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Development, Characterization and Validation of Trastuzumab-Modified Gold Nanoparticles for Molecularly Targeted Radiosensitization of Breast Cancer
by
Niladri Chattopadhyay
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmaceutical Sciences University of Toronto
© Copyright by Niladri Chattopadhyay 2012
Development, Characterization and Validation of TrastuzumabModified Gold Nanoparticles for Molecularly Targeted Radiosensitization of Breast Cancer Niladri Chattopadhyay Doctor of Philosophy Department of Pharmaceutical Sciences University of Toronto 2012
Abstract The overexpression of the human epidermal growth factor receptor-2 (HER-2) in 20-25% of human breast cancers was investigated as a target for development of a gold nanoparticle (AuNP) based radiosensitizer for improving the efficacy of neoadjuvant X-radiation therapy of the disease. HER-2 targeted AuNPs were developed by covalently conjugating trastuzumab, a Health Canada approved monoclonal antibody for the treatment of HER-2-overexpressing breast cancer, to 30 nm AuNPs. Trastuzumab conjugated AuNPs were efficiently internalized by HER2-overexpressing breast cancer cells (as assessed by darkfield microscopy and transmission electron microscopy) and increased DNA damage from X-radiation in these cells by more than 5-fold.
To optimize delivery of AuNPs to HER-2-overexpressing tumors, high resolution
microSPECT/CT imaging was used to track the in vivo fate of
111
In-labelled non-targeted and
HER-2 targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and non-specific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving ii
internalization in HER-2-positive tumor cells as compared to non-targeted AuNPs. This phenomenon could be attributed to Fc-mediated recognition and subsequent sequestration of trastuzumab conjugated AuNP by the reticuloendothelial system (RES). Blocking of the RES did not increase tumor uptake of either HER-2 targeted or non-targeted AuNPs. Following i.t. injection, our results suggest that Au-NTs redistribute over time and traffick to the liver via the ipsilateral axillary lymph node leading to comparable exposure as seen with i.v. administration. In contrast, targeted AuNPs are bound and internalized by HER-2-overexpressing tumor cells following i.t. injection, with a lower proportion of AuNPs redistributing to normal tissues. In vivo, the combination of HER-2 targeted AuNPs injected i.t. and X-radiation (11 Gy) yielded a 46% decrease in tumor size over a 4 month period in contrast to an 11.5% increase in tumor size for X-radiation treatment alone. Toxicology studies (evaluated through complete blood cell counts, by serum transaminase and creatinine measurements and by monitoring the body weight) demonstrated no apparent normal organ toxicity from the combination of HER-2 targeted AuNPs and X-radiation. These results are promising for the clinical translation of HER-2-targeted AuNPs for radiosensitization of tumors to X-radiation.
iii
Acknowledgments The PhD training is a long journey and it wouldn’t have been possible without the contributions of many individuals. I would like to extend my deepest thanks to: My mentor, Dr. Raymond Reilly for offering me the opportunity to train in his laboratory. As a doctoral student I could not have asked for a more enthusiastic and supportive mentor. Ray is an extraordinary teacher and a role model. The duration of my training in his laboratory has been one of great intellectual creativity for me. I have enjoyed the high standards of scientific research in his laboratory that have prepared me well for a career in pharmaceutical sciences. I would like to thank Dr. Jean-Philippe Pignol for his constant motivation and encouragement to dream big. I also thank him for making the time in his busy schedule to allow me to attend his clinic at Sunnybrook Hospital, Toronto. This unique opportunity allowed me to observe firsthand the clinical management of breast cancer as it stands today. This experience engrained in me, that despite great advances in medicine, how devastatingly life-altering a diagnosis of breast cancer is for a patient and her family. This experience served as a constant motivation and I hope the small contribution of this thesis will in some way further the ultimate goal of successfully treating cancer patients without increasing the inherent toxicity of the treatment itself. Dr. Reina Bendayan has been one of my intellectual mentors and has closely guided my training since I joined the department as a M.Sc. candidate. I am indebited to Reina for her efforts in my career development. I sincerely thank Dr. Shana Kelley who always offered to meet with me to discuss the project, shaping my training in pharmaceutical sciences and offering career advice. My mentors at the University of Mumbai nurtured my early interest in pharmaceutical science research and I am grateful for the encouragement and advice they have provided over the years. I also thank Dr. Paresh Thakkar for his constant support and guidance. My training in the multidisciplinary field of pharmaceutical sciences has required diverse expertise and I would like to thank my colleagues including Dr. Zhongli Cai, Dr. Humphrey Fonge, Dr. Conrad Chan, Dr. Jeff Leyton, Deborah Scollard, Eva Razumienko, Aisha Fasih, Yinka Akinlolu, Amy Boyle, Karen Lam and Eli Lechtman for their numerous contributions but also providing a wonderful work environment. In particular, I would thank Dr. Zhongli Cai for her contributions. Zhongli is an outstanding radiation biologist and has played an instrumental role in my PhD training. She has been a wonderful colleague and provided much needed encouragement during challenging phases of the PhD when failed experiments hugely outnumbered the successful ones! I am also grateful to Dr. Dena Taylor who has taught me how to effectively communicate complex scientific research. An extremely enriching experience during my graduate studies has been serving in the capacity of a lead residence counselor (don) at New College and 89 Chestnut Residences at the University of Toronto. I am eternally grateful to Dean Ann Yeoman for having the faith in me to undertake these responsibilities and the hundreds of undergraduate students who made it a truly enjoyable mentorship experience (especially Elmsley House and Floor 20). The close friends I have made iv
during this time including Salima Jaffer, Tarun Bablani and Anath Lionel will always be cherished. I thank Tammy Chan and Carla Serpe in the graduate office for helping me navigate through the various deadlines and administrative requirements for completion of the PhD degree. The professorial and administrative staff at the Leslie Dan Faculty of Pharmacy have greatly assisted in various capacities during my doctoral training and I acknowledge their assistance. I am grateful to the University of Toronto (Connaught Fellowship, 2007–2009), United States Department of Defence Breast Cancer Research Program (predoctoral fellowship grant # W81XWH-08-1-0519, 2008–2012), the Canadian Institutes of Health Research (Vanier Canada Graduate Scholarship, 2009–2012) and the Ara Mooradian Foundation for providing financial support. I also acknowledge funding from the Canadian Society of Pharmaceutical Sciences (CSPS), Association of Faculties of Pharmacy of Canada (AFPC), American Association of Pharmaceutical Sciences (AAPS) and American Association of Cancer Research (AACR) for providing various travel grants and bursaries for attending conferences which have greatly broadened my training in pharmaceutical sciences. My family has always been my greatest supporter. I thank my parents, Prashantkumar and Kanchan Chattopadhyay. It is because of the sacrifices they have made that I have had the opportunity to consistently pursue world class education. Your wisdom has helped me stay focused through the many uncertainities of graduate school. I thank my sister, Tansa Chattopadhyay and brother-in-law Yoshit Rastogi for providing crucial support as I transitioned to a new country for graduate studies. My nieces Tasha and Thea Rastogi complete my family in Canada. Finally, I thank my best friend and wife, Soumya, for her unbounded love. You have been a pillar of strength, listening to my fears, comforting me in my worries and supporting my aspirations. Without you, completing the Ph.D. would not have been possible.
v
Dedicated to my parents, Prashantkumar and Kanchan Chattopadhyay and my wife, Soumya Chattopadhyay
vi
Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Dedication ...................................................................................................................................... vi Table of Contents .......................................................................................................................... vii List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Abbreviations ................................................................................................................... xvi List of Appendices ........................................................................................................................ xx Chapter 1: INTRODUCTION ..................................................................................................... 1 1.0 Breast Cancer ...................................................................................................................... 2 1.1 Incidence and etiology ........................................................................................................ 2 1.2 Locally Advanced Breast Cancer (LABC) ......................................................................... 3 1.2.1 Management of LABC ............................................................................................ 3 1.2.2 Radiation therapy .................................................................................................... 8 1.3 Human Epidermal Growth Factor Receptor-2 (HER-2) ................................................... 11 1.3.1 HER-2 expression in normal tissues and role in normal human development ..... 11 1.3.2 How is HER-2 overexpressed? ............................................................................. 11 1.3.3 Detection of HER-2 overexpression ..................................................................... 12 1.4 Immunotherapy of HER-2-overexpressing Tumours with Trastuzumab ......................... 12 1.4.1 Endogenous antibodies versus recombinant antibodies ........................................ 12 1.4.2 Recombinant monoclonal antibody trastuzumab (Herceptin) .............................. 14 1.5 Neoadjuvant Radiation Therapy for LABC ...................................................................... 14 1.6 Adverse effects of Radiation Therapy of Breast Cancer .................................................. 15 vii
1.7 Challenges and Opportunities for Improving Response to Neo-adjuvant Radiation Therapy of LABC ............................................................................................................. 16 1.8 What is Nanotechnology? ................................................................................................. 17 1.8.1 Application of nanotechnology to improving the radiation treatment of LABC .. 17 1.9 Gold Nanoparticles ........................................................................................................... 19 1.9.1 History of the applications of gold in medicine .................................................... 19 1.9.2 Synthesis of AuNPs .............................................................................................. 19 1.9.3 Strategies for conjugation of antibodies to AuNPs ............................................... 20 1.9.4 Pharmacokinetics and biodistribution of AuNPs .................................................. 24 1.9.5 Toxicity ................................................................................................................. 26 1.9.6 Clinical Status ....................................................................................................... 28 1.10 AuNPs as a Radiosensitizer .............................................................................................. 29 1.10.1 History of radiosensitization with high atomic (Z) materials ............................... 29 1.10.2 Advantages of AuNPs ........................................................................................... 30 1.10.3 Characteristics of an ideal radiosensitizer............................................................. 30 1.11 Development of AuNP Based Radiosensitizers ................................................................ 31 1.11.1 Importance of radiation energy ............................................................................. 31 1.11.2 Importance of size ................................................................................................. 32 1.12 Challenges in Successful Clinical Translation of AuNPs ................................................. 34 1.12.1 Rapid elimination by the RES............................................................................... 34 1.12.2 Mechanism of nanoparticle uptake by RES .......................................................... 35 1.12.3 PEGylation ............................................................................................................ 36 1.12.4 Analytical techniques for studying the biodistribution of AuNPs ........................ 37 1.13 Hypothesis of the Thesis ................................................................................................... 40 1.14 Goals and Objectives ........................................................................................................ 40
viii
Chapter 2: DESIGN AND CHARACTERIZATION OF HER-2 TARGETED GOLD NANOPARTICLES FOR ENHANCED X-RADIATION TREATMENT OF LOCALLY ADVANCED BREAST CANCER ................................................................... 42 2.0Abstract ............................................................................................................................... 44 2.1 Introduction ....................................................................................................................... 45 2.2 Materials and Methods ...................................................................................................... 46 2.2.1 A] PEGylation of Trastzumab .............................................................................. 46 2.2.2 B] Characterization of PEGylated Trastuzumab................................................... 46 2.2.3 C] Conjugation of PEGylated Trastuzumab to AuNPs......................................... 50 2.2.4 D) HER-2 targeting: Evaluation of Internalization by Darkfield Microscopy .... 52 2.2.5 Clonogenic Assay ................................................................................................. 52 2.2.6 Evaluation of Trastuzumab-AuNP Enhanced and X-Radiation Induced DNA Double-Strand Breaks ........................................................................................... 53 2.2.7 Statistical Analysis ................................................................................................ 54 2.3 Results ............................................................................................................................... 54 2.3.1 Characterization of PEGylated Trastuzumab........................................................ 54 2.3.2 Immunoreactivity of PEGylated Trastuzumab ..................................................... 54 2.3.3 Conjugation of PEGylated and Immunoreactive Trastuzumab to AuNPs............ 58 2.3.4 HER-2 targeting: Evaluation of Internalization by Darkfield Microscopy .......... 63 2.3.5 Evaluation of Trastuzumab-PEG-AuNP Enhanced and X-Radiation Induced DNA Double Strand Breaks.................................................................................. 63 2.4 Discussion ......................................................................................................................... 63 2.5 Conclusion ........................................................................................................................ 69 Chapter 3: ROLE OF ANTIBODY MEDIATED TUMOR TARGETING AND ROUTE OF ADMINISTRATION IN NANOPARTICLE TUMOR ACCUMULATION IN VIVO ....................................................................................................................................... 71 3.0 Abstract ............................................................................................................................. 73 3.1 Introduction ....................................................................................................................... 74 3.2 Materials and Methods ...................................................................................................... 78 ix
3.2.1 Synthesis of SH-PEG2k-Bn-DTPA ....................................................................... 78 3.2.2
111
In-labelling SH-PEG2k-Bn-DTPA .................................................................... 78
3.2.3 Preparation of 111In-labelled AuNP ...................................................................... 78 3.2.4 Breast Cancer Cells and Tumor Xenograft Mouse Model.................................... 79 3.2.5 MicroSPECT/CT Imaging and Biodistribution .................................................... 79 3.2.6 Pharmacokinetic Evaluation ................................................................................. 80 3.2.7 Tumor Immunohistochemistry and Autoradiography .......................................... 81 3.2.8 Ex Vivo -Transmission Electron Microscopy (TEM) .......................................... 81 3.2.9 Ex-vivo Inductively Coupled Plasma Mass Spectrometry (ICP-MS) .................. 82 3.2.10 Statistical Analysis ................................................................................................ 82 3.3 Results and Discussion ..................................................................................................... 82 3.3.1 MicroSPECT/CT Imaging, Biodistribution and Pharmacokinetics ...................... 82 3.3.2 Mechanism of Redistribution of AuNPs from Tumor to the Systemic Circulation............................................................................................................. 89 3.3.3 Quantification of Mass of AuNPs in Tissues Using ICP-MS and VOI analysis. . 90 3.3.4 Transmission Electron Microscopy (TEM) .......................................................... 92 3.4 Conclusions ....................................................................................................................... 97 Chapter 4: MOLECULARLY TARGETED GOLD NANOPARTICLES ENHANCE THE RADIATION RESPONSE OF BREAST CANCER CELLS AND TUMOR XENOGRAFTS TO X-RADIATION ................................................................................. 100 4.0 Abstract ........................................................................................................................... 102 4.1 Introduction ..................................................................................................................... 103 4.2 Materials and Methods .................................................................................................... 104 4.2.1 Preparation of HER-2 targeted AuNPs ............................................................... 104 4.2.2 Breast Cancer Cells and Tumor Xenograft Mouse Model.................................. 105 4.2.3 X-ray Irradiation and Dosimetry ......................................................................... 105 4.2.4 Clonogenic and Immunofluorescence γ-H2AX Assays ..................................... 107 x
4.2.5 In vivo X-radiation Dose Selection ..................................................................... 107 4.2.6 In vivo Evaluation of Radiosensitization Properties of AuNPs .......................... 108 4.2.7 Normal-Tissue Toxicity of Au-T ........................................................................ 108 4.2.8 Statistical Comparisons ....................................................................................... 109 4.3 Results ............................................................................................................................. 109 4.3.1 X-ray Irradiation and Dosimetry ......................................................................... 109 4.3.2 Clonogenic and Immunofluorescence γ-H2AX Assays ..................................... 109 4.3.3 In vivo X-radiation Dose Selection ..................................................................... 111 4.3.4 In vivo Evaluation of Radiosensitization Properties of AuNPs .......................... 115 4.3.5 Normal Tissue Toxicity of Au-T Combined with X-Radiation .......................... 115 4.4 Discussion ....................................................................................................................... 116 4.5 Conclusion ...................................................................................................................... 121 Chapter 5: DISCUSSION AND FUTURE DIRECTIONS .................................................. 122 5.1 Thesis Conclusion and Summary of Findings ............................................................... 123 5.2 Future Directions ............................................................................................................ 127 5.2.1 Future Direction 1: development of HER-2 targeted AuNPs using trastuzumab (Fab) fragments ................................................................................................... 127 5.2.2 Future Direction 2: use of HER-2 targeted-111In labeled AuNPs as a novel therapeutic agent ................................................................................................. 127 5.2.3 Future Direction 3: monitoring response by microPET imaging........................ 129 References ................................................................................................................................... 133 Appendix A: ................................................................................................................................ 165
xi
List of Tables Table 2.1 Number of 123I-Labeled Trastuzumab-PEG-OPSS Molecules Conjugated Per AuNP. 60 Table 2.2 Dynamic Light Scattering (DLS) Analysis of AuNPs Modified with Varying Concentrations of Trastuzumab-PEG-OPSS ................................................................................ 62 Table 3.1 Tumor and Normal Tissue Uptake in MDA-MB-361 Tumor-Bearing Mice 48 Hours After Intravenous and Intratumoral Injection (expressed as % of injected dose/gram of tissue).87 Table 4.1 Normal Tissue Toxicity of Au-T combined with X-Radiation ................................... 117 Table 5.1 Quantification of aldehydes generated per trastuzumab molecule by varying the concentration of trastuzumab ...................................................................................................... 171 Table 5.2 Quantification of aldehydes generated per trastuzumab molecule by varying the concentration of sodium periodate, reaction time or reaction temperature................................. 172 Table 5.3 Quantification of NH2-PEG-Fluorescein5000 molecules conjugated to oxidised trastuzumab ................................................................................................................................. 175
xii
List of Figures Fig. 1.1 Concept of enhancement of X-radiation therapy with radiosensitizing compounds ....... 18 Fig. 2.1 Step-wise reactions for development of trastuzumab-PEG-AuNP and illustration of HER-2-mediated cellular internalization of these targeted AuNPs .............................................. 48 Fig. 2.2 SDS-PAGE analysis of trasutuzumab-PEG conjugates................................................... 55 Fig. 2.3 Size-exclusion HPLC chromatogram of trastuzumab-PEG conjugates. ......................... 56 Fig. 2.4 Assessment of immunoreactivity of trastuzumab-PEG conjugates towards HER-2 receptors using flow cytometry ..................................................................................................... 57 Fig. 2.5 Assessment of stability of AuNPs using dynamic light scattering (DLS) and UVspectroscopy .................................................................................................................................. 59 Fig. 2.6 Evaluation of trastuzumab conjugation to AuNPs using transmission electron microscopy (TEM) ........................................................................................................................ 61 Fig. 2.7 Evaluation of internalization of AuNPs into HER-2 overexpressing SK-BR-3 breast cancer cells using darkfield microscopy ....................................................................................... 64 Fig. 2.8 Evaluation of DNA double-strand break (DSB) induction in SK-BR-3 cells using the γH2AX assay .................................................................................................................................. 65 Fig. 3.1 Schematic representation of assembly of 111In-radiolabeled targeted and non-targeted AuNPs ........................................................................................................................................... 76 Fig. 3.2 Synthesis scheme for the bifunctional PEG based radiometal chelator (SH-PEG2k-BnDTPA). .......................................................................................................................................... 77 Fig. 3.3 MicroSPECT/CT images of MDA-MB-361 HER-2-overexpressing human breast cancer tumor bearing mice injected with 111In-radiolabeled HER-2 targeted or non-targeted AuNPs by intravenous (i.v.) or i.v. with pre-blocking of RES (by prior administration of GdCl3 and irrelavant anti-CD20 monoclonal antibody rituximab) or intratumoral administration or i.v. administration of 111In-PEG-SH ................................................................................................... 83 xiii
Fig. 3.4.Liver, Spleen and Kidney uptake 111In-radiolabeled HER-2 targeted or non-targeted AuNPs in MDA-MB-361 tumor bearing mice upon i.v. administration with or without prior injection of 10 mg/kg GdCl3 for deactivation of macrophages and irrelevant anti-CD20 monoclonal antibody ..................................................................................................................... 84 Fig. 3.5 Pharmacokinetics of 111In-radiolabeled HER-2 targeted or non-targeted AuNPs in healthy CD-1 nude mice ............................................................................................................... 86 Fig. 3.6 Autoradiography and immunohistochemistry images of tumor sections following intratumoral injection of 111In-radiolabeled HER-2 targeted or non-targeted AuNPs. ................. 91 Fig. 3.7 Comparison of the biodistribution data of Au (µg/g) in spleen, liver and tumor obtained by gamma counting and subsequent ICP-MS analysis for elemental Au at 48 h after i.v. or intratumoral administration of 111In-radiolabeled HER-2 targeted or non-targeted AuNPs......... 94 Fig. 3.8 Ex-vivo Transmission Electron Micrographs (TEM) of HER-2-overexpressing MDAMB-361 xenograft tumor cells following i.t. injection of 111In-radiolabeled HER-2 targeted or non-targeted AuNPs. ..................................................................................................................... 96 Fig. 3.9 Schematic of the biodistribution pattern observed for 111In-radiolabeled HER-2 targeted or non-targeted AuNPs on i.v or i.t administration. ...................................................................... 99 Fig. 4.1 Experimental set-up for delivery of X-radiation using a preclinical microirradiator .... 106 Fig. 4.2 Quality control of X-radiation for in vitro and in vivo experiments using Gafchromic® EBT2 dosimetry films. ................................................................................................................ 110 Fig. 4.3. Clonogenic and γ-H2AX assays assessing the radiosensitization effects of HER-2 targeted or non-targeted AuNPs on HER-2 overexpressing MDA-MB-361 human breast cancer cells ............................................................................................................................................. 112 Fig. 4.4 Tumor growth index vs. time and body weight index vs. time plots for athymic mice implanted subcutaneously with HER-2-overexpressing MDA-MB-361 human breast cancer xenografts and treated with 0, 2, 6 or 15 Gy of 100 kVp X-radiation ........................................ 113
xiv
Fig. 4.5 Tumor growth index vs. time and body weight index vs. time plots for athymic mice implanted subcutaneously with HER-2-overexpressing MDA-MB-361 human breast cancer xenografts and treated with 11 Gy of 100 kVp X-radiation in the presence of i.t injected HER-2targeted AuNPs or X-radiation alone or pre-treatment with HER-2-targeted AuNPs alone or no X-radiation. ................................................................................................................................. 114 Fig. 5.1 Experimental set-up for brachytherapy with i.t injection of
111
In-radiolabeled HER-2-
targeted AuNPs ........................................................................................................................... 130 Fig. 5.2 Tumor growth index and body weight index of athymic mice implanted subcutaneously with HER-2-positive MDA-MB-361 xenografts and treated with i.t injected 111In-radiolabeled HER-2-targeted AuNPs. ............................................................................................................. 131 Fig. 5.3 Standard curve of the Purpald assay for measuring aldehydes.. ................................... 170 Fig. 5.4 SDS PAGE analysis of trastuzumab-PEG conjugates prepared by reacting oxidised trastuzumab with 10 to 50 fold mole excess of NH2-PEG-SH3000. ............................................. 173 Fig. 5.5 SDS PAGE analysis of trastuzumab-PEG conjugates prepared by reacting oxidised trastuzumab with 100 to 1000 fold mole excess of NH2-PEG-SH3000. ....................................... 174 Fig. 5.6 Size exclusion column chromatograms of purification of NH2-PEG-Fluorescein5000 conjugated to trastuzumab from excess fluorescein. .................................................................. 177 Fig. 5.7 Size exclusion HPLC chromatogram of trastuzumab-PEG conjugates prepared by reaction of oxidised trastuzumab with 100 fold mole excess of NH2-PEG-SH5000 ................... 178 Fig. 5.8 Size exclusion HPLC chromatogram of trastuzumab-PEG conjugates prepared by incubating with increasing mole ratios (5 to 500) of NH2-PEG-SH5000. .................................... 179
xv
List of Abbreviations ρ
density
%ID/g
percent injected dose per gram
18
[18f]-2-fluorodeoxyglucose
F-FDG
ADCC
antibody-dependent cellular cytotoxicity
AIs
aromatase inhibitors
ALT
alanine aminotransferase
ATCC
american type culture collection
AUC
area under the curve
AuNP
gold nanoparticle
Au-T
targeted gold nanoparticles
Au-NT
non-targeted gold nanoparticles
BCS
breast conserving surgery
BWI
body weight index
CBC
complete blood counts
CDC
complement-dependent cytotoxicity
CDRs
complementary determining regions
CEP17
chromosome 17 centromere
Cl
clearance
Cr
creatinine
CT
computed tomography
CVAP
cyclophosphamide, vincristine, adriamycin and prednisone
DAPI
6-diamidino-2-phenylindole
DEF
dose enhancement factor xvi
DDIW
double deionised water
DLS
dynamic light scattering
DLU
digital light units
DSBs
double strand break
DTPA
diethylenetriaminepentaacetic acid
ECD
extracellular domain
EC
electron capture
EDC
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
EGFR
epidermal growth factor receptor
EPR
enhanced permeation and retention
ER
estrogen receptor
ELISA
enzyme linked immunosorbent assay
eV
electron volt
Fab
antigen binding fragment
Fc
fragment crystallizable
FISH
fluorescence in-situ hybridisation
γ-H2AX
gamma histone 2ax
Gy
gray
HAMA
human anti-mouse antibody
Hb
hemoglobin
HER-2
human epidermal growth factor receptor-2
Ig
immunoglobulin
IHC
immunohistochemistry xvii
ICP-AES
inductively coupled plasma atomic emission spectroscopy
ICP-MS
inductively coupled plasma mass spectroscopy
i.t
intratumoral
i.v
intravenous
LABC
locally advanced breast cancer
LET
linear energy transfer
LN
lymph node
mAb
monoclonal antibody
MBq
mega bequerel
MeV
mega electron volt
MW
molecular weight
NSABP
national surgical and bowel project
NHS
n-hydroxysuccinimde
OPSS
orthopyridyldisulfide
PBS
phosphate buffer saline
pCR
pathologically complete response
PE
photoelectron
PEG
polyethylene glycol
PET
positron emission tomography
p.i.
post injection
Plt
platelet
PMT
photomultiplier tube
RBC
red blood cells
RES
reticuloendothelial system
rhTNF
recombinant human tumor necrosis factor xviii
ROI
region of interest
SD
standard deviation
SDS-PAGE
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
SEM
standard error of mean
SF
surviving fraction
SLN
sentinel lymph node
SPECT
single photon emission computed tomography
STTARR
spatio-temporal targeting and amplification of radiation response
SWNT-PEG5400-RGD
single walled carbon nanotubes modified with arginineglycine-aspartic acid
TEM
transmission electron microscopy
TGI
tumor growth index
Tr
retention time
U.S. FDA
united states food and drug administration
V1
volume of the central compartment
Vss
volume at steady state
WBC
white blood cells
xix
List of Appendices Appendix A Site-specific conjugation (oxidation of carbohydrate residues on the Fc domain of mAbs)
165
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1
Chapter 1
INTRODUCTION
2
1.0 Breast Cancer 1.1
Incidence and etiology Breast cancer is a major health problem affecting Canadian women. The Canadian
Cancer Society estimates in 2012 that 22,700 will be diagnosed with breast cancer and despite advanced medical care 5,100 will die of it (1). It is estimated that on average 62 Canadian women will be diagnosed with breast cancer every day. The wide spread pervasiveness of this disease is further realized from statistics indicating that 1 in 9 women is expected to develop breast cancer during her lifetime and one in 29 will die of it. The breast cancer incidence rate in Canada rose steadily from the early to late 1980s (1). In the 1990s Canada implemented wide spread breast cancer screening programs which resulted in an increase of breast cancer cases diagnosed (1). In Ontario, breast cancer is the most frequently diagnosed type of cancer and an estimated 9,100 women will be diagnosed with breast cancer this year (1). The risk of developing breast cancer has been attributed to multiple factors, including increasing age, family history, exposure to female reproductive hormones (both endogenous and exogenous), dietary factors, benign breast disease, and environmental factors. The majority of these factors contribute only a small to moderate increase in risk for any individual woman (2). It has been estimated that approximately 50% of women who develop breast cancer have no identifiable risk factor beyond increasing age and female gender (2). The importance of age as a breast cancer risk factor is sometimes overlooked. The incidence of breast cancer increases with advancing age. The disease is rare before age 30 (
