The effect of vaccination of TMVs expressing GPI-mIL-12 and GPI-mGM-CSF on a colon cancer model Open Access

Yamanaka, Satoshi (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/kw52j853m?locale=en
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Abstract

Colorectal cancer is currently treated by surgical removal of the affected tissue. Surgery is often unsuccessful, and chemotherapy is administered to help prevent post-surgical relapse. Immunotherapy provides an alternative for the systemic administration of toxic chemicals; its effects are localized to the tumor and can be personalized for each patient. This study explored the development of active immunotherapy against the CT 26 murine colon cancer model using tumor membrane vesicles (TMVs) expressing IL-12 and GM-CSF cytokines. The naturally soluble cytokines were modified with glycosylphosphatidylinositol (GPI)-anchors to incorporate them onto membrane surfaces by a novel protein transfer method. Vaccination with CT26 TMVs expressing GPI-mIL-12 and GPI-mGM-CSF was unable to provide antitumor immunity to any significant extent than compared to unvaccinated mice. None of the treatments with TMVs expressing GPI-mIL-12 alone, GPI-mGM-CSF alone, or both GPI-mIL-12 and GPI-mGM-CSF were able to induce complete tumor rejection. Future studies will need to increase the dosage of TMVs or increase the amount of GPI-IL-12 and GPI-GM-CSF incorporation onto the TMVs in order to prepare a more immunogenic vaccine capable of inducing tumor rejection in a therapeutic setting.

Table of Contents

Introduction

Colorectal cancer................................................................................ 1

Tumor membrane vesicles...................................................................... 2

Protein transfer method......................................................................... 4

IL-12................................................................................................. 5 GM-CSF............................................................................................. 6

CT26 murine colon cancer model............................................................. 7

Methods

Cell cultures........................................................................................ 8

Mouse model....................................................................................... 8

GPI-anchored protein purification............................................................ 9

Protein transfer.................................................................................. 10

CT26 tumor membrane vesicles............................................................. 10

In vivo experiments............................................................................. 11 Results

Surface protein characterization of the CT26 cell line................................ 12

Purification of GPI-mIL-12 and GPI-mGM-CSF............................................14

GPI-mIL-12 and GPI-mGM-CSF incorporation onto sheep RBCs..................... 15

GPI-mIL-12 and GPI-mGM-CSF incorporation onto CT26 TMVs..................... 17

Live CT26 cell challenge....................................................................... 19

Vaccination with CT26 TMVs................................................................. 21

Vaccination with CT26 TMVs expressing GPI-mIL-12 and GPI-mGM-CSF......... 24

Production of Anti-CT26 Antibodies by Vaccinated Mice.............................. 26

Discussion

CT26 immunoescape through expression of surface molecules....................... 31

Membrane incorporation of GPI-proteins via protein transfer......................... 32

Limited efficacy of TMV vaccines in prophylactic setting.............................. 33

Lack of correlation between antibody response and tumor size...................... 35

Conclusion................................................................................................... 37 References................................................................................................... 39

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