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PF-06804103, a site-specific anti-HER2 antibody-drug conjugate for the treatment of HER2-expressing breast, gastric, and lung cancersEdmund I. Graziani1+, Matthew Sung2+?, Dangshe Ma2 , Bitha Narayanan2, Kimberly Marquette3 , Sujiet Puthenveetil1, L. Nathan Tumey1, Jack Bikker1, Jeffrey Casavant1, Eric M. Bennett3, Manoj B. Charati2, Jonathon Golas2, Christine Hosselet2, Cynthia M. Rohde4, George Hu4, Magali Guffroy4, Hadi Falahatpisheh4, Martin Finkelstein4, Tracey Clark5, Frank Barletta6, Lioudmila Tchistiakova3, Judy Lucas2, Edward Rosfjord2, Frank Loganzo2, Christopher J. O’Donnell1, Hans-Peter Gerber2, Puja Sapra2?1Pfizer Inc., World Wide Medicinal Chemistry (Groton, CT), 2Pfizer Inc., Oncology Research & Development (Pearl River, NY), 3Pfizer Inc., BioMedicine Design (Cambridge, MA), 4Pfizer Inc., Drug Safety Research & Development (Pearl River, NY), 5Pfizer Inc., BioMedicine Design (Groton, CT), 6Pfizer Inc., BioMedicine Design (Pearl River, NY)+ Co-first authors? Co-Corresponding authors Co-Corresponding Authors Contact Information:Matthew Sung, Ph.D.Principal Scientist, Targeted Therapeutics UnitOncology Research and DevelopmentPfizer Worldwide Research & Development401 N. Middletown Road, Pearl River, NY 10965Tel: +1 (845) 602-3731Email: sungms@Puja Sapra, Ph.D.Vice President and CSO, Targeted Therapeutics UnitOncology Research and DevelopmentPfizer Worldwide Research & Development401 N. Middletown Road, Pearl River, NY 10965Tel: +1 (845) 602-3389Email: Psapra111@Running title: PF-06804103, a novel site-specific anti-HER2 ADCDisclosure of Potential Conflicts of InterestAll authors are either employees and/or shareholders of Pfizer Inc., a publicly traded company.SUPPLEMENTARY INFORMATIONMaterials & MethodsCell linesMDA-MB175-VII, MDA-MB-468, MDA-MB-453 and MDA-MB361-DYT2 were grown in MEM (Gibco, USA) supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 2% MEM vitamins. BT474-M1 and JIMT-1 were grown in DMEM (Gibco, USA) supplemented with 10% FBS, 1% sodium pyruvate. All other cell lines were grown in RPMI media (Gibco, USA) with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, glucose and HEPES. The cells were maintained under standard conditions (37°C in humidified atmosphere containing 5% CO2). All cell lines used for in vivo tumor model studies were authenicated by STR DNA profiling and confirmed mycoplasma free before injection in mice. Cell lines were typically kept under 30 passages from thaw for experiments described in this report.Antibody generation and characterizationThe crystal structure of human IgG1 (publicly available at Sondermann et al., 2000, Nature 406:267-273; PDB code 3DO3, 10.2210/pdb3do3/pdb) was used to predict, using structural modeling, the positions where the reactive cysteines should be introduced for optimal conjugation with a sulfhydryl reactive agent. The crystal complex of the Fc domain of human IgG1 (PDB code 3DO3, 10.2210/pdb3do3/pdb) was obtained from the RCSB protein databank and prepared for visualization and modeling in Discovery Studio (Accelrys Inc., San Diego, CA). The individual side chains were mutated to cysteine and minimized using the Mutate Residue feature in Discovery Studios according to manufacturer’s instructions. The side chain solvent accessibility of the mutated residue was calculated, as was the residue pKa, using the method of Spassov and Yan (2008, Protein Sci. 17(11):1955–1970). Based on this analysis, Lys-290, Lys-334 and Lys-392 (EU numbering system as set forth in Kabat et al. 1991, NIH Publication 91 – 3242, National Technical Information Service, Springfield, VA) were selected as positions in the Fc-region for introducing reactive cysteines into the IgG1-Fc region for site-specific conjugation of vc0101. Sites to engineer reactive cysteines were selected in the Kappa light chain constant region to expand diversity of positions for site-specific conjugation and to enable conjugation of 4 toxic payloads per antibody by combining engineered Kappa regions with select single Fc-region cysteine mutants. Property predictions were performed on several Kappa domain crystal structures, and positions giving optimal property predictions on multiple structures (2R8S and 1N8Z; Ye et al., 2008, Proc. Natl. Acad. Sci.USA 105:82-87 and Cho et al., 2003, Nature 421:756-760, respectively) were preferred. Each position was examined in each crystal structure by first mutating the position to cysteine and predicting the rotamer with SCWRL4 (G. G. Krivov, M. V. Shapovalov, and R. L. Dunbrack, Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins, 2009), then by predicting the cysteine side chain pKa using methods such as those described in, inter alia, Spassov and Yan, 2008, Protein Sci. 17:1955-1970) and side chain solvent accessibility using Discovery Studio 3.0 (Accelrys, Inc., San Diego, CA). Lysine at position 183 (Kabat numbering system) was selected and engineered to introduce a reactive cysteine for site-specific conjugation.PCR mutagenesis was performed as follows to replace native amino acids in the PT-Her2 antibody with reactive cysteines. Sense and anti-sense mutagenic oligonucleotides harboring the individual cysteine mutations as well as forward and reverse human IgG1 constant region flanking primers were synthesized at Integrated DNA Technologies, Inc (Park Coralville, Iowa). PCR reaction 1 contained one hundred nanograms (ng) of PT-Her2 antibody encoding plasmid DNA, 100 pmoles forward flanking primer oligonucleotide, 100 pmoles anti-sense mutagenic oligonucleotide, 1 μl Vent? polymerase (New England Biolabs Inc., Ipswich, Massachusetts), 25 ?l 2x HN PCR buffer (EPICENTRE? Biotechnologies, Madison, WI) and H2O to bring the volume of the reaction to 50 ?l. Similarly, PCR reaction 2 was made by mixing 100 ng PT-Her2 antibody encoding plasmid DNA, 100 pmoles sense mutagenic oligonucleotide, 100 pmoles reverse flanking primer oligonucleotide, 1 ?l Vent? polymerase, 25 ?l 2x HN PCR buffer and adding H2O to bring the volume of the reaction to 50 ?l. The PCR parameters for reactions 1 and 2 were 95°C for 1 minute, 63°C for 1 minute, 72°C for 1 minute for 25 cycles and then 10 minutes at 72°C. The final PCR reaction was done by mixing 1 μl each of PCR reactions 1 and 2, 100 pmoles each of the forward and reverse flanking primer oligonucleotides, 1 ?l Vent? polymerase, 25 ?l 2x HN PCR buffer and H2O to bring the volume of the reaction to 50 ?l. The final PCR reaction parameters were the same as used for reactions 1 and 2. The human IgG1 and Cys-183 variants harboring the individual engineered cysteine residues were joined to the PT-Her2 heavy and light chain variable region, respectively, using T4 DNA Ligase (New England Biolabs Inc., Ipswich, Massachusetts) and nucleic acid was sequence confirmed.Conjugation of anti-HER2 antibody with vc0101 and smcc-DM1Site-specific cysteine conjugation. A 500 mM TCEP solution (50 to 100 molar equivalents) was added to the antibody (5 mg) such that the final antibody concentration was 5-15 mg/mL in PBS containing 20 mM EDTA. After allowing the reaction to stand at 37o C for 2.5 hour, the antibody was buffer exchanged into PBS containing 5 mM EDTA using a gel filtration column (PD-10 desalting column , GE Healthcare). The resulting antibody (5-10 mg/mL) in PBS containing 5 mM EDTA was treated with a freshly prepared 50 mM solution of DHA in 1:1 PBS/EtOH (final DHA concentration = 1 mM – 4 mM) and allowed to stand at 4° C overnight.The antibody/DHA mixture was buffer exchanged into PBS containing 5 mM EDTA (pH of the equilibration buffer adjusted to ~7.0 using phosphoric acid) and concentrated using a 50 kD MW cutoff spin concentration device. The resulting antibody in PBS (antibody concentration ~5-10 mg/ml) containing 5 mM EDTA was treated with 5-7 molar equivalents of 10 mM maleimide payload (PF-06424469) in DMA. After standing for 1.5-2.5 hours, the material was buffer exchanged (PD-10). Purification by SEC was performed (as needed) to remove any aggregated material and remaining free payload.Conventional Cysteine Conjugation: Trastuzumab was dialyzed into Dulbecco's Phosphate Buffered Saline (DPBS, Lonza). The dialyzed antibody was diluted to 15 mg/mL with PBS containing 5 mM 2, 2', 2", 2"'-(ethane-1, 2-diyldinitrilo)tetraacetic acid (EDTA), pH 7. The resulting antibody was treated with 2-3 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mM in distilled water) and allowed to stand 37 °C for 1-2 hours. Upon cooling to room temperature, dimethylacetamide (DMA) was added to achieve 10% (v/v) total organic. The mixture was treated with 8-10 equivalents of the appropriate linker-payload as a 10 mM stock solution in DMA. The reaction was allowed to stand for 1-2 hours at room temperature and then buffer exchanged into DPBS (pH 7.4) using GE Healthcare Sephadex G-25 M buffer exchange columns per manufacturer's instructions. Material used for succinimide ring hydrolysis was immediately carried into the protocol outlined in condition C. Material that was intended to remain ring-closed was purified by size exclusion chromatography (SEC) using GE AKTA Explorer system with GE Superdex200 column and PBS (pH 7.4) eluent. Final samples were concentrated to ~5 mg/mL protein, filter sterilized, and checked for loading using the MS conditions outlined below.Hydrophobicity Evaluation of Site Specific ConjugatesCompounds were prepared for hydrophobic interaction chromatography (HIC) analysis by diluting a 30 uL sample (at approximately 1 mg/mL ADC) with 30 uL of 2M K2HPO4 (pH 8.5). The samples were analyzed using an Agilent 1200 HPLC with a TSK-GEL Butyl NPR column (4.5x35 mm, 2.5 μm). About 60 uL of sample was injected and a gradient method was run as follows: A: 1M K2HPO4 (pH 8.5); Mobile phase B: water; T=0 min. 90% A; T=40 min., 0% A; and T=50 min, 0% A.In vitro cytotoxicity studies In vitro potency and specificity was determined following procedures previously described. Cells seeded in 96-well plates were dosed the following day with 4-fold serial dilutions of the ADCs. Cells were incubated for 96 hours in a humidified 37?C/5% CO2 incubator. Cell Titer Glo Solution (Promega, Madison, WI) was added to the plates and absorbance measured on a Victor plate reader (Perkin-Elmer, Waltham, MA) at wavelength 490 nm. IC50 values were calculated using a four-parameter logistic model with XLfit (IDBS, Bridgewater, NJ). Immunohistochemistry studiesHER2 expression was determined immunohistochemically using 2 detection methods. Five micrometer-thick formalin-fixed, paraffin embedded tissue sections were deparaffinized in xylene substitute and rehydrated with graded alcohols to distilled water. For HER2 staining using a rabbit monoclonal antibody, the sections were heated in a pressure cooker (Retriever 2100, Electron Microscopy Sciences, Hatfield, PA) in EDTA buffer pH 8.0 (ThermoFisher Scientific, Waltham, MA, Catalog Number 15575020) and cooled to room temperature. Endogenous peroxidase activity was inactivated with Peroxidazed 1 (Biocare Medical, Concord, CA, Catalog Number PX968) for 10 minutes. Non-specific protein interactions were blocked for 10 minutes with Background Punisher (Biocare Medical, Catalog Number BP974). Sections were incubated with a rabbit monoclonal anti-HER2 antibody (Cell Signaling Technologies, Danvers, MA, Catalog Number 2165, Clone 29D8; final concentration 0.264 ?g/mL) for one hour, washed in Tris-buffered saline (TBS) and incubated with SignalStain? Boost IHC Detection Reagent (Cell Signaling Technologies, Catalog Number 8114) for 30 minutes. Sections were washed in TBS and chromogenic detection of tissue slides was developed with 3,3’-diaminobenzidine tetrahydrochloride (DAB)(DAB+ Substrate, Agilent Technologies, DAKO, Santa Clara, CA, Catalog Number GV82511-2) for 5 minutes. To stop the development of DAB, sections were washed in distilled water. The HercepTest (Agilent Technologies, DAKO, Catalog Number K520421-5) was also utilized as per the manufacturer’s instructions to detect HER2 expression. Finally, all stained slides were briefly counterstained with Hematoxylin (Biocare Medical, Catalog Number CATHE), washed in tap water, dehydrated in graded alcohols, cleared in xylene, and coverslipped with Permount? Mounting Medium (ThermoFisher Scientific, Catalog Number SP15-500). For all immunostains, a rabbit immunoglobulin G served as a negative control. All stained slides were scanned using an Aperio AT2 slide scanner (Leica, Wetzlar, Germany). Additional primary antibodies used were anti-phospho-Histone H3 (#9701, Cell Signaling Technologies; 0.13 mg/mL), and control rabbit IgG. Toxicity and Toxicokinetic Assessments of Conventional and Site-Specific HER2 ADC in Rats and Non-Human PrimatesAll animal studies were approved by the Pfizer Institutional Animal Care and Use Committee according to established guidelines.Rats: Male Sprague-Dawley rats (n=5-6/group) were administered a single intravenous (IV) bolus injection of conventional anti-HER2 vc0101 conjugate (HER2-vc0101) or the site-specific conjugate, PF-06804103, at doses up to 30 mg/kg/dose, and then monitored for 14 days to assess toxicity and exposure. Rats were euthanized via isoflurane inhalation followed by exsanguination and necropsied on Day 15. Repeat-dose toxicity was evaluated in male Sprague-Dawley rats (n=5-6/group) after IV bolus administration of 1, 3, or 10 mg/kg HER2-vc0101 or 3, 10, or 30 mg/kg PF-06804103 once every 3 weeks for a total of 3 doses. Rats were euthanized as described above and necropsied on Day 46, 3 days after the last dose. Assessments in both single and repeat-dose studies included clinical observations, body weights, food consumption, clinical pathology parameter measurement, toxicokinetic (TK) analysis, and macroscopic and microscopic tissue pathology evaluations. Microscopic tissue findings were graded on a scale of 1 to 5 as minimal, mild, moderate, marked, or severe.To investigate the potential contribution of prematurely released payload in circulation on the observed toxicities and to better understand the mechanism of bone marrow toxicity associated with vc0101 conjugates, male Sprague-Dawley rats (controls: n=3; test article groups: n=6) were IV infused with Aur0101 (PF-06380101) continuously for 72 hours in a manner modeled to mimic the slow release of the payload from the ADC at an ADC dose of 3 (51.5 ?g/kg Aur0101 [PF-06380101] in total) or 10 mg/kg/dose (171.5 ?g/kg Aur0101 [PF-06380101] in total). Concurrent control groups were administered 0, 3, or 10 mg/kg/dose of HER2-vc0101 via IV bolus injection. Rats were euthanized as described above and necropsied on Day 4. In a separate study to determine if the stability of the ADC correlated with the degree of bone marrow toxicity observed, male rats (n=3/group) were administered a single IV bolus dose at 10 mg/kg/dose of HER2-vc0101 or PF-06804103. Animals were euthanized as described above on Day 4. Assessments included mortality, clinical observations, hematology and clinical chemistry parameters on Day 4, TK, and macroscopic and bone marrow microscopic tissue evaluation.Cynomolgus monkeys: HER2-vc0101 and PF-06804103 were evaluated in Mauritian cynomolgus monkeys to determine their toxicity and toxicokinetic profiles. Male and female cynomolgus monkeys (1/sex/group for exploratory toxicity studies and 3-5/sex/group for a good laboratory practice [GLP] toxicity study) were administered test article via IV bolus injection of HER2-vc0101 at 3 or 5 mg/kg or PF-06804103 at 3, 6, 9, or 12 mg/kg once every 3 weeks (Days 1, 20, and 41 or Days 1, 22 and 43) . On Day 44 or 46 (3 days after the third dose) animals were euthanized using sodium pentobarbital administration followed by exsanguination and then necropsied. Assessments included clinical observations, body weights, qualitative food consumption, clinical pathology parameters, TK, and macroscopic and microscopic tissue pathology evaluations. Additionally, in the GLP toxicity study, assessment of ophthalmic, electrocardiographic, and echocardiographic parameters were conducted. Microscopic tissue findings were graded on a scale of 1 to 5 as minimal, mild, moderate, marked, or severe.Quantitation of ADC and Total Ab in Rats and Cynomolgus monkeyQuantitation of total antibody (conjugated and unconjugated mAb) and ADC (mAb with at least one drug molecule conjugated) concentrations in plasma collected from Sprague Dawley rat or Cynomolgus monkey following a single bolus administration of the ADC was achieved using a Gyrolab? (Warren, NJ) workstation with fluorescence detection.? Isolation and detection of total antibody (either conjugated or not conjugated to the linker payload) and ADC (mAb conjugated to at least on linker payload) concentrations from biological matrix was carried out with streptavidin coupled micro columns located on Bioaffy?200 CDs within the Gyrolab? workstation.? Biotinylated labeled reagents from different sources were used for capturing onto the CD.? CDs were centrifuged to remove excess reagent and then washed with wash buffer followed by further centrifugation.?? Plasma calibration standards, quality control samples and study samples were all diluted to a minimum required dilution (MRD) and loaded onto the CDs.? Samples were further diluted with assay diluent if required.? The micro columns were again washed with wash buffer and Alexa Fluor 647 labeled reagents from different sources added for detection.? The CDs were centrifuged to remove excess reagent and then washed with wash buffer followed by further centrifugation.? Fluorescence of analyte was measured using a laser embedded within the workstation.? All data was processed and pharmacokinetic parameters were calculated using Watson v7.4 LIMS with a 5-parameter 1/Y*2 weighting.UPLC-MS/MS Analysis of Free Payload (Aur0101) in Serum A standard curve containing Aur0101 (PF-06380101) was prepared in control rat serum before extraction with protein precipitation method. Sample (including dilution) or standards were combind with acetonitrile and internal standard of PF-06691147. Chromatography was performed on a Waters Acquity UPLC System (Milford, MA). Separation was achieved with a MacMod ACE C18 column (2.1*30mm, 4 ?m), and a gradient of 5 mM ammonium acetate (Mobile Phase A) and acetonitrile (Mobile Phase B) at a flow rate of 0.35 mL/min. An initial mobile phase composition of 35% B was ramped to 95% in 3 minutes, held at 95% and then returned to initial 35% B for re-equilibration. Data was collected on an AB Sciex API5500 (QTRAP) mass spectrometer (Foster City, CA, USA) using positive Turbo IonSpray? electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. Typical source conditions, heated capillary temperature, gas1, gas2, and curtain gas were set at 500?C, 40, 40 and 10 respectively. Transitions for PF-06380101 and internal standard PF-06691147 were m/z 743.6 ? 188 for PF-06380101 and m/z 751.6 ? 188 for PF-06691147 respectively. Data acquisition and processing was carried out with Analyst software version 1.5.2. (Applied Biosystems/MDS Sciex, Canada).Statistical analysisStatistical analysis for in vitro potency assays were done using the four-parameter analysis. In vivo efficacy data was analyzed using student T-test.Therapeutic Index (TI) CalculationCalculation of Tumor Static Concentration (TSC)TSC was defined as the concentration of PF-06804103 where tumor growth and death rates are equal and tumor volume remains unchanged. This PK/PD derived parameter combines the growth pattern information and the drug effect, providing insight on the efficacy of the ADC. See equation 1 for TSC calculation. An 80% confidence interval on TSC was calculated using parametric bootstrap by resampling from the estimated parameters using a log-normal distribution.(1) TSC= kgEx? × kC50n × 1- V0Vmaxkkmax × 1+kgExkg?×V0φ 1φ- kgEx ×?1?-?V0Vmax1nCalculation of TITo calculate the preclinical TI, the average concentration (Cavg) of PF-06804103 was calculated in cynomolgus monkey at the highest non-severly toxic dose (HNSTD) and divided by the TSC. The area under the curve at 504 h (AUC0-504h) was used to calculate the average concentration. See Equations 2 - 3.(2)Cavg at HNSTD= ADC AUC0-504h504(3) TI= Cavg at HNSTDTSCAntibody Amino Acid Sequence of PF-06804103The complete amino acid sequence of the mature heavy and light chains of PF-06804103 are shown below. Variable regions are shown in bold and underlined, constant regions are in regular non-underlined font, IMGT CDRs are highlighted (yellow), and Kabat CDRs are in green font. The engineered cysteins mutations (lysine to cysteine) are shown in bold, non-underlined and red font in both heavy (position 307, Kabat numbering; position 290 EU Index of Kabat numbering) and light (position 183, Kabat numbering) chains. Heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR50IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG100GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK150DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT200YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP250KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQYN300STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ350VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV400LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG448Light chainDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS50ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ100GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV150DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQG200LSSPVTKSFNRGEC214Synthesis of ‘Aur0101’ payload and ‘vc0101’ linker payload Auristatin-0101 (PF-06380101) is a synthetic analog of the auristatin dolastatin 10. A detailed synthesis route for PF-06380101 has been previously described (Compound #20a, Maderna et al. J Med Chem. 2014). For the anti-HER2 ADC PF-06804103 described in this manuscript, PF-06380101 was modified on the N-terminus to include the ‘mcValCit-PABC’ linker (creating the ‘vc0101’ linker payload) to enable conjugation to the engineered cysteines of the anti-HER2 monoclonal antibody (WO2017/093844, PC072091).Supplementary Figure LegendsFigure S1. In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model. N87 xenograft tumors were established in mice as described in the Materials and Methods and were treated intravenously four times every four days (q4dx4) with PBS as vehicle [blue] or 4 DAR anti-HER2 ADC at 0.3 mg/kg [red], 1 mg/kg [green] or 3 mg/kg [purple]. Data shown is the mean ± SEM of tumor size on each measurement day. Figure S2. Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models. Representative tumors from each in vivo model were collected at study staging size (tumor volume = ~200-300 mm3), formalin-fixed and paraffin-embedded. Each tumor was stained with an anti-HER2 detection antibody to assess HER2 protein quantity and distribution in each model. The blue scale bar represents 200 microns in each image. ADDIN SUPPLEMENTARY DATAFigure S1. In vivo efficacy of site-specific anti-HER2 vc0101 4 DAR ADCs in N87 xenograft tumor model. N87 xenograft tumors were established in mice as described in the Materials and Methods and were treated intravenously four times every four days (q4dx4) with PBS as vehicle [blue] or 4 DAR anti-HER2 ADC at 0.3 mg/kg [red], 1 mg/kg [green] or 3 mg/kg [purple]. Data shown is the mean ± SEM of tumor size on each measurement day. Figure S2. Immunohistochemical evaluation of HER2 expression in vivo cell line and patient-derived xenograft models. Representative tumors from each in vivo model were collected at study staging size (tumor volume = ~200-300 mm3), formalin-fixed and paraffin-embedded. Each tumor was stained with an anti-HER2 detection antibody to assess HER2 protein quantity and distribution in each model. The blue scale bar represents 200 microns in each image. ADDIN ................
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