To increase the antitumor efficacy, in the present study, we proposed several settings of
matrix metalloproteinase (
MMP)-2/9-oriented combinations that comprise the
MMP-2/9-targeting fusion
protein dFv-LDP and the
MMP inhibitor doxycycline (DOX) in association with EGFR/HER2-bispecific fusion
protein Ec-LDP-Hr, its enediyne-energized analogue Ec-LDP-Hr-AE, and
gemcitabine (GEM). The expressions of various fusion
proteins were detected by western blot analysis. Proliferation and migration inhibition of cells were determined by MTT and Transwell assay, respectively. The binding capability of dFv-LDP and Ec-LDP-Hr to
cancer cells was examined by ELISA, cell immunofluorescence coimmunoprecipitation and confocal assays. Animal experiments were set to investigate the antitumor efficacy of various combinations against
colorectal carcinoma HCT-15 xenograft in athymic mice. These two targeting
proteins dFv-LDP and Ec-LDP-Hr had strong binding capabilities and antiproliferation effects on various
cancer cell lines. Enhanced therapeutic efficacy in vivo was observed in the
MMP-2/9-targeting fusion
protein dFv-LDP integrated combinations including: i) dFv-LDP and Ec-LDP-Hr, ii) dFv-LDP and enediyne-energized fusion
protein Ec-LDP-Hr-AE, iii) dFv-LDP and Ec-LDP-Hr-AE plus DOX, and iv) dFv-LDP and GEM plus DOX against
colorectal cancer HCT-15 xenograft in athymic mice. In setting iii, DOX (20 mg/kg), dFv-LDP (20 mg/kg) and Ec-LDP-Hr-AE (0.3 mg/kg) alone suppressed
tumor growth by 35, 49.7 and 67.5%, respectively. The combination of dFv-LDP and Ec-LDP-Hr-AE was 75.1%. Furthermore, this combination plus DOX showed stronger efficacy with an inhibitory rate of 82.7%. In setting iv, the combination of dFv-LDP and GEM suppressed
tumor growth by 66.3%. Notably, the
tumor inhibitory rate of the dFv-LDP/GEM/DOX combination reached 85.5%, producing initial shrinkage after the first administration. The
MMP-2/9-oriented combination strategy that employs the
MMP-2/9-targeting antibody-based fusion
protein and the small molecular inhibitor DOX as the basic composed agents may enhance antitumor efficacy in association with the EGFR/HER2-targeting fusion
protein and GEM. This multiple targeting approach may be useful for enhancing antitumor efficacy against
colorectal cancer.