Adjuvating Melanoma TMVs By Incorporating GPI-anchored Immunostimulatory Molecules To Induce B16F10 Melanoma Tumor Suppression Open Access

Amaram, Nikhil Kanth (2013)

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The body's immune system makes up the first line of defense against foreign pathogens and abnormal host cells as well. However, this system is not perfect and at times either of these two antagonists can evade elimination. In the case of a foreign pathogen, it may be called a disease or sickness, but for abnormal cells it is called a cancer. Many studies have tried utilizing the immune system to prime it against these cancers and help the body eliminate a tumor on its own. Our research lab has designed a new way of approaching tumor vaccines. If successful, a primary tumor could be removed from a patient and tumor membrane vesicles (TMVs) could be made from them. In addition, the lab has also created ISMs linked to a glycosylphophatidylinositol (GPI) anchor which allows these ISMs to incorporate even soluble cytokines into the membranes. This study aimed to test this combined vaccination model using both GPI-anchored ISMs and TMVs created from B16F10 cells, a popular model for melanoma. Successful incorporation of GPI-anchored IL-12 and GM-CSF was shown onto TMVs and were able to induce higher tumor protection than unmodified TMV in a prophylactic experiment. One mechanism for tumor-based immunity was found in TMVs incorporated with GPI-anchored GM-CSF through the use of antibody production in response to the vaccine. However, mice vaccinated with GPI-anchored IL-12 and GPI-anchored IL-12 and GM-CSF showed the greatest tumor prevention and are possible targets for a therapeutic vaccine using the same vaccination design.

Table of Contents

Table of Contents

Abstract iv Introduction 1-12

Elements of the Immune System 2

Immunosurveillance on Cancerous Phenotypes 4

Methods of Evasion By Tumor Cells 5

Systemic Cytokine Administration and Costimulation Blockades 7

Vaccines Using Antigens Presenting Cells 8

Immunostimulatory Molecule Gene Transfected Vaccines 9

Protein Transfer of Immunostimulatory Molecules 10

Experimental Design 13-16

Materials and Methods 17-26

Cell culture 17

Fluorescence-activated cell sorting (FACS) analysis 17

Affinity chromatography of GPI-ISMs 18

Dot blot Analysis 20

Western blot analysis 21

Polyvinylpyrrolidone mediated concentration 22

TMV preparation 23

BCA assay for protein concentrations 23

B16F10 live cell mouse challenge 24

B16F10 TMV Immunization experiment to determine concentration of TMV to inject. 24

GPI-ISM incorporation onto B16F10 TMVs 25

Modified B16F10 TMV prophylactic vaccination mouse challenge 25

Serum antibody assay 26

Results 27-42 Discussion 43-49 Conclusion 50 References 51-62

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