Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide 89 Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide 124 I. Methods: To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant aHER2 Fab fused with 200 Pro, Ala, and Ser (PAS 200 ) residues was either conjugated with 124 I via an iodination reagent or coupled with deferoxamine (Df) and complexed with 89 Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1 nu mice bearing HER2-positive human tumor xenografts. Results: 89 Zr⋅Df-Fab-PAS 200 and 124 I-Fab-PAS 200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt 124 I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for 89 Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Conclusion: Both 89 Zr-and 124 I-labeled versions of aHER2 Fab-PAS 200 allowed PET tumor imaging with high contrast. With its residualizing radiometal, the tracer 89 Zr⋅Df-Fab-PAS 200 showed better in vivo stability and higher tumor uptake.