Targeting HER2 and FGFR-positive cancer cells with a bispecific cytotoxic conjugate combining anti-HER2 Affibody and FGF2

Breast cancer remains a significant global health challenge, necessitating the development of effective targeted therapies. This study aimed to create bispecific targeting molecules against HER2 and FGFR1, two receptors known to play crucial roles in breast cancer progression. By combining the high-affinity Affibody Z_HER2:2891 and a modified, stable form of fibroblast growth factor 2 (FGF2), we successfully generated bispecific proteins capable of simultaneously recognizing HER2 and FGFR1. Two variants were designed: Af_HER2-sFGF2 with a shorter linker and Af_HER2-lFGF2 with a longer linker between the HER2 and FGFR1-recognizing proteins. Both proteins exhibited selective binding to HER2 and FGFR1, with Af_HER2-lFGF2 demonstrating simultaneous binding to both receptors. Af_HER2-lFGF2 exhibited superior internalization compared to FGF2 in FGFR-positive cells and significantly increased internalization compared to Af_HER2 in HER2-positive cells. To enhance their therapeutic potential, highly potent cytotoxic agent MMAE was conjugated to the targeting proteins, resulting in protein-drug conjugates. The bispecific Af_HER2-lFGF2-vcMMAE conjugate demonstrated exceptional cytotoxic activity against HER2-positive, FGFR-positive, and dual-positive cancer cell lines that was significantly higher compared to monospecific conjugates. These data indicate the beneficial effect of simultaneous targeting of HER2 and FGFR1 in precise anticancer medicine and contribute valuable insights into the design and potential of bispecific targeting molecules for breast cancer treatment.