A bispecific protein rG7S-MICA recruits natural killer cells and enhances NKG2D-mediated immunosurveillance against hepatocellular carcinoma

Abstract

MHC class I-related chain A (MICA) is a principal immunoligand of the natural killer (NK) cell receptor NK group 2, member D (NKG2D) and plays a key role in NK cell-mediated immune recognition. Shedding of MICA from tumor cells leads to immunosuppression. To reconstitute the immunosurveilance function of NK cells, we constructed a fusion protein rG7S-MICA and explored its potential anti-tumor activity against hepatocellular carcinoma (HCC). rG7S-MICA consists of human MICA and a single-chain antibody fragment (scFv) targeting the tumor-associated antigen cluster of differentiation 24 (CD24). In vitro, rG7S-MICA engaged both NK cells and CD24⁺ human HCC cells, and triggered NK cell-mediated cytolysis. Furthermore, in CD24⁺ HCC-bearing nude mice, rG7S-MICA specifically targeted to the tumor tissue, where it effectively recruited NK cells and induced the release of cytokines, and showed superior anti-tumor activity. In conclusion, rG7S-MICA provides a new approach for HCC-targeting immunotherapy and has attracting potentials for clinical applications.

Introduction

Hepatocellular carcinoma (HCC) is the third most common cancer and the second leading cause of cancer-related death worldwide [1]. Many clinical chemotherapy trials attempting to treat advanced HCC have been abandoned due to severe adverse effects and poor survival benefit [2], [3]. HCC remains an incurable disease with short median survival after diagnosis [4], and is unsuitable for systemic therapy, especially cytotoxic drugs [5]. In recent years, therapeutic antibodies including bevacizumab (Avastin) [6], [7] and cetuximab [8], which show better therapeutic effects than chemotherapy, have shown some promise. However, as single agents these antibodies have limited efficacy against HCC [9]. Treatment of HCC still presents serious challenges such as poor prognosis, severe toxicity, low response rate and high recurrence rate [10]. As a novel therapeutic regimen, immunotherapy focuses on overcoming the limitations of conventional treatments. Cluster of differentiation 24 (CD24) is a potential tumor-initiating cell marker impacting patients' clinical outcome, and it is critical in multistage of tumorigenesis and tumor progression which provides prospect for HCC targeted therapy [11], [12], [13].

Natural killer (NK) cell is the first line of immune defense against tumor cells [14]. While T cells are the major histocompatibility complex (MHC) restricted and need to be activated before recognizing their targets, NK cell-based immunotherapy directly targets the ligands expressed on the tumor cell surface and provides anti-tumor response against HCC by harnessing the power of innate immune [15], [16]. The activity of NK cells depends on the balance between a family of activating and inhibitory receptors [14], [17], [18]. As an activating receptor, NKG2D mediates the anti-tumor activity of NK cells through binding to ligands such as MHC class I-related chain A (MICA)/MICB which are induced in cancer cells but generally not expressed in the normal cells [19], [20]. Unfortunately, the shedding of MICA increases serum levels of soluble MICA and hinders the recognition of HCC by NKG2D receptor, resulting in tumor immune escape [21], [22], [23].

In a previous study, we generated a mouse anti-CD24 monoclonal antibody (G7mAb) and a hybridoma derived scFv (rG7S) with well-established targeting efficacy in HCC xenografted mouse models [24]. To recruit NK cells against HCC, we designed a fusion protein (rG7S-MICA) consisting of human MICA extracellular domains and the single chain antibody fragment rG7S. Upon binding to CD24, the MICA portion was expected to promote the recognition of tumor cells by NK cells and to enhance NK cell-mediated cytotoxicity [25], [26].

In this study, we aimed to evaluate the targeting and anti-tumor activity of rG7S-MICA against HCC. Our results showed that rG7S-MICA directed NK cells to accumulate in the tumor and induced the release of cytokines, resulting in significantly increased anti-tumor activity in HCC-bearing nude mice.