Engineering Armored CAR T Cells to Secrete Functional VIPR Antagonist 公开

Abstract

While CAR T cells have proven to be an effective treatment option for cancers the therapy still faces many roadblocks, such as solid cancers. One such solid tumor is pancreatic ductal adenocarcinoma (PDAC), which is resistant to many treatments, and expected to increase by over two-fold in the next ten years.25 The tumor microenvironment (TME) of PDAC is highly immunosuppressive and T cell infiltration is sparse. The VIP/VIPR axis has been studied as a targetable immune checkpoint pathway where antagonization of VIPR presents a possible strategy to increase T cell infiltration into the PDAC TME. This project presents the use of a type of armored CAR T cell that can secrete a VIP receptor (VIPR) antagonist to help retain T cell function in the immunosuppressive cancer microenvironment of PDAC. Here, we use an engineered CAR T cell that secretes the VIPR antagonist bound to Gaussia luciferase to monitor the ability of the armored CAR T cell to secrete the antagonist into the extracellular compartment as well as to validate its binding ability through measuring luminescence. Using these methods, we were able to detect Gaussia luciferase secreted into the extracellular space. We also confirmed that the binding of the VIPR antagonist to the VIPR was not disrupted by Gaussia. The peptide was also validated to be the correct size through a western blot. The results strongly support the hypothesis that our engineered armored CAR T cells can secrete the VIPR antagonist and that the antagonist can bind to T cells. With this confirmation, further studies could analyze if the secreting CAR T cells can help increase T cell infiltration in PDAC models, and Gluc can be used to monitor localization in mouse models. 

Table of Contents

Introduction 1

CAR T Cells 1

Figure 1 2

Problems with Solid Tumors 3

Armored CAR T Cells 4

Figure 2 4

Pancreatic Cancer 5

Figure 3 6

Vasoactive Intestinal Peptide 7

Figure 4 8

Engineering Armored CAR T Cells to Secrete VIPR Antagonist 8

Figure 5 9

Figure 6 10

Figure 7 11

Figure 8 12

Materials and Methods 13

Experimental Conditions 13

Transfection into H29 and Transduction into 293galv9 13

Generation of CAR T cells 14

Validating Peptide Secretion Through Luminescence Assay 15

Using Luminescence to Detect Binding 16

Nickel Purification and Western Blot 17

Results 18

Figure 9 18

Figure 10 19

Figure 11 20

Discussion 21

Transduction of CAR T Cells 21

Luminescence Assay 22

Binding Assay 24

Western Blot 26

Future Directions 28

Conclusion 28 

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Immunology Health Sciences, Oncology
关键词	Pancreatic Ductal Adenocarcinoma Immunotherapy Luminescence Vasoactive Intestinal Peptide Peptide Inhibition PDAC Cancer CAR T Cell Immunology Cell Culture Checkpoint Inhibitor Armored CAR Pancreatic Cancer Immune Checkpoint Gaussia Luciferase
Committee Chair / Thesis Advisor	Rafiq, Sarwish, Emory University
Committee Members	Waller, Edmund, Emory University Salaita, Khalid, Emory University

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