PEGylated Recombinant Human Hyaluronidase PH20 Research Articles

PEGylated recombinant human hyaluronidase (PEGPH20) pre-treatment improves intra-tumour distribution and efficacy of paclitaxel in preclinical models

BackgroundScarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs.MethodsWe investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan (hyaluronic acid; HA), one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry.ResultsPre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model.ConclusionRemodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.

Abstract B38: Increasing tumor-infiltrating CD8+ T cell response and checkpoint inhibitor efficacy by enzymatic reduction of tumor hyaluronan in a murine syngeneic pancreatic cancer model

Abstract Inducing an efficient CD8+ T-cell-mediated antitumor immune response at the tumor site is critical for successful immunotherapy. The trafficking and activation of these effector CD8+ T cells to tumors are often inhibited by various immunosuppressive mechanisms in the tumor microenvironment (TME). Hyaluronan (HA), a glycosaminoglycan that accumulates in the TME of many solid tumors, is associated with rapid tumor progression, poor prognosis and increased immunosuppression. We have previously demonstrated in preclinical models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) enhances anti-Programmed death-ligand 1 (anti-PD-L1) efficacy and increases access of immune cells to the tumor (Clift, AACR 2017 poster #641). In this study, we aimed to extend these findings to a murine syngeneic pancreatic cancer model to explore the pan-tumor potential of combining PEGPH20 and anti-PD-L1 to improve anti-tumor immune response. To test this hypothesis in a pancreatic tumor model, Pan02 murine pancreatic adenocarcinoma cells were engineered to over-express hyaluronan synthase-3 (Pan02/Has3). Peritibial Pan02/Has3 tumors accumulated 10-fold higher levels of HA (1668 ng/mg) compared with Pan02 parental tumors (166 ng/mg) as determined by HA ELISA. HA accumulating Pan02/Has3 tumors were treated with combinations of PEGPH20 and/or anti-PD-L1, and tumor growth was monitored. Treatment with PEGPH20 (1mg/kg, biweekly) resulted in nearly complete removal of HA at 24hrs, the time point at which anti-PD-L1 treatment (5mg/kg, biweekly) was initiated. Tumor burden growth was reduced by 50% with combination of PEGPH20 and anti-PD-L1 versus 21% with PEGPH20 alone (p<0.01) or 5% with anti-PD-L1 alone (p<0.01). To elucidate the underlying mechanism, tumor infiltrating immune cells were analyzed by flow cytometry. We observed a significant increase in the numbers of cytotoxic CD8+ tumor infiltrating lymphocytes (TILs) in tumors treated with PEGPH20 alone compared with the vehicle group. Similarly, tumors treated with PEGPH20 in combination with anti-PD-L1 contained higher numbers of CD8+ TILs, CD4+ TILs, and Natural killer (NK) cells, and had increased CD8:Treg ratios compared to anti-PD-L1 treatment alone. Taken together, our data suggest that tumor HA accumulation may act as a barrier to immune cell access in a mouse model of pancreatic cancer, and that enzymatic reduction of HA can facilitate access of CD8+ T cells and enhance anti-PD-L1 efficacy. These findings are consistent with prior studies and support the ongoing clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors to improve anti-tumor efficacy in HA-accumulating tumors. Citation Format: Benjamin Thompson, Trevor Kimbler, Barbara Blouw, Renee Clift, Yujun Huang, Sanna Rosengren, Curtis B. Thompson, Jisook Lee. Increasing tumor-infiltrating CD8+ T cell response and checkpoint inhibitor efficacy by enzymatic reduction of tumor hyaluronan in a murine syngeneic pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B38.

Abstract 2740: PEGylated recombinant hyaluronidase PH20 (pegvorhyaluronidase alfa PEGPH20) converts HA-rich tumors from resistant to sensitive to anti-PD-L1 immunotherapy in murine syngeneic breast cancer models

Abstract Hyaluronan (HA) is an extracellular glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumors. In preclinical studies, enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the TME, reduces tumor interstitial fluid pressure, decompresses tumor blood vessels, and facilitates delivery of chemotherapeutics. PEGPH20-mediated HA degradation increases anti-PD-L1 efficacy in HA-rich EMT-6 tumors and enhances CD8+ T-cell infiltration in tumors. Cancer patients can be resistant to anti-PD-L1; strategies to increase T cell infiltration may lead to anti-PD-L1 effectiveness in nonresponders. We hypothesized that PEGPH20 may convert HA-rich tumors with low T cell infiltration from resistant to sensitive to anti-PD-L1 immunotherapy. 4T1 breast cancer cells transduced with HAS3 to generate HA-rich 4T1/HAS3 cells averaged ~2.9×104 CD8+ T cell/g tumor on day 18 post-tumor inoculation and were resistant to anti-PD-L1 monotherapy. PEGPH20 treatment of 4T1/HAS3 tumors significantly increased CD8+ T cell infiltration to ~7.2×104/g tumor (p=0.0007) and 4T1/HAS3 tumors became sensitive to anti-PD-L1 treatment. PEGPH20 + anti-PD-L1 led to 64.0% tumor growth inhibition (TGI; p<0.0001 vs vehicle + isotype antibody [IgG2b]) vs PEGPH20 alone (10.7% TGI) or anti-PD-L1 alone (-5.1% TGI). PEGPH20 significantly increased infiltration of CD4+ T cells and NK cells, and decreased the percentage of CD11b+GR1+ myeloid derived suppressive cells among CD45+ cells, vs vehicle + IgG2b in 4T1/HAS3 tumors. EMT-6 tumors were sensitive to anti-PD-L1 when starting tumor size was small (~133 mm3) but became resistant with larger starting tumor size (~325 mm3). PEGPH20 + anti-PD-L1 converted larger EMT-6 tumors to sensitive to anti-PD-L1 with 64.9% TGI (p=0.049 vs vehicle + IgG2b), whereas neither PEGPH20 alone nor anti-PD-L1 alone significantly inhibited tumor growth. Small EMT-6 tumors (~136 mm3) were resistant to a lower dose of anti-PD-L1; however, PEGPH20 + low-dose anti-PD-L1 converted EMT-6 tumors from resistant to sensitive to low-dose anti-PD-L1 (65.4% TGI, p<0.0001 vs vehicle + IgG2b). Imaging studies using fluorescence-labeled anti-PD-L1 antibody showed PEGPH20 enhanced the accumulation of anti-PD-L1 in TME. Our data suggest that PEGPH20-mediated HA removal increases infiltration of lymphocytes and immunotherapeutic antibody accumulation in HA-rich tumors. PEGPH20 converts HA-rich tumors from resistant to sensitive to anti-PD-L1. Clinical trials are ongoing to evaluate PEGPH20 + anti-PD-L1 in patients with cholangiocarcinoma/gallbladder cancer (NCT03267940) and gastric cancer (NCT03281369), and to evaluate PEGPH20 + anti-PD-1 in patients with non-small cell lung cancer and gastric cancer (NCT02563548). Citation Format: Renee Clift, Xiaoming Li, Barbara Blouw, Curtis B. Thompson, Yujun Huang. PEGylated recombinant hyaluronidase PH20 (pegvorhyaluronidase alfa PEGPH20) converts HA-rich tumors from resistant to sensitive to anti-PD-L1 immunotherapy in murine syngeneic breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2740.

Abstract 3036: PEGylated hyaluronidase increases tumor uptake of 89Zr-DFO-HuMab-5B1 (MVT-2163) in a CA19-9 positive hyaluronan-accumulating pancreatic cancer model

Abstract Hyaluronan (HA) accumulates in the extracellular matrix of many solid tumors, and is associated with poor prognosis in pancreatic ductal adenocarcinoma. In non-clinical models, enzymatic degradation of HA with PEGylated recombinant human hyaluronidase PH20 (PEGPH20) has been shown to remodel the tumor stroma, reduce tumor interstitial fluid pressure, and expand tumor blood vessels, resulting in enhanced delivery of therapeutic and imaging agents, such as monoclonal antibodies. 89Zr-DFO-HuMab-5B1 (MVT-2163) is a targeted ImmuoPET imaging agent for CA19-9 positive malignancies currently in clinical evaluation. CA19-9 plays a role in tumor adhesion and metastasis, and is an independent prognostic indicator of cancer survival. CA19-9 is expressed in pancreatic and other cancers, including small cell lung and colon. In this study, we aimed to show the effects of PEGPH20 on the biodistribution of MVT-2163 in a CA19-9 positive HA-rich human pancreatic tumor xenograft model. Nude mice were peritibially implanted on the right hind limb with 5x106 human pancreatic tumor cells engineered to overexpress hyaluronan synthase 3 (BxPC3/HAS3). Tumor volumes were measured by MR imaging, and mice were staged when average tumor size reached 320 mm3. The control group received intravenous (IV) vehicle, and the test group received PEGPH20 (IV, 1 mg/kg). Both control and test groups also received MVT-2163 (IV, 3 mg/kg) 24 hours later. PET images were captured from 2 to 120 hours post MVT-2163, and region of interest (ROI) of tumor and liver were analyzed (N=6/group). After the final PET image, tumors and livers were harvested for ex vivo gamma counting. Ex vivo gamma counting demonstrated improved MVT-2163 accumulation in animals treated with PEGPH20, with a 50.9% increase in counts in the excised tumors. ROI analysis of the tumors showed increased tumor SUV of 8.0, 9.4, and 24.1% at 72, 96, and 120 hours post-injection, respectively, with PEGPH20 compared to vehicle. ROI analysis of liver regions showed an 11.4 to 26.4% reduction in SUV in the PEGPH20 group compared to control. Ex vivo counts of liver tissue confirmed the PET signal reduction with PEGPH20. Analysis of tumor-to-liver ratios showed average increases of 34.1, 35.7, and 58.5% at 72, 96, and 120 hours, respectively, post MVT-2163 injection in PEGPH20 treated mice. In summary, as measured by SUV, PEGPH20 increased both the tumor uptake and the tumor-to-liver ratios of MVT-2163 in a CA19-9 positive xenograft mouse model of HA accumulating pancreatic cancer. Ex vivo analysis confirmed in vivo results. Taken together, the increased tumor uptake and the decreased liver uptake support further investigation into the potential clinical utility for the combination of PEGPH20 and MVT-2163. Citation Format: Jonah Rainey, Paul Maffuid, Wolfgang W. Scholz, Jack Ostrowski, H Toni Jun, Paul Resnick, Xiaoming Li, Jesse D. Bahn, Susan Zimmerman, Kelly Chen, Barbara Blouw, Curtis B. Thompson, Daniel C. Maneval, David W. Kang. PEGylated hyaluronidase increases tumor uptake of 89Zr-DFO-HuMab-5B1 (MVT-2163) in a CA19-9 positive hyaluronan-accumulating pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3036.

774TiP - Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma (PDA)

774TiP - Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma (PDA)

Abstract CT066: Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma

Abstract Background: Poor outcome in pancreatic ductal adenocarcinoma (PDA) is associated partly with stromal hyaluronan (HA) accumulation, which may compromise chemotherapy access to tumors. In animal models, PEGPH20 degrades HA in tumors. Key endpoint data from a phase 2 study showed that PEGPH20 plus chemotherapy improved efficacy over chemotherapy alone in tumors retrospectively identified to accumulate HA (“HA-High”). The objectives of this phase 3 study are to compare efficacy and safety of standard-dose nab-paclitaxel (NAB) and gemcitabine (GEM) combined with either PEGPH20 or placebo in patients with HA-High, previously untreated, Stage IV PDA. Primary endpoints are progression-free survival (PFS) and overall survival (OS). Secondary endpoints are objective response rate, duration of response, and safety. Methods: 420 patients ≥18 years with untreated HA-High metastatic PDA, ECOG PS 0-1 will be randomized (stratified by geographic region: North America/Europe/Other) 2:1 to NAB 125 mg/m2 + GEM 1000 mg/m2 + PEGPH20 3.0 μg/kg or to placebo. Patients with HA-High tumors will be prospectively identified by the co-developed VENTANA HA RxDx Assay, which indicates HA in the extracellular matrix. HA-High status (indicating patients who may achieve clinical benefit) was determined by Halozyme to be ≥50% staining based on clinical outcome data from a phase 2 study. Treatment will be provided in 4 week cycles (Wk 13, Wk 4 rest) until disease progression, unacceptable toxicity, death, or consent withdrawal. PEGPH20 or placebo will be given twice weekly (Cycle 1) then weekly (Cycles 2+), NAB + GEM once weekly for all cycles. Dexamethasone will be given before and after PEGPH20 administrations to reduce treatment related musculoskeletal symptoms and enoxaparin will be given to minimize thromboembolic events. Tumor response will be assessed by an independent central imaging vendor using RECIST v1.1. Adverse events will be graded per NCI CTCAE v4.03. An independent Data Monitoring Committee will evaluate safety and data for PFS and OS. Trial initiated Q12016 (EudraCT 2015-004068-13; NCT02715804). Citation Format: Lei Zheng, Andrew E. Hendifar, Michele Reni, William P. Harris, Michel Ducreux, Andrea J. Bullock, Pippa G. Corrie, Tara Seery, Dimitrios Chondros, Eric Van Cutsem. Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma [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 CT066. doi:10.1158/1538-7445.AM2017-CT066

Global ph 3, randomized, double-blind, placebo-controlled study of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) + nab-paclitaxel & gemcitabine in pts with previously untreated, hyaluronan-high, stage iv pancreatic ductal adenocarcinoma

Global ph 3, randomized, double-blind, placebo-controlled study of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) + nab-paclitaxel & gemcitabine in pts with previously untreated, hyaluronan-high, stage iv pancreatic ductal adenocarcinoma

Hyaluronan assessment in tumor microenvironment using new affinity histochemistry assay and scoring method.

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.

715TiP - Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma

715TiP - Global phase 3, randomized, double-blind, placebo-controlled study evaluating PEGylated recombinant human hyaluronidase PH20 (PEGPH20) plus nab-paclitaxel and gemcitabine in patients with previously untreated, hyaluronan (HA)-high, stage IV pancreatic ductal adenocarcinoma

Abstract 2463: PEGPH20 increases the anticancer activity of standard chemotherapy combinations, vincristine (VIN) and D actinomycin (DACT), in a Wilms’ xenograft model

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.

Abstract 283: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances pemetrexed antitumor efficacy in a human nonsquamous NSCLC xenograft model

Abstract 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 <9%. Staining of A549/HAS3 tumors showed increased accumulation of HA as compared with the parental A549 xenografts. 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.

Abstract P1-03-09: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances efficacy of eribulin mesylate (HALAVEN®) in triple negative breast cancer xenografts

Abstract Hyaluronan (hyaluronic acid, HA), a glycosaminoglycan found in tissue throughout the body, overaccumulates in the tumor microenvironment (TME) of many non-hematologic malignancies, including breast cancer. HA overaccumulation in breast cancer patients correlates with tumor progression and decreased survival (Tammi 2008). Pegylated recombinant human hyaluronidase PH20 (PEGPH20), an investigational therapeutic agent entering Phase 3 clinical development in pancreatic cancer, enzymatically removes HA from the TME. In preclinical animal models, PEGPH20-mediated HA degradation is associated with remodeling of the tumor stroma, reduction of tumor pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy (Thompson 2010, Provenzano 2012, Jacobetz 2013). Accordingly, preclinical studies investigated the combination of PEGPH20 with eribulin mesylate (ERI, HALAVEN®), a microtubule dynamics inhibitor with a novel mechanism of action (Towle 2001, Jordan 2005), currently approved for treatment of certain patients with advanced breast cancer. NCr nu/nu mice were inoculated subcutaneously with human triple-negative breast cancer (TNBC) HCC1806 or HCC1806/HAS3 cells; the latter subline was engineered to accumulate high HA levels, confirmed by immunohistochemistry, via overexpression of hyaluronan synthase 3 (HAS3). When tumors reached ∼350 mm3, animals were randomly assigned to four treatments groups: vehicle, ERI (0.7 mg/kg, IV, QW), PEGPH20 (37.5 µg/kg, IV, BIW), or ERI plus PEGPH20. In the parental HCC1806 model, addition of PEGPH20 did not significantly change the antitumor effects of ERI. In contrast, combining PEGPH20 with ERI in the HCC1806/HAS3 model increased the antitumor effects of ERI by 27% (94.5% vs. 119.7% TGI, ERI alone vs. ERI+PEGPH20, respectively; p=0.05) and resulted in 6 of 7 complete tumor regressions. In a complementary study in HCC1806/HAS3 tumors evaluating ERI pharmacokinetics with and without PEGPH20, mice were assigned to three treatments groups: ERI (0.5 mg/kg, IV), simultaneous ERI plus PEGPH20 (37.5 µg/kg, IV); or ERI plus PEGPH20 predosed 24 h prior to ERI. Animals were sacrificed at 0.5, 1, 4, 24, 48, 72 and 96 h post ERI dose, and ERI levels in tumor, muscle, plasma and liver were subsequently analyzed by liquid/liquid extraction and LC-MS/MS chromatography. Simultaneous administration of ERI and PEGPH20 increased ERI maximum tumor concentration (Cmax) slightly and approximately doubled ERI tumor exposure (AUC); whereas the 24 h pretreatment with PEGPH20 approximately doubled ERI Cmax and increased ERI AUC more than two-fold. No significant differences in plasma ERI levels were observed between groups, and no significant differences in ERI levels in liver or muscle tissue were observed between groups. Taken together, these data suggest that PEGPH20-mediated HA removal significantly increases both ERI tumor concentrations and antitumor effectiveness in an HA-high TNBC model. A clinical phase 1b/2 clinical trial is planned to evaluate PEGPH20 plus ERI in first-line HER2-negative metastatic breast cancer. Citation Format: Bahn JD, DesJardins C, Condon KB, Fathallah A, Zimmerman S, Maneval DC, Littlefield BA, Thompson CB. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances efficacy of eribulin mesylate (HALAVEN®) in triple negative breast cancer xenografts. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-09.

Abstract 2547: PEGPH20 enhances chemotherapy in patient-derived and traditional cell-derived xenograft NSCLC models

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

Abstract B86: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances nab-paclitaxel plus gemcitabine efficacy in human pancreatic cancer xenografts

Abstract Hyaluronan (HA) over-accumulation in the extracellular matrix (ECM) of many solid tumors is associated with tumor progression and poor prognosis. Accordingly, an HA-degrading enzyme, PEGPH20, is being developed to deplete tumor-associated HA in the ECM. In preclinical animal models, enzymatic removal of ECM HA with PEGPH20 is associated with remodeling of the tumor stroma, reduction of tumor interstitial fluid pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy. As pancreatic ductal adenocarcinoma (PDA) has been identified as a cancer type that accumulates high levels of HA, and the combination of albumin-bound paclitaxel (nab-paclitaxel, NAB) and gemcitabine (GEM) has been shown to improve survival in patients with metastatic pancreatic cancer (Von Hoff 2013), preclinical studies were conducted using human PDA xenograft models to investigate whether PEGPH20 enhanced the anti-tumor activity of NAB and GEM. In brief, nude mice were inoculated with human BxPC3 PDA cells, or with HA-high BxPC3/HAS3 cells, a cell line engineered to over-express HA, adjacent to the right tibial periosteum. Tumor growth was monitored via ultrasonography. When tumors reached ~350 mm3, mice were staged into 8 treatment groups: (1) vehicle control; (2) PEGPH20 alone, 4.5 mg/kg; (3) NAB alone, 10 mg/kg; (4) GEM alone, 180 mg/kg; (5) NAB plus PEGPH20; (6) GEM plus PEGPH20; (7) NAB plus GEM; and (8) NAB plus GEM plus PEGPH20. Vehicle, PEGPH20 and/or NAB was administered intravenously starting on study day 0, and then dosed twice weekly for four weeks. GEM was administered intraperitoneally starting on study day 1 and then dosed weekly for three weeks. Histology of tumors confirmed HA removal in all PEGPH20 treated groups. In the parental BxPC3 model, the addition of PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 15% (81% vs. 66%, respectively) and extended median survival time (MST) by >31% (68d vs. 52d, respectively); whereas in the HA-high BxPC3/HAS3 model, PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 34% (104% vs. 70%) and extended MST by >125% (72d vs. 32d, respectively). As Jacobetz et al. (2013) have shown that PEGPH20 treatment increases the intratumoral delivery of GEM in genetically engineered mouse PDA models; we evaluated whether PEGPH20 also improves intratumoral accumulation of paclitaxel in the BxPC3/HAS3 model vs. NAB alone. The co-administration of PEGPH20 with NAB increased intratumoral paclitaxel accumulation by >43% one hour following a single administration. Further, PEGPH20 enhanced NAB plus GEM mediated reduction in the PDA serum biomarkers CA19-9 and CEA in both models. As KRAS mutations occur in 90% of non neuro-endocrine pancreatic tumors (Thomas 2007), and BxPC3 cells are KRAS WT, we repeated these studies in KRAS mutant/HA-low AsPC1 xenografts, and engineered KRAS mutant/HA-high AsPC1/HAS3 PDA xenografts. Mice were inoculated and tumor growth was monitored as described above. Animals were then staged into 8 groups and then dosed when tumors reached ~350 mm3. The PEGPH20 dose in these studies was reduced to 37.5 μg/kg to approximate dosing in ongoing clinical trials (NCT01839487). As predicted by tumor HA level, the addition of PEGPH20 did not increase the anti-tumor efficacy or survival in the HA-low parental AsPC1 model. In the HA-high AsPC1/HAS3 model, the addition of PEGPH20 increased the anti-tumor efficacy of NAB plus GEM by 25% (78% vs. 53%, respectively) and extended MST by >31% (74d vs. 48d, respectively). Taken together, the data suggest that PEGPH20-mediated HA removal significantly increases the anti-tumor efficacy of NAB plus GEM in HA-high mouse models of PDA. Clinical trials are currently ongoing to evaluate PEGPH20 plus NAB plus GEM in stage IV patients with metastatic pancreatic cancer (NCT01839487). Citation Format: Ryan J. Osgood, James F. Skipper, Jessica A. Cowell, Yanling Chen, Li Zhu, Michael E. Bledsoe, Susan J. Zimmerman, David W. Kang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances nab-paclitaxel plus gemcitabine efficacy in human pancreatic cancer xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B86.

Abstract P4-04-25: Naked mole rat hyaluronan synthase 2 displays similar effects as human hyaluronan synthase 2 and promotes tumor growth in a mouse xenograft model

Abstract Accumulation of hyaluronan (HA) is correlated with poor prognosis in many human cancer including breast cancer and pancreatic cancer. Hyaluronan synthase 2 (HAS2) is one of the three HA synthase genes (HAS1-3) found in mammals. Up-regulation of the HAS2 gene has been observed in breast cancer patients; and HAS2 expression correlates with the incidence of metastasis and lower rate of overall patient survival (Okuda et al 2012). Interestingly, a recent study suggested that the naked mole rat (nmr) HAS2 enzyme may function differently than the HAS2 proteins from mice and humans, and its expression had a tumor suppressor effect (Tian et al 2013). The nmrHAS2 protein sequence was distinct from other mammals in the cytoplasmic loop where the active site resides, which led to the hypothesis that nmrHAS2 may contribute to the production of extremely high molecular weight HA species. Furthermore, nmrHAS2 is required for the resistance of nmr skin fibroblasts to malignant transformation. In this study, we examined whether the nmrHAS2 gene can also render human cells resistant to tumor progression. We have previously observed that HA level in the pancreatic cancer model AsPC1 can be modulated by the over-expression of the human HAS3 protein (Osgood et al 2014). To compare and contrast the effect of nmrHAS2 and huHAS2 on HA production and tumor growth, nmrHAS2 and huHAS2 AsPC1 models were engineered using a recombinant lentivirus encoding the nmrHAS2 and huHAS2 genes respectively. Over-expression of nmrHAS2 and huHAS2 both significantly increased the HA level in AsPC1 cells. Both nmrHAS2 and huHAS2 promoted the growth of AsPC1 in an intramuscular xenograft tumor model. Ex vivo analysis of tumor xenografts showed that nmrHAS2 and huHAS2 AsPC1 tumors contain elevated levels of HA, and the size range of the HA in the nmrHAS2 tumors is similar to that in the huHAS2 AsPC1 tumors. Therefore, nmrHAS2 functions similarly as huHAS2 gene and promotes tumor growth in the human pancreatic tumor model AsPC1.

Abstract 3646: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances cetuximab efficacy in BxPC-3/HAS3 human pancreatic cancer xenografts

Abstract Hyaluronan (HA) over-accumulation in the extracellular matrix (ECM) of many solid tumors is associated with tumor progression and poor prognosis (Tammi 2008). In preclinical animal models, enzymatic removal of ECM HA with pegylated recombinant human hyaluronidase PH20 (PEGPH20) is associated with remodeling of the tumor stroma, reduction of tumor interstitial fluid pressure, expansion of tumor blood vessels and facilitated delivery of chemotherapy (Thompson 2010, Jacobetz 2012, Provenzano 2012). Additionally, epidermal growth factor receptor (EGFR), a tyrosine kinase essential for cell division and tumor growth, has been implicated in multiple epithelial malignancies and is over expressed in ∼60% of human pancreatic carcinomas (Frolov 2007). Cetuximab (CET), a chimeric monoclonal antibody (mAb), targets EGFR preventing tyrosine kinase mediated phosphorylation and subsequent signal transduction (Enrique 2012). As pancreatic ductal adenocarcinoma (PDA) has been identified as a cancer type that expresses high levels of HA (∼87%; Jiang 2010), studies were conducted to evaluate PEGPH20 enhancement in anti-tumor activity of CET in an EGFR positive HA-overexpressing PDA BxPC3/HAS3 xenograft model (Kultti 2013). In brief, NCr nu/nu mice were inoculated with PDA BxPC3/HAS3 cells adjacent to the tibial periosteum, and tumor growth was monitored with 3D high resolution ultrasonography. When tumors reached ∼200 mm3, mice were treated with: (1) vehicle control; (2) PEGPH20 alone, 1 mg/kg; (3) CET alone, 0.03 mg; (4) CET alone, 0.1 mg; (5) PEGPH20 plus CET, 0.03 mg; or (6) PEGPH20 plus CET, 0.1 mg. Vehicle control or PEGPH20 was given intravenously while CET was administered intraperitoneally starting on study day 0, and then dosed twice weekly for 3 weeks (BIWx3). At study termination, the average tumor growth inhibition (TGI) of CET (0.03 mg) was not significantly different from vehicle-treated animals; however, PEGPH20 alone (78%, p<0.05) and CET alone at 0.1 mg (61%, p<0.05) inhibited tumor growth. The addition of PEGPH20 to the 0.03 mg and 0.1 mg CET groups increased TGI to 88% (p<0.05) for both treatments, relative to vehicle. In a second study, when tumors reached ∼200 mm3, mice were treated with: (1) vehicle control; (2) PEGPH20 alone, 37.5 µg/kg (3 µg/kg human equivalent dose); (3) CET alone, 0.03 mg; or (4) PEGPH20 plus CET. Animals were dosed as described above. At study termination, the average TGI of CET alone was not significantly different from vehicle-treated animals; however, PEGPH20 alone significantly inhibited tumor growth (47%, p<0.05). The combination of PEGPH20 and CET increased TGI to 70% (p<0.05) relative to vehicle. In conclusion, PEGPH20 treatment of HA-overexpressing tumors potentiates the subsequent anti-tumor activity of mAbs, such as CET. Citation Format: Ryan J. Osgood, James F. Skipper, Susan Zimmerman, Rebecca C. Symons, Harold M. Shepard, Daniel C. Maneval, Curtis B. Thompson, David W. Kang. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances cetuximab efficacy in BxPC-3/HAS3 human pancreatic cancer xenografts. [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 3646. doi:10.1158/1538-7445.AM2014-3646

Abstract 4955: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumor perfusion in mouse xenografts and phase 1 cancer patients.

Abstract Pegylated recombinant human hyaluronidase PH20 (PEGPH20) is an investigational therapeutic agent under clinical development for use alone or in combination with other cancer therapies for the treatment of patients with non-hematologic malignancies that may accumulate hyaluronan (HA), a glycosaminoglycan that is a significant component of the extracellular matrix of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, decreases tumor interstitial pressure, increases vascular area, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. As dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a clinically translatable MRI perfusion protocol, we used DCE-MRI imaging to evaluate the effects of PEGPH20 treatment on tumor vascular permeability, vascular surface area and/or blood flow. In mice, prostate cancer PC3 cells were implanted adjacent to the right tibial periosteum. When tumors reached ∼500 mg, animals were staged into vehicle or PEGPH20 (4.5 mg/kg) treatment groups. Utilizing gadopentetate dimeglumine (Gd-DTPA) as a contrast agent, pre-treatment baseline DCE scans of the tumors were acquired. Animals were subsequently IV dosed with test article and post-treatment scans were acquired at 24 h. Initial area under the curve (IAUC) and Ktrans were calculated voxel-by-voxel using a 2-compartment PK model. Although vehicle alone did not increase or decrease Ktrans or IAUC, PEGPH20 treatment increased Ktrans (1/sec) and IAUC (mmol.s) by 74% and 78.7%, respectively. Concurrent to preclinical studies, DCE-MRI imaging was conducted pre- and post- PEGPH20 in 9 patients in a Phase 1 dose-escalation, safety, tolerability, PK/PD study of PEGPH20 treatment in patients with advanced solid tumors. Patients were treated with 1.6, 3 or 5 μg/kg PEGPH20 1-2/weekly. Baseline, day 1, day 2-5 and end-of-cycle (EOC) scans were acquired (w/ Gd-DTPA) in 3 patients. Images were fit on a pixel-by-pixel basis to a 2-compartment, 3-parameter, PK model and Ktrans and extracellular volume (Ve) calculated. Results suggest an early and rapid increase in tumor perfusion in target lesions, compared to baseline (mean ΔKtrans = 428%; N=3; Day 2-5 scans), as well as an expansion of the extracellular space with a reduction of these parameters towards baseline by EOC (mean ΔKtrans = 28% above baseline). Taken together, these studies suggest that PEGPH20 increases vessel permeability, blood flow and/or vessel surface area in HA-rich solid tumors. Additional preclinical and clinical studies, complete with DCE-MRI imaging, are ongoing to further evaluate PEGPH20 in the treatment HA-rich malignancies. Citation Format: Curtis B. Thompson, Patrick McConville, Ron Korn, Jenifer Baranski, Meridith Baugher, Deanne Lister, Erin Trachet, Jacob Y. Hesterman, Jack Hoppin, Joy Zhu, Daniel C. Maneval. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) increases tumor perfusion in mouse xenografts and phase 1 cancer patients. [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 4955. doi:10.1158/1538-7445.AM2013-4955

Abstract 3915: Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc.

Abstract A new investigational therapeutic agent, pegylated recombinant human hyaluronidase PH20 (PEGPH20) is a currently under clinical development for the treatment of tumors that accumulate hyaluronan (HA), a nonsulfated glycosaminoglycan and a major constituent of the extracellular matrix (ECM) of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival in multiple malignancies. Preclinical studies have demonstrated that sustained HA removal, accomplished with PEGPH20, inhibits tumor growth and enhances chemotherapeutic activity in HA-rich xenografts and genetically engineered mouse tumor models. To facilitate non-invasive clinical selection of patients with HA-rich malignancies, and confirm PEGPH20 enzymatic targeting of these malignancies, we have developed a soluble recombinant HA binding protein by fusing a tumor necrosis factor-stimulated gene-6 protein (TSG6) fragment, mutated at the heparin-binding site, with human IgG1 Fc (TSG6dHep-Fc). In preclinical proof-of-concept studies, recombinant TSG6dHep-Fc was labeled with the near-infrared fluorophore DyLight755 (TSG6dHep-FcDL755) and administered IV to the following xenograft peritibial athymic mouse models: HA-rich pancreatic BxPC3, HA-poor prostate Du145 and HA-rich prostate Du145/HAS2. TSG6dHep-FcDL755 HA binding was imaged via fluorescence (IVIS Lumina II, Caliper Life Sciences, Inc.). Systemic/dermal HA binding was weakly present, but easily removed via background subtraction to allow tumor intensity quantification. In the HA-rich xenograft models, pancreatic BxPC3 and prostate Du145/HAS2, TSG6dHep-FcDL755 strongly labeled the peritibial tumors, peaking at 2 days post administration and was completely absent at day 10 post administration. In contrast, the HA-poor prostate Du145 tumors were very weakly labeled, making background subtraction of systemic/dermal HA binding difficult. In separate experiments, mice were treated with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle prior to TSG6dHep-FcDL755 imaging. In all groups dosed with PEGPH20, TSG6dHep-FcDL755 signal intensity was extremely low and appeared to be limited to non-specific, whole body labeling. These results suggest that imaging with labeled TSG6dHep-Fc could be a useful tool for selecting patients with HA-rich malignancies, thereby enabling clinicians to noninvasively identify patients who might benefit from therapies that target HA and the ECM. Citation Format: Xiaoming Li, Lei Huang, Ge Wei, Ryan J. Osgood, Qiping Zhao, Ping Jiang, H. Michael Shepard, Daniel C. Maneval, Curtis B. Thompson. Tumor-targeted hyaluronan (HA) imaging with a recombinant HA binding protein: TSG6dHep-Fc. [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 3915. doi:10.1158/1538-7445.AM2013-3915

Abstract 5635: Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances Nab-Paclitaxel efficacy in BxPC-3 human pancreatic cancer xenografts

Abstract Hyaluronan (HA) accumulates in the extracellular matrix (ECM) of many solid tumors, including those of the prostate, colon, breast, stomach, ovary, and pancreas. This accumulation is associated with tumor progression and a negative clinical outcome. Accordingly, an HA-degrading enzyme, pegylated recombinant human hyaluronidase PH20 (PEGPH20), was developed to target tumor-associated HA in the ECM. Preclinical studies demonstrated that PEGPH20-mediated removal of HA from HA-rich xenograft tumors in mice decreased tumor interstitial fluid pressure and tumor water content resulting in a decompression of tumor vasculature, increased tumor vascular perfusion, tumor growth inhibition (TGI) and enhanced chemotherapeutic activity (Thompson 2010). We further characterized HA expression across multiple human tumor types and identified pancreatic ductal adenocarcinoma (PDA) as the cancer type with the most HA (∼87% express high levels). These observations coupled with a lack of curative therapy for PDA led us to evaluate alternative treatment strategies for PDA. Specifically, using the peritibial BxPC-3 human pancreatic cancer xenograft model, we investigated whether the antitumor activity of Nab-Paclitaxel was significantly enhanced with PEGPH20 treatment. Nude mice were inoculated with human PDA BxPC-3 cells adjacent to the right tibial periosteum, and tumor growth was monitored with ultrasonography. When tumors reached ∼400 mm3 (n≥8/group), mice were staged into 8 treatment groups: (1) vehicle control; (2) PEGPH20 monotherapy, 4.5 mg/kg; (3) Nab-Paclitaxel, 3 mg/kg; (4) Nab-Paclitaxel, 10 mg/kg; (5) Nab-Paclitaxel, 30 mg/kg; (6) Nab-Paclitaxel, 3 mg/kg, plus PEGPH20; (7) Nab-Paclitaxel, 10 mg/kg, plus PEGPH20; or (8) Nab-Paclitaxel, 30 mg/kg, plus PEGPH20. Vehicle or PEGPH20 ± Nab-Paclitaxel was administered intravenously starting on study day 0, and then dosed every third day for 15 days. At study termination, the average TGIs from animals treated with either PEGPH20 alone (12.2%), low dose (3 mg/kg) Nab-Paclitaxel (20.3%), or low dose Nab-Paclitaxel (3 mg/kg) plus PEGPH20 (25%) were not significantly different from vehicle-treated animals. However, Nab-Paclitaxel alone at both 10 mg/kg (61.5%, p<0.01) and 30 mg/kg (73.6%, p<0.01) inhibited tumor growth. High doses of Nab-Paclitaxel (30 mg/kg) ± PEGPH20 was associated with dose-related toxicity. The addition of PEGPH20 to the 10 mg/kg and 30 mg/kg Nab-Paclitaxel groups increased TGI to 71.7% (p<0.01), and 90.8% (p<0.01), respectively, relative to vehicle. These findings suggest that PEGPH20 significantly increases the anti-tumor efficacy of Nab-Paclitaxel in HA-rich PDA tumors at moderate to high Nab-Paclitaxel doses (10 and 30 mg/kg). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5635. doi:1538-7445.AM2012-5635

Abstract 2501: PEGylated human recombinant hyaluronidase (PEGPH20) enhances radiotherapy in hyaluronan-rich solid tumors

Abstract The accumulation of the glycosaminoglycan hyaluronan (HA) in the extracellular matrix (ECM) of solid tumors contributes to increased tumor interstitial fluid pressure IFP), localized edema and reduced intratumoral blood flow. We recently reported that sustained enzymatic depletion of tumor HA with a pegylated recombinant human hyaluronidase PH20 (PEGPH20) decreases tumor IFP and water content, while significantly increasing vascular perfusion within HA-rich prostate PC3 tumors (Thompson et al, 2010). As localized tissue hypoxia reduces the effectiveness of radiation therapy (Baumgartner et al, 1998), we hypothesized that PEGPH20 treatment would reduce tumor hypoxia, thereby radio-sensitizing tumor cells. To evaluate whether PEGPH20 sensitizes HA-rich tumors, radiotherapy resistant human prostate PC3 cells (2×106) were inoculated into the right tibia periosteum of nude mice (NCR nu/nu) and tumor growth monitored with a high resolution 3D ultrasound. When the tumors reached ∼800 mm3 (n ≥ 8/group), mice were staged into 4 treatment groups: (1) vehicle control; (2) mono-radiotherapy at 2.5 Gy, q3d; (3) PEGPH20 monotherapy, 4.5 mg/kg, i.v. q3d; or (4) PEGPH20 plus radiotherapy. To increase intra-tumoral blood flow, PEGPH20 was administrated 3 hours prior to radiation therapy. Histological evaluation of a subset of mice confirmed that HA was completely degraded 3 hours post- PEGPH20 treatment. At study Day 15, tumor growth inhibition (TGI) with radiation alone or PEGPH20 alone was 99.2% (p=0.009) and 71.4% (p=0.08), respectively, relative to vehicle treated animals. PEGPH20 plus radiotherapy inhibited tumor growth by 140.6% (p=0.0002), relative to vehicle treated animals. The mean tumor size of the PEGPH20 plus radiotherapy group was reduced to ∼550 mm3, with 3-out-of-8 partial regressors (i.e. tumor size decrease to 50% or less of tumor staging size). These findings suggest that PEGPH20-mediated HA removal, and subsequent increase in tumoral blood flow, may increase the efficacy of radiation therapy in hyaluronan-rich tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2501. doi:10.1158/1538-7445.AM2011-2501