Remodeling the Tumor Microenvironment …...twice per week by digital caliper, and tumor volumes calculated using the modiﬁed ellipsoid formula: 1/2 (length width 2 ). Tumorgrowth,measuredastumorvolume(mm

Tumor Biology and Immunology

Remodeling the Tumor MicroenvironmentSensitizes Breast Tumors to Anti-ProgrammedDeath-Ligand 1 ImmunotherapyRenee Clift1, Jennifer Souratha2, Sheryl A. Garrovillo3, Susan Zimmerman4,and Barbara Blouw3

Abstract

Hyaluronan removal in the tumor microenvironment improves immune cells and checkpoint inhibitor access to tumors.

Capillary Cancer cell Hyaluronan

Hyaluronan accumulation Hyaluronan degradation

Immune checkpoint inhibitor

Immunotherapies targeting immune checkpointinhibitors have changed the landscape of cancertreatment, however, many patients are resistant orrefractory to immunotherapy. The sensitivity oftumor cells to immunotherapy may be influencedby hyaluronan (HA) accumulation in the tumormicroenvironment (TME). Enzymatic degradationof HA by pegvorhyaluronidase alfa (PEGPH20;PVHA) remodels the TME. This leads to reducedtumor interstitial pressure and decompressedtumor blood vessels, which are both associatedwith increased exposure of tumor cells to chemo-therapy drugs. Here, we demonstrate PVHAincreased the uptake of anti-programmed death-ligand 1 (PD-L1) antibody in HA-accumulatinganimal models of breast cancer. The increasedlevels of anti-PD-L1 antibody were associated withincreased accumulation of T cells and natural killercells and decreased myeloid-derived suppressorcells. PD-L1 blockade significantly inhibited tumorgrowth when combined with PVHA, but not alone. Our results suggest that PVHA can sensitize HA-accumulating tumors toanti-PD-L1 immunotherapy.

Significance: These findings show removal of hyaluronan in the tumor microenvironment improves immune cells andcheckpoint inhibitors access to tumors.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/16/4149/F1.large.jpg.

IntroductionCancer immunotherapy using mAbs to target ligands

[programmed death-ligand 1 (PD-L1)], receptors [programmed

cell death protein 1 (PD-1), and cytotoxic T lymphocyte–associated protein 4 (CTLA-4)] of inhibitory immune check-points, which can be overexpressed by cancer cells, representsone of the biggest advances in cancer treatment in recent years.Durable responses with cancer immunotherapies have beenobserved in a variety of solid tumors (1). Two anti-PD-1 mAbs[pembrolizumab (KEYTRUDA,Merck&Co., Inc.) andnivolumab(OPDIVO, Bristol-Myers Squibb Company)] and three anti-PD-L1 mAbs [atezolizumab (TECENTRIQ, Genentech, Inc.), durva-lumab (IMFINZI, AstraZeneca, Inc.), and avelumab (BAVENCIO,EMD Serono, Inc.)], have been approved by the FDA for clinicaluse in multiple tumors, and an anti-CTLA-4 mAb [ipilimumab(YERVOY, Bristol-Myers Squibb Company)] has been approvedfor the treatment ofmetastaticmelanoma (2–4).However, severalcommon cancer types such as breast, prostate, and colon haveshown very low frequency of response to mAbs and there is anongoing effort to increase the effectiveness of cancer immuno-therapy (5). Antibodies targeting immune checkpoints regulate

1Department of Drug Delivery, Halozyme Therapeutics, Inc., San Diego,California. 2Translational Development, Halozyme Therapeutics, Inc., San Diego,California. 3Histology and Exploratory Biomarkers, Halozyme Therapeutics, Inc.,San Diego, California. 4Cell Biology, Halozyme Therapeutics, Inc., San Diego,California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Renee Clift, Halozyme Therapeutics, Inc., San Diego, CA92121. Phone: 858-704-8159; Fax: 858-704-8311; E-mail: [email protected]

Cancer Res 2019;79:4149–59

doi: 10.1158/0008-5472.CAN-18-3060

�2019 American Association for Cancer Research.

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immune responses at different biological levels and by differentmechanisms. These antitumor responses may be enhanced bymeans of combinatorial strategies that are intelligently designedand guided by preclinical models (6).

The extracellularmatrix (ECM) is an essential component of thetumor microenvironment (TME) and can prevent immune cellinfiltration, as well as promote immunosuppression and resis-tance to a checkpoint blockade (7, 8). Immune cell infiltration ofthe TME is important, as access of tumor-infiltrating lymphocytes(TIL), such asCD8þ andCD4þT cells andnatural killer (NK) cells,to tumors is a prerequisite for response to PD-L1 blockade (9). Inaddition, presence of TILs correlates with better patient outcomesfor individuals receiving various antitumor therapies (10, 11).Myeloid-derived suppressor cells (MDSC) are important compo-nents of the TME and mediate T-cell suppression, contributing toresistance to checkpoint inhibitors (12). Furthermore, increasedexpression of genes involved in ECM organization significantlycorrelates with response of melanoma tumors to anti-PD-1 ther-apy (13). Hyaluronan (HA) is a naturally occurring glycosami-noglycan that is distributed widely throughout connective, epi-thelial, and neural tissues (14). In cancer, deposition ofHA is seenearly in tumorigenesis and persists during tumor progression andmetastasis (15). Accumulation of HA in several solid tumorscorrelates with local invasion, presence of distant metastases, andpoor overall survival (16–21). In breast cancer, inhibition of HAsynthesis has been shown to suppress tumorigenicity in vitro andmetastatic bone lesions in vivo (22). A strong association has beenestablished between the level of HA around malignant cells,spreading of cancer, and patient outcomes in breast cancer (21).In terms of specific breast cancer subtypes, highly aggressive breastcancer cell lines such as MDA-MB-231 and HS-578T have beenreported to express high levels of hyaluronidase-2 and synthesizehigh amounts of HA (23). In addition, HA has been shown topromote tumor growth in triple-negative breast cancers and isassociated with HER2-positivity and reduced overallsurvival (24, 25).

Accumulation of HA in the TME has previously been shown toincrease tumor interstitial pressure, inducing vascular collapse,which was associated with a decreased exposure of tumor cells tochemotherapy. Conversely, removal of HA in the TME can reversethese physiologic characteristics and, ultimately, improve acces-sibility of the tumor cells to chemotherapy in various mousemodels of pancreatic ductal adenocarcinoma (15, 26–29). Fur-thermore, it was shown that depletion of HA can lead to anincreased accumulation of CD8þ T cells in tumors of a mousemodel of ovarian cancer (30).

Taken together, targeting the accumulation of HA by enzymat-ically inhibiting HA signaling or synthesis, and thereby degradingHAin theTME, is apromising therapeutic strategy (15, 16,31–34).Pegvorhyaluronidase alfa (PEGPH20; PVHA; Halozyme Thera-peutics, Inc.) is a novel, first-in-class biologic that enzymaticallydegrades tumor HA (33) and is in clinical development for thetreatment of HA-accumulating tumors (35). In animal tumormodels of pancreatic and ovarian cancer, PVHA has been shownto reduce tumor interstitial pressure, improve vascular perfusion,decrease hypoxia, and notably increase access of anticancer agentsinto the tumor (15, 30, 33, 36).

We hypothesized that enzymatic degradation of HA in the TMEbyPVHA inmousemodels of breast cancermay improve responseto immune checkpoint inhibition. The work described hereindicates that removal of HA creates a more immunogenic tumor

and points to potentially converting HA-accumulating breasttumors with low immune cell accumulation from resistant/refrac-tory to sensitive to anti-PD-L1 immunotherapy when used incombination with PVHA.

Materials and MethodsReagents

Anti-mouse PD-L1 (clone 10F.9G2), rat immunoglobulin G2b(IgG2b) isotype control (clone LTF-2), and anti-mouse CD8a(clone 2.43) were purchased from Bio X Cell and diluted in PBSbefore administration. PVHA and its vehicle were provided byHalozyme Therapeutics, Inc. and used as describedpreviously (33).

Cell linesMurine syngeneic 4T1 (CRL-2539 Lot 60770568) and EMT-6

(CRL-2755 Lot 63226370) mammary carcinoma cells wereobtained from the ATCC on September 24, 2015, and March18, 2016, respectively. The4T1 cell linewas genetically engineeredto express hyaluronan synthase 3 [HAS3 (4T1/HAS3)] by trans-duction of 4T1 with pLV-EF1a-hHAS3-IRS-Hyg (Lot 15C09)obtained from Biosettia, Inc. Cells were transduced for 6 hourswith 2.1� 107 IU/mL lentiviral stock supplementedwith 8 mg/mLPolybrene (Sigma-Aldrich, Inc., catalog no. TR-1003). After48 hours, transduced cells were selected using 150 mg/mL hygro-mycin B (Gemini Bio Products, catalog no. 400-123). Cell lineswere authenticated and confirmed to be of mouse origin with nomammalian interspecies orMycoplasma contamination. A geneticprofile was generated for the samples using a panel of shorttandem repeat markers for genotyping. Sample profiles wereconfirmed as identical to the genetic profile established for thesecell lines. The cell lines were maintained in RPMI1640 Medium(Mediatech, Inc.) supplemented with 10% FBS and 500 mg/mLhygromycin B at 37�C in a humidified environmentwith 5%CO2.

Syngeneic tumor studiesFemale BALB/cmice aged 6–8weeks (Taconic) were inoculated

orthotopically in mammary fat pads 5th or 10th, with 5 � 105

syngeneic EMT-6 breast carcinoma cells or 1 � 105 4T1 or4T1/HAS3 breast carcinoma cells. For treatment studies, micewere randomized into treatment groups when tumors reachedapproximately 100–150 mm3 (or as indicated size for largertumor experiments). Anti-PD-L1 (5 mg/kg, or 0.5 mg/kg forEMT-6 experiments) or its isotype control, rat IgG2b, was admin-istered by intraperitoneal injection at indicated dates.

PVHA (dosed at either 1, or 0.0375 mg/kg) or its vehicle(control), were administered by intravenous injection 24 hoursprior to anti-mouse PD-L1 or its isotype control. Specific toxicitystudies were not performed. PVHA treatment in the presence orabsence of anti-PD-L1 appeared tobewell tolerated. For depletionof CD8þ T cells, mice were injected intraperitoneally with 250 mgof CD8þ-depleting antibodies (clone 2.43, Bio X Cell) 1 daybefore and3days after tumor inoculation. Tumorsweremeasuredtwice per week by digital caliper, and tumor volumes calculatedusing the modified ellipsoid formula: 1/2 � (length � width2).Tumor growth,measured as tumor volume (mm3), assessed priorto study start with digital calipers and twiceweekly thereafter untilstudy termination. Tumor growth inhibition (TGI) was calculatedusing the formula [1 – (TB� TA)/(CB�CA)]� 100,where TB is theaverage tumor volume (mm3) of treatment group at study

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termination, TA the average tumor volume of treatment group atthe day of randomization and first treatment, CB the averagetumor volume of control group at study termination, and CA theaverage tumor volume of control group at the day of randomi-zation and first treatment.

All animal-related experiments were performed in full compli-ance with approved institutional protocols and in accordancewith guidelines established by the Animal Use and Care Admin-istrative Advisory Committee at Halozyme Therapeutics, Inc.Power calculation analyses based on pilot in vivo experimentswere performedbefore themain animal experiments todeterminemouse numbers and dosing regimens. Representative experi-ments presented in this article were repeated three times, verifyingresults.

Flow cytometrySpleens and tumors were harvested from tumor-bearing mice,

and single-cell suspensionswere prepared. Spleensweremanuallydisassociated, and tumors were enzymatically dissociated withLiberase DH (Sigma-Aldrich, Inc.) and DNase I(Sigma-Aldrich, Inc.) using gentalMACS Octo Dissociators (Mil-tenyi Biotec). Surface staining was performed with antibodiesagainst mouse CD8bþ, PD-L1, CD45, CD19, NKp46, CD3, CD4,CD8a, CD44, and PD-1 (all eBioscience). For intracellular stain-ing, cells were stained for surfacemarkers and permeabilized withFoxp3 fixation buffer set (eBioscience) according to the manu-facturer's directions. Fixed cells were stained with antibodiesagainst Granzyme B (Thermo Fisher Scientific) and Foxp3(eBioscience). All samples were acquired on Novocyte (ACEABioscience Inc.) and analyzed using NovoExpress software. Flowcytometry data were plotted using Prism Graphical Softwareversion 7 (GraphPad Software, Inc.).

Imaging study for fluorescence-labeled antibody uptake intumors

ADyLight 755 Labeling Kit, #84538 (Thermo Fisher Scientific),was used to label anti-PD-L1 antibody, clone 10F.9G2 (Bio XCell). The kit was designed for excitation/emission 754/776 nmlabeling of antibodies and proteins. The In Vivo Imaging System(Xenogen Corp.) was used to detect anti-PD-L1DL755

–expressingtumor cell uptake in 4T1/HAS3 and EMT-6 tumor-bearing mice.Animals were dosed at 5 mg/kg labeled anti-PD-L1 antibody.In vivo fluorescence and epi-illumination (from the top) imageswere acquired while mice were anesthetized with inhaled 2%isoflurane. After acquisition, images were opened as sequencefiles, set to the same scale (adjusting every image in the sequencesimultaneously), and analyzed/processed for contrast and bright-ness by Living Image 4.3.1 Software (PerkinElmer Inc.). Fluores-cence contrast was quantified by measuring the radiance withinidentical size ROI on the animal image.

IHCTo detect accumulation of HA, formalin-fixed, paraffin-

embedded tissue sections were stained with a biotinylated pro-prietary HA probe (HTI-601; ref. 37) provided by HalozymeTherapeutics, Inc. For CD8þ detection, anti-CD8þ (eBioscience)was used. Sections were counterstained with hematoxylin. Todetect CD8þ, sections were stained on the DISCOVERY ULTRA(Ventana Medical Systems) and visualized with the DISCOVERYChromoMap DAB Kit (Ventana Medical Systems).

Figure 1.

Levels of HA in PVHA-treated orthotopic murine breast tumors. A, Baselineand PVHA-treated tumor HA levels, at 24 hours posttreatment, asdetermined by HA ELISA. Tissue samples were digested with proteinase K,and HA levels quantified using Hyaluronan DuoSet ELISA (R&D Systems) andare expressed as HA ng/mg tissue weight. B, IHC staining of peritumoral HA(brown) on formalin-fixed, paraffin-embedded tissue sections incubated witha biotinylated proprietary HA probe (HTI-601) of tumors at 24 hoursposttreatment with vehicle or PVHA (0.0375 or 1 mg/kg). C,Quantificationof HA levels from IHC staining.

Pegvorhyaluronidase Alfa þ Anti-PD-L1 in Breast Tumors

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Whole-section digital scans were obtained using an AperioScanScope AT Turbo slide scanner, and representative micro-graphs were captured using an Aperio ImageScope version12.2.1.5005 (Leica Biosystems Inc.). A digital scoring algorithmwas used to quantify the HA content of tissue sections (AperioPositive Pixel Count V9, Leica Biosystems Inc.). For each tumor,the entire section was analyzed, apart from necrotic areas.Hyaluronan content was calculated as percentage of DAB- orHA-positive pixels over entire pixel count in the tissue.

Representative images were randomly chosen to reflect overallalterations in tissue staining/architecture of the respective experi-ments performed.

ELISAPortions of excised tumor tissues were incubated with protein-

ase K at 55�C to dissociate tumor tissue and digest proteins.Proteinase K enzyme was then heat inactivated by incubating at95�C for 30 minutes and resulting sample lysate was subjected toHA quantification using Hyaluronan DuoSet ELISA (R&D Sys-tems, Inc.) with a stringent quantitation range of 0.37–20 ng/mL.The concentration ofHAwas normalized by tissuewetweight andwas reported as HA ng/mg tissue weight.

Statistical analysesAll data presented asmean� SE of themean, as indicated in the

figure legend. Statistical analysis was carried out with Prism

Software (GraphPad Prism v7) and P < 0.05 considered statisti-cally significant. Multiple conditions and drug treatment condi-tions over time were compared using a parametric two-wayANOVA, followed by Tukey, unpaired two-tailedMann–Whitney,or Bonferroni post hoc tests. Survival curves were estimated by theKaplan–Meier method and compared using the log-rank test orWilcoxon test, whichever was appropriate. Specific analyses usedfor each experiment are described in figure legends.

Data availabilityAll other remaining data that support the findings of this study

are available within the article and Supplementary Data or areavailable from the corresponding author upon reasonablerequest.

ResultsPVHA reduced HA levels in mouse mammary tumor models

The effect of PVHA on intratumoral HA levels was investigatedin three tumor models: naturally HA-accumulating murineEMT-6 mammary carcinoma; naturally low HA–accumulatingmurine 4T1 mammary carcinoma; and 4T1/HAS3 cells trans-duced to over-accumulate HA. To confirm presence of HA in thedifferent models in vivo, cells from each line were inoculatedorthotopically into female BALB/c mice 6–8 weeks of age. Levelsof HA in resulting tumors were assessed by ELISA: HA-low (4T1)

Figure 2.

PVHA treatment increased uptake of anti-PD-L1 antibody in orthotopic murine breast cancer models. In-vivo fluorescence representative images ofanti-PD-L1DL755 expressing tumor cell uptake in 4T1/HAS3 (A) or EMT-6 (B). For both models, tumor-bearing mice at various timepoints are shown. PVHAwasadministered intravenously on study day �1 and anti-PD-L1 was administered intraperitoneally on study day 0. Target-to-background fluorescence ratio ofanti-PD-L1DL755 imaging studies using fluorescence-labeled anti-PD-L1 antibody showed PVHAþ anti-PD-L1 treatment enhanced the uptake of anti-PD-L1antibody in the TME of 4T1/HAS3 tumors on day 3 (P¼ 0.0018) and day 4 (P¼ 0.0122; A) and EMT-6 tumors on day 3 (P¼ 0.02) versus anti-PD-L1 alone (B).Symbols represent data from individual animals for days on study. Radiance scale of the color bar represents the fluorescence emission normalized to theillumination intensity. The tumor-associated fluorescence (target) to nontumor-associated fluorescence (nontarget) was quantified using identical size regionsof interest on the animal image, using Living Image 4.3.1 Software (PerkinElmer).

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361 � 130 ng/mg; HA-accumulating (EMT-6) 730 � 133 ng/mg;and HA over-accumulating (4T1/HAS3) 1,458 � 190 ng/mg.

PVHA treatment degraded HA in all three breast cancer modelscompared with vehicle controls, as shown in Fig. 1 by ELISA andIHC. Using ELISA to detect HA, PVHA dosed at 0.0375 mg/kgenzymatically reducedHA levels of EMT-6 tumor-bearing animalsby 64%, 650 � 51 ng/mg in controls to 233 � 95 ng/mg inPVHA-treated animals. PVHA also lowered 4T1HA-accumulatingtumors by 28%, 650 � 51 ng/mg in controls to 323 � 67 ng/mgin PVHA-treated animals. Animals bearing 4T1/HAS3 tumorswere treated with a higher dose of 1 mg/kg, resulting in areduction of 94% HA content by ELISA (HA content 1,647 �276 ng/mL in vehicle tumors and 95� 11 ng/mg in PVHA-treatedtumors; Fig. 1A). These results were corroborated by IHC analysis(Fig. 1B and C). These data demonstrate that the breast cancermodels tested accumulate HA, which can be depleted by PVHAtreatment.

PVHA treatment improved tumoral uptake of anti-PD-L1Next, we asked whether reduction in HA would lead to an

increased anti-PD-L1 accumulation in tumor-bearing mice trea-ted with PVHA. To this end, EMT-6 and 4T1/HAS3 tumor-bearinganimals were divided into two groups: one treated with controlbuffer, and the other with PVHA as described in Materials andMethods. After 24 hours, animals received an intraperitonealinjection of fluorescently labeled anti-PD-L1 antibody at 5 mg/kg(Fig. 2). At days 3 and4post PVHA treatment, significant increasesof intratumoral fluorescently labeled anti-PD-L1 antibody wereobserved in 4T1/HAS3 tumors (P¼0.0018, day 3 and P¼ 0.0122,day 4) and in EMT-6 tumors (P¼ 0.02, day 3) inmice treatedwithPVHA þ anti-PD-L1 relative to those treated with anti-PD-L1alone (Fig. 2A and B, respectively).

PVHA increased anti-PD-L1–mediated 4T1/HAS3 TGIWhile the mechanism by which removal of HA led to an

increased accumulation of labeled antibody as described aboveremains unknown, the data suggests anti-PD-L1 accumulation inthe tumor may be enhanced when given in combination withPVHA. To test whether this accumulation resulted in improvedefficacy of anti-PD-L1 treatment, 4T1/HAS3 tumor-bearing ani-mals were dosed with either anti-PD-L1 alone or in combinationwith PVHA. Monotherapy treatment of either PVHA (1mg/kg) oranti-PD-L1 (5 mg/kg) alone did not result in significant TGIcompared with controls (Fig. 3A). However, when anti-PD-L1was given in combination with PVHA (1 mg/kg), TGI increas-ed by 56% compared with anti-PD-L1 þ vehicle alone(P < 0.0001; Fig. 3). IHC staining of CD8 cells in these tumorsis shown in Fig. 3B. This phenomenon was also seen when4T1/HAS3 tumor-bearing animals were treated at a lower dose,0.0375 mg/kg PVHA þ anti-PD-L1 (5 mg/kg), which resulted in40% TGI compared with vehicle þ anti-PD-L1 (SupplementaryFig. S1A). These observations were recapitulated in the HA low-accumulating 4T1 model, albeit to a lesser extent; treatment withthe combination of PVHA (0.0375 mg/kg) þ anti-PD-L1(5 mg/kg) led to a 28% TGI compared with vehicle þ anti-PD-L1–treated animals (Supplementary Fig. S1B and S1C). To furtherinvestigate increased efficacy of anti-PD-L1 in combination withPVHA treatment, a survival study was performed. As shownin Fig. 3C, 4T1/HAS3 tumor-bearing animals treated with PVHA(1mg/kg)þ anti-PD-L1 (5mg/kg) increased survival, 15–22days,compared with animals treated with vehicle þ anti-PD-L1

Figure 3.

PVHA increased anti-PD-L1–mediated growth inhibition in 4T1/HAS3 tumors.A, 4T1/HAS3 tumor-bearing mice treated with vehicleþ IgG2b isotypecontrol, or anti-PD-L1 5 mg/kg once weekly i.p., or PVHA 1 mg/kg onceweekly i.v.þ IgG2b isotype control, or anti-PD-L1 5 mg/kg once weekly i.p.Vehicle or PVHAwas administered 24 hours prior to IgG2b or anti-PD-L1.B, Randomly selected representative images of IHC staining of CD8 cells(brown; scale bar, 500 mm) on formalin-fixed, paraffin-embedded tumorsections 24 hours after treatment with vehicle, PVHA (1 mg/kg), vehicleþIgG2b, PVHA (1 mg/kg)þ IgG2b, vehicleþ anti-PD-L1 (5 mg/kg), or PVHA(1 mg/kg)þ anti-PD-L1 (5 mg/kg) treatment. C, Kaplan–Meier survivalplot for PVHAþ anti-PD-L1–treated mice. Treatment regimen was thesame as described in A. �� , P < 0.0001.






Figure 4.

PVHA increased the density of T cells and NK cells and decreased the proportion of Gr1þMDSCs in 4T1/HAS3 orthotopic breast tumors. Intratumoral immune cellcomposition of 4T1/HAS3 tumor-bearing mice. The absolute numbers of CD8þ T cells (A), CD4þ T cells (B), and NK cells (C) were significantly increased intumors treated with PVHA (1 mg/kg weekly i.v.)þ anti-PD-L1 (5 mg/kg once weekly i.p.) compared with vehicle controls, 7 days post IgG2b or anti-PD-L1treatment.D, The percentage of CD11bþGr1þ/CD45 cells was significantly decreased in tumors treated with PVHA (1 mg/kg weekly i.v.)þ anti-PD-L1 (5 mg/kgonce weekly i.p.) compared with vehicle control and anti-PD-L1 treatment alone, and with PVHAmonotherapy compared with vehicle control, 7 days post IgG2bor anti-PD-L1 treatment. Vehicle or PVHAwas administered 24 hours prior to IgG2b or anti-PD-L1. 4T1/HAS3 tumor-bearing mice were subjected to CD8þ T-cellreduction using depleting antibodies prior to inoculation and beginning treatment with PVHAþ anti-PD-L1 treatment. The data suggest that CD8þ T cells arerequired for the efficacy response seen with PVHAþ anti-PD-L1 treatment. E. Tumors in mice receiving anti-CD8a grew similarly to those in the vehicle group.�� , P < 0.0001. F. Kaplan–Meier survival plot for the addition of anti-CD8a to PVHAþ anti-PD-L1–treated mice, dosed weekly. Vehicle or PVHAwasadministered 24 hours prior to IgG2b or anti-PD-L1.

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(P ¼ 0.0262), indicating PVHA could improve the tumor growthinhibitory effects of anti-PD-L1 treatment in tumors that accu-mulate HA and that may not be responsive to anti-PD-L1monotherapy.

Treatment with PVHA and anti-PD-L1 increased density of Tcells and NK cells in 4T1/HAS3 tumors

To determine whether the tumor growth inhibitory effects ofPVHA þ anti-PD-L1 in 4T1/HAS3 tumor-bearing animals wasassociated with changes in the presence of various immune cells,flow cytometry analysis was performed. The immune cell com-position of untreated parental 4T1 and 4T1/HAS3 tumors did notreveal significant differences in the density of immune cells(Supplementary Fig. S2A–S2J). Tumor-bearing animals weredivided into the four different treatment groups outlined inMaterials and Methods. One week after the last dose of anti-PD-L1 tumors were subjected to flow cytometry.

In 4T1/HAS3 tumor-bearing animals treated with PVHA(1 mg/kg) þ anti-PD-L1 (5 mg/kg), the number of immunestimulatory cells, such as CD8þ T cells and NK cells, was signif-icantly increased per gram tumor (Fig. 4A–D; Table 1), whereasthe percent of CD11bþ/Grþ cells was decreased, when comparedwith control-treated animals. The density of tumoral CD8þ T cellspresent was increased 5-fold comparing vehicle þ anti-PD-L1(2.9 � 104 cells/g tumor to 1.6 � 105 cells/g tumor;P ¼ 0.0093; Fig. 4A). When comparing the same tumors, NKcells increased 4-fold, from 2.1 � 104 to 8.1 � 104 cells/g tumor(P¼ 0.0093; Fig. 4C) andMDSCs Gr1þCD11bþ decreased 2-foldin density, from 4.1 � 101 to 1.9 � 101 cells/g tumor(P ¼ 0.014; Fig. 4D).

In addition to increased density of immune stimulatory cellswith the combination therapy, there was also a 3-fold increase inFoxp3þCD4þ T cells (from 8.2 � 104 to 2.8 � 105 cells/g tumor;P ¼ 0.0093; Fig. 4B). It cannot be ruled out that these cells mayhave had an immunosuppressive effect. However, TGI wasincreased in PVHA and PVHA þ anti-PD-L1–treated tumors asopposed to those treated with vehicle and vehicle þ anti-PD-L1(Supplementary Figs. S3A–S3G and S4A–S4K),indicating that theimmune stimulatory effects outweighed the immunosuppressiveeffects of the increased regulatory T cells.

The correlation of increased TGI and increased density of CD8þ

T cells in tumor-bearing animals, treated with the combinationregime versus anti-PD-L1 monotherapy, led us to hypothesizethat this observation of increased TGI may be caused by a CD8þ

T-cell–dependent response. To test this hypothesis, a CD8þ

depletion study was carried out using 4T1/HAS3 tumor-bearinganimals. Both, tumor volumes and survival were examined. Asdescribed in Materials and Methods, animals were injected with

CD8þ-depleting antibodies 1 day before and 3 days after tumorcell inoculation.Once tumors reachedavolumeof100–150mm3,tumor-bearing animals were randomized and treated with PVHA(1 mg/kg) þ anti-PD-L1 (5 mg/kg) once per week. Tumors ofanimals pretreated with anti-CD8þ followed by PVHA (1 mg/kg)þ anti-PD-L1 (5 mg/kg) showed a significant increase in tumorvolume compared with those animals that received controlIgG2b antibody and combination therapy (1,000 mm3� 100 vs.600 mm3 � 70, respectively; P < 0.0001; Fig. 4E). These observa-tions were recapitulated in the survival study, where animalspretreated with anti-CD8þ T cells exhibited a similar survival asvehicle control, 7–8 days, whereas IgG2b pretreated animalsdisplayed an increase to 15 days (P ¼ 0.010; Fig. 4F), suggestingthe increased tumor growth inhibitory effect of the combinationtreatment (PVHA þ anti-PD-L1) is CD8þ T-cell–dependent.

PVHA increased anti-PD-L1–mediated TGI in EMT-6 orthotopicbreast tumors

To test whether results seen in the HA-overexpressing4T1/HAS3 breast cancer model would repeat in the EMT-6 nat-urally HA-accumulating breast cancermodel, similar experimentsas described above were carried out.

Unlike in 4T1/HAS3, EMT-6 tumors treated at a similar startingvolume (�150 mm3) were sensitive to anti-PD-L1 (5 mg/kg)monotherapy, exhibiting a TGI of 54% (P < 0.0001) comparedwith the vehicle control-treated group (Fig. 5A). However, addingPVHA (0.0375mg/kg) increased the antitumor growth inhibitoryeffects of anti-PD-L1 by 46% compared with anti-PD-L1 mono-therapy (P ¼ 0.0069) and 79% compared with vehicle controls(P < 0.0001).

PVHA can overcome resistance to anti-PD-L1 monotherapytreatment in EMT-6 tumors

As shown above, the EMT-6 breast cancer model displayed aresponse to anti-PD-L1 monotherapy treatment, but the4T1/HAS3 model was resistant. To investigate whether PVHAcould overcome resistance to anti-PD-L1 treatment in the EMT-6 breast cancer model, we sought to develop an EMT-6 anti-PD-L1–resistantmodel via twomethods; a dose reduction of anti-PD-L1, as well as by treating tumors of larger volumes.

A limited dose finding study in EMT-6 tumor-bearingmice wasconducted to establish at which dose anti-PD-L1, as a monother-apy, was no longer efficacious. As shown in Supplementary Fig.S5, 0.5 mg/kg anti-PD-L1 treatment no longer led to significantTGI compared with vehicle controls. However, when anti-PD-L1at this dose was combined with PVHA (0.0375 mg/kg), TGIincreased to 57% compared with controls (Fig. 5B; P < 0.0001).

Table 1. PVHA increased the density of T cells and NK cells, and decreased the proportion of Gr1þ MDSCs, in 4T1/HAS3 tumors and EMT-6 tumors

Immune cell type, median absolute no. cells/g Vehicle þ IgG2b Vehicle þ anti-PD-L1 PVHA þ IgG2b PVHA þ anti-PD-L1

4T1/HAS3 TumorsCD8þ T cells 2.9 � 104 2.9 � 104 7.2 � 104 1.6 � 105

CD4þ T cells 8.3 � 104 8.2 � 104 2.8 � 105 2.8 � 105

NK cells 2.9 � 104 2.1 � 104 7.6 � 104 8.1 � 104

%CD11bþGr-1þ cells 4.2 � 101 4.1 � 101 2.1 � 101 1.9 � 101

EMT-6 TumorsCD8þ T cells 5.4 � 104 7.9 � 104 3.1 � 104 1.8 � 105

CD4þ T cells 7.7 � 104 9.2 � 104 5.8 � 104 1.7 � 105

NK cells 1.9 � 104 1.9 � 104 1.1 � 104 3.8 � 104

%CD11bþGr-1þ cells 3.9 � 101 2.9 � 101 3.5 � 101 2.5 � 101






It was recently shown in non–small cell lung cancer thatbaseline tumor size (ameasure of tumor load, bulk, and burden),is a negative prognostic factor in response to immune checkpointinhibitors (38). Along those lines, we executed similar experi-ments in tumor volumes that were roughly 3-fold larger(�325 mm3). Larger EMT-6 tumors appeared resistant to anti-PD-L1 (5 mg/kg) monotherapy (Fig. 5C). However, when PVHA(0.0375 mg/kg) was given in combination with anti-PD-L1(5 mg/kg), tumor growth was significantly inhibited comparedwith vehicle þ anti-PD-L1 (TGI 65%; P ¼ 0.0209; Fig. 5C, top).These data were further corroborated by a survival study. EMT-6tumor-bearing animals treated with anti-PD-L1 alone, at thelarger staging tumor volume, displayed a survival of 7 days.Survival increased in animals receiving PVHA (0.0375 mg/kg) þanti-PD-L1 (5 mg/kg) to 15 days (P ¼ 0.0262; Fig. 5C, bottom).

Taken together, these data suggest resistance to anti-PD-L1monotherapy in the EMT-6 model as a result of either using anonefficacious dose, or larger tumors, can be overcome by addingPVHA.

PVHAþ anti-PD-L1 increased density of T cells and NK cells inEMT-6 tumors

Equally as described for the 4T1/HAS3 breast cancer model,naturally HA-accumulating EMT-6 tumors were also analyzed viaflow cytometry. The immune cell composition of EMT-6 tumors,shown by study day and tumor size in SupplementaryFigs. S6A–S6M and S7A–S7G, respectively, did not reveal anysignificant differences in various immune cells over time.

EMT-6 tumors were subjected to flow cytometry 7 days aftertreatment on study day 12 (18 days post inoculation; Fig. 6A–D).Tumor-bearing animals treated with PVHA (0.0375 mg/kg) þanti-PD-L1 (5 mg/kg) had both CD8þ T cells and NK cellsincreased 2.2- and 2-fold, respectively, when compared withanti-PD-L1 alone (P ¼ 0.043 for CD8þ T cells; Fig. 6A andP ¼ 0.043 for NK cells; Fig. 6C; Table 1). In addition, a slightdecrease in percentage of CD11bþGr1þ MDSCs among CD45þ

immune cells in the PVHA þ anti-PD-L1 group was observedversus anti-PD-L1 treatment alone, although not significant(Fig. 6D; Table 1).

Similar to our observations in the 4T1/HAS3 model, weobserved an increase of regulatory T cells in the combinatorialtreatment regimen. Foxp3þCD4þ regulatory T cells displayed amild 1.8-fold increase when compared with anti-PD-L1 (P ¼0.008; Fig. 6B). While regulatory T cells have an immunosup-pressive effect, the increased TGI suggests the effect of increasedcytotoxic T cells, as well as NK cells, may have been a greatercontributor to response than the effect of increased numbers ofregulatory T cells.

Figure 5.

PVHA increased anti-PD-L1–mediated TGI in EMT-6 orthotopic breasttumors. A, EMT-6 tumor response to anti-PD-L1 (5 mg/kg) treatment wasincreased with PVHA (0.0375 mg/kg) þ anti-PD-L1 (5 mg/kg) treatment.Study length, 13 days (19 days postinoculation). Vehicle or PVHA wasadministered 24 hours prior to IgG2b or anti-PD-L1. PVHA treatment wasreceived on study days 0 and 5. Anti-PD-L1 treatment was received onstudy days 1 and 6. B, EMT-6 tumors treated with a lower dose(0.5 mg/kg) of anti-PD-L1 became sensitive to anti-PD-L1 treatment when

combined with PVHA. Study length, 13 days (19 days postinoculation).Vehicle or PVHA was administered 24 hours prior to IgG2b or anti-PD-L1.PVHA treatment was received on study days 0 and 7. Anti-PD-L1 treatmentwas received on study days 1 and 8. C. Larger EMT-6 tumors becamesensitive to anti-PD-L1 (5 mg/kg) treatment when combined with PVHA(0.0375 mg/kg) as shown by tumor volume measurements (top) and asurvival study (bottom). Study length, 7 days (18 days postinoculation).Vehicle or PVHA was administered 24 hours prior to IgG2b or anti-PD-L1.PVHA treatment was received on study day 0 and anti-PD-L1 treatmentwas received on study day 1. Corresponding Kaplan–Meier survival plot forPVHA þ anti-PD-L1–treated mice in larger EMT-6 tumors (bottom).

Clift et al.





In brief, the data indicates that in 4T1/HAS3, as well as inEMT-6models of breast cancer, PVHA increased the tumor growthinhibitory effects of anti-PD-L1, which correlated with anincreased number of CD8þ T cells, as well as NK cells, via removalof HA.

DiscussionBreast cancers are generally regarded as poorly immunogenic

andwith the exceptionof antibodies targetingHER2, demonstratepoor responses to immunotherapy (39). While the mechanismbehind these observations is unknown, there is evidence suggest-ing HA accumulates in the ECM of breast cancer; levels aresignificantly higher in serum of patients with metastatic breastcancer compared with healthy individuals (21, 30, 39). PVHA hasbeen reported to remove HA in tumors of pancreatic and ovarianpreclinical models (15, 29, 30, 33). While to date, there is nodescription of a clear relationship between a poor response toimmunotherapy and levels of HA in patients with breast cancer,we hypothesized that HA removal may improve efficacy ofimmune checkpoint inhibitors. This idea based on several linesof evidence showing, in preclinical models, enzymatic degrada-tion of HA can increase efficacy of various types of chemotherapyby reversing vascular collapse, as well as reducing interstitialpressure (32, 36).

We tested this hypothesis in three different orthotopic modelsof breast cancer EMT-6, 4T1, and 4T1/HAS3. In all three models,PVHA treatment resulted in removal of tumoral HA in vivo. Thisdepletion of HA was correlated with an increased anti-PD-L1accumulation, as shown with a fluorescently labeled anti-PD-L1antibody, although the kinetics of tumoral HA removal andpeak distribution of anti-PD-L1 did not precisely overlap (Fig. 2).This work does not describe the mechanism that underlies thisobservation and remains to be elucidated. Historical evidenceindicates PVHA increases tumor cell exposure to chemotherapyand antibodies via removal of HA, but it has not been confirmedwhether this is solely due to reductions in interstitialfluidpressureand changes in vessel patency (15, 29, 30, 33, 34, 36), or whetherother biochemical or genetic changes in the TME may also becontributing. Along those lines, further investigation may helpclarify whether tumoral HA depletion elicits changes in antigenexpression, which may influence anti-PD-L1 accumulation.

Increased accumulation of anti-PD-L1 in tumor-bearing micetreated with combination therapy correlated with increased TGIin 4T1/HAS3, as well as in the EMT-6 tumor model. Flowcytometry analysis revealed parallel increases in cytotoxic T cells

Figure 6.PVHA in combination with anti-PD-L1 increased the density of T cells and NKcells, and decreased the proportion of Gr1þMDSCs in EMT-6 orthotopicbreast tumors. Intratumoral immune cell composition of EMT-6 tumorsanalyzed 7 days post IgG2b or anti-PD-L1 treatment. The absolute numbersof CD8þ T cells (A), CD4þ T cells (B), and NK cells (C) were significantlyincreased in tumors treated with PVHA (0.0375 mg/kg weekly i.v.)þ anti-PD-L1 (5 mg/kg once weekly i.p.) compared with vehicle controls and PVHAand anti-PD-L1 monotherapy.D, The percentage of CD11bþGr1þ/CD45 cellswas decreased, although not significantly, in tumors treated with PVHAþanti-PD-L1 compared with vehicle control and PVHA alone. Vehicle or PVHAwas administered 24 hours prior to IgG2b or anti-PD-L1.






as well as NK cells. However, the density of regulatory T cells(Foxp3þCD4þ T cells) also increased, albeit to a lesser extent.Regulatory T cells are known to be immunosuppressive (40). Theactivity of tumoral immune cells was not explored, therefore wecannot rule out the possibility of immune suppression fromregulatory T cells. However, CD8þ T-cell depletion studiesshowed that the decreased tumor growth in the combinationtreatment was dependent on the presence of CD8þ T cells. Thismay suggest that PVHA potentially converts immune checkpointinhibitor–resistant tumors to sensitive tumors, by increasing thedensity of cytotoxic T cells.

This concept further supported by experiments in the EMT-6tumor model used to create anti-PD-L1–resistant tumors. Weattempted to create such a model by reducing the dose of anti-PD-L1 and treating larger tumors. It is unknown if thesemechanisms are responsible for resistance to immune check-point inhibitors in breast cancer. Recently it was shown thatbaseline tumor size correlated to resistance to anti-PD-L1treatment in non–small cell lung cancer (39). In both cases,PVHA was able to increase the tumor growth inhibitory effectsof anti-PD-L1, as measured by tumor volume, as well as animalsurvival.

Taken together, these data suggest that PVHA has the potentialto convert a resistant refractory tumor to sensitive to anti-PD-L1treatment. In addition, the data expands our understanding ofPVHA and can provide additional support for potentially mean-ingful clinical responses to treatment in HA-accumulatingtumors (32, 36, 41, 42).

Clinical trials evaluating PVHA þ anti-PD-L1 treatment inpatients with metastatic pancreatic ductal adenocarcinoma(Morpheus – Pancreatic Cancer; NCT03193190), gastric cancer(Morpheus – Gastric Cancer; NCT03281369), and cholangiocar-cinoma/gallbladder cancer (HALO 110-101; NCT03267940) areongoing. TheMorpheus studies are phase Ib/II trials investigatingmultiple immunotherapy-based combination treatments, includ-ing the combination of PVHA and atezolizumab. The HALO110-101 study is a phase I trial assessing PVHA with cisplatin

and gemcitabine with or without atezolizumab. All three clinicaltrials are currently enrolling patients.

Disclosure of Potential Conflicts of InterestR. Clift has ownership interest (including stock, patents, etc.) in

Halozyme Therapeutics. J. Souratha has ownership interest (including stock,patents, etc.) in Halozyme Therapeutics. B. Blouw is a senior scientist atand has ownership interest (including stock, patents, etc.) in HalozymeTherapeutics. S.A. Garrovillo has ownership (stock) in Halozyme Therapeutics.No potential conflicts of interest were disclosed by the other author.

Authors' ContributionsConception and design: R. Clift, J. Souratha, S. ZimmermanDevelopment of methodology: R. Clift, J. Souratha, S.A. Garrovillo,S. Zimmerman, B. BlouwAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Clift, J. Souratha, S.A. Garrovillo,S. Zimmerman, B. BlouwAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Clift, J. Souratha, S. Zimmerman, B. BlouwWriting, review, and/or revision of the manuscript: R. Clift, J. Souratha,S. Zimmerman, B. BlouwAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R. Clift, J. Souratha, S. ZimmermanStudy supervision: R. Clift

AcknowledgmentsThe authors thank and acknowledge YujunHuang for his contribution to this

study; Kelly Chen and Trevor Kimbler for assistance with in vitro work; GenaroRonquillo, Max Bersabe, James Skipper, and Clay Conway for their assistancewith in vivo studies; andMichael Jorge and Flavio Araiza for their assistance withHA ELISA processing. Medical writing support and editorial assistance wasprovided by Talya Underwood, MPhil, and Shaun W. Foley, BSc (Hons), ofParagon, Knutsford, United Kingdom, supported by Halozyme Therapeutics,Inc. This study was supported by Halozyme Therapeutics, Inc.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 28, 2018; revised January 22, 2019; accepted June 24,2019; published first June 27, 2019.

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2019;79:4149-4159. Published OnlineFirst June 27, 2019.Cancer Res Renee Clift, Jennifer Souratha, Sheryl A. Garrovillo, et al. Tumors to Anti-Programmed Death-Ligand 1 ImmunotherapyRemodeling the Tumor Microenvironment Sensitizes Breast

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Remodeling the Tumor Microenvironment …...twice per week by digital caliper, and tumor volumes calculated using the modiﬁed ellipsoid formula: 1/2 (length width 2 ). Tumorgrowth,measuredastumorvolume(mm