111In and 225Ac-Labeled Cixutumumab for Imaging and Alpha

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Subscriber access provided by Universiteit Utrecht Article 111In and 225Ac-labeled cixutumumab for imaging and alpha particle radiotherapy of IGF-1R positive triple negative breast cancer Viswas Raja Solomon, Elahe Alizadeh, Wendy Bernhard, Siddesh V. Hartimath, Wayne Hill, Rufael Chekol, Kris M Barreto, Clarence Ronald Geyer, and Humphrey Fonge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00542 • Publication Date (Web): 13 Sep 2019 Downloaded from pubs.acs.org on October 31, 2019

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1 2 3 111In and 225Ac-labeled cixutumumab for imaging and alpha particle radiotherapy of IGF- 4 5 6 1R positive triple negative breast cancer 7 8 9 10 Viswas Raja Solomon1, Elahe Alizadeh1, Wendy Bernhard2, Siddesh V. Hartimath1, Wayne 11 12 Hill2, Rufael Chekol1, Kris M. Barreto2, Clarence Ronald Geyer2*, Humphrey Fonge1,3* 13 14 15 1Department of Medical Imaging, University of Saskatchewan, College of Medicine, Saskatoon 16 17 SK, Canada 18 19 2Department of Pathology and Laboratory Medicine, University of Saskatchewan, College of 20 21 22 Medicine, Saskatoon SK, Canada 23 24 3Department of Medical Imaging, Royal University Hospital Saskatoon, Saskatoon SK, Canada 25 26 27 28 29 Authors for correspondence 30 31 *Humphrey Fonge, PhD 32 33 Email: [email protected] 34 35 Tel: 306-655-3353 36 37 Fax: 306 655 1637 38 39 40 *Clarence Ronald Geyer, PhD 41 42 Email: [email protected] 43 44 Tel: 306-966-2040 45 46 47 48 49 50 51 52 53 54 55 56 57 1 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 2 of 44

1 2 3 Abstract 4 5 6 Insulin growth factor receptor (IGF-1R) is overexpressed in many cancers of epithelial origin 7 8 9 where it confers enhance proliferation and resistance to therapies targeted at other receptors. Anti- 10 11 IGF-1R monoclonal antibodies have not demonstrated significant improvements in patient 12 13 outcomes in clinical trials. Humanized monoclonal antibody cixutumumab (IMC-A12) binds to 14 15 16 IGF-1R with low nM affinity. In this study cixutumumab was conjugated with p-SCN-Bn-DOTA 17 18 and radiolabeled with 111In or 225Ac for imaging or radiotherapy using a triple negative breast 19 20 cancer (TNBC) model SUM149PT. The antibody conjugate showed low nM affinity to IGF-1R 21 22 23 which was not affected by conjugation and radiolabeling procedures. Cixutumumab 24 25 immunoconjugates was effectively internalized in SUM149PT and was cytotoxic to the cells with 26 225 27 an EC50 of Ac-cixutumumab (0.02 nM) that was almost 5000-fold less than unlabeled 28 29 cixutumumab (95.2 nM). MicroSPECT imaging of SUM149PT xenograft showed the highest 30 31 32 tumor uptake occurred at 48 h post injection and was 9.9 ± 0.5 % injected activity per gram 33 34 (%IA/cc). In radiotherapy studies, we evaluated the effect of specific activity of 225Ac- 35 36 cixutumumab on efficacy following a tail vein injection of two doses (day 0 and 10) of the 37 38 39 investigation agent or controls. Cixutumumab (2.5 mg/kg) prolonged the survival of SUM149PT 40 41 tumor bearing mice with a median survival of 87 days compared to PBS control group (median 42 43 survival of 62 days). Median survival of high specific activity 225Ac-cixutumumab (8 kBq/µg, 225 44 45 225 46 nCi, 0.05 mg/kg) was 103.5 days compared to 122 days for low specific activity Ac- 47 48 cixutumumab (0.15 kBq/µg, 225 nCi, 2.5 mg/kg). Additionally, low specific activity 49 50 radioimmunoconjugate led to complete tumor remission in 2/6 mice. The data suggest that efficacy 51 52 of cixutumumab can be enhanced by radiolabeling with 225Ac at a low specific activity. 53 54 55 56 57 2 58 59 60 ACS Paragon Plus Environment Page 3 of 44 Molecular Pharmaceutics

1 2 3 Keywords: Insulin Growth Factor Receptor type I (IGF-1R); Alpha Particle Radiotherapy; 4 5 6 MicroSPECT Imaging; Triple Negative Breast Cancer; Cixutumumab 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 3 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 4 of 44

1 2 3 Introduction 4 5 1 6 Breast cancer is a leading cause of cancer-related deaths in women worldwide . Among the several 7 8 subtypes, 15–20% of diagnosed breast tumors are triple-negative breast cancer (TNBC) along with 9 10 BRCA germline mutations2, 3. Recent preclinical and population studies demonstrated a role for 11 12 insulin growth factor receptor (IGF-1R) signaling in TNBC. Amplification of IGF-1R is seen in 13 14 15 residual disease following neoadjuvant chemotherapy in patients with TNBC, suggesting a 16 17 potential contribution of IGF-1R signaling to chemotherapy resistance4. These findings 18 19 demonstrate that IGF-1R signaling promotes tumor progression in TNBC and that IGF-1R signal 20 21 22 inhibition may be a valuable target for TNBC imaging and radiotherapy. IGF-1R inhibition 23 24 impeded the proliferation of TNBC cells with high IGF-1R expression in vitro5, 6. IGF-1R tyrosine 25 26 kinase inhibitor (TKI) monotherapy was sufficient to reduce tumor size in a TNBC xenograft 27 28 7, 8 29 model . 30 31 Several small-molecule inhibitors and antibodies directed against the IGF-1R have been 32 33 developed and tested in phase I and II clinical trials9-11. Stable disease and objective response were 34 35 reported in patients treated with these new agents, but no relationship with IGF-1R expression and 36 37 38 response has been thoroughly investigated10, 11. Preclinical studies showed that IGF-1R expression 39 40 is necessary for antitumor activity of anti–IGF-1R antibodies12, 13. Therefore, patient selection for 41 42 IGF-1R–targeted therapy should be based on receptor expression. Thus far, studies on IGF-1R 43 44 45 expression have been performed retrospectively on tumor tissue sections by immunohistochemistry 46 47 (IHC). IHC cannot be used to measure IGF-1R expression in multiple lesions, especially in 48 49 metastatic setting because of its invasive nature. Furthermore, the expression of IGF-1R may change 50 51 52 over time. 53 54 55 56 57 4 58 59 60 ACS Paragon Plus Environment Page 5 of 44 Molecular Pharmaceutics

1 2 3 The encouraging preclinical data seen with anti-IGF-1R antibodies have not been 4 5 6 replicated in clinical trials. Clinical trials of anti-IGF-1R agents e.g. ganitumab, figitumumab, and 7 8 cixutumumab have all not shown significant benefits in patients and most of these trials have been 9 10 abandoned 14, 15. The failure of anti-IGF-1R inhibitors means other approaches aimed at targeting 11 12 this receptor could yield important benefits for patients. Our goal is to develop a theranostic pair 13 14 15 using 111In-labeled and alpha particle 225Ac-labeled anti-IGF-1R antibody. The characteristics of 16 225 17 Ac: t1/2 10.0 days; energy range of 4 αs is 6 – 8 MeV (cumulative emission of 28 MeV/decay) 18 19 decays with the emission of 5 αs, 3 β- disintegrations, a range of 50 – 80 μm in tissues and with a 20 21 22 linear energy transfer of up to 0.16 MeV/μm, makes this isotope ideal for radiotherapy, especially 23 24 in micrometastatic settings. Recently, improved clinical outcomes have been obtained using alpha 25 26 particle (e.g. 255Ac and 213Bi) labeled agents, particularly in prostate cancer and neuroendocrine 27 28 16, 17 29 tumors . 30 31 Cixutumumab is a fully human IgG1/λ monoclonal antibody directed against IGF-1R. 32 33 Cixutumumab binds IGF1R with high affinity and blocks interaction between IGF-1R and its 34 35 ligands, IGF-1 and –2, and induces internalization and degradation of IGF-1R18. Anti-tumor effects 36 37 38 of cixutumumab have been demonstrated in preclinical and clinical studies18-20. Currently, there 39 40 are five ongoing clinical trials evaluating the efficacy of cixutumumab as either a single agent or 41 42 in combination with other targeted therapies or chemotherapy agents18-21. For this purpose, we have 43 44 111 225 45 used cixutumumab (IMC-A12) to develop a In/ Ac-cixutumumab theranostic agent against 46 47 IGF-1R positive tumors. This is the first reported study of an anti-IGF-1R alpha particle 48 49 radioimmunotherapeutic. Our hypothesis is that 225Ac-cixutumumab will be more effective than 50 51 111 52 cixutumumab in IGF-1R positive cells/tumor. In-cixutumumab can be used for patient selection 53 54 and for monitoring of response to anti-IGF-1R treatments as well as for dosimetry estimation for 55 56 57 5 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 6 of 44

1 2 3 225Ac-cixutumumab radioimmunotherapy. We have evaluated the in vitro cytotoxicity and in vivo 4 5 6 therapeutic potential in a mouse model with IGF-1R positive xenograft. 7 8 9 10 Materials and Methods 11 12 General 13 14 15 16 Chemicals used in the conjugation, radiolabeling, and purification steps were American Chemical 17 18 Society reagent grade or better. Water and buffers were rendered metal-free by passing them 19 20 through a column of Chelex-100 resin, 200–400 mesh (Bio-Rad Laboratories, Inc.), and were 21 22 23 sterile-filtered through a 0.22 μm filter. The monoclonal antibody cixutumumab was purchased 24 25 from Creative Biolabs (Shirley, NY). Human IgG isotype control was purchased from Thermo 26 27 scientific (Louise, MO). Bifunctional chelating agent 2-(4-isothiocyanatobenzyl)-1,4,7,10- 28 29 tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) was obtained from 30 31 111 32 Macrocyclics (Plano, TX). In was purchased from Nordion as indium chloride (Kanata, ON). 33 34 225Ac (0.05 M HCl) was received from Oak Ridge National Laboratories as an actinium nitrate 35 36 (Oak Ridge, TN). All chemicals and reagents obtained from Sigma-Aldrich (St. Louise, MO) were 37 38 39 used without further purification. 40 41 42 43 Cell culture 44 45 46 Human breast cancer cell line MCF-7/Her18 that overexpresses IGF-1R was kindly provided by 47 48 Dr. Hung Mien-Chie (M.D. Anderson Cancer Center, Houston, TX). MCF-7/Her18 cells were 49 50 maintained in DMEM containing high glucose levels and 10% fetal bovine serum (FBS). Triple 51 52 negative human breast cancer cell line SUM149PT was purchased from Astrand Bioscience 53 54 55 (Detroit, MI). SUM149PT cells were maintained as monolayer in a culture flask containing Ham’s 56 57 6 58 59 60 ACS Paragon Plus Environment Page 7 of 44 Molecular Pharmaceutics

1 2 3 F-12 medium supplemented with 10% FBS (25 mL), hydrocortisone (1 mg/mL, 500 µL), and 4 5 6 insulin (10 mg/mL, 250 µL) at cultured at 37˚C in a humidified atmosphere with 5% CO2. Prior to 7 8 in vitro and in vivo use, cells were detached using trypsin-EDTA. 9 10 11 12 Conjugation and Quality Control of Immunoconjugates 13 14 15 Conjugation of antibodies was performed as reported earlier 22. The excess unreacted chelator was 16 17 removed by centrifugation using an Amicon Ultra-10K (Burlington, MA) molecular filtration 18 19 device. The resulting solution was filtered using a 0.22 μm Millipore filter and stored at -80°C for 20 21 111 225 22 labeling with In and Ac. Purity of cixutumumab, DOTA-cixutumumab, control IgG, and 23 24 DOTA-control IgG was determined using SEC-HPLC (Waters 2796 Bioseparations Module, 25 26 Waters 2487 Dual λ Absorbance Detector, XBridge® BEH 200A SEC 3.5 µm 7.8 x 300 mm 27 28 29 column, Waters Corporation). The UV-Detector was set at 220 and 280 nm and the solvent system 30 31 was PBS at a flow rate of 0.6 mL/min. Further, the size and purity was characterized by automated 32 33 electrophoresis (2100 Bioanalyzer system, Agilent). 34 35 Bioanalzyer 36 37 38 Binding kinetics between the antibodies and target proteins were measure with ForteBio Octet 39 40 RED384 (PALL Corporation). Antibodies were immobilized on Anti-human IgG Fc capture 41 42 sensors (Forte Bio: 18-5060) according to manufactures instructions. Briefly, sensors were 43 44 45 incubated in buffer for 120 seconds and then moved to wells containing 100 nM of antibody and 46 47 incubated for another 180 seconds. After immobilization, sensors were incubated in buffer again 48 49 for 120 seconds to establish a stable baseline. Antibodies were then exposed to 500 nM, 166 nM, 50 51 52 and 55 nM concentrations of Human IGF1R protein (Sino Biological: 10164-H08H) for 120 53 54 seconds to obtain the association curve. This was followed by incubation in buffer for another 300 55 56 57 7 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 8 of 44

1 2 3 seconds to obtain the dissociation curve. At the same time, empty sensors were exposed to the 4 5 6 same concentrations of target protein. This is used during analysis for subtraction of non-specific 7 8 binding. All reactions were performed with a shake speed of 1000 rpm at 30 °C in 1 × kinetics 9 10 buffer (Forte Bio: 18-5032). The equilibrium dissociation constant (KD) was obtained using a 1 to 11 12 1 model with global fitting. Data analysis and curve fitting was performed using Data Analysis 13 14 15 software 7.1.0.33 (Forte Bio). 16 17 18 Biolayer Interferometry 19 20 21 Binding kinetics between cixutumumab and DOTA-cixutumumab and IGF-1R was measured 22 23 using biolayer interferometry (BLI) using ForteBio Octet RED384 (PALL Corporation, CA). 24 25 26 Antibodies were immobilized on Anti-human FAB-CH1 sensors (18-5104, Forte Bio) according 27 28 to manufactures instructions. The equilibrium dissociation constant (KD) was obtained using a 1 29 30 to 1 binding model with global fitting. Data analysis and curve fitting was performed using Data 31 32 33 Analysis software 7.1.0.33 (Forte Bio). 34 35 36 37 Flow Cytometry 38 39 MCF-7/Her18 cells were treated with antibodies at twelve concentrations ranging from 1000 – 40 41 42 0.015 nM, incubated for 30 min at room temperature, and then cooled on ice for 15 min. Cells 43 44 were washed with PBS + 2% FBS and incubated with secondary antibody FITC-labeled Goat 45 46 F(ab’) fragment anti-human IgG (H + L) (1:50 ratio) (IM0839, Beckman Coulter) for 30 min at 47 2 48 49 4°C, and then washed again. Flow data was acquired using a Beckman Coulter Gallios flow 50 51 cytometer (Beckman Coulter) and analyzed by FlowJoV10.6. The EC50 was determined using 52 53 GraphPad Prism 6. 54 55 56 57 8 58 59 60 ACS Paragon Plus Environment Page 9 of 44 Molecular Pharmaceutics

1 2 3 Internalization of Immunoconjugates 4 5 6 MCF7/Her18 cells (10,000 per well) were plated on flat-bottom 96 well plates and incubated 12 h 7 8 prior to assay by using IncuCyte S3 Live cell imaging system (Essen BioScience, Ann Arbor, MI). 9 10 Briefly, cixutumumab immunoconjugates or control IgG isotype (4 µg/mL) mixed with IncuCyte 11 12 FabFluor reagent (Essen BioScience, Ann Arbor, MI) at a molar ratio of 1 to 3 in complete growth 13 14 15 media and incubated for 15 min at 37° C. FabFluor labeled antibody (50 µL) was then added 16 17 directly to cells (in 50 µL). Following addition of Ab/Fab complex, assay plates were immediately 18 19 placed in an IncuCyte® S3 live-cell imager. Images were captured every 2 hours (from 2 to 48 20 21 22 hours) using a 10× objective lens with phase contrast and fluorescence channel. During each 23 24 scanning, 5 images were acquired until the end of the experiment. Images were automatically 25 26 analyzed using the integrated IncuCyte software for phase confluence (measure of cell area) and 27 28 29 red fluorescence object area (index FabFluor labeled internalized antibody). Top Hat subtraction 30 31 was used to minimize any background fluorescence signal. 32 33 34 35 Radiolabeling and Quality Control of Radioimmunoconjugates 36 37 111 38 Radiolabeling of DOTA conjugated antibodies with InCl3 was performed as per the standard lab 39 40 protocol 22. After labeling, the reaction was monitored using iTLC strip with 100 mM sodium 41 42 citrate buffer (pH 5.0) as the mobile phase and analyzed using ScanRam (LabLogic, Brandon, FL). 43 44 111 45 In-labeled conjugates were purified using Amicon Ultra-4 centrifugal filters (10K, EMD 46 47 Millipore, Burlington, MA) with PBS. The purity of the radiolabeled immunoconjugates were 48 49 determined using size exclusion radio-HPLC and iTLC. A radiochemical purity (RCP) of more 50 51 52 than 95% was considered good for in vitro and in vivo experiments. 53 54 55 56 57 9 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 10 of 44

1 2 3 For radiolabeling with 225Ac, 225Ac-nitrate (1.0 MBq), previously reconstituted with 0.05 M 4 5 6 HCl (Optima grade, Fisher scientific) was added to a 1.5 mL microtube. To this was added L- 7 8 ascorbic acid (150 g/L; 10 µL), 150 mM ammonium acetate (pH 6.0, 25 µL), and DOTA- 9 10 cixutumumab (125 µg). The pH of the reaction was determined by spotting 1 µL of the reaction 11 12 mixture onto Hydrion pH paper (range, 5.0 – 9.0) (Sigma-Aldrich); pH of a typical reaction was 5.8. 13 14 15 The reaction mixture was incubated at 37 ◦C on a shaker at 650 RPM for 2 h. After this, a small 16 17 aliquot (0.5 µL) was spotted on a iTLC strip to determine the extent of incorporation of 225Ac onto 18 19 the protein using mobile phase of 50 mM sodium citrate (pH 5.2). Purification of the 225Ac- 20 21 22 cixutumumab was done using Amicon Ultra-4 centrifugal filters (10K, EMD Millipore, 23 24 Burlington, MA) with PBS. The radioactivity was detected using a flow through radioHPLC- 25 26 detector (Flow-RAM, Broomhill, UK). 27 28 29 The stability of the radioimmunoconjugates was investigated by adding 50 µL of 30 31 radiotracer to 1.0 mL of plasma or PBS, followed by incubation at 37 °C for up to 7 or 10 days (n 32 33 = 3). Aliquots of 2 – 5 µL were taken at different time points and analyzed for radiochemical purity 34 35 using iTLC. The immunoreactive fraction of 111In-cixutumumab was determined as described 36 37 38 previously23, 24. 39 40 41 42 In Vitro Cytotoxicity 43 44 45 In vitro cytotoxicity (EC50 values) of cixutumumab immunoconjugates was determined using 46 47 IncuCyte S3 Live-cell imager. (Essen BioScience, Ann Arbor, MI). Briefly, 3,000 – 5,000 48 49 (SUM149PT) cells were seeded 24 hours prior to treatment in a 96 well plates. The next day, the 50 51 ® 52 media was removed and washed with PBS. Cells were then incubated with IncuCyte Cytotox Red 53 54 reagent diluted in complete media (1×, Essen Bioscience Cat #4632) for 3 h before treatment. Cells 55 56 57 10 58 59 60 ACS Paragon Plus Environment Page 11 of 44 Molecular Pharmaceutics

1 2 3 were treated with different concentrations (500 – 0.2 nM) of cixutumumab, control IgG, 225Ac- 4 5 225 ° 6 cixutumumab and Ac-control IgG. The plate was incubated at 37 C for 30 min prior to imaging. 7 8 Live cell images were captured every 2 hours using a 10× objective lens using phase contrast and 9 10 fluorescence channel. During each scanning five images were acquired until the end of the 11 12 experiment. Images were processed and analyzed using IncuCyte S3 software. The red fluorescent 13 14 15 values were generated and EC50 values for individual compounds were calculated using GraphPad 16 17 prism 5.03. 18 19 20 21 22 Tumor Xenograft, MicroSPECT/CT Imaging and Biodistribution 23 24 All animal studies were approved by the University of Saskatchewan Animal Care and Use 25 26 Committee in accordance with the guidelines set forth in Use of Laboratory Animals (protocol # 27 28 29 20170084). Mice were housed under standard conditions in approved facilities with 12 h light/dark 30 31 cycles and given food and water ad libitum throughout the duration of the studies. Female nude 32 33 mice were purchased from the Charles River Laboratory (Sherbrooke, QC). For inoculation, 34 35 SUM149PT cells (5 x 107 cells/mL) were resuspended in a 1:1 mixture of phosphate-buffered 36 37 38 saline: Matrigel (BD Biosciences). Each mouse was injected in the right flank with 0.2 mL of the 39 40 cell suspension. Mice were used for imaging and biodistribution studies when the tumors reached 41 42 200 – 400 mm3. 43 44 45 Mice bearing SUM149PT xenograft received a tail vein injection of 10 ± 0.3 MBq (20.1 ± 46 47 0.5 µg; specific activity 0.5 MBq/µg) 111In-cixutumumab, or control 111In-control IgG. At 24, 48, 48 49 96, and 120 h post injection, SPECT images were acquired using Vector4CT scanner (MILabs, 50 51 52 Utrecht). The imaging and reconstruction parameters, and animal care during imaging was 53 54 described earlier 22. For quantification, CT and SPECT images were analyzed using PMOD 3.8 55 56 57 11 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 12 of 44

1 2 3 biomedical image analyzing software (PMOD, Switzerland). A 3D region-of-interest (ROI) was 4 5 6 manually drawn to encompass the radioactivity uptake in the organ of interest. Tracer uptake was 7 8 expressed as % IA/mL of tissue volume (% IA/cc). The result was reported as mean ± standard 9 10 deviation within each study group. After microSPECT imaging, mice were sacrificed at 144 h post 11 12 injection. Additional groups of mice were administered with the agents and sacrificed at 72 h post 13 14 15 injection. The activity was measured using gamma counter and activity in organs was expressed 16 17 as percentage injected activity per gram organ weight (% IA/g). 18 19 20 21 225 22 Ac-radioimmunotherapy 23 24 When xenografts average size of about 50 mm3, mice were randomized into 5 groups (n < 8 per 25 26 group). We evaluated the effectiveness of two doses of cixutumumab (2.5 mg/kg per dose), 225Ac- 27 28 225 29 cixutumumab (specific activity 8.0 kBq/µg, 225 nCi, 1 µg (~ 0.05 mg/kg) per dose), Ac- 30 31 cixutumumab (specific activity 0.15 kBq/µg, 225 nCi, 51 µg (2.5 mg/kg) per dose), isotype control 32 33 225Ac-control IgG (specific activity 0.15 kBq/µg, 225 nCi, 51 µg (2.5 mg/kg) per dose) per dose) 34 35 following a tail vein injection. Tumor growth was monitored by measuring the greatest length and 36 37 38 width using a digital caliper (tumor volume = length × width2/2). The study was terminated when 39 40 xenograft reached a volume ≥ 1500 mm3 and this was used to determine survival in the different 41 42 groups using Kaplan Meier curves. Individual body weight of mouse was recorded during the 43 44 45 quarantine (every other day) and experimental period. 46 47 48 49 Statistical Analysis 50 51 52 53 All data was expressed as the mean ± SD or SEM of at least 3 independent experiments. Statistical 54 55 comparisons between the experimental groups were performed by 1-way ANOVA with Bonferoni 56 57 12 58 59 60 ACS Paragon Plus Environment Page 13 of 44 Molecular Pharmaceutics

1 2 3 multiple comparison post hoc test (multiple-group comparison). Survival was described as median, 4 5 6 and survival curves were compared with the log-rank (Mantel-Cox) test. Graphs were prepared 7 8 and p values calculated by using GraphPad Prism (version 5.03; GraphPad, La Jolla, CA). P values 9 10 of less than 0.5 were considered significant. 11 12 13 14 15 Results 16 17 18 Conjugation and Quality Control 19 20 21 The conjugation of p-SCN-Bn-DOTA to cixutumumab or control IgG resulted in a clear solution 22 23 with no particulate matter or milky appearance. The purity of DOTA-cixutumumab and DOTA- 24 25 26 human-IgG were > 98% on HPLC with < 2% aggregates. Immunoconjugates were further 27 28 characterized for stability, binding to recombinant IGF-1R, aggregation, and size. Bioanalyzer 29 30 showed that cixutumumab and DOTA-cixutumumab were > 98% pure with molecular weights of 31 32 33 146.3, and 148.5 kDa, respectively (Fig. 1A). Antibodies were labeled with an average three 34 35 DOTA molecules per antibody when a chelator to antibody ratio of 16:1 was used in the 36 37 conjugation reaction. HPLC profiles were identical between conjugated and unconjugated 38 39 antibodies, showing > 95% purity, which was similar to the bioanalyzer results. 40 41 42 The effect of DOTA conjugation on cixutumumab binding to recombinant IGF-1R was 43 44 studied using biolayer interferometry. The binding constant (KD) for cixutumumab and DOTA- 45 46 cixutumumab was 2.2 ± 0.2nM and 1.6 ± 0.1 nM, respectively (Fig. 1B). Saturation binding of 47 48 49 immunoconjugates on MCF-7/Her18 cells was analyzed using flow cytometry. Median 50 51 Fluorescence Intensity (MFI) was converted into percent bound (Figs. 1C) and plotted against 52 53 concentration to calculate EC50 values of immunoconjugates for MCF7/Her18 cells. The estimated 54 55 56 EC50 values for cixutumumab and DOTA-cixutumumab were 5.2 ± 1.9 and 21.9 ± 5.9 nM, 57 13 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 14 of 44

1 2 3 respectively (Fig. 1C) and there was no statistical difference between cixutumumab and DOTA- 4 5 6 cixutumumab (p > 0.05). The control immunoconjugate did not show significant binding affinity 7 8 to MCF-7/Her18 cells at 100 nM concentration (Fig. S1). 9 10 11 12 Internalization of Immunoconjugates 13 14 15 A rapid time-dependent increase in red fluorescence was observed with cixutumumab antibodies 16 17 and not with the isotype control IgG or media control from the first time point (4 h) to endpoint 18 19 (48 h) of the assay (Fig. 1D). The red signal was observed in the cytosolic compartment of the 20 21 22 cells, consistent with the expected localization of the internalized antibody to lysosomes and 23 24 endosomes. The red object area (µm2/well) was used to quantify the specific signal. Internalization 25 26 rate of cixutumumab increased over time with the highest rate being observed at 48 h after 27 28 29 incubation (Fig. 1D & Fig. S2). 30 31 32 33 Radiolabeling and In vitro Cytotoxicity 34 35 36 The radiochemical yield of 111In-cixutumumab was 79.5 ± 3.8% at a specific activity of 0.5 37 38 225 39 MBq/µg. A radiochemical yield of > 95 % was obtained for Ac-cixutumumab, with a specific 40 41 activity of 8.0 kBq/µg. A radiochemical purity of >95% was obtained for 111In-cixutumumab after 42 43 purification. To investigate the stability of the radioimmunoconjugates 111In-cixutumumab and 44 45 225 46 Ac-cixutumumab were analyzed at different time periods by iTLC following storage at 37 ºC in 47 48 PBS or plasma. In PBS, over 90% of 111In-cixutumumab remained intact after 72 h storage at 37 49 50 ºC (Fig. S3A). Additionally, 111In-cixutumumab transchelated in mouse plasma following 51 52 incubation with 10% of the radiolabel lost at 48 h post incubation (Fig. S3A). 225Ac-cixutumumab 53 54 55 56 57 14 58 59 60 ACS Paragon Plus Environment Page 15 of 44 Molecular Pharmaceutics

1 2 3 was > 90% intact in PBS after 48 h with similar results seen after incubation in plasma (Fig. S3B). 4 5 111 6 The immunoreactive fraction of In-cixutumumab was 77% (Fig. S4). 7 8 In vitro cytotoxicity in SUM149PT cells was studied using Incucyte S3 Live cell imager. 9 10 The EC50 of each immunoconjugate was calculated with reference to untreated control cells (Table 11 12 1). The EC of 25Ac-cixutumumab (0.02 nM, 0.5 nCi/mL) in SUM149PT was almost 5000-fold 13 50 14 15 less than the EC50 of cixutumumab (95.6 nM). Control IgG had negligible cytotoxicity to these 16 17 cells with some non-specific cell killing due to 225Ac in the case of 225Ac-control IgG. 18 19 20 21 22 MicroSPECT/CT and Biodistribution 23 24 25 MicroSPECT imaging showed persistently high tumor uptake in SUM149PT xenograft (Fig. 2A). 26 27 ROI analysis at 4 time points is represented as %IA/cc (Fig. 2B). Differences in tumor uptake of 28 29 111In-cixutumumab and 111In-control IgG were observed over time (Fig. 2A and B). Uptake of 30 31 111 32 In-cixutumumab was visible as early as 24 h p.i. and peaked at around 48 h p.i., before clearing 33 34 thereafter (9.7 ± 0.4, 9.9 ± 0.5, 6.9 ± 0.8, and 6.2 ± 0.1 %IA/cc at 24, 48, 96, and 120 h p.i., 35 36 respectively; n = 3). Tumor uptake of 111In-control IgG in SUM149PT was low (4.5 ± 0.9, 4.6 ± 37 38 111 39 1.1, 3.8 ± 1.4, and 3.6 ± 1.4 %IA/cc at 24, 48, 96, and 120 h p.i., respectively; n = 3). In-control 40 41 IgG tumor uptake was significantly lower than 111In-cixutumumab at all the time points (24 to 120 42 43 h; 24 h, p < 0.001; 48 h, p < 0.001; 96 h, p < 0.01; 120 h, p < 0.05). Liver uptake of 111In- 44 45 46 cixutumumab in SUM149PT tumor-bearing mice displayed (9.3 ± 1.3, 7.3 ± 1.5, 4.1 ± 1.0 and 3.8 47 48 ± 0. 9 %IA/cc, n = 3) at 24, 48, 96 and 120 h p.i., respectively (Fig. 2B). Control 49 50 radioimmunoconjugate had higher uptake in the liver (Fig. 2C) and muscle (Fig. 2D). Highest 51 52 tumor-to-background was observed at 120 h p.i. for 111In-cixutumumab (11.5 ± 1.1), whereas 111In- 53 54 55 control IgG was 3.0 ± 0.5 (p < 0.001) (Fig. 2E). 56 57 15 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 16 of 44

1 2 3 Mice were euthanized at 72 and 144 h p.i. for the biodistribution. Tumors, blood, liver, and 4 5 6 spleen of the mice had significant radioactivity accumulation at 72 and 144 h p.i. (Fig. 3A and B) 7 8 as expected, since radiolabeled antibodies typically have long circulation half-lives and hepatic 9 10 clearance. Tumor uptake (5.4 ± 0.8 %IA/g) of 111In-cixutumumab at 72 h p.i. was higher (p < 11 12 0.001, Fig. 3A) from 111In-control IgG. 13 14 15 16 17 Efficacy of 225Ac-labeled Radioimmunoconjugates 18 19 20 We evaluated the effectiveness of two doses of cixutumumab, 225Ac-cixutumumab and isotype 21 22 225 23 control Ac-control IgG (Fig. 4A). There were no significant differences in the tumor volumes 24 25 (± SEM) at the start of treatment: cixutumumab 33.2 ± 5.1 mm3; 225Ac-cixutumumab (8.0 kBq/µg) 26 27 49.9 ± 10.5 mm3; 225Ac-cixutumumab (0.15 kBq/µg) 25.7 ± 7.2 mm3; 225Ac-control IgG 40.9 ± 28 29 8.7 mm3; and PBS treated 42.7 ± 11.6 mm3. Following the second dose (day-16) all 7/7 mice in 30 31 32 the cixutumumab group had significantly slower tumor growth as shown by 11.3-fold decrease in 33 34 tumor volume compared with PBS treated group. Complete remission was seen in 2/7 mice in this 35 36 group which lasted till the end of the study (day-180) (Fig. S5). The initial tumor response was not 37 38 3 39 sustained in 5/7 mice, which reached the humane endpoint (1500 mm ) between day-58 and day- 40 41 100. On day-16, 6/6 mice in the high specific activity 225Ac-cixutumumab (8.0 kBq/µg) group 42 43 showed a 7.6-fold decrease in tumor volume compared with PBS treated mice (Fig. S5). 1/6 mouse 44 45 46 had complete remission which lasted till day-180. Relapse was observed in the other mice in this 47 48 group. Complete remission that lasted till the end of the study was seen in 2/7 mice in the low 49 50 specific activity group 225Ac-cixutumumab (0.15 kBq/µg) (Fig. S5). Immediately following 51 52 administration of the second dose (day-16), average tumor volume in this group was 16.3-fold less 53 54 55 the PBS treated mice. Some non-specific tumor control (5.1-fold decrease compared with PBS 56 57 16 58 59 60 ACS Paragon Plus Environment Page 17 of 44 Molecular Pharmaceutics

1 2 3 treated mice) was in mice treated with 225Ac-control IgG treated group (Fig. S5). However, mice 4 5 6 in this group quickly reached study endpoint. There was no significant decrease in body weight in 7 8 all groups and no toxicity related deaths occurred in any treatment group (Fig. S6). 9 10 11 The Kaplan Meier survival curve of the different groups is shown in Fig. 4B. Median 12 13 survival (days) of mice in the PBS group was significantly lower than 225Ac-cixutumumab 8.0 14 15 225 16 kBq/µg group (p < 0.01, Log-rank (Mantel-Cox) Test) and Ac-cixutumumab 0.15 kBq/µg group 17 18 (p < 0.001, Log-rank (Mantel-Cox) Test). The difference between the cixutumumab and 225Ac- 19 20 cixutumumab 0.15 kBq/µg group survival curves were no statistically significant (p > 0.05, Log- 21 22 23 rank (Mantel-Cox) Test). The median survival of the PBS group was 52 days, while that of 24 25 cixutumumab group was 87.0 days. On the other hand, median survival of the 225Ac-cixutumumab 26 27 8.0 kBq/µg and 225Ac-cixutumumab 0.15 kBq/µg groups 103.5 and 122.0 days, respectively. The 28 29 improvement in survival seen using 225Ac-cixutumumab 0.15 kBq/µg was not statistically different 30 31 225 32 from Ac-cixutumumab 8.0 kBq/µg. 33 34 35 36 Discussion 37 38 39 There is overwhelming evidence that enhanced IGF-1R signaling occurs in cells that are intolerant 40 41 to anti-EGFR and anti-Her2 targeted therapies as well as chemotherapy25, 26. The discovery that 42 43 IGF-1R was implicated in most cancers and resistance to some targeted therapies was greeted with 44 45 46 a lot of excitement and many clinical trials of monoclonal antibodies and tyrosine kinase inhibitors 47 48 (TKIs) against IGF-1R were initiated, including ganitumab, figitumumab, and cixutumumab. 49 50 However, most of these agents failed to show any benefit in patients14, 15. Reasons for the failed 51 52 15 53 trials have been extensively reviewed and include; 1) Mutations in phosphoinositide 3-kinase 54 55 render cells resistant to targeting of the IGF-1R by constitutively activate Akt; 2) lack of IGF-1R 56 57 17 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 18 of 44

1 2 3 biomarkers and poor clinical trial design; and 3) Nuclear translocation of the IGF-1R. For example 4 5 6 in the recently failed trial of ganitumab in breast cancer (in patients that had become resistant to 7 8 tamoxifen), the obvious potential biomarker candidate of tumour expression of IGF-1R was not 9 10 measured at the time of enrolment. The determination of IGF-1R in tumor samples is usually done 11 12 by immunohistochemistry on biopsy samples. This procedure is invasive and sample collection is 13 14 15 not very reproducible. Our approach was therefore to develop a probe for non-invasive imaging of 16 17 IGF-1R and a 225Ac-labeled agent for targeted radiotherapy of IGF-1R positive tumors. To that end, 18 19 cixutumumab was conjugated with DOTA, which resulted in a slight decrease in the binding of the 20 21 22 immunoconjugate as shown by biolayer interferometry and flow cytometry. The resulting DOTA- 23 24 cixutumumab was labeled with 111In or 225Ac for microSPECT/CT imaging and alpha particle 25 26 therapy, respectively. While others have developed immunoSPECT/PET and fluorescent agents 27 28 27-30 225 29 against IGF-1R , there is no report in the literature of a Ac-labeled anti-IGF-1R alpha particle 30 31 therapeutic agent. IGF-1R is an internalizing antigen. Internalization studies showed 6 – 8.5-fold 32 33 higher internalization of the cixutumumab compared with control antibody. This attribute in addition 34 35 to the residualizing nature of 111In and 225Ac make radiolabeled derivatives of cixutumumab a good 36 37 38 candidate for anti-IGF-1R theranostics. 39 40 41 Some studies showed that the complex of 225Ac with DOTA is stable with less than 5% loss 42 43 of the radiolabel after 21 days31. In our experience, the complex is not very stable and results in a 44 45 225 46 significant loss of the radiometal over time. Up to 20% of Ac was lost from the complex over a 47 48 period of 10 days. This observation was confirmed by a recent study by Thiele et al 32 who showed 49 50 that just 90% of 225Ac-DOTA was intact one day after challenge with serum. Eighteen-membered 51 52 ring macrocylic H macropa forms more inert and stable complex with 225Ac32. Stability issues is 53 2 54 55 56 57 18 58 59 60 ACS Paragon Plus Environment Page 19 of 44 Molecular Pharmaceutics

1 2 3 likely to have implications on the efficacy and dosimetry/normal tissue toxicity of DOTA-conjugated 4 5 225 225 6 Ac radiopharmaceuticals, particularly the Ac-cixutumumab used in this study. 7 8 9 Heskamp et al. conjugated anti-IGF-1R antibody (R1507, Roche) with isothiocyanatobenzyl– 10 11 diethylenetriaminepentaacetic acid (SCN-DTPA) followed by radiolabeling with 111In30. Tumor 12 13 uptake of 111In-R1507 was evaluated in triple negative SUM149 xenograft following administration of 14 15 16 different specific activities (corresponding to 1 – 1,000 µg of antibody) of the radioimmunoconjugate. 17 18 Highest tumor uptake was observed when 1 µg of 111In-R1507 was administered, with tumor uptake 19 20 decreasing with increasing mass amounts of the administered antibody: 1 µg (38 ± 11 %IA/g) > 3 µg 21 22 23 (35 ± 6 %IA/g) > 10 µg (15 ± 4 %IA/g) > 300 µg (9 ± 1 %IA/g). This trend was suggestive of in vivo 24 25 blocking of IGF-1R binding sites of the tumor with increasing antibody mass. On the contrary, other 26 27 authors have shown that increasing the mass dose of the antibody can lead to increased non-specific 28 29 blocking, reduced interaction with FcγRs receptors (e.g. in the liver) and hence increased antibody 30 31 33, 34 32 circulation and tumor uptake . In addition, other factors such as tumor size and KD of the antibody 33 34 contribute to the absolute tumor uptake. The KD of R15017 was 0.1 nM while cixutumumab was 2.2 35 36 nM. The average tumor weight at the time of imaging in the present study was 410 mg compared with 37 38 39 112 mg in the Heskamp et al study. Smaller well vascularized tumors show higher uptake. Tumor 40 41 uptake following the administration of 20 µg 111In-cixutumumab in our microSPECT imaging studies 42 43 was lower compared to those of the Heskamp et al. study. A comparison of the Heskamp et al. study 44 45 46 with this should be interpreted with caution since the in vivo pharmacology of cixutumumab and R1507 47 48 can be significantly different. 49 50 51 We then investigated the anti-tumor effects of the radioimmunoconjugates. We studied the 52 53 in vitro cytotoxicity using IncuCyte S3 live-cell imaging with Cytotox® Red reagent, which allows 54 55 225 56 for real-time quantification of dead cells (Table 1). Ac-cixutumumab was almost 5000-fold 57 19 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 20 of 44

1 2 3 (IC50 95.6 nM vs 0.02 nM) more cytotoxic than cixutumumab to SUM149PT breast cancer cells. 4 5 225 6 This IC50 (0.5 nCi/mL) was similar to values obtained using other highly potent Ac-labeled 7 8 radioimmunconjugates 35, 36. We then studied the effectiveness of the unconjugated antibody 9 10 cixutumumab and 225Ac-cixutumumab at controlling the growth of SUM149PT xenograft. A 11 12 review of the literature shows that cixutumumab has been used in doses of 2.5 – 60 mg/kg in 13 14 15 preclinical and clinical studies37. In the present study, we initially opted for the lower dose, to stay 16 17 within acceptable toxicity profile, given that a maximum tolerated dose study was not performed. 18 19 Median survival (days) of cixutumumab (2.5 mg/kg) and 225Ac-cixutumumab (8 kBq/µg; 0.05 20 21 22 mg/kg) was 87.0 and 103.5 days, respectively, compared with 52 days for PBS treated. 23 24 Interestingly, mice administered with low specific activity agent 225Ac-cixutumumab (0.15 25 26 kBq/µg; 2.5 mg/kg) survived longer than the other groups with a median survival of 122.0 days. 27 28 29 This group also had complete remission in 2/6 mice. It is likely that the lower specific activity 30 31 agent (higher antibody mass) resulted in higher effective dose of the alpha particle delivered 32 33 compared with the higher specific activity administration. Many studies have shown that 34 35 increasing the mass of administered antibody (lower specific activity) results in enhanced 36 37 38 circulation half-life and higher tumor-to-background ratios for radiolabeled antibodies33, 39 40 especially for antibodies that have significant FCγR receptor interactions (hence higher non- 41 42 specific tissue accumulation). This, in additional to the antibody mediated cytotoxic/cytostatic 43 44 225 45 effects likely led to improved survival in the low specific activity Ac-cixutumumab (0.15 46 47 kBq/µg; 2.5 mg/kg) group compared with the higher specific activity group. In several in vitro 48 49 and in vivo models cixutumumab has been shown to bind to IGF-1R thereby leading to decrease 50 51 52 receptor phosphorylation and receptor degradation as well as induction of antibody-dependent 53 54 cell-mediated cytotoxicity (ADCC)37. 55 56 57 20 58 59 60 ACS Paragon Plus Environment Page 21 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 Conclusion 7 8 111In-cixutumumab specifically accumulated in triple-negative SUM149 xenografts which allowed 9 10 for the in vivo visualization of IGF-1R using immunoSPECT. We also described for the first time 11 12 an alpha particle labeled anti-IGF-1R antibody 225Ac-cixutumumab. The best therapeutic outcome 13 14 15 was seen in lower specific activity 225Ac-cixutumumab (0.15 kBq/µg; 2.5 mg/kg) 16 17 immunoconjugate. Given the fact that up to 60 mg/kg of cixutumumab has been administered in 18 19 clinical studies, future in vivo studies will focus on the determining the optimal specific activity 20 21 22 for radioimmunotherapy studies. This work shows the potential to enhance the efficacy of anti-IGF- 23 24 1R antibodies by radiolabeling with a high linear energy transfer alpha particle. Enhanced efficacy 25 26 can be obtained with the use of a more stable chelator for the alpha emitter (225Ac). 27 28 29 30 31 Acknowledgement and Funding 32 33 This study was funded by a grant from the Sylvia Fedoruk Centre (J2013-128) and the Canadian 34 35 Breast Cancer Foundation (No. 300030). 225Ac used in this research was supplied by the Isotope 36 37 38 Program within the Office of Nuclear Physics in the Department of Energy’s Office of Science. 39 40 41 42 Conflict of Interest 43 44 45 All authors declare they have no conflicts of interest. 46 47 48 49 50 Supporting Information 51 52 Supporting information of the binding of cixutumumab to cells by flow cytometry (Fig. S1), 53 54 internalization studies (Fig. S2), stability of 111In/225Ac-cixutumumab in plasma and saline (Fig. 55 56 S3), immunoreactive fraction determination (Fig. S4), tumor volumes of individual mouse in all 57 21 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 22 of 44

1 2 3 the groups (Fig. S5) and their respective body weight (Fig. S6) is available in supplementary 4 5 material. 6 7 8 9 10 11 12 a 13 Table 1. EC50 values of antibodies immunoconjugates on SUM149PT cells 14 15 EC50 (nM) EC50 (nCi/mL) 16 17 Cixutumumab 95.6 ± 1.0 NA 18 225Ac-cixutumumab 0.02 ± 0.004 0.5 ± 0.1 19 Control IgG 340.1 ± 1.5 NA 20 225Ac-control IgG 13.8 ± 0.52 16.0 ± 0.6 21 22 Values are the mean of triplicates of at least two independent experiments. NA: Not applicable 23 24 25 26 Figure Legends 27 28 29 Fig. 1A-D. A) Bioanalyzer electropherograms of ladders, cixutumumab, and DOTA-cixutumumab 30 31 B) Binding of antibodies to recombinant IGF-1R using biolayer interferometry at different 32 33 concentrations (55, 166 and 500 nM). C) In vitro flow cytometry binding assay in IGF-1R positive 34 35 36 MCF-7/Her18 breast cancer cells. An 8- point curve allowed for the estimation KD of 37 38 cixutumumab and DOTA-cixutumumab. Percentage of bound was estimated from the relative 39 40 fluorescence intensity and plotted against concentration. A non-linear curve fitting was used to 41 42 43 estimate the KD values. D). Internalization of cixutumumab antibody conjugates in MCF- 44 45 7/Her18 cells. MCF-7/Her18 cells were treated with either IncuCyte® FabFluor labeled 46 47 cixutumumab antibody or IgG1 isotype control (4 μg/mL), HD phase and red fluorescence 48 49 images (10 ×) were captured every 2 h for 48 h. All data shown as a mean of 6 wells ± SEM. 50 51 52 53 54 55 56 57 22 58 59 60 ACS Paragon Plus Environment Page 23 of 44 Molecular Pharmaceutics

1 2 3 Fig. 2A-E. A) Representative microSPECT/CT fused images of a mouse bearing SUM149PT 4 5 111 111 6 xenograft after intravenous injection of In-cixutumumab and In-control IgG. Time-activity 7 8 curves of B) tumor, C) liver, D) muscle (c) and tumor/muscle ratios. Data are %IA/cc, expressed 9 10 as mean ± SD. 11 12 13 Fig. 3A & B. Biodistribution of 111In-cixutumumab, and 111In-control IgG in nude mice bearing 14 15 16 SUM149PT xenografts at A) 72 h B) 144 h post injection. Data are %IA/g, expressed as mean ± 17 18 SD. 19 20 21 Fig. 4A & B: Efficacy of two doses (day 0 and 14) of cixutumumab (2.5 mg/kg), 225Ac- 22 23 cixutumumab (225 nCi, 0.05 mg/kg, 8 kBq/µg), 225Ac-cixutumumab (225 nCi, 2.5 mg/kg, 0.15 24 25 225 26 kBq/µg), Ac-control IgG (225 nCi, 0.15 kBq/µg, 2.5 mg/kg) and PBS treated mice in IGF-1R 27 28 positive SUM149PT mouse model. A) Tumor growth curves and B) Kaplan–Meier survival 29 30 curves. Study endpoint occurred when xenograft volume reached 1500 mm3, or > 20% ulcerated. 31 32 33 34 35 36 References 37 38 1. Islami, F.; Torre, L. A.; Drope, J. M.; Ward, E. M.; Jemal, A. Global Cancer in Women: 39 40 41 Cancer Control Priorities. Cancer Epidemiol Biomarkers Prev 2017, 26, (4), 458-470. 42 43 44 2. Foulkes, W. D.; Stefansson, I. M.; Chappuis, P. O.; Begin, L. R.; Goffin, J. R.; Wong, N.; 45 46 Trudel, M.; Akslen, L. A. Germline BRCA1 mutations and a basal epithelial phenotype in 47 48 breast cancer. J Natl Cancer Inst 2003, 95, (19), 1482-5. 49 50 51 3. Lips, E. H.; Mulder, L.; Oonk, A.; van der Kolk, L. E.; Hogervorst, F. B.; Imholz, A. L.; 52 53 Wesseling, J.; Rodenhuis, S.; Nederlof, P. M. Triple-negative breast cancer: BRCAness 54 55 56 57 23 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 24 of 44

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 30 58 59 60 ACS Paragon Plus Environment Page 31 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Graphical Abstract 26 27 108x60mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 32 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 1A 26 27 108x60mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 33 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 1B 26 27 108x60mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 34 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 1C 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 35 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 1D 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 36 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 2A 26 27 108x60mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 37 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 2B 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 38 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 2C 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 39 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 2D 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 40 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 2E 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 41 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 3A 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 42 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 3B 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 43 of 44 Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 4A 26 27 27x15mm (600 x 600 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Molecular Pharmaceutics Page 44 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Fig. 4B 26 27 108x60mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment