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.

Hyaluronan removal in the tumor microenvironment improves immune cells and checkpoint inhibitor access to tumors. CapillaryCancer cell Hyaluronan Hyaluronan accumulation Hyaluronan degradation Immune checkpoint inhibitorImmunotherapies 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 deathligand 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.

DNA sequences corresponding to gene segments that code for the nonstructural protein, the matrix protein, and the hemagglutinin of influenza A virus [strain A/Udor/72 (H3N2)] were cloned in Escherichia coli pBR 322. Initially, positive and negative cDNA strands were prepared separately by reverse transcription. The positive strands of cDNA were transcribed from genomic RNA segments by using a specific dodecamer DNA sequence as a primer; the negative strands of cDNA were transcribed from cytoplasmic viral mRNA segments by using an oligo(dT) primer. DNA duplexes corresponding in size to the virus RNA segments were then purified, inserted into the plasmid DNA, and used for transformation of E. coli. The influenza virus-specific DNA sequences isolated from recombinant plasmid molecules were characterized by mapping restriction enzyme cleavage sites. In addition, the orientation of cloned DNA was determined with reference to the 3' terminus of viral RNA.The negative-stranded RNA genome of influenza A virus contains eight segments that vary from 0.2 to 0.9 X 106 daltons (1-4). Analysis of virion RNA from influenza A strains diverse in antigenic subtype indicates that all eight RNA segments contain a common sequence of 13 nucleotides at the 3' terminus and another common sequence of 12 nucleotides at the 5' terminus (5, 6). In the infected cell, these RNA segments are transcribed and polyadenylylated to generate the corresponding positive-stranded mRNA species (7,8). With the exception of viral RNA segment 8 which encodes two nonstructural proteins (NS1 and NS2), each RNA segment seems to code for a specific viral component and to be responsible for a specific viral function (9-11). The virion components include the polymerase proteins (P1, P2, P3), nucleoprotein (NP), matrix protein (M), and the two surface proteins, the hemagglutinin (H) and the neuraminidase (N) (12,13 (50 ,ug) was reverse transcribed under similar conditions by using 10 jig of oligo(dT) as primer instead of the DNA dodecamer. Incubation was carried out at 37°C for 1 hr, followed by heating at 70°C in 0.2 M NaOH for 30 min to remove RNA. After neutralization and phenol extraction the labeled cDNA products were purified by gel filtration on Sephadex G-50.Cloning Influenza Virus Gene Segments in Escherichia coli Plasmid pBR 322. Positive (+) cDNA strands transcribed from vRNA and negative (-) cDNA strands transcribed from mRNA, approximately 10 Mug each, were mixed and hybridized by heating at 68°C for 3 hr in 10 ml of 0.14 M phosphate, pH 6.8/1 mM EDTA/0.1% sodium dodecyl sulfate. The DNA duplexes containing influenza virus-specific sequences were selected by hydroxyapatite chromatography and separated according to size by electrophoresis in 3.5% polyacrylamide gel (acrylamide/bisacrylamide = 20:1) at 3.5 V/cm for 40 hr. Ten to 30 deoxycytidylyl residues were added to the 3' end of the influenza-specific DNA fragments eluted from the gel, by using terminal transferase (18 210The publication costs of this article were defrayed in part by p...

Hyaluronan (HA), a major extracellular matrix component in many solid tumors, has been proposed to contribute to tumor progression, and to play a complex role in T lymphocyte biology. Its depletion by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the tumor stroma, reduces intratumoral pressure, decompresses tumor blood vessels, and facilitates tumor drug delivery. However, the impact of HA removal on intra-tumoral immune responses and the efficacy of immune checkpoint inhibitors is unknown. To evaluate checkpoint blockade efficacy with PEGPH20, two mouse tumor cell lines, CT26 (colon) and MH194 (pancreatic, derived from spontaneous tumors in KrasLSL-G12D/+Trp53LSL-R172H/+Cre mice) were transduced with hyaluronan synthase-3 (HAS3) to generate syngeneic HA-high tumor models. For anti-CTLA4 studies, parental CT26 and CT26/HAS3 cells were implanted peritibially in Balb/C mice. While treatment with anti-mouse-CTLA4 alone (clone 9D9) inhibited tumor growth in CT26 tumors (37%), PEGPH20 alone did not significantly inhibit tumor growth or increase anti-CTLA4 efficacy. In contrast, tumor growth of CT26/HAS3 tumors was inhibited to a greater extent by the combination of PEGPH20 and anti-CTLA4 (79%) (PEGPH20 treatment 24h prior to anti-CTLA4 treatment), compared to anti-CTLA4 alone (60%, p = 0.002) or PEGPH20 alone (43%, p = 0.0001). Furthermore, gene expression of markers associated with immune suppression, such as IL10 and FoxP3, was higher in CT26/HAS3 than in CT26 tumors; suggesting an association between HA content and immune suppression. To evaluate the effect of PEGPH20 on tumor growth inhibition by PD-1 blockade, MH194/HAS3 cells were implanted peritibially in C57BL/6 mice along with immortalized pancreatic stellate cells. Growth of MH194/HAS3 tumors was significantly inhibited (33%, p = 0.049) by anti-mouse-PD-L1 antibody (clone 10F.9G2), and the addition of PEGPH20 (24h prior to anti-PD-L1) to anti-PD-L1 further enhanced tumor growth inhibition (79%, p <0.0001 to both anti-PD-L1 alone and PEGPH20 alone). Similar findings were obtained with anti-mouse-PD-1 (clone RMP1-14), where the tumor growth inhibition by anti-PD-1 (33%) was further enhanced by PEGPH20 (56%, p = 0.020 and 0.017, respectively, to anti-PD-1 alone and PEGPH20 alone). At 24h following injection, the dose of PEGPH20 used (37.5 μg/kg, the human equivalent dose) removed approximately 50% of HA from tumors as shown by immunohistochemistry and HA ELISA on tumor lysates. Finally, in separate studies, PEGPH20 enhanced labelled intratumoral anti-PD-L1 accumulation (2.6 fold, p = 0.006) in a SKOV3 ovarian xenograft model engineered to express HAS2. In conclusion, in HA-high tumors, PEGPH20 reduced HA, increased anti-PD-L1 accumulation, and significantly enhanced tumor growth inhibition induced by anti-CTLA4, anti-PD-L1, and anti-PD-1 antibodies. Citation Format: Sanna Rosengren, Renee Clift, Susan J. Zimmerman, Jennifer Souratha, Benjamin J. Thompson, Barbara Blouw, Xiaoming Li, Qiping Zhao, Michael Shepard, Dan C. Maneval, Christopher D. Thanos, Curtis B. Thompson. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances checkpoint inhibitor efficacy in syngeneic mouse models of cancer. [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 4886.

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.