Linkage with cathepsin B-sensitive dipeptide promotes the in vitro and in vivo anticancer activity of PEGylated tumor necrosis factor-alpha (TNF-α) against murine fibrosarcoma

Abstract

To improve the pharmacological profile of tumor necrosis factor alpha (TNF-α), we have synthesized a new PEGylated prodrug, PEG-vcTNF-α, using a cathepsin B-sensitive dipeptide (valine-citrulline, vc) to link branched PEG and TNF-α. PEG-modified TNF-α without the dipeptide linker (PEG-TNF-α) and unconjugated TNF-α were also tested as controls. It was found for the first time that TNF-α released from PEG-vcTNF-α was specifically dependent on the presence of cathepsin B. PEG-vcTNF-α induced higher cytotoxicity and greater apoptosis against L929 murine fibrosarcoma cells than PEG-TNF-α. Reversal of these effects by a cathepsin-B inhibitor confirmed that these effects were mediated by cathepsin B-specific release of TNF-α. In vivo pharmacokinetics studies demonstrated that the plasma stability of PEG-vcTNF-α was significantly increased compared to TNF-α. Finally, the improved anticancer efficacy of PEG-vcTNF-α and the distinct activities among the three formulations confirmed the positive contribution of both PEGylation and the dipeptide linkage to the improved drug-like properties of PEG-vcTNF-α. The results here indicate that linking proteins and PEG via the cathepsin B-sensitive dipeptide may be a promising strategy for developing protein therapeutics.

Growth factor conjugation: Strategies and applications

“…TNF‐α in vitro release from these conjugated species was regulated by cathepsin B and was inhibited by the presence of a cathepsin B inhibitor in vitro . Such pegylated TNF‐α species showed a greater in vitro and in vivo bioactivity in comparison with simply pegylated TNF‐α …”

“…Such pegylated TNF-a species showed a greater in vitro and in vivo bioactivity in comparison with simply pegylated TNF-a. 113,114 To actively target TNF-a, the protein was conjugated to a murine antibody called ZME-018, which targets a glycoprotein expressed on the majority of melanoma cells population. This conjugation reaction was accomplished by a heterobifunctional crosslinking reagent called N-succimindyl 3-(2-pyridyldithio)proprionate (SPDP).…”

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α (TNF-α) conjugate for therapeutic application

“…The method of preparation of TNF- conjugates has been describe [21]. Synthesized rPEG-TNF- and PEG-TNF- conjugates were each purified and separated into three fractions of various molecular weights by gel filtration chromatography (GFC) using a Sephacryl S-200HR column on a fast protein liquid chromatography system eluted with 0.1 mol L 1 phosphate buffer, pH 7.0.…”

“…Lysosomes were collected from liver homogenates by centrifugation using the metrizamide discontinuous gradient method with slight modifications [21]. All drug release studies were performed at 37°C.…”

“…This compound showed enhanced stability in human and mouse plasma and sensitivity to cathepsin B and rat liver lysosomes. Moreover, the conjugate showed potent anticancer activity against Meth-A solid tumors and was toxic in vitro to L929 cells [21]. Further studies are necessary to determine the anticancer activity of this conjugate.…”