Tumor-Associated Hyaluronan Limits Efficacy of Monoclonal Antibody Therapy

Published OnlineFirst December 15, 2014; DOI: 10.1158/1535-7163.MCT-14-0580

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Tumor-Associated Hyaluronan Limits Efﬁcacy of Monoclonal Antibody Therapy Netai C. Singha1,Tara Nekoroski1, Chunmei Zhao1, Rebecca Symons1, Ping Jiang1, Gregory I. Frost2, Zhongdong Huang1, and H. Michael Shepard1

Abstract

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

Introduction (12–14). High HA content contributes significantly to the protumorigenic impact of the tumor stromal compartment (15). Hyaluronan (HA) is an extracellular glycosaminoglycan com- Recent advances in targeted monoclonal antibody (mAb)– ponent of many human tissues, and HA accumulation occurs in a derived therapeutics have led to remarkable response and survival variety of human solid tumors (1–5). HA binds noncovalently to with reduced toxicity and have established mAbs as part of the the N-terminal globular domains of proteoglycans, such as aggre- anticancer armamentarium (16–18). Trastuzumab (targeting can or versican, and other hyaladherins or hyalectans forming a p185HER2) and cetuximab [targeting the epidermal growth factor complex network in the extracellular matrix (ECM; refs. 4–6). receptor (EGFR)] are approved for breast cancer, gastric cancer, Accumulation of HA in tumors is associated with malignancy and colon cancer, and other solid tumors (16). Trastuzumab and predictive of more aggressive disease (1–5). Elevation of HA levels cetuximab inhibit tumor growth in human cancers characterized in tumors, sometimes in combination with collagen, results in by elevated expression of HER2 and EGFR, respectively (16–20). blood vessel compression, increased interstitial fluid pressure Although trastuzumab and cetuximab have shown success in the (IFP), and decreased perfusion (7, 8). HA depletion from solid clinic, resistance (intrinsic and acquired) to these mAb therapies tumors with a tumor microenvironment (TME) containing high has been reported in many cases (17, 21). Multiple mechanisms of levels of HA (HAhigh) reverses these physiologic effects, resulting resistance to these mAbs have been described previously (17, 19– in reperfusion of the tumor vasculature, increased chemothera- 24). Recently, the role of the tumor ECM has been recognized as a peutic drug accumulation, and tumor growth inhibition (TGI) in potential contributor to therapeutic resistance (7, 25, 26). preclinical animal models (8–11). The HAhigh pericellular matrix One mechanism of efficacy for mAb therapies is antibody- may also protect malignant cells from immune cell surveillance dependent cell-mediated cytotoxicity (ADCC), which involves therapeutic antibody binding to antigen on the tumor cell surface, and subsequent recognition of the antibody Fc region by immune cell surface FcgIIIA (CD16) receptor, followed by target tumor cell 1 2 Halozyme Therapeutics, Inc., San Diego, California. Intrexon Corpo- killing by immune cells [predominantly natural killer (NK) cells; ration, San Diego, California. refs. 16, 17, 20, 22]. ADCC plays a key effector role in mAb-based Note: Supplementary data for this article are available at Molecular Cancer therapy by direct killing of target tumor cells, possibly leading to Therapeutics Online (http://mct.aacrjournals.org/). tumor antigen presentation and induction of tumor antigen- Corresponding Author: H. Michael Shepard, Halozyme Therapeutics, Inc., 11388 directed T-cell response (16, 17, 20, 27–30). In this report, we Sorrento Valley Road, San Diego, CA 92121. Phone: 858-794-8889; Fax: 858- describe experiments suggesting a novel mechanism of resistance 704-8311; E-mail: [email protected] to ADCC in vitro and in vivo, mediated by an HAhigh pericellular doi: 10.1158/1535-7163.MCT-14-0580 matrix and its reversal by depletion of HA using a pegylated 2014 American Association for Cancer Research. recombinant human PH20 (PEGPH20).

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Materials and Methods Particle exclusion assay and movie Cancer cells were grown in 96- or 48-well plates (2,000–5,000 RPMI and McCoy 5A medium, Accutase, and human AB serum cells/well) for 2 days. On the day of the assay, cells were labeled were from Mediatech. Aggrecan (Elastin Products), calcein AM with calcein AM, and then treated with 1 mg/mL aggrecan for 1 and G418 (Sigma-Aldrich), cetuximab (Prodigy), trastuzumab hour, followed by incubation with antibody PEGPH20 for 1 (Prodigy), and recombinant human interleukin (IL)-2 (rhIL2; hour at 37 C. Human NK cells (0.5–1 106) were added and Peprotek) were purchased from commercial sources. Murine allowed to settle for 15 to 20 minutes before microscopic pho- anti-human CD16 and murine immunoglobulin (Ig) G1 isotype tography. One thousand cells were seeded on an eight-chambered control antibodies were from eBioscience. MDA-MB-231, SKOV3, glass slide and a movie was generated from images captured every and SKBR3 cell lines were purchased from the American Type 30 seconds at 32 C. All images were captured using a Nikon Eclipse Culture Collection, MDA-MB-231/Luc cell line was from Caliper TE200U inverted microscope (Nikon) attached with a SPOT Lifescience Inc., and cell lines MDA-MB-231/Luc/HAS2 (over- camera (SPOT imaging solutions) with a 40 objective lens. expressing luciferase and HAS2 genes), SKOV3/HAS2, and SKBR3/HAS2 were made by genetic engineering (incorporating Immunofluorescence and HA ELISA HAS2 gene using retroviral infection followed by G418 antibiotic Cells were incubated for 1 hour with aggrecan (1 mg/mL) selection) as described previously (the cell lines have not been followed by T-AF488 (4 mg/mL), or C-AF488 (10 mg/mL) authenticated; ref. 9). HAS2 gene overexpression was determined PEGPH20 (1,000 U/mL) for 1 to 2 hours in a humidified incu- by qPCR for HAS2 mRNA (Supplementary Fig. S1A). MDA-MB- bator with 5% CO2 at 37 C in the growth medium, followed by 231 cells were cultured in RPMI with 10% FBS. SKBR3 and SKOV3 fluorescent microscopic imaging using a 60 objective lens. HA cells were cultured in McCoy 5A with 10% FBS. Peripheral blood ELISA was performed following the manufacturer's protocol mononuclear cells (PBMC) were isolated from fresh healthy (Hyaluronan duoSet; R&D Systems). donor buffy coats (San Diego Blood Bank) using Histopaque- 1077 (Sigma-Aldrich) gradient centrifugation and stored (10 Immunohistochemistry and HA staining in human tumors 6 10 PMBC/vial) in liquid nitrogen. Human NK cells were expand- Four human breast cancer tissue arrays, including 58 invasive ed by culturing PBMCs as previously described (31). ductal carcinoma, four intraductal carcinoma, three invasive lobular carcinoma, and four of other tumor type with HER2 Antibody-dependent cell-mediated cytotoxicity expression status, were purchased from US BioMax. The arrays high To study HA pericellular matrix-mediated resistance to were stained for HA using biotinylated HA-binding protein fol- ADCC and its reversal by PEGPH20, we first established trastu- lowed by horseradish peroxidase and 3,30-diaminobenzidine zumab-dependent NK cell–mediated ADCC with adherent (DAB) detection. The array slides were scanned with a ScanScope 3þ SKBR3 (HER2 ) cells in culture. Target cells (10,000–15,000 CS (Aperio Technologies, Inc.) using bright field imaging at 20 cells/well) were seeded in 96-well plates and allowed to grow for 2 magnification, and analyzed for HA distribution using pixel count days to form a 70% to 80% confluent adherent monolayer. On the algorithm of the Spectrum program (Aperio Technologies). HA day of assay, monolayer cells were labeled with calcein AM (as staining was scored as high (HAhigh), medium (HAmedium), and described by the manufacturer), followed by treatments with HA- low (HAlow) corresponding to strong positive pixel area of 25%, binding proteoglycan aggrecan (1 mg/mL final concentration, 1 10% to 25%, and <10%, respectively (9). The intensity threshold hour at 37 C), PEGPH20 (1,000 U/mL), and cetuximab, trastu- was established using HA staining of positive control tumors, zumab, or human IgG1 isotype in succession (each for 1 hour at where the strong positivity is consistent from batch to batch. 37C). The antibody-treated cells were then incubated with ex vivo–expanded human NK effector cells (at NK to target cell ratio Ex vivo imaging of tumors of 15:1) for 5 hours (37 C with 5% CO2). At the end of incuba- Athymic nude mice (Taconic) were maintained in the animal tion, the number of live cells was determined by measuring facility of Halozyme Therapeutics, Inc. The animal use protocol fl calcein AM uorescence (excitation: 488 nm; emission: 537 nm) was reviewed and approved by Halozyme's Institutional Animal e on a SpectraMax M2 plate reader (Molecular Devices). In the Care and Use Committee. Female, 5- to 7-week-old nude mice ADCC blocking assay, NK cells were pretreated with mouse anti- (NCr-nu/nu) were inoculated intramuscularly into pretibial space human CD16 or mouse IgG1 isotype (5 mg/mL) for 30 minutes with 5 106 SKOV3/HAS2 cells. When tumor sizes reached 300 before being added to the antibody-loaded target cells. The mm3, mice (5/group) were treated intravenously (single dose) fi statistical signi cance of differences in the target cell killing under with NK-PKH26 cells (5 106 cells/mouse) and T-AF488 (0.75 t different conditions was calculated using the Student test. mg/kg) with or without PEGPH20 (40 mg/kg). Tumors were harvested 48 hours after dosing and imaged with ChemiDoc MP Flow cytometry Bio-Rad System (Bio-Rad). Images were quantified using Image- Cells were harvested and washed with fluorescence-activated Pro Analyzer 7.0 software (Tektronix). NK cells were labeled with cell sorting (FACS) buffer (PBS with 5% FBS) and labeled with PKH26 (Sigma Chemicals; Sigma-Aldrich) following the manu- fluorescent-labeled antibody for 1 hour at 4 C. Alexa Fluor 488 facturer's protocol. conjugated to trastuzumab (T-AF488) and cetuximab (C-AF488) was made following the manufacturer's protocol (Invitrogen) In vivo TGI and tested for HER2 or EGFR binding by live cell imaging. The mice were inoculated intramuscularly with 5 106 – Fluorescein isothiocyanate (FITC) conjugated anti-CD44 and SKOV3/HAS2 cells. When tumor sizes reached 200 mm3, mice – Alexa Fluor 488 conjugated anti-CD24 were from BioLegend. (6/group) were dosed intravenously twice weekly with ex vivo– Flow cytometry was performed on a BD FACS scan machine expanded human NK cells (8 106 cells/mouse) and trastuzu- (Becton Dickinson). mab (0.2 mg/kg) with and without PEGPH20 (40 mg/kg). Tumor

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size was measured weekly using ultrasound imaging (Vevo 2100 (compare Fig. 1D with Fig. 1E; Supplementary Movie S1). Imaging System; VisualSonics). Statistical analysis of difference in PEGPH20 treatment removed the HA-rich pericellular matrix, tumor volume between the control and treatment groups 1 day allowing NK cells to access the target tumor cells (Fig. 1F and after the last treatment dose was performed using one-way Supplementary Movie S1). We also observed NK cell synapse with ANOVA, with a P value less than or equal to 0.05 (P 0.05) tumor cells following PEGPH20 treatment, resulting in morpho- considered statistically significant (32). logic changes in the tumor cells, indicating initiation of NK cell– mediated killing of the target tumor cells (Fig. 1G). Overall, HAS2 Results overexpression led to formation of an HAhigh pericellular matrix coat capable of preventing access of NK cells to tumor cells in cell Simultaneous occurrence of high HA level and EGFR or HER2 culture, and this physical restriction was removed by HA deple- expression in human tumors tion following PEGPH20 treatment. Extracellular HA accumulation occurs in a wide variety of cancers [e.g., breast, prostate, colorectal, gastric, and head and HAhigh pericellular matrix inhibits ADCC in vitro neck squamous cell carcinoma (HNSCC)], and high levels of HA We conducted ADCC studies to test the functional potential of surrounding tumor and/or stromal cells are associated with HA coats in preventing NK cell–mediated tumor cell lysis. During epithelial–mesenchymal transition, host–tumor interactions, and ADCC, antibody-dependent synapse between NK cells and target other protumorigenic processes (1–5). We investigated the fre- tumor cells is essential for tumor cell killing (16, 20, 22). Mono- quency of occurrence of accumulation of high HA levels in þ þ layer-adherent SKBR3 parental and SKBR3/HAS2 cells were incu- primary HER23 breast adenocarcinoma tumors and EGFR bated in the presence of aggrecan to study the effect of an HAhigh HNSCC tumors using biotinylated HA-binding protein. Twen- þ þ pericellular matrix coat on ADCC. In the presence of NK cells, ty-four of 63 EGFR HNSCC samples (38%) and 14 of 24 HER23 trastuzumab-dependent targeted killing of SKBR3/HAS2 cells was breast tumor samples (58%) were found to be HAhigh (Table 1). þ consistently lower than that of parental SKBR3 cells (Fig. 2A), This finding indicates that a substantial fraction of HER23 or þ suggesting that an HA coat was mediating resistance to antibody- EGFR tumors exhibit an HAhigh phenotype, and provides a dependent NK cell-mediated cytotoxicity of HAhigh tumor cells. rationale for exploring the role of pericellular matrix–associated The difference in ADCC between SKBR3/HAS2 and SKBR3 cells HA accumulation in resistance to anticancer therapies such as appeared to be trastuzumab concentration-dependent, with the trastuzumab or cetuximab. biggest difference observed at 40 ng/mL trastuzumab with 46% cytotoxicity in HAS2-transfected cells versus 72% in untransfected HAhigh pericellular matrix inhibits NK cell access to tumor cells in vitro cells (Fig. 2A). Smaller differences between SKBR3/HAS2 and þ þ SKBR3 were observed at 0.8 and 2,000 ng/mL trastuzumab (Fig. Parental HER23 SKBR3 and SKOV3, and EGFR MDA-MB- 2A). A similar trend in resistance to antibody-dependent NK cell– 231 breast cancer cells with low levels of HA (HAlow) were mediated cytotoxicity was observed with MDA-MB-231/Luc/ transfected with a retrovirus carrying human HA synthase 2 HAS2 breast tumor cells in the presence of cetuximab, with (HAS2) cDNA to generate HAS2-overexpressing SKBR3/HAS2, reduced killing of MDA-MB-231/Luc/HAS2 cells compared with SKOV3/HAS2, and MDA-MB-231/Luc/HAS2 cell lines. These parental MDA/MB/231 cells (Fig. 2B). This is not related to the HAS2-overexpressing cell lines secreted higher levels of HA com- differences in HER2 or EGFR surface expression between trans- pared with parental cells in culture (Supplementary Fig. S1B). fected and nontransfected cell populations (Supplementary Fig. Human HAS2 gene overexpression did not alter HER2, EGFR, S2) or HA–aggrecan complex interference in trastuzumab or CD44, or CD24 levels on cell surface (Supplementary Fig. S2). cetuximab binding to HER2 or EGFR on SKBR3/HAS2 or In the presence of aggrecan, an HA-binding proteoglycan, MDA-MB-231/Luc/HAS2 cells, respectively (Fig. 2C). In addition SKBR3/HAS2 (HAhigh) cells formed enlarged pericellular matrices to resistance to ADCC, the HA-rich pericellular matrix also in vitro (Fig. 1A) compared with parental SKBR3 cells (Fig. 1B), showed a basal level (non-mAb-mediated) resistance to NK which, in turn, restricted the access of human NK cells to tumor cell–mediated cytotoxicity (to a lesser extent than resistance to cells in culture. The pericellular matrix surrounding SKBR3/HAS2 ADCC) in SKBR3/HAS2 and MDA-MB-231/Luc/HAS2 tumor cells cells was diminished by PEGPH20 treatment, allowing increased (Fig. 2A and B, control). Pretreatment of NK cells with mouse anti- proximity of NK cells to tumor cells as shown in Fig. 1C, where the CD16 antibody blocked its ADCC activity on SKBR3 cells (Sup- NK cells can be seen closely surrounding the tumor cells. This plementary Fig. S3), suggesting that the ADCC observed on finding was further extended to HAhigh MDA-MB-231/Luc/HAS2 adherent SKBR3 cells is CD16 dependent. The observed resistance cells. Compared with parental MDA-MB-231 cells, MDA-MB- of HAS2-overexpressing tumor cells to ADCC and basal NK cell– 231/Luc/HAS2 cells formed enlarged pericellular matrix coats, mediated cytotoxicity supports the observation that an HAhigh which inhibited access of human NK cells to tumor cells pericellular matrix on the tumor cell surface interferes with functional access of NK cells to target tumor cells, and partially 3þ Table 1. HA distribution in EGFR-positive HNSCC and HER2 breast protects the tumor cells from NK cell–mediated cytotoxicity. adenocarcinoma tissues HA-positive incidence HA depletion by PEGPH20 enhances in vitro ADCC of HAhigh HNSCC (n ¼ 63) Breast (n ¼ 24) HA score EGFRþ (%) HER23þ (%) tumor cells high Low 11/63 (17) 1/24 (4) PEGPH20 treatment depleted the HA pericellular matrix of Medium 28/63 (44) 9/24 (38) SKBR3/HAS2 cells and MDA-MB-231/Luc/HAS2 cells, resulting in High 24/63 (38) 14/24 (58) increased access of NK cells to tumor cells (Fig. 1). To study NOTE: HA staining was scored as high, medium, and low corresponding to the effect of PEGPH20 on ADCC, calcein AM-labeled adherent strong positive pixel area of 25%, 10%–25%, and <10%, respectively (9). tumor cells were incubated with PEGPH20 and trastuzumab or

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A B C

SKBR3/HAS2 SKBR3 SKBR3/HAS2 D E F

Figure 1. HAhigh pericellular matrix inhibits human NK cell interaction with malignant tumor cells. Human NK cell exclusion by pericellular matrix of HAhigh SKBR3/HAS2 cells (A) versus HAlow parental SKBR3 (B). C, PEGPH20 treatment removed MDA-MB-231/Luc/HAS2 MDA-MB-231 MDA-MB-231/Luc/HAS2 pericellular matrix of SKBR3/HAS2. Pericellular matrix–mediated NK cell –PEGPH20 +PEGPH20 exclusion by MDA-MB-231/Luc/HAS2 (HAhigh) cells (D) versus parental G MDA-MB-231 (HAlow) cells (E). F, depletion of HAhigh pericellular matrix of MDA-MB-231/Luc/HAS2 cells by PEGPH20 treatment. Tumor cells (green) and NK cells (small round cells) are shown. G, synapse formation between NK cells and MDA-MB-231/ Luc/HAS2 following HA depletion by PEGPH20 treatment. PEGPH20 was added at time zero (0 minutes) and images were captured every 30 seconds. Images at 0, 20, 40, and 60 0 min 20 min minutes are shown. NK cell–tumor cell synapse is indicated by red arrow.

40 min 60 min

cetuximab then incubated with NK cells to induce ADCC. HA ADCC inhibition. As expected, PEGPH20 only caused a small depletion following PEGPH20 treatment resulted in a 40% increase in cetuximab-mediated ADCC of HAlow parental MDA- increase in trastuzumab-dependent NK cell–mediated killing of MB-231 cells (Fig. 3D and Supplementary Table S1). The increase SKBR3/HAS2 cells (Fig. 3A and Supplementary Table S1). Con- in ADCC on SKBR3/HAS2 (HAhigh) and MDA-MB-231/Luc/HAS2 versely, a much smaller increase of trastuzumab-mediated ADCC (HAhigh) cells following PEGPH20 treatment appeared to be of parental HAlow SKBR3 cells resulted following PEGPH20 antibody concentration dependent, with the increase in ADCC treatment (Fig. 3B and Supplementary Table S1). Pretreatment diminished at a higher antibody concentration (Fig. 3A and C). of NK cells with PEGPH20 showed no effect on NK cell–mediated This is probably due to a greater number of antibody-bound ADCC, suggesting that the observed increase in ADCC on SKBR3/ receptors on target cells at higher antibody concentration, which HAS2 following PEGPH20 treatment is not due to altered NK results in effective cell killing despite fewer encounters with NK effector function caused by PEGPH20 (Supplementary Fig. S4A). cells. The effect of limited NK cell accessibility, mediated by an In HAhigh MDA-MB-231/Luc/HAS2 cells, PEGPH20 treatment HAhigh pericellular matrix, is likely to be most sensitive at inter- also resulted in a signiﬁcant increase in cetuximab-dependent mediate antibody concentration. The observed increase in ADCC ADCC (Fig. 3C and Supplementary Table S1), further supporting following PEGPH20 treatment in HAhigh tumor cells is supported the observation that an HAhigh pericellular matrix plays a role in by a moderate increase in basal NK cell–mediated cytotoxicity by

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A B ** ** *** ***

** *** ** *** Figure 2. *** HAhigh pericellular matrix inhibits ADCC on tumor cells and HA– aggrecan complex does not interfere with mAb binding to HER2 or EGFR surface receptors. A, dose-dependent trastuzumab-mediated ADCC of monolayer SKBR3 (HAlow) and SKBR3/HAS2 (HAhigh) cells. B, cetuximab-mediated ADCC of monolayer MDA-MB-231 (HAlow)and C Aggrecan Aggrecan and PEGPH20 MDA-MB-231/Luc/HAS2 (HAhigh) cells. t tests were performed to compare responses of parental and HAS2- overexpressing cells. , P < 0.05; , P < 0.005; bars, SEM; n ¼ 5. C, trastuzumab or cetuximab binding to HER2 on SKBR3/HAS2 or EGFR on MDA-MB-231/Luc/HAS2, respectively, T-AF488 in the presence of aggrecan (left) and aggrecan and PEGPH20 (right). Cells SKBR3/HAS2 were incubated with aggrecan to generate HA–aggrecan complex followed by incubation with Alexa Fluor 488–conjugated trastuzumab (T-AF488) or cetuximab (C-AF488) antibody with or without PEGPH20. Antibody binding was detected using ﬂuorescent live cell imaging. C-AF488 MDA-MB-231/Luc/HAS2

PEGPH20 (Fig. 3A and C, control), suggesting NK access inhibi- imaging. HA depletion in SKOV3/HAS2 tumors following tion in HAhigh tumor cells. PEGPH20 treatment was determined by immunohistochemistry (IHC) and ELISA to confirm PEGPH20 hyaluronidase activ- HA depletion increased antibody and NK cell accessibility to ity in vivo. PEGPH20 caused approximately 60% HA removal in HAhigh xenograft tumors SKOV3/HAS2 tumors (Supplementary Fig. S5A and S5B). In Sensitization of drug-resistant cancer cells to chemothera- contrast to the in vitro experiment (Supplementary Fig. S4B), peutic agents by hyaluronidase treatment suggests higher drug PEGPH20 treatment significantly increased tumor-associated exposure to tumor cells following HA depletion (33). Similarly, trastuzumab levels in animals dosed with T-AF488 (Supple- HER2 receptor in HAhigh tumors can be masked by pericellular/ mentary Fig. S5C and S5D). Similarly, HA depletion by stromal HA (34, 35); however, in our in vitro experiments, HA– PEGPH20 treatment resulted in increased trastuzumab levels aggrecan complex did not interfere with trastuzumab or cetux- in tumors of animals concomitantly dosed with T-AF488 and imab binding to HER2 or EGFR in HAhigh SKBR3/HAS2 or NK-PKH26 (Fig. 4A; top middle vs. right). Quantitation of the MDA-MB-231/Luc/HAS2 cells, respectively (Fig. 2C). We fur- fluorescence signal intensity showed an approximately 2-fold ther investigated whether PEGPH20 treatment would improve increase in trastuzumab levels (Fig. 4B). To our surprise, trastuzumab accessibility to HAhigh tumors in vivo.Weuseda PEGPH20 treatment showed no statistically significant increase HAS2-overexpressing HER2-positive ovarian cancer SKOV3 in tumor-associated NK levels in animals dosed with NK- (SKOV3/HAS2) tumor xenograft model in the in vivo studies. PKH26 in the absence of antibody(SupplementaryFig.S5E As observed with SKBR3/HAS2, trastuzumab binding to HER2 and S5F). However, PEGPH20 treatment increased access of NK was not altered in SKOV3/HAS2 in the presence of aggrecan in cells to tumors in the animals dosed with T-AF488 plus NK- vitro (Supplementary Fig. S4B). SKOV3/HAS2 tumor-bearing PKH26 (Fig. 4A; bottom middle vs. right). NK cell infusion in mice were dosed intravenously with T-AF488, PKH26-labeled the PEGPH20/NK cell/trastuzumab–treated tumors increased NK cells (NK-PKH26), or T-AF488 plus NK-PKH26 with or by 3-fold compared with the trastuzumab/NK cell–treated without PEGPH20, and tumors were harvested 48 hours after tumors without PEGPH20 (Fig. 4C). Trastuzumab or NK cells dosing to assess trastuzumab and NK cell accessibility by ex vivo were not detected in kidney, muscle, or heart of the mice treated

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Figure 3. PEGPH20 enhanced mAb-mediated ADCC of HAhigh tumor cells. PEGPH20 increased trastuzumab-mediated ADCC on SKBR3/HAS2 (A) and cetuximab-mediated ADCC on MDA- MB-231/Luc/HAS2 cells (C). PEGPH20 showed modest effect on trastuzumab-mediated ADCC on SKBR3 (HAlow) cells (B) and cetuximab-mediated ADCC on MDA-MB-231 (HAlow) cells (D). t tests were performed to compare control and PEGPH20-treated samples for each treatment group. , P < 0.05; , P < 0.005; bars, SEM; n ¼ 5.

with T-AF488, NK-PKH26, or T-AF488 plus NK-PKH26 with mab and NK cell–induced TGI, which is likely a consequence of or without PEGPH20 (Fig. 4D and Supplementary Fig. S6) increasing trastuzumab and NK cell exposure to tumor cells in despite the fact that both muscle and heart have substantial HA SKOV3/HAS2 tumors (Fig. 5). content (36). Taken together, these data show that HA depletion by PEGPH20 increased trastuzumab and NK cell accessibility to HAhigh xenograft tumors in the SKOV3/HAS2 tumor Discussion model, probably as a result of tumor vasculature decompres- The majority of HA is localized in tumor ECM, where it binds sion (3, 8–11). with multiple hyaladherins or hyalectans (5, 6, 37, 38). Versican is predominantly expressed in HAhigh tumors (5, 37, 38) and has HA depletion from HAhigh tumors following PEGPH20 limited commercial availability. Aggrecan is a proteoglycan with treatment increased TGI by increasing ADCC structure composition similar to versican and binds to HA efﬁ- Higher antibody and NK cell accessibility to HAhigh tumors ciently and was used in the in vitro studies reported here. High following HA depletion and stromal remodeling by PEGPH20 levels of HA accumulation in primary human tumors were found þ treatment suggests that PEGPH20 may enhance ADCC in vivo, in a substantial percentage of HER23 breast tumor tissues and þ leading to improved TGI. We tested this possibility in the EGFR HNSCC tissues, and these ﬁndings are consistent with a þ SKOV3/HAS2 tumor model. In this tumor model, NK cell previous report that high levels of HA is associated with HER23 exposure to tumor peaked at day 2 and NK cells were detectable breast cancer tissues (1). HAhigh glioma cells and mouse sarcoma within tumor up to day 5 after dosing with NK-PKH26 (Fig. cells have been previously shown to exhibit pericellular matrix– 5A). SKOV3/HAS2 tumor-bearing mice were treated twice dependent resistance to CTL-mediated cytotoxicity in vitro (12– weekly with trastuzumab (0.2 mg/kg), human NK cells (8 14). Thus, we hypothesized that the pericellular matrix of HAhigh 106 cells/mouse), and PEGPH20 (40 mg/kg) individually tumor cells may form a physical barrier capable of interfering with or in combinations to study the effect of PEGPH20 on anti- access of antibody and immune cells to target tumor cells. In this body-dependent NK cell–mediated TGI. The trastuzumab/NK study, we investigated PEGPH20-sensitive pericellular matrix– combination treatment group showed higher TGI (41%) com- mediated inhibition of trastuzumab- or cetuximab-dependent pared with the trastuzumab (22%) or NK treatment groups ADCC targeting HAhigh solid tumors. (24%; Fig. 5B), suggesting the requirement of a human NK In alignment with our hypothesis, exogenous HAS2-overex- supplement for optimal tumor sensitivity. Treatment with pressing tumor cells formed an enlarged pericellular matrix that PEGPH20 alone showed 46% TGI (Fig. 5B). The triple com- limited access of NK cells to tumor cells, and depletion of HA bination of PEGPH20/NK cell/trastuzumab treatment resulted following PEGPH20 treatment allowed NK cell access to tumor in 68% TGI (Fig. 5B). These in vivo data demonstrate that HA cells. These results suggest that an HAhigh pericellular matrix may depletion following PEGPH20 treatment enhanced trastuzu- contribute to the innate resistance of tumors to host immune

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A Vehicle – PEGPH20 +PEGPH20 T-AF488

Figure 4. HA depletion by PEGPH20 concomitantly enhanced trastuzumab and NK cell accessibility to HAhigh tumors. NK-PKH26 Fluorescently labeled trastuzumab and NK cells (T-AF488 and NK-PKH26) were used for detection of trastuzumab and NK cell T-AF488 and NK-PKH26 delivery to tumors. A, representative ex B C vivo images of SKOV3/HAS2 tumors 70 70 ** showing trastuzumab (green) and NK cell ** (red) accessibility in PEGPH20-untreated 60 60 versus PEGPH20-treated tumors 48 hours 50 50 after dosing. Boundary of vehicle-treated tumor is shown. Quantiﬁcation data of 40 40 trastuzumab (B) and NK cell (C) 30 30 accessibility to SKOV3/HAS2 IM tumors. t tests were performed to compare control 20 20 and PEGPH20-treated samples. , P < Fluorescence intensity Fluorescence intensity 0.05; bars, SEM; n ¼ 5. D, representative 10 Fluorescence intensity 10 images showing biodistribution of 0 0 trastuzumab and NK cells in HER2-positive Vehicle T-AF488 and T-AF488 and Vehicle T-AF488 and T-AF488 and high HA tumors, and kidney, muscle, and NK-PKH26 NK-PKH26 NK-PKH26 NK-PKH26 heart. Samples from mice concomitantly and PEGPH20 and PEGPH20 treated with T-AF488 and NK-PKH26 in the presence or absence of PEGPH20 are D T-AF488 and NK-PKH26 T-AF488 and NK-PKH26 and PEGPH20 shown. Tumor Kidney Muscle Heart Tumor Kidney Muscle Heart

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surveillance and promote tumor cell survival. This conclusion is trastuzumab and NK cell) access to HAhigh tumors. Furthermore, further supported by (i) the observed resistance of HAS2-over- neither exogenously administered trastuzumab nor NK cells were expressing tumor cells to ADCC, and (ii) a dramatic increase of present at detectable levels in normal tissues even in the presence ADCC by PEGPH20 treatment in HAhigh tumor cells in vitro. of PEGPH20, suggesting that the effect of PEGPH20 on trastu- Although HAhigh tumor cells were reported to mask HER2 from zumab and NK cells is predominantly tumor-specific and HAhigh binding to trastuzumab (34–35), we have not detected HA/ tumor-specific activity of PEGPH20 would likely contribute to an aggrecan–mediated HER2 or EGFR receptor masking in HAS2- improved therapeutic index as a result of pharmaceutical activity overexpressing cells in vitro. Limited access of NK cells to tumor on the HAhigh (high-IFP) tumors. These results demonstrate an cells by the HAhigh pericellular physical barrier likely contributed HA-dependent TME-mediated inhibition of antibody and NK cell to the observed resistance of HAS2-overexpressing tumor cells to access to HAhigh tumors, which suggests that these tumors may be ADCC in vitro. Restricted therapeutic antibody access to tumor as sensitized to ADCC following PEGPH20 treatment. Indeed, HA well as masking of receptors by HA may also contribute to depletion by PEGPH20 resulted in higher TGI in the triple- resistance to ADCC in other HAhigh tumor cells. combination group (PEGPH20/NK cells/trastuzumab) compared HA depletion from HAhigh tumors has been shown to be with the double-combination group (trastuzumab/NK cells) in effective in increasing intratumoral exposure of chemotherapeutic the SKOV3/HAS2 tumor model. This is consistent with previous agents, suggesting an HA-dependent limitation of drug access in reports that PEGPH20 can enhance TGI of HAhigh tumors when HAhigh tumors mediated by compression of blood vesicles by combined with chemotherapy in several preclinical animal mod- intratumoral pressure (3, 8–11). Here, we demonstrate that HA els (8–11). The observed statistically significant increase in TGI in depletion by PEGPH20 increased large-size therapeutics (both the PEGPH20/NK cells/trastuzumab group compared with NK

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Singha et al.

A 70 ** contribute to resistance to mAb therapy (34, 35). Thus, enhanced 60 ** tumor access of NK cells and antibody, as well as increased 50 antibody binding to exposed target receptors, can be achieved by depletion of stromal HA with PEGPH20 treatment. 40 ** ** Growing evidence indicates that HA provides a protumorigenic 30 environment and exogenous hyaluronidase administration 20 showed signiﬁcant antitumor activity in HA-overexpressing

Fluorescence intensity – 10 tumors (3, 7 11, 15). Conversely, increased hyaluronidase activity has been detected in tumor tissues and/or serum from several 0 – 24 h 48 h 72 h 96 h 120 h primary and metastatic cancers (3 5, 39, 40). Although hyaluron- Time idase expression (predominantly associated with hyaluronidase NK-PKH26 NK-PKH26 and PEGPH20 1, which has very little enzymatic activity at physiologic activity) in the tumor has been shown to potentially increase metastasis in 1,100 some preclinical animal models (3, 39, 40), PEGPH20 treatment B Control did not increase metastasis in an HAhigh tumor xenograft model 1,000 NK cells and PEGPH20 in combination with gemcitabine reduced meta-

900 Trastuzumab ** stasis burden in a KPC mouse model (3, 11). Although, the possibility of increasing metastasis following HA depletion by ) ** 3 800 PEGPH20 PEGPH20 cannot be ruled out by the current studies, available

700 Trastuzumab and NK cells literature indicates that an increase in metastatic potential by PEGPH20 treatment is unlikely (3, 11). Additional investigation 600 Trastuzumab and PEGPH20 and NK is required to understand the protumorigenic and/or prometa- cells ** 500 static role of hyaluronidase and it is likely tumor type dependent. Tumor volume (mm In our mouse model, if some tumor cells are liberated from HAhigh 400 tumor by PEGPH20, they will likely be sensitized to effector 300 immune cell–mediated elimination. Despite the breakthroughs brought to cancer treatment by 200 9 16 22 30 mAbs, innate tumor resistance to drug remains a major problem Days (17, 19, 20, 23, 24). Targeted mAb therapy for human solid tumors relies on binding of the mAb to tumor cell surface target Figure 5. receptors followed by intracellular signaling attenuation to sen- HA depletion by PEGPH20 improved NK cell and antibody-dependent TGI in – HAhigh tumors. A, NK persistence in SKOV3/HAS2 tumor. SKOV3/HAS2 sitize tumor cells, direct tumor cell killing by FcgIIIA (CD16) tumor-bearing mice were dosed with NK-PKH26 cells (5 106), trastuzumab expressing immune cells, and mAb-mediated host immune (0.75 mg/kg) and PEGPH20 (40 mg/kg), and tumors were harvested at response activation. Success of mAb therapies in the clinic is indicated time points for ex vivo imaging under the same exposure limited by several underlying mechanisms including underap- condition. , P < 0.05; n ¼ 4. B, tumor growth curves showing groups of preciated TME-mediated host immune response suppression, SKOV3/HAS2 tumor-bearing mice treated with IgG control, trastuzumab, NK þ such as reduced malignant cell killing by CD8 CTLs and/or NK cell, PEGPH20, trastuzumab/NK cell, and trastuzumab/NK cell/PEGPH20. The triple-combination trastuzumab/NK cell/PEGPH20 treatment increased cells, and properties of some immune cells harboring protumor TGI over the double combination trastuzumab/NK cell group. The animal activities (25). In addition, tumor cells have been shown to use model was generated as described in Materials and Methods. TGI was multiple immunosuppressive mechanisms to avoid host immune T T C C T calculated as [1 ( n 0)/( n 0)] 100% ( n, average tumor volume in attack, including tumor-induced impairment of antigen presen- n T the treatment group at day after the last dose of PEGPH20; 0, average tation (41, 42), secretion of immunosuppressive and growth tumor volume in the treatment group at day 0 before treatment; Cn, factors (e.g., TGFb and IL10) in the TME (43, 44), ampliﬁcation average tumor volume in the control group at day n after the last dose of of receptor tyrosine kinases (45), upregulation/expression of vehicle; C0, average tumor volume in the control group at day 0 before treatment; ref. 8). TGI for trastuzumab, NK cell, PEGPH20, trastuzumab/NK negative costimulatory signaling molecules (CTLA-4 binding cell, and trastuzumab/NK cell/PEGPH20 versus control was 22%, 24%, 46%, CD80 and/or CD86, PD-L1 and/or PD-L2) by tumor cells (46, 41%, and 68%, respectively. t tests were performed to compare control with 47), inhibition of dendritic cell differentiation and maturation other treatments. , P < 0.05; bars, SEM; n ¼ 6. (44, 46), and increased inﬁltration of regulatory T-cells (48, 49). Here, we demonstrate that an HAhigh tumor ECM enables forma- cells/trastuzumab is rather modest in our tumor model. This is tion of a protective pericellular matrix coat capable of inhibiting likely caused by tumor heterogeneity and a low NK cell to target access of NK cells to tumor cells in vitro as well as antibody and NK cell number ratio in vivo and unlikely to be caused by limited cell access in vivo, suggesting a novel mechanism for cancer cells to PEGPH20 availability into the tumor, because PEGPH20 has a escape detection and clearance by antibody- and NK cell-medi- serum half-life of 10.3 hours, which is dramatically higher than ated ADCC. rhuPH20 (t1/2 < 5 minutes), and HA removal from tumors by The role of the TME in tumor progression and metastasis PEGPH20 is sustained for up to 72 hours (3, 8). Both stromal cells supports the approach of targeting one or more TME components and tumor cells may provide HA to the TME, and our results for cancer therapeutics (7, 15, 50). HA in the TME is a promising demonstrate that an HAhigh TME contributes to limiting the access target for cancer therapeutics due to its role in creating high of NK cells and therapeutic antibody into the tumor. Although, intratumoral pressure resulting in blood vessel compression and while we have not observed reduced antibody binding to HAhigh development of hypoxia, epithelial–mesenchymal transition, tumor cells in vitro, receptor masking in HAhigh tumors in vivo may tumor progression and metastasis, multidrug resistance, and

530 Mol Cancer Ther; 14(2) February 2015 Molecular Cancer Therapeutics

Stromal HA Elevation Limits mAb-Mediated Cancer Therapy

escape from immune system surveillance. Increasing tumor expo- Writing, review, and/or revision of the manuscript: N.C. Singha, T. Nekoroski, sure of therapeutic mAbs and immune cells by depleting HA from P. Jiang, G.I. Frost, Z. Huang, H.M. Shepard the TME is a therapeutic strategy with great potential. Our data Administrative, technical, or material support (i.e., reporting or organizing high in data, constructing databases): T. Nekoroski show that an HA TME contributes to inhibition of ADCC Study supervision: P. Jiang, Z. Huang vitro and in vivo that can be reversed by HA depletion following Other (histology processing and IHC): R. Symons PEGPH20 treatment, and demonstrates the potential beneficial effect of PEGPH20 treatment together with mAb-based therapies Acknowledgments for HAhigh solid tumors. The authors thank Susan Zimmerman, Gina Wei, and Qiping Zhao for their contributions in cell lines, retroviral vector, and retrovirus generation; Grace Lee Disclosure of Potential Conflicts of Interest for in vivo experiments; Ryan Osgood, Robert Connor, and Calvin Vu for assistance with in vivo studies; Curtis Thompson for critical suggestions in article G.I. Frost has ownership interest in and is a consultant for Halozyme preparation; and Daniel C. Maneval for article review. The authors acknowledge Therapeutics, Inc. No potential conflicts of interest were disclosed by the other the pioneering work of Robert Stern, Markku Tammi, and Robert Kerbel on role authors. of HA biology in cancer. Authors' Contributions Conception and design: T. Nekoroski, P. Jiang, G.I. Frost, Z. Huang, H.M. Grant Support Shepard All work in this study was funded by Halozyme Therapeutics, Inc. Development of methodology: N.C. Singha, T. Nekoroski, C. Zhao, G.I. Frost, The costs of publication of this article were defrayed in part by the payment of Z. Huang page charges. This article must therefore be hereby marked advertisement in Acquisition of data (provided animals, acquired and managed patients, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. provided facilities, etc.): N.C. Singha, T. Nekoroski, C. Zhao Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Received July 21, 2014; revised November 25, 2014; accepted December 1, computational analysis): N.C. Singha, T. Nekoroski, C. Zhao, P. Jiang, Z. Huang 2014; published OnlineFirst December 15, 2014.

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532 Mol Cancer Ther; 14(2) February 2015 Molecular Cancer Therapeutics

Tumor-Associated Hyaluronan Limits Efficacy of Monoclonal Antibody Therapy

Netai C. Singha, Tara Nekoroski, Chunmei Zhao, et al.

Mol Cancer Ther 2015;14:523-532. Published OnlineFirst December 15, 2014.

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