Labeling of Anti-SAS1B with Zirconium-89 as a Novel Immune-PET Probe for Uterine Cancer Imaging

Overexpression of Sperm Acrosomal SLLP1 Binding protein (SAS1B) in various cancer types, including uterine cancer cells, was discovered a few years ago, and different monoclonal antibodies (anti-SAS1B) that specifically bind to SAS1B antigens were developed. Labeling of these antibodies with radionuclides can provide an opportunity for imaging and radioimmunotherapy. The objective of this study was to label anti-SAS1B (SB5) with Zirconium-89 ( 89 Zr) for PET imaging and determine its biodistribution. Anti-SAS1B (SB5) antibody was labeled with 89 Zr indirectly using the chelator desferrioxamine B (DFO), which is currently a best linker for 89 Zr. The antibody, SB5, was first conjugated to DFO with a ratio of 1:5 and then labeled with 250 μCi of 89 Zr. Results of PET imaging in mouse-bearing uterine cancer tumor showed a limited uptake. The bio-distribution study matched the PET imaging and con-firmed the uptake by the tumor, and the accumulation in bones. In conclusion, labeling of anti-SAS1B could provide an effective way of uterine cancer detection and treatment progression.


Introduction
Molecular imaging has had a transformative impact on academic research, industry, and the clinical field [1]. Molecular imaging can also provide quantitative information, which is important in monitoring cancer progression over time or treatment response [2]. The positron emission tomography (PET) is one of the molecular imaging modalities that has provided non-invasive and quantitative physiological data relating to disease pathophysiology, receptor expression, enzyme activity, and cellular metabolism [3] [4]. PET imaging depends on radionuclide decay by positron emission [5]. Radionuclides are chemically attached to molecular ligands designed to trace biochemical phenomena in vivo, such as the expression of surface antigen on cancer cells [6] [7]. Uterine cancers are considered aggressive cancers [8] and frequently have a terminal prognosis because of the lack of early diagnosis and effective treatment options [9]. According to American Cancer Society statistics, the estimation of new uterine cancer cases for 2020 was about 66,000, and the deaths related to uterine cancer were about 13,000 [10]. During the last decade, overexpression of SAS1B protein on the cell surface in many types of tumors, including uterine tumor, was discovered by Dr. John C, Herr and coworkers, who also developed different monoclonal antibodies (anti-SAS1B) against this protein and demonstrated specific binding to SAS1B [11]. Because of their sensitivity and specificity and biological half-live Monoclonal antibodies have been used for more than two decades to target cancer cells [12] [13]. Furthermore, for their potential treatment, antibodies have been loaded with radioisotopes to serve as a vehicle taking them specifically to their target [14] [15]. This advantageously prevents the systemic effects of radiation on the normal organs [5]. In addition, based on the radionuclide's properties, labeled antibodies with some radionuclides can also provide imaging capability, which is useful for cancer diagnosis and treatment monitoring [16]. Early diagnosis and treatment assessment is crucial for cancer therapy [17].
Pharmacokinetics studies have shown that intact antibodies typically have a half-life of about 3 -4 days [18]. Therefore, this requires the use of radionuclides with long-hour's half-lives. Among PET imaging radionuclides, 89 Zr is attractive, because it has been safely used in the clinic for many years, and it has several advantages, including a long half-life of 78.4 hours, which matches the biological half-lives of the antibodies [19] [20]. In addition, 89 Zr decays low average positron energy of 395 keV, which provides high-resolution PET images. Based on these properties [21], 89 Zr was selected as radio nuclei to label SB5 antibody for generating a novel tracer (probe) to obtain quality images, and its tumor uptake.

Material and Methods
[ 89 Zr]-Oxalate was produced in-house at VCU hospital on a PET-Trace Cyclotron (Sofie Biosciences, Richmond, VA, USA) using the 89 Y (p, n) 89 Zr reaction. The DFO chelator was purchased from Macrocyclics. Uterine cancer cell lines SNU539 and Anti-SAS1B antibodies were provided by late Dr. John C, Herr at UVA. Female nude mice, 3 weeks old, were obtained from the National Cancer Institute in Bethesda.

Conjugation of Anti-SAS1B (SB5) with DFO
AntiSAS1B and Zirconium-89 were linked using DFO, because it is the most prominent chelator of Zr 4+ . The DFO is a hexadentate siderophore containing three hydroxamate groups for chelating metals such as 89 Zr and a primary amine tail for conjugation to antibodies. Several principle preclinical studies have been conducted using DFO to label antibodies with 89 Zr; however, this labelling caused accumulation of free 89 Zr [22]. To resolve this issue, many studies have attempted to improve the linkage between DFO and the antibody [23], while others concentrated on improving the chelator itself [24]. During preliminary experiments a 1:5 molar ratio SB5 to DFO was found optimal for the reaction and these ratios was used for conjugating SB5 with DFO in 500 μl of phosphate-buffered saline solution (PBS), and the pH was adjusted to 8.8 -9.0 using 0.1 M sodium bicarbonate. The mixture was incubated at 37°C and at a rotating speed of 300 rpm for 60 minutes.

SB5-DFO Characterization
The conjugation yield of SB5-DFO was monitored using HPLC (High-performance liquid chromatography with ultraviolet detector) with a size exclusion column (SEC) (Superdex 200 10/300 column GE Healthcare, Piscataway, NJ USA) at room temperature. The mobile phase consisted of 0.01 M phosphate buffer and 0.14 M NaCl, pH 7.4 at a flow rate of 0.5 mL min. UV detection was accomplished at 280 nm using a Waters 2489 UV (10-mm, 11-μL analytical flow cell).

Radiochemistry
High-performance Liquid Chromatography Analysis: Zirconium-89 was received as [ 89 Zr]-oxalate. Therefore, conversion of [ 89 Zr]-oxalate to [ 89 Zr]-chloride using 1M HCl was the first step before labeling anti-SAS1B (SB5). The reaction yield was monitored by HPLC. The HPLC was connected to a radio detector to detect the radioactivity signal of the labeled antibody. This is one of the methods used to determine the radiochemical purity of radiolabeled antibodies. The

PET/CT Imaging
The uptake of radiolabeled antibodies in tumor by the tumor-associated antigens and it's the distribution in other organs was quantitatively measured by [ 89 Zr]-PET. A female nude mouse was injected via tail vein with 250 μCi of [ 89 Zr]-DFO-SB5. After intervals of 2 hours, 24 hours, and 72 hours, the mice were anesthetized with 2% isoflurane inhalation anesthesia. The mice were positioned in PET/CT system, and images were obtained. The region-of-interest (ROI) was analyzed with the percent injected dose/gram of tissue (%ID/g). The animal was imaged by both PET and CT. The CT images provided the anatomical background for the distribution of the radioactivity. Biodistribution The biodistribution of [ 89 Zr]-DFO-SB5 in animals was determined in different organs after injection and animal sacrifice. Six nude mice bearing SNU539 tumor xenografts were divided into two groups for treatment with labeled-antibodies via tail vein injection. The first group of 3 mice was injected with a solution of [ 89 Zr]-DFO-SB5. The other groups of 3 mice were injected with the non-specific labeled antibody [ 89 Zr]-DFO-3A4 (as negative control). The mice were sacrificed, blood and tissues were collected from each animal and weighed, and radioactivity of 89 Zr in each sample was determined by gamma-counting on a Perkin Elmer Wallac Wizard 1470. The radioactivity percent of injected dose per gram %ID/g of each organ was calculated. The organs were collected include blood, heart, lung, liver, gallbladder, spleen, kidney, tumor, femur, and muscle.
First, confirm that you have the correct template for your paper size. This template has been tailored for output on the custom paper size (21 cm * 28.5 cm). High-Performance Liquid Chromatography (HPLC) results showed a high reaction yield of DFO-SB5 cold conjugation (Figure 1). The peak of conjugated-DFO-SB5 was at 24 minutes retention time. The small peak at retention time 37.12 represents free DFO. The area under the peak represents the conjugation yield of DFO to SB5. As the HPLC chromatograph showed, about 92% of DFO was conjugated to SB5.

Characterization of Conjugated Antibody-DFO-SB5
First, confirm that you have the correct template for your paper size. This template has been tailored for output on the custom paper size (21 cm * 28.5 cm). High-Performance Liquid Chromatography (HPLC) results showed a high reaction yield of DFO-SB5 cold conjugation ( Figure 1). The peak of conjugated-DFO-SB5 was at 24 minutes retention time. The small peak at retention time 37.12 represents free DFO. The area under the peak represents the conjugation yield of DFO to SB5. As the HPLC chromatograph showed, about 92% of DFO was conjugated to SB5.

Radio-HPLC Chromatography
Radio-HPLC was used to determine the radiolabeling yield of SB5. The radioactive signal of 89 Zr was validated using peaks on a UV-HPLC ( Figure 2). The Figure 1. Characterization of DFO-SBF conjugation reaction. Conjugation reaction (20 ul) was separated using an HPLC system as described in the text above. The chromatogram shows a 92% conjugation yield B5. radio-HPLC chromatograph showed that almost 70% of radioactive signal matched the UV signal of DFO-SB5. Few small peaks possibly represent the SB5 fragments.

Thin-Layer Chromatography (Radio-TLC) of [ 89 Zr-DFO-SB5]
The radio-instant thin layer of chromatography showed a high radioactive signal of labeled SB5 with a very small signal representing free 89 Zr or [ 89 Zr]-DFO. Almost 97% radioactivity was recovered with the DFO-SB5 conjugate (peak 1) and only a small fraction (3%) of radioactivity was incorporated into SB5 fragments (peak2) (Figure 3).

PET/CT Imaging
The animal was imaged by both PET and CT. The CT image provided the anatomical background for the distribution of the radioactivity. As the image showed (Figure 4), the vast majority of radioactivity appeared to be clearing   from the blood, spleen, liver, and other organs. It is also observed that the right flank bearing tumor showed radioactive accumulation, whereas the left flank did not show any uptake. This confirms that the 89 Zr-lablled SB5 antibody bound to its receptor, SAS1B. In addition, activity was also noticed in the bone, which is likely due to the detachment of 89 Zr from DFO and uptake by the bones After 72 hours, biodistribution results showed that about 6% of radioactivity was in the blood whereas major of radioactivity was present in the liver, spleen, and kidney (Figure 6(a)). The non-specific antibody 3A4 (negative control antibody) also showed similar distribution with a low uptake (Figure 6(b)).
Data showed about 1% radioactivity of [ 89 Zr]-DFO-SB5 was found in the tumor (Green bars), whereas non-specific uptake of radioactivity was present in blood, liver, and spleen. Excessive radioactivity in kidney showed clearance of the radioactivity. Non-specific labeled antibody [ 89 Zr]-DFO-3A4 (blue) also showed distribution in other organs but to a lesser extent than that of [ 89 Zr]-DFO-SB5. However, the uptake of [ 89 Zr]-DFO-3A4 was lower than that of [ 89 Zr]-DFO-SB5.

Discussion
The PET/CT imaging is a non-invasive technique that can be used to gain knowledge for cancer progression, metastasis [25]. In this study, conjugation of anti-SAS1B (SB5) with the chelator DFO was performed before labeling with 89 Zr according to a published protocol [26]. The results showed greater than 90% conjugation yield of SB5 with DFO. HPLC with a size exclusion column was used to evaluate the cold conjugation yield. The conjugation solution of anti-SAS1B with  were harvested and the radioactivity was measured using gamma-counting as described in the text above. The data is reported as radioactivity percent of injected dose per gram (%ID/g). DFO separately was injected into the HPLC at the beginning of the reaction to identify the retention time of each one, based on their molecular weight. Since the antibody has a higher molecular weight than the chelator, the antibody retention time was 24 minutes, which is less than the retention time of the chelator DFO was at 37 minutes. When the cold conjugation was injected, the HPLC chromatograph showed two peaks. The area under the peaks represented the amount of antibody and the chelator in the conjugation reaction. Therefore, HPLC was used to quantitatively monitor the conjugation yield. The area under the peaks reflected the degree of conjugation efficiency.
Once reaction parameters are optimized and validated by radio-HPLC, a second chromatographic method, radio-TLC, was used to evaluate [ 89 Zr]-DFO-SB5 labelling. This offers a quick and straightforward approach based on the migration of radiolabeled antibodies versus the migration of non-antibody radioactivity on the TLC plate. This method was adopted for rapid evaluation once being validated by radio-HPLC. This is done by scanning the entire plate using faster radiation detection. Labeled and the chromatograph showed about 80% of SB5 was labeled with 89 Zr. Following the successful radiolabeling of SB5 with different yields, the radiolabeled antibodies were purified from non-antibody radioactivity. That resulted in a 97% yield (Figure 3).
We used CT and PET images to measure biodistribution. In general, PET/CT images of [ 89 Zr]-DFO-SB5 uptake reflect the biodistribution profile, including some uptake in tumors ( Figure 5). The bone uptake is indicative of 89 Zr detaching from the radio-conjugate and accumulation in bone. This observation has been reported in other studies [27] [28] [29]. The overall biodistribution of [ 89 Zr]-DFO-SB5 (specific for SAS1B) and [ 89 Zr]-DFO-3A4 (presumed to be non-specific for SAS1B) is similar across several tissues, including blood. The SNU tumor uptake was slightly higher for [ 89 Zr]-DFO-SB5 than for [ 89 Zr]-DFO-3A4, although the significance of this difference was not determined.
It has been shown by western blot that SNU cells express more SAS1B antigen on their surface compared to 3A4-SAS1B expression level [11]. It is also worth noting that the uptake of [ 89 Zr]-DFO-3A4 is 2 -3 times lower than that of [ 89 Zr]-DFO-SB5 in both liver and spleen at 72 hours post-injection.
Eventually, a ligand that is both octadentate and oxygen-rich is believed to be the most stable Zr 4+ chelator since it would be able to incorporate all eight coordination sites of zirconium [22]. In theory, this novel high stability Zr 4+ ligand would minimize the uptake of liberated Zr 4+ in the bone and other non-targeted tissues. To date, the design, synthesis, and evaluation of such a Zr 4+ chelate require further research.

Conclusion
This is a preliminary study to conjugate 89 Zr radioactive tracer to anti-SAS1B. Our data showed limited success validating the conjugation and stability of tracer labelling with the antibody. However, it was of some concern that the radioac-tivity was also detected in other organs, this may be due to detachment of 89 Zr from the antibody. Further experiments are required to design more stable radioactive tracer labelling using other chelators such as HOPO [30] [31] [32].