e23196 Background: Enzymatic degradation of hyaluronan (HA) by PEGylated recombinant human hyaluronidase PH20 (PEGPH20) induces a remodeling of the tumor microenvironment that impacts tumor behavior and response to therapy. Ventana Medical Systems, Inc., (VMSI) and Halozyme Therapeutics co-developed the VENTANA HA RxDx Assay and a scoring method to evaluate HA in solid tumors to potentially predict which patients might better respond to the addition of PEGPH20 to cancer treatments. Methods: Using Halozyme’s novel recombinant HA binding probe altered to include a rabbit Fc region, VMSI developed a highly specific and sensitive affinity histochemistry assay that binds to HA in tissues. Histological features and staining patterns of 106 samples from Halozyme’s 109-202 Phase 2 study of PEGPH20 combined with nab-paclitaxel plus gemcitabine (AG) vs. AG alone in patients with previously untreated stage IV pancreatic ductal adenocarcinoma (PDA) with clinical outcome data were analyzed to develop a scoring method evaluating HA accumulation. Results: A novel scoring algorithm focused solely on the extracellular matrix (ECM) was developed. An ECM HA staining ≥ 50% of the entire tumor surface at any intensity defines an HA-High PDA. We recommend that a 0.05 cm x 0.2 cm uninterrupted fragment of tumor cells and associated stroma obtained using needle gauges of 16 to 20 G as the minimally required amount of tissue to assess HA in PDA. Based on verification studies performed by VMSI, including an inter-reader precision study with a 94% agreement rate, VMSI scoring method proved to be trainable, reproducible, and transferable to pathologists, supporting an IDE approval for selecting patients in the Phase 3 HALO 109-301 study. Conclusions: Performance of the VENTANA HA RxDx Assay, a robust affinity histochemical assay, has been successfully verified in PDA. Two Halozyme clinical studies in PDA (Phase 2 HALO 109-202 Stage 2 & ongoing Phase 3 HALO 109-301) are expected to validate the VMSI HA scoring method in selecting patients who might better respond to the addition of PEGPH20 to cancer treatments. Current Phase 1/2 studies are exploring the utility of the VENTANA HA RxDx Assay and the pantumor potential of PEGPH20.

322 Background: Pancreatic ductal adenocarcinoma (PDA) is characterized by marked stromal fibrosis and hyaluronan (HA) accumulation. Degradation of stromal HA using PEGylated recombinant human hyaluronidase (PEGPH20) in combination with anti-cancer therapeutics has demonstrated increased efficacy in preclinical models. This study used an exploratory prototype HA assay to assess HA and to explore the association of HA status with clinical and pathological variables. Methods: Sixty-four PDA samples from 49 patients treated with gemcitabine and nab-paclitaxel were stained for HA using a prototype histochemical binding assay. The tumor extracellular matrix staining for HA at any intensity above background as a proportion of the total tumor surface area was recorded. Cases were categorized as HA-high (HA score of ≥50%) or HA-low (HA score of <50%). Subgroup analyses were also performed in paired pre- and post-chemotherapy samples. Results: Twenty-six of 49 (53%) patients were determined to be HA-high. HA-high status was significantly associated with pN1 (positive node) status (p<0.001) and well/moderate differentiation (p<0.001). No correlation of HA status was observed with sex, race, primary tumor location, pT (tumor) stage, lymph-vascular invasion, pathologic stage, or initial CA19-9 levels. No trend in HA status was observed comparing pre- and post-chemotherapy specimens (n=5). Conclusions: HA status was significantly associated with nodal stage and grade.

Correction to: British Journal of Cancer (2018) 118, 156–167; doi: 10.1038/bjc.2017.327; published online 26 September 2017 There was an error in the description of the pharmacodynamics within the ‘Materials and methods’ section of this manuscript, and with the reference cited Printz et al (2017). The correct description is shown below:

Abstract Hyaluronan (HA), which accumulates in the tumor microenvironment of many solid tumors, is associated with tumor progression and negative clinical outcomes. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content, resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic activity. Accordingly, the HA degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), is currently being evaluated in clinical trials with selected treatment regimens in several malignancies, including metastatic pancreatic adenocarcinoma, gastric cancer, breast cancer and non-small cell lung cancer. Advances in treatment for pediatric Wilms’ tumor patients have significantly increased survival, but salvage chemotherapy for relapsed pediatric patients remains challenging. To evaluate the role of HA in this disease, 19 Wilms’ tumor histological specimens were surveyed for HA status using a novel probe (Jadin 2014). 79% (15/19) of the samples stained strongly for HA when compared to non-tumor kidney tissue, prompting additional preclinical studies. In brief, a human Wilms’ tumor cell line, WT-CLS1, was transduced with hyaluronan synthase-3 (HAS3). The subsequent WT-CLS1/HAS3 cells produced more HA and WT-CLS1/HAS3 xenograft tumors grew significantly faster than parental WT-CLS1 tumors. To evaluate the efficacy of PEGPH20 in combination with Wilms’ tumor chemotherapy (CTX) combinations, WT-CLS1/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors reached ∼250 mm3, mice were staged into treatment groups: vehicle control, PEGPH20, vincristine (VIN) plus Dactinomycin (DACT), and PEGPH20+VIN+DACT at different dose/frequency combinations. Average tumor growth inhibition (TGI) and overall survival for PEGPH20+VIN+DACT-treated mice were superior to TGI and survival of VIN+DACT treatment alone. In separate studies WT-CLS1/HAS3 tumor-bearing mice were staged into three groups: vehicle control, low dose PEGPH20 (0.0375 mg/kg, iv) and high dose PEGPH20 (1 mg/kg, iv). Animals were administered the hypoxyprobe pimonidazole (60 mg/kg, ip) three hours prior to sacrifice, and whole tumors were removed and processed via immunofluorescence. High dose PEGPH20 reduced this measure of tumor hypoxia by 5% (p = 0.023). Taken together, these data demonstrate that HA accumulation in Wilms’ tumor is common, and treatment with PEGPH20 hyaluronidase can increase CTX efficacy and reduce tumor hypoxia in a preclinical model of this disease. These results support further studies with PEGPH20 in combination with chemotherapy in preclinical models of Wilms’ tumor and suggest investigation in this pediatric patient population may be warranted. Citation Format: Jessica Cowell, Susan J. Zimmerman, Mathieu Marrela, Ping Jiang, Peter J. Houghton, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms’ xenograft model. [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 2463.

248 Background: Improved understanding of TME in CCA may allow for development of novel therapeutics targeting stromal components. Hyaluronic acid (HA) is a polysaccharide that has been found in the TME of a number of solid tumors characterized by desmoplastic stroma, such as pancreatic cancer. Elevated HA levels were prognostic in these malignancies. HA prevalence and association with clinic-pathological features in CCA is unknown. Methods: A retrospective study was conducted using clinically annotated formalin-fixed paraffin embedded tissue from resected intrahepatic CCA samples. Specimens were analyzed for HA, scored using a positive pixel count algorithm (Aperio Imagescope) and positivity was calculated as % of total pixel count. Clinical variables were examined by chi-square or ANOVA F-test. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan-Meier method and compared via log-rank test. Multivariate analysis was conducted using Cox proportional hazards regression models. Results: 142 patient samples were stained for HA. HA was identified in all samples (100%), with a range of HA positivity of 32.8 – 91.8%. Median HA value was 67.1% and 95% of patients had HA positivity ≥ 45%. Factors associated with higher than median HA ( > 67%) included perineural invasion (PNI) (40% vs. 21%, p = 0.011), necrosis (67% vs. 41%, p = 0.003), lymph node positivity (32% vs. 14%, p = 0.027), and small tumor size ( < 6.8 cm vs. > 8.4cm, p = 0.014). Stratification of HA staining by median positivity value was not an independent predictor of survival based on multivariate analysis. OS for patients with HA > 67% was 40.1 months (mo) vs 48.5 mo with HA ≤ 67%, p = 0.537. PFS for patients with HA > 67% was 14.4 mo vs. 25.7 mo with HA ≤ 67%, p = 0.177. Conclusions: Prevalence of HA staining in resected CCA is higher than seen in many solid tumors and is ubiquitous in our study. Absence of statistically significant association with OS and PFS might be attributable to ubiquitous prevalence, narrow signal range, and association with known negative prognostic factors. Further pre-clinical and clinical evaluation would be warranted.

Introduction: Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that has been shown to accumulate at high levels in several cancers, including non-small cell lung cancer (NSCLC). The accumulation of HA in NSCLC is associated with a more aggressive phenotype and shortened overall survival. In a prevalence analysis using tissue microarrays of archival human specimens (N = 194) stained by affinity histochemistry, we demonstrated that 55% of adenocarcinomas, 74% of squamous cell carcinomas and 83% of large cell carcinomas accumulated HA. The engineered enzyme PEGylated recombinant human hyaluronidase PH20 (PEGPH20) removes HA from the tumor microenvironment. PEGPH20-based therapies are being evaluated in clinical studies of several solid tumors, including NSCLC (NCT02346370, NCT02563548). Herein, we evaluated the hypothesis that PEGPH20 combination therapy could enhance the efficacy of pemetrexed (PEM), a folate antimetabolite approved by FDA as a first-line treatment in combination with cisplatin, against locally advanced and metastatic NSCLC in patients with non-squamous histology. Methods: We evaluated PEGPH20 plus PEM in a NSCLC xenograft model. First, the human NSCLC adenocarinoma cell line A549 was transduced with hyaluronan synthase-3 (HAS3) to generate the A549/HAS3 cell line. Next, A549/HAS3 cells were inoculated adjacent to the tibial periosteum of nude mice and tumor growth was monitored via ultrasonography. When tumors size reached ∼250 mm3, mice were staged into six groups: (1) vehicle; (2) PEGPH20, (3) PEM daily, (4) PEM weekly, (5) PEGPH20+PEM daily or (6) PEGPH20+PEM weekly. Vascular volume, hypoxia and tumor growth inhibition (TGI) were assessed using conventional methods. Affinity histochemical staining for HA was performed on formalin-fixed paraffin-embedded tissue sections after harvest. Results: A549/HAS3 tumors grew faster than parental A549 tumors in vivo. PEGPH20 monotherapy significantly reduced tumoral HA, increased vascular volume and reduced hypoxia in tumors. Further, TGI with PEGPH20+PEM, weekly or daily, was 29.7 or 31.1%, respectively, whereas in either PEGPH20 alone or PEM alone, both were Conclusion: These data suggest that addition of PEGPH20 to pemetrexed increases the efficacy of PEM and support further investigation of PEGPH20 plus PEM treatment in NSCLC tumors of non-squamous histology that accumulate HA. Citation Format: Renee Clift, Jessica A. Cowell, Susan J. Zimmerman, Mathieu Marella, Ping Jiang, Arnold B. Gelb, Michael J. LaBarre, Daniel C. Maneval, Curtis B. Thompson, Xiaoming Li. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model. [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 283.

AbstractPurpose: This phase Ib study evaluated the safety and tolerability of PEGylated human recombinant hyaluronidase (PEGPH20) in combination with gemcitabine (Gem), and established a phase II dose for patients with untreated stage IV metastatic pancreatic ductal adenocarcinoma (PDA). Objective response rate and treatment efficacy using biomarker and imaging measurements were also evaluated.Experimental Design: Patients received escalating intravenous doses of PEGPH20 in combination with Gem using a standard 3+3 dose-escalation design. In cycle 1 (8 weeks), PEGPH20 was administrated twice weekly for 4 weeks, then once weekly for 3 weeks; Gem was administrated once weekly for 7 weeks, followed by 1 week off treatment. In each subsequent 4-week cycle, PEGPH20 and Gem were administered once weekly for 3 weeks, followed by 1 week off. Dexamethasone (8 mg) was given pre- and post-PEGPH20 administration. Several safety parameters were evaluated.Results: Twenty-eight patients were enrolled and received PEGPH20 at 1.0 (n = 4), 1.6 (n = 4), or 3.0 μg/kg (n = 20), respectively. The most common PEGPH20-related adverse events were musculoskeletal and extremity pain, peripheral edema, and fatigue. The incidence of thromboembolic events was 29%. Median progression-free survival (PFS) and overall survival (OS) rates were 5.0 and 6.6 months, respectively. In 17 patients evaluated for pretreatment tissue hyaluronan (HA) levels, median PFS and OS rates were 7.2 and 13.0 months for “high”-HA patients (n = 6), and 3.5 and 5.7 months for “low”-HA patients (n = 11), respectively.Conclusions: PEGPH20 in combination with Gem was well tolerated and may have therapeutic benefit in patients with advanced PDA, especially in those with high HA tumors. Clin Cancer Res; 22(12); 2848–54. ©2016 AACR.

Therapeutic efficacy of monoclonal antibodies (MAbs) against solid tumor targets, like trastuzumab (anti-HER2) and cetuximab (anti-EGFR), have been used successfully to treat cancer, despite the many physical barriers impeding their access to the malignant cell surface 1 . For example, only about 50% of HER2 3+ patients have a durable response to therapy with trastuzumab 2 . Extracellular matrix (ECM)-mediated inhibition may be among the mechanisms of resistance to MAb therapy of solid tumors. Aberrant accumulation of hyaluronan (HA), a major component of the ECM in many tumors, is associated with poor prognosis and treatment-resistance in multiple malignancies 3-5 . We investigated HA-dependent pericellular matrix-mediated inhibition to ADCC in HA high human cancer cells in vitro and in vivo . We observed high levels of tumor associated HA (HA 3+ ) in >50% of HER2 3+ breast adenocarcinoma and ∼40% of EGFR + head and neck squamous cell carcinoma (HNSCC) primary tumors. Human hyaluronan synthase 2 (HAS2)-overexpressing breast cancer cells formed an HA high pericellular matrix, which inhibited both natural killer (NK) cell access to tumor cells and ADCC in vitro . Hyaluronan depletion by PEGPH20, a pegylated recombinant human PH20 hyaluronidase currently in clinical study for pancreatic cancer, increased NK cell access to HAS2-overexpressing breast cancer cells and greatly enhanced trastuzumab- or cetuximab-dependent ADCC. Trastuzumab and NK cell accessibility to HAS2-overexpressing tumors was enhanced following HA-depletion by PEGPH20. In an in vivo ADCC-based efficacy study, PEGPH20 treatment in combination with trastuzumab and NK cells enhanced tumor growth inhibition. This work describes a novel tumor microenvironment (TME)-dependent mechanism of inherent resistance to therapeutic antibody-mediated ADCC in vitro and in vivo , and furthermore shows that ADCC can be enhanced by hyaluronan depletion. These results may help to explain as to why tumors with high levels of HA are more aggressive, and suggest potential benefits of PEGPH20-mediated HA depletion in combination with therapeutic antibodies like trastuzumab or cetuximab in the treatment of HA high solid tumors. References: 1. J. Christiansen, A. K. Rajasekaran, Biological impediments to monoclonal antibody–based cancer immunotherapy. Mol Cancer Ther. 3 , 1493-1501 (2004). 2. H. M. Shepard, C. M. Brdlik, H. Schreiber, Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family. J. Clin. Invest. 118 , 3574-3581 (2008). 3. A. Kultti, X. Li, P. Jiang, C. B. Thompson, G. I Frost, H. M. Shepard Therapeutic Targeting of Hyaluronan in the Tumor Stroma. Cancers 4 , 873-903 (2012). 4. R.K. Jain, Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol. 31 , 2205-18 (2013). 5. R. K. Boregowda, H. N. Appaiah, M. Siddaiah, S. B. Kumarswamy, S. Sunila, K. N. Thimmaiah, K. Mortha, B. Toole, S. D. Banerjee, Expression of hyaluronan in human tumor progression. J. Carcinog. 5 , 2 (2006). Citation Format: Netai C Singha, Tara Nekoroski, Chunmei Zhao, Rebecca Symons, Ping Jiang, Gregory Frost, Zhongdong Huang, H Michael Shepard. Hyaluronan (HA) depletion sensitizes HA high tumors to antibody-dependent cell-mediated cytotoxicity [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-02.

Abstract Hyaluronan (HA) accumulates in the tumor microenvironment (TME) of many solid tumors, including prostate, colon, breast, stomach, ovary, pancreas and lung, and HA accumulation is associated with tumor progression and a negative clinical outcome. Accordingly, the HA degrading enzyme pegylated recombinant human hyaluronidase PH20 (PEGPH20) was developed to allow systemic therapy and enable treatment of HA-high tumors. Preclinical studies have demonstrated that PEGPH20-mediated HA removal from HA-rich xenograft tumors in mice decreases tumor interstitial fluid pressure and water content; resulting in decompression of tumor vasculature, increased tumor perfusion and enhanced chemotherapeutic (CTX) activity. As non-small cell lung cancer (NSCLC) is the most common cancer worldwide and is characterized by high levels of HA (∼30%), we aimed to evaluate PEGPH20 enhancement of anti-tumor activity of NSCLC CTXs. Specifically, using both cell derived xenograft (CDX) models, peritibial H2170/HAS3 human NSCLC xenografts, and patient derived xenograft (PDX) models, we investigated CTX tumor growth inhibition (TGI)+/- PEGPH20. Nude mice were inoculated with either H2170/HAS3 cells adjacent to the tibial periosteum or patient derived tumor fragments of human NSCLCs implanted subcutaneously, and tumor growth was monitored via ultrasonography or caliper, respectively. When tumors reached ∼400 mm3, mice were staged into four groups: (1) vehicle; (2) PEGPH20, (3) CTX(s), or (4) PEGPH20 + CTX(s). PEGPH20 alone depleted tumor HA in both CDX and PDX models. In CDX studies, PEGPH20 increased tumor perfusion, reduced hypoxia and decreased HIF-1α expression (p<0.05). A summary table of the TGI data is shown below. The average TGI for PEGPH20 plus CTX(s) was superior to all CTX(s) alone, suggesting that enzymatic depletion of TME HA increases tumor sensitivity to NSCLC CTXs, likely due to increased CTX access into the TME. PEGPH20 clinical trials in NSCLC are currently in development. Summary of TGIsGroup (n≥5/group)CDX%TGI w/ docetaxelCDX%TGI w/ nab-paclitaxel + carboplatinPDX%TGI w/ docetaxelPDX%TGI w/ gemcitabine + cisplatin(p-value vs. V)Vehicle (V)----PEGPH20 (P)28.5 (p<0.05)11.5 (p>0.05)44.4 (p<0.01)29.3 (p>0.05)CTX(s)19.8 (p>0.05)68.6 (p<0.0001)52.5 (p<0.001)38.4 (p>0.05)P + CTX56.1 (p<0.001)93.5 (p<0.0001)>100 (p<0.0001)76.6 (p<0.01) Citation Format: Jessica A. Cowell, Xiaoming Li, Ping Jiang, Susan Zimmerman, Rebecca Symons, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. PEGPH20 enhances chemotherapy in patient-derived and traditional cell-derived xenograft NSCLC 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 2547. doi:10.1158/1538-7445.AM2015-2547

AbstractDespite tremendous progress in cancer immunotherapy for solid tumors, clinical success of monoclonal antibody (mAb) therapy is often limited by poorly understood mechanisms associated with the tumor microenvironment (TME). Accumulation of hyaluronan (HA), a major component of the TME, occurs in many solid tumor types, and is associated with poor prognosis and treatment resistance in multiple malignancies. In this study, we describe that a physical barrier associated with high levels of HA (HAhigh) in the TME restricts antibody and immune cell access to tumors, suggesting a novel mechanism of in vivo resistance to mAb therapy. We determined that approximately 60% of HER23+ primary breast tumors and approximately 40% of EGFR+ head and neck squamous cell carcinomas are HAhigh, and hypothesized that HAhigh tumors may be refractory to mAb therapy. We found that the pericellular matrix produced by HAhigh tumor cells inhibited both natural killer (NK) immune cell access to tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Depletion of HA by PEGPH20, a pegylated recombinant human PH20 hyaluronidase, resulted in increased NK cell access to HAhigh tumor cells, and greatly enhanced trastuzumab- or cetuximab-dependent ADCC in vitro. Furthermore, PEGPH20 treatment enhanced trastuzumab and NK cell access to HAhigh tumors, resulting in enhanced trastuzumab- and NK cell–mediated tumor growth inhibition in vivo. These results suggest that HAhigh matrix in vivo may form a barrier inhibiting access of both mAb and NK cells, and that PEGPH20 treatment in combination with anticancer mAbs may be an effective adjunctive therapy for HAhigh tumors. Mol Cancer Ther; 14(2); 523–32. ©2014 AACR.

359 Background: Poor outcome in pancreatic cancer (PDA) has been associated with tumor stroma limiting access of chemotherapy drugs. PEGPH20 (PEG), PEGylated recombinant human hyaluronidase, which depletes hyaluronan (HA) in tumors, has demonstrated anti-tumor activity in preclinical PDA models. In a KPC model of PDA, PEG + gemcitabine (Gem) significantly prolonged survival compared to Gem alone. In Phase 1 PEG monotherapy studies, the MTD was 3µg/kg. The most common adverse events (AEs) were musculoskeletal events. Methods: This was a phase 1b study to determine the recommended phase 2 dose of PEG + Gem in patients (pts) with previously untreated Stage IV pancreatic cancer. PEG was given at 1, 1.6, or 3µg/kg IV twice weekly Wks 1–4 and weekly Wks 5–7, followed by 1 wk rest. Gem was given at 1000mg/m2IV once weekly for Wks 1–7, then 1 wk rest. Thereafter, PEG + Gem were given once weekly for 3 wks in 4-wk cycles. Dexamethasone was given pre and post PEG doses. Due to evolving SOC, the study was discontinued before initiation of the phase 2 randomization. Results: Twenty-eight pts were enrolled in the study. The majority of the patients (89%) had metastatic sites in the liver. Four, 4 and 20 pts received PEG at 1, 1.6 and 3µg/kg, respectively. The most common AEs related to PEG were muscle spasm (54%), myalgia (39%), arthralgia (29%), peripheral edema (29%), fatigue (25%), and extremity pain (18%). Median progression free survival (PFS) and overall survival (OS) were assessed and were 154 and 200 days, respectively. In an exploratory analysis, tumor biopsies from 17 pts were evaluated for HA levels (HAhigh or HAlow). 6 pts were determined to have HAhigh tumors and 11 pts had HAlow tumors. The median PFS and OS for HAhigh pts were 219 days (95% CI: 159-276) and 395 days (95% CI: 210-578). For HAlowpts median PFS and OS were 108 (95% CI: 14-163) and 174 days (95% CI: 34-293). Conclusions: PEG + Gem is generally well tolerated in advanced pancreatic cancer and shows promising clinical activity, especially in pts with HAhigh tumors. ClinicalTrials.gov Identifier: NCT01453153.

300 Background: PEGPH20 (PEG), a PEGylated recombinant human hyaluronidase, has anti-tumor activity as a single agent and in combination with chemotherapy in preclinical models. A Phase 1b study of PEG + Gem (P+G) in patients (pts) with advanced PDA showed good anti-tumor activity, particularly in pts with HAhigh tumors (ECCO 2013). In this study, we investigated pharmacodynamic (PD) markers including plasma(soluble) HA (sHA), dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and 18Fluorodeoxyglucose positron emission tomography (PET/CT) to explore additional correlates for PEG activity. Methods: 28 pts with stage IV PDA were treated with PEG at 1, 1.6, or 3µg/kg IV twice weekly for Wks 1-4 and Wks 5-7, followed by 1 wk rest, plus Gem at 1000 mg/m2 IV once weekly for Wks 1-7, then 1 wk rest. Thereafter, P+G was given once weekly for 3 wks in 4-wk cycles. Serial plasma samples were collected and analyzed in a quantitative assay for PEG and sHA. Exploratory imaging by DCE-MRI was performed on s...

Abstract Background: Hyaluronan (HA), a glycosaminoglycan distributed in the extracellular matrix, is frequently upregulated in cancer and correlates with disease progression. Administration of a first-in-class PEGylated recombinant human hyaluronidase (PEGPH20) depletes HA, unmasks HA around tumor cells and decreases tumor interstitial fluid pressure, increasing accessibility to systemically administered agents in preclinical models. PEGPH20 recently received Orphan Drug designation in pancreatic ductal adenocarcinoma (PDA) in the USA. We report the development and analytical validation of a novel prototype assay for a companion diagnostic to select patients with PDA for molecularly targeted therapies with PEGPH20. Design: A recently described biotinylated recombinant immunoadhesin (HTI-601, Jadin 2014) was adapted for use in an immunohistochemistry-based assay on formalin-fixed paraffin-embedded tissue. Sensitivity, specificity, and within-laboratory precision studies were performed in a research mode and then at a central laboratory on a validation set of approximately 200 tumor and normal tissues under GCP conditions. Both pathologist scoring and operator-assisted image analysis (positive pixel count for strong positive pixels) were evaluated. Results: Analytical sensitivity studies identified an optimal probe dilution of 0.417 μg/mL on an immunostainer based on dynamic range in 4 human tumor xenografts containing differential levels of HA. The frequency of high HA observed was 62.7% of archival PDA (N = 75). During analytical specificity, 5.1% of 75 PDA and 3 normal adjacent tissue samples had faint or focal staining after pre-digestion with recombinant PH20 hyaluronidase. No cross-reactivity was identified in 99 normal human tissues across a panel of histotypes; only 1 (1.0%) colon sample had staining of macrophages. Within-laboratory precision among three observers’ annotations had average%CVs as follows: between day 4.8%, between run 12.2%, and repeatability 16.6%. Conclusion: The sensitivity, specificity and within-laboratory precision of the prototype assay using image analysis are acceptable. The frequency of high HA expression is similar to other studies in the literature. Cross-reactivity was focal and faint so is unlikely to interfere. Subset analysis showed contributions to%CV to be higher for HAlow than HAmedium or HAhigh samples as expected. These findings warrant further evaluation of HTI-601 staining on biopsies for PEGPH20-associated therapies in clinical studies. Citation Format: Arnold B. Gelb, Ping Jiang, Laurence Jadin, Daniel C. Maneval, H. Michael Shepard. Development and analytical validation of a novel assay for tissue detection of hyaluronan in the tumor microenvironment to select patients for molecularly targeted pancreatic cancer therapies. [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 576. doi:10.1158/1538-7445.AM2015-576

Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.

Abstract Pancreatic cancer is one of most deadly cancers with a 5-year survival rate of 6%. Accumulation of hyaluronan (HA) is found in about 87% of human pancreatic adenocarcinomas, and removal of HA suppresses tumor growth in HA-rich preclinical models. In a transgenic pancreatic cancer mouse model (LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx-1-Cre, KPC), removal of HA by pegylated human recombinant PH20 hyaluronidase (PEGPH20) inhibits tumor growth and increases survival in combination with gemcitabine compared to gemcitabine monotherapy. In this study, we explored the role of HA synthesizing (HAS) enzymes HAS2 and HAS3 and HA accumulation in pancreatic cancer tumor growth and remodeling of tumor microenvironment. HAS2 and HAS3 were overexpressed in BxPC3 human pancreatic cancer cells using lentiviral vectors. Stable HAS2 and HAS3 overexpressing pancreatic cancer cell lines secreted more HA to culture medium and produced larger pericellular HA matrices than parental BxPC3 cells. In vivo, overexpression of HAS2 or HAS3 led to an increase in BxPC3 xenograft tumor growth (peritibial i.m. tumor model) compared to parental cells. Interestingly, overexpression of HAS3 was more effective to enhance tumor growth than overexpression of HAS2. In addition, massive accumulation of extracellular HA was found in HAS3 overexpressing tumors while HAS2 overexpressing tumors contained both extracellular and intracellular HA. Treatment with PEGPH20 removed the majority of extracellular HA and induced a 87% reduction of tumor volume in BxPC3 HAS3 model (p<0.001) but had weaker effect on BxPC3 HAS2 (33%, p<0.001) and BxPC3 tumors (36%, p<0.01). Accumulation of extracellular HA was associated with enriched tumor stroma, loss of membranous E-cadherin and accumulation of cytoplasmic β-catenin in pancreatic cancer cells, suggesting HA-induced epithelial-mesenchymal transition (EMT). Removal of HA by PEGPH20 reversed the remodeling of the tumor stroma and induced translocation of E-cadherin and β-catenin to the plasma membrane.Translocation of E-cadherin was also observed in the KPC pancreatic tumors after PEGPH20 treatment. In conclusion, accumulation of extracellular HA by HAS3 overexpression favors tumor growth and leads to a strong response to PEGPH20 in a pancreatic cancer xenograft model. Deposition of extracellular HA is associated with optimization of the tumor microenvironment and EMT. Depletion of HA by PEGPH20 reverses changes in the tumor stroma and induces translocation of epithelial markers to the plasma membrane. Citation Format: Anne Kultti, Chunmei Zhao, Susan Zimmerman, Ryan J. Osgood, Yanling Chen, Rebecca Symons, Ping Jiang, Curtis B. Thompson, David A. Tuveson, Gregory I. Frost, H Michael Shepard, Zhongdong Huang. Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2014-4844

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β -catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β -catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.

4010 Background: PEGPH20 is a PEGylated version of human recombinant hyaluronidase. In preclinical studies, PEGPH20 depleted pancreatic cancers of their high hyaluronan (HA) content. In a genetically-engineered murine model of PDA, PEGPH20 + gemcitabine (Gem) significantly prolonged survival compared to Gem alone. In Ph1 PEGPH20 monotherapy studies, the MTD was 3.0 μg/kg. The most common AEs were musculoskeletal events (MSEs). Methods: This was a dose-escalation study to find the recommended Phase 2 dose (RP2D) of PEGPH20 in combination with Gem in patients (pts) with Stage IV previously untreated pancreatic cancer. Pts received PEGPH20 at 1, 1.6, or 3 μg/kg IV twice a week for Wks 1-4, weekly for Wks 5-7, then 1 wk rest. Dose escalation was based on safety. Gem was given at 1000 mg/m2 IV once a week for Wks 1-7, then 1 wk rest. Thereafter, PEGPH20 + Gem were given once a week for 3 wks in 4-wk cycles. Dexamethasone was given pre and post PEGPH20 doses. Results: Of the 28 pts enrolled, the majority had a Karnofsky performance status of 80%, and 85%/19%/26% of pts had liver/lung/visceral metastases. The median age was 58 yrs. Four pts received PEGPH20 at 1 μg/kg, 4 at 1.6 μg/kg, and 20 at 3 μg/kg. The RP2D was 3 μg/kg. Treatment duration ranged from 1-274 days; 5 pts remain on study. Treatment was generally well tolerated. Ten pts had 1 Gem dose reduction, 2 pts had 1 PEGPH20 dose reduction (3 to 1.6 µg/kg), but no pt had a DLT. The most common PEGPH20-related AEs were MSEs (25% Gr1; 18% Gr2) and fatigue (21% Gr1; 11% Gr2). Objective response was assessed by an independent central radiologist using RECIST 1.1. Of the 21 pts evaluable for efficacy, 7 had partial response (PR) for an overall response rate (ORR) of 33%, and 9 had stable disease for ≥ 2 mo. Tumor biopsies from 12 pts were evaluable for HA staining. HA was high in 9 and low in 3. Of the 9 with high HA staining, 5 had PR (56% ORR); HA data were not available for the other 2 PR pts. PK results show dose-dependent exposure consistent with data from PEGPH20 monotherapy studies. Conclusions: PEGPH20 in combination with Gem is generally well tolerated in advanced pancreatic cancer and shows promising efficacy, especially in pts with high intratumoral HA content. Clinical trial information: NCT01453153.

e15005 Background: Enzymatic degradation of hyaluronan (HA) is a novel strategy to target the desmoplastic stroma of pancreatic cancer. PEGPH20, a pegylated form of recombinant human hyaluronidase PH20, is an investigational drug in clinical trials. Preclinical studies demonstrate that sustained HA removal by PEGPH20 inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. Ph1 PEGPH20 monotherapy studies show increased tumor perfusion by DCE-MRI, metabolic partial responses by FDG-PET, and stromal remodeling in tumor biopsies from selected advanced cancer patients (pts). Methods: This was a dose-escalation study to find the recommended Ph2 dose of PEGPH20 in combination with Gem in pts with Stage IV previously untreated pancreatic cancer. Pts received Gem at 1000 mg/m2 IV qwk for Wks 1-7 plus PEGPH20 at 1, 1.6, or 3 μg/kg IV twice a week for Wks 1-4 and qwk for Wks 5-7. Wk 8 was a rest week. Thereafter, PEGPH20 + Gem were given qwk for 3 wks in 4-wk cycles. Serial plasma samples were collected and hyaluronidase activity measured by an ultrasensitive assay to assess PEGPH20 exposure. Plasma HA catabolites were measured by quantitative HPLC to assess PD. Results: 28 pts were enrolled. Plasma PEGPH20 concentrations were proportional to dose, and kinetics were well-characterized by a 2-compartment PK model. Estimates for clearance (0.5-2 mL/hr/kg) were consistent with long t1/2 (1-2 days) previously seen with single-dose PEGPH20 monotherapy. Wks 1 and 4 PK profiles were similar, suggesting no changes to PEGPH20 clearance mechanisms after multiple doses or effects of Gem on PEGPH20 exposure. Most pretreatment plasma HA levels were <1 μg/mL and increased in a time- and dose-dependent manner after dosing. Circulating HA concentration was >500 µg/mL in several pts given 3 μg/kg PEGPH20. Conclusions: PEGPH20 plasma levels can be predicted using a linear PK model and circulating HA catabolites can be used as a quantitative measure of PEGPH20 PD. Results are consistent with the mechanism of action of hyaluronidase and support further study of PEGPH20 with anticancer agents. Clinical trial information: NCT01453153.

Abstract Accumulation of Hyaluronan (HA), an important glycosaminoglycan of the extracellular matrix (ECM), has been detected in about 25% of human tumors, and 56% of breast cancers1-2. HA is produced by malignant and stromal cells and acts to cross-link ECM proteins, contributing to the desmoplastic phenotype. Aberrant accumulation of HA in tumors has been associated with more aggressive malignancy1-2. We have observed that >50% of human epidermal growth factor receptor 2 (HER2) triple-positive breast tumors have a high accumulation of HA (HA3+). Monoclonal antibodies (MAbs) have been developed successfully as part of the anticancer armamentarium. However, even in cases where the target of the antibody is stably and abundantly expressed, efficacy often falls below expectations. In this work, we present a novel HA-dependent ECM-mediated mechanism of resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) and this resistance can be reduced by PEGPH20 (a pegylated human PH20) treatment. Human hyaluronan synthase 2 (HAS2)-overexpressing tumor cells formed an HA-rich pericellular matrix which can restrict human NK cells from accessing tumor cells. This HA-dependent ECM-mediated physical barrier in HAS2- overexpressing tumor cells contributed to resistance to trastuzumab/cetuximab-dependent ADCC. Removal of HA from ECM by PEGPH20 treatment sensitized HAS2-overexpressing tumor cells to trastuzumab/cetuximab-mediated ADCC in vitro. HAS2-overexpressing ovarian cancer cell SKOV3 showed more aggressive growth pattern (median survival on 42 days) compared to parental SKOV3 (median survival of 61 days) in an intra-peritoneal xenograft model. In the same model with HAS2-overexpressing SKOV3, PEGPH20 in combination with trastuzumab and NK cell treatment (to simulate ADCC in vivo) resulted in 70% tumor growth inhibition (TGI) in comparison to 40% TGI with trastuzumab and NK cell treatment, indicating that PEGPH20 enhanced in vivo ADCC by trastuzumab and NK cell. Our results demonstrated a mechanism of high HA-content pericellular matrix-mediated resistance to MAb-mediated ADCC. These results may help explain why tumors with high levels of HA (HA3+ phenotype) are more aggressive, and suggest that removal of HA by PEGPH20 treatment may be an effective combinatorial therapy together with anti-cancer MAbs. 1. Kultti A, Li X, Jiang P, Thompson C.B., Frost G.I., Shepard H.M. 2012. Therapeutic targeting of Hyaluronan in the tumor stroma. Cancers, 4:873-903; doi:10.3390/cancers4030873. 2. Sironen, R.K., Tammi, M., Tammi, R., Auvinen, P.K., Anttila, M., and Kosma, V.M. 2011. Hyaluronan in human malignancies. Exp. Cell. Res. 317:383-391. doi: 10.1016/j.yexcr.2010.11.017 Citation Format: Netai C. Singha, Tara Nekoroski, Susan Zimmerman, Chunmei Zhao, Ping Jiang, Robert Connor, Gregory I. Frost, Zhondong Huang, Michael H. Shepard. Hyaluronan-rich ECM contributes to resistance to antibody-dependent cell-mediated cytotoxicity in solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4999. doi:10.1158/1538-7445.AM2013-4999

Abstract The tumor microenvironment is crucial for cancer cell survival and spreading. The glycosaminoglycan hyaluronan (HA) is accumulated in 50% of malignant breast cancer tumors and its accumulation correlates with poor survival of breast cancer patients. HA is synthesized at the cell surface by HA synthase enzymes (HAS1-3) and is extruded to the extracellular space where HA molecules can be attached to the cell surface via interactions with its receptors or HAS proteins. HA can also interact with its binding proteins and be incorporated into surrounding ECM. However, the origin and exact functions of HA in breast cancer are still unclear. The aim of this study was to explore the role of HA in the tumor microenvironment of breast cancer, especially in the interaction of tumor and stromal cells in vitro and in vivo. First, interaction of breast cancer cells and stromal cells were studied in mono- and co-cultures. Human bone marrow-derived mesenchymal stem cells (MSCs) and breast cancer-associated fibroblasts (CAFs) synthesized high amounts of HA, while this was the case for <5% MDA-MB-468 breast cancer cells. In co-culture with MSCs or CAFSs, MDA-MB-468 and MDA-MB-231 cells formed distinct pericellular HA coats. Similar HA coats were observed after addition of exogenous FITC-labeled high molecular weight HA (1,2 MDa) to MDA-MB-468 and MDA-MB-231 cell cultures. Interestingly, binding of FITC-labeled HA was not efficiently blocked by unlabeled HA below 500 kDa. In co-cultures, the high molecular weight HA coats around MDA-MB-468 and MDA-MB-231 cells were prevented by antibody blockade of the HA receptor CD44, indicating that formation of HA coats is CD44-mediated. Knockdown of CD44 by shRNA also inhibited the formation of HA coats when FITC-HA was added to the cultures or when breast cancer cells were co-cultured with MSCs. MSCs also increased proliferation and migration of MDA-MB-468 (parental/Luc) cells, analyzed by luciferin and Transwell migration assays, respectively. MDA-MB-468 cell proliferation was slightly inhibited by removal of HA with pegylated human recombinant hyaluronidase PH20 (PEGPH20), and migration towards exogenous HA could be inhibited by CD44 knockdown. Importance of HA coats around breast cancer cells was also studied in vivo using MDA-MB-468 cells over-expressing HAS3 which forms 4.7-fold larger HA coats than parental MDA-MB-468 cells. MDA-MB-468 HAS3 cells exhibited much enhanced in vivo growth compared to MDA-MB-468 cells, and tumor growth of MDA-MB-468 HAS3 xenografts was inhibited up to 85% by PEGPH20. The results suggest that HA in tumor microenvironment, produced by tumor or stromal cells, provides growth benefit for breast cancer cells via promoting their proliferation and migration. Both phenomena seem to be mediated by CD44, which highlights the importance of HA-CD44 interaction in the growth of breast cancer. Citation Format: Anne Kultti, Susan Zimmerman, Lei Huang, Yanling Chen, Jessica Cowell, Rebecca C. Symons, Laurence Jadin, Ping Jiang, Gregory I. Frost, Michael Shepard, John Huang. The role of hyaluronan-CD44 interaction in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2013-511