Evaluation of Novel Prostate-Specific Membrane Antigen-Targeted Near-Infrared Imaging Agent for Fluorescence-Guided Surgery of Prostate Cancer

Prostate cancer continues to present significant medical challenges affecting a sizable portion of the male population. According 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 undergoing 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 posterolateral 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 posterolateral 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 prostatectomies in the United States are performed robotically) due to its minimally invasive and fast recovery process (1–2 days) when compared with open surgery (10–12 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, better methods for assessing negative tumor margins and affected lymph nodes are needed.

In response to this unmet clinical demand, fluorescence-guided surgery (FGS) has emerged as a technique that uses fluorescence to highlight cancerous cells and guide surgeons to resect tumors in real time. Currently, while the field is still in its infancy, the industry is slowly developing better dyes that selectively accumulate in prostate cancer with improved tumor-to-back ground ratios (TBRs). FDA-approved indocyanine green (ICG) has been used in prostatectomy to detect prostate cancer tissues, lymph nodes, and vascularization of prostate (11). However, ICG has shown significant limitations with respect to sensitivity, specificity, poor TBR, and higher liver as well as GI tract uptake due to the non-targeted nature of the molecule (12, 13). In order to overcome deficiencies of nontargeted NIR dyes, tumor-specific NIR agents that target biomarkers solely expressed on cancer cells have been evaluated in preclinical stages. One example is prostate-specific membrane antigen (PSMA) that is overexpressed on prostate cancer cells in >90% of the prostate cancer patient population (14). Examination of postprostatectomy specimens has shown that the expression level of PSMA correlated not only with tumor grade, pathologic stage, PSA level, and aneuploidy but also with BCR (15). PSMA is also expressed in neovasculature of solid tumors developed in organs such as liver, lung, breast, colon, renal, brain, sarcoma, gastric, and oral (16, 17). It allows internalization of PSMA-targeted agents into an endosomal compartment, thereby maneuvering PSMA as an excellent biomarker for use in FGS (18). Therefore, antibodies or small-molecule ligands-targeted NIR agents that are specific for PSMA are being currently evaluated in the preclinical research stages (19–26). Because each of these molecules has their own limitations, we developed a PSMA-targeted NIR agent (referred to herein as OTL78) with superior optical, pharmacokinetic (PK), and biological properties for use in FGS. In this paper, we describe the synthesis and characterization, optical properties, preclinical evaluation in cancer cell in culture and in subcutaneous and orthotopic tumor models, accomplishment of negative surgical tumor margins using FGS, and PK properties of OTL78. We then provide preclinical evidence of its remarkable safety profile in mice.

For OTL78 relative affinity (IC₅₀), 22Rv1 or PC3 cells were plated into a T75 flask and allowed to form a monolayer over 48 hours. After trypsin digestion, released cells were transferred into centrifuge tubes (1 × 10⁶ cells/tube) and centrifuged. Spent medium in each tube was replaced with 100 nmol/L DUPA-FITC in the presence of increasing concentration (0.001 nmol/L–10 μmol/L) of OTL78 in fresh medium (0.5 mL). After incubating for 30 minutes at 4°C, cells were rinsed with culture medium (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 cell bound fluorescence was quantified using a flow cytometer. The relative affinities were calculated using a plot of percent cell bound fluorescence versus the log concentration of OTL78 using GraphPad Prism 6.

For OTL78 binding affinity, 22Rv1 or PC3 cells were seeded into 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 replaced with fresh medium containing increasing concentration of OTL78 and incubated for 30 minutes at 4°C. After rinsing with fresh medium (2 × 1.0 mL) and saline (1 × 1.0 mL), cells were lysed with 1% SDS in saline (1.0 mL), and cell bound fluorescence was analyzed using a fluorometer (Cary Eclipse, Agilent Technologies). The binding affinity (K_d) was calculated using a plot of percent cell bound fluorescence versus concentration using GraphPad Prism 6.

22Rv1, LNCaP, or PC3 cells (50,000 cells/well in 1 mL) were seeded into poly-d-lysine microwell Petri dishes and allowed cells to form monolayers over 12 hours. Spent medium was replaced with fresh medium containing OTL78 (100 nmol/L), and cells were incubated for 1 hour at 37°C or 4°C. After rinsing with fresh medium (2 × 1.0 mL) and saline (1 × 1.0 mL), fluorescence images were acquired using an epimicroscopy.

Seven-week-old male nu/nu mice were inoculated subcutaneously with 5.0 × 10⁶ 22Rv1, LNCaP, PC3, or A549 cells/mouse in 50% high concentrated (HC) matrigel with RPMI1640 medium on the shoulder. Growth of the tumors was measured in perpendicular directions every 2 days using a caliper (body weights were monitored on the same schedule), and the volumes of the tumors were calculated as 0.5 × L × W² (L = longest axis and W = axis perpendicular to L in millimeters). Once tumors reached approximately 300 to 400 mm³ in volume, animals (3–5 mice/group) were intravenously injected with appropriate dose of OTL78 in saline.

For orthotopic tumors, 2 × 10⁵ 22Rv1 cells/mouse in 10% HC matrigel with RPMI1640 medium were surgically implanted in the prostate of 7-week-old male SCID mice. Briefly, 7-week-old male SCID mice were given 1% to 5% isoflurane for anesthesia and subcutaneous injection of 5 mg/kg meloxicam preoperatively for analgesia. The mice were placed dorsal side up and washed above the prostate with a chlorhexidine scrub to ensure a sterile area for incision. After an insertion was made using scalpel through the skin, the peritoneal lining was lifted to make a small incision using a scissor and widened using forceps. Dorsal lobes were exteriorized and gently stabilized with a wet (PBS) cotton swab. 22Rv1 cells (in 10 μL of 10% HC-matrigel) were injected to the prostate using a 28-gauge needle. After placing the prostate back into the peritoneum, the abdominal wall was sutured, the body wall was closed using 3–0 or 4–0 vicryl and the skin was closed using staples. Animals were monitored until they were used for the studies. After 1 month, the animals were administered with OTL78 (10 nmol/L in 100 μL saline per mouse), euthanized after 2 hours by CO₂ asphyxiation, and imaged using the AMI image system.

For whole-body imaging and biodistribution studies, animals were euthanized after 2 hours of administration of OTL78 by CO₂ asphyxiation. For time-dependent studies, animals were imaged under anesthesia using isoflurane. Imaging experiments were then performed using IVIS or AMI image systems. Following whole-body imaging, animals were dissected and selected tissues were analyzed for fluorescence activity using IVIS or AMI image system and region of interest (ROI) of the tissues were calculated using Living Image 4.0 software or AMIView Image Analysis Software.

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

Seven-week-old male nu/nu mice were inoculated subcutaneously with 5.0 × 10⁶ 22Rv1 cells/mouse in 50% HC matrigel with RPMI1640 medium on the shoulder. Growth of the tumors was measured as previously described. After 1 month, the animals were mixed and divided into two groups (n = 5 mice/group). Two hours after administering OTL78 (10 nmol in 100 μL saline per mouse), animals were given 1% to 5% isoflurane for anesthesia and imaged using the AMI image system. After an insertion was made using scalpel through the skin, surgical removal of the tumors was performed either following conventional technique (e.g., visualization under white light or palpation) or with the aid of fluorescence (FGS: debulking of visible tumors under conventional method followed by resection of residual fluorescence tissues under image-guided method). After the surgery, the skin was closed using staples and imaged the mice using AMI image system. After imaging, the residual fluorescent tissues from selected mice of the conventional surgery group and tissues samples from the tumor beds of selected mice of the FGS group were submitted for pathologic (IHC) analysis. Response to surgical treatment was monitored for over 30 days by imaging using the AMI image system 2 hours after injecting OTL78 (10 nmol/mouse) and by measuring the growth of the tumor volume using a caliper. Any animals with tumor volume ≥1,000 mm³ were euthanized. Tumor-free survival of the mice was documented as a percentage of survival versus time using GraphPad Prism 6. IHC studies were done as explained in the Safety studies in Supplementary Data and Methods.

For serum clearance, 10 nmol/L of OTL78 was administered to male nude mice (n = 3 mice) as a single bolus intravenous injection. Blood was collected at regular intervals (0–90 minutes), and serum-bound OTL78 was quantified by measuring the fluorescence using IVIS imager. The half-life of OTL78 was calculated as a percentage of serum-bound fluorescence versus time using GraphPad Prism 6.

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

Note: Procedures for in vitro binding of DUPA-FITC, human serum binding studies, safety study, and tolerability studies can be found in the Supplementary Data and Methods.

In an effort to improve limitations in current clinical practice of radical prostatectomy and tumor-specific imaging agents that are in the preclinical stages, OTL78 was assembled using: (i) a high-affinity PSMA-targeting ligand (coined DUPA; refs. 19, 26), (ii) a rationally designed 14 atoms long polyethylene glycol–dipeptide linker, and (iii) an inexpensive NIR dye (<$400/g) named S0456 (see Supplementary Fig. S1A for the chemical structure). The dipeptide consisting of phenylalanine-tyrosine was designed to fit to the contours and chemistry of the tunnel accessing the binding pocket of the PSMA protein (27). Upon conjugation of DUPA–PEG–dipeptide to S0456, the dipeptide not only improved the binding affinity of OTL78 but also enhanced the fluorescence of S0456 by ≥2 at the same concentration (see Supplementary Fig. S1B showing excitation and emission spectra of OTL78 at 1 μmol/L and S0456 at 1 μmol/L in 1 mL of PBS obtained using fluorometer). Synthesis of OTL78 was conducted as shown in the Supplementary Scheme 1 by following procedure in the Supplementary Materials and Methods. OTL78 was obtained as a dark green solid with >99% purity at 774 nm wavelengths and with ∼82% yield. It was characterized using ¹H- and ¹³C-NMR, HPLC, and HRMS (Supplementary Figs. S2 and S3).

In an effort to evaluate the specificity of OTL78 for prostate cancer, PSMA expression in LNCaP, 22Rv1, PC3 (three human prostate cancer cell lines), and A549 (a human alveolar basal epithelial carcinoma cell line), as a negative control, was first examined by flow cytometry. PSMA expression was highest in LNCaP followed by 22Rv1 and negligible in PC3 and A549 (Supplementary Fig. S1C). These results agreed with the number of PSMA molecules per LNCaP, 22Rv1, and PC3 cells reported by Wang and colleagues (28). Due to moderate PSMA expression levels and better tumorigenic capacity with low necrosis, 22Rv1 was selected as the primary cell line to characterize OTL78.

The affinity of OTL78 for PSMA was first screened by competing with DUPA-FITC. The absolute binding affinity (K_d) and specificity of DUPA-FITC for PSMA (K_d = 6 nmol/L; Supplementary Fig. S1D) was first established using PSMA⁺22Rv1 and PSMA-negative PC3 cells as described in the SI Materials and Methods. OTL78 was able to compete with DUPA-FITC for PSMA on 22Rv1 cells with IC₅₀ of 7 nmol/L (Supplementary Fig. S1E). The affinity and specificity of OTL78 was then evaluated by incubating increasing concentrations of OTL78 with either 22Rv1 or PC3 cells and analyzing for cell bound fluorescence by fluorometer. OTL78 was able to bind to PSMA on 22Rv1 cells with very high affinity (K_d = 4.7 nmol/L), whereas it did not bind to PSMA-negative PC3 cells, confirming specificity of OTL78 to PSMA (Fig. 1B).

PSMA-mediated internalization of OTL78 was next evaluated by incubating OTL78 with 22Rv1 and PC3 cells. Analysis of fluorescence microscopy images indicates that OTL78 was able to efficiently label 22Rv1 and LNCaP cells [Fig. 1C (i and ii)] but not PC3 cells [Fig. 1C (iii and vi)], indicating PSMA-mediated uptake of OTL78. Fluorescence was detected throughout the cytoplasm of 22Rv1 and LNCaP cells at 37°C. Moreover, we also observed that OTL78 is highly concentrated and entrapped in the certain regions of 22Rv1 and LNCaP cells. Labeling of 22Rv1 and LNCaP cells with OTL78 in the presence a nuclear staining dye (DAPI) at 4°C was also conducted to decrease the endocytosis and recycling of PSMA [Supplementary Fig. S1F (i and ii)]. Epifluorescence images from this study indicated that OTL78 binds to PSMA on the cell surface. Therefore, we assume that OTL78 first binds to PSMA on the cell surface and then it undergoes receptor-mediated endocytosis. We further assume that OTL78 is entrapped in the acidic endosomes within prostate cancer cells.

The ability of OTL78-mediated imaging of prostate cancer was next established by conducting a series of experiments in mouse models. First, the optimal dose for tumor imaging was determined by administering increasing concentrations of OTL78 (0.3–120 nmol/mouse) to mice bearing 22Rv1 tumor xenografts followed by ex vivo tissue biodistribution analysis. The IVIS image analysis obtained at 2-hour time point indicated that OTL78 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 by administering 10 nmol/L of OTL78 to mice bearing subcutaneous 22Rv1, LNCaP, PC3, or A549 tumor xenografts followed by conducting whole-body imaging and ex vivo tissue biodistribution using either IVIS or AMI image systems. Both studies demonstrated that OTL78 accumulated predominantly in PSMA-expressing 22Rv1 (Fig. 2A–D and Fig. 3A–D) and LNCaP (Fig. 3B–E) tumors, with no substantial fluorescence activity in other tissues except kidneys. Although tumor accumulated fluorescence was not seen in PC3 and A549 tumors at higher threshold (Fig. 2B and C and E and F), uptake of OTL78 was observed in both tumors at lower threshold (Fig. 2E and F, bottom). Although fluorescence intensities of PC3 and A549 tumors were ∼6-fold less compared with 22Rv1 tumors (Fig. 2A–D), fluorescence accumulation in PC3 and A549 tumors was higher than rest of the tissues except kidneys and skin (Supplementary Fig. S5A–S5B). We therefore assume that the observed fluorescence in PC3 and A549 tumors may be due to accumulation of OTL78 via PSMA in the neovasculature of PC3 and A549 solid tumors. This further suggests that OTL78 will be able to detect tumors with low PSMA expression levels. OTL78 also had a significant kidney uptake due to high PSMA expression in murine kidneys and clearance of OTL78 through the kidneys. More importantly, fluorescence in the kidneys was clearly visible in whole-body images collected from AMI imager demonstrating penetrating ability of OTL78 to locate buried PSMA⁺ tissues. We assume that observed skin uptake may be due to nonspecific uptake of S0456 moiety of the OTL78 molecule. Although skin uptake clears within 4 to 5 hours, skin will not be interfered with open or robotic surgery because the camera will be directly focusing to the prostate in both techniques.

We then examined the ability of OTL78 to detect primary tumors in the prostate and regional metastasis in seminal vesicles. In that case, 22Rv1 cells were surgically implanted in the prostate of SCID mice as described in the Supplementary Materials and Methods. Once tumors grew, the animals were imaged using AMI image system 2 hours after administration of 10 nmol/L of OTL78. Orthotopic imaging studies also demonstrated that OTL78 mainly accumulated in prostate tumors with no fluorescence observed in other tissues except kidneys (Fig. 3C–F and Supplementary Fig. S5C and S5D). Moreover, OTL78 was able to detect local regional metastasis in seminal vesicles in the presence of primary tumor (Fig. 3G and Supplementary Fig. S5D), indicating ability of OTL78 to locate tumors and lymph nodes that are buried under the prostate.

Following biodistribution studies, specificity of OTL78 for PSMA was quantitated by calculating TBR. In both subcutaneous and orthotopic tumor models, OTL78 displayed excellent TBR (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 model compared with subcutaneous model may be due to: (a) higher accumulation 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 tissue boundaries was evaluated using ImageJ software analysis. The whole-body image of mice injected with 10 nmol/L of OTL78 was acquired as fluorescence in a gray scale and either a line or box (Supplementary Fig. S6B) was drawn to quantitate the fluorescence to be defined in the tumor boundaries. As shown in Supplementary Fig. S6C and S6D, OTL78 was able to define tumor boundaries precisely with a TBR of 5:1, suggesting its capability to guide surgeons to accurately detect the tumor margins (acceptable TBR for image-guided surgery is considered to be >1.5; ref. 29).

The ability of OTL78 to guide surgeons to excise all cancerous tissues with negative tumor margins was next investigated by performing image-guided surgery in tumor-bearing mice. Briefly, 10 nmol/L of OTL78 was administered into mice bearing 22Rv1 tumor xenografts, and a comparative study was conducted by performing surgeries under conventional (e.g., visualization under white light or palpation) or fluorescence-guided technology (i.e., debulking of visible tumors under conventional method followed by resection of residual fluorescence tissues under image-guided method) at 2-hour time point. Preoperative fluorescence images of tumor-bearing mice demonstrated that OTL78 was able to localize 22Rv1 tumors with high contrast within 2 hours (Fig. 4A, first column; Supplementary Fig. S7). Postoperative fluorescence imagers indicated presence of residual fluorescence in the tumor bed of the conventional cohorts, whereas no significant fluorescence was observed in the FGS cohorts (Fig. 4A, middle column; Supplementary Fig. S7). Pathologic analysis of residual fluorescent tissues from the conventional surgery confirmed that the fluorescence is due to cancer cells (Fig. 4A and B, middle). More importantly, no residual tumors were identified in tissues from tumor margin/bed from the FGS cohorts (Fig. 4B, right column). Following surgeries, BCR of the cancer was assessed by monitoring animals for over a month using fluorescence imaging. As anticipated, only the conventional surgery cohort had recurrence at the primary tumor site, and no sign of BCR was observed in the FGS cohort during the study (Fig. 4A and Supplementary Fig. S7). As shown in the survival curve (Fig. 4C), the FGS cohorts survived during the study with no BCR, whereas all mice in the conventional surgery group had to be euthanized within 3 weeks. Although the observed BCR rate is higher than the reported values for human and mice (6, 30), this proof-of-concept study highlights the importance of excising all cancerous tissues with negative tumor margins to improve the quality of life and life expectancy of the patient.

Having demonstrated in vivo specificity, we then evaluated the PK profile of OTL78 in animal models as described in the Materials and Methods. OTL78 was able to generate excellent tumor images within 1 hour of postinjection, with TBR ratios remaining excellent throughout the experiment time of 48 hours (Fig. 5A and Supplementary Fig. S8). Moreover, time-dependent tumor clearance studies demonstrated that ∼50% of fluorescence was retained in the tumor at 48 hours after injection (Fig. 5B), suggesting that OTL78 fluorescence was indeed entrapped in the tumor. OTL78 was excreted mainly through the kidneys, and fluorescence of the kidneys became negligible after 8 hours (Fig. 5B). We also elected to monitor the skin clearance due to observed high fluorescence in the skin at the 2-hour time point. Although skin uptake may not affect the outcome of open or robotic-assisted FGS, as shown in Fig. 5B, OTL78 cleared from the mice skin between 4 and 5 hours of postinjection.

Finally, serum clearance of OTL78 was examined by injecting 10 nmol/mouse intravenously, collecting blood samples at regular intervals (0–90 minutes), and measuring serum-bound OTL78 using AMI imager. OTL78 reached a peak concentration in the circulation ∼10 minutes after injection and cleared with a serum half-life of ∼17 minutes (Fig. 5C). Human serum binding studies conducted using LC/MS analysis also indicated that OTL78 has very low affinity (32%) for human serum proteins (Supplementary Fig. S9). Taken together, OTL78 clears rapidly from nonmalignant tissues to allow tumor visualization within 1 to 2 hours of administration, avoiding the requirement for a prolonged hospital stay.

Motivated by the specificity and PK properties described above, the safety profile of OTL78 was then evaluated using ex vivo and in vivo models. The acute maximum tolerance dose of OTL78 was initially determined by injecting 6 μmol/mouse (600× of normal dose) to healthy Balb/c mice. Body weights and clinical observations were monitored during the study, and histopathologic analysis on selected tissues was then conducted on day 14 of postinjection. The animals were active after administration of OTL78 and behaved normally throughout the study. As shown in Fig. 6A, body weights over the course of the study remained unchanged (<5% increase), suggesting that OTL78 is not grossly toxic to the animals. Moreover, no obvious pathologic changers were detected in hematoxylin and eosin (H&E) staining conducted on any of the tissues (Fig. 6B and Supplementary Fig. S10). No noticeable toxicities were also noticed in clinical pathology analysis on blood samples collected from mice injected with OTL78 (6 μmol/mouse).

Possible OTL78-related hypersensitivity in human was next examined using basophil activation assay. Drug-related hypersensitivity occurs mainly due to immune response caused by crosslinking of immunoglobulin E (IgE) expressed on mast cells and basophils (Fig. 6D) resulting in activation and subsequent degranulation to release vasoactive amines, prostaglandins, and cytokines (31). Because crosslinking of IgE can be due to aggregates, concentration-dependent UV spectrometric studies were conducted to determine higher order aggregates of OTL78. As shown in Fig. 6C, there were no noticeable higher order aggregates observed with OTL78, whereas the positive control (i.e., OTL38; ref. 32) exhibited concentration-dependent aggregation peak at λ_max ∼700 nm at 75 μmol/L in saline.

Because basophils are readily available from blood samples when compared with tissue-resident mast cells, we then evaluated drug-related hypersensitivity due to monomer and low order aggregates (if present) of OTL78 using basophil activation test in human blood samples as described in the Materials and Methods section. Briefly, 75 μmol/L of OTL78 was first added to a tube containing whole blood from donors and stimulating buffer. After labeling with anti-CCR3 (CD193)–phycoerythrin and anti-CD63–CD203c–PE-DY647, the percentage of activated basophils was quantitated using flow-cytometric analysis (33). As shown in Fig. 6E and Supplementary Table S1, no obvious differences in percentage activated basophils were seen between the OTL78-treated sample and negative control, resulting in a stimulated index of 1, whereas stimulated index is defined as the ratio of percentage of basophil activation by the allergen: percentage of basophil activation by background and stimulated index ≥ 2 considered as positive response (33). However, when similar assays were conducted using fMLP (a nonspecific basophil activator) or anti-FcϵR antibody, positive response of 73.5% (stimulated index = 29.5) or 6.49% (stimulated index = 2.6) was observed.

The objective of the study was to introduce a prostate cancer–specific NIR agent that could assist surgeons to conduct radical prostatectomies with negative tumor margins. Conventional prognostic modalities such as visual inspection and palpation may lead to BCR as a result of incomplete tumor removal or to erectile dysfunction and/or urinary incontinence due to nerve damages. Thus, we developed a PSMA-targeted NIR imaging agent (OTL78) that: (i) binds to PSMA⁺ tumors with high affinity and specificity; (ii) allows use of subnanomolar concentration to visualize small tumors; (iii) clears rapidly from PSMA-negative tissues with half-life of 17 minutes, allowing for real-time FGS within 1 to 2 hours of postinjection; (iv) retains tumor fluorescence 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, OTL78 can 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 the clinic for use in radical prostatectomy, several PSMA-targeted NIR dyes are currently under preclinical development (20–25). Unfortunately, antibody-targeted dyes such as J591-ICG have taken ∼2 days to obtain acceptable tumor contrast in PSMA-transfected prostate cancer tumor xenograft model (20). The observed slow clearance from the nonmalignant tissues and longer optimal imaging time necessitate a prolonged hospital stay or multiple hospital visits, leading to higher cumulative cost. On the other hand, NIR dyes are often nonspecifically conjugated to antibodies using 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 to undergo retro-Michael reaction (β-elimination) and oxidation, leading to release thiol and maleimide adducts that eventually lead to poor TBR (34, 35). Therefore, production cost of these antibody–NIR conjugates can be higher when compared with small 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, the development of an NIR dye that maintains the properties of an ideal fluorescent contrast agent can be challenging. Although investigators claimed to observe acceptable tumor contrast within 6 hours, IR800CW-YC-27 (a small-molecule ligand-targeted IR800CW) has taken ∼20 hours to obtain optimal tumor images in PSMA-transfected prostate cancer tumor xenograft model and 72 hours to clear from nontargeted tissues (22). This will also be a cause for an extended hospital stay and to higher cumulative cost. Furthermore, IR800CW-YC-27 has also demonstrated a substantial amount of nonspecific fluorescence uptake in PSMA-negative tumor xenograft. On the other hand, IR800CW is an expensive asymmetric NIR dye, which could cost $30,000/gram, whereas the cost of S0456 is <$400/gram. Moreover, as established in the literature, IR800CW has additional disadvantages over other NIR dyes: (i) hydrolysis of N-hydroxysuccinimide ester during the synthesis and (ii) formation of unwanted enamine byproduct due to replacement of 4-hydroxybenzensulfonate during the reaction with ligand-linker-amine (i.e., in the presence of amines; ref. 36). Formation of undesired byproducts may result to perform complex purifications, higher production cost, and longer waiting period for clinical translation.

Due to its high affinity and specificity for PSMA-expressing tumors, rapid clearance from PSMA-negative healthy tissues, excellent safety profile, and amenability to synthesize in gram scale at low cost, OTL78 has proven to be a clinical candidate to yield sharp tumor boundaries with negative tumor margins within 1 to 2 hours of infusion during radical prostatectomy. With its recent entry into IND-enabling studies, OTL78 has the potential to become the first PSMA-targeted NIR agent to enter into the clinic for use in fluorescence-guided radical prostatectomy.

No potential conflicts of interest were disclosed.

Conception and design: S.A. Kularatne

Development of methodology: S.A. Kularatne, M. Thomas, P. Gagare, A.K. Kanduluru

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Thomas, C.H. Myers, C.J. Crian

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.A. Kularatne, M. Thomas, C.H. Myers, P. Gagare

Writing, review, and/or revision of the manuscript: S.A. Kularatne, M. Thomas, P. Gagare, A.K. Kanduluru

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Thomas, A.K. Kanduluru

Study supervision: S.A. Kularatne, M. Thomas

Other (performed surgical procedures and post-surgical care of animals): C.J. Crian

Other (surgical removal of the subcutaneous tumors): B.N. Cichocki

The authors thank Xin Liu at Purdue Drug Discovery Center for insights on docking images. The authors also thank Dr. Tiffany Lyle at Purdue University College of Veterinary Medicine for insights on pathologic analysis and Dr. Gert J. Breur for insights on orthotopic tumor implantations.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.