In vivo comparison of two anti-PSMA mAb Fab' conjugates containing branched-chain PEG derivatives with Fab' and F(ab')₂

Objectives Our previous studies demonstrated that conjugating an anti-PSMA Fab' fragment with branched-chain polyethyleneglycol (PEG) derivatives (MW~4-8kDa) dramatically reduced kidney concentrations. However, tumor targeting was not evaluated in that study. The objective of this investigation was to prepare two Fab'-PEG conjugates and compare their biodistributions with Fab' and F(ab')₂ in mice bearing human prostate cancer (C4-2B) xenografts. The two Fab'-PEG conjugates differed only in whether a closo-decaborate(2-) moiety, used for ²¹¹At labeling, was part of the PEG conjugation reagent or was conjugated in a second step.

Methods Two maleimido-PEG conjugation reagents were synthesized, one incorporating a closo-decaborate(2-) moiety. The maleimido-PEG reagents were conjugated with the Fab' fragment via thiol groups produced by DTT reduction. For the Fab' conjugated with the PEG reagent without a closo-decaborate(2-) moiety, a second conjugation was conducted with an isothiocyanato-closo-decaborate(2-) reagent. The Fab' conjugates were characterized by SE-HPLC, SDS-PAGE and IEF. The Fab'-PEG conjugates and F(ab')₂ were labeled with ¹²⁵I. Fab' was labeled with ¹³¹I for dual-label injections.

Results Gamma counts of tissues indicated that: (1) blood concentrations of both Fab'-PEG conjugates were higher than Fab', but lower than F(ab')₂, (2) both Fab'-PEG conjugates had decreased kidney concentrations from that of Fab', and the Fab'-PEG without closo-decaborate(2-) conjugated had the lowest kidney concentrations, (3) both Fab'-PEG conjugates with closo-decaborate(2-) had higher liver concentrations than Fab' or F(ab')₂, and (4) the Fab'-PEG conjugate with closo-decaborate(2-) on the PEG reagent had tumor concentrations similar to F(ab')₂.

Conclusions The Fab'-PEG conjugates had similar tissue clearance, but higher tumor concentrations were obtained with the Fab'-PEG conjugate containing the closo-decaborate(2-) on the PEG reagent. High liver concentrations obtained with the Fab'-PEG conjugates indicates reagent modification is needed.

Research Support We thank the National Institutes of Health (CA113431) for funding this research