Evaluation of the Anti-Fibrotic Activity of PCBP2 siRNA in Primary Hepatic Stellate Cells and Discovery of Anti-PD-L1 Peptide and Nanobody for Immunotherapy
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There are two major research objectives in this dissertation. Our laboratory recently discovered a small interfering RNA (siRNA) that silences the poly (rC) binding protein 2 (PCBP2) gene. It is hypothesized that silencing of the PCBP2 gene in hepatic stellate cells (HSCs) leads to the reversal of the accumulated type I collagen in fibrotic liver. The first research objective is therefore to evaluate whether the PCBP2 siRNA can reverse the alcohol- and cytokine-induced profibrogenic effects on HSCs. The second research objective is to discover programmed death ligand 1 (PD-L1) specific peptides and nanobodies using phage display. These PD-1/PD-L1 checkpoint inhibitors can be potentially used as immunotherapy agents for cancer. Chapter 1 introduces the background of the dissertation research and presented the Statement of the Problems and Objectives. Chapter 2 reviews the mechanisms of liver fibrogenesis and cancer immunotherapy using checkpoint inhibitors. Chapter 3 details our investigation of the anti-fibrotic effect of the PCBP2 siRNA in rat primary HSCs and HSC-T6 cells. The α-complex protein-2 (αCP2), encoded by the PCBP2 gene, is responsible for the accumulation of type I collagen in fibrotic liver. In this dissertation, we aimed to silence the PCBP2 gene using a siRNA to reverse the alcohol- and cytokines-induced pro-fibrogenic effects on HSCs. Primary rat HSCs and the HSC-T6 cell line was used as fibrogenic models to mimic the initiation and perpetuation stages of fibrogenesis, respectively. Our laboratory recently discovered a PCBP2 siRNA, which can efficiently silence the expression of αCP2 and reduce the stability of type I collagen mRNA in HSC-T6 cells. In this dissertation, we investigated the effects of PCBP2 siRNA on cell proliferation and migration. Expression of type I collagen in primary HSCs was analyzed using quantitative real-time PCR and western blot. In addition, we evaluated the effects of PCBP2 siRNA on apoptosis of the HSCs. Our results showed that PCBP2 siRNA reversed the alcohol- and cytokine-induced multiple pro-fibrogenic effects on primary rat HSC and HSC-T6 cells. The PCBP2 siRNA also reversed the alcohol- and cytokine-induced accumulation of type I collagen as well as cell proliferation and migration. Moreover, the combination of LY2109761, a TGF-β1 inhibitor, and PCBP2 siRNA exhibited a synergistic inhibition effect on the accumulation of type I collagen in HSCs. We therefore concluded that silencing of PCBP2 using siRNA could be a potentially therapeutic strategy for alcoholic liver fibrosis. Chapter 4 discusses our discovery of several anti-PD-L1 peptides using a phage display peptide library. PD-L1 is overexpressed on a variety of cancer cells, and programmed cell death protein 1 (PD-1) is expressed on T cells. The interaction between PD-L1 and PD-1 negatively regulates the immune responses of T cells, leading to escape of cancer cells from the attack of T cells. Blocking the PD-L1/PD-1 interaction is therefore a promising strategy to treat cancers by restoring the immune activity of T cells. The FDA has approved several monoclonal antibodies targeting PD-1 or PD-L1 for various cancer immunotherapies. However, large size (150 kDa) of antibodies limits their tumor penetration, especially in solid tumors. There has been a growing interest in developing low-molecular weight checkpoint inhibitors, such as peptides, in the past few years. Using a novel phage biopanning, we discovered several PD-L1-specific peptide antagonists to block the PD-1/PD-L1 interaction. The peptide candidate CLP002 exhibited the highest binding affinity to PD-L1 with an equilibrium dissociation constant (KD) of 366 nM. The apparent KD values of CLP002 to PD-L1-positive human cancer cell lines MDA-MB231 and DU145 are 212.9 nM and 184.1 nM, respectively. CLP002 efficiently blocked 85% of the PD-1/PD-L1 interactions with an IC₅₀ of 2.17 µM. We verified that CLP002 restored T cell proliferation and prevented T cell apoptosis in vitro, when T cells were co-cultured with cancer cells. More importantly, we demonstrated that CLP002 is highly specific for PD-L1, thus minimizing its potential off-target effects in the body. According to the results of the in vivo anti-tumor activity study, the CLP002 peptide inhibited tumor growth and increased the survival of the CT26 tumor-bearing mice, suggesting the CLP002 peptide represents a promising drug candidate for cancer immunotherapy. Chapter 5 describes our work towards discovering anti-PD-L1 nanobodies for cancer immunotherapy using a phage display of single-domain antibody (sdAb) library. Nanobody (also named sdAb or VHH) is an antibody fragment composed of a single variable domain of the heavy chain on heavy chain only antibody. Nanobody is the smallest antibody fragment (14 kDa) that maintains the similar antigen-binding affinity. We discovered seven anti-PD-L1 nanobodies that blocked the PD-1/PD-L1 interaction. Among them, the nanobody CLV3 showed the highest binding affinity to PD-L1 with a KD value of 12.37 nM. The nanobody CLV3 also exhibited the highest blockade of the PD-1/PD-L1 interaction with an IC50 of 32.3 nM. CLV3 inhibited tumor cell proliferation by binding to PD-L1 on the cancer cell surface and blocking the PD-1/PD-L1 interaction between cancer cells and T cells. Antitumor activity of the nanobody CLV3 was evaluated in a CT26 xenograft mouse model. The nanobody CLV3 significantly inhibited tumor growth and increased the survival of the tumor-bearing mice. The nanobody CLV3 is therefore a promising PD-L1 inhibitor that can be used for cancer immunotherapy.
Table of Contents
Introduction -- Review of Literature -- Silencing of αCP2 Reverse the Alcohol-and Cytokine-induced Fibrogenesis in Hepatic Stellate Cells -- Discovery of Small Anti-PD-L1 Peptide for Cancer Immunotherapy -- A Novel Nanobody Targeting PD-L1 for Cancer Immunotherapy -- Summary and Conclusions -- Appendix
Ph.D. (Doctor of Philosophy)