Induction of Anti-Tumor Immunity in an Immune Checkpoint Inhibitor Resistant Murine Lung Cancer Pubblico

Abstract

FDA approved immune checkpoint inhibitors (ICIs), such as monoclonal antibodies for PD-1 and CTLA-4, have shown efficacy in abrogating tumor growth, as well as in improving survival and prognosis in a variety of preclinical and clinical studies. However, only a small fraction of the patient population is responsive to immune checkpoint blockade. One of the reasons suggested to account for this selective efficacy of immune checkpoint blockade therapy is the lack of pre-existing anti-tumor immunity in these patients.

In this study, we hope to generate a de novo, T cell mediated anti-tumor immune response in an ICI resistant murine Lewis Lung Carcinoma model through Tumor Membrane Vesicle (TMV) based vaccines. We further enhance the TMV mediated anti-tumor immune response by "stepping on the gas" using various vaccine adjuvants such as TLR agonists to induce DC maturation and enhance activation of tumor specific T cells. Lastly, we combine TMVs, adjuvants and ICI therapy to create "TMV+ Adjuvant+ ICI" vaccines to induce protection against tumor challenge and/or abrogation of existing tumors in vivo. Our results in the LL/2 model suggest that this tumor model is resistant to both checkpoint blockade and/or TMV vaccines adjuvated with common synthetic adjuvants.

Table of Contents

Introduction ............................................................................................................ 1

Tumor Associated Antigens in Cancer Immunotherapy .................................. 2

The Cancer Immunity Cycle: The dynamics of an ideal

anti-tumor response following vaccination with a TAA-

carrying cancer vaccine .................................................................................. 3

Purpose and Hypothesis........................................................................................ 8

Intervention #1: Delivering Tumor Associated Antigens

through Tumor Membrane Vesicles for the initiation

of the Cancer-Immunity Cycle .............................................................................. 9

Intervention #2: Enhancing APC maturation and antigen

presentation by co-administration of TMVs with vaccine

adjuvants .............................................................................................................. 13

TLR Agonists as Adjuvants: Initiating the adaptive

immune response through activation of the innate

immune system........................................................................................ 21

.

Monophosphorylated Lipid A ........................................................ 23

Poly IC .......................................................................................... 24

Imiquimod .................................................................................... 24

CpG .............................................................................................. 24

Alhydrogel (Alum) ......................................................................... 25

MF59/ AddaVax ............................................................................ 27

Quil A/Saponin ............................................................................. 29

Intervention #3: "Releasing the breaks" of the cancer

immunity cycle: Preventing immunosuppression in the

immuno-tumor interactosome by using immune checkpoint

inhibitors - monoclonal antibodies against PD-1 and CTLA-4 .............................. 31

Immunosuppression mediated by CTLA-4 .......................................... ..... 32

Immunosuppression mediated by PD-1 ................................................... 34

Initiating an anti-tumor immune response in an immune checkpoint

inhibitor resistant murine Lewis Lung Carcinoma ................................................ 37

Potential TAAs for Lewis Lung Carcinoma? : A brief review of

Lewis Lung Carcinoma based immunotherapy .................................................... 39

Experimental Design ............................................................................................ 41

Materials and methods ......................................................................................... 44

Results ................................................................................................................. 51

Results from Experiment 1: Characterization of the LL/2

cell line through flow cytometry ................................................................ 51

Results from Experiment 2: Characterization of LL/2

tumor growth in vivo ................................................................................. 54

Results from Experiment 3: Production and

Characterization of LL/2 cell pellet derived and tumor

tissue derived TMVs ................................................................................. 61

Results from Experiment 4: Determining the LL/2 TMV

Dose for vaccine studies .......................................................................... 65

Results from Experiment 5: "TMV+ Adjuvant" Prophylactic

vaccination study ...................................................................................... 70

Results from Experiment 6: "TMV + ICI" Therapeutic

vaccination study ...................................................................................... 80

Results from Experiment 7: "TMV + Adjuvant + ICI" Study ...................... 87

Results from Experiment 8: In Vitro treatment of LL/2 cells ..................... 100

with IFN-γ

Discussion ............................................................................................................ 102

Conclusion ............................................................................................................ 107

References ........................................................................................................... 108

List of Figures

Figure 1: The Cancer-Immunity Cycle .................................................................. 4

Figure 2: Protein Transfer of GPI-proteins onto TMVs ......................................... 12

Figure 3: TLR Signaling ........................................................................................ 19

Figure 4: aCTLA-4 antibodies can rescue T cell activation ............................. ..... 33

Figure 5: Immunosuppression mediated by PD-1 and

PD-L1/PD-2 interactions ....................................................................................... 35

Figure 6: Antigenic profile of LL/2 cells ................................................................. 52

Figure 7: LL/2 tumor growth in vivo ...................................................................... 55

Figure 8: Subcutaneous LL/2 tumors up to 2 cm

in diameter may not metastasize to the lungs ...................................................... 58

Figure 9: Tumor challenge dose was ascertained by

inoculating varying amounts of LL/2 cells in vivo. ................................................. 60

Figure 10: Preparation of LL/2 cell derived TMVs from cell

pellet or tumor derived TMVs from fresh or frozen tumor tissue ........................... 62

Figure 11: Antigen profile of LL/2 tumor tissue derived TMVs

and LL/2 cell pellet derived TMVs ......................................................................... 63

Figure 12: Negative staining of TMVs was performed and

images were acquired using transmission electron microsccopy .................... ..... 64

Figure 13: Prophylactic Vaccination with LL/2 TMVs does

not protect against tumor challenge ...................................................................... 67

Figure 14: Immunization with LL/2 TMVs does not induce

antibodies against LL/2 ........................................................................................ 69

Figure 15: Prophylactic Vaccination with "TMV+Adjuvant"

vaccines does not protect against tumor challenge or enhance survival. ............. 73

Figure 16: Prophylactic Vaccination with "TMV+Adjuvant"

vaccines does not protect against tumor challenge or enhance survival .............. 75, 76

Figure 17: Prophylactic vaccination with "TMV + Adjuvant"

vaccines does not induce an anti-LL/2 antibody response ................................... 78

Figure 18: Vaccination with both LL/2 tumor tissue derived TMVs

and "TMV+ Adjuvant Vaccines" does not prevent LL/2 tumor

induced splenomegaly .......................................................................................... 79

Figure 19: Therapeutic Vaccination with ICI and "TMV + ICI" Vaccines ............... 82

.

Figure 20: Therapeutic Vaccination with ICI and "TMV + ICI" Vaccines ............... 84, 85

.

Figure 21: Therapeutic vaccination with ICIs and "TMV + ICI" vaccines

does not reduce splenomegaly in LL/2 tumor bearing mice .................................. 86

Figure 22: "TMV + Adjuvant + ICI" vaccines do not protect

against tumor challenge nor induce regression of tumors

in vivo, nor enhance survival ................................................................................. 89

Figure 23: Vaccination with "TMV+ Adjuvant+ ICI" vaccines

does not protect from tumor challenge or regress established tumors.................. 91 - 93

Figure 24: Vaccination with "TMV+ MPL (20ug)" and

"TMV+ Addavax (50uL) does not induce an antibody response ........................... 95

Figure 25: DTH response is not generated in vaccinated mice ............................ 96

Figure 26: Cytokine levels in sera of mice immunized with

"TMV + Adjuvant" vaccines.................................................................................... 98

Figure 27: "TMV + Adjuvant + ICI" vaccines do not reduce

splenomegaly in tumor bearing mice..................................................................... 100

Figure 28: MHC I expression does not increase upon treatment with IFN- γ........ 101

List of Tables

Table 1: The properties of vaccine adjuvants used in this study........................... 14

Table 2: The properties and dosage of adjuvants selected to

make "LL/2 TMV + Adjuvant" Vaccines................................................................. 72

Table 3: A summary of adjuvants selected to make

"LL/2 TMV + Adjuvant + ICI" Vaccines.................................................................. 88

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Biology, General Health Sciences, Oncology Health Sciences, Immunology
Parola chiave	Translational Oncology Cancer-Immunity Cycle Cancer Adjuvants Vaccine Adjuvants Tumor Membrane Vesicles Therapeutic Cancer Biology Prophylactic Lewis Lung Carcinoma TMV Checkpoint Blockade Cancer Vaccine Immune Checkpoint Inhibitors Anti-tumor immunity Vaccine C57BL/6
Committee Chair / Thesis Advisor	Selvaraj, Periasamy, Emory University
Committee Members	Gilbert-Ross, Melissa, Emory University Shanmugam, Mala, Emory University

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