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Supplementary figures and methods
Supplementary Figure S1. LY6E is overexpressed in a subset of breast
cancers (A) Relative expression of LY6E gene expression in two different
breast cancer subtypes. (B) Metabric analysis of LY6E expression in
molecularly defined breast cancer subclasses. Table shows fold change and
statistical significance for LY6E expression in basal-like cancers versus other
breast cancer subtypes. P-values were calculated by t-test on log2transformed probe intensities, then adjusted with the Benjamin-Hochberg
method.
Supplementary Figure S2. LY6E transcript expression in normal human
tissues is shown. LY6E expression detected from normal tissue RNA using
qPCR analysis (Origene array) is represented as fold change over the
expression of GAPDH and compared to LY6E transcript expression in the
xenograft efficacy models depicted in Fig. 6 and in Supplementary Fig. S4.
Supplementary Figure S3. Endocytosis of anti-LY6E antibody 9B12. (A) Live
ovarian cancer cells (KURAMOCHI) were pre-incubated with either no
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inhibitors (none), lipid raft disruptor, 1 mM Methyl-β- Cyclodextrin (MBC)
or inhibitor of clathrin mediated endocytosis, 25 μM Chlorpromazine (CPZ);
followed by a 30 minute incubation with 5 μg/mL of anti-LY6E antibody
9B12 at 370C. Cells were then fixed, permeabilized and co-stained for LY6E
(9B12 antibody) in conjunction with lysosomal marker LAMP1. Detection
reagents, Alexa 488- conjugate (green) and Cy3- conjugate (red) were used
to detect anti-LAMP1 and 9B12 antibodies respectively. Colored images
overlapping LY6E and LAMP1 staining are presented in the middle panel
(Merge). White scale line indicates 20 M. (B) Clathrin mediated
Endocytosis of Transferrin. Live KURAMOCHI ovarian cancer cells were preincubated with either vehicle control (none), Methyl-β- Cyclodextrin
(MBC) or chlorpromazine (CPZ) and then incubated with transferrin directly
conjugated to Alexa 555 (red). Cells were then fixed, permeabilized and costained for lysosomal marker LAMP1; which was detected with Alexa 488–
labeled secondary antibody (green). Colored images with overlapping
Transferrin and LAMP1 staining are presented in the middle panel (Merge).
White scale line indicates 20 M.
Supplementary Figure S4. LY6E ADC activity in MAXF-1162, a HER2+/ T2
DM1 resistant breast cancer model. (A) Photomicrograph illustrating
heterogeneous 1+/2+ LY6E expression in MAXF-1162 xenografts. Bar
denotes scale (100m). T-DM1 (B) and anti-LY6E ADC (C) efficacy against
MAXF 1162, a HER2- positive patient derived breast tumor model. Mice
were administered a single IV injection of either vehicle, T-DM1, anti-LY6E
antibody 9B12, either as unconjugated or as ADC (LY6E), control ADC (Ctrl)
at mg/kg doses indicated in parentheses. Average tumor volumes with
standard deviations were determined from 8-10 animals per group and
depicted on the Y-axis whereas day of study was depicted on the X-axis.
Supplementary Table S1. Table lists the relative LY6E protein expression
detected by IHC in various normal tissues using the GEN-93-8-1 antibody
Expression scores signify no staining (0) or weak (1+), moderate (2+), and
strong (3+) LY6E expression. Definitions are described further in the
Methods section.
Supplementary Table S2. Anti-LY6E antibody 9B12 affinity to human and
cynomolgus monkey LY6E. Affinity of 9B12 was determined by Scatchard
analysis on exogenous LY6E expressed in transiently transfected HEK-293
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cells.
Supplementary Table S3. The correlation of LY6E copy number to antiLY6E ADC killing in a panel of ovarian cancer cell lines is shown. Sensitivity
of the cell lines to free MMAE drug is also depicted.
Supplementary Table S4. LY6E and GAPDH specific primer and probe sets
used for transcript analysis and LY6E specific siRNAs are listed.
Supplementary Table S5. Detailed percent tumor growth inhibition data for
xenograft efficacy studies presented in Fig. 6 and in Supplementary Fig. S4B.
AUC is defined as Area under the fitted tumor volume curve as calculated
using a linear mixed effects model (see Methods). AUC/Day %TGI (lower,
upper) is the percentage AUC/Day (on the original mm3 volume scale)
reduction compared to control based on the fitted curves, with upper and
lower limits (of the 95% uncertainty interval) for only those days where all
treatment groups still have some animals present. This means the control
group must always be 0 and that the TGI calculation is based on data up to
the lowest day in the Last Day column. Groups that reduce growth or cause
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shrinkage will have a positive value. Any groups for which the tumors
increased faster than the control will have negative values. The formula for
AUC/Day %TGI is the following: AUC/Day %TGI = [1-(AUC/Day Treatment ÷
AUC/Day Control)] X 100. TTP 2X is defined as time (in days) to progression
to twice the average tumor volume value at time 0. TTP 5X is defined as
time (in days) to progression to five times the average tumor volume value
at time 0. PR, CR are defined as partial and complete response, where a PR
is defined as at least 50% shrinkage from Day 0 in tumor volume and CR is
shrinkage to 0 (on the original scale) at any point during the study. Values
are calculated from the raw, not fitted data. “NA” indicates that the tumor
did not progress.
Supplementary Table S6. Statistical analyses of differences between tumor
growth in test groups versus vehicle control group in xenograft studies
presented in Fig 6 and Supplementary Fig. 4B, as calculated by GraphPad
Prism software v6.0e.
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Supplementary protocols:
Affinity binding assays (Scatchard analysis): HEK-293 cells were seeded in
150-cm dishes (BD; Franklin Lakes, NJ) at 15  106 cells/dish. The next day,
cells were transfected with plasmid DNA encoding human or cynomolgus
monkey LY6E, using Lipofectamine 2000 (Invitrogen) as described by the
manufacturer. At approximately 48 hours post-transfection, cells were
harvested for use in binding assays to determine affinity of 9B12 antibody
to LY6E.
Anti-LY6E antibody 9B12 was iodinated using the Iodogen method. The
radiolabeled 9B12 antibody was purified from free 125I-Na by gel filtration
using a NAP-5 column and had a specific activity of 29.3 Ci/g.
Competition reaction mixtures of 50 L containing a fixed concentration of
iodinated antibody and decreasing concentrations of unlabeled antibody
were placed into 96-well plates. HEK-293 cells transiently expressing
recombinant human or cynomolgus monkey LY6E were detached from
plates using Sigma Cell Dissociation Solution and were washed with binding
buffer (DMEM with 2% FBS, 50 mM HEPES, pH 7.2, and 0.1% sodium azide).
The washed cells were added at an approximate density of 150,000 cells in
0.2 mL of binding buffer to the 96-well plates containing the 50-L
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competition reaction mixtures. The final concentration of the iodinated
antibody in each competition reaction with cells was 50 pico Moles and the
final concentration of the unlabeled antibody in the competition reaction
with cells varied, starting at 1000 nM and then decreasing by 1:2-fold
dilution for ten concentrations, and included a zero-added, buffer-only
sample. Competition reactions with cells for each concentration of
unlabeled antibody were assayed in triplicate. Competition reactions with
cells were incubated for 2 hours at room temperature. After the 2-hour
incubation, the competition reactions were transferred to a Millipore
Multiscreen filter plate and washed four times with binding buffer to
separate the free from bound iodinated antibody. The filters were counted
on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical
Sciences; Wellesley, MA). The binding data were evaluated using New
Ligand software (Genentech), which uses the fitting algorithm of to
determine the binding affinity of the antibody (1).
1.
Munson PJ, Rodbard D. Ligand: a versatile computerized approach for
characterization of ligand-binding systems. Analytical biochemistry 1980; 107:22039.
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