Anti-CD20 diabodies for targeting and imaging lymphoma

Objectives One issue with random radiolabeling of small antibody fragments such as diabodies (scFv dimers, 55 kDa), is loss of immunoreactivity. Increasing the avidity of a diabody (Db) by multimerization, offer the prospect of improved tumor uptake while retaining rapid blood clearance, as well as immunoreactivity.

Methods Diabodies in V_L-V_H orientation, derived from rituximab, containing 8-, 5-, 3- or 1-residue linker were generated. The 5-residue linker variant was also modified to contain a C-terminal cysteine residue. The diabodies were characterized in vitro. Two of the diabody variants (Db-8 and Cys-Db) were radiolabeled with ¹²⁴I and evaluated by microPET imaging at 8 hours after administration in mice bearing CD20 positive tumors.

Results SDS-PAGE revealed that the Cys-Db migrated as a covalent bound scFv-dimer at 55 kDa, whereas the other four Db-variants migrated as monomers of about 25 kDa. All variants showed binding to CD20 positive cells. Size exclusion chromatography revealed that all Db variants retained as scFv-dimers. Tumor uptake at 8 hours was 3.9(±1.1)% ID/g for ¹²⁴I-Db-8 and 2.2(±0.6) for ¹²⁴I-Cys-Db which was significantly higher (P < 0.05) that that in the negative control tumor. The positive to negative tumor ratios were 1.7 for ¹²⁴I-Db-8 and 2.2 for ¹²⁴I-Cys-Db.

Conclusions Shortening the linker did not promote multimerization of rituximab scFv fragment suggesting the influence of sequence and orientation of the variable domains. However, specific tumor targeting was achieved suggesting the potential clinical utility of these molecules for imaging CD20-positive lymphomas.