Cell Death and Survival

Near-Infrared Photoimmunotherapy TargetingProstate Cancer with Prostate-SpecificMembraneAntigen (PSMA) AntibodyTadanobu Nagaya, Yuko Nakamura, Shuhei Okuyama, Fusa Ogata,Yasuhiro Maruoka, Peter L. Choyke, and Hisataka Kobayashi

Abstract

Prostate-specific membrane antigen (PSMA) is a membraneprotein that is overexpressed manifold in prostate cancer andprovides an attractive target for molecular therapy. Near-infraredphotoimmunotherapy (NIR-PIT) is a highly selective tumor treat-ment that employs an antibody-photoabsorber conjugate (APC).Here,wedescribe the efficacyofNIR-PIT, using a fullyhuman IgG1

anti-PSMA monoclonal antibody (mAb), conjugated to thephotoabsorber, IR700DX, in a PSMA-expressing PC3 prostatecancer cell line. Anti-PSMA-IR700 showed specific binding andcell-specific killing was observed after exposure of the cells to NIRlight in vitro. In the in vivo study, anti-PSMA-IR700 showed hightumor accumulation and high tumor–background ratio. Tumor-bearing mice were separated into 4 groups: (i) no treatment; (ii)100 mg of anti-PSMA-IR700 i.v.; (iii) NIR light exposure; (iv) 100mg of anti-PSMA-IR700 i.v., NIR light exposure was administered.

Thesewereperformedeveryweek for up to3weeks. Tumor growthwas significantly inhibited by NIR-PIT treatment compared withthe other control groups (P < 0.001), and significantly prolongedsurvival was achieved (P < 0.0001 vs. other control groups). Morethan two thirds of tumorswere curedwithNIR-PIT. In conclusion,the anti-PSMA antibody is suitable as an APC for NIR-PIT. Fur-thermore, NIR-PITwith the anti-PSMA-IR700 antibody is a prom-ising candidate of the treatment of PSMA-expressing tumors andcould be readily translated to humans.

Implications: NIR-infrared photoimmunotherapy (NIR-PIT)using a fully human anti-PSMA-IR700 conjugate showed poten-tial therapeutic effects against a PSMA-expressing prostate cancerthat is readily translated to humans.Mol Cancer Res; 15(9); 1153–62.�2017 AACR.

IntroductionProstate cancer is the most common cancer in men, with

161,360 estimated new cases and it is the third leading cause ofcancer-related death amongmen in theUnited States, with 26,730deaths estimated in 2017 (1). Localized prostate cancer typically istreated with surgery or radiation, and recurrent disease can becontrolled temporarily with radiation often combined withandrogen ablation (2). However, many prostate cancers eventu-ally become hormone refractory and then rapidly progress (3).Hormone-refractory or androgen-independent metastatic pros-tate cancer is usually lethal. Therefore, it is important that theclinical trajectory of prostate cancer be intercepted as early in thecourse of the disease as possible (4, 5). Newmolecularly targetedtherapies are urgently needed for this task.

Monoclonal antibody (mAb) therapies have shown consider-able value in the treatment of many malignant tumors (6–9). Asmonotherapy,mAbs can either block receptors needed for growth

or induced antibody-dependent complement-mediated cytotox-icity (ADCC). Antibodies can also be used to deliver drugs ortherapeutic radioisotopes. However, for antibodies to be mosteffective, a distinct antigen must be commonly expressed on thecancer cell surface.

Prostate-specific membrane antigen (PSMA) is a well-estab-lished cell membrane marker of prostate cancer and therefore is aplausible target for molecular therapy. PSMA is a type 2 integralmembrane glycoprotein, also known as glutamate carboxypepti-dase II (GCPII) or folate hydrolase 1 (FOLH1; refs. 10–12) and itwas originally discovered in the androgen-dependent LNCaphuman prostatic adenocarcinoma cell line (13). PSMA isexpressed in nearly all prostate cancers, and expression is highestin poorly differentiated, metastatic, and hormone-refractory cases(14–17).

Compared with other therapies, antibody-directed photother-apy has several advantages over conventional therapies because itis minimally invasive and can be used repeatedly without limi-tation of the total dose or treatment resistance (18). In the past,antibodies were conjugated to very hydrophobic photosensitizersin conventional photodynamic therapy (PDT). As a consequenceof their hydrophobicity, these conjugates tended to benonspecificand limited by toxicity.

Near-infrared photoimmunotherapy (NIR-PIT) is a newlydeveloped cancer treatment that employs a highly targetedmAb-photoabsorber conjugate (APC). The photoabsorber,IRDye700DX (IR700, silica-phthalocyanine dye), is a highlyhydrophilic dye, differentiating it from prior hydrophobic dyesused in PDT (19). The first-in-human phase I trial of NIR-PIT in

Molecular Imaging Program, Center for Cancer Research, National CancerInstitute, NIH, Bethesda, Maryland.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Hisataka Kobayashi, National Cancer Institute, Bldg. 10,Rm B3B69, 10 Center Dr., MD 20892-1088. Phone: 301-435-4086; Fax: 301-402-3191; E-mail: kobayash@mail.nih.gov

doi: 10.1158/1541-7786.MCR-17-0164

�2017 American Association for Cancer Research.

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patientswith inoperable head andneck cancer targeting EGFRwasapproved by the FDA, and started in June 2015 (https://clinicaltrials.gov/ct2/show/NCT02422979) andfinished inAugust 2016.In this trial, patients were injected with cextuximab–IR700 con-jugate, (referred to as RM1929 in the study), that binds to targetEGFR molecules on the cell membrane of head and neck cancers.About 24 hours later, the tumor is exposed to NIR light by meansof a laser at a wavelength of 690 nm that is absorbed by IR700.NIR-PIT induces nearly immediate necrotic cell death rather thanapoptotic cell death that is most commonly induced by othercancer therapies (20).

NIR-PIT has been shown to be effective with a variety ofdifferent antibodies but has not been previously tested with fullyhuman anti-PSMA antibody (21–26). In this study, we investi-gated anti-PSMA antibody-IR700 as a candidate APC for NIR-PIT.Using a PSMA-expressing PC3 cell line in vitro binding, in vivotumor accumulation and intratumoral distribution were evalu-ated. NIR-PIT was then performed with anti-PSMA-IR700 in vitroand repeated NIR-PIT was performed in a tumor-bearing mousemodel in vivo.

Materials and MethodsReagents

Water soluble, silica-phthalocyanine derivative, IRDye 700DXNHS ester was obtained from LI-COR Biosciences. A fully humanIgG1 anti-human prostate-specific membrane antigen (PSMA)was kindly provided by Progenics Pharmaceuticals, Inc. patent;US 8114965 B2; ref. 27). All other chemicals were of reagentgrade.

Synthesis of IR700-conjugated anti-PSMAConjugation of dyes with mAb was performed according to a

previous report (19). In brief, anti-PSMAmAb (1.0mg, 6.7 nmol)was incubated with IR700 NHS ester (65.1 mg, 33.3 nmol) in 0.1mol/L Na2HPO4 (pH 8.6) at room temperature for 1 hour. Themixture was purified with a Sephadex G25 column (PD-10; GEHealthcare). The protein concentration was determined withCoomassie Plus Protein Assay Kit (Thermo Fisher Scientific Inc)by measuring the absorption at 595 nmwith UV-Vis (8453 ValueSystem; Agilent Technologies). The concentration of IR700 wasmeasured by absorption at 689 nm to confirm the number offluorophore molecules per mAb. The synthesis was controlled sothat an average of two IR700 molecules was bound to a singleantibody. We abbreviate IR700 conjugated to anti-PSMA mAb asanti-PSMA-IR700. As a quality control for the conjugate, weperformed SDS-PAGE. Conjugate was separated by SDS-PAGEwith a 4%–20% gradient polyacrylamide gel (Life Technologies).A standard marker (Crystalgen Inc.) was used as a protein molec-ular weightmarker. After electrophoresis at 80 V for 2.5 hours, thegel was imaged with a Pearl Imager (LI-COR Biosciences) using a700-nm fluorescence channel. We used diluted anti-PSMA anti-body as a control. The gel was stainedwithColloidal blue stainingto determine the molecular weight of conjugate.

Cell culturePC3-PSMAþ (PC3pip) cell line generated by transduction of

PC3 cells using VSV-G pseudotyped lentiviral vector expressinghuman PSMA and a control blank vector–transfected PC3 cellline, PC3-PSMA- (PC3flu; refs. 28, 29) were used for PSMA-targeting studies with anti-PSMA-IR700. Both cells were estab-

lished at the Cleveland Clinic Foundation. A luciferase stablyexpressed PC3pip cell line was also established with the trans-fection of pGL4.51 luc2/CMV/Neo Vector (Promega). High lucif-erase expression was confirmed with 10 passages. We abbreviatethis cell line as PC3pip-luc. Cells were grown in RPMI1640 (LifeTechnologies) supplemented with 10% FBS and 1% penicillin/streptomycin (Life Technologies) in tissue culture flasks in ahumidified incubator at 37�C in an atmosphere of 95% air and5% carbon dioxide.

Flow cytometryTo verify in vitro anti-PSMA-IR700 binding, fluorescence from

cells after incubation with anti-PSMA-IR700 was measuredusing a flow cytometer (FACSCalibur, BD Biosciences) andCellQuest software (BD Biosciences). PC3flu and PC3pip-luccells (2� 105) were seeded into 12-well plates and incubated for24 hours. Medium was replaced with fresh culture mediumcontaining 3 mg/mL of anti-PSMA-IR700 and incubated for 6hours at 37�C. To validate the specific binding of the conjugatedantibody, excess antibody (30 mg) was used to block 3 mg of anti-PSMA-IR700.

Fluorescence microscopyTo detect the antigen-specific localization and effect ofNIR-PIT,

fluorescence microscopy was performed (BX61; Olympus Amer-ica, Inc.). Ten-thousand PC3flu and PC3pip-luc cells were seededon cover-glass–bottomeddishes and incubated for 24hours. Anti-PSMA-IR700 was then added to the culture medium at 3 mg/mLand incubated for 6 hours at 37�C. After incubation, the cells werewashedwith PBS. The filter set to detect IR700 consisted of a 590–650 nm excitation filter, a 665–740 nm band-pass emission filter.Transmitted light DIC images were also acquired.

In vitro NIR-PITThe cytotoxic effects of NIR-PIT with anti-PSMA-IR700 were

determined by flow cytometric propidium iodide (PI; LifeTechnologies) staining, which can detect compromised cellmembranes. A total of 2 � 105 PC3flu and PC3pip-luc cellswere seeded into 12-well plates and incubated for 24 hours.Medium was replaced with fresh culture medium containing 3mg/mL of anti-PSMA-IR700 and incubated for 6 hours at 37�C.After washing with PBS, PBS was added, and cells were irradi-ated with a red light-emitting diode (LED), which emits light at690 � 20 nm wavelength (L690-66-60; Marubeni America Co.)at a power density of 50 mW/cm2 as measured with an opticalpower meter (PM 100, Thorlabs). Cells were scratched 1 hourafter treatment. PI was then added in the cell suspension (final2 mg/mL) and incubated at room temperature for 30 minutes,followed by flow cytometry. Each value represents mean� SEMof five experiments.

For bioluminescence imaging (BLI), either 2� 105 PC3pip-luccells were seeded into 12-well plates or 2 � 107 PC3pip-luc cellswere seeded onto a 10-cm dish; both were preincubated for 24hours. After replacing the medium with fresh culture mediumcontaining 3 mg/mL of anti-PSMA-IR700, the cells were incubatedfor 6 hours at 37�C in a humidified incubator. After washingwith PBS, phenol-red–free culturemediumwas added. Then, cellswere exposed with a LED or a NIR laser which emits light at 690�5 nm wavelength (BWF5-690-8-600-0.37; B&W TEK INC.). Theoutput power density in mW/cm2 was measured with an opticalpower meter (PM 100, Thorlabs). For luciferase activity, 150 mg/mL D-luciferin–containing media (Gold Biotechnology) was

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administered toPBS-washed cells 1hour afterNIR-PITwhichwereanalyzed on a BLI system (Photon Imager; Biospace Lab). Regionsof interest (ROI) were placed on each entire well, and the lucif-erase activity (photons/minute) was then calculated using M3Vision Software (Biospace Lab).

Animal and tumor modelsAll in vivo procedures were conducted in compliance with the

Guide for the Care and Use of Laboratory Animal Resources(1996), USNational Research Council, and approved by the localAnimal Care and Use Committee. Six- to 8-week-old femalehomozygote athymic nude mice were purchased from CharlesRiver (NCI-Frederick). During the procedure, mice were anesthe-tized with inhaled 3%–5% isoflurane and/or via intraperitonealinjection of 1 mg of sodium pentobarbital (Nembutal SodiumSolution, Ovation Pharmaceuticals Inc.). To determine tumorvolume, the greatest longitudinal diameter (length) and thegreatest transverse diameter (width) were measured with anexternal caliper. Tumor volumes were based on caliper measure-ments and were calculated using the following formula; tumorvolume¼ length�width2� 0.5. Bodyweight was alsomeasured.Mice were monitored daily for their general health. The presenceof skin necrosis or toxicity attributable to the anti-PSMA-IR700was evaluated with the observation of skin color and generalhealth, including weight loss and appetite loss. Tumor volumesweremeasured three times aweekuntil the tumor volume reached2,000 mm3, whereupon the mice were euthanized with inhala-tion of carbon dioxide gas.

In vivo fluorescence imaging studiesPC3pip-luc cells (2� 106) were injected subcutaneously in the

right dorsum of the mice. Tumors were studied after they reachedvolumes of approximately 50 mm3. Serial ventral and dorsalfluorescence images of IR700 were obtained with a Pearl Imagerusing a 700-nm fluorescence channel before and 0, 0.5, 1, 2, 3, 4,5, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 hours afterintravenous injection of 100 mg of anti-PSMA-IR700 via the tailvein. Pearl Cam Software (LI-COR Biosciences) was used foranalyzing fluorescence intensities. ROIs were placed on the tumorand liver. ROIs were also placed in the adjacent nontumor regionas background (left dorsum and lower abdomen). Average fluo-rescence intensity of each ROI was calculated. TBRs (fluorescenceintensities of target/fluorescence intensities of background) werealso calculated (n ¼ 10).

In vivo NIR-PITPC3pip-luc cells (2� 106) were injected subcutaneously in the

right dorsum of the mice. Tumors were studied after they reachedvolumes of approximately 50 mm3. To examine the therapeuticeffect of in vivo NIR-PIT on PC3pip-luc tumors, tumor-bearingmice were randomized into 4 groups of at least 10 animals pergroup for the following treatments: (i) no treatment (control); (ii)100 mg of anti-PSMA-IR700 i.v., no NIR light exposure (anti-PSMA-IR700 i.v.); (iii) NIR light exposure only, NIR light expo-sure was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day2 (NIR light exposure); (iv) 100 mg of anti-PSMA-IR700 i.v., NIRlight exposure was administered at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 after injection (NIR-PIT). These therapies wereperformed every week for up to 3 weeks. Serial fluorescenceimages, as well as white light images, were obtained using a PearlImager with a 700-nm fluorescence channel.

For in vivo BLI, D-luciferin (15 mg/mL, 200 mL) was injectedintraperitoneally and the mice were analyzed on a BLI system(Photon Imager) for luciferase activity (photons/minute). ROIswere set on the entire tumors to quantify the luciferase activities.ROIs were also placed in the adjacent nontumor region as back-ground. Average luciferase activity of each ROI was calculated. Toclarify therapeutic effect luciferase activity of the tumor beforeNIR-PIT set to 100%.

Histologic analysisTo detect the antigen-specific microdistribution in the tumor,

fluorescence microscopy was performed. Tumor xenograftswere excised from mice without treatment, 24 hours afterinjection of anti-PSMA-IR700 (anti-PSMA-IR700 i.v.) and 24hours after NIR-PIT. Fluorescence images, as well as white lightimages, of extracted tumors were obtained using a Pearl Imagerwith a 700-nm fluorescence channel. Then, extracted tumorswere frozen with optimal cutting temperature (OCT) com-pound (SAKURA Finetek Japan Co.) and frozen sections (10-mm thick) were prepared. Fluorescence microscopy was per-formed using the BX61 microscope with the following filters:excitation wavelength 590 to 650 nm, emission wavelength665 to 740 nm long-pass for IR700 fluorescence. DIC imageswere also acquired. To evaluate histologic changes, lightmicroscopy study was also performed using Olympus BX61.Extracted tumors were also placed in 10% formalin and serial10-mm slice sections were fixed on glass slide with hematoxylinand eosin (H&E) staining.

Statistical analysisData are expressed as means � SEM from a minimum of five

experiments, unless otherwise indicated. Statistical analyseswere carried out using GraphPad Prism version 7 (GraphPadSoftware). For multiple comparisons, a one-way ANOVA fol-lowed by the Tukey correction was used. The cumulativeprobability of survival based on volume (2,000 mm3) wereestimated in each group with a Kaplan–Meier survival curveanalysis, and the results were compared with use of the log-ranktest. Student t test was used to compare the treatment effectswith that of control. A P of <0.05 was considered statisticallysignificant.

ResultsIn vitro characterization of PC3flu and PC3pip-luc cell lines

As defined by SDS-PAGE, anti-PSMA-IR700 and nonconjugat-ed control anti-PSMAmAb showed an identicalmolecularweight,around 150 kDa, and fluorescence intensity was confirmed in theband of anti-PSMA-IR700 (Fig. 1A). After 6-hour incubation withanti-PSMA-IR700, PC3pip-luc cells showed high fluorescencesignal, which was confirmed with flow cytometry (Fig. 1B) andfluorescence microscopy (Fig. 1C). On the other hand, PC3fulcells showed no fluorescence signal. Fluorescence signal inPC3pip-luc cells was completely blocked by adding excess anti-PSMA mAb (Fig. 1C), indicating that anti-PSMA-IR700 specifi-cally binds to PSMA on the cells.

In vitro NIR-PITImmediately after exposure, NIR light exposure induced

cellular swelling, bleb formation, and rupture of vesicles

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Figure 1.

Confirmation of PSMAexpression as a target for NIR-PIT in PC3 cells, and evaluation of in vitroNIR-PIT.A,Validation of anti-PSMA-IR700by SDS-PAGE (left, colloidalblue staining; right, fluorescence). Diluted anti-PSMA mAb was used as a control. B, Expression of PSMA in PC3flu and PC3pip-luc cells was evaluated byFACS. After 6 hours of anti-PSMA-IR700 incubation, PC3pip-luc cells showed high fluorescence signal. Fluorescence in PC3pip-luc cells was completely blocked byadding excess anti-PSMA. On the other hand, PC3flu cells showed no fluorescence signal. C, DIC and fluorescence microscopy images of PC3flu and PC3pip-luccells after incubation with anti-PSMA-IR700 for 6 hours. High fluorescence intensities were shown in only PC3pip-luc cells. Necrotic cell death was observed uponexcitation with NIR light (after 15 minutes) in PC3pip-luc cells. Scale bars ¼ 20 mm. D, Bioluminescence imaging (BLI) of a 10-cm dish demonstrated thatluciferase activity in PC3pip-luc cells decreased in a NIR-light dose-dependentmanner. E, Luciferase activity in PC3pip-luc cells wasmeasured, which also decreasedin a NIR-light dose-dependent manner (n ¼ 5; �P < 0.05 vs. untreated control; ��P < 0.01 vs. untreated control, by Student t test). F, Membrane damage ofPC3flu cells induced byNIR light exposurewasmeasuredwith the dead cell count usingpropidium iodide (PI) staining. Nomembranedamagewas observed in PC3flucells after NIR light exposure. G, Membrane damage of PC3pip-luc cells induced by NIR-PIT was measured with the dead cell count using PI staining, whichincreased in a light dose-dependentmanner (n¼ 5; �� ,P<0.01, vs. untreated control, by Student t test). Therewas no significant cytotoxicity associatedwithNIR lightexposure alone in the absence of anti-PSMA-IR700 and with anti-PSMA-IR700 alone without NIR light exposure.

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representing necrotic cell death was observed in PC3pip-luccells (Supplementary Video). Most of these morphologicchanges were observed within 15 minutes of light exposure(Fig. 1C), indicating rapid induction of necrotic cell death. Onthe other hand, PC3flu cells showed no obvious change afterNIR light exposure. Bioluminescence showed a decrease ofluciferase activity in a light dose-dependent manner (Fig.1D). BLI also showed significant decreases of luciferase activityin NIR-PIT–treated cells (Fig. 1E). On the basis of incorporationof PI, although no NIR-PIT effects were shown in PC3flu cells(Fig. 1F), percentage of cell death increased in a light dose-dependent manner in PC3pip-luc cells (Fig. 1G). Over 80% ofPC3pip-luc cells died when exposed to 8 J/cm2 of NIR light.There was no significant cytotoxicity associated with NIR lightexposure alone in the absence of anti-PSMA-IR700 and withanti-PSMA-IR700 alone without NIR light exposure.

In vivo fluorescence imaging studiesThe fluorescence intensity of anti-PSMA-IR700 in PC3pip-luc

tumor showed high intensities within 1 day after anti-PSMA-IR700 injection but this decreased gradually over the following

days (Fig. 2A and B). On the other hand, target-to-backgroundratio (TBR) of anti-PSMA-IR700 in tumor and liver was highimmediately after APC injection, followingwhich the TBR did notchange for several days (Fig. 2C). TBR of anti-PSMA-IR700 washigh in tumor due to specific APC binding to PSMA expressingPC3pip-luc cells, while TBR was high in liver likely due tononspecific accumulation of anti-PSMA–IR700 conjugate as theliver is not known to express PSMA. To obtain the maximaltherapeutic effect, the tumor fluorescence caused by binding ofthe APC should be high in tumor and low in background. Tumorfluorescence was high after APC injection, while fluorescencesignal of background including liver decreased beginning 9 hoursafter APC injection. Thus,weused 1dayof incubationwithAPC toget the maximal difference between tumor and backgroundnormal tissue.

In vivo NIR-PITThe treatment and imaging regimen is shown in Fig. 3A. One

day after injection of anti-PSMA-IR700, the tumors showedhigher fluorescence intensity than did the tumor with no anti-PSMA-IR700. After exposure to 50 J/cm2 of NIR light, IR700

Figure 2.

In vivo fluorescence imaging of PC3pip-luc tumor. A, In vivo anti-PSMA-IR700 fluorescence real-time imaging of tumor-bearing mice (right dorsum). The tumorshowed high fluorescence intensity after injection and the intensity was gradually decreased over days. Most of the excess agent was excreted to the urineimmediately after injection. ROIs were placed on the tumor and liver, then ROIs were also placed in the adjacent nontumor region as background (left dorsum; bluecircle and lower abdomen; green circle). B, Time course of NIR fluorescence signal of IR700 in tumors and livers (n ¼ 10). The IR700 fluorescence intensityof tumor and liver showed high intensities within 1 day after anti-PSMA-IR700 injection but this decreased gradually over days. C, Time course of NIR fluorescencesignal of TBR in tumors and livers (n ¼ 10). TBR of tumor and liver showed high within four days after anti-PSMA-IR700 injection, then the TBR was graduallydecreased over the following days.

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Figure 3.

In vivo effect ofNIR-PIT for PC3pip-luc tumor.A,NIR-PIT regimen. Fluorescence andbioluminescence imageswere obtained at each time point as indicated.B, In vivofluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT. The tumor treated by NIR-PIT showed decreasing IR700 fluorescence afterNIR-PIT. C, In vivo BLI of tumor bearing mice in response to NIR-PIT. Before NIR-PIT, tumors were approximately the same size and exhibited similar BLI signals. Thetumor treated by NIR-PIT showed decreasing luciferase activity after NIR-PIT. D, Quantitative luciferase activity (before NIR-PIT is set to 100) showed asignificant decrease in NIR-PIT tumors (n� 10, ��P < 0.001 vs. other groups, by Tukey test with ANOVA). Luciferase activity of tumor in other control groups showedan increase due to rapid tumor growth. E, Tumor growth was significantly inhibited in the NIR-PIT treatment group with anti-PSMA-IR700 (n � 10; �� , P < 0.001vs. other control groups, Tukey test with ANOVA). F, Significantly prolonged survival was observed in the NIR-PIT treatment group with anti-PSMA-IR700 (n� 10;�� , P < 0.0001 vs. other control groups, by log-rank test).

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fluorescence signal of the tumor decreased due to dying cellsand partial photobleaching, while the IR700 fluorescencegradually decreased over the following days in tumors receiv-ing anti-PSMA-IR700 but not NIR light exposure (Fig. 3B).NIR-PIT resulted in decreases in bioluminescence (Fig. 3C).Luciferase activity significantly decreased after NIR-PIT (P <0.0001 vs. other control groups; Fig. 3D). In contrast, lucifer-ase activity of tumor in other control groups showed anincrease due to rapid tumor growth. Tumor growth was sig-nificantly inhibited in the NIR-PIT treatment group comparedwith the other control groups (P < 0.001; Fig. 3E), andsignificantly prolonged survival was achieved in the NIR-PITgroup (P < 0.0001 vs. other control groups; Fig. 3F). Surpris-ingly, more than two thirds of tumors were cured with thisregimen of NIR-PIT. No significant therapeutic effect wasobserved in the control groups, including those receivinganti-PSMA-IR700 i.v. only or in mice receiving NIR light

exposure only. There was no skin necrosis or toxicity attrib-utable to the anti-PSMA-IR700 in any group.

Histological analysisThe treatment and imaging regimen is shown in Fig. 4A. High

fluorescence intensity was shown in tumors 24 hours after anti-PSMA-IR700 injection compared with that in control tumors.The majority of fluorescence signal in tumors disappeared 24hours after NIR-PIT in resected tumor (Fig. 4B). In frozenhistologic specimens, high fluorescence intensity was alsoshown in tumors 24 hours after anti-PSMA-IR700 injection,and the signal decreased 24 hours after NIR-PIT (Fig. 4C). H&Estaining of NIR-PIT–treated PC3pip-luc tumors revealed diffusenecrosis and microhemorrhage, with scattered clusters of livebut damaged tumor cells, while no obvious damage wasobserved in the tumor receiving only anti-PSMA-IR700 but nolight (Fig. 4D).

Figure 4.

Invivo histologicfluorescencedistribution andhistologicNIR-PIT effect.A,The regimenofNIR-PIT and imaging is shown.B,Fluorescence images of resected PC3pip-luc tumors. White light images (top) and IR700 fluorescence image (bottom). High fluorescence intensity was shown in PC3pip-luc tumor 24 hours afterinjection of anti-PSMA-IR700, but the fluorescence decreased 24 hours after NIR-PIT. C, Differential interference contrast (DIC) and fluorescence microscopyimages of PC3pip-luc tumor xenografts. High fluorescence intensity was shown in PC3pip-luc tumor 24 hours after injection of anti-PSMA-IR700, but thefluorescence decreased 24 hours after NIR-PIT. Scale bars, 100 mm.D, Resected tumor stained with H&E. A few scattered clusters of damaged tumor cells were seenwithin a background of diffuse cellular necrosis and microhemorrhage after NIR-PIT, while no obvious damage was observed after anti-PSMA-IR700 alonewith NIR light exposure. White scale bars, 100 mm; black scale bars, 20 mm.

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DiscussionPSMAis theprototypic cell-surfacemarker of prostate cancer and

provides an attractive target for mAb targeted therapies. The firstmAb that received FDA approval for clinical use in prostate cancerwas capromab pendetide, a murine, 111In-labeled, mAb (7E11)directed to PSMA (13, 30). However, results were disappointing asthe mAb targeted an internal domain of PSMA (31, 32). SeveralmAbs targeting the external domain of PSMA are currently under-going clinical investigation in prostate cancer. Although there wassomepotential clinical utility of radiolabeledmAb for imaging anddiagnostic purposes (33), therapeutic radioisotopic labeling of themAb have proven disappointing (7, 34).

The conjugate anti-PSMA–IR700proved tobe an effective agentfor treating a PSMA-expressing prostate cancer model with NIR-PIT. Unlike other mAb-mediated therapies, NIR-PIT with anti-PSMA–IR700 led to rapid cell death in vitro and tumor growthreduction and survival improvement in vivo. Thus, anti-PSMA-IR700 could be an effective platform for NIR-PIT in PSMA-expressing tumors. IR700 conjugation minimally alters the anti-bodydue to the small size andhydrophilic nature of the IR700dye(19). As a result, these APCs show high target accumulation withminimal distribution in normal tissue and minimal binding tonontarget-expressing cells. The effectiveness of NIR-PIT has beendemonstrated in a variety of different tumor typeswith a variety ofantibodies (19, 21–26, 35).

The anti-PSMA antibody–IR700 conjugate achieved adequatetumor TBRs as shown in Fig. 2, indicating that it may be practicalfor clinical application during surgical, endoscopic, or trans-needle procedures because of its high TBR on the PSMA-expres-sing tumors. Efficient binding and distribution of the antibody inthe tumor are important for APCs to be effective as agents for NIR-PIT. This also holds for antibody–toxin or antibody–drug con-jugates since, to be effective, the drugs and toxins must beinternalized after cell binding.Our results showed that anti-PSMAantibody bound to PSMA on cells specifically and was internal-izedwithin 6 hours of incubation in PSMA-expressing cancer cellsas shown in Fig. 1. These results suggest that anti-PSMA antibodyhas favorable characteristics for an antibody–drug conjugate.

Rapid and massive cancer cell killing adjacent to tumor vesselswith NIR-PIT leads to an immediate increase in vascular perme-ability after therapy (36–38). The delivery of various nano-sizedor macromolecular drugs into a NIR-PIT–treated tumor increasesup to 24-fold compared with that in a control nontreated tumorimmediately after the initial NIR light exposure, a phenomenonthat is known as the super enhanced permeability and retention(SUPR) effect induced byNIR-PIT (36–38). After the first NIR-PIT,circulating APCs can enter the treated tumor bed with greaterpermeability and penetration than prior to NIR-PIT. There, theybind homogeneously to the surviving fraction of cancer cells (39).Therefore, the second exposure to NIR light can further enhancetherapeutic effects of NIR-PIT (40). Thus, we chose the currenttherapeutic regimen with a single injection of the APC and twolight exposures to maximize therapeutic effectiveness.

Fractionated dosing of the APC with repeated light exposure isalso likely to increase effectiveness. For instance, in a modelutilizing EGFR-targeted NIR-PIT, repeated dosing of the APC andNIR light dosing was reported to improve the therapeutic effectcompared with a single shot approach (40, 41). Therefore, weinvestigated this regimen of repeated APC andNIR light exposure.This proved to be an effective therapy for treating a PSMA-

expressing prostate cancer model. Looking forward to NIR-PITuse in humans, splitting the APC dose and using repeated lightexposures will reduce toxicity of drugs because NIR light exposureby itself is harmless. Therefore, repeated NIR-PIT will optimizeefficacy without increasing toxicity.

In cancer therapy, anticancer drugs often fail because hetero-geneous cellular density and vascularity, increased interstitialpressure, andother structural barriers imposedby the extracellularmatrix prevent the drug from reaching its target in sufficientconcentrations to be effective (42, 43).Moreover, naturally occur-ring tumors usually are phenotypically and functionally hetero-geneous (44, 45). RepeatedNIR-PIT with additional various typesof APCor delivery of higher doses of nontargeted anticancer drugsby taking advantage of the SUPR effect are good strategies toimprove the therapeutic effect of cancer (38).

Another aspect of NIR-PIT is its immunogenic nature. Cellstreated with NIR-PIT undergo rapid volume expansion leading torupture of the cell membrane and extrusion of cell contents intothe extracellular space (20). Thus, NIR-PIT induces nearly imme-diate immunogenic cell death rather than apoptotic cell deathwhich is induced by most other cancer therapies (46–48). As theimmune effects of NIR-PIT are currently unknown it is difficult toknow whether this will be of benefit to patients, although it isanticipated that immunogenic cell death will augment the ther-apeutic effect of NIR-PIT as it selectively kills target cells whilenontarget cells immediately adjacent, including effector immunecells, show no toxic effects (19). In one specific example, targetingof local regulatory T cells with NIR-PIT (35) induced a rapidantitumor immune activation and regression of the PIT-treatedtumor. Moreover, NIR-PIT could enable activated cytotoxic effec-tor cells to attack other tumors of a similar nature located distantfrom the NIR-PIT–treated lesion.

While NIR-PIT shows highly selective cytotoxicity, and NIRlight exposure can be easily applied to superficial tumors, anobvious limitation is the inability to deliverNIR light to the tumorlocated deep in the tissue such as bone. Skin, fat, and other organswill absorbNIR light before it reaches the tumor. There are severalpotential solutions to this problem. For instance, NIR lightexposure could be delivered to a tumor and to adjacent structureswhile the tissues are still exposed during a surgical resection, thustreating residual tumor in the tumor margin or in regional lymphnodes. Such procedures have been proposed in the past with PDT(49, 50); however, we believe that NIR-PIT would be much moreeffective with lower toxicity than PDT. Alternatively, fiber opticlight probes could be placed within or nearby tumor usingendoscopes, laparoscopes, catheters, or image-guided percutane-ous needles. This would enable lesions such as prostate cancerrecurrences or regional lymphnodes to be effectively treated as thedepth of penetration of NIR light is at least 2 cm. For instance, it isentirely feasible that locally recurrent prostate cancer adjacent tothe urethra could be treated with NIR-PIT via a transparenttransuretheral catheter.

ConclusionThe fully human anti-PSMA antibody–IR700 conjugate

showed accumulation in PSMA-expressing prostate cancer cells.Subsequent NIR-PIT using anti-PSMA–IR700–induced remark-able therapeutic responses after repeated NIR-PIT cycles in aPSMA-expressing prostate cancer and cured more than two thirdof cancers. Thus, NIR-PIT utilizing PSMA as the targeting antigen

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for the anti-PSMA-IR700 might be successful new treatmentmodality for prostate cancer especially at diagnosis, initial treat-ment and at the time of locoregional recurrence in the pelvis.

Disclosure of Potential Conflicts of InterestNo potential conflict of interest were disclosed.

Authors' ContributionsConception and design: T. Nagaya, P.L. Choyke, H. KobayashiDevelopment of methodology: H. KobayashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Nagaya, Y. Nakamura, S. Okuyama, F. Ogata,H. KobayashiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Nagaya, S. Okuyama, Y. Maruoka, P.L. Choyke,H. Kobayashi

Writing, review, and/or revision of the manuscript: T. Nagaya, P.L. Choyke,H. KobayashiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P.L. Choyke, H. KobayashiStudy supervision: P.L. Choyke, H. Kobayashi

Grant SupportAll authors were supported by the Intramural Research Program of the NIH,

NCI, Center for Cancer Research.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 27, 2017; revised May 3, 2017; accepted June 1, 2017;published OnlineFirst June 6, 2017.

References1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin

2017;67:7–30.2. Klein EA, Kupelian PA. Localized prostate cancer: radiation or surgery?Urol

Clin North Am 2003;30:315–30.3. Denmeade SR, Isaacs JT. A history of prostate cancer treatment. Nat Rev

Cancer 2002;2:389–96.4. Gulley J, DahutWL. Chemotherapy for prostate cancer: finally an advance!

Am J Ther 2004;11:288–94.5. PetrylakDP, TangenCM,HussainMH, Lara PN Jr, Jones JA, TaplinME, et al.

Docetaxel and estramustine compared with mitoxantrone and prednisonefor advanced refractory prostate cancer. N Engl J Med 2004;351:1513–20.

6. CunninghamD,Humblet Y, Siena S, Khayat D, BleibergH, Santoro A, et al.Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004;351:337–45.

7. Deb N, Goris M, Trisler K, Fowler S, Saal J, Ning S, et al. Treatment ofhormone-refractory prostate cancer with 90Y-CYT-356 monoclonal anti-body. Clin Cancer Res 1996;2:1289–97.

8. Hurwitz H, Fehrenbacher L, NovotnyW, Cartwright T, Hainsworth J, HeimW, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin formetastatic colorectal cancer. N Engl J Med 2004;350:2335–42.

9. Leget GA, Czuczman MS. Use of rituximab, the new FDA-approvedantibody. Curr Opin Oncol 1998;10:548–51.

10. Barinka C, Sacha P, Sklenar J, Man P, Bezouska K, Slusher BS, et al.Identification of the N-glycosylation sites on glutamate carboxypeptidaseII necessary for proteolytic activity. Protein Sci 2004;13:1627–35.

11. Chen Y, Chatterjee S, Lisok A, Minn I, Pullambhatla M,Wharram B, et al. APSMA-targeted theranostic agent for photodynamic therapy. J PhotochemPhotobiol B 2016;167:111–6.

12. Huang X, Bennett M, Thorpe PE. Anti-tumor effects and lack of side effectsin mice of an immunotoxin directed against human and mouse prostate-specific membrane antigen. Prostate 2004;61:1–11.

13. Horoszewicz JS, Kawinski E, Murphy GP. Monoclonal antibodies to a newantigenic marker in epithelial prostatic cells and serum of prostatic cancerpatients. Anticancer Res 1987;7:927–35.

14. Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specificmembrane antigen expression in prostatic intraepithelial neoplasia andadenocarcinoma: a study of 184 cases. Cancer 1998;82:2256–61.

15. Israeli RS, Powell CT, Corr JG, Fair WR, Heston WD. Expression of theprostate-specific membrane antigen. Cancer Res 1994;54:1807–11.

16. Sweat SD, Pacelli A,MurphyGP, BostwickDG. Prostate-specificmembraneantigen expression is greatest in prostate adenocarcinoma and lymph nodemetastases. Urology 1998;52:637–40.

17. Wright GL Jr, Grob BM, Haley C, Grossman K, Newhall K, Petrylak D, et al.Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. Urology 1996;48:326–34.

18. Wilson BC, Patterson MS. The physics, biophysics and technology ofphotodynamic therapy. Phys Med Biol 2008;53:R61–109.

19. Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, KobayashiH. Cancer cell-selective in vivo near infrared photoimmunotherapy target-ing specific membrane molecules. Nat Med 2011;17:1685–91.

20. Ogawa M, Tomita Y, Nakamura Y, Lee MJ, Lee S, Tomita S, et al.Immunogenic cancer cell death selectively induced by near infraredphotoimmunotherapy initiates host tumor immunity. Oncotarget 2017;8:10425–36.

21. Hanaoka H, Nakajima T, Sato K, Watanabe R, Phung Y, Gao W, et al.Photoimmunotherapy of hepatocellular carcinoma-targeting Glypican-3combined with nanosized albumin-bound paclitaxel. Nanomedicine2015;10:1139–47.

22. Nagaya T, Nakamura Y, Sato K, Harada T, Choyke PL, Hodge JW, et al. Nearinfrared photoimmunotherapy with avelumab, an anti-programmeddeath-ligand 1 (PD-L1) antibody. Oncotarget 2017;8:8807–17.

23. Nagaya T, Nakamura Y, Sato K, Harada T, Choyke PL, Kobayashi H. Nearinfrared photoimmunotherapy of B-cell lymphoma. Mol Oncol 2016;10:1404–14.

24. Nagaya T, Nakamura Y, Sato K, Zhang YF, Ni M, Choyke PL, et al. Nearinfrared photoimmunotherapy with an anti-mesothelin antibody. Onco-target 2016;7:23361–9.

25. Sato K, Choyke PL, Kobayashi H. Photoimmunotherapy of gastric cancerperitoneal carcinomatosis in a mouse model. PLoS One 2014;9:e113276.

26. Watanabe R, Hanaoka H, Sato K, Nagaya T, Harada T, Mitsunaga M, et al.Photoimmunotherapy targeting prostate-specific membrane antigen: areantibody fragments as effective as antibodies? J Nucl Med 2015;56:140–4.

27. MaD,HopfCE,Malewicz AD,DonovanGP, Senter PD,GoeckelerWF, et al.Potent antitumor activity of an auristatin-conjugated, fully human mono-clonal antibody to prostate-specific membrane antigen. Clin Cancer Res2006;12:2591–6.

28. Chang SS, Reuter VE, Heston WD, Bander NH, Grauer LS, Gaudin PB. Fivedifferent anti-prostate-specificmembrane antigen (PSMA) antibodies con-firm PSMA expression in tumor-associated neovasculature. Cancer Res1999;59:3192–8.

29. Liu C, Hasegawa K, Russell SJ, Sadelain M, Peng KW. Prostate-specificmembrane antigen retargeted measles virotherapy for the treatment ofprostate cancer. Prostate 2009;69:1128–41.

30. Kahn D, Williams RD, Seldin DW, Libertino JA, Hirschhorn M, DreicerR, et al. Radioimmunoscintigraphy with 111indium labeled CYT-356for the detection of occult prostate cancer recurrence. J Urol 1994;152:1490–5.

31. Liu H, Moy P, Kim S, Xia Y, Rajasekaran A, Navarro V, et al. Monoclonalantibodies to the extracellular domain of prostate-specific membraneantigen also react with tumor vascular endothelium. Cancer Res 1997;57:3629–34.

32. Troyer JK, Beckett ML, Wright GL Jr. Location of prostate-specific mem-brane antigen in the LNCaP prostate carcinoma cell line. Prostate 1997;30:232–42.

33. Wong WW, Schild SE, Vora SA, Ezzell GA, Nguyen BD, Ram PC, et al.Image-guided radiotherapy for prostate cancer: a prospective trial ofconcomitant boost using indium-111-capromab pendetide (ProstaScint)imaging. Int J Radiat Oncol Biol Phys 2011;81:e423–9.

34. Kahn D, Austin JC, Maguire RT, Miller SJ, Gerstbrein J, Williams RD. Aphase II study of [90Y] yttrium-capromab pendetide in the treatment of

www.aacrjournals.org Mol Cancer Res; 15(9) September 2017 1161

Near-Infrared Photoimmunotherapy Targeting PSMA

on May 28, 2020. © 2017 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst June 6, 2017; DOI: 10.1158/1541-7786.MCR-17-0164

men with prostate cancer recurrence following radical prostatectomy.Cancer Biother Radiopharm 1999;14:99–111.

35. Sato K, Sato N, Xu B, Nakamura Y, Nagaya T, Choyke PL, et al. Spatiallyselective depletion of tumor-associated regulatory T cells with near-infra-red photoimmunotherapy. Sci Transl Med 2016;8:352ra110.

36. Kobayashi H, Watanabe R, Choyke PL. Improving conventional enhancedpermeability and retention (EPR) effects; what is the appropriate target?Theranostics 2013;4:81–9.

37. Sano K, Nakajima T, Choyke PL, Kobayashi H. Markedly enhanced per-meability and retention effects induced by photo-immunotherapy oftumors. ACS Nano 2013;7:717–24.

38. Sano K, Nakajima T, Choyke PL, Kobayashi H. The effect of photoimmu-notherapy followed by liposomal daunorubicin in amixed tumormodel: ademonstration of the super-enhanced permeability and retention effectafter photoimmunotherapy. Mol Cancer Ther 2014;13:426–32.

39. Nagaya T, Nakamura Y, Sato K, Harada T, Choyke PL, Kobayashi H.Improved micro-distribution of antibody-photon absorber conjugatesafter initial near infrared photoimmunotherapy (NIR-PIT). J ControlRelease 2016;232:1–8.

40. MitsunagaM, Nakajima T, Sano K, Choyke PL, Kobayashi H. Near-infraredtheranostic photoimmunotherapy (PIT): repeated exposure of lightenhances the effect of immunoconjugate. Bioconjug Chem 2012;23:604–9.

41. Nagaya T, Sato K, Harada T, Nakamura Y, Choyke PL, Kobayashi H. Nearinfrared photoimmunotherapy targeting EGFR positive triple negative

breast cancer: optimizing the conjugate-light regimen. PLoS One 2015;10:e0136829.

42. Hambley TW, Hait WN. Is anticancer drug development heading in theright direction? Cancer Res 2009;69:1259–62.

43. Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat RevCancer 2006;6:583–92.

44. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells withina malignant tumor. Science 1977;197:893–5.

45. Nowell PC. Mechanisms of tumor progression. Cancer Res 1986;46:2203–7.

46. Kerr JF. Shrinkage necrosis: a distinct mode of cellular death. J Pathol1971;105:13–20.

47. Willingham MC. Cytochemical methods for the detection of apoptosis.J Histochem Cytochem 1999;47:1101–10.

48. Ziegler U, Groscurth P. Morphological features of cell death. News PhysiolSci 2004;19:124–8.

49. Hiroshima Y, Maawy A, Zhang Y, Sato S, Murakami T, Yamamoto M, et al.Fluorescence-guided surgery in combination with UVC irradiation curesmetastatic human pancreatic cancer in orthotopic mouse models. PLoSOne 2014;9:e99977.

50. Pass HI, Temeck BK, Kranda K, Thomas G, Russo A, Smith P, et al.Phase III randomized trial of surgery with or without intraopera-tive photodynamic therapy and postoperative immunochemo-therapy for malignant pleural mesothelioma. Ann Surg Oncol 1997;4:628–33.

Mol Cancer Res; 15(9) September 2017 Molecular Cancer Research1162

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2017;15:1153-1162. Published OnlineFirst June 6, 2017.Mol Cancer Res   Tadanobu Nagaya, Yuko Nakamura, Shuhei Okuyama, et al.   Prostate-Specific Membrane Antigen (PSMA) AntibodyNear-Infrared Photoimmunotherapy Targeting Prostate Cancer with

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