Therapeutic Vascular and Immune Normalization in the Melanoma Microenvironment Using STING Agonists
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Title Page Therapeutic Vascular and Immune Normalization in the Melanoma Microenvironment Using STING Agonists by Manoj Chelvanambi Bachelor of Science, Texas Christian University, 2015 Submitted to the Graduate Faculty of the School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2021 Committee Membership Page UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Manoj Chelvanambi It was defended on March 12, 2021 and approved by Robert J. Binder, Ph.D., Professor, Department of Immunology, University of Pittsburgh Lisa H. Butterfield, Ph.D., Adjunct Professor, Department of Microbiology and Immunology, University of California, San Francisco Louis D. Falo Jr., M.D., Ph.D., Professor, Department of Dermatology, University of Pittsburgh Simon C. Watkins, Ph.D., Professor, Department of Cellular Biology, University of Pittsburgh Dissertation Director: Walter J. Storkus, Ph.D., Professor, Department of Dermatology, University of Pittsburgh ii Copyright © by Manoj Chelvanambi 2021 iii Abstract Therapeutic Vascular and Immune Normalization in the Melanoma Microenvironment Using STING Agonists Manoj Chelvanambi, PhD University of Pittsburgh, 2021 CD8+ T-cells are indispensable for immune-mediated rejection of solid cancers. Hence, the conditional enhancement of intratumoral T-cell content and/or function defines a preferred outcome for successful immunotherapies. Activated anti-tumor CD8+ T-cells rely on functional blood vessels for their efficient trafficking to, and extravasation into, the tumor parenchyma. Indeed, pathologic progression of solid tumors is closely associated with the development of structurally and functionally abnormal tumor blood vessels which impede T-cell infiltration into cancer lesions. In this regard, therapeutic dosing of anti-angiogenic interventional strategies fortifies or reprograms tumor blood vessels (or vascular normalization) to significantly improve intratumoral CD8+ T-cell infiltration. Intriguingly, agonists of Stimulator of Interferon Genes (STING), which evolved from a class of anti-angiogenic agents, have recently demonstrated significant clinical promise for their ability to enhance CD8+ T-cell recruitment into tumors but whether therapeutic changes to the tumor vasculature underlies successful immune-mediated tumor control remain only partially resolved. Indeed, in this thesis, I demonstrate that intratumoral administration of STING agonist ADU S-100 induces vascular normalization (i.e., improved vascular perfusion, enhanced pericyte coverage and increased endothelial activation) and enhances tumor infiltration by immune cells, specifically, CD8+ T-cells and CD11c+ dendritic cells (DC). iv STING activation also increases local production of pro-inflammatory cytokines/chemokines that sponsor the development of high endothelial venules (HEV) and HEV-associated tertiary lymphoid structures (TLS) within the therapeutic melanoma tumor microenvironment (TME). HEV/TLS formation with STING agonism was further linked to evidence of local T-cell cross- priming by tumor-resident antigen presenting cells (APC) within the tumor microenvironment (TME), with the therapeutic tumor infiltrating lymphocyte (TIL) repertoire exhibiting enrichment in T cell clonotypes found in the periphery as well as those detected only within the TME. These vasculature-centric underpinnings for the efficacy of STING agonist-based interventions provide enthusiasm for improved translational value of future combinational cancer immunotherapies that seek to integrate these agents. v Table of Contents Acknowledgements ..................................................................................................................... xv 1.0 Introduction ....................................................................................................................... 1 1.1 Epidemiology of melanoma .......................................................................................... 1 1.1.1 Risk factors ................................................................................................................ 1 1.1.1.1 Extrinsic risk factor: ultraviolet radiation ..................................................... 1 1.1.1.2 Host risk factors ................................................................................................ 3 1.1.1.2.1 Nevi ................................................................................................................. 3 1.1.1.2.2 Genetic mutations ......................................................................................... 3 1.1.1.2.2.1 CDKN2A.................................................................................................. 3 1.1.1.2.2.2 BRAF ....................................................................................................... 4 1.1.1.2.2.3 PTEN........................................................................................................ 5 1.1.2 Types of Melanoma ................................................................................................... 5 1.1.2.1 Superficial Spreading Melanoma (SSM) ........................................................ 5 1.1.2.2 Nodular Melanoma (NM) ................................................................................. 6 1.1.2.3 Lentigo Maligna Melanoma (LMM) ............................................................... 6 1.1.2.4 Acral Lentiginous Melanoma (ALM).............................................................. 6 1.1.2.5 Desmoplastic Melanoma (DM) ........................................................................ 7 1.2 Current treatment options for patients with melanoma ........................................... 7 1.2.1 Chemotherapy ........................................................................................................... 7 1.2.1.1 Dacarbazine ....................................................................................................... 7 vi 1.2.2 Immunotherapy......................................................................................................... 9 1.2.2.1 Cytokine therapy ............................................................................................... 9 1.2.2.2 Cellular therapy .............................................................................................. 10 1.2.2.2.1 DC Vaccines ................................................................................................. 10 1.2.2.2.2 Adoptive cell therapy (ACT) ...................................................................... 11 1.2.2.2.3 Engineered T-cell therapies ....................................................................... 13 1.2.2.2.3.1 Chimeric Antigen Receptor T-cells (CAR-T cells): ........................... 13 1.2.2.2.3.2 T-Cell Receptor Transgenic T-cells (TCR-T cells):........................... 14 1.2.2.3 Immune checkpoint blockade antibodies...................................................... 15 1.2.2.3.1 Anti-CTLA4................................................................................................. 15 1.2.2.3.2 Anti-PD-1 ..................................................................................................... 16 1.3 Challenges in current treatments for melanoma ..................................................... 17 1.3.1 The tumor microenvironment: an immune desert .............................................. 17 1.3.1.1 T-cell exclusion in targeted therapy .............................................................. 18 1.3.1.2 Melanoma exclusion of adoptively transferred T-cells................................ 19 1.3.1.3 Exclusion in immune checkpoint blockade................................................... 20 1.3.2 The tumor-vascular endothelial barrier ............................................................... 21 1.3.2.1 Pathologic properties of the tumor vasculature ........................................... 21 1.3.3 Therapeutic reprogramming of the solid tumor vascular network to improve T- cell infiltration ..................................................................................................................... 23 1.3.3.1 Normalizing existing tumor vasculature ....................................................... 23 1.3.3.1.1 Targeting VEGF signaling for vascular normalization (VN) ................. 25 1.3.3.1.2 Vaccines targeting tumor blood vessel antigens to promote VN ............ 26 vii 1.3.3.1.3 Metronomic chemotherapy for VN ........................................................... 28 1.3.3.2 Therapeutic induction of a specialized vasculature in the TME for enhanced clinical benefit. ............................................................................................... 29 1.3.3.2.1 High endothelial venules and tertiary lymphoid structures ................... 29 1.3.3.2.2 SLOs vs TLSs .............................................................................................. 31 1.3.3.2.3 Types of TLS ............................................................................................... 31 1.3.3.2.4 Cellular mediators of HEV and/or TLS neogenesis in cancer ................ 32 1.3.3.2.4.1 DCs ......................................................................................................... 32 1.3.3.2.4.2 NK cells and effector T-cells ................................................................ 32 1.3.3.2.4.3 Removal of Tregs .................................................................................