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Precision Medicine and Imaging Evaluation of Novel Prostate-Specic Membrane Antigen-Targeted Near-Infrared Imaging Agent for Fluorescence-Guided Surgery of Prostate Cancer Sumith A. Kularatne 1 , Mini Thomas 1 , Carrie H. Myers 1 , Pravin Gagare 1 , Ananda K. Kanduluru 1 , Christa J. Crian 2 , and Brandy N. Cichocki 2 Abstract Purpose: The ability to locate and remove all malignant lesions during radical prostatectomy leads not only to prevent biochemical recurrence (BCR) and possible side effects but also to improve the life expectancy of patients with prostate cancer. Fluorescence-guided surgery (FGS) has emerged as a technique that uses uorescence to highlight cancerous cells and guide surgeons to resect tumors in real time. Thus, development of tumor-specic near-infrared (NIR) agents that target biomarkers solely expressed on prostate cancer cells will enable to assess negative tumor margins and affected lymph nodes. Experimental Design: Because PSMA is overexpressed in prostate cancer cells in >90% of the prostate cancer patient population, a prostate-specic membrane antigen (PSMA)- targeted NIR agent (OTL78) was designed and synthesized. Optical properties, in vitro and in vivo specicity, tumor-to- background ratio (TBR), accomplishment of negative surgical tumor margins using FGS, pharmacokinetics (PKs) properties, and preclinical toxicology of OTL78 were then evaluated in requisite models. Results: OTL78 binds to PSMA-expressing cells with high afnity, concentrates selectively to PSMA-positive cancer tis- sues, and clears rapidly from healthy tissues with a half-time of 17 minutes. It also exhibits an excellent TBR (5:1) as well as safety prole in animals. Conclusions: OTL78 is an excellent tumor-specic NIR agent for use in uorescence-guided radical prostatectomy and FGS of other cancers. Introduction Prostate cancer continues to present signicant medical chal- lenges affecting a sizable portion of the male population. Accord- ing to the American Cancer Society, over 164,690 men will be diagnosed with prostate cancer in the United States during 2018, leading to over 29,430 deaths (1). Prostate cancer also has a major impact on the US economy with cumulative treatment costs estimated at $10 billion/year (2). Radical prostatectomy remains the primary therapeutic modality for patients with localized prostate cancer (3, 4). With more than 90,000 patients undergo- ing prostatectomies in every year in the United States (5), 32% to 38% of them have biochemical recurrence (BCR) within 5 years (6). In fact, 20% to 48% of men with prostate cancer leave the surgery room with positive tumor margin that directly correlates to BCR and cancer management (7, 8). Therefore, it is important to excise all cancerous tissues with negative tumor margins to improve the quality of life and life expectancy of the patient. Although removal of malignant tissues completely depends on accuracy of prognosis, there are major limitations in current standard of care for accomplishing negative tumor margins in radical prostatectomy. In prostate cancer, the most common sites for BCR and positive tumor margins are known to be the pos- terolateral prostate and prostatic apex (9). These are the areas closely associated with nerves responsible for erectile function and urinary control. Because erectile dysfunction and urinary incontinence are the major possible side effects of prostatectomy, surgeons may tend to preserve tissues and nerves around pos- terolateral prostate and prostatic apex to maintain quality of life of the patient (9). Consequently, the caution exercised in sparing these areas may lead to disease tissues being left behind. The standard practice for prostatectomy relies on visual inspection and palpation during classic open surgery. Because the naked eye is more often limited in its ability to differentiate cancer cells versus healthy cells, visual localization of tumor cells and margins using abnormal color and/or morphology is not reliable (10). Moreover, the naked eye cannot detect smaller and early-stage tumors, especially tumors obscured under other healthy tissues. Palpation, on the other hand, lacks the sensitivity to feel and distinguish the texture of cancerous versus healthy tissue. Because robotic surgery has gained popularity (i.e., >80% of all prosta- tectomies in the United States are performed robotically) due to its minimally invasive and fast recovery process (12 days) when compared with open surgery (1012 days), use of palpation as a diagnostic tool is in decline (3, 4). Moreover, complete removal of affected lymph nodes using conventional visual or palpation methods is unreliable as they often look or feel normal. Therefore, 1 On Target Laboratories, West Lafayette, Indiana. 2 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, Indiana. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Sumith A. Kularatne, On Target Laboratories, 1281 Win Hentschel Boulevard, West Lafayette, IN, 47906, Phone: 765-588-4547; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-18-0803 Ó2018 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 177 on August 7, 2020. © 2019 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst September 10, 2018; DOI: 10.1158/1078-0432.CCR-18-0803

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Precision Medicine and Imaging

Evaluation of Novel Prostate-Specific MembraneAntigen-Targeted Near-Infrared Imaging Agentfor Fluorescence-Guided Surgery of ProstateCancerSumith A. Kularatne1, Mini Thomas1, Carrie H. Myers1, Pravin Gagare1,Ananda K. Kanduluru1, Christa J. Crian2, and Brandy N. Cichocki2

Abstract

Purpose: The ability to locate and remove all malignantlesions during radical prostatectomy leads not only toprevent biochemical recurrence (BCR) and possible sideeffects but also to improve the life expectancy of patientswith prostate cancer. Fluorescence-guided surgery (FGS) hasemerged as a technique that uses fluorescence to highlightcancerous cells and guide surgeons to resect tumors in realtime. Thus, development of tumor-specific near-infrared(NIR) agents that target biomarkers solely expressed onprostate cancer cells will enable to assess negative tumormargins and affected lymph nodes.

Experimental Design: Because PSMA is overexpressed inprostate cancer cells in >90% of the prostate cancer patientpopulation, a prostate-specific membrane antigen (PSMA)-

targeted NIR agent (OTL78) was designed and synthesized.Optical properties, in vitro and in vivo specificity, tumor-to-background ratio (TBR), accomplishment of negative surgicaltumormargins using FGS, pharmacokinetics (PKs) properties,and preclinical toxicology of OTL78 were then evaluated inrequisite models.

Results: OTL78 binds to PSMA-expressing cells with highaffinity, concentrates selectively to PSMA-positive cancer tis-sues, and clears rapidly fromhealthy tissueswith a half-time of17 minutes. It also exhibits an excellent TBR (5:1) as well assafety profile in animals.

Conclusions: OTL78 is an excellent tumor-specific NIRagent for use in fluorescence-guided radical prostatectomyand FGS of other cancers.

IntroductionProstate cancer continues to present significant medical chal-

lenges affecting a sizable portion of the male population. Accord-ing to the American Cancer Society, over 164,690 men will bediagnosed with prostate cancer in the United States during 2018,leading to over 29,430 deaths (1). Prostate cancer also has amajorimpact on the US economy with cumulative treatment costsestimated at $10 billion/year (2). Radical prostatectomy remainsthe primary therapeutic modality for patients with localizedprostate cancer (3, 4). With more than 90,000 patients undergo-ing prostatectomies in every year in the United States (5), 32% to38% of them have biochemical recurrence (BCR) within 5 years(6). In fact, 20% to 48% of men with prostate cancer leave thesurgery room with positive tumor margin that directly correlatesto BCR and cancer management (7, 8). Therefore, it is importantto excise all cancerous tissues with negative tumor margins toimprove the quality of life and life expectancy of the patient.

Although removal of malignant tissues completely depends onaccuracy of prognosis, there are major limitations in currentstandard of care for accomplishing negative tumor margins inradical prostatectomy. In prostate cancer, the most common sitesfor BCR and positive tumor margins are known to be the pos-terolateral prostate and prostatic apex (9). These are the areasclosely associated with nerves responsible for erectile functionand urinary control. Because erectile dysfunction and urinaryincontinence are the major possible side effects of prostatectomy,surgeons may tend to preserve tissues and nerves around pos-terolateral prostate andprostatic apex tomaintainquality of life ofthe patient (9). Consequently, the caution exercised in sparingthese areas may lead to disease tissues being left behind. Thestandard practice for prostatectomy relies on visual inspectionand palpation during classic open surgery. Because the naked eyeis more often limited in its ability to differentiate cancer cellsversus healthy cells, visual localization of tumor cells andmarginsusing abnormal color and/or morphology is not reliable (10).Moreover, the naked eye cannot detect smaller and early-stagetumors, especially tumors obscured under other healthy tissues.Palpation, on the other hand, lacks the sensitivity to feel anddistinguish the texture of cancerous versus healthy tissue. Becauserobotic surgery has gained popularity (i.e., >80% of all prosta-tectomies in the United States are performed robotically) due toits minimally invasive and fast recovery process (1–2 days) whencompared with open surgery (10–12 days), use of palpation as adiagnostic tool is in decline (3, 4).Moreover, complete removal ofaffected lymph nodes using conventional visual or palpationmethods is unreliable as they often look or feel normal. Therefore,

1On Target Laboratories, West Lafayette, Indiana. 2Department of VeterinaryClinical Sciences, Purdue University, West Lafayette, Indiana.

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

Corresponding Author: Sumith A. Kularatne, On Target Laboratories, 1281 WinHentschel Boulevard, West Lafayette, IN, 47906, Phone: 765-588-4547;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0803

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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better methods for assessing negative tumor margins and affectedlymph nodes are needed.

In response to this unmet clinical demand,fluorescence-guidedsurgery (FGS) has emerged as a technique that usesfluorescence tohighlight cancerous cells and guide surgeons to resect tumors inreal time. Currently, while the field is still in its infancy, theindustry is slowly developing better dyes that selectively accumu-late in prostate cancer with improved tumor-to-back groundratios (TBRs). FDA-approved indocyanine green (ICG) has beenused in prostatectomy to detect prostate cancer tissues, lymphnodes, and vascularization of prostate (11). However, ICG hasshown significant limitations with respect to sensitivity, specific-ity, poor TBR, and higher liver as well as GI tract uptake due to thenon-targeted nature of the molecule (12, 13). In order to over-come deficiencies of nontargeted NIR dyes, tumor-specific NIRagents that target biomarkers solely expressed on cancer cells havebeen evaluated in preclinical stages. One example is prostate-specific membrane antigen (PSMA) that is overexpressed onprostate cancer cells in >90% of the prostate cancer patientpopulation (14). Examination of postprostatectomy specimenshas shown that the expression level of PSMA correlated not onlywith tumor grade, pathologic stage, PSA level, and aneuploidy butalso with BCR (15). PSMA is also expressed in neovasculature ofsolid tumors developed inorgans such as liver, lung, breast, colon,renal, brain, sarcoma, gastric, and oral (16, 17). It allows inter-nalization of PSMA-targeted agents into an endosomal compart-ment, thereby maneuvering PSMA as an excellent biomarker foruse in FGS (18). Therefore, antibodies or small-molecule ligands-targeted NIR agents that are specific for PSMA are being currentlyevaluated in the preclinical research stages (19–26). Because eachof these molecules has their own limitations, we developed aPSMA-targeted NIR agent (referred to herein as OTL78) withsuperior optical, pharmacokinetic (PK), and biological propertiesfor use in FGS. In this paper, we describe the synthesis andcharacterization, optical properties, preclinical evaluation in can-cer cell in culture and in subcutaneous and orthotopic tumormodels, accomplishment of negative surgical tumor marginsusing FGS, and PK properties of OTL78. We then provide pre-clinical evidence of its remarkable safety profile in mice.

Materials and MethodsIn vitro binding

For OTL78 relative affinity (IC50), 22Rv1 or PC3 cells wereplated into a T75 flask and allowed to form a monolayer over 48hours. After trypsin digestion, released cells were transferred intocentrifuge tubes (1 � 106 cells/tube) and centrifuged. Spentmedium in each tube was replaced with 100 nmol/L DUPA-FITCin the presence of increasing concentration (0.001 nmol/L–10 mmol/L) of OTL78 in freshmedium (0.5mL). After incubatingfor 30minutes at 4�C, cells were rinsedwith culturemedium (2�1.0 mL) and saline (1� 1.0 mL) to remove any unbound DUPA-FITC. Cells were then resuspended in saline (0.5 mL), and cellbound fluorescence was quantified using a flow cytometer. Therelative affinitieswere calculatedusing aplot of percent cell boundfluorescence versus the log concentration of OTL78 usingGraphPad Prism 6.

For OTL78 binding affinity, 22Rv1 or PC3 cells were seededinto a T75 flask and allowed to form a monolayer over 48 hours.After trypsin digestion, cells were transferred into centrifuge tubes(1 � 106 cells/tube) and centrifuged. The medium was replacedwith freshmedium containing increasing concentration ofOTL78and incubated for 30 minutes at 4�C. After rinsing with freshmedium (2 � 1.0 mL) and saline (1 � 1.0 mL), cells were lysedwith 1% SDS in saline (1.0 mL), and cell bound fluorescence wasanalyzed using a fluorometer (Cary Eclipse, Agilent Technolo-gies). The binding affinity (Kd) was calculated using a plot ofpercent cell bound fluorescence versus concentration usingGraphPad Prism 6.

Confocal microscopy22Rv1, LNCaP, or PC3 cells (50,000 cells/well in 1 mL) were

seeded into poly-D-lysinemicrowell Petri dishes and allowed cellsto form monolayers over 12 hours. Spent medium was replacedwith fresh medium containing OTL78 (100 nmol/L), and cellswere incubated for 1 hour at 37�C or 4�C. After rinsing with freshmedium (2 � 1.0 mL) and saline (1 � 1.0 mL), fluorescenceimages were acquired using an epimicroscopy.

Whole-body imaging and tissue biodistributionSeven-week-old male nu/nu mice were inoculated subcutane-

ously with 5.0� 106 22Rv1, LNCaP, PC3, or A549 cells/mouse in50% high concentrated (HC) matrigel with RPMI1640 mediumon the shoulder. Growth of the tumors was measured in perpen-dicular directions every 2 days using a caliper (body weights weremonitored on the same schedule), and the volumes of the tumorswere calculated as 0.5 � L � W2 (L ¼ longest axis and W ¼ axisperpendicular to L in millimeters). Once tumors reached approx-imately 300 to 400 mm3 in volume, animals (3–5 mice/group)were intravenously injected with appropriate dose of OTL78 insaline.

For orthotopic tumors, 2� 105 22Rv1 cells/mouse in 10%HCmatrigel with RPMI1640 medium were surgically implanted inthe prostate of 7-week-old male SCID mice. Briefly, 7-week-oldmale SCID mice were given 1% to 5% isoflurane for anesthesiaand subcutaneous injection of 5mg/kgmeloxicampreoperativelyfor analgesia. The mice were placed dorsal side up and washedabove the prostate with a chlorhexidine scrub to ensure a sterilearea for incision. After an insertion was made using scalpelthrough the skin, the peritoneal lining was lifted to make a smallincision using a scissor and widened using forceps. Dorsal lobes

Translational Relevance

We developed a prostate-specific membrane antigen(PSMA)-targeted near-infrared (NIR) imaging agent (OTL78)that: (i) binds to PSMAþ tumors with high affinity andspecificity, (ii) allows to use subnanomolar concentration tovisualize small tumors, (iii) clears rapidly fromPSMA-negativetissues with half-life of 17 minutes, (iv) retains tumor fluo-rescence for over 48 hours, allowing visualization throughoutFGS, and (v) allows to accomplish negative surgical tumormargins, (vi) has an excellent safety profile in animals. OTL78has proven to be a clinical candidate to yield sharp tumorboundarieswithnegative tumormarginswithin 1 to 2hours ofinfusion during radical prostatectomy. With its recent entryinto investigational new drug–enabling studies, OTL78 has apotential to become the first PSMA-targeted NIR agent to enterinto the clinic for use in fluorescence-guided radicalprostatectomy.

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were exteriorized and gently stabilized with a wet (PBS) cottonswab. 22Rv1 cells (in 10 mL of 10%HC-matrigel) were injected tothe prostate using a 28-gauge needle. After placing the prostateback into the peritoneum, the abdominal wall was sutured, thebody wall was closed using 3–0 or 4–0 vicryl and the skin wasclosedusing staples. Animalsweremonitoreduntil theywere usedfor the studies. After 1month, the animalswere administeredwithOTL78 (10 nmol/L in 100 mL saline per mouse), euthanized after2 hours by CO2 asphyxiation, and imaged using the AMI imagesystem.

For whole-body imaging and biodistribution studies, animalswere euthanized after 2 hours of administration ofOTL78 by CO2

asphyxiation. For time-dependent studies, animals were imagedunder anesthesia using isoflurane. Imaging experimentswere thenperformed using IVIS or AMI image systems. Following whole-body imaging, animals were dissected and selected tissues wereanalyzed for fluorescence activity using IVIS or AMI image systemand region of interest (ROI) of the tissues were calculated usingLiving Image 4.0 software or AMIView Image Analysis Software.

For ImageJ analysis, whole-body imaging was acquired ingray scale and processed in ImageJ software. Either a line acrossthe tumor or box around the tumor was drawn to define thefluorescence to be quantitated. The tumor-to-muscle ratio wasanalyzed using a plot of the fluorescence gray value versusdistance.

Tumor surgeriesSeven-week-old male nu/nu mice were inoculated subcutane-

ously with 5.0� 106 22Rv1 cells/mouse in 50%HCmatrigel withRPMI1640 medium on the shoulder. Growth of the tumors wasmeasured as previously described. After 1 month, the animalsweremixed and divided into two groups (n¼ 5mice/group). Twohours after administering OTL78 (10 nmol in 100 mL saline permouse), animals were given 1% to 5% isoflurane for anesthesiaand imaged using the AMI image system. After an insertion wasmade using scalpel through the skin, surgical removal of thetumors was performed either following conventional technique(e.g., visualization under white light or palpation) or with the aidof fluorescence (FGS: debulking of visible tumors under conven-tional method followed by resection of residual fluorescencetissues under image-guided method). After the surgery, the skinwas closed using staples and imaged the mice using AMI imagesystem. After imaging, the residual fluorescent tissues from select-ed mice of the conventional surgery group and tissues samplesfrom the tumor beds of selected mice of the FGS group weresubmitted for pathologic (IHC) analysis. Response to surgicaltreatment was monitored for over 30 days by imaging using theAMI image system 2 hours after injecting OTL78 (10 nmol/mouse) and by measuring the growth of the tumor volume usinga caliper. Any animals with tumor volume �1,000 mm3 wereeuthanized. Tumor-free survival of themice was documented as apercentage of survival versus time using GraphPad Prism 6. IHCstudies were done as explained in the Safety studies in Supple-mentary Data and Methods.

PK studyFor serum clearance, 10 nmol/L of OTL78 was administered to

male nude mice (n ¼ 3 mice) as a single bolus intravenousinjection. Bloodwas collected at regular intervals (0–90minutes),and serum-bound OTL78 was quantified by measuring the fluo-rescence using IVIS imager. The half-life of OTL78 was calculated

as a percentage of serum-bound fluorescence versus time usingGraphPad Prism 6.

For tissue clearance, 10 nmol/L of OTL78 was administered tomale nude mice bearing 22Rv1 tumors (n ¼ 5 mice/group) as asingle bolus intravenous injection. Animals were euthanized at 2,4, 8, 24, and 48-hour time points, and selected tissues wereanalyzed using IVIS imager. The tissue clearance was determinedas a percentage of tissue-bound fluorescence versus time usingGraphPad Prism 6.

Note: Procedures for in vitro binding of DUPA-FITC, humanserumbinding studies, safety study, and tolerability studies canbefound in the Supplementary Data and Methods.

ResultsDesign and synthesis of OTL78

In an effort to improve limitations in current clinical practice ofradical prostatectomy and tumor-specific imaging agents that arein the preclinical stages, OTL78 was assembled using: (i) a high-affinity PSMA-targeting ligand (coined DUPA; refs. 19, 26), (ii) arationally designed 14 atoms long polyethylene glycol–dipeptidelinker, and (iii) an inexpensive NIR dye (<$400/g) named S0456(see Supplementary Fig. S1A for the chemical structure). Thedipeptide consisting of phenylalanine-tyrosine was designed tofit to the contours and chemistry of the tunnel accessing thebinding pocket of the PSMA protein (27). Upon conjugation ofDUPA–PEG–dipeptide to S0456, the dipeptide not onlyimproved the binding affinity of OTL78 but also enhanced thefluorescence of S0456 by �2 at the same concentration (seeSupplementary Fig. S1B showing excitation and emission spectraof OTL78 at 1 mmol/L and S0456 at 1 mmol/L in 1 mL of PBSobtained using fluorometer). Synthesis of OTL78 was conductedas shown in the Supplementary Scheme 1 by following procedurein the Supplementary Materials and Methods. OTL78 wasobtained as a dark green solid with >99% purity at 774 nmwavelengths and with �82% yield. It was characterized using1H- and 13C-NMR, HPLC, and HRMS (Supplementary Figs. S2and S3).

Evaluation of in vitro affinity and specificity of OTL78In an effort to evaluate the specificity of OTL78 for prostate

cancer, PSMA expression in LNCaP, 22Rv1, PC3 (three humanprostate cancer cell lines), and A549 (a human alveolar basalepithelial carcinoma cell line), as a negative control, was firstexamined by flow cytometry. PSMA expression was highest inLNCaP followed by 22Rv1 and negligible in PC3 and A549(Supplementary Fig. S1C). These results agreed with the numberof PSMAmolecules per LNCaP, 22Rv1, and PC3 cells reported byWang and colleagues (28). Due to moderate PSMA expressionlevels and better tumorigenic capacity with low necrosis, 22Rv1was selected as the primary cell line to characterize OTL78.

The affinity ofOTL78 for PSMAwasfirst screened by competingwith DUPA-FITC. The absolute binding affinity (Kd) and speci-ficity of DUPA-FITC for PSMA (Kd ¼ 6 nmol/L; SupplementaryFig. S1D) was first established using PSMAþ22Rv1 and PSMA-negative PC3 cells as described in the SI Materials and Methods.OTL78was able to compete with DUPA-FITC for PSMA on 22Rv1cells with IC50 of 7 nmol/L (Supplementary Fig. S1E). The affinityand specificity of OTL78 was then evaluated by incubatingincreasing concentrations of OTL78 with either 22Rv1 or PC3cells and analyzing for cell bound fluorescence by fluorometer.

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OTL78 was able to bind to PSMA on 22Rv1 cells with veryhigh affinity (Kd ¼ 4.7 nmol/L), whereas it did not bind toPSMA-negative PC3 cells, confirming specificity of OTL78 toPSMA (Fig. 1B).

PSMA-mediated internalization of OTL78 was next evaluatedby incubating OTL78 with 22Rv1 and PC3 cells. Analysis offluorescence microscopy images indicates that OTL78 was ableto efficiently label 22Rv1 and LNCaP cells [Fig. 1C (i and ii)] butnot PC3 cells [Fig. 1C (iii and vi)], indicating PSMA-mediateduptake of OTL78. Fluorescence was detected throughout thecytoplasm of 22Rv1 and LNCaP cells at 37�C. Moreover, we alsoobserved that OTL78 is highly concentrated and entrapped in thecertain regions of 22Rv1 and LNCaP cells. Labeling of 22Rv1 andLNCaP cells with OTL78 in the presence a nuclear staining dye(DAPI) at 4�Cwas also conducted to decrease the endocytosis and

recycling of PSMA [Supplementary Fig. S1F (i and ii)]. Epifluor-escence images from this study indicated that OTL78 binds toPSMA on the cell surface. Therefore, we assume that OTL78 firstbinds to PSMA on the cell surface and then it undergoes receptor-mediated endocytosis. We further assume that OTL78 isentrapped in the acidic endosomes within prostate cancer cells.

Evaluation of in vivo efficacy and specificity of OTL78The ability of OTL78-mediated imaging of prostate cancer was

next established by conducting a series of experiments in mousemodels. First, the optimal dose for tumor imaging was deter-mined by administering increasing concentrations of OTL78(0.3–120 nmol/mouse) to mice bearing 22Rv1 tumor xenograftsfollowed by ex vivo tissue biodistribution analysis. The IVIS imageanalysis obtained at 2-hour time point indicated that OTL78

Figure 1.

In vitro binding and specificity of OTL78. A, Excitation (Ex) and emission (Em) spectra of OTL78. B, Dose-dependent binding of OTL78 to PSMAþ 22Rv1 cellsand PSMA� PC3 cells in culture (n ¼ 2). C, Binding and internalization of OTL78 to (i) 22Rv1, (ii) LNCaP, or (iii) PC3 (fluorescence image), and (iv) PC3(DIC image) cells by epifluorescence (epi) microscopy. Note: OTL78 is highly concentrated in the acidic endosomes of 22Rv1 and LNCaP cells. DIC, deferentialinterference contrast images.

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provided excellent TBR at dose range between 1 and 30 nmol/mouse with the best TBR occurring at �3 to 10 nmol/mouse(Supplementary Fig. S4A–S4B).

We next evaluated in vivo tumor specificity of OTL78 byadministering 10 nmol/L ofOTL78 tomice bearing subcutaneous22Rv1, LNCaP, PC3, or A549 tumor xenografts followed byconductingwhole-body imaging and ex vivo tissue biodistributionusing either IVIS or AMI image systems. Both studies demonstrat-ed that OTL78 accumulated predominantly in PSMA-expressing22Rv1 (Fig. 2A–Dand Fig. 3A–D) and LNCaP (Fig. 3B–E) tumors,with no substantial fluorescence activity in other tissues exceptkidneys. Although tumor accumulated fluorescence was not seenin PC3 and A549 tumors at higher threshold (Fig. 2B and C andE and F), uptake of OTL78 was observed in both tumors atlower threshold (Fig. 2E and F, bottom). Although fluorescenceintensities of PC3 and A549 tumors were �6-fold less comparedwith 22Rv1 tumors (Fig. 2A–D), fluorescence accumulation in

PC3 and A549 tumors was higher than rest of the tissues exceptkidneys and skin (Supplementary Fig. S5A–S5B). We thereforeassume that the observed fluorescence in PC3 and A549 tumorsmay be due to accumulation of OTL78 via PSMA in the neovas-culature of PC3 and A549 solid tumors. This further suggests thatOTL78 will be able to detect tumors with low PSMA expressionlevels. OTL78 also had a significant kidney uptake due tohigh PSMA expression in murine kidneys and clearance ofOTL78 through the kidneys. More importantly, fluorescence inthe kidneys was clearly visible in whole-body images collectedfrom AMI imager demonstrating penetrating ability of OTL78 tolocate buried PSMAþ tissues. We assume that observed skinuptake may be due to nonspecific uptake of S0456 moietyof the OTL78 molecule. Although skin uptake clears within 4 to5 hours, skin will not be interfered with open or robotic surgerybecause the camera will be directly focusing to the prostate inboth techniques.

Figure 2.

In vivo efficacy and specificity of OTL78 in subcutaneous tumor models using the IVIS image system. Representative fluorescence images from the IVIS imagershowing mice bearing (A) 22Rv1 (n ¼ 5 mice/group), (B) PC3 (n ¼ 5 mice/group), and (C) A549 (n ¼ 3 mice/group) tumors 2 hours after administering10 nmol/L of OTL78. Tissue biodistribution analysis of the same mice with (D) 22Rv1, (E) PC3, and (F) A549 tumors at 2 hours after injection. Note: �, Representativefluorescence images of PC3 and A549 after lowering threshold to � 1 � 108 [(p/sec/cm3/sr)/(mW/cm2)].

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We then examined the ability of OTL78 to detect primarytumors in the prostate and regionalmetastasis in seminal vesicles.In that case, 22Rv1 cells were surgically implanted in the prostateof SCID mice as described in the Supplementary Materials andMethods. Once tumors grew, the animals were imaged using AMIimage system 2 hours after administration of 10 nmol/L ofOTL78. Orthotopic imaging studies also demonstrated thatOTL78 mainly accumulated in prostate tumors with no fluores-cence observed in other tissues except kidneys (Fig. 3C–F andSupplementary Fig. S5C and S5D). Moreover, OTL78 was able todetect local regional metastasis in seminal vesicles in the presenceof primary tumor (Fig. 3G and Supplementary Fig. S5D), indi-cating ability ofOTL78 to locate tumors and lymphnodes that areburied under the prostate.

Following biodistribution studies, specificity of OTL78 forPSMA was quantitated by calculating TBR. In both subcutaneousand orthotopic tumor models, OTL78 displayed excellentTBR (Supplementary Fig. S6A) ranging from 19:1–25:1 (tumor:

muscle), 11:1–14:1 (tumor:lung), 11:1–15:1 (tumor:liver), 14:1–23:1 (tumor:heart), 19:1 (tumor:intestine), 11:1–20:1 (tumor:spleen), 4:1 (tumor:prostate), and 4:1–10:1 (tumor:skin).Observed better TBRs, especially tumor:skin, in orthotopic modelcompared with subcutaneous model may be due to: (a) higheraccumulation of OTL78 due to better tumor angiogenesis and (b)less nonspecific skin uptake of NIR dye moiety in SCID mice.

Finally, the ability of OTL78 to define the tumor/healthy tissueboundaries was evaluated using ImageJ software analysis. Thewhole-body image ofmice injected with 10 nmol/L ofOTL78wasacquired as fluorescence in a gray scale and either a line or box(Supplementary Fig. S6B) was drawn to quantitate the fluores-cence to be defined in the tumor boundaries. As shown inSupplementary Fig. S6C and S6D, OTL78 was able to definetumor boundaries precisely with a TBR of 5:1, suggestingits capability to guide surgeons to accurately detect the tumormargins (acceptable TBR for image-guided surgery is consideredto be >1.5; ref. 29).

Figure 3.

In vivo efficacy and specificity of OTL78 in orthotopic and subcutaneous tumor models using the AMI image system. Representative fluorescence imagesfrom the AMI image system showing mice bearing (A) 22Rv1 subcutaneous (n ¼ 3 mice/group), (B) LNCaP subcutaneous (n ¼ 3 mice/group), and (C)22Rv1 orthotopic (n ¼ 5 mice/group) tumors 2 hours after administering 10 nmol/L of OTL78. Tissue biodistribution analysis of the same mice with(D) 22Rv1, (E) LNCaP, and (F) 22Rv1 tumors at 2 hours after injection. Note: �Primary tumor is in the prostate in G; K, kidneys; PT, primary tumor; SC,secondary tumor; and SV, seminal vesicle.

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FGS using OTL78The ability of OTL78 to guide surgeons to excise all cancerous

tissues with negative tumor margins was next investigated byperforming image-guided surgery in tumor-bearing mice. Briefly,10 nmol/L of OTL78 was administered into mice bearing 22Rv1tumor xenografts, and a comparative study was conducted byperforming surgeries under conventional (e.g., visualizationunder white light or palpation) or fluorescence-guided technol-ogy (i.e., debulking of visible tumors under conventionalmethodfollowed by resection of residual fluorescence tissues underimage-guided method) at 2-hour time point. Preoperative fluo-rescence images of tumor-bearingmice demonstrated that OTL78was able to localize 22Rv1 tumors with high contrast within 2hours (Fig. 4A, first column; Supplementary Fig. S7). Postoper-ative fluorescence imagers indicated presence of residual fluores-cence in the tumor bed of the conventional cohorts, whereas nosignificant fluorescence was observed in the FGS cohorts (Fig. 4A,middle column; Supplementary Fig. S7). Pathologic analysis ofresidual fluorescent tissues from the conventional surgery con-firmed that the fluorescence is due to cancer cells (Fig. 4A and B,middle). More importantly, no residual tumors were identified intissues from tumor margin/bed from the FGS cohorts (Fig. 4B,right column). Following surgeries, BCR of the cancer wasassessed by monitoring animals for over a month using fluores-

cence imaging. As anticipated, only the conventional surgerycohort had recurrence at the primary tumor site, and no sign ofBCRwas observed in the FGS cohort during the study (Fig. 4A andSupplementary Fig. S7). As shown in the survival curve (Fig. 4C),the FGS cohorts survived during the study with no BCR, whereasall mice in the conventional surgery group had to be euthanizedwithin 3weeks. Although the observed BCR rate is higher than thereported values for humanandmice (6, 30), this proof-of-conceptstudy highlights the importance of excising all cancerous tissueswith negative tumormargins to improve the quality of life and lifeexpectancy of the patient.

Evaluation of PK properties of OTL78Having demonstrated in vivo specificity, we then evaluated the

PK profile of OTL78 in animal models as described in theMaterials and Methods. OTL78 was able to generate excellenttumor images within 1 hour of postinjection, with TBR ratiosremaining excellent throughout the experiment time of 48 hours(Fig. 5A and Supplementary Fig. S8). Moreover, time-dependenttumor clearance studies demonstrated that�50% of fluorescencewas retained in the tumor at 48 hours after injection (Fig. 5B),suggesting that OTL78 fluorescence was indeed entrapped in thetumor. OTL78 was excreted mainly through the kidneys, andfluorescence of the kidneys became negligible after 8 hours

Figure 4.

Comparison of surgeries performed under conventional and fluorescence-guided techniques. A, Representative fluorescence images of tumor beds of mice beforeand after surgically removing 22Rv1 tumor xenografts by conventional (n ¼ 5 mice/group) or fluorescence-guided (n ¼ 5 mice/group) techniques. Mice wereadministered with OTL78 (10 nmol/mouse) 2 hours before imaging with AMI image system. B, Representative H&E staining of 22Rv1tumor (left column) aftersurgical resection, the residual fluorescent tissues after conventional surgery showing positive tumor margins (middle column), and tumor bed tissues after FGSshowing negative tumor margins. C, Survival curve of the same mice (n ¼ 5 mice/group) over 30 days. Growth of tumors was monitored during the study,and any animal with tumor volume �1,000 mm3 were euthanized.

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(Fig. 5B). We also elected to monitor the skin clearance due toobserved high fluorescence in the skin at the 2-hour time point.Although skin uptake may not affect the outcome of open orrobotic-assisted FGS, as shown in Fig. 5B, OTL78 cleared from themice skin between 4 and 5 hours of postinjection.

Finally, serum clearance of OTL78 was examined by injecting10 nmol/mouse intravenously, collecting blood samples at reg-ular intervals (0–90 minutes), and measuring serum-boundOTL78 using AMI imager. OTL78 reached a peak concentrationin the circulation �10 minutes after injection and cleared with aserum half-life of �17 minutes (Fig. 5C). Human serum bindingstudies conducted using LC/MS analysis also indicated thatOTL78 has very low affinity (32%) for human serum proteins(Supplementary Fig. S9). Taken together, OTL78 clears rapidlyfrom nonmalignant tissues to allow tumor visualization within 1to 2 hours of administration, avoiding the requirement for aprolonged hospital stay.

Evaluation of safety profile of OTL78Motivated by the specificity and PK properties described above,

the safety profile of OTL78 was then evaluated using ex vivo and

in vivomodels. The acute maximum tolerance dose of OTL78 wasinitially determined by injecting 6 mmol/mouse (600� of normaldose) to healthy Balb/c mice. Body weights and clinical observa-tions were monitored during the study, and histopathologicanalysis on selected tissues was then conducted on day 14 ofpostinjection. The animals were active after administration ofOTL78 and behaved normally throughout the study. As shownin Fig. 6A, body weights over the course of the study remainedunchanged (<5% increase), suggesting that OTL78 is not grosslytoxic to the animals. Moreover, no obvious pathologic changerswere detected in hematoxylin and eosin (H&E) staining con-ducted on any of the tissues (Fig. 6B and Supplementary Fig.S10). No noticeable toxicities were also noticed in clinical pathol-ogy analysis on blood samples collected from mice injected withOTL78 (6 mmol/mouse).

Possible OTL78-related hypersensitivity in human was nextexamined using basophil activation assay. Drug-related hyper-sensitivity occurs mainly due to immune response caused bycrosslinking of immunoglobulin E (IgE) expressed on mast cellsand basophils (Fig. 6D) resulting in activation and subsequentdegranulation to release vasoactive amines, prostaglandins, and

Figure 5.

PK profile of OTL78. A, Representative time-dependent whole-body fluorescence images over white light images of a mouse bearing 22Rv1 tumor afterinjecting 10 nmol/L of OTL78 and image with IVIS imager at different time intervals (n ¼ 5 mice/group). B, Clearance of OTL78 from tumor, kidney, and skin fromtime-dependent biodistribution analysis. C, Determination of half-life of OTL78 by time-dependent serum analysis. Error bar, SD (n ¼ 3 mice/group). K, kidneys.

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cytokines (31). Because crosslinking of IgE can be due to aggre-gates, concentration-dependent UV spectrometric studies wereconducted to determine higher order aggregates of OTL78. Asshown in Fig. 6C, therewere nonoticeable higher order aggregatesobserved with OTL78, whereas the positive control (i.e., OTL38;ref. 32) exhibited concentration-dependent aggregation peak atlmax �700 nm at 75 mmol/L in saline.

Because basophils are readily available from blood sampleswhen comparedwith tissue-residentmast cells, we then evaluateddrug-related hypersensitivity due to monomer and low orderaggregates (if present) of OTL78 using basophil activation testin human blood samples as described in the Materials andMethods section. Briefly, 75 mmol/L of OTL78 was first added toa tube containing whole blood from donors and stimulating

buffer. After labeling with anti-CCR3 (CD193)–phycoerythrinand anti-CD63–CD203c–PE-DY647, the percentage of activatedbasophils was quantitated using flow-cytometric analysis (33). Asshown in Fig. 6E and Supplementary Table S1, no obviousdifferences in percentage activated basophils were seen betweenthe OTL78-treated sample and negative control, resulting in astimulated index of 1, whereas stimulated index is defined as theratio of percentage of basophil activation by the allergen: per-centage of basophil activation by background and stimulatedindex � 2 considered as positive response (33). However, whensimilar assayswere conducted using fMLP (a nonspecific basophilactivator) or anti-FceR antibody, positive response of 73.5%(stimulated index ¼ 29.5) or 6.49% (stimulated index ¼ 2.6)was observed.

Figure 6.

Safety profile of OTL78. A, Assessment of bodyweight change after administering 6 mmol/L (i.e., 600� of normal dose) of OTL78 to healthy balb/c mice and (B)representative H&E staining of kidney and prostate of mouse injected with 6 mmol/L of OTL78 at 14 days after injection (n ¼ 5 mice/group). C, UV spectraof OTL78 showing no aggregates, whereas the positive control (OTL38) demonstrating >50% higher aggregates at 75 mmol/L concentration in saline. D,Possible mechanism for drug-related hypersensitivity reactions due to activation of basophils and mast cells. E, Evaluation of drug-related hypersensitivity inhuman blood samples using basophil activation assay by flow cytometry. fMLP: N-formylmethionyl-leucyl-phenylalanine is a nonspecific cell activator, anti-FceR: ahigh affinity monoclonal antibody binding to IgE, CCR3 (CD193): specific biomarker on basophils, CD63 and CD203c: receptors that upregulated uponactivation of basophils, PE: phycoerythrin, background: negative control, and CD63–CD203c–PE-DY647þ/CCR3-PEþ (Q2) cell population considered as the positiveresponse for basophil activation.

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DiscussionThe objective of the study was to introduce a prostate cancer–

specific NIR agent that could assist surgeons to conduct radicalprostatectomies with negative tumor margins. Conventionalprognostic modalities such as visual inspection and palpationmay lead to BCR as a result of incomplete tumor removal or toerectile dysfunction and/or urinary incontinence due to nervedamages. Thus,we developed a PSMA-targetedNIR imaging agent(OTL78) that: (i) binds to PSMAþ tumors with high affinity andspecificity; (ii) allows use of subnanomolar concentration tovisualize small tumors; (iii) clears rapidly from PSMA-negativetissues with half-life of 17 minutes, allowing for real-time FGSwithin 1 to 2 hours of postinjection; (iv) retains tumor fluores-cence for over 48 hours, allowing visualization throughout FGS;(v) allows to accomplish negative surgical tumor margins; and(vi) has an excellent safety profile in animals. Moreover, OTL78can be readily synthesized in multigram quantities at low cost(<$500/gram) with high purity and excellent yield.

Although tumor-specific NIR dyes have not yet entered theclinic for use in radical prostatectomy, several PSMA-targeted NIRdyes are currently under preclinical development (20–25). Unfor-tunately, antibody-targeted dyes such as J591-ICG have taken�2days to obtain acceptable tumor contrast in PSMA-transfectedprostate cancer tumor xenograft model (20). The observed slowclearance from the nonmalignant tissues and longer optimalimaging time necessitate a prolonged hospital stay or multiplehospital visits, leading to higher cumulative cost. On the otherhand, NIR dyes are often nonspecifically conjugated to antibodiesusing Lys or Cys sites that lead to heterogeneous chemical entities,resulting in variable affinities, efficacies, PK, and safety profiles.Moreover, it is well established that the Cys-S-maleimide (i.e.,thio–ether) bond is unstable during the circulation and tends toundergo retro-Michael reaction (b-elimination) and oxidation,leading to release thiol and maleimide adducts that eventuallylead to poor TBR (34, 35). Therefore, production cost of theseantibody–NIR conjugates can be higher when compared withsmall molecular ligands. In contrast, small-molecule ligands(Mr >0.5 Da) penetrate solid tumors rapidly, clear from PSMA-negative tissues in < 2 hours, show high TBR, are easy to synthesis,and are stable during the synthesis and storage.

Despite all the advantages of small-molecule ligands have, thedevelopment of an NIR dye that maintains the properties of anideal fluorescent contrast agent can be challenging. Althoughinvestigators claimed to observe acceptable tumor contrast within6 hours, IR800CW-YC-27 (a small-molecule ligand-targetedIR800CW) has taken�20 hours to obtain optimal tumor imagesin PSMA-transfected prostate cancer tumor xenograft model and72 hours to clear fromnontargeted tissues (22). This will also be acause for an extended hospital stay and to higher cumulative cost.Furthermore, IR800CW-YC-27 has also demonstrated a substan-

tial amount of nonspecific fluorescence uptake in PSMA-negativetumor xenograft. On the other hand, IR800CW is an expensiveasymmetric NIR dye, which could cost $30,000/gram, whereasthe cost of S0456 is <$400/gram. Moreover, as established in theliterature, IR800CW has additional disadvantages over other NIRdyes: (i) hydrolysis of N-hydroxysuccinimide ester during thesynthesis and (ii) formation of unwanted enamine byproduct dueto replacement of 4-hydroxybenzensulfonate during the reactionwith ligand-linker-amine (i.e., in the presence of amines; ref. 36).Formation of undesired byproducts may result to perform com-plex purifications, higher production cost, and longer waitingperiod for clinical translation.

Due to its high affinity and specificity for PSMA-expressingtumors, rapid clearance from PSMA-negative healthy tissues,excellent safety profile, and amenability to synthesize in gramscale at low cost, OTL78 has proven to be a clinical candidate toyield sharp tumorboundarieswithnegative tumormarginswithin1 to 2 hours of infusion during radical prostatectomy. With itsrecent entry into IND-enabling studies,OTL78has thepotential tobecome the first PSMA-targeted NIR agent to enter into the clinicfor use in fluorescence-guided radical prostatectomy.

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

Authors' ContributionsConception and design: S.A. KularatneDevelopment of methodology: S.A. Kularatne, M. Thomas, P. Gagare,A.K. KanduluruAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Thomas, C.H. Myers, C.J. CrianAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.A. Kularatne, M. Thomas, C.H. Myers, P. GagareWriting, review, and/or revisionof themanuscript: S.A. Kularatne,M. Thomas,P. Gagare, A.K. KanduluruAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Thomas, A.K. KanduluruStudy supervision: S.A. Kularatne, M. ThomasOther (performed surgical procedures and post-surgical care of animals):C.J. CrianOther (surgical removal of the subcutaneous tumors): B.N. Cichocki

AcknowledgmentsThe authors thank Xin Liu at Purdue Drug Discovery Center for insights on

docking images. The authors also thank Dr. Tiffany Lyle at Purdue UniversityCollege of VeterinaryMedicine for insights onpathologic analysis andDr.Gert J.Breur for insights on orthotopic tumor implantations.

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.

ReceivedMarch 12, 2018; revised July 13, 2018; accepted September 5, 2018;published first September 10, 2018.

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2019;25:177-187. Published OnlineFirst September 10, 2018.Clin Cancer Res   Sumith A. Kularatne, Mini Thomas, Carrie H. Myers, et al.   Prostate CancerNear-Infrared Imaging Agent for Fluorescence-Guided Surgery of Evaluation of Novel Prostate-Specific Membrane Antigen-Targeted

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