Structure and characterization of the CD38-bispecific protein. (A) Schematic of the 028-Fc-C825 bispecific (anti-CD38 × anti-Y-DOTA) Fc fusion gene. An anti-human CD38 028 scFv gene and an yttrium-DOTA capturing C825 disulfide-stabilized scFv (ds-scFv) gene were fused to the human immunoglobulin G1 Fc fragment at the amino and carboxyl ends, respectively. An NLG was incorporated between the Fc and C825 ds-scFv domains, as shown. Relevant restriction enzymes for cloning and linearization are indicated (schematic not drawn to scale). (B) SDS-PAGE analysis of the 028-Fc-C825 fusion protein. Bispecific 028-Fc-C825 fusion polypeptides were expressed in CHO-DG44 cells, where they spontaneously formed dimers via the hinge regions and were secreted into the growth medium. The purification fractions and the 028-Fc-C825 fusion protein (5 µg) were analyzed by electrophoresis on a 4% to 20% 2-morpholinoethanesulfonic acid SDS-PAGE gel (Invitrogen). Lane 1, SeeBlue Plus2 marker proteins in kilodaltons (Invitrogen); lane 2, culture supernatant; lane 3, protein A column flow-through; lane 4, wash; lane 5, the nonreduced 028-Fc-C825 fusion protein (samples boiled); lane 7, the monomeric 028-Fc-C825 fusion protein (samples boiled and reduced with 2-mercaptoethanol); lane 6 is empty. The gel was stained with Coomassie blue. (C) Sandwich enzyme-linked immunosorbent assay demonstrating concentration-dependent binding of the CD38 (028-Fc-C825) bispecific protein (red) to the Y-DOTA ligand. A 96-well plate was coated with 70 µL of the bovine serum albumin (BSA)–Y-DOTA conjugate (1 µg/mL in PBS) and then blocked with 200 µL of 2% BSA in PBS buffer. After washing, the wells were treated with 100 µL of bispecific protein at 16 µg/mL followed by serial dilution as indicated. The plate was further treated with horseradish peroxidase (HRP)–anti-human Fc Ab followed by 3,3′,5,5′-tetramethylbenzidine (TMB). Controls demonstrate that binding to Y-DOTA is dependent on the C825 portion of the bispecific protein: the positive control, CD20 2H7-Fc-C825 bispecific (blue), shows binding to Y-DOTA; the negative control, fusion protein-Fc without C825 (black), does not. (D-E) Bifunctional binding assays of the CD38 (028-Fc-C825) bispecific protein demonstrate targeted binding to CD38⁺ cells and ligand capture of Y-DOTA–biotin. (D) CD38⁺ target cells (H929) or (E) CD38⁻ control cells (U266) (0.5 × 10⁶) were incubated in 40 µL of HBSS–2% FBS buffer containing 1 µg of biotin–Y-DOTA ligand and 2 µg of either CD38 (red and blue) or CD20 (green) bispecific proteins, or no protein (purple) for 30 minutes at 4°C. For CD38-blocking controls (blue), cells were preincubated for 30 minutes in buffer containing 40 µg of anti-CD38 Ab. Cells were finally washed and resuspended in 40 µL of buffer plus 2 µL of phycoerythrin-SA, incubated 30 minutes at 4°C, washed 3 times, resuspended in 500 µL of PBS buffer containing 1% formaldehyde, and analyzed by flow cytometry. Bsp., bispecific; DHFR, dihydrofolate reductase; FP, fusion protein; Neg., negative; PCMV, (cytomegalovirus) promoter; Pos., positive; SP, signal peptide.