Bispecific Fynomer-antibody fusion proteins targeting two epitopes on HER2.

Abstract

2575 Background: Fynomers are small binding proteins (7 kDa) derived from the SH3 domain of Fyn kinase. They can be engineered to yield specific and high-affinity binding domains to target proteins of interest. In the past, we have isolated Fynomers with excellent physico-chemical properties binding to 16 different antigens. One important application of the Fynomer technology represents the genetic fusion of a Fynomer to an antibody to provide bispecific fusion proteins. Methods: Using phage display technology we have isolated Fynomers binding to tumor-associated antigens. After genetic fusion of these Fynomers to antibodies of interest the resulting bispecific proteins were evaluated in vitro and in vivo for their antitumoral activity. Results: The fusion of Fynomers to the antibodies of interest did not alter the favorable biophysical properties of the antibodies. First, the Fynomer-antibody fusion proteins could be purified with the same high yields from the supernatant of transiently transfected CHO cells as the unmodified antibodies (in the range of 100 mg/L). Second, the purified fusion proteins were monomeric and showed no signs of aggregation even after four months of storage at 4 °C or -20 °C in PBS as determined by size exclusion chromatography. Third, the Fc-mediated effector functions of antibodies were not altered by their fusion with Fynomers. Antibody-dependent cellular cytotoxicity (ADCC) was maintained, and the affinity to the neonatal Fc-receptor (FcRn) was similar as observed for the unmodified antibody. In addition, the bispecific Fynomer-antibody fusion proteins demonstrated excellent growth inhibition of tumor cells in vitro and high efficacy in vivo in tumor xenograft mouse models. Conclusions: These encouraging preclinical results indicate that the bispecific Fynomer-antibody fusions have highly promising properties with a great potential for further preclinical and clinical development.