The study aimed to evaluate a PET tracer ⁸⁹Zr-CTB006 for detecting DR5 expression status quantitatively and non-invasively from animals to patients, and validate the benefit from CTB006 therapy in patients with tumor ⁸⁹Zr-CTB006 uptake. The clinical trial was an open-label, single site, prospective study (figure 1) and registered on Chinese Clinical Trail Registry (ChiCTR2100042022). The objective was to determine the safety, dosimetry, biodistribution and the tumor uptake of ⁸⁹Zr-CTB006, and investigate the potential of the ⁸⁹Zr-CTB006 PET/CT to detect DR5 expression and screen DR5 overexpression patients with gastrointestinal cancers. All patients provided written informed consents.

The Colo205 cell line, N87 cell line were purchased from American type culture collection. ⁸⁹Zr was produced and purified by the Nuclear Medicine Department of Beijing Cancer Hospital. The CTB006 monoclonal antibody, Balb/c mice, mice-bearing Colo205 xenografts and Sp2/0 xenografts, mice-bearing CS225 and CS263 patient-derived tumor xenografts (PDX) were provided by Sinotau (Beijing, China). All procedures in studies involving animals were performed in accordance with the ethical standards of the institutional and/or national research committee.

⁸⁹Zr was coupled with CTB006 via the bifunctional metal ion chelate deferoxamine in solution with pH 7.0 for 1 hour. Then a PD-10 column was used to generated ⁸⁹Zr-CTB006 with radiochemical purity (RP) of >95.0%. Quality control was conducted using radioactive thin-layer chromatography (Bioscan, IAR-2000, Washington, District of Columbia, USA) and high-performance liquid chromatography (Agilent, Lake Forest, California, USA) in a standard protocol.

A serial dilution of DR5 positive Colo205 cells (1×10⁷/ mL, 5×10⁶/ mL, 2.5×10⁶/ mL, 1.25×10⁶/ mL, 0.625×10⁶/ mL, 0.31×10⁶/ mL) and DR5 negative N87 cells (1×10⁷/ mL), BGC803(1×10⁷/ mL) were incubated for 1 hour with 4 µg/mL (7.4 Bq) at room temperature. After incubation, cells were centrifuged and the activity in the cell pellet was measured in a gamma counter.

Female mice-bearing Colo205 xenografts (DR5 positive model of human colorectal cancer) and Sp2/0 xenografts (DR5 negative model of human myeloma) were divided into experimental group (n=4), blocking group(n=4), control group(n=4) and free ⁸⁹Zr group (n=2). For PET/CT imaging, 3.7MBq ⁸⁹Zr-CTB006 was injected per mouse for the experiment group, blocking group, control group while 30 mg/kg CTB006 was coinjected for the blocking group, and ⁸⁹Zr was injected per mouse for the free ⁸⁹Zr group. Static PET/CT scans were performed for 10 min at 1 hour, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 312 hours after injection with the SNPC-103 micro-PET/CT (Pingsheng, China). After reconstruction, the images were processed by image processing software PMOD (Zürich, Switzerland). Mice-bearing CS225 and CS263 xenografts, two PDX established from a lung cancer patient with high DR5 expression (with RNAscope score of 4) and a patient with colorectal cancer with medium DR5 expression (with RNAscope score of 3), respectively, were obtained. Static PET/CT scans were performed for 30 min 14 days after administration of 7.4MBq ⁸⁹Zr-CTB006 per mouse (n=3). After scanning, the original data were reconstructed and processed by image processing software. The mice were sacrificed to obtain major organs, which were weighed by balance and measured by the gamma count to calculate the %ID/g value.

For the prospective clinical trial, patients with pathological confirmed gastrointestinal cancers who would undergo surgical operation, with an Eastern Cooperative Oncology Group performance status ≤2, were eligible. Other inclusion criteria were an age between 18–75 years; a life expectancy of at least 3 months; and adequate liver and renal function. Patients who were pregnant, allergic to research drugs, unable to tolerate the whole examination, or suffering from claustrophobia were excluded.

All patients underwent ¹⁸F-flurodeoxyglucose (¹⁸F-FDG) PET/CT and ⁸⁹Zr-CTB006 PET/CT after 52.77±11 MBq ⁸⁹Zr-CTB006 injection. The first 6 patients underwent ⁸⁹Zr-CTB006 PET/CT at 2 hours, 24 hours, 48 hours, 72 hours, 120 hours after injection of ⁸⁹Zr-CTB006 with Siemens Biograph mCT Flow 64 scanner (Erlangen, Germany). Others underwent ⁸⁹Zr-CTB006 PET/CT at 2 hours, 48 hours, 72 hours after the ⁸⁹Zr-CTB006 injection. PET images were acquired from head to upper thigh with scan speeds of 0.8 mm/s, 0.5 mm/s, 0.4 mm/s, 0.3 mm/s and 0.2 mm/s at 2 hour, 24 hours, 48 hours, 72 hours/96 hours, 120 hours, respectively, and were reconstructed using the ordered-subset expectation maximization method. Images were interpreted by two experienced nuclear medicine physicians who were aware of the patient’s history. The maximum standardized uptake value (SUV_max) of the tumors was measured. The uptake ratio of the tumor to the adrenal glands (SUV_ratio) was calculated with the use of SUV_max of the tumor and SUV_mean of the adrenal glands.

SUV_mean in main organs based on the whole-body uptake of ⁸⁹Zr-CTB006 was evaluated. Volume of interests were drawn on the main organs including the brain, lung, heart, liver, spleen, kidneys, muscle, and bone on the PET/CT images to measure their activity concentration with the IntelliSpace Portal workstations (Philips, Netherlands). Values of standard male/female organ masses were taken from the Organ Level Internal Dose Assessment (OLINDA/EXM) software (V.2.2, HERMES Medical Solutions, Canada) and the human organ dosimetry was estimated.

An mRNA in situ hybridization (ISH) technique named RNAscope was used to investigate the DR5 expression in the tumor lesions. RNAscope 2.5 HD Brown Detection Kit (Advanced Cell Diagnostics, Hayward, California, USA) and RNAscope Probes for DR5 mRNA were applied, according to the manufacturer’s instruction.14 Probes are hybridized for 2 hours and followed by a cascade of signal amplification for enhancing the signal. According to the signal number in the cells, the staining results were scoring into five levels (score 0–4, online supplemental table 1). Each sample was quality controlled with a probe specific to the house keeping PPIB mRNA for positive control and a probe specific to bacterial dapB gene for negative control.

All patients underwent the measurement of vital signs (blood pressure, heart rates, respiratory rate, and temperature), clinical laboratory testing (blood routine examination, routine urinalysis, hepatic and renal function examination) and electrocardiography before and 72 hours after ⁸⁹Zr-CTB006 administration.

Descriptive statistics included median or mean and SD. Comparisons between groups were done using the one-samples t-test and relationship between two groups were done using pearson correlation analysis. The receiver operating characteristic (ROC) curves were computed for SUV_max and SUV_ratio for evaluating the ability to detect DR5 expression status and the optimum cut-off value was obtained with Yoden index. Statistical analysis was performed with the IBM SPSS software (V.20.0; IBM).

CTB006 was efficiently radiolabeled with ⁸⁹Zr and passed the quality control for clinical application (online supplemental table 2). The average radiolabeling efficiency and RP are 96.9%±0.5% (n=21）and over 97.4% (n=21), respectively. ⁸⁹Zr-CTB006 showed the highest binding affinity for Colo205 cells while the binding affinities for N87 and BGC803 cells were lower. The dissociation constant (K_d) of ⁸⁹Zr-CTB006 was 65.24 nmol, as calculated by a one site saturation binding affinity test (online supplemental figure 1G).

Female Balb/c mice were injected with 5.18 MBq ⁸⁹Zr-CTB006. The biodistribution results showed high uptake in the heart, liver, lung, spleen, kidney and blood. The uptake in the marrow increased slowly while the uptake in the other organs decreased with time.

Mice with Colo205 and Sp0/2 tumors were injected with 3.7 MBq ⁸⁹Zr-CTB006 for imaging via the tail vein. The Colo205 tumor in the experimental group showed obvious uptake of ⁸⁹Zr-CTB006 and had the highest uptake at 168 hours after administration (%ID/g: 12.75±3.81) (online supplemental figure 1). The experimental group showed significantly higher uptake in tumors than the blocking group (%ID/g: 12.75±3.81 vs 8.21±1.16, p<0.05), the control group (%ID/g: 12.75±3.81 vs 4.34±0.55, p<0.01) and the free ⁸⁹Zr group (%ID/g: 12.75±3.81 vs 3.88±1.93, p<0.01).

PDX model mice were injected with 7.4 MBq ⁸⁹Zr-CTB006 via the tail vein and PET/CT was performed at 168 hours after injection. The images showed that uptake of the tracer in CS225 tumors was significantly higher than that in CS263 tumors (p=0.037) (figure 2). The RNAscope scores of CS225 and CS263 tumors were 4 and 3, respectively, which was consistent with the uptake of ⁸⁹Zr-CTB006. There was no significant difference in the biodistribution in normal organs between the two groups.

In the clinical study, 21 patients (14 males, 7 females), including 12 patients with colorectal cancers and 9 patients with gastric cancers with a mean age of 58 years (range, 35–75), were enrolled from April 2019 to October 2019 (table 1). One patient withdrew for personal reasons after the first ⁸⁹Zr-CTB006 PET/CT scan.

The pathology results obtained through surgical operation or endoscopy showed that there were 18 adenocarcinomas and 3 adenocarcinomas with signet ring cell carcinoma. The tumor tissues of 18 patients who underwent surgical operation were obtained and DR5 expression levels were detected by RNAscope. The RNAscope scores of ten patients were 0; those of three patients’ were 1, two patients’ scores were 2; two patients’ scores were 3, and one patient’s score was 4.

A similar biodistribution pattern of ⁸⁹Zr-CTB006 was observed in all patients and was in line with the metabolism of ⁸⁹Zr labeled antibodies in vivo. The SUV_mean of the main organs of the first 6 patients was measured to investigate the biodistribution in humans (figure 3). At 2 hours, most of the activity on the PET images was concentrated in the heart and major arteries. The liver and spleen also showed high uptake but lower uptake than the blood. At 24 hours, the tracer in the blood began to clear and the signal was reduced, as was the case in the spleen and liver. At 48 hours and 72 hours, the tracer continued clearing from the blood, and the signals in the blood, liver and spleen were decreased. The kidney, brain, lung, muscle showed low uptake. Some patients experienced earlier high uptake in the intestine at 48 hours and 72 hours, which influenced the imaging of intestinal tumors. Strikingly, throughout the whole examination from 2 to 120 hours, the adrenal glands sustained high accumulation and remained more stable than other organs such as the liver and spleen.

Except for the tumors in two patients, one with diffuse-type, poorly differentiated adenocarcinoma with signet ring cell carcinoma and one with early gastric cancer, which showed no obvious uptake of ¹⁸F-FDG, the other 18 patients showed different levels of uptake in tumors on ¹⁸F-FDG PET/CT. The two ¹⁸F-FDG negative patients also showed no uptake of ⁸⁹Zr-CTB006. The other patients showed different levels of uptake of ⁸⁹Zr-CTB006 with a mean SUV_max of 6.63±3.29 (range, 1.8–13.8) and the heterogeneity of uptake in tumors was more obvious than that of ¹⁸F-FDG. At 24 hours, the tumor was distinguishable but had low contrast to the background. The uptake of tumors peaked at 48 hours in most patients and was still visible at 120 hours. Optimal tumor visualization was generally obtained at 72 hours after injection with the highest tumor background ratio. The highest SUV_max of 13.8 in the tumor was observed in a patient with colon cancer (No.7). Uptake in colorectal cancers was significantly higher than that in gastric cancers (SUV_max 48 hours: 7.61±3.19 vs 3.96±1.56, p=0.008; SUV_max 72 hours: 7.61±3.04 vs 3.96±1.82, p=0.007) (figure 4C,D). There was a significant positive correlation between the SUV_max of tumors on ⁸⁹Zr-CTB006 PET/CT at 72 hours and that on ¹⁸F-FDG PET/CT (R: 0.785, p<0.001) (online supplemental figure 2B).

Uptake of ⁸⁹Zr-CTB006 was also observed in most metastatic lesions but varied in different sites. The liver metastasis had the highest uptake in a female patient in her early 60s with sigmoid colon cancer (no.7), showing uneven uptake of ⁸⁹Zr-CTB006 with an SUV_max of 8.8 (72 hours) (figure 5B–D). A gastric cancer female patient in her early 40s (no. 16) with perineal and bone metastasis showed high uptake in the metastatic lesions, and the bone metastasis showed higher uptake on ⁸⁹Zr-CTB006 PET/CT than on ¹⁸F-FDG PET/CT (online supplemental figure 3). The uptake of ⁸⁹Zr-CTB006 at 48 hours in the metastatic lesions was significantly lower than that in the primary lesions in the operation cohort (SUV_max: 6.97±3.14 vs 4.22±2.45, p=0.0135), but not at 72 hours (SUV_max: 5.18±2.63 vs 3.52±2.19, p=0.0704) (online supplemental figure 4A,B).

The uptake of ⁸⁹Zr-CTB006 in tumors at 48 hours was significantly higher in gastric cancer patients with stage III–IV disease than in those with stage I–II disease(SUV_max: 4.82±1.63 vs 2.2, p<0.0001). Nevertheless, uptake in colorectal cancer patients with stage III–IV disease was significantly lower than that in patients with stage I–II disease (SUV_max: 6.09±2.25 vs 10.48±2.96, p=0.015) (online supplemental figure 4C). No difference in uptake of ⁸⁹Zr-CTB006 was observed between different degrees of differentiation or different pathological types of gastric cancers or colorectal cancers.

The SUV_max of the primary tumors in patients with colorectal cancers who had received radiotherapy or chemotherapy was significantly lower than that in patients without treatment on the 48 hours ⁸⁹Zr-CTB006 PET/CT (SUV_max: 4.83±0.51 vs 8.94±3.28, p=0.005) (online supplemental figure 2C).

The tumor uptake of ⁸⁹Zr-CTB006 in patients with RNAscope scores of 3–4 was significantly higher than that in patients with scores of 0–2 (SUV_max 48 hours: 12.50±1.47 vs 5.49±2.26, p<0.001; 72 hours: 9.30±2.00 vs 4.41±2.00, p=0.001) (figures 4A,B and 6). The tumor uptake of ¹⁸F-FDG was also significantly higher in patients with RNAscope scores of 3–4 than in those with scores of 0–2 (SUV_max: 17.55±3.90 vs 9.60±6.05, p=0.046) (online supplemental figure 2D).

The adrenal glands of all patients showed stable uptake during the whole examination, and patients with higher tumor uptake than adrenal glands were more likely to have DR5 overexpression (figure 4E). ROC curves were obtained by comparing DR5 expression status with SUV_max at 48 hours, 72 hours and SUV_ratio (ratio of SUV_max of tumor vs SUV_mean of the adrenal glands) at 48 hours on ⁸⁹Zr-CTB006 PET/CT (figure 4F). The areas under the curve were 0.976, 0.905 and 0.9091, respectively, with p<0.05 (figure 4F). An SUVmax of 9.3 at 48 hours and 6.3 at 72 hours could be used to discriminate DR5 expression status of tumors both with a sensitivity and specificity of 100% and 92.9%, respectively. When a ratio of 1.38 was used to discriminate the DR5 expression status of tumors, the sensitivity and specificity of the model were 100% and 78.6%, respectively.

There were no related adverse events or clinically detectable pharmacological effects in any of the patients. No significant changes were observed in vital signs, or laboratory tests or ECG. The dosimetry estimated from OLINDA showed that the organs receiving the highest absorbed doses were the liver and spleen with mean values of 1.42 and 1.17 mGy/MBq, respectively (online supplemental table 3). The mean effective dose was 0.349 mSv/MBq (1.29 rem/mCi).