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Supplementary Information for Damelin, Bankovich, et al.
Supplementary Figure Legends
Supplementary Figure 1. Analysis of EFNA4 copy number in breast and hepatocellular
carcinoma. (A) EFNA4 copy number in 709 and 1,861 breast tumor samples from TCGA and
METABRIC, respectively. The gray box represents the 25th-75th percentiles, the error bars
demarcate the 10th-90th percentiles, and the individual dots fall below the 10th or above the 90th
percentile. (B) EFNA4 copy number analysis of 1,138 ovarian tumor (OVCA) samples from
TCGA dataset. The gray box represents the 25th-75th percentiles, the error bars demarcate the
10th-90th percentiles, and the individual dots fall below the 10th or above the 90th percentile. (C)
Correlation of EFNA4 mRNA level (log2 transformed intensity value, the intensity value was
transformed using Rosetta’s variance stabilization algorithm ) versus DNA copy number (log2
ratio; Tumor vs Normal) from 204 patients in the ACRG dataset. EFNA4 mRNA expression
correlates with copy number in all 3 datasets: ACRG is shown, correlation coefficient = 0.56 (p ~
0); TCGA correlation coefficient = 0.50 (p ~ 0); correlation coefficient = 0.50 (p ~ 0); and
Samsung correlation coefficient = 0.38 (p = 3E-09). (D) EFNA4 copy number in hepatocellular
carcinoma (HCC) tumor samples from 3 datasets: ACRG (n = 310), Samsung (n = 272) and
TCGA (n = 212).
Supplementary Figure 2. Characterization of PF-06647263 conjugates. (A) Representative
BIAcore data assessing hE22 mAb affinity for EFNA4 protein is shown. (B) Western
immunoblot of lysates of (1) 293T-EFNA4 cells and (2) parental 293T cells, with hE22 antiEFNA4 mAb or anti--actin mAb after resolution by non-reducing gel electrophoresis. (C) Flow
cytometry of hE22 or control (non-binding) mAb with 293T vs. 293T-EFNA4 cells. MCF =
mean channel fluorescence. (D) Capillary isoelectric focusing (cIEF) characterization of the
drug:antibody ratio (DAR) for a typical batch of the ADC PF-06647263 is shown in parallel with
the unconjugated mAb trace. In a typical batch, 76% of the ADC molecules had DAR of 3, 4 or
5, with no detectable amount of DAR = 1 or unconjugated (DAR = 0) species. Note that the
cIEF method cannot distinguish between a conjugation event and a deamidation event (of
glutamine to glutamate or asparagine to aspartate), which is why a small fraction of the
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unconjugated mAb appears to be DAR 1, and why the ADC overall DAR is slightly
overestimated. (E) Representative BIAcore data assessing the ADC PF-06647263 affinity for
EFNA4 protein is shown, along with (F) a summary of hE22 and PF-06647263 on and off rates
contributing to the affinity (KD) determination.
Supplementary Figure 3. Biomarkers of PF-06647263 activity in breast PDX tumors.
Tumors were harvested as indicated after one administration of 1 mg/kg PF-06647263.
Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections with antihIgG mAbs to detect ADC (brown staining, upper panels) and anti-γH2A.X mAbs to detect
DNA damage (brown staining, lower panels).
Supplementary Figure 4. Characterization of EFNA4 affinity for EphA and EphB
receptors.
Binding of monomeric EFNA4 to immobilized Eph receptors was assessed by
BIAcore. The Eph receptor, (A) EphA2 was demonstrated to have the highest affinity for
EFNA4, whereas (B) EphA10 had the lowest affinity for EFNA4. (C) A summary of EFNA4
binding of Eph receptor family members shows differential affinity within a 14-fold range and
cross-reactivity with both EphA and EphB receptors.
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Supplementary Materials & Methods
Flow cytometry and FACS
All analyses and cell isolations were performed using freshly dispersed cell suspensions.
Antibody staining was performed for 30 minutes at 4°C at a density of 1x107 cells/mL using the
antibodies noted above, anti-human CD24 (BioLegend, Clone ML5), and anti-human CD324
(BioLegend, Clone 67A4).
In all experiments, cells staining positively for murine lineage
markers (H-2Kd & murine CD45) were excluded from further analysis. Dead cells were also
excluded using the viability dye DAPI, and cell clumps and doublets were excluded using
doublet discrimination gating. Cell isolation and tumor initiation data shown is representative of
more than 10 individual experiments with 5 TNBC PDX tumor models and 20 individual
experiments with 10 ovarian cancer PDX tumor models. Furthermore, personnel performing the
cell isolation were different from those implanting isolated tumor cells, thus essentially blinding
them as to what was being implanted. Cell isolation and tumor initiation studies are summarized
in Supplementary Tables 1 & 2. Cellular phenotype and viability of FACS-purified cells was
confirmed by serial flow cytometric analysis after isolation by FACS and prior to injection for
tumorigenicity studies. Purity was typically > 99%.
Anti-EFNA4 antibody binding specificity determination by ELISA
Human or mouse EFNA4-Fc protein was captured on an ELISA plate with donkey anti-human
IgG (Fc specific) recognizing the human Fc portion of the recombinant EFNA4 protein. Human
and mouse EFNA4-Fc (369-EA-200 & 569-A4-200, respectively; R&D Systems) were captured
at 400ng/mL. anti-EFNA4 mAb clones E22 and E91 were then added to the washed wells at
1ug/mL to test for binding to either antigen. Antibody binding was detected by anti-mouse IgG
secondary and read out with TMB substrate to confirm cross-reactivity patterns of these
antibodies.
Antibody binding to EFNA family members was determined by direct ELISA using
EFNA1-His (10882-H03H-50, Sino Biological), EFNA3-huFc (359-EA-200, R&D Systems),
EFNA4-huFc (369-EA-200, R&D Systems) and EFNA5-huFc (374-EA-200, R&D Systems)
coated on BioOne Microlon plates (Greiner) in PBS overnight at 4°C. Biotin-labeled hE22 was
incubated on the plate for 2.5h at RT and Streptavidin-HRP (Jackson Immuno Research) was
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then used to develop signal from biotin labeled antibody in the absence of interfering human IgG
signal. Binding data for hE22 recapitulates the known affinity difference of these antibodies for
their target, EFNA4.
EFNA4 protein expression determination
Tumor tissues were flash frozen on dry ice/ethanol.
Protein Extraction Buffer (Biochain
Institute, Inc.) was added to the thawed tissues, which were then pulverized using a TissueLyser
system (Qiagen GmbH). Lysates were cleared by centrifugation (20,000 x g, 20 minutes, 4°C)
and the total protein concentration in each lysate was quantified using bicinchoninic acid.
Protein lysates were stored at -80°C until assayed. Normal tissue lysates used for expression
comparison to tumor included adrenal, artery, brain, breast, colon, esophagus, eye, heart, kidney,
liver, lung, ovary, pancreas, skin, spleen, stomach and trachea (Asterand). The standard curve
and quantification assay were conducted with the MesoScale Discovery (MSD) platform ELISA.
EFNA4 protein concentrations were determined by interpolating the values from a standard
protein concentration curve that was generated using purified recombinant EFNA4 protein. The
MSD assay was performed with two anti-EFNA4 mAbs, E91.4 (capture) and sulfo-tagged E47.3
(detection).
Plates were read on an MSD Sector Imager 2400 using an integrated software analysis program.
Values were divided by total protein concentration to yield nanograms of EFNA4 per milligram
of total lysate protein.
In vitro cytotoxicity assays
HEK293T-EFNA4 cells were generated by transfecting HEK293T cells with a lentiviral vector
driving expression of human EFNA4. Cells were cultured in DMEM (high glucose), Lglutamine, 10% fetal bovine serum (FBS) and penicillin/streptomycin. For the cytotoxicity
assay, HEK293T-EFNA4 cells were plated at 2000 cells per well in a 96-well plate. Following
adherence for 24 hours, the cells were treated with biotin-labeled mouse or humanized antibody
premixed with dStreptavidin-ZAP (Advanced Targeting Systems, #IT-27). These solutions were
then added to the cells at a 10x dilution and the cells were cultured for an additional 72 hours,
after which cell viability was assessed with CellTiter Glo (Promega, #G7572) on a Victor5 plate
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reader (Perkin Elmer). Results were reported in relative light units (RLU) normalized to a no
antibody control.
For ADC cytotoxicity, HEK293T-EFNA4 and HEK293T parental cells were plated into a
clear flat-bottom tissue culture plate (BD Falcon, #353072) at 500 cells per well in 180 µL of
culture media. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On the
following day PF-06647263 or control ADC were added at a 10-point dilution curve in triplicate.
The plate was incubated for 96 hours, and cell viability was then measured with MTS (Promega,
#G5430) according to the manufacturer’s instructions. The concentration that effected cell
viability by 50% (EC50) relative to untreated cells was calculated by logistic non-linear
regression using GraphPad Prism.
Antibody affinity measurements
The affinity of E22 mAb and the ADC PF-06647263 for human EFNA4 were determined by
surface plasmon resonance (SPR) using a BIAcore 2000 (GE Healthcare). An anti-human
antibody capture kit was used to immobilize capture antibodies on a CM5 biosensor chip. Prior
to each antigen injection cycle, humanized antibodies at a concentration of 2 µg/mL were
captured on the surface with a contact time of 2 min and a flow rate of 5 µL/min. The captured
antibody loading from baseline was constant at 80-120 response units. Following test article
capture and 1 min baseline, monomeric human EFNA4 was flowed over the surface at
concentrations of 50, 25, 12.5 nM for a 2 min association phase followed by a 2 min dissociation
phase at a flow rate of 5 µL/min. Following each cycle, the anti-human capture surface was
regenerated with 30 seconds contact time of 3M MgCl2 at 10 µL/min.
Biacore data was
processed by initially subtracting a control IgG surface from the specific antibody binding
surface. The response data was then truncated to the association and dissociation phase. The
resulting response curves with three different antigen concentrations were used to fit a 1:1
Langmuir binding model and to generate an apparent affinity by the calculated k on and koff
kinetics constants. All data analysis steps were completed in BiaEvaluation Software 3.1 (GE
Healthcare).
The binding affinities of EFNA4 to Eph receptors were also measured by BIAcore. An
anti-human antibody capture kit was used to immobilize recombinant Eph receptors on a CM5
biosensor chip. Prior to each antigen injection cycle, Eph receptors at the concentration of 2
5
μg/mL were captured on the surface with a contact time of 2 min and a flow rate of 5 µL/min.
The captured antibody loading from baseline was constant at 150-260 response units. Following
receptor capture and 1 min baseline, monomeric human EFNA4 was flowed over the surface at
concentrations of 400, 241, 145, 87, 53, 32, 19 and 12 nM for a 2 min association phase followed
by a 2 min dissociation phase at a flow rate of 10 µL/min. Following each cycle, the anti-human
capture surface was regenerated with 30 seconds contact time of 3M MgCl2 at 10 µL/min.
Biacore data was processed by initially calculating an average of the response at
equilibrium (Req). Req was then plotted versus concentration (M) and the steady state affinity
fit was used to calculate the affinity and theoretical Rmax. In all cases, at least 5 different
analyte concentrations were used and all data analysis steps were completed in BiaEvaluation
Software 3.1 (GE Healthcare).
Antibody bioconjugation
Purified hE22 mAb was incubated with AcBut-N-acetyl γ-calicheamicin dimethyl hydrazide
OSu at pH 8.3 with high agitation for 5 minutes. The ADC was purified on a Butyl Sepharose-4
Fast Flow HIC column (GE Healthcare) to achieve a narrow distribution of DAR, averaging ~
4.6. Characterization of the ADC was performed by hydrophobic interaction chromatography
(HIC; TSKgel Butyl-NPR, Tosoh Bioscience), size exclusion chromatography (SEC; Acquity
UPLC BEH200 SEC, Waters), reverse phase high-performance liquid chromatography (HPLCRP; Zorbax 300SB-CN, Agilent), and capillary isoelectric focusing (cIEF; iCE280,
ProteinSimple). The control ADC consisted of a non-binding mAb of the same isotype that was
conjugated in the same manner as hE22.
PF-06647263 and the control ADC exhibited
comparable DARs and loading distributions.
Immunofluorescence
HEK293T-EFNA4 and HEK293T parental cells were seeded on poly-D-lysine-coated glass
slides and cultured for 18 hours. Cells were then treated for 4 hours with 0.3 μg/mL PF06647263, control ADC or unconjugated hE22. Cells were washed, fixed with 4%
paraformaldehyde, permeablized with 1% Triton-X100, incubated for 1 hour with anti-histone γH2A.X (Millipore #05-636), washed and incubated for 30 minutes with AlexaFluor-4886
conjugated secondary antibody and DAPI, and then washed and protected with mounting
medium prior to cover-slipping. Cells were visualized with a Zeiss LSM510 confocal
microscope, and data presented is representative of three independent experiments.
Immunohistochemistry
Tumors were harvested at the indicated time-points after one administration of 1 mg/kg PF06647263, fixed in formalin and embedded in paraffin (FFPE). Sections were stained with 2.4
μg/mL anti-huIgG (Cell Signaling #3443-1) or 1.8 μg/mL anti-γ-H2A.X (Cell Signaling 2577S),
and slides were scanned on an Aperio AT2. For digital image analysis, the region of healthy
viable tissue was classified while necrotic regions and irrelevant tissues were excluded.
Individual cells within viable regions were scored based on user-defined parameters (i.e.
membrane hIgG staining or intracellular γ-H2A.X staining), and the percentage of markerpositive cells in the viable region was reported.
Gene expression and statistics
For whole transcriptome data, tumor cell subpopulations were isolated by FACS and
immediately pelleted and lysed in Qiagen RLTplus RNA lysis buffer (Qiagen, Inc.). The lysates
were then stored at -80°C until used. Upon thawing, total RNA was extracted using the Qiagen
RNeasy isolation kit (Qiagen, Inc.) following vendor’s instructions and quantified on the
Nanodrop (Thermo Scientific) and a Bioanalyzer 2100 (Agilent) again using the vendor’s
protocols and recommended instrument settings. Total RNA from normal kidney, pancreas,
ovary, melanocytes, lung, liver, heart, and colon was obtained from Clontech, Ambion and/or
Asterand was also obtained. The resulting total RNA preparation was suitable for genetic
sequencing and analysis. mRNA obtained was prepared for whole transcriptome sequencing
using an Applied Biosystems SOLiD 3.0 (Sequencing by Oligo Ligation/Detection) next
generation sequencing platform (Life Technologies), starting with 5 ng of total RNA per sample.
The data generated by the SOLiD platform mapped to 34,609 genes from the human genome,
was able to detect EFNA4, and provided verifiable measurements of EFNA4 levels in most
samples. Sequencing reads were mapped to regions defined by exon boundaries within
transcripts for each gene and the total number of reads was counted. Counts are then used as a
measure of relative abundance using the metric Reads Per Kilobase of exon per Million reads
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mapped (RPKM).3 EFNA4 RPKM expression in individual samples is plotted and the Geometric
Mean is indicated by the red horizontal bar. Statistics reflect group comparisons using a twotailed unpaired t-test.
For microarray data, 1-2 µg of whole tumor total RNA was derived as described above.
Samples were analyzed using the Agilent SurePrint GE Human 8x60 v2 microarray platform,
which contains 50,599 biological probes designed against 27,958 genes and 7,419 lncRNAs in
the human genome. Standard industry practices were used to normalize and transform the
intensity values to quantify gene expression for each sample. The normalized intensity of
EFNA4 expression in each sample is plotted and the Geometric Mean is indicated by the red
horizontal bar.
Statistics reflect group comparisons using a two-tailed unpaired t-test in
GraphPad Prism.
To assess gene expression and EFNA4 copy number variation in primary patient
specimens using publically available datasets, several large scale genomic datasets were
surveyed, including TCGA for breast (n = 400 specimens: 108 normal adjacent, 95 TNBC and
197 Other BRCA) and ovarian cancer4,5 and METABRIC for breast cancer6, and ACRG and
Samsung for hepatocellular carcinoma.7,8
Breast cancer subtype sample classification was
derived from the TCGA clinical sample annotation. Level 3 data from TCGA was used for copy
number analysis. Correlations were analyzed using GraphPad Prism.
For those figures with unpaired data, unpaired two-tailed t-tests were deemed most
appropriate, and the calculated variance of all two-tailed unpaired t-tests were determined to be
significant (p-value < 0.001). All data also appeared to have a normal distribution.
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