Effect of chelator conjugation level and injection dose on tumor and organ uptake of 111In-labeled MORAb-009, an anti-mesothelin antibody

Abstract

Introduction Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin and the effect of MORAb dose on the biodistribution of 111In labeled MORAb-009. Methods We used nude mice bearing A431/K5 tumor as a mesothelin-positive tumor model and A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5, and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed-mesothelin in the circulation, the biodistribution studies were performed after the i.v. co-injection of the 111In labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses, 0.2, 2, and 30 μg of MORAb-009. Results The tumor uptake in A431/K5 tumor was 4 times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin-mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in biodistribution of the 111In label. The 111In labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake, and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30 μg dose produced higher tumor uptake than the 0.2 and 2 μg doses whereas the 30 μg dose produced lower liver and spleen uptakes than the 0.2 μg dose. Conclusion This study demonstrates that the number of chelate conjugation and the injected dose are two important parameters to achieve high tumor and low non-target organ uptake of 111In labeled MORAb-009. This study also suggests that the injected dose of mAb could be individualized based on the tumor size or the blood level of shed-antigen in a patient to achieve the ideal tumor-to-organ radioactivity ratios.

Tumor and organ uptake of 64Cu-labeled MORAb-009 (amatuximab), an anti-mesothelin antibody, by PET imaging and biodistribution studies

“…The BD and PET imaging studies of 64 Cu-amatuximab in a mouse model of A431/H9 tumors demonstrated that the tumor uptake and blood retention of 64 Cu-amatuximab was inversely proportional to the tumor size whereas the liver uptake was positively correlated to the tumor size (Fig.2), similar to the results obtained previously with 111 In-amatuximab using A431/K5 tumors [14]. The PET imaging and uptake data from the ROI analysis of the images best illustrated the effect of amatuximab dose (2 vs. 60 μg) on the blood retention and the uptake of 64 Cu-amatuximab in tumor and major organs (Fig.4).…”

“…Since shed mesothelin can act as a decoy preventing anti-mesothelin antibodies from targeting mesothelin-expressing tumors, we undertook our current research to find an optimum dose of amatuximab to neutralize the shed mesothelin and improve the tumor uptake of the antibody. We found in a nude mouse model of A431/K5 tumor (mesothelin-positive human epidermoid carcinoma cells) that the tumor uptake and blood retention of 111 In labeled amatuximab was inversely correlated to the tumor size, whereas the liver and spleen uptakes showed a positive correlation with tumor size [14]. However, we did not correlate the tumor and major organ uptakes with the concentration of circulating shed mesothelin since no methods to determine the shed mesothelin were available at the time the study of 111 In-labeled amatuximab was performed.…”

“…In this study we used 64 Cu to label MORAb-009. Amatuximab 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 [14, 15]. Mesothelin, an immunogenic 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 [16, 17].…”

“…Mesothelin is internalized actively into the cell’s cytosol and is shed from the tumor cell surface, generating soluble mesothelin in the tumor’s interstitial space, which can also be detected in circulation [18, 19]. We previously conjugated MORAb-009 with a bifunctional chelator, CHX-A″, for radiolabeling with 111 In [14]. Previously, 111 In-CHX-A″-MORAb-009 has been evaluated preclinically and in clinical studies [17, 20].…”

ImmunoPET with Anti-Mesothelin Antibody in Patients with Pancreatic and Ovarian Cancer before Anti-Mesothelin Antibody–Drug Conjugate Treatment

“…By recognizing the existence and extent of heterogeneity, PET imaging of a tumor-specific target adds valuable information for individualized treatment decisions. (45)(46)(47)(48). Moreover, an antibody targeting MSLN was recently conjugated to quantum dots encapsulated in micelles to detect human tumor xenografts in mice (49).…”