INOM EXAMENSARBETE BIOTEKNIK,AVANCERAD NIVÅ, 30 HP

, STOCKHOLM SVERIGE 2016

Purification of bi- and multispecific cancer therapeutic affinity proteins and C-terminally modified anti-HER3 affibody imaging agents

CHARLES DAHLSSON LEITAO

KTHSKOLAN FÖR BIOTEKNOLOGI

www.kth.se

Contents1 Background 2

1.1 HER-Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 HER-Targeted Cancer Therapeutics and Diagnostics . . . . . . . . . . . . . 2

2 Introduction 3

3 Materials and Methods 43.1 SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2 MALDI-TOF MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.3 Analysis of Available Bispecific Constructs . . . . . . . . . . . . . . . . . . 53.4 Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.5 HSA Affinity Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.6 Subcloning of genes for the imaging agents and Z3-ABD-Z3-Z2 . . . . . . 63.7 IEC purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.8 IMAC Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.9 RP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.9.1 Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.9.2 Analytical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Results and Discussion 8

5 Conclusion and Outlook 11

6 Acknowledgements 11

7 Appendix 13

Abstract

Affibody molecules are small protein scaffolds that have been engineered to bind to a variety of targets with di-verse therapeutic and diagnostic applications. In this study, an array of affibody containing therapeutic constructs,targeting HER2 and HER3, and diagnostic anti-HER3 imaging agents have been purified in preparation for subse-quent cancer cell assays and imaging studies in tumour-bearing mice respectively. Herein, the workflow for severalpurification techniques is delineated.

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1 BACKGROUND

1.1 HER-Biology

The epidermal growth factor receptor (EGFR) familyconsists of the four structurally related transmembranereceptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3(ErbB3) and HER4 (ErbB4), each with an extracellu-lar domain associated with dimerization, and an in-tracellular tyrosine kinase domain [1]. Activity of allreceptors, except HER2, is regulated by the epider-mal growth factor (EGF) family. Upon ligand bind-ing, the receptors adopt a conformation that enableshomo- or heterodimerization, activating the intrinsickinase domain resulting in trans- or autophosphory-lation of tyrosine residues situated on the kinase do-main. The residues initiate signalling cascades of var-ious biochemical pathways collectively culminating incellular proliferation, survival and migration, includingRas/Raf/MAPK, PI3K/Akt and PLCγ/PKC, by serv-ing as docking and concomitant activation sites for cy-toplasmic signal transducers that mediate downstreamtransforming effects [2].

HER2 is constitutively prone to heterodimerize withthe other HER receptors without ligand-induced con-formational change [3]. In addition to this, it pos-sesses the strongest catalytic kinase activity and is thepreferred heterodimerization partner for all other HERfamily members [4]. As a result of this, genetic aberra-tions and amplification of this receptor strongly poten-tiate downstream signalling and have been associatedwith various cancers and poor prognosis [5, 6]. Con-sequently, HER2 has been immensely scrutinized as apotential therapeutic and diagnostic target.

HER3 has a functionally impaired tyrosine kinasedomain, unlike the other HER family members, butadopts a dimerization enabled conformation upon stim-ulation by its natural ligands, neuregulin I (heregulin)and neuregulin II, forming an active heterodimeric sig-nalling unit with any of the other receptors [1]. HER3forms together with HER2 the most potent oncogenicunit in the HER family of receptors, pertaining to ac-tivation of the transforming PI3K/Akt signalling path-way mediated by phosphorylated HER3 [5, 7]. More-over, HER3 has been implicated in the resistance toHER2-targeted tyrosine kinase inhibitor (TKI) therapy[8]. Additionally, treatments targeting the extracellulardomain of HER2, such as trastuzumab, might be nul-lified through the bypass activation of PI3K/Akt via

EGFR mediated HER3 phosphorylation [9]. In fact,HER3 is pivotal in the activation of the PI3K/Akt path-way and attempts at shutting down this signalling axisby HER3-ligand inhibition could prove effective [10].Overexpression of HER3 in tumours has also been asso-ciated with a shorter survival time of patients comparedto overexpression of HER2 [11].

1.2 HER-Targeted Cancer Therapeutics andDiagnostics

A plethora of HER-family targeted therapeutics for awide range of malignancies, that are the product ofHER receptor aberrations, have been developed in re-cent years [12, 13]. In most cases, the drugs, rangingfrom small molecules to antibodies, inhibit or attenu-ate the activity of the receptor or the downstream sig-nalling, for instance, by inhibiting the intracellular ty-rosine kinase domain, preventing receptor dimerizationor by blocking ligand binding [12]. For example, am-plification of HER-receptors and mutation-driven con-stitutive autophosphorylation are distinct mechanismsfor conveying an abundance of pro-survival signals andtherefore require different treatment approaches.

Complex diseases, such as cancer, are multifacetedwith intricate and overlapping signalling networks. Aconsequence of this is the resistance to treatment bythe compensatory upregulation and activation of al-ternative signalling pathways, or by intrinsic alterna-tions of receptors, which poses a problem for targetedtreatments [14]. Combination therapies can enhance,through synergistic effects, the treatment compared toconventional single receptor targeted therapies, andpossibly circumvent resistance, by blocking the signalof e.g. both HER2 and HER3 in HER2 amplified breastcancer [7].

Alternatively, these properties can be combined intoa single therapeutic agent by utilizing a bi- or mul-tispecific format, with the potential to bind two ormore targets simultaneously. This concept has been in-vestigated with bifunctional antibodies, combining in-hibitory functions of HER1/HER3 [15], HER1/HER2[16], and HER2/HER3 [17], surpassing the inhibitoryfunction of monospecific antibodies, and a myriad ofother bispecific antibodies used for both therapy and di-agnostics [18].

A previous study has combined engineered vari-ants of the small non-immunoglobulin based disulfide-independent affibody scaffold into a bispecific format

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targeting HER2 and HER3 [19]. The smaller size of thisalternative scaffold has advantages over antibodies interms of superior tissue penetration and less complexproduction with reduced cost. However, the drawbackis the rapid plasma clearance due to its small size, thisproblem was addressed in the study by introducing analbumin binding domain (ABD) to retain the protein inblood by allowing it to associate with circulating humanserum albumin (HSA). The concept of affibodies as ther-apeutic and diagnostic agents is the main focus of thisstudy and will be discussed in greater detail.

Combining anti-HER2 and anti-HER3 therapeuticsin a bispecific format would be interesting to evalu-ate potential superior anti-oncogenic effects and ther-apy resistance circumvention, for reasons previouslydiscussed. Furthermore, this format could facilitatethe localization to tumours for treatments targetingmembrane-bound proteins that are expressed to a lesserextent, e.g. amplified HER2 on tumours can localizea bispecific protein targeting HER2 and HER3, where-upon the anti-HER3 functionality can exert its therapeu-tic function on the less abundant HER3 and vice versa.

These targeted treatments necessitate, preferentiallyrapid and non-invasive, anti-HER diagnostic agentswith high resolution for phenotypic determination andmonitoring to allow for patient stratification [20]. Thiscan be achieved by exploiting the small size of the afore-mentioned affibody scaffold, which takes advantage ofthe fast plasma clearance to improve the resolution andrate of the tumour imaging. By labelling the affibody,targeting a particular receptor, with a detectable ele-ment, tumours expressing that receptor can be visual-ized. This has been done with anti-HER2 affibody vari-ants labelled with technetium-99m [21]. Ultimately, thisconcept could be developed even further into a therag-nostic format.

2 INTRODUCTION

Affibodies are small (58 amino acids), stable and versa-tile three-helical bundle proteins with many biotechno-logical and therapeutic applications [22]. The affibodyscaffold is based on the so called Z-domain, which is de-rived from the membrane-bound protein A, originallyfound in Staphylococcus aureus. It is used by the bacte-rial cell to evade the immune system during infectionby binding to the heavy-chain Fc region of IgG, thusnullifying its effector function. ABD-Derived Affinity

Protein (ADAPT) is another small three-helical protein,derived from the albumin binding domain of strepto-coccal protein G, with properties similar to affibodies[23]. The intrinsic affinity for albumin increases the re-tention of the scaffold in blood, which is an advantagewhen considering therapeutic applications as the pro-longed effective concentration reduces the dose requiredand the number of drug applications, which diminishesdiscomfort and economical strain for the patients [24].Reversely, for diagnostic purposes, the rapid clearanceof unbound proteins is preferred [25] to achieve hightumour-to-organ ratios with examination of the resultsshortly after injection, in which case the albumin bind-ing propensity of ADAPT is undesirable. The inclusionof ADAPT, binding to both albumin and an additionaltarget simultaneously, in a bispecific construct abolishesthe need for additional domains, such as ABD, to extendthe in vivo half-life, while still retaining the propertiesassociated with small molecules.

The first part of this study is concerned with an ar-ray of bivalent/bispecific and multispecific constructs.The bivalent constructs are comprised of affibody andADAPT domains targeting HER2 that are intercon-nected by varying repeats (1-3) of a glycine and serinecontaining linker (G4S). The future prospect is to evalu-ate the effect of the linker-length on cancer cell growthinhibition by a bivalent construct targeting the dimer-ization domain of HER2. Additionally, bivalent and bis-pecfic constructs targeting HER2 and HER3 and multi-specific constructs targeting a range of different cancer-associated cell surface proteins are also included in thisstudy, for a complete list, see table 1. The affibodieshave in previous studies been selected by various dis-play techniques from a combinatorial protein library byrandomization of 13 solvent-exposed residues on helicesone and two, to bind to HER2 [26, 27] and HER3 [28].ADAPT molecules with affinities for HSA as well as anadditional binding affinity for HER2 [29] and HER3 [30]have previously been generated. A list of the affibodyand ADAPT variants used in this study is presentedin table 2. Both the anti-HER3 affibody and ADAPTvariants compete for binding to HER3 with the nat-ural ligand NRG-β1, suggesting therapeutic potential.In fact, the anti-HER3 affibody variant has previouslydemonstrated anti-proliferative effects on breast cancercell lines [31]. The epitopes recognized by the anti-HER2affibody ADAPT variants differ. Notably, the ADAPTvariant competes with trastuzumab for binding.

The second part of this study is concerned with

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Table 1: The proteins involved in the study and their respective theoretical masses, based on the isotopically averaged molecularweights. The isoelectric point (pI), based on pKa of individual amino acids, is included for its relevance in deciding on purificationstrategy for the imaging agents. The domains of the octamer and hexamer are interconnected by a G4S linker and ZVEGFR2(a)and ZVEGFR2(b) are affibodies against two different epitopes of VEGFR2. The affibody and ADAPT variants used are presentedin table 2.

Bivalent/Bispecific Multispecific Imaging agents

Construct Mw (Da) Construct Mw (Da) Construct Mw (Da) C-terminus Mw (Da) pI

A2-(G4S)1-Z2 12809 A3-(G4S)1-Z3 12671 Octamer1 52706 -GGEC 6740.5 5.31A2-(G4S)2-Z2 13124 Z3-(G4S)1-A3 12671 Hexamer2 38746 -GGGC 6668.4 6.62A2-(G4S)3-Z2 13439 A3-(G4S)1-Z2 12731 Z3-ABD-Z3-Z2 25953 -GGSC 6698.4 6.62Z2-(G4S)1-A2 12809 Z2-(G4S)1-A3 12731 -GGKC 6739.5 8.12Z2-(G4S)2-A2 13124 -QAPKC (WT) 6793.6 8.12Z2-(G4S)3-A2 13439 -QAPK (H6-WT) 7644.5 8.41

1 A3-ZEGFR-Z3-ZVEGFR2(a)-ZVEGFR2(b)-A2-ZIGF1R-Z2-Cys2 A3-ZEGFR-ZVEGFR2(a)-ZVEGFR(b)-A2-ZIGF1R-Cys

the production and purification of four C-terminallymodified derivatives of the previously generatedZHER3:08698 variant [32] as well as both the wild-type(denoted WT) and His6-tagged WT (denoted H6-WT),listed in table 1. The C-terminal modifications are de-signed to function as peptide chelators to allow for coor-dination of a radionuclide. The C-terminal variants areof the kind -GGXC, where X is either Lysine (K), Glycine(G), Serine (S) or Glutamic acid (E). Each variant will beemployed in a different study conducted in mice to in-vestigate their potential use as diagnostic tumour imag-ing agents. This has already been done using anti-HER2affibodies, with the same array of C-terminal modifica-tions [21]. For this purpose, a minimum of 1 mg of eachC-terminally modified imaging agent, and 3 mg of bothWT and H6-WT, must be produced and purified with afinal purity of at least 95%.

Table 2: The affibody (Z) and ADAPT (A) vari-ants used in this study.

Abbr. Full name Ref.

Z2 ZHER2:02891 [27]

Z3 ZHER3:05417 [28]

A2 ABDErbB2-1 [29]

A3 ABDErbB3-3 [30]

3 MATERIALS AND METHODS

3.1 SDS-PAGEThe amount of protein on SDS-PAGE gels throughoutthe study was, whenever possible, approximated to be1-5 µg for optimal visualization. The gels used wereeither Mini-PROTEAN® TGX gels (BioRad Systems) orNuPAGE® Bis-Tris gels (ThermoFisher Scientific), andthe following protocol was always employed. A totalvolume of 20 µl containing reduction and loading buffer,appropriate amount of protein (1-5 µg) and Milli-Q wa-ter was applied to each well. The samples were heatedto near boiling temperature (95°C) for 5 min to increasethe rate of reduction. For the Mini-PROTEAN® TGXgels, 1X TGS (Tris, Glycine, SDS) was used as runningbuffer (BioRad Systems), and for the NuPAGE® Bis-Trisgels, 1X MES SDS (MES, Tris, SDS, EDTA) was used(ThermoFisher Scientific). The electrophoresis programused was 140 V, 400 mA, 45 min for the TGX gels, and200 V, 400 mA, 30 min for the Bis-Tris gels. Standard al-bumin ladder (BioRad) was always used as a molecularweight reference (97, 66, 45, 30, 20.1, 14.4 kDa).

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3.2 MALDI-TOF MS

A 4800 MALDI TOF/TOF™ Mass Analyser (AppliedBiosystems) was systematically used to verify the pres-ence of target proteins throughout the purification pro-cess. Additionally, it was used as a preliminary ver-ification of mass for the bispecific constructs and theimaging agents. The matrix used was α-Cyano-4-hydroxycinnamic acid (Bruker Daltonics), which is suit-able for low to mid-range protein masses.

3.3 Analysis of Available Bispecific Con-structs

The concentration of the constructs was determined us-ing a bicinchoninic acid assay (BCA assay) with tripli-cates made for each. SDS-PAGE was used to determineif any cleaved products had been accumulated over thestorage period. MALDI-TOF MS was used to verify themass of the specific constructs. From the BCA assay, itwas concluded that the amounts of five constructs (Z3-A3, Z2-A3, Z2-3-A2, Hexamer and Octamer) were insuf-ficient for cell assays, thus more had to be produced.

3.4 Protein Expression

Available BL21(DE3)* expression strain E. coli harbour-ing isolated pET26b(+) plasmids containing the genesfor Z2-3-A2, Hexamer and Octamer and TOP10 E. coliharbouring pET26b(+) for Z3-A3 and Z2-A3 were in-oculated into 10 ml TSB+Y medium with 50 µg/mlkanamycin (KM) and incubated at 37°C overnight.

The plasmids produced from TOP10 cells were ex-tracted using QIAprep Spin Miniprep Kit (Qiagen), ac-cording to protocol, and the concentrations were mea-sured to be 96.1 ng/µl for Z3-A3 and 50.6 ng/µl for Z2-A3, using a NanoDrop 1000 spectrophotometer (Ther-moFisher Scientific). BL21(DE3)* cells were transformedwith the extracted plasmids using heat shock treatment.5 µl of plasmid was added to 1 ml BL21(DE3)* cells andincubated on ice for 20 min. The cells were heat-shockedat 42°C for 45 sec and returned to incubation on ice for5 min. 400 µl TSB was added to each tube and incu-bated at 37°C in a rotamixer for 60 min. 100 µl and400 µl from each tube were transferred to TBAB platescontaining KM and incubated at 37°C overnight. Non-transformed heat-treated cells was included as a nega-tive control. 5 ml TSB+Y+KM cultures were inoculatedwith single colonies and incubated at 37°C overnight.

5 ml from the five starter cultures were inoculatedinto 500 ml TSB+Y+KM and incubated at 37°C untilan OD600 of ~0.8 was reached, by which time pro-tein expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG), from Apollo Scientific,for a final concentration of 1 mM, followed by cultiva-tion overnight at 24°C. The cells were subsequently har-vested by centrifugation at 5000xg and 4°C for 10 minand the pellets frozen at -20°C.

3.5 HSA Affinity Purification

The albumin affinity of ADAPT and ABD enabled affin-ity purification of the construct using HSA as ligand.

The pellets were resuspended in 40 ml TST buffer(pH 8) and the cells were mechanically lysed usingfrench press (Baseclear) three times for each sample. Thelysates were centrifuged at 17,000xg and 4°C for 10 minand the supernatant filtered (0.45 µm). The columnswere prepared using HSA-coupled agarose matrix (10ml column volume) and pulsed with 30 ml Milli-Q, 30ml 0.5 M HAc (pH 2.8) and 60 ml TST. The columnswere equilibrated with 75 ml TST followed by the ad-dition of the protein sample (approximately 40 ml). Thecolumn was washed with 75 ml TST followed by 50 ml 5mM NH4Ac (pH 5.5) and the target proteins were elutedby 15 ml HAc into 1 ml fractions. Before storage, thecolumns were pulsed with 30 ml HAc, 30 ml NH4Ac,60 ml TST and filled with 20% ethanol. The absorbance(280 nm) of the eluate fractions was measured and theprotein containing fractions were pooled.

Z2-3-A2 was lyophilized but exhibited extensive ag-gregation when resuspended in Phosphate BufferedSaline (PBS). Therefore, instead of freeze-drying, thebuffer was changed to PBS buffer using disposable PD-10 columns (GE Healthcare) for the other constructs(Octamer, Hexamer, Z2-A3, Z3-A3). Fractions of 2.5ml protein solution was applied to the PD-10 columnand eluted with 3.5 ml PBS. The constructs were sub-sequently aliquoted and frozen at -20°C. Attempts atsolubilizing the aggregated Z2-3-A2 included adding L-Arginine to a concentration of 50 mM and increasing thevolume. The residual aggregates were spun down andthe clear supernatant was aliquoted and frozen.

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3.6 Subcloning of genes for the imagingagents and Z3-ABD-Z3-Z2

Production of the anti-HER3 imaging agents was ini-tially attempted using pUC57 expression vectors. How-ever, low to no protein expression was observed us-ing an E. coli BL21(DE3)* strain for any of the imagingagents. Previously successful production and purifica-tion of bi-specific constructs using a pET26b(+) expres-sion vector rationalized the subcloning of the affibodygenes from pUC57 to pET26b(+).

Reverse primers, annealing to the 3′ end, were de-signed for each protein gene and ordered (IntegratedDNA Technologies). The same forward primer wasused for all except for H6-WT and -GGEC. The primerscomprised specific overlap sequences, NdeI and XhoIrestriction sites on the forward and reverse primer re-spectively and an overhang with a length of six nu-cleotides, see table 5 in appendix.

The gene inserts for the imaging agents and Z3-ABD-Z3-Z2 on pUC57 were amplified by touchdown PCR us-ing the designed primers and Phusion® High-FidelityDNA Polymerase (ThermoFisher Scientific). AdditionalPCR products could be observed from the amplificationof the Z3-ABD-Z3-Z2 insert. The correct Z3-ABD-Z3-Z2amplicon with the size of 700 bp was isolated by excis-ing a preparative 1% agarose gel followed by purifica-tion using QIAquick Gel Extraction Kit (Qiagen)

The amplified inserts were purified using QIAquickPCR Purification Kit (Qiagen), according to protocol,and digested overnight at 37°C using XhoI and NdeI re-striction enzymes (New England Biolabs), both lackingstar activity. The concentrations were measured withNanoDrop. Digestion protocol for 1 µg of DNA was ob-tained from NEBCloner [33]. Following digestion, theinserts were again purified to remove the overhang se-quences.

The pET26b(+) vector backbone was generated bydigestion using XhoI and NdeI. The digested backbonewas separated from circular plasmid and insert by elec-trophoresis on a DNA recovery 1% agarose gel at 140 Vfor 30 min. The digested vector band was excised fromthe gel and the backbone was recovered using QIAquickGel Extraction Kit, according to protocol, and the con-centration measured.

Ligation was performed using 50 ng of vector back-bone, 15x molar excess of insert (30 ng for the imagingagents and 87.5 ng for Z3-ABD-Z3-Z2), T4 DNA ligase,5 µl of T4 DNA ligase buffer (New England Biolabs) in a

total reaction volume of 50 µl. The reaction was allowedto proceed for 2 hours. The vectors containing each in-sert were sent for sequencing using a promoter specificforward primer (T7 promoter) and the insert specific re-verse primer.

KCM-competent TOP10 cells were transformed, ac-cording to the aforementioned protocol, using 8 µl of lig-ated plasmid for each construct and 2 µl of KCM solu-tion. Five colonies for each imaging agent and Z3-ABD-Z3-Z2 were picked, except for ZHER3-WT, for whichonly four colonies were available. Each colony wasdipped in 20 µl of Milli-Q and then transferred to a newTBAB KM plate.

Colony PCR was performed on the in-solutioncolonies using DyNAzyme II DNA polymerase (Ther-moFisher Scientific) and the respective primers. TheTOP10 colonies verified to harbour plasmids with in-serts of correct size were cultured in 10 ml TSB+Y+KM.Subsequently, the plasmids were extracted using QI-Aprep Spin Miniprep Kit (Qiagen), according to pro-tocol. Additionally, freeze-stocks containing 200 µl ofeach culture mixed with 800 µl glycerol were made andstored at -80°C.

The extracted plasmids were used to transformBL21(DE3)* cells and the obtained colonies were cul-tured and induced to produce protein, as previously de-scribed. As with the TOP10 cells, a freeze-stock wasmade for the BL21(DE3)* cells. Following overnightproduction, the cells were harvested and the expressionanalysed with SDS-PAGE on intact cells. All imagingagents and Z3-ABD-Z3-Z2 were overexpressed, exceptfor -GGKC. For this reason, the five -GGKC coloniesobtained from the transformation underwent the samepreparatory process for protein production as previ-ously described, to determine if any exhibited suffi-cient expression of the target protein. All -GGKCcolonies harboured the correct insert as establishedby colony PCR. Furthermore, the plasmid from each-GGKC colony was sent for sequencing. The presenceof insoluble -GGKC contained in inclusion bodies wasinvestigated, by separating and comparing the solubleand insoluble proteins on SDS-PAGE. The -GGKC geneon pET26b(+) was ultimately ordered (BioBasic).

3.7 IEC purification

The first step purification of the non-tagged imagingagents was performed using ion exchange chromatogra-phy (IEC) on ÄKTAexplorer (GE Healthcare). The type

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of IEC was decided based on the pI, listed in table 1.ZHER3-WT and -GGKC have a pI of 8.12, thus cation ex-change was chosen using 20 mM MES (pH 5.5) as washbuffer, and 20 mM MES + 1 M NaCl (pH 5.5) as elutionbuffer. For the imaging agents with a pI of 5.31 and 6.62,anion exchange was chosen using 20 mM Tris (pH 8.6)as wash buffer and 20 mM, 1 M NaCl (pH 8.6) as elutionbuffer. All buffers were filtered (0.45 µm) and degassedbefore use.

A general procedure before IEC purification was em-ployed for all imaging agents. The frozen pellet was re-suspended in 10 ml of the appropriate wash buffer. Thecells were lysed by sonication. The lysates were cen-trifuged at 10,000xg and 4°C for 20 min. The supernatantwas heat-treated at 90°C for 10 min, followed by instantincubation on ice for 20 min. An additional 10 ml ofwash buffer was added before spinning down precipi-tated proteins at 10,000xg and 4°C for 20 min. Lastly, thesupernatant was filtered (0.45 µm).

The columns used for the cation and anion exchangechromatography were Resource S (sulphonate group asligand) and Resource Q (quaternary ammonium as lig-and) respectively (GE Healthcare). The flow-rate was4 ml/min and the linear elution buffer gradient wentfrom 0-50%, however, these parameters were in somecases optimized for maximized peak separation basedon pilot runs for each protein. Eluate fractions andflow-through were analysed with MS and SDS-PAGEto determine the peaks corresponding to the target pro-tein. Large amounts of protein were observed in theflow-through from the purification of WT, -GGGC, and-GGSC, possibly due to an overload of the column,hence a second round of purification was undertaken tosalvage as much protein as possible.

The correct fractions were pooled and the bufferchanged to NH4Ac (pH 7) using PD-10 columns fol-lowed by lyophilization. 500 µl from each imaging agentwas taken and freeze-dried separately with the purposeof checking for aggregation after resuspension and to beused in pilot RP-HPLC runs.

3.8 IMAC Purification

The lysate preparation was identical to the other imag-ing agents, described above. The IMAC column waspacked with ~3 ml TALON Metal Affinity Resin (Clon-tech) and pulsed with 30 ml Milli-Q followed by equi-libration with 40 ml wash buffer (50 mM Na2HPO4,500 mM NaCl, 15 mM imidazole, pH 8). The lysate sam-

ple (~20 ml) was applied to the column and impuritieswere removed with the wash buffer. A buffer containing50 mM Na2HPO4, 500 mM NaCl, 500 mM imidazole, pH8 was used to elute the proteins. The protein containingfractions were pooled and the buffer changed to NH4Ac(pH 7) with subsequent lyophilization.

3.9 RP-HPLC

3.9.1 Purification

ZHER3-WT (+FT), -GGEC, and -GGGC (+FT) were puri-fied with reversed-phase high-performance liquid chro-matography (RP-HPLC) on Agilent 1200 HPLC Systems(Agilent Technologies) using 0.1% trifluoroacetic acid(TFA) in water for washing and a 20-40% linear gradi-ent of 0.1% TFA in acetonitrile (ACN) for eluting. Theflow-rate was 3 ml/min for 30 min, with some opti-mization based on pilot chromatograms. The columnused was Zorbax 300SB-C18 Semi-prep, 9.4x250 mm, 5µm particle size (Agilent Technologies). The lyophilizedproteins were resuspended in 20% ACN. Two distinc-tive peaks were obtained that, according to MS analy-sis, corresponded to the same protein (Fig. 9a). Thiswas assumed to be the result of dimerization via theC-terminal cysteine. The peaks putatively correspond-ing to monomers and dimers were pooled separately.With this knowledge, -GGSC was reduced with 20 mMDithiothreitol (DTT), from Saveen & Werner, before pu-rification to obviate dimer formation. The monomerand dimer eluate fractions were freeze-dried and resus-pended in NH4Ac (pH 7.2). H6-WT was purified with-out any prior reduction since it lacks a C-terminal cys-teine.

3.9.2 Analytical

To evaluate the purity of each imaging agent, analyticalRP-HPLC was performed with a Zorbax 300SB-C18 An-alytical column, 4.6x150 mm, 3.5 µm particle size (Ag-ilent Technologies) using a 20-40% linear ACN + 0.1%TFA buffer gradient and a flow-rate of 0.9 ml/min for30 min. 10 µg from both the monomer and dimer frac-tions were pooled and reduced with 20 mM DTT for30 min at 37°C. Following reduction, an equal amountof 40% ACN + 0.1% TFA was added for a final concen-tration of 20%. The sample was filtered (0.45 µm) be-fore injection. This procedure noticeably decreased thepresumed dimer peak, but not completely, strongly sug-

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gesting the presence of dimers (Fig. 9b). In an attemptto entirely reduce the proteins, thus producing a soli-tary peak, a stronger reducing agent was used. Reduc-tion using 5 mM Tris(2-carboxyethyl)phosphine (TCEP),from Sigma-Aldrich, for 20 min was performed in anotherwise identical procedure prior to analytical RP-HPLC. Complete reduction using TCEP was achievedfor all imaging agents (Fig. 9c) with the exception of-GGEC, which still exhibited a minor presence ofdimers.

The monomer and dimer fractions of each con-cerned imaging agent were pooled, reduced with 20 mMTCEP for 30 min, and the buffer changed to NH4Ac(pH 5.5) to prevent redimerization and to remove thereducing agent. The absorbance (A280) of all imagingagents was measured and the proteins were distributedin 100 µg aliquots, freeze-dried and stored in -20°C.

4 RESULTS AND DISCUSSION

The concentration and mass measurements of the pre-viously produced constructs as well as the amounts andmass identity of the constructs produced and purified inthis study are summarized in table 3. The structural in-tegrity of the previously produced constructs was anal-ysed with SDS-PAGE (Fig. 2a). SDS-PAGE was alsoused to evaluate the purity level of the newly producedconstructs (Fig. 1 and 2b), as no HPLC-based purityanalysis was performed.

Figure 1: Purification of Z3-ABD-Z3-Z2. (1) Standard albuminladder (2) cell lysate (3) flow-through (4) TST wash (5) NH4Acwash (6) pooled eluate.

Concentration analysis of the available constructs,using both spectrophotometric measurement and BCA,revealed a tenfold lower concentration than what hadpreviously been reported (table 3), requiring additional

protein to be produced and purified for subsequent cellassays. SDS-PAGE did not reveal any cleaved products(Fig. 2a), however, the approximate masses of the pro-teins on the gel were not in concordance with the the-oretical masses (table 1), with bands slightly above the14.4 kDa mark. This was not corroborated by the MS re-sults (table 3), which indicated an actual mass closer tothe theoretical, with some deviations. These deviationscould possibly be due to the relatively poor precision ofthe instrument, with significant differences in measuredmass observed between runs (data not shown). How-ever, this seems to be more prominent for larger pro-teins, as the newly produced imaging agents exhibiteddifferences within the range isotopic distribution whilstthe newly produced Z3-ABD-Z3-Z2 exhibited a consid-erable mass difference of -37.3 Da, and also significantlylower peak signal (spectrum not shown), see tables 4and 3. This was especially noticeable for the octamer,which exhibited close to no signal and a significant massdifference of -256 and -182 Da for the previously andnewly construct respectively. Potential improvement inthis regard could be to use another more suitable matrixto facilitate desorption of these large proteins. A plausi-ble explanation for some of these mass deviations couldbe the oxidation of methionine to methonine sulphox-ide and cysteine to cysteine sulfenic acid, which read-ily occurs at the presence of atmospheric oxygen whenstored at 5°C [34]. This would make sense for the Z2-containing constructs, since only Z2 contains a methion-ine residue, which would explain the mass deviationsfor A2-1-Z2 and possibly Z2-3-A2. A3Z3 and Z3A3,with negligible deviations, do not contain methionine,which supports this theory. A more precise mass deter-mination would be achieved with ESI-MS, which wasdesired, but unfortunately the instrument was unavail-able for use.

Sequencing established that the cloned vectors har-boured the correct gene sequences for the imagingagents and Z3-ABD-Z3-Z2, except for some discrep-ancies observed in the sequences for WT and H6-WT.However, these could be dismissed after inspection ofthe sequencing chromatograms (Fig. 6 in appendix)and MS verification, which corroborated the correct se-quences. The sequencing of the plasmids correspondingto all five available -GGKC colonies were inconclusive.

8

Table 3: Summary of the analysis on the bi- and multispecific constructs. (1) Available amounts (mg) based on absorbances ob-tained from BCA. (2) Available amounts (mg) based on absorbances obtained from single spectrophotometric measurements. (3)Amounts (mg) produced and purified in this study. (4) Mass differences (Da) between theoretical and MALDI-TOF MS measure-ments, see theoretical masses in table 1. Mass differences of the newly produced constructs are in parenthesis.

A2-1-Z2 A2-2-Z2 A2-3-Z2 Z2-1-A2 Z2-2-A2 Z2-3-A2 A3-Z2 Z2-A3 Z3-A3 A3-Z3 Octamer Hexamer 3-A-3-2

(1) 0.47 0.55 0.57 0.61 0.38 0.04 2.58 - 0.12 0.53 0.32 0.11 -

(2) 0.55 0.25 0.28 0.49 0.38 0.08 2.17 - 0.07 0.34 0.12 0.3 -

(3) - - - - - 10.23 - 2.63 9.74 - 5.93 19.91 23.84

(4) 17.5 23.2 7.2 -5.5 -3.6 24.8 (-12) 0.76 9.9 (6.8) -0.04 (-2.7) 1.5 -256 (-182) -91.5 (-13.3) -37.3

(a) (b)

Figure 2: (a) SDS-PAGE on the previously produced and biotinylated (451.54 Da mass difference from those listed in table 1) bi-and multispecific constructs, (1) standard Albumin ladder (97, 66, 45, 30, 20.1, 14.4 kDa), (2) A2-1-Z2, (3) A2-2-Z2, (4) A2-3-Z2,(5) Z2-1-A2, (6) Z2-2-A2, (7) Z2-3-A2, (8) A3-Z2, (9) A3-Z3, (10) Z3-A3, (11) Hexamer, (12) Octamer. (b) New production of non-biotinylated (451.54 Da mass difference) bi- and multi-specific constructs (1) Standard albumin ladder (2) Octamer (3) Hexamer(4) Z2-3-A2, (5) Z2-A3 (6) Z3-A3.

Figure 3: SDS-PAGE page on intact cells to analyse proteinexpression. The overexpressed bands correspond to affibodydimers. (1) Standard albumin ladder, (2) WT, (3) H6-WT, (4)-GGGC, (5) -GGEC, (6) -GGKC, (7) -GGSC, (8) Z3-ABD-Z3-Z2.

The genes for the imaging agents and Z3-ABD-Z3-Z2 were successfully cloned into pET26b(+) and exhib-ited overexpression of the correct proteins following cul-tivation and induction using IPTG, except for -GGKC(Fig. 3). This was perplexing since all kanamycin se-

lected colonies obtained from transformation of the lig-ated vector insert harboured circular plasmids with thecorrect insert size, based on results from colony PCR.None of these five colonies overexpressed -GGKC (Fig.4), nor did they contain any -GGKC trapped in inclu-sion bodies (data not shown). The gene in pET26b(+)was instead acquired from BioBasic, which successfullyexpressed when induced in BL21(DE3)* cells.

A summary of the protein amounts obtained afterIEC and RP-HPLC purification as well as the puritylevel of each imaging agent and MALDI-TOF MS deter-mined masses are presented in table 4. The amount ofthe -GGGC obtained after HPLC was comparably low(0.153 mg), entailed by the low amounts obtained fol-lowing AIEX purification which could be a consequenceof the ambiguous chromatogram (Fig. 7c in appendix)and low expression, indicated somewhat by a relativelyfaint band on SDS-PAGE (Fig. 3). A second batch of-GGGC was produced and instead purified with CIEX

9

Table 4: Summary of the purification results for the imaging agents concerning the amounts obtained after IEC and RP-HPLCpurification, the A280 purity level revealed by analytical HPLC, and the difference in mass from MALDI-TOF MS measurementscompared to the the theoretical values presented in table 1. The values in parenthesis are the amounts obtained from the purifi-cation of flow-through from the first batch. The results from the second batch is presented on the second row for each concernedconjugate.

ConjugatePurifiedusing

Amount(mg)

Amount afterHPLC (mg)

A280

Purity (%)∆ Mw (Da)

CIEX 4.88 (5.43) 1.1 95 -3.2WT

CIEX 5.45 3.2 100 -1.5

AIEX 0.99 (2.35) 0.15 - --GGGC

CIEX 1.4 1.1 97.2 -1

AIEX 9.8 0.85 95 -2.5-GGEC

CIEX 1.39 1.04 95.5 -2.6

-GGSC AIEX 1.36 (4.44) 1.37 100 -1.6

-GGKC CIEX 2.82 1.94 100 -1

WT-His6 IMAC 9.9 5.45 98.1 -4.7

following reduction using TCEP, resulting in a solitarypeak with a 280 nm purity level of higher than 95% (Fig.8c in appendix), revealed by subsequent HPLC purifica-tion. Although the expression was still low (1.4 mg afterCIEX), a satisfactory final amount of 1.1 mg was avail-able following HPLC purification. A cation exchangerwas also used for a second batch of -GGEC (pI 5.31) withsimilar results as -GGGC (Fig. 8b in appendix). No-tably, a low pH buffer was required for CIEX purifica-tion of -GGEC (MES pH 4.6), but owing to the stabilityof affibodies at low pH, this approach could prove to bemore effective. The amount of WT obtained from thefirst batch was insufficient as well (1.1 mg) with the re-quirement of 3 mg. A second production batch with thesame purification strategy as the first, with the exceptionof TCEP reduction, resulted in 3.2 mg (table 4). The totalamount of protein acquired after both batches exceededthe requirement of 1 mg for the C-terminally modifiedimaging agents, and 3 mg for WT and H6-WT, with atleast 95% purity determined by analytical HPLC.

The salient differences between the two types of ionexchangers, apparent from the AIEX chromatograms infigure 7 and the CIEX chromatograms in figure 8, accen-tuates the advantage of utilizing a cation exchanger forpurification, at least when concerned with the affibodymolecules studied here, and presumably also other vari-ants. The peak corresponding to the overexpressed pro-tein is easily discerned and protein identity determina-tion by SDS-PAGE or MS analysis can in these cases

often be omitted, additionally allowing for immediatepooling of primary eluate fractions and potential puri-fied flow-through.

Figure 4: SDS-PAGE on intact cells showing the lysate fromfive cultivations corresponding to five different colonies ob-tained from transformation after subcloning of the -GGKC in-sert. No expression of the target protein can be seen.

The protein retention could vary between CIEX andAIEX for each conjugate, one possibly eluting morein the flow-through or during the column wash thanthe other, however, any protein collected in the flow-through can easily be reapplied and salvaged in an ad-ditional purification run. Moreover, the amount of pro-tein remaining in the column after gradient elution waslow for CIEX compared to AIEX, which can be seen infigures 7 and 8.

A significant portion of the protein amount fromIEC purification was obtained from purifying the flow-through in the case of AIEX (table 4). The high retention

10

of undesired proteins and nucleic acids, not prominentfor CIEX, presumably saturates the binding capacity ofthe column, resulting in low retention of the target pro-tein.

Plausibly, the presence of negatively charged nu-cleotides associates with the more clustered chro-matogram observed from anion chromatography, as op-posed to the more unambiguous cation exchange chro-matogram. Additionally, there are generally more acidicthan alkaline proteins produced by E. coli strains [36],hence fewer proteins are subject to retention by a cationexchanger. It might be good practise to assume a sub-stantial loss of target protein in the flow-through andthus always reapply for one or two additional purifica-tion runs.

The rationale behind heat-treatment lies in thepropensity of affibodies to effectively and rapidly re-fold following denaturation. In fact, its three helicalbundle structure endows it with one of the fastest fold-ing kinetics currently known [35]. The instant and dra-matic temperature change will cause bulkier proteinsto aggregate and precipitate. Meanwhile, the affibodymanages to re-fold and remain soluble. It is highly ef-fective, removing a majority of the proteins present inthe bacterial lysate (Fig. 5). This level of purity might besufficient to omit ion exchange purification, and poten-tially achieve higher than 95% purity solely using HPLCpurification, thus increasing the protein yield.

Figure 5: Effectiveness ofheat-treatment. A majorityof the lysate proteins (lane2) are affected and can beremoved (lane 3). A smallportion of proteins, includ-ing the affibody (strongband), remain soluble. Theaffibody shown here is H6-WT

The imaging agents produced and purified in thisstudy were meant to coordinate a radionuclide usingthe chelating properties of the C-terminal residues, how-ever, initial reports based on the first production batch,suggest an intrinsic inability of ZHER3 to properly doso. This is surprising considering the success of a pre-vious imaging study using chelating anti-HER2 affibod-ies with the same C-terminal modifications. Instead, theimaging agents will be coupled to an external chelator

via biotin-maleimide functionalization. The C-terminalsequence is known to affect the labelling efficacy andfunction of the chelator and by extension the biodistri-bution. Even though the concept of a peptide chela-tor was unsuccessful in the case of the anti-HER3 affi-body variant, which is useful information in and of it-self, the great potential for imaging agents based on af-fibody molecules warrants great excitement for futureanti-HER3 diagnostics regardless of the labelling ap-proach.

5 CONCLUSION AND OUTLOOK

The purification workflow for the imaging agents wasoptimized throughout the study, with the most notewor-thy adjustments being the use of a cation over an anionexchanger, whenever possible, and the use of a strongreducing agent (TCEP) prior to every purification step.Additionally, the required amount and purity were at-tained for all of the imaging agents.

Unfortunately, due to time limitations, the investi-gation of the bi- and multispecific constructs could notbe finalized and was limited to concentration measure-ments, mass identity determination and stock replenish-ment by production and purification of those in scarceamounts. No kinetics or secondary structure analysiswas performed on the newly produced constructs. Ac-quisition of new such data on the old as well as the newconstructs might be warranted due to the extent of thestorage period, before commencing cell assays.

Although the potential use of the C-terminal endsof anti-HER3 affibody variants as peptide chelatorswas abrogated, exciting results await with regard tothe newly conceived approach for anti-HER3 affibodyimaging agents.

6 ACKNOWLEDGEMENTS

I would like to sincerely thank my two amazing super-visors, Tarek Bass and Stefan Ståhl, who have been im-mensely helpful and considerate throughout the entireproject. I would also like to thank my previous supervi-sor, John Löfblom, who presented me with this fantasticopportunity. Lastly, I would like to voice my exuberantappreciation for everyone at the department and my fel-low thesis workers who collectively created a wonderfulatmosphere.

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7 APPENDIX

Table 5: Primer sequences for the imaging agents and Z3-ABD-Z3-A2. Colour notation: Overhangsequence (purple), NdeI restriction site (red), XhoI restriction site (green).

GGE Forward 5’-CGTTGTCATATGGCCGAAGC-3’GGE Reverse 5’-ACAACGCTCGAGTTAACACTCG-3’

WT-His6 Forward 5’-CGTTGTCATATGCACCATCATCA-3’WT-His6 Reverse 5’-ACAACGCTCGAGCTCGAGTCATTTGGG-3’General forward 5’-CGTTGTCATATGGCTGAAGCCAAGTACGCAAAG-3’

GGG Reverse 5’-ACAACGCTCGAGTTAGCATCCTCCCCCCTGG-3’GGK Reverse 5’-ACAACGCTCGAGTTAGCATTTCCCACCCTGACTATC-3’GGS Reverse 5’-ACAACGCTCGAGTTAGCAACTTCCCCCCTGGG-3’WT Reverse 5’-ACAACGCTCGAGTCAACATTTAGGCGCTTGGGAATC-3’

3-A-3-2 Reverse 5’-ACAACGCTCGAGTCAACACTTCGGTGCTTGGCTG-3’

13

(a) (b)

Figure 6: Sequencing results analysed with Geneious (Biomatters). Deviations from expectedsequences. (a) ZHer3-WT with an erroneous representation of Serine (TCC) instead of the correctTyrosine (TAC) and (b) WT-His6 with an apparent insertion AAG(A)TT. However, this frame-shifting insertion would introduce a stop-codon a few bases downstream. Both proteins exhibitthe correct mass when analysed with MS.

(a) -GGSC (AIEX) (b) -GGEC (AIEX)

(c) -GGGC (AIEX) (d) -WT (CIEX)

Figure 7: Representative ion-exchange chromatograms for the first batch: ZHer3-WT, -GGGC,-GGEC, and -GGSC.

14

(a) WT (CIEX) (b) -GGEC (CIEX)

(c) -GGGC (CIEX) (d) -GGGC (CIEX)

Figure 8: Representative CIEX chromatograms from the second batch of WT, -GGEC and -GGGCas well as from the purification of -GGKC.

15

(a) (b)

(c)

Figure 9: Analytical HPLC chromatograms showing the effect of (a) no reduction (b) DTT reduc-tion (c) TCEP reduction.

16