Positron Emission Tomography Imaging of Tumor Angiogenesis with a ^61/64Cu-Labeled F(ab′)₂ Antibody Fragment

Abstract

The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')2 fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after 61/64Cu-labeling. TRC105-F(ab')2 of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-F(ab')2 (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of > 75% (specific activity: ~115 GBq/μmol). PET imaging revealed rapid tumor uptake of 64Cu-NOTA TRC105-F(ab')2 in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7 %ID/g at 0.5, 3, 16, 24, and 48 h post-injection respectively; n = 4). Since tumor uptake peaked at 3 h post-injection, 61Cu-NOTA-TRC105-F(ab')2 also gave good tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')2 fragment led to more rapid tumor uptake (which peaked at 3 h post-injection) than radiolabeled intact antibody (which often peaked after 24 h post-injection), which may allow for same day immunoPET imaging in future clinical studies.

Influence of molecular size on tissue distribution of antibody fragments

“…5 However, even molecules larger than 70 kDa have been reported to accumulate in kidney to a limited extent. [6][7][8] Another factor that could influence these high BC values for the kidney is the distribution of the free label to the kidney. However, since we find increasing BC values with decreasing size of antibody fragments irrespective of the labeling, this effect may not contribute significantly.…”

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

“…S3). Medium-sized fragments (100 kDa) such as anti-CD105 F(ab')2 have been shown to clear through both renal and hepatic pathways [35]. Since we have increased the diabody size from 50 kDa to roughly 90 kDa through the addition of a SpyTag/SpyCatcher, we expected a similar clearance profile.…”

Preparation of (177Lu-DOTA)n-PAMAM-[Nimotuzumab-F(ab’)2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancers Treatment

“…Nimotuzumab was fragmented using pepsin with a similar method as reported by Hao Hong, et al, [11]. Termination of the fragmentation process, unlike reported by R.D.…”

“…MAb fragments have been reported to have better tumor penetration, more rapid blood clearance, lower retention time in non-target tissues and less immunogenicity. Uptake by mAb fragments is eight time faster than by intact monoclonal antibodies, so imaging can be performed on the same day as the administration of radioimmunoconjugate [11].…”