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Supplemental Materials and Methods
Analysis of TCGA and GTex RNA sequencing databases
The levels of PRLR gene expression were assessed in publicly available RNAseq databases. To assess the levels of PRLR expression in breast cancer and
matched normal breast tissues, the breast cancer dataset from The Cancer
Genome Atlas was used (TCGA Network, Nature 490, 61-70, 2012). Data from
the Genotype-Tissue Expression (GTEx) project was used for the assessment of
PRLR expression in a panel of normal tissues (Lonsdale et al, Nature Genetics
45, 580-85, 2013). Raw data from these databases were converted to FASTQ
format and then aligned to the human genome model B37.3 using the Array
Studio Omicsoft Sequence Aligner (Omicsoft Corporation, North Carlolina) (Hu et
al, Bioinformatics 28(14):1933-4, 2012). Normalized gene expression is
quantified in FPKM (Fragments Per Kilobase of transcript per Million mapped
reads) units using the RSEM algorithm (RNA-Seq by Expectation Maximization)
with UTR trimming (Li et al, BMC Bioinformatics 12:323, 2011).
Flow Cytometry
The relative expression of PRLR on the surface of cell lines was assessed
via flow cytometric analysis. Cells were incubated with REGN2878 as a primary
antibody followed by detection with an Allophycocyanin (APC)-labeled goat
F(ab’)2 anti-human IgG secondary antibody. Briefly, cells were washed in PBS
containing 1% fetal calf serum (PBS/FCS buffer). 1x106 cells per well were added
in 100 μL of PBS/FCS buffer to triplicate rows of a 96-well plate. Cells were
incubated with 10μg/mL primary staining reagent for 30 minutes at 4°C.
Following a washing step with PBS/FCS buffer, cells were incubated on ice with
10μg/mL of PE-labeled anti-human IgG antibody for 30 minutes. Cells were
washed thrice in PBS/FCS buffer prior to analysis on a flow cytometer (Accuri C6
Cytometer). Cells without any addition of antibody and cells incubated with the
secondary antibody alone were used as controls for non-specific staining.
For Quantitative Flow Cytometry, PRLR antibody was conjugated with
Alexa Fluor 647 (A647) according to standard protocols. A Quantum™ Alexa 647
MESF kit (Bangs Laboratories) was then used to determine the number of
surface receptors per cell according to the manufacturer’s instructions. First,
A647-conjugated standard beads with known Molecules of Equivalent Soluble
Fluorophore (MESF) were run on the cytometer to generate a standard curve of
MESF vs A647 mean fluorescence intensity (MFI). The A647-conjugated PRLR
antibody was then bound to Simply Cellular® standard beads with known
numbers of anti-human IgG (or known Antigen Binding Capacity, ABC) to
determine the MFI of the PRLR antibody at binding saturation. The MESF of
PRLR antibody MFI at saturated binding was then identified from the standard
curve and divided by the bead ABC number to give the Fluorescence to Protein
Ratio (F/P) for the A647-conjugated PRLR antibody. The A647-conjugated PRLR
antibody (0-100nM) was then bound to 1 x 105 cells to determine the PRLR
antibody MFI at saturated binding. The MESF of PRLR antibody MFI at saturated
binding was again identified from the standard curve and the number of surface
receptors per cell is given by dividing the MESF by the F/P ratio.
Cytotoxicity assays
Cells were seeded into triplicate rows of collagen-coated clear bottom 96well plates in a volume of 100 μL/well at 5-6x103 and incubated overnight at 37oC
and 5% CO2. Serial dilutions of REGN2878-DM1, REGN2878 control ADC were
generated and added to the cells. Cells were incubated for 72 hours at 37oC and
5% CO2. Untreated control wells were included to determine maximal viability or
background signal after lysis with digitonin. To measure cell numbers, cells were
fixed and stained for 20 minutes on ice with 4% formaldehyde containing 3 μg/mL
Hoechst DNA stain. Following a final wash, Hoechst-labeled nuclei were imaged
on the ImageXpress Micro XL System and counted using the Columbus Image
Data Storage and Analysis System. Cell viability was calculated based on the
assay-specific signal (Hoechst DNA stain-positive nuclei count or luminescence)
as follows:
experimental signal – background signal (digitonin lysis)
% viability = maximum signal (untreated) – background signal (digitonin
x100
lysis)
Data were analyzed using a four-parameter logistic equation over a 10-point
response curve (direct cell count-based assay) or over an 11-point response
curve (metabolic activity-based assay) using GraphPad Prism software and IC50
values, defined as the concentration of test article required to achieve 50%
reduction in cell viability, were calculated. Cytotoxicity was defined as the inverse
of viability. The percentage of cytotoxicity was calculated as follows: %
cytotoxicity = 100 – % cell viability.
Generation of T47Dv11 variant cells
Initially, 10x106 parental T47D cells were implanted subcutaneously (SC)
into the left flank of female C.B.-17 SCID mice (Taconic, Hudson NY)
supplemented with a 90-day release 1.7 mg estrogen pellet (Innovative Research
America). T47D tumors of 500 mm3 were excised, cut into 3 mm fragments and
subsequently implanted into the left flank of separate female C.B.-17 SCID mice
via trocar. Mice were monitored until a large (~2000 mm 3) tumor was observed
that was then disaggregated into a single cell suspension. The cells from this
tumor (named T47D variant from mouse 11, or T47Dv11) were then cultured in
vitro and expanded. Flow cytometry confirmed that T47Dv11 cells retained PRLR
expression at similar levels to the parental cells.
Pharmacokinetic analysis of REGN2879-DM1 in T47DvII tumor bearing
mice.
Serum levels of Total Antibody (REGN2878-DM1 and unconjugated
REGN2878) and Total Conjugated Drug (Only REGN2878-DM1) in T47Dv11
tumor bearing mice were measured by ELISA. Mice bearing T47DvII xenografts
(~180 mm3) received REGN2878-DM1 at 1, 2.5, 5 and 15 mg/kg. Blood was then
collected at 3 hours and 3, 7, 14, 21 and 28 days post administration. Serum was
collected at each time-point. Six dilutions per sample ranging from 1:100-
1:2,430,000 were used with fresh REGN2878-DM1 used as a standard. Total
Antibody levels were measured by binding to captured mouse anti-human IgG Fc
antibody and Total Conjugated Drug levels were measured by binding to
captured anti-DM1 antibodies. Bound Total Antibody or Total Conjugated Drug
were then detected using mouse anti-human IgG Fc HRP conjugated antibody.
ELISAs were developed using chemiluminescent substrate.