RT Journal Article
SR Electronic
T1 HSA Domain III as a protein scaffold with defined serum pharmacokinetics
JF Journal of Nuclear Medicine
JO J Nucl Med
FD Society of Nuclear Medicine
SP 1582
OP 1582
VO 50
IS supplement 2
A1 Vania  Kenanova
A1 Tove Olafsen
A1 Felix Bergara
A1 Anna Wu
YR 2009
UL http://jnm.snmjournals.org/content/50/supplement_2/1582.abstract
AB 1582 Objectives Our objective is to generate a protein scaffold of small molecular size, controlled serum persistence and architecture suitable for modification. When attached to tumor targeting agents with transient serum persistence, the scaffold would provide improved pharmacokinetics and residues for functional group conjugation. Methods We engineered several small (23 or 11 kDa), non-immunogenic scaffolds, based on HSA Domain III (DIII). HSA DIII binds the recycling FcRn receptor, thereby extending the serum persistence of albumin. To modulate the serum half life of the scaffold, we individually mutated conserved histidines H535, H510 and H464, and expressed the DIIIa and DIIIb half domains. To evaluate the effect of mutations, fusion proteins consisting of the anti-CEA diabody (Db) and either the HSA DIII wild type (WT), H535A, H510A or H464A variants were generated, labeled with 124I and injected in xenografted mice that were imaged serially by microPET/CT. Results Image analysis of the Db-DIIIs and Db at 4, 20, 28, 44 and 51h showed segregation of kinetics at 28h. The 4h tumor-to-soft tissue ratios were 2.4 (WT, H535) and 2.5 (H510A, H464A, Db), reaching 4.5 (WT), 4.9 (H535A), 5.2 (H510A), 7.6 (H464A) and 25.1 (Db) at 51h. Biodistribution confirmed the clearance order from slowest to fastest: Db-DIII WT>H535A>H510A>H464A>Db with 4.0, 2.0, 1.8, 1.6, 0.08 %ID/g of blood radioactivity at 51h, respectively. Conclusions Imaging demonstrated that HSA DIII can extend the serum persistence of the Db, while retaining tumor targeting. H464 is most crucial for FcRn binding, followed by H510 and H535, with DIIIa and DIIIb participating in the FcRn interaction. By mutating the DIII scaffold, we can dial serum kinetics suitable for imaging or therapy applications. Research Support 2008 SNM Postdoctoral Molecular Imaging Scholar Award and NIH grant CA 086306