Prostate Specific Membrane Antigen is a Potential Anti-Angiogenic Target in Adrenocortical Carcinoma

Michael JP Crowley, MSc,1,2*, Theresa Scognamiglio, MD3*, Yi-Fang Liu, MD,3, David A Kleiman, MD,1, Toni Beninato, MD,1, Anna Aronova, MD,1, He Liu, MD,4, Yuliya S Jhanwar, MD,5, Ana Molina, MD,6, Scott T Tagawa, MD,4,6, Neil H Bander4, MD, Rasa Zarnegar1, MD, Olivier Elemento2,7, PhD, and Thomas J Fahey III1, MD

1New York Presbyterian Hospital - Weill Cornell Medical College, Department of Surgery; 2Weill Cornell Medical College- Institute for Computational Biomedicine, Department of Physiology and Biophysics; 3New York Presbyterian Hospital - Weill Cornell Medical College, Department of Pathology and Laboratory Medicine; 4New York Presbyterian Hospital -Weill Cornell Medical College, Department of Urology; 5New York Presbyterian Hospital -Weill Cornell Medical College, Department of Nuclear Medicine; 6New York Presbyterian Hospital -Weill Cornell Medical College, Department of Medicine; 7 HRH Prince Alwaleed Bin Talal Bin Abdulaziz Al-Saud Institute for Computational Biomedicine, Weill Cornell Medical College

Context: Adrenocortical carcinoma (ACC) is a rare tumor type with a poor prognosis and few therapeutic options.

Objective: Assess prostate specific membrane antigen (PSMA) expression as a potential novel therapeutic target for ACC.

Design: Expression of PSMA was evaluated in benign and malignant adrenal tumors and one patient with metastatic ACC.

Setting: This study took place at a tertiary referral center.

Patients: Fifty adrenal samples were evaluated, including: 16 normal adrenal glands (NML), 16 adrenocortical adenomas (AA), 15 primary ACC and 3 ACC metastases.

Main Outcome Measures: Demographics, PSMA expression levels via qPCR and IHC and whole body PET-CT standardized uptake values for one patient.

Results: qPCR demonstrated an elevated level of PSMA in ACC relative to all benign tissues (p<0.05). IHC localized PSMA expression to the neovasculature of ACC and confirmed overexpres- sion of PSMA in ACC relative to benign tissues both in intensity and percentage of vessels stained (78% of ACC, 0% of NML and 3.27% of adenoma- associated neovasculature, p<0.001). Those with >25% PSMA positive vessels were 33 times more likely to be malignant than benign (Odds ratio, p<0.001). Whole body PET-CT imaging showed targeting of anti-PSMA Zr89J591 to 5/5 of the patient’s multiple lung masses with an average measurement of 3.49±1.86cm and a SUV of 1.4+0.65 relative to blood pool at 0.8 SUV.

Conclusions: PSMA is significantly overexpressed in ACC neovasculature when compared to normal and benign adrenal tumors. PSMA expression can be used to image ACC metastases in vivo and may be considered as a potential diagnostic and therapeutic target in ACC.

doi: 10.1210/jc.2015-4021

Abbreviations:

THE JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM

JCEM

EARLY RELEASE:

ENDOCRINE

SOCIETY

A drenocortical carcinoma (ACC) is an uncommon and aggressive cancer arising in the cortex of the adrenal gland. The incidence of ACC has been estimated at 0.7- 2 cases per 1 million persons accounting for 0.02% of all cases of carcinoma per year (1, 2, 3). The 5-year survival rate for patients with stage I-III disease is 55%-80%, and 13% for stage IV. Furthermore, even with surgical resec- tion 75%-85% of patients will experience a relapse (1, 2, 3). Currently, most tumors are discovered incidentally at a fairly advanced stage, which in part accounts for the poor prognosis.

Recently, the First International Randomized Trial in Locally Advanced and Metastatic Adrenocortical Carci- noma treatment (FIRM-ACT, 2, 3), suggested that pa- tients with advanced disease (radiologically confirmed with no possibility of surgical resection) eligible for che- motherapy who received etoposide, doxorubicin and cis- platin (EDP) plus mitotane had a significant increase in objective tumor response (23.2% vs 9.2%), and percent- age of patients without progression at 12 months (26.1% vs 7.2%) than those treated with streptozotocin and mi- totane. Crossover was permitted and no overall survival advantage was seen. Although these data show some im- provement for the management of ACC, essentially all patient’s tumors progress on mitotane and combination chemotherapy only delays progression to 6 months me- dian with the cost of some toxicity; more effective methods of both detection and treatment are still needed (1, 2, 3).

In an effort to identify novel potential targets for ther- apy in ACC, we performed mRNA-Sequencing on a set of 21 samples, comprised of normal adrenals (NML), adre- nocortical adenomas (AA) and ACCs and focused our analysis on differentially expressed transmembrane pro- teins (data unpublished). This analysis highlighted PSMA as the most upregulated gene in ACC relative to all benign adrenal tissues. Prostate specific membrane antigen (PSMA, also known as FOLH1, GCPII, or NAALADase) is a 100 kDa type II transmembrane glycoprotein receptor heterogeneously expressed at low levels in normal prostate secretory epithelium; in contrast, it is expressed homog- enously at much higher levels in prostate carcinoma (4, 5) and by the endothelium of most solid tumor neovascula- ture, including but not limited to: gastric, colorectal, blad- der, liver, melanoma, lung, pancreatic, breast, and renal cell carcinomas (4, 5, 6, 7). Here we demonstrate that PSMA mRNA and protein expression is elevated in ACC compared to benign adrenal tissues and show that it can serve as a target for ACC in vivo.

Materials and Methods

Patient Selection

A retrospective review of a prospectively maintained tissue bank was performed in order to identify patients who had un-

dergone surgery for an adrenocortical tumor at a single academic tertiary referral center between January 1994 and December 2014. Only tumors arising from within the cortex of the adrenal gland from patients greater than 18 years of age were included in this study. Adrenal medullary tumors and metastases from other primary sites to the adrenal were excluded. The pathological diagnosis of ACC was confirmed, utilizing the criteria of Weiss modified by Aubert, with any sample scoring 3 or higher being considered as malignant (8). Demographic, clinical and patho- logic data were collected for all patients from electronic medical records. 31 adrenocortical tumors were included in this study: 15 ACC (12 conventional, 3 oncocytic) and 16 AA. NML tissue samples were collected from 16 patients, 10 with matched ade- noma and 6 patients who did not have matching neoplastic tissue (NML from adrenals resected in radical nephrectomies). Fur- thermore, of the 12 conventional ACCs, 3 possessed metastases to the omentum, bone and diaphragm, which were also included. All studies were performed with approval from the Institutional Review Board (IRB) at New York Presbyterian Hospital-Weill Cornell Medical Center.

RNA Extraction, Purification and Quantitative Polymerase Chain Reaction

mRNA was extracted from 21 (7 NML, 7 AA and 7 ACC) fresh frozen tissue samples using the RNeasy kit (Qiagen, Venlo, Netherlands). mRNA quality was ensured by utilizing a Bioana- lyzer (Agilent Technologies,Santa Clara, CA), and an RNA In- tegrity Number (RIN) of ≥ 7 was required for qPCR. Comple- mentary DNA (cDNA) synthesis was performed using the SuperScript™M First-Strand Synthesis system following the man- ufacturer’s protocol (Invitrogen, Carlsbad, CA). qPCR was per- formed utilizing the TaqMan Universal PCR system in accor- dance with the manufacturer’s protocol. PSMA (Applied Biosystems, Cat no. Hs00379515_m1) was measured in tripli- cate and normalized relative to the housekeeping gene ß-gluco- ronidase (Applied Biosystems, Cat no. Hs00939627_m1). Gene expression values were calculated according to the -AACT method.

Immunohistochemical staining for CD34 and PSMA Antigens

In addition to the 21 samples submitted for RNA-Seq and confirmed via qPCR, an expanded set of samples was analyzed to validate PSMA protein expression. These included 8 ACCs, 9 AAs and 9 NMLs. In order to assess the expression of PSMA within the vascular endothelium we additionally stained for an endothelial cell marker, CD34, in order to identify the vasculature.

Immunohistochemical staining was performed as previously described (4, 5). Briefly, 5 um-thick sections were mounted on glass slides, deparafinized by Histo-Clear (National Diagnostics, Atlanta, GA), and rehydrated. Slides were stained for CD34 (1: 50, clone QBEnd/10; Biogenex, Freemont, CA) and PSMA (1:20, clone 3E6; cat. no. N1611; DAKO).

Sections undergoing CD34 staining were pressure-cooked for 1 minute in 0.01M citrate retrieval solution (pH 6.0). Sections undergoing PSMA staining were incubated in Target Retrieval Solution (pH 9.0. DAKO, Carpinteria, CA) at 95-99℃ for 30 minutes. Endogenous peroxidase activity was blocked with Per- oxidase Block (Envision System kit, DAKO) for 5 minutes.

Sections undergoing PSMA staining were incubated for 60

minutes with anti-PSMA antibody (cat. no. S0809; DAKO). Slides were further treated with antimouse Ig-HRP labeled sec- ondary antibodies for 60 minutes, followed by diaminobenzi- dine (DAB) solution (cat. no. K4006; DAKO) for 8 minutes. Lastly, slides were counterstained with Harries Modified He- matoxylin (1:100, Fisher Scientific, Pittsburgh, PA).

Sections undergoing anti-CD34 staining were incubated se- quentially with primary antibody, postprimary (equivalent to secondary antibody), polymer (equivalent to tertiary antibody), diaminobenzidine (DAB) and hematoxylin for 5, 25, 15, 25, 10 and 5 minutes respectively. Finally the stained slides were dehy- drated and mounted in Cytoseal™M XYL (Richard-Allan Scien- tific, Kalamazoo, MI). Appropriate positive and negative con- trols were prepared.

Intratumoral Microvessel Density (MVD) and Staining Intensity

Intratumoral microvessel count values were calculated with the method described by Weidner et al (9, 10, 11, 12). In brief,

slides were stained with hematoxylin and eosin and CD34. Using hematoxylin and eosin stained slides the 3 areas (“hot-spots”) within each slide possessing the highest concentration of mi- crovessels were identified. Subsequent microvessel counts using CD34 were performed in each of these “hot-spots”. The mean count of these areas for each stain was recorded (Figure 2B). Major vessels and areas bordering necrosis or hemorrhage were not considered. The following were considered single, countable microvessels: highlighted endothelial cells, endothelial cell clus- ters, and clearly identifiable vessels crossing the section. Staining intensity (I) was graded on a 0-3 scale as follows: 0-no staining, 1-weak, 2-moderate, and 3-strong. The expression of PSMA in tumor vessels was evaluated by identifying the same “hot-spots” utilized in the microvessel density calculation. For the purposes of percentage calculations MVD as determined by PSMA was then compared with MVD as determined by CD34, which was considered to be expressed on 100% of the microvasculature on each slide. These values were then placed into quartiles (0%=0, 0%-25%=1, 26%-50%=2, 51%-75%=3, >75 = 4) and

Figure 1. PSMA quality control (QC). Figure 1A. Contains a boxplot of the RNA-Seq expression values with an overlaid stripchart for normal adrenal cortex, AA and ACC. Figure 1B. A boxplot of PSMA QPCR expression values, with an overlaid stripchart (P < . 05). Figure 1C. The microvessel density of adrenal tissues as determined by CD34 counts per square millimeter visualized by a boxplot with overlaid stripchart. Figure 1D. Representative images of H&E, CD34 and PSMA staining in NML, AA, and ACC. Figure 1E and F, the percent of Composite PSMA scoring intensity in the initial 21 samples and of all samples, respectively, were separated into those scoring none to weak (1-4) and moderate to strong (5-7) for NML (white), AA (Gray) and ACC (Black).

Adrenal PSMA Expression by mRNA-Seq

Adrenal PSMA Expression by qPCR

Adrenal Microvessel Density

Fragments Per Kilobase Million

400

Fold Change (2^-ddct)

CD34+ Vessels/mm

300

200

100

NML

ACC

NML

ACC

Adrenal Tissue Types

NML

ACC

Adrenal Tissue Types

NML

ACC

Percent PSMA Staining in Initial Cohort

120

H&E

100

PSMA Score (%)

ONML ☐

DAA ☐

ACC

CD34

None/Weak

Moderate/High

Composite Staining Score in all Samples

120

100

PSMA Score (%)

ONML ☐

PSMA

DAA ☐

ACC

None (0)

Weak (1-4)

Moderate

High (7)

(5-6)

added to the average PSMA intensity (same scale as CD34) to get a composite PSMA staining score (Figure 1C). A PSMA score of 0, 1-4, 5-6, or 7 was considered to represent no staining, weak staining, moderate staining, or strong staining, respectively. All immunohistochemical analyses were performed by an endocrine pathologist (TS).

Imaging of a Patient with Metastatic Adrenocortical Carcinoma

A patient with nonresectable metastatic ACC was consented and enrolled in the 177Lu Radiolabeled Monoclonal Antibody HuJ591-GS (177 Lu-J591) study in patients with nonprostate solid tumors. The patient was injected with 3.98 mCi (147.3 MBq) of Zirconium 89 (Zr89) J591 and whole body PET-CT

(WB-PET-CT) was performed. Follow up staging 1 8F FDG (fluo- rodeoxyglucose) PET-CT was performed 3 months later.

Statistical Analysis

All p-values for comparisons were calculated using linear re- gression, Fisher’s exact test, Wilcoxon sum rank test, Odds ratio, or student’s t test, as appropriate and as indicated in the text below. Data are presented as percentages, medians, ranges, means and standard deviations, in accordance with the distri- bution. A p-value < 0.05 was considered to be significant. All analyses were performed in R (R Development Core Team, Vi- enna, Austria). All statistical analyses were performed by a mem- ber of the Institute for Computational Biomedicine at Weill Cor- nell Medical College (OE).

Figure 2. PSMA activity in metastatic ACC. Figure 1A, B and C. H&E and PSMA staining of an liver, omental and bone metastasis at 4x with 40x insets, respectively. Figure 1D. Axial CT (top left), Axial Zr89 J591 PET (top right), fused PET and CT (bottom left) and Maximum intensity projection (MIP) whole body image (bottom right) demonstrating radiotracer uptake associated with left upper lobe (seen on MIP) and right middle and lower lobe intrathoracic masses.

H&E

PSMA

5.00 mm

5.0sp

SFOV 50.0 cm

-82.00

W: 400

L: 40

HYBRID_CT Transaxials

PT Transaxials

5.0sp

SFOV 50.0 cm

-82.00

W: 500 L: 750

Results

Patient Demographics

A total of 50 samples were analyzed in this study, which included 16 NML, 16 AA,15 primary ACC and 3 metas- tases. Of the collected patient and tumor demographics, both tumor size and mass were found to be statistically significant between ACC and benign tissues (Table 1) (P < .001, and P = . 01, student’s t test). However, gender, age and functional status were not found to be different be- tween the two categories.

Validation of PSMA Gene Expression

mRNA from the same 21 frozen tumor samples (7 NMLs, 7 AAs and7 ACCs), that were submitted for RNA- Sequencing, were utilized to evaluate PSMA expression (Figure 1A & 1B). PSMA gene expression assessed via qPCR was on average 9 and 10 fold higher in the ACCs when compared with the NML and AA samples, respec- tively (Figure 1B, P < . 01, P <. 01).

Microvessel Densities Calculated With CD34

Whole sections were stained and examined, demon- strating reactivity in the neovasculature only (data not shown). CD34 was used to quantify microvessel density (Figure 1C, 1D). Microvessel densities measured using CD34 were 121 (43-208) v/mm2, 79 (23-202) v/mm2, and 29 (17-124) v/mm2 for NML, AA and ACC, respec- tively (Figure 1C). NML and AA had a significantly higher microvessel density than ACC (P < . 001, P = . 027, re- spectively). NML and AA were not significantly different from each other (P = . 076).

PSMA Expression on Microvasculature and Staining Intensity

Utilizing CD34 as an endothelial cell marker, we sought to establish the percent PSMA positivity in the neovascu- lature and assess the potential utility of PSMA as a pre- dictor of malignancy. The percentage of PSMA-positive vessels and PSMA staining intensity in the discovery set

were found to be significantly less in both NML (0 ± 0%, I= 0, P <. 001) and AA (1.8±5.5%,I=0.1, P <. 001) when compared to ACC (78.2 ± 30.1%, I = 2.7). In con- trast, there was no difference between AA and NML for either parameter. Percentile scores were converted into quartiles and summed with the average intensity (these values were rounded to the nearest whole number), yield- ing a composite PSMA staining score. Scores were grouped into four categories, none (0), weak (1-4), mod- erate (5-6) and strong (7). In the initial cohort of 21 sam- ples, 100% of NML and AA were grouped in the none category, 13% of the ACCs stained weakly, 43% stained moderately and 42% stained strongly (Figure 1E). The ACCs demonstrated a significantly higher PSMA staining than both NML and AA (Fisher’s Exact Test, P < . 001). Furthermore, in an expanded cohort of samples including 16 NMLs, 16 AAs and 15 ACCs (Figure 1F), 100% of NML and 87.5% of AA were categorized as none and 12.5% of AAs were weakly positive. In contrast, 44% and 43% of ACCs stained within the high and moderate groups respectively, while only 13% stained weakly. Ad- ditionally, 3 of the 15 ACC samples were oncocytic vari- ants, and were grouped within the moderate and strong groups with an average of 87% PSMA positive vessels. There was no difference between AA and NML. These differences were found to be true regardless of the func- tional status of the tumor. Furthermore, functional AA and ACC did not differ from nonfunctional AA and ACC for either parameter. In order to further assess whether PSMA expression correlated with aggressive clinicopath- ologic features we compared PSMA expression with size, mass, large vessel extension, mitotic counts, fuhrman nu- clear grade, each of the individual Weiss criteria, compos- ite Weiss score, and the presence of both preoperative and postsurgical metastases. We found no correlation between PSMA composite score and any feature of malignancy (Supplemental Table 1).

We next examined the role of PSMA as a predictor of malignancy. Those samples with > 25% PSMA positive

Table 1. Patient demographics and histopathologic features of adrenal tumors grouped by tumor type.
Total n = 47	Benign		Malignant	p-value
Total n = 47	Normal n = 16	Adenoma n = 16	Adrenocortical Carcinoma n = 15	p-value
Age (Mean/Range)	50.1 (22-76)	42.8 (22-80)	35.4 (21-54)	N/S
Sex (Male:Female)	9:7	7:9	8:7	N/S
Size cm (Mean ± Stdev)	-	3.00 ± 1.40	13.69 ± 6.6	<0.001
Mass g (Mean + Stdev)	-	18.50 ± 9.48	566.475 ± 630.86	0.01
Functional	-	6	9	N/S
Weiss Score 0 or 1	-	15	0	<0.001
Weiss Score 2	-	1	0	N/S
Weiss Score ≥ 3	-	0	15	<0.001

vessels were 33 times more likely to be malignant than benign (Odds ratio, P < . 001). Utilizing the PSMA com- posite scores from the initial cohort (21 samples) we es- tablished a cutoff score of 4 (moderate). When this cutoff was applied to the expanded cohort of 26 samples (8 NML, 8 AA and 7 ACC) it correctly predicted benignancy and malignancy in all cases. For both the initial and ex- panded cohorts a cutoff score of 4 was 100% specific and 100% sensitive.

Metastatic Immunohistochemical Staining and In Vivo Patient Imaging With Zr89-J591Whole Body PET-CT

Three of the patients in this study developed metastases and returned to WCMC-NYPH. The metastatic lesions from the liver, omentum and bone (Figure 2A, 2B, 2C, respectively) were stained for PSMA. All three metastases demonstrated high levels of PSMA staining, of differing intensities, both in the primary tumor (data not shown) and their respective metastases (Figure 2A, 2B, 2C). One patient was subsequently enrolled in the 177 Lu-J591 trial at WCMC-NYPH. WB-PET-CT demonstrated targeting of Zr89-J591 to the patient’s multiple lung masses (Figure 2D). Nodules measuring > 1cm in at least one dimension were considered, and the blood pool standardized uptake value (SUV) was utilized as a baseline for Zr89-J591 up- take (measured at 0.8 SUV). 5 nodules met these criteria with an average measurement of 3.49 ± 1.86cm and a SUV of 1.4 ± 0.65 by Zr89-J591 imaging (Supplemental Table 2). When measured 3 months later by FDG-PET, nodules were found on average to be 3.53 ± 1.47cm (Sup- plemental Table 2).

Discussion

PSMA, a transmembrane glycoprotein receptor has been previously identified and investigated as a potential target therapeutic target in prostate cancer and other solid tu- mors. Here we have confirmed that PSMA is highly over- expressed at both the mRNA and protein levels in ACC as compared to NML and AA. Furthermore, as in other solid tumors, PSMA expression appears to be isolated to the tumor neovasculature.

In our study, 87% of ACCs were found to have a mod- erate to high expression of PSMA, whereas none of either the NML or AA did. Furthermore, the lowest ACC PSMA composite score was 4, whereas the highest benign score did not exceed 2. Our data suggests that adrenal tumors with > 25% positivity were 33 times more likely to be an ACC as opposed to a benign adenoma, and a composite IHC PSMA score of 4 was 100% sensitive and specific for

carcinoma. Previous studies have highlighted PSMA pos- itive carcinomas that have since gone on to participate in Phase I clinical trials for PSMA imaging in nonprostatic solid tumors. In a clinical trial reported by Milowski et al when formalin fixed paraffin embedded tissue samples possessed strong PSMA staining there was additionally a strong positive signal from the J591 imaging and an ad- ditional study demonstrated 94% of soft tissue metastases with In111-J591 uptake (7, 13, 14, 15, 16). These findings are in line with our own data from metastatic samples wherein, metastases were found to be strongly positive, and furthermore, when one of these patients was imaged with Zr89-J591, the metastases were found to demon- strate strong uptake in vivo. Taken together, these data suggest that strong IHC staining via the 3E6 anti-PSMA antibody correlates well with the imaging findings in vivo utilizing the humanized J591 anti-PSMA antibody. This raises the possibility that J591-PET imaging of adrenal masses could also serve as a novel preoperative diagnostic tool in ACC.

As PSMA has been reported to be present in the endo- thelium of tumor vessels, we utilized a baseline vascular density score, via CD34 staining, to determine the extent of PSMA staining. Using the vascular marker CD34, we found a significant reduction in vascular density in ACC when compared with both NML and AA. This is in sur- prising contrast to most malignant tumors in which an increased level of vascularization has been documented when compared to normal tissue (9, 10, 11, 12). However, two other groups have examined the microvascular den- sity in adrenal tumors and normal adrenal tissue and have reported similar observations. Bernini et al (17) and Diaz- Cano et al (18) studied a total of 118 tumors and also found a significant decrease in total vessel density in ACC compared to NA and AA regardless of functional status of the tumor. Conversely, Xu et al (19) studied 44 tumors and reported an increase in microvascular density of ACC rel- ative to benign tumors. Regardless of microvessel density in ACC, it is clear that PSMA is expressed in a high per- centage (78.2%) of microvessels in ACC.

The main limitation of this study could be considered to be a small sample size. Although a total of only 47 spec- imens were examined, we believe that our findings are unlikely to be due to technical error or chance given the consistent differences seen with regard to PSMA at both the RNA and protein level. Furthermore, the positive staining of metastatic samples, and in vivo uptake of PSMA in a patient with nonresectable metastatic disease strongly suggests that PSMA could be considered as a po- tential therapeutic target in ACC.

In summary, PSMA is markedly overexpressed in ACCs and expression appears to be highly specific to the micro-

vasculature of ACC. This can be visualized in metastatic disease via WB-PET-CT with Zr89-J591. Thus, PSMA may be a good candidate for directed biologic therapy for these tumors. The precise function of PSMA in the devel- opment of the neovasculature in solid tumors is unknown and must be further investigated to delineate its role in tumor angiogenesis. However, given the recent success of the anti-PSMA mAb J591 antibody in a phase I & II clin- ical trials (13, 14, 15, 16), coupled with a need for viable alternatives for the treatment and imaging of ACC, ex- panding current clinical trials of anti-PSMA antibody to include ACC seems reasonable and even promising.

Acknowledgments

Address all correspondence and requests for reprints to: Email: tjfahey@med.cornell.edu. Corresponding Author’s Mailing Ad- dress: 525 East 68th Street, Starr 8, NY, NY, 10 065.

This work was supported by Grants or Fellowships: Dancers Care Foundation.

*These two authors contributed equally to the writing of this manuscript

Disclosure Statement: MJPC, TS, YFL, DAK, TB, AA, HL, YSJ, AM, STT, RZ, OE & TJF declare that the research was conducted in the absence of any commercial or financial rela- tionships that could be construed as a potential conflict of in- terest. NHB is an inventor on patents that are assigned to Cornell Research Foundation (“CRF”) for the J591 antibody described in this article. NHB is a paid consultant to and holds equity in BZL Biologics, LLC, the company to which the patents were licensed by CRF for further research and development.

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