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Translation and Development of Molecular Imaging Probes for
Detecting Response of Breast Cancer to Trastuzumab
By
Karen Lam
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Pharmaceutical Sciences
University of Toronto
© Karen Lam, 2017
ii
ABSTRACT
Translation and Development of Molecular Imaging Probes for Detecting Response of
Breast Cancer to Trastuzumab
Karen Lam
Doctor of Philosophy, 2017
Graduate Department of Pharmaceutical Sciences
University of Toronto
The human epidermal growth factor receptor 2 (HER2) is overexpressed in up to 20% of breast
cancers (BC) and confers an aggressive phenotype and a poor prognosis. Trastuzumab
(Herceptin®) is a humanized anti-HER2 monoclonal antibody approved for immunotherapy of
HER2-positive BC, however, many eligible patients do not derive benefit and better predictors
of response are needed. SPECT/CT imaging with 111In-labeled pertuzumab has previously been
shown to sensitively detect trastuzumab-mediated HER2 downregulation as a marker of
response to trastuzumab therapy in HER2-overexpressing mouse xenografts. To advance this
agent to the clinic, a kit for the preparation of 111In-BzDTPA-pertuzumab injection was
developed under good manufacturing practices and tested against and consistently met quality
specifications, including labeling efficiency, purity, sterility and endotoxins. Biodistribution,
pharmacokinetic and radiation dosimetry studies of 111In-labeled pertuzumab in non-tumour
bearing mice revealed no abnormal uptake in organs, slow elimination from the blood
(t½β=228.2 h), and a projected total body radiation absorbed dose of 0.05 mSv/MBq. In an acute
toxicity study of normal mice administered 23-times the planned clinical radioactivity dose
iii
(scaled), blood analyses revealed only slight changes in hemoglobin and serum creatinine levels;
no differences in body weight between control and radiotracer injected groups, and no
histopathological abnormalities of tissues were noted. A Phase I clinical trial was approved by
Health Canada and initiated. A PET probe, 64Cu-NOTA-pertuzumab F(ab')2 was constructed to
similarly detect HER2 changes in response to trastuzumab and was expected to have a lower
radiation absorbed dose due to the faster elimination of F(ab')2 fragments and shorter half-life of
64Cu. The total body radiation absorbed dose projected for 64Cu-NOTA-pertuzumab F(ab')2 in
humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to 111In-BzDTPA-pertuzumab.
PET/CT showed specific accumulation of 64Cu-NOTA-pertuzumab F(ab')2 in SK-OV-3
xenografted mice. Image analysis of mice treated with trastuzumab showed 2-fold reduced
uptake of radioactivity in BT-474 xenografts injected with 64Cu-NOTA-pertuzumab F(ab')2 after
1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3
xenografts after 3 weeks of trastuzumab, consistent with tumour response and resistance,
respectively. These results demonstrate that radiolabeled pertuzumab-based imaging probes are
able to detect response to trastuzumab therapy.
iv
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to Dr. Raymond Reilly for the opportunity
to pursue my doctoral studies in his laboratory. I am especially thankful for his kind support and
positive attitude which drove me to continue my studies and overcome challenges. Dr. Reilly’s
expertise, experience and guidance were invaluable to my work and professional growth.
I wish to thank the members of my advisory committee, Dr. Gang Zheng, Dr. Martin
Yaffe, and Dr. Micheline Piquette-Miller for their encouragement and advice. Thank you for
challenging me to think critically about my research.
I would like to thank my past and present colleagues in Dr. Reilly’s laboratory,
especially Dr. Conrad Chan, Ms. Deborah Scollard, and Dr. Zhongli Cai for sharing their
knowledge and friendship. I am grateful to the wonderful friends I made within the Reilly lab as
well as in neighbouring labs for supplying wit and camaraderie, especially Dr. Eva Sulatycki
(Razumienko), Dr. Simmyung Yook, Noor Alsaden, Dr. Sina Eetezadi, and Sohyoung Her.
I wish to extend my deepest gratitude to my family: my sisters Janet, Wilmar and
Carman; to my brothers-in-law, Winston, Shawn and David; and to my parents, Tommy and
Lau Kuen, for their unwavering love and support throughout my Ph.D. studies and my life.
Thank you for encouraging me to pursue my goals.
Finally, a very special thank you to Michael Dunne whose love, patience, and care have
made hard times easier and good times better. Thank you for believing in me, sharing your
wisdom, and supporting all of my hopes and dreams.
v
TABLE OF CONTENTS
ABSTRACT .................................................................................................................................. ii ACKNOWLEDGMENTS ............................................................................................................ iv
TABLE OF CONTENTS .............................................................................................................. v
LIST OF TABLES ...................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... x
LIST OF ABBREVIATIONS .................................................................................................... xiii CHAPTER 1: Introduction ............................................................................................................ 1
1.1 Incidence, diagnosis and management of breast cancer ...................................................... 3
1.1.1 Breast cancer epidemiology and etiology ..................................................................... 3
1.1.2 Detection, diagnosis and staging of breast cancer ........................................................ 4
1.1.3 Treatment of breast cancer............................................................................................ 5
1.2 Trastuzumab in the management of HER2-positive breast cancer ...................................... 7
1.2.1 HER2 biology and clinical significance ....................................................................... 7
1.2.2 Monoclonal antibody trastuzumab (Herceptin®) ......................................................... 8
1.2.3 Clinical trials with trastuzumab .................................................................................. 10
1.2.4 Evaluation of HER2 positivity for trastuzumab therapy ............................................ 12
1.2.5 Mechanisms of action of trastuzumab ........................................................................ 17
1.2.6 Trastuzumab resistance ............................................................................................... 18
1.3 Pertuzumab in the management of HER2-positive breast cancer ..................................... 19
1.4 Trastuzumab emtansine (T-DM1) in the management of HER2-positive breast cancer ... 20
1.5 Molecular imaging ............................................................................................................. 21
1.5.1 Single photon emission computed tomography (SPECT) .......................................... 22
1.5.2 Positron emission tomography (PET) ......................................................................... 26
1.6 Approaches to imaging HER2 overexpression with PET and SPECT .............................. 29
1.6.1 Antibodies ................................................................................................................... 30
1.6.2 Antibody fragments .................................................................................................... 34
1.6.3 Affibodies ................................................................................................................... 39
1.6.4 Peptides ....................................................................................................................... 41
1.7 Translating novel molecular imaging probes to Phase I clinical trials .............................. 42
1.7.1 Roadmap to clinical translation of novel molecular imaging probes ......................... 44
1.7.2 Radiopharmaceutical formulation .............................................................................. 47
1.7.3 Good manufacturing practices (GMP)........................................................................ 50
1.7.4 Preclinical pharmacology and toxicology studies ...................................................... 54
vi
1.7.5 Clinical trial design and human ethics approval ......................................................... 56
1.7.6 Regulatory agency submission ................................................................................... 58
1.8 Hypotheses ......................................................................................................................... 61
1.9 Specific aims ...................................................................................................................... 61
1.10 Thesis organization .......................................................................................................... 62
CHAPTER 2: Kit for the Preparation of 111In-Labeled Pertuzumab Injection for Imaging Response of HER2-Positive Breast Cancer to Trastuzumab (Herceptin) ................................... 64
2.0 Abstract .............................................................................................................................. 66
2.1 Introduction........................................................................................................................ 67
2.2 Materials and methods ....................................................................................................... 69
2.2.1 Raw materials ............................................................................................................. 69
2.2.2 Pharmaceutical quality buffers .................................................................................. 70
2.2.3 Kit formulation ........................................................................................................... 71
2.2.4 Kit quality testing ....................................................................................................... 72
2.2.5 111In-BzDTPA-pertuzumab injection ......................................................................... 73
2.2.6 Stability in plasma ...................................................................................................... 74
2.2.7 Imaging and biodistribution studies............................................................................ 75
2.2.8 Statistical analysis ....................................................................................................... 76
2.3 Results ............................................................................................................................... 76
2.3.1 Raw materials and pharmaceutical quality buffers ..................................................... 76
2.3.2 Kit formulation .......................................................................................................... 77
2.3.3 111In-BzDTPA-pertuzumab injection ......................................................................... 86
2.3.4 Stability in plasma ...................................................................................................... 88
2.3.5 Imaging and biodistribution studies............................................................................ 90
2.4 Discussion .......................................................................................................................... 93
2.5 Conclusions ....................................................................................................................... 96
CHAPTER 3: Preclinical Pharmacokinetics, Biodistribution, Radiation Dosimetry and Acute Toxicity Studies Required for Regulatory Approval of a Clinical Trial Application for a Phase I/II Clinical Trial of 111In-BzDTPA-Pertuzumab ........................................................................ 97
3.0 Abstract .............................................................................................................................. 99
3.1 Introduction...................................................................................................................... 101
3.2 Materials and methods ..................................................................................................... 103
3.2.1 Radiopharmaceutical preparation ............................................................................. 103
3.2.2 Pharmacokinetic and biodistribution studies ............................................................ 103
3.2.3 Internal radiation dosimetry projections ................................................................... 104
vii
3.2.4 Acute toxicology ....................................................................................................... 105
3.2.5 Statistical analysis ..................................................................................................... 106
3.3 Results ............................................................................................................................. 107
3.3.1 Pharmacokinetic and biodistribution studies ............................................................ 107
3.3.2 Internal radiation dosimetry projections ................................................................... 111
3.3.3 Acute toxicology ....................................................................................................... 114
3.4 Discussion ........................................................................................................................ 118
3.5 Conclusion ....................................................................................................................... 121
CHAPTER 4: Development and Preclinical Studies of 64Cu-NOTA-Pertuzumab F(ab')2 Fragments for Imaging Changes in Tumor HER2 Expression Associated with Response to Trastuzumab by PET/CT ........................................................................................................... 122
4.0 Abstract ............................................................................................................................ 124
4.1 Introduction...................................................................................................................... 125
4.2 Materials and methods ..................................................................................................... 127
4.2.1 Cells lines and tumour xenografts ............................................................................ 127
4.2.2 Pertuzumab F(ab')2 fragments .................................................................................. 128
4.2.3 Preparation of 64Cu-NOTA-pertuzumab F(ab')2 ....................................................... 129
4.2.4 HER2 binding and trastuzumab-mediated HER2 internalization ............................. 130
4.2.5 Biodistribution, pharmacokinetic and radiation dosimetry studies .......................... 131
4.2.6 MicroPET/CT imaging studies ................................................................................. 133
4.2.7 Statistical analysis ..................................................................................................... 134
4.3 Results ............................................................................................................................. 135
4.3.1 64Cu-NOTA-pertuzumab F(ab')2 fragments .............................................................. 135
4.3.2 Trastuzumab-mediated HER2 internalization .......................................................... 138
4.3.3 Biodistribution, pharmacokinetic and radiation dosimetry studies .......................... 138
4.3.4 MicroPET/CT imaging studies ................................................................................. 144
4.4 Discussion ........................................................................................................................ 148
CHAPTER 5: ............................................................................................................................. 152
5.1 Summary of key findings ................................................................................................ 153
5.2 Future directions .............................................................................................................. 157
APPENDICES ........................................................................................................................... 163
APPENDIX A: ...................................................................................................................... 164
REFERENCES .......................................................................................................................... 165
viii
LIST OF TABLES
Table Title Page
1.1 Physical Properties of Single Photon Emitting Radionuclides Used in Gamma Camera Imaging
24
1.2 Physical Properties of Positron Emitting Radionuclides Used in PET Imaging
27
1.3 Properties of Radiolabelled Antibodies and Antibody Fragments 36
2.1 Quality testing of kits for the preparation of 111In-BzDTPA-pertuzumab injection
83
2.2 Stability testing at 4 months post-manufacturing of kits for the preparation of 111In-BzDTPA-pertuzumab injection for key quality parameters
85
2.3 Quality testing of 111In-BzDTPA-pertuzumab injection 87
2.4 Tumour and normal-tissue distribution of radioactivity in CD1 nude mice implanted subcutaneously with MDA-MB-361 human breast cancer xenografts at 72 h p.i. of 111In-BzDTPA-pertuzumab
92
3.1 Radiation absorbed dose projections for 111In-BzDTPA-pertuzumab in humans
112
3.2 Body weights of Balb/c mice administered a single i.v. dose of 111In-BzDTPA-pertuzumab, BzDTPA- pertuzumab, or Sodium Chloride Injection USP
115
4.1 Chelate:protein substitution levels under different reaction conditions for NOTA conjugation of pertuzumab F(ab')2
137
ix
Table Title Page
4.2 Tumour and normal tissue distribution at 24 h post-injection of increasing mass amounts of 64Cu-NOTA-pertuzumab F(ab')2 fragments in mice with SK-OV-3 human xenografts
142
4.3 Radiation absorbed dose projections for 64Cu-NOTA-pertuzumab F(ab')2 fragments in humans
143
x
LIST OF FIGURES
Figure Title Page
1.1 Algorithm for evaluation of HER2 protein expression by IHC assay of a breast cancer specimen.
14
1.2 Algorithm for evaluation of HER2 gene amplification by ISH of a breast cancer specimen using a single-signal (HER2 gene) assay (single-probe ISH).
15
1.3 Algorithm for evaluation of HER2 gene amplification by ISH of a breast cancer specimen using a dual-signal (HER2 gene) assay (dual-probe ISH).
16
1.4 Structures of an intact IgG antibody and antibody fragments. 35
1.5 The “roadmap” demonstrating the four steps in the translational bridge phase to advance novel molecular imaging agents from preclinical studies to Phase I clinical trial.
45
1.6 The radiopharmaceutical formulation step includes formulation of a kit and final radiopharmaceutical as well as establishment of specifications and quality control assays for raw materials, intermediates (including the kit) and final radiopharmaceutical.
48
1.7 Preclinical pharmacology and toxicology studies to advance a novel molecular imaging agent to Phase I clinical trial.
55
1.8 The clinical trial design and human ethics approval step for advancing a novel molecular imaging agent to Phase I trial.
57
1.9 The final step in advancing a novel molecular imaging agent to Phase I clinical trial is regulatory agency submission which includes completion of a CTA (Canada) or IND application (U.S.).
59
xi
Figure Title Page
2.1 Size-exclusion HPLC (SE-HPLC) analysis of BzDTPA-pertuzumab and pertuzumab using ultraviolet (UV) detection at 280 nm.
79
2.2 SDS-PAGE analysis of pertuzumab and BzDTPA-pertuzumab under reducing conditions or non-reducing conditions on a 4-20% Tris HCl gradient minigel.
80
2.3 Direct (saturation) receptor-binding curve for the binding of 111In-BzDTPA-pertuzumab (prepared from kit lot 11R015) to SK-BR-3 human breast cancer cells.
81
2.4 Relationship between BzDTPA substitution level and binding affinity (Ka) of 111In-BzDTPA-pertuzumab for HER2 or the maximum number of receptors (Bmax) of HER2-positive SK-BR-3 human breast cancer cells.
82
2.5 In vitro stability of 111In-BzDTPA-pertuzumab in human plasma and 0.1 M sodium bicarbonate buffer pH 8.2 at 37°C as determined by SE-HPLC over a 5-day period.
89
2.6 Posterior whole-body microSPECT/CT images of athymic mice implanted s.c. with MDA-MB-361 human breast cancer xenografts.
91
3.1 Elimination of radioactivity from the blood in Balb/c mice injected i.v. with 111In-BzDTPA-pertuzumab.
108
3.2 Biodistribution of 111In-BzDTPA-pertuzumab in normal Balb/c mice at selected times up to 166 hours post-i.v. injection.
109
3.3 Blood chemistry of mice administered 111In-BzDTPA-pertuzumab or unlabeled BzDTPA-pertuzumab at 23 and 10 times the planned dose on a MBq/kg or mg/kg basis, respectively, or in control mice administered Sodium Chloride Injection USP.
116
xii
Figure Title Page
4.1 SDS-PAGE analysis of unconjugated F(ab')2 and NOTA-F(ab')2, and SE-HPLC analysis of 64Cu-NOTA-pertuzumab F(ab')2.
136
4.2 Radioactivity vs. time curve for the elimination of 64Cu-NOTA-F(ab')2 from the blood of non tumour-bearing Balb/c mice after i.v. injection.
140
4.3 Normal tissue distribution of radioactivity at selected times up to 48 h post-injection of 64Cu-NOTA-F(ab')2 in non-tumour bearing mice.
141
4.4 Whole-body microPET/CT images of mice with SK-OV-3 HER2-overexpressing xenografts at 24 h post-injection with 64Cu-NOTA-pertuzumab F(ab')2 fragments.
146
4.5 MicroPET/CT images of 64Cu-NOTA-pertuzumab F(ab')2 fragments in mice with subcutaneous BT-474 BC or SK-OV-3 ovarian cancer xenografts at baseline and at 1 and/or 3 weeks after commencing treatment with trastuzumab.
147
xiii
LIST OF ABBREVIATIONS
%ID/g percent injected dose per gram
17-DMAG 17-dimethylaminoethylamino-17-
demothoxygeldanamycin
18F-FDG [fluorine-18]-fluoro-2-deoxy- D-glucose
64Cu copper-64
65Zn zinc-65
111In indium-111
114mIn indium-114 metastable
ACS American Chemical Society
ADC antibody-drug conjugate
ADCC antibody-dependent cellular cytotoxicity
Akt protein kinase B
ALT alanine aminotransferase
BC breast cancer
BCS breast conserving surgery
Bmax maximum number of receptors
BzDTPA 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic
acid
CBC complete blood counts
CDRs complementarity determining regions
COA certificate of analysis
CT computed tomography
xiv
CTA clinical trial application
DCIS ductal carcinoma in situ
DTT dithiothreitol
ECD extracellular domain
EGF epidermal growth factor
EGFR epidermal growth factor receptor
ER estrogen receptor
FDA Food and Drug Administration
FISH fluorescence in situ hybridization
FSA flow scintillation analyzer
GMP good manufacturing practices
Hb hemoglobin
Hct hematocrit
HER1 human epidermal growth factor receptor 1
HER2 human epidermal growth factor receptor 2
HER3 human epidermal growth factor receptor 3
HER2 human epidermal growth factor receptor 4
HERA Herceptin Adjuvant (clinical trial)
HR hazard ratio
HRP horseradish peroxidase
IB investigator’s brochure
IHC immunohistochemistry
i.p. intraperitoneal
xv
i.v. intravenous
IDC invasive ductal carcinoma
IGF-1R insulin-like growth factor 1 receptor
ILC invasive lobular carcinoma
IND investigational new drug
ISH in situ hybridization
ITLC-SG instant thin layer-silica gel chromatography
Ka affinity constant
Kd dissociation constant
LCIS lobular carcinoma in situ
LAL limulus amebocyte lysate
LOR line of response
MAPK mitogen-activated protein kinase
mAb monoclonal antibody
MIRD medical internal radiation dose
Mr relative molecular mass
MRI magnetic resonance imaging
mRNA messenger ribonucleic acid
MTA materials transfer agreement
NMR nuclear magnetic resonance
NOTA 1,4,7-triazacyclononane-1,4,7-triacetate
NSB non-specific binding
OCOG Ontario Clinical Oncology Group
xvi
OLINDA organ level internal radiation dose assessment
ORR objective response rate
PBS phosphate buffered saline
pCR pathologic complete response
PET positron emission tomography
PFS progression-free survival
p.i. post-injection
PI3K phosphoinositide 3-kinase
PLT platelet counts
PR progesterone receptor
PTEN phosphatase and tensin homolog
PVDF polyvinylidene fluoride
QIS-B quality information summary – biologics
QIS-R quality information summary – radiopharmaceuticals
RBC red blood cells
RCP radiochemical purity
SB specific binding
SCr serum creatinine
scFv single chain variable fragment
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis
SE-HPLC size-exclusion high-performance liquid chromatography
SLNB sentinel lymph node biopsy
xvii
SOP standard operating procedure
SPECT single photon emission computed tomography
SUV standardized uptake value
TB total binding
T-DM1 trastuzumab emtansine
TGI tumour growth index
TTF time-to-treatment failure
USP United States pharmacopeia
VOI volume of interest
WBC white blood cells
1
CHAPTER 1
INTRODUCTION
2
Section 1.7 of this chapter represents a reprint of: “Reilly RM, Lam K, Chan C, Levine MN.
Advancing novel molecular imaging agents from preclinical studies to first-in-humans Phase I
clinical trials in academia – a roadmap for overcoming perceived barriers. Bioconjug Chem.
2015 Apr 15;26(4):625-32.” Copyright © 2015 American Chemical Society.
3
1.1 Incidence, diagnosis and management of breast cancer
1.1.1 Breast cancer epidemiology and etiology
Breast cancer (BC) is the most commonly diagnosed cancer in North American women
(excluding non-melanoma skin cancer), and follows lung cancer as the second most fatal cause
of cancer-related deaths. It is estimated that 25,000 women in Canada and over 240,000 women
in the United States (U.S.) are diagnosed with breast cancer each year (1,2). Although female
breast cancer mortality has decreased 44% from 1986 to 2015 in Canada and similarly in the
U.S. due to early detection and advances in treatment, 18% of women diagnosed with breast
cancer will eventually die from the disease. The lifetime probability of developing breast cancer
in Canada in 2015 is currently 1 in 9 for women. Recognized risk factors for developing breast
cancer include age (including age at menarche and menopause, and age at first pregnancy),
estrogen exposure, radiation exposure, family history and genetic predisposition (e.g. mutations
in BRCA1/BRCA2 genes) and lifestyle factors such as obesity, alcohol intake, and physical
inactivity (3,4).
The female breast is comprised of fibrous and adipose tissue, and a gland containing 16
to 20 lobes, which in turn consist of up to 40 lobules. The lobules contain alveoli which are cells
responsible for generating milk during lactation. Lactiferous tubules carry secreted milk from
the lobules and connect to a lactiferous duct that flows to the nipple for discharge (5). The
majority of breast cancer cases present as invasive ductal carcinoma (IDC) or invasive lobular
carcinoma (ILC) which account for 75% and 10% of breast cancer cases, respectively (6). BC is
thought to arise as either the result of random mutations to any breast epithelial cell, with
genetic and epigenetic alterations selected over time leading to tumour progression, or through a
select few stem and progenitor cells that can initiate and maintain tumour development (7).
4
Ductal BC has been proposed to evolve from normal epithelium to flat epithelial atypia, to
atypical ductal hyperplasia (ADH), progressing to ductal carcinoma in situ (DCIS) and
advancing to IDC. Atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS)
have been suggested to be the non-obligate precursors to ILC (7,8).
1.1.2 Detection, diagnosis and staging of breast cancer
BC is detected by mammography screening or presentation of clinical symptoms (mass)
(9). Diagnosis is based on clinical examination, imaging, and pathology of a biopsy sample. The
clinical examination includes manual palpation of the breasts and locoregional lymph nodes,
metastatic disease assessment, medical and family history, clinical chemistry and menopausal
status. Imaging involves bilateral mammography, ultrasound of the breast and regional lymph
nodes and in some cases magnetic resonance imaging (MRI). Cardiac imaging may be required
prior to certain treatments involving trastuzumab and/or anthracyclines. Biopsy specimens are
obtained through fine-needle aspiration, core biopsy or surgical excision and histologically
evaluated for type, grade, and molecular biomarkers for subtype classification (9). Histologic
type may define the BC as in situ (not invasive) or invasive, carcinoma, and ductal or lobular (or
other). Other tumours such as sarcomas, lymphomas and metastases can also be found in the
breast. Histology is also used to assign tumour grade which is based on the differentiation of the
cells and indicates the rapidity of cancer growth and probability of spreading.
Immunohistochemical assessment for estrogen receptors (ER), progesterone receptors (PgR) and
human epidermal growth factor receptor 2 (HER2) classifies BCs into molecular subtypes which
confer prognostic and predictive information (10). The most well characterized molecular
subtypes include luminal A (ER/PgR+, HER2-), luminal B (ER/PgR+, HER2+ or high Ki67),
HER2-positive (ER/PgR-, HER2+), and basal-like (ER/PgR-, HER2-). ER+ BCs exhibit the best
5
prognosis whereas basal-like (loosely corresponding to triple-negative) tumours confer the worst
outcome of the subtypes (6). The prognosis for HER2+ tumours has improved since the
introduction of trastuzumab therapy and other HER2-targeted agents (11). Gene expression
profiling such as MammaPrint (Agendia, Amsterdam, the Netherlands), Oncotype DX
Recurrence Score (Genomic Health, Redwood City, CA), Prosigna (Nanostring Technologies,
Seattle, WA) and Endopredict (Myriad Genetics, Salt Lake City, Utah), may be performed to
supplement prognostic information and predict the benefit of adjuvant chemotherapy.
Following diagnosis, BC disease stage is determined. Staging is used to describe the
extent of the cancer taking into account the size and characteristics of the original tumour and
the degree of spread in the body. Staging defines prognosis, guides appropriate treatment and
allows for comparisons of treatment outcomes. BCs are staged according to the TNM system
developed by the American Joint Committee on Cancer (AJCC) which takes into account the
size of the primary tumour (T) and extent of lymph node (N) and metastatic (M) involvement
(12). The earliest stage of cancer is in situ disease and is designated Stage 0 (Tis, N0, M0),
whereas the most advanced stage of BC is metastatic disease and is denoted Stage IV (any T,
any N, M1). The stages between 0 and IV include IA, IB, IIA, IIB, IIIA, IIIB, and IIIC. Survival
declines with increasing stage (12,13). Staging may be aided by chest X-rays, computed
tomography (CT), positron emission tomography (PET) scans, bone scans, and/or sentinel
lymph node biopsies (SLNB) (9).
1.1.3 Treatment of breast cancer
The choice of therapeutic regimen takes into account disease stage, molecular subtype,
age and general health of the patient, and patient preferences. BC treatment options include
surgery, radiation, chemotherapy, hormone therapy and targeted therapies. Treatment for
6
women with Stage 0 BC (non-invasive, in situ carcinoma) includes breast conserving surgery
(BCS, also known as lumpectomy) alone, BCS followed by radiation, or mastectomy (14), the
latter two procedures share equivalent survival outcomes (15). Hormone therapy (e.g.
tamoxifen) may be recommended to those women with ER+ DCIS.
Early BC includes Stages I, II and IIIA which include cancers less than 2 cm with no
nodal involvement, and cancers greater than 2 cm which may have spread to the axillary lymph
nodes. Surgical intervention with either mastectomy or BCS with radiation, followed by
chemotherapy, hormonal therapy and/or targeted therapy is the usual standard of care. Axillary
lymph nodes may be removed after a positive SLNB conducted at the time of surgery.
For locally advanced BCs (Stage III, manifested as tumours that are greater than 5 cm or
have spread to the chest wall, skin, or many lymph nodes but not to other organs), neoadjuvant
chemotherapy with anthracyclines or taxanes is usually given to decrease the size of an
inoperable primary tumour (Stage IIIB or IIIC) to one that permits mastectomy, or may be given
to decrease the primary tumour size to allow for BCS in the case of operable (Stage IIIA)
tumours (9,16). Neoadjuvant targeted therapy (e.g. trastuzumab) may also be given in HER2+
BC or hormonal therapy in the case of ER+ BC. Radiation therapy may be given prior to
surgery if the tumour does not respond to neoadjuvant chemotherapy; otherwise, patients are
treated with radiation following surgery. Chemotherapy, hormone therapy and targeted therapy
are continued after surgery and radiation if not all cycles of neoadjuvant therapy were completed
prior to surgery. An alternative treatment approach for operable Stage IIIA tumours is
mastectomy, followed by adjuvant chemotherapy and radiotherapy (16).
In metastatic breast disease (Stage IV), the cancer has spread to other organs of the body,
and prognosis is poor. Care focuses on palliation of symptoms and prolonging life rather than
7
cure. Chemotherapy, hormonal and targeted therapies and radiotherapy are commonly used for
managing metastatic BC (17).
1.2 Trastuzumab in the management of HER2-positive breast cancer
1.2.1 HER2 biology and clinical significance
HER2, is one of four tyrosine kinase receptor members of the human epidermal growth
factor receptor (ErbB) protein family. Other ErbB receptor family members include HER1 (also
referred to as EGFR or ErbB1), HER3 (ErbB3) and HER4 (ErbB4) (18). The common structure
between the four HERs includes a ligand binding extracellular region consisting of four domains
(I, II, III, and IV), a single transmembrane helix, and an intracellular C-terminal tail with
tyrosine autophosphorylation sites (19). HER2 is a 185 kDa protein and is encoded by a gene on
band q21 of chromosome 17 (20). Low levels of HER2 are expressed on cell membranes of
epithelial cells in the gastro-intestinal, respiratory, and urinary tract, and in the skin, breast and
placenta (21). HER2 is also expressed in the heart and is critical for heart development and
function in adults (22). HERs and their ligands are important for development, proliferation,
differentiation and homeostasis in mammals.
At least twelve ligands are recognized to bind HERs including epidermal growth factor
(EGF) and neuregulins. Except in the case of HER2, ligand binding induces a conformational
change in the receptors which exposes the dimerization arm and activates their kinase activity
upon dimerization (19). No known ligand exists for HER2 and its dimerization arm is
constitutively active, which may account for it being the preferred dimerization partner for the
three other HERs (19). Dimerization may occur between two same receptors
(homodimerization) or between two different HER members (heterodimerization) to activate
tyrosine kinase activity and initiate signaling cascades that promote proliferation and
8
differentiation (18). HER2 has also been shown to dimerize with the insulin-like growth factor I
receptor (IGF-IR) (23).
In the early 1980s, the HER2 gene (also known as neu or c-erbB-2) was discovered
following transfection studies with DNA obtained from ethylnitrosourea-induced rat
neuroblastomas (24). It was subsequently shown that this gene shares homology with the erb-B
(HER2) gene and encodes for a 185 kDa protein that is similar to the epidermal growth factor
(EGF) receptor (25). Slamon et al. subsequently demonstrated that 18-30% of BCs have
evidence of HER2 gene amplification and HER2 protein overexpression, which is correlated
with larger tumour size, lymph node involvement, a shorter time to relapse and lower overall
survival (20,26). Indeed, BC cells may contain up to 100 copies of the HER2 gene per cell (27)
and accordingly, the cell membrane of BC cells may express as many as 500 thousand to 2
million receptors per cell compared to approximately 20 thousand receptors per cell on most
normal epithelial cells (28). The overexpression of constitutively active HER2 increases the
frequency of dimerization events leading to potent mitogenic and transforming responses in
cells (28). The poor prognosis in BC patients associated with overamplification of the HER2
gene and overexpression of HER2 protein has made this an intensely studied therapeutic target
in BC.
1.2.2 Monoclonal antibody trastuzumab (Herceptin®)
The first HER2-targeted therapy for BC was developed by Genentech Inc. (San
Francisco, CA, USA) as a 150-kDa recombinant humanized monoclonal antibody (mAb) known
as trastuzumab, or Herceptin® (29). Trastuzumab contains the complementarity determining
regions (CDRs) of the murine mAb, mumAb4D5, which were inserted into human variable and
immunoglobulin G1 (IgG1) constant domains to limit immunogenicity (29). Trastuzumab binds
9
to the juxtamembrane region of the extracellular domain of HER2 (domain IV) with a binding
affinity (Kd) of 0.1 nM (29,30).
Early investigations with the original murine mumAb4D5 demonstrated that HER2-
overexpressing BC cell lines SK-BR-3, MDA-MB-361 and MDA-MB-175-VII treated with 2.5
µg/mL of mumAb4D5 were significantly growth-inhibited by 54%, 42% and 48% whereas no
anti-proliferative effects were seen against BC cells that lacked or had low HER2 expression
(31). Similarly, treatment of HER2-overexpressing SK-BR-3 human BC cells with the
humanized antibody, trastuzumab, at a concentration of 8 µg/mL reduced cell proliferation from
100% to 54% (29). Furthermore, the clonogenic growth of HER2-overexpressing breast cancer
cells was inhibited in a dose-dependent manner using concentrations of trastuzumab ranging
from 0.1-10 nM and this effect was enhanced up to 67% by the addition of the chemotherapeutic
agent, paclitaxel (32).
The first in vivo investigation of trastuzumab’s anti-tumour activity was reported by
Tokuda et al. in which they showed that a single intravenous (i.v.) dose of trastuzumab (36
mg/kg) significantly inhibited the growth of HER2-overexpressing 4-1ST gastric tumours in
immune deficient mice but did not eradicate the tumours (33). The antitumour effect of multiple
trastuzumab dosing on human BC xenografts was subsequently reported by another group (32).
In mice bearing BT-474 HER2-overexpressing xenografts, dose dependent antitumour activity
was observed with trastuzumab doses up to 1 mg/kg. Complete eradication of tumours occurred
in 30-50% of mice treated with 1-30 mg/kg without toxicity. The effect of adding paclitaxel to
trastuzumab on reducing tumour volume was superior to trastuzumab or paclitaxel alone.
Another study investigating trastuzumab and chemotherapy combinations found significant
reductions in MCF7/HER2 tumour volumes in mice treated with trastuzumab combined with
10
doxorubicin, etoposide, vinblastine, cyclophosphamide, or methotrexate compared to either
agent alone (34). Numerous clinical studies have since been launched to investigate trastuzumab
combined with chemotherapy (35).
1.2.3 Clinical trials with trastuzumab
Following Phase I studies in which dose-limiting toxicity was not reached with
trastuzumab at doses of up to 500 mg (36), the toxicity and efficacy of trastuzumab was then
evaluated in Phase II trials for patients with HER2-overexpressing metastatic BC. In a study of
46 patients that received a loading dose of 250 mg of single agent trastuzumab followed by 10
weekly doses of 100 mg each, an objective response rate (ORR) of 11.6% was achieved and
32.6% of patients had stable disease that lasted a median of 5.1 months (37). Trastuzumab was
well tolerated and not immunogenic. In a study of 222 women with HER2-positive metastatic
BC who had progressed following one or two chemotherapy treatment regimens, an ORR of
15% was achieved following monotherapy with a trastuzumab loading dose of 4 mg/kg
followed by weekly maintenance doses of 2 mg/kg (38). The median durations of response and
overall survival were 9.1 and 13 months, respectively. Cardiac dysfunction occurred in 4.7% of
the patients and was the major adverse event. Many of the patients that had experienced cardiac
dysfunction had previously been treated with anthracyclines, suggesting that trastuzumab may
have exacerbated previous myocardial damage caused by anthracyclines.
In contrast to these two trials in which metastatic patients had received extensive prior
anticancer therapy, another trial evaluated the safety and efficacy of first-line, single-agent
trastuzumab in HER2-overexpressing metastatic BC (39). Patients received a loading dose of 4
mg/kg followed by weekly doses of 2 mg/kg or an 8 mg/kg loading dose followed by weekly
doses of 4 mg/kg. The ORR was 26% with no benefit of administering the higher dose. The
11
ORR was 35% in patients identified with high (3+) HER2 overexpression by
immunohistochemistry (IHC) and 34% for those with HER2 gene amplification by fluorescence
in situ hybridization (FISH) (see next section). This was almost 2-fold greater than the ORR that
was reported in previous trials (37,38) and may be due to the inclusion of patients with moderate
HER2 overexpression (2+) in the previous trials which are less responsive to trastuzumab.
Cardiac dysfunction occurred in 2% of patients, but these patients had pre-existing heart
conditions and did not require cardiac drugs after discontinuing trastuzumab.
In a pivotal phase III trial, 469 women with HER2-positive metastatic BC were
randomly assigned to receive chemotherapy alone (paclitaxel or an anthracycline plus
cyclophosphamide) or chemotherapy plus trastuzumab (11). Compared to those patients
receiving only chemotherapy, patients receiving the combination of chemotherapy and
trastuzumab experienced a significant increase in time to disease progression (median, 7.4 vs.
4.6 months), a greater rate of objective response (50% vs. 32%), an extended duration of
response (median, 9.1 vs. 6.1 months), and longer overall survival (median survival 24.1 vs.
20.3 months). An unexpected adverse effect of trastuzumab was cardiotoxicity in 27% of
patients receiving trastuzumab with an anthracycline plus cyclophosphamide compared to 13%
of patients receiving trastuzumab and paclitaxel. This later led to the recommendation that
trastuzumab not be administered concurrent with anthracyclines and investigations of various
combinations of trastuzumab with chemotherapies such as taxanes and vinorelbine have since
shown promise (35). These studies contributed to the 1998 U.S. Food and Drug Administration
(FDA) approval and 1999 Health Canada approval of first-line trastuzumab combined with
paclitaxel for HER2-positive metastatic BC.
12
Trastuzumab was also U.S. FDA and Health Canada approved in 2006 for use in the
adjuvant setting (Stages I-III) based on several pivotal studies. Combined data from the National
Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 and the North Central Cancer
Treatment Group (NCCTG) trial N9831 revealed that the addition of 1 year of trastuzumab to a
regimen of paclitaxel following doxorubicin and cyclophosphamide treatment significantly
reduced the number of recurrences and deaths due to BC (hazard ratio [HR] = 0.48) (40). The
disease-free survival at 4 years was 67.1% for the control chemotherapy-treated group and
85.3% for the group that also received trastuzumab. In the Herceptin Adjuvant (HERA) trial,
disease-free survival was significantly improved in women with early stage BC who received 1
year of trastuzumab following adjuvant chemotherapy versus chemotherapy alone (HR = 0.54)
(41). Subsequently, patients in the control chemotherapy alone arm were allowed to cross over
and receive adjuvant trastuzumab. Follow-up 2 years later mirrored the initial results showing
significant improvement in disease-free survival in women given 1 year of trastuzumab
following adjuvant chemotherapy versus those that only received observation after
chemotherapy (HR = 0.54) (42).
1.2.4 Evaluation of HER2 positivity for trastuzumab therapy
HER2-positivity is predictive of response to trastuzumab (43). Guidelines have therefore
been established by the American Society of Clinical Oncology (ASCO) and the College of
American Pathologists (CAP) for assessing HER2 status in all patients presenting with invasive
BC to determine eligibility for trastuzumab-containing therapeutic regimens (44). HER2-
positivity is defined on the basis of HER2 protein overexpression or HER2 gene amplification.
HER2 protein overexpression is evaluated by immunohistochemistry (IHC) of biopsied tumour
tissue and scored on a 4-point scale of increasing receptor expression, where 0 and 1+ are
13
HER2-negative, 2+ is considered equivocal and 3+ is positive (Figure 1.1). A score of 3+ is
defined as membrane staining that is complete, intense, and present in >10% of tumour cells and
2+ is defined as staining that is incomplete and/or weak/moderate and present in >10% of
tumour cells, or, staining that is intense and present in ≤10% of tumour cells (44). HER2 gene
amplification is measured with “single-probe” or “dual-probe” in situ hybridization (ISH) in
which scoring is based on absolute HER2 copy number in the former case and the ratio of HER2
to chromosome centromere 17 (HER2:CEP17) in the latter case (Figures 1.2 and 1.3). A
positive ISH score is defined as ≥6 HER2 copies or a HER2:CEP17 ratio ≥2. ISH results are
considered equivocal if HER2 gene copy numbers fall between 4.0 to 6.0 and/or if the
HER2:CEP ratio is ≤2.0. Equivocal results must be retested for positivity using an alternative
test on the same specimen or using the same assay on a new specimen. Several approved test
kits exist for HER2 testing such as HercepTest (DAKO) and Pathway (Ventana) for IHC assays,
PathVysion (Abbott) and INFORM (Ventana) for fluorescence ISH (FISH), SPoT-Light
(Invitrogen) for chromogenic ISH, and EnzMet (Ventana) for silver enhanced ISH (43).
14
Figure 1.1. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2)
protein expression by immunohistochemistry (IHC) assay of the invasive component of a breast
cancer specimen. Although categories of HER2 status by IHC can be created that are not
covered by these definitions, in practice they are rare and if encountered should be considered
IHC 2+ equivocal. ISH, in situ hybridization. NOTE: the final reported results assume that there
is no apparent histopathologic discordance observed by the pathologist. (*) Readily appreciated
using a low-power objective and observed within a homogeneous and contiguous invasive cell
population. Reprinted with permission from Wolff AC (44).
15
Figure 1.2. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) gene
amplification by in situ hybridization (ISH) assay of the invasive component of a breast cancer
specimen using a single-signal (HER2 gene) assay (single-probe ISH). Amplification in a
single-probe ISH assay is defined by examining the average HER2 copy number. If there is a
second contiguous population of cells with increased HER2 signals per cell, and this cell
population consists of more than 10%of tumour cells on the slide (defined by image analysis or
visual estimation of the ISH or immunohistochemistry [IHC] slide), a separate counting of at
least 20 nonoverlapping cells must also be performed within this cell population and also
reported. Although categories of HER2 status by ISH can be created that are not covered by
these definitions, in practice they are rare and if encountered should be considered ISH
equivocal (see Data Supplement 2E). NOTE: the final reported results assume that there is no
apparent histopathologic discordance observed by the pathologist. (*) Observed in a
homogeneous and contiguous population. Reprinted with permission from Wolff AC (44).
16
Figure 1.3. Algorithm for evaluation of human epidermal growth factor receptor 2 (HER2) gene
amplification by in situ hybridization (ISH) assay of the invasive component of a breast cancer
specimen using a dual-signal (HER2 gene) assay (dual-probe ISH). Amplification in a dual-
probe ISH assay is defined by examining first the HER2/CEP17 ratio followed by the average
HER2 copy number. If there is a second contiguous population of cells with increased HER2
signals per cell, and this cell population consists of more than 10% of tumour cells on the slide
(defined by image analysis or visual estimation of the ISH or immunohistochemistry [IHC]
slide), a separate counting of at least 20 nonoverlapping cells must also be performed within this
cell population and also reported. NOTE. The final reported results assume that there is no
apparent histopathologic discordance observed by the pathologist. (*) Observed in a
homogeneous and contiguous population. (†) See Data Supplement 2E from Wolff et al. (44) for
more information on these rare scenarios. Reprinted with permission from Wolff AC (44).
17
1.2.5 Mechanisms of action of trastuzumab
Several mechanisms of action have been proposed for trastuzumab. Since trastuzumab
contains the framework of a consensus human IgG1, the Fc domain of the antibody can recruit
mononuclear cells (natural killer cells) via Fcγ receptors to lyse the target cells through a
process called antibody-dependent cell-mediated cytotoxicity (ADCC) (45). Indeed, ADCC was
observed in 51Cr-labeled SK-BR-3 cells treated with 10 or 100 ng/mL of trastuzumab but not in
WI-38 normal lung epithelial cells, which express 100-fold lower levels of HER2 than SK-BR-3
cells, as measured by the radioactivity released following incubation with and lysis by human
blood mononuclear cells (29).
Trastuzumab may also act by down-regulating the cell surface expression of HER2.
Hudziak et al. showed that HER2 was degraded more rapidly in SK-BR-3 cells exposed to
mumAb4D5, reducing the half-life of HER2 on the cell membrane from 7 h to 5 h (31). This
down-regulation of HER2 may be mediated by endocytosis through ubiquitination by c-Cbl,
resulting in degradation (46).
Preclinical studies have suggested that trastuzumab induces the cyclin-dependent kinase
inhibitor p27Kip1 which promotes cell-cycle arrest (47). Additionally, by binding to HER2,
trastuzumab may interfere with HER2-mediated activation of the phosphoinositide 3-
kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling
pathways. For example, Nagata et al. showed that trastuzumab-HER2 binding can inhibit
tyrosine Src signaling and increase the tumour suppressor protein phosphatase and tensin
homolog (PTEN). This in turn disrupts the PI3K/Akt pathway and diminishes cell growth and
survival (48). Angiogenesis has also been reported to be blocked in tumour-bearing mice by
trastuzumab (49). Furthermore, HER2 may shed its extracellular domain (ECD) via proteolysis
18
to leave a truncated 95-kDa isoform (p95HER2) with constitutive kinase activity. The binding
of trastuzumab to HER2 reduces this ECD shedding and prevents constitutive ligand-
independent oncogenic signaling (30).
1.2.6 Trastuzumab resistance
As discussed in Section 1.2.3, trastuzumab has significantly improved the prognosis of
breast cancer patients with HER2-positive disease. However, fewer than 50% of patients with
metastatic HER2-positive BC respond to trastuzumab therapy (11) due to “primary” or
“inherent” resistance to the drug. Of responding patients, about 70% progress within a year (50),
implying that patients develop a “secondary” or “acquired” resistance to trastuzumab. Why or
how inherent or acquired resistance occurs is not fully understood, but one postulated
mechanism of resistance is truncation of cell surface HER2. As mentioned earlier, HER2 may
undergo proteolysis to produce the truncated p95HER2 isoform with constitutive activity and as
a result, HER2 no longer possesses the binding site for trastuzumab (51). Signaling initiated by
upregulated expression of other tyrosine kinase receptors such as HER3, IGF-1R, and c-Met
may compensate for reduced signaling by HER2 (52). Deficiency in PTEN, an inhibitor of the
PI3K signaling pathway, has previously been proposed as a mechanism of resistance based on
preclinical and limited clinical studies, however, recent clinical data show no association
between trastuzumab sensitivity and PTEN-negative tumours (53). The upregulated expression
of mucins has also been proposed as a mediator of trastuzumab resistance by maintaining
persistent HER2 activation through MUC1 and masking of trastuzumab binding to HER2 by
MUC4 (54).
The mechanism by which trastuzumab resistance occurs and evolves still requires
elucidation. Nonetheless, better ways of identifying those patients who would most likely
19
respond or when a patient has ceased responding are needed so that potentially more effective
alternative therapies can be provided to the patient.
1.3 Pertuzumab in the management of HER2-positive breast cancer
Following the success of trastuzumab, Genentech Inc. developed a second generation
anti-HER2 antibody, pertuzumab (Perjeta®) (55), which received U.S. FDA approval in 2012
and Health Canada approval in 2013 for the treatment of HER2-overexpressing metastatic BC in
combination with trastuzumab. Pertuzumab was also U.S. FDA approved for neoadjuvant
treatment of early stage BC in 2013. Pertuzumab acts by binding to domain II of HER2 and in
effect, sterically blocks receptor dimerization and signaling (19). Since pertuzumab is
constructed from the same human antibody framework as trastuzumab with the exception of the
CDRs (29), it is also able to mediate ADCC.
Early in vitro and preclinical studies have shown that pertuzumab disrupts the formation
of HER2-HER1 and HER2-HER3 heterodimers in breast and prostate cancer lines (56), and
inhibits tumours of the breast (56), prostate (56,57), lung (58), ovaries (59), and colon (60).
Trastuzumab and pertuzumab bind to different epitopes of HER2 (domain IV and domain II,
respectively) and have complementary mechanisms of action, and thus delivery of these agents
in combination showed enhanced antitumour efficacy in HER2-positive breast and non-small
cell lung cancer xenografts in mice (61).
The clinical efficacy of pertuzumab was demonstrated in a Phase II trial in which the
addition of pertuzumab to a trastuzumab plus docetaxel regimen increased progression-free
survival by 6.1 months (62). The pivotal Phase III CLEOPATRA trial showed that median
overall survival was increased by 15.7 months by the addition of pertuzumab to trastuzumab and
docetaxel (63) and led to the approval of pertuzumab for metastatic HER2-positive BC.
20
The U.S. FDA and Health Canada approvals of the use of pertuzumab in the neoadjuvant
setting was based on results from the Phase II NeoSphere and TRYPHAENA clinical trials
which showed a superior pathological complete response rate with the addition of pertuzumab to
a trastuzumab plus docetaxel regimen with low rates of cardiac side effects (64,65).
Although pertuzumab is yet another triumph for patients with HER2-overexpressing BC,
most deaths in the CLEOPATRA trial were due to BC progression (63). Challenges remain in
developing better treatments and in identifying those patients who may benefit most from the
combination of pertuzumab and trastuzumab.
1.4 Trastuzumab emtansine (T-DM1) in the management of HER2-positive
breast cancer
Antibody-drug conjugates (ADCs) consist of a monoclonal antibody linked to cytotoxic
drugs via chemical linkers, which minimize the toxicity to normal tissues conferred by cytotoxic
drugs due to the highly specific targeting enabled by the antibody (66). Trastuzumab emtansine
(T-DM1) is a novel ADC that received U.S. FDA and Health Canada approval in 2013 as a
single agent for the treatment of patients with HER2-positive, metastatic BC who had previously
been treated with trastuzumab and a taxane (67). T-DM1 consists of approximately 3.5 DM1
molecules, which are a derivative of maytansine, conjugated to trastuzumab by the MCC (4-[N-
maleimidomethyl] cyclohexane-1-carboxylate) linker. T-DM1 is degraded within cancer cells
where DM1 is released. DM1 binds to tubulin and inhibits microtubule assembly, leading to cell
cycle arrest and apoptotic cell death (68).
Phase II clinical trials conducted in patients with HER2-positive metastatic BC who had
or had not received prior treatment with trastuzumab combined with chemotherapy and were
administered 3.6 mg/kg every 3 weeks of T-DMI showed an ORR of 26-64% (69,70). Response
21
rates were notably higher in patients whose tumours were HER2 3+ by IHC or exhibited HER2
gene amplification by FISH, or if HER2 mRNA was greater than the median HER2 mRNA
expression of the overall patient population (8.9). In another study, progression-free survival
(PFS) was 14.2 months for patients treated with T-DM1 versus 9.2 months for patients treated
with trastuzumab and doxetaxel (70). In the pivotal Phase III EMILIA trial, T-DM1 increased
PFS by 3.2 months and overall survival by 5.8 months in patients with HER2-positive advanced
BC compared to those treated with the standard therapy of lapatinib and capecitabine for
trastuzumab-resistant BC (71). While these represent significant improvements in the outcome
of patients with HER2-positive BC, it is clear that considering the relatively short PFS, as with
trastuzumab, patients acquire resistance. Novel ways of identifying these mechanisms of
resistance as they emerge are needed.
1.5 Molecular imaging
Molecular imaging enables imaging of specific molecules within a living system using
contrast agents that augment our understanding of disease and disease processes (72). Imaging
modalities that are capable of providing functional and molecular information using contrast
agents include MRI, CT, optical imaging, ultrasound, and nuclear medicine imaging including
positron emission tomography (PET) and single photon emission computed tomography
(SPECT). SPECT and PET offer high sensitivity using intravenously administered
concentrations of imaging probes in the nanomolar or picomolar range (73). Furthermore,
SPECT and PET are able to detect radioactivity from deep tissue whereas in optical and
ultrasound imaging, detection of signals from deeper tissues is limited. Tracer uptake can be
quantified with SPECT and PET imaging and thus provide quantitative information on tracer
distribution and target expression. SPECT imaging has been used in the oncology setting for
22
various diagnostic purposes including the localization of primary and metastatic somatostatin
receptor-expressing neuroendocrine tumours following an injection with 111In-pentetreotide
(Octreoscan™) (74), the identification of bone metastases using 99mTc-methylene diphosphonate
(MDP) (75), and the detection of metastatic lymph node involvement in prostate cancer using
111In-capromab pendetide (76). PET imaging with [fluorine-18]-fluoro-2-deoxy- D-glucose (18F-
FDG) is valuable for staging and restaging cancer, detecting recurrence, and monitoring
response to treatment to some regimens (77). SPECT and PET imaging will be discussed in the
following sections.
1.5.1 Single photon emission computed tomography (SPECT)
SPECT imaging is based on the detection of single photons emitted by radionuclides
such as γ-rays arising from isomeric transition, and X-rays arising from electron capture or
internal conversion to generate a series of 2-dimensional images (slices) that are then
reconstructed to create a 3-dimensional image (78). The γ-rays emitted from within the patient
are detected by a gamma camera with two detector heads which rotate around the patient. The
detector heads consist of a large scintillation crystal [most often NaI(Tl)] which converts γ-
photons into light and is affixed to a lead collimator. Collimation isolates individual gamma
photons directly originating from a source in the patient and reduces processing of scattered or
degraded photons. This reduces noise and increases spatial resolution but attenuates the majority
of incoming photons and therefore greatly reduces sensitivity (79). The visible light is received,
converted into electrons and amplified by photomultiplier tubes to produce pulses. An X, Y
positioning circuit sums up the output from the array of photomultiplier tubes to produce X and
Y pulses that are in direct proportion to the X, Y coordinates of the point of interaction of the γ-
rays which results in an image of the radioactivity distribution within the patient. Pulses are
23
further electronically sorted and processed for display (80). Following acquisition of a series of
planar images obtained 360o around the patient, these are reconstructed by filtered back
projection or by iterative algorithms to produce a 3-dimensional dataset. Ideally, radionuclides
with gamma photon energies that fall within 100-200 keV are most desirable due to the ability
to adequately penetrate through tissue and yet be efficiently collimated and also detected by the
NaI(Tl) scintillation crystal (78). Examples of single photon emitting radioisotopes used for
SPECT imaging are provided in Table 1.1.
24
Table 1.1
Physical Properties of Some Single Photon Emitting Radionuclides Used in SPECT
Radionuclide Physical half-life Energies of imageable
X and γ rays (keV)
Abundance of
imageable X and γ
rays (%)
Gallium-67 (67Ga) 3.26 days 93
185
300
40
24
16
Technetium-99m (99mTc) 6.01 hours 140 89
Indium-111 (111In) 2.83 days 172
247
90
94
Iodine-123 (123I) 13.2 hours 159 84
Iodine-131 (131I) 8.04 days 364 82
Adapted from Zanzonico P (78).
25
Spatial resolution is defined as the ability to clearly delineate two neighbouring sources
and is expressed as the full-width-at-half-maximum (FWHM). For SPECT imaging, spatial
resolution is at least an order of magnitude poorer than that of CT and MRI (81). Spatial
resolution has typically been 5-10 mm for clinical SPECT systems and is limited by technology
such as collimator design, especially the diameter of the holes in the collimator, and is often a
trade-off with maximizing the sensitivity. Smaller collimator hole size results in increased
rejection of scattered γ-photons which increases spatial resolution but reduces the number of γ-
photons reaching the crystal, and therefore reduces sensitivity. Spatial resolution also decreases
with increasing distance between the patient and the detector. However, advances in hardware
such as multi-pinhole collimators have increased γ-photon sensitivity (82) and the newest
clinical SPECT scanners offer spatial resolutions as low as 3 mm (83). Finer spatial resolutions
are needed for imaging small animals such as mice, and their organs, which are several orders of
magnitude smaller than humans. Dedicated small-animal SPECT systems using multi-pinhole
collimators have been developed that offer spatial resolutions of <1 mm FWHM. Accurate
quantitation of radioactivity uptake in target tissues remains a challenge for SPECT due to
scatter and because it is necessary to know the exact depth within the body where the
radioactive decay originated in order to correct for attenuation by overlying tissues. Still,
systems and software continue to advance with better corrections for γ-photon scatter and
attenuation (82). Additionally, the development of a SPECT/CT combined imaging modality
allows the co-registration of molecular features visualized using radiopharmaceutical probes by
SPECT with the precise anatomical information supplied by CT.
26
1.5.2 Positron emission tomography (PET)
In PET, a positron emitted by a radionuclide interacts with an electron in tissues and is
annihilated to generate two 511-keV γ-photons that propagate in virtually opposite directions.
Scintillation crystal detectors with attached photomultiplier tubes are arranged in a 360-degree
ring around the patient and the two γ-photons are detected by two opposite detectors in
coincidence, then the data is collected from many angles around the patient’s body and, as in
SPECT, analysed by a computer using filtered back projection or iterative algorithms to
reconstruct the image (80). The scintillation crystals used for detection in PET are usually
bismuth germanate (BSO), cerium-doped gadolinium oxyorthosilicate (GSO[CE]), cerium-
doped lutetium oxyorthosilicate (LSO[Ce]) and cerium-doped lutetium-yttrium oxyorthosilicate
(LYSO[CE]) which have greater stopping power for the 511-keV γ-photons than NaI(Tl)
scintillators due to their higher mass density and effective atomic number (78). Examples of
positron emitting isotopes used for PET and their properties are listed in Table 1.2.
27
Table 1.2
Physical Properties of Some Positron Emitting Radionuclides Used in PET Imaging
Radionuclide Physical half-
life
Maximum
positron energy
(MeV)
Positron
branching ratio
(%)
Positron
range in
water (mm)
Carbon 11 (11C) 20.4 min 0.96 99 0.4
Fluorine-18 (18F) 1.83 h 0.64 97 0.2
Copper-64 (64Cu) 12.7 h 0.58 19 0.2
Gallium-68 (68Ga) 1.14 h 1.90 88 1.2
Zirconium-89 (89Zr) 78.4 h 0.90 23 0.4
Adapted from Zanzonico P (78).
28
In order for an event to be registered, the two annihilation photons must be detected
simultaneously (within nanoseconds) to create a line of response (LOR). During a PET scan,
several million coincidence events are recorded producing many intersecting LORs providing
information on the spatial location of radiopharmaceutical uptake in the body. The coincidence
detection of photon events negates the need for physical collimation in PET and results in a
sensitivity that is up to three orders of magnitude greater than that achieved with SPECT (78).
This greater sensitivity results in improved image quality (signal-to-noise ratios) and may
shorten the scan time (79). Due to the generally shorter half-lives of positron emitters compared
to single photon emitters, higher amounts of radioactivity may be injected without increasing
radiation doses to normal tissues and consequently, this may also increase sensitivity.
Alternatively, lower amounts of radioactivity may be injected to achieve the same sensitivity but
with a lower radiation dose to the patient.
In contrast to SPECT, attenuation correction is easily achieved with PET owing to the
fact that attenuation depends only on the overall probability of both 511-keV photons reaching 2
opposite detectors. The result is that the attenuation correction factor depends only on the total
thickness of the attenuation medium, independent of the depth of the source. In contrast, due to
the single-photon emission nature of SPECT, attenuation changes depend on the point of
emission (79). A transmission CT scan can be used to calculate the attenuation correction
factors at different positions in the body as well as provide anatomic information for co-
registration with the PET image. As a result, quantitation of radioactivity uptake is more
straightforward and accurate with PET imaging.
The spatial resolution of PET is limited by 2 inherent physics factors: positron range and
photon non-collinearity. Positron range refers to the average distance that a positron travels in a
29
medium before it encounters an electron and is annihilated. As a result, the origin of the γ-
photon is not the position of the radionuclide but some distance from it (84). Positron range is
directly dependent on the energy of the positron whereby higher energy positrons travel further.
Photon non-collinearity is the slight deviation from the 180° trajectories travelled between the
two annihilation photons which results in resolution blurring that is dependent on the ring
diameter of the detector. This results in a typical spatial resolution of approximately 5 mm for
clinical PET scanners (78). The use of smaller detector elements and smaller diameters of
detector rings in small-animal PET scanners results in spatial resolutions of 1-2 mm.
1.6 Approaches to imaging HER2 overexpression with PET and SPECT
Despite significant advances in personalized treatment options for HER2-positive BC,
only up to 50% of patients in Phase 3 clinical trials responded to trastuzumab and chemotherapy
(11) and most patients who initially respond acquire resistance within a year (85). It has also
been proposed that some patients with BC classified as HER2-negative may also receive benefit
from trastuzumab (8).
The intra- and intertumoural heterogeneity of tumours (86), challenges associated with
IHC such as the preservation of specimen integrity during fixation and subjectivity in slide
scoring, and equipment/cost associated with FISH, limits IHC and ISH techniques in selecting
the most responsive candidates for trastuzumab therapy (87). Furthermore, monitoring response
to trastuzumab therapy by taking serial biopsies for IHC or ISH would not be practical.
Therefore, serial molecular imaging of HER2 status at the commencement of treatment and as
the disease progresses may provide a sensitive, feasible approach to monitoring the
effectiveness of trastuzumab in individual patients and detecting early in the course of treatment
those patients who require alternative therapies due to tumour resistance (88).
30
1.6.1 Antibodies
There is a small but growing collection of clinical studies that have evaluated the ability
of radiolabeled antibodies to target and image HER2. One strategy employs radiolabeled
antibodies specific for HER2. The first such approach conducted in the clinic was reported by
Behr et al. who proposed that the therapeutic efficacy and cardiotoxicity of trastuzumab may be
attributed to HER2-mediated specific uptake of the drug in the tumour and heart, respectively,
and that this uptake may be visualized by indium-111 labeled trastuzumab (111In-trastuzumab)
by SPECT imaging (89). The study showed that in the 11 patients with tumour uptake of 111In-
trastuzumab visualized by SPECT, objective responses were experienced whereas only 1 of the
9 women without labeled trastuzumab uptake responded. Furthermore, images of 6 of the 7
patients that experienced cardiotoxicity had myocardial uptake of 111In-trastuzumab whereas no
heart uptake was seen on those patients without this adverse effect, suggesting that baseline
SPECT imaging with this agent could predict therapeutic efficacy and cardiotoxicity. The ability
of 111In-trastuzumab to detect HER2-positive lesions in patients with metastatic BC was
confirmed by a subsequent clinical study reported by Perik et al. but the value of 111In-
trastuzumab in predicting cardiotoxicity was not supported (90). The effective radiation dose
was later reported to be 0.19 mSv/MBq, corresponding to 28.5 mSv for a 150 MBq dose (91).
The sensitivity of detecting HER2-positive lesions by imaging using radiolabeled antibodies
may be improved by taking advantage of the 10-100 fold higher γ-photon detection sensitivity
of PET. Indeed, a clinical study by Dijkers et al. reported that 89Zr-trastuzumab showed
excellent tumour uptake, high spatial resolution and good signal-to-noise- ratio with image
quality that was not achievable by their previous SPECT scans with 111In-trastuzumab (92).
They demonstrated that a higher 50 mg mass dose of 89Zr-trastuzumab enabled better tumour
31
imaging than a 10 mg dose in trastuzumab-naive patients, whereas those patients already
receiving trastuzumab only required a 10 mg dose. However, in approximately half of the
patients, the PET scans revealed no uptake of 89Zr-trastuzumab in some lesions that had been
identified by CT or MRI. Biopsies were not available for all lesions and no comparison of
tumour uptake with pathological HER2 status was done to rule out if lack of uptake was due to
HER2-negativity or competition for binding to HER2 with therapeutic trastuzumab. For an
administered radioactivity dose of 37 MBq, the effective radiation dose was 18 mSv. Despite
these limitations, 89Zr-trastuzumab was applied in a novel way in another study to identify
patients unlikely to benefit from T-DM1. 89Zr-trastuzumab was administered to patients and
imaged by PET/CT. In addition, PET scans were obtained with 18F-FDG at baseline, and again
with 18F-FDG after one cycle of trastuzumab emtansine (T-DM1) in patients with HER2-
positive metastatic BC who had received at least one line of prior therapy (93). Those patients
who had a negative HER2 PET image and exhibited stable disease or progression on the 18F-
FDG PET scan (i.e. no metabolic response) after one cycle did not respond to T-DM1 (negative
predictive value of 100%). Patients with positive HER2 PET imaging and a reduction of 18F-
FDG uptake of at least 15% (metabolic response) did respond to T-DM1 (positive predictive
value of 100%). Furthermore, the combination imaging was able to distinguish patients with a
median time-to-treatment failure (TTF) of 2.8 months from those with a TTF of 15 months.
In contrast to the high mass dose (50 mg) used with 89Zr-trastuzumab imaging, and in an
effort to reduce the radiation dose elicited by the long half-life and high γ-ray energy of 89Zr
[909 keV with a 99.0% branching ratio (94)], a clinical study conducted in Japan administered
only 86.2 µg and 126 MBq of 64Cu-DOTA-trastuzumab to patients and this group was able to
visualize primary BC and metastatic lesions in the brain (95). The effective dose was 4.5 mSv, a
32
4-fold lower dose than that deposited by 89Zr-trastuzumab. However, lesions around the liver,
heart, and blood were difficult to detect due to the sequestration of the radiolabeled antibodies in
the liver combined with the long circulation half-life of antibodies. Therefore, increasing the
mass dose may be helpful, or using radiolabeled antibody fragments may be a better approach to
more quickly clearing radioactivity from the liver and blood. Similar to the 89Zr-trastuzumab
study, another study reported that PET imaging with a 50 mg dose of 64Cu-DOTA-trastuzumab
enhanced tumour uptake and reduced liver uptake by 75-80% (96). The radiation dose was 12
mSv, comparable to 11 mSv for 18F-FDG.
While clinical reports of imaging HER2 expression in BC patients are limited, there
exists a broad array of preclinical studies evaluating radiolabeled antibodies to image HER2.
The first preclinical imaging of HER2 expression was described by Saga et al. who showed
improved tumour targeting and retention of 111In-labeled anti-HER2 antibody (111In-SV2-61r)
compared to the same antibody labeled with 125I (125I-SV2-61r), due to the residualizing nature
of radiometals (97). In Balb/c nude mice xenografted with human HER2 gene-transfected NIH-
3T3 cells, a maximum tumour uptake of 15 %ID/g and tumour-to-blood (T/B) ratio of 5.6 were
achieved at 48 h post-injection (p.i.) for 111In-SV2-61r whereas the tumour uptake and T/B ratio
for 125I-SV2-61r were 5.7 %ID/g and 1.1. This study revealed the feasibility of
radioimmunodetection of HER2 and subsequently launched a variety of studies examining other
anti-HER2 antibodies including trastuzumab labeled with single photon emitters such as 111In
(98-101), 131I (102), and 177Lu (103), and positron emitters such as 86Y (98,104), 64Cu (105,106),
124I (107), 89Zr (108,109). Most studies demonstrated high uptake in HER2-overexpressing
tumour xenografts ranging from 12.6 to 66.9 %ID/g and T/B ratios of 1.15 to 6.6 at time points
33
from 24-144 h p.i. High tumour accumulation was obtained due to the retention of the
radiometals by tumour cells as well as the long circulation time of the antibodies.
While imaging tumour HER2 expression with radiolabeled trastuzumab is feasible, a
probe that does not compete with trastuzumab for the binding site of HER2 would enable more
sensitive imaging of changes in HER2 expression while a patient is treated with trastuzumab.
The data from the aforementioned clinical study investigating 111In-trastuzumab for predicting
trastuzumab cardiotoxicity by Perik et al. were reanalyzed and the results suggested that
concomitant therapeutic trastuzumab administration reduced 111In-labeled trastuzumab uptake
by about 20% (91). To probe for HER2 more sensitively in the presence of therapeutic
trastuzumab, McLarty et al. developed 111In-labeled pertuzumab to target HER2 as a means of
monitoring response to trastuzumab therapy since pertuzumab and trastuzumab bind to different
epitopes of HER2 (110). This study demonstrated that SPECT/CT imaging was able to detect a
2-fold decrease in tumour uptake of 111In-labeled pertuzumab as early as three days after the
start of trastuzumab therapy. Following 3 weeks of trastuzumab, tumour uptake of 111In-labeled
pertuzumab decreased 4.5-fold relative to phosphate buffered saline (PBS)-treated mice. This
was associated with the elimination of HER2 positivity as determined with IHC and nearly
complete eradication of viable tumour cells as determined by hematoxylin and eosin staining of
excised tumour tissue.
Although high tumour uptake can be achieved with radiolabeled antibodies due to their
long circulation times, this may result in accumulation of radioactivity in the blood and liver
which can mask HER2-positive lesions within areas of highly perfused organs (e.g. nodal
metastases) and within the liver (95,96). Furthermore, the long circulation times of mAbs
require radioisotopes with longer half-lives to match which may lead to increased radiation
34
doses. Smaller molecular weight agents such as antibody fragments or affibodies that clear more
quickly from the blood and most normal tissues (except the kidneys) than intact antibodies may
serve to address these issues.
1.6.2 Antibody fragments
Antibody fragments include Fab (25 kDa) and F(ab')2 (110 kDa) prepared by enzymatic
digestion of intact immunoglobulins, as well as engineered antibody fragments including Fab,
single chain variable fragments (scFv; 25 kDa), diabodies (55 kDa), minibodies (80 kDa), and
scFv with fragment crystallizable (scFv-Fc; 105 kDa). A comparison of intact IgG antibody and
antibody fragments is provided in Table 1.3 and Figure 1.4. Antibody fragments confer more
rapid blood clearance properties due to their reduced size but may also result in lower tumour
uptake. The fast blood clearance properties of radiolabeled small antibody fragments result in
maximum T/B ratios to be reached quicker than radiolabeled intact antibodies. This allows
tumour imaging to occur at very early time points (on the order of a few hours p.i.) and therefore
allows for radioisotopes with shorter half-lives (t½) such as 68Ga (t½ = 68 mins) to be used.
Antibody fragments have also demonstrated improved tumour penetration and more
homogeneous tissue distribution than intact antibodies (111).
Smith-Jones et al. demonstrated faster blood clearance and higher T/B ratios for 111In-
labeled trastuzumab F(ab')2 compared to the intact antibody at 24 h p.i. in BT-474 HER2-
overexpressing human BC xenografts in nude athymic mice (10.0 vs. 3.4, respectively) (105).
High contrast PET images of BT-474 tumours in mice were achieved at only 3 h after
administration of 68Ga-labeled trastuzumab F(ab')2 fragments. Tang and colleagues also
demonstrated high T/B ratios (25:1) for 111In-labeled trastuzumab Fab fragments in athymic
35
Figure 1.4. Structures of an intact IgG antibody and antibody fragments.
36
Table 1.3
Properties of Radiolabelled Antibodies and Antibody Fragments
Antibody Molecular
Weight
(kDa)
Blood
elimination
half-life
Tumour
uptake
(%ID/g)
Optimal
tumour/blood
ratio
References
Intact IgG 150 1-3 weeks 15-50% 96-168 h (103,108,110,
112)
F(ab')2 110 10-12 h 10-20% 24-72 h (105,113-115)
Fab 25 5 h 2-10% 24-72 h (115-117)
scFv 25 0.5-2 h 6% 1-4 h (112,118)
scFv-Fc 105 12 d 15-50% 72-120 h (112,119,120)
minibody 80 5-11 h 5-30% 8-48 h (112)
diabody 55 3-7 h 6-10% 4-24 h (112,121,122)
37
mice with BT-474 xenografts at 72 h p.i. (116). The ability of 99mTc-trastuzumab Fab to image
HER2-overexpressing xenografts was also evaluated in athymic mice bearing BT-474
xenografts (123). The T/B ratios for 111In-trastuzumab Fab and 99mTc-trastuzumab Fab at 24 h
p.i. were 4.2 and 3.2, respectively, lower than that achieved by 68Ga-trastuzumab F(ab')2, which
may be due to decreased avidity of the monovalent Fab for binding HER2 (105,116,123). The
studies investigating 111In- and 99mTc-trastuzumab Fab also reported increased kidney uptake
(>50 %ID/g at 24 h p.i.) relative to intact antibodies, a property frequently observed with
imaging agents smaller than 60 kDa, due to glomerular filtration and subsequent reabsorption
into renal proximal tubules (124). The residualizing nature of the radiometals, 111In and 99mTc,
also contributes to renal accumulation whereby the renal metabolite, radiometal-chelate-ε-amino
lysine, is only slowly cleared (125).
The ability to intraoperatively detect tumour margins in HER2-positive BC using a hand-
held γ-probe following injection of 111In-labeled trastuzumab Fab fragments was evaluated by
Holloway et al. in a Phase I clinical trial (126). Using an injected dose of 74 MBq, low
intraoperative γ-probe counts in tumours were observed and imaging of tumour uptake and
delineating tumour margins was not feasible. Higher injected activities or using radiolabeled
F(ab')2 fragments that have higher avidity for binding HER2 may improve intraoperative
detection of tumour margins.
Bispecific radioimmunoconjugates composed of trastuzumab Fab fragments and
heregulin-β1 or EGF to image HER2-HER3 or HER2-EGFR dimers, respectively, or each
receptor on its own were investigated by Razumienko et al. (127,128). Uptake in HER2/HER3-
positive BT-474 human BC xenografts and HER2/EGFR-positive 231-H2N human BC tumours
were about 7 %ID/g in CD1 athymic mice and SPECT/CT imaging revealed good tumour
38
visualization but with high kidney accumulation. These bivalent radioimmunoconjugates may
impart more avid binding and possibly allow visualization of heterodimerized receptors.
To achieve more rapid blood clearance and a higher T/B ratio at an earlier time point
than that achieved with 68Ga-DOTA-trastuzumab F(ab')2 (105), 68Ga-labeled anti-HER2 scFv
using the desferrioxamine chelator (Df) was developed and evaluated for its ability to monitor
response to 17-dimethylaminoethylamino-17-demothoxygeldanamycin (17-DMAG), a heat
shock protein chaperone inhibitor involved in HER2 degradation (118). 68Ga-Df-anti-HER2
scFv displayed faster blood clearance and a higher T/B ratio compared to the 68Ga-trastuzumab
F(ab')2 (1.1 vs. <1 at about 3 h p.i.). The probe was able to monitor changes in HER2 expression
following 17-DMAG therapy however, 68Ga-Df-anti-HER2 scFv binds to the same epitope of
trastuzumab and would therefore be less sensitive for monitoring response to trastuzumab.
HER2-overexpressing tumours were imaged by PET/CT with the radiolabeled minibody,
68Ga-NOTA-2Rs15d, which binds to an epitope on HER2 that is different than that bound by
trastuzumab. Biodistribution analysis revealed a relatively modest uptake of 4.3 %ID/g in
HER2-overexpressing tumours, however, the fast blood clearance kinetics yielded a very high
T/B ratio of 28.5 (129) at 1 h p.i. Similar results were observed with the SPECT/CT analogue,
99mTc-2Rs15d (130). Dosimetric analysis from mouse data of 68Ga-NOTA-2Rs15d yielded a
projected effective dose of 4 mSv for a 185 MBq injected amount in a human, which is lower
than the dose for a standard 18F-FDG PET scan (7 mSv for 370 MBq of administered activity)
(129). In a Phase I clinical trial of PET/CT imaging with 68Ga-NOTA-2Rs15d, 13 of 15 primary
tumours were visualized at 60-90 min p.i. with standardized uptake values (SUVmean) ranging
from 0.7-11.8 and distant metastases with SUVmean ranging from 3.1-6.0. The SUVmean is a
measure of relative tissue uptake and is calculated by dividing the radioactivity concentration in
39
an organ by the whole body concentration of the injected radioactivity (injected dose divided by
the body weight of the patient). The effective dose was low and was 4.6 mSv for an injection of
107 MBq. Thus, in summary, preclinical and clinical studies reveal that radiolabeled anti-HER2
antibodies and their fragments are promising strategies for imaging HER2 expression in
tumours.
1.6.3 Affibodies
Another approach to imaging HER2 expression in tumours is to use radiolabeled
affibodies, which have demonstrated excellent tumour targeting and pharmacokinetic properties.
Affibodies are 58-amino acid proteins that are derived from the B-domain of the
immunoglobulin-binding region of staphylococcal protein A (131). Their small size (~6.5 kDa)
enables rapid blood clearance and good tissue penetration. Randomization of 13 amino acid
positions in the binding surface of the domain scaffold has generated combinatorial phagemid
libraries from which affibody molecules with affinities towards a variety of cell surface antigens
can be selected by phage display. The first HER2-directed affibody, ZHER2:4, bound to HER2
with an affinity of 50 nM and at a binding site distinct from that of trastuzumab (132). Using
affinity maturation, a second generation affibody molecule, ZHER2:342, was developed that
showed a binding affinity of 22 pM, a >2,200-fold increase in affinity compared to the ZHER2:4
(133). The tumour uptake in Balb/c nude mice with SK-OV-3 human ovarian cancer xenografts
at 4 h p.i. was 9% injected dose per gram of tissue (%ID/g), a 4-fold improvement compared to
the parent affibody. Tumour uptake in Balb/c nude mice with SK-OV-3 xenografts increased up
to 23 %ID/g at 1 h p.i. after site-specific DOTA-chelator conjugation and 111In radiolabeling,
however kidney uptake was also very high with an uptake of 243 %ID/g at 1 h p.i. (134).
Imaging of HER2-overexpressing tumour xenografts in mice has successfully been achieved
40
with other affibody variants with different radioisotopes such as 68Ga-DOTA-ZHER2:342 (135),
57Co-DOTA-ZHER:2395-Cys (136), 18F-FBEM-ZHER2:342 and 18F-FBO-ZHER2:477 (137).
In a Phase I clinical trial, the detection of HER2-positive lesions using PET/CT and
SPECT imaging with 68Ga- and 111In-DOTA-ZHER2:342-pep2 (ABY-002), respectively, was
evaluated in three patients with recurrent, HER2-positive metastatic BC (138). Most lesions
detected by 18F-FDG PET imaging were also detected by PET/CT and SPECT imaging with
68Ga- and 111In-DOTA-ZHER2:342-pep2. A lymph node or adrenal metastasis was not detected by
111In-ABY-002 in one patient due to high overlying radioactivity in the kidney, and a liver
metastasis was not detected by 68Ga-ABY-002 in another patient due to high liver background.
The high liver uptake was not expected since liver uptake was low in preclinical studies with
non-tumour-bearing and tumour-bearing mice. The authors hypothesized that HER2 expression
in the liver led to specific uptake of the probe. A second generation radiolabeled affibody, 111In-
ABY-025 was able to differentiate between HER2-positive and HER2-negative tumours in
patients but did not detect all small metastatic lesions that had been detected by PET using 18F-
FDG and were HER2-positive by IHC or FISH (139). The effective absorbed dose was reported
to be 0.15 mSv/MBq. The authors hypothesized that imaging with PET would be better able to
detect small lesions due to the higher spatial resolution that it offers, however current clinical
SPECT scanners can offer better spatial resolution than PET. Most recently, a study of PET
imaging in women with metastatic BC using 68Ga-ABY-025 demonstrated that most HER2-
positive and HER2-negative lesions could be distinguished and changes in HER2 status over
time could be detected (140). Normal liver uptake was reduced when a higher mass dose of
radiolabeled affibody (427 µg) was administered compared to a lower mass dose (78 µg). The
use of PET allowed accurate quantitation of radioactivity uptake into tumours and the authors
41
were able to propose a SUV cut-off of 6 to discriminate between HER2-positive from HER2-
negative lesions. The effective absorbed dose was 0.028-0.030 mSv/MBq depending on whether
a high or low affibody dose was administered, corresponding to 6.0 and 5.6 mSv for a 200-MBq
high and low affibody dose, respectively (141). Radiolabeled affibodies are a promising strategy
for sensitively detecting HER2 status and could be used to monitor response to trastuzumab
therapy since they do not compete for the same binding site on HER2 as trastuzumab.
1.6.4 Peptides
Aside from affibodies, short peptides that bind to HER2 have also been investigated for
tumour imaging. The phage display-selected HER2-targeting peptide, KCCYSL, was
radiolabeled with 111In using the DOTA chelator and a Gly-Ser-Gly spacer between the peptide
and chelator. Biodistribution studies in SCID mice bearing HER2-overexpressing ovarian
OVCAR-3 xenografts showed peak tumour uptake of 0.5 %ID/g at 1 h p.i. but a T/B ratio of
only 2.2 at this time point (142). However, the T/B ratio of 111In-KCCYSL in HER2-
overexpressing MDA-MB-435 BC tumours reached 5.1 after 2 h and the tumour uptake was 0.7
%ID/g (143). Tumour specificity was demonstrated by a significant decrease in tumour uptake
when unlabeled DOTA(GSG)-KCCYSL was administered 15 minutes prior to 111In-KCCYSL.
Similarly, another anti-HER2 peptide, AHNP ([Cys6-Cys12]H-
GAGGYCDGFYACYMDVCONH2), was radiolabeled with 99mTc for tumour targeting of
transfected HER2-overexpressing mouse fibroblast NIH-3T3 (T6-17) tumours. Ex vivo
biodistribution studies revealed that tumour uptake was low (0.2 %ID/g at 3 h p.i.) as well as the
T/B ratio (0.4) (144). Generally, the results of radiolabeled anti-HER2 peptide studies have been
less successful than affibodies and antibody based imaging agents. However, interest remains in
radiolabeled peptides, as well as with antibody fragments and affibodies, due to their potential to
42
confer lower radiation absorbed doses to normal organs by clearing from the circulation faster
and using shorter lived radioisotopes that match the biological half-lives of antibody fragments.
In the next section, strategies to translate promising molecular imaging agents to the clinic are
described.
1.7 Translating novel molecular imaging probes to Phase I clinical trials
Molecular imaging, especially single photon emission computed tomography (SPECT)
and positron emission tomography (PET), is a powerful tool for detecting cancer and for
characterizing the biological properties of tumours. Revealing tumour biology in an individual
patient could aid in the optimal selection of molecularly targeted (i.e. “personalized”) cancer
therapies (88). Molecular imaging can also trace the delivery of anticancer agents to tumours
(92) and probe their mechanisms of action which may predict tumour response (105,110).
Furthermore, the downstream effects of treatment on tumour viability can be probed by
molecular imaging which reports on the effectiveness of treatment (145,146). Thus, there has
been rapid growth in radiopharmaceutical research aimed at the discovery of novel molecular
imaging agents for cancer. A Pubmed (http://www.ncbi.nlm.nih.gov/ pubmed) search combining
the terms “molecular imaging” and “SPECT” and “cancer” yielded almost 1000 publications.
Interrogating the terms “molecular imaging” and “PET” and “cancer” yielded an additional
2500 publications. However, within these “hits”, selecting the article type as “Clinical Trial”
revealed only 24 reports (2.4%) of SPECT probes and 95 reports (3.8%) of PET probes that
were formally investigated in clinical trials in humans. Radiolabeled mAbs represent one class
of molecular imaging agents that are increasing attention due to the success of antibody-based
cancer therapy (147). However, searching the ClinicalTrials.gov registry
(https://clinicaltrials.gov/ct2/home) by combining the terms “SPECT” and “cancer” and
43
“monoclonal antibodies” revealed only 8 clinical trials of tumour imaging with radiolabeled
mAbs. Interrogating ClinicalTrials.gov for the terms “PET” and “cancer” and “monoclonal
antibodies” identified another 6 trials of PET imaging with radiolabeled mAbs. Taken together
with the PubMed search data, it is evident that only a small proportion of molecular imaging
agents studied preclinically for tumour imaging have been advanced to clinical trials in patients.
The barriers preventing translation of promising molecular imaging agents from the
“bench to the bedside” are not clear. One barrier may be financial. A report published almost 10
years ago estimated that the cost of development of a new diagnostic imaging agent from
preclinical studies to regulatory approval was $100−200 million, while the market for a
“blockbuster” imaging agent was only $200−400 million, making it difficult to recoup develop-
ment costs (148). It was further estimated that about a decade was required for a new imaging
agent to reach regulatory approval and be marketed (149). However, these represent the total
costs and length of time for clinical development of an imaging agent from preclinical studies
through all stages of clinical trials (Phase I to III) to final regulatory approval and marketing.
Based on our experience, we have found that novel molecular imaging probes can be advanced
to first-in-humans Phase I trials in academia at a much lower cost (about $1 million) and
requiring only a few years following completion of preclinical imaging studies. If successful,
the results of these Phase I trials may “de-risk” further development by the radiopharmaceutical
industry and encourage investment which should accelerate and expand the portfolio of probes
reaching the clinic for the benefit of cancer patients.
To achieve translation of a novel molecular imaging agent to Phase I clinical trial in
academia requires a very good understanding of the steps required to advance an imaging probe
from a “molecular entity” studied in mouse tumour xenograft models to a “molecular imaging
44
radiopharmaceutical” that meets the expected high quality and safety standards for human
investigation. Radiopharmaceutical scientists may perceive major barriers to clinical translation
in academia including a lack of expertise and insufficient resources. Unfortunately, only a few
articles in the literature inform on the processes for advancing novel molecular imaging probes
to human studies (108,139,150,151). The following sections will describe the approach taken
within an academic radiopharmaceutical research laboratory at the University of Toronto to
overcome these perceived barriers. To illustrate, a description of the steps taken to advance
111In-labeled pertuzumab (111In-BzDTPA-pertuzumab; see Chapter 2), a novel probe for
detecting response of HER2-positive BC to treatment with trastuzumab (Herceptin) from
preclinical studies (110) to a Phase I clinical trial in patients with metastatic HER2-positive BC
(PETRA; ClinicalTrials.gov identifier: NCT01805908).
1.7.1 Roadmap to clinical translation of novel molecular imaging probes
The four major steps in the “roadmap” to clinical translation (Figure 1.5) for 111In-
labeled pertuzumab or for any molecular imaging probe are (i) radiopharmaceutical formulation,
(ii) preclinical pharmacology and toxicology studies, (iii) clinical trial design and human ethics
approval, and (iv) regulatory agency submission and approval.
45
Figure 1.5. The “roadmap” demonstrating the four steps in the translational bridge phase to
advance novel molecular imaging agents from preclinical studies to Phase I clinical trial.
46
The first three steps are closely linked and should not be considered in isolation. For
instance, the injected radioactivity and mass amount to be administered to humans in the Phase I
trial must be selected first in order to design the radiopharmaceutical formulation. Moreover,
regulatory agencies require preclinical toxicology studies at scaled multiples of the proposed
human injected radioactivity and mass amounts for the Phase I trial and using the actual
radiopharmaceutical formulation to be administered in the trial. The injected radioactivity
amount to be selected is dependent in part on the radiation dosimetry in order to minimize the
radiation absorbed dose to patients from imaging studies in the trial. The radiation dosimetry in
humans is projected from preclinical biodistribution and pharmacokinetic studies that are
conducted at an injected radioactivity amount and mass amount that is scaled from the proposed
human dose. Previously published literature with analogous probes that have been evaluated in
humans are helpful to guide the selection of the injected radioactivity and mass amounts. In the
case of 111In-BzDTPA-pertuzumab, a dose of 5 mg was selected for the PETRA trial based on
the range of mass amounts (10−100 mg) that were previously employed for other 111In-labeled
mAbs (90,152,153). The PETRA trial protocol required three SPECT/CT imaging studies: (i) a
baseline study prior to commencing trastuzumab treatment, (ii) a study at 1 week to evaluate
trastuzumab-mediated HER2 downregulation compared to baseline, and (iii) an imaging study at
one month to evaluate further decreases in HER2 expression and/or possible therapeutic
response to trastuzumab. Each imaging study required a separate administration of the radio-
pharmaceutical. Most previously reported imaging studies with 111In-labeled mAbs employed a
single injected radioactivity amount of 150−185 MBq. In order to minimize the radiation dose to
patients for three administrations of 111In-BzDTPA-pertuzumab, a lower injected radioactivity
amount of 111 MBq was chosen. Formulation of the radiopharmaceutical and preclinical
47
pharmacology and toxicology studies proceeded based on the administration of 111 MBq (5 mg)
of 111In-BzDTPA-pertuzumab in the PETRA trial.
1.7.2 Radiopharmaceutical formulation
In order to advance 111In-BzDTPA-pertuzumab to Phase I clinical trial, the first step in
the roadmap was radiopharmaceutical formulation (Figure 1.6). A radiopharmaceutical kit is an
attractive formulation for preparing novel molecular imaging agents for human studies because
(i) many quality control tests can be performed in advance of preparing the final
radiopharmaceutical for patients, (ii) the kits are stable and can be stored and labeled only when
needed for a patient study, and (iii) the kits utilize radiometal chelation chemistry that robustly
provides high labeling efficiency (≥90%) and does not require post-labeling purification. For
imaging purposes, a labeling efficiency ≥90% for a kit (equivalent to final radiochemical purity)
is considered acceptable by regulatory agencies, whereas therapeutic applications of
radiopharmaceuticals may require higher radiochemical purity (≥95%). These properties of kit
formulations simplify radiopharmaceutical preparation and reliably ensure high quality for
patient studies. There are several examples of kit formulations reported in the literature for
preparation of molecular imaging probes (154-157).
A kit for preparation of 111In-BzDTPA-pertuzumab was designed that consisted of a
unit-dose vial containing 5.0 mg of pertuzumab modified with 2-(4-isothiocyanatobenzyl)-
diethylenetriaminepentaacetic acid (p-SCN-BzDTPA) formulated in 0.5 mL of ammonium
acetate buffer, pH 6.0.22 p-SCN-BzDTPA was used instead of DTPA dianhydride previously
employed for preclinical studies since this chelator provides a more stable 111In complex and
avoids cross-linking of the mAb (158). The mean labeling efficiency of the kits with 111In
(110−150 MBq) was 95.8 ± 2.7%. Other quality control parameters for the radiopharmaceutical
48
Figure 1.6. The radiopharmaceutical formulation step includes formulation of a kit and final
radiopharmaceutical as well as establishment of specifications and quality control assays for raw
materials, intermediates (including the kit) and final radiopharmaceutical. The stability of the kit
and final radiopharmaceutical need to be evaluated by testing against specifications over time,
and this data is used to establish expiry times. At least three independent lots of kits and final
radiopharmaceutical are manufactured and tested against specifications to assure that these will
be reliably met.
49
kit included protein concentration, volume, pH, appearance, BzDTPA substitution level, purity,
and homogeneity. HER2 immunoreactivity was evaluated for each lot of kits and did not need to
be measured for each lot of the final radiopharmaceutical product. For a full description of the
specifications and assays for these parameters, see Chapter 2 (159). Testing for sterility was
performed by the USP Sterility Test at the clinical microbiology laboratory at Mount Sinai
Hospital (Toronto, ON, Canada). The USP Bacterial Endotoxins Test was performed within the
laboratory using a commercially available colorimetric limulus amebocyte lysate (LAL) assay
(QCL-1000 End point Chromogenic LAL Assay, Lonza, Walkersville, MD). Quality
specifications for 111In-BzDTPA-pertuzumab included limits for total radioactivity and
radioactivity concentration, specific activity, pH, radiochemical purity (>90%), radionuclidic
purity, appearance, and sterility (retrospective). Retrospective USP sterility testing was
performed on a randomly selected sample (5%) of lots of 111In-BzDTPA-pertuzumab after
radioactive decay for 30 days. Retrospective sterility testing assures that the radiopharma-
ceutical administered to patients was sterile, but importantly that the method for its aseptic
preparation will reliably result in a sterile product. Endotoxins testing was not routinely
performed on the final radiopharmaceutical but a validation study was conducted by testing
several pilot lots of 111In-BzDTPA-pertuzumab for endotoxins to demonstrate that the product
would meet USP requirements for bacterial endotoxins.
Stability testing was performed on one randomly selected vial from at least 3
independent lots of kits and final radiopharmaceutical by testing these lots monthly against the
established specifications which included protein concentration, purity and homogeneity, pH,
clarity and color, labeling efficiency or radiochemical purity but not sterility and apyrogenicity.
These studies showed that the kits were stable when stored at 4 °C, and a 4-month expiry was
50
assigned. 111In- BzDTPA-pertuzumab was stable up to 24 h at room temperature (20°C) but an 8
h expiry was assigned since this was adequate to allow shipping to the clinical trial site for
patient administration.
1.7.3 Good manufacturing practices (GMP)
Good Manufacturing Practices (GMP) are often perceived as a barrier to advancing
novel molecular imaging probes to Phase I clinical trials in academia. There is frequently a
misconception that GMP is focused only on the environment for pharmaceutical manufacturing,
when in fact, GMP is a much broader quality assurance system that documents in detail the
production of a pharmaceutical from raw materials through intermediates to final product, as
well as the assays and specifications that have been implemented to ensure its quality (160).
Regulatory agencies usually do not apply full GMP requirements for novel molecular imaging
agents to be investigated in a Phase I trial, but do require that many components of GMP are in
place to ensure the quality and safety of the radiopharmaceutical for these first-in-humans
studies. Raw materials used in the preparation of a novel molecular imaging agent for clinical
investigation need to be pharmaceutical quality. Examples of raw materials include buffer salts
and acids, radiometal chelators, mAbs or peptides, sterile water and normal saline, glass vials,
and the radioisotope (e.g., 111In). Pharmaceutical quality is most easily assured by purchasing
pharmacopeial grade (e.g., USP) materials whenever available. Particularly important is to
employ Sterile Water for Injection USP or Sodium Chloride Injection USP for formulation of
any pharmaceutical buffers or for preparing kits or the final radiopharmaceutical. Since trace-
metal contamination may decrease the efficiency of radiometal labeling, it is important to purify
all pharmaceutical buffers by cation exchange chromatography on a column of Chelex-100 resin
(BioRad, Hercules, CA). In the case where no pharmacopeial grade material is available,
51
American Chemical Society (ACS) grade chemicals may be used but a high purity (>95%)
should be selected. For any nonpharmacopeial raw materials, it is necessary to obtain an
individual lot Certificate of Analysis (COA) from the supplier that certifies a high level of purity
and lists and provides limits for trace impurities present. In addition, raw materials should be
identity tested on receipt by analytical methods (e.g., NMR or testing for key functional groups)
to ensure that the correct material has been received. Macrocyclics, Inc. (Dallas, TX) provides
custom GMP synthesis of radiometal chelators, but in our experience, chemical grade chelators
with high purity accompanied by an individual lot COA and identity testing on receipt are
sufficient to meet regulatory requirements.
Small molecule precursors or peptide raw materials used to prepare molecular imaging
agents may be synthesized in-house but require high purity (>95%) and full analytical
characterization. Radioimmunoimaging agents present special challenges since regulatory
agencies often require that the manufacturing process be fully elaborated including complete
descriptions of any host cell banks and expression vectors used. Recombinant mAbs must also
be tested for adventitious virus and endotoxins contamination (161). In our experience, we have
found that these challenges are most easily overcome by using a mAb that is already an
approved pharmaceutical product. In such cases, the manufacturer may provide a letter to permit
the regulatory agency to consult the manufacturing information on file for the mAb. The rapid
growth in cancer immunotherapy has generated many pharmaceutical quality mAbs that
recognize a wide range of molecular targets that could be developed as novel molecular imaging
agents (147). A Materials Transfer Agreement (MTA) needs to be established between the
university and the company that describes the conditions for transfer of the material, including
any intellectual property protection considerations. It may be possible for the university to
52
negotiate shared intellectual property in the MTA for any improvements in the material such as
development into a novel molecular imaging agent. 111In-BzDTPA-pertuzumab was prepared
from pertuzumab (Perjeta; Hoffman La Roche, Mississauga, ON, Canada) which is an approved
pharmaceutical mAb used for the treatment of HER2-positive metastatic BC (162). The
manufacturer provided a letter of authorization to Health Canada to access the information on
file for pertuzumab and provided the raw material through a MTA to prepare 111In-BzDTPA-
pertuzumab for the PETRA trial.
Quality control assays and specifications for concentration, pH, clarity and color, and
sterility and apyrogenicity must be established for all pharmaceutical buffers used to prepare the
kit and final radiopharmaceutical. In some cases, pharmacopeial assays may be adapted to assay
the concentrations of buffer salts (e.g., sodium phosphate, acetate, or bicarbonate). Kits and the
final radiopharmaceutical must be terminally sterilized, usually by filtration through a 0.22 μm
filter. The integrity of the filter is tested using the “bubble point test” which involves passing air
through the filter after use and assuring that there is strong resistance. Kits must be tested for
quality parameters (see section 1.7.2) including protein concentration, volume, pH, appearance,
chelator substitution level, purity and homogeneity, and immunoreactivity or receptor-binding.
Sterility and apyrogenicity are determined by the USP Sterility Test and USP Bacterial
Endotoxins Test, respectively, to ensure that the product is pharmaceutically acceptable for
injection. The final radiopharmaceutical is tested for radiochemical purity (≥90%) and several
other key quality parameters including radioactivity concentration, pH, and sterility (performed
retrospectively on a randomly selected 5% sample of vials). The specifications and assays for
pharmaceutical buffers, kits, and 111In-BzDTPA-pertuzumab are outlined in Chapter 2. Standard
operating procedures (SOPs) should be developed to provide written detailed processes for
53
manufacturing and quality control of pharmaceutical buffers, kits, and the final
radiopharmaceutical. Complete records on raw materials, intermediates (including kits), and the
final radiopharmaceutical that are traceable by a lot numbering system must be maintained to
investigate any quality issues. A lot release and recall procedure should be in place. In Canada,
the results of quality control testing of radiopharmaceutical kits must be FAXed to Health
Canada with a request for authorization of individual lot release prior to use for
radiopharmaceutical preparation for patients in the trial.
The environment for kit and radiopharmaceutical manufacturing should meet GMP
standards for air quality (160). Sterile products must be prepared in a grade A air environment in
a room with a minimum of grade C air. Grade A air contains <3520 particles per m3 with
diameter ≥0.5 μm and <20 particles with diameter ≥5 μm. A Class A laminar air flow cabinet
(biosafety cabinet) provides grade A air since the High Efficiency Particulate Air (HEPA) filter
removes all particles >0.45 μm. Ideally, the cabinet should be located in a “clean room” which
has a HEPA-filtered air supply that meets at least grade C. At operation, i.e., during kit or
radiopharmaceutical preparation, grade C air contains <3 520 000 particles per m3 with diameter
≥0.5 μm and <29 000 particles per m3 with diameter ≥5 μm. These limits are 10-fold lower
when there are no operations taking place (i.e., at rest). When no clean room is available, a
dedicated room for pharmaceutical manufacturing in a clean facility may provide grade C air,
but this requires testing by a certified air quality testing service to ensure that these standards are
met. The biosafety cabinet requires testing and recertification annually. The cabinet and any
equipment or supplies placed in the cabinet should be disinfected with 70% alcohol and sterile
plasticware and syringes should be used for pharmaceutical formulation. Nonsterile equipment
must be sterilized by autoclaving or gas sterilization. Equipment used in kit or
54
radiopharmaceutical preparation ideally should be dedicated to avoid contamination, and should
be maintained in a high state of cleanliness and in very good operating condition and regularly
calibrated. Individuals preparing radiopharmaceuticals should be qualified and trained in sterile
product preparation, and should wear a clean lab coat, face mask, and head covering whenever
conducting manufacturing operations.
1.7.4 Preclinical pharmacology and toxicology studies
The next steps in the roadmap to clinical translation are preclinical pharmacology and
toxicology studies (Figure 1.7). Normal tissue biodistribution studies and evaluation of the
pharmacokinetics of elimination from the blood are used to predict the radiation absorbed doses
to humans for the Phase I trial. Biodistribution and pharmacokinetic studies for 111In-BzDTPA-
pertuzumab were performed in groups of four non-tumour-bearing Balb/c mice at several time
points up to 7 days post-injection. This data was used to estimate the cumulative radioactivity in
each source organ (A, Bq × h) which was then applied to predict the radiation absorbed doses
(D; mSv) to target organs in humans using the Organ Level INternal Dose Assessment
(OLINDA) software. OLINDA employs the Medical Internal Radiation Dose (MIRD)
formalism which estimates target organ doses as D= A× S; where S is the dose (mSv/Bq × h) to
a target organ per unit of cumulative radioactivity in a source organ (163).
Acute toxicity studies were performed in groups of 10 female, non-tumour-bearing mice
administered 111In-BzDTPA-pertuzumab at 10-times the planned injected mass amount for
patients in the PETRA trial and at 23-times the planned injected radioactivity amount scaled
from the human to the mouse on a mg/kg or MBq/kg basis, respectively. Body mass was
measured every few days over a 15-day period. Other measurements included complete blood
cell counts (CBC), hemoglobin, hematocrit, serum creatinine, and serum alanine amino-
55
Figure 1.7. Preclinical pharmacology and toxicology studies to advance a novel molecular
imaging agent to Phase I clinical trial. These include evaluation of the normal tissue
biodistribution at several time points and determination of the pharmacokinetics of elimination
from the blood. The acute toxicity of the molecular imaging agent is studied at multiples of the
proposed human dose and include evaluation of any adverse effects on the hematological
system, liver, kidneys or other normal organs. The radiation absorbed doses in humans are
projected from preclinical biodistribution data using OLINDA dosimetry software.
56
transferase (ALT) at 2 days and 15 days post-injection. Mice were sacrificed and a
comprehensive panel of tissues were collected after 15 days that were then examined
histopathologically by a clinical pathologist. The pathologist provided a detailed written report.
The controls for toxicity studies of 111In-BzDTPA-pertuzumab were groups of 10 mice adminis-
tered normal saline or unlabeled BzDTPA-pertuzumab. These studies demonstrated that there
were no serious toxicities associated with the administration of 111In-BzDTPA-pertuzumab at
multiples of the planned human injected radioactivity and mass amounts. Regulatory agencies
may require toxicology studies to be performed in a nonrodent as well as a rodent species, but
this was not required in this case by Health Canada for 111In-BzDTPA-pertuzumab since this
was prepared from an approved pharmaceutical product (pertuzumab). The main purpose of the
toxicology studies was to assess if the toxicity of pertuzumab was increased by labeling with
111In. In the CTA to Health Canada, all available literature on previous preclinical and clinical
studies of pertuzumab that documented its safety at therapeutic doses and which greatly
exceeded the injected mass amount planned for the PETRA trial was provided.
1.7.5 Clinical trial design and human ethics approval
The next step in the roadmap to clinical translation is the design of the Phase I clinical
trial protocol and informed consent document as well as obtaining human ethics approval
(Figure 1.8). To design a Phase I trial for 111In-BzDTPA-pertuzumab as well as conduct the trial
under Good Clinical Practices, we partnered with the Ontario Clinical Oncology Group (OCOG;
http://www.ocog.ca) at McMaster University (Hamilton, ON, Canada). OCOG is an academic
clinical trials organization that provides a multidisciplinary team of oncologists, biostatisticians,
clinical trialists, human ethics and regulatory affairs specialists, information technology
programmers, clinical research coordinators, and data monitoring and management assistants.
57
Figure 1.8. The clinical trial design and human ethics approval step for advancing a novel
molecular imaging agent to Phase I trial. Consultation with a biostatistician estimates the patient
sample size and statistical power of the trial to test the hypothesis. This phase includes design of
the clinical trial protocol and informed consent documents, and application for human ethics
approval.
58
OCOG collaborates with radiopharmaceutical scientists, nuclear medicine physicians,
medical physicists, and oncologists to conduct clinical trials of innovative molecular imaging
agents for cancer. In collaboration with our group, OCOG designed a Phase I trial (PETRA;
Clinical-Trials.gov identifier: NCT01805908) to study SPECT/CT imaging with 111In-BzDTPA-
pertuzumab to predict the response of patients with metastatic HER2-positive BC to treatment
with trastuzumab combined with chemotherapy. In addition, OCOG submitted an application
for human ethics approval for the trial to the Ontario Cancer Research Ethics Board (OCREB)
which is a province-wide review board for clinical trials in cancer patients. Finally, OCOG
provided regulatory support for the CTA submission to Health Canada for the trial and
functioned as the liaison between Health Canada and our group as well as the trial investigators.
Forming a partnership with interested and committed oncologists is essential to advance a novel
molecular imaging agent to Phase I clinical trial. Partnering with an academic clinical trials
organization such as OCOG assures that GCP are incorporated into the design and conduct of
the trial.
1.7.6 Regulatory agency submission
The final step in the roadmap to clinical translation is regulatory agency submission
(Figure 1.9). In Canada, a Clinical Trial Application (CTA) must be submitted to Health
Canada. Guidelines for compiling a CTA are available on the Health Canada Web site
(http://www.hc-sc.gc.ca). A CTA is analogous to an Investigational New Drug (IND)
submission to the U.S. Food and Drug Administration (FDA) and the process for
radiopharmaceuticals in the U.S. has been published (164). Overviews of the U.S. FDA
regulations for PET radiopharmaceuticals (21 CFR part 212) are also available (150,151,165).
59
Figure 1.9. The final step in advancing a novel molecular imaging agent to Phase I clinical trial
is regulatory agency submission which includes completion of a CTA (Canada) or IND
application (U.S.) that is supported by information on the chemistry & manufacturing of the
agent, results of preclinical pharmacology and toxicology studies, clinical trial protocol and
informed consent, and the investigator brochure (IB). Regulatory agency review normally takes
30 days.
60
The USP provides further guidance on production of PET radiopharmaceuticals for human use
(USP Chapter 823) (166). The CTA for a Phase I clinical trial in Canada requires three
supporting modules: (i) chemistry and manufacturing, (ii) preclinical pharmacology and
toxicology, and (iii) clinical trial protocol, investigator’s brochure (IB), and informed consent.
Information in these modules is compiled from the steps to clinical translation described (Figure
1.4). The IB is a product monograph for an investigational agent that summarizes the product
information for the trial investigators. This information includes details of the formulation, the
radioactivity and mass amount to be administered, radiation dosimetry, summaries of all
previous preclinical or clinical studies, and any precautions regarding possible adverse effects
from the investigational agent. In addition to the three Supporting Information modules, in
Canada several forms must be completed for the CTA including the Drug Submission
Application (Form HC3011), Clinical Trial Site Information form, and Quality Information
Summary − Radiopharmaceuticals (QIS-R). In the case of biologics such as radiolabeled mAbs,
the Quality Information Summary − Biologics (QIS-B) form must also be completed. The QIS-
R and QIS-B forms summarize the standards and specifications for all steps in the manufacture
of the radiopharmaceutical from raw materials through intermediates including the kit, to the
final radiopharmaceutical. These forms further provide information on quality testing results for
all lots of the kit and final radiopharmaceutical to date, including any pilot formulation
development batches. Following submission, Health Canada reviews the CTA and will provide
a “No Objection Letter” (NOL) within 30 days if the application is deemed satisfactory. Upon
receipt of the NOL, the trial may proceed. In some cases, Health Canada requests additional
information during the CTA review that must be provided in a timely manner, often within 48 h.
For the 111In-BzDTPA-pertuzumab CTA, the NOL for the PETRA trial was received within the
61
30 day review period on February 14, 2013. There are similar processes for review and approval
of IND applications by the U.S. FDA including a 30-day review period (http://www.fda. gov).
Health Canada and the U.S. FDA both provide an opportunity for pre-CTA/IND meetings to
discuss the requirements for individual agents.
1.8 Hypotheses
The hypotheses of this thesis were:
1) The development of a radiopharmaceutical kit comprised of pertuzumab conjugated to
the DTPA-metal chelator can be labeled with 111In and consistently meet established
quality specifications and will demonstrate favourable preclinical safety properties that
will support the advancement of 111In-labeled pertuzumab to a Phase I/II clinical trial in
humans.
2) PET/CT imaging with pertuzumab F(ab')2 fragments labeled with the positron emitting
isotope 64Cu will specifically localize to HER2-overexpressing tumours and detect
changes in HER2 expression in tumours that are associated with a good response to
treatment with trastuzumab while resulting in a reduced radiation absorbed dose
compared to 111In-labeled pertuzumab.
1.9 Specific aims
To test these hypotheses, the specific aims were:
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1) To design and manufacture several lots of a radiopharmaceutical quality kit composed of
pertuzumab conjugated to DTPA for labeling with 111In and evaluate the kit and final
111In-labeled pertuzumab radiopharmaceutical against established specifications for
quality.
2) To determine the pharmacokinetic, biodistribution and toxicity profile, and radiation
dosimetry of 111In-labeled pertuzumab prepared from the kit in vivo in non-tumour
bearing mice.
3) To evaluate the specificity of HER2 tumour uptake of 64Cu-pertuzumab F(ab')2
fragments and determine if trastuzumab–mediated HER2 downregulation associated
with tumour response could be detected by PET/CT imaging using 64Cu-pertuzumab
F(ab')2 fragments in SK-OV-3 ovarian cancer and BT-474 BC xenografts.
4) To evaluate the pharmacokinetic and biodistribution profile, and radiation dosimetry of
64Cu-pertuzumab F(ab')2 fragments in non-tumour bearing mice for comparison to 111In-
labeled-pertuzumab.
1.10 Thesis organization
The studies addressing the above specific aims are described in Chapters 2-4 of the
thesis. Chapter 2 describes the formulation of a kit to prepare 111In-BzDTPA-pertuzumab and
the results of quality control tests compared to established specifications. The ability of 111In-
BzDTPA-pertuzumab prepared from the kit to specifically image HER2 expression in tumours
63
by SPECT/CT is also shown. Chapter 3 presents the blood pharmacokinetics, normal tissue
biodistribution and acute toxicity profile, and predicts the radiation absorbed doses from 111In-
BzDTPA-pertuzumab in humans based on the biodistribution in non-tumour bearing mice.
Chapter 4 demonstrates that tumour uptake of 64Cu-pertuzumab F(ab')2 fragments is specific to
HER2 and that HER2 downregulation mediated by trastuzumab is detected by PET/CT imaging
using 64Cu-NOTA-pertuzumab F(ab')2. The pharmacokinetics and radiation dosimetry of 64Cu-
NOTA-pertuzumab F(ab')2 in non-tumour bearing mice is also reported. In Chapter 5, the
research findings are summarized and some future research is proposed.
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CHAPTER 2
Kit for the Preparation of 111In-Labeled Pertuzumab Injection for Imaging
Response of HER2-Positive Breast Cancer to Trastuzumab (Herceptin)
65
This chapter represents a reprint of: “Lam K, Scollard DA, Chan C, Levine MN, Reilly RM. Kit
for the Preparation of 111In-Labeled Pertuzumab Injection for Imaging the Response of HER2-
Positive Breast Cancer to Trastuzumab (Herceptin). Appl Radiat Isot. 2014 Oct 23;95C:135-
142.” Reprinted with permission. Copyright 2014 Elsevier Ltd.
All experiments and analyses of data were carried out by Karen Lam. MicroSPECT/CT was
performed with technical assistance from Deborah A. Scollard and Dr. Conrad Chan.
66
2.0 Abstract
We previously reported that 111In-labeled pertuzumab imaged trastuzumab (Herceptin)-mediated
changes in HER2 expression preclinically in breast cancer tumours. To advance 111In-labeled
pertuzumab to a Phase I/II clinical trial, a kit was designed for preparing this agent in a form
suitable for human administration. Unit-dose kits containing pertuzumab modified with 2-(4-
isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (BzDTPA) were prepared that labeled
to high efficiency (>90%) with 111In and met specifications for pharmaceutical quality. The kits
were stable for 4 months and the final radiopharmaceutical was stable for 24 h. Imaging studies
demonstrated high and specific uptake in HER2-positive tumours in mice using this clinical kit
formulation.
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2.1 Introduction
We previously reported that 111In-labeled pertuzumab was a sensitive imaging probe for
detecting response of human epidermal growth factor-2 (HER2)-positive breast cancer (BC)
xenografts in mice to treatment with trastuzumab (Herceptin; Roche) (110). HER2 is
overexpressed in 15-20% of cases of BC and is associated with a poor prognosis and an
aggressive phenotype (26). Trastuzumab, a humanized IgG1 monoclonal antibody (mAb)
combined with chemotherapy has improved the overall survival and disease-free progression of
patients with HER2 positive BC (167-169). Pertuzumab (Perjeta; Roche) is a second-generation
humanized IgG1 HER2 mAb that inhibits receptor dimerization (55). Pertuzumab was recently
approved for treatment of HER2 positive metastatic BC based on the results of the Phase III
CLEOPATRA trial which demonstrated improved survival with the addition of pertuzumab to
trastuzumab and docetaxel (162). The Neosphere trial further demonstrated that pertuzumab
combined with trastuzumab and docetaxel was effective for treatment of early stage BC (64).
Since pertuzumab binds to dimerization domain II of HER2 while trastuzumab binds to domain
IV and there is no interference in the binding of pertuzumab caused by trastuzumab (55), we
reasoned that 111In-labeled pertuzumab could probe decreased HER2 expression in BC caused
by trastuzumab, which is one of its mechanisms of action (85). Such an approach is not possible
with other reported HER2 imaging probes such as 111In, 64Cu or 89Zr-labeled trastuzumab since
these compete with trastuzumab for HER2 binding (90,95,96). Our results revealed that
microSPECT/CT imaging with 111In-labeled pertuzumab detected decreased HER2 expression
caused by trastuzumab as soon as 3 days after commencing treatment in tumour-bearing mice
and prior to any change in tumour size. Imaging at 21 days revealed almost complete loss of
HER2 signal due to tumour eradication (110). Imaging the response of HER2 positive BC to
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trastuzumab is important because ex vivo testing of tumour biopsies for HER2 overexpression
using existing guidelines does not always predict response to treatment (44). Only 1 in 2
patients with metastatic HER2 positive BC responded to treatment with trastuzumab combined
with chemotherapy in a Phase III trial, indicating that many patients had tumours that were
intrinsically resistant (170). Moreover, almost all patients who initially benefit from
trastuzumab-containing regimens acquire resistance within a year (85). 111In-pertuzumab may be
a valuable theranostic probe that could aid in selecting patients for trastuzumab-containing
regimens as well as monitoring their response to treatment.
We recently initiated a Phase I/II clinical trial to investigate imaging with 111In-labeled
pertuzumab for detecting response of patients with metastatic HER2 positive BC to trastuzumab
combined with chemotherapy (PETRA trial; ClinicalTrials.gov identifier NCT01805908). In
order to advance 111In-labeled pertuzumab to this first-in-humans trial, it was necessary to
design and formulate a kit for routine and robust preparation of the radiopharmaceutical under
GMP conditions and establish specifications and quality assays to assure its suitability for
patient administration. In this report, we illustrate the systematic process that we applied in our
radiopharmaceutical research laboratory at the University of Toronto to construct a clinical
quality kit for the preparation of 111In-labeled pertuzumab (111In-BzDTPA-pertuzumab)
injection. The kits were approved by Health Canada as part of a Clinical Trial Application (CTA
#160445). To validate the utility of 111In-BzDTPA-pertuzumab prepared using this kit
formulation, we further studied its ability to image HER2 positive MDA-MB-361 human BC
xenografts in nude mice by microSPECT/CT.
69
2.2 Materials and methods
2.2.1 Raw materials
Sodium bicarbonate USP (NaHCO3) and ammonium acetate (NH4OAc) ACS (C2H7NO2;
≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sterile Water for Irrigation
USP and Sodium Chloride for Irrigation USP were purchased from Baxter (Toronto, ON,
Canada). 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA;
≥94%] was purchased from Macrocyclics, Inc. (Dallas, TX, USA). All other chemicals and
reagents were purchased in analytical ACS grade with a purity >95%. Sterile, apyrogenic Type
1 glass vials (5 or 30 mL) were obtained from Omega Laboratories, Ltd. (Montreal, QC,
Canada). 111InCl3 (>3.7 GBq/mL; <0.1% 114mIn and 65Zn) was purchased from Nordion (Kanata,
ON, Canada). Certificates of actual lot analysis were obtained from the vendors. The identities
of NaHCO3 and NH4OAc were tested by USP methods. Pertuzumab was provided through a
Materials Transfer Agreement by Genentech, Inc. (South San Francisco, CA, USA). The
identity and purity of pertuzumab were determined by size-exclusion high-performance liquid
chromatography (SE-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)/Western blot. Size-exclusion HPLC (SE-HPLC) was conducted on a BioSep
SEC-s4000 column (Phenomenex, Inc., Torrence, CA, USA) eluted with 100 mM sodium
phosphate (NaH2PO4) buffer (pH 7.0) at a flow rate of 0.8 mL/min and fitted with a diode array
detector (PerkinElmer, Woodbridge, ON, Canada) monitoring at 280 nm. SDS-PAGE was
performed on a 4-20% Tris HCl gradient minigel (Bio-Rad Laboratories, Mississauga, ON,
Canada) under non-reducing and reducing [dithiothreitol (DTT)] conditions and stained with
Coomassie R-250 brilliant blue. Western blot was conducted by transferring electrophoresed
proteins onto a polyvinylidene fluoride (PVDF) membrane (Immun-Blot, Bio-Rad, Hercules,
70
CA, USA) and probing with a goat anti-human IgG (Fab specific) HRP immunoconjugates
(Sigma-Aldrich). Diamidobenzidine/0.03% H2O2 (Sigma-Aldrich) was used to detect bands.
The specification for the identity of pertuzumab was: one major band on SDS-PAGE that
corresponds to a protein with Mr of ~170 kDa under non-reducing conditions, and two bands
that correspond to proteins with Mr of ~50 kDa and 25 kDa under reducing conditions. These
bands must be immunopositive for human IgG by Western blot analysis. The specification for
the purity of pertuzumab was: one major peak by SE-HPLC analysis that accounts for >90% of
the total area of all chromatographic peaks. The identity of p-SCN-Bn-DTPA was confirmed by
proton NMR (Varian Mercury 400 MHz).
2.2.2 Pharmaceutical quality buffers
Sterile 100 mM NaHCO3 buffer (pH 8.2) in Sodium Chloride for Irrigation USP was
prepared. Sterile 100 mM NH4OAc buffer (pH 6.0) was prepared by diluting 1 M NH4OAc
buffer (pH 6.0) with Sterile Water for Irrigation USP. Trace metals were removed from buffers
by passage through a 10 mL column of Chelex-100 cation exchange resin (BioRad,
Mississauga, ON, Canada) pre-hydrated for 1 h in Sterile Water for Irrigation USP. 1 N HCl and
glacial acetic acid (Sigma-Aldrich) were used to adjust the pH of NaHCO3 and NH4OAc
buffers, respectively. Buffers were sterilized by filtration through a 0.22-µm Millex-GS filter
(EMD Millipore, Billerica, MA, USA) into 30-mL glass vials and stored at 2-8°C. All buffers
were tested for sterility by the USP Sterility Test. The concentration of NaHCO3 was assayed by
titration with 0.1 N sulfuric acid according to the USP method. The concentration of NH4OAc
was calculated based on the weight incorporated into the solution as no USP assay method was
available. Clarity and color were assessed by holding a vial of buffer against a light and a dark
background.
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2.2.3 Kit formulation
Pertuzumab was diluted with 100 mM NaHCO3 buffer (pH 8.2) and concentrated to <2.0
mL using an Amicon Ultra-15 Centrifugal Filter Device with a nominal 30-kDa cut-off (EMD
Millipore, Billerica, MA, USA). The solution was centrifuged at 5000 × g for 10 min and the
filtrate discarded. Dilution and ultrafiltration were repeated a total of 4 times. The retentate
containing buffer-exchanged pertuzumab was recovered into a sterile polypropylene tube
(Sarstedt, Montreal, QC, Canada). The pertuzumab concentration was measured by its
absorbance at 280 nm and adjusted to 17.5 mg/mL by addition of 0.1 M NaHCO3 buffer (pH
8.2), then transferred to a sterile 10 mL Reacti-Vial (Pierce Chemical Co., Rockford, IL, USA).
Pertuzumab was derivatized with BzDTPA by adding a 10-fold molar excess of a 10 mg/mL
solution of p-SCN-Bn-DTPA in 100 mM NaHCO3 buffer (pH 8.2). The reaction mixture was
vortexed for 10 sec, and allowed to incubate at room temperature for 1.5 h. An aliquot (12 µL)
of the reaction mixture was removed for measurement of BzDTPA conjugation efficiency.
Excess BzDTPA was then separated from BzDTPA-pertuzumab on a sterile PD-10 column (GE
Healthcare Life Sciences) eluted with 100 mM NaHCO3 buffer (pH 8.2). The eluate was
collected as 24 × 0.5 mL fractions into sterile polystyrene tubes (VWR International,
Mississauga, ON, Canada). Tubes containing partially purified BzDTPA-pertuzumab (fractions
3-7) were pooled into an Amicon Ultra-15 Centrifugal Filter Device with a nominal 30-kDa cut-
off and diluted with 100 mM NH4OAc buffer (pH 6.0). The solution was centrifuged at 5000 ×
g for 10 min and the filtrate discarded. A total of 4 dilution and ultrafiltration steps were
performed. The retentate was recovered into a sterile polypropylene tube (Sarstedt). The
concentration of BzDTPA-pertuzumab was determined spectrophotometrically and the solution
diluted to a final concentration of 10 mg/mL by addition of 100 mM NH4OAc buffer (pH 6.0).
72
Finally, the solution was sterilized by passing through a 0.22-µm Millex-GV filter (EMD
Millipore) into a sterile 5-mL glass vial (Omega Laboratories). Aliquots (0.5 mL; 5.0 mg) were
aseptically dispensed into sterile 5-mL glass vials using a 1-mL syringe in a laminar air flow
hood to yield unit-dose kits. Kit vials were stored at 2-8°C.
2.2.4 Kit quality testing
The purity and homogeneity of BzDTPA-pertuzumab were evaluated by SDS-PAGE and
SE-HPLC and the concentration was assayed spectrophotometrically at 280 nm. The pH was
measured using 4.5-7.5 range pH paper (pHydrion, Micro Essential Laboratory, Brooklyn, NY,
USA). Clarity and colour were assessed by examination of a kit vial against a light and dark
background. The volume contained in each vial was measured by weight assuming a density of
1 g/cm3. The BzDTPA substitution level was determined by incubating 0.9 MBq of 111InCl3
(Nordion, Kanata, ON, Canada) with 10-μL of unpurified BzDTPA-pertuzumab reaction
mixture for 30 min. This sample was then analyzed by instant thin-layer chromatography-silica
gel (ITLC-SG; Pall Life Sciences, Ann Arbor, MI, USA) developed in 100 mM sodium citrate
(pH 5.0). 111In-BzDTPA-pertuzumab remains at the origin (Rf = 0.0) while 111In-BzDTPA
migrates to an Rf = 0.6-0.7 and free 111In migrates to the solvent front (Rf = 1.0). The fraction of
radioactivity at the origin (111In-BzDTPA-pertuzumab) was multiplied by the molar ratio used
for conjugation (10:1) to calculate the BzDTPA substitution level.
The affinity of BzDTPA-pertuzumab (labeled with 111In) for binding HER2 was assessed
in a direct (saturation) radioligand binding assay using SK-BR-3 human BC cells (1-2 × 106
HER2/cell) as previously reported (110). Briefly, increasing concentrations of 111In-BzDTPA-
pertuzumab (0.07 to 300 nM) were incubated with 1 × 106 SK-BR-3 cells in 1.5-mL Eppendorf
tubes in phosphate buffered saline (PBS) in the presence [non-specific binding (NSB)] or
73
absence [total binding (TB)] of 61 μM of unlabeled pertuzumab. The tubes were incubated at
4°C for 3.5 h with intermittent shaking. The tubes were centrifuged and the supernatant and cell
pellet separated and measured in a γ-counter. Specific binding (SB) was calculated by
subtracting NSB from TB and plotted against the free concentration of 111In-BzDTPA-
pertuzumab (nmols/L). The curve was fitted to a one-site receptor-binding model using Prism
version 4.0 software (GraphPad, San Diego, CA, USA) and the affinity constant (Ka = 1/Kd) and
maximum number of receptors (Bmax) were estimated. To determine whether the number of
BzDTPA chelators attached to pertuzumab had an effect on the binding of 111In-BzDTPA-
pertuzumab to HER2, the Ka and Bmax were plotted versus BzDTPA substitution level for each
lot of kit produced. The sterility and apyrogenicity of the kits were respectively assessed by the
USP Sterility Test and USP Bacterial Endotoxins Tests (QCL-1000 Endpoint Chromogenic
LAL Assay, Lonza, Walkersville, MD, USA).
The labeling efficiency of the kits was determined by adding 111InCl3 to achieve a
specific activity of 22-30 MBq/mg, incubating for 30 min at room temperature, and then
determining the percentage of 111In-BzDTPA-pertuzumab by ITLC-SG. The labeling efficiency
of the kits using 111InCl3 that was produced up to 7 days prior to use was also determined in
order to test if in-growth of the decay product of 111In (stable 111Cd) affected the labeling
efficiency. The stability of the kits stored at 2-8°C was evaluated by repeating selected key
quality control tests at monthly intervals up to 4 months.
2.2.5 111In-BzDTPA-pertuzumab injection
111In-BzDTPA-pertuzumab was prepared by aseptically decapping a kit vial in a laminar
air flow hood and adding 111InCl3 to achieve a specific activity of 22-30 MBq/mg (110-150
MBq/kit). Decapping of the vial was required since the volume of 111InCl3 solution was small
74
due to the high concentration (>3.7 GBq/mL) requiring use of a micropipette and sterile tip.
Following an incubation period of 30 min at room temperature, Sodium Chloride Injection USP
was added to a final volume of 2.0 mL. The radiopharmaceutical was drawn up into a lead
glass-shielded 3 mL syringe with attached needle and sterilized by filtration through a 0.22-μm
Millex-GV filter into a 5-mL glass vial. The amount of radioactivity dispensed into the vial was
measured using a dose calibrator (Capintec Model CRC-15R, Ramsey, NJ, USA). The
radiochemical purity (RCP) was evaluated by ITLC-SG developed in 100 mM sodium citrate
(pH 5.0) and by SE-HPLC using a flow scintillation analyzer (FSA) (PerkinElmer Model
Radiomatic 610TR). The pH was measured using pH paper. Selected lots were randomly
selected for testing for endotoxins and sterility. The stability of 111In-BzDTPA-pertuzumab
injection at 4°C was assessed by performing the RCP analysis up to 24 h after preparation.
2.2.6 Stability in plasma
Transchelation of 111In from 111In-BzDTPA-pertuzumab to transferrin in human plasma
in vitro was determined by SE-HPLC. 111In-BzDTPA-pertuzumab was diluted in human plasma
(Sigma-Aldrich) to a radioactivity concentration of 18.5 MBq/mL. A negative control consisted
of 111In-BzDTPA-pertuzumab diluted with 100 mM NaHCO3 buffer pH 8.2 to 18.5 MBq/mL.
The samples were incubated at 37°C for up to 5 d. Aliquots of 17 µL of each sample were
analyzed in duplicate by SE-HPLC daily using a BioSep SEC-s4000 column (Phenomenex, Inc.,
Torrence, CA, USA) eluted with sodium phosphate buffer (100 mmol/L; pH 7.0) at a flow rate
of 0.8 mL/min. Peaks were monitored by use of a FSA detector (PerkinElmer Model Radiomatic
610TR).
75
2.2.7 Imaging and biodistribution studies
Female athymic CD1 nu/nu mice (Charles River Laboratories, Wilmington, MA, USA)
were implanted with a 0.72 mg, 60-d sustained release 17β-estradiol pellet (Innovative Research
of America, Sarasota, FL, USA). Mice were inoculated subcutaneously (s.c.) 24 h later with 1.1
× 107 (0.1 ml) HER2 positive MDA-MB-361 human BC cells [5.4 ± 0.7 × 104 receptors/cell
(171)] suspended in serum free Leibovitz’s L-15 medium mixed with 0.1 mL Matrigel (BD
Biosciences, Mississauga, ON, Canada). After 8 weeks, one group of tumour-bearing mice
(n=4) was injected i.v. (tail vein) with 111In-BzDTPA-pertuzumab (37 MBq; 13.5 µg) prepared
using the kit formulation for combined imaging and biodistribution studies. A second group of
tumour-bearing mice was injected with 111In-BzDTPA-pertuzumab (5 MBq; 13.5 µg) for
biodistribution studies only. Some mice received 1 mg of unlabeled pertuzumab 24 h before
injection of 111In-BzDTPA-pertuzumab to block HER2 in order to assess the specificity of
tumour uptake. MicroSPECT/CT imaging was performed at 72 h p.i. of 111In-BzDTPA-
pertuzumab using a NanoSPECT/CT tomograph (Bioscan, Washington, DC, USA) equipped
with 4 NaI scintillation detectors fitted with 1.4-mm multi-pinhole collimators [full-width half-
maximum (FWHM) ≤ 1.2 mm]. A total of 24 projections were acquired in a 256 × 256 matrix
with a minimum of 70,000 counts per projection. Anesthesia was induced and maintained by
inhalation of 2% isoflurane in O2. Cone-beam CT images were obtained (180 projections, 1
s/projection, 45 kVp) prior to the microSPECT images. Coregistration of microSPECT and CT
images was achieved using InvivoScope software (Bioscan). Immediately following
microSPECT/CT imaging, mice were sacrificed, tumour, blood and samples of normal tissues
were collected, weighed and their radioactivity was measured in a γ-counter. Tumour and
normal-tissue uptake were expressed as percent injected dose per gram (%ID/g) and as tumour-
76
to-normal tissue (T/NT) ratios. Animal studies were conducted in accordance with Canadian
Council on Animal Care (CCAC) guidelines under a protocol (No. 989.15) approved by the
Animal Care Committee at the University Health Network.
2.2.8 Statistical analysis
Statistical analysis was performed using Student’s t-test (P<0.05) for comparisons and
with Pearson’s correlation coefficient for associations. All analyses were performed on SPSS
version 17.0 (IBM, Armonk, NY, USA).
2.3 Results
2.3.1 Raw materials and pharmaceutical quality buffers
SE-HPLC analysis of pertuzumab using UV detection demonstrated one major peak with
a retention time (tR) of 11.5 min that accounted for >98% of all chromatographic peaks (Figure
2.1). SDS-PAGE analysis of pertuzumab under non-reducing conditions displayed one major
band corresponding to a protein with a Mr of ~170 kDa (Figure 2.2, lane 1). This band was
positive by Western blot when probed with a goat anti-human Fab-specific antibody (results not
shown). When pertuzumab was analysed under reducing conditions, two major bands
corresponding to proteins with Mr of ~50 kDa and ~25 kDa were found, representing the IgG
heavy and light chains, respectively (Figure 2.2, lane 3). Based on these results, pertuzumab raw
material met the specifications established for identity and purity. Proton NMR (500 MHz)
spectra confirmed the identity of p-SCN-Bn-DTPA and was in agreement with the reported 1H
spectrum (172).
Seven lots of 100 mM NaHCO3 buffer (pH 8.2) were prepared and all passed
specifications for concentration (95-105 mM), and pH (8.18 – 8.22). Four lots of 100 mM
NH4OAc buffer (pH 6.0) were prepared but this buffer was not assayed for NH4OAc due to the
77
unavailability of a USP assay. The pH was within specifications (5.58 – 6.02). All lots of buffers
were clear, particulate-free and colorless and passed the USP Sterility Test.
2.3.2 Kit formulation
Nine lots of kits for the preparation of 111In-BzDTPA-pertuzumab injection were
prepared and all passed quality specifications for protein concentration, volume, pH, clarity and
color, sterility, apyrogenicity, BzDTPA substitution level, purity and homogeneity, labeling
efficiency with 111In and HER2 binding (Table 2.1). SDS-PAGE analysis under non-reducing
conditions revealed one major band corresponding to a protein with an approximate Mr of 150
kDa (Figure 2.2, lane 2), representing BzDTPA-pertuzumab which dissociated into the heavy
and light IgG chains under reducing conditions (Figure 2.2, lane 4). SE-HPLC analysis showed
a single peak with a tR of 11.5 min that corresponded to the tR expected for BzDTPA-
pertuzumab and there were no apparent impurities (Figure 2.1). An average substitution level of
5.0 ± 1.0 moles of BzDTPA per mole of pertuzumab was achieved (Table 2.1). All lots of kits
met the specification for labeling efficiency with 111In (>90%; Table 2.1). The labeling
efficiency of the kits was not affected by the in-growth of stable 111Cd in the 111InCl3 solution.
The mean RCP of the kits with 111InCl3 that was 7 d post-production was 98.0 ± 1.0% (n=4).
The mean Ka and Bmax values for binding to HER2 were 2.3 ± 1.8 × 108 L/mol and 1.2 ± 0.5 ×
106 sites per cell, respectively (Figure 2.3 and Table 2.1). Pearson correlation analysis (Figure
2.4) revealed no significant correlation between BzDTPA substitution level and Ka (r = 0.124, P
= 0.751) or Bmax (r = 0.144, P = 0.711). Each manufacturing campaign yielded five to six kits.
Three of the 4 lots manufactured were tested for stability and these met specifications for
selected key quality assays for up to 4 months (Table 2.2). No changes in quality parameters
were observed with respect to pH, color or clarity, SDS-PAGE or HPLC profiles, protein
78
concentration, or HER2 immunoreactivity during this period. The mean labeling efficiency for
the 9 lots immediately after production was 95.8 ± 2.7%. The three lots of kits that underwent
stability testing had a mean labeling efficiency of 97.1 ± 0.4% (P = 0.491) at 4 months post-
manufacture.
79
Figure 2.1. Size-exclusion HPLC (SE-HPLC) analysis of BzDTPA-pertuzumab (red line; tR =
11.5 min) and pertuzumab (grey line; tR = 11.6 min) using ultraviolet (UV) detection at 280 nm.
111In-BzDTPA-pertuzumab (blue line; tR = 12.7 min) was monitored with a flow scintillation
analyser (FSA) radioactivity detector. The UV detector and FSA detector are aligned in
sequence and a delay between the UV and radioactivity signals results in a 1.2 min difference in
tR.
80
Figure 2.2. SDS-PAGE analysis of pertuzumab and BzDTPA-pertuzumab under reducing
conditions (lanes 1 and 2, respectively) or non-reducing conditions (lanes 3 and 4, respectively)
on a 4-20% Tris HCl gradient minigel. MW = molecular weight markers. Gels were stained with
Coomassie R-250 Brilliant Blue. The amount of protein loaded was 10 µg.
81
Figure 2.3. Direct (saturation) receptor-binding curve for the binding of 111In-BzDTPA-
pertuzumab (prepared from kit lot 11R015) to SK-BR-3 human breast cancer cells showing total
binding (TB), non-specific binding (NSB) in the presence of an excess of pertuzumab and
specific binding (SB) obtained by subtraction of NSB from TB. The Ka and Bmax values derived
from the specific binding (SB) curves were 1.5 × 108 L/mol and 1.9 × 106 receptors/cell,
respectively.
82
Figure 2.4. Relationship between BzDTPA substitution level and binding affinity (Ka) of 111In-
BzDTPA-pertuzumab for HER2 (A) or the maximum number of receptors (Bmax) of HER2-
positive SK-BR-3 human breast cancer cells (B).
83
Table 2.1
Quality testing of kits for the preparation of 111In-BzDTPA-pertuzumab
injection
Test Parameter Specification Resultsa
Protein Concentration 9.5 – 10.5 mg/ml 10.1 ± 0.3
mg/ml
Volume 0.45 – 0.55 ml 0.50 ± 0.01 ml
pH 5.5 – 6.5 6.0 ± 0.2
Appearance Clear, colorless, particulate-free Pass
Sterility Passes USP XXXIV Test Pass
Endotoxins Passes USP XXXIV Test Pass
BzDTPA 2 – 7 BzDTPA/pertuzumab 5.0 ± 1.0
SDS-PAGE Major band at Mr=170 kDa under
non-reducing conditions; Bands at
Mr=25 and 50 kDa under reducing
conditions
Pass
SE-HPLC 1 major peak at tR = ± 0.2 mins
of tR for pertuzumab peak
Pass
HER2 Binding Ka = 0.3 – 7.0 × 108 L/mol
Bmax = 0.3 – 2.5 × 106 sites/cell
Ka = 2.3 ± 1.8 ×
108 L/mol
Bmax = 1.2 ± 0.5
× 106 sites/cell
Labeling Efficiency ≥ 90% 95.8 ± 2.7%
84
a Results of analysis of 9 separate lots of kits. Numerical values shown represent
mean ± S.D.
85
Table 2.2
Stability testing at 4 months post-manufacturing of kits for the preparation of 111In-
BzDTPA-pertuzumab injection for key quality parameters
Test Parameter Specification Resultsa
Protein Concentration 9.5 – 10.5 mg/ml 10.2 ± 0.2 mg/ml
pH 5.5 – 6.5 6.0 ± 0.0
Appearance Clear, colorless, particulate-free Pass
Sterility Passes USP XXXIV Test Pass
SDS-PAGE Major band at Mr=170 kDa under
non-reducing conditions; Bands at
Mr=25 and 50 kDa under reducing
conditions
Pass
SE-HPLC 1 major peak at tR = ± 0.2 mins
of tR for pertuzumab peak
Pass
HER2 Binding Ka = 0.3 – 7.0 × 108 L/mol
Bmax = 0.3 – 2.5 × 106 sites/cell
Ka = 1.9 ± 0.9 × 108 L/mol
Bmax = 1.3 ± 0.3 × 106
sites/cell
Labeling Efficiency ≥ 90% 97.1 ± 0.4%
a Results of analysis of 3 separate lots of kits. Numerical values shown
represent mean ± S.D.
86
2.3.3 111In-BzDTPA-pertuzumab injection
Seventeen lots of 111In-BzDTPA-pertuzumab injection were prepared (Table 2.3). Two
lots had specific activities outside the specified range due to over or under addition of 111InCl3.
All other lots met specifications for specific activity, pH, radiochemical purity, radionuclidic
purity, clarity and colour, and sterility. Three lots of 111In-BzDTPA-pertuzumab were selected
for testing for bacterial endotoxins and met USP standards. SE-HPLC using a FSA detector
showed one major peak at tR = 12.7 min with no apparent radiochemical impurities (Figure 2.1).
The FSA detector is in sequence with the UV diode array detector, and there is a delay of 1.2
min between the two signals being recorded using an eluate flow rate of 0.8 mL/min. In
addition, the flow cell for the FSA detector is larger than for the UV detector, which results in
peak broadening for the radioactivity signal compared to the UV signal. The SE-HPLC results
agreed with the ITLC-SG analyses for RCP. All lots tested maintained RCP over 24 h when
stored at 2-8°C with a mean RCP of 96.3 ± 1.1% at the time of preparation and 96.5 ± 0.8% at
24 h post-labeling (n = 3, P = 0.347). The expiry was set to 8 h since patients were expected to
be injected with 111In-BzDTPA-pertuzumab within this time period.
87
Table 2.3
Quality testing of 111In-BzDTPA-pertuzumab injection
Test Specification Results a
Specific radioactivity 22 – 30 MBq/mg 25.6 ± 1.7 MBq/mgb
pH 5.5 – 6.5 6.0 ± 0.1
Radiochemical purity ≥ 90% 96.4 ± 1.7%
Radionuclidic purity > 99.9% (<0.1% 114mIn or 65Zn) Pass
Appearance Clear, colorless, particulate-free Pass
Sterility (retrospective) Passes USP XXXIV Test Pass
a Results of analysis of 17 separate lots of the radiopharmaceutical. Numerical values represent
mean ± S.D.
b Two lots contained a specific radioactivity that was outside the specified range and were not
included in the mean value (n = 15).
88
2.3.4 Stability in plasma
SE-HPLC was used to evaluate the radiochemical stability of 111In-BzDTPA-pertuzumab
in human plasma or in 0.1 M sodium bicarbonate buffer pH 8.2 up to 5 d at 37°C. 111In
remained bound to BzDTPA-pertuzumab over this time period, with no loss observed to
transferrin in plasma, and minimal release of 111In in sodium bicarbonate buffer pH 8.2 (Figure
2.5).
89
Figure 2.5. In vitro stability of 111In-BzDTPA-pertuzumab in human plasma and 0.1 M sodium
bicarbonate buffer pH 8.2 at 37°C as determined by SE-HPLC over a 5-day period. The data are
normalized to day 0.
90
2.3.5 Imaging and biodistribution studies
To study the tumour and normal tissue localization properties of 111In-BzDTPA-
pertuzumab prepared using the kit formulation, microSPECT/CT images were obtained at 72 h
p.i. in CD1-nude mice bearing s.c. HER2 positive MDA-MB-361 human BC xenografts. The
images revealed intense tumour uptake with only modest uptake in normal organs (Figure
2.6A). Pre-injection of a 100-fold excess of unlabeled pertuzumab (1 mg) at 24 h before 111In-
BzDTPA-pertuzumab significantly reduced tumour radioactivity (Figure 2.6B), demonstrating
that uptake of 111In-BzDTPA-pertuzumab was HER2 specific. The tumour and normal-tissue
uptake of 111In-BzDTPA-pertuzumab and T/NT ratios at 72 h after injection were quantified ex
vivo by γ-counting (Table 2.4). In agreement with the microSPECT/CT images, the tumour
uptake was high (34.5 ± 18.4 %ID/g) and was significantly decreased by 5-fold (6.6 ± 1.0
%ID/g; P < 0.05) by pre-administration of unlabeled pertuzumab. The highest normal tissue
uptake of radioactivity was found in the spleen, blood and liver (5.9 ± 5.3, 5.9 ± 4.1 and 5.0 ±
1.2 %ID/g, respectively). T/NT ratios were highest for muscle (41.2 ± 20.5) and lowest for liver
(7.6 ± 4.7).
91
Figure 2.6. Posterior whole-body microSPECT/CT images of athymic mice implanted s.c. with
MDA-MB-361 human breast cancer xenografts (white arrow) at 72 h p.i. of 111In-BzDTPA-
pertuzumab (37 MBq; 13.5 µg) without (A) or with (B) preinjection of 1 mg of unlabeled
pertuzumab. Tumour uptake was decreased by 5-fold by pre-blocking with pertuzumab. Images
were adjusted independently to most clearly visualize the tumour and normal tissues in each
panel.
92
Table 2.4
Tumour and normal-tissue distribution of radioactivity in CD1 nude mice implanted
subcutaneously with MDA-MB-361 human breast cancer xenografts at 72 h p.i. of 111In-
BzDTPA-pertuzumab
Not Blockeda Blockeda
Organ %ID/g T/NT %ID/g T/NT
Blood 5.9 ± 4.1 11.9 ± 12.4 8.0 ± 1.3 0.8 ± 0.2
Heart 1.8 ± 0.9 19.1 ± 3.9b 1.7 ± 0.2 3.9 ± 0.6
Lungs 3.6 ± 2.4 11.0 ± 3.3b 3.9 ± 0.8 1.7 ± 0.3
Liver 5.0 ± 1.2 7.6 ± 4.7 3.7 ± 0.3 1.8 ± 0.2
Spleen 5.9 ± 5.3 10.3 ± 7.3 3.3 ± 1.6 2.3 ± 1.0
Kidneys 3.6 ± 0.8 9.0 ± 4.0b 3.7 ± 0.5 1.8 ± 0.3
Small Intestine 1.2 ± 0.4 32.0 ± 20.1 1.0 ± 0.2 7.1 ± 1.5
Large Intestine 1.1 ± 0.2 31.5 ± 18.4b 1.0 ± 0.3 7.2 ± 1.8
Muscle 0.9 ± 0.4 41.2 ± 20.5 0.7 ± 0.2 10.1 ± 1.9
Tumour 34.5 ± 18.4b 6.6 ± 0.5
a Groups of 4 tumour-bearing mice were intravenously administered 111In-BzDTPA-pertuzumab
(13.5 µg) without (not blocked) or with (blocked) pre-injection of 1 mg of unlabeled
pertuzumab. Values are expressed as mean ± SD.
b Statistically significant difference (P<0.05) compared to blocked group.
93
2.4 Discussion
In this report, we describe for the first time a kit formulation for the preparation of 111In-
labeled pertuzumab injection (111In-BzDTPA-pertuzumab), a novel imaging agent for detecting
response of BC to treatment with trastuzumab (110). These kits were approved by Health
Canada as part of a Clinical Trial Application for a Phase I/II trial 111In-BzDTPA-pertuzumab in
patients with metastatic HER2 positive BC (PETRA trial; ClinicalTrials.gov identifier
NCT01805908). We describe a systematic approach to the manufacture of these kits prepared
under GMP conditions in an academic research environment that assures the quality of all raw
materials, intermediates and finished product. Kit formulations are ideal for preparing mAb-
based theranostics labeled with relatively short-lived radiometals such as 111In since key quality
parameters including immunoreactivity and sterility and apyrogenicity can be tested prior to
preparation of the final radiolabeled product and they routinely provide high labeling efficiency
(>90%) without the need for further purification. We have previously designed analogous kits
for 111In-labeling of human epidermal growth factor (hEGF) (155) or trastuzumab Fab fragments
(156).
An important consideration in designing the kit formulation for 111In-BzDTPA-
pertuzumab was to select the dose of radioactivity and mass of pertuzumab required for tumour
imaging in patients. Previous imaging studies with other 111In-labeled mAbs have used a
radioactivity dose of 150-185 MBq and protein mass doses ranging from 10 to 100 mg
(90,152,153). However, multiple administrations of 111In-BzDTPA-pertuzumab injection were
planned for the PETRA trial in order to monitor response to trastuzumab treatment. Thus,
patients receive a baseline image prior to treatment with trastuzumab and chemotherapy, then
follow-up images at one week and at 4 weeks to evaluate changes in tumour HER2 expression
94
probed by 111In-BzDTPA-pertuzumab. Each imaging study requires a separate administration of
111In-BzDTPA-pertuzumab. Preclinical radiation dosimetry studies (173) predicted that 111In-
BzDTPA-pertuzumab would deliver a whole body radiation absorbed dose of 0.05 mSv/MBq.
In order to minimize the radiation absorbed dose, the administered dose of 111In-BzDTPA-
pertuzumab was set to 111 MBq. This was predicted to deliver a total whole body dose of 17
mSv for three administrations which is lower than the dose (27 mSv) from a single
administration of 111In-capromab pendetide (ProstaScint®), a radiolabeled mAb used clinically
to image prostate cancer (76). The mass dose was set to 5 mg. However, Dijkers et al. found that
a 10 mg mass dose (37 MBq) of 89Zr-labeled trastuzumab was suboptimal for imaging HER2
positive tumours in BC patients by positron-emission tomography (PET) due to hepatic
clearance, but a 50 mg dose provided good tumour visualization (92). 111In-BzDTPA-
pertuzumab has not been previously administered to humans, therefore it is not known if these
higher mass doses are required. To study these mass effects, increasing doses (5, 15 or 50 mg)
will be studied in the PETRA trial by supplementing 111In-BzDTPA-pertuzumab (111 MBq; 5
mg) with unlabeled pertuzumab.
Pertuzumab was conjugated to p-SCN-Bn-DTPA, a bifunctional chelator for 111In that
provides a more stable radiometal complex than DTPA dianhydride which was used previously
(110) and does not cause cross-linking since it has only one functional group for conjugation to
antibodies. SE-HPLC (Figure 2.1) and SDS-PAGE (Figure 2.2) analyses of 111In-BzDTPA-
pertuzumab confirmed no cross-linking of pertuzumab. The stability of the
radioimmunoconjugate was confirmed by the absence of transchelation of 111In from 111In-
BzDTPA-pertuzumab to transferrin in human plasma (Figure 2.5). There were 5.0 ± 1.0 moles
of BzDTPA substituted per mole of pertuzumab. This chelate to protein substitution level
95
increased the Kd by 4-fold (Figure 2.3) compared to 111In-labeled pertuzumab modified with
DTPA dianhydride or previously reported for unmodified pertuzumab (Kd = 8.3 ± 6.5 nM vs.
2.0 ± 1.0 nM and 1.8 ± 1.1 nM, respectively) (103,110). Modest decreases (<10-fold) in
immunoreactivity have not been associated with decreased tumour localization in mouse
xenograft models for other radiolabeled mAbs (174). Interestingly, in the manufactured kit lots,
up to 7 BzDTPA moieties were conjugated to pertuzumab with no apparent trend towards
decreased binding affinity (Kd) or the maximum number of HER2 recognized on SK-BR-3 cells
(Figure 2.4). Although there are 9 lysines within the variable heavy (VH) and variable light (VL)
chains of pertuzumab that present ε-amino groups for reaction with p-SCN-Bn-DTPA, only two
of these lysines are in the complementarity determining regions (CDRs) (55). Since there are
about 80 lysine residues in total in an IgG molecule (175), this suggests that pertuzumab may be
modified with DTPA mostly outside the CDR which preserves its immunoreactivity. Indeed
microSPECT/CT imaging and biodistribution studies showed high (>34% i.d./g) tumour uptake
and high tumour/blood ratios (12:1) at 72 h p.i. of 111In-BzDTPA-pertuzumab prepared using
the kit formulation, in mice engrafted s.c. with HER2 positive MDA-MB-361 human BC
xenografts (Table 2.4 and Figure 2.6). Tumour uptake was HER2 specific since it was decreased
by 5-fold by pre-administration of an excess of pertuzumab. Despite the higher Kd values for
111In-labeled pertuzumab modified with DTPA dianhydride, tumour uptake in mice with MDA-
MB-361 xenografts was identical (34% i.d./g) (110). The tumour uptake of 111In-BzDTPA-
pertuzumab was also comparable to that reported for 177Lu-labeled pertuzumab (20% i.d./g) at
72 h p.i. in mice with HER2 positive SK-OV-3 human ovarian cancer xenografts (103).
The kits reproducibly provided very high labeling efficiency with 111In (>95%) measured
by both ITLC or SE-HPLC. Labeling with 111In that had been produced 7 d prior to its use did
96
not affect the labeling efficiency of the kits despite having a higher proportion of stable 111Cd
decay product than freshly produced 111In to compete for chelation sites. This may be
attributable to the relatively low specific activity used (22 MBq/mg). ITLC was selected for
radiochemical purity testing of the final 111In-BzDTPA-pertuzumab injection since it is more
convenient and rapid to use on the day of patient administration. The kits met specifications for
at least 4 months stored at 2-8 °C and the 111In-BzDTPA-pertuzumab was stable for at least 24 h
at 2-8 °C, but the expiry was set to 8 h since patients are expected to receive this agent in this
time period. Previous kits for labeling EGF or trastuzumab Fab fragments with 111In have
demonstrated similar stabilities (155,156).
2.5 Conclusions
A kit for the preparation of 111In-BzDTPA-pertuzumab injection suitable for patient
administration for imaging the response of HER2 positive BC to treatment with trastuzumab
was formulated. Quality parameters and assays were established to assure the suitability of the
kits for preparation of this agent for a first-in-humans Phase I/II clinical trial in BC patients
(PETRA trial). The kits were approved by Health Canada. MicroSPECT/CT imaging studies in
mice engrafted with HER2 positive BC xenografts demonstrated high and specific tumour
uptake, confirming the excellent tumour imaging properties of this clinical kit formulation of
111In-BzDTPA-pertuzumab.
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CHAPTER 3:
Preclinical Pharmacokinetics, Biodistribution, Radiation Dosimetry and
Acute Toxicity Studies Required for Regulatory Approval of a Clinical Trial
Application for a Phase I/II Clinical Trial of 111In-BzDTPA-Pertuzumab
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This chapter represents a reprint of: “Lam K, Chan C, Done SJ, Levine MN, Reilly RM.
Preclinical pharmacokinetics, biodistribution, radiation dosimetry and acute toxicity studies
required for regulatory approval of a Clinical Trial Application for a Phase I/II clinical trial of
111In-BzDTPA-pertuzumab. Nucl Med Biol. 2015 Feb;42(2):78-84.” Copyright 2014 Elsevier
Inc.
All experiments and analyses of data were carried out by Karen Lam except for the
histopathological analysis of excised tissues performed by Dr. Susan J. Done.
99
3.0 Abstract
Introduction: 111In-BzDTPA-pertuzumab is a novel imaging probe for detecting changes in
HER2 expression in BC caused by treatment with trastuzumab (Herceptin). Our aim was to
evaluate the pharmacokinetics, normal tissue biodistribution, radiation dosimetry and acute
toxicity of 111In-BzDTPA-pertuzumab in non-tumour bearing mice in order to obtain regulatory
approval to advance this agent to a first-in-humans Phase I/II clinical trial.
Methods: Biodistribution and pharmacokinetic studies were performed in non-tumour bearing
Balb/c mice injected i.v. with 111In-BzDTPA-pertuzumab (2.5 MBq; 2 μg). The cumulative
number of disintegrations per source organ derived from the biodistribution data was used to
predict the radiation absorbed doses in humans using OLINDA/EXM software. Acute toxicity
was studied at two weeks post-injection of 111In-BzDTPA-pertuzumab (1.0 MBq, 20 μg) with
comparison to control mice injected with unlabeled BzDTPA-pertuzumab (20 μg) or Sodium
Chloride Injection USP. The dose of 111In-BzDTPA-pertuzumab corresponded to 23-times the
human radioactivity dose and 10-times the protein dose on a MBq/kg and mg/kg basis,
respectively. Toxicity was assessed by monitoring body mass, complete blood cell count (CBC),
hematocrit (Hct), hemoglobin (Hb), serum creatinine (SCr) and alanine aminotransferease
(ALT) and by histopathological examination of tissues at necropsy.
Results: 111In-BzDTPA-pertuzumab exhibited a biphasic elimination from the blood with a
distribution half-life (t1/2α) of 3.8 h and an elimination half-life (t1/2β) of 228.2 h. The
radiopharmaceutical was distributed mainly in the blood, heart, lungs, liver, kidneys and spleen.
The projected whole-body radiation absorbed dose in humans was 0.05 mSv/MBq
100
corresponding to a total of 16.8 mSv for three separate administrations of 111In-BzDTPA-
pertuzumab (111 MBq) planned for the Phase I/II trial. There were slight changes in Hb and SCr
levels associated with administration of multiples of the human dose in healthy Balb/c mice but
no histopathological abnormalities were noted in any tissues. There were no significant
differences in body mass between mice injected with 111In-BzDTPA-pertuzumab or control
mice.
Conclusion: Preclinical studies predicted that 111In-BzDTPA-pertuzumab is safe to administer
to humans at a dose of 111 MBq (5 mg). The radiopharmaceutical exhibited preclinical
pharmacokinetic, biodistribution and radiation dosimetry properties suitable for advancement to
a first-in-humans clinical trial.
Advances in knowledge and implications for patient care: The results of these studies supported
the regulatory approval by Health Canada of 111In-BzDTPA-pertuzumab for a Phase I/II clinical
trial of imaging the response of patients with metastatic BC to treatment with trastuzumab
combined with chemotherapy (PETRA trial; ClinicalTrials.gov identifier: NCT01805908).
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3.1 Introduction
Breast cancer (BC) patients are selected for treatment with the human epidermal growth
factor receptor 2 monoclonal antibody, trastuzumab (Herceptin) based on tumour HER2 status
assessed by immunohistochemical staining (IHC) for HER2 protein expression, or by in situ
hybridization (ISH) for HER2 gene copy number (44). Tumours are HER2-positive if there is
intense (3 +) IHC staining and/or a dual-probe HER2/CEP17 gene copy ratio ≥ 2.0 or a single
HER2 gene copy number of ≥ 6.0 signals/cell (44). While tumour HER2 status is valuable to
identify patients who would likely respond to trastuzumab, the pivotal Phase 3 trial of
trastuzumab combined with chemotherapy reported in 2001 (11) revealed that only 1 in 2
patients with HER2-positive BC benefited from this treatment. Moreover, it is recognized that
almost all BC patients who initially respond to trastuzumab-containing regimens acquire
resistance within a year (85). Molecular imaging could provide a useful theranostic tool to
monitor the effectiveness of trastuzumab in individual patients and identify early in the course
of treatment those patients who require alternative treatments due to tumour resistance (88). One
of the proposed mechanisms of action of trastuzumab is to promote HER2 internalization, thus
decreasing the surface HER2 density on tumour cells (176). Thus, probing these changes in
tumours by molecular imaging could be a promising strategy to distinguish responders from
non-responders.
Pertuzumab is a humanized IgG1 monoclonal antibody which binds to domain II on
HER2, interfering with receptor dimerization, whereas trastuzumab binds to domain IV (55).
There is no interference in the binding of pertuzumab to HER2 caused by trastuzumab, and
pertuzumab has been combined in clinical trials with trastuzumab for treatment of HER2-
positive BC yielding improved patient outcome (64,162). We previously reported that
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microSPECT/CT imaging with 111In-labeled pertuzumab detected trastuzumab-mediated
decreased HER2 expression in s.c. human BC xenografts in mice with changes in the tumour
uptake of this probe detected as early as 3 days after initiating trastuzumab treatment and prior
to any changes in tumour size (110). Furthermore, images at 21 days revealed dramatically
reduced tumour uptake of 111In-labeled pertuzumab which corresponded to almost complete
tumour eradication by trastuzumab. 111In-labeled pertuzumab may provide a novel theranostic
imaging probe for monitoring the response of BC patients to trastuzumab-containing regimens,
particularly since there is no interference in the binding of 111In-labeled pertuzumab to HER2 on
BC cells caused by trastuzumab treatment (110). This prompted us to design a Phase I/II clinical
trial (PETRA trial; ClinicalTrials.gov identifier NCT01805908) of SPECT/CT imaging with
111In-labeled pertuzumab to detect changes in tumour HER2 expression in patients with
metastatic BC treated with trastuzumab combined with chemotherapy. In this trial, patients will
receive a baseline image (pre-therapy) and follow-up images at one week and at 4 weeks post-
initiation of treatment, with changes in the tumour uptake of 111In-labeled pertuzumab correlated
with response to treatment at a 3 month endpoint. In order to advance 111In-labeled pertuzumab
from preclinical studies to this first-in-humans clinical trial, we formulated a unit dose kit for the
preparation of the radiopharmaceutical under Good Manufacturing Practices (GMP) which will
be reported separately. In the current article, we report the results of preclinical studies to assess
the pharmacokinetics, normal tissue distribution and radiation dosimetry of 111In-BzDTPA-
pertuzumab, as well as its acute toxicity administered to non-tumour bearing mice at multiples
of the proposed human dose for the PETRA trial. These studies which were required for
regulatory approval of this new radiopharmaceutical were included in a Clinical Trial
Application (CTA) that was approved by Health Canada (#160445). Since these studies were
103
conducted within our academic radiopharmaceutical research laboratory at the University of
Toronto, we believe that this report is instructive for imaging scientists interested to advance
novel theranostic imaging probes from preclinical studies to early phase clinical trials.
3.2 Materials and methods
3.2.1 Radiopharmaceutical preparation
111In-labeled pertuzumab (111In-BzDTPA-pertuzumab) was prepared from a kit
containing 5.0 mg (10 mg/mL) of pertuzumab (Genentech, Inc., South San Francisco, CA,
USA) modified with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-
BzDTPA). The radiopharmaceutical was sterilized by filtration through a 0.22-μm Millex-GV
filter. The radiochemical purity of 111In-BzDTPA-pertuzumab was ˃95% assessed by instant
thin-layer chromatography-silica gel (ITLC-SG; Pall Life Sciences, Ann Arbor, MI, USA)
developed in 100 mM sodium citrate buffer, pH 5.0. All kits used to prepare 111In-BzDTPA-
pertuzumab met specifications for protein concentration, volume, pH, clarity and colour,
sterility and apyrogenicity, BzDTPA substitution level, purity, HER2 immunoreactivity and
labeling efficiency (˃90%) with 111In.
3.2.2 Pharmacokinetic and biodistribution studies
Groups of 4 female Balb/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were
injected i.v. (tail vein) with 2.5 MBq (2 μg) of 111In-BzDTPA-pertuzumab. Mice were
euthanized at selected times up to 166 h after injection by cervical dislocation under anesthesia
with isoflurane in O2 and blood was immediately collected by cardiac puncture. For phar-
macokinetic studies, 50 μL of blood from each mouse was transferred to γ-counting tubes and
the radioactivity measured in a γ-counter (Wizard Model 1480, Perkin Elmer, Woodbridge, ON,
Canada), along with a standard of the injected dose. Blood concentrations of radioactivity were
104
calculated as the percent injected dose per mL (%ID/mL) and were plotted vs. the time p.i. The
resulting elimination curve was fitted to a two-compartment pharmacokinetic model using
Scientist® Ver. 2.01 software (MicroMath Scientific Software, Saint Louis, MO, USA).
Standard pharmacokinetic parameters were estimated. For biodistribution studies, tissues
including the blood were collected at the time of sacrifice, weighed, and counted in a γ-counter
along with a standard of the injected dose. The mean body weight of the mice was 17.7 ± 0.8 g
(n = 32). Tissue radioactivity was expressed as the percent injected dose per gram (%ID/g) and
percent injected dose per organ (%ID/organ). Previously measured standard organ weights for
the blood, heart, lung, liver, kidney, spleen, stomach, intestines, brain and ovaries of Balb/c
mice were used to calculate the %ID/organ values from %ID/g values (177). Organ weights for
the muscle, skin and bone were obtained from a standard mouse model (178).
3.2.3 Internal radiation dosimetry projections
The area under the curve (AUC) from 1 to 166 h (AUC1–166h; Bq × h) for the
radioactivity vs. time curve derived for each organ (not corrected for radioactive decay) from
the biodistribution studies was integrated using Prism® version 4.0 software (GraphPad
Software, Inc., La Jolla, CA, USA). The AUC0–1h from the time of injection to the first data
point (1 h) was calculated using the Trapezoidal Rule (179). Elimination of radioactivity beyond
the last measured data point (166 h p.i.) was assumed to be by radioactive decay only and the
AUC166h–∞ was estimated by dividing the amount of radioactivity (Bq) at this final time point by
the decay constant for 111In (1.03 × 10−2 h−1). The number of disintegrations per source organ
(N, Bq × h/Bq administered) was calculated by dividing the total AUC0h–∞ (Bq × h) by the
injected dose (Bq). The values for each organ obtained for mice were used to predict the
radiation absorbed doses in humans (mSv/Bq) using the OLINDA/EXM 1.0 computer program
105
(163). The radionuclide contaminants, 114mIn and 65Zn, were assumed to be present at a
maximum of 0.1% each in 111In (Nordion specifications). The contribution to the total radiation
absorbed dose from each radiocontaminant up to 166 h was obtained by multiplying the AUC0–
166h by 0.1%. The contribution from each radiocontaminant to the AUC166h–∞ was obtained by
multiplying the 111In radioactvity per organ at 166 h (Bq) by 0.1% and dividing this value by the
decay constant for 114mIn or 65Zn (5.83 × 10−4 h−1 or 1.18 × 10−4 h−1, respectively), thus
assuming that elimination beyond 166 h was only by radioactive decay. N (Bq × h/Bq
administered) was determined by dividing the total AUC0h–∞ (Bq × h) for 114mIn or 65Zn by 0.1%
of the injected dose of 111In (Bq). The values obtained were entered into the OLINDA/EXM 1.0
computer program, and the projected radiation absorbed doses from these radiocontaminants
were included in the estimation of the total radiation absorbed doses from 111In-BzDTPA-
pertuzumab.
3.2.4 Acute toxicology
Groups of 10 female Balb/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were
injected i.v. (tail vein) with 111In-BzDTPA-pertuzumab (1.0 MBq; 20 μg), unlabeled BzDTPA-
pertuzumab (20 μg), or an equivalent volume of Sodium Chloride Injection USP (100 μL).
These doses represented 10 and 23 times the human dose for the PETRA trial on a mg/kg basis
and MBq/kg basis, respectively. Body weight was measured prior to administration of these
treatments (day 0) and then again after 3, 7, 10 and 15 days. The mean body weight of the mice
on day 0 was 17.9 ± 0.8 g (n = 30). A two week acute toxicology study is required by Health
Canada for radiopharmaceuticals intended to be administered as a single dose to humans in a
Phase I clinical trial (180). For radiopharmaceuticals based on a pharmaceutical that has
received approval for clinical evaluation (e.g. pertuzumab), Health Canada does not normally
106
require toxicological evaluation in a non-rodent species, since these data have been previously
submitted by the manufacturer (a letter to Health Canada to permit access to the Drug Master
File for pertuzumab was provided by Roche). At the completion of the study, mice were
euthanized by cervical dislocation under isoflurane anaesthesia in O2. Blood samples were
obtained by cardiac puncture and collected (~ 50 μL) into EDTA coated microcapillary tubes
(Sarstedt, Montreal, QC, Canada) for hematology analyses or collected (~ 200 μL) into serum
(clot activating) microcapillary tubes (Sarstedt) for serum biochemistry analyses including
alanine aminotransferase (ALT) and creatinine (SCr) levels. Hematology analyses were
conducted using a Hemavet 950 FS instrument (Drew Scientific, Dallas, TX, USA) and included
leukocyte (WBC), erythrocyte (RBC), and platelet counts (PLT), as well as hematocrit (Hct) and
hemoglobin (Hb) concentrations. In addition, samples of 12 tissues (lungs, heart, stomach,
colon, pancreas, liver, kidney, ovaries, uterus, mammary gland, spleen, bone marrow) were
collected at necropsy, fixed in formalin, embedded in paraffin, and sectioned. All sections were
stained with hematoxylin and eosin and examined by light microscopy by a clinical pathologist
(S.D.). All studies were conducted in accordance with the Canadian Council on Animal Care
(CCAC) guidelines under a protocol (No. 989.15) approved by the Animal Care Committee at
the University Health Network.
3.2.5 Statistical analysis
A paired Student’s t-test (P <0.05) was used for body weight comparisons in toxicity
studies. The nonparametric Mann–Whitney U-test (P<0.05) was used to test for statistical
significance for biochemistry and hematology analyses. All analyses were performed using
SPSS version 17.0 (IBM, Armonk, NY, USA).
107
3.3 Results
3.3.1 Pharmacokinetic and biodistribution studies
111In-BzDTPA-pertuzumab exhibited biexponential elimination from the blood in non-
tumour bearing Balb/c mice following i.v. (tail vein) administration (Figure 3.1). The
distribution half-life (α-phase) was 3.8 h. The elimination phase half-life (β-phase) was 228.2 h.
The volume of distribution of the central compartment (V1) was 2.3 mL (128 mL/kg) and the
volume of distribution at steady state (Vss) was 4.6 mL (259 mL/kg). The V1 and Vss volumes
would correspond to volumes in a 50 kg standard adult female of 6.4 L and 12.9 L, respectively.
The systemic clearance (CLs) was 0.014 mL/h (0.8 mL/h/kg). The CLs values in mice would
correspond to 40 mL/h in a 50 kg adult female. The concentration of radioactivity in tissues
(%ID/g) at selected times post-i.v. injection of 111In-BzDTPA-pertuzumab (2.5 MBq; 2 μg) to
non-tumour bearing Balb/c mice is shown in Figure 3.2A. The total amount of radioactivity in
these organs (%ID/organ) is shown in Figure 3.2B. The highest concentration of radioactivity
was found in the blood (up to 50.4 ± 3.6 %ID/g) and lungs (up to 12.6 ± 4.0 %ID/g) at 1 h p.i.
but these concentrations decreased over a 166 h period to 12.4 ± 0.9 %ID/g and 4.5 ± 0.7
%ID/g, respectively. The next highest concentrations of radioactivity occurred in the heart,
spleen and kidneys (10.7 ± 2.2, 10.4 ± 1.8 and 9.7 ± 2.0 %ID/g at 1 h p.i., respectively). There
was <10 %ID/g for all other organs at all time points. The greatest proportion of the injected
dose per organ was found in the blood (104.3 ± 6.6 %ID at 1 h p.i. which decreased to 25.7 ±
2.0 %ID at 166 h post-injection). The muscle sequestered the next greatest amount of the
injected dose (13.5 ± 5.1 at 1 h p.i.), which was likely due to the large proportion of body mass
represented by this tissue (63% of body weight) (178).
108
Figure 3.1. Elimination of radioactivity from the blood in Balb/c mice injected i.v. (tail vein)
with 111In-BzDTPA-pertuzumab (2.5 MBq; 2 μg). Values shown are the mean ± SD (n = 4). The
curve was fitted to a 2-compartment pharmacokinetic model using Scientist® Ver. 2.01
software. The α-phase half-life was 3.8 h and the β-phase half-life was 228.2 h.
109
Figure 3.2. Biodistribution of 111In-BzDTPA-pertuzumab in normal Balb/c mice at selected
110
times up to 166 hours post-i.v. (tail vein) injection expressed as percent injected dose per gram
(% ID/g) of tissue (A) and as percent injected dose per organ (%ID/organ) (B). Bars represent
mean ± SD.
111
3.3.2 Internal radiation dosimetry projections
The radiation absorbed dose estimates predicted for humans from i.v. administration of
111In-BzDTPA-pertuzumab to mice are shown in Table 3.1. The organs that would receive the
highest radiation absorbed doses are the intestines, kidneys and liver (0.29, 0.21, and 0.18
mSv/MBq, respectively). The estimated total body dose would be 0.05 mSv/MBq, which
corresponds to a whole body dose of 5.6 mSv for a 111 MBq administered dose planned for the
PETRA Phase I/II clinical trial.
112
Table 3.1
Radiation absorbed dose projections for 111In-BzDTPA-pertuzumab in humans
Organ Radiation absorbed dose (mSv/MBq) a
Brain 0.02
Breasts 0.02
Gallbladder wall 0.14
Small intestine 0.12
Stomach wall 0.15
Upper large intestine 0.50
Heart wall 0.11
Kidneys 0.33
Liver 0.23
Lungs 0.07
Muscle 0.05
Ovaries 0.15
Pancreas 0.09
Spleen 0.18
Total body 0.05
a Radiation absorbed dose projections in humans were based on the cumulative
number of disintegrations in source organs in mice (N) obtained from biodistribution
studies and were estimated using OLINDA Ver. 1.0 software. This assumes that the
organ biodistribution of 111In-BzDTPA-pertuzumab in humans will be the same as
113
that in mice. A maximum of 0.1% of each of the radionuclide impurities 114mIn and
65Zn in 111In was considered in estimating the total radiation absorbed doses.
114
3.3.3 Acute toxicology
There were no significant decreases in body weight observed for non-tumour bearing,
healthy Balb/c mice administered Sodium Chloride Injection USP, unlabeled BzDTPA-
pertuzumab (20 μg) or 1.0 MBq (20 μg) of 111In-BzDTPA-pertuzumab (Table 3.2). The dose of
111In-BzDTPA-pertuzumab injected represented 23-fold and 10-fold higher radioactivity and
protein doses, respectively than planned for the PETRA Phase I/II clinical trial scaled on a
MBq/kg and mg/kg basis. Results from hematology and biochemistry analyses at 15 days are
shown in Figure 3.3. The Hb concentration in mice injected with 111In-BzDTPA-pertuzumab
(104.1 ± 4.4 g/L) was slightly but significantly lower than in mice receiving unlabeled
BzDTPA-pertuzumab (112.1 ± 3.4 g/L; P<0.01) or Sodium Chloride Injection USP (111.1 ± 5.6
g/L; P<0.01). SCr was modestly but significantly higher in mice that received 111In-BzDTPA-
pertuzumab (71.3 ± 12.7 μmol/L) than in mice receiving unlabeled BzDTPA-pertuzumab (49.9
± 17.1 μmol/L) (P<0.05). No other significant differences were observed for hematology and
biochemistry parameters. There were no morphological abnormalities found in any of the 12
tissues (lungs, heart, stomach, colon, pancreas, liver, kidney, ovaries, uterus, mammary gland,
spleen, bone marrow) examined histopathologically by light microscopy in mice administered
111In-BzDTPA-pertuzumab, unlabeled BzDTPA-pertuzumab, or Sodium Chloride Injection
USP.
115
Table 3.2
Body weights of Balb/c mice administered a single i.v. dose of 111In-BzDTPA-pertuzumab, BzDTPA-
pertuzumab, or Sodium Chloride Injection USPa
Normalized body weight at selected times post-administrationb:
Days post-administration 111In-BzDTPA-
pertuzumab BzDTPA-pertuzumab
Sodium Chloride
Injection USP
3 1.05 ± 0.02 1.03 ± 0.02 0.99 ± 0.02c
7 1.05 ± 0.02 1.03 ±0.02 1.03 ± 0.02
10 1.04 ± 0.01 1.05 ± 0.03 1.02 ± 0.01
15 1.07 ± 0.02 1.07 ± 0.02 1.03 ± 0.02
a Mice were intravenously administered 1.0 MBq (20 µg) of 111In-BzDTPA-pertuzumab, an equivalent
mass of unlabeled BzDTPA-pertuzumab or an equivalent volume (100 µL) of Sodium Chloride injection
USP. b Normalized body weight is expressed as mean ratio ± SD of the weight at the selected time point
divided by the initial body weight on day 0 (n=10). c No statistically significant decreases in body weight were observed at any of the selected times between
any of the three treatments.
116
117
Figure 3.3. Complete blood cell counts, hemoglobin, hematocrit and serum creatinine and
alanine aminotransferase levels in groups of 7-10 mice administered 111In-BzDTPA-pertuzumab
or unlabeled BzDTPA-pertuzumab at 23 and 10 times the planned dose on a MBq/kg or mg/kg
basis, respectively for a Phase I/II clinical trial or in control mice administered Sodium Chloride
Injection USP. WBC = white blood cells; RBC = red blood cells; PLT = platelets; Hb =
hemoglobin; HCT = hematocrit; SCr = serum creatinine; ALT = alanine aminotransferase; 111In-
Pmab = 111In-BzDTPA-pertuzumab; BzDTPA-Pmab = unlabeled BzDTPA-pertuzumab; NaCl =
Sodium Chloride Injection USP. Horizontal lines represent the median values. *Statistically
significant difference (P<0.05; Mann Whitney U test).
118
3.4 Discussion
In this report, we describe for the first time the preclinical pharmacokinetics,
biodistribution, radiation dosimetry and toxicity of 111In-BzDTPA-pertuzumab, a novel
theranostic imaging probe for HER2-positive BC prepared from a kit formulation. These studies
were required by Health Canada for regulatory approval of a Clinical Trial Application for a
Phase I/II clinical trial of 111In-BzDTPA-pertuzumab (PETRA; ClinicalTrials.gov identifier
NCT01805908). Following i.v. (tail vein) injection in Balb/c mice, 111In-BzDTPA-pertuzumab
was eliminated biexponentially from the blood with α- and β-phase half-lives of 3.8 h and 9.5
days, respectively (Figure 3.1). The α-phase half-life was comparable to that reported for
unlabeled pertuzumab (2.4–7.2 h) administered to CD-1 mice at doses of 3, 30, or 90 mg/kg [6].
The relatively short α-phase half-life of 111In-BzDTPA-pertuzumab was not due to instability of
the radiometal complex as the p-SCN-BzDTPA chelator is known to provide a very stable 111In
complex (181). The β-phase half-life of 111In-BzDTPA-pertuzumab was shorter than unlabeled
pertuzumab (11.4–15.7 days). This may be due to the lower protein dose administered (0.1
mg/kg) for 111In-BzDTPA-pertuzumab in our study compared to unlabeled pertuzumab (3–90
mg/kg). A Phase I clinical trial revealed that pertuzumab administered at a low dose (0.5 mg/kg)
was eliminated more rapidly than at higher doses of 2.0–15.0 mg/kg (182). It is also possible
that the charges introduced into pertuzumab by conjugation to BzDTPA may have contributed
to its faster elimination. Each BzDTPA group complexed with 111In imparts one negative charge
while uncomplexed BzDTPA provides 2–3 negative charges (183). Anionic charges promote the
elimination of monoclonal antibodies in mice (124).
Biodistribution studies of 111In-BzDTPA-pertuzumab revealed that the highest
concentrations (% ID/g) of radioactivity were present in the blood, heart, lungs, liver, kidneys
119
and spleen (Figure 3.2). Interestingly, the radioactivity concentrations in the blood at 70 h p.i. of
111In-BzDTPA-pertuzumab (18% ID/g) in non-tumour bearing Balb/c mice were about 3-fold
higher than previously reported by us for 111In-labeled pertuzumab in athymic mice implanted
s.c. with HER2-positive MDA-MB-361 human BC xenografts (110). Similarly, the uptake of
radioactivity in the heart, lungs and kidneys was about 2-fold higher for 111In-BzDTPA-
pertuzumab in non-tumour bearing Balb/c mice than in tumour-bearing mice. The tumour
uptake of 111In-labeled pertuzumab in the previous study was very high (>34% ID/g) which may
have contributed to the more rapid elimination from the blood (110). The uptake of 111In-
BzDTPA-pertuzumab in the liver and spleen of Balb/c mice was similar to that in tumour-
bearing mice (110). Binding to Fc receptors is likely responsible for the spleen and liver uptake
of 111In-BzDTPA-pertuzumab (184). Persson et al. (185) reported very high tumour uptake (~
55% ID/g) of 177Lu-labeled pertuzumab in mice implanted s.c. with HER2-positive SK-OV-3
human ovarian cancer xenografts. The normal organ distribution pattern of 177Lu-pertuzumab
was similar to 111In-BzDTPA-pertuzumab with the highest concentrations found in the blood,
heart, lungs, liver, spleen and kidneys. The blood concentration of 177Lu-pertuzumab in mice
bearing SK-OV-3 tumours at 24 h was about 14% ID/g, whereas the blood concentration for
111In-BzDTPA-pertuzumab at this time point was about 22% ID/g (Figure 3.2A). The lower
blood concentration for 177Lu-pertuzumab may be caused by uptake into SK-OV-3 tumours
which is not a factor in non-tumour bearing mice administered 111In-BzDTPA-pertuzumab. The
study by Persson et al. (185) is the only other report of the biodistribution of radiolabeled
pertuzumab in mice.
Radiation absorbed doses predicted for humans were based on the biodistribution of
111In-BzDTPA-pertuzumab and were calculated using OLINDA software (Table 3.1) (163). It
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was predicted that a 50 kg female human would receive a whole body dose of 0.05 mSv/MBq,
which corresponds to 5.6 mSv for an administered dose of 111 MBq of 111In-BzDTPA-
pertuzumab planned for the PETRA trial. These radiation dosimetry projections assumed that
the organ biodistribution (% ID/ organ) of 111In-BzDTPA-pertuzumab in a human would be the
same as that in a mouse and that the cumulative uptake and elimination of radioactivity from
organs would be the same. These assumptions are often made in predicting the radiation
absorbed doses for first-in-humans studies of new radiopharmaceuticals (186). Projected doses
may vary with patient mass and calculations may be scaled to reflect this (187,188). Since three
administrations of 111In-BzDTPA-pertuzumab (111 MBq each) were planned for the PETRA
trial to image tumour HER2 expression prior to commencing treatment with trastuzumab and
chemotherapy, and at 1- and 4 weeks after starting treatment, the total whole body radiation
absorbed dose would be 16.8 mSv. For comparison, a single administration of 111 MBq of
111In-capromab pendetide (Prostascint®) for imaging prostate cancer delivers a whole body
radiation absorbed dose of 16 mSv (76). The whole body dose for a single administration of 18F-
2-fluorodeoxyglucose (18FDG) for positron-emission tomography (PET) is 7–14 mSv (189).
Acute toxicology studies at 15 days p.i. of 111In-BzDTPA-pertuzumab in healthy Balb/c
mice administered 10-fold the protein dose (on a mg/kg basis) or 23-fold the radioactivity dose
(on a MBq/kg basis) planned for the PETRA trial revealed no clinically significant toxicity
(Figure 3.3 and Table 3.2). Hb concentrations were slightly (7%) lower in mice injected with
111In-BzDTPA-pertuzumab than in control mice injected with unlabeled BzDTPA-pertuzumab
or mice receiving Sodium Chloride Injection USP. However, there were no differences in RBC,
WBC or PLT counts or Hct between mice administered 111In-BzDTPA-pertuzumab, unlabeled
BzDTPA-pertuzumab or Sodium Chloride Injection USP. SCr levels in mice injected with 111In-
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BzDTPA-pertuzumab were modestly higher than in mice that received unlabeled BzDTPA-
pertuzumab but were not significantly elevated compared to mice receiving Sodium Chloride
Injection USP. Since histological examination of a comprehensive panel of tissues by a clinical
pathologist revealed no abnormalities in any of the groups of mice, we conclude that no
clinically significant toxicities were associated with administration of 111In-BzDTPA-
pertuzumab at multiples of the planned human dose, providing a wide margin of safety. Health
Canada did not require toxicology testing in a non-rodent species since 111In-BzDTPA-
pertuzumab was prepared based on the approved drug, pertuzumab (Perjeta®; Roche).
3.5 Conclusion
Preclinical studies predicted that 111In-BzDTPA-pertuzumab would be safe to administer
to humans at a dose of 111 MBq (5 mg). The radiopharmaceutical exhibited preclinical
pharmacokinetic, biodistribution and radiation dosimetry properties suitable for advancement to
a first-in-humans clinical trial. Health Canada approval of a Clinical Trial Application was
granted for a Phase I/II trial of 111In-BzDTPA-pertuzumab (PETRA; ClinicalTrials.gov
identifier NCT01805908) for imaging the response of patients with metastatic BC to treatment
with trastuzumab combined with chemotherapy.
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CHAPTER 4:
Development and Preclinical Studies of 64Cu-NOTA-Pertuzumab F(ab')2
Fragments for Imaging Changes in Tumor HER2 Expression Associated with
Response to Trastuzumab by PET/CT
123
This chapter represents a preprint of: “Lam K, Scollard DA, Chan C, Reilly RM. Development
and preclinical studies of 64Cu-NOTA-pertuzumab F(ab')2 fragments for imaging changes in
tumor HER2 expression associated with response to trastuzumab by PET/CT. MAbs. In Press
2016.”
All experiments and analyses of data were carried out by Karen Lam. MicroPET/CT was
performed with technical assistance from Dr. Conrad Chan.
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4.0 Abstract
We previously reported that microSPECT/CT imaging with 111In-labeled pertuzumab detected
decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated
with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to
PET/CT by constructing F(ab')2 fragments of pertuzumab modified with NOTA chelators for
complexing 64Cu. The effect of the administered mass (5-200 µg) of 64Cu-NOTA-pertuzumab
F(ab')2 was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian
cancer xenografts. Biodistribution studies were performed in non-tumour bearing Balb/c mice to
predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on
mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-
insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no
significant effects of the administered mass of 64Cu-NOTA-pertuzumab F(ab')2 on tumour or
normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold
reduction compared to 111In-labeled pertuzumab. MicroPET/CT images revealed specific tumour
uptake of 64Cu-NOTA-F(ab')2 at 24 or 48 h p.i. in mice with SK-OV-3 tumours. Image analysis
of mice treated with trastuzumab showed 2-fold reduced uptake of 64Cu-NOTA-pertuzumab
F(ab')2 in BT-474 tumours after 1 week of trastuzumab normalized to baseline, and 1.9-fold
increased uptake in SK-OV-3 tumours after 3 weeks of trastuzumab, consistent with tumour
response and resistance, respectively. We conclude that PET/CT imaging with 64Cu-NOTA-
F(ab')2 detected changes in HER2 expression in response to trastuzumab while delivering a
lower total body radiation dose compared to 111In-labeled pertuzumab.
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4.1 Introduction
The human epidermal growth factor receptor-2 (HER2) is overexpressed in 15-20% of
breast cancers (BC) and confers a poor prognosis (20,87,190). Treatment with trastuzumab
(Herceptin, Roche), a humanized IgG1 anti-HER2 monoclonal antibody (mAb) combined with
chemotherapy improves patient outcome in the adjuvant and metastatic settings in patients who
have BC that is defined as HER2-positive either by immunohistochemistry (IHC) or in situ
hybridization (ISH) analyses (11,40,41). Guidelines have been established to define tumour
HER2 positivity using these techniques (44). Despite the establishment of trastuzumab as the
standard-of-care for treatment of HER2-positive BC, clinical trials revealed that only 1 in 2
patients with HER2-positive tumours responded to trastuzumab combined with chemotherapy
(11) and most responding patients acquire resistance within a year (176). It has also been
proposed that some patients with BC classified as HER2-negative may also receive benefit from
trastuzumab (191).
Molecular imaging which includes single photon emission computed tomography
(SPECT) and positron emission tomography (PET) provides a sensitive tool to non-invasively
assess tumour phenotype at any location in the body, as well as to monitor response to targeted
cancer therapies (88). One proposed mechanism of action of trastuzumab is to induce HER2
internalization, thus reducing the density of HER2 on tumour cells available for receptor
dimerization and oncogenic signaling (192). Probing changes in HER2 expression in tumours
could be a promising strategy to discriminate responders from non-responders to trastuzumab
treatment. Pertuzumab is a humanized IgG1 mAb which binds to domain II on HER2 and
hinders receptor dimerization (55). Since the HER2 binding domain of pertuzumab is distinct
from that of trastuzumab (domain IV) and pertuzumab has a different mechanism of action than
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trastuzumab (52), these mAbs have been combined to improve patient outcome (64,162). We
previously reported that microSPECT/CT imaging with 111In-labeled pertuzumab sensitively
detected changes in HER2 expression in MDA-MB-361 human BC xenografts in athymic mice
following treatment with trastuzumab, since the binding of the imaging probe to HER2 is not
affected by trastuzumab binding (110). Decreased HER2 expression was detected by imaging as
early as 3 days after commencing trastuzumab treatment, and images at 21 days demonstrated
significantly lower tumour uptake of 111In-labeled pertuzumab associated with almost complete
tumour eradication. Our group has launched a Phase I/II clinical trial (PETRA trial;
ClinicalTrials.gov identifier NCT01805908) investigating SPECT/CT imaging with 111In-
labeled pertuzumab to detect changes in tumour HER2 expression in patients with metastatic
BC treated with trastuzumab and chemotherapy. The clinical formulation and translational
preclinical studies that were required to advance this imaging agent to clinical trial are reported
elsewhere (159,173).
Our aim in the current study was to develop an analogous positron-emitting imaging
probe based on pertuzumab to detect trastuzumab-mediated HER2 internalization that would
extend these promising findings to PET, and potentially reduce the radiation dose associated
with the three administrations of 111In-labeled pertuzumab required in the PETRA clinical trial
protocol. The predicted combined total body radiation dose for these three imaging studies
performed at baseline, 1 week and 4 weeks after commencing treatment with trastuzumab and
chemotherapy was 17 mSv, based on an administered amount of 111 MBq of 111In-labeled
pertuzumab for each study (0.05 mSv/MBq) (173). PET is 100-fold more sensitive than SPECT
and yields high resolution images that are more accurately quantitated (79). 64Cu is an attractive
positron-emitter for labeling pertuzumab since it is produced in a biomedical cyclotron (193),
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emits a moderate energy positron [0.7 MeV (19%)] that provides good intrinsic spatial
resolution (0.7 mm), and is strongly complexed by macrocyclic chelators such as 1,4,7-
triazacyclononane-1,4,7-triacetate (NOTA) that are easily conjugated to antibodies (158). Due
to the short half-life of 64Cu (t1/2 = 12.7 h), it is necessary to employ mAb fragments [e.g. Fab or
F(ab′)2] which are taken up by tumours but rapidly eliminated from the blood and most normal
tissues to provide high tumour:blood ratios within the useful lifetime of the radionuclide [up to
48 h post-injection (p.i)]. The short half-life of 64Cu combined with the rapid elimination of
mAb fragments is expected to minimize the radiation dose for the imaging procedure. We report
here the synthesis and characterization of 64Cu-NOTA-pertuzumab F(ab')2 and its first
evaluation for detecting changes in HER2 expression and response to treatment in athymic mice
with trastuzumab-sensitive BT-474 human BC xenografts and trastuzumab-resistant SK-OV-3
human ovarian cancer xenografts. We further studied the effect of increasing the administered
mass of 64Cu-NOTA-pertuzumab F(ab')2 on tumour and normal tissue uptake, and projected the
radiation absorbed doses in humans based on its pharmacokinetics of uptake and elimination
from normal organs in mice.
4.2 Materials and methods
4.2.1 Cells lines and tumour xenografts
SK-BR-3 human breast cancer (BC) cells and SK-OV-3 human ovarian cancer cells,
both expressing 1-2 × 106 HER2/cell (171,194) were cultured in RPMI 1640 (Sigma-Aldrich
#R8758) supplemented with 10% fetal bovine serum (FBS, Life Technologies #12484028). BT-
474 human BC cells (1-2 × 106 HER2/cell) (123) were grown in Dulbecco Modified Eagle
Medium (University Health Network) supplemented with 10% FBS. All cells were cultured at
37°C/5% CO2. Female NOD/SCID mice (Ontario Cancer Institute) were inoculated
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subcutaneously (s.c.) with 1 × 107 SK-OV-3 or BT-474 cells in 200 µL of serum free medium or
a 1:1 mixture of Matrigel (Corning #CACB354234) and medium. Mice were implanted with a
0.72 mg 60 days sustained release 17β-estradiol pellet (Innovative Research of America #SE-
121) at 24 h prior to inoculation of BT-474 cells, required for growth of these tumours in
NOD/SCID mice.
4.2.2 Pertuzumab F(ab')2 fragments
Pertuzumab F(ab')2 fragments were generated by pre-equilibrating 625 µL of
immobilized pepsin slurry (Thermo Scientific #20343) with 20 mM sodium acetate trihydrate
buffer, pH 4.5 (500 µL) and then adding 10 mg of pertuzumab IgG (Mr ≈ 148 kDa; Genentech)
prepared in 1 mL of the same buffer. The mixture was incubated at 37°C on an end-over-end
mixer for 5.5 h and then centrifuged at 1000 × g for 5 min. The supernatant was collected and
the resin was rinsed twice by resuspending in 750 µL phosphate buffered saline (PBS, pH 7.4),
centrifuging again, collecting and pooling the washes, and then passing the pooled volume
through a Millex-GV PVDF 0.22 µm filter (EMD Millipore #SLGV033SL). Completion of
digestion of IgG to F(ab')2 was assessed by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) on a 4-20% Tris HCl gradient gel (Bio-Rad #456-1093) stained
with Coomassie G-250 stain (Bio-Rad #161-0786). If digestion was not complete, the pooled
supernatant was applied to a NAb Protein A agarose spin column (Thermo Scientific #89956)
equilibrated with PBS (pH 7.4), and then incubated for 10 min at room temperature (RT) on an
end-over-end mixer to bind IgG. The column was centrifuged at 1000 × g for 1 min and the
flow-through containing the F(ab')2 was collected. The column was washed twice by adding 1
mL of PBS, centrifuging, and collecting the wash fractions. Pertuzumab F(ab')2 fragments were
evaluated for purity by SDS-PAGE on a 4-20% gel stained with Coomassie G-250 blue dye.
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Additionally, size-exclusion high performance liquid chromatography (SE-HPLC) was
conducted on a BioSep SEC-S2000 column (Phenomenex) eluted with 0.1 M NaH2PO4 buffer
(pH 7.0) at a flow rate of 0.8 mL/min and monitored with a diode array UV detector at 280 nm
(PerkinElmer). F(ab')2 fragments of non-specific human IgG (hIgG) from human serum (Sigma-
Aldrich #I4506) were similarly prepared and analysed for purity.
4.2.3 Preparation of 64Cu-NOTA-pertuzumab F(ab')2
Pertuzumab F(ab')2 were buffer-exchanged and concentrated to 2.5, 5 or 10 mg/mL in
0.1 M NaHCO3, pH 9.0, in an Amicon Ultra device (Millipore #UFC503096; Mr cut-off = 30
kDa). Protein concentration was determined spectrophotometrically [E280nm = 1.5 (mg/mL)-1cm-
1]. F(ab')2 (2.5, 5 or 10 mg/mL) were modified with NOTA for complexing 64Cu by reaction
with a 5-fold or 10-fold molar excess of 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-
1,4,7-triacetic acid (p-SCN-Bn-NOTA; Macrocyclics #B605) for 2 h at RT. The p-SCN-Bn-
NOTA was dissolved in 5 µL DMSO, incubated for 10 min at 37°C to facilitate dissolution, and
then diluted to a concentration of 1.5 mg/mL in 0.1 M NaHCO3, pH 9.0. NOTA-pertuzumab
F(ab')2 were purified from unconjugated NOTA by ultracentrifugation with 0.1 M NaCH3CO2
buffer, pH 5.5, in an Amicon Ultra device (Mr cut-off = 30 kDa). NOTA-pertuzumab F(ab')2
concentration was determined with the Bradford Assay using Pierce Coomassie Plus Assay
Reagent (Thermo Scientific #23238). The NOTA substitution level of the F(ab')2 fragments was
measured by labeling an aliquot of the unpurified conjugation mixture with 64Cu, then
determining the proportion of 64Cu-NOTA-pertuzumab F(ab')2 vs. 64Cu-NOTA by instant thin-
layer silica gel chromatography (ITLC-SG; Agilent Technologies #SGI0001) and multiplying
this fraction by the molar ratio used in the reaction. An alternative spectrophotometric assay to
measure and verify substitution level was also developed (Appendix A). For this assay, the UV
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absorbance of the NOTA-F(ab')2 conjugate was measured at 280 nm. The absorbance
contribution from the F(ab')2 was determined by comparing the known protein concentration of
F(ab')2 (Bradford Assay) to a standard curve of UV absorbance at 280 nm vs. F(ab')2
concentration. The difference in absorbance between the NOTA-F(ab')2 conjugate and F(ab')2
was attributable to NOTA. NOTA concentration was derived by reference to a standard curve of
UV absorbance at 280 nm vs. NOTA concentration. The ratio of NOTA concentration to F(ab')2
concentration yielded the substitution level. Purity and homogeneity of the optimized NOTA-
pertuzumab F(ab')2 conjugates were assessed by SDS-PAGE and SE-HPLC as described above.
Radiolabeling of NOTA-pertuzumab F(ab')2 with 64Cu was achieved by incubation for 1 h at
40°C with 64CuCl2 (Washington University, St. Louis, MO) to achieve a specific activity of 370
kBq/µg for general procedures or 2.6 MBq/µg for imaging studies. 64Cu-NOTA-pertuzumab
F(ab')2 was purified in an Amicon Ultra device (Mr cut-off = 30 kDa). The final radiochemical
purity was determined by ITLC-SG developed in 0.1 M sodium citrate, pH 5.0 and by SE-HPLC
using a Flow Scintillation Analyzer (FSA) radioactivity detector (PerkinElmer). The Rf values
for 64Cu-NOTA-F(ab')2 on ITLC were 0.0 and those for 64Cu-NOTA and free 64Cu were 0.8-0.9
and 1.0, respectively. F(ab')2 fragments of non-specific hIgG were similarly modified with
NOTA and labeled with 64Cu. Trace metals were removed from all buffers using Chelex-100
cation-exchange resin (Bio-Rad #142-2832).
4.2.4 HER2 binding and trastuzumab-mediated HER2 internalization
The binding of 64Cu-NOTA-pertuzumab F(ab')2 to HER2 on SK-BR-3 cells (1-2 × 106
HER2/cell) was determined by saturation radioligand binding assays (171). Increasing
concentrations of 64Cu-NOTA-pertuzumab F(ab')2 (0.07 to 300 nM) were incubated with 1 × 106
SK-BR-3 cells in 1.5 mL microcentrifuge tubes in PBS in the presence [non-specific binding
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(NSB)] or absence [total binding (TB)] of 61 µM of unlabeled pertuzumab. The tubes were
incubated at 4°C for 3.5 h with occasional shaking, then centrifuged at 420 × g for 5 min and the
supernatant and cell pellet separated and measured in a γ-counter. Specific binding (SB) was
calculated by subtracting NSB from TB and plotted vs. the unbound concentration of 64Cu-
NOTA-F(ab')2. The resulting curve was fitted to a 1-site receptor-binding model by Prism Ver.
4.0 software (GraphPad) and the dissociation constant (Kd) and maximum number of
receptors/cell (Bmax) estimated.
Single concentration radioligand binding assays with 64Cu-NOTA-pertuzumab F(ab')2
were performed to assess trastuzumab-mediated HER2 internalization in SK-OV-3 or BT-474
cells. Briefly, 4 × 105 SK-OV-3 or 3 × 105 BT-474 cells were seeded in 6-well plates and
cultured overnight. The medium was removed and the cells incubated at 37°C for 24 h with
trastuzumab (14 µg/mL) in 2 mL of fresh medium or medium alone. The medium was again
removed and rinsed with PBS, pH 7.3. The cells were then incubated with 10 nM 64Cu-NOTA-
pertuzumab F(ab')2 in the absence or presence of a 50-fold excess of pertuzumab in PBS, pH 7.3
for 3 h at 4°C to measure TB and NSB. The medium was removed, and the cells were rinsed
twice with PBS, pH 7.3 and then solubilized in 100 mM NaOH. The solubilized cells were
transferred to γ-counting tubes and the cell-bound radioactivity measured in a γ-counter. SB was
calculated by subtracting NSB from TB. The percent change in HER2 expression was calculated
by comparing the SB of trastuzumab treated cells to the SB of untreated cells.
4.2.5 Biodistribution, pharmacokinetic and radiation dosimetry studies
To evaluate the effect of the administered mass of 64Cu-NOTA-F(ab')2 on tumour and
normal tissue uptake, SK-OV-3 tumour-bearing mice were injected i.v. (tail vein) with 5, 50,
100, or 200 µg labeled with 1-3 MBq of 64Cu. Mice were sacrificed at 24 h post-injection (p.i.),
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and tumours and selected normal tissues were collected, weighed and counted, along with a
standard of the injected radioactivity, in a γ-counter. Tumour and normal tissue uptake were
expressed as percentage injected dose per gram (%ID/g). The 24 h time point was chosen based
on a previous study with 111In-DOTA-trastuzumab F(ab')2 that showed maximal tumour uptake
was achieved at 24 h p.i. (105).
The radioactivity in the blood and normal tissues at 1, 3, 6, 24, and 48 h post-injection
was determined in groups of 4 non-tumour bearing female Balb/c mice (Charles River)
following i.v. injection of 64Cu-NOTA-pertuzumab F(ab')2 (50 µg; 2-4 MBq). In addition, the
pharmacokinetics of elimination of 64Cu-NOTA-pertuzumab F(ab')2 from the blood of Balb/c
mice were determined by plotting the radioactivity (%ID/mL) vs. time post-injection, and fitting
the curve to a two-compartment pharmacokinetic model using Scientist® Ver. 2.01 software
(MicroMath Scientific Software). Standard pharmacokinetic parameters were estimated.
Radiation absorbed doses to normal organs were estimated as previously described
(173). Briefly, the dose to target organs was estimated as D = N × DF, where N is the number of
disintegrations (Bq × h/Bq) in a source organ, and DF is the Dose Factor using the RAdiation
Dose Assessment Resource (RADAR) formalism. The number of disintegrations (N0-48h) from 0
h to 48 h p.i. of 64Cu-NOTA-pertuzumab F(ab')2 was estimated by first integrating the area-
under-the-curve (AUC) for the radioactivity (not corrected for decay) vs. time plot for each
source organ. The AUC from 48 h to infinity (N48h-∞) was estimated by dividing the
radioactivity in the source organ at 48 h by the decay constant for 64Cu (0.05457 h-1), thus
assuming further elimination only by radioactive decay. The total AUC (N0-∞) was the sum of
N0-48 h and N48h-∞ and was then divided by the injected amount of radioactivity (Bq) to yield the
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total number of disintegrations for input into OLINDA/EXM 1.0 radiation dosimetry software
for prediction of the radiation absorbed doses to target organs in humans (163).
All animal studies were conducted under a protocol (no. 4336.0) approved by the
Animal Care Committee at the University Health Network in accordance with Canadian Council
on Animal Care guidelines.
4.2.6 MicroPET/CT imaging studies
For microPET/CT imaging, groups of 3-4 mice with s.c. SK-OV-3 tumours were
injected i.v. with 64Cu-NOTA-pertuzumab F(ab')2 (50 µg; 10-11 MBq) or 64Cu-labeled non-
specific hIgG F(ab')2 [64Cu-NOTA-hIgG F(ab')2; 50 µg; 6-10 MBq]. To further assess the
specificity of tumour uptake, some groups of mice received an intraperitoneal (i.p.) injection of
1 mg of pertuzumab 24 h prior to 64Cu-NOTA-pertuzumab F(ab')2. Mice were sedated with
isoflurane and imaged with a microPET tomograph (Siemens MicroPet Focus 220) and CT
scanner (GE Locus Ultra) at 24 and 48 h p.i. MicroPET images were acquired with a 350-650
keV window for 20-75 min with a coincidence timing window of 6 ns. Image reconstruction
was achieved using ordered subset expectation maximization (OSEM), followed by a maximum
a posterior probability reconstruction algorithm with no correction for attenuation or partial-
volume effects. The full width at half maximum (FWHM) resolution of the microPET was 1.6
mm. Following microPET imaging, mice were immediately transferred to an eXplore Locus
Ultra Preclinical CT scanner (GE Healthcare) for a whole body CT scan using routine
acquisition parameters (80 kVp, 50 mA, and voxel size of 154 × 154 × 154 µm). MicroPET and
CT images were processed using Inveon Research Workplace software (Siemens). Immediately
after CT imaging, mice were sacrificed and the 48 h biodistribution determined as described
above.
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To study the utility of microPET/CT imaging with 64Cu-NOTA-pertuzumab F(ab')2 to
detect HER2 internalization in tumours associated with response to treatment with trastuzumab
(110), groups of 3-6 athymic mice bearing SK-OV-3 or BT-474 tumour xenografts were
injected with 64Cu-NOTA-pertuzumab F(ab')2 (50 µg; 7-13 MBq). Images were obtained at 24 h
p.i. to obtain baseline tumour uptake of 64Cu-NOTA-pertuzumab F(ab')2. Mice were then treated
with trastuzumab administered intraperitoneally (i.p.) 2 days later using a loading dose of 4
mg/kg followed by weekly doses of 2 mg/kg for 2 weeks, diluted in normal saline to a volume
of 100 µL. We previously found that i.p. administration of 111In-labeled trastuzumab provides
70% bioavailability relative to i.v. (tail vein) injection in athymic mice (195). Assessment of
HER2 internalization was performed by repeating the injection of 64Cu-NOTA-pertuzumab
F(ab')2 at 1 week and 3 weeks and re-acquiring microPET/CT images at 24 h p.i. Tumour
response to trastuzumab was assessed by weekly measurements of tumour length and width
using calipers and calculating tumour volume as V = length × width2 × 0.5. A tumour growth
index (TGI) was derived by dividing the measured tumour volume by the intitial volume prior to
trastuzumab treatment. Quantification of tumour uptake on the pre- and post-treatment images
was performed by region-of-interest (ROI) analysis using Inveon Research Workplace software
(Siemens) and tumour uptake was expressed as %ID/g.
4.2.7 Statistical analysis
Results were expressed as mean ± SD and tested for statistical significance using a one
way ANOVA (F-test) or two-sided Student’s t-test and SPSS version 17.0 software (IBM). The
level of significance was set at P<0.05. Response to trastuzumab treatment as measured by
tumour uptake of 64Cu-NOTA-pertuzumab F(ab')2 over time was compared by a paired t test.
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4.3 Results
4.3.1 64Cu-NOTA-pertuzumab F(ab')2 fragments
F(ab')2 fragments were obtained in high purity (>90%) by pepsin digestion of
pertuzumab IgG as shown by SE-HPLC analysis which demonstrated a single peak (not shown)
and by SDS-PAGE analysis (Figure 4.1A), which revealed one major band associated with a
protein with Mr ≈ 110 kDa. The chelate substitution level achieved for F(ab')2 concentrations of
2.5, 5, or 10 mg/mL reacted with a 5 or 10-fold molar excess of p-SCN-Bn-NOTA are shown in
Table 4.1. NOTA-pertuzumab F(ab')2 exhibited a single major band on SDS-PAGE analysis
(Figure 4.1B). The reaction using 2.5 mg/mL of F(ab')2 protein and a 10-fold molar excess of
NOTA yielded a substitution level of 4.1 ± 1.9 NOTA/F(ab')2, and these reaction conditions
were used for all subsequent experiments. Following purification by ultrafiltration, the final
radiochemical purity of 64Cu-NOTA-pertuzumab F(ab')2 was 85-95% by both ITLC-SG and SE-
HPLC (Figure 4.1C). 64Cu-NOTA-F(ab')2 demonstrated saturable binding to SK-BR-3 cells with
Kd and Bmax values of 2.6 ± 0.3 nM and 0.9 ± 0.3 × 106 receptors/cell, respectively. These values
were similar to those reported by our group for 111In-BzDTPA-pertuzumab (Kd = 2.0-5.3 nM,
Bmax = 1.2-1.3 ×106 receptors/cell) (110,159).
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Figure 4.1. SDS-PAGE analysis of unconjugated F(ab')2 (A) and NOTA-F(ab')2
immunoconjugate (B). A protein ladder for standard molecular weights (MW) is also shown.
(C) SE-HPLC analysis with ultraviolet (UV) detection at 280 nm of 64Cu-NOTA-pertuzumab
F(ab')2 fragments [retention time (tR) = 8.5 mins; red line] and the corresponding chromatogram
obtained by radioactivity detection using a flow scintillation analyser (FSA) detector (tR = 9.8
mins; blue line). The offset for the radiochromatogram relative to UV detection corresponds to
the time interval required for eluate to flow from the UV to FSA detector which are in sequence.
The larger flow cell of the FSA detector causes peak broadening compared to the UV detector.
The chemical and radiochemical purity of 64Cu-NOTA-pertuzumab F(ab')2 fragments by SE-
HPLC analysis was >95%.
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Table 4.1
Chelate:protein substitution levels under different reaction conditions for
NOTA conjugation of pertuzumab F(ab')2
F(ab')2 protein concentration (mg/mL)
Molar ratio of
NOTA:F(ab')2
2.5 5.0 10.0
5:1 2.1 ± 0.0 3.6 ± 0.6 3.3 ± 0.8
10:1 4.1 ± 1.9 6.9 ± 1.8 6.5 ± 1.8
Values shown are mean ± SD (n = 4).
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4.3.2 Trastuzumab-mediated HER2 internalization
The binding of 64Cu-NOTA-pertuzumab F(ab')2 by BT-474 cells was significantly
reduced to 67.0 ± 8.1% (P<0.05) following exposure to trastuzumab (14 µg/mL) at 37°C for 24
h compared to untreated cells, which was set at 100%. Trastuzumab exposure significantly
reduced the binding of 64Cu-NOTA-pertuzumab F(ab')2 to SK-OV-3 cells to 85.5 ± 3.8%
compared to untreated cells (P<0.05). Trastuzumab exposure significantly decreased the binding
of 64Cu-NOTA-pertuzumab F(ab')2 to trastuzumab-sensitive BT-474 cells by 1.3-fold compared
to trastuzumab-resistant SK-OV-3 cells (P<0.05).
4.3.3 Biodistribution, pharmacokinetic and radiation dosimetry studies
The tumour and normal tissue uptake of increasing mass amounts of 64Cu-NOTA-
pertuzumab F(ab')2 (5, 50, 100 or 200 µg) at 24 h p.i. in NOD/SCID mice bearing HER2-
overexpressing SK-OV-3 tumour xenografts are shown in Table 4.2. The greatest uptake for all
amounts was observed in the kidneys (52.4-65.6 %ID/g), followed by the spleen (7.4-11.9
%ID/g) and liver (7.8-10.9 %ID/g), but no statistically significant differences were observed
between groups receiving different masses of 64Cu-NOTA-pertuzumab F(ab')2 (P = 0.357, 0.173
and 0.191, respectively). There appeared to be slightly lower tumour uptake for the 200 µg
administered amount (5.8 ± 1.3 %ID/g) compared to 5 µg (8.2 ± 2.6 %ID/g), 50 µg (9.8 ± 5.1
%ID/g or 100 µg (8.2 ± 2.1 %ID/g), but these differences were not significant (P=0.210).
Tumour/blood (T/B) ratios for 5, 50, 100 or 200 µg amounts of 64Cu-NOTA-pertuzumab F(ab')2
were 14.4 ± 5.14, 18.6 ± 7.4, 15.7 ± 0.8, and 11.2 ± 1.9, respectively. Tumour/muscle (T/M)
ratios for 5, 50, 100 or 200 µg amounts of 64Cu-NOTA-pertuzumab F(ab')2 were 22.4 ± 6.6, 25.1
± 12.7, 23.7 ± 5.6, and 15.6 ± 5.7, respectively. There were no significant differences in T/B
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(P=0.205) or T/M ratios (P=0.405) for different masses of 64Cu-NOTA-pertuzumab F(ab')2. An
administered amount of 50 µg was selected for subsequent imaging and biodistribution studies.
The elimination of radioactivity from the blood of non-tumour bearing Balb/c mice
following i.v. (tail vein) injection of 64Cu-NOTA-pertuzumab F(ab')2 was fitted to a 2-
compartment model (Figure 4.2). The distribution half-life (t1/2α) was 1.3 h and the elimination
half-life (t1/2β) was 10.4 h. The volume of distribution of the central compartment (V1) was 4.0
mL, the volume of distribution at steady-state (Vss) was 9.6 mL, and the systemic clearance was
1.6 mL/h. Biodistribution studies (Figure 4.3) showed normal tissue uptake similar to that in
NOD/SCID tumour bearing mice (Table 4.2). The greatest accumulation of radioactivity was
found in the kidneys with lower uptake in the liver and spleen, with the maximum uptake at 3 h
p.i. (81.0 ± 21.3, 8.4 ± 2.3 and 7.4 ± 2.4 %ID/g, respectively), decreasing by 2-fold at 48 h p.i.
(45.0 ± 4.0, 4.7 ± 0.5, 3.2 ± 0.5 %ID/g; Figure 4.3). The uptake and elimination of radioactivity
from normal tissues in mice after i.v. injection of 64Cu-NOTA-pertuzumab F(ab')2 was used to
project the radiation absorbed doses in human female adults. These estimates revealed that the
kidneys would receive the highest radiation dose (1 mSv/MBq), followed by the lower large
intestine, and liver (Table 4.3). The estimated whole body equivalent dose was 0.015 mSv/MBq.
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Figure 4.2. Radioactivity vs. time curve for the elimination of 64Cu-NOTA-F(ab')2 from the
blood of non tumour-bearing Balb/c mice after intravenous (tail vein) injection. The curve was
fitted to a two-compartment model with i.v. bolus input using Scientist Ver. 2.01 software
(MicroMath).
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Figure 4.3. Normal tissue distribution of radioactivity at selected times up to 48 h post-injection
of 64Cu-NOTA-F(ab')2 in non-tumour bearing Balb/c mice.
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Table 4.2
Tumour and normal tissue distribution at 24 h post-injection of increasing mass
amounts of 64Cu-NOTA-pertuzumab F(ab')2 fragments* in mice with
subcutaneous SK-OV-3 human ovarian cancer xenografts
Percent injected dose/g (%ID/g)
Tissue 5 µg 50 µg 100 µg 200 µg
Blood 0.6 ± 0.0 0.5 ± 0.2 0.5 ± 0.2 0.5 ± 0.1
Heart 2.3 ± 0.3 2.2 ± 0.4 2.0 ± 0.2 1.9 ± 0.3
Lungs 2.1 ± 0.2 2.1 ± 0.4 1.9 ± 0.2 2.0 ± 0.3
Liver 10.7 ± 1.3 10.9 ± 3.3 9.3 ± 2.1 7.8 ± 1.3
Kidneys 60.0 ± 4.7 65.6 ± 16. 2 54.8 ± 3.9 52.4 ± 12.8
Spleen 11.9 ± 0.9 11.5 ± 5.8 9.0 ± 1.4 7.4 ± 0.4
Stomach 2.8 ± 2.9 1.2 ± 0.2 1.1 ± 0.1 1.1 ± 0.1
Intestines 2.1 ± 0.1 2.0 ± 0.4 1.7 ± 0.2 1.8 ± 0.4
Muscle 0.4 ± 0.0 0.4 ± 0.2 0.4 ± 0.1 0.4 ± 0.1
Bone 1.9 ± 0.2 1.6 ± 0.5 1.6 ± 0.2 1.4 ± 0.2
Skin 1.0 ±0.4 1.0 ± 0.6 1.5 ± 1.0 1.5 ± 0.8
Tumour 8.2 ± 2.6 9.8 ± 5.1 8.2 ± 2.1 5.8 ± 1.3
Values shown are mean ± SD (n = 4).
*Mice were intravenously administered 1-3 MBq of 64Cu-NOTA-pertuzumab F(ab')2 fragments.
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Table 4.3
Radiation absorbed dose projections for 64Cu-NOTA-pertuzumab F(ab')2
fragments in humans
Organ Equivalent dose (mSv/MBq)
Brain 0.001
Breasts 0.002
Gallbladder wall 0.015
Small intestine 0.011
Lower large intestine 0.282
Heart wall 0.019
Kidneys 1.070
Liver 0.092
Lungs 0.010
Muscle 0.009
Ovaries 0.025
Pancreas 0.016
Spleen 0.068
Total body 0.015
*Radiation absorbed dose projections in humans were based on the cumulative number of
disintegrations (N) in source organs in mice obtained from biodistribution studies and were
estimated using OLINDA Ver. 1.0 software. This assumes that the relative organ biodistribution
of 64Cu-NOTA-pertuzumab F(ab')2 in humans will be the same as that in mice.
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4.3.4 MicroPET/CT imaging studies
Representative microPET/CT images of 64Cu-NOTA-pertuzumab F(ab')2 in mice bearing
SK-OV-3 tumours at 24 h p.i. (Figure 4.4A) and 48 h p.i. (Figure 4.4B) showed accumulation in
the tumour but low normal tissue uptake with the exception of the kidneys. Images were
comparable between the two time points. Tumour uptake was visibly diminished in mice that
received excess unlabeled pertuzumab 24 h prior to 64Cu-NOTA-pertuzumab F(ab')2 (Figure
4.4C) or that were injected with non-specific 64Cu-NOTA-hIgG F(ab')2 (Figure 4.4D).
Biodistribution studies at 48 h (Figure 4.4E) revealed a significant 2.2- or 3.1-fold lower tumour
uptake for mice receiving excess pertuzumab or injected with 64Cu-NOTA-hIgG F(ab')2
compared to mice injected with 64Cu-NOTA-pertuzumab F(ab')2 (3.9 ± 1.0 and 2.7 ± 0.5 %ID/g
vs. 8.4 ± 3.4, respectively P<0.05). These results demonstrated that 64Cu-NOTA-pertuzumab
F(ab')2 accumulated specifically in HER2-positive SK-OV-3 tumour xenografts.
MicroPET/CT images of mice bearing BT-474 human BC xenografts revealed
diminished uptake of 64Cu-NOTA-F(ab')2 at 5 days after administration of a loading dose of
trastuzumab (4 mg/kg) compared to baseline images (Figure 4.5A). VOI analyses of the 1-week
time point images revealed a significant decrease in normalized tumour uptake compared to
baseline (0.5 ± 0.4, P<0.05, Figure 4.5B). Tumour size, measured with calipers, decreased
following trastuzumab treatment and mirrored the VOI analysis results (TGI = 0.6 ± 0.2,
P<0.05, Figure 4.5C). It was not possible to determine changes in tumour volume at week 3 in
mice with BT-474 tumours, since only 4 mice were available for measurement of tumour
dimensions and these mice exhibited complete response to trastuzumab which precluded tumour
measurement. Images of mice bearing SK-OV-3 tumours and treated with trastuzumab (4 mg/kg
loading dose then 2 mg/mg weekly for 2 weeks) showed an apparent increase in radiotracer
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uptake at week 1 and 3 (Figure 4.5D). VOI analysis showed up to 1.9-fold increased tumour
uptake of 64Cu-NOTA-F(ab')2 compared to baseline following trastuzumab treatment, but these
values did not reach significance due to variability in this small groups size (1.7 ± 0.7, P=0.137
and 1.9 ± 1.4, P=0.754, Figure 4.5E). Similarly, SK-OV-3 xenografts clearly exhibited a strong
trend towards increased tumour volume over time, despite trastuzumab treatment, but these
values did not reach significance due to variability (TGI = 2.2 ± 1.1, P=0.109 at 1 week, 16.5 ±
11.3, P=0.124 at 3 weeks, Figure 4.5F).
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Figure 4.4. Whole-body microPET/CT images of mice with subcutaneous SK-OV-3 HER2-
overexpressing human ovarian cancer xenografts at 24 h post-injection (p.i.) (A) or 48 h p.i. (B)
of 64Cu-NOTA-pertuzumab F(ab')2 fragments. (C) Images obtained at 24 h p.i. of 64Cu-NOTA-
pertuzumab F(ab')2 with pre-administration of 1 mg of pertuzumab 24 h prior to
radiopharmaceutical injection. (D) Images obtained at 24 h p.i. with 64Cu-labeled nonspecific
hIgG F(ab')2. Tumour xenografts are indicated by the green circle. Also visualized are the
kidneys (blue arrowhead) and bladder/urine (white arrowhead). The HER2 specificity of tumour
uptake of 64Cu-NOTA-pertuzumab F(ab')2 was confirmed by biodistribution studies at 48 h p.i.
(E) showing a significant decrease in tumour uptake of the radiopharmaceutical in mice pre-
administered excess unlabeled pertuzumab to block HER2 or injected with 64Cu-labeled non-
specific hIgG F(ab')2 (*P < 0.05).
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Figure 4.5. (A) MicroPET/CT images at 24 h post-injection (p.i.) of 64Cu-NOTA-pertuzumab
F(ab')2 fragments in NOD/SCID mice with subcutaneous BT-474 human breast cancer
xenografts at baseline and at 1 week after commencing treatment with trastuzumab. (B) The
corresponding changes in BT-474 tumour uptake [percent injected dose/g (%ID/g) normalized
to baseline] of 64Cu-NOTA-pertuzumab F(ab')2 and (C) tumour growth index (TGI). (D)
MicroPET/CT images at 24 h p.i. of 64Cu-NOTA-pertuzumab F(ab')2 fragments in NOD/SCID
mice with subcutaneous SK-OV-3 human ovarian cancer xenografts at baseline and at 1 week
and 3 weeks after commencing trastuzumab treatment. (E) The corresponding changes in SK-
OV-3 tumour uptake of 64Cu-NOTA-pertuzumab F(ab')2 and (F) TGI. Significant differences
compared to baseline values are indicated (*P < 0.05).
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4.4 Discussion
We describe here the construction and characterization of 64Cu-NOTA-pertuzumab
F(ab')2 fragments for PET/CT imaging of the response of HER2-positive tumours to treatment
with trastuzumab. MicroPET/CT with 64Cu-NOTA-pertuzumab F(ab')2 fragments detected
changes in tumour HER2 expression in athymic mice engrafted with s.c. BT-474 human BC
tumours at 1 week after commencing trastuzumab therapy and this was associated with a good
response to treatment with trastuzumab (Figure 4.5A-C). BT-474 tumour xenografts are
sensitive to trastuzumab (32). In contrast, SK-OV-3 human ovarian cancer xenografts which
overexpress HER2 but are trastuzumab-resistant (196), demonstrated a 1.9-fold increased uptake
of 64Cu-NOTA-pertuzumab F(ab')2 at 1 and 3 weeks after commencing trastuzumab treatment,
and this was associated with continued and rapid tumour growth (Figure 4.5D-F). The poor
response of SK-OV-3 cells to trastuzumab has been attributed to the absence of the tumour
suppressor protein Ras homolog member-1, leading to constitutively phosphorylated MAPK
(197). These results agree with those previously reported by our group for trastuzumab-sensitive
MDA-MB-361 human BC xenografts in athymic mice in which microSPECT/CT imaging using
111In-labeled pertuzumab revealed decreased uptake of the imaging probe within 3 days after
starting trastuzumab treatment, with almost complete disappearance of tumour accumulation
visualized by imaging at 3 weeks post-treatment and which was associated with tumour
eradication (110).
To optimize tumour imaging with 64Cu-NOTA-pertuzumab F(ab')2, we studied the effect
of increasing the administered mass over the range of 5 µg to 200 µg, corresponding to a human
mass dose of 5 mg to 400 mg, scaled by body weight (25 g for a mouse vs. 50 kg for a human
female). Dijkers et al. reported that a 50 mg mass amount of 89Zr-labeled trastuzumab was
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optimal for tumour imaging in patients with HER2-positive BC who had not received
trastuzumab (92). Similarly, Mortimer et al. found that pre-administration of 45 mg of
trastuzumab prior to 64Cu-labeled trastuzumab (5 mg) for PET decreased liver uptake in patients
with HER2-positive BC, but did not diminish tumour uptake (96). We did not find a significant
effect of increasing the mass dose of 64Cu-NOTA-pertuzumab F(ab')2 on tumour or normal
tissue biodistribution at 24 h p.i. in athymic mice with s.c. SK-OV-3 tumour xenografts (Table
4.2). Wong et al. similarly reported that there was no difference in tumour or liver uptake at 48
h p.i. of 3 µg or 15 µg of 86Y-CHX-A''-panitumumab F(ab')2 in athymic mice with s.c. EGFR-
positive LS174T human colon cancer xenografts (113). In contrast, van Dijk et al. found that
mass amounts of 50 µg or more of 111In-cetuximab F(ab')2 administered to mice with s.c. FaDu
squamous cell carcinoma xenografts decreased tumour uptake compared to 10 µg or less, but no
differences in liver uptake were noted (114). The inability to identify a mass effect on liver
uptake of 64Cu-NOTA-pertuzumab F(ab')2 may be related to poor recognition of F(ab')2 by FcRn
receptors (198). Tumour uptake was moderately high up to 100 µg of 64Cu-NOTA-pertuzumab
F(ab')2 (Table 4.2) and there was a trend towards lower tumour uptake at 200 µg. Thus, lower
protein amounts may be beneficial and a 50 µg dose was subsequently used for imaging studies.
Tumour uptake of 64Cu-NOTA-pertuzumab F(ab')2 (6-10 %ID/g; Table 4.2) in SK-OV-3
ovarian cancer xenografts at 24 h p.i. was about 3-fold lower than we previously reported for
111In-labeled pertuzumab IgG (34.5 ± 9.2 %ID/g) in MDA-MB-361 human BC xenografts at 72
h p.i.(110). Nonetheless, tumour uptake was specific, since blocking with an excess of
pertuzumab significantly reduced accumulation by 3.7-fold (Figure 4.4C) and non-specific 64Cu-
NOTA-hIgG F(ab')2 exhibited 2.2-fold significantly lower uptake (Figure 4.4D). Lower tumour
uptake of 64Cu-NOTA-pertuzumab F(ab')2 than 111In-labeled pertuzumab IgG was likely due to
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faster elimination of 64Cu-NOTA-pertuzumab F(ab')2 from the blood (t1/2β = 10.4 h; Figure 4.2)
compared to 111In-labeled pertuzumab IgG (t1/2β=228.2 h) (173). Tumour uptake of 64Cu-
NOTA-pertuzumab F(ab')2 in SK-OV-3 xenografts was comparable to other radiolabeled F(ab')2
fragments (7-20 %ID/g) (105,113,199). SK-OV-3 tumours were imaged by microPET/CT at 24
or 48 h p.i. of 64Cu-NOTA-pertuzumab F(ab')2 (Figure 4.4A, B).
The uptake and elimination of 64Cu-NOTA-pertuzumab F(ab')2 by normal organs in non-
tumour bearing Balb/c mice were studied to predict the radiation absorbed doses in humans. The
kidneys exhibited the highest normal organ uptake (>80 %ID/g at 3 h p.i. decreasing by 2-fold
at 48 h p.i.; Figure 4.3). These results were comparable to the kidney uptake found in athymic
mice with SK-OV-3 tumours (65 %ID/g; Table 4.2). Kidney uptake was also similar to that
reported by Smith-Jones et al. for 111In-trastuzumab F(ab')2 in athymic mice with BT-474
human BC xenografts (65 %ID/g at 24 h p.i. decreasing to 45 %ID/g at 48 h p.i) (105).
However, renal uptake of radiolabeled F(ab')2 may be dependent on the protein amino acid
sequence, which is identical for pertuzumab and trastuzumab except for the complementarity-
determining regions (CDRs) (29), since 64Cu-NOTA-panitumumab F(ab')2 exhibited much
lower kidney uptake (<6 %ID/g at 24 h p.i.) in NOD/SCID mice with PANC-1 pancreatic
cancer xenografts (200). Kidney uptake of antibody fragments is thought to be due to charge
interactions of the filtered proteins with renal tubules (201). Radiation doses projected for
administration of 64Cu-NOTA-pertuzumab F(ab')2 to humans revealed that the kidneys would
receive the highest dose (1 mSv/MBq) while the total body dose would be 0.015 mSv/MBq
(Table 4.3). The total body dose for 64Cu-NOTA-pertuzumab F(ab')2 is reduced by 3.3-fold
compared to 111In-labeled pertuzumab IgG (0.05 mSv/MBq) (173), but the dose to the kidneys is
increased by 3-fold (1 mSv/MBq vs. 0.33 mSv/MBq). Since Phase I clinical trials of 64Cu-
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labeled trastuzumab for PET imaging of HER2-positive BC have used administered amounts as
low as 115 MBq (95), we project that the total body dose for a single injection of 64Cu-NOTA-
pertuzumab F(ab')2 would be 1.7 mSv at this amount, and for the three administrations used for
the baseline, 1 week and 4 weeks imaging studies in the PETRA trial, would be 5.1 mSv. This
compares to 17 mSv for 111In-labeled pertuzumab, thus the radiation dose to patients would be
reduced by more than 3-fold. The dose to the kidneys assuming three administrations of 64Cu-
NOTA-pertuzumab F(ab')2 (115 MBq each) would be 115 mSv, which is not clinically
significant, since there is <5% risk of renal dysfunction at kidney radiation doses, which are
200-fold higher than projected for 64Cu-NOTA-pertuzumab (Fab')2 (202). In conclusion, we
have demonstrated that 64Cu-NOTA-pertuzumab F(ab')2 fragments specifically target HER2 on
BC tumour xenografts in NOD/SCID mice and that microPET/CT can detect changes in HER2
expression associated with response to trastuzumab treatment. Organ absorbed doses associated
with 64Cu-NOTA-pertuzumab F(ab')2 fragments in humans are projected to be lower than those
of 111In-BzDTPA-pertuzumab with the exception of the kidneys and lower large intestine.
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CHAPTER 5:
Summary and Future Directions
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5.1 Summary of key findings
The overall conclusions of the research described in this thesis are:
1) A kit for the preparation of 111In-BzDTPA-pertuzumab was manufactured and
reproducibly met established quality specifications suitable for preparing this
radiopharmaceutical for a Phase I/II clinical trial in humans.
2) 111In-BzDTPA-pertuzumab prepared from the kit demonstrated favourable preclinical
pharmacokinetic, biodistribution, toxicity and radiation dosimetry properties that
supported advancement of this agent to a Phase I/II clinical trial.
3) PET/CT imaging with 64Cu-NOTA-pertuzumab F(ab')2 in athymic mice bearing human
breast cancer xenografts demonstrated specific tumour accumulation and detected
changes in tumour HER2 expression which differentiated continued tumour growth or
regression.
4) The total body radiation absorbed dose (mSv/MBq) of 64Cu-NOTA-pertuzumab F(ab')2
to humans predicted from biodistribution studies in mice was 3.3-fold lower than that
associated with 111In-BzDTPA-pertuzumab.
Chapter 2 described the formulation of a kit for the preparation of 111In-labeled
pertuzumab suitable for human administration to support the advancement of a Phase I/II
clinical trial evaluating the ability of 111In-labeled pertuzumab to image trastuzumab-mediated
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changes in HER2 expression. Nine lots of the kit containing BzDTPA-pertuzumab were
manufactured and reproducibly passed quality control specifications that tested for protein
concentration, volume, pH, appearance, sterility, endotoxins, chelator substitution level, purity
and homogeneity, HER2 binding, and radiolabeling efficiency (≥ 90%). The kit was shown to
be stable for at least 4 months post-manufacturing as assessed by monthly quality control
testing. 17 lots of the final radiopharmaceutical, 111In-BzDTPA-pertuzumab injection, prepared
from the kit were also tested against and passed quality specifications for specific radioactivity,
pH, radiochemical purity, radionuclidic purity, appearance and sterility. Furthermore, raw and
intermediate materials used in manufacturing the kit were assayed and passed for identity and
quality. The stability of 111In-BzDTPA-pertuzumab injection in plasma was assessed by SE-
HPLC and showed no loss of 111In to transferrin. Although not required for the Clinical Trial
Application to Health Canada, an imaging study with SPECT/CT was conducted showing high
and specific tumour uptake of 111In-BzDTPA-pertuzumab injection (34.5 ± 18.4 %ID/g). The
results of these studies were included in the chemistry, manufacturing and controls component
of a Clinical Trial Application, which received Health Canada approval. This chapter
demonstrates how GMP can be implemented in an academic laboratory and serves as an
example to other laboratories wishing to translate their molecular imaging probes to the clinic.
Chapter 3 evaluated the pharmacokinetics, normal tissue distribution, radiation
dosimetry and acute toxicity of 111In-BzDTPA-pertuzumab to advance this agent to a Phase I/II
clinical trial. Blood pharmacokinetics were determined in non-tumour bearing Balb/c mice
administered i.v. with 111In-BzDTPA-pertuzumab, and results showed biphasic elimination with
a distribution half-life of 3.8 h and an elimination half-life of 228.2 h. Biodistribution analysis
revealed that the radiopharmaceutical was distributed mainly in the blood, heart, lungs, liver,
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kidneys, and spleen. Estimated whole-body radiation absorbed dose to humans was 0.05
mSv/MBq, corresponding to a total of 16.8 mSv for three separate injections of 111In-BzDTPA-
pertuzumab (111 MBq) planned for the Phase I/II trial. To assess acute toxicity, non-tumour
bearing mice were injected with a dose of 111In-BzDTPA-pertuzumab that corresponded to 23-
times the planned human radioactivity dose and 10-times the planned protein dose on a MBq/kg
and mg/kg basis, respectively. Slight changes in Hb and SCr levels were observed with
administration of multiples of the human dose in healthy Balb/c mice but no histopathological
abnormalities were noted in any tissues. No significant differences in body mass between mice
injected with 111In-BzDTPA-pertuzumab or control mice administered Sodium Chloride
Injection USP were observed. The results of these studies supported the regulatory approval by
Health Canada to investigate 111In-BzDTPA-pertuzumab in a Phase I/II clinical trial. As
discussed in Section 1.7, a myriad of preclinical studies evaluating novel molecular imaging
probes have been developed but only a handful of these probes have entered first-in-human
clinical trials. Few novel molecular imaging probes may be translated to the clinic because
investigators do not completely understand the data that needs to be generated and presented to
regulatory authorities. This chapter contributes to the molecular imaging research field by
outlining the preclinical studies required by regulatory authorities to bring a radiolabeled
molecular imaging agent to the clinic. A toxicity study conducted in a nonrodent species was not
required by Health Canada to advance this agent since 111In-BzDTPA-pertuzumab was prepared
from an approved pharmaceutical product (pertuzumab). This suggests that predicating a
molecular imaging agent on an approved pharmaceutical may reduce the number of tests/studies
required for a Clinical Trial Application which may be one translation approach for academic
institutions that are more limited in resources.
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Chapter 4 describes the development and evaluation of a second generation imaging
probe, 64Cu-NOTA-pertuzumab F(ab')2, for detecting HER2 changes in response to trastuzumab
using PET/CT imaging. The effect of increasing mass (5-200 µg) of 64Cu-NOTA-pertuzumab
F(ab')2 was investigated since clinical studies have demonstrated that normal tissue (particularly
liver) accumulation of radiolabeled antibodies can be reduced by increasing the mass dose of the
radiopharmaceutical. No significant effect of mass was seen on the biodistribution of 64Cu-
NOTA-pertuzumab F(ab')2 in SK-OV-3 tumour-bearing mice suggesting that the strategy of
increased mass doses used with radiolabeled intact antibodies to improve biodistribution by
binding FcRn receptors in normal tissues is less useful for modulating the normal tissue
distribution of F(ab')2. In SK-OV-3 tumour bearing mice, tumours were visualized by
microPET/CT at 24 h and 48 h p.i. Images revealed uptake in tumours was low when a non-
specific 64Cu-labeled hIgG F(ab')2 was injected (2.7 ± 0.5 %ID/g) and tumour uptake of 64Cu-
NOTA-pertuzumab F(ab')2 was blocked by 1 mg of preadministered unlabeled pertuzumab (2.7
± 0.5 vs. 8.4 ± 3.4%ID/g), demonstrating that tumour uptake of 64Cu-NOTA-pertuzumab
F(ab')2 was specific. The predicted whole-body radiation absorbed dose of 64Cu-NOTA-
pertuzumab F(ab')2 in humans was 0.015 mSv/MBq, based on biodistribution studies in non-
tumour bearing mice. This dose was 3.3-fold lower than the total body dose for 111In-BzDTPA-
pertuzumab. Phase I clinical trials of 64Cu-labeled trastuzumab for PET imaging of HER2-
positive BC have administered radioactivities as low as 115 MBq (95) and the projected total
body dose for a single injection of 64Cu-NOTA-pertuzumab F(ab')2 fragments would be 1.7 mSv
at this radioactivity amount, which is lower than the projected total body dose of 5.1 mSv for a
111 MBq injection of 111In-BzDTPA-pertuzumab. PET/CT imaging and analysis of mice treated
with trastuzumab showed 2-fold decreased uptake of 64Cu-NOTA-pertuzumab F(ab')2 in BT-474
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tumours after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in
SK-OV-3 tumours after 3 weeks of trastuzumab, consistent with tumour response and
resistance, respectively. These results suggest that PET/CT imaging using 64Cu-NOTA-
pertuzumab F(ab')2 may be useful during trastuzumab therapy for detection of a therapeutic
response. The advantage of this PET probe over the 111In-BzDTPA-pertuzumab probe for
SPECT is the generally reduced radiation absorbed doses to organs due to the faster clearing of
the F(ab')2 fragments probe as well as the shorter half-life of the 64Cu isotope. Imaging with
64Cu-NOTA-pertuzumab F(ab')2 can also take advantage of the higher sensitivity and
quantification abilities of PET over SPECT.
5.2 Future directions
Current clinical studies investigating radiolabeled imaging probes are in Phase I and
therefore have mostly focused on testing the feasibility of imaging HER2-positive lesions
(89,92,95,96). In order for radiolabeled imaging probes to monitor response to HER2-targeted
therapies, future studies should seek to define a quantitative relationship between tumour uptake
(e.g. SUV) and response, as has been established with 18F-FDG to monitor response to
chemotherapy (203). An increase of >25% in 18F-FDG SUV is classified as progressive
metabolic disease and a decrease of >25% compared to a baseline imaging study is considered a
metabolic response. Gebhart et al. combined this quantitative 18F-FDG imaging parameter with
HER2-positivity shown by 89Zr-trastuzumab PET imaging to establish thresholds that had a
PPV and NPV of 100% each for selecting patients who would/would not benefit from the
antibody-drug immunoconjugate (ADC), trastuzumab-DM1 (T-DM1) (93). Therefore, such
quantitative analyses will be crucial for turning these molecular imaging probes into meaningful
predictive tools of response to HER2-targeted therapies.
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The imaging probes 111In-BzDTPA-pertuzumab and 64Cu-NOTA-pertuzumab F(ab')2
described in this thesis are currently proposed as molecular imaging tools to predict and monitor
response to trastuzumab therapy. The advantage of these probes over radiolabeled trastuzumab-
based probes is that pertuzumab does not compete with trastuzumab for binding to HER2 since
it binds to a different epitope, and hence the rationale for combining these two antibodies
therapeutically for treatment of HER2-positive BC. Since the 2012 U.S. FDA and Health
Canada approval of pertuzumab to be optionally added to a trastuzumab regimen for HER2-
overexpressing BC, the utility of imaging with a pertuzumab-based probe may not be feasible,
since the presence of therapeutic pertuzumab may interfere with tumour uptake (63). In the
Phase I/II PETRA clinical trial, this issue was circumvented by delaying pertuzumab therapy
such that pertuzumab therapy was initiated with the second cycle of trastuzumab (21 days after
the first trastuzumab dose). This strategy allows ample time to conduct a baseline and at least
one post-trastuzumab follow-up imaging scan to monitor trastuzumab response without denying
patients pertuzumab therapy. McLarty et al.’s study in MDA-MB-361 tumour-bearing mice
demonstrated that the reduction in tumour uptake of 111In-BzDTPA-pertuzumab 3 days after
trastuzumab treatment predicted tumour regression at 3 weeks. In Chapter 4, I showed that a
reduction in tumour uptake of 64Cu-NOTA-pertuzumab F(ab')2 mirrored the tumour volume
regression in BT-474 xenografts one week after trastuzumab treatment, and a trend in increased
uptake of 64Cu-NOTA-pertuzumab F(ab')2 at one week in SK-OV-3 trastuzumab resistant
tumours reflected tumour progression at 3 weeks. These results suggest that a single imaging
follow-up study after baseline imaging may be enough to detect tumour response. Future clinical
studies investigating probes for detecting response to trastuzumab should optimize the interval
159
between trastuzumab administration and follow-up radionuclide imaging that would show
reduced tracer uptake as an indicator of early response.
These pertuzumab-based imaging probes identify HER2-positive lesions and thus could
be used to select patients for various HER2-targeted therapies including trastuzumab, T-DM1 or
pertuzumab. T-DM1, an ADC, retains the therapeutic mechanisms of action of trastuzumab,
while delivering a cytotoxic agent to HER2-overexpressing tumour cells in which it is bound
and internalized. As mentioned in Chapter 1, clinical studies with T-DM1 have demonstrated
that many patients continue to progress with this therapy. Resistance to T-DM1 is thought to
relate to mechanisms that do not allow for adequate accumulation of the DM1 metabolite in
cancer cells, thus meaning the drug is unable to achieve the necessary concentrations to evoke
cell death (204). Low HER2 expression and poor internalization of the HER2-T-DM1 complex
have been proposed as possible mechanisms of resistance, among others (204-206). The ability
of T-DM1 to target and internalize HER2 is driven by trastuzumab and thus, serial imaging with
pertuzumab-based probes to detect whether HER2 expression decreases (HER2 internalization)
following T-DM1 administration, could provide useful information for identifying resistance. T-
DM1 therapy studies in mice with HER2-overexpressing tumour xenografts that are sensitive
and resistant to trastuzumab and imaged with 111In-BzDTPA-pertuzumab or 64Cu-NOTA-
pertuzumab F(ab')2 could be conducted.
It was shown in Chapter 4 that 64Cu-NOTA-pertuzumab F(ab')2 was accumulated and
retained in the kidneys. This resulted in a higher radiation absorbed dose to the kidneys than to
other organs. Future studies could attempt to reduce this relatively high kidney uptake. Renal
retention has been attributed to the positive charges of the radioimmunoconjugates that interact
with the negatively charged surface of proximal tubular cells and result in electrostatic
160
interactions and reabsorption by endocytosis (124). One approach to reducing renal uptake is to
administer cationic amino acids prior to i.v. administration of the radioimmunoconjugates. Behr
et al. demonstrated that i.p. and oral administrations of arginine or lysine prior to injection of
111In-, 99mTc, 188Re, 88Y, or 125I- labeled Fab' and F(ab')2 fragments in mice bearing human GW-
39 colon carcinoma xenografts reduced renal uptake and radiation absorbed dose to the kidneys
by up to 80% without affecting tumour uptake of the radioimmunoconjugates (201). In a Phase I
clinical study using a cross-over design, patients with somatostatin receptor-positive
neuroendocine tumours were infused with lysine and arginine prior to administration of 86Y-
DOTA(0)-d-Phe(1)-Tyr(3)-octreotide and imaged by PET to obtain biodistribution data for
calculating absorbed doses to tissues (207). An infusion of 26.4 g of lysine and arginine resulted
in a 21% decrease in renal uptake of 86Y-DOTA(0)-d-Phe(1)-Tyr(3)-octreotide which reduced
the radiation absorbed dose to the kidney. Therefore, preclinical studies investigating the effect
of preadministration of lysine and/or arginine, using a potential regimen of 4 x 2000 µg/g body
weight of the mouse at 30 min pre-, and 1 , 2 and 3 h post-injection (based on the study by Behr
et al.), on biodistribution of 64Cu-NOTA-pertuzumab F(ab')2 could be performed to improve the
clinical utility of this probe.
The NOTA chelator used to attach 64Cu to the pertuzumab F(ab')2 fragments may also
partly contribute to the high kidney retention. In a study comparing 64Cu-NOTA-TRC105 (a
chimeric antibody that targets CD105) and 64Cu-DOTA-TRC105 in mice with 4T1 tumours,
kidney uptake for the DOTA conjugate was higher than for the NOTA conjugate at 24 h p.i.;
however, at 48 h p.i., the renal uptake of the NOTA conjugate was unchanged whereas the
kidney uptake of the DOTA conjugate decreased to levels lower than those with the NOTA
conjugate, demonstrating the persistent retention of radioactivity in the kidney when NOTA was
161
used as the chelator (208). This may be due to higher in vivo stability of NOTA over the DOTA
chelator leading to radioactivity retention in the renal tubular cells. Free 64Cu accumulates in the
liver after being carried from the blood by albumin and transcuprin (209). Dissociation of 64Cu
from DOTA in this study is supported by the 4-fold higher liver uptake of the DOTA conjugate
at 24 h p.i. compared to the NOTA conjugate. Furthermore, whereas 4T1 tumour accumulation
of the conjugates is comparable at 24 h p.i., tumour uptake appears to decrease by
approximately a third with the DOTA conjugate at 48 h p.i. whereas no change in uptake is
observed with the NOTA conjugate, suggesting loss of 64Cu from DOTA. Future studies could
try novel and improved 64Cu chelators for attaching to pertuzumab F(ab')2 fragments for
improved biodistribution. For example, the DOTA derivative, DOTHA2, which bears N-methyl-
hydroxamic acid donor groups as pendant arms, has demonstrated rapid 64Cu chelation within 5
min at room temperature in a wide range of concentrations, pH and counterions (210). It was
recently demonstrated that in a biodistribution study comparing 64Cu-DOTHA2- and 64Cu-
NOTA-PEG-RM26 (a peptide targeting gastrin-releasing peptide receptors in prostate cancer),
kidney uptake at 60 min p.i. with the DOTHA2 conjugate was reduced by 2-fold compared to
that with the NOTA conjugate (211). Similar comparative studies could be conducted with 64Cu-
labeled pertuzumab F(ab')2.
Lastly, testing of 64Cu-labeled pertuzumab F(ab')2 in the clinic would be interesting
given that PET/CT imaging of this probe could be quantitative, and therefore could help to
establish tumour uptake thresholds that delineate trastuzumab responders from non-responders
Most of studies required for a CTA to translate this probe to the clinic have already been
conducted. A study investigating the acute toxicity of 64Cu-labeled pertuzumab F(ab')2 in mice
would need to be performed. A kit formulated under GMP would also need to be developed.
162
Molecular imaging has the potential to revolutionize breast cancer management
strategies by helping to optimize drug development (imaging of drug effects) and facilitating
patient stratification for targeted therapies. This thesis is a small contribution to further
extending the reach of the nuclear medicine field in oncology. Numerous promising novel
molecular imaging agents are currently being studied preclinically; their clinical translation will
be facilitated by the improved understanding of the regulatory hurdles that must be overcome in
order to introduce these agents into clinical oncology practice.
163
APPENDICES
164
APPENDIX A:
UV Assay to measure NOTA substitution on pertuzumab F(ab’)2 fragments
Substitution level can be measured by preparing standard curves of UV absorbance at 280 nm
vs. increasing concentrations of (A) pertuzumab F(ab')2 and, (B) NOTA. The absorbance
contribution from F(ab')2 can be subtracted from the absorbance of the conjugate as determined
by the Bradford assay and referencing a standard curve (A). The remaining absorbance
contribution can be compared to a standard curve (B) to give the concentration of NOTA. The
ratio of NOTA concentration to F(ab')2 concentration gives the substitution level.
A
B
165
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