PNA-MEDIATED AFFIBODY PRETARGETING

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PNA-MEDIATED AFFIBODY

PRETARGETING

Hanna Tano

Supervisors: Kristina Westerlund,

Amelie Eriksson Karlström

Examiner: Amelie Eriksson

Karlström

Master Thesis

KTH

School of Biotechnology

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ABSTRACT

Affibody molecules are small (6-7 kDa), alternative scaffold proteins which can be engineered to bind to disease associated targets with high affinity. Affibody molecules have previously been utilized for imaging of breast cancer overexpressing the human epidermal growth factor receptor 2 (HER2). Utilization of HER2-binding Affibody molecules have rarely been studied for therapeutic applications, mainly due to high level of reabsorption of Affibody molecules in kidneys. A previously examined pretargeting strategy was applied in which ZHER2:342 conjugated to a PNA based recognition tag is

injected in a first step, and a complementary PNA probe carrying a toxic agent is injected in the second step. Pretargeting was applied in order to improve tumor to kidney uptake ratio. In this project, dimeric (ZHER2:342)2 affibody molecules were produced and characterized with the hypothesis of achieving a

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ACKNOWLEDGEMENTS

I would like to express my appreciation to the following:

Kristina – for your brilliant supervising, help and support ever since day one of this master project. Thank you for your patience and for sharing your extensive knowledge with me, you have taught me a lot!

Amelie – for accepting me to this project and for creating a creative and positive working atmosphere with your kind and tolerant approach to all members in your research group. I think you are a wonderful leader.

Mohamed, Vladimir and Anna – for generously letting me come for a study visit in Uppsala, and for taking care of me that day.

Wojtek – for all your support.

Jakob - for critically reading this report and coming with very helpful feedback.

Last, but not least, I would like to thank all people at floor 3, AlbaNova, whom in any way helped me through this project.

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INTRODUCTION

Background

Cancer

Cancer has for a very long time been a global health problem. Cancer can be caused by various factors and there is no overall reason to why cancer is developed. Breast cancer is the most common cancer type among women, corresponding to approximately 29 % of new cases of cancer, and about 26 % of all women dying from cancer suffered from breast cancer. [1] The tyrosine kinase receptor family; human epidermal growth factor receptor (HER) is involved in processes promoting cellular growth, proliferation and differentiation, hence playing an important role in the development of cancer. The HER family consists of four members; HER1/EGFR, HER2, HER3 and HER4. Overexpression of HER2 (sometimes referred to as ErbB2) is associated with breast cancer (15-30 % of all breast cancer cases), ovarian cancer (20-30 % of all ovarian cancer cases) and bladder cancer. [2] Breast cancer with overexpression of HER2 (i.e. HER2-positive breast cancer) is often aggressive and the prognosis is usually poor. [3] [4] HER2 receptors normally exist as monomers on the cell-surface, however they possess the ability of forming homo- or heterodimers, which activates intracellular downstream signalling cascades promoting cellular proliferation and differentiation, which both are crucial factors in the development of cancer. [5]

Chemotherapy has been revolutionary in terms of improving and prolonging lives of cancer patients. However, the combination of toxicity and lack of target specificity of chemotherapeutics often leads to extensive side effects. Targeted radiotherapeutics for treatment of cancer have been developed, where cytotoxicity is mediated by radionuclides. By attaching radionuclides to molecular targets binding specifically to cancer associated epitopes, cytotoxic radioactivity can be delivered directly and specifically to the malignant tumor. After accumulation of radionuclides in the malignant tumor, the cytotoxic radioactivity can be spread to malignant cells in proximal tumor tissue by cross-fire effect. [6] The potential of delivering cytotoxicity to tumor cells by cross-fire effect depends on the energy of the radioisotope. Difficulty in delivering the required radioactivity to tumors has previously been limiting the use of radiotherapeutics. Solid tumors such as breast cancer, are more radioresistant than lymphomas, and thus requires additional exposure to radioactivity in order to be treated successfully. [7]

Baxxin (131_{I-tositumomab and unlabeled tositumomab) and Zevalin (}90_{Y-ibritumomab tiuxetan and}

unlabeled rituximab) [8] are examples of drugs developed to treat non-Hodgkins lymphoma. Tositumomab, ibritumomab and rituximab are all monoclonal antibodies (mAb) and treatment with Baxxin and Zevalin is often referred to as conventional radioimmunotherapy (RIT). RIT is based on injection of mAb, containing a site binding to a disease associated molecular target. The mAb is further directly attached to a radionuclide. [9] [7]

Affibody molecules

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are exposed to different targets. Helices 1 and 2 constitute the binding surface of Affibody molecules. [10]

One of the most studied Affibody molecules is the HER2-binding Affibody molecule ZHER2:342, binding

to a different epitope than the mAbs currently used in immunotherapy treatment of HER2-positive breast cancer, Trastuzumab and Pertuzumab. [12] There are some disadvantages of using mAb in diagnostics and treatment of solid tumors. The large size of mAbs (approximately 150 kDa [7]) causes slow blood clearance and poor tumor penetration, entailing radioactive damage to normal tissue, when carrying cytotoxic radionuclides. Requirement for additional radioactive exposure for treatment of solid tumors stresses the importance of using protein complexes with a fast clearance from blood when used in RIT. The small size, high affinity and thermal stability of Affibody molecules, make them an interesting platform which could be utilized in the process of improving pharmacokinetics in RIT.

Figure 1 The characteristic three-helix bundle of Affibody molecules is illustrated. Helices 1, 2 and 3 (behind) of

the Affibody molecule are shown. The N- and C-termini of the Affibody molecule are marked with N and C, respectively, in the figure. Amino acid positions randomized when creating Affibody libraries are denoted with stars.

Pretargeting

The strategy of pretargeting involves a two-step process where the targeting agent labelled with a recognition tag, is injected in the first step. After a waiting period, where the mAb accumulates in tumor and clears from blood, a radiolabelled agent is injected, containing a recognition tag complementary to the recognition tag on mAb. This technique enables construction of smaller radioactive components, which allows for fast biodistribution, and quick tumor accumulation. The large size of the mAb results in a long waiting time before injection of the second agent. By substituting mAb with smaller molecules, e.g. Affibody molecules, improved tumor penetration and shorter waiting times between the first and second injection are enabled. [9]

PNA

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proteases. Furthermore, PNA is non-immunogenic, hence does not trigger an immune system reaction. PNA has the ability of binding with high affinity and specificity to complementary sequences of PNA, mRNA and DNA, and the absence of charge repulsion between the backbones of PNA results in a higher binding strength of complementary strands. [14] The non-charged backbone further gives the characteristics of a lower cellular uptake, which could be useful in some medical purposes, such as pretargeting. [13]

Figure 2 Structure of PNA versus DNA structure. The backbone of PNA is non-charged, similarly to peptides.

PNA shares the four different nucleobases with DNA, and has the capability to form hybridized complexes to complementary DNA strands.

Sortase A

Sortases are transpeptidases existing naturally in almost all gram-positive bacteria. Their function is to covalently anchor surface proteins to the peptidoglycan layer. [15] Sortase A is one variant of sortases which origins from Staphylococcus aureus. The recognition site for Sortase A is located in the C-terminal of the target protein and consists of the amino acid sequence LPXTG, where X corresponds to an arbitrary amino acid. Cleavage is conducted in the recognition site between threonine and glycine, and subsequent ligation occurs with substrates containing an N-terminal oligoglycine sequence. [16] Sortase A is a popular tool for labelling of proteins and for immobilization of proteins to solid supports. Sortase A is beneficial for these purposes due to its high specificity, but also due to the feasibility to recombinantly or synthetically introduce the LPXTG sequence and N-terminal glycines to almost any protein. However, the reaction kinetics of Sortase A is slow, and large quantities of the enzyme are usually required to successfully accomplish a conjugation reaction. Improvement of reaction kinetics has been performed by yeast display, where point mutations in Sortase A were introduced and evaluated. An improved variant of Sortase A, containing three point mutations; P94S, D160N and K196T was evolved, henceforth referred to as Sortase A*. [17]

Previous research

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filtration in kidneys. [18] [19] Affibody molecules are cleared from blood by kidney excretion. Previous studies with Affibody molecules have shown that Affibody molecules have a tendency to have high renal reabsorption, which may result in kidney tissue damage if the Affibody molecule is carrying toxicity. One approach to solve this problem is pretargeting. According to previous research, the pretargeting strategy with PNA probes resulted in a tumor-to-kidney radioactive ratio of 2, when using a waiting time of 4 hours between the first and second injection. [20] One requirement in order for the pretargeting therapy to be efficient, is that the targeting agent is accessible for binding of the secondary agent and not internalized after binding to HER2 receptors on tumor cells. However, if the targeting agent is retained in the kidney, as shown for the Affibody molecule, the protein should preferably be internalized by the cells in the proximal tubuli in kidneys, to reduce the risk of binding the secondary agent, which might result in kidney tissue damage.

Project Aim

In order to improve the tumor-to-kidney ratio, two dimeric HER2-binding Affibody molecules (ZHER2:342)2, were produced and evaluated with the hypothesis that dimeric Affibody molecules will have

stronger affinity to the HER2-receptor, due to avidity effects. By creating stronger affinity for dimeric Affibody molecules, the dissociation rate might be slower than the dissociation of monomeric ZHER2:342.

A decreased dissociation rate would decrease the risk of obtaining free circulating Affibody molecules carrying cytotoxicity, exposing normal cells to radioactivity. A slower dissociation rate would also be useful when optimizing the procedure of injection of Affibody molecule and the secondary labelled probe, e.g. slower dissociation rate allows for longer waiting time between the first and second injection, which might result in increased tumor-to-kidney ratio.

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Figure 3 Illustration of the two-step process of pretargeting. HER2-binding Affibody/PNA conjugate is injected

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METHODS AND MATERIALS

Design of dimeric anti-HER2 Affibody molecule

A plasmid containing a synthetic gene coding for the monomeric ZHER2:342 protein was used as template

for creating dimers of the HER2-binding Affibody molecule. ZHER2:342 has previously been engineered

to carry a C-terminal peptide extension (Table 1) containing a GS-region providing flexibility, a Sortase A* recognition site (SR), and a His-6 tag utilized for purification. [9] The extension is attached to the C-terminal of helix 3 of the Affibody molecule, ensuring minimal steric hindrance in recognition site for binding between ZHER2:342 and the HER2-receptor. The dimers, (ZHER2:342)2, have a similar design, but

with the original amino acid sequence of ZHER2:342 repeated twice. The linker length used in previous

studies of dimeric Affibody molecules has been 15 amino acids, or undisclosed due to intellectual properties. [21] [22] [23] Two different Affibody dimers with 2 and 13 amino acid linkers ((ZHER2:342)2

-2/13 linker) (Figure 4) were produced in order to study the role of linker length required to enable slower dissociation rate after binding to HER2.

Figure 4 HER2-binding Affibody dimers with 13 amino acid linker (left) and 2 amino acid linker (right). Table 1 Amino acid sequences, molecular weights and number of amino acids for the different Affibody

molecules. Letters given in italics correspond to the original amino acid sequence of ZHER2:342. Blue letters “VD” corresponds to the cleavage site of restriction enzyme AccI. Amino acid sequence “GSGSGS”, illustrated in green, provides flexibility and improves reachability for Sortase A*. LPETGG, given in red, and H6, given in purple corresponds to the Sortase A* recognition site and hexahistidine tag for IMAC purification, respectively. GS and VDGSGSGSGSGGG correspond to the 2 and 13 amino acid spacers connecting the two monomeric ZHER2:342, illustrated in light blue and light green, respectively.

Protein Sequence Molecular

weight [Da]

Number of amino acids Z-HER2:342-SR-H6 VDNKFNKEMR NAYWEIALLP NLNNQQKRAF IRSLYDDPSQ

SANLLAEAKK LNDAQAPKVDGSGSGSLPET GGHHHHHH

8729 78 (Z-HER2:342)2-2 linker- SR-H6 VDNKFNKEMR NAYWEIALLP NLNNQQKRAF IRSLYDDPSQ

SANLLAEAKK LNDAQAPK GS VDNKFNKEMR

NAYWEIALLP NLNNQQKRAF IRSLYDDPSQ SANLLAEAKKL NDAQAPKVDGSGSGSLPET GGHHHHHH

15561 138

(Z-HER2:342)2-13 linker- SR-H6

VDNKFNKEMR NAYWEIALLP NLNNQQKRAF IRSLYDDPSQ SANLLAEAKK LNDAQAPKVD GSGSGSGSGG

GVDNKFNKEMR NAYWEIALLP NLNNQQKRAF

IRSLYDDPSQ SANLLAEAKKL NDAQAPKVDGSGSGSLPET GGHHHHHH

16379 149

Production of (Z

HER2:342

)

2

-2/13 linker-SR-H

6

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restriction sites for NdeI and AccI, obtained from plasmid pAY430-ZHER2:342. Amplified segments were

ligated together with DNA ligase, hence creating a construct coding for two ZHER2:342, separated by

BamHI restriction site (coding for glycine and serine), and with restriction sites for NdeI and AccI in its 5’ and 3’ ends. The construct was subcloned into a pAY430 vector containing genes coding for VD-[GS]3-LPETGG-H6 and kanamycin resistance. Expression of Zwt-VD-[GS]3-LPETGG-H6 was under

control of T7 promoter. Ligation and subcloning steps were monitored by PCR, gel electrophoresis and digestion by enzymes (NdeI and HindIII HF), in order to confirm success of ligation and subcloning. Expression plasmid for (ZHER2:342)2-13 linker-SR-H6 was constructed from pAY430-ZHER2:342, which was

PCR amplified together with primers containing a sequence coding for six amino acids, and restriction sites for NdeI and BamHI. A plasmid pAY430 containing the sequence coding for G3-ZHER2:342-Cys-

ZHER2:342-SR-H6 was double digested with NdeI and BamHI, removing the sequence coding for cysteine

and one ZHER2:342. The PCR-amplified product was subcloned into the vector, creating a construct coding

for ZHER2:342-13 amino acids- ZHER2:342-SR-H6 (i.e. (ZHER2:342)2-13 linker-SR-H6). The linker consists of

VDGSGSGSGSGGG, promoting linker flexibility due to the large proportion of small and polar amino acids. Final sequences were sent for sequencing (Seqlab-Microsynth) to confirm correct DNA-sequences were obtained.

Plasmid pAY430-(ZHER2:342)2-2/13 linker-SR-H6 was transformed into Escherichia Coli (E. coli)

BL21(DE3) chemically competent cells (Life Technologies) and cultivation was performed in complex medium, tryptic soy yeast extract (TSB + y) in presence of kanamycin. Protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 between 0.6 – 0.8. Cultures

were incubated at 25℃ with constant shaking at 150 rpm. Harvesting was performed by centrifugation after approximately 20 hours after induction. IMAC binding buffer (25 mM NaH2PO4, 150 mM NaCl,

10 mM imidazole, pH 7) was used to resuspend cell pellets, and cell lysis was achieved by sonication (2.0 s pulse, 2 min 30 s run). Centrifugation was performed in order to remove cell debris prior to IMAC purification. Talon® Metal Affinity Resin (Clontech Laboratories Inc., Takara Bio Company) was utilized for IMAC purification of (ZHER2:342)2-2/13 linker-SR-H6. IMAC binding buffer was used for

equilibration and washing, while elution was performed by IMAC elution buffer (25 mM NaH2PO4, 150

mM NaCl, 300 mM imidazole, pH 7).

Purified (ZHER2:342)2-2/13 linker-SR-H6 was dehydrated by lyophilization, resuspended, and transferred

to a PD-10 gel filtration column (GE Healthcare) with the purpose of changing storage buffer to Sortase A ligation buffer (50 mM Tris base, 150 mM NaCl, 10 mM CaCl2, pH 7.5), and removing salts and

imidazole prior to Sortase A-mediated ligation to HP15. [24] Purity and identity of (ZHER2:342)2-SR-H6

was verified by SDS-PAGE and Matrix Assisted Laser Desorption Ionisation Time of Flight (MALDI ToF) mass spectrometry.

All enzymes used for production of (ZHER2:342)2-2/13 linker-SR-H6 were purchased from New England

Biolabs.

Production of Sortase A*

Genes coding for Sortase A* with point mutations P94S/D160N/K196T (Novagen), were cloned into the plasmid pET21d. Plasmid pAY430-SortaseA*, containing genes coding for ampicillin resistance, T7 RNA promotor system, and a C-terminal His6 tag enabling IMAC purification, was transformed into

E.coli BL21(DE3) chemically competent cells. Cultivation was performed in complex medium TSB +

y, in presence of ampicillin. Protein expression was induced by addition of 1 mM IPTG, triggering transcription of the lac-operon, at OD600 between 0.6 – 0.8. Cultures of Sortase A* were incubated in

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and cell pellets were dissolved in IMAC binding buffer. Sonication was used in order to lyse the cells (1.0 s pulse for 2 min 30 s). Samples were put on ice during sonication to not overheat thermosensitive Sortase A*. Cell debris was removed by centrifugation prior to IMAC purification. Talon® Metal Affinity Resin was used to purify Sortase A*, which later was eluted with IMAC elution buffer. Buffer was changed to Sortase A storage buffer using Vivaspin 10 kMw (GE Healthcare). Through this procedure, Sortase A* was additionally concentrated to 1 mM and stored in -80℃.

Design of PNA probes

The PNA based probes used in this project are based on the earlier reported PNA pretargeting probes HP1 and HP2 [9]. The PNA conjugation probes contain three N-terminal glycines required for Sortase A-mediated ligation. All PNA probes contain a DOTA ( 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelator. The macrocyclic DOTA chelator forms stable complexes with radiometals, hence enabling attachment of such for imaging or therapeutic purposes. [7] Radiometals such as 111_{In or} 68_{Ga attached to the primary PNA probe are suitable for imaging. The ability to perform imaging is}

particularly important when characterising pharmacokinetics of (ZHER2:342)2-2/13 linker-SR-HP15, when

optimising dosing, and when determining which patients would benefit from pretargeted therapy. Patients lacking overexpression of the HER2 receptor would be spared the treatment and thus also potential side effects. DOTA chelator attached to the secondary probe, enables labelling with radiometals such as beta emitter 177_{Lu or}212_{Bi, which could be useful for treatment of small tumors. [25]}

The secondary probes are further functionalized with tyrosine (Y), enabling direct radioiodination. [26] Direct iodination with e.g. 131_{I has previously been utilized in RIT.}131_{I is a beta emitter and the labelling}

chemistry of 131_{I is simple.}131_{I is also a gamma emitter, thus it can be utilized for imaging. [27] AEEA}

((2-(2-aminoethoxy)ethoxy)aceticacid) was introduced to increase solubility and to prevent steric hindrance.

Synthesis and purification of PNA probes

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procedure was repeated 3 times in order to remove PNA from the resin. DOTA chelators and AEEA were purchased from CheMatech and Sigma Aldrich, respectively.

Synthesized PNA probes were lyophilized, dissolved in 0.1 % TFA, 10 % ACN in MilliQ water, and filtered (pore size 0.45 µm, diameter 4 mm, Millex-HV PVDF filter) prior to purification with RP-HPLC on a semi preparative column (5 µm, 9.4 x 250 mm Zorbax 300SB-C18, Agilent Technologies), using a

gradient increasing content ACN from 10% to 95% ACN in water in 17.5 min. Total running time was 20 min, and a column temperature of 70°C was used in order to obtain maximal separation of potential impurities. Molecular weights were verified using MALDI ToF mass spectrometry with α-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The purity of purified PNA probes were later evaluated with RP-HPLC using analytical column (3.5 µm, 4.6 x 150 mm Zorbax 300SB-C18, Agilent Technologies)

with column temperature 70°C. Samples were injected and exposed for a gradient increasing from 0-50 % ACN with 0.1% TFA, in a time span of 30 min.

Conjugation of HP15 to (Z

HER2:342

)

2

-2/13 linker

After confirming purity and identity of HER2-binding Affibody molecules, and the PNA probe HP15, conjugation of Affibody to HP15 was performed by Sortase A-mediated ligation. A molar ratio of 2.5:1 of (ZHER2:342)2-2/13 linker to PNA probe was used in order to minimize the amount of non-conjugated

Affibody molecules. The reaction proceeded in Sortase A ligation buffer. Ni(II)Acetate was added in equimolar amount to the Affibody molecules in order for the Ni2+_{to bind to His}

6 tag hence inhibiting

the backwards reaction. Sortase A* was added before incubation of reaction for 30 min in 37℃. The reaction was stopped by addition of 0.1% TFA in H2O. Ni(II)Acetate was removed by adding 50 mM

EDTA to the reaction. EDTA was removed in PD-10 exchange column, and the sample was subsequently lyophilized and dissolved in IMAC buffer without imidazole. The conjugation product was purified with IMAC followed by RP-HPLC. Conjugation product was dissolved in 0.1% TFA in 10% ACN in MilliQ water prior to RP-HPLC purification. Semi preparative column Zorbax C18 and a

gradient ranging from 10 % ACN to 40 % ACN in 33.5 min were used for the purification. Column temperature was set to 70°C for all runs to improve separation. Molecular weight (Table 2) and purity of purified conjugates were confirmed with MALDI ToF and SDS-PAGE.

Table 2 Molecular weights of conjugates of HP15 to the different Affibody molecules.

Conjugate Molecular weight [Da] Extinction coefficients at 260 nm [M-1_cm-1_]

(Z-HER2:342)2-2 linker-SR-HP15 20088.8 153800 (Z-HER2:342)2-13 linker-SR-HP15 20906.9 153800

Z-HER2:342-SR-HP15 13256.8 153800

Binding analysis with Surface Plasmon Resonance

Two separate binding analysis experiments were performed using Biacore 3000 (GE Healthcare), one in which the HER2 receptor was immobilized to Biacore chip surface, and (ZHER2:342)2-13 linker-SR-H6

was injected over the surface. In the other experiment, ZHER2:342-SR-HP15 was immobilized to the chip

surface and the complementary PNA probe HP18 was acting as analyte, injected and flown over chip surface.

All Biacore experiments were performed at 25℃ using carboxymethylated dextran-coated gold sensor chips (CM5). Phosphate-buffered saline with Tween (PBS-T) (10 mM Na2HPO4, 150 mM NaCl, 0.005%

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immobilization of HER2-receptor/ZHER2:342-SR-H6 with N-hydroxysuccinimide (NHS) and

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) (Amine Coupling Kit, BR-1000-50, GE Healthcare). The flow rate used was 50 µl/min during the experiments, and 1 M ethanolamine (Amine Coupling Kit, BR-1000-50, GE Healthcare) was used as deactivation solution.

Prior to immobilization, response levels of HER2-Fc (Recombinant Human HER2-Fc, Sino Biological Inc.) and ZHER2:342-SR-HP15 were calculated and fixed, aiming for an immobilization level

corresponding to a maximum response ranging from 50 to 200 resonance units (RU) (Table 3). For all experiments, one channel in the chip was left as a reference surface, prepared with only activation of NHS and EDC, followed by deactivation with ethanolamine. Analytes were injected with concentrations ranging from 3.2 nM to 200 nM and 11.3 nM to 725 nM, in case of ZHER2:342-SR-H6 and HP18,

respectively. Samples were diluted in PBS-T and filtered prior to injection. Injection of 250 µl of sample was followed by association times of 5 min, and dissociation times of 40 min and 20 min for binding analysis of ZHER2:342-SR-H6 and HP18, respectively. Dissociation time for ZHER2:342-SR-H6 was

prolonged to 80 min for the highest concentration (200 nM) in order to determine the dissociation rate constant. Regeneration of ZHER2:342-SR-H6 was performed with a 25 µl injection of 10 mM HCl, followed

by 20 µl 10 mM NaOH. Regeneration of the sensor surface after binding of HP18 was performed by injection of 25 µl 25 mM NaOH. Chips were stabilized in PBS-T for 20 min between injections. Data obtained from Biacore measurements were fitted to a 1:1 Langmuir model for further determination of kinetic constants for the reactions. All samples were filtered with 0.45 µm, 4 mm diameter, Millex-HV filter prior to injection in Biacore.

Table 3 Applied conditions in Biacore measurements.

Ligand RU of ligand Analyte Association time [min] Dissociation time [min] Regeneration conditions ZHER2:342-SR-HP15 200 HP18 5 20 25 mM NaOH HER2-Fc 750 (ZHER2:342)2-SR-H6 5 40 10 mM HCl + 10 mM NaOH HER2-Fc 850 (ZHER2:342)2-SR-H6 (conc. 200 nM) 5 80 10 mM HCl + 10 mM NaOH

Spectroscopic characterisation with Circular Dichroism

Circular dichroism (CD) was used to determine secondary structures of (ZHER2:342)2-13 linker-SR-H6 and

ZHER2:342-SR-HP15:HP18. Solutions of 50 µM of each component were prepared by dissolving

lyophilized samples in 10 mM KPi, 10 mM KCl, pH 7.4. When analysing ZHER2:342-SR-HP15:HP18,

solutions of ZHER2:342-SR-HP15 and HP18 were heated to 95°C for 5 min, and a fixed volume of both

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RESULTS

Conjugation of HP15 to (Z

HER2:342

)

2

-2/13 linker-SR-H

6

Production of (ZHER2:342)2-2/13 linker-SR-H6 was monitored by SDS-PAGE (Figure 5) and MALDI ToF

(data not shown). According to results obtained from SDS-PAGE analysis, dimers (ZHER2:342)2-2/13

linker-SR-H6 are pure following purification with IMAC, giving rise to one distinct band (gel A and gel

B). Molecular weights of (ZHER2:342)2-2/13 linker-SR-H6 agrees with expected molecular weights when

examined with MALDI ToF. Conjugation of HP15 to (ZHER2:342)2-13 linker-SR-H6 (Gel B) generates

one distinct band corresponding to approximately 30 kDa according to reference ladder (LMW SDS-marker kit, GE Healthcare), suggesting successful purification with RP-HPLC. The dimer complexes (ZHER2:342)2-13/2 linker-SR-H6 have theoretical molecular weights of 20.9 kDa and 15.6 kDa

respectively. Previous experiments indicated that monomeric ZHER2 runs as a larger protein in

SDS-PAGE. SDS-PAGE analysis of (ZHER2:342)2-2 linker-SR-HP15 subsequent to purification with

RP-HPLC, resulted in several visible bands (marked in orange and blue arrows, Gel A). Hybridization of HP18 to the complex gave rise to band corresponding to increase in molecular weight in a similar pattern. Running buffer was changed from reducing buffer to urea buffer in order to denaturate potential secondary structure of the PNA, hence forcing PNA to migrate through the gel according to molecular weight. Change of running buffer gave rise to different bands on gel (Gel C). However, potential contaminations are still visible.

Figure 5 SDS-PAGE gels of (ZHER2:342)2-2/13 linker-SR-H6, (ZHER2:342)2-2/13 linker-SR-HP15 and (ZHER2:342)2 -2/13 linker-SR-HP15:HP18. Different running buffers were applied for the gels. Reducing buffer was used for all samples in gel A and B, while urea buffer was used for all samples in gel C.

Products from Sortase A-mediated ligation were purified using RP-HPLC (Figure 6). Unconjugated HP15 is eluted approximately by 18% ACN, (ZHER2:342)2-2/13 linker-SR-HP15 is eluted approximately

at 34% ACN. By-products were formed in the reaction and not successfully removed in IMAC purification prior to analysis with RP-HPLC. The by-products are eluted at approximately 35% ACN. Peaks were collected and molecular weights were confirmed with MALDI-ToF (data not shown). Elution of unconjugated HP15 and (ZHER2:342)2-2/13 linker-SR-HP15 were primarily monitored by

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Peaks corresponding to unconjugated HP15 and (ZHER2:342)2-13/2 linker-SR-HP15 were collected for

future conjugation and analysis.

Figure 6 Representative chromatogram from RP-HPLC purification of the product from Sortase A-mediated

ligation of HP15 to (ZHER2:342)2-2/13 linker-SR-H6. Red arrow marks the peak corresponding to (ZHER2:342)2 -SR-HP15.

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Purification of PNA hybridization probe

The chromatogram after 2 iterative purifications of HP18 using RP-HPLC is shown in Figure 8A. Purity of HP18 was determined with MALDI-ToF and RP-HPLC using the analytical column Zorbax C18.

Chromatogram obtained from analytical RP-HPLC after two repeated purifications by RP-HPLC suggests high purity (Figure 8B). Spectrum from MALDI-ToF contains a peak corresponding to the expected m/z (Figure 8C).

Figure 8 Chromatograms of HP18 from RP-HPLC analysis and spectrum of HP18 from analysis with

MALDI-ToF. A – Chromatogram after two iterative RP-HPLC purifications of HP18 with the semi-preparative column Zorbax C18. B – Chromatogram of RP-HPLC analysis of purified HP18 using the analytical column Zorbax C18. C – MALDI-ToF spectrum of purified HP18. The spectrum contains the peak corresponding to expected molecular weight, assuming z=1.

Biacore binding analysis

Biacore analysis of hybridization of HP18 to immobilized ZHER2:342-SR-HP15 (Figure 9) resulted in an

association rate constant of ka=4.2∙ 104 M-1s-1. Dissociation rate constant, kd, requires a longer

dissociation phase between injections in order to be estimated. Dissociation time of 25 minutes resulted in a decrease in barely 1 RU, which is not sufficient for estimation of kd. Chi2 value for the measurement

was 0.272. Reproducibility of the result was not tested due to degradation of chip surface as a result of harsh regeneration conditions.

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Figure 9 Sensorgram from Biacore analysis of the hybridization of HP18 to immobilized ZHER2:342-SR-HP15. Rate constant of association was estimated to ka=4.2∙104 M-1 s-1. Rate constant of dissociation, kd, was too slow to be determined during this experiment.

Analysis of binding of (ZHER2:342)2-13 linker-SR-H6 to immobilized HER2 receptor were repeated at two

separate occasions (representative sensorgram is illustrated in Figure 10). Two separate rate association constants were determined; ka=2.1∙105 M-1 s-1 and ka=1.6∙105 M-1 s-1. Chi2 values were calculated to

0.741 and 0.592. Dissociation rate constants could not be determined during these experiments due to too slow dissociation of (ZHER2:342)2-13 linker-SR-H6 from HER2 receptor. After a dissociation time of

40 minutes, a decrease of approximately 1 RU was achieved, corresponding to a decrease of 2 %, which is not enough for estimation of kd. Two separate runs were performed to estimate the dissociation rate

constant for the reaction (Figure 11). Dissociation rate constants were estimated to kd=1.5∙10-5 s-1 and

kd=1.6∙10-5 s-1. Equilibrium dissociation constant, KD, for binding of (ZHER2:342)2-13 linker-SR-H6 to

HER2 receptor was estimated to KD=86 pM (Table 4).

Table 4 Kinetic parameters obtained from binding analysis with Biacore.

Immobilized protein

Analyte Association rate constant, ka [M-1 s-1] Dissociation rate constant, kd [s-1] Chi2 _Mean ZHER2:342 -SR-HP15 HP18 4.2∙104 _0.272 HER2-Fc (ZHER2:342)2-13 linker-SR-H6 1.6∙105 _{- 0.592} HER2-Fc (ZHER2:342)2-13 linker-SR-H6 2.1∙105 _{- 0.741} _1.8∙105 HER2-Fc (ZHER2:342)2-13 linker-SR-H6 - 1.5∙10-5 _0.378 HER2-Fc (ZHER2:342)2-13 linker-SR-H6 - 1.6∙10-5_0.328 _1.6∙10-5

Figure 10 Representative sensorgram of Biacore measurement of binding of (ZHER2:342)2-13 linker-SR-H6 to immobilized HER2 receptor. Association rate constant for current run was estimated to ka=2.08∙105 M-1 s-1.

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Figure 11 Sensorgram from Biacore analysis of binding of (ZHER2:342)2-13 linker-SR-H6 to immobilized HER2 receptor. The dissociation rate is very slow, after 80 min of dissociation, kd for the reaction could be estimated.

Spectroscopic characterisation: Circular dichroism

Secondary structures of (ZHER2:342)2-SR-H6 and ZHER2:342-SR-HP15 hybridized to HP18 were investigated

by CD spectroscopy measurements (Figure 12). CD spectrum of (ZHER2:342)2-SR-H6 shows the

characteristic pattern of an alpha-helix (Figure 12A), with negative peaks of large amplitude at approximately 210 nm and 220 nm, and positive peak of large amplitude at approximately 195 nm. CD spectrum of ZHER2:342-SR-HP15 hybridized to HP18 suggests existence of alpha helical structure, with

positive peak at 195 nm, and negative peaks around 210 nm and 220 nm. It is further possible to distinguish the hybridization of HP18 to ZHER2:342-SR-HP15, due to the presence of peaks at 240 nm and

270 nm (Figure 12B).

Figure 12 CD spectroscopy analysis of (ZHER2:342)2-13linker-SR-H6 (A) and ZHER2:342-SR-HP15:HP18 (B). Wavelength was varied from 195 nm to 260 nm and 300 nm, respectively.

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DISCUSSION

The aim of this project was to produce two HER2-binding dimeric Affibody molecules with the hypothesis of increasing the affinity for HER2 receptor due to avidity effects. In this project, affinity for (ZHER2:342)2-13 linker-SR-H6 to HER2 receptor was evaluated. Conducted experiments generated some

promising data. However, there are still experiments to be performed in order to evaluate the binding characteristics of dimeric Affibody molecules.

The short amino acid linker in (ZHER2:342)2-2 linker might reduce the risk of crosslinking the HER2

receptors on the tumor cell surface, hence reducing risk of triggering internalization. This would mainly be due to the limited reachable distance because of the short linker. Even if the (ZHER2:342)2-2 linker

protein would not be able to bind two receptors simultaneously, the apparent affinity could still be improved due to a higher local concentration of the HER2-binding domains. Furthermore, the small size of (ZHER2:342)2-2 linker could theoretically lead to a somewhat faster clearance from blood. However,

steric hindrance might be a problem when using (ZHER2:342)2-2 linker. Additionally, tumor penetration

could be affected by the linker length. (ZHER2:342)2-2 linker could result in a less flexible and “more

globular” shaped protein, which could reduce the tumor penetration for the HER2-binding Affibody-PNA conjugate. The 13 amino acid linker could circumvent these potential problems. However, (ZHER2:342)2-13 linker might be a target for proteases, increasing the risk of degradation in blood.

SDS-PAGE analysis of (ZHER2:342)2-2 linker-SR-HP15 indicated impurities even after HPLC

purification. The complex resulted in two separate bands, both when analysed as (ZHER2:342)2-2

linker-SR-HP15, and when analysed hybridized to HP18 (Figure 5). Evaluation of (ZHER2:342)2-2

linker-SR-HP15 in Biacore measurements resulted in poor quality data. Difficulties of immobilizing (ZHER2:342)2-2

linker-SR-HP15 to chip surface in Biacore experiments was also experienced. This summed up to a decision of proceeding the project focusing on (ZHER2:342)2-13 linker-SR-HP15.

Results from purification of HP18 suggest that the PNA probe is highly pure, when analysing chromatograms obtained from RP-HPLC semi preparative column, and analytical column. Programs used for the analysis were long (40 min) and a column temperature of 70℃ was used, promoting separation of impurities. Hence, any potential impurities from incomplete PNA synthesis should be visible. By products from incomplete PNA synthesis would probably be specifically troublesome to purify using RP-HPLC, due to their similarity in hydrophobicity. Spectrum of HP18 obtained from MALDI ToF contain one peak corresponding to the expected m/z. However, other peaks are also present in the spectrum. None of these peaks can be derived to incomplete coupling reactions in the PNA synthesis.

Kinetics for binding of ZHER2:342 to HER2 has previously been estimated, where the rate reaction

constants were estimated to approximately ka= 4.8∙106 M-1 s-1 and kd=1.1∙10-4 s-1. [28] The study of the

new dimeric protein suggests a slower binding of (ZHER2:342)2-13 linker-SR-H6 to the HER2 receptor. In

vivo studies are required in order to determine if the reduced association rate will have a negative impact on the radionuclide pretargeting therapy. The equilibrium dissociation constant for interaction of (ZHER2:342)2-13 linker-SR-H6 with HER2 was estimated to KD=86 pM, indicating a decreased affinity

compared to the literature value for binding of ZHER2:342 to HER2 receptor (22 pM). [28][29] However,

the literature value KD=22 pM was obtained when using HER2-ECD, and not HER2-Fc receptors, hence

the values are not fully comparable. The estimated affinity for the interaction with the dimeric Affibody molecule (KD=86 pM) indicates increased affinity if comparing to the interaction of ZHER2:342

-SR-HP1:HP2 to the HER2 receptor, which has an equilibrium constant of KD=212 pM. [9] Concentration

of (ZHER2:342)2-13 linker-SR-H6 was determined by Nanodrop measurement at 280 nm. The samples

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13 linker-SR-HP15:HP18 binding to the HER2 receptor have to be conducted before being able to compare the affinity to ZHER2:342-SR-HP1:HP2.

Results obtained from secondary structure analysis using CD spectroscopy shows that ZHER2:342-SR-H6

has the secondary structure of an alpha-helix. Further, the results show that hybridization of HP18 to ZHER2:342-SR-HP15 is feasible, indicating that hybridization of PNA probes is not disturbed by

conjugation of HP15 to the monomeric ZHER2:342.

Future Work

Further experiments are planned to be conducted after the end of this project. Due to restrictions in time span and lack of materials, not all planned experiments could be performed on time. Biacore experiment with HER2 immobilized to chip surface and (ZHER2:342)2-13 linker-SR-HP15:HP18 functioning as

analyte, will be conducted in order to confirm that hybridization of HP15 and HP18 is not disturbing binding of the complex to the HER2 receptor. There is also of interest to perform a two-step Biacore experiment where HER2 is immobilized to the chip surface and (ZHER2:342)2-13 linker-SR-HP15 is used

as analyte in the first step, and HP18 is injected and flown over chip surface in a second step. This experiment is important to confirm retained affinity between the HER2 receptor and (ZHER2:342)2-13

linker-SR-HP15.

CONCLUSION

In this thesis, Affibody molecules (ZHER2:342)2-2/13 linker-SR-H6 have been successfully produced and

purified. The Affibody molecules have further successfully been conjugated to a previously synthesized based probe, HP15, with Sortase A-mediated ligation using Sortase A*. Furthermore, the PNA-based probe HP18 have been manually synthesized with solid phase synthesis, and purified with RP-HPLC. Analysis from analytical and semi-preparative RP-HPLC suggests high purity of HP18. Results obtain from SPR analysis indicates decreased affinity for the dimeric Affibody (ZHER2:342)2-13 linker

binding to the HER2 receptor, compared to the literature value of the monomeric Affibody ZHER2:342, but

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PNA-MEDIATED AFFIBODY PRETARGETING