Induction of anti-tumor immunity by vaccine immunotherapy in murine lung cancer models Öffentlichkeit

Abstract

Lung cancer is the leading cause of cancer deaths in the world.Despite the development of novel treatments in the recent years such as immune checkpoint inhibitor (ICI) therapy, the prognosis of lung cancer patients is still poor. The patient-to-patient heterogeneity is the major problem in treating lung cancer, which results in differential responses to ICI therapy. In this study, two projects were designed to induce the anti-tumor immunity by using vaccine immunotherapy or foreign protein transfection to introduce novel antigen presentation in ICI resistant murine lung cancer models. In the first project, we developed a vaccine immunotherapy prepared by tumor membrane vesicle (TMV), which express tumor-specific antigens, further modified to express immunostimulatory molecules (ISMs). We investigated the efficacy of the ISM-incorporated TMV vaccine (TMVV) in combination with ICI therapy in a partially ICI resistant murine lung cancer model. We demonstrate that prophylactic vaccination of TMV adjuvanted with IL-12 and B7-1 prevents the CMT-167 tumor growth and helps the mice develop immunological memory. In a therapeutic setting, TMVV immunotherapy induced primary tumor regression in 40% of the mice and significantly reduced the metastatic tumor burden. Moreover, stimulation of splenocytes with TMVV induces IFN- gsecretion, which can upregulate the level of MHC I and MHC II on CMT-167 cells. We also find that prophylactic TMVV immunotherapy stimulates MHC II-dependent CD4+ T cells-mediated cytotoxic activity, while CD8+ T lymphocytes are indispensable for a complete effect of the vaccination. In the second project, we transfected a fully ICI resistant murine lung cancer model with a foreign antigen, red fluorescent protein (RFP) and investigated its response to the vaccine immunotherapy. Our findings suggest that the expression of RFP in LL/2 cells induces tumor immunogenicity by increasing TILs and inhibiting tumor-induced G-CSF and MDSC levels. In addition, prophylactic TMV immunotherapy primes the immune system and boosts the anti-tumor immune response in LL/2-RFP-tumor bearing mice by further inducing TILs and inhibiting immunosuppressive G-CSF and MDSC levels. Overall, our studies provide insight into using immunotherapy to regulate novel antigen presentation for treating ICI resistant lung cancer.

Table of Contents

Table of Contents

Project I - Immunotherapy with TMV vaccine induces CD4+ T cell mediated antitumor immunity in a partially ICI resistant murine lung cancer model

INTRODUCTION                                                                                                                        1

MATERIALS AND METHODS                                                                                                    5

Cell culture.......................................................................................................................... 6

IFN-gtreatment in vitro....................................................................................................... 6

Tumor model and animals ................................................................................................. 6

Prophylactic vaccination study........................................................................................... 7

Therapeutic vaccination study ........................................................................................... 7

In vivo cell depletion .......................................................................................................... 7

Cytokine assays................................................................................................................... 8

Enzyme-linked immunosorbent assay (ELISA)................................................................. 8

Lung metastasis assay......................................................................................................... 9

Cytotoxic T lymphocyte (CTL) assay.............................................................................. 10

Cellular phenotyping of immune infiltrates...................................................................... 10

Flow cytometry ................................................................................................................ 11

BCA protein assay............................................................................................................ 11

Statistical analysis ............................................................................................................ 12

RESULTS                                                                                                                                      13

Prepare and verify incorporation of GPI-ISMs onto TMVs............................................. 14

Characterization of CMT-167 TMVs .............................................................................. 15

Prophylactic TMVV immunotherapy prolongs the survival and induces immunological memory in CMT-167 tumor bearing mice         17

Therapeutic TMVV immunotherapy inhibits CMT-167 primary and metastatic tumor growth       18

CD4+ T cells and NK cells are involved in anti-tumor effect induced by TMVV immunotherapy in CMT-167-tumor bearing mice        20

TMVV immunotherapy induces robust CTL activities..................................................... 22

TMVV immunotherapy alters the tumor microenvironment of the CMT-167 tumors ... 23

DISCUSSION                                                                                                                                27

REFERENCES                                                                                                                              30

Project II - Expression of red fluorescent protein increases TILs while inhibits tumor-induced G-CSF and MDSC in a fully ICI resistant murine lung cancer model

INTRODUCTION                                                                                                                       31

MATERIALS AND METHODS                                                                                                 34

Cell culture........................................................................................................................ 35

Cell proliferation assay..................................................................................................... 35

IFN-gtreatment in vitro.................................................................................................... 35

Single cell cloning............................................................................................................. 36

Tumor model and animals ............................................................................................... 36

Prophylactic vaccination study ........................................................................................ 37

In vivocell depletion ........................................................................................................ 37

Cell preparation from various organs or tissues............................................................... 37

Lung metastasis assay....................................................................................................... 38

Cytokine assays................................................................................................................ 39

Enzyme-linked immunosorbent assay (ELISA)............................................................... 39

Flow cytometry ................................................................................................................ 40

Statistical analysis ............................................................................................................ 41

RESULTS                                                                                                                                     42

Purify and verify RFP expression in LL/2-RFP NEG and LL/2-RFP POS cells............. 43

Cell surface antigens of LL/2 and LL/2-RFP cells in culture........................................... 44

Transfection with RFP expression inhibits tumorigenicity of LL/2 tumor cells.............. 45

The expression of RFP in LL/2 cells induces tumor immunogenicity by inhibiting tumor induced G-CSF and MDSC levels       47

LL/2-RFP tumor growth is inhibited by CD4+ T cells, CD8+ T cells, and NK cells in vivo            49

Prophylactic TMV immunotherapyinduces immune cell infiltration in the LL/2 tumors but does not protect mice from tumor cell challenge.......................................................................................................................................... 50

Prophylactic TMV vaccination inhibits primary and metastatic tumor growth and induces tumor immunogenicity in the mice challenged with LL/2-RFP tumor cells ...................................................................................................... 52

DISCUSSION                                                                                                                               54          

REFERENCES                                                                                                                              59

Table of Figures

Project I

Figure 1. Anti-PD-1 antibody therapy reduces primary tumor growth of CMT-167 cells in mice    4

Figure 2. TMV preparation and incorporation of ISMs on the TMVs via a GPI-anchor to generate TMVVs         15

Figure 3. Surface markers of CMT-167 cells and TMVs............................................................. 16

Figure 4. Prophylactic TMV or TMVV immunotherapy prolongs the survival of CMT-167 tumor bearing mice.  18

Figure 5. Therapeutic vaccination of TMVV inhibits CMT-167 tumor growth and significantly reduces lung metastasis.19

Figure 6. CD4+ T cells, CD8+ T cells, and NK cells are involved in the anti-tumor response induced by the TMV vaccine immunotherapy       21

Figure 7. Induction of tumor antigen specific CTL response in mice survived from prophylactic TMVV vaccination.      23

Figure 8. Prophylactic TMVV immunotherapy induces MHC II-restricted CD4 T cell-mediated killing of target CMT-167 tumor cells by activation of eosinophils and macrophages................................................................................................... 25

Project II

Figure 1. Immunotherapy of TMV, MPL adjuvant, anti-PD-1 antibody, or their combinations do not protect against LL/2 tumor in mice.          33

Figure 2. RFP expression is maintained in vivo............................................................................ 44

Figure 3. Cell surface markers of LL/2-RFP NEG and POS cells .............................................. 45

Figure 4. RFP expression in LL/2 cell line decreases its tumorigenicity.................................... 46

Figure 5. The expression of RFP in LL/2 cells induces tumor immunogenicity by inhibiting tumor induced G-CSF and MDSC levels    48

Figure 6. LL/2-RFP tumor growth is inhibited by CD4+ T cells, CD8+ T cells, NK cells in vivo   50

Figure 7. TMV immunotherapy induces immune cell infiltration in the LL/2 tumors but does not protect mice from tumor cell challenge          51

Figure 8. TMV vaccination inhibits primary and metastatic tumor growth and induces tumor immunogenicity in the mice challenged with LL/2-RFP tumor cells..................................................................................................................................... 53

Figure 9. Schematic representations of RFP and TMV-mediated anti-tumor immune response in LL/2 tumor model        57

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, General
Stichwort	Oncology Immunotherapy Cancer Biology Vaccine immunotherapy
Committee Chair / Thesis Advisor	Dr. Ramireddy Bommireddy, Emory University Dr. Periasamy Selvaraj, Emory University
Committee Members	Dr. Rita Nahta, Emory University Dr. Melissa Gilbert-Ross, Emory University

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