Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides.

Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185(HER-2) single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185(HER-2) and on SK-OV-3 cells. The (99m)technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.