drugs may have

different conjugation sites (Wang 2005a; Wakankar 2010).

By using an IgG-binding protein, for which the binding site to the antibody is known, the

antibody can be site-specifically targeted for labeling or immobilization. There are membrane

proteins on certain bacteria that have evolved to bind antibodies as a way to avoid the immune

system, such as protein A on Staphylococcus aureus, protein G on streptococci strains and

protein L on Peptostreptococcus magnus. Protein A consists of five homologous domains, E, D,

A, B, and C, all with individual binding ability to the Fc-region of an antibody. Domain B was

further engineered (as described in Chapter 1) to obtain the obligate Fc-binder domain Z. For

certain applications, the reversible, non-covalent interaction between the IgG-binding protein

and the antibody is not stable enough and by instead covalently attaching the protein to the

antibody, a stabile site-specific modification of the antibody can be performed. Covalently

attaching an IgG-binding domain to an antibody with the photoactivable probe


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benzoylphenylalanine (BPA) has previously been shown using both protein A- and protein G-

based methods (Jung 2009; Konrad 2011; Yu 2013). The photoactivable probe will after

photoactivation form a radical that in turn can react with a nearby amino acid, forming a

covalent bond, a process that has been used in interaction mapping studies with proteins,

peptides, carbohydrates and nucleotides (Dorman 1994).

Paper V – Site-specific photoconjugation of antibodies using chemically synthesized IgG-binding domains

Previous studies have shown that by introducing a photoactivable probe into the Z domain, the

IgG-binding domain could be covalently attached to rabbit IgG without impairing the affinity of

the antibody (Konrad 2011). The study of paper V focused on improving the conjugation

efficiencies for a wider panel of antibodies, mainly those commonly used in diagnostics and

therapy. For this reason mouse IgG1, IgG2A and IgG2B, commonly used for in vitro diagnostic

applications, but also the different subclasses of human IgG were included in this study. Four

different variants of the Z domain were synthesized to investigate the effect on conjugation

efficiency from the position of the probe as well as the length of the linker to the photoactivable

probe (Figure 20).

Figure 20: Schematic figure of the benzophenone-labeled Z variants. A) Z5BBA in ribbon illustration, with BBA conjugated to the ε-amino group of lysine in position 5. B) Z32BPA in ribbon illustration, highlighting the chemical structure of BPA in position 32.

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The synthesized variants were incubated and allowed to bind to human IgG1, IgG2 and IgG4 as

well as mouse IgG1, IgG2A and IgG2B in a molar excess to optimize antibody binding, prior to the

photoconjugation reaction. Conjugated antibodies were evaluated using SDS-PAGE in reducing

conditions, separating the unconjugated heavy chain from the conjugated heavy chain, which has

a slightly higher molecular weight (Figure 21). The ratio of conjugated heavy chain to

unconjugated chain was then used to calculate the conjugation efficiency for each antibody and

Z-variant, e.g. with a conjugation efficiency of 50%, there is on average one domain Z per

antibody, and as the antibody has two heavy chains two Z domains on each antibody will result in

100% conjugation efficiency.

Figure 21: Schematic representation of the antibody photoconjugation. The conjugated heavy chain is separated from the free heavy chain when analyzed by reducing SDS-PAGE.

The conjugation efficiencies of each Z variant varied for the different antibody species and

subclasses with no conjugation seen for two different mouse IgG2B antibodies. For mouse IgG1,

the difference was prominent, as the conjugation efficiency increased from 1% to 66% by

introducing a linker to the photoactivable probe in position 5 (Table 4). On the other hand, the

linker length in position 5 did not have any influence on the conjugation efficiency for mouse

IgG2A, whereas moving the photoactivable probe to position 32 with a short linker reduced the

conjugation efficiency to a great extent, from 50% to 6%, for this antibody. After evaluating the

different Z variants, one additional variant was synthesized where the photoactivable probe was

introduced at both positions, with the best performing linker length for each position, with the

aim of generating a conjugation reagent that could target a wider panel of antibody species. The

resulting double-labeled variant Z5BBA32BPA, could efficiently label human IgG1 with retained

conjugation yield, and the other two variants tested with somewhat lower conjugation efficiency.


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Table 4: Conjugation efficiencies for the different antibody subclasses and subtypesa

a The conjugation efficiency is presented as a mean from two separate SDS-PAGE gels for each antibody.

Even though the photoconjugation yield in other studies has been shown to be increased with a

photoactivable probe having higher conformational flexibility (Kawamura 2008), the variations

in conjugation efficiencies for the different antibody subclasses tested in this study were not

directly correlated with the increased flexibility caused by a longer linker length. Instead, the

variation in conjugation efficiency may arise due to differences in the conjugation site, i.e. the

changes in the sequence and in the binding site for the different antibody subclasses and species.

The photoactivable probe can in its excited state react with otherwise unreactive

C-H-bonds in side chains of Leu, Arg, Lys and Met of the protein (Dorman 1994), and especially

Met has been reported to be the preferred amino acid for benzophenone conjugation, almost

acting as a magnet (Wittelsberger 2008).

Sequence alignment of regions in the Fc-fragment involved in the binding of the Z domain

indicates that there are differences in the availability of Met within a range of 10-15 Å from the

selected position, a range where the benzophenone probe has been reported to be reactive. For

mouse IgG1 there is a Met in position 309, at a distance of 16.6 Å to position 5 of the Z-variant,

i.e. at a relatively long distance for the photoconjugation to occur, which can explain the drastic

increase in photoconjugation efficiency when introducing the benzophenone with a long, more

flexible linker. On the other hand, the distance from the Met in position 309 to position 32 in

domain Z is only 9 Å, and in this case the Z-variant with a shorter linker to the benzophenone

resulted in higher conjugation efficiency to mouse IgG1. Similar correlation with distance to

closest Met and linker length was seen for mouse IgG2A where Z32BPA showed lower conjugation

efficiency than Z32BBA.The distance from position 32 to Met in position 252 was 13 Å and to Met

in position 314 was 15 Å, and this longer distance might explain the improved conjugation yield

for the longer more flexible linker. The distances were measured from backbone to backbone in a

Anna Perols

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crystal structure of human IgG1 and domain B, and might be somewhat different for the Z-

variants and for the mouse subclasses.

Figure 22: Ribbon illustration of part of the complex between the human IgG1 Fc (pdb entry 1FC2) and the B domain, from which the Z domain is derived. A) Showing the distances (in Å) between the residue 5 in the B domain and selected residues in the Fc-fragment and B) showing the corresponding distances for position 32 of the B domain to the same positions in the Fc-fragment. (C) Sequence alignment of loop regions in CH2 and CH3 having different Met residues in human IgG1, mouse IgG1, and mouse IgG2A.

Conclusions and future outlook Site-specific labeling of antibodies is of great importance for therapeutic applications such as in

antibody-drug conjugates where the conjugation chemistry has been shown to affect the

pharmacokinetic properties (Webb 2013). Furthermore, it has also been shown to be of

importance in different in vitro diagnostic applications, where a heterogeneously labeled

antibody could have implications on quantitative readout (Vashist 2012) or influence protein-

protein interactions studies using high-sensitivity fluorescence techniques as fluorescence

correlation spectroscopy (FCS) (Bacia 2006). In this study, a novel Z-variant Z32BPA was

synthesized with improved conjugation efficiency for the therapeutic important human IgG1

subclass resulting in one Z domain per antibody on average. Additionally, by increasing the

linker length and introducing more flexibility for the photoactivable probe, a Z variant with


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improved conjugation efficiency for mouse IgG1, a commonly used antibody subclass in many

biotechnological applications, was developed.

Paper VI – Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions

In this study, the work from paper V was continued and the Z-variant Z32BPA, developed for

increased photoconjugation efficiency to human IgG1 antibody was used. In order to label

antibodies in a fast and selective way, a bioorthogonal reaction handle was introduced to the

Z-variant. There are different strategies that can be utilized for selective labeling of proteins in a

complex environment (as discussed in Chapter 3), where click chemistry has grown popular. For

that purpose, Z32BPA was further functionalized with either an alkyne or an azide at the

N-terminus for efficient antibody labeling using Cu(I)-catalyzed click reactions. Additionally, a

variant where a dibenzoazacyclooctyne-maleimide derivative was conjugated to Z32BPA-Cys-Gly,

resulting in DBCO-Z32BPA 3, was synthesized for antibody labeling using strain-promoted click

chemistry. As a reference protein, a biotinylated variant of Z32BPA was also prepared, biotin-

Z32BPA (4) (Figure 23). Thanks to the small size of the IgG-binding domain, it is amenable for

production by chemical protein synthesis which allows for incorporation of the photoactivable

probe, but it also enables introduction of building blocks for click chemistry.

Figure 23: Representation of the synthesized variants for both Cu(I)-catalyzed as well as strain-promoted click chemistry and the click chemistry derivatives of biotin.

Anna Perols

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In order to verify the Cu(I)-catalyzed click reactions, alkyne-PEG5-Z32BPA 1 and azide-Z32BPA 2

were mixed with either azide-PEG3-biotin 6 or alkyne-PEG4-biotin 5 (2 equiv.) in the presence of

the catalyst Cu(II)SO4 and the reducing agent sodium ascorbate. For alkyne-Z32BPA, conjugated

product was seen already after 5 min and the reaction seemed to be complete at 30 min,

however, a competing reaction resulting in a side product with mass difference of -38 Da reduced

the amount of product formed. For azide-Z32BPA, the reaction was faster and only the product

with no side products was seen by MALDI-TOF MS already after 5 min, and verified by HPLC

(Figure 24). DBCO-Z32BPA was also allowed to react with azide-biotin without catalyst in a Cu(I)-

free reaction, and product was seen already after 5 min and at 30 min no starting material was

detected, showing a complete reaction for the strain-promoted click reaction as well.

Figure 24: Analysis of Cu(I)-click reaction with azide-Z32BPA. A) Analyzed using HPLC, showing the overlay of HPLC traces for azide- Z32BPA (black) and the reaction at 5 min (red) and 10 min (green) at 220 nm. B) The MALDI-TOF MS spectra of azide-Z32BPA and of an aliquot of the reaction after 5 min.


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Functionalized Z-variants 1-4 were covalently conjugated to trastuzumab, with comparable

conjugation efficiencies as unmodified Z32BPA, around 40-45%, resulting in an average of nearly

one Z-domain per antibody.

Figure 25: SDS-PAGE of trastuzumab conjugated with the Z-variant alkyne-Z32BPA, trastuzumab-1, (lane 1), DBCO-Z32BPA, trastuzumab-3, (lane 2), azide-Z32BPA, trastuzumab-2, (lane 3), biotin-Z32BPA, trastuzumab-4, (lane 4), unmodified Z32BPA, trastuzumab-Z32BPA, (lane 5) and unconjugated trastuzumab as reference (lane 6). HC= heavy chain, HC+Z=heavy chain with the Z domain covalently attached, and LC= light chain.

Furthermore, conjugated antibodies were site-specifically biotinylated using the introduced

chemical handle in Z32BPA either by a Cu(I)-catalyzed or a strain-promoted click reaction. The

Cu(I)-catalyzed click reaction for conjugated antibodies was performed with 5 equiv. of azide-

PEG3-biotin 6 or alkyne-PEG4-biotin 5 for 30 min in the presence of Cu(I), and the strain-

promoted click reaction was performed correspondingly with 6 omitting the catalyst. The

efficiency of the click reaction and the degree of biotinylation, was estimated using western blot

where small aliquots of the labeled antibodies (100 ng) were analyzed using streptavidin-HRP

and chemiluminiscent detection. The reference protein biotin-Z32BPA (4) was conjugated to the

antibody, resulting in trastuzumab-4, and was loaded in different amounts (25 ng, 50 ng and 100

ng) to be used as a marker for estimating the click cycloaddition yield for trastuzumab

conjugated with the different Z variants 1-3. As conjugation efficiencies for the different Z

variants were comparable (Figure 25), the signal from trastuzumab-4 in western blot would

correspond to a click cycloaddition with 100% yield.

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Figure 26: Western blots of biotinylated trastuzumab using streptavidin-HRP in a chemiluminescent readout. A) Evaluation of click reactions for trastuzumab-3, trastuzumab-1 and trastuzumab-Z32BPA (control) with azide-PEG3-biotin 6. Trastuzumab-4 was loaded in different amount as a marker. B) Evaluation of click reaction of trastuzumab-2 and trastuzumab-Z32BPA (control) with alkyne-PEG4-biotin 5. Trastuzumab-4 was loaded in different amounts as a marker.

For the Cu(I)-catalyzed click reactions, trastuzumab-1 showed a high degree of biotinylation, but

lower than trastuzumab-4 loaded in the same amount indicating that the reactions are not

complete (Figure 26A). However, for trastuzumab-2 the degree of biotinylation was low (Figure

26B), even though the reaction for azide-Z32BPA alone was complete within 5 min. Similarly, low

biotinylation yield was also seen for the strain-promoted click chemistry, where trastuzumab-3

was allowed to react with azide-PEG3-biotin 6 (Figure 26A), even though the the strain-

promoted reaction with DBCO-Z32BPA resulted in a complete reaction within 15 min.

Conclusions and future outlook The use of antibodies as targeted therapy has increased during the last years, and has shown

promising results in oncology, but also for immunological and cardiovascular targets (Nelson

2010b; Sliwkowski 2013). In addition to the use of naked antibodies as therapeutics, antibodies

have also emerged as conjugated antibody-targeted therapy, where the antibody is used for

delivery of potent cytotoxic compounds (Alley 2010; Zolot 2013). Furthermore, a controlled way

of labeling will also reduce the heterogeneity associated with conjugation strategies targeting

lysines, as selection of conjugation chemistries and conjugation sites are important for the

performance of the conjugated antibody (Webb 2013). Strategies for producing homogenous

ADCs have been developed, such as THIOMABS (Junutula 2008), where similar efficacy and a


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higher tolerability was proposed for the site-specific DM1-labeled trastuzumab in comparison to

the corresponding heterogeneously labeled antibody (Strop 2013; Webb 2013).

In this study a human IgG1 antibody has been site-specifically labeled with a Z domain

functionalized with a chemical handle for either Cu(I)-catalyzed or strain-promoted click

chemistry. In the Cu(I)-catalyzed click reaction, product formation differed when the reaction

was performed with the Z-variant alone or conjugated to the antibody. This was especially seen

for azide-Z32BPA 2 which was the best performing variant when only the Z-variant was used but

resulted in low biotinylation yield when conjugated to trastuzumab. The small azido-group was

coupled to the N-terminus of Z32BPA without the spacer arm present in alkyne-PEG5-Z32BPA 1.

The low yield of detected biotinylated trastuzumab-2 could either be due to that the azide is not

accessible in the click-reaction or due to sterical hindrance in the detection step using the

streptavidin-enzyme conjugate.

In this method for site-specific labeling of human IgG1, a chemical handle has been introduced to

the IgG-binding domain using solid phase peptide synthesis, and with the variety of available

click reactive probes such as fluorophores or PET-percursors, the Z-variants presented here can

be used as a versatile antibody labeling strategy.

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Concluding remarks and future outlook

The influence of site-specificity in protein labeling has been extensively discussed in this thesis,

both for the smaller affinity proteins Affibody molecules, as well as for the larger antibodies.

These affinity proteins both can bind their targets with high affinity, but due to the large

difference in size, 7 kDa and 150 kDa respectively, they are better suited for different

applications. Naturally produced by B-cells, antibodies are part of the immune system and will

activate the complement system or engage effector cells by Fcγ-receptor interactions upon

antigen encountering. The Fc-region is also responsible for the prolonged half-life in serum by

binding to the neonatal Fc-receptor. The half-life extension could be an advantage for certain

applications, as in therapy where the effect is improved both by the prolonged circulation time

and the recruitment of effector cells. (Panowski 2014) For example, the monoclonal antibody

cetuximab (Erbitux®) targeting the epidermal growth factor receptor (EGFR) mediates its anti-

tumor activity by ADCC (Kimura 2007). However, for in vivo diagnostic applications, such as

molecular imaging, the long circulation time and the retention in non-targeted tissues are not

beneficial as the background will increase and the contrast is lowered (Kaur 2012). In contrast to

antibodies, Affibody molecules do not have the Fc-region responsible for these effector functions

or the prolonged half-life. The low molecular weight will increase the clearance rate, as the small

affinity protein will be eliminated by glomerular filtration, with a half-life in humans of less than

30 min (Baum 2010) compared to 21 days for full-length antibodies, as well as improving the

tissue penetration. The optimal tumor-to-organ ratio is typically achieved within hours for the

smaller Affibody molecules, and imaging can be performed at the same day as administration,

whereas the optimal tumor-to-organs ratio for an antibody is not reached until 2-4 days.

In papers I-IV, it was shown that small differences in the design of the radiotracer greatly

influenced the uptake in both tumor and non-targeting organs. These findings pursued the

research in tracer design and radiolabeling strategy in order to understand what mechanisms

that give rise to the different findings. For the purpose of improving the molecular imaging

properties for Affibody molecules, novel bi-functional chelators were introduced and labeled with

the radionuclide 111In. As seen in paper III, the use of the 111In-NOTA radiolabeled conjugate

resulted in an increased hepatic uptake compared to the other conjugates used in the study,

possibly due to the positive net charge of the radionuclide-chelator complex in the N-terminus of

the protein. Similar results have been seen for Affibody molecules with a positive charge N-

terminally (Tran 2008), but incorporation of a triglutamyl spacer in a synthetic Affibody

molecule radiolabeled using oxime ligation showed a reduced hepatic uptake (Rosik 2013). By


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identification of trends that could influence the biodistribution, such as charge distribution, the

tracer design could be improved.

The efforts to improve the HER2-targeting Affibody molecules for molecular imaging have given

an understanding on how different macrocyclic chelators could influence the biodistribution and

hence the tumor-targeting properties. These small differences in the sensitivity could be crucial

for finding small HER2-expressing lesions or for following changes in treatment responses for

patients using HER2-targeting therapeutics. The clinical relevance of Affibody molecules was

further established in a study by Baum et al., where HER2-expressing lesions were detected in

patients with recurrent metastatic breast cancer using PET and SPECT (Baum 2010). For the

second generation Affibody molecule scaffold where the biophysical properties had been

improved, such as increased hydrophilicity, Sörenen et al. showed a first-in-human study with

efficient tumor detection and a reduced hepatic uptake in comparison to the study performed

with the first generation Affibody scaffold (Sörensen 2014).

The use of the larger antibodies in the clinics has had a large effect on the medical care of many

diseases including cancer. The effectiveness of antibodies targeting tumor associated antigens

could potentially be even further improved by conjugation with a cytotoxic compound, and the

antibodies can be used for targeted delivery of the drug (Sievers 2013). Site-specific labeling has

been shown to eliminate the heterogeneity often associated with traditional antibody labeling,

and increases the therapeutic window (Webb 2013). The strategy described in papers V-VI for

site-specific labeling relies on the inherent IgG-binding properties of the Z-domain. The binding

site in the region between CH2 and CH3 on the Fc-fragment is far from the antigen binding site

and does not seem to affect the antigen binding properties. How the covalent attachment of the

Z domain to the antibody affects the effector functions or the activation of the complement

system has however not yet been elucidated. The binding site for FcγR varies among the different

subtypes, where some receptors seem to compete with protein A binding (Wines 2000). The

binding site for the neonatal Fc-receptor is overlapping with the binding site for protein A in the

CH2-CH3 region (Roopenian 2007), and how this binding affects the half-life of site-specifically

labeled antibodies using the Z-domain remains to be investigated. However, mutations in the

CH2-CH3 region of the antibody have been shown to significantly reduce the binding to the

Fc-receptor, reducing the half-life (Kim 1999; Knowles 2012). Alternative Ig-binding domains

with binding sites distinct from the Fc-receptor binding sites, such as protein L, which binds

certain subtypes of kappa light chains could also be used. However, protein L is considerably

larger than the Z-domain with a molecular weight of 76 kDa and can be expected to be more

difficult to produce by solid phase peptide synthesis. Protein G also binds Igs (Björck 1984), with

the main binding site in the CH2-CH3 region of IgGs, but with an additional binding site identified

in the Fab-fragment (Graille 2000), binding to the CH1 domain, the protein also has a binding

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site not overlapping with either antigen binding or Fc-receptors. The small size of the protein G

derived Ig-binding domain (6 kDa), makes it amenable for production using solid phase

synthesis, however the binding would preferably be directed to either the Fc-fragement or the

Fab-fragment for a controlled site-specific binding.

Taken together, the labeling chemistry and label positioning in protein labeling of Affibody

molecules have been shown to greatly affect the behavior in in vivo applications, such as

molecular imaging. For the larger antibody, a site-specific labeling technique was established

without impairing the antigen binding properties. Both the Affibody molecule and its parental

molecule, the IgG-binding domain can readily be produced using solid phase synthesis, which

enables site-specific modifications. Labeling could either be performed during synthesis such as

the introduction of chelators, or by introducing chemical handles for labeling using

bioorthogonal conjugations. Based on the work summarized in this thesis, both the choice of

labeling chemistry and labeling site have been shown to be important parameters for affinity

protein labeling for both in vitro and in vivo applications.


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Acknowledgements

The work of this thesis was supported by ProNova VINN excellence centre for Protein Technology, Swedish Governmental Agency for Innovation Systems, Swedish Research Council and EU’s Seventh Framework Programme for Research (FP7).

This work would not have been possible without the support and and help from my supervisors, present and former co-workers and collaborators. Thank you!

~ Först och främst vill jag tacka min handledare Amelie för all hjälp, input, korrekturläsande och framförallt pepp under dessa år! Du har alltid tagit dig tid för både större och mindre funderingar, och styrt mig åt rätt håll när jag inte riktigt har vetat vart jag ska rikta mitt fokus! Även om det ibland har varit tufft så har du hittat de saker som gör att projekten går framåt!

Även ett stort tack till min bihandledare PerÅke, för att du tar dig tid (och plats på kontoret) för frågor och diskussioner, men även för ditt engagemang i mina projekt! Din kunskap är guld värd! Även ett stort tack för en bra sightseeing av Rhen, även om det var smärre hinder i vägen!

Jag vill även tacka Sophia för möjligheten att göra exjobbet hos dig, det var en väldigt bra introduktion till forskning. Jag började och slutade i (nästan) samma projekt! Även ett stort tack till Anna K för att du var en superb ex-jobbshandledare!

Tack även övriga PIs på plan 3 för att ni gör det till en så fantastisk plats att arbeta på!

Anna and Vladimir, you are always so warm and friendly! Thank you for being so generous with your knowledge in molecular imaging, and for a very fruitful collaboration! Also Hadis for your biodistribution experiment demonstration (we passed with excellence, right?) and Mohammed, Jonna and Jennie, for nice collaboration.

Till alla fantastiska nuvarande och tidigare kollegor i gruppen Chemical Protein Engineering. Peter för den gruppkänsla du införde med morgonfikat, luncherna och korsordslösandet vid fredagsölen! Ett stort tack för allt du lärt mig (även inom forskning!) Gruppen blev tom utan dig! Joel, både som skrivbordgrannar i Rookie-rummet (visst var det väl inte bara trångt!), på konferenser och i labbet har du varit en klippa! Daniel, grymt kul att du började i vår grupp! Utöver excelmallar och syntes-mek har vi hunnit med ett par roliga fredagsöl, både i Köpenham och på Isla de Mujeres! (utöver de i lunchrummet förstås) Nima, för att du är en inspirations-källa med ditt målinriktade fokus. Kristina, en fantastisk rumskompis i Mexico och en härlig vän! Tack för alla kaffepauser och för att du tog dig tid med min avhandling under en jobbig tid! Andréas, från labbass på TBI i Linköping och nu i samma grupp på KTH, vem hade kunnat ana det? Alltid lika kul att snacka hus& ölbryggning med dig. Även ett stort tack till Melina, min härliga exjobbare, så fantasktiskt kul att du stannade kvar (och att vi fick ihop manuskriptet!) Anders, välkommen till gruppen och till skrivrummet! Alexis, thank you for the help producing the NODAGA-variant. Även stort tack till alla exjobbare som tillhört gruppen.

Camilla, från första veckan på exjobbet har vi labbat, fikat och lunchat ihop, så mycket kul vi haft! Det har blivit många sena kvällar, både på jobbet och utanför, och en fantastisk vecka på Manhattan! Ullis, du finns alltid där för en kaffe, en pratstund eller bara för en kram. Så uppskattad! Helena, Basia, Johanna och Maja, jag saknar er! Snyggrummet blir liksom bara ”rummet” utan er! När ska vi allihopa ta nästa syjunta (dricka vin och skvallra)?

Anna Perols

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Ken, ett norrländskt hjärta av guld! Det har varit grymt kul att dela skrivrum med dig. Tarek, tack för introduktionen till en ny värld en solig söndag i LA... Sorry att du fick vänta på fönsterplatsen på kontoret! ;) Lunchgubbsen; Andreas, tack för att jag får äran att luncha med dig! Även ett stort tack för bra input vid Biacoren. John, ett extra stort tack för granskningen av avhandlingen! (Men även för uppdateringar om Djurgårdens senaste förluster...) Johan, du har alltid lika bra koll på antikroppar, även om segling, öl och mat är klart roligare samtalsämnen!

Lisa och Hanna, finaste hotellrumskompisar, två konferenser hann vi med tillsammans! Vi gjorde både NY&Boston samt LA med bravur! Sara K, finaste skånska synterska (titeln delas med Anna K)! Du gör livet så mycket roligare i både i synteslabbet och i mellangården! Tove, du är bra på det mesta med ditt fika brukar vara ngt extra. Filippa och Magda, den bästa labbkvarten (med inbyggt takdropp) innan mellangården lockade över mig! Feifan, I miss you over there! Anna-Luisa, en ängel som tog min diskvecka under mitt avhandlingsskrivandet! Tjejerna på labbet, vi har haft skoj, både på stand-up och på labbet.

Även ett stort tack till alla tidigare kollegor på plan 3 för det varma välkomnadet när jag började som doktorand, och för all hjälp genom åren! Erik V, Sebastian och Johan N för många bra tips och trix! Tack Emma och Roxana för all disk, men även ett stort tack till Emma och LanLan för all hjälp med beställningar. Även ett stort tack till ProFab för att jag fått låna MassSpec:en otaliga gånger och Immunotech för hjälp med framkallning av mina blottar. Post-it-lappar är grejen!

Ett stort tack till Bo Jansson för bra bra diskussioner och input i ProNova-samarbetet, samt för era antikroppar från Bioinvent. Även tack till Niklas på Mabtech, samt Sarah och Malin på Genovis och GeccoDots för ett bra samarbete inom ProNova-projekten.

Även Daniel Rönnlund, Lei Xu, Evangelos Sisamakis samt Jerker Widengren för samarbetet inom EU-projektet FLUODIAMON. I would also like to thank the group of prof. Claus Seidel at Heinrich-Heine Universität for the nice collaboration in the EU-project FLUODIAMON.

Ett stort tack till Pelle på Affibody AB för all hjälp vid CD- och VTM-mätningarna!

Till världen utanför labbet. Sara, ett fantastisk vän som alltid funnits där för mig, och ett bra bollplank (som kanske inte alltid säger det man vill höra...) Bodil, tack för all hjälp med avhandlingen och för mycket skoj under alla dessa år! Maria, utekvällar och utlandsresor är numer utbytta till långpromenader och spahelger, en bra förändring! Vad vore livet utan bra vänner?

Till min familj. Världens bästa Mamma, som fixar och ställer upp, när jag bett om hjälp och alla gånger jag inte gjort det. Jag säger det inte ofta nog, stort tack för allt du gör! Min kloka fina Pappa, som du sliter, du är värd all uppskattning! Ni är båda en obegränsad källa av support, jag älskar er! Bästa syster-yster! Du är nog den som har stöttat mig mest genom åren. Tack även Elin, ditt leende värmer mosters hjärta!

Björkarna, en bättre extrafamilj får man leta efter! Eva och Lennart, med varmt hjärta blev jag välkomnad till familjen, och det är fantastiskt skönt att bara ha er några minuter bort oavsett om det är för att låna saker eller att bjuda in sig på middag.

Och så slutligen, Joakim. Tack för att du funnits där de senaste 10 åren och för att du påminner mig om ingenting är omöjligt! Tack för att du är den du är. Du är fantastisk, jag älskar dig!


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