Induction of Anti-Tumor Immunity in an Immune Checkpoint Inhibitor Resistant Murine Lung Cancer Open Access
Puravankara Menon, Ashwathi (2017)
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
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