Enhancing the detection sensitivity and blood circulation time for cytokines using a non-antibody protein scaffold

Abstract

Cytokines are key signaling molecules to mediate and regulate inflammatory responses in the immune system. They mediate the activity and proliferation of immune cells for effective immune responses. Based on these attributes, cytokines have been used as immunological biomarkers for diagnosis of tumor or chronic inflammatory autoimmune diseases. Therefore, precise and sensitive detection of cytokine is essential for early diagnosis to increase therapeutic efficacy. In addition, cytokines have therapeutic potential in cancer treatment because it has an ability to directly stimulate the proliferation and cytotoxic functions of NK cells and T cells. However, due to the extremely short half-life of cytokine in the blood, there are many limitations in real medical application. Thus, there have been many efforts to extend the half-life of cytokines by modifying with Fc region of antibody, polyethylene glycol (PEG) and all that sort of things. In chapter 1, we present a sensitive immunoassay platform using DNA-tethered gold nanoparticles and repebody to detect tumor necrosis factor alpha (TNF-α) which is a representative inflammatory cytokine. The DNA-tethered AuNPs were reacted with two different zinc finger proteins which had been genetically fused to a TNF-α-specific protein binder and alkaline phosphatase, respectively. Functionalized gold nanoparticles were used as a signal generator for colorimetric immunoassay of TNF-α. The functionalized gold nanoparticle-based immunoassay system has been confirmed high reproducibility and sensitivity, having 160-fold enhanced detection limit compared with conventional system. In chapter 2, we developed human interleukin 15 (hIL15) which can efficiently activate NK cells and T cells with a dramatically increased half-life by fusing the human serum albumin specific protein binders (rHSA) to maximize treatment efficacy. To maintain the biological function of each part, rHSA and hIL15 were fused into one considering structure-based protein engineering. We provide evidence supporting the improved pharmacokinetics of hIL15 through in vivo test, and the result showed a nearly 40-fold longer half-life. Furthermore, anti-tumor activity of rHSA-hIL15 were also proved in two types of melanoma xenograft mouse model. In this study, we successfully develop the immunoassay platform for the sensitive cytokine detection and the cancer immunotherapeutics for the effective extension of half-life, based on the use of non-antibody protein scaffold. Our results suggest that the cytokines which have both potentials, biomarkers or therapeutics, could be utilized to particular purposes by utilizing protein engineering for the development of detection system or therapeutics.