Preparation of RGD4C fused anti-TNFα nanobody and inhibitory activity on triple-negative breast cancer in vivo

Abstract

Aims: Triple-negative breast cancer (TNBC) is not sensitive to current endocrine treatments, so new treatment strategies need to be explored. Based on previous antitumour studies on anti-TNFα nanobody, we designed a novel fusion nanobody to enhance antitumour activity of the anti-TNFα nanobody in TNBC.

Main methods: The RGD4C contains RGD sequence, which is the smallest recognition unit binding to the αvβ3 receptor on tumour cell membranes and involved in tumour cell adhesion, proliferation, and metastasis. RGD4C was fused to anti-TNFα nanobody to investigate the antitumour activity in vitro and in vivo.

Key findings: The antitumour effects of fusion nanobody V-L-R-H could effectively bind to αvβ3 and inhibit cell migration and proliferation of MDA-MB-231, which had satisfying purification efficiency and approving antigen or receptor binding activity. V-L-R-H could inhibit the TNFα-mediated PI3K/AKT/NF-κB signal pathway and integrin αvβ3 correlative FAK focal adhesion signal pathway. Mouse xenograft tumour experiments showed that the V-L-R-H could inhibit tumour proliferation and metastasis; reduce the TNFα, HIFα, Ki67, and CD31 concentrations in tumour; and inhibit the process of epithelial-mesenchymal transition.

Significance: The fusion nanobody enhanced antitumour activity of the anti-TNFα nanobody on TNBC. It provided a reference for the design of dual functional fusion proteins and development of tumour treatment strategies of antagonistic TNFα and αvβ3, and a new therapeutic strategy and research direction for the treatment of TNBC.

Keywords: Epithelial-mesenchymal transition; Fusion nanobody; RGD4C; TNFα; Triple-negative breast cancer; αvβ3.