Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas

Accepted Manuscript Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas Jarrod D. Predina, MD MTR, Andrew D. Newton, MD, Courtney Connolly, BA, Ashley Dunbar, BA, Michael Baldassari, BA, Charuhas Deshpande, MD, Edward Cantu, III, MD, Jason Stadanlick, PhD, Sumith A. Kularatne, PhD, Philip Low, PhD, Sunil Singhal, MD PII:

S1525-0016(17)30532-4

DOI:

10.1016/j.ymthe.2017.10.016

Reference:

YMTHE 4491

To appear in:

Molecular Therapy

Received Date: 5 August 2017 Accepted Date: 20 October 2017

Please cite this article as: Predina JD, Newton AD, Connolly C, Dunbar A, Baldassari M, Deshpande C, Cantu III E, Stadanlick J, Kularatne SA, Low P, Singhal S, Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas, Molecular Therapy (2017), doi: 10.1016/j.ymthe.2017.10.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C EP TE D

M AN US C

RI PT

ACCEPTED MANUSCRIPT

Identification of a Folate Receptor-Targeted Near-Infrared Molecular Contrast Agent to Localize Pulmonary Adenocarcinomas Running Title: Targeted Intraoperative Imaging for Lung Cancer

RI PT

Jarrod D. Predina, MD MTR1,*, Andrew D. Newton, MD2,3, Courtney Connolly, BA1,3, Ashley Dunbar, BA1,3, Michael Baldassari, BA1,3, Charuhas Deshpande, MD4, Edward Cantu, III, MD3,5, Jason Stadanlick, PhD1,2, Sumith A. Kularatne, PhD6, Philip Low, PhD7, Sunil Singhal, MD1,2

SC

1. Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104

M AN U

2. Division of Thoracic Surgery, Department of Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 3. Department of Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 4. Pulmonary and Mediastinal Pathology, Department of Clinical Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104

TE D

5. Division of Cardiac Surgery, Department of Surgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 6. On Target Laboratories, West Lafayette, IN 47906 7. Department of Chemistry, Purdue University, West Lafayette, IN 479067

EP

* Corresponding Author. 6 White building; 3400 Spruce St. Philadelphia, PA 19104. [email protected]. Ph: 215-662-4767. Fax: 215-615-6562.

AC C

Potential Conflicts of Interest: PL is on the Board of Directors at On Target Laboratories, producers of the study drug. SAK is the Vice President of Research and Design at On Target Laboratories. Key words: pulmonary adenocarcinoma, surgery, intraoperative imaging, folate receptor alpha Word Count (Main Document): 8509 Figures/Tables/Videos: 6/2/1

Supplemental Tables/Figures: 1/5

1

ACCEPTED MANUSCRIPT

Abstract: Non-small cell lung cancer (NSCLC) is the number one cancer killer in the United States. Despite

RI PT

attempted curative surgical resection, nearly 40% of patients succumb to recurrent disease. High recurrence rates may be partially explained by data suggesting that 20% of NSCLC patients harbor synchronous disease that is missed during resection. In this report, we describe the use of a novel

SC

folate receptor-targeted near infrared contrast agent (OTL38) to improve intraoperative localization of NSCLC during pulmonary resection. Using optical phantoms, fluorescent imaging with OTL38

M AN U

was associated with less autofluorescence and greater depth of detection compared to traditional optical contrast agents. Next, in in vitro and in vivo NSCLC models, OTL38 reliably localized NSCLC models in a folate receptor-dependent manner. Before testing intraoperative molecular imaging with OTL38 in humans, folate receptor-alpha expression was confirmed to be present in 86% of pulmonary adenocarcinomas upon histopathologic review of 100 human pulmonary resection

TE D

specimens. Lastly, in a human feasibility study, intraoperative molecular imaging with OTL38 accurately identified 100% of pulmonary adenocarcinomas and allowed for identification of

EP

additional subcentimeter neoplastic processes in 30% of subjects. This technology may enhance the surgeon’s ability to identify NSCLC during oncologic resection and potentially improve long-term

AC C

outcomes.

2

ACCEPTED MANUSCRIPT

Introduction: Non-small cell lung cancer (NSCLC) is the most common cancer killer in the United States and

RI PT

accounts for more than 160,000 deaths each year.1 During resection, limitations in both visualization and tactile feedback make localization of small pulmonary nodules and ground-glass opacities (GGOs) challenging. In fact, recent reports suggest that nearly 10-20% of NSCLC patients harbor

SC

synchronous local disease that is routinely missed by the surgeon during resection.2 These statistics may partially explain why up to 40% of NSCLC patients suffer disease recurrence within 5 years of

M AN U

oncologic resection.3

Current techniques to improve upon a surgeons’ reliance on tactile and visual methods to find pulmonary nodules include intraoperative ultrasound, radionucleotide imaging, wire localization and

TE D

intraoperative marking by bronchoscopy or CT.4 These techniques, however, have major limitations. First, they require a priori knowledge about the location of the nodule.4 Second, these methods carry morbidity and include risks of hemothorax, pneumothorax and air embolus. Finally, these approaches

EP

fail to identify synchronous disease or evaluate margin status.

AC C

In this era of miniaturization of intraoperative devices and advances in the chemistry of novel molecular contrast agents, intraoperative molecular imaging (IMI), also known as fluorescence guided surgery, has emerged as an alternative technique to localize small nodules and assess for synchronous malignancies during cancer surgery. This approach incorporates systemic delivery of optical contrast agents which accumulate in tumors and allows for real-time disease localization using calibrated lighting and camera systems. These methods have been found safe and effective for a wide range of common neoplasms including central nervous system tumors, abdominal cancers, and soft3

ACCEPTED MANUSCRIPT

tissue malignancies.5-10 In these reports, IMI has been found to improve complete resection, lymph node identification, and margin assessment.

RI PT

Our group has been focused on applying this approach to the NSCLC patient. To this end, we have initiated several Phase I trials (NCT02651246, NCT02653612, NCT01778920) involving optical contrast agents that can aide the thoracic surgeon during NSCLC resection.4, 11-13 Our initial studies

SC

with IMI for pulmonary malignancies involved systemic delivery of a non-targeted near infrared (NIR) contrast agent, indocyanine green (ICG).14 Intraoperative imaging with ICG was both safe and

M AN U

effective in detecting nodules as small as 2mm. Although excellent sensitivity and depth of detection was appreciated, specificity was lacking due to spillage from invasive nodules and non-specific

TE D

contrast uptake.

To improve specificity, we evaluated a targeted contrast agent (EC17) which bound the folate receptor-α (FRα), emitted in the visible range, and could be used safely in humans.12 FRα targeting

EP

was chosen based on literature demonstrating its high degree of upregulation in NSCLC and successes with multi-targeted antifolate drugs such as Pemetrexed.15,

16

In our earlier studies, IMI

AC C

using a targeted probe was associated with >85% accuracy in identifying pulmonary adenocarcinomas. Although this contrast agent improved accuracy of lung cancer surgeries, we identified several technical challenges including autofluorescence from benign tissue and a limited depth of detection (common with visible-range contrast agents).12

Due to these shortcomings,

investigations with EC17 were aborted.

Based on our previous experiences with ICG and EC17, our group has developed a NIR agent that 4

ACCEPTED MANUSCRIPT

selectively targets the FRα (OTL38), thus combining advantages of a NIR fluorophore (depth of detection and low autofluorescence) with the high specificity of FRα-targeting.9 In this report, we describe our translational experiences involving IMI with OTL38 in NSCLC. We provide optical and

RI PT

preclinical data demonstrating utility of molecular imaging with OTL38. We also report the results of a pilot study involving 5 human subjects who received OTL38 prior to pulmonary resection. This work suggests that IMI with OTL38 may be an effective tool to enhance the thoracic surgeon’s

SC

ability to locate malignant disease during pulmonary resection.

M AN U

Results:

Optical properties of OTL38 are superior to alternative intraoperative molecular imaging probes: The study drug, OTL38, was developed to improve upon previously evaluated agents. First, utilizing whole lung specimens from human cadaveric donors (n=5), imaging with OTL38 was compared to

TE D

three traditional optical contrast agents (two visible-range agents—FITC and PPIX; one NIR agentICG) with respect to autofluorescence and depth of fluorescence detection. As seen in Figure 1a, imaging of visible-range contrast agents, FITC and PPIX, was associated with observable levels of

EP

background autofluorescence from benign lung parenchyma (note green discoloration in Figure 1a). Autofluorescence was 14.8 AU (sd, 3.5AU) and 25.3AU (sd, 4.5AU) when utilizing FITC and PPIX

AC C

parameters, respectively (Figure 1a, 1b). In comparison, autofluorescence was significantly reduced when imaging NIR compounds; autofluorescence associated with ICG parameters was 2.7AU (sd, 2.1 AU) and with OTL38 was 3.9AU (sd, 2.7AU). These autofluorescence values were nearly five-fold lower than with FITC parameters (p<0.01) and ten-fold lower than with PPIX parameters (p<0.01); (Figure 1a, 1b). Autofluorescence was similar between ICG and OTL38 (p=0.68).

5

ACCEPTED MANUSCRIPT

Next, we focused on the second potential advantage of NIR-based imaging approaches: depth of penetration. We noted that visible-range contrast agents were associated with rapidly declining signal, and no signal was observed deeper than 1mm. In contrast, both NIR agents displayed

RI PT

fluorescence which was detected at the deepest tested level of 10mm (p=0.007) (Figure 1a, 1c). At all depths, we observed significantly improved TBRs with NIR agents as compared to visible-range agents (p<0.001). Although similar at the pleural surface, OTL38 displayed superior TBRs to ICG at

M AN U

SC

other tested depth levels (p<0.001).

Together, these data confirmed that imaging with OTL38 is associated with minimal background autofluorescence and superior depth of target detection with respect to other commonly utilized

TE D

optical contrast agents.

OTL38 binds models of human NSCLC and is proportional to FRα expression: Next, we sought to determine if OTL38 would bind human NSCLC models in vitro. Given a paucity

EP

of data involving FRα expression in human NSCLC models, we characterized expression levels within 5 commonly utilized human NSCLC cell lines (A549, L55, ChaGo-K-1, H2170, and H1264).

AC C

After immunostaining samples with the anti-FRα monoclonal antibody (mAb) 26B3.F2, we observed FRα expression in 4 NSCLC cell lines, with the most prominent patterns in A549, L55 and ChaGoK-1 (Figure 2). Moderate expression was observed in the H2170 line, and absent expression in the H1264 line.

Using FRα expression as a baseline, we next analyzed in vitro OTL38 binding to NSCLC models by flow cytometry and fluorescent microscopy. By flow cytometry, high levels of fluorescence were 6

ACCEPTED MANUSCRIPT

observed with the A549, L55 and ChaGo-K-1 models, while moderate fluorescence was observed in H2170 (Figure 2, Supplemental Table 1). Low levels of NIR fluorescence were observed in the H1264 model. By fluorescent microscopy, the strongest NIR signal was similarly observed in A549,

RI PT

L55 and Chago-K-1; moderate signal was associated with the H2170 line, and absent fluorescence within the H1264 model (Figure 2). For both flow cytometry and fluorescent microscopy, fluorescence of NSCLC models correlated to FRα staining observed by immunohistochemistry (IHC)

SC

(p=0.02).

and in a FRα-dependent manner.

M AN U

Taken together, these in vitro results suggested that OTL38 binds human NSCLC models effectively

OTL38 accumulates in FRα expressing xenografts:

TE D

To determine whether OTL38 accumulates in FRα expressing tumors in vivo, we evaluated mice bearing A549 subcutaneous xenografts. Mice were administered OTL38 via tail vein injection at varying dosing levels (n=3 per dosing level), and then imaged periodically over 8 days. In all groups,

EP

we appreciated strong background fluorescence immediately following injection (Figure 3a, 3b). At the lowest dosing levels (0.0125 mg/kg and 0.025mg/kg), fluorescence was predominantly observed

AC C

in the tumor and the kidney (due to native FRα expression in the kidney as well as the renal excretion of this drug) 17 by 8 hours.

Peak tumor-to-background fluorescence ratios (TBR) were observed at 24 hours after injection in both the 0.0125 mg/kg and 0.025mg/kg dosing levels, with higher TBRs being noted in mice receiving OTL38 at 0.025 mg/kg (3.04 vs 2.25; p=0.01). Mice receiving higher doses required a longer washout period, with clear demarcation of tumor and renal fluorescence not appearing until 7

ACCEPTED MANUSCRIPT

day 4 in mice receiving 0.050 mg/kg and day 6 in mice receiving 0.250 mg/kg. After an adequate washout period, peak TBRs within the 0.050 mg/kg and 0.250 mg/kg dosing groups were, 2.42 and 2.89, respectively. These values were similar to those observed in the animals dosed at 0.025mg/kg

of 0.025mg/kg with imaging occurring at 24 hours.

RI PT

(p=0.32). This data suggested that the most effective and efficient dosing protocol involved delivery

SC

After establishing dosing parameters, we sought to evaluate OTL38 biodistribution. Five mice bearing A549 xenografts were delivered 0.025mg/kg of OTL38. After 24 hours, mice were

M AN U

euthanized and the fluorescence of multiple organs was obtained. The highest levels of fluorescence were observed in kidneys (211; sd 17 AU) and A549 tumors (147; sd 16 AU) (Figure 3c, 3d); with low background (<15 AU) observed in in other organs. To confirm FRα specific tumor uptake, fluorescence of A549 flank xenografts were compared to NCIN87 flank xenografts in mice

TE D

undergoing the same dosing protocol. Minimal fluorescence (<15 AU) was observed in mice bearing NCIN87 tumors (Figure 3c, 3d).

EP

Given high tumor fluorescence and low signal from the benign lung (Figure 3d), we hypothesized that OTL38 could accumulate within orthotopic A549 pulmonary xenografts and display strong

AC C

fluorescence twelve days after establishing pulmonary nodules, OTL38 was delivered via retrobulbar injection at 0.025mg/kg. Upon evaluation, we appreciated strong tumor fluorescence within tumors and low background from lung parenchyma (Figure 3d); mean TBR of pulmonary nodules was 2.9 (sd, 0.6). An average of 3.3 nodules (sd, 1.3 nodules) were identified in each mouse, with a mean size of 2.4mm (sd, 0.7mm). Nodules were confirmed to be A549 xenografts by H&E and FRα IHC (Supplementary Figure 2).

8

ACCEPTED MANUSCRIPT

High levels of FRα are present in surgically resectable pulmonary adenocarcinoma: Based on encouraging preclinical data, we aimed to translate our work to humans with resectable NSCLC. We were particularly interested in pursuing patients with pulmonary adenocarcinoma;

RI PT

however, we noted that previous data describing FRα expression in pulmonary adenocarcinomas were ascertained from non-surgical cohorts 15, 18. Thus, before pursuing IMI with OTL38 in a clinical trial, we evaluated FRα expression within a purely surgical cohort. To this end, histologic samples

SC

from 100 pulmonary adenocarcinoma patients who underwent pulmonary resection were immunostained using the anti-FRα Mab 26B3.F2 and scored by a pulmonary pathologist to determine

M AN U

FRα expression.

Of the 100 pulmonary adenocarcinoma specimen analyzed, 86 demonstrated FRα expression (1+, 2+ or 3+) (Figure 4, Table 1). Twenty-four specimen (24%) were found to have a 1+ staining pattern,

TE D

35 (35%) had 2+ staining patterns, and 27(27%) had 3+ staining. Sixty-two (72%) of the 86 tumors showing FRα expression demonstrated overexpression (2+ or 3+). FRα staining heterogeneity was appreciated in 54 of the specimen with staining positivity (Figure 4). On multivariate analysis, FRα

EP

expression level was noted to be independent of several patient and histopathologic variables (patient age, patient gender, history of smoking, cancer stage, utilization of neoadjuvant chemotherapy,

AC C

smoking status and race; p>0.05) (Table 1).

These data confirmed a high level of FRα expression in patients with surgically resectable pulmonary adenocarcinoma and supported investigation of OTL38 in surgical patients. Furthermore, the ubiquitous FRα expression pattern observed within this cohort suggested that this agent may be broadly applicable to patients with resectable pulmonary adenocarcinoma.

9

ACCEPTED MANUSCRIPT

OTL38 Accumulates in FRα expressing pulmonary adenocarcinomas: To evaluate feasibility of OTL38 based IMI in humans, a pilot study involving 10 subjects with pulmonary nodules was executed. Ten subjects (n=6 female) with a mean age of 67.7 years (range, 48

RI PT

to 79 years) were enrolled after meeting inclusion criteria. All subjects had a diagnosis of a solitary pulmonary nodule, with subjects 1, 2 and 3 having a preoperative diagnosis of pulmonary adenocarcinoma. Preoperative CT and PET were obtained in all subjects and mean tumor size was

SC

determined to be 2.8 cm (range, 1.2 to 5.6cm) and mean standardized uptake value (SUV) was 5.2

M AN U

(range, 1.6 to 11.8). Complete subject and tumor characteristics are provided in Table 2.

Subjects received an average of 2.21 mg (range, 1.48 to 3.29 mg) of the study drug 3.8 hours (range, 2.9 to 5.6 hours) prior to resection and imaging (Table 2). No adverse events were observed during drug delivery, intraoperatively or postoperatively. On average, molecular imaging added 9 minutes

TE D

(range, 7 to 16 minutes) to the case duration (noted in Supplemental Figure 3).

EP

In 8 of 10 subjects (80%), in situ fluorescence was appreciated through the pleural surface with a mean TBR 3.4 of (range, 2.7 to 4.2) (Figure 5, Supplemental Video 1). Eight of 8 (100%)

AC C

fluorescent nodules were found to be pulmonary adenocarcinoma with FRα expression. The two nonfluorescent nodules were found to be a squamous cell carcinoma and a pulmonary hamartoma with absent FRα expression (Figure 5). Upon histopathologic review of specimens, OTL38 accumulation was confirmed to be primarily within tumors and in proportion to FRα expression patterns (Figure 6).

10

ACCEPTED MANUSCRIPT

In three subjects, IMI with OTL38 revealed subcentimeter nodules that were not identified on review of preoperative imaging or with traditional intraoperative techniques (finger palpation and visualization). First, in Subject 1, in addition to the previously known left upper lobe nodule, several

RI PT

synchronous subcentimeter adenocarcinomas were identified in the left lower lobe (TBR=2.4) (Supplemental Figure 4). Based on these findings, the operative plan changed from a planned left upper lobectomy to a wedge resection of each nodule. Further, identification of the synchronous

SC

nodules upstaged the subject from Stage IA (T1N0) to Stage IIIA (T4N0), and resulted in chemotherapy being offered to this subject. Next, in Subject 5, the preoperatively identified left lower

M AN U

lobe nodule was non-fluorescent (squamous cell carcinoma); however, during completion lobectomy an incidental left lower lobe nodule measuring 0.8cm was identified (TBR = 2.7) (Supplemental Figure 4). On final pathology, this nodule was found to be adenocarcinoma in situ. Finally, in Subject 9, in addition to a known right upper lobe nodule, a 0.6cm nodule was located in the right

(T4N0).

TE D

lower lobe. These findings upstaged the patient’s disease from Stage IA (T1N0) to Stage IIIA

EP

A final observation involved Subjects 3 and 6. Both subjects had preoperative imaging which revealed pulmonary nodules without PET avidity. During resection with IMI, however, both tumors

AC C

displayed high levels of fluorescence (TBR=2.7 and 2.6). Pathologic evaluation confirmed that that these nodules were indeed invasive pulmonary adenocarcinomas with FRα expression.

These findings suggest that IMI with OTL38 is safe and feasible. Additionally, these preliminary data suggest IMI may improve accurate identification of subcentimeter pulmonary adenocarcinomas that may otherwise be undetectable.

11

ACCEPTED MANUSCRIPT

Discussion: Lung cancer is the most common cancer killer in the United States, and accounts for more than 160,000 deaths each year.1 Surgery is the mainstay of therapy for early stage disease, however,

RI PT

surgery is challenging due to the issue of locating small pulmonary nodules. In this report, we provide preclinical and translational evidence that intraoperative molecular imaging utilizing the novel FRα-targeted NIR contrast agent, OTL38, can improve upon this clinical problem. We

SC

demonstrate that OTL38 displays superior optical properties when compared to commonly utilized optical imaging agents. We establish that OTL38 binds FRα expressing NSCLC models in both in

M AN U

vitro and in vivo models. Lastly, we provide histologic and clinical trial data supporting feasibility in subjects with pulmonary adenocarcinoma. This work provides an encouraging platform for future studies to investigate the role of IMI with OTL38 in patients undergoing resection for pulmonary

TE D

adenocarcinoma.

The FRα is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein that is typically found only in a limited subset of polarized epithelial cells under benign tissues, namely the kidney

EP

and choroid plexus.19 Aberrant FRα expression has been described in several malignancies, most notably ovarian cancer, invasive pulmonary adenocarcinoma and adenocarcinoma-spectrum lesions 18

This pattern of distribution has made the FRα an attractive molecular target for

AC C

in the lung.15,

diagnostic and therapeutic development. Much of this data has been drawn from cohorts which include a significant number of non-surgical candidates with advanced disease or significant comorbidity. In order to obtain FRα expression data more generalizable to surgical candidates, we reviewed

clinicopathologic data involving specimens of 100

patients

with

pulmonary

adenocarcinoma who underwent pulmonary resection. We observed FRα expression in 86% of pulmonary adenocarcinoma specimens, with overexpression (defined as 2+ or 3+ staining patterns) 12

ACCEPTED MANUSCRIPT

noted in 62% of analyzed specimens. These results are in accord with previously published data involving mixed surgical and non-surgical patients which demonstrate FRα expression in 70-80% of

RI PT

pulmonary adenocarcinomas, and overexpression in 60%.15, 18

Prior to this report, there have been no data evaluating the relationship of patient or histopathologic variables to pulmonary adenocarcinoma FRα expression. Upon review of our cohort, we found that

SC

no variable (patient age, patient gender, history of smoking, cancer stage, utilization of neoadjuvant chemotherapy, smoking status and race) significantly correlated to FRα expression. This pervasive

M AN U

FRα expression pattern suggests that OTL38 may be useful in most resectable pulmonary adenocarcinoma patients. This contrasts with other molecular targets which have been skewed towards specific patient populations. For example, the ALK fusion gene is more commonly found in younger patients without smoking histories while the EGFR mutation which is more frequently

TE D

present in females without significant smoking histories.20

Although the concept of intraoperative molecular imaging has previously been proposed, there

EP

remain no targeted drugs approved by the Food and Drug Administration (FDA) for this indication. The lack of successful translation can be explained by several short comings associated with previous

AC C

agents. For example, trials involving a prior FRα-targeted optical imaging involved the visible-range contrast agent, EC17 (folate-FITC)—although excellent specificity was noted, IMI with EC17 was associated with background autofluorescence and poor depth of target detection.12 High levels of autofluorescence associated with visible range fluorophores are a result of naturally occurring fluorophores (eg elastin, porphyrins and collagen) which have broad excitation and emission wavelengths that overlap the emission wavelengths.21 Naturally occurring NIR fluorophores are far less common, thus probes emitting in these wavelengths provide a promising alternative. The second 13

ACCEPTED MANUSCRIPT

major advantage of NIR imaging probes is an association with superior depth of detection. This is based on known electromagnetic properties which state that longer wavelengths (lower energy) have

RI PT

enhanced tissue penetration.

To overcome hurdles of autofluorescence and poor depth of detection while maintaining high levels of specificity, OTL38 incorporates folate conjugated to the NIR fluorescent dye, S0456 (chemical

SC

formula: C61H63N9Na4O17S4 (Tetrasodium salt); molecular weight: 1414.42 Da).22 Before evaluating OTL38 in controlled in vitro and in vivo models, we examined the optical properties associated with

M AN U

OTL38 and compared to those of the traditional imaging fluorophores FITC, PPIX and ICG. We observed that OTL38 and ICG (NIR compounds) were associated with significantly less autofluorescence than the visible-range contrast agents, FITC and PPIX. The second advantage of imaging with NIR molecular imaging, superior depth of detection, was similarly confirmed in this

TE D

study. Fluorescence with OTL38 was observed as deep as 1cm while signals of the other three tested agents quickly degraded with depth. In fact, signal from FITC and PPIX were not observed deeper than the most superficial locations.

Although we confirmed decreases in autofluorescence and

EP

improved depth of signal detection with NIR agents, other optical properties were not fully evaluated. For example, one would have expected more rapid decay of signal with increasing depths. The lack

AC C

of this trend is likely explained by the use of human cadaveric lungs, which display heterogeneous pathology patterns, as our optical phantom.

Although improved optical properties of OTL38 were noted, proof-of-principle data was required prior to translation to human lung cancer patients. To this end, we first sought to explore the ability of OTL38 to bind FRα expressing models of NSCLC in vitro. Interestingly, upon review of published literature, only the H2170 human NSCLC cell line had data pertaining to FRα expression.23 Given the 14

ACCEPTED MANUSCRIPT

paucity of data surrounding FRα expression in human NSCLC models, we first characterized FRα expression in 5 human NSCLC models using anti-FRα immunohistochemistry protocols as described by O’Shannessy and colleagues.15 Each of the examined NSCLC models expressed FRα, but signal

RI PT

was most robust in the A549 (bronchoalveolar carcinoma), L55 (adenocarcinoma) and ChaGo-K1(adenocarcinoma) models. Interestingly, H2170 which was previously described as FRα expressing NSCLC model, displayed only moderate expression levels. Using this information as a baseline, we

SC

confirmed that in vitro OTL38 binding to human NSCLC lines was proportional to FRα expression

M AN U

levels.

We similarly appreciated strong in vivo binding of OTL38 to FRα expressing NSCLC xenografts. After brief washout periods, high levels of tumor specific fluorescence could be appreciated within flank xenografts. We observed no increase in TBRs with increased drug dosages. These findings

TE D

were also observed when evaluating OTL38 in orthotopic xenograft models of NSCLC where we reproducibly detected nodules within the 1 to 2mm range. In addition to tumor specific fluorescence, strong fluorescence was also observed from the kidney for as long as 7 days after drug delivery in the

EP

highest dosing cohorts. This most likely the result of known levels of FRα expression within the nephron.19 Despite high background from the kidney, we hypothesized that this would not impact a

AC C

translational application to thoracic malignancies given large distances between the human kidney and thorax.

In a pilot study involving 10 human subjects receiving OTL38 (0.025mg/kg) prior to pulmonary resection, we appreciated both feasibility and utility of IMI with OTL38. Although the optimal time to imaging was 24 hours in mice, we delivered the study drug 3-6 hours prior to resection in our human trial. There were two reasons why we chose to image using these parameters. First, our past 15

ACCEPTED MANUSCRIPT

studies involving previous iterations of folate receptor-targeted agents demonstrated that human pharmacokinetics tend to be faster than those observed in mice—we believe this is probably due to a number of physiologic variables, including the fact that human tumors display significantly different

RI PT

biology than artificial xenograft models commonly used for preclinical evaluation. Second, and most importantly, we felt that imaging several hours prior to surgery would substantially increase feasibility of intraoperative molecular imaging. We appreciated TBRs near 2.0 in tumor-bearing mice

SC

as early as 3 hours after drug infusion. Data from a number of previous studies demonstrates that a TBR of 2.0 is adequate for reliable tumor identification.24 We felt a minor sacrifice in TBR was

M AN U

worth the dramatic increase in the translational practicality of this technology when utilizing these delivery parameters. By imaging only a few hours after infusion, we were able to deliver the drug in the preoperative holding area while the patient completed preoperative anesthesia tasks. If drug delivery was 24 hours prior surgery, the patients would be required to make an additional trip to the

TE D

hospital for drug delivery—this would pose a major inconvenience as patients frequently travel great distances to obtain care at tertiary care centers such as ours. Despite delivering the drug closer to resection, we observed reproducible and easily identifiable tumor fluorescence patterns which

AC C

EP

allowed for dependable tumor localization.

Although additional data is currently being generated, these preliminary results are nevertheless encouraging. First, we observed no adverse events. This safety profile echoes low toxicity observed with other optical contrast agents currently undergoing Phase I/II investigation. For example, 5aminoleuvolinic acid/PPIX25, BLZ-100, Folate-FITC12, Bevacizumab-IRDye80026 and CetuximabIRDye80010 have displayed almost exclusively Grade I/II toxicities. This provides important patient safety advantages over other localization techniques such as percutaneous wire placement, lymph

16

ACCEPTED MANUSCRIPT

node mapping and fluoroscopic localization.4 Additionally, unlike other targeted fluorophores that which are delivered up to a week prior to resection, OTL38 appears to be effective even when administered just hours prior to resection in the preoperative holding area. Finally, dye spillage was

RI PT

not noted as in our previous work with ICG.

Eight of 8 pulmonary adenocarcinomas accumulated OTL38 and displayed fluorescence. Two non-

SC

fluorescent nodules were encountered; however, these were found to be a squamous cell cancer and a

M AN U

pulmonary hamartoma with absent FRα expression. The higher than anticipated rates of fluorescence within pulmonary adenocarcinomas (100% fluorescent vs 86% FRα expressing by histologic review) may be due to random error from a small sample size. Alternatively, FRα expression rates within pulmonary adenocarcinomas may be underestimated due to a sampling error inherent to our analysis which was based on assessment of 5µm sections. These results will require further evaluation in future studies. Regardless,

TE D

successful accumulation of OTL38 within FRα-expressing nodules supports the provided preclinical data and

EP

corroborates results involving previous FRα-targeted agents such as EC17.9, 12

In addition to preoperatively identified nodules, IMI with OTL38 allowed the surgeon to identify 3

AC C

subcentimeter nodules (two adenocarcinomas and one adenocarcinoma in situ) which were undetectable during resection by both finger palpation or visualization. On review of preoperative imaging, these disease foci were similarly undetectable. As described in our results, localization of synchronous disease influenced both intraoperative planning and post-operative clinical decisions (namely adjuvant therapy). Detection of synchronous disease in 30% of our cohort is somewhat higher than previous reports suggesting that 8-9% of NSCLC patients harbor synchronous disease that is missed during minimally invasive pulmonary resection.2 Further, this high rate of localization 17

ACCEPTED MANUSCRIPT

is encouraging given reported failure rates of nodule localization during minimally invasive lobectomy which approach 60% in some series.27 As additional patients are enrolled, we hope to better understand the reproducibility of synchronous disease identification and implications to patient

RI PT

outcomes.

Although not evaluated in our series, FRα expression has also been documented in 20-40% of

SC

pulmonary squamous cell carcinomas.15, 18 The high prevalence of the FRα within the two dominant

M AN U

NSCLC histologies makes it an attractive target for NSCLC molecular probe development, particularly in comparison to other molecular targets such as the EGFR mutation and ALK fusion gene which are only found in 20-30% and less than 10% of NSCLC patients, respectively.20 These results suggest that FRα-targeted IMI may be a useful adjunct in most surgical candidates with pulmonary adenocarcinoma, and perhaps up to a third of those patients with other NSCLC variants.

TE D

For those NSCLC patients without tumors demonstrating FRα expression, pre-clinical and phase I evaluations involving alternative IMI agents targeting the EGFR mutation and the prostate specific

EP

membrane antigen (upregulated in 70% of early lung cancers 28) are underway.10, 29

AC C

Although results using OTL38 are promising, several limitations are acknowledged. First, as noted previously, OTL38 was 100% accurate in identifying pulmonary adenocarcinomas. However, this data does not provide information applicable to other pulmonary malignancies in which FRα expression is less common. Second, utilization of a NIR agent, such as OTL38, allowed for deeper detection of tumor deposits when compared to visible-range agents; however, we were unable to reliably detect lesions deeper than 3cm in our clinical experiences. This may be a result of poor tissue penetration at these depths or, alternatively, high amounts of scatter which is often associated NIR 18

ACCEPTED MANUSCRIPT

imaging parameters. Additional techniques, such as photoacoustic imaging, may provide additional approaches to detect deeper nodules and is currently being investigated in the setting of metastatic melanoma. Third, the use of this probe to identify lymph node metastasis is an exciting opportunity

RI PT

which was not evaluated in our early human experiences. This may prove challenging as lymph node constituents, namely macrophages, express the FRβ and may bind OTL38 and result in false positive

SC

signal.30 Despite these concerns, we plan to evaluate feasibility of this application in future studies.

In summary, we demonstrate that the FRα-targeted NIR optical contrast agent, OTL38, integrates

M AN U

advantages of prior IMI agents. In both in vitro and in vivo models of NSCLC, OTL38 binds flank and orthotopic NSCLC models in a FRα dependent manner. Lastly, we demonstrate feasibility and utility of IMI with OTL38 in patients with pulmonary adenocarcinoma. Given the high prevalence of the FRα in pulmonary adenocarcinomas and other NSCLC histologies, OTL38 may become a

TE D

powerful tool for the surgical oncologist. We are currently further evaluating this technology in a

Methods:

AC C

Study Drug: OTL38

EP

Phase II clinical trial (NCT02602119).

OTL38 (chemical formula: C61H63N9Na4O17S4 (Tetrasodium salt); molecular weight: 1414.42 Da) is a folate analogue conjugated to the NIR fluorescent dye, S0456 (Supplemental Figure 1). OTL38 maximally excites at a wavelength of 774-776 nm and has a peak emission of 794-796nm.31 OTL38 (>96% purity) was obtained via collaboration with Philip Low, PhD (Purdue University, West Lafayette, IN) and On Target Laboratories (West Lafayette, Indiana). OTL38 was synthesized and manufactured at Aptuit in compliance with Good Manufacturing Practices. OTL38 was stored at 19

ACCEPTED MANUSCRIPT

−20°C in vials containing 6 mg OTL38 free acid in 3 mL water. Before utilization, the frozen vials

RI PT

were thawed, vortexed, and then diluted with 0.9% NaCl, 5% dextrose or culture media.

Cell lines

The human nasopharyngeal carcinoma cell line, KB, has known high levels of FRα expression and

SC

served as a positive control in experiments.32 The human gastric cancer cell line, NCIN87, was obtained from the American Type Culture Collection (Manassas, VA, USA) and was used as a

M AN U

negative control in experiments given negligible FRα expression. A range of human lung cancer cell lines were obtained from our laboratory and utilized: L55, ChaGo-K-1, H1264, (adenocarcinoma); H2170 (squamous cell carcinoma); and A549 (bronchoalveolar carcinoma). Cell lines were maintained in vitro using media containing RPMI, 10% fetal bovine serum (FBS), 2 mmol/L

TE D

glutamine, and 5 Ag/mL penicillin/streptomycin. Cell lines were regularly tested and maintained negative for Mycoplasma spp.

EP

A surrogate human model to measure autofluorescence and depth of fluorescent signal detection To evaluate autofluorescence and depth of detection associated with various optical contrast agents, a

AC C

model was developed using fresh human lungs from transplant donors (courtesy of Edward Cantu, MD—Hospital of the University of Pennsylvania). Specimens (n=5) were obtained under a University of Pennsylvania Institutional Review Board approved protocol. In addition to OTL38, three commonly utilized optical agents were tested: 5-aminoleuvolinc acid/protoporphyrin IX (PPIX) (Sigma-Adrich®, P8293, molecular weight 562.62; λex 405nm/λem 635nm), fluorescein isothiocyanate (FITC) (Sigma-Aldrich®, 3326-32-7, molecular weight 389.38; λex 495nm/λem 519nm); and indocyanine green (ICG) (Akorn®, 17478-701-02, molecular weight 774.96; λex 805nm/λem 825nm). 20

ACCEPTED MANUSCRIPT

Individual dyes were diluted to a concentration of 1x10-5 M, then infused into a 1.7mL Eppendorf tube. Target fluorescence and background autofluorescence was analyzed using the FluoCam imaging system as previously described.12 To confirm uniform impact of Eppendorf tubes on mean

well plate (described within Supplemental Figure 5).

RI PT

fluorescence intensity, fluorescence intensity was compared to imaging using a black-bottomed 96-

SC

Eppendorf tubes were inserted into pulmonary parenchyma at varying depths from the pleural surface

M AN U

using a calibrated needle with markings. In order to image FITC, a light emitting diode (LED) emitting at 468-482nm was used for fluorophore excitation and emission was selectively captured using a band-pass filter (512-542nm). An LED emitting at 382-407nm was used to excite PPIX, and fluorescence emission was detected using 610nm long-pass filter. Finally, for OTL38 and ICG, an LED emitting between 730-750nm was used for fluorophore excitation, and 770nm long-pass filter

TE D

was utilized to select for emission signal.

EP

Detection of in vitro binding of OTL38 to NSCLC by flow cytometry and fluorescent microscopy Cell lines were cultured in 6 well plates with folate deficient RPMI media (Gibco, 1818588)

AC C

supplemented with 10% FBS, L-glutamine and Penicillin/Streptomycin for 24 hours. Cells were then incubated in media spiked with OTL38 (1µM) for 4 hours. Next, cells were then stained with an antiEPCAM (CD326) antibody conjugated to the FITC fluorophore (Biologend, San Diego, CA). OTL38 cellular binding was assessed by flow cytometry using an LSR Fortessa X-20 (BD Biosciences, San Diego, CA), specifically utilizing red laser excitation (640nm) and detection within the APC-Cy7 range (720nm to 840nm). A minimum of 10,000 events were recorded, and experiments were completed in triplicate. Samples were analyzed using FlowJo software (Ashland, OR). 21

ACCEPTED MANUSCRIPT

After flow cytometry, the remaining cells were then prepared on a slide using a CytospinTM 4 Cytocentrifuge (ThermoFisher Scientific, Waltham, MA). Slides were examined with an Olympus®

RI PT

IX51 fluorescent microscope equipped with a FITC specific filter set (Chroma®, 49012) and a NIR specific filter set (Chroma®, 49030). Microscope settings (gain, offset, and exposure time) were

M AN U

(Calgary, Canada). Analyses were completed in triplicate.

SC

maintained for all samples. Unicolor fluorescent images were merged using iVision Software

Small animal tumor models:

Female immunodeficient mice (Crl:NU(NCr)-Foxn1nu) were purchased from Charles River Laboratories. All mice were maintained in pathogen-free conditions and used for experiments at ages

TE D

8 week or older. Recognized principles of laboratory animal care (NIH publication No.85-23, revised 1985) were followed and the Animal Use Committees of the Children’s Hospital of Philadelphia and the University of Pennsylvania approved all protocols. To establish flank xenografts, 1x106 cells for

EP

were suspended in 50µL PBS mixed with 50µL Matrigel (Corning®, 354234), then injected subcutaneously into the flank of mice. Orthotopic pulmonary xenografts were established by injecting

AC C

1x106 cells (suspended in 100 PBS) via tail vein. Seven days prior to molecular imaging, mice were placed on folate deficient chow (Harlan Laboratories Inc, Indianapolis, IN).

In vivo small animal imaging: Mice bearing flank xenografts measuring 250±50 mm3 were randomized to intravenous OTL38 delivery at 5 dosage levels: 0.0mg/kg, 0.0125 mg/kg, 0.025 mg/kg, 0.050 mgk/kg and 0.25 mg/kg (n=3 per dosing level). After tail vein injection, fluorescence of tumor and background were recorded 22

ACCEPTED MANUSCRIPT

at several time points using the Pearl Trilogy In Vivo Imaging System (LiCor, Lincoln, NE). OTL38 fluorescence was obtained using the “800nm Channel” which utilizes an excitation light source of 785nm and emission detection at 820nm. Mean fluorescence of regions of interest (ROI) were

RI PT

delineated over tumor sites and compared to surrounding soft tissue (background) to create a target to background ratio (TBR).

SC

After determining optimized dosing and time parameters using flank xenografts, mice bearing orthotopic pulmonary xenografts (n=10) were intravenously administered OTL38 at a dose of

M AN U

0.025mg/kg via retrobulbar injection. Retrobulbar drug delivery was chosen as mice developed tumors at the site of tail vein injection (used for establishment of pulmonary xenografts). After 24 hours, mice were euthanized and lungs were removed and imaged on the Pearl Trilogy In Vivo

TE D

Imaging System as described. Suspicious nodules were analyzed by H&E and FRα immunostaining.

Assessing FRα expression in human NSCLC by immunohistochemistry: Under a University of Pennsylvania Institutional Review Board approved protocol, histologic

EP

specimen from 100 consecutive pulmonary adenocarcinoma resections were obtained from the Hospital University of Pennsylvania’s Lung Biobank. Samples were prepared and immuno-stained

AC C

for the FRα using the anti-FRα monoclonal antibody (Mab) 26B3.F2 (Biocare Medical, BRI4006KAA). Once stained, a certified pulmonary pathologist manually scored specimen using an established scoring system ranging from 0 to 3+ as previously described.15 Briefly, a score of 0 corresponded with absence of staining; 1+ equaled faint staining on luminal borders; 2+ equaled moderate staining on apical and sometimes lateral borders and 3+ indicated strong circumferential staining. The tumor was considered positive when more than 10% of malignant cells were positively stained. Overexpression of FRα was defined as a score of 2+ or 3+. Using a multivariate model, 23

ACCEPTED MANUSCRIPT

several patient and clinicopathologic variables were analyzed to determine correlation with FRα expression: patient age, patient gender, patient race (Caucasian, African American, other), cancer

RI PT

stage, smoking status (previous/current or never smoker), and preoperative chemotherapy.

Pilot Study of IMI with OTL38 in subjects undergoing pulmonary resection:

A pilot study of IMI with OTL38 was approved by the University of Pennsylvania Institutional

SC

Review Board. All subjects (n=10) provided informed consent and were recruited. Subjects had previously underwent CT scanning with 1 mm slice thickness that was reviewed by a specialized

M AN U

thoracic radiologist to confirm the presence of a pulmonary nodule and identify other suspicious nodules. Subjects also underwent preoperative positron-emission tomography (PET). Subjects were asked to maintain a low-folate diet for 7 days prior to resection. On the day of pulmonary resection, study participants received 0.025 mg/kg of intravenous OTL38 3 to 6 hours prior to resection. During

TE D

minimally invasive pulmonary resection, surgeons utilized white-light (also known as brightfield imaging) and finger palpation through port-site incisions to confirm the lesion in the lobe of interest. Nodules were then imaged using an optimized NIR imaging system (Iridium, Visionsense,

EP

Philadelphia, PA) consisting of an 10mm, 30o thoracoscope; an excitation laser of 785nm; and emission filters selecting for light ranging from 800nm to 835nm. Additionally, surgeons evaluated

AC C

the remainder of the thorax using white-light thoracoscopy and IMI to inspect the ipsilateral lung for additional nodules. Total time required for imaging was recorded.

All specimens underwent pathologic examination by a specialized lung pathologist. The presence of FRα expression was determined by FRα immunohistochemistry as described above. In addition, resected specimens were evaluated using a NIR microscopic scanner (Odyssey, LiCor, Lincoln, NE)

24

ACCEPTED MANUSCRIPT

to assess patterns of OTL38 accumulation.

Statistics:

RI PT

For in vitro assays and in vivo murine studies, at least 5 samples were utilized per group unless noted. Post hoc image analysis was performed to quantify the amount of fluorescence using region of interest (ROI) software within ImageJ (National Institute of Health; http://rsb.info.nih.gov/ij). A

SC

background fluorescence level was similarly obtained, and target- or tumor-to-background (TBR) was calculated. For preclinical (in vitro and murine data), results are expressed as mean (standard

M AN U

deviation) unless otherwise noted. Within our pilot study, given the small number of subjects (n=5), data are presented as mean (range) unless otherwise noted. All comparisons were made using Stata Statistical Software: Release 14 (College Station, TX: StataCorp LP). A p-value of 0.05 or less was

Acknowledgements:

TE D

considered statistically significant.

We would like to thank On Target Laboratories for providing the study drug, OTL38. SS was

EP

supported by the NIH (R01 CA193556). JDP was supported by a grant from the American Philosophical Society, the NIH (F32 CA210409) and (NCI LRP Award), and the Association for

AC C

Academic Surgery Research Grant.

Author Contributions: JDP and SS obtained data, analyzed data, interpreted data, prepared manuscript and designed studies. AN obtained data, analyzed data, interpreted data, and prepared manuscript. CC, AD, MB, CD and JS, CC, EC SAK and PL interpreted data and prepared manuscript.

25

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

26

ACCEPTED MANUSCRIPT

References:

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

RI PT

SC

5.

M AN U

4.

TE D

3.

EP

2.

Siegel, RL, Miller, KD, and Jemal, A (2017). Cancer Statistics, 2017. CA Cancer J Clin 67: 7-30. Cerfolio, RJ, and Bryant, AS (2008). Is palpation of the nonresected pulmonary lobe(s) required for patients with non-small cell lung cancer? A prospective study. J Thorac Cardiovasc Surg 135: 261-268. Kelsey, CR, Marks, LB, Hollis, D, Hubbs, JL, Ready, NE, D'Amico, TA, et al. (2009). Local recurrence after surgery for early stage lung cancer: an 11-year experience with 975 patients. Cancer 115: 5218-5227. Keating, J, and Singhal, S (2016). Novel Methods of Intraoperative Localization and Margin Assessment of Pulmonary Nodules. Semin Thorac Cardiovasc Surg 28: 127-136. Tipirneni, KE, Warram, JM, Moore, LS, Prince, AC, de Boer, E, Jani, AH, et al. (2016). Oncologic Procedures Amenable to Fluorescence-guided Surgery. Ann Surg. Tipirneni, KE, Rosenthal, EL, Moore, LS, Haskins, AD, Udayakumar, N, Jani, AH, et al. (2017). Fluorescence Imaging for Cancer Screening and Surveillance. Mol Imaging Biol. Marbacher, S, Klinger, E, Schwyzer, L, Fischer, I, Nevzati, E, Diepers, M, et al. (2014). Use of fluorescence to guide resection or biopsy of primary brain tumors and brain metastases. Neurosurg Focus 36: E10. Lee, JY, Thawani, JP, Pierce, J, Zeh, R, Martinez-Lage, M, Chanin, M, et al. (2016). Intraoperative Near-Infrared Optical Imaging Can Localize Gadolinium-Enhancing Gliomas During Surgery. Neurosurgery 79: 856-871. Hoogstins, CE, Tummers, QR, Gaarenstroom, KN, de Kroon, CD, Trimbos, JB, Bosse, T, et al. (2016). A Novel Tumor-Specific Agent for Intraoperative Near-Infrared Fluorescence Imaging: A Translational Study in Healthy Volunteers and Patients with Ovarian Cancer. Clin Cancer Res 22: 2929-2938. Rosenthal, EL, Warram, JM, de Boer, E, Chung, TK, Korb, ML, Brandwein-Gensler, M, et al. (2015). Safety and Tumor Specificity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer. Clin Cancer Res 21: 3658-3666. Keating, JJ, Kennedy, GT, and Singhal, S (2015). Identification of a subcentimeter pulmonary adenocarcinoma using intraoperative near-infrared imaging during video-assisted thoracoscopic surgery. J Thorac Cardiovasc Surg 149: e51-53. Kennedy, GT, Okusanya, OT, Keating, JJ, Heitjan, DF, Deshpande, C, Litzky, LA, et al. (2015). The Optical Biopsy: A Novel Technique for Rapid Intraoperative Diagnosis of Primary Pulmonary Adenocarcinomas. Ann Surg 262: 602-609. Okusanya, OT, DeJesus, EM, Jiang, JX, Judy, RP, Venegas, OG, Deshpande, CG, et al. (2015). Intraoperative molecular imaging can identify lung adenocarcinomas during pulmonary resection. J Thorac Cardiovasc Surg 150: 28-35 e21. Okusanya, OT, Holt, D, Heitjan, D, Deshpande, C, Venegas, O, Jiang, J, et al. (2014). Intraoperative near-infrared imaging can identify pulmonary nodules. Ann Thorac Surg 98: 1223-1230. O'Shannessy, DJ, Yu, G, Smale, R, Fu, YS, Singhal, S, Thiel, RP, et al. (2012). Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget 3: 414-425. Parker, N, Turk, MJ, Westrick, E, Lewis, JD, Low, PS, and Leamon, CP (2005). Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 338: 284-293.

AC C

1.

27

ACCEPTED MANUSCRIPT

23.

24.

25.

26.

27.

28.

29.

30. 31.

32.

RI PT

22.

SC

21.

M AN U

20.

TE D

19.

EP

18.

Low, PS, and Antony, AC (2004). Folate receptor-targeted drugs for cancer and inflammatory diseases. Adv Drug Deliv Rev 56: 1055-1058. Boogerd, LS, Boonstra, MC, Beck, AJ, Charehbili, A, Hoogstins, CE, Prevoo, HA, et al. (2016). Concordance of folate receptor-alpha expression between biopsy, primary tumor and metastasis in breast cancer and lung cancer patients. Oncotarget 7: 17442-17454. Xia, W, and Low, PS (2010). Folate-targeted therapies for cancer. J Med Chem 53: 68116824. Cagle, PT, Zhai, QJ, Murphy, L, and Low, PS (2013). Folate receptor in adenocarcinoma and squamous cell carcinoma of the lung: potential target for folate-linked therapeutic agents. Arch Pathol Lab Med 137: 241-244. Monici, M (2005). Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Annu Rev 11: 227-256. Kelderhouse, LE, Chelvam, V, Wayua, C, Mahalingam, S, Poh, S, Kularatne, SA, et al. (2013). Development of tumor-targeted near infrared probes for fluorescence guided surgery. Bioconjug Chem 24: 1075-1080. Lin, J, Spidel, JL, Maddage, CJ, Rybinski, KA, Kennedy, RP, Krauthauser, CL, et al. (2013). The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity. Cancer Biol Ther 14: 1032-1038. Predina, JD, Okusanya, O, A, DN, Low, P, and Singhal, S (2017). Standardization and Optimization of Intraoperative Molecular Imaging for Identifying Primary Pulmonary Adenocarcinomas. Mol Imaging Biol. Stummer, W, Tonn, JC, Mehdorn, HM, Nestler, U, Franz, K, Goetz, C, et al. (2011). Counterbalancing risks and gains from extended resections in malignant glioma surgery: a supplemental analysis from the randomized 5-aminolevulinic acid glioma resection study. Clinical article. J Neurosurg 114: 613-623. Koch, M, de Jong, JS, Glatz, J, Symvoulidis, P, Lamberts, LE, Adams, A, et al. (2016). Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer. Cancer Res. Suzuki, K, Nagai, K, Yoshida, J, Ohmatsu, H, Takahashi, K, Nishimura, M, et al. (1999). Video-assisted thoracoscopic surgery for small indeterminate pulmonary nodules: indications for preoperative marking. Chest 115: 563-568. Wang, HL, Wang, SS, Song, WH, Pan, Y, Yu, HP, Si, TG, et al. (2015). Expression of prostate-specific membrane antigen in lung cancer cells and tumor neovasculature endothelial cells and its clinical significance. PLoS One 10: e0125924. Kularatne, SA, Venkatesh, C, Santhapuram, HK, Wang, K, Vaitilingam, B, Henne, WA, et al. (2010). Synthesis and biological analysis of prostate-specific membrane antigen-targeted anticancer prodrugs. J Med Chem 53: 7767-7777. Shen, J, Putt, KS, Visscher, DW, Murphy, L, Cohen, C, Singhal, S, et al. (2015). Assessment of folate receptor-beta expression in human neoplastic tissues. Oncotarget 6: 14700-14709. van Dam, GM, Themelis, G, Crane, LM, Harlaar, NJ, Pleijhuis, RG, Kelder, W, et al. (2011). Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat Med 17: 1315-1319. De Jesus, E, Keating, JJ, Kularatne, SA, Jiang, J, Judy, R, Predina, J, et al. (2015). Comparison of Folate Receptor Targeted Optical Contrast Agents for Intraoperative Molecular Imaging. Int J Mol Imaging 2015: 469047.

AC C

17.

28

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

29

ACCEPTED MANUSCRIPT

Figures:

Figure 1: Optical properties of OTL38 are superior to those of FITC, PPIX and ICG. Contrast

RI PT

agents (1x10-5M) was suspended in a 1.7mL Eppendorf tube. Tubes were then inserted into pneumonectomy specimen at variable depths (0mm, 1mm, 5mm and 10mm). Representative data, which was obtained using a specimen from a deceased 72-year-old male with no smoking history. (a)

SC

Fluorescent overlay images were obtained using the FloCam imaging system. (b) Autofluorescence

M AN U

of pulmonary parenchyma was quantified under lighting conditions specific to each optical agent. (c) Target-to-background ratios (TBRs) were calculated using ROI software for each imaging agent with respect to depth from the pleural surface. Legend: ns-not significant or p>0.05; *-p<0.05; **-p<0.01;

TE D

***- p<0.001

Figure 2: OTL38 binds human NSCLC models in vitro, and is proportional to FRα expression. In vitro FRα expression and OTL38 binding potential was evaluated for several human NSCLC lines;

EP

KB (a known high FRα expressing nasopharyngeal carcinoma model) was used as positive control while NCIN87 (a gastric carcinoma with low FRα expression) was used as a negative control. Left

AC C

column: Representative flow cytometry tracings of cells after exposure to OTL38-spiked media (1µM) for 4 hours. Mean fluorescence intensity (MFI) of OTL38 exposed cells correspond to blue histogram; unstained cells were used as a baseline (red histogram). Middle column: Cells co-cultured with OTL38 were examined by fluorescent microscopy (green pseudocoloration). Cells were counterstained with an anti-EPCAM antibody conjugated to FITC fluorophore (red pseudocoloration). Right column: Representative images of cell lines that were immunostained for FRα using the anti-FRα monoclonal antibody (Mab) 26B3.F2. 30

ACCEPTED MANUSCRIPT

Figure 3: OTL38 accumulates in FRα expressing NSCLC xenografts: Mice bearing A549 flank xenografts were administered OTL38 at increasing dosing levels then imaged with the Pearl Trilogy

RI PT

in vivo Imaging System. (a) Representative images of mice at various times after intravenous drug delivery. (b) Tumor-to Background Ratios (TBRs) were obtained for each dosing level and plotted over time from drug delivery. (c) Twenty-four hours after delivery of OTL38 at 0.025 mg/kg, mice

SC

bearing A549 and NCIN87 were euthanized to determine drug biodistribution. Fluorescence of organs and tumors were obtained using the Pearl Trilogy. (d) Bar graph demonstrating fluorescence

M AN U

of flank A549, NCIN87 and kidneys are provided. (e) Mice with established orthotopic pulmonary A549 xenografts (12 days) were imaged 24 hours after receiving OTL38 at 0.025mg/kg. **-p<0.01

TE D

Figure 4: Staining Patterns and Intensities for FRα Expression in Pulmonary Adenocarcinoma. Representative staining of pulmonary adenocarcinoma specimen scored as (a) 0, no staining; (b) 1+,

EP

weak staining; (c) 2+ moderate staining; (d) 3+ strong staining.

Figure 5: OTL38 results in fluorescence in human FRα expressing pulmonary adenocarcinomas.

AC C

Representative data from first 5 subjects enrolled in a pilot study involving IMI with OTL38 (0.025 mg/kg). Approximately 4 hours after intravenous delivery, subjects underwent minimally invasive pulmonary resection (VATS). Preoperative CT (Column 1) and PET (Column 2) scans are provided. Intraoperative brightfield (Column 3) and fluorescent overlay views (Column 4) during VATS resection. H&E (Column 5) and FRα IHC (Column 6) of resected tumors.

31

ACCEPTED MANUSCRIPT

Figure 6: OTL38 accumulates within pulmonary adenocarcinomas in areas of FRα expression: Representative histopathologic analysis (Subject 3) of resected pulmonary adenocarcinoma. Wholesection images were obtained and evaluated using H&E staining, FRα IHC and a NIR microscopic The tumor (outlined in dash marks) demonstrated strong fluorescence,

RI PT

scanning (top row).

particularly in areas of FRα expression. We noted increased fluorescence in areas of strong FRα (*second row), and moderate levels in those areas with less intense expression (**-third row). Normal

SC

pulmonary parenchyma displayed negligible fluorescence (***-bottom row).

* - Area of tumor with strong FRα expression, ** - Area of tumor with moderate FRα expression,

AC C

EP

TE D

M AN U

*** - normal lung parenchyma.

32

ACCEPTED MANUSCRIPT

Tables: Table 1: Multivariate model exploring FRα expression with respect to patient/histopathologic characteristics in 100 patients who underwent resection of pulmonary adenocarcinoma.

FRα Staining by IHC 0

1+

2+

3+

n (%)

n (%)

n (%)

n (%)

Odds Ratio (95%CI)

pvalue

12 (26.7) 15 (27.3)

0.71 (0.35-1.48)

0.374

Gender Male (n=45) Female (n=55)

6 (13.3) 8 (15.6)

7 (15.6) 17 (30.9)

20 (44.4) 15 (27.3)

<50 yrs (n=18) 51-60 yrs (n=36) 61-70 yrs (n=37) >71 yrs (n=9)

1 (5.5) 7 (19.4) 5 (13.5) 1 (11.1)

2 (11.1) 7 (19.4) 14 (37.8) 1 (11.1)

7 (38.9) 16 (44.4) 11 (29.7) 1 (11.1)

8 (44.5) 6 (16.7) 7 (18.9) 6 (66.7)

0.82 (0.42-1.52)

0.531

IA (n=28) IB (n=26) IIA (n=11) IIB (n=9) IIIA (n=18) IIIB (n=8)

3 (10.7) 4 (15.4) 2 (18.2) 1 (11.1) 2 (11.1) 2 (25.0)

3 (10.7) 10 (38.5) 1 (9.1) 2 (22.2) 7 (38.9) 1 (12.5)

13 (46.4) 8 (30.8) 6 (54.6) 2 (22.2) 3 (16.7) 3 (37.5)

9 (32.1) 4 (15.4) 2 (18.0) 4 (44.4) 6 (33.3) 2 (25.0)

0.865 (0.751.39)

0.865

21 (23.8) 3 (25.0)

29 (32.9) 6 (50.0)

25 (28.4) 2 (16.7)

1.20 (0.42-3.42)

0.721

2.21 (0.42-11.69)

0.350

0.95 (0.67-1.32)

0.750

Neoadjuvant Chemotherapy

M AN U

No (n=5) Yes (n=95)

0 (0.0) 14 (14.7)

1 (20.0) 23 (24.2)

2 (40.0) 33 (34.7)

2 (40.0) 25 (26.3)

Caucasian (n=47) African American (n=37) Other (n=16)

4 (8.5) 9 (24.3)

13 (27.7) 8 (21.6)

17 (36.2) 10 (27.0)

13 (27.6) 10 (27.0)

1 (6.3)

3 (18.8)

8 (50.0)

4 (25.0)

AC C

Race

13 (14.7) 1 (8.3)

EP

No (n=88) Yes (n=12)

TE D

Stage

SC

Age

Smoking History

RI PT

Variable

33

ACCEPTED MANUSCRIPT

Table 2: Clinical and Histopathologic Characteristics of NSCLC Subjects Involved in a Pilot Study of IMI with OTL38

79

F

2

78

M

LUL

RUL

Size (cm)

SUV by PET

Stage

Total OTL38 Delivered (mg)

Time (hours)

Adverse Event

AC

2.5

8.9

IIIA*

2.58

3.3

AC

4.3

2.8

IB

2.16

3

67

F

RUL

AC

1.5

1.6

IA

4

71

M

RUL

AC

3.5

11.8

IIIA

77

F

LLL

SCC

1.7

8.7

IA

6

58

F

LUL

AC

1.2

1.7

IA

7

48

F

LLL

AC

2.0

4.3

8

72

M

RLL

Pulmonary Hamartoma

5.6

3.0

2.18

4.8

1.54

no

no

no

no

Impact of IMI with OTL38

yes/3.6

Identification of several additional subcentimeter adenocarcinomas within the Left Lower Lobe (Sup Figure 3). This finding upstaged subject from Stage IA (T1N0) to Stage IIIA (T4N0). Upstaging altered operative plan and need for postoperative adjuvant chemotherapy.

yes/4.2

None

yes/2.7

Preoperative PET scan was negative; however, IMI with OTL38 showed strong fluorescence (Supplemental Video 1). Final Pathology revealed AC with FRα expression.

yes/3.1

None Known SCC did not fluoresce (Figure 5); however, IMI identified a 0.8cm synchronous Adenocarcinoma in situ (Sup Fig 3) in the Left Lower Lobe. PET scan was negative; however, IMI with OTL38 showed strong fluorescence. Final Pathology revealed AC with FRα expression.

5.6

no

no/1.1

2.22

3.5

no

Yes/2.6

IA

2.72

2.8

no

yes/2.4

None

2.4

n/a

2.14

4.5

no

no/1.4

None

62

F

RUL

AC

3.4

6.1

IIIA*

1.48

3.0

no

yes/2.7

Identification of additional 0.6cm adenocarcinoma within the Right Lower Lobe. This finding upstaged subject from Stage IA (T1N0) to Stage IIIA (T4N0). Upstaging impacted operative plan and postoperative adjuvant therapy course.

10

65

M

LUL

AC

2.5

4.1

IIA

3.29

2.9

no

yes/3.1

None

AC C

9

EP

TE D

5

5.0

1.79

Fluorescent/ TBR

RI PT

1

Histology

SC

Gender Location Age (years)

M AN U

ID

* subject upstaged after additional nodules identified with use of IMI SUV-Standardized Uptake Value LUL-Left Upper Lobe, RUL-Right Upper Lobe, LUL-Left Upper Lobe, LLL-Left Lower Lobe, AC-Adenocarcinoma, SCCSquamous Cell Carcinoma

34

ACCEPTED MANUSCRIPT

Video 1: Real-time intraoperative molecular imaging with OTL38 in a human subject with

RI PT

pulmonary nodule (Subject 3): Computed tomography demonstrating 1.5cm right upper lobe nodule. During resection, high levels of fluorescence were observed within the tumor. Upon final histopathologic evaluation, the nodule was confirmed to be a pulmonary adenocarcinoma

AC C

EP

TE D

M AN U

SC

with FRα expression (See Figure 6 for full analysis).

35

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Recently published in Molecular Therapy, Predina et al. (2017) explore folate receptor-targeted intraoperative molecular imaging in preclinical models and in humans with non-small cell lung cancer.