Michael Shepard

Abstract 4886: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances checkpoint inhibitor efficacy in syngeneic mouse models of cancer

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 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.

Abstract 5583: Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells

Extracellular adenosine can be generated from ATP and/or ADP through the concerted action of the ectoenzymes CD39 and CD73. Adenosine can bind different type-1 purinergic receptors eliciting potent cytoprotective and immunosuppressive effects. We previously focused on chronic lymphocytic leukemia (CLL), a disease characterized by the progressive expansion of a mature population of B lymphocytes, showing that a subset of patients with a CD73+ clone possess a poor prognosis. This patient subset can actively generate adenosine, which favors leukemic cell survival, synthesis of immunosuppressive cytokines, while inhibiting T lymphocyte proliferation. ADA2 is a human enzyme that catalyzes the conversion of adenosine into inosine. We hypothesized that depletion of high adenosine levels from the tumor microenvironment through the administration of exogenous ADA2 could be therapeutically relevant to limit tumor protection and immunosuppression. Several engineered PEG-ADA2 variants possessed improved enzyme activity and pharmacokinetics compared to wild-type ADA2. Inhibition of adenosine receptor activation was evaluated by measuring intracellular cAMP concentrations in T lymphocytes purified from healthy donors. Adenosine depletion was observed with increasing concentrations of PEG-ADA2-R222Q/S265N and significantly reduced cAMP levels. PEG-ADA2 variants were then tested on CD73+ CLL lymphocytes to determine their effects on extracellular adenosine and inosine. Treatment of leukemic B cells with PEG-ADA2-R222Q/S265N and PEG-ADA2-R222Q/S265N/K374D resulted in complete removal of high levels of adenosine, as measured by HPLC. Conversely, PEG-ADA2-E182T, which possesses significantly attenuated catalytic activity, was less effective in reducing adenosine. The functional effects of adenosine depletion on CLL cell survival were analyzed following treatment with the chemotherapeutic, DNA-damaging agent etoposide that robustly induces apoptosis within 16 hours. Addition of exogenous adenosine to cultures of purified CD73+ CLL cells significantly rescued cells from etoposide-induced apoptosis. However, when these primary leukemic cells were pretreated with PEG-ADA2-R222Q/S265N or PEG-ADA2-R222Q/S265N/K374D, the cell viability rate was significantly decreased, abrogating the cytoprotective effects of adenosine. On the contrary, PEG-ADA2-E182T had a minimal effect, suggesting enzymatic depletion of adenosine is critical to observe these effects. Similar effects were observed by the PEG-ADA2 variants on a CLL cell line expressing CD73+. These preliminary results suggest that enzymatic depletion of extracellular adenosine following treatment with PEG-ADA2 is a relevant approach to counteract the cytoprotective effects of adenosine, warranting further development of PEG-ADA2 as a possible approach to treat CLL. Citation Format: Sara Serra, Cinzia Bologna, Luz Londono, Lin Wang, Michael Shepard, Sanna Rosengren, Christopher Thanos, Silvia Deaglio. Pegylated adenosine deaminase 2 (PEG-ADA2) abrogates the cytoprotective effects of adenosine against chronic lymphocytic leukemia cells [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 5583. doi:10.1158/1538-7445.AM2017-5583

Abstract 1240: Evaluating clinically relevant pharmacological agents in a rat ambulation model to ameliorate PEGylated recombinant hyaluronidase PH20 (PEGPH20)-mediated musculoskeletal adverse events

Some cancers accumulate high levels of the glycosaminoglycan hyaluronan (HA) within the tumor microenvironment (TME), and this HA accumulation is associated with rapid tumor progression and poor clinical outcomes. As preclinical data have demonstrated that enzymatic degradation of HA, via intravenous (IV) administration of PEGylated recombinant human hyaluronidase PH20 (PEGPH20), increases therapeutic efficacy in HA-accumulating tumors, several clinical trials evaluating PEGPH20 in combination with anti-cancer therapies are ongoing. The most advanced clinical trial (Phase 3) is evaluating PEGPH20 plus Abraxane® (nab-paclitaxel)/gemcitabine (AG) in patients with stage IV untreated pancreatic ductal adenocarcinoma (PDA)(NCT02715804). The safety profile of PEGPH20/Abraxane/gemcitabine (PAG) was generally similar to AG alone in an interim analysis of the Phase 2 data, with the exception of a potential imbalance of thromboembolic events (TEs), and an increased incidence of musculoskeletal events (MSEs) in the PAG arm, with the predominant MSE being muscle spasms (55.4% PAG vs. 1.6% AG, all grades)(Bullock, ASCO 2016). In an effort to find pharmacological agent(s) that might decrease MSEs in PAG-treated patients, a rat locomotor activity assay was developed. In brief, locomotor activity of male Sprague Dawley rats was measured with an automated infrared photobeam monitoring apparatus (Hamilton/Kinder®, San Diego) during the 12h dark cycle when rats are normally active. Any PEGPH20-mediated decrease in rat locomotor activity was interpreted as MSE-like effects. In response to PEGPH20 (1 mg/kg) the locomotor activity of male rats was reduced by ~75% for the first 2 dark cycles post-treatment (48h), and then gradually returned to vehicle baseline levels by days 4-5. FDA approved drugs used clinically to alleviate pain were subsequently tested for their ability to ameliorate the effects of PEGPH20 on locomotor activity. Agents included anti-inflammatories, anti-spasmodics, neuropathic pain medications, neuroleptics, diuretics, anti-hypertensives, anti-fibromyalgics and opioids. The steroid dexamethasone, currently being used in the Phase 2 and Phase 3 PDA clinical trials to manage MSEs, was the positive control. As a class, nonsteroidal anti-inflammatory drugs (NSAIDs), including ketorolac, ketoprofen, diclofenac, and piroxicam, increased rat ambulation by ~40% on the first night following PEGPH20 treatment compared to PEGPH20 alone. Mobility in rats treated with NSAIDs/PEGPH20 increased ~60% on the second night compared to PEGPH20 alone. No other drug classes increased rodent ambulation. These data suggest that NSAIDs may be useful in partially alleviating the MSEs experienced by patients receiving PEGPH20. Citation Format: Li Ma, Marc R. Azar, Michael Shepard, Ya Huang, Piet A. Vervaet, Daniel C. Maneval, Curtis B. Thompson, Rudy Paladini. Evaluating clinically relevant pharmacological agents in a rat ambulation model to ameliorate PEGylated recombinant hyaluronidase PH20 (PEGPH20)-mediated musculoskeletal adverse events [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 1240. doi:10.1158/1538-7445.AM2017-1240

Abstract B32: A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors

The epidermal growth factor (EGF) signaling pathway relies on recognition by its receptor, EGFR, and subsequent downstream signaling by the KRAS and BRAF proteins to relay proper proliferative, migratory, and angiogenic functions. Cancers with activating KRAS or BRAF mutations are resistant to EGFR targeting agents and correspond to a significant unmet medical need. We hypothesized that an anti-EGFR antibody-drug conjugate (ADC) could be active against KRAS or BRAF mutated tumors, due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, we wished to engineer an antibody with enhanced specificity towards EGFR in the tumor microenvironment (TME) and attenuated binding to EGFR in normal tissue. This was achieved by screening a library of antibody variants (based on cetuximab) in a spatially addressed manner for binding to a recombinant version of the EGFR extracellular domain (EGFRECD) in two separate ELISA reaction conditions. High affinity binding to the EGFRECD was desired in the first condition, which approximated the physicochemical properties of the TME (acidic pH, high lactic acid concentration, 25% human serum). In the second assay condition, which approximated mAb binding to EGFRECD in normal tissue (neutral pH, low lactic acid concentration, 25% human serum), attenuated binding affinity was desired. We identified a lead mAb variant, cMab-1501, which possessed several fold reduced binding to EGFRECD in the neutral pH, low lactic acid condition, when compared to EGFRECD binding in the low-pH, high lactic acid, assay condition. To evaluate enhanced specificity for binding to EGFR in vivo, cMab-1501 was compared to cetuximab for binding to both human donor foreskin xenografts and human A431 tumor xenografts, using a DyLight 755 conjugated version of each antibody, and subsequent fluorescence detection with a Caliper IVIS system. cMab-1501 and cetuximab demonstrated relatively comparable binding towards human A431 tumor xenografts in vivo. In addition, cetuximab bound relatively equally between human tumor xenografts and human skin grafts. However, no binding to EGFR in the human skin graft was detected for cMab-1501 over all days measured; suggesting that cMab-1501 was highly specific for binding to EGFR in the TME. We next generated an cMab-1501 based ADC (antibody-drug conjugate), via maleimide chemistry carrying a protease cleavable valine-citrulline-p-aminobenzyloxycarbonyl monomethylauristatin E (vcPAB-MMAE) cytotoxic moiety, forming a cMab-1501-vcPAB-MMAE conjugate. Both the conjugated and un-conjugated versions of cMab-1501 were rapidly internalized by EGFR positive MDA-MB-231M tumor cells over several hours. In tumor xenograft models, the TME-specific anti-EGFR ADC demonstrated complete tumor regressions against two human EGFR overexpressing tumor types, MDA-MB-231M (TNBC, KRAS G13D) and HT-29 (CRC, BRAF V600E). In both in vivo models, tumors were resistant to treatment by cetuximab. These data suggest that it is possible to engineer a monoclonal antibody with enhanced specificity for its target within the TME and that an ADC-based approach could be utilized as potential treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Bob Veneziale, Lei Huang, Xiaoming Li, Qiping Zhao, Chunmei Zhao, Ryan Osgood, Jessica Cowell, Sanna Rosengren, Jason Parise, Ge Wei, Kim Phan, Robert Connor, Steve Rowe, Gilbert Keller, Gregory Frost, Dan Maneval, Curtis Thompson, Michael Shepard, Christopher Thanos. A tumor microenvironment specific EGFR targeting antibody-drug conjugate promotes regression in KRAS or BRAF mutant tumors. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B32.

Abstract 1217: Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors

Cancers with downstream activating KRAS or BRAF mutations in the EGFR pathway are resistant to EGFR targeting agents such as cetuximab and correspond to a significant unmet need. We hypothesized that an anti-EGFR ADC could be effective against KRAS or BRAF mutated tumors due to the cytotoxic mechanism of the ADC warhead. In an effort to eliminate the known dermal toxicity associated with anti-EGFR therapy, and to mitigate potential toxicities associated with treatment by an anti-EGFR ADC, a mAb was engineered with increased tumor microenvironment (TME) specificity for EGFR. The lead mAb demonstrated undetectable in vivo binding to human donor foreskins grafted onto nude mice, while binding to human A431 tumor xenografts with similar intensity to cetuximab (P 99.7%). Approximately 70% of this compound was rapidly internalized by human tumor cells grown in vitro over 4 hours, overlapping the internalization kinetics of the unconjugated mAb. HTI-1511 was evaluated for efficacy against two human EGFR overexpressing tumor models, MDA-MB-231M (triple-negative breast cancer, KRAS-G13D) and HT-29 (colorectal cancer, BRAF-V600E), and dosed at 5, 10, and 15 mg/kg, (qw, IV). A clear dose dependent anti-tumor response was observed with complete tumor regressions observed at the 15 mg/kg dose in both models, which were resistant to treatment by cetuximab. In addition, HTI-1511 was well-tolerated at 2 and 8 mg/kg in a cynomolgus monkey toxicity study (n = 3 per group), with limited dermal findings that were comparable with the vehicle control group. No adverse findings were observed at either dose. HTI-1511 showed a high degree of circulating stability in cynomolgus monkeys, and lacked in vivo degradation and instability that was observed in a control ADC conjugated using maleimide chemistry. HTI-1511 demonstrated significantly attenuated binding to FcγRIIa, FcγIIb, FcγIIIa 158V, and FcγIIIa 158F receptors, but not attenuated binding to FcγR1, in a FACS based assay format specific for each receptor, suggesting that HTI-1511 might have improved tolerability due to lack of binding by FcγRII-III receptors, possibly due steric hindrance from the PEG side chain. Thus, HTI-1511 holds promise as a potentially safe and effective treatment of EGFR overexpressing tumors with KRAS or BRAF mutations. Citation Format: Lei Huang, Bob Veneziale, Mark Frigerio, George Badescu, Xiaoming Li, Qiping Zhao, Jesse Bahn, Jennifer Souratha, Ryan Osgood, Chunmei Zhao, Kim Phan, Jessica Cowell, Sanna Rosengren, Jason Parise, Martin Pabst, Mathew Bird, William McDowell, Gina Wei, Curtis Thompson, Antony Godwin, Michael Shepard, Christopher Thanos. Preclinical evaluation of a next-generation, EGFR targeting ADC that promotes regression in KRAS or BRAF mutant tumors. [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 1217.

Abstract 1472: Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors

Adenosine is an endogenous immunosuppressant that binds to adenosine receptor checkpoints and protects tissue from immune-mediated rejection. Abnormally high adenosine levels (up to 100-fold greater than other tissues) contribute to a highly immunosuppressive tumor microenvironment (TME). We hypothesized that adenosine deaminase 2 (ADA2), a human enzyme that catalyzes the deamination of adenosine, could be administered at therapeutic levels to deplete high levels of TME adenosine and stimulate anti-tumor immune activity. Recombinant wild-type ADA2 (wtADA2) was cleared extremely rapidly from circulation (t1/2 = 69 min, 7.5 mg/kg iv, n = 9 mice), rendering it unsuitable for therapeutic testing. Therefore, a series of variants was designed to attenuate the heparin binding properties of ADA2 to improve bio-distribution and conjugated with 20K PEG to improve pharmacokinetics (PK). The variant PEG-ADA2-K374D displayed 94% less binding to heparin compared to wtADA2, enzymatic activity comparable to wtADA2, and 33-fold improved PK (t1/2 = 2,256 min); and consistently induced at least 60% (p 40-fold increase). This variant induced a maximum TGI of 69% (p suggest that engineered PEG-ADA2 variants induce significant tumor growth inhibition activity in several syngeneic solid tumor models, validating enzymatic depletion of high TME adenosine levels as novel immunotherapeutic approach to treat solid tumors. Citation Format: Lin Wang, Jessica Cowell, Sanna Rosengren, Lei Huang, Xiaoming Li, Qiping Zhao, Jennifer Souratha, Mathieu Marella, Barbara Blouw, Keri Cannon, Chunmei Zhao, Kim Phan, Curtis Thompson, Michael Shepard, Christopher Thanos. Enzymatic depletion of adenosine by pegylated, engineered adenosine deaminase 2 (PEG-ADA2): A novel immunotherapeutic approach to treat solid tumors. [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 1472.

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

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. References: Okuda H, Kobayashi A, Xia B, Watabe M, Pai SK et al. Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal Cells. Cancer Research 72:537-47, 2012. Tian X, Azpurua J, Hine C, Vaidya A, Myakishev-Rempel M et al. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature 499:346-349, 2013. Osgood RJ, Skipper JF, Cowell JA, Chen Y, Zhu L et al. Pegylated recombinant human hyaluronidase PH20 (PEGPH20) enhances Nab-Paclitaxel plus gemcitabine efficacy in human pancreatic cancer xenografts. AACR Special Conference Pancreatic Cancer 2014. Citation Format: Chunmei Zhao, Susan Zimmerman, Calvin T Vu, Michael Shepard, Zhongdong Huang. Naked mole rat hyaluronan synthase 2 displays similar effects as human hyaluronan synthase 2 and promotes tumor growth in a mouse xenograft model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-25.

Abstract 511: The role of hyaluronan-CD44 interaction in breast cancer.

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

Abstract 4999: Hyaluronan-rich ECM contributes to resistance to antibody-dependent cell-mediated cytotoxicity in solid tumors.

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

Abstract B35: Hyaluronan (HA) accumulation in tumors predicts response to pegylated rHuPH20 hyaluronidase (PEGPH20) in human tumors: A biomarker strategy.

Increasing evidence suggests that the tumor microenvironment (TME) is a potential source of novel anti-tumor targets. Extracellular matrix components in the TME include the matrix glycosaminoglycan, hyaluronan (HA), which accumulates in 20–30% of solid tumors. HA functions to increase water content of HA-rich stroma, thereby creating high tumor interstitial fluid pressure (IFP) characteristic of many tumors. Cells engineered to overexpress hyaluronan synthase form tumors more readily in mouse models, and HA accumulation has been associated with disease progression and poor prognosis in human cancers. Enzymatic degradation of HA with hyaluronidase can reduce IFP and facilitate delivery of anti-tumor agents. We have previously presented data that demonstrate the therapeutic potential of PEGPH20 in tumor xenografts derived from a spectrum of human malignancies. Here we highlight the utility of HA detection methods that use aggrecan-derived HA binding region (HABR). Accumulation of HA was measured in tumor tissues from 14 different xenografts and compared with anti-tumor activity of PEGPH20. When cell line-derived tumors were classified according to HABR staining intensity as HA+1,2,3 (low to high staining), the anti-tumor activity of PEGPH20 correlated with higher accumulation of HA in tumor specimens (r=0.8). To more rigorously test the hypothesis that HA-depletion is an effective candidate therapeutic approach for cancer, we utilized non-small cell lung cancer (SCC) patient tumor explants which were characterized for HA accumulation in advance of implantation into immunodeficient mice. Three SCC explants (HA+1,2,3) were treated with PEGPH20 and tumor growth was measured over a 2-week interval. Tumor growth inhibition corresponded to HA accumulation status, with the most dramatic reduction (97%; 4/10 regressions) observed in the HA+3 SCC explants. We have extended this work and detected tumor HA in pre- and post-treatment tumor biopsies collected from two patients enrolled in a Phase 1 study with PEGPH20. In both sets of specimens, a reduction in HA accumulation was demonstrated following PEGPH20 treatment. These results provide a rationale to assess tumor HA staining as a promising biomarker for clinical investigation with PEGPH20 for the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B35.