Th-1 Cytokine and Antibody Mediated Immunity against HER Family Expressing Breast Cancer ...

171 Pages·2016·3.99 MB·English

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Th-1 Cytokine and Antibody Mediated Immunity against HER Family Expressing
Breast Cancer Cells

A dissertation submitted to
Kent State University in partial
fulfillment of the requirements for the
degree of Doctor of Philosophy

By
Prachi Namjoshi

May 2016
© Copyright
All rights reserved
Except for previously published materials
Dissertation written by
Prachi Namjoshi
B. Pharm., AISSMS College of Pharmacy, India, 2008
M.S., NIPER, India, 2010
PhD., Kent State University, 2016

Approved by
Dr. Gary Koski, Chair, Doctoral Dissertation Committee
Dr. Derek Damron, Members, Doctoral Dissertation Committee
Dr. Colleen Novak,
Dr. Mark James,
Dr. John Johnson,

Accepted By
Dr. Ernest Freeman Director, School of Biomedical Sciences
Dr. James L. Blank Dean, College of Arts and Science

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TABLE OF CONTENTS

Appendix 1: List of Figures………………………………………………………………v
Appendix 2: List of Tables………………………………………………..……….….....ix
Appendix 3: List of abbreviations used…………………………………………………..x
Acknowledgements………………………………………………..…………………....xii

Chapter 1: General Introduction………………………………………………………….1
1.1. A brief historical perspective on cancer immunotherapy…..……………………….1
1.2. Contemporary immunotherapeutic approaches..……………………………………2
1.3. Apoptosis: a common death mechanism initiated by multiple effectors of
anti-tumor immunity including Th1 cytokines ………………………………..…..20
1.4. The rational design of a therapeutic breast cancer vaccine……………………...…25
1.5. Statement of objective and specific aims………………………………………..…41

Chapter 2: To study the effect of archetypical Th 1 cytokines TNF-α and IFN-γ on
various murine and human breast cancer cell lines that varies in their expression of
HER-family members …………………………………………………………………...43
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2.1. Introduction……………………………………………………………………...…43
2.2. Materials and Methods……………………………………………………………..47
2.3. Results……………………………………………………………………………...56
2.4. Discussion………………………………………………………………………..…78

Chapter 3: Identification of linear B-Cell (antibody) Epitopes within the Extracellular
Region of Human HER-3 and characterization of the in vitro biological activity of
murine antisera on a HER-3-expressing breast cancer cell line.…………………...……85
2.1. Introduction………………………………………………………………………..85
2.2. Materials and Methods…………………………………………………………….94
2.3. Results…………………………………………………………………………..…98
2.4. Discussion…………………………………………………………………………115

Chapter 4: Discussion and Future directions…………………………………………122
4.1. Discussion………………………………………………………………………….122
4.2. Future directions…………………………………………………………………...126

Bibliography…………………………………………………………………...………130
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APPENDIX 1: List of Figures

Figure 1: Mode of action of antibodies……………………………………………………4
Figure 2: Adoptive T cell therapy…………………………………………………………8
Figure 3: DC based immunotherapy………………………………………………………9
Figure 4: T helper cell subsets and their effector cytokines……………………………..15
Figure 5: Schematic representation of Tumor necrosis factor receptor-1 (TNFR-1)
signaling pathway and IFN-γ induced JAK/STAT signaling pathway………………….19
Figure 6: Schematic representation of extrinsic and intrinsic apoptotic pathways…...…23
Figure 7: Structure and ligands of HER receptor family……………………………...…28
Figure 8: RTK signaling……………………………………………………………....…30
Figure 9: Immunohistochemical staining of lymphocytes infiltrating into breast
following DC vaccine in DCIS patients……………………………………………....…35
Figure 10: Immunohistochemical staining of HER-2 on DCIS before and after
vaccination…………………………………………………………………………....…36
Figure 11: Mode of action of CD8pos (CTLs) cells…………………………………...…38
Figure 12: Mode of action of CD4pos Th cells………………………………………..….40
Figure 13: Determination of optimal cytokine concentrations……………..………...…56
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Figure 14: Effect of Th1 cytokines on metabolic activity of murine breast
cancer cell lines……………………………………………………………………….…58
Figure 15: Effect of Th1 cytokines on proliferation of murine breast cancer
cell lines…………………………………………………………………………………60
Figure 16: Photo microscopic study of Th1 cytokine-treated murine breast
cancer cells………………………………………………………………………………62
Figure 17: Cytokine-sensitive and insensitive cell lines express comparable
levels of Th1 cytokine receptors………………………………………………………...63
Figure 18: Annexin-PI apoptosis assay to determine induction of apoptosis
by Th1 cytokines……………………………………………………………………...…65
Figure 19: TUNEL assay to determine induction of apoptosis by Th1 cytokines………67
Figure 20: Th1 cytokine-induced activation of caspase-3………………………...……69
Figure 21: Th1 cytokines alter HER-family expression on murine and human
breast cancer cells…………………………………………………………………….…71
Figure 22: Loss of HER-2 expression is associated with apoptosis……………….……73
Figure 23: Caspase 3 agonist induces apoptosis in breast cancer cell likes………….…75
Figure 24: Caspase 3 agonist induces loss of HER-family surface expressio………..…76
Figure 25: Caspase 3 inhibitor blocks Th1 induced loss of HER-2 expression……...…77
Figure 26: Schematic overview of HER-3 structure……………………………….…...90
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Figure 27: A three-cell system is required for the generation of antigen specific
antibody response……………………………………………………………………..…92
Figure 28: Illustration of 4 subdomains of HER-3 ECD the associated
peptide pools………………………………………………………………………….…99
Figure 29: Recognition of vaccinating peptides by sera pools from immunized
Balb/c and C57bl/6 mice…………………………………………………………….…102
Figure 30: Recognition of vaccinating peptides by peptide pool antisera generated in
Balb/c and C57bl/6 mice……………………………………………………………….103
Figure 31: Recognition of HER-3 ECD by peptide pool antisera generated in
Balb/c and C57bl/6 mice……………………………………………………………….105
Figure 32: Recognition of native HER-3 on MDA-MB-468 breast cancer
cell by peptide pool antisera from vaccinated Balb/c mice…………………………….107
Figure 33: Capacity of individual peptides from pools VI and XI to independently
induce anti-peptide antibodies in Balb/c mice.………………………………………....109
Figure 34: Recognition of HER3 ECD by antisera against individual peptides
from pools VI and XI generated in Balb/c mice………………………………………..110
Figure 35: Recognition of native HER-3 on MDA-MB-468 breast cancer
cell by sera 27 and sera 55 of Balb/c mice……………………………………………...111
Figure 36: Incubation with HER-3-reactive peptide antisera does not induce
changes in metabolic activity in HER3pos breast cancer line……………..………….…113
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Figure 37: Anti-HER-3 peptide antisera suppresses metabolic activity of
HER-3pos breast cancer cells when combined with Th1 cytokines………………….…114
Figure 38: Mechanistic model…………………………………………………………128

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Appendix 2: List of Tables

Table 1: FDA approved mAbs for use in oncology Mode of action of
antibodies……………………………………………………………………………….…6
Table 2: Clinical applications of cytokines in cancer patients…………………………...11
Table 3: Aminoacid sequence of HER-3 ECD peptides 1-63…………………………..100

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Appendix 3: List of Abbreviations

ADCC Antibody dependent cellular cytotoxicity
APC Antigen presenting cell
BH Bcl-2 homology
CD Cluster of differentiation
CDC Complement dependent cytotoxicity
CR Complete response
DC Dendritic cell
DCIS Ductal carcinoma in situ
DD Death Domain
DED Death effector domain
DISC Death-inducing signaling complex
EGF Epidermal growth factor
FADD Fas activated death domain
GAS Gamma activated sequence
GM-CSF Granulocyte–macrophage colony-stimulating factor
HER Human epidermal growth factor receptor
IFN Interferon
IL Interleukin
IRF IFN response factor
JAK Janus activated kinase
mAb Monoclonal antibody
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