Background: The targeting of antigen expressed on the surface of tumor cells by an antigen-specific mAb can be hampered by soluble shed antigen in the blood circulation and the interstitial space of tumor because it can act as a decoy to the tumor targeting of mAb, even though its shed level in the circulation provides information on tumor stage and tumor treatment efficacy. Mesothelin, a membrane glycoprotein of 40 kDa, is over-expressed in many epithelial cancers (almost 100% of all pancreatic adenocarcinomas and mesotheliomas, in > 65 % of ovarian adenocarcinomas, and in many non-small cell lung cancers) and has limited expression in normal tissues. Mesothelin is internalized actively into the cells cytosol and also is shed from the cell surface, generating soluble mesothelin in the blood circulation and in the tumors interstitial space. We previously radiolabeled MORAb-009 with In-111, and In-111-labeled MORAb-009 has been evaluated in preclinical and clinical studies. MORAb-009 is a high affinity chimeric (mouse/human) antibody that shares 82.6% amino acid sequence identity to a human IgG1κ and is directed against mesothelin. Objectives: In the past year, we developed a method to radiolabel amatuzimab (MORAb-009) with Cu-64 and investigated the effect of the injection dose of MORAb-009, the tumor size and the level of circulating shed mesothelin on the uptake of the Cu-64-labeled antibody in mesothelin-positive tumor and organs by biodistribution and positron emission tomography (PET) imaging studies. Positron emission tomography (PET) offers high resolution and sensitivity combined with the unique ability to measure tissue concentrations of radioactivity in three dimensions. Methods: 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to MORAb-009 and labeled with Cu-64Cl2 in 0.25M acetate buffer, pH 4.2. The resulting Cu-64-NOTA-MORAb-009 was purified with a PD-10 column. The immunoreactivity of Cu-64-NOTA-MORAb-009 was 80.1+/-10.7 % (N=3). To investigate the effect of dose or the effect of tumor size, the biodistribution (BD) was performed in groups of nude mice (n=5) with mesothelin-expressing A431/H9 tumors (range, 80 to 300 mm3) one day after iv injection of Cu-64-MORAb-009 (10 microCi) containing a total MORAb-009 dose of 2, 30, or 60 microg. The BD and PET imaging were also performed 3, 24 and 48 h after injecting a total dose of 30 microg (10 microCi for BD), and 2 or 60 microg (300 microCi for PET), respectively. For the BD studies, the animals were euthanized at 3, 24, and 48 h by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Blood and various organs were removed and weighed, and their decay corrected radioactivity counts were measured with a gamma-counter (Wallac, Inc., Perkin-Elmer, Inc., Boston, MA). The percentage of injected dose per gram (% ID/g) of the blood or each organ was calculated and normalized to a 20-gram mouse. For PET imaging studies, longitudinal 15 min static PET scans were performed on athymic mice (n=5) using a Siemens Inveon micro PET scanner (Siemens Preclinical Solutions, Knoxville, TN) at 3, 24, and 48 h post-injection (p.i.). The mice were euthanized after the imaging session. The images were reconstructed with a 3-dimensional ordered-subset expectation maximization/maximum a posteriori (OSEM3D/MAP) algorithm, with no attenuation or scatter correction. The reconstructed pixel size was 0.77 0.77 0.79 mm on a 128 128 159 imaging matrix. Tumors were manually segmented and each image analysis was performed using ASIPRO software (provided by Siemens, v6.8.0.0) on decay-corrected whole-body images. To characterize the accumulation of the probe in tumors, the region-of-interest (ROI) were drawn manually on individual tumor area, liver, spleen, muscle, and heart. The %ID/g was calculated for mice at 3, 24, and 48 h p.i.. Activity concentration (cps/voxel) was determined by maximum pixel value and was then converted to microCi/cc using a cross calibration factor. Activity concentration (microCi/cc) was divided by injected dose to get the image derived percent injected dose (%ID/g) assuming the tissue density as 1 g/ml. Results: Comparing the results of the BDs from three different injection doses, the major difference was shown in the uptake (% ID/g) of the radiolabel in tumor, liver and blood at 24 h p.i.. The tumor uptake (9.0+/-2.6 for 2 microg vs.17.7+/-4.4 %ID/g for 30 microg, p=0.02 and 16.4+/-4.6 %ID/g for 60 microg, p=0.02) and blood retention of the radiolabel (5.1+/-1.0 for 2 microg vs.12.9+/-3.7 %ID/g for 30 microg, p=0.06 and 10.0+/-0.7 %ID/g for 60 microg, p<0.003) from 30 and 60 microg doses were greater than those from 2 microg dose, whereas the liver uptake (30.8+/-5.6 for 2 microg vs. 16.2+/-3.2 %ID/g for 30 microg, p<0.002 and 15.9+/-3.3 %ID/g for 60 microg, p<0.002) was smaller. The BD studies also demonstrated a positive correlation between tumor size (or the level of shed mesothelin in blood) and liver uptake of the radiolabel for each co-injected MORAb-009 dose. However, there was a negative correlation between tumor size (or the shed mesothelin level) and tumor uptake and between tumor size and blood retention. These findings were confirmed by the PET imaging study. The PET images visualized tumors as early as 3 h p.i. for both 2 and 60 microg MORAb-009 dose. At this time, the major radioactivity was shown in heart (blood pool), liver and spleen for both 2 and 60 microg MORAb-009 injections and the radioactivity signal was also present in the lower abdomen especially for 2 microg injection, indicating that a significant percentage of the injected dose was excreted via both the hepatobiliary and renal systems for 2 microg injection. At 24 and 48 h p.i., the radioactivity signal in the tumor remained relatively unchanged compared to that at 3 h p.i. for 2 microg whereas the tumor signal at later time points was much stronger than that at 3 h p.i. for 60 microg, indicating that this higher injection dose is advantageous for the tumor visualization by PET. Both transverse and coronal PET images clearly displayed tumor uptake that was diffused into the center of the tumors for both 2 and 60 microg MORAb-009 injections. The tumor and organ uptake values were determined by the region-of-interested analysis (ROI) on PET images. Comparing the effect of two different injection doses on tumor, blood and organ accumulations of the radiolabel, the radioactivity of 2 microg injection cleared more rapidly from the blood than that of 60 microg injection (34.4+/-4.4 vs. 33.7+/-1.8 %ID/g at 3 h, 5.4+/-1.3 vs. 13.1+/-1.5 %ID/g, P<0.001 at 24 h, and 2.4+/-0.5 vs. 7.6+/-2.6 %ID/g, p<0.01 at 48 h). This difference in blood clearance was reflected in a higher tumor uptake (25.4+/-6.5 vs. 13.2+/-1.8 %ID/g, p=0.01) and lower liver uptake (21.9+/-3.3 vs. 31.5+/-6.5 %ID/g, p=0.03) for 60 microg injection at 24 h p.i. than for 2 microg injection. The tumor-to-liver ratio for 60 microg showed a value > 1 at 24 and 48 h p.i., whereas the ratios for 2 microg dose at the same time points was < 1, indicating that it is feasible to visualize tumors in the upper abdomen with the higher injection dose. Conclusion: Our studies demonstrated that the injection of a MORAb-009 dose capable of saturating the shed mesothelin in blood and extracellular space could provide a beneficial effect in maximizing tumor uptake while maintaining minimum liver and spleen uptakes, and in helping the radiolabel penetrate deeply into the tumor core. Our studies using a nude mouse model of A431/H9 tumor demonstrated that a 30 to 60 fold molar excess (30 to 60 microg) of MORAb-009 to the steady state concentration (4 nM) of shed mesothelin in blood could adequately neutralize the shed mesothelin in blood and extracellular space.
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