H. Michael Shepard

Phase Ib Study of PEGylated Recombinant Human Hyaluronidase and Gemcitabine in Patients with Advanced Pancreatic Cancer

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

Accumulation of extracellular hyaluronan by hyaluronan synthase 3 promotes tumor growth and modulates the pancreatic cancer microenvironment.

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

Novel splice variants derived from the receptor tyrosine kinase superfamily are potential therapeutics for rheumatoid arthritis

IntroductionDespite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF) – a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins – many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis.MethodsTo identify novel splice variants, we performed RT-PCR using an mRNA pool representing major human tissue types and tumors. Novel ASV were identified by alignment of each cloned sequence to its respective genomic sequence in comparison with full-length transcripts. To test whether these ASV have biologic activity, we characterized a subset of them for ligand binding, and for efficacy in an animal model of arthritis. The in vivo study was accomplished using adenoviruses expressing secreted ASV.ResultsWe cloned 60 novel human ASV from 21 genes, encoding cell surface receptors – many of which are known to be important in the regulation of angiogenesis. The ASV were characterized by exon extension, intron retention and alternative exon utilization. Efficient expression and secretion of selected ASV – corresponding to VEGF receptor type 1, VEGF receptor type 2, VEGF receptor type 3, angiopoietin receptor Tie1, Met (receptor for hepatocyte growth factor), colony-stimulating factor 1 receptor, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, Kit, and RAGE – was demonstrated, together with binding to their cognate ligands. Importantly, ASV derived from VEGF receptor type 1 and Tie1, and to a lesser extent from VEGF receptor type 2 and fibroblast growth factor receptor 1, reduced clinical signs of arthritis in vivo. The reduction was paralleled by decreased joint inflammation and destruction.ConclusionThe present study shows that unique ASV derived from receptors that play key roles in angiogenesis – namely, VEGF receptor type 1 and, for the first time, Tie1 – can markedly reduce arthritis severity. More broadly, our results demonstrate that ASV are a source of novel proteins with therapeutic potential in diseases in which angiogenesis and cellular hyperplasia play a central role, such as rheumatoid arthritis.

Tumor-Associated Hyaluronan Limits Efficacy of Monoclonal Antibody Therapy

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

Recombinant Human Hyaluronidase PH20 Does Not Stimulate an Acute Inflammatory Response and Inhibits Lipopolysaccharide-Induced Neutrophil Recruitment in the Air Pouch Model of Inflammation

Hyaluronidase (Hyal) and low m.w. hyaluronan (LMW HA) fragments have been widely reported to stimulate the innate immune response. However, most hyaluronidases used were purified from animal tissues (e.g., bovine testis Hyal [BTH]), and contain endotoxin and other unrelated proteins. We tested a highly purified recombinant human Hyal (rHuPH20) and endotoxin-free HA fragments from Mr 5,000 to 1,500,000 in the rodent air pouch model of inflammation to determine their potential for stimulation of the innate immune response. Exogenous LMW HA fragments (average Mr 200,000) failed to induce either cytokine/chemokine production or neutrophil infiltration into the air pouch. Challenging the air pouch with LPS or BTH stimulated production of cytokines and chemokines but rHuPH20 did not, suggesting that neither PH20 nor generation of LMW HA fragments in situ stimulates cytokine and chemokine production. LPS and BTH also induced neutrophil infiltration into the air pouch, which was not observed with rHuPH20 treatment. Endotoxin-depleted BTH had much reduced proinflammatory activity, suggesting that the difference in inflammatory responses between rHuPH20 and BTH is likely due to endotoxin contaminants in BTH. When rHuPH20 was dosed with LPS, the induction of cytokines and chemokines was the same as LPS alone, but neutrophil infiltration was inhibited, likely by interrupting HA–CD44 interaction. Our results indicate that neither rHuPH20 nor its directly generated HA catabolites have inflammatory properties in the air pouch model, and rHuPH20 can instead inhibit some aspects of inflammation, such as neutrophil infiltration into the air pouch.

Breaching the Castle Walls: Hyaluronan Depletion as a Therapeutic Approach to Cancer Therapy.

Hyaluronan (HA) has many functions in the extracellular milieu of normal and diseased tissues. Disease-associated HA accumulation has been shown to predict a worsened prognosis in cancer patients, with tumors having a high-extracellular HA content (HA-high) being more aggressive than their HA-low counterparts. HA-high tumor aggressiveness is derived from the specialized biomechanical and molecular properties of the HA-based assembly of HA binding proteins and the growth-promoting factors that accumulate in it. Biophysical characteristics of an HA-high tumor microenvironment include high tumor interstitial pressure, compression of tumor vasculature, and resulting tumor hypoxia. Within the tumor cell membrane, HA receptors, primarily CD44 and RHAMM, anchor the HA-high extracellular network. HA–CD44 association on the tumor cell surface enhances receptor tyrosine kinase activity to drive tumor progression and treatment resistance. Together, malignant cells in this HA-high matrix may evolve dependency on it for growth. This yields the hypothesis that depleting HA in HA-high tumors may be associated with a therapeutic benefit. A pegylated form of recombinant human hyaluronidase PH20 (PEGPH20) has been deployed as a potential cancer therapeutic in HA-high tumors. PEGPH20 can collapse this matrix by degrading the HA-assembled tumor extracellular framework, leading to tumor growth inhibition, preferentially in HA-high tumors. Enzymatic depletion of HA by PEGPH20 results in re-expansion of the tumor vasculature, reduction in tumor hypoxia, and increased penetration of therapeutic molecules into the tumor. Finally, HA-depletion results in reduced signaling via CD44/RHAMM. Taken together, HA-depletion strategies accomplish their antitumor effects by multiple mechanisms that include targeting both biophysical and molecular signaling pathways. Ongoing clinical trials are examining the potential of PEGPH20 in combination with partner therapeutics in several cancers.

Response to Chauhan et al.: Interstitial Pressure and Vascular Collapse in Pancreas Cancer—Fluids and Solids, Measurement and Meaning

Chauhan et al. suggest that vascular collapse and hypoperfusion in pancreatic ductal adenocarcinoma (PDA) are caused by solid stress (SS) (Chauhan et al., 2014) instead of the elevated interstitial fluid pressure (IFP) associated with high extravascular concentrations of hyaluronan (Provenzano et al., 2012). We appreciate their attention to our work and the opportunity to clarify underlying mechanisms. Chauhan et al. make four important claims, to which we respond.

Characterization of a Novel Recombinant Hyaluronan Binding Protein for Tissue Hyaluronan Detection

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

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

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 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

Antagonism of the human epidermal growth factor receptor family controls disease severity in murine collagen-induced arthritis.

OBJECTIVE To evaluate the therapeutic potential of the human epidermal growth factor receptor (HER) family inhibitor, herstatin, in an animal model of arthritis. METHODS Constructs of herstatin and modified tissue plasminogen activator (tPA)-herstatin were expressed in HEK 293T cells, and secreted protein was analyzed by Western blotting. Tissue PA-herstatin adenovirus (Ad-tPA-Her) was prepared, and titers established. Gene expression of Ad-tPA-Her was determined by polymerase chain reaction using HeLa cells. Pharmacokinetics of gene and protein expression in vivo in liver tissue and serum samples were confirmed via intravenous administration of Ad-tPA-Her. Clinical signs of disease were monitored in arthritic DBA/1 mice after therapeutic administration of Ad-tPA-Her, and histologic analysis of hind foot specimens was performed. RESULTS Native herstatin was not secreted in supernatants, while modified tPA-herstatin was detected in abundance. HeLa cells stably expressed the tPA-herstatin gene when infected with virus. Additionally, tPA-herstatin gene and protein expression was observed over time in mice treated with virus. Importantly, Ad-tPA-Her, when administered therapeutically to arthritic mice, controlled clinical and histologic signs of disease and reduced the number of joints with severe damage. CONCLUSION Our results support the notion that the human epidermal growth factor receptor family has a role in the progression of collagen-induced arthritis. The novel tPA-herstatin fusion protein could be used as an effective therapeutic tool for control of inflammatory disorders involving an angiogenic component.