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

Marcus Fischer, Wan-Ching Yen, Chun Zhang, Randall Henner, Fiore Cattaruzza, Tracy Tang, Pete Yeung, Tanuka Biswas, John Lewicki, Austin Gurney, Ann M. Kapoun and Timothy Hoey

OncoMed Pharmaceuticals Inc., Redwood City, California, 94063

SUMMARY1. A subset of ovarian patient-derived xenograft tumors are responsive to the WNT antagonist ipafricept and combination of ipafricept with Taxol2. Responsive tumors have reduced levels of WNT target genes post-therapy3. Ipafricept reduces expression of WNT signaling molecule LEF1 and reduces the ovarian cancer stem cell frequency4. Anti-tumor activity of ipafricept is enhanced by pre-dosing ipafricept prior to Taxol5. Targeting the WNT pathway in ovarian cancer may benefit a subset of patients

Preclinical PDX Models Have Identified Ovarian Cancers Responsive and Non-Responsive to Ipafricept

RESULTSABSTRACTOvarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. The Wnt/β-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 consist-ing 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 ovarian cancer. 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 signifi-cantly 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 β-catenin protein levels were reduced by ipafric-ept 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 clini-cally.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.

The Wnt/β-catenin signaling pathway, which signals through the family of FZD receptors and several co-receptors, plays an important role in controlling cell differentiation, self- renewal, and maintenance of cancer stem cells.

Antagonizing the WNT pathwat has demonstrated activity in Ovarian Cancer

Ipafricept is a first-in-class fusion protein consisting of the extracellular ligand-binding domain of the FZD8 receptor and the Fc domain of a human IgG1 antibody. Ipafricept binds Wnt ligands and prevents them from binding to FZD receptors.

Ipafricept (FZD8-fc, OMP-54F28) was generated at OncoMed Pharmaceuticals, Inc.

The tumor biopsies were provided from Cooperative Human Tissue Network. Tumor xeno-grafts were initiated and propagated at OncoMed Pharmaceuticals, in NSG and NOD/SCID mice.

For efficacy studies, treatments were initiated when tumors reached 100-200 mm3.

For Limiting Dose Dilution (LDD) studies, single cell suspensions were depleted of H2KD/CD45 murine cells, diluted to appropriate cell doses and injected subcutaneously into NOD/SCID mice. Mice were followed up for up to three months. Cancer stem cell frequency was determined using L-Calc Version 1.1 software program (StemCell Technologies, Inc., Vancouver, Canada).

In Vivo and Gene Expression data is illustrated as mean ± S.E.M.

Responders to ipafricept + Taxol

qRT-PCR of ipafricept + Taxol Normalized to Control Treatment Group

FZD8extracellular

(Wnt binding)domain

ImmunoglobulinFc domain

Domain Structure

Isolate tumors post-treatment Transplant 100, 1000 cells (n=10) Grow 78 days without treatment Calculate CSC Frequency based

on tumor take rate

Ipafricept Reduces the Cancer Stem Cell Frequency in Ovarian Cancer

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Combinatorial Treatment Enhanced by Antagonizing the WNT Pathway with Ipafricept Prior to Taxanes

Serial Passage of Treated Tumor Cells

0 28 56 84 112 140 1680

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Serous carcinoma OMP-OV19

Serous carcinoma OMP-OV40

INTRODUCTION

MATERIALS and METHODS

Control mAb Taxolipafricept ipafricept+ Taxol

WNT Signaling Molecule LEF1Modulated by Ipafricept in Responsive Tumors

ipafricept responsive tumor : Serous carcinoma OMP-OV40

ipafricept non-responsive tumor : Serous carcinoma OMP-OV63

0.5 1 1.5 2.0 Relative Quantity

Color Key

* = 10% FDR

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Serous carcinoma OMP-OV19 ipafricept day 1 + Taxol day 3

individual measurements

FZD1FZD2FZD3FZD4FZD5FZD6FZD7FZD8FZD10LRP5LRP6WNT2BWNT4WNT5AWNT6WNT7BWNT11AXIN2DKK3LEF1MYC

OMP-OV19 OMP-OV38 OMP-OV40

Ipafricept Downregulates WNT Pathway Genes in Ovarian Tumors

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Serous carconima OMP-OV40

Serous carcinoma OMP-OV38

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Serous carcinoma OMP-OV27

Serous carcinoma OMP-OV16

Serous carcinoma OMP-OV63

Non-Responders to ipafricept + Taxol

Control mAbipafriceptTaxolipafricept + Taxol

Control ipafricept Taxol ipafricept + Taxol0.00

0.01

0.02

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(1/142) (1/104) (1/43) (1/737)

Frequency of Ovarian Cancer Stem Cells

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