Vasoactive Intestinal Peptide Antagonist Enhances T cell Proliferation and Synergizes with PD-1 Antibodies to Promote Anti-Melanoma T cell Immune Responses 公开

Abstract

Antagonism of vasoactive intestinal peptide (VIP) signaling is a novel approach to reverse immune check-point inhibitory pathways. Treatment with a peptide antagonist to VIP (VIPhyb) has been shown to enhance protective immune responses to mCMV and leukemia in murine models. While treatment with VIPhyb has reduced tumor burdens in human glioblastoma, lung cancer, and breast cancer engrafted in immune-deficient mice treated with concomitant chemotherapy, a stimulatory effect of VIPhyb on anti-cancer immunity to solid tumors has not been described. We hypothesized that VIPhyb treatment would enhance T cell activation and cytotoxicity in response to melanoma, which can be highly immunogenic. We describe herein a dose-dependent enhancement of in vitro T cell proliferation in response to stimulation with anti-CD3 antibodies following the addition of VIPhyb, and reversal of the suppressive effect of exogenous VIP peptide on T cell proliferation. VIPhyb synergistically enhanced T cell proliferation in vitro when combined with anti-PD1 antibodies. 

To test the effect of blocking VIP-signaling on anti-cancer immune responses to solid tumors, mice bearing B16 melanoma tumors were treated with daily injections of VIPhyb. VIPhyb-treated mice had more effector CD8+ T cells compared with saline-treated controls, but single-agent VIPhyb treatment did not have a significant effect on the kinetics of tumor growth. Combining treatment with VIPhyb and PD-1 antibodies in mice with established melanoma, we found significantly enhanced suppression of tumor growth and improved survival compared with mice treated with either inhibitor alone or saline-treated controls. VIPhyb-and anti-PD1 antibody treatment synergistically enhanced T cell mediated immunity as assessed by increased numbers of effector CD8+ T cells. 

Notably, VIPhyb treatment did not inhibit melanoma growth, invasiveness, or change expression of immunological surface ligands in vitro. We hypothesize that treatment of mice with VIPhyb induced adaptive immunity against melanoma by down-regulating co-inhibitory pathways and stimulating T cell survival through inhibition of NF-kB signaling leading to increased numbers of cytotoxic CD8+ T cells. Thus, blocking signaling through the VIP receptor represents a new strategy to induce anti-tumor immunity in solid tumor immune-oncology. 

Table of Contents

Introduction & Background__________________________________________1 

Melanoma and Clinical Management……………………………………...……..2 

Immunotherapy for the Treatment of Melanoma……...…………………...……..3 

Vasoactive Intestinal Peptide……….……………………………………...……...7 

Antagonist to Vasoactive Intestinal Peptide..……………………………...……...8 

B16 Murine Melanoma Model System………………….………………..……...10 

Experimental Hypothesis……………...…………………………………...…….11 

Materials and Methods_____________________________________________12 

Cell Lines…………………………...……………………………………….…...13 

B16 Cell Line Transfection………………………………………………….…...13 

B16 Phenotypic Analysis…..……….…………………………………….……...13 

Cell Viability Assay………..…………….....……………………………….…...14 

Transwell Migration Assay………………………………………….…....……...14 

Co-Culture Assay...……………………………….……………………........…...15 

Western Blot……………....…………………..……………………..………......16 

T cell Proliferation Assay...…………………..……………………..……….......16 

Mice……………...………………………………………………………….…...16 

VIPhyb Treatment......……………………………………………………...…....17 

VIPhyb Nanoparticle.....…………………………………………………...…….17 

Bioluminescent Imaging...………….……………………………………...…….17 

Flow Cytometry………....………….……………………………………............18 

Immunohistochemistry.....………….…………………………………….....…...18 

Results _________________________________________________________19 

VIPhyb Has No Effect on B16F1 Tumor Growth and Survival Despite Different

Routes of Administration………………………………………………………..21 

VIPhyb Administered Peritumorally Enhances CD8+ T cell Proliferation and

Non-Significantly Activates an Adaptive Immune Response in a B16F1 Model.23 

VIPhyb Alone Does Not Enhance Splenocyte Proliferation and T cell Expansion

in a B16F1 and B16F10 Co Culture Unless Combined with an Anti-PD-1 Antibody……………………………………………………………………...….25 

VIPhyb Synergizes with an Anti-PD-1 Antibody to Reduce Tumor Burden…....27 

VIPhyb and VIPhyb-NP Increases T cell Proliferation In Vitro….……………..29 

B16F1 and B16F10 Express PD-L1, PD-L2, MHCI, and VPAC2, but do not

Respond to VIPhyb……………………………………………………………...30 

Discussion ______________________________________________________33 

References ______________________________________________________37 

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, General Health Sciences, Oncology Health Sciences, Immunology
关键词	VIPhyb Oncology Melanoma T cell Immunotherapy Immunooncology PD-1 Checkpoint blockade Adaptive immunity Cancer immunotherapy Cancer Biology Immunology Vasoactive Intestinal Peptide
Committee Chair / Thesis Advisor	Waller, Edmund, Emory University
Committee Members	Thomas, Susan, Georgia Tech Pollack, Brian, Emory University

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