Active Tumoral/Tumor Environmental Dual-Targeting by Non-Covalently Arming with Trispecific Antibodies or Dual-Bispecific Antibodies on Docetaxel-Loaded mPEGylated Nanocarriers to Enhance Chemotherapeutic Efficacy and Minimize Systemic Toxicity.

Purpose This study was aimed at developing the trispecific antibodies (anti-EGFR/anti-FAP/anti-mPEG, TsAb) or dual bispecific antibodies (anti-EGFR/anti-mPEG and anti-FAP/anti-mPEG) docetaxel (DTX)-loaded mPEGylated lecithin-stabilized micelles (mPEG-lsbPMs) for improving the targeting efficiency and therapeutic efficacy. Methods mPEG-lsbPMs were simply prepared via thin film method. The trispecific antibodies or bispecific antibodies bound the mPEG-lsbPMs by anti-mPEG Fab fragment. The formulations were characterized by DLS and TEM; in vitro and in vivo studies were also conducted to evaluate the cellular uptake, cell cytotoxicity and therapeutic efficacy. Results The particle sizes of mPEG-lsbPMs with or without the antibodies were around 100 nm; the formulations showed high encapsulation efficiencies of 97.12%. The TsAb and dual bispecific antibodies were fabricated and demonstrated their targeting ability. Two EGFR-overexpressed cell lines (HT-29 and MIA PaCa-2) were co-cultured with FAP-overexpressed WS1 cells (HT-29/WS1; MIA PaCa-2/WS1) to mimic a tumor coexisting in the tumor microenvironment. Cellular binding study revealed that the binding of anti-FAP micelles to three co-culture ratios (4:1, 1:1, and 1:4) of HT-29/EGFR to WS1/FAP was significantly higher than that for TsAb micelles and dual (1:1) micelles, and the binding of those targeting antibodies to WS1/FAP and MIA PaCa-2/EGFR was equally efficacious resulting in a similar binding amount of the TsAb and dual BsAbs (1:1) with the co-culture of MIA PaCa-2/EGFR and WS1/FAP at a 1:1 ratio. Antitumor efficacy study showed that treatment with DTX-loaded mPEG-lsbPMs modified with or without BsAbs, dual BsAbs (1:1), and TsAbs was enhanced in inhibiting tumor growth compared with that for Tynen® while showing fewer signs of adverse effects. Conclusion Active targeting of both tumors and TAF-specific antigens was able to increase the affinity of DTX-loaded mPEG-lsbPMs toward tumor cells and TAFs leading to successive uptake by tumor cells or TAFs which enhanced their chemotherapeutic efficacy against antigen-positive cancer cells.