Tumor-Specific Zr-89 Immuno-PET Imaging in a Human Bladder Cancer Model

Purpose Tumor-specific molecular imaging is an important tool for assessing disease burden and treatment response. CA19.9 is an important tumor-specific marker in several malignancies, including urothelial carcinoma. [89Zr]DFO-HuMab-5B1 (MVT-2163) is a CA19.9-specific antibody-based construct that has been validated in preclinical animal models of lung, colorectal, and pancreatic malignancies for positron emission tomography (PET) imaging and is currently in a phase I trial for pancreatic cancer (NCT02687230). Here, we examine whether [89Zr]DFO-HuMab-5B1 may be useful in defining urothelial malignancies. Procedures Surface expression of CA19.9 was confirmed in the human bladder cancer line HT 1197. The radioimmunoconjugate [89Zr]DFO-HuMab-5B1 was injected into mice bearing HT 1197 xenografts, and followed by PET imaging, ex vivo experiments including biodistribution, histology and autoradiography, and analysis of blood samples for shed antigen levels were performed. Results [89Zr]DFO-HuMab-5B1 specifically accumulates in HT 1197 engrafted tumors when imaged with PET. Ex vivo biodistribution of organs and autoradiography of engrafted tumors confirm our construct’s specific tumor binding. The target antigen CA19.9 was not found to be shed in vitro or in vivo. Conclusions [89Zr]DFO-HuMab-5B1 can be used to delineate urothelial carcinomas by PET imaging and may provide tumor-specific information prior to, during, and after systemic therapies.


Introduction
This year, there will be an estimated 79,030 new cases of and 16,870 deaths from urothelial carcinoma in the USA [1].
While the majority of patients with bladder cancers present in early stages, those with locally advanced or metastatic disease have a 5-year survival rate of only 5 % [2]. First-line therapy for these patients is combination chemotherapy with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) or doublet therapy with gemcitabine plus cisplatin. These treatments have been shown to result in both higher overall response rates as well as improved overall survival [3]. In addition to standard cytotoxic therapy, the recent approval of antibodies targeting an immune checkpoint for patients with metastatic disease reaffirms the importance of host immunity in bladder cancer, especially since therapy for early-stage disease has long included an immune modulator, Bacillus Calmette-Guerín (BCG) [4]. Furthermore, novel therapeutic interventions targeting key oncogenic pathways specifically altered in urothelial carcinoma expanded our therapeutic armamentarium and allow for precision tailoring of treatments for individual patients [5,6].
Taking tumor-specific features and devising therapeutic interventions that exploit differences between cancer and normal tissues optimizes the therapeutic ratio and is the ultimate goal of any treatment. This approach is also paramount for engineering tumor-specific molecular imaging agents, which can (1) help define micrometastatic disease, (2) provide additional important biological information about the tumor beyond glucose uptake (e.g., standard 2-deoxy-2-[ 18 F]Fluoro-D-glucose ([ 18 F]FDG) PET), and (3) allow for monitoring of treatment response. Additional value can be derived from successful molecular imaging agents, such as PET radioimmunoconjugates, because once proven successful for immno-PET imaging, they can be modified for radiolabeling with radiotherapeutic isotopes such as Lu-177, Y-90, Pb-212, or Ac-225.
Carbohydrate antigen 19.9 (CA 19.9) is a common tumor-associated antigen of hepatobiliary, colorectal, lung, and bladder cancers [7]. Serum CA19.9 is significantly increased in 35-58 % of patients with known urothelial carcinoma and is significantly increased in 9 70 % of muscle-invasive cases versus non-muscle-invasive cancers, making a potentially important prognostic marker [2,[7][8][9]. While several serum and cytology markers for urothethial cancers have been identified, they lack the sensitivity needed for appropriate screening [10]. In fact, serum CA 19.9 suffers similar limitations. To address these limitations, we have previously demonstrated the value in being able to image this antigen in situ. Our group has taken HuMab-5B1 (MVT-5873), a CA19.9-specific human antibody, and confirmed the feasibility and efficacy of targeting CA19.9 for both imaging and therapy in pancreatic cancer models [7,11,12]. Here, we demonstrate the utility of our immuno-PET probe, [ 89 Zr]DFO-HuMab-5B1 (MVT-2163), which, like serum CA19.9, can confirm the presence of CA19.9expressing human bladder cancer cells, but, unlike serum CA19.9, also identifies the specific anatomical location of these lesions in situ, in vivo, and in real time, and unlike [ 18 F]FDG PET, is tumor specific.

Cell Culture
HT 1197 and BxPC3 cells (ATCC, Manassas, VA) were grown according to standard methods described by supplier. All media were purchased from the Media Preparation Facility at Memorial Sloan Kettering Cancer Center.

Immunofluorescence Staining
HT 1197 cells were plated on 8-well chamber slides at a density of 2 × 10 5 per well and incubated overnight. Chambers were washed with PBS three times. Cells were fixed with 4 % paraformaldehyde for 10 min on ice, then washed with PBS three times, and incubated with blocking buffer [1 % bovine serum albumin in phosphate-buffered saline (PBS)] for 5 min. After three washes with PBS, slides were incubated with fluorescently labeled HuMab-5B1 antibody for 20 min at 40°C. Following incubation, cells were washed five times with PBS, counterstained with DAPI for 15 min, and washed with dH 2 O. Gasket and rubber seal were removed and slide was rinsed with dH 2 O. Samples were covered with Mowiol mounting media followed by a coverslip, and the slides were stored at − 20°C prior to imaging.

Immunoreactivity Measurements
The immunoreactivity of the Zr-89-labeled construct, [ 89 Zr]DFO-HuMab-5B1, was determined using BxPC3, a human pancreatic cancer cell line which highly expresses CA19.9, via a previously reported method [15,16]. Data were background-corrected and the ratio of the total to bound (total/bound) radioactivity was plotted against the inverse of the normalized cell concentration (1/normalized cell concentration) for the linear regression analysis.

Murine Subcutaneous Xenograft Models
Female athymic homozygous nude mice, strain Crl:NU(NCr)-Foxn1 nu (Charles River Laboratories, Wilmington, MA), age between 6 and 8 weeks, were xenografted subcutaneously with 5 × 10 6 HT 1197 cells, suspended in 150 μl of a solution containing a 1:1 mixture of Matrigel (Corning, Corning, NY) and cell culture medium. Tumors were grown to a size of approximately 150 mm 3 post-implantation before imaging.

Biodistribution
The acute biodistribution of [ 89 Zr]DFO-HuMab-5B1 was determined using the same HT 1197 subcutaneous (right flank, 150 mm 3 ) model as used for PET imaging (athymic, nude mice). Mice tumor volumes were measured prior to imaging using a Peira TM900 tumor-measuring device, and the mice were separated into groups with similar mean tumor volumes before being injected via the lateral tail vein (52.3 ± 1.0 μCi/10 μg). At 48 and 120 h post-injection, mice (n = 5) were euthanized. Thirteen tissues including the tumor were collected. Each sample was weighed and a Wizard 2 automatic gamma counter calibrated for Zr-89 was used to measure the corresponding radioactivity. The counts from each sample were decay-and background-corrected, and counts were converted into activity by using a calibration curve generated from Zr-89 standards of known activity. The % ID/g was calculated by normalizing data to the total activity injected into the corresponding animal.

Autoradiography and Histology
Tumors of euthanized animals were excised, submerged in Tissue-Plus OCT compound (Scigen, Gardena, CA), and frozen on dry ice. Series of 10 μm tissue sections were cut and placed in a film cassette beneath a phosphor-imaging plate for 48-72 h at − 20°C (BASMS-2325; Fujifilm, Tokyo, Japan), which was then read on a Typhoon 7000IP plate reader (GE Healthcare, Chicago, IL) at 25 μm pixel resolution. Several sequential sections were submitted to the Molecular Cytology Core Facility at MSKCC for hematoxylin and eosin and HuMab-5B1 (0.5 μg/ml) staining. Image analysis was performed with ImageJ (https://imagej.nih.gov/ij/).

Microscopy
A Zeiss Axioplan2 fluorescence microscope connected to a CCD camera and equipped with a motorized stage (Prior Scientific Instruments, Rockland, MA) was used to acquire microscopic images. MetaMorph software (Molecular Devices, Sunnyvale, CA) was used to control the microscope as well as to generate a montage of the entire tumor from the captured image frames. Photoshop CS6 software (Adobe Systems, McLean, VA) was used for post-acquisition processing.
Assessing Shedding Status of CA19.9 Whole blood was collected from mice xenografted with HT 1197 cells at several time points post tumor cell inoculation (n = 4 per time point) via retro-orbital bleeds with capillary tubes. Blood was transferred immediately to 1.5 ml microcentrifuge tubes and allowed to clot for 1 h at RT. Gross clot was removed and remaining sample spun down at 1000×g × 10 min. Supernatant was collected and stored at − 20°C until ELISA (Affymetrix no. BC1017, Santa Clara, CA) was performed per manufacturer protocol. Positive controls of predetermined CA19.9 concentration and media from T175 flasks containing confluent HT 1197 were also assayed.

Statistical Analysis
Quantitation of results was performed with Prism software (Version 7.0, GraphPad software, La Jolla, CA). An unpaired, two-tailed Student's t test was used to analyze the data. In all cases, a 95 % confidence level (P G 0.05) was considered to represent a statistical difference in the data.

HuMab-5B1 Binds to Human Bladder Cancer Line
To confirm presence of CA19.9 on human bladder cancer, flow cytometry and immunofluorescent studies of cells incubated with fluorescently labeled HuMab-5B1 were performed. Flow cytometry histogram (Fig. 1a) and immunofluorescence imaging (Fig. 1b) demonstrate abundant expression of CA19.9 on HT 1197 cell lines as evidenced by binding of fluorescently labeled HuMab-5B1. In contrast, when cells are incubated with fluorescently labeled isotype control IgG, no significant binding is observed (Fig. 1).

Autoradiography and Microscopy Show In Situ Target Engagement
Tumors were harvested and sections were assessed for H&E, autoradiography, and CA19.9 staining. Results demonstrate heterogeneous distribution of activity across the entire tumor section coincident the glandular structures, and diffuse staining for CA19.9 (Fig. 4).

Sera of HT 1197-Engrafted Animals Do Not Demonstrate Evidence of Shed Antigen
To determine whether HT 1197 cells shed CA19.9, serum samples from mice engrafted with HT 1197 cells assessed at days 20 (n = 4) and 45 (n = 4) after engraftment and of media from confluent flasks containing HT 1197 (HT media) were

Discussion
The ability to better delineate tumors based on tumorspecific markers is paramount to clinical decision-making. As we discover new targets (e.g., receptors, enzymes) and deploy methods to generate imaging probes to these molecules, we can contribute to the refining of tumor  classification beyond existing paradigms, and, hopefully, help personalize treatment choices accordingly.
Cystoscopy is most useful for early diagnosis of bladder cancer. Approximately 70-80 % of newly diagnosed patients present superficial bladder tumors, and the prognosis of these patients depends largely on the grade of the tumor (https://www.cancer.gov/types/bladder/hp/bladder-treatment-pdq). The majority of deaths from bladder cancer are due to high-grade disease where cancer cells invade the muscular wall of the bladder and spread to other parts of the body. Patients with high-grade disease are typically staged with a CT scan of the chest, abdomen, and pelvis, and patients with suspected bone metastases undergo a bone scan.
While [ 18 F]FDG PET is also utilized to identify and monitor local or metastatic tumors of various histological types, including bladder cancer, intravesicular lesions are difficult to assess with this modality because [ 18 F]FDG is excreted renally and accumulates in the bladder. As such, [ 18 F]FDG PET is more commonly employed in the metastatic setting, exploiting preferential glucose uptake in rapidly dividing cells. It is well known that normal tissues can also take up [ 18 F]FDG, especially following surgical procedures, in the setting of inflammation or physiologic rapid cell turnover. Accordingly, developing tumor-selective functional imaging can provide valuable information about tumor aggressiveness and propensity for dissemination, which is typically unavailable with standard imaging.
We have previously reported on the synthesis of our CA19.9targeting immuno-PET construct, 89 Zr-DFO-HuMab-5B1, and its ability to specifically target human pancreatic, colorectal, and lung cancers in a xenograft animal model system [7,11,12,18]. Here, we show that HuMab-5B1 binds to human bladder cancer cells in vitro, and PET imaging with [ 89 Zr]DFO-HuMab-5B1 demonstrates tumor localization in vivo. Tumor localization was further confirmed ex vivo via organ biodistribution and tumor autoradiographic studies. A majority of bladder cancers express CA19.9, and this work provides a new potential PET imaging approach for visualizing invasive bladder cancers expressing high levels of CA19.9 in patients [8]. While prior studies demonstrated utility of imaging bladder cancer-selective agents, to our knowledge, the antigen CA19.9 has not been previously used as a target for PET imaging of this disease [19].
Notably, our studies indicate that, while we are able to get excellent localization in bladder tumors, there is also accumulation in the bones of our experimental animals. This is a wellknown, previously described drawback of the Zr-89-DFO complex, and we and others are investigating alternative Zr-89 chelation strategies to mitigate this phenomenon [20][21][22]. Uptake of [ 89 Zr]DFO-HuMab-5B1 in CA19.9 overexpressing murine xenograft models of bladder cancer (HT 1197) was high by PET visualization and by ex vivo biodistribution, with peak uptake at 120 h p.i. of 31.8 ± 20.2 %ID/g. Autoradiography and immunohistochemical staining has confirmed co-localization in HT 1197 tumor xenografts of the radioactive [ 89 Zr]DFO-HuMab-5B1 construct with high CA19.9 expression.
Most bladder cancers are transitional cell carcinomas arising from transitional epithelium, a specialized mucous membrane. Serum levels of CA19.9 are moderately increased in patients with advanced and metastatic bladder cancer [9,10]. It is well documented that the CA19.9 antigen in serum is displayed on mucin; however, not all mucins are shed from the cell surface [23,24]. Notably, heterogeneity of antigen shedding is observed with various cell lines, and we find that the HT 1197 cell line neither sheds CA19.9 in in vitro nor in vivo [7].
Target shedding or cleavage from tumor, as can occur with several tumor-associated antigens that are subsequently detected with serum assays, can in principle diminish in situ tumor localization, degrade signal-to-noise in imaging studies, and diminish the therapeutic ratios when utilizing therapeutic radionuclides. While it is important to assess the shedding status of this  antigen in our model system, we understand that individual patient tumor types may differ in CA19.9 shedding. However, herein lies the advantage of the HuMab-5B1 PET imaging agent: HuMab-5B1 is highly internalized and can localize to tumor despite circulating shed antigen [11]. Additionally, the hurdle of targeting the tumor tissues in the presence of shed CA19.9 in the serum is easily overcome by preloading with unmodified 5B1 [17]. Thus, the PET imaging agent can provide information about tumor location and extent of metastasis which cannot be gathered from the serum biomarker alone. Tumor-specific PET imaging could be used for staging and selection of precision treatment regimens of patients. Moreover, fluorescence-guided surgery is gaining increased interest and it may be worthwhile to examine whether fluorescent dye-labeled HuMab-5B1 could be used to guide partial cystectomy [25].
Studies are currently ongoing to assess our construct in orthotopic models of this cell line and assess the contribution of the microenvironment on antigen targeting. Concurrently, [ 89 Zr]DFO-HuMab-5B1 is being evaluated in an ongoing clinical trial in patients with pancreatic cancer or other CA19-9-positive malignancies (clinicaltrials.gov, NCT02687230). Our current study confirms that the [ 8 9 Zr]DFO-HuMab-5B1 radioimmunoconjugate can detect human urothelial cancer, adding preclinical rationale for utility in this aggressive cancer type, especially in patients with elevated CA19.9 levels. We believe that this construct can serve as an important diagnostic tool for assessing tumor-specific responses prior to, during, and after systemic therapies and has the potential to guide the development of targeted bladder cancer therapies.