Cellular screening of Trop2 expression
Western blotting results of pancreatic cancer cell lines showed that T3M-4 and BXPC-3 had high Trop2 expression level, while PaTu8988, MiaPaCa-2, PANC-1, AsPC-1 and HPNE had low level (Fig. 1a). The semi-quantitative analysis of Trop2 divided by β-actin showed that the expression level of Trop2 in T3M-4 was higher than BXPC-3. Therefore, T3M-4 with higher Trop2 expression level and PaTu8988 with low level were selected for the evaluation of cell-antibody binding ability.
Antibodies binding kinetics
The binding kinetics of IMB1636 to Trop2 was measured by BLI. IMB1636 was loaded onto anti-human IgG Fc biosensors at 10 μg/mL and the Trop2 concentration was 1.56 nM, 3.12 nM, 6.25 nM, 12.50 nM, and 25 nM respectively. IMB1636 had strong binding affinity to Trop2 antigen according to the results of BLI. The association rate constant Kon is 6.08×105 Ms-1, the dissociation rate constant Koff is 1.59×10-3 s-1, and the corresponding equilibrium dissociation constant KD is 2.61×10-9 M (Fig. 1b).
Cellular binding ability
Shown by flow cytometry, T3M-4 cells had significant signal shift after incubating with IMB1636 at a concentration of 10 μg/mL, compared to non-specific IgG, FITC-labeled secondary antibody-only, cells-only groups, and PaTu8988 cell line (Fig. 1c). The result indicated that IMB1636 had a specific binding capacity with Trop2 on the cell surface of T3M-4. We finally chose T3M-4 and PaTu8988 cell lines to construct subcutaneous positive and negative pancreatic cancer tumor models.
Radiolabeling of 64Cu/177Lu-labeled IMB1636
IMB1636 was first conjugated with NOTA and DOTA, and then labeled with 64Cu and 177Lu used for immunoPET imaging and RIT experiments respectively (Fig. 1d). The radiochemical purity of 64Cu-NOTA-IMB1636 and 177Lu-DOTA-IMB1636 exceeded 95% and the range of specific activity was about 10 ~ 20 MBq/mg.
ImmunoPET imaging and biodistribution of 64Cu-NOTA-IMB1636
The maximum intensity projection (MIP) images showed that 64Cu-NOTA-IMB1636 in T3M-4 positive group (n = 4) could clearly display the tumor morphology at 4 h post injection (p.i.), and the tumor radioactive uptake gradually accumulated with time. The tumor uptake of 64Cu-NOTA-IMB1636 in T3M-4-blocked group (n=3) slightly increased, while PaTu8988 negative group (n = 4) was similar with background (Fig. 2a).
We further delineated the ROI and quantitatively analyzed the radioactive uptake of tumor, heart (blood), liver and kidney (Fig. 2b). The peak tumor uptake of 64Cu-NOTA-IMB1636 in T3M-4 was about 8.95 ± 1.07 %ID/g at 48 h p.i., which had significant differences withT3M-4-blocked and PaTu8988 groups (3.56 ± 0.18 %ID/g, 2.08 ± 0.19 %ID/g; P < 0.001). And there was significant difference between tumor uptake of 64Cu-NOTA-IMB1636 in T3M-4 and T3M-4-blocked groups (P = 0.003). The radioactive uptake of the heart (blood), liver and kidney gradually decreased with time. The liver uptake of 64Cu-NOTA-IMB1636 in T3M-4 was lower than T3M-4-blocked group (P = 0.004), and there was no statical difference between T3M-4-blocked and PaTu8988 group. There were no significant differences among the three groups in heart (blood) and kidney (P >0.05).
After the terminal time point imaging, the mice were euthanized for ex vivo biodistribution analysis (Fig. 2c). The tumor uptake of 64Cu-NOTA-IMB1636 in T3M-4, T3M-4-blocked, and PaTu8988 was 9.95 ± 0.47 %ID/g, 4.00 ± 0.52 %ID/g, and 1.45 ± 0.25 %ID/g respectively, which had significant differences among three groups (P < 0.001) and consistent with the in vivo PET imaging results. The radioactive uptake of blood, heart, spleen, and kidney in T3M-4-blocked group was higher than T3M-4 (P < 0.05), which may be caused by higher tumor uptake of 64Cu-NOTA-IMB1636 in T3M-4 group. There were no statistical differences in lung, stomach, intestine, pancreas, bladder, muscle, bone, brain, and skin among three groups (P > 0.05).
RIT of 177Lu-labeled IMB1636
The Trop2-overexpresing T3M-4 mice were photographed at 4 h, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days after a single-time tail vein injection, which visually displayed the changes of tumor size in 5 groups (Fig. 3a). Meanwhile, tumor volume and body weight of mice were measured. High-177Lu-IMB1636 group demonstrated strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62 % at 14 days p.i., significantly smaller than other groups (low-177Lu-IMB1636 = 172.89 ± 30.03 %, 177Lu-only = 391.57 ± 70.11 %, IMB1636-only = 282.84 ± 49.98 %, and PBS = 402.41 ± 66.06 %; P = 0.000 ~ 0.047; Fig. 3b). The tumor volume of high-177Lu-IMB1636 groups reached the maximum at 4 days p.i. (109.57 ± 6.19 %) and then gradually decreased with time, which may be related to the large molecular weight and the long blood circulation cycle of intact antibody. There was no statistically significant difference in tumor volume changes between the 177Lu-only and PBS groups (P = 0.866), but both were significantly different from the IMB1636-only group (P = 0.009 and 0.006).
The body weight of tumor-bearing mice in all groups showed a downward trend (Fig. 3c). The IMB1636-only group had the smallest change about 95.19 ± 6.46 % at 14 days, and had significant differences with high-177Lu-IMB1636, 177Lu-only, and PBS groups (89.98 ± 4.72 %, 90.34 ± 3.82 %, and 91.31 ± 10.37 %; P = 0.023, 0.016 and 0.027, respectively). There were no statistical differences among high-, low-177Lu-IMB1636, 177Lu-only, and PBS groups (P > 0.05).
SPECT imaging and distribution of 177Lu-labeled IMB1636
We performed in vivo SPECT imaging in high-177Lu-IMB1636 and 177Lu-only groups while tumor size monitoring (Fig. 4a). The high-177Lu-IMB1636 group could show tumor morphology at 2 days p.i., while the 177Lu-only group couldn’t display the tumor throughout. The radioactive uptakes of liver were high and gradually decreased over time.
Biodistribution analysis of high-, low-177Lu-IMB1636 and 177Lu-only groups were performed after the terminal imaging timepoint (Fig. 4b). The tumor radioactive uptake of high- and low-177Lu-IMB1636 groups was 5.72 ± 0.68 %ID/g and 4.04 ± 0.76 %ID/g respectively (P = 0.005). The tumor uptake of 177Lu-only was only 0.65 ± 0.52 %ID/g, which had significant difference with high- and low-177Lu-IMB1636 groups (P < 0.001). The liver and spleen radioactive uptake of 177Lu-only group was 31.52 ± 4.23 %ID/g and 31.02 ± 1.92 %ID/g respectively, which was significant different from high- and low-177Lu-IMB1636 groups (P < 0.001). There were no statistical differences in heart, lung, intestine, pancreas, bladder, brain, and skin among three groups (P > 0.05). SPECT imaging enabled visualization and dynamic monitoring of the RIT therapeutic efficacy of 177Lu-labeled IMB1636.
18F-FDG PET imaging
The high-177Lu-IMB1636 and 177Lu-only groups of RIT experiments underwent 18F-FDG PET imaging at 14 days p.i. (n = 3). The MIP images showed that the tumor of 177Lu-only group had higher 18F-FDGuptake than high-177Lu-IMB1636 (Fig. 4c). Quantitative ROI analysis was performed of tumors, the 18F-FDGuptake value of the high-177Lu-IMB1636 group was 2.74 ± 0.50 %ID/g and 177Lu-only was 4.19 ± 0.67 %ID/g, representing the RIT reduced the glucose metabolism of tumors, but there was no significant statistical difference between the two groups (P > 0.05, Fig. 4d).
Biodistribution and safety evaluation of 177Lu-DOTA-IMB1636 in normal mice
Normal BALB/c nude mice were euthanized for biodistribution at 4 hours, 4 days, 7 days, 10 days, and 14 days after the injection of 177Lu-DOTA-IMB1636 (~11.1 MBq, n = 3). Biodistribution results (Fig. 5a) showed that the radioactive uptake of blood, heart, lung, kidney, stomach, intestine, pancreas, bladder, muscle, brain, and skin gradually decreased with time, and the liver and spleen reached the peak at 7 days and 10 days p.i. (23.40 ± 0.26 %ID/g and 19.74 ± 1.29 %ID/g, respectively).
The hematological indexes of WBC, RBC, PLT and HGB gradually decreased within 4 days but recovered to normal level within 14 days, indicating that high-dose 177Lu-DOTA-IMB1636 had a certain myelosuppressive effect in the short term (Fig. 5b). The body weight changes ranging from 96.83 ± 4.49 % to 101.67 ± 2.36 %, and reached a minimum about 96.83 ± 4.49 % at 8 days p.i. (n = 3, Fig. 5c). These results suggesting no significant long-term blood toxicity of 177Lu-DOTA-IMB1636.
H&E and IHC staining were performed in major organs of T3M-4 and PaTu8988 tumor models (Fig. 6). The H&E staining results showed that there were no differences in heart, liver, kidney, lung, and stomach. There were different degrees of hemosiderin deposition in the spleen of 177Lu-only, high- and low-177Lu-IMB1636 goups, which may be the reason for the decrease in WBC and PLT p.i.. 177Lu-only group developed intestinal mucosal sloughing and submucosal telangiectasis, indicating that 177Lu may be more metabolized through intestine and resulting in radiation enteritis compared to other groups. The tumor H&E staining of high- and low-177Lu-IMB1636 groups had hyperchromatic nucleolus and hypochromatic cytoplasm, indicating that 177Lu-DOTA-IMB1636 may induce apoptosis through RIT. The Trop2 IHC staining of high-, low-177Lu-IMB1636 and IMB1636-only groups were lighter than that in other groups, illustrating the therapeutic effect of IMB1636 targeting Trop2. The tumor IHC staining of PaTu8988 showed almost no expression of Trop2, which was also consistent with the western blotting, flow cytometry and in vivo immunoPET imaging results.