An EGFR/CD13 bispecific fusion protein and its enediyne-energized analog show potent antitumor activity

Targeting to two or more objectives simultaneously has been pursued as a strategy to potentially increase the efficiency and selectivity of targeted drugs to certain cancers. In this study, an epidermal growth factor receptor (EGFR)/CD13-targeting, bispecific fusion protein ER(Fv)–LDP–NGR consisting of an anti-EGFR single-chain variable fragment (scFv), an apoprotein (LDP) of lidamycin (LDM), and a tri-CNGRC (Cys–Asn–Gly–Arg–Cys) peptide against CD13 was constructed, and then an enediyne-energized analog ER(Fv)–LDP–NGR–AE was generated by integration with an enediyne chomophore (AE) derived from LDM. The apoprotein LDP was used as a ‘scaffold’ to connect the scFv fragment and the tri-CNGRC peptide and also served as a specific ‘carrier’ for the extremely potent cytotoxic enediyne chromophore of LDM. Compared with its monospecific counterparts, ER(Fv)–LDP and LDP–NGR, the bispecific fusion protein ER(Fv)–LDP–NGR showed higher affinity to EGFR/CD13-overexpressed tumor cells. Determined by the MTT assay, the bispecific, enediyne-energized ER(Fv)–LDP–NGR–AE showed highly potent cytotoxicity to EGFR/CD13-overexpressed MCF-7 cells, with an IC50 value of 3.4×10 mol/l, whereas for the EGFR-overexpressed A431 cells, the IC50 value was 2.2×10 mol/l. For MCF-7 cells, the bispecific ER(Fv)–LDP–NGR–AE was more potent in cytotoxicity than the corresponding monospecific energized fusion proteins. In athymic mice models, the bispecific fusion protein ER(Fv)–LDP–NGR presented stronger inhibitory activity than the monospecific ER(Fv)–LDP and LDP–NGR. For the enediyne-energized fusion proteins, ER(Fv)–LDP–NGR–AE significantly inhibited the growth of EGFR/CD13-overexpressed MCF-7 xenograft and EGFR-overexpressed A431 xenograft by 86.3 and 81.4%, respectively. In addition, the bispecific ER(Fv)–LDP–NGR–AE showed much higher efficacy than its monospecific analogs ER(Fv)–LDP–AE and LDP–NGR–AE in both MCF-7 and A431 xenograft models. The results show that EGFR/CD13 bitargeting effectively improved the antitumor efficacy. Both the bispecific fusion protein and its enediyne-energized analog are highly effective in athymic mice bearing xenografts, and the latter exerts more marked efficacy. Generation of a pair of bispecific antibody-based therapeutics and its corresponding antibody–drug conjugate simultaneously may be a feasible strategy for the development of new targeted drugs for cancer therapy.