Download Alewine et al., MCT-14-0132 Efficacy of RG7787, a Next Generation

Alewine et al., MCT-14-0132
Efficacy of RG7787, a Next Generation Mesothelin-targeted Immunotoxin, Against Triplenegative Breast and Gastric Cancers
Christine Alewine, Laiman Xiang, Takao Yamori, Gerhard Niederfellner, Klaus Bosslet, and
Ira Pastan
SUPPLEMENTARY MATERIAL
Alewine et al., MCT-14-0132
Supplementary Methods
Analysis of TCGA Data Set for MSLN expression. We used data from The Cancer Genome Atlas
(TCGA) breast cancer cohort and sorted patients according to their ER, PR and HER2 status as
annotated in the clinical covariates that come with the patient data. MSLN expression levels
(log2 RPKM) are based on the RNASeq data of the associated tumor biopsy from the patients.
All data was downloaded from the publicly available TCGA data portal and represents original
expression values and pathology assessments provided by TCGA.
Alewine et al., MCT-14-0132
Supplementary Figure Legends
Supplementary Figure 1. Mesothelin is expressed in TNBC specimens in the TCGA data set.
Patients in the TCGA data set were grouped according to their ER, PR and HER2 status (based
on clinical annotation available in the data set). MSLN expression is shown on the y-Axis as
log2 RPKM values. For most groups (e.g. ER/PR positive but HER2 negative patients in the
lower right corner), MSLN expression in the majority of samples is below an RPKM of 1
(log2=0) and therefore relatively low. In contrast, high MSLN expression is evident in a
considerable number of triple negative breast cancer patients.
Supplementary Figure 2. Immunotoxin action is target-specific. LMB11 is an immunotoxin
that uses the same PE24 fragment as RG7787 fused to an anti-CD22 Fab fragment. CD22 is a B
cell differentiation antigen that is not expressed by epithelial malignancies. HCC70 (A) or
MKN28 (B) cells were treated with the indicated concentrations of RG7787 or LMB11, then
assessed 72 hours later for cell viability by WST-8 assay. Viability for vehicle-treated cells was
normalized to 1.0, and that for cycloheximide (a positive control which reliably produces nearly
100% cell kill) was normalized to 0. Each data point represents measurements of six treated
wells. (C) Athymic nude mice were xenografted with MKN28 cells then treated with vehicle,
LMB11 (2.5 mg/kg) or SS1P (0.4 mg/kg, maximum safe dose in mice) at the time points
indicated by the orange arrows.
Supplementary Figure 3. Combination treatment with paclitaxel and RG7787 prolongs tumors
response. Athymic nude mice bearing MKN28 gastric cancer tumors were treated with
paclitaxel (50 mg/kg) on days indicated by the long navy arrows and with vehicle or RG7787
(2.5 mg/kg) on time points indicated by orange arrows. Tumor volume tracings for each
individual mouse in the experiment are shown.
Supplementary Figure 4. Paclitaxel and RG7787 have no synergy in vitro. HCC70 cells were
treated with the indicated concentrations of paclitaxel followed 12 hours later by RG7787 and
assessed for cell viability by WST-8 assay 72 hours post paclitaxel administration. Viability for
vehicle-treated cells was normalized to 1.0, and that for cycloheximide (a positive control which
reliably produces nearly 100% cell kill) was normalized to 0. Each data point represents
measurements of six treated wells.