Common gamma-chain (ϒc) cytokine-based fusion proteins forapplications in immunotherapy Open Access

Ng, Spencer (2016)

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Cytokines are protein messengers that can induce immune cells to activate, proliferate, differentiate, and engage in their effector functions. They may also directly inhibit any of these processes and instruct immune cells to undergo senescence and cell death. One family of cytokines with a wide array of immunomodulatory properties is known as the common gamma-chain (γc) group of cytokines. Comprising of interleukins-2, 4, 7, 9, 15, and 21, the γc cytokines are so named because they share the use of a common gamma-chain receptor (CD132). Each of these cytokines uses a ligand-specific alpha-chain (αc) for binding in addition to the γc, creating a heterdimeric receptor. IL-2 and IL-15 also share the use of a common beta-chain (βc, CD122), to form a heterotrimeric receptor. IL-2 was the first of these cytokines to be discovered as a T cell growth factor and has since become the only cytokine approved by the Food and Drug Administration (FDA) for use in cancer immunotherapy. Despite their immunostimulatory capabilities, γc cytokine monotherapy has had mediocre success in clinical trials for cancer, chronic viral infections, and autoimmune ailments. In order to improve upon the efficacy of γc cytokine therapy, we have modified them by creating fusion proteins consisting of a γc cytokine and transforming growth factor-beta (TGF-β) antagonists (FIST fusion family) for cancer immunotherapy. The rationale for the fusion of a TGF-β antagonist to γc cytokines is the observation that TGF-β secreted by tumors can actively suppress the immune response and dampen the effect of cytokine-based therapies. In particular, we find that the fusion of IL-15 to a TGF-β antagonist (FIST-15) potently stimulates natural killer cell-mediated anti-tumor immunity. We further explore the effects of FIST-15 in the setting of acute viral infection and a model of hepatic fibrosis. A second family of fusion proteins consisting of γc cytokines and granulocyte/monocyte-colony stimulating factor (GMCSF; GIFT fusion family) were similarly designed to augment immunological responses against cancer. However, the fusion of GMCSF to IL-15 resulted in a protein (GIFT-15) with immunosuppressive properties. We have found that GIFT-15 acts primarily on B cells, converting them to a regulatory phenotype (Bregs). We demonstrate that Bregs may be therapeutically exploited in autoimmune conditions, where inappropriate activation of the immune response results in pathology. Taken together, we find that modification of γc cytokines by creating fusions with TGF-β receptor antagonists or GMCSF can result in the formation of new proteins with unique immunobiological properties. The use of such γc-derived fusion proteins can be used to enhance the efficacy of cytokine-based immunotherapies.

Table of Contents

Chapter 1: Introduction 1

1.0.0 Overview of common gamma-chain cytokines 2

1.1.0 Cancer immunology 5

1.1.1 Natural history of cancer and the immune system 6

1.1.2 Anti-tumor effector functions of the immune system 11

1.1.3 Tumor immune-evasion strategies 22

1.1.4 γc cytokines in cancer immunotherapy 31

1.1.5 Transforming growth factor-beta (TGF-β) and the anti-tumor immune response 42

1.2.0 Overview of granulocyte colony-stimulating factor (GMCSF) and common gamma-chain interleukin fusion proteins (GIFTs) 45

1.2.1 GIFT-2 48

1.2.2 GIFT-15 50

1.2.3 GIFT-21 53

1.2.4 GIFT-4 54

1.2.5 GIFT-7 55

1.2.6 GIFT-9 57

1.2.7 GIFT Summary 57

1.2.8 Table Legend and Table 58

Chapter 2: Fusion of interleukin-15 (IL-15) to the sushi domain of the IL-15 receptor-alpha and dimeric TGF-β receptor (FIST-15) in cancer immunotherapy 60

2.1.0 Abstract 61

2.2.0 Introduction 62

2.3.0 Materials and Methods 64

2.4.0 Results 69

2.5.0 Discussion 75

2.6.0 Figure Legends 83

2.7.0 Figures and Supplemental Figures 91

Chapter 3: Alternative applications of FIST-15 103

3.1.0 FIST-15 in murine cytomegalovirus (MCMV) infection 104

3.1.1 Introduction 104

3.1.2 Materials and Methods 105

3.1.3 Results 107

3.1.4 Discussion 109

3.1.5 Figure Legends 114

3.1.6 Figures 117

3.2.0 FIST-15 in carbon tetrachloride (CCl4) induced liver fibrosis 121

3.2.1 Introduction 121

3.2.2 Materials and Methods 124

3.2.3 Results 125

3.2.4 Discussion 126

3.2.5 Figure Legends 128

3.2.6 Figures 130

Chapter 4: GIFT-15 induced regulatory B cells (GIFT-15 Bregs) promote the development of CD4+CD25+FoxP3+ regulatory T cells (Tregs) and CD4+FoxP3- type-1 regulatory T cells (Tr-1) in murine experimental autoimmune encephalomyelitis (EAE) 132

4.1.0 Abstract 133

4.2.0 Introduction 134

4.3.0 Materials and Methods 137

4.4.0 Results 141

4.5.0 Discussion 148

4.6.0 Figure Legends 153

4.7.0 Figures and Supplemental Figures 158

Chapter 5: Discussion 177

5.1.0 Summary 178

5.2.0 Discussion 180

5.3.0 Future Directions 193

5.4.0 Conclusion 199

5.5.0 Figure Legends 199

5.6.0 Figures 201

References 202

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