Development of a covalent site-specific antibody labeling strategy by the use of

photoactivable Z domains

Anna Konrad

Royal Institute of Technology

School of Biotechnology

Stockholm 2012

© Anna Konrad Stockholm 2012

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

Printed by E-print Oxtorgsgatan 9-11 SE-111 57 Stockholm Sweden

ISBN 978-91-7501-329-9 TRITA-BIO Report 2012:9 ISSN 1654-2312

Anna Konrad (2012): Development of a covalent site-specific antibody labeling strategy by the use of photoactivable Z domains. School of Biotechnology, Royal Institute of Technology (KTH), Sweden ISBN 978-91-7501-329-9

Abstract

The joining of two molecular functions or the strategy of adding functions to proteins has been

tremendously important for the development of proteins as tools in research and clinic.

Depending on the intended application, there are a wide variety of functions that can be added

to a proteins. In clinical applications drugs are a commonly conjugated to antibodies and in

research adding reporter groups such as biotin, enzymes or fluorophores is a routine procedure.

The chemistries and methods most often used suffer from drawbacks such as lack of stringency,

which could lead to undesired effects on the protein. Many site-specific methods of labeling of

antibodies require modification or insertion of handles in the antibody recombinantly, before

labeling can be performed.

The core of this thesis is the development of a strategy for covalent specific labeling of

antibodies by exploiting the site specific binding of the Z domain to Protein A. Photoreactive Z-

domains were produced by solid phase peptide synthesis, which provides the opportunity to

insert a photoreactive amino acid and a reporter biotin at specific positions in the domain. The

inherited binding to the Fc-part of the antibody in combination with the incorporated

photoreactive amino acid, BPA, is used for site-specific interaction, and thereafter, covalent

coupling to the antibody. The exposure with the appropriate wavelength of light enables the

formation a covalent linkage between the Z domain and the antibody. The biotinylated

photoactivable domains were subsequently used to site-specifically label a number of different

types of antibodies, polyclonal rabbit IgG, monoclonal human IgG1 and monoclonal mouse

IgG2a, and thereafter the antibodies was employed in a variation of applications. The

photolabeling procedure of antibodies by the use of photoactivable Z domains has proven to be

successful and could serve as a valuable tool in several applications.

Keywords: antibody labeling, site-specific, covalent, SPPS, Z domain , BPA © Anna Konrad

ListofPublications

This thesis is based upon the following three papers, which are included in the appendix.

I. Anna Konrad, Amelie Eriksson Karlström and Sophia Hober.

Covalent Immunoglobulin labeling through a photoactivable synthetic Z domain.

Bioconjug Chem. 2011 Dec 21;22(12):2395-403.

II. Sandra Andersson, Anna Konrad, Nikhil Ashok, Fredrik Pontén, Sophia Hober and Anna

Asplund.

Antibodies biotinylated using a synthetic Z-domain provide stringent in situ protein

detection. Manuscript.

III. Anna Konrad, Josefin Anfelt, Sandra Andersson, Amelie Eriksson Karlström and Sophia

Hober. Optimization of an antibody labeling strategy through the use of a photoactivable

synthetic Z domains. Manuscript.

RelatedPublications

Vernet E, Konrad A, Lundberg E, Nygren PA, Gräslund T. Affinity-based entrapment of the

HER2 receptor in the endoplasmic reticulum using an affibody molecule. J Immunol Methods.

2008 Sep 30;338(1-2):1-6

Tegel H, Steen J, Konrad A, Nikdin H, Pettersson K, Stenvall M, Tourle S, Wrethagen U, Xu L,

Yderland L, Uhlén M, Hober S, Ottosson J. High-throughput protein production--lessons from

scaling up from 10 to 288 recombinant proteins per week. Biotechnol J. 2009 Jan;4(1):51-7.

Contents ABBREVIATIONS......................................................................................................................... 1

INTRODUCTION........................................................................................................................... 3

ANTIBODIES ................................................................................................................................ 7 STRUCTURE ................................................................................................................................ 7 POLYCLONAL, MONOCLONAL OR RECOMBINANT ANTIBODIES ........................................................ 9

PROTEIN A................................................................................................................................. 11 Z DOMAIN .................................................................................................................................. 12

MODIFICATION OF PROTEINS................................................................................................. 15 INCORPORATION OF UNNATURAL AMINO ACIDS INTO PROTEINS .................................................... 16 BENZOYLPHENYLALANINE – BPA – A PHOTO REACTIVE AMINO ACID............................................. 18

PRESENT INVESTIGATION ...................................................................................................... 21 COVALENT IMMUNOGLOBULIN LABELING THROUGH A PHOTOACTIVABLE SYNTHETIC Z

DOMAIN (PAPER I) ..................................................................................................................... 23 ANTIBODIES BIOTINYLATED USING A SYNTHETIC Z-DOMAIN PROVIDE STRINGENT IN SITU

PROTEIN DETECTION (PAPER II) ................................................................................................. 24 OPTIMIZATION OF AN ANTIBODY LABELING STRATEGY THROUGH THE USE OF

PHOTOACTIVABLE SYNTHETIC Z DOMAINS (PAPER III).................................................................. 25 CONCLUDING REMARKS ............................................................................................................. 26

ACKNOWLEDGEMENTS........................................................................................................... 27

REFERENCES............................................................................................................................ 31

1

Abbreviations

ADC – antibody drug conjugate

Boc – t-butyloxycarbonyl

BP – benzophenone

BPA – benzoylphenylalanine

CDR – complementarity determining region

DNA – deoxyribonucleic acid

E. coli – Escherichia coli

Fab – fragment antigen binding

Fc – fragment crystallizable

Fmoc – fluorenylmethyloxycarbonyl

HSA – human serum albumin

IgG – immunoglobulin G

IHC – immunohistochemistry

mRNA – messenger ribonucleic acid

scFv – single chain fragment variable

SPA – staphylococcal protein A

SPPS – solid phase peptide synthesis

SPR – surface plasmon resonance

tRNA – transfer ribonucleic acid

2

3

Introduction

Everyday around the world a vast number of researchers and hospital personnel strive to

answer questions regarding disease with the help of different methods. The dependence and

need for reliable tools in research and medicine are great and a considerable contribution has

been made through the technical revolution within the field of biotechnology. Any progress made

within method development increases the prospect of understanding disease and biology better

and serves the common good.

Timelines describing the milestones of Modern Biotechnology often start with the term

biotechnology being coined by the Hungarian engineer Karl Ereky in 1917, defining it as ”all

lines of work by which products are produced from raw material with the aid of living things” (1).

However it was in the 1960s that a microbiologist Carl Göran Hedén introduced the term in a

sense that is regarded as modern biotechnology, meaning that it stands on the ground of the

scientific fields of microbiology, biochemistry and chemical engineering (1). In a review from

2002 Rita Colwell reflects over the history of biotechnology and the time after the

deoxyribonucleic acid (DNA) structure being solved in the 1950s: “The field of biotechnology has

progressed from the world of science fiction to the world of science present”, meaning that

during the later part of the twentieth century the field progressed tremendously (2). In fact in the

mid 1970s the research community felt that the technical advances were faster than the ability to

make ethical considerations regarding what the technique could lead to. Therefore they decided

to temporarily put all genetic engineering on hold. This voluntary moratorium was held until

laboratory guidelines were established at the Asilomar Conference in 1975 (3, 4). A few

highlights in the history of biotechnology are discoveries that today are taken for granted and

used in laboratories everyday worldwide, a few of these will be mentioned in short.

The understanding of genetic material and structure of the DNA molecule was a significant step

in history. Avery and coworkers suggested that DNA was the factor responsible for making

bacteria transform (5) and, inspired by Rosalind Franklin (6, 7), Watson and Crick published the

4

description of the helical structure of the DNA-molecule (8). Gaining knowledge of genetic

material led to the discovery of diseases that are result of a mutation at the genetic level, sickle

cell anemia being one example, which was shown by Pauling in 1949 (9). Further discoveries in

the field, for example the replication process of DNA (10-12) and the isolation of DNA modifying

enzymes, such as DNA polymerase and restriction enzymes (13-15), made it possible to

perform genetic engineering. This was indeed important for Boyer and Cohen, who

demonstrated the first recombinant DNA transformed into bacteria in 1973 (16). This resulted in

the recombinant production of human insulin helping people around the world that suffer from

diabetes (17). Finally, in the 1980s the polymerase chain reaction (PCR) was developed, that

allowed for exponential amplification of DNA (18) and made genetic cloning into routine work.

Two events in biotechnological history that paved the way for the use of immunoglobulins, or

antibodies, as biotherapeutics and research reagents is the developed technique of producing

monoclonal antibodies by Köhler and Milstein, for which they were rewarded the Nobel prize in

1984 (19), and also the discovery of the gene rearrangement of antibodies described by

Tonegawa (20). The combination of many of the above mentioned techniques with selection

systems, e.g phage display (21), made it possible to create and select human antibodies or

fragments thereof in vitro (22, 23) making them able to recognize targets relevant from a clinical

or a research perspective.

In the early 1990s we entered the “age of omics”, when the techniques of DNA sequencing were

enough developed to render the Human Genome Project possible. During 2000 a draft of the

human DNA sequence was declared finished (24, 25). The knowledge about the genome set the

stage for exploration of the human proteome. Where, when and how much of the human

proteins are expressed in our bodies are tremendously important, since proteins are involved in

every process in our cells, i.e. from constructing the cell to its function. Several initiative have

been started to investigate human proteins and some used affinity reagents (26, 27). During

2003 in Sweden, a large-scale project was initiated for the mapping of the human proteome by

using antibodies to visualize expression and localization of the proteins. A valuable source of

reagents to almost all human proteins has been created as well as a publically available Human

Protein Atlas on the web (www.proteinatlas.org) (28-30).

5

Today an exceptional amount of antibodies has been produced worldwide, both as therapeutics

and as reagents in research and diagnostics. Up to date approximately 168 000 reviewed

antibodies to human targets can be found which is covering 84% of all human genes (31). The

success of the use of antibodies as affinity reagents in clinic and research depends on the ability

of the antibody to bind its target selectively and thereby being a reliable reporter system. The

work this thesis rests upon is the field of labeling of antibodies and the focus has been

exploration of possibilities of a new site-specific and covalent labeling system.

6

7

Antibodies

Antibodies are proteins that are part of the adaptive immune system where their significant

function is recognition of foreign molecules. The amazing trait of antibodies was employed

unintentionally as early as in the 1890s when passive immunization first came in use and serum

from animals actively immunized with diphteria toxin was exploited as antitoxins (32). In 1948

Astrid Fagreus could show that white blood cells, or more specifically plasma cells a kind of

mature B-cells, were the cells producing antibodies in the body (33). As previously mentioned

antibodies form a group of molecules with enormous impact in the area of biotechnology and

speculations on its promise in medicine and trends in the development of tools based on

antibodies are featured in many articles (34-37).

Structure

The antibodies are constituted of four peptide chains, two longer identical heavy chains, and two

shorter identical light chains (Figure 1). These four chains order themselves by the help of

disulfide bonds and non-covalent interactions in a way that gives the antibody a characteristic Y-

shape. The two “arms” on the Y corresponds to one light chain and a part of one heavy chain,

forming the fragment antigen binding (Fab) and the rest of the two heavy chains interact forming

the fragment crystallizable (Fc), the “stem” of the Y (38, 39). The cleavage of the antibody into

fragments with the protease papain divides the antibody into distinct parts with different

functions; the Fab fragment mediates the antigen binding whilst the Fc part mainly conducts

effector functions such as binding and activating the complement cascade in the immune

response (39, 40). Furthermore, the light and heavy chains have a constant region, composed of

the Fc- and a part of the Fab-fragment, and a variable region, which is positioned at the tip of the

Fab fragment (38). The diversity in the variable region that is responsible for binding to other

molecules, the so-called molecular recognition of the antibody, is located in the complementarity

determining regions (CDR’s), which present immense variety given the limitation at gene level.

The immune system’s ability to produce a pool of antibodies with high variety and thus

increasing the probability to defend us against antigens originating from invading

microorganisms is essential. Nature has solved this by varying the length and composition of the

8

CDRs in the antigen-binding site through shuffling and recombination of gene segments of B-

cells (38). Two other occurrences that contribute to the variation found in the antigen-binding site

of antibodies are somatic hypermutation, which introduces point mutations in the loops in the

CDRs and affinity maturation, which introduces single mutations in a later stage of B-cell

maturation (38). Immunoglobulins are divided into different subclasses/isotypes depending on

the functional properties of the antibody and structure of the Fc-part of the antibody, IgG, IgA,

IgD, IgE, IgM. The subtype most commonly found in sera is IgG (38).

Figure 1.The structure of an IgG-molecule (antibody) and single chain fragment variable (scFV)

represented in a schematic illustration. An antibody is stabilized by several disulphide bonds and it

consists of two identical heavy chains (C, in turquoise) and two identical light chains (L, in purple). In the

variable domains (V) the complementarity determining regions (CDRs) can be found which are

responsible for the antigen binding. The heavy chain includes three constant domains (CH1-3) and the

variable chain (VL) includes one. An scFv constitutes of the variable domains of an antibody, which are

genetically joined by a peptide linker.

9

Fragments of antibodies, Fab-fragments and single chain fragments (ScFv) being two examples,

are frequently used in different applications today (39). However, the use of antibodies as a

whole molecule are central to the work this work. Therefore a short description of three main

production routes of obtaining antibodies will be described.

Polyclonal,MonoclonalorRecombinantAntibodies

In order to acquire polyclonal antibodies animals are immunized with an antigen and a pool of

antibodies recognizing the antigen can be obtained. The pool typically contains antibodies that

recognize distinct epitopes and that bind to different parts of the antigen because the antibodies

come from different B-cells. An advantage with polyclonal production of antibodies is that the

process is rather uncomplicated and less expensive than monoclonal production. However, a

disadvantage is that the source of antibodies is not renewable, as another immunization with the

same antigen will not give the same pool of antibodies (28). The ability of a polyclonal antibody

pool of recognizing several epitopes could be beneficial for some applications were an apparent

risk of a single epitope to be inaccessible could be found, such as immunohistochemistry (28,

41). Affinity purification, employing the antigen as ligand to retrieve the target-specific antibodies

from the polyclonal sera, so called monospecific antibodies (28) is beneficial, although the use of

Protein A or Protein G as generic, immunoglobulin-binding ligands is a widely applied strategy

for obtaining a pure antibody sample (42).

As mentioned, a great achievement in the history of biotechnology was the creation of

hybridomas. By fusing mouse myeloma cells with antibody producing B-cells from mice

immunized with an antigen, immortality of the antibody producing cell and unlimited production

of identical antibodies were achieved (19). The feature of the monoclonal antibody binding a

single epitope is an advantage in applications as immunotherapy and diagnostics where

minimizing the risk of crossreactivity is important (43). However, when antibodies have been

developed for biotherapeutics, issues regarding the rodent origin has been raised since they

may evoke an immune response when administered to humans (44). This has given strong

incentives for driving the development of antibody production to a recombinant route. Isolation of

cDNA (complementery DNA) in combination with genetic engineering has given progress such

as; chimerization, where the variable domains and antigen binding are of mouse origin whilst the

10

constant regions are of human origin (45, 46), or humanization processes, where only the CDRs

originate from the mouse clone and are grafted upon a human framework giving almost a

complete human antibody (47). Moreover, efforts have been made to provide expression

systems for production of antibodies or antibody fragments produced in non-mammalian cells for

instance Escherichia coli (E. coli) (48, 49), and through the employment of synthetic libraries and

selection systems antibodies and fragments can thereof be isolated (39, 50).

11

ProteinA

Several pathogenic strains of bacteria have evolved a strategy to evade the host immune

response by expressing cell wall proteins with the ability to bind host proteins (51). Various

streptococcal and staphylococcal strains, as well as other strains, have been found with these

features (52, 53).

The first cell wall-exposed protein to be characterized was staphylococcal Protein A (SPA) and

in 1966 Forsgren and Sjöquist could show that the interaction between SPA and antibodies was

not a specific antigen-antibody binding event, which was the belief at the time. They showed that

the interaction of SPA and the antibody was non-immune in the sense that it took place in other

parts than the binding site of the antibody (54). The sequencing and the expression of the gene

led to the insight that the protein was a single polypeptide chain, composed of five homologous

domains that share the IgG-binding feature (Figure 2) (55). The domains were denoted E, D, A,

B and C each composed of approximately of 58 amino acids (55). Further studies of SPA

revealed other distinct parts of the protein; in the N-terminal part of the protein a signal

sequence was found and at the C-terminal part there is a region that is responsible for binding to

the bacteria’s cell wall (55, 56).

In 1981 Deisenhofer published the result from an X-ray crystallography study of the B domain

binding the Fc part of a human antibody, and it showed that helix 1 and helix 2 of the B domain

were structured in an antiparallel fashion and responsible for binding to the CH2 and CH3

domains of Fc (57). In this first study the third helix seemed unstructured but NMR studies of the

B domain binding an Fc fragment confirm a three-helical bundle structure also in the binding

state (58, 59). There are eleven proposed amino acids of the IgG-binding domain that take part

in binding to IgG and the characteristics of the binding are mainly hydrophobic (57-60). In

addition, the IgG-binding domains of SPA exhibit binding to the Fab regions of immunoglobulin

and the interface is not overlapping with the site for the Fc binding. The disclosure of the crystal

structure of the complex with the D domain binding to the Fab region of human IgM showed that

12

residues of helices 2 and 3 were involved in the interaction. Also, it was shown that the

interaction with the variable heavy chain of the antibody was mainly of polar character (61).

The ability to bind IgG has given SPA expansive use as a tool in biotechnology; it is frequently

used in many different applications whereof affinity purification of IgG molecules is the most

prevalent. Nevertheless, the detection and quantification of antibodies on cell surfaces and

depletion of IgG in serum samples are other applications were the ability to bind the Fc part

antibodies is exploited (42, 62).

Zdomain

The combination of the small size and the ability to fold and retain the IgG binding feature, that

the domains of SPA possess, makes them suited for use in many applications (42). The

sequence of the B domain exhibits the attribute of being the consensus sequence of the IgG-

binding domains found in SPA (55), and it was also the most studied domain. Therefore the B

domain was a natural choice for the creation of an engineered variant, the Z domain. Two

positions in the amino acid sequence were altered to obtain the Z domain; to facilitate cloning an

AccI site was introduced in the N-terminal of the domain resulting in a replacement of an alanine

residue by valine. Also, for chemical stabilization of the protein a glycine-to-alanine substitution

was performed, which also resulted in loss of binding to the Fab region (61, 63, 64). Jendeberg

and colleagues investigated the structure of the Z domain and when comparing it to the B

domain they found that the Z domain displayed an unaltered binding towards the hinge region of

the Fc-part of immunoglobulin. Moreover, it retains the structure of the B-domain, a three-helical

bundle with the helices placed in an antiparallel fashion (65, 66). Furthermore a comparison of

the kinetics of the B- and Z-domains interacting with Fc has been done and they were found to

be almost identical (51, 67).

13

Figure 2. A schematic illustration of staphylococcal protein A. Five homologous domains A-E shares the

immunoglobulin-binding feature. S is a signal peptide and the X and M domains are cell wall anchoring

parts. The three-helical bundle that constitutes the Z domain was engineered from the B domain.

Like SPA the Z domain has been used for protein purification, and the domain has also been

use as a fusion tag for purification of recombinant target proteins, by IgG capture (68). Another

application that the Z domain has been used for is site-directed capture or immobilization of

antibodies on cells or beads (69, 70). Efforts have been made to further improve and engineer

the Z domain which as an example has resulted in an alkali stabilized variant that is used

commercially as ligand in a resin for purification of antibodies or Fc-fused proteins (71;

MabSelect SuRe, GE Healthcare). The Z domain has also been the subject of the construction

of library with the intention to change the specificity from Fc to other molecules, i.e. creation of

so-called affinity ligands. In the library 13 positions in helix 1 and helix 2 in the domain are

randomized to create a novel interaction surface (72).

S DE A B C X M

Z

SPA

Immunoglobulin binding domains

14

As mentioned, among the advantages of the Z domain is the fact that it is small (6.7 kDa), and it

has a stable three-dimensional structure and also the capacity to refold (42). These features are

beneficial when performing chemical peptide synthesis, which the Z domain has proven suited

for, and thereby render the introduction of synthetic groups possible, extending the usability of

the domain (73).

15

Modificationofproteins

In the beginning of the year of 1990 a new journal saw the light, Bioconjugate Chemistry. In the

editorial of the first issue Claude Meares stated that the rationale for the new journal was that

bioconjugate chemistry is a growing field with rapid development and has a potential for

scientific breakthrough (74), and indeed the need for reliable conjugation strategies are still

found both in research and clinical applications. The term bioconjugate chemistry is described

as the joining of two different molecular functions chemically or biologically and the importance

of the strategy to add functions to proteins has been tremendous in the development of proteins

as tools in research and clinic. There are numerous functions that proteins can be provided with;

groups that either increase or decrease the immunogenicity of proteins, drugs, reporter groups,

enzymes and fluorescent groups, are a few examples. In a review in the first issue of

Bioconjugate Chemistry the history of chemical modifications of proteins are discussed and how

protein modification from the past were sprung out from organic chemistry with methods like

acetylation, iodination, deamination and reaction with formaldehyde (75). Today there are plenty

of commercially available kits and reagents that serve different purposes, but they predominantly

conjugate functional molecules to specific side chains in the protein.

The use of a specific kind of reactive group in the protein is the most common strategy when

modifying proteins and the predominant choice is to target the primary amines, preferably the ε-

amino group situated on lysine (76). Lysine is an abundant amino acid in proteins and it is a

good nucleophile over pH 8, which provides efficiency to the reaction. However, since a reactive

lysine can appear anywhere within the amino acid sequence the risk of influencing the proteins

active site is quite substantial. The thiol group in cysteine is also considered as highly reactive

and has the favorable ability to react at neutral pH, but cysteines are rather uncommon and

when they do appear in proteins they are often forming disulphide bridges and the reduction of

these might influence the stability of the protein. Nevertheless targeting a naturally occurring or

inserted cysteine provides a specificity to the modification of the protein that is beneficial in

some applications (77). Carboxylic acids that can be found in the side chains of amino acids

16

aspartic acid and glutamic acid can occasionally be subject to conjugations of reporter groups

although they display relatively low reactivity in water which is considered a disadvantage (76).

The introduction of specific reactive groups into the protein for conjugation of reporter groups

can be done on a genetic level and/or by the use of enzymes, and several elegant methods in

this field have been developed. Intein-mediated ligation is a method were the protein to be

modified is expressed recombinantly in fusion with an intein-chitin binding domain and during the

binding of the construct to chitin a cleavage can be performed and a peptide is ligated to the

protein (78). The holoenzyme synthetase BirA covalently attaches a biotin to the ε-amino group

on a specific lysine found in a substrate when enzyme and substrate are expressed in E. coli

and a site-specific modification can be obtained (79, 80). The Sortase family of enzymes found

in several gram positive bacteria are enzymes that covalently attach proteins with a specific

motif to cell wall proteins containing a poly-glycine peptide sequence and the system has been

exploited as a method for in vitro modification of recombinant proteins such as biotinylation of

scFv and PEGylation of proteins containing the motif for the enzyme (81, 82).

In the development of antibody-drug conjugates (ADC) as a therapeutic agent the general idea

is to utilize the antibody as targeting probe and the drug as payload to carry the therapeutic

effect. For this strategy to be successful the conjugation method used must be reliable since it is

vital that the drug stays bound to the antibody, minimizing risk for unspecific tissue damage. It

has been shown that the linker and the conjugation site influences the in vivo stability and

efficacy of ADCs (83, 84), which presents a strong incentive for the continuation of progress in

the field of covalent specific modification of proteins and antibodies.

Incorporationofunnaturalaminoacidsintoproteins

The capability to insert another amino acid than the 20 natural amino acids is most certainly a

powerful tool, thus the option to incorporate different functions or handles into the protein

appears. The possibility to introduce an orthogonal reactivity means that specificity in

conjugation can be obtained. There are two main routes to incorporate unnatural amino acids

into the amino acid sequence of proteins, recombinantly or through peptide synthesis.

17

In the cell when a protein is being synthesized the translation machinery reads the messenger

ribonucleic acid (mRNA) and the appropriate transfer ribonucleic acid (tRNA) charged with a

specific amino acid binds the corresponding triplet codon to insert the amino acid into the

growing peptide chain. The enzyme aminoacyl-tRNA synthetase is responsible for charging the

tRNA with the corresponding amino acid. This natural, protein production system in the cell was

exploited in the lab of Peter Schultz at the end of the 1980s and a new method was developed

(85). The employment of site-directed mutagenesis for the introduction of an amber stop codon

in combination with a corresponding chemically amino-acylated tRNA were proven to site-

specific introduce unnatural amino acids into proteins (85). In the beginning of the century

further steps were taken in the ambition of expanding the genetic code and progress was made

by genetical engineering of tRNAs to make them orthogonal to the bacteria’s own tRNA but still

specific for one, in this case, unnatural amino acid (86-88). A system for selection and

identification of aminoacyl-tRNA mutants specific for unnatural amino acids of choice has made

it possible to site-specific introduce functional groups into recombinant proteins to a high level of

efficiency and usability (89). Proteins, especially antibodies and fragments thereof, have been

modified through the use of orthogonal aminoacyl-tRNA synthetase/tRNA pairs (90, 91) and

approximately 70 unnatural amino acids has been added to proteins by the translational

machinery of E. coli, yeast and mammalian cells (92). Of special interest for the work presented

in this thesis, is the introduction of photoreactive groups such as benzoylphenylalanine and

azidophenylalanine that has successfully been incorporated into recombinant proteins produced

in E.coli (93, 94).

The ability to perform chemical synthesis and create shorter peptides developed during the first

half of the twentieth century (95, 96), but a true scientific revolution occurred when Bruce

Merrifield introduced a solid-phase method to synthesize peptides (97, 98). Until then, the

synthesis of peptides was performed in solution and even though at the time a biologically active

protein like oxytocin (99) was successfully produced there were disadvantages with poor

solubility, slow reaction rates and purification from byproducts being the main issues. Another

improvement Merrifield was responsible for was the use of t-butyloxycarbonyl (Boc) as a

temporary protecting group and as a result the synthesis could be performed under milder

reaction conditions. In 1964 Merrifield published the synthesis of the peptide bradykinin and

18

proved the increase of efficiency using the new chemistry (95, 100). Even milder reaction

conditions could be obtained by the introduction of the fluorenylmethyloxycarbonyl (Fmoc) group

and the development of Fmoc chemistry, which is today considered to be the main choice of

chemistry in solid phase peptide synthesis (SPPS) (95, 101, 102).

During the process of solid phase peptide synthesis the amino acid sequence grows from the C-

terminal to the N-terminal by one amino acid at the time. The synthesis starts with the first amino

acid being coupled to the solid support, which usually is a polystyrene resin. Every cycle in the

process contains the step of first cleavage of the protecting group of the amine situated on the

growing chain, then an activated amino acid, where a good leaving group has been placed on

the carboxyl group of the amino acid, is allowed to react with the amino group creating a peptide

bond. As a final step the protecting groups of the side chains and the release of the peptide from

the resin are performed in one step.

In the field of SPPS the development of different orthogonal protection strategies of side chains

gives a great benefit since it provides the opportunity to incorporate functional groups in a

specific manner. When using Fmoc as temporary protecting group, which is base-labile, the side

chains are protected with groups that are cleaved off with strong acid and a common orthogonal

protecting group that can be used on amines is the weakly acid-labile 4-methyltrityl (Mtt) group.

Other examples of groups that provide orthogonality in Fmoc chemistry are the allyloxycarbonyl

(Allloc) group, which is cleaved by palladium catalysis and the thiol-labile dithiasuccinoyl (Dts)

group (103-105).

Benzoylphenylalanine–BPA–aphotoreactiveaminoacid

Photoactivable probes have mostly been used in the field of mapping protein-ligand binding and

drug-target identification and they can be found in quite a variety and a number of these are

commercially available. The photo reactive groups mainly function through two routes. Either

they form a new covalent bond, photoaffinity labeling, upon irradiation or a special bond is

cleaved, photodeprotection (106). The strategy when performing protein-protein interaction

studies is to produce variants of a protein with a photolabile amino acid incorporated at different

19

positions. Followed by allowing the protein to bind its interaction partner and when exposed to

light of appropriate wavelength a covalent bond might be created and different methods will be

applied to analyze the interaction (107).

Benzophenone (BP) is a photophore that is one of the most commonly used photoactivable

probes since it is considered to be efficient, stable and can be handled in ambient light (108).

Another important feature related to BP is that its activation requires wavelengths of 350-360 nm

(109), which is above the wavelength regarded as damaging for proteins. Moreover it primarily

reacts with C-H bounds rather than water, which also is a great advantage (110). The structure

of BP is regarded bulky and to better serve its purpose it is preferably used in a hydrophobic

interaction site. However upon irradiation, if no weak C-H bound is able to react with the

activated triplet state of the benzophenone group it will relax to its ground state (109, 110). As

part of the synthetic amino acid benzoylphenylalanine (BPA), BP can be incorporated in a

peptide during solid phase peptide synthesis (108, 111).

20

21

PresentInvestigation

The work presented in this thesis focuses on the development of a strategy for covalent and

specific labeling of antibodies. Furthermore, the analysis of the method developed and the

investigation of the labeled antibodies in several applications have been performed. A

photoreactive reagent was developed and produced using solid phase peptide synthesis and

subsequently used to label different variants of antibodies. The thesis rests upon three papers,

in paper I the development of the technology, the production of the reagent and the specific

labeling of antibodies are presented. In paper II the functionality of the photolabeled antibodies

are compared with antibodies covalently labeled by another method. The antibodies were used

as a detection tool for proteins and to visualize protein targets in tissue using

immunohistochemistry. In order to investigate the possibility to obtain a higher signal and a

higher sensitivity in the use of the photolabeled antibodies, different variants of the photoreactive

reagents are compared and discussed in paper III.

The modification of proteins and covalent conjugation of other molecules covalently to proteins

are a frequent operation in biotechnology and most often the incentive is to add a feature to be

exploited in a particular application. The method and the chemistry utilized to enable the actual

conjugation ought to be efficient and leave the binding site of the protein unaffected. The most

commonly used method to couple reporter groups to proteins is to address the ε-amino group

situated on lysines in the protein by the use of NHS-esters (76). This method is indeed efficient,

but the absence of control of level and location of the conjugation could lead to an undesired

influence of the proteins active site since a protein often contain several lysines. Site-specific

covalent attachment of reporter groups to antibodies is important and there are many

applications both in the development of reagents for research and therapeutics for clinic where

this is highly desired. Antibodies are often modified by labeling with different parts of reporter

systems, such as fluorophores, biotin, haptens or enzymes, which makes them useful tools in

research and diagnostics, and conjugated with drugs for use in therapy. The greatest advantage

when employing a site-specific conjugation is the high likelihood for the binding site to be

preserved.

22

The method that has been developed in this thesis, aims for site-specific labeling of antibodies

by exploiting a modified Z domain incorporated with a photoreactive group. The Z domain has

been produced using SPPS that enables the incorporation of BPA as a synthetic amino acid in

specific position within the domain. The BPA should preferably be located in the vicinity of the

interaction site with the antibody. The strategy that has been established in this thesis makes

use of the Z domains natural binding to the Fc-part of the antibody. This inherited activity is

combined with the crosslinking features of the incorporated BPA and by exposure of light of a

defined wavelength a covalent linkage between the Z domain and the antibody will be formed

(Figure 3). Furthermore, taking advantage of orthogonal strategy, reporter groups can be

inserted at side chains of specific amino acids. In the first study a biotin was introduced at the C-

terminus of the Z domain. This reporter group was utilized in a number of different detection

methods. In the third study the reagent was extended with an additional biotin. By the use of

biotinylated photoreactive Z domains, several types of antibodies, polyclonal rabbit IgG,

monoclonal human IgG1 and monoclonal mouse IgG2a, have been site-specifically labeled and

analyzed.

Figure 3. An illustration of the developed strategy for covalent site-specific labeling of antibodies through

the use of a photoactivable Z domain. A Z domain inserted with a BPA molecule (blue round shape) and a

biotin binding the Fc domain of an antibody.

23

CovalentImmunoglobulinLabelingthroughaPhotoactivableSyntheticZdomain(PaperI) A strategy for site-specific labeling of antibodies was developed and a production route for a

photoreactive reagent is presented in paper I. Previously Jung and colleagues has reported the

utilization of an IgG-binding protein with an incorporated photoreactive group for attachment to

antibodies. By recombinant production of a C2 domain from streptococcal Protein G, modified

with two incorporated cysteines, a covalently attachment of a BP-molecule by maleimide

chemistry was made. Thereby, a photoreactive procedure could be used for covalent

modification of antibodies. It was demonstrated that this approach could be used for directed

antibody immobilization onto a surface (112). Our approach for covalent modification of

antibodies was to, by SPPS, produce two different Z domains with the photoactivable amino acid

BPA incorporated at position 5 in one variant and position 18 in the other variant. The variant

with BPA in position 5 displayed similar affinity to human IgG as the parental molecule, proving

that the insertion of the bulky group of BPA in the binding site of the Z domain had limited effect

on the affinity. The insertion of the amino acid BPA at position 18 in the Z domain proved to

have a large negative effect on the binding of the domain to human IgG since the affinity of that

variant was considerably lower than that of the wildtype domain. It was shown that the variant

with BPA in position 5 could successfully covalently couple to antibodies. However, the variant

with BPA in position 18 could not obtain any covalent attachment between the domain and the

antibody. Furthermore in paper I, it was shown that antibodies of various types and origin were

covalently and site-specifically labeled with the biotinylated Z domain. The photolabeled

antibodies were analyzed using the bead array Luminex system (113, 114). Two different set-

ups were used, both with antigen attached to beads but also with a sandwich set-up were

capture antibodies were coupled to the beads and incubated with antigen and finally detection

were made with photo-coupled detection antibodies and fluorescent streptavidin. Furthermore,

Western blot was successfully employed and show that the biotinylated photolabeled antibodies

could be used for detection in this application. Moreover, this technology was used to prove that

the covalent attachment is site-specific and occurs at the Fc part of the antibody when the

biotinylated Z domain with BPA is used. Further evidence on the specificity of the photolabeling

procedure was shown when samples containing IgG and human serum albumin were analyzed

24

using Western blot and Luminex. In these experiments it was shown that no biotinylation of

albumin occured. Surface Plasmon Resonance (SPR) technology was used to prove that the

procedure of photolabeling of the antibodies does not impair the binding of the corresponding

antigen. Hence, the conclusion of this is that the strategy for site-specific covalent labeling of

antibodies presented in paper I was successfully performed and tested in different applications.

AntibodiesbiotinylatedusingasyntheticZ‐domainprovidestringent in situ proteindetection(PaperII)

Protein detection in situ is of importance since the location of a protein in a cell is essential for

understanding its function. Immunohistochemistry and immunofluorescence microscopy are

methods that can be applied to establish protein location. Both methods rely on the use of

antibodies and their specificity and therefore it is imperative that the antibody or the reporter

system exclusively conducts specific binding events. For the detection of a binding antibody, a

secondary antibody, directed towards the primary antibody is often used. This is a concept that

works efficiently, but it would be of great advantage to reduce the number of experimental steps

by omitting the secondary antibody. This could be accomplished by directly link the primary

antibody to the reporter group. There are different techniques available for covalent labeling of

antibodies. Hence, we decided to compare the labeling technology using the photoactivable Z

domain with another commercially available technology, Lightning Link. In this project the

antibodies were applied in antibody-based protein profiling using immunohistochemistry. In total

13 antibodies were used in the comparison and labeled with the different systems. The obtained

staining patterns from tissue sections were compared to unconjugated antibodies, using a

secondary antibody for detection. It was found that the staining pattern from all 13 antibodies

labeled with the photoactivable Z domain were in concordance with the pattern from

unconjugated antibodies. As a comparison 3 out of 13 antibodies labeled with Lightning Link

produced staining patterns similar to those of unconjugated antibodies. Tissue microarrays with

18 formalin paraffin-embedded human tissues were used in the staining procedure with the

differently biotinylated antibodies. In conclusion, the method of antibody labeling of the

photoactivable Z domain proved to be stringent and specific.

25

Optimization of an antibody labeling strategy through the use ofphotoactivablesyntheticZdomains(PaperIII)

The method of labeling antibodies by the use of a photoactivable Z domain was further

developed. Antibodies biotinylated using the photoactivable Z domain has proven to be

successful in many different applications. However, in some applications question marks were

raised regarding the number of biotins and also the availability of the biotin for binding of

streptavidin influencing the sensitivity of the method. Therefore, two new variants of the

photoactivable Z domain were produced using SPPS to exploit the opportunity to insert the

photoreactive amino acid, BPA, in position 5 and biotin groups in a specific manner. First variant

was made with a biotin coupled to position 58 but with a PEG-linker in between the lysine in the

domain and the biotin. The second variant, compared to the first variant, had an additional biotin

with linker incorporated in position 39, where a serine had been exchange with lysine, i.e. one

biotin in each end of the three-helix bundle domain. The new variants were compared with the

original photoreactive reagent, the Z domain with one biotin coupled to a lysine in position 58..

The biotins in these three variants were used to immobilize the Z domains on a Neutravidin chip

and the affinities to antibodies were analyzed using surface plasmon resonance (SPR). Different

types of IgG was flown over the surface and the conclusion were made that the variant with two

biotins had a higher apparent affinity than the variants with only one biotin incorporated into

them when immobilized to a surface. The three variants were photo-coupled to several types of

antibodies and analyzed using the Luminex system utilizing beads that had been coupled with

corresponding antigen and the detection were made with fluorescently labeled streptavidin. The

result implied that antibodies coupled with the variant with two biotins provided a 100 % higher

signal in Luminex. However, the PEG linker that was incorporated between the protein domain

and the biotin does not affect the signal noticeably. Hence, the PEG-linker does not influence

the availability of the biotin. Moreover, the labeled antibodies were used in

immunohistochemistry and a stronger and more evident staining pattern was obtained with the

variant containing two biotins than when using a single biotin. Hence, a Z-variant with two biotins

gives a higher sensitivity and provides a higher signal compared to Z-version with a single biotin

in the application analyzed.

26

Concludingremarks

The modification of proteins, in particular antibodies, is increasingly important since the

exploration of proteins and antibodies as tools in research, diagnostics and therapeutics expand.

The functionalities that are most often of interest to combine with the protein or antibody depend

on the intended use of the system. Fluorophores, enzymes, haptens and drugs are all examples

of regular functionalities added to proteins or antibodies. The chemistry most often used could

suffer from lack of specificity of the labeling and thereby risk of influencing the binding site. To

increase the specificity of labeling a cysteine could be employed. However, there may be a

drawback, in vivo instability and the release of the conjugate as a consequence (84). There is a

need for new conjugations strategies since in some cases the methods normally applied do not

meet the requirements for antibodies used as tools or drugs.

The development and investigation of the strategy for covalent site-specific labeling of

antibodies using photoactivable Z domains is the foundation for this licentiate thesis. The SPPS-

produced photo-reactive reagents have been used to covalently biotinylate several types of

antibodies. The first study was done with a photoactivable Z domain containing one biotin and

the photolabeled antibodies was proved to operate successfully in a number of applications. The

metod of labeling antibodies by the use of the photoactivable Z domain was compared to a

commercially available kit for biotinylation of antibodies in the application of protein profiling, by

immunohistochemistry. It was found that the staining by the Z-labeled antibodies was in

concordance with the control staining and the performance of the system was satisfactory. To

further enhance the system the Z domain was equipped with a second biotin group. Indeed

antibodies coupled with the Z domain containing two biotins showed a higher signal when

binding its antigen on beads and also a stronger staining in IHC. The photoactivable Z domains

have proven to covalently and specifically label antibodies successfully and shown to be a

valuable tool for many biotechnological applications, in research, diagnostics and also potentially

for use in therapeutics.

27

Acknowledgements

Det är mer än en handfull gånger under årens lopp som jag skänkt denna delen av

avhandlingen en tanke eftersom jag tillhör den känslosamma (sentimentala) sorten. Och nu när

jag väl ska nämna er som har gjort detta arbete möjligt, är ni så många, och det är jag verkligen

tacksam för. Tack till alla er som jag inte nämner men ni är många som ler i korridoren, hjälper

till i labbet eller vid kopiatorn eller helt enkelt delar en trevlig stund vid lunchbordet.

Min handledare Sophia, det har varit otroligt lärorikt att jobba i din grupp och jag har fått

chansen att utveckla så mycket under denna tiden. Jag är riktigt stolt över det arbete vi gjort.

Men ibland vet jag inte om jag ska önska dig ett lugnare arbetsliv eller en snabbare cykel.

Amelie, som bihandledare har du varit närvarande och hjälpt mig mycket, inte minst med alla

knepiga syntes-grejer.

ProNova Vinn Excellence center for Protein Technology för finansiering.

I ProNova vill jag tacka Amelie och Per-Åke som styrt upp hela centret men också alla övriga

medarbetare inom affinity-gruppen, Feifan, Peter, Mahya, Afshin och Anna P. Jag vill också

rikta ett stort tack till de företagsrepresentanter som deltagit i ZBPA-projektet som jag haft

kontakt med; Niklas Ahlborg (Mabtech), Mats Inganäs (Gyros), Åke Danielsson (GE), Bo

Jansson (Bioinvent), Simon Fredriksson och Mats Gullberg (O-link) och Henrik Johannesson (Atlas Antibodies). Jag har verkligen värdesatt att få ta del av ert yrkeskunnande.

Jag vill tacka övriga PIs, och Stefan för att det alltid känns som om du lyssnar och stöttar.

Hjältar lite mer bakom kulisserna som styr upp administrationen Inger, Kristina, Mona och

Christelle. Emma Ö för att du gjort livet i labbet så mycket enklare med grym ordning i

diskrummet och avlastning av inköp.

Jag är verkligen tacksam för den granskning och hjälpt jag fått med avhandlingen; Tobbe,

Andreas, Amelie och Basia.

28

Mina riktigt grymma ex-jobbare, Anna P, Josefin och Sara, jag gillar att ni alla stannade kvar,

för jag låtsas att jag har en liten del i det. Tack för era insatser i projekten. Anna Torell Holm för

det lilla gästspelet i C2-projektet.

Tack Tobbe, för att jag fick göra ett fantastiskt roligt ex-jobb i din grupp. John, Johan N och

Magdalena för ovärderlig hjälp med SPR. Ett stort tack för all hjälp med Luminex, Jochen, Maja

och Ulrika. Gustav som kört och pratat mycket MS med mig.

Sandra, Nikhil, Anna A och Fredrik för ett lyckat samarbete. Tack för de fina bilderna Sandra.

Tack till alla som jag delat grupptillhörighet med och haft både vetenskapliga och andra roliga

diskussioner med och mycket skojiga aktiviteter, Sophias grupp; Cilla, Tove A, Johanna,

Hanna, Karin, Margareta, Johan, ToveBo, Sara, Cajsa, Jenny OT, Henrik W, Micke och

Amelies grupp; Anna P, Joel, Peter, Daniel, Nima och Kristina.

Jenny OT, du anställde mig i Proteinfabriken, tack för det förtroendet. Tack till Hanna och

Johanna, det var otroligt roligt att vara foing med er ”på den gamla goda tiden” . Tack också till

Lanlan, Pawel, Asif, Ulla, Kattis, Jens, Hero, Lasse, Anna B, Louise och övriga Profabbare.

Ett tack också till Mathias och alla andra i HPA för årliga träffar nära och långt bort och alla

roliga människor som gör den fantastiska atlasen. Ett speciellt tack till immunotech för generös

hjälp med WB och till alla i molbio som accepterat mina intrång i gruppen med jämnmod.

Tack till alla er jag delat skrivrum med och som verkligen gjort stor skillnad i vardagen, Emma,

Tove A, Cecilia, Pawel, Mårten, Julia, Johan, Charlotte, Magdalena, Burcu, Chi, Filippa,

Andreas och nu på slutet, nya kompisar som jag inte riktigt hunnit lära känna, Aman, Ali och

Pavan.

Det är mycket och många som jag kommer att minnas med glädje från livet på plan 3, alla

trevliga reskamrater under flertalet konferenser som i soliga Portugal; Basia, Anna P, Johan N.

Joel, Amelie, Sophia med sina fina pojkar Marcus och Jonathan. Det stora tjej-gänget i

Boston; Nina, Basia, Camilla, Anna P, Lisa, Johanna, Hanna L och i underbara Bologna;

Tove A, ToveBo och Sophia. Slutligen John och Stefan i San Diego när vi hängde med

Andreas, Caroline och Elin. Stor kram till dig Sverker, det var kul att starta upp

Socialförvaltingen med dig och tack till alla som fixat/fixar aktiviteter för andra på avdelningen.

Smarta och underbara Basia, lika knäpp som jag, tack för all hjälp med avhandlingen och alla

skratt. Alla som druckit fredagsöl med DN-kryss, och sköna öl-snubben, Peter, vi har handlat öl

29

och labbat tillsammans, det var grymt. Härliga och viktiga små stunder som kaffe, lunch, frukost

och/eller AW; Nina, Emma, Julia, Basia, Helena, Cilla, Maja, Johanna, Tove A och flera

andra fina personer.

Mina kära Kittel-tjejer, Emma, Nina, Julia, Tove och Torun, den årliga skid-resan till Kittel är

helig för mig. Det en sådan grym höjdpunkt på året och förbaskat roligt. Nån dag ska jag fanimej

klara mig hyfsat utanför pisten också.

Lena du kommer som en frisk fläkt och delar generöst med dig av dina upplevelser, resor och

om stort och smått. Helt underbart, jag är uppfylld flera dagar efteråt och är tacksam för att du

kom “med i gänget” även om det var lite sent, som i sjuan typ. Självklart jag hänger med på en

träningsresa framöver.

Ulla och Jenny, det känns som om jag känt er hela livet. Verkligen magiskt, tack för att ni finns

och för luncher, stöd och framförallt alla skratt. Ni vill mig så väl och jag önskar er allt gott som

finns på jorden.

Beda, du är som en syster och med dig delar jag i princip hela min barndom och ungdom och

utan dig hade jag aldrig kommit i tid en enda gång under högstadiet. Tack för alla fina minnen

från Österlen och i mormors trädgård med våra familjer.

Mina nära vänner och Kajsas gudföräldrar varma, fina Monki, och påhittiga, långa Rillo. Tack

för alla resor, helger på Rådmansö och allt vi delat genom många, många år. Ni finns alltid där

och ni är fantastiska gudföräldrar, världens bästa.

Familjen Frank-Bille, Petra, Magnus & Robert, er trio är så oerhört viktig får vår trio. Inte för mitt

liv hade jag kunnat föreställa allt vi nu delat när vi träffades på föräldrautbildningen. Minnen på

flera nivåer, resor och skidåkning med alla de äventyren, men framförallt alla middagar och

samtal i våra respektive kök under årens lopp. Jag ser framemot att dela minst lika många

äventyr till i livet.

Till härliga och fina kusin Noak med familj, det är verkligen bra att ni finns och tack Noak för att

du livar upp oss lite när vi ses. Tack också till min svärmor Ann-Mari, jag känner så ofta hur du

hejar på mig, tack för ditt stöd och till Peter, Anni, Anders, Martin, Christina, Perka, Linnea

och Henrik som accepterat mig så gott det går trots ”svår” dialekt.

30

Min kära, älskade mamma du har klarat av så mycket och kämpat så hårt, jag hade önskat att

du hade fått bli mer belönad. Det är inspirerande att tänka på hur förbaskat duktig du var på ditt

jobb och hur du jonglerade allt långt innan termen “livspussel” var uppfunnen. Tack för att du

alltid hjälpt till när vi behövt det.

Min mentor, Eva-Britt, du har väglett och coachat mig till ett fantastiskt liv, ibland har det känts

som du räddat livet på mig, tack.

Kajsa och Staffan, ni betyder allt. Kajsa min smarta, vackra, roliga och älskade unge, jag kan

inte tänka mig livet utan att få vara din mamma. Staffan min älskling, jag är så tacksam för att vi

har levt ihop i 19 år och mer än nånsin känner jag hur mycket vi båda vill fortsätta leva ihop. Jag

älskar er.

31

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