2.2 Differential expression and prognosis of MUC18 in GEPIA2
The internet database GEPIA2 (Gene Expression Profiling Interactive Analysis 2) was used to assess MUC18 gene expression in several types of tumors and normal tissues by matching TCGA normal and GTEx data. Survival maps, survival curves for overall survival (OS) and disease-free survival (DFS) were generated based on the gene expression patterns of 33 different cancers using GEPIA2 through log-rank and Mantel–Cox tests, and the group cutoff was based on the median (cutoff-high: 50%, cutoff-low: 50%). The hazard ratio (HR) was calculated based on the Cox PH model, and the confidence interval (CI) was 95%. Special survival graphs with log-rank p values were generated using the “Survival Analysis” module in GEPIA2. The results were considered to be statistically significant if p < 0.05. The data were downloaded from http://gepia2.cancer-pku.cn/\#analysis.
2.3 DFO conjugation and89Zr-labeling of IP150
The MUC18-specific mAb IP150 was conjugated to DFO-NCS (Innochem) via lysine residues. DFO was prepared at 10 mg/mL (20 μL) in neat DMSO and mixed with 0.1 M Na2CO3 (pH 9.0, 200 μL). Then, 500 μg of mAb was added and incubated at 37 °C for 60 min. The nonconjugated chelate was removed by size exclusion chromatography using metal-free PBS as the eluent and a PD-10 column (Cytiva). The products were collected into five centrifuge tubes (500 µL). Then, the concentration was determined by a NanoDrop, and the average molecular weight of the mAb (20 µL) before and after the reaction was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
The 89Zr-IP150 probe was labeled by reacting89Zr-oxalate and DFO-IP150 at 37 ℃ for 60 min (Figure 1A), and 89Zr-oxalate (48.1 MBq; 125 µL in 1 M oxalic acid) was mixed with HEPES buffer (1375 µL, pH 7.0). Then, 200 µL of DFO-IP150 (200 µg) was added to the mixed liquor. After incubating at room temperature for 60 minutes, the reaction mixture was purified on a PD-10 column using metal-free PBS as the eluent. Similar to DFO conjugation, 89Zr-IP150 was collected in five centrifuge tubes (500 µL). Before and after purification, 3 μL of sample was spotted on a TLC silica gel strip and developed in 0.05 M citric acid (pH 5.0)/saturated EDTA as the eluent, after which the radiolabeling rate and radiochemical purity were analyzed by BIOSCAN. Two hundred microliters of 89Zr-IP150 in 5% HSA (Baxter) was incubated at 4 ℃ to assess the in vitro stability by analyzing the radiochemical purity every 24 h.
2.4 Binding properties of IP150 and DFO-IP150 to the