Pegvorhyaluronidase Alfa Research Articles

Investigating the contribution of hyaluronan to the breast tumour microenvironment using multiparametric MRI and MR elastography.

Hyaluronan (HA) is a key component of the dense extracellular matrix in breast cancer, and its accumulation is associated with poor prognosis and metastasis. Pegvorhyaluronidase alfa (PEGPH20) enzymatically degrades HA and can enhance drug delivery and treatment response in preclinical tumour models. Clinical development of stromal-targeted therapies would be accelerated by imaging biomarkers that inform on therapeutic efficacy in vivo. Here, PEGPH20 response was assessed by multiparametric magnetic resonance imaging (MRI) in three orthotopic breast tumour models. Treatment of 4T1/HAS3 tumours, the model with the highest HA accumulation, reduced T1 and T2 relaxation times and the apparent diffusion coefficient (ADC), and increased the magnetisation transfer ratio, consistent with lower tissue water content and collapse of the extracellular space. The transverse relaxation rate R2 * increased, consistent with greater erythrocyte accessibility following vascular decompression. Treatment of MDA-MB-231 LM2-4 tumours reduced ADC and dramatically increased tumour viscoelasticity measured by MR elastography. Correlation matrix analyses of data from all models identified ADC as having the strongest correlation with HA accumulation, suggesting that ADC is the most sensitive imaging biomarker of tumour response to PEGPH20.

Blood-based extracellular matrix biomarkers as predictors of survival in patients with metastatic pancreatic ductal adenocarcinoma receiving pegvorhyaluronidase alfa

BackgroundExtensive extracellular matrix (ECM) remodeling is a hallmark of metastatic pancreatic ductal adenocarcinoma (mPDA). We investigated fragments of collagen types III (C3M, PRO-C3), VI (PRO-C6), and VIII (C8-C), and versican (VCANM) in plasma as biomarkers for predicting progression-free survival (PFS) and overall survival (OS) in patients with mPDA treated with pegvorhyaluronidase alfa, a biologic that degrades the ECM component hyaluronan (HA), in a randomized phase 2 study (HALO109-202).MethodsHALO109-202 comprised a discovery cohort (Stage 1, n = 94) and a validation cohort (Stage 2, n = 95). Plasma ECM biomarkers were analyzed by ELISAs. Univariate Cox regression analysis and Kaplan–Meier plots evaluated predictive associations between biomarkers, PFS and OS in patients treated with pegvorhyaluronidase alfa plus nab-paclitaxel/gemcitabine (PAG) versus nab-paclitaxel/gemcitabine (AG) alone.ResultsPFS was improved with PAG vs. AG in Stage 1 patients with high C3M/PRO-C3 ratio (median cut-off): median PFS (mPFS) 8.0 vs. 5.3 months, P = 0.031; HR = 0.40; 95% CI 0.17–0.92). High C3M/PRO-C3 ratio was validated in Stage 2 patients by predicting a PFS benefit of PAG vs. AG (mPFS: 8.8 vs. 3.4 months, P = 0.046; HR = 0.46; 95% CI 0.21–0.98). OS was also improved in patients with high C3M/PRO-C3 ratio treated with PAG vs. AG (mOS 13.8 vs 8.5 months, P = 0.009; HR = 0.35; 95% CI 0.16–0.77). Interestingly, high C3M/PRO-C3 ratio predicted for a PFS benefit to PAG vs. AG both in patients with HA-low tumors (HR = 0.36; 95% CI 0.17–0.79) and HA-high tumors (HR = 0.20; 95% CI 0.06–0.69).ConclusionsThe C3M/PRO-C3 ratio measuring type III collagen turnover in plasma has potential as a blood-based predictive biomarker in patients with mPDA and provides additional value to a HA biopsy when applied for patient selection.Trial registration: NCT01839487. Registered 25 April 2016

Abstract IA-08: Clinical advances in pancreas adenocarcinoma

Abstract IA-08: Clinical advances in pancreas adenocarcinoma

Abstract 2206: Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model

Abstract The extracellular matrix (ECM) structure heavily influences tumor growth, invasion and various steps of the cancer-immunity cycle. Hyaluronan (HA), a major component of ECM, is a glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumor types, and HA accumulation can restrict T cell infiltration and promote immunosuppression. We have demonstrated in preclinical tumor models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (Pegvorhyaluronidase alfa, PEGPH20) facilitates delivery of chemotherapeutics to tumors and enhances efficacy of current immunotherapy strategies including checkpoint inhibitors. In this study, we hypothesized that degradation of HA with PEGPH20 treatment may remodel the TME to improve the efficacy of oncolytic reovirus therapy (pelareorep) and checkpoint blockade therapy. To test this hypothesis, EMT6 murine breast tumor cells, which spontaneously accumulate HA and are sensitive to oncolytic activity of pelareorep were implanted orthotopically into the mammary fat pad of BALB/c mice. In this model, PEGPH20 treatment resulted in a reduction of HA measured by immunohistochemistry and induced tumor growth inhibition (30% TGI). Pelareorep plus anti-PD-L1 treatment also resulted in anti-tumor activity (44% TGI), and flow cytometric analysis of tumor infiltrating immune cells in pelareorep + anti-PD-L1-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor infiltrating lymphocytes (TILs, p=0.0004), a decrease in regulatory T cells (T reg, p<0.0001) and an increase in CD8:Treg ratio (p=0.0018), indicating a robust antitumor immune response. Combination of PEGPH20 with pelareorep + anti-PD-L1 resulted in enhanced anti-tumor activity (71% TGI) compared to either PEGPH20 (30%TGI) or pelareorep + anti-PD-L1 (44% TGI). PEGPH20 in combination with pelareorep + anti-PDL1 also extended survival (median survival time, MST- 30 days, p<0.0001) in comparison with PEGPH20 alone (MST-21 days) or pelareorep + anti-PD-L1 (MST-22 days). Our data demonstrate the potential for matrix modeling through degradation of HA as a strategy to improve oncolytic reovirus + anti-PD-L1 immunotherapy. These findings are consistent with prior preclinical studies and support clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors in HA-accumulating tumors. Citation Format: Jisook Lee, Grey A. Wilkinson, Trevor Kimbler, Barbara Blouw, Curtis B. Thompson. Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2206.

Abstract A46: Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model

Abstract T-cell migration and infiltration in solid tumors is a critical step for maximizing the efficacy of many cancer immunotherapies. Hyaluronan (HA) is a glycosaminoglycan that accumulates in the extracellular matrix (ECM) of a range of solid tumors, including pancreatic ductal adenocarcinoma (PDA). PEGylated recombinant human hyaluronidase PH20, pegvorhyaluronidase alfa (PEGPH20; PVHA), is a novel, first-in-class biologic that enzymatically degrades HA in the tumor microenvironment (TME) and is in clinical development in combination with chemotherapy for the treatment of HA-accumulating tumors. Previously, in a murine PDA tumor model, we demonstrated that accumulation of HA restricts T-cell infiltration and promotes immunosuppression. In the present study, we hypothesized that degradation of HA by PEGPH20 would increase access of CD8+ T cells to the tumor. To test this hypothesis, Pan02 murine PDA cells were engineered to overexpress hyaluronan synthase 3 (Pan02-HAS3) and were implanted orthotopically into the pancreas. In Pan02-HAS3 tumors, HA accumulation was 53-fold higher (2606 ng/mg) compared with Pan02 parental tumors (50 ng/mg) as determined by HA ELISA. Degradation of HA in Pan02-HAS3 tumors with intravenous PEGPH20 as a single agent inhibited tumor growth by 58% at a dose of 0.0375 mg/kg and 73% at a dose of 1 mg/kg. Flow cytometric analysis of tumor-infiltrating immune cells in PEGPH20-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor-infiltrating lymphocytes and natural killer (NK) cells versus control tumors. However, no changes in CD8+ T-cell proliferation were detected, as measured by Ki67 expression or bromodeoxyuridine (BrdU) incorporation. Degradation of HA in the TME was also associated with an increased frequency of CD103+ dendritic cells and a decreased frequency of immunosuppressive CD206+ tumor-associated macrophages. These data support prior reports that accumulation of HA in the TME restricts T-cell infiltration and promotes immunosuppression, and that PEGPH20 treatment may facilitate access of CD8+ T cells and NK cells into tumors. These findings are consistent with prior studies and support the ongoing clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors to improve antitumor efficacy in HA-accumulating tumors. Citation Format: Jisook Lee, Trevor Kimbler, Curtis Thompson, Benjamin J. Thompson. Degradation of hyaluronan from the tumor extracellular matrix increases infiltration of CD8+ T cells and natural killer cells in an orthotopic pancreatic tumor model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A46.

Abstract A81: Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia

Abstract Hyaluronan (HA) is a naturally occurring glycosaminoglycan that accumulates in the tumor microenvironment of several cancer types, including pancreatic ductal adenocarcinoma (PDA), where it is an independent negative predictor of survival. An ongoing phase 3 study is evaluating pegvorhyaluronidase alfa (PEGPH20; PVHA), a first-in-class biologic that enzymatically degrades HA in the tumor microenvironment, in combination with gemcitabine and nab-paclitaxel in metastatic PDA. Recently, we reported that HA-accumulation can promote tumor hypoxia; however, the effects this may have on tumor-infiltrating immune cells are unknown. Transduction of syngeneic murine PDA cells (Pan02) with hyaluronan synthase 3 (HAS3) generates tumors (Pan02-HAS3) containing high levels of HA, and a higher frequency of TAMs with an immunosuppressive phenotype (CD206-high MHCII-low), relative to tumors generated by empty vector-transduced cells (Pan02-EV). Given the association of this TAM phenotype with tumor hypoxia, we assessed the relationship between tumor hypoxia and the TAM phenotypes of Pan02-HAS3 and Pan02-EV tumors. To confirm whether the effects seen in Pan02-HAS3 tumors require HA synthesis, we also generated tumors expressing an enzymatically inactive HAS3 mutant (Pan02-HAS3 D215A). Tumor-bearing mice were injected with pimonidazole hydrochloride 2 hours prior to harvest and dissociation of tumor tissue; subsequent flow cytometric analysis of tumor-infiltrating immune cells included an antibody against pimonidazole hydrochloride adducts (HP-1) to detect cells residing in hypoxic zones. A greater mean percentage of TAMs isolated from Pan02-HAS3 peritibial tumors had high levels of HP-1 staining (HP-1+) compared with TAMs isolated from either Pan02-EV or Pan02-HAS3 D215A tumors (18.2%, 4.2% and 3.9%, respectively). Furthermore, HP-1+ cells were enriched among CD206-high MHCII-low TAMs compared with their MHCII-high counterparts in Pan02-HAS3 tumors (38.7% vs 11.9%). Likewise, more HP-1+ cells were seen among dendritic cells (DCs) isolated from Pan02-HAS3 tumors compared with either Pan02-EV or Pan02-HAS3 D215A tumors (6.9%, 1.3% and 0.8%, respectively). Similar findings were obtained in orthotopic tumors. In contrast, no consistent differences in the percentages of HP-1+ CD8 T cells or NK cells were observed across the three tumor types in this study. Finally, exposure of cultured macrophages to hypoxia recapitulated some of the changes in gene expression observed in TAMs sorted from Pan02-HAS3 tumors, including increased expression of Arg1 and Nos2. These data suggest that tumor HA accumulation may promote an immunosuppressive TAM phenotype by creating tumor hypoxic zones that are subsequently infiltrated by TAMs, but not T or NK cells. Citation Format: Trevor Kimbler, Jessica Cowell, Jisook Lee, Benjamin J. Thompson. Hyaluronan-dependent skewing of tumor-associated macrophage (TAM) phenotype in a murine pancreatic tumor model is associated with increased tumor hypoxia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A81.

The growth of a xenograft breast cancer tumor model with engineered hyaluronan-accumulating stroma is dependent on hyaluronan and independent of CD44.

Hyaluronan accumulation in the tumor microenvironment is associated with poor prognosis in several solid human cancers. To understand the role of stromal hyaluronan in tumor progression, we engineered 3T3HAS3, a hyaluronan-producing fibroblast cell line, by lentiviral transduction of Balb/c 3T3 cells with the human hyaluronan synthase 3 (HAS3) gene. 3T3HAS3 cells significantly enhanced tumor growth when co-grafted with MDA-MB-468 cells in nude mice. Immunohistochemical analysis of the xenograft tumors showed that MDA-MB-468 cells were surrounded by hyaluronan-accumulating stroma, closely resembling the morphology observed in human breast cancer specimens. Tumor growth of MDA-MB-468 + 3T3HAS3 co-grafts was greatly reduced upon hyaluronan degradation by lentiviral transduction of a human hyaluronidase gene in 3T3HAS3 cells, or by systemic administration of pegvorhyaluronidase alfa (PEGPH20). In contrast, the growth of the co-graft tumors was not inhibited when CD44 expression was reduced or ablated by small hairpin RNA-mediated CD44 knockdown in MDA-MB-468 cells, CD44 CRISPR knockout in 3T3HAS3 cells, or by grafting these cells in CD44 knockout nude mice. Collectively, these data demonstrate that tumor growth of an engineered xenograft breast cancer model with hyaluronan-accumulating stroma can be dependent on hyaluronan and independent of CD44.

506P - Efficacy and safety of pegvorhyaluronidase alfa (PEGPH20; PVHA) and pembrolizumab (pembro) combination therapy in patients (Pts) with stage III/IV non-small cell lung cancer (NSCLC)

BackgroundImmunotherapy is a promising treatment option for advanced NSCLC; however, its efficacy may be limited by accumulation of hyaluronan (HA). PEGPH20 degrades tumor-associated HA and increases the efficacy of chemo- and immuno-therapeutic agents in animal models. Here we report efficacy and safety results of PEGPH20 plus pembro in a cohort of pts with Stage IIIB/IV NSCLC in the 2-cohort, phase Ib dose escalation (DE) and expansion (DX) HALO 107-101 study (NCT02563548). MethodsThis analysis included pts with advanced NSCLC and ≥1 prior platinum-based chemotherapy (DE: 1.6 µg/kg and 2.2 µg/kg of PEGPH20, 2 pts per dose) and pts with HA-high NSCLC (HA staining ≥25% of tumor surface), previously untreated or treated with 1 prior platinum-based chemotherapy (DX: 17 pts). PEGPH20 was given on days 1, 8, and 15 and pembro (DE = 2 mg/kg; DX = 200 mg) on day 1 of 21day cycles. Pts received enteric-coated aspirin (81 mg daily) for thromboembolism prophylaxis. Primary endpoints included: DE: doselimiting toxicity, recommended phase 2 dose (RP2D); DX: objective response rate (ORR). Secondary endpoints included: ORR (DE only), disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and safety. ResultsMean pt age was 68 years; 76% of pts had an ECOG PS 1, and 52% were women. The RP2D dose of PEGPH20 was 2.2 μg/kg. A total of 17 of 21 enrolled pts had evaluable tumor responses: 4 had a partial response, 8 had stable disease, and 5 had progressive disease. ORR was 24% and DCR 71%. PFS rates at 6, 12, and 18 months were 25%, 0%, and 0%, respectively, and OS rates were 62%, 47%, and 31%, respectively. Any-grade treatment-emergent adverse events (TEAEs) were reported in all pts, the most common events being muscle spasms (71%), myalgia (43%), nausea (38%), fatigue (29%), and peripheral edema (29%). Grade ≥3 TEAEs were reported in 57% of pts; 38% of pts experienced ≥1 serious adverse event (AE). There was 1 (5%) treatment-emergent thromboembolic AE. ConclusionsThe combination of PEGPH20 with pembro has an acceptable safety profile that is consistent with results from other PEGPH20 clinical trials and demonstrates activity in pts with locally advanced or metastatic NSCLC. Clinical trial identificationNCT02563548. Editorial acknowledgementMedical writing support provided by Miriam Cohen, PhD, of Paragon Medica, Knutsford, UK, supported by Halozyme Therapeutics Inc. Legal entity responsible for the studyHalozyme Therapeutics Inc. FundingHalozyme Therapeutics Inc. DisclosureE. Bertino: Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy: Takeda. V. Lin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Halozyme Therapeutics Inc. V. Kuruvadi: Full / Part-time employment: Halozyme Therapeutics Inc. T. Heineman: Leadership role, Shareholder / Stockholder / Stock options: Halozyme Therapeutics Inc. All other authors have declared no conflicts of interest.

Remodeling the Tumor Microenvironment Sensitizes Breast Tumors to Anti-Programmed Death-Ligand 1 Immunotherapy.

Immunotherapies targeting immune checkpoint inhibitors have changed the landscape of cancer treatment, however, many patients are resistant or refractory to immunotherapy. The sensitivity of tumor cells to immunotherapy may be influenced by hyaluronan (HA) accumulation in the tumor microenvironment (TME). Enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA) remodels the TME. This leads to reduced tumor interstitial pressure and decompressed tumor blood vessels, which are both associated with increased exposure of tumor cells to chemotherapy drugs. Here, we demonstrate PVHA increased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulating animal models of breast cancer. The increased levels of anti-PD-L1 antibody were associated with increased accumulation of T cells and natural killer cells and decreased myeloid-derived suppressor cells. PD-L1 blockade significantly inhibited tumor growth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors to anti-PD-L1 immunotherapy. SIGNIFICANCE: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells and checkpoint inhibitors access to tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.

Abstract 97: The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models

Abstract The accumulation of hyaluronan (HA) in the solid tumor microenvironment (TME) can be associated with poor prognosis in several human cancers. Accumulation of HA in the TME can increase interstitial pressure that compresses local blood vessels and impairs the access of anti-cancer therapy. In preclinical models, enzymatic degradation of HA by pegvorhyaluronidase alfa (PEGPH20; PVHA), a PEGylated form of recombinant human hyaluronidase PH20 (rHuPH20), is associated with decreased tumor interstitial pressure, improved tumor vascular perfusion, and increased access and anti-tumor efficacy of cytotoxic and immunotherapies. The functional effect of HA degradation on the TME has been mostly characterized in animal tumor models. Here, we describe the utilization of 3D tumor spheroid models to evaluate the effects of HA degradation on the cytotoxic effect of anti-cancer therapies. A number of commonly used human colorectal cancer (CRC) cell lines were assessed for HA accumulation and tumor spheroid morphology: HCT 116, HT-29 parental, and HT-29/HAS3 (HT-29 cells engineered to over-express hyaluronan synthase 3). CRC 3D tumor spheroid models were developed to evaluate the uptake and cytotoxic activity of liposome-encapsulated doxorubicin (DOX) using the IncuCyte® S3 Live-Cell Analysis System. The effect of HA degradation on DOX uptake and cytotoxic activity was initially tested with rHuPH20 treatment in all three models, and further evaluated with PVHA treatment in HT-29 parental and HT-29/HAS3 models. A dose-dependent uptake and cytotoxic activity of DOX was observed in HCT 116 tumor spheroids. HA degradation after rHuPH20 treatment enhanced DOX uptake by an average of 6.1% (up to 7.7%) and cytotoxicity by an average of 37.9% (up to 58.2%) over the 5-day duration of the experiment compared with DOX alone. Dose-dependent uptake and cytotoxic activity of DOX in HT-29/HAS3 tumor spheroids were enhanced with rHuPH20 treatment to a greater extent than in HT-29 parental tumor spheroids. Specifically, rHuPH20 treatment led to an average increase in DOX uptake of 9.2% (up to 14.6%) with HT-29/HAS3 spheroids and 2.6% (up to 5.1%) with HT-29 parental spheroids. The addition of rHuPH20 resulted in an average increase in DOX cytotoxicity of 101% (up to 227.6%) with HT-29/HAS3 spheroids and 24.9% (up to 63.9%) with HT-29 parental spheroids. Enhanced uptake and cytotoxic activity of DOX was also observed with PVHA treatment in HT-29 and HT-29/HAS3 models. These data corroborate our previous findings with in vivo tumor models, in which the combination of PVHA-mediated HA degradation and other anti-cancer therapies display enhanced anti-tumor activity compared with either agent alone. Further, 3D tumor spheroid models may be used to evaluate the effect of extracellular matrix remodeling such as HA degradation and serve as a bridge between 2D cell-based assays and in vivo tumor models. Citation Format: Feng Gao, Susan Zimmerman, Kelly Chen, Daniel C. Maneval, Chunmei Zhao. The effect of hyaluronan degradation on the uptake and cytotoxic activity of liposome-encapsulated doxorubicin in 3D tumor spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 97.

Dynamic Contrast-enhanced MRI Detects Responses to Stroma-directed Therapy in Mouse Models of Pancreatic Ductal Adenocarcinoma.

The dense stroma underlies the drug resistance of pancreatic ductal adenocarcinoma (PDA) and has motivated the development of stroma-directed drugs. Our objective is to test the concept that dynamic contrast-enhanced (DCE) MRI using FDA-approved contrast media, an imaging method sensitive to the tumor microenvironment, can detect early responses to stroma-directed drug. Imaging studies were performed in three mouse models exhibiting high desmoplastic reactions: the autochthonous PDA in genetically engineered mice (KPC), an orthotopic model in syngeneic mice, and a xenograft model of human PDA in athymic mice. An investigational drug, PEGPH20 (pegvorhyaluronidase alfa), which degrades hyaluronan (HA) in the stroma of PDA, was injected alone or in combination with gemcitabine. At 24 hours after a single injection of PEGPH20, Ktrans , a DCE-MRI-derived marker that measures how fast a unit volume of contrast media is transferred from capillaries to interstitial space, increased 56% and 50% from baseline in the orthotopic and xenograft tumors, respectively, compared with a 4% and 6% decrease in vehicle groups (both P < 0.05). Similarly, after three combined treatments, Ktrans in KPC mice increased 54%, whereas it decreased 4% in controls treated with gemcitabine alone (P < 0.05). Consistently, after a single injection of PEGPH20, tumor HA content assessed by IHC was reduced substantially in all three models while drug delivery (measured by paclitaxel accumulation in tumor) was increased by 2.6-fold. These data demonstrated a DCE-MRI marker, Ktrans , can detect early responses to stroma-directed drug and reveal the sustained effect of combination treatment (PEGPH20+ gemcitabine).

Plasma hyaluronan is a predictive marker for pegvorhyaluronidase alfa (PEGPH20; PVHA) response in a phase II study of pancreatic ductal adenocarcinoma (PDAC).

404 Background: Hyaluronan (HA) can be a constituent of the tumor microenvironment (TME). Accumulation of HA in the TME may result in elevated interstitial pressure, vascular compression, hypoxia, and reduced drug delivery and immune cell access. Pegvorhyaluronidase alfa (PEGPH20; PVHA) enzymatically degrades HA, potentially increasing access and antitumor efficacy of cytotoxic and immuno-therapies. Liquid biopsies may provide a non-invasive approach to predict therapeutic efficacy of PVHA. Methods: Total plasma HA was analyzed as a pharmacodynamic biomarker for PVHA activity in HALO 109-202 (NCT01839487), a Phase 2, open-label, two-stage randomized study of PVHA + nab-paclitaxel + gemcitabine (PAG) vs AG only, in previously untreated patients with Stage IV metastatic PDAC. HA was measured with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) disaccharide assay using baseline plasma samples from Stage 1 (n = 132) and Stage 2 (n = 128) of the study. Results: Lower baseline plasma HA values were significantly correlated with survival outcomes, predicting progression-free survival (PFS) benefit in PVHA-treated patients in Stages 1 and 2, and overall survival (OS) benefit in PVHA-treated patients in Stage 2 (Table). Conclusions: These results support further exploration of a liquid biopsy-based companion diagnostic (CDx) for predicting the therapeutic efficacy of PVHA. [Table: see text]

Why HALO 301 Failed and Implications for Treatment of Pancreatic Cancer.

Survival rates for pancreatic cancer (PC) remain dismal. Current standard of care treatment regimens provide transient clinical benefit but eventually chemoresistance develops leading to poor outcomes. PC is a relatively chemoresistant tumor and one of the explanations for this is attributed to desmoplasia that impedes drug delivery. Based on this, stromal modifying agent such as Pegvorhyaluronidase alfa (PEGPH20) was developed and investigated in phase I-III studies. Although phase I-II studies showed promising results in patients with high hyaluronic acid (HA) expressing tumors, the phase III HALO 301 study failed to miss it’s primary endpoint and further development of PEHPH20 is halted. This failure implies that targeting desmoplasia alone is not sufficient and other intrinsic factors such as lack of significant neoantigens, low tumor mutational burden, and epithelial to mesenchymal transition may be at play. It is also important to consider that although the tumor stroma may be a physical barrier hampering drug delivery, it may also have protective effects in restraining tumor growth and progression. Further studies in molecular biology to better characterize the complex interaction between the microenvironment and cancer cells are warranted.

311P - Early results from an open-label phase 1b/II study of eribulin mesylate (EM) + pegvorhyaluronidase alfa (PEGHP20) combination for the treatment of patients with HER2-negative, high-hyaluronan (HA) metastatic breast cancer (MBC)

311P - Early results from an open-label phase 1b/II study of eribulin mesylate (EM) + pegvorhyaluronidase alfa (PEGHP20) combination for the treatment of patients with HER2-negative, high-hyaluronan (HA) metastatic breast cancer (MBC)

PO-264 Preclinical studies with pegvorhyaluronidase alfa (PEGPH20) in combination with FOLFIRINOX (FFX) chemotherapy in models of pancreatic ductal adenocarcinoma

IntroductionHyaluronan (HA) accumulates in the tumour microenvironment (TME) of many solid tumours and has been associated with tumour progression and negative clinical outcomes. Pegvorhyaluronidase alfa (PEGPH20, P) is a novel biologic that enzymatically degrades HA. In preclinical models, P-mediated enzymatic degradation of HA is associated with decreased tumour interstitial fluid pressure, increased tumour perfusion, and increased access and anti-tumour efficacy of cytotoxic and immunotherapies. As pancreatic ductal adenocarcinoma (PDA) has been identified as a cancer type that accumulates high levels of HA, here we evaluated whether P alters the activity of the cytotoxic components of FOLFIRINOX (FFX), a common therapeutic regimen for advanced PDA, on human and murine PDA cells. We also evaluated p+FFX in vivo anti–tumour activity in BxPC3/HAS3, a HA accumulating human PDA model.Material and methodsFor in vitro experiments, fluorouracil, irinotecan and oxaliplatin were evaluated individually and in combination in both 2D culture and 3D tumour spheroids. For in-vivo evaluation, mice were inoculated with BxPC3/HAS3 cells adjacent to the right tibial periosteum and tumour growth was monitored via ultrasonography. When tumours reached ~230 mm3, mice (n=8/group) were staged into treatment groups: 1) vehicle; 2) 0.0375 mg/kg P BIW; 3) low FFX - 30 mg/kg leucovorin, 15 mg/kg fluorouracil, 30 mg/kg irinotecan, and 1.2 mg/kg oxaliplatin QW; 4) high FFX - 65 mg/kg leucovorin, 32.5 mg/kg fluorouracil, 65 mg/kg irinotecan, and 2.6 mg/kg oxaliplatin QW; 5) p+low FFX; and 6) p+high FFX. FFX was administered 24 hour after P.Results and discussionsPDA cells were more sensitive to chemotherapy treatment in 2D than 3D culture. The TME-modifying enzyme P did not show an effect on chemotherapy sensitivity, whether tested as individual or combined cytotoxics. In the BxPC3/HAS3 model, P increased the anti-tumour efficacy of low FFX 4-fold (56.3% vs. 11.3% tumour growth inhibition [TGI], respectively) and extended median survival time (MST) by >36% (32d vs. 23.5d, respectively), whereas P increased the efficacy of high FFX by 61% (64.3% vs. 39.8% TGI, respectively) and extended MST by 12.5% (36d vs. 32d, respectively). A maximum 5% BW loss was observed following the initial FFX dose.ConclusionTaken together, the results suggest that P does not affect the cytotoxic activity of FFX components on PDA cells in culture, and that P-mediated HA degradation improves the anti-tumour efficacy of FFX (especially suboptimal doses) in a preclinical PDA model.

Abstract 1743: Rationale for evaluating PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) in patients with hyaluronan (HA)-accumulating colorectal cancer

Abstract Colorectal cancer is the third most common cancer, and the third most common cause of cancer-related death, among both men and women in the United States. To address this unmet clinical need, ongoing efforts have focused on classifying different subtypes of colorectal cancer, with the goal of identifying therapies with the highest likelihood of success for a given tumor subtype. One marker that has been associated with poor clinical outcome in multiple solid tumor types, including colorectal cancer, is the accumulation of the glycosaminoglycan hyaluronan (HA), which, in preclinical tumor models, increases interstitial fluid pressure, compresses tumor vasculature, and restricts the access of therapeutics. PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20), which enzymatically degrades HA, is currently being evaluated in combination with existing therapies in clinical trials for HA-accumulating pancreatic, gastric, non-small cell lung, and bile duct cancers. Here, we review the available data for PEGPH20 as published by the authors to evaluate the potential utility of PEGPH20 in treating colorectal cancer. We first studied PEGPH20 in two HA-accumulating murine colon tumor models: CT26 transduced with hyaluronan synthase-3 (HAS3) and MC38 (Charles River Laboratories). In CT26-HAS3 tumors, PEGPH20 monotherapy induced 43% tumor growth inhibition and was associated with tumor blood vessel decompression. In combination with anti-CTLA4 treatment, PEGPH20 further enhanced tumor growth inhibition (79%, p≤0.002 vs anti-CTLA4 alone and PEGPH20 alone). In MC38 tumors, combination of PEGPH20 with anti-PD-L1 treatment significantly increased infiltration of CD8+ T cells (2.8-fold, p&amp;lt;0.05) and NK cells (3-fold, p&amp;lt;0.0001) compared with anti-PD-L1 alone. We then investigated the prevalence of HA accumulation in human colorectal cancer. To do this, we histologically analyzed 138 patient tumor specimens for stromal content and presence of HA. In 38 samples (28%), ≥50% of the tumor stromal area stained positive for HA. Additionally, Phase 1 studies with single agent PEGPH20 included several subjects with colorectal cancer. Pharmacodynamic measurements before and after treatment demonstrated increased tumor perfusion as measured by median volume transfer constant (Ktrans), decreased tumor metabolic activity as measured by 18F-FDG-PET/CT in one patient, and decreased tumor HA following PEGPH20 administration. Collectively, these data warrant further investigation of the potential for PEGPH20 to benefit patients with HA-high colorectal cancer. Citation Format: Renee Clift, Benjamin J. Thompson, Darin Taverna, Barbara Blouw, Jisook Lee, Curtis B. Thompson, Daniel C. Maneval. Rationale for evaluating PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) in patients with hyaluronan (HA)-accumulating colorectal cancer [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 1743.

Abstract 1747: Hyaluronan (HA) accumulation restricts CD8+ T cell numbers and skews tumor-associated macrophage (TAM) phenotype in mouse syngeneic pancreatic tumors

Abstract The glycosaminoglycan hyaluronan (HA) is abundant in the tumor microenvironment (TME) of diverse solid tumor types, and tumor accumulation of HA is associated with poor patient outcome. We previously demonstrated, in multiple HA-rich mouse syngeneic tumor models, that enzymatic degradation of HA using PEGylated recombinant human hyaluronidase PH20 (pegvorhyaluronidase alfa; PEGPH20) enhanced tumor growth inhibition and increased tumor accumulation of T and NK cells when combined with a checkpoint inhibitor. However, the ability of HA accumulation in the TME to alter the composition of tumor-infiltrating immune cells has not been well characterized. Here, we addressed this question by performing a detailed immune phenotypic analysis of HA-poor and HA-rich tumors using a mouse syngeneic pancreatic tumor model. To achieve this, we transduced Pan02 cells with a lentivirus encoding hyaluronan synthase 3 (HAS3, Pan02-HAS3) or empty vector (EV, Pan02-EV). After peritibial implantation, Pan02-HAS3 tumors contained 13.4-fold higher levels of HA than Pan02-EV tumors, as determined by HA ELISA. Both variants grew with similar kinetics to the Pan02 parental line. Flow cytometric analysis of tumor-infiltrating immune cells in four independent and identical studies revealed an average 6.2-fold reduction in the number of CD8+ T cells in Pan02-HAS3 tumors compared with Pan02-EV. Likewise, numbers of CD4+ T cells and NK cells were reduced by an average of 4.8- and 2.9-fold, respectively, in Pan02-HAS3 tumors compared with Pan02-EV. In contrast, we did not observe consistent differences in the numbers of tumor-associated macrophages (TAMs), neutrophils/granulocytic myeloid-derived suppressor cells (G-MDSC), monocytes/monocytic (M)-MDSC, or dendritic cells (DCs) between the two tumor variants. However, within the TAM population, cells displaying an “M2-like” phenotype (CD206high MHCIIlow) were present at an average 10.5-fold higher frequency in Pan02-HAS3 tumors compared with Pan02-EV. Similarly, the percentage of DCs that were CD103+ CD11b- was reduced by 1.4-fold in Pan02-HAS3 tumors compared with Pan02-EV. Together, our data indicate that HAS3-driven HA accumulation in Pan02 mouse syngeneic tumors induces several aspects of immunosuppression: it restricts the numbers of tumor-infiltrating lymphocytes, and skews TAMs and DCs toward immunosuppressive phenotypes. These findings are consistent with our previous work, which demonstrated that combining PEGPH20 with anti-PD-L1 increased tumor-infiltrating lymphocyte numbers and reduced the frequency of CD206high MHCIIlow TAMs in Pan02-HAS3 tumors. Our results support the ongoing clinical evaluation of PEGPH20 in combination with immunotherapy in HA-rich tumor indications (NCT02563548 and NCT03193190). Citation Format: Benjamin Thompson, Trevor Kimbler, Jisook Lee, Chunmei Zhao, Kelly Chen, Renee Clift, Curtis B. Thompson, Sanna Rosengren, Daniel C. Maneval. Hyaluronan (HA) accumulation restricts CD8+ T cell numbers and skews tumor-associated macrophage (TAM) phenotype in mouse syngeneic pancreatic tumors [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 1747.

PO-263 Assessment of the accumulation of hyaluronan in the tumour microenvironment (TME) of solid tumours

IntroductionHyaluronan (HA), a primary component of the extracellular matrix, can accumulate in a variety of solid malignancies, where histological analyses of patient tumour tissues demonstrated that HA levels may correlate with patient outcomes. HA accumulation in the TME may be associated with poor prognosis. Preclinical studies demonstrated elevated TME HA levels increased tumour interstitial pressure, reduced tumour blood flow, and resulted in poor penetration of anticancer agents and immune cells. A PEGylated recombinant human hyaluronidase, pegvorhyaluronidase alfa (PEGPH20), depolymerizes HA, increases tumour vascular perfusion, and may increase access and anti-tumour efficacy of cytotoxic and immunotherapies.Material and methodsPegvorhyaluronidase alfa is being evaluated in clinical studies for pancreatic, breast, lung, cholangiocarcinoma, and gastric cancers. To broaden the spectrum of possible tumour types that may benefit from pegvyorhyaluronidase alfa, we assessed the prevalence and accumulation of HA in 3633 human histological samples in 22 solid tumour types. HA levels were measured using tissue microarrays (TMAs) stained with a proprietary HA probe, HTI–601, a recombinant immunoadhesin containing a modified HA–specific link domain from TNF-Stimulated Gene 6 protein (TSG–6) and the Fc portion of human IgG1. The resulting HA probe was biotinylated for use in an affinity-based histochemistry method. HA staining was evaluated with research-grade histological image analysis software. HA levels associated with distinct tumour compartments (stroma or tumour cells) were assessed. HA accumulation associated with immune infiltrates present in the TMA cores was also evaluated.Results and discussionsDistribution of HA accumulation across compartments fell into 4 main arrangements:- For desmoplastic tumour indications (pancreatic ductal adenocarcinoma, cholangiocarcinoma), the stroma was associated with high percentage of surface area stained with HTI-601 relative to the tumour cells;- Comparable distribution of HA staining in both tumour cells and stroma (gall bladder and glioblastoma);- Sub-clusters of HA staining within a specific tumour type (NSCLC non-squamous);- Distribution bias towards increased or decreased HA intensity staining (head and neck excluding nasopharyngeal cavities and renal cell carcinoma, respectively).ConclusionUnderstanding HA prevalence and accumulation in solid tumours may potentially identify malignancies that may benefit from pegvorhyaluronidase alfa therapy targeting HA.

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&amp;lt;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&amp;lt;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.

Efficacy of pegvorhyaluronidase alfa (PEGPH20) plus FOLFIRINOX in a preclinical model of human pancreatic ductal adenocarcinoma.

e16244Background: Hyaluronan (HA) accumulates in the solid tumor microenvironment, and is associated with poor drug penetration and negative clinical outcomes. Pegvorhyaluronidase alfa (P) is a nov...