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PF-06804103, a site-specific anti-HER2 antibody-drug conjugate for the treatment of HER2-expressing breast, gastric, and lung cancers
Edmund 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: [email protected]
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: [email protected]
Running title: PF-06804103, a novel site-specific anti-HER2 ADC
Disclosure of Potential Conflicts of Interest
All authors are either employees and/or shareholders of Pfizer Inc., a publicly traded company.
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SUPPLEMENTARY INFORMATION
Materials & Methods
Cell lines
MDA-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 characterization
The 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
2
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.,
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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 µl 2x HN PCR buffer (EPICENTRE® Biotechnologies, Madison, WI) and H2O to bring the
volume of the reaction to 50 µl. 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 µl Vent® polymerase, 25 µl 2x HN PCR buffer and
adding H2O to bring the volume of the reaction to 50 µl. 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 µl Vent®
polymerase, 25 µl 2x HN PCR buffer and H2O to bring the volume of the reaction to 50 µl. 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.
4
Conjugation of anti-HER2 antibody with vc0101 and smcc-DM1
Site-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
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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 Conjugates
Compounds 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 37C/5% CO2 incubator. Cell Titer Glo
Solution (Promega, Madison, WI) was added to the plates and absorbance measured on a Victor
6
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 studies
HER2 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
7
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 Primates
All 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
8
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 µg/kg Aur0101
[PF-06380101] in total) or 10 mg/kg/dose (171.5 µg/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
9
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 monkey
Quantitation 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
10
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 µm), 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 500ºC, 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).
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Statistical analysis
Statistical 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) Calculation
Calculation 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 × kC 50n×(1−
V 0
V max)
(k kmax ×(1+( kgEx
kg×V 0)
φ
)1φ−kgEx ×(1− V 0
V max))
1n
Calculation of TI
To 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.
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(2) Cavg at HNSTD=ADC AUC0−504h
504
(3) TI=Cavg at HNSTDTSC
Antibody Amino Acid Sequence of PF-06804103
The 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 chain
EVQLVESGGG LVQPGGSLRL SCAAS GFNIK DT Y IH WVRQA PGKGLEWVA R 50IYPT NG YT RY A DS VKG RFTI SA DT SK NT AY LQ MNS LRAE D T AVYYC SR WG 100G DG FYA M DY W GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK 150DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT 200YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP 250KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTCPREEQYN 300STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 350VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 400LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 448
Light chain
DIQ M TQSPSS LSASVGDRVT ITC RAS QDV N
T A VA WYQQKP GKAPKLLIY S 50
AS FLYS GVPS RFSGSRSGTD FTLTISSLQP EDFATYYC QQ HYTTPPT FGQ 100GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 150DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSCADYEKHK VYACEVTHQG 200LSSPVTKSFN RGEC 214
Synthesis of ‘Aur0101’ payload and ‘vc0101’ linker payload
13
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).
14
Supplementary Figure Legends
Figure 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.
15
SUPPLEMENTARY DATA
Figure 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.
16
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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.
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