Purpose: Bi-specific antibody (BsAb) is an emerging novel format of antibody. We aimed to develop the natural killer (NK) cell receptor NK group 2, member D (NKG2D)-mediated, immune surveillance system. In this system, the NKG2D ligand MHC class I-related chain A (MICA) was fused with BsAb, which targeted a cluster of differentiation 24 (CD24), a tumor-initiating cell marker that is over-expressed on hepatocellular carcinoma (HCC).
Methods: The Homo MICA extracellular domains (hMICA) were fused to the end of the heavy chain of cG7 with the flexible pentapeptide (Gly-Gly-Gly-Gly-Ser; G4S), which formed the cG7-MICA that was further identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB). The targeting specificity was characterized using the Surface Plasmon Resonance (SPR) technology and a flow cytometry assay. Furthermore, the design of BsAb cG7-MICA that targeted CD24 and NKG2D was proven to enhance antibody-dependent, cell-mediated cytotoxicity (ADCC) in vitro by the CytoTox 96 Nonradioactive Cytotoxicity assay. Degranulation and a cytokine production assay of NK cells demonstrated that NK cells were activated effectively by cG7-MICA. Further, in HCC-bearing nude mice, the anti-tumor effects of cG7-MICA combined with sorafenib were verified again.
Results: We purified cG7-MICA successfully, and it has a high affinity. In vivo, cG7-MICA recruited NK cells to the tumor site and improved the anti-tumor efficacy of sorafenib. cG7-MICA also activated NK cells to release interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), and it increased the CD107a expression on the surface of the NK cells in vitro.
Conclusion: NK cells play a major role in the natural, innate immune system, and they have the function of identifying and killing target cells. cG7-MICA remodels the function of MICA molecules to activate NK cells, which provides a possible strategy for HCC-targeting immunotherapy.
Keywords: BsAb; HCC; MICA; NK92; NKG2D; Sorafenib.