Schematic figure of FGFR2 mutations identified in endometrioid endometrial tumors.

<p>Blue diamonds indicate each instance of a mutation in the Washington University School of Medicine cohort. Mutations are numbered relative to <i>FGFR2</i>b (NP_075259.2). Mutations at 6 codons (S252, P253, Y376, C383, N550, K660) comprise >90% of all mutations identified.</p

Potential utility of <i>FGFR2</i> mutation status as an adverse prognostic factor to affect clinical decision-making.

<p>The decision tree is adapted from 2011 National Comprehensive Cancer Network guidelines using FIGO 2009 staging. BT = brachytherapy; RT = radiation therapy.</p

Patient Demographics and Clinicopathologic Characteristics.

<p>Patient Demographics and Clinicopathologic Characteristics.</p

Hazard ratio (HR) and 95% confidence interval (CI) for cohort of 386 Stage I/II cases.

<p>*For DFS, the multivariate model included Stage 1C, II, Grade 2 and 3.</p><p>**For OS, the multivariate model included age, FIGO Stage 1C, II, Grade 2 and Grade 3.</p>a<p>FGFR2 adjusted for KRAS in addition to covariates above.</p>b<p>KRAS adjusted for FGFR2 in addition to covariates above.</p

CHO-CAR cells expressing membrane-anchored TR3 variants induce cell-cell contact-dependent target cell death.

<p><b>(A)</b> CHO-CAR cells were infected with the indicated Ad5-based viruses using MOIs that were determined to achieve equivalent transduction efficiencies during prior titration experiments. Infection of the cells with Ad5-eYFP (MOI 1000), Ad5-TR3GPIeYFP (MOI 5000) and Ad5-TR3DAFeYFP (MOI 8750) did indeed result in comparable transduction efficacies gauged on the proportion of YFP-positive cells using flow cytometry. <b>(B)</b> Western Blot analysis of infected CHO-CAR cell lysates confirmed the molecular weights of TR3GPI (~65 kDa) and TR3DAF (~130 kDa). <b>(C)</b> Functional TR3 activity was assessed employing a co-culture experiment with Jurkat suspension cells. Two hours later, and compared to the control (GFP), large numbers of apoptotic bodies were noted only on cells expressing the membrane-anchored TR3 variants (asterisks), indicative of widespread apoptosis induction. <b>(D)</b> Graphic representation of the overlay assay shown in (C), with specific cell death of ~64% mediated by expression of TR3GPIeYFP and ~74% by TR3DAFeYFP on the CHO-CAR effector cells determined by FACS analysis. <b>(E)</b> Blocking experiment using anti-TRAIL antibody to demonstrate mechanism of cell-cell contact-dependent mechanism of cell death. The contact-dependent killing capacity of TR3GPI-expresssing CHO-CARs (Ad5-TR3GPIeYFP) co-cultured with Jurkat suspension cells was dose-dependent and was completely abolished at the highest concentration of TRAIL-blocking antibody (> 600 ng).</p

Exploring human MSCs to serve as cellular carriers for TR3-based cancer therapy.

<p><b>(A)</b> In a first characterization step, we aimed at verifying a set of surface markers present on undifferentiated human adipose-derived mesenchymal stem cells by flow cytometry. MSCs from an individual donor were stained with monoclonal antibodies directed against CD90, CD 166, CD105, CD49d and CD73. Based on the respective, homogeneous biomarker expression profiles, we could confirm that all of our generated stable cell lines were indeed MSCs. <b>(B)</b> A panel of Ad5 vectors was evaluated for gene transfer efficiency on human MSCs. Among the variants tested were: Ad5-GFP (wild-type), which expressed the marker luciferase and green-fluorescence protein; Ad5/PK4GFP, an Ad5-based vector containing a pK4 motif at the C-terminus; Ad5/3, a variant featuring a chimeric fiber protein knob domain derived from Ad3; Ad5RGD, an Ad5 vector containing a heterologous RGD motif in the HI loop of the fiber knob; Ad5pk7-RGD, a double-modified Ad5 vector containing arginine-glycine-aspartate (RGD) motif in the HI loop and a pK7 motif at the C-terminus; and Ad5pK7 is an Ad5 vector containing a pK7 motif at the C-terminus (courtesy Contreras et al 2003). This screening effort resulted in the identification of Ad5pK7 giving rise to the highest transduction efficacy among all other tested Ad5 variants using a representative and randomly chosen MSC line. <b>(C)</b> In order to demonstrate broader applicability of the pK7 fiber knob as a more universal delivery vehicle to transduce human MSCs, we tested additional adipose-derived MSC lines. Compared to an Ad5 wild-type control, Ad5pK7 was capable of infecting a broad range of MSC lines with nearly 100% transduction efficiency. <b>(D)</b> A dose-escalation curve confirms the enhanced infectivity rate of Ad5pK7 compared to control Ad5.</p

Hazard Ratio (HR) and 95% Confidence Interval (CI) for Cohort of 466 Endometrioid Endometrial Cancers.

<p>*For DFS, the multivariate model included Stage 1C, II, III/IV, grade 2 and 3.</p><p>**For OS, the multivariate model included age, FIGO stage 1C, II, III/IV, grade 2 and grade 3.</p>a<p>FGFR2 adjusted for KRAS in addition to covariates above.</p>b<p>KRAS adjusted for FGFR2 in addition to covariates above.</p

Adenovirus transduction of CHO-CAR cells to produce soluble, biologically active TR3 variants.

<p><b>(A)</b> In order to confirm efficient virus production, CHO-CAR cells were infected with increasing multiplicities of infections. The eYFP signal was used as an indicator of expression level of the respective biologics. All viruses transduced the reporter cell in a dose-dependent fashion. <b>(B)</b> Western Blot analysis of supernatant from CHO-CAR-infected cells (MOI 10000) confirmed production and molecular weights of TR3 and Meso64TR3 at ~61 kDa and ~65 kDa, respectively. <b>(C)</b> In order to produce secreted TR3 drugs for functional activity testing, plateau-reaching MOIs were used for the respective virus preparation. These were subsequently confirmed by flow cytometry to ensure that infection rates resulted in equivalent production of the respective biologics (Ad5-eYFP, MOI 5000 [control]; Ad5-TR3eYFP, MOI 10000; Ad5-Meso64TR3eYFP, MOI 3000). <b>(D)</b> Cell killing profiles of TR3 and Meso64TR3 at increasing drug volumes were established on MUC16-deficient T cell leukemia cell line Jurkat. Supernatant of cells infected with Ad5-eYFP was used as a control. Please note that both drugs induced a dose-dependent cell death overlapping response curves, consistent with their known activity profiles. ***, P < 0.0002. <b>(E)</b> The same cell death determination as in (D) using identical drug volumes was performed in MUC16-positive OVCAR3 cells. Please note the much enhanced activity profile of the MUC16-targeted cancer drug Meso64TR3 relative to parental TR3. NS, not significant; ***, P < 0.0002. <b>(F)</b> In order to confirm extrinsic pathway involvement as the cause cell death induction, Jurkat cells were treated with a constant amount of TR3 and Meso64TR3 in the presence of Z-VAD-FMK, a pan-caspase inhibitor. Z-VAD-FMK completely eliminated the killing capacity of both biologics. ***, P < 0.0002.</p

Ad5pK7 adenovirus can be “armed” with secreted forms of TR3-based cancer therapeutics.

<p><b>(A)</b> Adipose-derived MSCs from patient #15 were infected with Ad5pK7 carrying TR3eYFP and Meso64TR3eYFP genomes using an MOI of 5000. Representative images were taken and show expression of eYFP fluorescence (original magnification 10x). <b>(B)</b> In order to produce secreted TR3 drugs for functional activity testing, plateau-reaching MOIs were used for the respective virus preparation (MSC #20, MOI 2500). These were subsequently confirmed by flow cytometry to ensure that infection rates resulted in equivalent production of the respective biologics. <b>(C)</b> Cell killing profiles of TR3 and Meso64TR3 at increasing drug volumes were established on MUC16-deficient T cell leukemia cell line Jurkat. Supernatant of cells infected with Ad5-eYFP was used as a control. Please note that both drugs induced a dose-dependent cell death overlapping response curves, consistent with their known activity profiles. NS, not significant. <b>(D)</b> The same cell death determination as in (C) using identical drug volumes was performed in MUC16-positive OVCAR3 cells. Please note the much enhanced activity profile of the MUC16-targeted cancer drug Meso64TR3 relative to parental TR3. NS, not significant; *, P < 0.04.</p

Schematic representation of native TRAIL and recombinant TR3 variants used in this study.

<p><b>(A)</b> Illustration of the key structural features of wild-type, native TRAIL and a recombinant TR3 form anchored to the membrane via carboxyl-terminal incorporation of a transmembrane domain (TR3-TM). Please note the vastly different domain architecture/stoichiometry of the trimers between TRAIL (1:1) and TR3-TM (1:3). <b>(B)</b> Bicistronic adenoviral vector design in which TR3 expression correlates with the expression level of yellow fluorescent protein (eYFP) via Internal Ribosome Entry Site (IRES)-mediated cotranslation of the mRNA. <b>(C)</b> Schematic representation of two membrane-anchored TR3 variants are depicted: TR3GPI, in which the GPI-encoding sequence of human DAF servers as anchors signal and TR3DAF, in which the entire mature form of human DAF serves as an anchor unit. The short consensus repeats (SCRs) of DAF are indicated (1–4) and serve as a molecular spacer relative to the plasma membrane. <b>(D)</b> Secreted variants TR3 (parental) and Meso64TR3 are shown, the latter representing a MUC16-targeted TR3 trimer, in which only the 64 amino-terminal amino acids of mesothelin were used as delivery moiety to the MUC16 biomarker (including an N-terminal FLAG tag, not shown).</p