CD46 targeted ²¹²Pb alpha particle radioimmunotherapy for prostate cancer treatment

Hydrochloric acid (Trace Metal Grade) was purchased from Fisher Chemical. Pb-resin™ (100–150 μm) was purchased from Eichrom Technologies LLC (Lisle, IL USA). The fully human CD46-targeted monoclonal antibody, YS5, was produced and purified as described previously [15]. The radiolabeling chelator, p-SCN-Bn-DFO (1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)- 6,11,17, 22- tetraazaheptaeicosine] thiourea) (catalog No. B-705), and p-SCN-Bn-TCMC (S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane) (catalog No. B-1005) were purchased from Macrocyclics, Inc. ⁸⁹Zr oxalate was purchased from the Cyclotron Laboratory at University of Wisconsin, Madison (Madison, WI). The ²²⁴Ra/²¹²Pb generators used for these studies were purchased through the Isotopes Program, US Department of Energy, Oak Ridge National Laboratory (ORNL; Oak Ridge, TN USA). All other chemical reagents and materials were the highest grade available from Sigma-Aldrich and used as received without further purification (unless otherwise described). Radioactivity measurements of samples from the in vitro and in vivo experiments were performed on a calibrated Capintec CRC® -55tR Dose Calibrator (Florham Park, NJ) with dial setting at 101 or 158 at equilibrium, or the calibrated Hidex Automatic Gamma Counters using an energy window centered on the main gamma peak from ²¹²Pb at 238.6 keV. Radioactivity analyses were corrected for radioactive decay. (Note: Gamma counters are used for measuring lower activity levels in cell and biodistribution studies; dose calibrators are mostly used for assessing moderate-high activity levels. Different models of dose calibrators differ with respect to chamber gas pressure, chamber wall thickness and material, resulting in different output responses, especially for ²¹²Pb in equilibrium with progeny. It is recommended to verify the validity of the dial setting on each system as described by Napoli et al. [22] and follow the manufacturer’s instruction). Size-exclusion HPLC were performed for radiolabeled YS5 on Agilent Technologies 1260 Infinity system (Santa Clara, CA, USA) equipped with the software, Laura, from PETLablogic Inc. (LabLogic Systems, Inc., Tampa, FL, USA), Zenix-C-SEC-300 column (3 µm, 300 Å, 4.6 × 300 mm), UV detector, and a Flow-Ram radio-HPLC detector (NaI detector). UV absorbance for the TCMC assay was measured on a Nanodrop Spectrophotometer (Thermo Scientific Inc., Waltham, USA).

The apparent K_D value of ²¹²Pb-TCMC-YS5 binding to the CD46-expressing mCRPC cell line PC3 was determined by a saturation binding assay with concentrations at 5, 10, 25, 50, and 100 nM as previously described [20]. Radioactivity bound on tumor cells were measured on the calibrated Hidex Automatic Gamma Counters by using an energy window centered on the main gamma peak from ²¹²Pb at 238.6 keV.

CDX studies were performed using 5–7 week old male athymic nude mice (Nu/Nu Nude mice, Charles River, Wilmington, MA). PDX studies were performed using 6–8 weeks old male NSG mice (NOD/SCID/IL-2Rγ^−/−) (The Jackson Laboratory, Bar Harbor, ME). Animal studies were approved by the University of Virginia Institutional Animal Care and Use Committee and performed in compliance with guidelines from the Public Health Service Policy and Animal Welfare Act of the United States.

Approximately 3–5 weeks after tumor implantation, animals with PC3 xenografts reaching 100 mm³ in volume were anesthetized by isoflurane inhalation. For ⁸⁹Zr-DFO-YS5 PET imaging, 3.70–5.55 MBq (100–150 µCi, 30 µg/mouse) of ⁸⁹Zr-DFO-YS5 in saline was administered through the tail vein, and animals were imaged at 24, 72, and 120 h post injection, with a 20-min acquisition time by using microPET/CT Albira Trimodal PET/SPECT/CT Scanner (Bruker Corporation). PET imaging data were acquired in list mode and reconstructed using Albira Software Suite provided by the manufacturer. The resulting image data were then normalized to the administered activity to parameterize images in terms of %IA/g. Imaging data were viewed and processed using an open source Amide software. CT images were acquired following PET, and the CT data were used for attenuation correction for PET reconstruction, and anatomic reference.

To determine the safe dose for therapy study, a dose escalation study was performed in nude mice without tumor to determine toxicity. Groups of nude mice (n = 5–7/group) were given doses of ²¹²Pb-TCMC-YS5 at 0, 0.185 MBq, 0.37 MBq, 0.74 MBq, and 1.85 MBq (0, 5, 10, 20, and 50 µCi) per mouse. Mice were monitored for health status, signs of stress and body weight twice weekly. Mice were sacrificed when loss of body weight was more than 20%, or if the mice showed visible signs of distress, including lack of appetite, excessive lethargy, or formation of sores and rashes.

To evaluate the potential toxicity of ²¹²Pb-TCMC-YS5, hematological analysis and clinical chemistry were performed as reported [5]. CD-1 mice (Charles River Laboratories, Wilmington, MA) were administered with 0.74 MBq (20 µCi) of ²¹²Pb-TCMC-YS5 as study group. The control group received no treatment. Blood was collected via the orbital sinus into EDTA coated tubes and analyzed immediately using the Heska Element HT5 hematology analyzer (Barrie, Ontario, Canada). For blood chemistry, plasma was separated from blood by centrifugation (12,000 rpm, 10 min, at 4 °C) and was either studied immediately or stored at -80 °C for later analysis. Biomarkers for organ function such as albumin, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using enzyme-linked immunosorbent assay (ELISA) kits (ELISA kits for albumin, blood urea nitrogen, creatinine, alanine aminotransferase were purchased from MyBioSource, San Diego, CA; and ELISA kit for aspartate aminotransferase were purchased from Abcam, Boston, MA).

For the therapeutic study, mice bearing PC3 tumors about 100 mm³ in volume were given a single i.v. dose of ²¹²Pb-TCMC-YS5 at 20 µCi (740 kBq) (n = 5) or the unlabeled YS5 antibody (n = 7) or no treatment (n = 4). Animal number per group was based on past experience with prostate cancer models, not power calculation. Graft failures (i.e., no palpable tumor) at the time of randomization were excluded from the study. Tumor dimensions were measured and recorded 3 times weekly with a digital caliper, and tumor volumes were calculated using the following modified ellipsoid equation: volume = 0.5 × length x width x width [21]. Tumor growth was calculated as mean percent change in volume relative to the initial volume at the time of dosing. Animal survival data was analyzed by Kaplan–Meier analysis (GraphPad Prism 9). Body weight was measured twice weekly, and the mice were monitored for signs of distress. Mice were sacrificed if any one of the situations occurred: tumors reached 2.0 cm in diameter, loss of body weight more than 20%, or if the mice showed visible signs of distress, including lack of appetite, excessive lethargy, or formation of sores and rashes. The same health/humane endpoint applies to all our animal studies (subcu- and ortho-CDXs and PDX).

PC3-luc cells were derived from PC3 cells transfected with the firefly luciferase expressing plasmid from Addgene (Ubc.Luc.IRES.Puro, RRID:Addgene 33307) [25]. PC3-luc cells (3 × 10⁵) were injected into the murine prostate in 30 µL of PBS, as previously described [26‐28]. Tumor growth was monitored weekly by in vivo bioluminescent imaging (BLI) using a Lago X (Spectral Instruments Imaging, Tucson, AZ, USA). Images were acquired 10 min after intraperitoneal injection of 3 mg d-Luciferin Potassium Salt (Gold Biotechnology, Inc., St Louis MO, USA) in 200 µL 0.1 M phosphate buffered saline. Signals were quantified using Aura (Spectral Instruments Imaging) with the manual ROI tool to determine the amount of photons emitted for a given time.

For the therapeutic study, 30 days after PC3-luc cells were inoculated and tumor engraftment were confirmed by in vivo BLI, the mice were given a single i.v. dose of ²¹²Pb-TCMC-YS5 at 0.74 MBq (20 µCi) (n = 4) or the unlabeled YS5 antibody (n = 5). Tumor growth was monitored by in vivo BLI. Animal survival data was analyzed by Kaplan–Meier analysis.

PDX mice with subcutaneously grafted tumor (Cat#: TM00298 PR1996) were purchased from The Jackson Laboratory (Bar Harbor, ME USA). Animal number per group was based on past experience, not power calculation. When tumors size reaches about 100 mm³, mice were given a single i.v. dose of ²¹²Pb-TCMC-YS5 at 0.74 MBq (20 µCi) (n = 6), 0.37 MBq (10 µCi) (n = 8) or the unlabeled YS5 antibody (n = 4), each along with non-binding human IgG (0.5 mg per mouse) for blocking Fc receptors. The PDX is carried in NSG mice that are known to have a high background antibody binding compared with nude mice used in the CDX study, which can be reduced by injection of Fc blocking agents [29]. To confirm the tumor targeting in these PDX model, ⁸⁹Zr-DFO-YS5 PET imaging was performed using microPET/CT Albira Trimodal PET/SPECT/CT Scanner (Bruker Corporation) 3 days post i.v. injection of 3.70 – 5.55 MBq (100 – 150 µCi, 30 µg/mouse) of ⁸⁹Zr-DFO-YS5 in saline along with non-binding human IgG (0.5 mg per mouse) for blocking Fc receptors. In the blocking control group, the cold YS5 antibody was administered 2 days prior to ⁸⁹Zr-DFO-YS5 injection. Tumor dimensions were measured twice weekly with a digital caliper. Tumor growth was calculated as mean percent change in volume relative to the initial volume at the time of dosing. Animal survival data was analyzed by Kaplan–Meier analysis. Body weight and signs of distress were assessed twice weekly.

The chemistry for ²¹²Pb radiolabeling of the YS5 antibody was performed as shown in Fig. 1A. The ²¹²Pb radiolabeling process was adopted from the Pb-resin pre-concentration and column extraction method described previously, with modifications [24]. This process simplifies the handling of the isotope, and shortens the whole process to only 1.5 – 2 h with a labeling yield around 70%. The radiochemistry purity of the final ²¹²Pb-TCMC-YS5 was > 98% by size-exclusion HPLC (Fig. 1B). On average each YS5 antibody bears about 2 TCMC molecules as determined by a spectrophotometric assay [23] (Supplemental Fig. S1). The apparent binding affinity (apparent K_D) ²¹²Pb-TCMC-YS5 to PC3 cells was determined by an in vitro cell binding assay and found to be 21.7 ± 7.3 nM (Fig. 1C).

We performed a dose escalation study to identify the safe dose range using non-tumor bearing nude mice. The study included 4 dose groups (n = 5 per group) of 0.185 MBq (5 μCi), 0.37 MBq (10 μCi), 0.74 MBq (20 μCi), 1.85 MBq (50 μCi) and one control group receiving “cold” YS5 (radioactivity defined as 0 μCi, n = 7). As shown in Fig. 2A, at the highest dose of 1.85 MBq (50 μCi), there was 80% death and profound loss of body weight within 10 days. Other groups receiving 0.185 MBq (5 μCi), 0.37 MBq (10 μCi), or 0.74 MBq (20 μCi) exhibit dose-dependent body weight loss within the first two weeks followed by a rebound and stabilization throughout the investigation period and have a 100% survival rate (Fig. 2B). The control group showed gain of body weight in a slow but steady manner and no death. From this data, the tolerated dose range is 0.185—0.74 MBq (5 – 20 μCi), and 0.74 MBq (20 μCi) was chosen as the dose for therapeutic study in tumor-bearing mice.

We performed additional studies, blood cell count and blood chemistry, to evaluate both acute (day-14 post treatment) and long-term (day-90 post treatment) toxicity in mice receiving 0.74 MBq (20 μCi) of ²¹²Pb-TCMC-YS5. As shown in Fig. 3A and Supplemental Table S1, there is no significant difference between treatment vs. control group on either day-14 and Day-90 for any of the cell types studied, except for neutrophil that showed a significant increase on day-14 for the treatment group, suggesting an acute but not long-term neutrophil reaction to treatment. The blood chemistry analysis from albumin, creatinine, BUN, AST, ALT were in the normal range and exhibit no significant difference between the treatment vs. the control group (Fig. 3B and Supplemental Table S2). Those data suggest that 0.74 MBq (20 μCi) of ²¹²Pb-TCMC-YS5 is well tolerated and an appropriate dose to use for therapy study.

We first studied anti-tumor efficacy of ⁸⁹Zr-DFO-YS5 in the PC3 subcu-CDX model. We showed, by PET imaging (Fig. 4A) and biodistribution (Fig. 4B), that ⁸⁹Zr-DFO-YS5 targeted the PC3 tumor in vivo, with similar tumor uptake and tumor/non-target ratios compared with other prostate cancer CDX models in our previous study [21]. We performed a single dose efficacy study of ²¹²Pb-TCMC-YS5 using the PC3 subcu-CDX. A significant tumor inhibition in all treated mice was observed (100%, a complete response): tumor reduction began within the first week with a volume decrease up to 70%, and the inhibitory effect lasted over 40 days post administration of ²¹²Pb-TCMC-YS5 (Fig. 5A, with individual mouse data shown in Fig. 5B). Tumor growth resumed in 3/5 mice after 40 days post treatment (Supplemental Fig. S2). Only a transient body weight reduction was observed over the duration of the experiment (Fig. 5C), consistent with the result of the dose escalation study in non-tumor bearing mice. All mice recovered to their initial weights within 2 weeks post treatment. 80% mice in the treatment group survived 55 days post treatment (Fig. 5D). In contrast, the control groups (cold YS5 antibody and untreated) showed rapid tumor growth (Fig. 5A) and 100% death within 37 days (Fig. 5D). There is no significant difference between the two controls. There is a significant difference between the study and control groups (p = 0.001).

We further studied the therapeutic effect of a single dose ²¹²Pb-TCMC-YS5 in an intraprostate orthotopic mCRPC CDX (Fig. 6A). The PC3 cell line expressing the firefly luciferase reporter gene was used to create the ortho-CDX model. As shown in Fig. 6B, tumor growth, as measured by BLI (representative images shown in Supplemental Fig. S3), was significantly inhibited up to 70% and the duration of inhibition was over 60 days for the study group treated with 0.74 MBq (20 µCi) ²¹²Pb-TCMC-YS5, while tumors in the control group continuously grew. Body weight remained steady for the treatment group for the duration of the study (141 days post treatment) (Fig. 6C). Survival data is shown in Fig. 6D. The mice in the study group survived up to 141 days post treatment (end of experiment) with no death within the first 60 days post treatment. In contrast, no mice survived for the control group 36 days post treatment. The median survival was 100.5 days for the study group, which is significantly longer than the control group that is only 23 days (p = 0.005).

To broaden clinical applicability, we performed therapy study using a prostate cancer PDX model. We first used PET imaging with ⁸⁹Zr-DFO-YS5 to confirm tumor targeting by YS5 in vivo. The result showed a clear evidence of tumor targeting by YS5 (Fig. 7A) with tumor uptake above 20%IA/g from PET images (Fig. 7B). Uptake in other organs and tissues was minimal, consistent with our previous study using different PDX models [21]. We next studied the therapeutic efficacy of ²¹²Pb-TCMC-YS5. The PDX tumor was implanted subcutaneously, allowing ready tumor size measurement by caliper. We studied two doses, 0.74 MBq (20 μCi), and the lower dose of 0.37 MBq (10 μCi). As shown in Fig. 8A (normalized tumor volume) and Supplemental Fig. S4 (actual tumor volume of individual PDX), both doses inhibited tumor growth, with the higher dose of 0.74 MBq (20 μCi) showing a longer duration of inhibition. Body weight remained steady for the two treatment groups (0.37 MBq (10 μCi) and 0.74 MBq (20 μCi)) for the duration of the study (Fig. 8B). The median survival was at 34 days, 56 days and 99 days respectively for the control, 0.37 MBq (10 μCi), and 0.74 MBq (20 μCi) groups (Fig. 8C), showing a significant improvement over the control for both treatment groups (p < 0.0001 for the two study groups vs. the control group; p < 0.001 between the two study groups).