MANUSCRIPT Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, Anna Orlova1*, Tarek Z. Bass2*, Christna Aterby1, Maria Rosestedt1, Lindvi Gudmundsdoter3, Charles Dahlsson Leitao2, Fredrik Y. Frejd3,4, John Löflom2, Vladimir Tolmachev4 and Stefan Ståhl2 *These authors contributed equally to this work.

ABSTRACT A number of monoclonal antbodies targetng HER3 are currently under clinical investgaton as poten- tal cancer therapeutcs. We have earlier generated high afnity (low picomolar) afbody molecules targetng HER3. These are small, 58 amino acid, non-immunoglobulin based scafold proteins that have proved suitable for tumor targetng applicatons, previously primarily for molecular imaging purposes. Our high afnity HER3-binding afbody molecule has demonstrated to have ant-proliferatve capacity on HER3-positve tumor cells. When formated as a bivalent construct, in which the two afbody moietes are fanking a small albumin-binding domain (ABD), we have recently demonstrated that tumor growth could be delayed in mice for HER3-positve xenografs. In this study, we have modifed the construct further and reduced the size. In a comparatve study, we evaluated safety, the capacity to delay tumor growth in mice with BxPC-3 xenografs, and mouse survival. Our novel construct was compared to the HER3-specifc monoclonal antbody seribantumab (MM-121), presently in clinical development. They were found to be equally potent in their therapeutc efects and in their safety profle. We conclude that this format of bivalent HER3-binding afbody molecules seems promising for further evaluaton as candidate therapeutcs for treatment of HER3-overexpressing tumors.

Introducton ing successful preclinical evaluation, several HER3-tar- geting antibodies, such as (RG7116) [5], Te use of monoclonal antibodies or their derivatives to elgemtumab (LJM176) [6], (U31287) [7], improve survival of patients with disseminated cancer is seribantumab (MM-121) [8], and the bispecifc anti- today established. Several mechanisms of action are uti- bodies duligotumab (EGFR and HER3, MEHD7945A) lized, including delivery of cytotoxic payloads (e.g. toxins, [9], MM-111 (HER2 and HER3) [10] and drugs or radionuclides) and recruitment of the immune (MM-141, IGF-1R and HER3) [11], are currently under c system. An important type of mechanism of action is linical evaluation and show in most cases encouraging re- based on prevention of mitogenic signaling through criti- sults. For example, a Phase II trial demonstrated that the cal transmembrane receptors. Currently, a major group of treatment with seribantumab plus paclitaxel signifcantly cancer-associated antibody targets belong to the tyrosine prolongs progression-free survival of ovarian cancer pa- kinase receptor family I (the human epidermal growth tients with high heregulin expression, compared with pa- factor receptor (HER) family). Clinically successful an- clitaxel alone [12]. tibodies, such as , , , Despite clinical success, the size, solubility and stability ado- and target EGFR limitations of monoclonal antibodies have urged research- or HER2, i.e. members of the HER family. Relatively re- ers to search for complementing strategies. Binding pro- cently, the importance of another member of this family, teins based on small non-immunoglobulin scafolds are HER3, as a cancer-associated target has also been recog- considered as a valuable alternative or complement to anti- nized [1,2]. Although the of HER3 is not bodies for imaging and therapy of cancer [13]. fully functional, heterodimers of HER3 with EGFR and especially HER2 provide extremely potent mitogenic sig- Afbody molecules are small (6.5 kDa) engineered scaf- naling [3]. Furthermore, there are accumulated evidences fold proteins [14], which can be selected for high afnity that HER3 expression and its signaling is associated with to cancer-associated targets. Tey possess excellent prop- resistance to anti-cancer therapy [4]. erties for tumor targeting [15]. First clinical studies with HER2-targeting afbody molecules demonstrated that Discoveries in tumor biology have been translated into de- these proteins are non-toxic, non-immunogenic and ca- velopment of therapeutic anti-HER3 antibodies. Follow- pable of target-specifc accumulation in tumors [16, 17]. Author afliatons: 1Division of Molecular Imaging, Department of Medicinal Chem- We have earlier reported the selection of anti-HER3 af- istry, Uppsala University, SE-751 83, Uppsala; 2Division of Protein Technology, School fbody molecules [18] and further afnity maturation to 3 of Biotechnology, KTH-Royal Insttute of Technology, SE-106 91 Stockholm; Afbody low picomolar afnity [19]. It was demonstrated that the AB, SE-171 63 Stockholm; 4Department of Immunology, Genetcs and Pathology, Uppsala University, SE-752 85, Uppsala, Sweden anti-HER3 afbody molecules inhibited heregulin-pro- Corresponding authors: Vladimir Tolmachev, Department of Immunology, Genetcs moted cancer cell growth in vitro by suppressing hereg- and Pathology, Uppsala University, SE-75285, Uppsala, Sweden. Email: vladimir. ulin-induced HER3 phosphorylation [20]. In the devel- [email protected] opment of a therapeutic agent, fast renal excretion of the Stefan Ståhl, Division of Protein Technology, School of Biotechnology, KTH-Royal afbody molecule should be prevented. Tis is a common Insttute of Technology, SE-106 91 Stockholm. Email: [email protected] limitation of scafold proteins for therapeutic application. 1 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab A We have earlier demonstrated that a fusion with an albu- ABD min binding domain (ABD) prolonged residence of an an- Z Z 20 kDa ti-HER2 afbody molecule in blood [21]. ABD is a scaf- HER3 HER3 fold protein (46 amino acid residues, MW ≈ 5.2 kDa) that has been engineered to bind human serum albumin with B femtomolar afnity [22,23]. A multimeric construct con- sisting of two HER3-binding afbody molecules fanking an ABD was previously designed and validated in vitro [24]. Recently, we evaluated the biodistribution and in vivo targeting properties of ZHER3:08699-(S4G)4-ABD035- (S4G)4-ZHER3:08699 (designated 3A3) [25]. It was shown that the growth of a HER3-expressing BxPC-3 pancreat- ic cell line was inhibited by 3A3 in vitro. Studies in mice MM-121 using 111In-labeled 3A3 revealed that incorporation of an ABD extended the residence time in blood compared with 155 kDa monomeric HER3-targeting unit. HER3-specifc uptake of 111In-labeled 3A3 in HER3-expressing xenografts was further demonstrated [25]. A pilot therapy study demon- strated signifcantly slower growth of xenografts in mice treated with 3A3 compared with PBS-treated control. However, survival could not be measured as the ethical permit allowed only four weeks of treatment because of Figure 1. Schematic representation of the 3A3s fusion protein and the risk for potential unknown side-efects [25]. the MM-121 with molecular masses indicated. Te aim of this study was to compare in vivo efcacy and safety in targeted therapy treatment of HER3-expressing (19.7 kDa) was generated according to the same principle; xenografts using a bivalent anti-HER3 ABD-fused af- ZHER3:08698-G4S-ABD094-G4S-ZHER3:08698, hereinafter fbody construct and anti-HER3 antibody seribantumab. denoted 3A3s. Te novel construct difers in that (1) short- Te afbody construct was re-designed for this study. er linkers between the binding moieties were used, that (2) Te albumin-binding domain ABD035 was replaced by a a deimmunized variant of ABD [26,27] was introduced, de-immunized ABD094 variant having retained high af- and that (3) a diferent but very similar HER3-binding fnity for serum albumin of diferent species [26, 27]. For afbody molecule ZHER3:08698 was chosen [19]. Te same HER3-targeting, the alternative ZHER3:08698 clone [19] size-matched non-HER3-binding construct as used pre- was utilized, which in monomeric form has demonstrat- viously, denoted TAT [24,25] was included as control. HER3:08699 ed better tumor localization than Z [28]. To re- Te bivalent fusion proteins 3A3s and TAT were produced duce size, which potentially could improve penetration of in E. coli BL21*DE3 cells (Novagen, Madison, WI) and 4 4 tumor tissue, the (S G) linkers between the protein do- purifed as described by us previously [25]. Briefy, afnity mains were replaced by shorter G4S linkers, resulting in chromatography on an anti-ABD-Sepharose matrix (Af- molecular weight reduction from 22.5 to 19.7 kDa. Te fbody AB, Solna, Sweden) was followed by reverse phase HER3:08698 4 094 4 HER3:08698 new construct Z -G S-ABD -G S-Z HPLC and size-exclusion chromatography. Finally, the was designated 3A3s. endotoxin content was analyzed (APL AB, Stockholm) and found to sufciently low for in vivo studies (<0.2 en- To validate the design, we investigated if the length of dotoxin units/mg). linkers infuenced the capacity of 3A3 to inhibit phos- phorylation of HER3 in vitro. Binding kinetics of 3A3s to Te monoclonal antibody seribantumab was produced in human HER3 and albumin as well as their murine coun- Chinese hamster ovary cells and purifed via afnity chro- terpart were measured. Te capacity of 3A3s to inhibit matography (Evitria, Zürich, Switzerland). Its purity was HER3 phosphorylation and growth of HER3 expressing confrmed using analytical size exclusion chromatography cells in vitro was confrmed, and in vivo xenograft growth (Superdex 200 10/300 GL) on ÄKTA Explorer (both GE inhibition was compared with the inhibition obtained with Healthcare, Uppsala, Sweden) seribantumab. Te infuence of treatment of HER3-ex- To confrm that the shorter linkers did not impair the pressing xenografts with 3A3s and seribantumab on mice function of the 3A3 fusion proteins, two diferent 3A3 survival was investigated, and the safety of 3A3s treatment constructs with diferent linker lengths were compared in was furthermore assessed. their ability to inhibit heregulin-induced signaling in an in vitro assay using MCF-7 cells (ATCC HTB-22). MCF-7 Materials and Methods cells were trypsinated and seeded at 25,000 cells per well Design, producton and characterizaton of the bivalent in an EnSpire-LFC microplate (Perkin Elmer, Waltham, afbody constructs. We have previously published the MA, cat no 6055408) and allowed to grow for 20 h at 37°C generation and characterization of a bivalent HER3-tar- in complete medium (RPMI 1640 medium with L-glut (Lonza, Basel, Switzerland) supplemented with sodium geting afbody construct, ZHER3:08699-(S4G)4-ABD035- pyruvate (Lonza), non-essential amino acids (Lonza), pen- (S4G)4-ZHER3:08699, (22.5 kDa), denoted 3A3 [25]. In the present study, a very similar but slightly smaller construct icillin/streptomycin (Lonza) + 10% fetal calf serum (FCS) (Gibco Life Technologies, Carlsbad, CA)). Te cells were 2 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab washed once with Hanks balanced salt solution (HBSS; lected and adjusted to the maximal OD reached by wells Sigma-Aldrich, Darmstadt, Germany, cat no H9269). 100 containing no afbody constructs. µl HBSS was added per well and the plates were incubat- Phospho-HER3 ELISA ed at ambient temperature for 1.5 h inside the EnSpire . BxPC-3 cells were seeded in 6-well Instrument (Perkin Elmer) to be used for assay readout. plates (2 000 000/well) and allowed to grow for 8 h at 37°C Te ability of the two 3A3 constructs with diferent linker in complete medium. Te cells were then starved in me- length to block heregulin-induced signaling was analyzed dium containing 0.5% FBS for 24 h. Te ZHER3 afbody by mixing the proteins in a 5-fold dilution series with a molecule, 3A3s (ZHER3-ABD-ZHER3) and TAT (ZTaq- fxed concentration of heregulin (1 nM) in a fnal assay ABD-ZTaq, negative control) were added to the wells volume of 130 µl/well. Te redistribution of dynamic mass (200 nM) and incubated for 10 minutes at 37°C. Further, upon addition of stimuli was recorded by an EnSpire In- heregulin (4 nM) was added to the wells and incubated for strument every 60 s for 1 h. Te half maximal inhibitory 10 minutes at 37°C. A well containing cells and heregulin concentrations (IC50) were determined from the dose re- was included as a positive control. All samples were treated sponse curves. in duplicates. Te media was removed and the cells were washed twice with 1.4 ml ice-cold PBS, and 0.4 ml 1x lysis Retained afnity of the 3A3s to murine ErbB3 and human bufer (1% NP-40, 20 mM Tris (pH 8.0), 137 nM NaCl, HER3 (Sino Biological, Beijing, China) was verifed on 10% glycerol, 2 mM EDTA, 1 mM activated sodium or- a BiaCore T200 system (GE Healthcare) using a CM5 thovanadate, 10 µg/ml Aprotinin, 10 µg/ml Leupeptin) sensor chip with immobilized murine and human serum was added to each well. Te cell plates were kept on ice albumin (both Sigma-Aldrich) according to a previously and the cells were detached with a cell scraper, transferred published protocol [24]. Both bivalent binders were cap- to Eppendorf tubes on ice, lysed in a sonicator bath for 45 tured on the surface, 3A3s at 100 nM and TAT at 25 nM seconds and centrifuged for 10 minutes at 14 000 rpm. Te for 12 s to allow the ABD to bind to the immobilized sur- supernatant from each tube was collected and used in the face with both target binding afbody moieties available phospho-HER3 ELISA. for interaction. After the capture step dilutions of murine ErbB3 and human HER3 were injected at concentrations DuoSet IC ELISA (R&D Systems, Minneapolis, MN) between 25 and 250 nM for 150 s for association and for detection of phosphorylated HER3 was used according 1800 s for dissociation. Separately, 3A3s and TAT were to manufacturer’s instructions as described below. 96-well injected at concentrations between 1 and 25 nM to deter- plates were coated with anti-HER3 capture antibody at 4 mine binding to albumin. Flow rates were kept at 10 µl/ µg/ml in PBS overnight at room temperature. Te plates min with PBST running bufer in all experiments and 10 were washed and blocked with blocking bufer (1% BSA, mM hydrochloric acid to regenerate the chip-surface. A 0.05% NaN3 in PBS, pH 7.2-7.4) for 2 h at room tem- 1:1 binding model was used to determine the afnity to perature. After washing, a 1:1 mixture of ”diluent #12” (1% albumin. A model allowing for a heterogeneous ligand was NP-40, 20 nM Tris (pH 8.0), 137 nM NaCl, 10% glycerol, used to ft data to determine the afnities towards murine 2 mM EDTA, 1 mM activated sodium orthovanadate) and and human ErbB3/HER3. cell lysate was added to each well in triplicates and incu- bated for 2 h at room temperature. Te plate was further Te 3A3s fusion protein was subjected to circular dichro- washed and incubated for 2 h at room temperature with ism at 195-250 nm and 20°C using a Jasco J-810 spec- anti-phospho-tyrosine-HRP, diluted 1:2000 in ”diluent tropolarimeter ( Jasco, Tokyo, Japan) in a cell with an op- #14” (20 mM Tris, 137 mM NaCl, 0.05% Tween 20, 0.1% tical path-length of 1 mm. In addition, the ellipticity at BSA, pH 7.2-7.4). Te plate was washed and substrate 221 nm was measured for each Afbody molecule while solution (1:1 mixture of Color Reagent A (H2O2) and heating the sample from 20 to 90°C (5°C/min). Color Reagent B (Tetramethylbenzidine), R&D Systems) was added. Te reaction was stopped with stop solution (2 Cell proliferaton assay . Cells for in vitro and in vivo M H2SO4) and the plate was analyzed in an Emax mi- experiments were purchased from American Type Tis- croplate reader (Molecular Devices, Sunnyvale, CA) at 450 sue Culture Collection (ATCC, Manassas, VA). Te cell nm. line BxPC-3 (human pancreatic carcinoma) was cultured in RPMI media (Life Technologies) supplemented with In vitro HER3 binding specifcity. In vitro specifcity ex- 10% fetal bovine serum (FBS, Life Technologies) and periment was performed with the aim to confrm binding maintained at 37°C and 5% CO2. Trypsin-EDTA (0.25% specifcity of 3A3s and to check cross reactivity of 3A3s trypsin, 0.02% EDTA in PBS) (Biochrom, Berlin, Germa- afbody construct and seribantumab. BxPC-3 cells (106 ny) was used to detach cells. cells/dish) were seeded the day before experiment in 6 well plates. Tis experiment was performed in triplicate. Te cell proliferation assays was performed as previous- Cells were incubated with 0.1 nM of 111In-3A3 in media ly described [25]. Cells were incubated for 6 days with (preparation was described in detail previously [25]. Addi- 0.05 to 900 nM of either bivalent afbody construct or tional cell dishes were pre-treated for 15 min with 50 nM no construct. BxPC-3 cells are autocrine for heregulin of 3A3s, seribantumab or bevasizumab (Avastin, Roche, [11], which was therefore not added to the cells. Cell sur- Basel, Switzerland), as a non-specifc control. Cells were vival was measured by adding 10 µl of cell counting kit 8 incubated for 1 h at ambient temperature and harvested (CCK8) solution (Sigma-Aldrich) and further incubated using Trypsin-EDTA. Cell associated radioactivity was at 37°C for 6 h before measuring the optical density at 450 measured using an automated gamma well counter (3-inch nm with a Tecan Sunrise microplate reader (Tecan Group, NaI(Tl) detector, 2480 Wizard2, PerkinElmer). Männedorf, Switzerland). OD measurements were col- 3 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab

Determinaton of dosage. A pharmacokinetics-pharmaco- carbonate water (diluted 1:5 from saturated solution) for 1 dynamics (PK/PD) simulation approach was used to deter- minute. Te slides were dehydrated in graded ethanol and mine the dosage regimen for the afbody molecule. A sim- lastly cover-slipped (PERTEX, Histolab) using an auto- ulation of the exposure levels of seribantumab and afbody mated glass cover slipper (CV5030, Leica). Te slides were molecule was performed using a one-compartment model scanned using the automated scanning system Aperio XT in the PK/PD simulation software Maxsim2 (Fraunhofer– (Aperio Technologies, Vista CA). Slides were examined by Chalmers Research Centre Industrial Mathematics, Goth- a pathologist. enburg, Sweden). Te dose for seribantumab was based on Statstcal analysis. data published by Schoeberl et al [29]. PK-parameters for Obtained values are presented as av- the 3A3s were adopted from in house data and the dose erage with standard deviation if not stated otherwise. Data level was iterated in order to achieve similar levels of expo- were assessed either by an unpaired, two-tailed t-test for sure for the antibody and afbody construct. comparison of two groups or by one-way ANOVA with Bonferroni correction for multiple comparisons. Log-rank Mouse xenograf studies. Tree groups (n=10) of female test for trend was used for multiple comparison of survival Balb/c nu/nu mice were subcutaneously implanted with in treated and control groups. GraphPad Prism (version 6 5×106 cells BxPC-3 per animal. Treatment was started 7 for Windows GraphPad Software) was used in order to de- days after tumor inoculation with 600 µg either 3A3s or termine signifcant diferences (p<0.05). seribantumab in 150 µl PBS. Te control group was treated Results and discussion with equal volume of PBS. Injections were done intraperi- toneally, 3 times per week, and treatment continued until Design, producton and in vitro characterizaton of the the mice reached humane end point: tumor’s size exceeded HER3-binding afnity protein 1 cm3, ulcerated, necrotic or infected tumor, animal weight . Te aim of this study was loss over 15% of initial weight, weight loss exceeded 10% to compare two diferent HER3-targeting afnity pro- within one week, debilitation reached BS2 in Body Con- teins, the 20 kDa afbody-based 3A3s fusion protein and dition Scoring, or animals conceded as sick according to a monoclonal antibody seribantumab [8] currently in clin- Guidelines for Pain and Distress in Laboratory Animals ical development (Fig. 1), for their ability to reduce tumor from US National Institute of Cancer. Mice were con- cell growth. Te 3A3s fusion protein [ZHER3:08698-G4S- trolled twice per week. Te tumors were measured using ca- ABD094-G4S-ZHER3:08698] comprises two HER3-binding lipers and volumes were calculated using ellipsoid formula. afbody units ZHER3:08698 [19], fanking a 46 amino acid albumin binding domain, ABD094 [26, 27]. Immunohistochemistry. At the time of euthanasia, tumors (together with eventual metastases), kidneys and livers were In order to confrm that the shorter G4S linkers of 3A3s excised and preserved in 37% formaldehyde until parafn- did not negatively infuence the performance as compared ized. Te tissues were subsequently embedded in parafn. to the previously described 3A3 construct [25] having four Haemotoxylin and Eosin (HE) and immunohistochemical S4G linkers fanking ABD, the constructs ability to inhib- (IHC) staining and slide scanning were performed at the it heregulin-induced signaling was analyzed in an in vit- Swedish SciLifeLab facilities. HE staining (Haemotoxy- ro cell assay (Fig 1S). Two 3A3s variants, difering only in lin (Mayers’ Histolab, Stockholm, Sweden), Eosin (0.2%, the length of the linkers, with either short (G4S) or longer Histolab)) of 4-µm sections was carried out followed by de- (G4S)4 linkers were compared. Te results showed that the hydration and mounting in Pertex (Histolab). IHC stain- two constructs had a very similar inhibitory capacity. Te ing was performed in accordance with protocols described obtained IC50 values for 3A3 with one or four G4S linkers elsewhere [30]. In brief, 4-µm sections collected on Super- were 0.7 nM and 0.6 nM, respectively. Te efect of link- Frost Plus slides were deparafnized in xylene, re-hydrated er length between the afbody- and ABD moieties was in graded alcohols, blocked for endogenous peroxidase, and indeed marginal, and the variant with shorter linker was subjected to heat-induced antigen retrieval using PT mod- thus chosen for further studies since a reduced size may ule bufer 1 (pH 6, Termo Fisher Scientifc) in a Decloak- have advantages in terms of penetration of tumor tissue. ing Chamber (Biocare Medical, Pacheco, CA) at 125°C Te same size-matched fusion protein TAT, binding the for 4 minutes. Automated IHC was performed using a non-mammalian target Taq polymerase, that was included LabVision Autostainer 480S (Termo Fisher Scientifc). in our previous study [25] was used as a control protein for Primary antibody monoclonal mouse anti-human HER3 cell proliferation studies. (Clone DAK-H3-IC, 0.2 mL, DAKO, Carpinteria, CA) was diluted in UltraAb Diluent (Termo Fisher Scientifc), Both bivalent fusion proteins were expressed in E. coli, and and applied to the slides for 30 min at room temperature. recovered by afnity chromatography and reversed phase Te slides were further incubated with the secondary re- HPLC to a purity exceeding 98%, (Fig. 2S A). Te mo- agent (anti-rabbit/mouse horse reddish peroxidase-conju- lecular masses as measured by mass spectrometry (Fig. 2S gated UltraVision; Termo Fisher Scientifc) for 30 min at B), 19,709 Da for 3A3s, and 22,126 Da for TAT, were in a room temperature. Following the washing steps, the slides very good agreement with the expected theoretical values, were developed for 5 min using the avidin-biotin perox- 19,709 Da and 22,124 Da, respectively. Te monoclonal idase staining technique (Vector Elite; Vector Laborato- antibody seribantumab was produced in CHO cells and ries, Burlingame, CA) using 3,3-diaminobenzidine as the recovered by afnity chromatography. Te purity was as- substrate. Slides were counterstained in Mayers hematox- sessed using analytical size exclusion chromatography and ylin (01820, Histolab) for 5 minutes using the Autostainer was found to be 96% (Fig. 2S C). XL (Leica, Wetzlar, Germany), and then rinsed in lithium For investigation of the thermal stability, the 3A3s afbody

4 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab

A KD1 = 4.8 ± 3.3 nM fusion protein was subjected to circular dichroism spec- KD2 = 0.4 ± 0.5 nM HER3 troscopy. Te melting temperature of 3A3s was determined HER3 100 ZHER3 by measuring the ellipticity at 221 nm while heating the Z

] HER3 ABD proteins from 20 to 90°C. Te obtained melting curve cor- [- HSA responded to a Tm value of 65°C (Figure S3A), which is in the same range as the original HER3-specifc afbody 50 molecules [24]. Additionally, spectra at wavelengths rang- ing from 195-250 nm were obtained before and after the variable temperature measurement in order to assess the Response units reversibility of the unfolding after heat treatment (Fig- 0 ure S3B). Te results showed that the 3A3s fusion protein 0 1000 2000 maintained the alpha-helical content and completely re- Time [s] folded after heating to 90°C, as shown by the overlap be- tween spectra generated before and after heating. C KD = 3.4 ± 0.5 nM Binding to HER3, mErbB3 and serum albumin was ver-

30 Z ifed by surface plasmon resonance (SPR) and the best ft HER3 ] ZHER3

[- ABD for the HER3 and mErbB3 interactions was observed us-

HSA ing a heterogeneous ligand-binding model (Fig. 2). Tis is likely due to that the afnity for HER3 is infuenced by the 15 orientation of the afbody molecules, i.e. the N- or C-ter- minal fusion afecting the binding diferently. Hence 3A3s has a distinct KD value towards HER3 for both its N- Response units and C-terminal HER3 binding moiety, 4.8±3.3 nM and 0 0.4±0.5 nM (Fig. 2). Te afnities for mErbB3 for 3A3s 0 1000 2000 are 35±26 nM and 146±114 nM. Te afnity for HSA was Time [s] determined to 3.4±0.5 nM and for MSA 2.4±0.4 nM (Fig. B 2). Te afnities for the serum albumins are likely infu- KD1 = 35 ± 26 nM enced by the fusion protein context and were lower than KD2 = 146 ± 114 nM previously reported [22]. 40 ]

[- 111In-labeled 3A3s demonstrated target-specifc bind- mErB3HER3 mErB3 ing to HER3 expressing BxPC-3 cells. Cells pre-treated ZHER3

ZHER3 with non-labeled 3A3s had signifcantly lower (p < 0.0001) ABD 20 radioactivity uptake than control cells (Fig. 3A). It was HSA also demonstrated that pre-treatment of cells with molar equivalent concentration of seribantumab signifcantly (p Response units < 0.001) decreased cellular uptake of radioactivity, demon- 0 strating that both therapeutic anti-HER3 proteins com- 0 1000 2000 pete for the same epitope of HER3. Pre-treatment of cells Time [s] with non-targeting mAb (bevasizumab) did not infuence cellular uptake of radioactivity (Fig. 3A). D KD = 2.4 ± 0.4 nM Te efect of 3A3s on HER3-signaling was demonstrated 80 ZHER3 in a phosphorylation assay. Te level of HER3 phospho- Z ] HER3 ABD

[- rylation was analyzed using phosphor-tyrosine-specifc MSA detection antibodies in a sandwich ELISA (Fig. 3B). Te pancreatic cancer cell line, BxPC-3 with the estimated 40 HER3 expression of 12±2 × 103 [31], was used in this assay. Te cells were stimulated by addition of heregulin, which resulted in signifcant increase in HER3 tyrosine phos- Response units phorylation levels (p < 0.005), as compared to cells with- 0 out addition of heregulin. Cells that were treated with both 0 750 1500 heregulin and anti-HER3 monomeric or dimeric afbody Time [s] (ZHER3 or 3A3s) demonstrated HER3 phosphorylation levels that were similar as for heregulin-unstimulated cells Figure 2. Results for the SPR afnity determinations. In both (p < 0.001). Similar mechanism of action was reported for cases, HSA was immobilized on the chip to capture 3A3s, followed by 1:2 dilution series of both human (A) and murine (B) ErbB3 seribantumab [8]. TAT was included as a negative control, ranging from 25 to 250 nM, (C) immobilized HSA with subsequent and as expected, it did not afect the HER3 phosphoryla- injections of 1.6 to 25 nM of 3A3s, (D) immobilized MSA with tion, which remained at the same level as heregulin stim- subsequent injections of 1.6 to 25 nM of 3A3s. ulated cells. A cell growth inhibition experiment was performed to as- sess the efect of treatment with the 3A3s afbody con- 5 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab

A B p < 0.05), respectively. Xenograft volumes were signifcant- ly smaller (p < 0.05) in both treated groups than in control 100 100 group but there was no diference between treated groups (Fig. 5A). Median survival was also signifcantly longer 50 50 for treated groups than for control group: control 41 days, seribantumab-treated 54 days and 3A3s-treated 60 days (p normalized signal [%] normalized

normalized cellular uptake [%] cellular uptake normalized 0 0 < 0.0001). Tere was no signifcant diference in median

TAQ HER3 survival between treated groups (p = 0.45). no Hrg 4 nM Hrg non-blocked 50 nM 3A3s Histological analysis of the excised xenografts showed a 50 nM seribantumab50 nM bevasizumab 4 nM + 200 nM4 nM 3A3s + 200 nM4 nMZ + 200 nM Z poorly diferentiated tumor with solid growth pattern and Figure 3. In vitro characterization of 3A3s. A. In vitro specifcity test. BxPC-3 cells were incubated with 1 nM of 111In-3A3 with presence of variable areas of necrosis. Between the three pre-incubation with 50 nM of 3A3s, MM-121 or non-HER3-spe- groups no relevant diferences were observed neither in cifc mAb (bevasizumab). Cell associated uptake was normalized growth pattern nor areas of necrosis (Fig. S6). to uptake of cells without pre-treatment. Data are presented as an average for 3 dishes ± SD. B. Inhibition efcacy. HER3 phosphoryl- Te immunohistochemical analysis of the HER3 receptor ation was quantifed in BxPC-3 cells treated with 4 nM heregulin expression in the xenografts demonstrated a distinct mem- and compared with phosphorylation in cells in presence of 200 branous staining in most tumor cells of all three groups, nM 3A3s, ZHER3, or ZTaq. Phosphorylation in non-treated cells independent of treatment (Fig. 5C). Tere was a tendency was used a control. Te signal was normalized to the signal in 4 that the central areas of the solid tumor islands showed a nM heregulin treated cells. Data are presented as an average for more intense expression than cells located in the periphery 9 dishes ± SD. Un-paired t-test was used to determine signifcant near the stroma. Tis phenomenon was more pronounced diference (** p < 0.01, *** p < 0.001). in treated xenografts. No obvious diference in staining pattern was observed in kidney and liver tissue between struct on the in vitro proliferation rate of BxPC-3. Cells treated and untreated mice. were incubated for 6 days with concentrations of 3A3s To conclude, our bivalent HER3-targeting 3A3s afbody and TAT, ranging from 0.05 to 900 nM. Cell growth was construct was equally potent in terms of decreased tumor determined by cell count-determination. BxPC-3 cells are growth and improved survival in xenografted mice, to the autocrine for the HER3 ligand heregulin and the concen- monoclonal antibody seribantumab, presently undergoing tration of heregulin is hence unknown. Te presence of clinical investigations as a HER3-targeting therapeutic. 3A3s resulted in a signifcantly reduced cell proliferation, Tese results are indeed encouraging, and although the with a clear dose-response efect (Fig. 4) similar to the re- mode of action for 3A3s is not completely investigated, the sults reported previously for the bivalent afbody construct biological activity is probably the result of several distinct 3A3 [25]. Te control protein TAT showed only small ef- mechanisms. In addition to blocking heregulin-induced fects on cell growth. signaling, it has previously been reported that bi- and tri- Tumor growth inhibiton in xenografed mice. Te dosage valent HER3-specifc afbody constructs downregulate for seribantumab was based on data published by Schoe- HER3 surface expression in diferent cancer cell types berl et al [29]. To determine the dosage regimen for the af- [32], which the immunohistochemistry results obtained fbody construct, a pharmacokinetics-pharmacodynamics in this study supports. If speculating, the bivalent 3A3s simulation approach was used using a one-compartment might also trap two kinase-defcient HER3 receptors in model. A simulation of the exposure levels of seribantum- a non-signaling dimer confguration, preventing them to ab and afbody molecule was performed, which suggested form signaling heterodimers with for example HER2 or that 600µg/dose of the afbody construct and 600µg/dose for seribantumab, 3 times/ week for 12 weeks was suf- cient to achieve a similar level of exposure (Fig. S4). 1

An in vivo therapy experiment was performed in female TAT mice bearing HER3 expressing BxPC-3 xenografts. Ter- 0.5 apy started at day 7 after xenograft implantation and the experiment reached a humane end point at day 68. Mice 3A3s were visually controlled and weighed twice per week. Ex- 0 terior, general conditions, behavior, and body-scoring were Normalized cell count [-] similar in both treated groups to that in the control group. Body weight did not difer between the three groups (Fig. 0.1 1 10 100 1000 S5A). All animals were euthanized when xenograft vol- Concentration [nM] ume reached 1 cm3. Pathological examination of liver and Figure 4. Cell proliferation of the BxPC-3 pancreatic cancer cell kidney tissue of euthanized animals did not show any dif- line analyzed by measuring cell growth inhibitory efects of the ferences between control and treated groups (Fig. S5B). bivalent fusion protein 3A3s (flled triangles) and TAT control Xenograft growth was slower in both treated groups than construct (flled squares) incubated at concentrations ranging from control, with doubling times of 13±1 days for 3A3s, 12±1 0.05 to 900 nM for 144 h. Cell viability was measured using a for seribantumab and 10±1 for control group (diference CCK8-kit. Maximum inhibition was set to the lowest absorbance between 3A3s group and control group was signifcant, signal of 3A3s treated cells.

6 in the periphery near the stroma (black arrows). showed amore intense expression than the cells that were located Te central areas (yellow arrows) of the solid tumor islands observed between treated and control groups in liver and kidneys. euthanized at the humane end point. No relevant di ferences were xenografs, livers and kidneys .Tissues were from excised animals pression of 4µmsections of para fnated tissue samples of BxPC-3 in control group. C.Immunohistochemical staining of HER3ex Median survival in treated groups was signifcantly longer than (p <0.05).B. Survival until euthanasia criteria were reached. control group were signifcantly bigger than in the treated groups Measured xenograf’s volume. From day 20,tumor volumes in the 33%). Data are presented asan average for 10 animals ±SD. A. PBS. Curves stopped when 3mice in agroup were euthanised (30- times per week, animals in control group were injected with neat injected i.p. with 600µgof 3A3sor MM121in 100µlof PBS 3 to humane end point (xenograf’s volume over 1cm3).Mice were planted at day 0and treatment was started at day 7and continued nu/nu female mice bearing BxPC-3xenografs. Mice were im Figure 5. C A Results of the in vivo HER3-targeting therapy in Balb/c Control seribantumab 3A3s Tumor size [mm3 ] 1000 1500 500 0 0 Evaluatng thetherapeutc potental ofadimericHER3-bindingafbody construct incomparison withamonoclonalantbody, seribantumab 7 treatment start control seribantumab ZHER3-ABD-ZHER3 BxPC-3 xenograft 14 21 Time [d] Time 28 35 42 49 56 - - B

Survival [%] 100 50 Liver 0 0 olds.f cifc antibodiesoralternative sca mabs. 2016;8:1195–209. 2. Malm M, Frejd FY, Ståhl S, Löfblom J. Targeting HER3 using mono- and bispe 2014;10:1006–12.Research; Nature Biol. Chem Nat Her3. pseudokinase the of targeting cological 1.Xie T,SM,Lim Westover KD, ME,Dodge D, Ercan SB, Ficarro al. et Pharma References analysis. sistance withtheIHC assistance in protein fcation puri and Patrick Micke for as technical for acknowledged VINNOVA is Olsson from Anders Dr.(AO). grant Cancer ESCAPE the and ogy 02353 the (VT)), Wallenberg Center for Protein Technol Council Research (grants 621-2012-5236 (SS), Swedish 2015-02509 (AO) and 2015- the (VT)), CAN2015/350 (SS),CAN2016-463(AO)and CAN2014-474 (grants ty Socie Cancer Swedish the by supported was work Tis Acknowledgements on studies potentialasreceptortheir therapeutic agents. trapping for receptors kinase tyrosine other targeting constructs afbody bivalent new of generation encourage also results Te mechanisms. exact the detail in termine de EGFR.However,to needed are investigations further 7 14 21 28 35 Time [d] Time 42 49 56 63 Kidney 70 treatment start control seribantumab ZHER3-ABD-ZHER3 77 7 ------Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab

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Supplementary informaton

30

20

10 Response [pM] Response

0 0.001 0.01 0.1 1 10 100 1000 Concentration [nM] Figure S1. Infuence of linker length. Te two diferent 3A3 constructs primarily difering in their linker lengths were com- pared in their ability to inhibit heregulin-induced signaling in an in vitro assay using MCF-7 cells. Te assay was performed utilizing a label free system and the results are plotted as recorded response, picomolar (pM) response shif versus concen- tration. Te half maximal inhibitory concentrations (IC50) were determined from the dose response curves. A

400

300

200 mAU [-] mAU

100

0 0 10 20 30 40 50 60 Time [min] B 4 x 10 19709.58 8

6

Counts [-] Counts 4

2

0 5000 10000 15000 20000 25000 30000 35000 40000 Deconvoluted mass [Da] C

150

100 mAU [-] mAU

50

0 0 10 20 30 40 Volume [ml] Figure S2. Purity of the compounds. Characterization of bivalent afbody construct 3A3s and monoclonal antibody MM-121. A. Te purity of the 3A3s fusion protein was determined using an analytical column Zorbax 300B-C18 (Agilent Technologies) on a 1200 series RP-HPLC (Agilent Technologies)). B. Te molecular mass of 3A3s was confrmed using a 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies). C. Te purity of the MM121 monoclonal antibody (Evitra AG) was analyzed using analytical SEC (Superdex 200 10/300 GL, GE Healthcare).

9 Evaluatng the therapeutc potental of a dimeric HER3-binding afbody construct in comparison with a monoclonal antbody, seribantumab A -8 -12 -16 -20 -24 Circular [mdeg] dichroism 20 30 40 50 60 70 80 90 Temperature [°C] B 50

25

0

-25

Circular [mdeg] dichroism 200 210 220 230 240 250 260 Wavelength [nm] Figure S3. Analysis of heat stability and refolding using circular dichroism (CD) spectroscopy. A. Variable temperature measurement (VTM) spectrum obtained at 221 nm while heating the sample from 20 to 91°C. B. Overlay of CD spectra before and afer VTM.

A Seribantumab B ZHER3-ABD-ZHER3 600µg dose 600µg dose 3 doses/week 3 doses/week 2.5 day intervall 2.5 day intervall 1 Half-life 3 days 1 Half-life 19h

0.1 0.1

0.01 0.01

0.001 0.001 Concentration [µM] Concentration Concentration [µM] Concentration

0.0001 0.0001 0 50 100 0 50 100 Time [d] Time [d]

Figure. S4. Determination of dosage using a one-compartment level. Simulation of antibody and afbody exposure levels performed using a one-compartment model in a PK/PD simulation sofware. Te graphs display the exposure levels plotted as µmol/L versus time.

10 control seribantumab 3A3s 200 µm presented asan average for 10animals ±SD. of 3A3sor MM-121in 100µlof PBS 3times per week, animals in control group were injected with PBS alone. Data are Figure S5. observed between treated and control groups. livers and kidneys. Tissues were from excised animals euthanized at the humane end point. No relevant diferences were Figure S6. BxPC-3 xenograft Haemotoxylin and Eosin (HE)staining of 4µmsections of parafnated tissue samples of BxPC-3xenografs, Monitoring of mice weights in the HER3-targeting therapy experiment. Mice were injected i.p. with 600µg Evaluatng thetherapeutc potental ofadimericHER3-bindingafbody construct incomparison withamonoclonalantbody, seribantumab Weight [g] 10 20 0 0 300 µm 20 control seribantumab ZHER3-ABD-ZHER3 Liver Time [d] Time 40 1 mm 60 Kidney 11