In vivo targeting of malignant melanoma by (125)iodine- and (99m)technetium-labeled single-chain Fv fragments against high molecular weight melanoma-associated antigen

Monoclonal antibodies (MAbs) against high-molecular-weight melanoma-associa ted antigen (HMW-MAA) have been used irt vivo to target melanoma, More rece ntly, single chain Fv (scFv) antibody fragments against HMW-MAA have been d escribed that may improve melanoma targeting. However, there have been few ill vivo studies with antimelanoma scFvs because these have proved difficul t to label with isotopes (e.g., Tc-99m) suitable for imaging. We have gener ated a series of scFvs against HMW-MAA by chain shuffling and antibody phag e selection on melanoma cells. In preliminary experiments we identified one scFv (RAFT3) as suitable for ill vivo melanoma targeting. Direct radiolabe ling of RAFT3 scFv with Tc-99m was simple, yielding a radiochemical purity of >90%, The label remained stable for 24 h in vitro. I-125- and Tc-99m-lab eled RAFT3 scFv were tested in a nude mouse xenograft model for human melan oma and were compared with the parent MAb LHM2 and its F(ab')(2) Fragment v ersus nonmelanoma-specific MAb and scFv. RAFT3 scFv accumulated specificall y in the tumor and showed greater tumor specificity compared with LHM2 with Faster pharmacokinetics (t(1/2)alpha, 8 min; t(1/2)beta, 189 min; and t(1/ 2)alpha, 37 min; t(1/2)beta, 384 min, respectively) and reduced background in liver, lung, and spleen. Nonspecific accumulation of Tc-99m-labeled RAFT 3 scFv in the kidney was high but tumor:normal tissue ratios were better co mpared with I-125-labeled RAFT3 scFv and LHM2 F(ab')(2). Overall, tumor-tar geting efficiency at equivalent time points was scFv > IgG > F(ab')(2) in g ood agreement with previously described scFvs engineered for Tc-99m labelin g. We discuss the potential use of RAFT3 scFv for imaging and therapy of me tastatic melanoma.