IN DEGREE PROJECT BIOTECHNOLOGY,SECOND CYCLE, 30 CREDITS

, STOCKHOLM SWEDEN 2019

The synthesis and analysis of a bombesin analogue for radiotherapy of prostate cancer

ÁBEL NAGY

KTH ROYAL INSTITUTE OF TECHNOLOGYSCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

1

THE SYNTHESIS AND ANALYSIS OF A

BOMBESIN ANALOGUE FOR

RADIOTHERAPY OF PROSTATE CANCER

Ábel Nagy

Supervisor: Amelie Eriksson Karlström

Co-supervisor: Hanna Tano Examiner: Yves Hsieh

Master Thesis

KTH School of Engineering Sciences in Chemistry, Biotechnology and Health

Department of Protein Science, Alba Nova

May 2019

2

Table of content

Table of content ........................................................................................................................ 2

Abstract ..................................................................................................................................... 4

Key words ................................................................................................................................. 4

Introduction ............................................................................................................................... 6

Prostate cancer ................................................................................................................................... 6

Peptide radiopharmaceuticals and radionuclide therapy .................................................................... 6

Bombesin receptors ............................................................................................................................ 9

Half-life of protein therapeutic and albumin binding domain .......................................................... 10

Solid phase peptide synthesis ........................................................................................................... 12

Materials and Methods ............................................................................................................ 15

Design of bombesin analogue RM26 ............................................................................................... 15

Design of ABD molecule ................................................................................................................. 16

Synthesis of ABD ............................................................................................................................. 17

Synthesis of bombesin RM26 analogue ........................................................................................... 19

Purification of ABD and bombesin RM26 analogue ........................................................................ 20

Conjugation of ABD and bombesin RM26 analogue ....................................................................... 20

Purification of the conjugate ............................................................................................................ 20

Recombinant expression of ABD035 ............................................................................................... 21

Binding analysis with Surface Plasmon Resonance ......................................................................... 22

Results ..................................................................................................................................... 23

Synthesis and purification of ABD .................................................................................................. 23

Synthesis and purification of bombesin RM26 ................................................................................ 24

Conjugation of ABD and bombesin RM26, purification of the conjugate ....................................... 26

3

Production of ABD035 ..................................................................................................................... 28

Binding analysis with Surface Plasmon Resonance ......................................................................... 29

Discussion ............................................................................................................................... 31

Future perspectives .................................................................................................................. 34

Acknowledgements ................................................................................................................. 34

References ............................................................................................................................... 34

4

Abstract

Targeted radionuclide therapy is becoming a widely used cancer treatment strategy. By

radiolabeling receptor-specific peptides, cancer cells overexpressing the receptor can be

selectively targeted, and the cytotoxic radionuclide can be delivered to the target cell or tissue

for therapeutic or diagnostic purposes. Bombesin analogues have been previously developed

and utilized to target the gastrin-releasing peptide receptor (GRPR), a receptor commonly

overexpressed in prostate cancer cells. The RM26 analogue derived from the native bombesin

is an antagonistic ligand of GRPR and a possible candidate for targeted radiotherapy.

Prolonging the half-life of the molecule is an important aspect of developing a new protein

therapeutic. Using albumin binding domain (ABD) for this purpose is an emerging strategy in

recent years. ABD is able to bind to serum albumin and thus remains in the blood circulation

for a long period of time. It is also a scaffold for protein engineering efforts and by coupling

receptor-specific ligands to ABD, the target-specific binding along with extended in vivo half-

life can be achieved. In this project, an RM26 analogue with a PEG linker and ABD with a

DOTA chelator for future radiolabeling were synthesized with solid phase peptide synthesis

(SPPS), conjugated, purified by RP-HPLC and analyzed by mass spectrometry. The binding

properties of the conjugate were evaluated by SPR-based biosensory studies, and further

experiments are planned for the testing the product and its potential application in radionuclide

therapy.

Riktad radioterapi är en allt vanligare metod för behandling av cancer. Genom att radioinmärka

receptor-specifika peptider kan dessa selektivt levereras till tumörceller som uttrycker

receptorn. Radioterapi kan användas för diagnostik eller terapi, beroende på kopplad

radionuklid. Bombesinanaloger har utvecklats och använts för att selektivt binda

gastrinfrisättande peptidreceptor (gastrin-releasing peptide receptor, GRPR), en receptor som

ofta är överuttryckt i prostatacancer. Bombesinanalogen RM26, som har sitt ursprung från

nativt bombesin, är en antagonist till GRPR och kan möjligen användas för riktad radioterapi

av prostatacancer.

Vid utvecklingen av nya proteinläkemedel är halveringstiden i serum en viktig aspekt. En

nyligen utvecklad strategi för att förlänga halveringstiden i serum är fusion av det

tumörspecifika proteinet till en albumin-bindande domän (ABD). ABD binder till albumin, och

således kan fusionsproteinet bevaras i blodcirkulationen under en längre tid.

5

I detta projekt, har både RM26 med en PEG-linker, och ABD med en DOTA kelator

syntetiserats med fastfaspeptidsyntes (solid phase peptide synthesis, SPPS). RM26-PEG och

DOTA-ABD har därefter konjugerats, renats med RP-HPLC och analyserats med mass-

spektrometri. Bindning till albumin har utvärderats med ytplasmonresonans (surface plasmon

resonance, SPR). Vidare studier planeras för att utvärdera peptid-proteinkonjugatet och dess

potential för riktad radioterapi.

Key words

Prostate cancer, radionuclide therapy, bombesin, gastrin-releasing peptide receptor, albumin

binding domain, SPPS, RM26 analogue

6

Introduction

Prostate cancer

Prostate cancer (PC) is the second most frequently diagnosed type of cancer after lung cancer

in men with almost 1.3 million new cases in 2018 and it remains one of the leading causes of

cancer-related death [1]. Screening and diagnosing prostate cancer in an early stage is important

in order to reduce mortality and prevent death through treatment. Since prostate cancer is

generally a slow growing malignancy, it is common that symptoms do not arise in the early

stages of the cancer development and the clinical diagnosis occurs too late, when treatment is

already problematic. Current methods for screening PC are based on a prostate-specific antigen

(PSA) blood test and a physical prostate examination. PSA is a widely studied serine protease

and the elevated level of PSA has been the indicator of PC for a long time. However, it has

been shown that elevated levels of PSA along with the enlargement of prostate glands can be

the result of other factors such as prostatitis or benign prostatic hyperplasia, making PSA-based

tests unspecific and unreliable for screening [2]. Overall, the currently used methods for

detecting PC are insufficient on their own, and it remains challenging to conveniently screen

and diagnose PC.

As for many types of cancer, metastasis complicates the treatment of PC. It commonly spreads

to the bone and soft tissue and the extent of the cancer determines the therapy. The therapy to

be applied considers the metastasis of the tumor and assesses how far the cancer has spread. As

the cancer progresses the properties of the tumor change. The tumor transitions from an

androgen-responsive growth to a hormone-refractory cancer, which further complicates the

type of therapy to be issued. When the cancer is diagnosed in an early stage and in a localized

tumor form, the surgical removal of the prostate gland and radiation therapy can be a solution

as a treatment. For late stage PC when the cancer has already metastasized, androgen

deprivation therapy (ADT), as a first-line treatment, and testosterone suppression offer the best

treatment.

Peptide radiopharmaceuticals and radionuclide therapy

Conventional treatments for cancer such as chemotherapy or ADT for prostate cancer have been

revolutionary in terms of improving and prolonging lives of patients. However, these therapies

7

are often associated with adverse side effects and toxicity resulting from the lack of target

specificity. Nowadays, therapeutics and therapies selectively targeting cancer cells have

become a widely used strategy in cancer treatment. One of the emerging strategies is

radionuclide cancer therapy and the use of radiopharmaceuticals, a strategy that utilizes the

combination of receptor-specific peptide ligands targeting cancer cells and selective delivery

of the cytotoxic radionuclide in order to achieve successful treatment or diagnostic.

There is a large variety of short peptides, often referred to as regulatory peptides, throughout

our body that regulates a number of important biological processes. Many peptides are known

to function as neurotransmitters, hormones and other chemical messengers, but they can also

act as highly active stimulators or inhibitors of different pathways [3]. These peptides typically

target and bind to membrane-associated receptors, most commonly G-protein coupled

receptors. In regard to cancer studies, it has been well described and studied that these receptors

are often abundant and overexpressed on different tumor cells. This makes these receptors and

their binding peptide ligands ideal candidates for targeting tumor cells. By radiolabeling these

receptor-specific peptides and proteins, they can be selectively delivered to the tissues and cells

that are overexpressing the given receptor. Radionuclides can be both used for imaging and

therapeutic purposes by directing radio-emitting, cytotoxic particles to the cancer cells. In

nuclear medicine different positron emission tomography (PET) and single photon emission

computed tomography (SPECT) methods are used as imaging modalities, to detect

radiopeptides labelled with radionuclides (most commonly 65Ga or 111In).

The structure of a peptide-based radioconjugate is very important for receptor binding and

discriminating between normal and target cells. A radioligand usually consists of three parts: a

receptor-binding peptide, a linker and chelating agent. The receptor-binding peptide is in charge

of target recognition; it is usually an agonist or antagonist analogue of the native ligand,

modified in a way to achieve higher affinity or selectivity. Introducing a spacer between the

peptide and the radionuclide contributes to the pharmacological properties and the behavior of

the radioconjugate. It could prevent the interference of the chelator with the binding site and

increase the efficiency of the binding. The chelator agents such as DOTA or NOTA are used

for coordinating metal ions and radionuclide labeling. Choosing the right components of a

radioconjugate is an important part of the design of these molecules, since they can affect the

outcome of receptor recognition, binding etc. [4]

8

Figure 1 Schematic structure of a radioconjugate.

The first peptide-based imaging agent, a somatostatin analogue named 111In-DTPA0-octreotide,

was introduced for targeting somatostatin receptors and it has been applied to the imaging of

Neuroendocrine Tumors (NETs) [5]. Since the success of somatostatin analogues, other

receptors have been in the center of attention such as GRPR, targeted by bombesin analogues,

the glucagon-like peptide receptor (GLP-1R) and other G-protein coupled receptors.

Radiopeptides have a number of advantages over other frequently used pharmaceutical

molecules such as antibodies and other therapeutic proteins. One of the biggest advantages is

the small size of these molecules and a number of beneficial characteristics can be explained

by their small size. They have excellent pharmacokinetic properties for molecular imaging, they

are able to penetrate the tumors very efficiently and they have high receptor binding affinity

[3],[6]. Also due to their small size the clearance from the blood and non-target tissues is rapid.

There are techniques such as solid-phase peptides synthesis (SPPS) that make it easy to produce

molecules with the desired characteristics in a short period of time and in an automated fashion.

The radiolabeling of the peptides is also possible with the addition of a chelating agent at the

C- or N-terminus of the peptide. From a therapeutic point of view radiopeptides have very few

side effects and they are less immunogenic compared to other protein therapeutics.

Radionuclide-labelled molecules are already making their way into the drug market or clinical

trials and pharmaceutical companies are expanding their push into radiotherapeutics. A good

example of the increased interest for radiolabelled drugs is Novartis, which has recently bought

the commercial rights to Lutathera, a peptide-based 177Lu- labelled therapy already on the

market. Novartis also made a huge investment to acquire Endocyte, a similar drug targeting

prostate cancer [7].

Chelator withradionuclide

Biomolecule ReceptorLinker

9

Bombesin receptors

One of the most studied cancer-associated receptors for targeted diagnostics or therapy is the

gastrin-releasing peptide receptor (GRPR), which has been in the focus of researchers along

with its ligand peptides and analogues. GRPR is G-protein coupled glycoprotein in the cell

membrane and it is overexpressed in a variety of cancers like prostate cancer, small cell lung

cancer, breast cancer, colon and many more, indicating a crucial part in malignant growth.

GRPR is activated by a group of ligands called bombesin-like peptides (BLP), a peptide family

consisting of members of different origin, found in both amphibians and mammals. In mammals

BLPs regulate through receptor binding a number of physiological functions of the

gastrointestinal tracks and nervous system, such as hormone release, but they also play role in

smooth muscle contraction, modulation of metabolism and most importantly cell proliferation

in cancer cells [8], [9]. The first member of the BLPs to be described was bombesin (BN), a 14

amino acid peptide originally discovered and isolated from the skin of amphibian species

Bombina bombina [10]. Later on, a 27 amino acid long human BN homolog, gastrin-releasing

peptide (GRP) with corresponding biological function was identified along with the third

member of the BLPs, a decapeptide named neuromedin B (NMB). All three peptides are

structurally highly conserved and show high homology in the carboxy-terminal amino acids

(see Table 1). With the exception of one amino acid, the carboxy-terminal decapeptide of GRP

is identical to that of BN, and NMB only differs in two amino acid positions in the same region

of GRP and BN. BN receptors can be divided into four subtypes according to the binding of its

native ligand: GRP receptor (GRPR), neuromedin B receptor (NMBR), and bombesin receptor

subtype 3 (BB3) and subtype 4 (BB4). It is known that bombesin binds with high affinity to

GRPR and NMBR, but the natural ligand of BB3 and BB4 is as of now unknown. BB3 is

expressed in mammals while BB4 is present only in amphibians [11]. These receptors are

members of the G-protein receptor superfamily meaning that the intracellular domain is coupled

to a G-protein and the agonist binding of BLP peptides activates several signal mechanisms

including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), tyrosine

phosphorylation of focal adhesion kinase (FAK) and calcium mobilization, overall eliciting cell

proliferation and growth [12],[13].

10

Table 1 Bombesin receptors and their corresponding ligands.

Receptor Native peptide ligand

Sequence of corresponding ligand Origin

NMBR NMB Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2 Mammalian

GRPR GRP

Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-

Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2

Mammalian

BB4 BN[Leu13] Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

Amphibian BN[Phe13] Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH2

BB3 Not identified Mammalian

Peptide agonists targeting receptors overexpressed on tumor cells has been the most common

type of ligand in nuclear medicine and in radionuclide therapy. The biggest advantage of agonist

binding is the following internalization and cellular uptake of the ligand, which contributes to

accumulation of radioactivity inside the cells, thus helping the imaging and therapeutic

purposes. Antagonists are inhibiting their targets thus not triggering internalization. Recently

however, a somatostatin antagonist has been found more effective in terms of biodistribution

than its agonist counterpart, displaying higher in vivo tumor uptake [14]. This finding has

initiated further research on antagonists and similar results have been found for bombesin

antagonists. Another advantage of using an antagonist is that the endocrinological and other

side effects observed in studies that were caused by agonist receptor binding could possibly be

avoided.

Half-life of protein therapeutic and albumin binding domain

One of the most important properties of therapeutic biomolecules is their half-life, a

pharmacokinetic parameter indicating the time that takes for the drug molecule to be removed

from the circulation of the patient. Longer half-life can lead to less frequent drug administration

or smaller doses making drug usage more convenient for the patients. Thus, for the

pharmaceutical industry extending the half-life of their therapeutic agent is a very important

aspect of drug development process.

11

The metabolic fate of protein therapeutics, similarly to other endogenous proteins, is complex;

they are metabolized by different catabolic mechanisms. One of the most significant pathways

is proteolysis, the degradation of proteins and peptides into free amino acids with the help of

proteolytic enzymes. As the liver is considered the center of metabolism, hepatic elimination is

the largest contributor of protein elimination. However, enzymes responsible for proteolysis are

actively present throughout our whole body and proteolysis is not restricted to metabolically

active tissues and organs such as liver and kidney, but can also occur in the blood. Another way

of protein elimination is receptor-mediated endocytosis, an intracellular degradation pathway

following the receptor-mediated uptake of the proteins. While most proteins are not subject to

renal or biliary excretion, for some proteins it plays an important role in protein clearance.

Smaller size proteins up to 40-50 kDa are especially exposed to renal filtration or biliary

excretion [15]. In the kidney plasma is filtered through the glomerular capillary and the complex

structure of the glomerular filtration barrier (GBM) prevents the passing of most proteins. The

fenestrated endothelium and slit diaphragm of the capillary presents a physical barrier for

macromolecules along with an anionic barrier provided due to proteoglycans of the endothelial

cells.

When it comes to designing a protein therapeutic to improve its half-life, not only the size of

the molecule but other physicochemical properties should be taken into account [16]. Most

techniques aim to extend the half-life of protein drugs by increasing the hydrodynamic volume

of the drug to avoid early clearance, while other methods have been utilizing the extraordinarily

long half-life of serum albumin and IgG molecules. The 2-4 weeks long half-life of these two

molecules is explained by their ability to bind to the neonatal Fc receptor (FcRn) during the

cellular uptake [17],[18]. By binding to FcRn in the early endosome, albumin and IgG will be

recycled and released back into the plasma, unlike non-receptor bound protein that is degraded

in the lysosome. Constructing Fc-fusion proteins, albumin fusion proteins, and conjugates with

the purpose of serum albumin binding in the plasma are already widely used techniques to

increase the half-life of protein therapeutics [19].

The discovery that many gram-positive bacteria are able bind to serum proteins (mostly

albumin) through their special proteins expressed on their surface have made them an

interesting subject for research and a promising opportunity to use bacterial proteins for

prolonging half-life of therapeutics. For the bacteria, this binding serves as a strategy to avoid

the detection of the host immune system and a way to promote growth and virulence as well as

access to nutrients of the host [20], [21]. There is a large variety of surface serum binding

12

proteins, collectively called albumin-binding proteins, and the structural domain that they have

in common is called albumin-binding domain (ABD) and it is responsible for mediating the

binding to albumin. One of the most studied surface proteins is streptococcal protein G (SPG)

derived from certain streptococcal strains. This large complex protein contains three

immunoglobulin-binding domains (C1-C3) and three albumin-binding regions (ABD1-3) [22].

The three ABD domains are homologs and ABD3 (referred to as G148-ABD, named after the

streptococcal strain G148) has been studied the most. The 46 amino acid domain folds into a

left-handed anti-parallel three helix bundle, and this particular motif can also be found in several

other proteins due to its energetically and functionally beneficial properties.

ABD is a great subject and a suitable scaffold for additional protein engineering efforts that aim

to introduce new or improved properties for the molecule. Large libraries with potential new

variants have been created and screened. One of the new variants ABD035 has shown an

increased affinity to HSA along with other beneficial properties [23], [24]. The idea of using

ABD and protein engineered variants to extend the half-life of protein products has gained

attention in research and is getting more popular in the pharmaceutical industry as well.

Figure 2 A - Structure of albumin binding domain. B - ABD binds to serum albumin, red circle indicates ABD. Figures from PDB database, PDB IDs: 1GJT and 1TF0

Solid phase peptide synthesis

Synthetic peptides have been important tools in biochemical research for a long time. With the

help of these molecules, protein-protein interactions and receptor binding of naturally occurring

peptides or analogues can be studied, among many other applications. The introduction of solid

N

C

A B

13

phase peptide synthesis (SPPS) by Bruce Merrifield in 1963 was a breakthrough in peptide

chemistry and synthesis [25]. This new method enabled the development of simplified and less

time-consuming techniques compared to the previously available solution-based strategies for

peptide production. The main principle of SPPS is that the assembly of the peptide from its

amino acid building blocks takes place on a solid, insoluble support, usually on resin beads.

This setup is stable to the repetitive chemical reaction steps during the synthesis and allows the

use of large excess of reagents that can drive the coupling reactions to completion. The use of

the solid phase also allows the application of washing steps to separate the growing peptide

from side products and reagents in the same reaction vessel and without transferring material.

This system also makes the automation of the synthesis process possible; instruments are now

available that are capable of automatically performing every step required to produce a

particular peptide, with a pre-programmed amino acid sequence.

The general process of SPPS includes repetitive cycles of formation of peptide bonds between

the amino acid building blocks. These steps include coupling, deprotection, capping and

protecting group cleavage. As the first step, an Na-protected amino acid is coupled to a resin

(polymeric material modified with hydroxy- or amine-functionalized anchoring linkers) via its

a-carboxyl group. In order to facilitate the formation of the peptide bond, the a-carboxyl group

has to be activated to help the nucleophilic attack by the a-amino group of the other residue

and to generate a good leaving group. The classically used reagent for activation of carboxyl

group is N,N’-dicyclohexylcarbodiimide (DCC). Activating the amino acid with DCC results

in the formation of O-acylisourea, which then can acylate the a-amino group of the other

residue (resin or amino acid). Since the application of DCC can lead to other side reactions, the

addition of alcohols with low pKa such as HOBt, HOAt, has been used to produce active esters

of the protected amino acid. These activated esters are less reactive than O-acylisourea, but can

still efficiently acylate the amino group. Another group of phosphonium and uranium coupling

reagents based on the generation of HOBt esters, HOAt esters, acyl bromides etc., has become

widely popular along with DCC. These are BOP, PyBOP, TBTU, etc. Diisopropylcarbodiimide

(DIC) has been used instead of DCC in SPPS, because while dicyclohexylurea, a byproduct of

DCC is almost insoluble in organic solvents, the urea from DIC remains in solution. When it

comes to the temporary protection of the a-amino group, two strategies have been used, namely

Boc/benzyl and Fmoc/tBu approach. The difference between the two strategies is in the removal

conditions. The temporary Boc protecting group on the amino function of the amino acid is acid

14

labile, easily removable by a neat trifluoroacetic acid (TFA) or a TFA in dichloromethane

(DCM) washing step. In this strategy it is important that the protecting groups on the side chains

are stable to TFA and the benzyl group is widely used in combination with Boc. The benzyl-

based protecting groups are cleaved at the end of the synthesis by super strong acids such as

HF. The Fmoc/tBu method is based on the base-labile property of Fmoc, which is stable to

acids but can be cleaved by bases such as piperidine in DMF. The side chain protecting groups

consist of tert-butyl groups which are stable to base but are cleaved by moderately strong acids

such as TFA in the final cleavage step.

Figure 3 General scheme of solid phase peptide synthesis.

A

oNH

R1

Fmoc

X

oNH

R1

Fmoc

Loading of the resin

X

oH2N

R1

Removal of Fmoc, temporaryNalpha protecting group

X

o

R1

A

oNH

R2

Fmoc

Coupling of the nextactivated amino acid

R2

o

NH

X

o

R1

R2

o

NH

HN

o

R3

NH

Fmoc

n

NHFmoc

X

o

R1

R2

o

NH

HN

o

R1

H2N

n

n cycles

SPPS cycle

Linker

Linker

Linker

Linker

Linker

Linker

Fmoc

Side chain protectinggroup

Support, Resin bead

Fmoc, temporary Nalpha protecting group

A Activating group

15

Materials and Methods

Design of bombesin analogue RM26

Several bombesin analogues have been developed as GRPR antagonists with the purpose of

selective binding to GRPR. The bombesin analogue used in this project is based on the earlier

reported bombesin RM26 analogue [26]. This molecule has been designed in a way to improve

its binding and other properties compared to the native bombesin molecule. It has been shown

that the C-terminal end of bombesin (residues 6-14) is responsible for receptor recognition and

binding affinity, thus most analogues designed for receptor binding purposes are based on this

part of bombesin. The RM26 variant contains three modifications of the truncated bombesin

(6-14), at the following amino acid positions: D-Phe6, Sta13 and Leu14. The replacement of Leu

with statine at the 13th position was suggested by the findings from a study where analogues

containing a statyl residue were evaluated and where it was shown that the presence of an

additional hydroxy group in this position of the peptide could play an important role for receptor

affinity [26]. It has also been discovered that antagonistic receptor binding can be achieved by

modifying the amide bond between residues 13 and 14 or by deleting the C-terminal amide

function. This explains the introduction of Leu at the 14th position [27].

In the construct designed for this project, a (PEG)4 linker was introduced in the N-terminal of

the peptide to provide a spacer between the receptor-binding ligand and the ABD molecule used

for half-life extension. A terminal cysteine was then coupled to (PEG)4 at the N-terminal end

of bombesin for the purpose of crosslinking to ABD. The thiol group of Cys and the

chloroacetyl group introduced in ABD are responsible for the chemoselective conjugation and

the formation of a thioether bond between ABD and the bombesin analogue RM26. In this

project another construct was also synthesized and used, differing in the N-terminal residue. In

one construct cysteine was present, and in the second, mercaptopropionic acid. Similarly to

cysteine, mercaptopropionic acid contains a nucleophilic thiol group and forms a thioether bond

with haloacetyl functional groups.

16

Table 2 Sequences of different bombesin constructs.

Bombesin (1-14):

Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

Bombesin RM26 analogue (D-Phe6, Sta13, Leu14) bombesin (6-14):

D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2

Bombesin analogues for ABD conjugation:

Cys-(PEG)4-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2

Mercaptopropionic acid-(PEG)4-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2

Design of ABD molecule

For the purpose of extending the half-life of bombesin analogue RM26, a modified version of

the albumin binding domain variant ABD035 was used in my project. These modifications

include the attachment of a DOTA chelator to the N-terminus and the addition of a chloroacetyl

functional group to the side chain of the Lys14 residue, for the purpose of radiolabelling and

bombesin conjugation, respectively. DOTA chelators form stable complexes with radiometals

(in our case 117Lu), thus enabling the attachment of such radiolabels for imaging or therapeutic

purposes. 117Lu is a beta-emitting isotope commonly used for radionuclide therapy and imaging.

The amino acid sequence of the construct for solid phase peptide synthesis can be seen in Table

3. During SPPS, the underlined amino acid residues were double-coupled due to the bulky side-

chain protecting groups on these amino acids. The side chain of lysine at the 14th position was

protected with a special 4-methyltrityl (Mtt) protecting group in order to be able to deprotect it

separately from the other amino acids. Mtt can be cleaved off in mild acidic conditions, in

conditions where the other side chain protecting groups remain on the side chains. After

synthesis, the Mtt group is deprotected and a chloroacetyl group is coupled to the side chain of

the Lys14 residue. This chloroacetyl group will react and form a thioether bond with the thiol

group on the N-terminal end of the bombesin analogue.

17

Table 3 Sequences of different ABD constructs.

ABD wild type (46 aa):

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP

ABD035 variant:

LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP

ABD for bombesin conjugation and 177Lu labelling:

DOTA-LAEAKVLANRELDK(Mtt)YGVSDFYKRLINKAKTVEGVEALKLHILAALP

Another construct was also designed and synthesized for this project, where the difference to

the original construct was the addition of a bromoacetyl group instead of the chloroacetyl group.

The presence of a bromide halogen predicts a more reactive functional group and a more

efficient conjugation reaction with the cysteine or mercaptopropionic residue of the bombesin.

Figure 4 Process of conjugation of bombesin analogue and ABD molecule.

Synthesis of ABD

The ABD molecule was produced by solid phase peptide synthesis with a fully automated

synthesis instrument (Biotage®-Initiator Alstara). The amino acids were Fmoc-protected and

as a solid support Rink amide ChemMatrix resin (Biotage®) was used. Amino acids were

HN

O

HS

NH2

OO

4

OO

Cl

N

NN N

O

O

NH

O

OH

O

HO

OH

HN

O

NH2

O O

O

S

N

NN N

O

O

NH

O

OH

O

HO

OH

O

4

DOTA chelator

ABD chloroacetyl group

cysteine PEG4 linker bombesinRM26

RadiolabeledABD-bombesin conjugate

18

purchased from Advanced Chemtech, Novabiochem and Sigma Aldrich. For a scale of 0.1

mmol peptide, 200 mg of resin with a resin loading of 0.5 mmol/g was used. A 5 times molar

excess of amino acid was used in each cycle. For the coupling reaction DIC/Oxyma reagents

with the presence of DIEA were used. Double coupling steps were applied for Asn9, Lys14,

Tyr15, Tyr21, Arg23 and Asn26 due to their bulky side chain protection. The temporary

protecting group Fmoc was cleaved with 20% piperidine in NMP. The non-reacted amino

groups were capped with acetic anhydride after each round of coupling to avoid the production

of unwanted side products. After the synthesis was completed, a microcleavage was performed.

A few resin beads from the reaction vessel were treated with 100 µl of 95:2.5:2.5 TFA/TIS/H2O

and incubated for 1 hour at room temperature to cleave the peptide from the resin. Following

the microcleavage MALDI-ToF mass spectrometry (ion source: matrix-assisted laser

desorption/ionization, mass analyzer: time-of-flight) was performed, with alpha-cyano-4-

hydroxycinnamic acid (CHCA) as a matrix, to make sure that a peptide with correct molecular

weight was produced.

Table 4 Amino acid side chain protecting groups.

Amino acid side chain protecting groups:

Arginine Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-

sulfonyl)

Asparagine Trt (trityl)

Aspartic acid OtBu (tert-butyl ester)

Glutamine OtBu

Histidine Trt

Lysine Boc (tert-butyloxycarbonyl) or Mtt (4-methyltrityl)

Serine tBu (tert-butyl)

Threonine tBu

Tyrosine tBu

Amino acids without side chain protection:

alanine, glycine, leucine, isoleucine, phenylalanine, proline and valine.

19

Conjugation of DOTA chelator to ABD was conducted in a similar fashion as the synthesis of

the ABD molecule, but in a separate step on the SPPS instrument. The reason for this was to

first make sure that the ABD synthesis went well before conjugating DOTA to the molecule.

After the conjugation of DOTA, the Mtt-protected Lys14 was manually deprotected by

treatment of the peptide-resin with a solution of DCM/TFA/TIS 94:1:5. The 2-minute

deprotection steps were repeated 10 times, or until the reaction solution stopped turning yellow

and the Ninhydrin test showed blue color. The Ninhydrin or Kaiser test is a good indicator of

free amines; the ninhydrin in the reaction mixture forms a blue product when amines are

present. The deprotected Lys residues were acylated with chloroacetic acid or bromoacetic acid

(10 equivalents) in the presence of DCC (5 equivalents) as an activator and DIEA (10

equivalents) as a base to deprotonate the amino group and make it nucleophilic. The reaction

mixture was incubated for 1 hour at room temperature. The chloroacetylation was repeated if

the Ninhydrin test showed that the reaction was not efficient enough (usually repeated 3 times).

The final cleavage from the resin was performed with TFA/TIS/H2O 95:2.5:2.5 mixture,

incubated with the resin for 3 hours at RT. The crude peptide was extracted with diethyl

ether/H2O 1:1. The water phase containing the peptide was then lyophilized. After each step

(DOTA conjugation, chloroacetylation/bromoacetylation of Lys14 etc.) the correct product was

verified with MALDI-ToF measurements.

Synthesis of bombesin RM26 analogue

Bombesin RM26 analogue was produced by solid phase peptide synthesis with a fully

automated synthesis instrument (Biotage®-Initiator Alstara) with the same setup and chemistry

as for ABD, introduced in the previous paragraph. After the synthesis of the first nine amino

acid, the PEG4 linker and the N-terminal cysteine or mercaptopropionic acid (respectively for

the two different construct) were coupled to the RM26 molecule in separate steps. As a last step

in the synthesis, a final piperidine deprotection was performed in order to reverse possible

acylation of the statine side chain. After the final cleavage from the resin and the extraction, the

correct product was verified with MALDI-TOF.

20

Purification of ABD and bombesin RM26 analogue

After synthesis, cleavage and extraction of the two crude peptides ABD and bombesin RM26,

the samples were lyophilized and then dissolved in 20% B (A: 0.1% TFA/H2O, B: 0.1%

TFA/CH3CN and filtered (pore size 0.45 µm, diameter 4 mm, Millex-HV PVDF filter) before

purification. Reversed-phase HPLC purification was performed on a semi preparative column

(5 µm, 9.4 x 250 mm Zorbax 300SB-C18, Agilent Technologies) using a gradient of 20-60% B

mobile phase. The total running time was 30 min with a flow rate of 3 ml/min and 70 °C column

temperature was applied in order to maximize the degree of separation. The peaks were

collected and analyzed by MALDI-TOF mass spectrometry to identify the correct product. The

fractions containing the correct product were pooled and lyophilized.

Conjugation of ABD and bombesin RM26 analogue

The purified ABD and bombesin RM26 were conjugated through an alkyl thioester formation

between the bromoacetyl/chloroacetyl and thiol functional groups on the two molecules. The

reaction was performed in a ligation buffer containing EDTA (10 mM) in PBS (60%, pH 8) and

a co-solvent of either CH3CN (40%) or DMSO (40%). The pH of the reaction was set to 8 –

8.5 with NaOH (5%) and different incubation temperatures were applied (room temperature

and 37 °C). A molar excess of 2 of the bombesin analogue was used in order to accelerate the

rate of conjugation. The total peptide concentration was approximately 2 mg/ml. To avoid the

formation of disulfide-linked RM26 dimers and to reduce dimers already present in the solution

(through to their cysteine or mercaptopropionic acid residues), TCEP (tris(2-

carboxyethyl)phosphine) (5 µl 200 mM TCEP for every 100 µl 0.2-1 mg/ml protein) was added

to RM26 prior to the conjugation reaction.

Purification of the conjugate ABD-bombesin conjugate was purified on a semi-preparative column with reversed-phase

HPLC. A gradient of 20-60% of B mobile phase was used in a time span of 30 min at 70 °C.

Along with the conjugate, the unconjugated bombesin and ABD peaks were also collected and

reused in later conjugation reactions.

21

Recombinant expression of ABD035

For recombinant expression of ABD035 a plasmid was used, containing the sequence for

ABD035 linked with a His-6 tag for purification. In this construct a C-terminal cysteine was

present on ABD.

Plasmid containing the sequence for ABD035 was transformed into Escherichia coli BL21

chemically competent cells and cultivation was performed in complex medium (tryptic soy

yeast extract TSB + Y) in the presence of kanamycin at 37 °C. Protein expression was induced

when the culture concentration reached OD600 0.6-0.8 right before the exponential phase, by

addition of 1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG). The culture was then

incubated overnight at room temperature with constant shaking at 150 rpm and harvested by

centrifugation (2500 RCF for 5 min) on the next day. The pellet was resuspended in IMAC

binding buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole), and the cells were lysed by

sonication (2 min 30 s run with 1 s pulse at 40% amplitude). Sonication was followed by

centrifugation (4000 RPM for 10 min) in order to remove cell debris before IMAC

(Immobilized Metal Affinity Chromatography) purification. The IMAC purification was

carried out on Cobalt Resin matrix (Thermo Scientific) packed in an empty PD-10 column

cartridge, and it was calibrated with IMAC binding buffer. Then the sample was loaded on the

column and washed with IMAC washing buffer (20 mM Tris, 300 mM NaCl, 30 mM imidazole)

and finally eluted with IMAC elution buffer (20 mM Tris, 300 mM NaCl, 300 mM imidazole).

Collected sample was buffer was exchanged from IMAC elution buffer to PBS on PD-10

Desalting column (GE Healthcare). The purity and identity of ABD was verified with SDS-

PAGE. The cysteine residue present on the C-terminus of this construct had to be masked in

order to avoid the formation of dimers. After the addition of TCEP (5 µl 200 mM TCEP for

every 100 µl 0.2-1 mg/ml protein) to the sample to reduce dimers already present in the solution,

iodoacetamide was used to selectively alkylate the thiol group of cysteine. 5 µl of 0.4 M

iodoacetamide was added to every 100 µl of 0.2-1 mg/ml protein solution (pH 8-8.5) and the

reaction mixture was incubated for 30 min in dark at room temperature. Finally, the purified

ABD035 was lyophilized and then dissolved in 20% B for RP-HPLC purification. For the

purification the same conditions were used as described for the purification of ABD-DOTA-

Cl.

22

Binding analysis with Surface Plasmon Resonance

The binding of ABD-RM26 conjugate to HSA was analyzed by SPR (Surface Plasmon

Resonance)-based biosensor analysis using a Biacore 8k (GE Healthcare) instrument. In the

experiment, HSA was immobilized on the surface of the biosensor chip (CM5

carboxymethylated dextran-coated gold sensor chip) and ABD-RM26 conjugate was injected

and flown over the surface. As a positive control recombinant ABD035 was used. HSA was

immobilized to the surface with a covalent amine coupling reaction, where the carboxyl groups

on the chip were activated with the mixture of NHS (N-hydroxysuccinimide) and EDC (N-

ethyl-N’-(dimethylaminopropyl) carbodiimide) (Amine Coupling Kit, GE Healthcare). The

HSA ligand was diluted with sodium acetate buffer (pH 4.5) to set the correct pH for

immobilization. After coupling, 1 M ethanolamine was used as deactivation solution to block

all remaining activated sites. As a running buffer for the binding experiments, phosphate-

buffered saline with Tween (PBS-T) (10 mM Na2HPO4, 150 mM NaCl, 0.005% Tween 20, pH

7.4) was used with a flow rate of 30 µl /ml.

The sensor chip is divided into several channels and experiments were performed on channels

in pair, one channel on the chip as a reference surface, prepared with only activation of NHS

and EDC, followed by the deactivation with ethanolamine, and the other surface with the

immobilized ligand. Analytes were injected at concentrations of 500 nM, 1000 nM and 5000

nM for the ABD-RM26 conjugate and at 1000 nM for ABD035. After injecting the samples,

the association time was 120 sec and the dissociation time was 300 sec. Regeneration of the

chip surface was performed with 10 mM HCl between each cycle (for 30 sec).

23

Results

Synthesis and purification of ABD

The synthesis of ABD was monitored by MALDI-ToF analysis. The mass spectrometric data

(Figure 5 A) shows that mainly one product was synthesized, and the molecular weight agrees

with the expected molecular weight of the 46 amino acid ABD (5110 Da). The successful

conjugation of DOTA and chloroacetylation/bromoacetylation was also verified with MALDI-

ToF (data not shown). After the final cleavage from the resin, ABD-DOTA-Cl and ABD-

DOTA-Br was extracted with diethyl ether/H2O 1:1 to remove impurities and by products

(Figure 5 B).

Figure 5 A - MALDI-ToF spectrum after SPPS of ABD (expected MW is 5110 Da, measured MW is 5093 Da, small difference between the two value is due to instrument calibration). B - MALDI- ToF spectrum of ABD-DOTA-Cl after final cleavage and extraction (expected MW is 5573 Da, measured 5556 Da). C - MALDI- ToF spectrum of ABD-DOTA-Cl after RP-HPLC purification (expected MW is 5573 Da, measured 5555 Da). Peaks are corresponding to the expected molecular weights and m/z values, assuming z=1.

The lyophilized product was then purified with RP-HPLC in order to further remove impurities

and by products produced during the synthesis of ABD or in later reactions (chloroacetylation

and bromoacetylation). ABD-DOTA-Cl is eluted around 45 % B and the peak containing the

right product was collected and analyzed with MALDI-ToF (Figure 5 C). The elution of ABD

was monitored at 220 and 280 nm wavelength. As the chromatogram of ABD shows (Figure 6)

the impurities and by products from the synthesis were eluting very close to the required product

resulting in a difficult purification process.

Acquired: \...\2019.01.24. ABD.T2D

1999.0 3004.2 4009.4 5014.6 6019.8 7025.0

Mass (m/z)

2.0E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

TOF/TOF™ Linear Spec #1[BP = 5092.3, 20398]

5092.9644

3424.0645

4356.4043 5130.9746

5163.11182545.32183445.49833025.5522 4949.7559

2629.5530 3181.44632248.0063 3586.4978 4935.60643123.2644 3714.2275 4226.61332133.7920 2614.6069 5775.52154892.2930 5256.58743655.98882124.0928 4410.43512841.1360

5794.0098

AB Sciex TOF/TOF™ Series Explorer™ 136

C:\Documents and Settings\Administrator\My Documents\Abel\190207_dotaABD-clextra.T2DPrinted: 14:45, February 08, 2019

2998.0 3803.6 4609.2 5414.8 6220.4 7026.0

Mass (m/z)

3.9E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

TOF/TOF™ Linear Spec #1[BP = 5555.8, 39451]

5555.8696

5631.7803

3426.7104

5211.9282

5612.7852

5514.9395

5427.6631

5441.2427

5356.2925 5707.2612

4900.17243589.3750 5173.72363271.17434305.2886 4828.71243183.8342 5759.87943793.1555

4700.75154149.41064418.45263396.4392 3751.65723125.7085

AB Sciex TOF/TOF™ Series Explorer™ 136

C:\Documents and Settings\Administrator\My Documents\Abel\190205_dotaABDfraction2.T2DPrinted: 10:55, February 05, 2019

2999 3805 4611 5417 6223 7029

Mass (m/z)

9.1E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

TOF/TOF™ Linear Spec #1[BP = 5554.1, 90918]

5554.6689

5608.9644

5537.9937

5496.7612

5465.72023425.6409 5421.2153 5700.8545

3522.2388 4417.7319

A B C

24

Figure 6 Chromatogram of crude ABD-DOTA-Cl from RP-HPLC purification. Blue arrow indicates the peak corresponding to ABD-DOTA-Cl. The chromatogram can be used to estimate the yield of ABD synthesis by integration of the peaks. Despite the lack of baseline separation, the crude purity is estimated to be around 60%.

Synthesis and purification of bombesin RM26

Following the SPPS production of the bombesin RM26 analogue, a PEG4 linker and an N-

terminal cysteine or mercaptopropionic acid residue were coupled to the molecule, resulting in

the peptides RM26-PEG4-Cys and RM26-PEG4-Merc. The molecular size and identity of the

final products were analyzed with MALDI-ToF. The measured molecular weight of both

RM26-PEG4-Cys and RM26-PEG4-Merc were corresponding to the calculated molecular

weights (1464.8 Da and 1449.7 Da respectively) as seen in Figure 7 A (data for RM26- PEG4-

Merc is not shown).

min12 14 16 18 20 22

mAU

0

200

400

600

800

1000

DAD1 A, Sig=220,4 Ref=off (Abel\2019-02-12 12-03-27 ABD.D)DAD1 E, Sig=280,4 Ref=off (Abel\2019-02-12 12-03-27 ABD.D)PMP1, PMP1D, Solvent Ratio B (2019-02-12 12-03-27 ABD.D)

Data File C:\Users\Public\Documents\ChemStation\1\Data\Abel\2019-02-12 12-03-27 ABD.D

Sample Name: ABD

HPLC_3 5/7/2019 1:26:09 PM SYSTEM

Page

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Signal 220 nm

Signal 280 nm

CH3CN + 0,1% TFA [%]

ABD-DOTA-Cl

25

Figure 7 A - MALDI-ToF spectrum of crude RM26-PEG4-Cys (expected MW is 1465 Da, measured MW is 1460 Da). B - MALDI- ToF spectrum of RM26-PEG4-Cys after RP-HPLC purification (expected MW is 1465 Da, measured MW is 1462 Da). Peaks are corresponding to the expected molecular weights and m/z values, assuming z=1.

The by-products and impurities from the synthesis reaction were removed and the product was

purified with RP-HPLC. Peaks containing RM26-PEG4-Cys or RM26-PEG4-Merc were eluting

approximately at 50 % B (Figure 8), and after collecting the fraction, it was analyzed and

confirmed with MALDI-ToF (Figure 7 B). The two main by-products seen on the

chromatogram could also be identified with MALDI-ToF. One of the side products has a

molecular weight corresponding to RM26 (without the PEG4 linker and N-terminal Cys) and

the other has a molecular weight corresponding to the RM26-PEG4-Cys analogue with an

additional acetyl group.

Figure 8 Chromatogram of crude RM26-PEG4-Cys from RP-HPLC purification. Blue arrow indicates the peak corresponding to RM26-PEG4-Cys. After the integration of the peaks, the crude purity of the product can be estimated to 60%.

AB Sciex TOF/TOF™ Series Explorer™ 136

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403.0 772.6 1142.2 1511.8 1881.4 2251.0

Mass (m/z)

2.4E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

TOF/TOF™ Linear Spec #1[BP = 1460.7, 23922]

1460.6667

1503.1357

916.5265

861.1051

1443.8442874.9556

1514.3497

1483.3485

1485.8547890.8217

728.70681344.3312

1557.3757853.28681398.31541152.1337695.8777 1542.3073

1197.5088900.6254 1357.2838734.2388 1599.3071619.5723

1832.5813

AB Sciex TOF/TOF™ Series Explorer™ 136

C:\Documents and Settings\Administrator\My Documents\Abel\190211_bombesinHPLC.T2DPrinted: 18:29, February 11, 2019

330 1045 1760 2475 3190 3905

Mass (m/z)

1.0E+5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

TOF/TOF™ Linear Spec #1[BP = 1459.2, 100000]

1462.5654

1482.8063

1514.8483

1492.84902920.3076

1498.8850

1649.7825

1382.5240

1520.4115

1607.21261152.8350 2999.5134620.4596836.7731 1314.5302 2084.4998

min16 18 20 22 24 26 28

Norm.

0

500

1000

1500

2000

2500

DAD1 A, Sig=220,4 Ref=off (Abel\2019-02-11 11-37-04 Bombesin.D)DAD1 E, Sig=280,4 Ref=off (Abel\2019-02-11 11-37-04 Bombesin.D)PMP1, PMP1D, Solvent Ratio B (2019-02-11 11-37-04 Bombesin.D)

Data File C:\Users\Public\Documents\ChemStation\1\Data\Abel\2019-02-11 11-37-04 Bombesin.D

Sample Name: Bombesin

HPLC_3 5/7/2019 1:24:20 PM SYSTEM

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Signal 220 nm

Signal 280 nm

CH3CN + 0,1% TFA [%]

RM26-PEG4-Cys

A B

26

Conjugation of ABD and bombesin RM26, purification of the conjugate

ABD and the bombesin RM26 analogue were conjugated in different reaction conditions and

with different combination of the constructs. ABD-DOTA-Br was coupled with both RM26-

PEG4-Cys and RM26-PEG4-Merc in separate reactions using different co-solvents and reaction

conditions and the same was performed with ABD-DOTA-Cl and the two RM26 constructs

(summarized in Table 3).

The best results for conjugation were achieved with the combination of ABD-DOTA-Br and

RM26-PEG4-Merc with CH3CN as co-solvent. The following data and results are focusing on

the coupling reaction of RM26-PEG4-Merc (further discussed in the Discussion chapter).

Table 3 Coupling reactions between ABD and RM26 analogues. The results of conjugation tests are categorized as “+++” for an efficient reaction, “++” or “+” for a less efficient reaction, and “-“ if you do not see any reaction.

Reaction partner 1 Reaction partner 2 Co-solvent Reaction

condition (temp.)

Result of

conjugation

ABD-DOTA-Cl RM26-PEG4-Cys CH3CN or DMSO RT -

ABD-DOTA-Cl RM26-PEG4-Cys CH3CN or DMSO 37 °C -

ABD-DOTA-Cl RM26-PEG4-Merc CH3CN or DMSO RT ++

ABD-DOTA-Cl RM26-PEG4-Merc CH3CN or DMSO 37 °C ++

ABD-DOTA-Br RM26-PEG4-Cys CH3CN or DMSO RT +

ABD-DOTA-Br RM26-PEG4-Merc CH3CN or DMSO RT +++

The following MALDI-ToF spectra are presenting the coupling reaction after 24 h incubation

times.

27

Figure 9 MALDI-ToF spectrum of the conjugation reaction of ABD-DOTA-Cl and RM26-PEG4-Merc (conjugate expected MW is 6986 Da, measured MW is 6967 Da). Peaks are corresponding to the expected molecular weights and m/z values, assuming z=1.

The difference in the coupling efficiency of ABD-DOTA-Cl and ABD-DOTA-Br is visible in

Figure 10. While coupling RM26 to bromoacetylated ABD resulted in a near complete reaction

(Figure 10 B), chloroacetylated ABD was unable to react with in its entirety with the bombesin

analogue (Figure 10 A).

Figure 10 A - MALDI-ToF spectrum of the conjugation reaction of ABD-DOTA-Cl to RM26-PEG4-Merc. B - MALDI-ToF spectrum of the conjugation reaction of ABD-DOTA-Br to RM26-PEG4-Merc. In this case there is not one distinct peak for the remaining unconjugated ABD-DOTA-Br, probably due to the reactivity of the bromide. Peaks are corresponding to the expected molecular weights and m/z values, assuming z=1.

From the chromatogram of the purification (Figure 11), we can see that there is a distinct peak

for the RM26 analogue that was used in excess during the conjugation reaction, and a clear

peak corresponding to the ABD-RM26 conjugate. It is also visible that most of the ABD has

reacted with the bombesin analogue, indicated by the smaller ABD peak. The fractions

AB Sciex TOF/TOF™ Series Explorer™ 136

C:\Documents and Settings\Administrator\My Documents\Abel\190502_ABD-Cl-BN-Merc_conj.T2DPrinted: 11:03, May 02, 2019

998.0 2403.6 3809.2 5214.8 6620.4 8026.0

Mass (m/z)

1.0E+5

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

TOF/TOF™ Linear Spec #1[BP = 1443.7, 100000]

1446.9902

2887.7236

6967.08351466.6144 5557.6855

1427.6364

5521.02881486.4355

1476.1056 5539.90877006.57713427.2793 5597.72171516.2622 2919.6501

7063.53613504.8564 4072.45431277.3518 5353.9497 6581.91312095.8452

AB Sciex TOF/TOF™ Series Explorer™ 136

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4998.0 5605.4 6212.8 6820.2 7427.6 8035.0

Mass (m/z)

4.2E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

TOF/TOF™ Linear Spec #1[BP = 6968.4, 42329]

6967.1606

5558.0317

5521.2588

7007.3071

5540.0171

6921.97755597.9487

7062.20025478.2925

7108.61185700.8101 6581.40535160.7744

AB Sciex TOF/TOF™ Series Explorer™ 136

C:\Documents and Settings\Administrator\My Documents\Abel\190402_conjugation\190402_BN-merc-ABD-Br-conjugation-ACN4.T2DPrinted: 13:41, April 02, 2019

4998.0 5604.8 6211.6 6818.4 7425.2 8032.0

Mass (m/z)

1.5E+4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

TOF/TOF™ Linear Spec #1[BP = 6963.7, 14549]

6964.0640

5768.4609

5697.2046

5519.0068

5478.9336

6982.5811

7009.97225555.27056920.1211

5751.08457062.55475815.9341

6874.0986

A B

RM26-PEG4-Merc

RM26 dimer

ABD-DOTA-Cl ABD-RM26 conjugate

ABD-DOTA-Cl ABD-RM26 conjugate

ABD-RM26 conjugate

ABD-DOTA-Br

28

containing the conjugate and the unconjugated ABD and RM26 analogue (including the dimers)

were both collected. The ABD-RM26 conjugated was lyophilized for upcoming experiments

and the unreacted ABD and RM26 were reused for new conjugation reactions.

Figure 11 Chromatogram of ABD-RM26 conjugation reaction from RP-HPLC.

Production of ABD035

After the recombinant production and IMAC purification of ABD035, an SDS-PAGE analysis

(Figure 11) was performed to study the purity and identity of the product. The results show that

the IMAC purification was successful, giving rise to one distinct band corresponding to

approximately 7 kDa according to the reference ladder (LMW, Low Molecular Weight ladder).

Further MALDI-ToF analysis (data not shown) confirmed that the molecular weight of the

produced protein is 6835 Da as expected for this ABD035 construct. The alkylation of the C-

terminal cysteine was performed with the addition of iodoacetamide after the IMAC

purification. Some impurities remaining in the sample after IMAC purification were removed

by subsequent RP-HPLC purification of the protein.

min13 14 15 16 17 18 19 20 21 22

Norm.

0

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1000

1500

2000

2500

DAD1 A, Sig=220,4 Ref=off (Abel\2019-05-02 ABD-BN conjugation_12-15-44.D)DAD1 E, Sig=280,4 Ref=off (Abel\2019-05-02 ABD-BN conjugation_12-15-44.D)PMP1, PMP1D, Solvent Ratio B (2019-05-02 ABD-BN conjugation_12-15-44.D)

Data File C:\Users\P...ts\ChemStation\1\Data\Abel\2019-05-02 ABD-BN conjugation_12-15-44.D

Sample Name: ABD-BN conjugation

HPLC_3 5/7/2019 1:21:47 PM SYSTEM

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Signal 220 nm

Signal 280 nm

CH3CN + 0,1% TFA [%]

RM26-PEG4-Merc

ABD-RM26 conjugate

BN dimer

ABD-DOTA-Cl conjugate

29

Figure 12 SDS-PAGE of ABD035. The red arrow indicates the expected product (MW is 6835 Da).

Binding analysis with Surface Plasmon Resonance

SPR-based biosensor analysis was performed with a Biacore instrument on a gold biosensor

chip with immobilized HSA ligand on the surface. The immobilization resulted in a ligand level

of 1824.6 RU indicating a high ligand density. Injection of different concentrations of the

conjugate and the positive control ABD035 shows that the binding response and association

rate are concentration-dependent (Figure 13 and 14). The kinetics of the binding were not

calculated due to the lack of data points and few concentrations used in the experiments.

However, the main goal of the binding analysis was to compare the binding profile of ABD035

and the RM26-ABD conjugate.

LMW

ladd

er

LMW

ladd

er

Crude p

rotein

Purif

ied pr

otein

66

30

20,1

14,4

97

40

kDa

30

Figure 13 Sensorgram from Biacore biosensor analysis of ABD-RM26 conjugate binding to immobilized HSA on the surface.

Figure 14 Sensorgram from Biacore biosensor analysis of ABD035 and ABD-RM26 conjugate binding to HSA immobilized on the surface.

-40

0

40

80

120

160

200

240

280

320

-80 0 80 160 240 320 400 480 560

Res

pons

e (R

U)

Time (s)

ABD-RM26 1000 nM

-20

0

20

40

60

80

100

120

-80 0 80 160 240 320 400 480 560

Res

pons

e (R

U)

Time (s)ABD035 1000 nM

ABD035 1000 nM

ABD-RM26 1000 nM

ABD-RM26 1000 nM

ABD-RM26 500 nM

ABD-RM26 500 nM

31

Discussion

The main goal of my thesis project was to synthesize a high-affinity bombesin analogue for

GRPR-targeted radiotherapy of prostate cancer. As the title of my project suggests my work

could be divided into two main parts, the synthesis and the analysis of the molecule. During the

time of my project, I successfully synthesized two molecule constructs and could successfully

complete the conjugation of the two molecules. However, despite the fact that the

characterization of the conjugate has started and data from SPR binding measurements are

promising, there are still more experiments to be conducted in order to fully evaluate the

molecule. This will involve further binding assays, in vitro cell studies and in vivo animal

experiments with the radiolabelled conjugate, performed in collaboration with a research group

at Uppsala university.

As a first step of the project, the synthesis of RM26 and ABD were completed without any

major problems. The results show that the SPPS synthesis was successful, and the right

molecules with the expected molecular weight were produced with only a few by-products

present after the synthesis. Due to the larger size of ABD (46 amino acid) the synthesis was

expected to be challenging, but as seen in the RP-HPLC purification chromatogram, the correct

peptide was achieved as the main product. In the case of the RM26 analogue, a shorter peptide

with only 9 amino acids and PEG4 linker and an N-terminal Cys or Merc, the synthesis was

expected to be less problematic. However, as shown on the HPLC chromatogram (Figure 8)

two main side products could be identified after the synthesis. One of the side products is RM26

(without the PEG4 linker and N-terminal Cys) and the other appears to be the RM26-PEG4-Cys

analogue with an additional acetyl group. Since the PEG4-Cys (or PEG4-Merc) parts of the final

molecule were coupled in separate steps after the completion of the RM26 peptide, the

accumulation of the first side product could possibly be avoided with a more efficient

conjugation reaction (increased amino acid loading concentrations or double coupling step).

These by-products and impurities, however, were successfully removed with diethyl

ether/water extraction and the following RP-HPLC purification. Despite the program used for

RP-HPLC purification (gradient elution, 30 min run time, 70 °C column temperature) for the

best degree of separation, the by-products from incomplete ABD synthesis demonstrated

similar elution to ABD, due to their similar hydrophobicity, making the separation of ABD

challenging. In the case of the RM26 analogues, the chromatograms from the purification and

MALDI-ToF data suggest that the correct products can be isolated with high purity.

32

The synthesis and purification of ABD and RM26 were followed by the conjugation reaction.

One of the early finding that led us to change the construct of RM26 analogue was that the

originally designed RM26 with an N-terminal cysteine showed very little reactivity in the

coupling reactions. This resulted in replacing cysteine with mercaptopropionic acid, an

organosulfur compound containing a carboxyl group for forming a peptide bond with RM26-

PEG4 and a thiol group for the coupling to ABD.

Regarding the ABD constructs, we can conclude that bromoacetylated ABD proved to be more

efficient and more reactive in the coupling reactions over chloroacetylated ABD. This can be

explained by the increasing reactivity of halogens within the group moving from chloride to

bromide. To increase the conjugation rate when using chloroacetylated ABD, the reaction

mixture was incubated at 37 °C. Incubation at higher temperature in order to drive the reaction

was not necessary when using the ABD-DOTA-Br construct, as the coupling reaction almost

went to completion when using a molar excess of the RM26 analogue. It is worth mentioning

that heating the reaction mixture during conjugation may increase the rate of competing side

reactions, so this makes the more reactive ABD-DOTA-Br a better candidate for future

conjugations. During conjugation experiments, two different co-solvents, CH3CN and DMSO,

were used and their influence on the efficiency of the conjugation was compared, but no major

difference was found between the two solvents. RP-HPLC purification of the conjugate resulted

in a high purity ABD-RM26 product and the unconjugated RM26 and ABD could be recovered

and reused in later conjugations. The obtained data showed that in a conjugation containing a

molar excess of RM26-PEG4-Merc, paired with ABD-DOTA-Br, the reaction went to near

completion; almost all ABD formed conjugates with the excess RM26. This could not be said

when combining ABD-DOTA-Cl with RM26-PEG4-Merc, when the reaction despite the long

incubation time (24 h) and 37 °C did not go to completion. These results suggest that for the

conjugation of these two molecules bromoacetylated ABD and RM26 with an N-terminal

mercaptopropionic acid residue is the best combination in terms of conjugation efficiency.

Biacore measurements were aiming to investigate the binding ability of RM26-ABD conjugate

to HSA and comparing it to ABD035 and thus predicting the potential of the conjugated ABD

for extending the half-life of RM26 in vivo. In single injection studies, injecting a high

concentration (5000 nM) of the conjugate, the sensorgram (Figure 12) suggests a strong binding

to HSA with fast association time and very low dissociation rate. We can see that the binding

response is concentration-dependent. The rate of association is also dependent on the injected

concentration as shown in Figure 13: the higher the concentration, the faster the association is.

33

When comparing the binding of the conjugate to ABD035, it can be seen that the RM26-ABD

conjugate displays a slower association rate compared to ABD035 with the same concentration

and that the overall binding response of the conjugate is also slightly lower. The difference in

binding profile and slower association can be explained by the fact that coupling the RM26

peptide and introducing the DOTA chelator to ABD slightly affect the overall binding propertie

of the conjugate. Another possible explanation could be that the determined concentration for

the conjugate and ABD035 was slightly off.

However, the off-rate is similar for the conjugate and ABD035 indicating a strong binding to

HSA. The kinetic parameters of the conjugate were not determined due to the limited number

of data points and the few concentrations used in the experiments.

Overall, we can state that the conjugated ABD domain fulfils its function to bind HSA with

high affinity, thus potentially prolonging the in vivo half-life of RM26.

34

Future perspectives

There are further experiments and measurements planned for the future, since the original

project plan was not completed/executed in its entirety. For the characterization of the ABD-

RM26 conjugate other experiments, such as flow cytometry, CD and cell studies will be

conducted as a next step. After larger scale synthesis and production of the conjugate, the

product will be sent to collaborators in Uppsala university for radiolabeling with 117Lu. The

radiolabeled molecule will then be a subject of cell studies, where its receptor binding ability

will be evaluated, followed by in vivo animal studies.

Acknowledgements

First of all, I would like to thank Amelie for the opportunity to do my master thesis project in

her group and thank you for being a great supervisor, helping me and giving advice when I

needed. It has been a great experience so far and hopefully it will continue for some time.

I would like to express my gratitude to Hanna, my co-supervisor, who has been around helping

me from the beginning and for patiently showing me everything and answering all my questions

along the way. I have learnt a lot, you are a great teacher.

Last but not least, I would like to thank the rest of the Synthesis group members for the

additional support, and all the people on floor 3 at Alba Nova.

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