Fiore Cattaruzza

WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death

WNT antagonism displays marked synergy with taxane chemotherapy and reverses taxane-induced enrichment of cancer stem cells. The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/β-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.

Abstract 2576: Enhanced antitumor efficacy by sequential application of Wnt pathway antagonists in combination with taxanes

The Wnt/beta-catenin pathway, which signals through the Frizzled (FZD) receptor family and several co-receptors, has long been implicated in cancer. We have previously demonstrated that inhibition of Wnt/beta-catenin signaling by vantictumab (anti-Fzd7, OMP-18R5) or ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth, decreases tumorigenicity and induces differentiation in solid tumors. The anti-tumor effect of our Wnt antagonists is most evident in combination with chemotherapeutic agents. We sought to determine if the anti-tumor effect of Wnt pathway inhibitors varied with different chemotherapeutic agents. We compared the growth inhibitory effect of vantictumab and ipafricept with either taxanes (paclitaxel and nab-paclitaxel) or with DNA synthesis inhibitors (gemcitabine and carboplatin) in patient-derived tumor xenografts. We observed enhanced anti-tumor activity when combining vantictumab or ipafricept with nab-paclitaxel or paclitaxel compared to the combination with gemcitabine or carboplatin in pancreatic ductal carcinoma and serous ovarian cancer xenograft models. Histologic analysis in a pancreatic ductal carcinoma indicated that nab-paclitaxel increased mitotic cells and beta-catenin levels. Importantly, the addition of vantictumab to nab-paclitaxel reversed the nab-paclitaxel-induced increase in mitotic cells and beta-catenin expression. A potential mechanism to account for these results involves the observation that Wnt/beta-catenin signaling is under cell cycle control and peaks at the G2/M phase. Taxanes inhibit microtubule function and block the cell cycle at G2/M. In contrast, other chemotherapeutic agents, such as platinum compounds and nucleoside analogs, inhibit DNA synthesis and block cell proliferation at S phase. Our findings suggest that combination of Wnt blockade with chemotherapeutic agents, such as taxanes, that induce G2/M arrest may resulted in enhanced anti-tumor activity. The optimal synergy of anti-Wnt plus taxane combination occurs when the antibody was applied prior to taxane. Further analyses in serous ovarian tumors reveal that pre-treatment with ipafricept resulted in dysregulated beta-cetenin localization within giant multi-nucleated cells and up-regulation of genes associated with negative regulators of G1 progression. Our work provides evidence for the enhanced anti-tumor effect of Wnt pathway inhibitors in combination with taxanes and highlights the importance of preclinical examination to identify the most efficacious combination therapy regimens and the timing of antibody action for Wnt antagonists in combination with taxanes for optimal treatment efficacy. Citation Format: Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Fiore Cattaruzza, Tracy Tang, Pete Yeung, John Lewicki, Austin Gurney, Timothy Hoey. Enhanced antitumor efficacy by sequential application of Wnt pathway antagonists in combination with taxanes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2576. doi:10.1158/1538-7445.AM2015-2576

Abstract 4233: Wnt pathway antagonist ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth and reduces tumor-initiating cell frequency in ovarian patient-derived xenograft models

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. The Wnt/beta-catenin pathway, which signals through the Frizzled (FZD) receptor family and several co-receptors, has long been implicated in cancer. We have developed ipafricept (FZD8-Fc, OMP-54F28), a recombinant fusion protein consisting of the ligand-binding domain of FZD8 and a human IgG1 Fc fragment. This fusion protein blocks Wnt signaling induced by multiple Wnt family members by binding and sequestering WNT. Using minimally passaged ovarian patient-derived xenograft tumors (PDX), we demonstrate that ipafricept is efficacious in combination with chemotherapy in four of eight ovarian cancer PDX tumors examined. Utilizing an in vivo serial transplantation assay, we quantified a reduction of the tumor initiating cell frequency by ipafricept in combination with paclitaxel. Additionally, we have discovered that pre-treatment with ipafricept several days prior to paclitaxel therapy enhances the activity of both agents when compared to delivering the drugs simultaneously. The anti-tumor effect observed is directly associated with a modulation of Wnt pathway gene sets. In responsive tumors, we discovered that a large number of WNT target genes were significantly down-regulated by ipafricept (e.g, AXIN2, LRP5/6, and FZD8). Conversely, in non-responsive tumors, these genes were either unchanged or up-regulated by the combination therapy. Histologic analysis revealed that total beta-catenin protein levels were reduced by ipafricept alone and in combination with paclitaxel in responsive tumors but were unchanged in non-responsive tumors. We are using these tumors to develop biomarkers that can be used clinically. Our data demonstrates the potential therapeutic benefit of targeting Wnt signaling in ovarian cancer. A Phase 1b clinical trial is currently examining ipafricept in combination with paclitaxel and carboplatin in patients with recurrent platinum-sensitive ovarian cancer. Citation Format: Marcus M. Fischer, Wan-Ching Yen, Chun Zheng, Randall Henner, Fiore Cattaruzza, Tracy Tang, Pete Yeung, Tanuka Biswas, John Lewicki, Austin Gurney, Ann M. Kapoun, Timothy Hoey. Wnt pathway antagonist ipafricept (FZD8-Fc, OMP-54F28) inhibits tumor growth and reduces tumor-initiating cell frequency in ovarian patient-derived xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4233. doi:10.1158/1538-7445.AM2015-4233

RSPO3 antagonism inhibits growth and tumorigenicity in colorectal tumors harboring common Wnt pathway mutations

Activating mutations in the Wnt pathway are a characteristic feature of colorectal cancer (CRC). The R-spondin (RSPO) family is a group of secreted proteins that enhance Wnt signaling and RSPO2 and RSPO3 gene fusions have been reported in CRC. We have previously shown that Wnt pathway blockers exhibit potent combinatorial activity with taxanes to inhibit tumor growth. Here we show that RSPO3 antagonism synergizes with paclitaxel based chemotherapies in patient-derived xenograft models (PDX) with RSPO3 fusions and in tumors with common CRC mutations such as APC, β-catenin, or RNF43. In these latter types of tumors that represent over 90% of CRC, RSPO3 is produced by stromal cells in the tumor microenvironment and the activating mutations appear to sensitize the tumors to Wnt-Rspo synergy. The combination of RSPO3 inhibition and taxane treatment provides an approach to effectively target oncogenic WNT signaling in a significant number of patients with colorectal and other intestinal cancers.

Abstract 3129: Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts

Background: The WNT/ β-catenin signaling pathway has been shown to play a key role in both normal development and tumorigenesis (Polakis, 2007; MacDonald et al., 2009). We have developed a monoclonal antibody, vantictumab, that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, including pancreas, breast, colon and lung. Furthermore, our studies showed that vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012). Material and methods: We set out to identify a predictive biomarker for the response to vantictumab in pancreatic cancer patients by analyzing mRNA-seq gene expression data from 14 patient-derived xenograft (PDX) models. These 14 minimally passaged pancreatic xenograft tumors were tested in vivo and their responses to vantictumab, in combination with the current SOC gemcitabine and nab-paclitaxel were established. Samples from these experiments were collected for Pharmacodynamic (PD) biomarker analysis. We utilized a two-sample Welch9s t-test to identify genes that can distinguish between responders and non-responders and the K-nearest neighbor (KNN, Altman 1992) algorithm for classification. A leave-one-out cross-validation was used to measure area under the ROC curve (Fawcett et al., 2006, AUC), accuracy (ACC), positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the model. Results: PD biomarker analysis confirmed inhibition of genes in Wnt and stem cell pathways by vantictumab in combination with gemcitabine as well as gemcitabine plus nab-paclitaxel. The selected 3-gene signature comprising TGFB3, IGF2 and SMO achieved the best performance (AUC = 0.875, ACC = 0.93, PPV = 0.91, NPV = 1, sensitivity = 1, specificity = 0.75) in the 14 PDX pancreatic tumor models. In addition, a strong correlation between the gene signature biomarker and the ratio of tumor inhibition (RTI) in the pancreatic xenograft experiments was observed. The identified 3-gene biomarker was used to predict the response to vantictumab in combination with gemcitabine and nab-paclitaxel in three additional pancreatic PDX tumor models. The efficacy in the three models was successfully predicted by the biomarker. Conclusions: The 3-gene biomarker is being evaluated in a Phase 1b study of vantictumab in combination with gemcitabine and nab-paclitaxel in previously untreated stage IV pancreatic cancer (NCT02005315). Citation Format: CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Claire Guo, Alayne Brunner, Min Wang, Belinda Cancilla, Austin Gurney, Rainer Brachmann, John Lewicki, Tim Hoey, Ann M. Kapoun. Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3129.

Abstract 4652: Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors

Background: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers, the leading cause of cancer-related deaths. Notch signaling has been shown to play an important role in lung cancer initiation and progression. Delta-like ligand 4 (DLL4) activates the Notch pathway and is important for cancer stem cell (CSC) survival. Demcizumab (OMP-21M18) is a humanized IgG2 anti-DLL4 antibody currently being tested in a Phase 2 trial in combination with pemetrexed and carboplatin for first-line treatment of patients with NSCLC. Previously, OMP-21M18 in combination with its mouse anti-DLL4 surrogate has been shown to inhibit tumor growth, decrease cancer stem cell frequency, and cause dysfunctional sprouting of new vessels resulting in an anti-angiogenic effect in patient-derived tumor xenograft (PDX) models in breast, colon, ovarian, and pancreatic cancers. Here we show results from NSCLC PDX models. Methods and Results: Anti-DLL4 treatment was tested in a series of NSCLC PDX models. Because DLL4 inhibition has been shown to have effects on the tumor as well as the vasculature, the combination of OMP-21M18 (targeting human DLL4) and 21R30 (antibody targeting mouse DLL4) treatment in the PDX models was used to model demcizumab treatment in humans. Treatment with anti-DLL4 in combination with chemotherapy inhibited tumor growth in a series of NSCLC PDX models. Additionally, a tumorigenicity assay showed a decrease in the frequency of tumor-initiating cells following treatment with anti-DLL4 and chemotherapy. Gene expression analysis of tumor samples provided insights into the mechanism of action. Conclusions: Anti-DLL4 treatment in a panel of NSCLC PDX tumor models in vivo showed inhibition of tumor growth and a decrease in the frequency of tumor-initiating cells. Mechanism of action and gene expression analysis of these models treated with anti-DLL4 will be presented. These findings provide additional evidence supporting demcizumab as an effective treatment for NSCLC patients. Citation Format: Alayne Brunner, Fiore Cattaruzza, Wan-Ching Yen, Pete Yeung, Marcus Fischer, Belinda Cancilla, Gilbert O’Young, Raymond Tam, Yu-Wang Liu, Austin Gurney, John Lewicki, Tim Hoey, Min Wang, Ann M. Kapoun. Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4652.

Abstract A30: Predictive and pharmacodynamic biomarkers of vantictumab (OMP-18R5; anti-Frizzled) in non-small cell lung cancer

Background: Vantictumab is a monoclonal antibody that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, reduces tumor-initiating cell frequency (TIC) and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012). To target responsive patients and understand the mechanism of action of the drug, we set out to identify predictive and pharmacodynamic (PD) biomarkers of vantictumab in non-small cell lung cancer (NSCLC). Materials and methods: The response to vanticutmab was established from in vivo efficacy experiments including different treatment groups: control, vantictumab, paclitaxel and vantictumab in combination with paclitaxel. For combination treatment, same day dosing and sequential dosing (paclitaxel dosed 2 days after the antibody) were compared. Samples were collected for PD biomarker analysis. To identify a predictive biomarker for the response to vantictumab in NSCLC patients, gene expression data from 7 NSCLC patient derived xenograft (PDX) models was analyzed. We utilized support vector machine-recursive feature elimination (SVM-RFE, Guyon et al., 2002) to select genes and support vector machine (SVM) for classification. Results: Vantictumab showed significant tumor growth inhibition as a single agent as well as in combination with paclitaxel. The reduction of TIC and the antitumor efficacy of vantictumab were significantly enhanced with sequential dosing compared with same day dosing. These findings suggested that optimal synergy occurs using sequential dosing, likely due to enhanced blockade of cell cycle progression at mitosis. PD biomarker analysis confirmed inhibition of genes in Wnt, Notch, and stem cell pathways by vantictumab both as a single agent and also in combination with paclitaxel. Wnt pathway targets including AXIN2 and LEF1 were down-regulated significantly by vantictumab in both sequential dosing and same day dosing confirming the mechanism of action. From a series of 7 in vivo efficacy PDX experiments, LEF1 was identified as a predictive biomarker of vantictumab response and achieved the best performance with cross-validated positive predictive value (PPV) = negative predictive value (NPV) = sensitivity = specificity = 100%. Strong correlation was also observed between LEF1 gene expression and the ratio of tumor volume. Furthermore, LEF1 was able to successfully predict the response to vantictumab in 2 independent NSCLC PDX models. Prevalence estimation for LEF1 ranged from 35% to 50% based on public microarray datasets. LEF1 was also found to be significantly correlated with the response to vantictumab in combination with paclitaxel in 12 NSCLC PDX models (p = 0.0162), indicating LEF1 as a potential predictive biomarker of the response vantictumab as a single agent and in combination with SOC in NSCLC. Conclusions: A biomarker study for the pharmacodynamics and response to vantictumab was performed using a series of PDX NSCLC models. PD biomarkers were identified which confirmed the mechanism of action of vantictumab. LEF1 was identified as a predictive biomarker and is being evaluated in the Phase 1b study of vantictumab in combination with SOC in previously treated NSCLC: NCT01957007. Comprehensive PD and predictive biomarker data will be presented. Citation Format: CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Alayne Brunner, Min Wang, Belinda Cancilla, Rainer Brachmann, Tim Hoey, John Lewicki, Ann M. Kapoun. Predictive and pharmacodynamic biomarkers of vantictumab (OMP-18R5; anti-Frizzled) in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A30.

Abstract A030: Biomarker study of vantictumab plus paclitaxel in HER2- breast cancer patients

Introduction: We have developed a monoclonal antibody, vantictumab, that blocks canonical Wnt/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, and 8). This antibody inhibits the growth of several tumor types, including breast. Vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) agents (Gurney et al., 2012). To confirm the mechanism of action and to potentially target breast cancer patients most likely to respond to vantictumab, we undertook a biomarker study. Methods: We previously identified a 6-gene Wnt pathway-related signature, FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, based on microarray gene expression data from 8 BC patient-derived xenograft (PDX) models with established in vivo response to vantictumab plus SOC. This signature successfully predicted the response of 8 additional and independent PDX breast tumors. We further developed a qPCR Research Use Only (RUO) assay for the 6 genes for use on FFPE human breast tumor samples. This assay was evaluated in the phase 1b study of vantictumab in combination with paclitaxel in locally recurrent or metastatic HER2- breast cancer (NCT01973309) and the signature was refined using a Lasso model with overall survival as the outcome. A repeated 10 fold cross-validation was used to evaluate the performance of the gene signature. The association of the signature with progression-free survival (PFS) and overall survival (OS) was examined (n=40 patients). Furthermore, pharmacodynamic (PD) biomarker analyses were performed on tumor biopsies and hair follicles by comparing gene expression data from post-treatment time points versus baseline data (Affymetrix U133 plus 2 Microarrays). Results: A potential predictive 6-gene Wnt pathway biomarker was identified based on preclinical data and the biomarker was evaluated and refined in a phase 1b study of vantictumab in combination with paclitaxel in HER2- breast cancer. In the phase 1b study, AUC = 75% with repeated 10 fold cross-validation measuring the performance of the gene signature. Based on this analysis, two genes, RHOU and DKK1, were dropped from the preclinical gene signature, which was consistent with the feature ranking in the preclinical qPCR data. The refined 4-gene signature was significantly associated with both PFS and OS at a 50% percentile cut-off. In addition, analysis of PD biomarkers demonstrated that Wnt pathway target genes including AXIN2, LEF1, and CTNNB1 were downregulated while differentiation markers, e.g., KRT19 and Wnt pathway inhibitors, e.g., SFRP1, DKK3 were upregulated by vantictumab plus paclitaxel. Conclusions: We developed a 4-gene signature as a potential predictive biomarker for the response to vantictumab plus paclitaxel in HER2- breast cancer. PD biomarker analysis in tumors and hair follicles confirmed the mechanism of action of vantictumab in patient samples. Preliminary efficacy of vantictumab plus paclitaxel in the phase 1b study was encouraging, particularly in breast cancer patients positive for the 4-gene signature. Updated biomarker and PK/PD data from the phase 1b trial (NCT01973309) will also be presented. Citation Format: Chun Zhang, William R. Henner, Min Wang, Fiore Cattaruzza, Pete Yeung, Gilbert O9Young, Yuwang Liu, Gretchen Argast, Lu Xu, Shailaja Uttamsingh, John Lewicki, Ann M. Kapoun. Biomarker study of vantictumab plus paclitaxel in HER2- breast cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A030.

Abstract 599: Pharmacodynamic biomarkers for anti-TIGIT treatment and prevalence of TIGIT expression in multiple solid tumor types

TIGIT (T cell immunoreceptor with Ig ITIM domain) is a co-inhibitory receptor and its signaling axis inhibits T cell and Natural Killer (NK) cell activity in the healthy immune system. In tumors TIGIT is highly expressed on a subset of dysfunctional T and NK cells and of highly suppressive regulatory T cells (Treg). Loss of TIGIT signaling enhances NK cell activity, CD4+ T cell priming and CD8+ T cell effector functions, suggesting a role in anti-tumor immunity. We have developed an anti-TIGIT blocking antibody that shows potent antitumor efficacy in in multiple syngeneic mouse models, including CT26WT colon, B16F10 melanoma and 4T1 breast cancer. Dose-dependent (12.5-0.1 mg/Kg) single agent efficacy was demonstrated in established tumors, and pharmacodynamic (PD) biomarkers in blood and in tumors were identified. Consistent with TIGIT’s role as a co-inhibitory receptor, anti-TIGIT promoted a dose-dependent increase in activation of CD8+ and CD4+ tumor infiltrating leukocytes (TIL) and NK cells, as shown by increased staining of IFN-γ and CD69 by flow cytometry, when compared to controls. Anti-TIGIT also increased CD4 and CD8 T cell frequency in the tumor, measured by immunohistochemistry (IHC). Furthermore, anti-TIGIT caused an increase in splenic NK cell cytotoxicity, which correlated with dose and efficacy. To identify gene expression biomarkers in tumor and in blood, we used microarray analysis, and found similar immune gene changes between the two tissues. As expected, anti-TIGIT increased the expression of genes associated with CD8+ T cells, CD4+ T cells, and NK cells. Markers indicative of cytotoxic activity and Th1 response were also induced by anti-TIGIT. The microarray data were validated by qRT-PCR and results were consistent with flow cytometry and cytotoxicity results, and underlie the mechanism of action of anti-TIGIT. We have developed an IHC assay to evaluate TIGIT expression patterns in tumor and the associated stroma and TILs. Using this assay, we profiled 17 tumor types to evaluate patterns of TIGIT expression. Expression was high on immune cells in the stroma and on TILs in 8 tumor types, while it was generally low on tumor cells. TIGIT expression was also low on tumor cells in a panel of 27 PDX models. Consistent with the IHC results, analysis of 33 tumor types in the TCGA by RNA-Seq showed a good correlation of the expression levels of TIGIT and T cell markers, suggesting that TIGIT is mostly expressed on immune cells in tumors. In conclusion, we have identified PD biomarkers for anti-TIGIT in tumors and in surrogate tissues in syngeneic mouse models. Analysis of the biomarkers demonstrates activation of T cells and NK cells upon inhibition of TIGIT signaling. These biomarkers can be used in the clinic to demonstrate target engagement. In addition, we have profiled the expression of TIGIT in multiple solid tumor types and characterized prevalence of TIGIT-positive cells. Citation Format: Fiore Cattaruzza, Pete Yeung, Min Wang, Alayne Brunner, Erwan Le Scolan, Jennifer Cain, Gretechen Argast, Gilbert O9Young, YuWang Liu, Belinda Cancilla, Austin Gurney, Tim Hoey, John Lewicki, Ann Kapoun. Pharmacodynamic biomarkers for anti-TIGIT treatment and prevalence of TIGIT expression in multiple solid tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 599. doi:10.1158/1538-7445.AM2017-599

Abstract 404: Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors

R-Spondin (RSPO) proteins bind to LGR receptors and potentiate Wnt/β-catenin signaling. We have identified a therapeutic anti-RSPO3 antibody targeting the RSPO-LGR pathway. In preclinical studies, RSPO3 gene expression has shown correlation with anti-RSPO3 antibody efficacy in multiple solid tumor types. A qPCR-based RSPO3 assay has been developed as a predictive biomarker for response to the anti-RSPO3 antibody. In addition, RSPO gene fusions may play a role in the activation of Wnt signaling. A gene fusion detection workflow consisting of a RSPO3 CLIA assay, a RSPO3 RUO assay and next generation sequencing (NGS) has also been developed. We designed 6 qPCR-based assays for the RSPO3 CLIA assay development and 2 assays for the RUO assay. These assays were designed to span exon-exon junctions or target microarray probe set sequences. Amplification sensitivity and specificity were assessed for assay selection. The analytic performance of the candidate RSPO3 CLIA assay and quality control measures were established in a validation study. The validation study included: 1) performance specifications of the RSPO3 assay including analytical sensitivity, linearity, and precision, 2) determination of a reportable range, 3) establishment of a cut-off for the RSPO3 CLIA assay for patient selection, and 4) establishment of quality control procedures. 104 human cancer tissues and 24 independent patient-derived tumor xenografts (PDX) were used in these studies. To evaluate the fusion detection workflow, the RUO assay was performed on samples that tested above the CLIA assay cut-off. The delta Ct difference between the CLIA and RUO assays was calculated to identify potential fusions. The limit of quantification was established for the RSPO3 CLIA assay. The 95% reference interval was estimated to be (-2.44, 16.02) with 90% confidence interval for the lower bound (-3.45, -2.12) and upper bound (15.26, 16.57). The delta Ct cut-off for the RSPO3 CLIA assay was set based on sensitivity, specificity and prevalence. No statistically significant difference in the total variance across the tested samples was observed. A549 and OV56 were identified to be cell line controls with established acceptable delta Ct limits. Using NGS, RSPO3 fusions were identified in 6 PDX tumors with delta Ct RUO - delta Ct CLIA>7, including a novel fusion. This cut-off was further refined with NGS of 9 clinical samples. Prevalence of the RSPO3 expression and fusions will be presented. A qPCR based RSPO3 assay was developed and CLIA-validated for use as a potential predictive biomarker for response to anti-RSPO3 therapy. This RSPO3 CLIA assay, together with the fusion detection workflow, will be evaluated in a Phase 1a/b dose escalation study of anti-RSPO3 (OMP-131R10) in advanced solid tumors and in combination with FOLFIRI in metastatic colorectal cancer (NCT02482441). Citation Format: Chun Zhang, Yuwang Liu, Min Wang, Gilbert OYoung, Joy Kavanagh, Cheryl McFarlane, Fiore Cattaruzza, Pete Yeung, Jennifer Cain, Wan-Ching Yen, Marcus Fischer, Belinda Cancilla, Edwina Dobbin, Michelle McCarthy, Austin Gurney, Leonardo Faoro, John Lewicki, Tim Hoey, Ann M. Kapoun. Development of a RSPO3 CLIA-validated assay as a predictive biomarker for response to anti-RSPO3 antibody treatment in patients with solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 404.