Panitumumab modified with metal chelating polymers

(MCPs) complexing indium‐111 and lutetium-177 as

theranostics for pancreatic cancer

By

Sadaf Aghevlian

A thesis is submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences

University of Toronto

© Copyright by Sadaf Aghevlian, 2019

ii

Panitumumab modified with metal chelating polymers (MCPs) complexing indium-111

and lutetium-177 as theranostics for pancreatic cancer

Sadaf Aghevlian

Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences

University of Toronto

2019

Abstract

The epidermal growth factor receptor (EGFR) is overexpressed in more than 90% of pancreatic

cancer (PnCa) patients. A metal-chelating polymer (MCP) with on average 13 DOTA

(tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing the β-particle emitter,

177Lu and Auger electron-emitter, 111In and 10 polyethylene glycol (PEG) chains was conjugated

to monoclonal antibody, panitumumab to target EGFR on PANC-1 human PnCa cells. Linking

panitumumab to MCPs enabled dual labeling with 111In and 177Lu at a higher SA (55.9± 4.8

MBq/μg and 20.5± 4.2 MBq/μg) compared to panitumumab-DOTA (4.4± 0.3 MBq/μg and 2.2±

0.3 MBq/μg), with preserved EGFR binding (2.2 ± 0.6 nmol/L and 1.0 ± 0.4 nmol/L;

respectively) and comparable tumour (6.9 ± 1.3% ID/g and 6.6 ± 3.3%ID/g) and normal tissue

localization except for the liver uptake which was 3-fold higher for panitumumab-MCP at 72 h

post injection (p.i.) in NOD/SCID mice with s.c. PANC-1 xenografts as confirmed by

microSPECT/CT imaging. 177Lu-labeled radioimmunoconjugates (RICs) were more effective for

killing PANC-1 cells in vitro than 111In-RICs with no significant differences between

panitumumab-DOTA and panitumumab-MCP. For therapy, NOD/SCID mice or NRG mice with

s.c. PANC-1 tumors were administered three amounts (10 MBq; 10 g) of panitumumab-MCP-

111In or panitumumab-DOTA-111In separated by 3 weeks or a single amount (6 MBq; 10 g) of

panitumumab-MCP-177Lu or panitumumab-DOTA-177Lu, respectively. Tumour growth was

assessed by a tumour growth index (TGI). Panitumumab-MCP-111In or panitumumab-DOTA-

111In inhibited tumour growth in NOD/SCID mice (TGI at 43 days = 3.9 ± 0.3 and 3.0 ± 0.4,

respectively; P> 0.05) compared to normal saline and panitumumab treated mice (TGI = 9.8 ±

1.6, 9.9 ± 1.4, respectively; P>0.5). Similarly, panitumumab-MCP-177Lu and panitumumab-

iii

DOTA-177Lu inhibited tumour growth in NRG mice (TGI at 33 days = 2.5 ± 0.3 and 1.8± 0.3;

respectively; P> 0.05) compared to normal saline and panitumumab treated mice (5.8 ± 0.9 vs.

6.1 ± 2.7, respectively; P>0.05). 177Lu-RICs deposited more absorbed doses in tumours (2-4 fold)

and normal organs compared to 111In-RICs. Low absorbed doses in the normal organs for 111In

allow for dose escalation. Panitumumab conjugates labeled with 177Lu or 111In are promising RIT

agents for treatment of EGFR positive cancers.

iv

Acknowledgments

First and foremost I’d like to convey my utmost gratitude to my supervisor Dr. Raymond Reilly

for the lifetime opportunity to come to Canada and peruse my doctoral studies under his

supervision. I am sincerely thankful for not only his scientific contributions to develop and

complete my research project, but also for being such a wonderful person and great role model.

I would also like to thank the members of my advisory committee, Dr. Mitchell Winnik, Dr.

David Hedley, and Dr. Stephan Angers for their helpful suggestions, comments and

encouragements. Thank you for challenging me to think critically about my research.

I would like to thank my past and present colleagues, especially Dr. Conrad Chan, Dr. Zhongli

Cai, Dr. Yijie Lu, Ms. Deborah Scollard and Ms. Teesha Kemal for sharing their knowledge and

friendship.

I wish to extend my deepest gratitude to my family, my brother Sohrab, my parents Reza and

Maryam and my dear uncle Khalil for their unwavering love and support throughout my whole

life. I am forever blessed to have you in my life. And a special thank you to my husband

Mehrdad for his love, patience and care. Thank you for believing in me and supporting all of my

hopes and dreams.

v

Table of contents Contents

Abstract .............................................................................................................................................. ii

Acknowledgments............................................................................................................................. iv

Table of contents ................................................................................................................................ v

List of tables ...................................................................................................................................... ix

List of figures .................................................................................................................................... xi

List of abbreviations ....................................................................................................................... xiv

Chapter 1: Introduction ...................................................................................................................... 1

1. Introduction ................................................................................................................................... 2

1.1 Current status in the detection, diagnosis and treatment of pancreatic cancer ....................... 2

1.1.1 Pancreatic cancer epidemiology and etiology ................................................................. 2

1.1.2 Detection, diagnosis and staging of PnCa ....................................................................... 2

1.2 PnCa biology .......................................................................................................................... 5

1.2.1 The genomic landscape of PnCa ..................................................................................... 5

1.2.2 PnCa stem cells ............................................................................................................... 7

1.2.3 The tumour microenvironment ....................................................................................... 7

1.2.4 Circulating tumour cells .................................................................................................. 9

1.3 Treatment of pancreatic cancer ............................................................................................... 9

1.3.1 Surgery ............................................................................................................................ 9

1.3.2 Chemotherapy ............................................................................................................... 10

1.4 Diagnostic imaging modalities for PnCa .............................................................................. 19

1.4.1 Contrast-enhanced abdominal ultrasound ..................................................................... 19

1.4.2 MDCT ........................................................................................................................... 19

1.4.3 Endoscopic Ultrasound Guided Fine Needle Aspiration (EUS/EUS-FNA) ................. 20

1.4.4 MRI ............................................................................................................................... 20

1.4.5 Nuclear medicine imaging ............................................................................................ 21

1.4.6 Summary of clinical roles for imaging in PnCa ............................................................ 32

1.5 Therapeutic targets for PnCa ................................................................................................ 34

1.6 Radioimmunotherapy of PnCa by targeting EGFR .............................................................. 40

1.6.1 Types of radiation used in radioimmunotherapy .......................................................... 41

1.6.2 Clinical trials with RICs ................................................................................................ 55

1.7 Increasing the specific activity (SA) .................................................................................... 58

1.7.1 Dendrimer ..................................................................................................................... 59

1.7.2 Metal Chelating Polymer (MCP) .................................................................................. 62

vi

1.8 Hypothesis ............................................................................................................................ 65

1.9 Objective and Aims .............................................................................................................. 65

Chapter 2: Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111In

and 177Lu – An EGFR-Targeted Theranostic for Pancreatic Cancer ............................................... 66

2. Abstract ....................................................................................................................................... 68

2.1 Introduction .......................................................................................................................... 71

2.2 Materials and Methods ......................................................................................................... 73

2.2.1 Cell Culture and Tumour Xenografts ............................................................................ 73

2.2.2 Radioimmunoconjugates (RICs) ................................................................................... 74

2.2.3 Measurement of Hydrodynamic Radius ....................................................................... 76

2.2.4 EGFR Immunoreactivity ............................................................................................... 77

2.2.5 Stability of the RICs ...................................................................................................... 78

2.2.6 Imaging and Biodistribution Studies............................................................................. 79

2.3 Statistical Analysis ............................................................................................................... 79

2.4 Results .................................................................................................................................. 80

2.4.1 Radioimmunoconjugates ............................................................................................... 80

2.4.2 Hydrodynamic Radius................................................................................................... 84

2.4.3 EGFR Immunoreactivity ............................................................................................... 87

2.4.4 Radioimmunoconjugate Stability .................................................................................. 89

2.4.5 Biodistribution and Imaging Studies............................................................................. 93

2.5 Discussion ............................................................................................................................. 97

2.6 Conclusions ........................................................................................................................ 101

Chapter 3: Radioimmunotherapy of PANC-1 Human Pancreatic Cancer Xenografts in NRG

Mice with Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 177Lu 102

3. Abstract ..................................................................................................................................... 104

3.1 Introduction ........................................................................................................................ 106

3.2 Materials and Methods ....................................................................................................... 108

3.2.1 Cell culture and tumour xenografts ............................................................................. 108

3.2.2 Radioimmunoconjugates ............................................................................................. 108

3.2.3 In vitro cytotoxicity studies ......................................................................................... 108

3.2.4 Subcellular distribution and microdosimetry .............................................................. 109

3.2.5 Evaluation of normal tissue toxicity ........................................................................... 110

3.2.6 Radioimmunotherapy studies ...................................................................................... 111

3.2.7 Tumour and normal organ dosimetry.......................................................................... 111

3.3 Statistical analysis............................................................................................................... 112

vii

3.4 Results ................................................................................................................................ 112

3.4.1 In vitro cytotoxicity studies ......................................................................................... 112

3.4.2 Subcellular localization and microdosimetry .............................................................. 115

3.4.3 Evaluation of normal tissue toxicity ........................................................................... 119

3.4.4 Radioimmunotherapy studies ...................................................................................... 123

3.4.5 Tumour and normal organ dosimetry.......................................................................... 123

3.5 Discussion ........................................................................................................................... 128

3.6 Conclusion .......................................................................................................................... 132

3.7 Acknowledgements ............................................................................................................ 133

Chapter 4: Comparison of Auger Electron-Emitting 111In- or -Particle-Emitting 177Lu

Complexed to Panitumumab for Radioimmunotherapy of EGFR-Positive PANC-1 Human

Pancreatic Cancer Xenografts in NOD/SCID and NRG Mice ...................................................... 134

4. Abstract ..................................................................................................................................... 136

4.1 Introduction ........................................................................................................................ 138

4.2 Materials and methods ........................................................................................................ 139

4.2.1 Cell Culture and tumour xenografts ............................................................................ 139

4.2.2 Radioimmunoconjugates (RICs) ................................................................................. 140

4.2.3 Clonogenic Survival (CS) ........................................................................................... 140

4.2.4 Assessment of DNA damage ...................................................................................... 141

4.2.5 Subcellular distribution and Cellular Dosimetry ........................................................ 141

4.2.6 Evaluation of Normal Tissue Toxicity ........................................................................ 142

4.2.7 Radioimmunotherapy (RIT) Studies ........................................................................... 143

4.2.8 Tumour and normal organ dosimetry.......................................................................... 143

4.3 Statistical Analysis ............................................................................................................. 144

4.4 Results ................................................................................................................................ 144

4.4.1 Clonogenic Survival (CS) ........................................................................................... 144

4.4.2 Assessment of DNA damage ...................................................................................... 146

4.4.3 Subcellular distribution and cellular dosimetry .......................................................... 148

4.4.4 Evaluation of Normal Tissue Toxicity ........................................................................ 153

4.4.5 Radioimmunotherapy (RIT) studies............................................................................ 155

4.4.6 Tumour and Normal Organ Dosimetry ....................................................................... 157

4.5 Discussion ........................................................................................................................... 160

4.6 Conclusion .......................................................................................................................... 164

4.7 Acknowledgments .............................................................................................................. 164

Chapter 5: Summary and Future Directions .................................................................................. 165

viii

5 Thesis Conclusion and Summary of Findings .......................................................................... 166

5.1.1 Chapter 2 ..................................................................................................................... 167

5.1.2 Chapter 3 ..................................................................................................................... 168

5.1.3 Chapter 4 ..................................................................................................................... 169

5.1.4 Future directions ......................................................................................................... 171

6 Appendices ................................................................................................................................ 174

6.1 Appendix A: Therapeutic efficacy of the 111In- and 177Lu-labeled RICs side by side ....... 175

6.1.1 Appendix A1. Clonogenic survival ............................................................................. 176

6.1.2 Appendix A2: DNA damage assessment using γ-H2AX assay .................................. 177

6.1.3 Appendix A3: Absorbed dose in the nucleus of PANC-1 cells for 111In- or 177Lu-

RICs…………. ....................................................................................................................... 178

6.1.4 Appendix A4: Toxicity study ...................................................................................... 179

6.1.5 Appendix A5: RIT study with 177Lu-RICs .................................................................. 180

6.1.6 Appendix A6: RIT study with 111In-RICs ................................................................... 181

6.1.7 Appendix A7: Tumour and normal organs dosimetry ................................................ 182

6.2 Appendix B. Immunohistochemical staining of PANC-1 xenografts obtained from

NOD/SCID mice and NRG mice for characterization of the tumour microenvironment .......... 183

References ...................................................................................................................................... 185

ix

List of tables

Table 1-1. TNM staging system for PnCa. ..................................................................................... 4

Table 1-2. Physical properties of some positron emitting radionuclides used in PET imaging. .. 25

Table 1-3 Physical properties of some of radionuclide used in SPECT imaging. ........................ 30

Table 1-4. Potential molecular targets with frequency of genetic aberration on PnCa and their

targeted agents. ............................................................................................................................. 37

Table 1-5. Phase II-III clinical trials with targeted therapies in PnCa. ......................................... 39

Table 1-6. α-particle emitters for conjugation to mAbs for RIT of cancer. .................................. 42

Table 1-7. Auger electron emitters for conjugation to mAb for RIT of cancer. ........................... 46

Table 1-8. β-particle emitters for conjugation to mAb for RIT of cancer. ................................... 48

Table 1-9. Ongoing clinical trials for cancer treatment using 177Lu labeled RICs and

radiopeptides. ................................................................................................................................ 50

Table 2-1. Labelling Efficiency and Specific Activity of Panitumumab-MCP and Panitumumab-

DOTA with 111In or 177Lu. ............................................................................................................ 83

Table 2-2. Hydrodynamic radii and retention times for PEG and protein standards by size

exclusion chromatography ............................................................................................................ 86

Table 3-1. Absorbed doses in the nucleus of PANC-1 cells by panitumumab-MCP-177Lu ....... 118

Table 3-2. Cumulative radioactivity in source organs for panitumumab-MCP-177Lu injected i.v.

in NRG mice with s.c. PANC-1 tumour xenografts. .................................................................. 126

Table 3-3. Estimated radiation absorbed doses in the tumour and normal organs in NRG mice

with s.c. PANC-1 xenografts injected with panitumumab-MCP-177Lu ...................................... 127

Table 4-1. Cumulative radioactivity in the source component after incubation of PANC-1 cells

with 1.2 MBq of 111In-labeled RICs in the absence or presence of 50-fold excess unlabeled

panitumumab for 24 h at 37 ᵒC. .................................................................................................. 151

Table 4-2. Cumulative radioactivity in the source component after incubation of PANC-1 cells

with 1.2 MBq of panitumumab-DOTA-177Lu in the absence or presence of 50-fold excess

unlabeled panitumumab for 24 h at 37 ᵒC. .................................................................................. 152

Table 4-3. Estimated absorbed doses in the tumour and normal organs in mice with s.c. PANC-1

xenografts injected with 111In- or 177Lu-RICs. ............................................................................ 159

Table S 1. Absorbed dose in the nucleus following incubation of cells with 1.2 MBq of 111In-

RICs in the absence or presence of excess of unlabeled panitumumab for 24 h at 37oC………178

x

Table S 2. Absorbed dose in the nucleus following incubation of cells with 1.2 MBq of 177Lu-

RICs in the absence or presence of excess of unlabeled panitumumab for 24 h at 37oC …...…178

Table S 3. Estimated absorbed doses in the tumour and normal organs in mice with s.c. PANC-1

xenografts injected with 111In- or 177Lu-RICs based on biodistribution study at different time

points……………………………………………………………………………………………159

Table S 4. Staining of various antigens in PANC-1 tumour sections………………….…….…183

xi

List of figures

Figure 1-1. Signaling pathways with mutation in PnCa. ................................................................ 6

Figure 1-2. Methylation of deoxyuridine monophosphate to deoxythymidine monophosphate by

thymidylate synthase. .................................................................................................................... 11

Figure 1-3. The chemical structure of gemcitabine. ..................................................................... 13

Figure 1-4. Schematic structure of Nab-paclitaxel or Abraxane.. ................................................ 16

Figure 1-5. Schematic structure of Irinotecan liposome injection (ONIVYDE).. ........................ 18

Figure 1-6. PET imaging system and types of coincidences in PET. ........................................... 23

Figure 1-7. Composition of the SPECT camera. .......................................................................... 28

Figure 1-8. Potential targets in pancreatic tumourigenesis. .......................................................... 35

Figure 1-9. The chemical structure of 177Lu-DOTATATE ........................................................... 52

Figure 1-10. Types of radiation used in RIT................................................................................. 54

Figure 1-11. The protocol schema of RIT with90Y-hPAM4 . ....................................................... 56

Figure 1-12. Three main parts of a dendrimer. ............................................................................. 59

Figure 1-13. The structure of a dendrimer with multiple surface functional groups .................... 60

Figure 1-14. The structure of a metal chelating polymer. ............................................................. 63

Figure 2-1. Chemical structure of the hydrazino nicotinamide metal chelating polymer (HyNic-

MCP). ............................................................................................................................................ 75

Figure 2-2. Synthesis of panitumumab-MCP conjugates. . .......................................................... 81

Figure 2-3. Determination of hydrodynamic radius of panitumumab-DOTA, panitumumab-MCP

and unconjugated MCP. ................................................................................................................ 85

Figure 2-4. Binding of panitumumab-DOTA-177Lu or panitumumab-MCP-177Lu to EGFR

positive MDA-468 human breast cancer cells. ............................................................................. 88

Figure 2-5. In vitro stability of the RIVCs against transchelation to EDTA and in human plasma

at 37ᵒC. .......................................................................................................................................... 90

Figure 2-6. In vivo stability of the 111In-RICs in NOD/SCID mice up to 72 h p.i. ...................... 92

Figure 2-7. Tumour and normal tissue uptake with/without preinjection of 10 μg unlabeled

panitumumab in NOD-scid mice bearing s.c. PANC-1 xenografts at 72 h p.i. of panitumumab-

DOTA-111In or panitumumab-MCP-111In... .................................................................................. 94

xii

Figure 2-8. SPECT/CT images of NOD-scid mice with s.c. PANC-1 xenografts at 72 h p.i. of 37

MBq of panitumumab-DOTA-111In or panitumumab-MCP-111In without or with preinjection of

unlabeled panitumumab or non-specific RICs.. ............................................................................ 96

Figure 3-1. Clonogenic survival of PANC-1 cells exposed to panitumumab-MCP-177Lu (0.3-1.2

MBq, 2.5 nmol/L) and density of γ-H2AX foci induced in the nucleus of PANC-1 cells.. ....... 114

Figure 3-2. Subcellular distribution in PANC-1 cells after incubation with panitumumab-MCP-177Lu in the absence or presence of unlabeled panitumumab or with non-specific hIgG-MCP-177Lu. ........................................................................................................................................... 116

Figure 3-3. Hematology and serum biochemistry analysis of NRG mice administered 6.0 MBq

(10g) of panitumumab-MCP-177Lu or normal saline. .............................................................. 120

Figure 3-4. Body weight index and tumour growth index for tumour bearing NRG mice at

different times post i.v. injection of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu or non-

specific hIgG-MCP-177Lu, unlabeled panitumumab (10 μg) or normal saline.. ......................... 122

Figure 3-5.Radioactivity (MBq) in selected source organs at 24, 72, 120 and 168 h post i.v.

injection of 6.0 MBq (10 µg) of panitumumab-MCP-177Lu in NRG mice .............................. 124

Figure 3-6. Tumour and normal organ biodistribution of panitumumab-MCP-177Lu (MBq/g) in

NRG mice with s.c. PANC-1 human pancreatic cancer xenografts at selected times up to 168 h

post-injection............................................................................................................................... 125

Figure 4-1. Clonogenic survival of PANC-1 cells exposed to increasing radioactivity (0.3 MBq,

0.6 MBq or 1.2 MBq) of panitumumab-DOTA-177Lu, panitumumab-MCP-111In or panitumumab-

DOTA-111In (2.5 nmoles/L) or to unlabeled panitumumab-DOTA. ........................................... 145

Figure 4-2. Assessment of DNA DSBs in PANC-1 cells after incubation with 0.3 MBq, 0.6 MBq

or 1.2 MBq of of 177Lu- labeled panitumumab-DOTA or 111In-labeled panitumumab-DOTA or

panitumumab-MCP.. ................................................................................................................... 147

Figure 4-3. Subcellular distribution in PANC-1 cells after incubation with with panitumumab-

DOTA-177Lu with or without excess unlabeled panitumumab or with hIgG-DOTA-177Lu... .... 149

Figure 4-4. Hematology and serum biochemistry analyses of NOD/SCID mice at 14 d after i.v.

injection of 10.0 MBq of panitumumab-DOTA-111In, panitumumab-MCP-111In or normal saline

and NRG mice administered 6.0 MBq of panitumumab-DOTA-177Lu.. .................................... 154

Figure 4-5. Tumour growth index and body weight index in NOD/SCID mice with s.c. PANC-1

xenografts receiving three amounts (10.0 MBq; 10 µg) of panitumumab-DOTA-111In,

panitumumab-MCP-111In, unlabeled panitumumab (10 µg) or normal saline separated by 3

weeks or in NRG mice with s.c. PANC-1 human PnCa xenografts receiving a single injection of

panitumumab-DOTA-177Lu (6.0 MBq; 10 µg).. ......................................................................... 156

xiii

Figure 4-6. Radioactivity (MBq) in selected organs at 24, 72, 120 and 168 h post i.v. injection of

a single amount (6 MBq; 10 μg) of panitumumab-DOTA-177Lu in NRG mice or panitumumab-

DOTA-111In (10 MBq ; 10 μg) or panitumumab-MCP-111In (10 MBq ; 10 μg) in NOD/SCID

mice.. ........................................................................................................................................... 158

Figure S 1 Clonogenic survival of PANC-1 cells exposed to 0.3, 0.6 or 1.2 MBq of 177Lu- or 111In-labeled panitumumab-MCP, 177Lu- or 111In-labeled panitumumab-DOTA, or to unlabeled

immunoconjugates……….…………………………………………………………………….176

Figure S 2. Assessment of unrepaired DNA DSBs in PACN-1 cells exposed for 16 h to 0.3, 0.6

or 1.2 MBq 177Lu- or 111In-labeled panitumumab-MCP, 177Lu- or 111In-labeled panitumumab-

DOTA, or to unlabeled immunoconjugates ………………………………………...…………177

Figure S 3. Hematology and serum biochemistry analyses of NOD/SCID mice at 14 d after i.v.

injection of 10.0 MBq (10 μg) of panitumumab-MCP-111In, panitumumab-DOTA-111In or normal

saline and NRG mice administered with 6.0 MBq (10 µg) of panitumumab-MCP-177Lu,

panitumumab-DOTA-177Lu or normal saline…………………………………………..………179

Figure S 4. Body weight index and tumour growth index (BWI) in NRG mice with s.c. PANC-1

xenografts receiving single amount (6.0 MBq; 10 g) of panitumumab-MCP-177Lu,

panitumumab-DOTA-177Lu or non-specific hIgG-MCP-177Lu or in mice receiving unlabeled

panitumumab (10 g) or normal saline…………………………………………………………180

Figure S 5. Body weight index and tumour growth index (BWI) in NRG mice with s.c. PANC-1

xenografts receiving three amounts (10.0 MBq; 10 g) of panitumumab-MCP-111In,

panitumumab-DOTA-111In or non-specific hIgG-MCP-111In separated by 3 weeks or in mice

receiving unlabeled panitumumab (10 g) or normal saline………………………………...…181

Figure S 6. The expression of EGFR, CD31 and α-SMA on PANC-1 xenografts obtained from

NOD/SCID mice and NRG mice………………………………………………………………184

xiv

List of abbreviations

Auger electron AE

ALT Alanine transaminase

ATCC American type culture collection

AUC Area under the curve

Bq Becquerel

BSO Bismuth germanate

BWI Body weight index 11C Carbon-11

CA19-9 Carbohydrate antigen 19-9

CBC Complete blood count 64Cu Copper-64

CDKN2A Cyclin-dependent kinase inhibitor 2A

CEA Carcinoembryonic antigen

Ci Curie

COX-2 Cyclooxygenase 2

cps/Bq Counting rate per unit radioactivity

Cr Creatinine

Cs Cell survival

CT Computed tomography

CTCs Circulating tumour cells

Da Dalton

DLS Dynamic light scattering

DMEM Dulbecco's Modified Eagle's Medium

DNA Deoxyribonucleic acid

DOTA 1 4 7 10-tetraazacyclododecane tetraacetic acid

DOTA-NHS-ester 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N

hydroxysuccinimide ester

DOTA-TATE DOTA0-Tyr3-Octreotate

dps Disintegration per second

DSB DNA double strand breaks

dTMP Thymidine monophosphate

DTT Dithiothreitol

dUMP Methylates deoxyuridine monophosphate

EC Electron captures

EDTA Ethylenediaminetetraacetic acid

EGFR Epidermal Growth Factor Receptor

EPR Enhanced permeability and retention

EUS Endoscopic ultrasound

xv

EUS-FNA Endoscopic Ultrasound Guided Fine Needle Aspiration

eV Electron volt 18F Flourine-18 18F-FAZA 18F-fluoroazomycin arabinoside 18F-FDG 18-fluorodeoxyglucose 18F-FLT 3′-deoxy-3′-[18F] fluorothymidine

FBS Fetal bovine serum

FDA Food and Drug Administration

FGF Fibroblast growth factor

FOV Field of view

FWHM Full-width-at-half-maximum

67Ga Gallium-67

68Ga Gallium-68

GSO[CE] Cerium-doped gadolinium oxyorthosilicate

Gy Gray

HACA Human anti-chimeric antibodies

HASA Human anti-sheep antibodies

Hb Hemoglobin

Hcl Hydrogen chloride

Hct Hematocrit

HPLC High performance liquid chromatography

HSG Histamine-succinyl-glycine

HyNic-MCP Hydrazino-nicotinamide Metal Chelating Polymer

I.v. Intravenous 111In Indium-111 123I Iodine-123 131I Iodine-131

Ic Internal conversion

ID/g Injected dose per gram

IGF1R Insulinelike Growth Factor 1 receptor

IgG Immunoglobulin G

ITLC Instant thin layer chromatography 177Lu Lutetium-177

LET Linear Energy Transfer

LSO[Ce] Ceriumdoped lutetium oxyorthosilicate

LYSO[CE] Cerium-doped lutetium-yttrium oxyorthosilicate

mAb Monoclonal Antibody

MAP Mitogen-activated protein

MBq Mega Becquerel

MCNP Monte Carlo N-Particle

xvi

MDCT Multi-detector computerized tomography

MEK Extracellular Kinase

MMPI Matrix Metalloproteinases

MRI Magnetic resonance imaging

MTD Maximum tolerated dose

MUC-1 Mucin-1

MWCO Molecular weight cut off

NaHCO3 Sodium bicarbonate

NaI[Tl] Sodium iodide activated with thallium

NF-kB Nuclear factor-kB

NHS N-Hydroxysuccinimide

NLS Nuclear localizing sequence peptide

NTR Nitroreductase enzyme

OS Overall survival

p.i. Post injection

PAm Polyacrylamide

PanIN Pancreatic intraepithelial neoplasia

PAsp Polyaspartamide

PBS Phosphate-buffered saline

PD-1 Programmed cell death-1

PDGF Platelet derived growth factor

PE Plating efficiency

PEG Polyethylene glycol

PGlu Polyglutamide

PI-3K/AKT Phosphoinositide 3-kinase-Akt

PL Polylysine

PMT Photomultiplier tube

PnCa Pancreatic Cancer

PNETs Pancreatic neuroendocrine tumours

PSMA Prostate specific membrane antigen

RBC Red blood cell

RIT Radioimmunotherapy

ROS Reactive oxygen species

S.c Subcutaneously

SD Standard deviation

SDF Stromal derived factor

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SEC Size exclusion column

SF Survival Fraction

Shh Sonic Hedgehog

xvii

SMA Superior mesenteric artery

SMV Superior mesenteric vein

SPARC Secreted protein-acid rich in cysteine

SPECT Single-photon emission computed tomography 99mTc Tecnitium-99m

TCO Trans-cyclooctene

TGFβ-1 Transforming growth factor beta 1

TGI Tumour growth index

TS Thymidylate synthase

Tz Tetrazine

VEGF Vascular endothelial growth factor

WBC White blood cell 89Zr Zirconium-89

1

Chapter 1: Introduction

2

1. Introduction

1.1 Current status in the detection, diagnosis and treatment of pancreatic cancer

1.1.1 Pancreatic cancer epidemiology and etiology

Pancreatic cancer (PnCa) is 12th most commonly diagnosed cancer in Canada (1) but is the fourth

most common cause of cancer death in Canada and the United States with its incidence almost

equivalent to mortality and an overall 5-year survival rate lower than 5% (2). According to

Canadian cancer statistics in 2017, 5,500 Canadians were diagnosed with PnCa and 4,800 died of

this disease. Less than 50% of patients with PnCa survive beyond 3.9 months. The high mortality

relative to incidence reflects the poor prognosis of PnCa. The complex pathophysiology, late

stage diagnosis in more than 60% of cases and unresponsiveness to radiation and chemotherapies

are major barriers for treatment of this disease (3). Given the limited improvements in PnCa

prevention, detection and treatment, especially relative to the other major cancers, PnCa is

expected to become the third leading cause of cancer-related death in the coming years. By 2030,

the leading causes of cancer-related death are projected to be lung, liver, and pancreas for men,

and lung, breast, and pancreas for women (4).

1.1.2 Detection, diagnosis and staging of PnCa

PnCa is a group of heterogeneous diseases and includes cancer of the endocrine pancreas (islet

cell carcinoma, neuroendocrine carcinoma and carcinoid tumours) and exocrine (pancreatic

ductal adenocarcinoma and acinar) pancreas. Pancreatic neuroendocrine tumours (PNETs)

account for about 6% of all PnCa (5). This disease develops from the abnormal growth of

neuroendocrine cells that release hormones which may result in tumour-associated adverse

effects in patients. PNETs might be benign or malignant and they grow slower than exocrine

tumours. Depending on hormone production this disease can be functional or nonfunctional.

Functional types cause hormone-related symptoms, however the majority of PNETs are

nonfunctional resulting in late diagnosis.

Acinar cell carcinoma is a very rare form of pancreatic exocrine tumour which overproduces

measurable pancreatic lipase in the blood. Among pancreatic cancer pathologies, pancreatic

ductal adenocarcinoma (PDAC) accounts for approximately 90% of all cases (3). Pancreatic

3

adenocarcinoma is defined as neoplasia of epithelial cells lining the pancreatic duct (pancreatic

intraepithelial neoplasia, PanIN) and has glandular origin, glandular characteristics, or both.

The stage of the disease which describes the size and location at the time of diagnosis aids in

selecting the appropriate treatment for the patient. The TNM system (Table 1-1) is the most

widely used system for cancer staging based on 3 factors:

T: Size of the primary tumour and whether it has grown outside the pancreas.

o TX: Primary tumour cannot be assessed

o T0: No evidence of primary tumour

o Tis: Carcinoma in situ

o T1: Tumour limited to the pancreas, ≤2 cm in greatest dimension

o T2: Tumour limited to the pancreas, >2 cm in greatest dimension

o T3: Tumour extends beyond the pancreas but without involvement of the celiac

axis or the superior mesenteric artery

o T4: Tumour involves the celiac axis or the superior mesenteric artery

(unresectable primary tumour)

N: Spread to regional lymph nodes.

o NX; Regional lymph nodes cannot be assessed

o N0: No regional lymph node metastasis

o N1: Regional lymph node metastasis

M: Metastasise to other organs. The most common sites of PnCa spread are the liver,

peritoneum and lungs (6).

o M0: No distant metastasis

o M1: Distant metastasis

4

Table 1-1. TNM staging system for PnCa.

Stage Stage grouping

Pancreas Spread to

Inside *Outside Major blood

vessel

Lymph

Node

Distant

Organ

0 Tis, N0, M0

Involvement of the top layers

of pancreatic duct cells

(pancreatic carcinoma in situ

or PanIn III).

IA T1, N0, M0 Tumour ≤2 cm (T1) No (N0) No (M0)

IB T2, N0, M0 Tumour > 2 cm (T2) No (N0) No (M0)

IIA T3, N0, M0 Yes No (T3) No (N0) No (M0)

IIB T1-T3, N1, M0 The tumour is either confined to the pancreas

or growing outside the pancreas No (T3) Yes (N1) No (M0)

III T4, Any N, M0 Yes May be (T4) May be No (M0)

IV Any T, Any N, M1 Yes (M1)

*Outside means local invasion in this table.

5

1.2 PnCa biology

PnCa is characterized by having an extremely compact, dense and poorly vascularized stroma

which is responsible for limited drug delivery to cancer cells (7), which might be the main reason

for the failure of most new treatments evaluated in clinical trials. Generally, PnCa is composed

of tumour stroma, PnCa cells, and PnCa stem cells. While numerically small, PnCa stem cells

are considered to be resistant to chemotherapy and radiation therapy and their survival is likely

responsible for recurrence following treatment (8).

1.2.1 The genomic landscape of PnCa

Genetic analysis of PnCa has revealed that 67% to 100% of the tumours are genetically altered

(9). A PnCa cell may carry more than 60 genetic alterations (Figure. 1-1) which can be grouped

in 12 core signaling pathways (9).

6

Figure 1-1. Signaling pathways with mutation in PnCa [Adapted from Jones S (10)].

7

Mutations in KRAS, p16/cyclin-dependent kinase inhibitor 2A (CDKN21), TP53 and

SMAD4/DPC4 drives clonal expansion by conferring a selective growth advantage on PnCa

cells. This signature molecular profile which is frequently found in PanIN triggers neoplastic

transformation and tumour progression (11, 12). Generation of PnCa from pancreatic

inflammation is stimulated by Hedgehog signaling, Notch signaling, and cyclooxygenase 2

(COX-2) (13-15). COX-2 mediates prostaglandin synthesis which then triggers cell proliferation

and cytokine synthesis. Pro-inflammatory cytokines and reactive oxygen species (ROS)

associated with extensive inflammation activate apoptosis as a cellular protective mechanism as

well as proliferation to rebuild the loss of tissue. Enhanced proliferation in the presence of ROS

and other potential mutagens lead to accumulation of growth promoting mutations and confer a

selective growth advantage to individual cell clones (16). Nuclear factor-kB (NF-kB), the

prototypical proinflammatory signaling pathway is also considered as a driver of tumourigenesis

from inflammation (17). Important cancer-associated genes, such as cmyc, jun B Cyclin D1,

TP53 and vascular endothelial growth factor (VEGF) are under the control of this transcription

factor (16).

1.2.2 PnCa stem cells

In early stage PnCa, stem cells are the principal tumour compartment and numerically decrease

as the disease advances. The most important phenotypic characteristics of these cells are the

capacity for self-renewal and asymmetric division. PnCa stem cells in primary tumours is

associated with shorter overall survival, resistance to gemcitabine, enhanced metastatic potential

and tumour heterogeneity (18, 19). PnCa stem cells are very plastic with transition among

different states including from epithelial to mesenchymal states which may be involved in the

metastatic spread of PnCa (20, 21). The expression of unique targets in PnCa stem cells led to

many targeted therapeutic studies. Most of these targets belong to pathways such as Hedgehog,

Wnt and Notch, apoptotic pathway targets such as DR5 and nodal-activin Alk4/7 pathway (22-

25).

1.2.3 The tumour microenvironment

A characteristic of PnCa is the formation of a dense stroma, termed a desmoplastic reaction (26,

27). The pancreatic stellate cells (PSCs) (also known as myofibroblasts) are the principle source

8

of fibrosis and interact closely with PnCa cells to form the tumour (28). Growth factors such as

transforming growth factor beta 1 (TGFβ-1), platelet derived growth factor (PDGF), and

fibroblast growth factor (FGF), activates PSCs for secretion of different components of the

extracellular matrix including collagen and matrix-metalloproteinases through which they

regulate the reabsorption and turnover of the stroma (29). PSCs are also responsible for the poor

vascularization characteristics of PnCa (29, 30). Secreted protein-acid rich in cysteine (SPARC),

is a calcium-binding protein and is highly expressed in PSCs and stromal fibroblasts immediately

adjacent to PnCa cells, with an important role in promoting epithelial-to-mesenchymal transition

and invasion through matrix metalloprotease expression (31). It is hypothesized that SPARC

actively binds the albumin in nab-paclitaxel which is an albumin coated nanoparticle of

paclitaxel used for treatment of PnCa and further concentrates the drug in the tumour

contributing to stromal collapse (32). In patient derived PnCa xenografts in mice, nab-paclitaxel

eliminated the stroma, increasing the delivery of gemcitabine and was associated with high

antitumour activity (21). SPARC overexpressing PSCs are also responsible for radioprotection of

PnCa cells through β1-integrin signaling (33) and by induction of hypoxia by producing

abundant collagen in the tumour stroma (21).

In addition to being a mechanical barrier, the stroma is a dynamic compartment involved in the

process of tumour formation, progression, invasion, and metastasis (26, 27). Among the proteins

expressed by stromal cells, Cox-2, PDGF receptor, VEGF, stromal derived factor (SDF),

chemokines, integrins, SPARC and hedgehog pathway elements, have been associated with a

worse prognosis and resistance to treatment (34). Preclinical studies have shown that the

hedgehog inhibitors such as Saridegib (IPI-926) or enzymatic targeting of hyaluronic acid in the

stroma using PEGPH20 (pegvorhyaluronidase alfa) disrupt the stroma, increase vascular supply

and improve drug delivery (35-37). However the results obtained from the clinical trials haven’t

been promising thus far. The tumour microenvironment of PnCa is also immunosuppressive

inhibiting antitumour immunity (21). The cell surface molecule CD40 is a member of the tumour

necrosis factor receptor superfamily and is broadly expressed by immune cells, in particular B

cells, dendritic cells (DC), and monocytes, as well as other normal cells and some malignant

cells (38-40). Clinical studies have shown that treatment of patients with surgically incurable

PnCa using agonist CD40 antibody combined with gemcitabine led to tumour regression (40).

9

Activation of CD40 in T cells stimulates the infiltration of tumour macrophages that deplete the

cancer stroma (40).

1.2.4 Circulating tumour cells

Circulating tumour cells (CTCs) are tumour cells that have acquired the ability to enter the

circulatory system. Frequent metastases of PnCa to the liver, lung and skeletal system reflects the

ability of these cells to detach from the primary tumour, and travel through the circulation to

distant organs, where they extravasate to establish metastases. In early stages of tumour

formation, tumourmalignant cells may be passively shed from the primary tumour in large

numbers and enter the blood circulation (41, 42). Furthermore transition of epithelial cells to a

mesenchymal phenotype and acquiring corresponding characteristics including motility,

invasiveness and resistance to apoptosis promotes detachment from the primary tumour (43).

There are different ways of CTC dissemination (44, 45) and the different sites of metastases that

also shed tumour cells (46, 47) results in considerable heterogeneity within the CTC population.

Most CTCs do not have the ability to form distant tumours, and only 0.01% of CTCs survive (48,

49) and have the characteristics of cancer stem cells (50).

1.3 Treatment of pancreatic cancer

PnCa exhibits an aggressive biological phenotype characterized by early invasion of surrounding

structures and rapid metastatic spread and resistance to radiation therapy and/or chemotherapy

(51). More than 80% of PnCa patients present with unresectable disease and one third of these

patients have locally advanced PDAC while the remainder have distant metastases (52).

Chemotherapy remains the only option for locally advanced or metastatic disease. Unfortunately

the response of patients is quite limited to chemotherapy due to both intrinsic and acquired

resistance to chemotherapeutic agents, therefore there is an urgent need for novel therapeutic

strategies.

1.3.1 Surgery

The Whipple procedure, or Kausch-Whipple procedure also known as pancreaticoduodenectomy

or pancreatoduodenectomy, is performed to remove resectable tumours of the pancreas

(53). However, only 20% of patients are eligible for surgery (54) depending on the presence or

10

absence of a fat layer creating a barrier between the tumour and major vessels (55) the extent of

local tumour invasion, anatomical adjacency to, or involvement of blood vessels (56). Due to the

shared blood supply of organs in the proximal gastrointestinal system, surgical removal of the

head of the pancreas, also necessitates removal of the duodenum, proximal jejunum, gallbladder,

and, occasionally, part of the stomach. The success of the Whipple procedure also depends on

the surgical expertise and general performance status of patients (54).

Computed tomography (CT) is usually used to determine whether or not surgical resection is

possible, however sometimes it only becomes apparent during the surgery that the tumour cannot

be removed without damaging vital tissues. Following surgery the existence of residual cancer

cells at the margins of removed tissue is examined microscopically by a pathologist (56).

Unfortunately the presence of microscopic deposits of cancer stem cells which may continue to

repopulate the tumour and aid in its invasion are not evident microscopically (34, 57). Treatment

of a post-operative complications which is not caused by the cancer itself is an issue for 30–45%

of patients. Difficulty in emptying the stomach is the most common complication of surgery

(55).

1.3.2 Chemotherapy

Patients who are not eligible for surgery receive chemotherapy in order to extend life or

improve its quality (55). For borderline resectable tumours neoadjuvant chemotherapy or

chemoradiotherapy may be used to reduce the size of tumour to enable surgery. After a recovery

period of one to two months following surgery, adjuvant chemotherapy is offered to patients.

There are currently five Food and Drug Administration (FDA) approved chemotherapy drugs for

the treatment of PnCa: fluorouracil (5-FU), gemcitabine (Gemzar), FOLFIRINOX, albumin-

bound paclitaxel (ABRAXANE), and irinotecan liposome injection (ONIVYDE).

1.3.2.1 5-Fluorouracil

5-Fluorouracil (5-FU) principally acts as a thymidylate synthase (TS) inhibitor. Thymidylate

synthase methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate

(dTMP) (Figure 1-2.). Interrupting the action of this enzyme blocks the synthesis of the

pyrimidine thymidine, which is a nucleoside required for DNA replication. Depletion of dTMP

in rapidly dividing cancer cells causes cell death.

11

Figure 1-2. Methylation of deoxyuridine monophosphate (dUMP) to produce deoxythymidine

monophosphate (dTMP) by thymidylate synthase (TS). 5-FU inhibits TS.

12

The chemotherapy drugs most commonly used in conjunction with radiation therapy are 5-FU

and gemcitabine. 5-FU is used most often since there is more experience using this drug in

combination with radiation and there are fewer side effects (58).

1.3.2.2 Gemcitabine

Gemcitabine (Gemzar) is a nucleoside analog of pyrimidine in which the hydrogen atoms on the

2' carbon of deoxycytidine are replaced by fluorine atoms (Figure.1-3). Gemcitabine was

approved by the U.S. FDA for treatment of PnCa in 1997 after it was shown to increase the

median survival of patients with advanced PnCa by 5 weeks compared to 5-FU which was the

standard treatment for unresectable PnCa at the time (59).

13

Figure 1-3. The chemical structure of gemcitabine.

14

In the clinical trial conducted by Burris et. al 126 patients with advanced symptomatic PnCa

were randomized to receive either gemcitabine 1,000 mg/m2 weekly × 7 weeks followed by 1

week of rest, then weekly × 3 weeks for 4 cycles thereafter (63 patients), or to 5-FU 600 mg/m2

once weekly (63 patients). The median survival was 5.65 and 4.41 months for gemcitabine-

treated and 5-FU-treated patients, respectively (P = 0.0025). The proportion of patients surviving

at 12 months was 18% for patients receiving gemcitabine and 2% for 5-FU. Treatment with

gemcitabibe was well tolerated (59).

1.3.2.3 FOLFIRINOX

FOLFIRINOX was recently approved as a more effective chemotherapy regimen than

monotherapy with gemcitabine for advanced and metastatic PnCa. This regimen is a combination

of four drugs that is effective but also associated with significant normal tissue toxicity:

5-FU

Folinic acid (leucovorin): a vitamin B derivative that reduces the side effects of 5-FU.

Irinotecan (Camptosar): a topoisomerase inhibitor, which prevents DNA from uncoiling and

duplicating.

Oxaliplatin (Eloxatin): a antineoplastic agent, which inhibits DNA repair and/or DNA

synthesis (60).

A randomized phase III clinical trial of 342 patients with metastatic PnCa conducted by Conroy

et al in 2011 (61) showed that patients on the FOLFIRINOX treatment (oxaliplatin, 85 mg/m2 of

body-surface area; irinotecan, 180 mg/m2; leucovorin, 400 mg/m2; and 5-FU, 400 mg/m2 given

as a bolus followed by 2400 mg/m2 given as a 46-hour continuous infusion, every 2 weeks) lived

4 months longer than patients receiving the standard gemcitabine treatment (1000 mg/m2 weekly

for 7 of 8 weeks and then weekly for 3 of 4 weeks). The median overall survival was 11.1

months in the FOLFIRINOX group as compared with 6.8 months in the gemcitabine group and

median progression-free survival was 6.4 months in the FOLFIRINOX group and 3.3 months in

the gemcitabine group (hazard ratio for disease progression, 0.47; 95% CI, 0.37 to 0.59;

P<0.001).

15

Due to the substantial side effects, FOLFIRINOX is only suitable for patients with good

performance status (62) meaning there is still much needed advancement in the systemic

treatment of PnCa.

1.3.2.4 Protein-bound paclitaxel (nab-paclitaxel)

In 2013, positive findings from the phase III MPACT trial led to the U.S. FDA approval of

nanoparticle albumin-bound paclitaxel (nab-paclitaxel or Abraxane) combined with gemcitabine

for treating late-stage PnCa as a less toxic (although less effective) alternative to FOLFIRINOX.

Nab-paclitaxel is a colloidal suspension of 130 nm particles homogenized in human serum

albumin that incorporate paclitaxel with better bioavailability compared to solvent-based

paclitaxel (63) (Figure 1-4.). Nab-paclitaxel binds to the albumin receptor (gp60) as well as

SPARC antigen expressed on cancer cells (64, 65). SPARC is a calcium-binding protein and

highly expressed in stromal fibroblasts immediately adjacent to PnCa cells, with an important

role in promoting epithelial-to-mesenchymal transition and invasion through matrix

metalloprotease expression (31).

16

Figure 1-4. Nab-paclitaxel or Abraxane. The formulation is prepared by high-pressure

homogenization of paclitaxel in the presence of serum albumin into a nanoparticle colloidal

suspension. The human albumin-stabilized paclitaxel particles have an average size of 130 nm.

17

A total of 861 patients with metastatic PnCa were randomly assigned to receive nab-paclitaxel

plus gemcitabine or gemcitabine alone. The median overall survival (OS) was significantly

longer for patients treated with nab-paclitaxel plus gemcitabine vs gemcitabine alone (8.7 vs 6.6

months). Long-term (>3-year) survivors were identified in the nab-paclitaxel plus gemcitabine

arm only (4%) (63).

1.3.2.5 Irinotecan liposome injection (ONIVYDE)

Irinotecan liposome injection (ONIVYDE) in combination with 5-FU and leucovorin, was

approved in October 2015 as treatment for metastatic PnCa that has progressed following

treatment with gemcitabine based therapy (Figure 1-5). The phase III NAPOLI-1 trial of this

regimen achieved a substantial improvement in 12-month OS compared to 5-FU and leucovorin

alone in patients with metastatic PnCa who had received prior gemcitabine chemotherapy (64).

18

Figure 1-5. Irinotecan liposome injection (ONIVYDE). Unilamellar lipid bilayer vesicles of

approximately 110 nm in diameter encapsulate 80,000 molecules of irinotecan in a gelated or

precipitated state, as sucrosofate salt. These liposomes release irinotecan slowly over time.

19

Currently, patients able to manage the side-effects receive FOLFIRINOX or nab-paclitaxel with

gemcitabine while those who cannot tolerate the side effects of this regimen receive gemcitabine

alone. Patients with metastatic PnCa who don’t respond to gemcitabine receive (ONIVYDE) in

combination with 5-FU and leucovorin. Clinical trials are actively being conducted for novel

adjuvant therapies, since new therapies introduced in the last few years have only increased

survival times by a few months (62).

1.4 Diagnostic imaging modalities for PnCa

Imaging plays an important role, not only for initial diagnosis and staging, but also for

determining the resectability and monitoring the effectiveness of treatment of PnCa (65-67).

Complete surgical resection of the tumour increases the survival of patients to 12-20 months,

however there is a high probability of relapse due to the highly adverse and aggressive nature of

PnCa (68, 69). Multi-detector computerized tomography (MDCT) is currently the imaging

modality of choice for evaluation of PnCa and determining its resectability (70).

1.4.1 Contrast-enhanced abdominal ultrasound

Contrast-enhanced abdominal ultrasound is a non-invasive and cost-effective imaging modality

for patients presenting with jaundice or abdominal pain (70). This method involves the

administration of intravenous contrast agents containing microbubbles of nitrogen or

perfluorocarbon. Pulses of ultrasound are transmitted into tissue using a probe, the sound echoes

off tissue interfaces and are recorded and displayed as an image. Different tissues have varying

degrees of sound wave reflection. Drawbacks of ultrasonography include limits on its field of

view, body habitus and its dependence on a skilled operator. Ultrasound has a 50%-90%

sensitivity for detecting PnCa (71-75).

1.4.2 MDCT

The most important pre-operative examination in patients with suspected PnCa is MDCT owing

to its very good spatial resolution and wide anatomic coverage, allowing local and distant disease

assessment (76). The capability to assess vascular involvement critically important for predicting

tumour resectability (77-83) is a major advantage of this imaging modality. The reported positive

20

predictive value, sensitivity, and specificity for predicting the resectability of PnCa are 89%,

100%, and 72%, respectively (84). Since MDCT may fail to depict small liver or peritoneum

metastases (80), for treatment monitoring MDCT may be used in conjunction with PET/CT (85,

86).

1.4.3 Endoscopic Ultrasound Guided Fine Needle Aspiration (EUS/EUS-FNA)

Endoscopic ultrasound guided fine needle aspiration (UES-FNA) combines imaging and tissue

sampling and on occasion collection of pancreatic cyst fluid. EUS-FNA is usually used to further

evaluate abnormal findings on CT, MRI or ultrasound. The EUS component involves the

insertion of an echoendoscope composed of a thin flexible tubular transducer into the

gastrointestinal (GI) tract. FNA uses a specialized needle that can be inserted through the wall of

the stomach or intestine into the pancreas allowing for sampling tissues of interest for

histological analysis.

As EUS offers excellent visualization of the pancreas from within the duodenum or stomach and

can produce high-resolution images of the pancreas, it has been considered one of the most

accurate methods for the detection of pancreatic focal lesions, especially in patients with small

tumours of 3 cm or less (70, 87, 88). Using EUS for guidance, a biopsy needle can be accurately

inserted into the area of interest in the pancreas. EUS-FNA helps to determine the nature of a

suspicious lesion, particularly if it may be malignant. EUS is an excellent test for examining an

array of pancreatic conditions including pancreatic masses, tumours, cysts, acute and chronic

pancreatitis, and auto immune pancreatitis (89). Absence of an identifiable mass lesion on EUS

rules out PnCa with almost 100% certainty (90). EUS may also have a role in preoperative

staging of PnCa for determining resectability but MDCT is the most accurate. Portal vein and

splenic vein invasion can be visualized with EUS. However, tumour involvement of the superior

mesenteric vein (SMV) and the superior mesenteric artery (SMA) are not reliably determined by

EUS (91).

1.4.4 MRI

The outstanding soft-tissue contrast of MRI confers an advantage for characterizing pancreatic

masses compared to CT. In most cases small tumours, a hypertrophied pancreatic head,

isoattenuating PnCa, and focal fatty infiltration of the parenchyma can be depicted by MRI but

21

not by CT (66). MRI with magnetic resonance cholangiopancreatography (MRCP) makes use of

heavily T2-weighted MRI pulse sequences (92). These sequences show high signal in static or

slow moving fluids within the gallbladder, biliary ducts and pancreatic duct, with low signal of

surrounding tissue. MRCP is a successful technique for delineating the pancreatic ductal system

and detecting ductal narrowing due to the presence of a small mass. Comprehensive

morphological information on the pancreas parenchyma and the pancreatic duct improve the

chance of tumour diagnosis at an early stage (68) and offer a problem-solving tool for patients

with non-cancerous pancreatic disease.

1.4.5 Nuclear medicine imaging

Nuclear medicine imaging non-invasively provides functional information at the molecular level

that augment our understanding of disease and disease processes by measuring the uptake and

turnover of target-specific radiotracers in tissue (93). Nuclear medicine imaging includes

positron emission tomography (PET) and single photon emission computed tomography

(SPECT). SPECT and PET offer high sensitivity using intravenously administered minimal

concentrations of imaging probes in the pico- to nanomolar range (94). Furthermore as opposed

to optical and ultrasound imaging with limited detection of signals from deeper tissues, SPECT

and PET are able to detect radioactivity from deep tissues. Quantitative information on

radiotracer distribution and target expression can be obtained by SPECT and PET imaging. PET

imaging with 18-fluorodeoxyglucose (18F-FDG) has been used for staging and restaging cancer,

detecting recurrence, and treatment response monitoring (95). Clinical SPECT imaging is of

great utility for diagnostic purposes such as the localization of primary and metastatic

somatostatin receptor-expressing neuroendocrine tumours using 111In-pentetreotide

(Octreoscan™) (96), the identification of bone metastases using 99mTc-methylene diphosphonate

(MDP) (97), and the staging of pelvic lymphadenectomy or the detection of metastatic lymph

node involvement in prostate cancer using 111In-capromab pendetide (98). The addition of

anatomic CT imaging to PET and SPECT functional imaging has further expanded the utility and

accuracy of nuclear medicine imaging (93). SPECT and PET imaging will be discussed in the

following sections.

22

1.4.5.1 PET imaging

In PET, collision of an electron with the positron emitted by radioisotope, results in annihilation

of the positron and creation of two anti-parallel 511 KeV -photons. Simultaneous detection of

these two -photons within a coincidence-timing window of a few nanoseconds by opposing

inorganic scintillation crystal detectors and affixed photomultiplier tubes (PMT) located in a

360-degree ring creates a line of response (LOR) through the point of decay (Figure 1-6). The

raw data is collected from many angles around the patient’s body and analysed by the scanner

computing unit and store as a list mode data from which the sinogram can be calculated and

reconstructed to PET image using filtered back projection or iterative algorithms (99, 100). The

scintillation crystals used for detection are usually bismuth germanate (BSO), cerium-doped

gadolinium oxyorthosilicate (GSO[CE]), ceriumdoped lutetium oxyorthosilicate (LSO[Ce]) and

cerium-doped lutetium-yttrium oxyorthosilicate (LYSO[CE]) which have greater stopping power

for the 511-keV γ-photons than sodium iodide activated with thallium, (NaI[Tl]) scintillators due

to their higher mass density and effective atomic number (101). During a PET scan, several

million coincidence events are recorded generating many intersecting LORs providing

information on the spatial location of radioactive decays in the body. The reconstruction of the

image in a PET system requires crossing many LORs (Figure 1-6B). A LOR can be formed by a

true coincidence, which occurs when both photons from an annihilation event are detected,

neither photon undergoes any form of interaction prior to detection, and no other event is

detected within the coincidence-timing window (102). If any of the photons from an annihilation

event scatters and changes its direction prior to detection, the resulting coincidence will be

scattered coincidence and represent a wrong LOR. Another type of coincidence is random

coincidence that occurs when two photons, not arising from the same annihilation event, impinge

the detectors within the coincidence-timing window of the system. Scatter and random

coincidences add a background to the true coincidence distribution, decreasing contrast and

causing the isotope concentrations to be overestimated (102).

23

Figure 1-6. PET imaging system and types of coincidences in PET. A) Opposing scintillation

detectors and affixed PMTs located in a 360-degree ring detect anti-parallel 511 KeV γ-photons

and create a LOR through the point of decay. Undesired LORs are crossed by the coincidence

processing unit prior to image processing. B) True coincidence; photons from an annihilation

event are detected without any prior interaction with the medium. Scattered coincidence; one of

the photons scatters and changes its direction prior to detection. Random coincidence;

simultaneous detection of two photons from different annihilation events by detectors. [Adapted

from Langner J (103)].

24

Spatial resolution for PET imaging: Spatial resolution is defined as the ability to clearly

delineate two neighbouring sources and is expressed as the full-width-at-half-maximum

(FWHM). It is empirically defined as the minimum distance between two points in an image that

can be detected by a scanner (104). The spatial resolution of PET is limited by factors such as

positron range, photon non-collinearity, detector size, and reconstruction methods (104, 105).

Positron range refers to the average distance that a positron travels in a surrounding medium

before it encounters an electron and is annihilated (106). This range is directly dependent on the

energy of the positron whereby higher energy positrons travel further. As a result, the origin of

the γ-photon is not the position of the radionuclide but some distance from it (107). Photon non-

collinearity is the angular deviation from the 180° (<0.5ᵒ C (108)) trajectories travelled between

the two annihilation photons (109) which results in resolution blurring that is dependent on the

ring diameter of the detector causing a misplacement of approximately 1 mm for a 50 cm ring

diameter and increasing to 2 mm for a 90 cm ring which is used for whole-body scanners (108).

The use of smaller detector elements and smaller diameters of detector rings in small animal PET

scanners results in spatial resolutions of 0.83 mm (105). The cut-off frequency of a filter defines

the frequency above which the noise is eliminated (110). In the filtered back projection

reconstruction method a filter with a too high cut off value introduces noise and thus degrades

spatial resolution (104).

In small animal PET systems the detector rings in the axial direction have been expanded,

therefore due to the large number of detector crystals ranging from 25,600 to 32,448 (111, 112)

the sensitivity of the small-animal PET systems is about 3 times that of a conventional PET

scanner (112). Examples of positron emitting isotopes used for PET and their properties are

listed in Table 1-2.

25

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

Flourine-18 (18F) 1.83 h 0.64 97 0.2

Copper-64 (64Cu) 12.7 h 0.58 19 0.2

Zirconium-89 (89Zr) 78.4 h 0.90 23 0.4

Gallium-68 (68Ga) 1.14 h 1.90 88 1.2

Adapted from Zanzonico P (101)

26

18F-FDG PET imaging: The increased utilization of glucose by tumours has been attributed to an

increased activity of membrane glucose transporters such as GLUT1 (113-115). PET scanning

with the radiotracer 18F-FDG relies on the increase rate of glycolysis. Like glucose, FDG is

transported by GLUT1 and phosphorylated by hexokinase, however, since FDG-6-phosphate is

not a substrate for the next enzyme (phosphohexose isomerase) in the glycolytic pathway, it

cannot proceed along the pathway (116). 18F-FDG-6-phosphate is trapped within the tumour cell

and imaged by PET. This imaging technique has moved into the clinical realm of diagnosis,

staging, and treatment planning (117-119). High accumulation of 18F-FDG in a lesion suggests

the presence of metabolically active cancer cells, although infiltration of inflammatory cells may

also cause 18F-FDG uptake. False negative PET scans has been reported in patients with elevated

blood glucose levels, which interfere with 18F-FDG uptake (91) or small lesions (low detection

sensitivity due to the spatial resolution limits of PET). There is still no consensus on whether

PET provides information beyond that obtained by contrast-enhanced CT (72) and even the

utility of PET for identifying metastases is controversial (91). Mertz et al. studied 35 patients

with presumed resectable PDAC with CT, EUS, and 18FDG-PET(120). They showed that

sensitivity for the detection of PnCa was higher for EUS (93%) and 18FDG-PET (87%) than for

CT (53%). 18FDG-PET diagnosed 7 of 9 cases of proven metastatic disease, whereas CT missed

4 of them. Two of three metastatic liver lesions suspected by CT were indeterminate for

metastases but 18FDG-PET confirmed metastases. In another study Wakabayashi et al. compared

the sensitivity and accuracy of tumour diagnosis for 18FDG-PET and CT in 53 patients with

proven primary PnCa (121). The sensitivity of 18FDG-PET, CT, were 92.5%, 88.7%;

respectively. 18FDG-PET was superior to CT in diagnosing distant disease (bone metastasis) but

for local staging, the sensitivity of CT was better than that of 18FDG-PET. However recent

studies have shown that integration of PET/CT into the diagnostic work-up is superior to

conventional imaging (MDCT, CT angiography, EUS) alone for tumour staging, especially

related to detection of distant metastases (sensitivity and specificity rates were 89% versus 56%

and 100% versus 95%, respectively) (91). Utility of 18F-FDG PET/CT in diagnosis, staging,

detection of liver metastasis and assessment of resectability and metabolic response of PnCa has

been studied in different clinical trials [reviewed in (122)]. Studies have shown that 18FDG-

PET/CT has an advantage in monitoring metabolic response, making it optimal in evaluation of

different kinds of treatments and is also a valuable tool to detect suspected recurrence (122).

27

Since 2011, 18F-FDG PET/CT has been approved by Cancer Care Ontario for PnCa patients who

have potentially resectable tumour (123).

PET imaging using 18F-fluoroazomycin arabinoside (18F-FAZA) has proven effective for

stratification of patients with hypoxic PnCa and to estimate perfusion and hypoxic fraction in

tumour (124). Owing to its lipophilicity, 18F-FAZA is diffused through the cell membrane,

reduced by nitroreductase enzyme (NTR) trapped in the cell (125). When the cell is well

oxygenated, this process is reversible and the tracer can freely flow back into the extracellular

environment. 3′-deoxy-3′-[18F] fluorothymidine (18F-FLT) is another imaging tracer that is

preferentially retained in proliferating cells (126). Intracellular thymidine kinase 1 (TK1),

catalyzes the phosphorylation of 18F-FLT to 18F-FLT-monophosphate which is trapped in cells

because of its negative charge (127, 128). The ability of 18F-FLT to scan primary PnCa using

PET is still under investigation in preclinical and clinical studies (129, 130).

1.4.5.2 Single-photon computed tomography (SPECT)

Single photons emitted by radionuclides such as γ-rays arising from isomeric transition, and X-

rays arising from electron capture or internal conversion are detected by a gamma camera which

rotates around the patient to generate a series of 2-dimensional images (slices) that are then

reconstructed to create a 3-dimensional image (101). The gamma camera (Figure 1-7) consists

of a collimator to isolate individual γ-photons directly originating from a source in the patient, a

large scintillation crystal [most often NaI(Tl)] which converts γ-photons into light, and

photomultiplier tubes (PMTs) to convert the received light into electrons and amplify the output

pulses. An X, Y position logic circuit sums up the output from the array of PMTs 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

further electronically sorted and processed for display (131). Following acquisition of a series of

planar images (also called projections) throughout the 360° rotation of the camera around the

patient, images are reconstructed by filtered back projection or by iterative algorithms to produce

a functional 3-dimensional distribution of a photon emitter radionuclide within the body (132).

28

Figure 1-7. Composition of the SPECT camera. Photons that pass through the collimators are

converted to light in scintillation crystal and subsequently amplified by photomultiplier tubes.

The output from the array of photomultiplier tubes is processed by a position logic circuit to

produce X and Y pulses that are in direct proportion to the X, Y coordinates of the interaction

point of the γ-rays. Pulses are further processed for display by filtered back projection or by

iterative algorithms to produce a functional 3-dimensional image using a data analysis computer.

29

It’s important to mention that collimators reduce 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 (106). Ideally, radionuclides with γ-photon

energies between 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 (101). Examples of single photon emitting radioisotopes used for SPECT imaging are

provided in Table 1.3

30

Table 1-3 Physical properties of some of radionuclide used in SPECT imaging.

Radionuclide Physical

half-life

Energies of imageable

X and γ-rays (KeV)

Abundance of imageable

X and γ-rays (%)

Indium-111 (111In) 2.83 d 172

247

90

94

Tecnitium-99m (99mTc) 6.01 h 140 89

Iodine-123 (123I) 13.2 h 159 84

Iodine-131 (131I) 8.04 d 364 82

Gallium-67 (67Ga) 3.26 day 93

185

300

40

24

16

Adapted from Zanzonico (101)

31

Spatial resolution for SPECT imaging: The most limiting factor for spatial resolution is the

diameter of the holes in a collimator. There is often a trade-off between spatial resolution and

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 of a clinical SPECT is 5-10 mm (133) which is at

least an order of magnitude poorer than that of CT and MRI (101). However, the newest clinical

SPECT scanners with multi-pinhole collimators (G-SPECT, MILabs) have increased γ-photon

sensitivity and offer spatial resolutions as low as 3 mm (134, 135). Small-animal SPECT systems

using multi-pinhole collimators have been developed that offer spatial resolutions of <1 mm

FWHM (136). This sub-millimeter resolution is required as organs are several orders of

magnitude smaller than humans. Spatial resolution also decreases with increasing distance

between the patient and the detector.

Sensitivity of SPECT vs PET: The sensitivity of a nuclear imaging system is basically based on

its photon-collection efficiency and can be reported as counting rate per unit radioactivity

(cps/Bq) in the scanner field of view (FOV) (112). In SPECT, the physical collimators are

needed in order to reject photons that are not within a small angular range otherwise the angle of

incidence will not be known leading to low geometric efficiencies. In PET, the coincidence

detection of photon events negates the need for physical collimation and results in a sensitivity,

which is typically 10 to 100-fold higher than SPECT (106). This greater sensitivity results in

improved image quality (signal-to-noise ratios) and may accelerate data acquisition time (106).

Furthermore, 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.

Temporal resolution of SPECT vs PET: The ability of an imaging instrument to frequently (over

a period of time) capture ‘acceptable’ images of an object in the FOV is defined as temporal

resolution. Temporal resolution is important for dynamic studies, in which several images are

obtained over a short period of time. Compared to spatial resolution, quantification of temporal

resolution is difficult and depends on the reconstruction algorithm being used. Since the temporal

resolution is very closely related to the sensitivity of imaging, PET has an advantage over

SPECT for dynamic studies.

32

Quantification of radioactivity in SPECT vs PET: In PET, attenuation depends only on the

overall probability of 2 anti-parallel 511-keV photons reaching the 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. Due to the single-photon emission nature of

SPECT, attenuation changes depend on the point of emission (106). 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. A transmission CT scan can calculate the

attenuation correction factors at different positions in the body as well as provide anatomic

information for co-registration with SPECT or PET image.

SPECT/CT is not commonly employed for detecting PnCa in the clinic, yet novel tracers may

promote its use in the future (137). Spanu et al. conducted a study in 104 patients with non-

functioning gastroenteropancreatic (GEP) tumour with overexpression of somatostatin receptor

to evaluate the usefulness of 111In-pentetreotide SPECT/CT in diagnosis and staging of GEP

compared to the conventional imaging procedures (CIP), such as CT, MRI and US (138). At both

CIP and SPECT/CT, 34/104 patients were classified as no evidence of disease and in 70/104

patients, neoplastic lesions were ascertained. At this stage, there was no significant difference in

the sensitivity and accuracy between SPECT/CT (91.4 and 94.2%, respectively) and CIP (71.4

and 80.8%, respectively). Out of 292 ascertained lesions (141 hepatic, 78 abdominal extra-

hepatic and 73 extra-abdominal), CIP detected 191/292 (65.4%) lesions in 50 patients, while

SPECT/CT detected 244/292 (83.6%) in 64 patient (p<0.0001). No false positive results were

found at both SPECT/CT and CIP. SPECT/CT sensitivity and accuracy were higher than CIP in

GEP grade 1 and grade 2, neuroendocrine carcinoma and mixed adeno-neuroendocrine

carcinoma patients, but significantly only for GEP grade 1. Another study showed that clinical

imaging of mesothelin-expressing PnCa in two patients with PDAC using an 111In-labeled

chimeric monoclonal antibody, amatuximab, produced a tumour to background ratio ≥1.2,

sufficient for distinguishing between tumour and normal tissue (139).

1.4.6 Summary of clinical roles for imaging in PnCa

In conclusion, MDCT is the preferred initial imaging modality for patients with a high suspicion

of PnCa, but MRI may also be used for this purpose (70). The most accurate method for

33

definitive diagnosis of PnCa is EUS and its combination with CT/MRI can successfully

determine tumour resectability. The presence of PnCa on imaging can be confirmed

histologically with EUS/EUS-FNA. PET scans can provide valuable information on metastatic

disease in PnCa, which changes the stage of the disease and the decision regarding resectability

(70). Clinical studies show that PET/CT has higher sensitivity and specificity (97.4% and 91.2%;

respectively) compared to CT (85.9% and 67.3%; respectively) for detecting metastatic disease

(140).

Small Animal PET and SPECT

PET and SPECT small animal imaging systems provide greater spatial resolution compared to

clinical PET and SPECT imaging systems and therefore they can be a translational research tool

to develop radiopharmaceuticals for both clinical PET and SPECT as well as

radioimmunotherapy. In vivo imaging bridges the gap between in vitro exploratory and in vivo

clinical research and facilitates the direct and fast clinical translation of newly developed

radiopharmaceuticals. There exists a broad array of preclinical studies evaluating imaging of

PnCa using animal imaging systems. High tumour accumulation of AMB8LK antibody targeting

ferritin labeled with 111In in mice with subcutaneous (s.c.) CAPAN-1 xenografts was shown by

SPECT/CT at 72 h post-injection (p.i.) with 23.6 ± 3.9% injected dose per gram (%ID/g) (141).

In another study, overexpression of integrin αvβ6, a member of the integrin family, in pancreatic

BxPC-3 xenografts was targeted using 99mTc-labeled integrin αvβ6-targeting peptide (99mTc-

HHK) and imaged by SPECT at 0.5 h p.i with an uptake of 0.88 ± 0.12 (%ID/g) (142). Liver

metastatic BxPC-3 tumour lesions in orthotopic models were clearly detected by SPECT/CT.

Targeting epidermal growth factor receptor (EGFR) using panitumumab is another approach to

target radioactivity to pancreatic tumour. Panitumumab fab fragments (F(ab')2) produced by

proteolytic digestion of panitumumab IgG labeled with 64Cu using NOTA has been studied for

PET imaging of NOD-scid mice engrafted subcutaneously or orthotopically with patient-derived

OCIP23 pancreatic tumours, or with s.c. PANC-1 human PnCa xenografts (143). Tumour uptake

of the tracer in the mentioned tumour models at 48 h p.i. was 12.0 ± 0.9% ID/g, 11.8 ± 0.9%

ID/g and 6.1 ± 1.1% ID/g; respectively and tumour to blood ratios were 5:1 to 9:1 for OCIP23

and 2.4:1 for PANC-1 tumours. All tumour xenografts were clearly imaged by microPET/CT at

24 or 48 h p.i. of 64Cu-NOTA-panitumumab F(ab')2. Lou et al. developed a bispecific

34

heterodimer for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic

tumours (144). An anti-tissue factor (TF) Fab was conjugated an anti-CD105 Fab, via the bio-

orthogonal "click" reaction between tetrazine (Tz) and trans-cyclooctene (TCO) and labeled with

64Cu. PET imaging of BXPC-3 (TF/CD105(+/+)) xenografts displayed significantly enhanced

tumour uptake (28.8 ± 3.2 %ID/g) at 30 h p.i., as compared with monospecific anti-TF Fab or

anti-CD105 Fab tracer (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; respectively) with a tumour to muscle

ratio of 75.2 ± 9.4. Tumour uptake of 64Cu-heterodimer in the orthotopic model was 17.1 ± 4.9

%ID/g at 30 h p.i. with a tumour to muscle ratio of 72.3 ± 46.7.

There is an increasing role for nuclear imaging in oncology. In general, PET and SPECT as non-

invasive functional imaging modalities can provide important information on the drug

development process (e.g pharmacokinetics and pharmacodynamics of novel therapeutics) by

utilizing radiotracers.

1.5 Therapeutic targets for PnCa

PnCa treatment continues to be a challenge because of its advanced stage at the time of

diagnosis, its aggressive nature, genetic instability and of course limited treatment options

available. With identification of various molecular pathways in PnCa tumourigenesis, potential

targets for drug development have been pursued with the use of monoclonal antibodies (mAbs)

and small-molecule inhibitors (9). Potential targets in pancreatic tumourigenesis that have been

studied thus far has been summarized in Figure 1-8.

35

Figure 1-8. Potential targets in pancreatic tumourigenesis.

36

Studies have explored some genetic mutations and altered expression patterns, which may serve

as potential targets for treatment of PnCa (Table 1-4). Immunohistochemistry studies have

shown that 73% of PaCa tumours carried Kirsten rat sarcoma (KRAS) mutation, 74% of them

expressed cyclooxygenase-2 (COX-2), 61% had high TOPO 1, and 44% had high SPARC

expression.

37

Table 1-4. Potential molecular targets with frequency of genetic aberration on PnCa and their

targeted agents [reviewed in (9)].

Target Frequency of Genetic

Aberration Targeted Agent

KRAS >90% (145) Tipifarnib, salirasib

VEGF

93% (146)

Bevacizumab, axitinib,

Sorafenib, aflibercept

Mesothelin 90%-100% (147, 148) SS1P, MORAb-009

MUC1 90% (149) Vaccine

COX-2 67%-90% (150-152) Celecoxib, apricoxib

Cholecystokinin/gastrin 95% (152) Gastrazole

Glycine-extended gastrin

55% (152) G17DT

Amidated gastrin

23% (152)

G17DT

Notch3 69%-74% (153)

Hedgehog 70% (154) Saridegib (IPI-926)

EGFR 43%-69% (155, 156) Erlotinib, cetuximab

IGF-1R

64% (157)

AMG-479,

NVP-AEW541

38

Although encouraging results have been demonstrated with multiple targeted therapies of PnCa

in preclinical studies, only erlotinib, an of epidermal growth factor receptor (EGFR) inhibitor,

showed a marginal survival benefit in a phase III clinical trial, when combined with gemcitabine

(9). Table 1-5 summarized all phases II/III clinical trials with targeted therapies in PnCa.

39

Table 1-5. Phase II-III clinical trials with targeted therapies in PnCa [reviewed in (9)].

Target Study Treatment Trial

Phase

No.

Patients

PFS MS 1-y Survival

EGFR GEM + erlotinib (158)

GEM

GEM + cetuximab (159)

GEM + cetuximab (160)

GEM

Phase III

Phase II

Phase III

285

284

41

372

371

3.75 mo

3.55 mo

3.8 mo

3.4 mo

3.0 mo

6.24 mo

5.91 mo

7.1 mo

6.3 mo

5.9 mo

23%

17%

31.7%

VEGF GEM + BEV (161)

GEM + BEV (162)

GEM + P

GEM + erlotinib + BEV (163)

GEM + erlotinib + P

GEM + sorafenib (164)

Sorafenib

GEM + axitinib (165)

GEM

GEM + axitinib (166)

GEM

GEM + aflibercept (167)

GEM + P

Phase II

Phase III

Phase III

Phase II

Phase II

Phase III

Phase III

52

279

256

306

301

37

15

69

34

314

316

271

275

5.4 mo

3.8 mo

2.9 mo

4.6 mo

3.6 mo

2.9 mo

2.3 mo

4.2 mo

3.7 mo

4.4 mo

4.4 mo

3.7 mo

3.7 mo

8.8 mo

5.8 mo

5.9 mo

7.1 mo

6.0 mo

6.5 mo

4.3 mo

6.9 mo

5.6 mo

8.5 mo

8.3 mo

6.5 mo

7.8 mo

29%

36.8%

23.5%

IGF-IR GEM + ganitumab (168)

GEM + conatumumab

GEM + P

Phase II

42

41

42

5.1 mo

4.0 mo

2.1 mo

8.7 mo

7.5 mo

5.9 mo

39%

20%

23%

MMPI GEM + marimastat (169)

GEM + P

BAY 12-9566 (170)

GEM

Phase III

Phase III

120

119

138

139

92.5 d

96 d

1.68 mo

3.5 mo

165.5 d

164 d

3.74 mo

6.59 mo

18%

17%

10%

25%

Gastrin G17DT responder (171)

G17DT nonresponder

Placebo

Gastrazole (172)

5-FU

Phase II

Phase III

49

27

75

53

42

176 d

63 d

83 d

7.9 mo

4.5 mo

33.3%

11.1%

MEK-1 Selumetinib (173)

Capecitabine

Phase II 37

32

2.1 mo

2.2 mo

5.4 mo

5 mo

COX-2 GEM + cisplatin + celecoxib

(174)

GEM + celecoxib (175)

GEM + irinotecan + celecoxib

(176)

Phase II

Phase II

Phase II

22

42

21

1.8 mo

5.1 mo

9 mo

5.8 mo

9.1 mo

18 mo

46% (6-mo)

80%

PFS: Progression-free survival MS: median survival

GEM: gemcitabine BEV: bevacizumab

P: placebo

40

1.6 Radioimmunotherapy of PnCa by targeting EGFR

EGFR is overexpressed on more than 90% of cases of PnCa in patients (177) and correlates with

more advanced disease, poor survival and the presence of metastases (178, 179). EGFR is the

first member of the human epidermal growth factor receptor family which also includes the

HER2, HER3 and HER4 receptors. Binding of EGF to the EGFR causes receptor

homodimerization or heterodimerization with other EGFR family members and initiates

mitogenic signaling pathways (180). EGFR-mediated cell signaling plays a major role in

promoting tumour proliferation, angiogenesis, metastasis, and evasion of apoptosis (181, 182).

Panitumumab or Vectibix is a U.S. FDA and Health Canada-approved fully human IgG2 mAb

that binds to the extracellular domain of the EGFR and competitively inhibits EGF binding to

EGFR. Panitumumab was initially approved for the treatment of refractory EGFR-expressing

metastatic colorectal cancer with disease progression however later on the lack of benefit with

anti-EGFR mAbs (panitumumab and cetuximab) in patients who carried KRAS mutations was

demonstrated by Amado et al. (182). Since PnCa has the highest incidence of activating KRAS

mutations of any cancer at over 90% (183-187), immunotherapy with panitumumab is expected

to be ineffective on PnCa cells. However, antibodies can serve as carriers for specific delivery of

cytotoxic agents such as radionuclides. For this purpose halogen radionuclides are incorporated

directly to antibodies, whereas other radionuclides are complexed to antibodies through

chelators. Both preclinical and clinical studies have demonstrated that radioimmunoconjugates

(RIC; antibodies labeled with radioisotopes), are more effective than therapy with naked

antibodies (187-190). The major reason might be because radioimmunotherapy (RIT) only relies

on targeting a cell-surface receptor/antigen to deliver a cytotoxic radionuclide payload to

tumours, whereas naked mAbs are intended to block downstream receptor-mediated growth

signaling pathways, which may be obviated or circumvented by compensatory upregulation of

alternative signaling mechanisms (e.g. KRAS mutation). This is of a great importance for PnCa

patients who have tumours that overexpress EGFR but carry KRAS mutation, therefore RIT of

PnCa with panitumumab may be a useful alternative strategy to treatment with naked anti-EGFR

mAbs.

41

1.6.1 Types of radiation used in radioimmunotherapy

Radiation can be divided into two classes: i) photon radiation or penetrating radiation such as X-

radiation and γ-rays and ii) particle radiation or non-penetrating radiation including alpha particle

(α-particle), beta particle (β-particle) and Auger electron. As opposed to photon radiation,

particle radiation deposits more energy and this characteristic makes them more efficient for

eradicating cancer cells. Basically these particles are characterized based on the amount of

energy deposited over the track length of the radiation in tissues (i.e. linear energy transfer:

LET). The emission of particles by radioisotope may be accompanied by the emission of gamma

photons (γ) allowing SPECT imaging of the delivery of the RIT agents to tumours, dosimetry,

and monitoring of therapeutic efficacy. However, γ emissions could contribute to normal tissue

toxicity due to their penetrating ability (191, 192).

1.6.1.1 Alpha-particle

An alpha-particle (α-particle) is a helium-4 (4He) nucleus consisting of two protons and two

neutrons which carries a +2 charge. Alpha particles are high energy (4-9 MeV) with high linear

energy transfer (LET; 50–230 keV/μm). Moreover, α-particles have a range of only 50–100 μm,

which restricts their coverage to 5–10 cell diameters. These properties make α-particles useful

for eradicating circulating malignant cells or small clusters of cells. Table 1-4 lists a number of

α-emitting radioisotopes of interest for RIT. Some α-emitters (e.g. 225Ac and 225Ra) decay to α-

emitting or β-emitting daughter products which creates “radionuclide generator systems”. Such

radionuclides deliver a higher radiation absorbed dose to tumours than radionuclides that decay

to stable elements or daughter products that emit radiation with lower relative biological

effectiveness (RBE) such as β-particles, X- or γ-radiation (190). RBE describes the effectiveness

of a particular type of radiation for causing an effect (e.g. cytotoxicity) compared to X- or γ-

radiation (191). Although the high RBE provided by the radionuclide generator concept is

desirable for RIT, the release and redistribution of the free daughter products to normal tissues

has been proposed as a limitation. Since the α-emitting daughters represent different chemistry

from their parents atoms they will not remain complexed to chelators substituted onto the mAb.

Furthermore the high recoil energy upon decay also promotes their release from the chelators

(191). Studies in non-human primates have shown that redistribution of 213Bi released from

42

225Ac-labeled anti-CD33 mAb HuM195 caused acute renal dysfunction following deposition of

α-radiation in the kidneys (192-194).

Table 1-6. α-particle emitters for conjugation to mAbs for RIT of cancer.

Radionuclide Production

method

α-particle

energy

(MeV)

Physical

half-life

(t1/2P)

Method for radiolabeling

225Ac 223U→225Ac 5.8 10 days Chelation by DOTA or NETA

211At 209Bi(α,2n)211At 7.4 7.2 h Radioastatination

212Bi 212Pb → 212Bi 6.0 61 min Chelation by DTPA or DOTA

213Bi 225Ac → 213Bi 6–8.4 46 min Chelation by DTPA or DOTA

212Pb 224Ra → 212Pb 6.0d 10.6 h Chelation by TCMC

227Th 227Ac → 227Th 5.9 18.7 days Chelation by DOTA

DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

NETA: 4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl-acetic

acid;

DTPA: diethylenetriaminepentaacetic acid; 212Pb decays by β-particle emission to 212Bi which emits an β-particle of this energy.

TCMC: 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1, 4, 7, 10-tetra-(2-carbamoyl methyl)-

cyclododecane.

43

Considering a RBE of 1 for β-radiation, The RBE range of 3 to 7 for α-particles ranges indicates

that at the same radiation absorbed dose biological effects of α-particles are 3 to 7 times greater

than for β-radiation (195). Nuclear DNA is the primary target of α-particles for cell death

induction. Radiation induced DNA double-strand breaks (DSBs), accumulate phosphorylated

histone 2A (gamma-H2AX) at the site, as the first step in recruiting and localizing DNA repair

proteins (196). Gamma-H2AX can be detected as a marker of DNA DSBs in vitro by

immunofluorescence probing. Hypoxia develops resistance to low LET radiation in cancer cells.

As opposed to low LET radiation that requires oxygen free radicals to indirectly damage DNA,

the induction of DSBs by α-particles is independent of tissue oxygenation (197, 198). Studies

showed that 213Bi-labeled EGFR mAb matuzumab, killed hypoxic CAL 33 squamous cell

carcinoma cells as effectively as normoxic cells in vitro (197).

1.6.1.2 Auger electrons

Auger electrons have relatively low energies ranging from a few eV to approximately 100 keV

(199). The range of Auger electrons in water is from a fraction of a nanometer to several

hundreds of micrometers and the range of LET is between 4 to 26 KeV/µm. Auger electrons

induce multiple lethal DNA DSBs in cancer cells, particularly if these electrons are released in

close proximity to nuclear DNA. Auger electrons are emitted by radionuclides that decay by

electron capture (EC) and/or internal conversion (IC). In these processes the proton-rich

nucleus of atom absorbs an inner atomic electron, usually from the K or L electron shell. The

vacancies created are filled by electrons decaying from higher shells creating subsequent

vacancies that move progressively towards the outer shells. These transitions are ultimately

accompanied by the emission of either a characteristic X-ray or low-energy Auger or IC electron

(191). The emission of on average 5 to 36 electrons following every EC and/or IC event

transiently imparts a positive charge on the decaying atom and deposits highly localized energy

surrounding the site of decay. The lethal DNA damaging effects of Auger electrons is dependent

on the position of radionuclide decay relative to the DNA (200). In order to exploit the high LET

of these electrons and achieve a high RBE, decay of Auger electron emitters should be in close

proximity to nuclear DNA (201-204). Dosimetric estimates showed that the Auger electron-

emitter, 125I deposits the highest radiation absorbed dose in the immediate vicinity of the decay

site (109 cGy/decaying atom) with increasing distance up to a few nanometers from the DNA

44

duplex (201, 202, 205, 206). When Auger electrons are emitted near but not within the DNA,

DSBs are caused indirectly by hydroxyl radical ( OH)-mediated ionizations, however electrons

still efficiently generates single-strand breaks (SSB) (207, 208). Some studies have exploited

nuclear translocation sequence (NLS) peptides for mediating nuclear transport of monoclonal

antibodies (mAbs) labeled with the Auger electron-emitter, 111In following receptor-mediated

internalization by cancer cells (209-211). Dosimetric estimates revealed that the mean lethal dose

of 125I conjugated to iododeoxyuridine (IUdR) which is incorporated directly into DNA is 7-fold

lower than X-radiation for killing mammalian cells (206). In contrast, the cytotoxic effects of the

Auger electron-emitters, 51Cr, 67Ga, 75Se, 125I, or 201Tl bound to the cell membrane (204, 212) or

located inside (201-204) or outside the cell were no different from low LET γ-radiation [i.e.

RBE < 2].

Pouget et al. studied the cytotoxicity of 125I when it’s localized in the nucleus (125I-labeled tat

peptides), cytoplasm (125I-labeled anti-EGFR mAb 225 or anti-HER2 trastuzumab mAbs) or

membrane (125I-labeled anti-carcinoembryonic antigen (CEA) 35A7 mAbs) of A431 epidermoid

cancer cells or SK-OV-3 ovarian cancer cells. Due to the DNA damaging properties of the Auger

electrons, nuclear 125I was the most cytotoxic agent, but membrane bound 125I proved more

effective for killing cells than 125I deposited in the cytoplasm (213). According to the critical

function of cell membrane in cell survival there seems to be no absolute need for internalization

of mAbs for Auger electron RIT, although nuclear localization enhances the potency of Auger

electrons. Ionizing radiation generates ceramide-enriched membrane microdomains which recruit

pro-apoptosis factors or interfere with the binding of growth factors and survival proteins that

trigger apoptosis (214-217).

Auger electron-emitters exhibit a localized “bystander effect” which promotes the apoptosis or

sometimes paradoxically stimulates the growth of non-targeted tumour cells that are proximal to

targeted cells (218-220). Co-inoculation of male Ncr nude mice with a mixture of unlabeled

LS174T human colon cancer cells and 125I-labeled cells demonstrated significant tumour growth

inhibition compared to inoculation of only unlabeled cells, whereas a mixture of unlabeled

and 123I-labeled cells exhibited enhanced tumour growth compared to inoculation of only

unlabeled cells (220). In a separate study, 123I demonstrated growth inhibitory effects on cancer

cells exposed to medium from cells that bound 123I-labeled metaiodobenzylguanidine (123I-

45

mIBG) and that decreased their clonogenic survival (CS) which is possibly mediated by release

of cell toxins from irradiated cells (218). Due to the nanometer range of Auger electrons, they

don’t have a cross-fire effect that can widely irradiate and affect non-targeted cells, however

emission on the cell surface of higher energy Auger electrons with a longer range can have a

more localized “cross-dose” effect in which targeted cells irradiate neighboring non-targeted

cells. This may overcome heterogeneity in tumour localization of the radiolabeled mAbs and

internalization by cancer cells (221). These cross-dose and bystander effects may contribute to

the effectiveness of Auger electron RIT for treatment of cancer (221).

Several Auger electron-emitting radionuclides have been studied for radiolabeling mAbs

(Table 1-7). There are several important factors that should be considered in selecting a

radionuclide for Auger electron RIT including: i) the energy of the emitted electrons, ii) the ratio

of penetrating forms of radiation (e.g. X-rays and γ-photons) compared to non-penetrating Auger

electrons (p/e ratio), iii) the physical half-life (t1/2p) of the radionuclide, and iv) the method for

radiolabeling the mAb (191). What determines the toxicity of a radioisotope is the average

electron energy not the total energy. If a radioisotope with high electron yield deposits a high

energy this energy would be distributed among a large number of electrons and the energy

portion of each electron would be decreased. For example 67Ga with a total electron energy of

34.4 keV but low electron yield (5.0 electrons/decay; Table 1-5) results in a higher average

electron energy compared to 111In or 125I, radionuclides which have total electron energies of

32.7 keV and 19.4 keV, but higher electron yield (14.9 and 15.1 electrons/decay,

respectively) (191, 222). A dose limiting factor for using Auger electron emitters is the emission

of penetrating forms of radiation. Although these forms of radiation especially γ-photons can be

exploited for dosimetry and monitoring tumour response by SPECT imaging, these may

contribute to normal organ toxicity, especially at the high doses of radioactivity required for

effective RIT.

46

Table 1-7. Auger electron emitters for conjugation to mAb for RIT of cancer.

Radionuclide Production method

Electron

yield per

decay

Total electron

energy (keV)

γ-emission (keV)

and abundance

half-life

(t1/2p)

Method for

radiolabelinga

125I 124Xe(n,γ)125Xe → 125I 25.8 19.4 36(7%) 57 days Radioiodination

123I 124Xe(n,2n)123Cs ⟶ 123Xe ⟶ 123I or 124Xe(p,pn)123Xe ⟶ 123I

15.1 27.6 159(83%) 13.2 h Radioiodination

111In 112Cd(p,2n)111In 14.9* 32.7 171 (90%);

245 (94%) 2.8 days

Chelation by

DTPA or DOTA

67Ga 68Zn(p,2n)67Ga 5.0 34.4

93 (36%);

185 (20%);

300 (16%)

3.3 days Chelation by DFO

or DOTA

99mTc 99Mo ⟶ 99mTc 5.1 16.3 140 (98%) 6.0 h Chelation by

HYNIC or N2S2

64Cu 64Ni(p,n)64Cu 4.0 18.2 d511 (19%) 12.7 h Chelation by

DOTA or NOTA

*Some studies reported 111In emits 8 electrons per decay (223).

DFO: desferrioxamine

HYNIC: hydrazinenicotinamide

N2S2: diamide dimercaptide

Energy of the two annihilation γ-photons resulting from 64Cu positron emission

NOTA: 1,4,7-triazacyclononane-N,N′,N″-triacetic acid.

47

1.6.1.3 β-particles

In neutron rich atoms, a neutron transforms into a proton by emitting an electron (β-particle).

The mass of the emitted β-particles is 1/200 of the mass of a proton or neutron. β-particles are

moderate to high energy electrons (0.50–2.30 MeV) that travel a relatively long range in

tissues (0.05–12.00 mm) with low LET of 0.1–1.0 keV/μm resulting in low deposition of energy

in tumour cells restricting the potency for killing these cells (224). The long path length of β-

particles however can overcome the heterogeneous distribution of antibodies in tumour tissue

through crossfire radiation but it also increases the bone marrow toxicity due to a cross-fire

effect from circulating radiolabeled mAbs. Furthermore, most of the energy of the β-particles is

deposited at the end of its track length, which may be outside the treatment volume of a single

cell or cluster of tumour cells. In contrast Auger electrons have a maximum range of 100 µm that

restrict their cross-fire to 10 cell diameters (225), and alpha particles deposit their energy over 5-

10 cell diameters which would represent a small cluster of tumour cells. As such, β-emitters are

not ideal for the treatment of small tumour burdens including micro-metastatic disease.

48

Table 1-8. β-particle emitters for conjugation to mAb for RIT of cancer.

Radionuclide Production Eβmax

(MeV)

γ-emission (keV) and

abundance Half-life

Method for

radiolabeling

90Y Sr (n, γ)90Y 2.28 - 2.7 days Chelation by DOTA

131I Th (n, γ) 0.81 364(81%) 8 days Radioiodination

177Lu 176Lu (n, γ)177Yb⟶ 177Lu 0.5 208(11%);

113(6%) 6.7 days Chelation by DOTA

186Re 185Re (n, γ)186Re 186W(p,n)186Re

1.08 137(9%);

122(12%) 3.8 days

Chelation by N3S,

HYNIC or N2S2

188Re 187W(n,γ)188W ⟶ 188Re 2.12 155(15%) 17 hours Chelation by N3S,

HYNIC or N2S2

67Cu 68Zn(p,2p)67Cu 0.58 184.6 (48%);

93.3(23%) 2.6 days

Chelation by DOTA or

NOTA

N3S: triaminemonothiol

HYNIC: hydrazinenicotinamide

N2S2: diamide dimercaptide

49

90Y and 131I are the most commonly used two radionuclides for RIT. 90Y delivers β particles with

5 times more energy to the tumour than 131I (Table 1-8); it also has a more favourable half-life

than 131I (2.5 days compared with 8 days). The longer path length of 90Y compared to 131I (5 mm

vs 0.88 mm) may be advantageous for treatment of larger tumours or those with poor antibody

penetration but it increases toxicity to the normal surrounding tissues. Non-tumour distribution

of uncomplexed 90Y has been reported to mainly be to the bone and liver, that of 131I is to the

thyroid. As opposed to 131I which also emits γ-rays, 90Y requires a surrogate γ-emitter (e.g. 111In)

to estimate dosimetry by imaging (226). Once radiolabeled antibody is internalized into the cells,

it undergoes lysosomal degradation. Radiolabeled amino acid (monoiodotyrosine) is rapidly

transported out of the cell whereas 90Y-chelator catabolites are not recognized by amino acid

transporter and remain trapped in the lysosome (227). Furthermore γ-radiation of 131I requires the

shielding of hospital personnel and restrictions following patient discharge. Currently 90Y-

ibritumomab tiuxetan (Zevalin) and previously, 131I-tositumomab (Bexxar) have been used for

RIT of patients with indolent B-cell non-Hodgkin lymphomas (NHL) by targeting CD20. Both

agents exhibit similar clinical outcomes but with unique clinical considerations and radiation

precautions due to the reasons discussed above (228).

Recently there has been a lot of interest in using 177Lu for RIT. Table 1-9 shows a list of

ongoing clinical trials found on ClinicalTrials.gov for treatment of cancer using 177Lu labeled

radioimmunoconjugates (RICs). 177Lu is a therapeutic reactor-produced radiometal that emits β-

particles [Eβ(max)=498 keV (78.6%), Eβ(max)=385 keV (9.1%) and Eβ(max)=176 keV

(12.2%)]. It is easily produced on a large scale and at low cost. The energy of β-particles emitted

by 177Lu is interemediate between that of 131I and 90Y offering high tumour toxicity and reduced

toxicity to surrounding organs and to the bone marrow. Low abundance γ-ray [Eγ=113 keV

(6.4%) and Eγ=208 keV (11%)] emitted by 177Lu allows for SPECT imaging without the

complication associated with high abundance γ-ray emitted by 131I [Eγ=364 keV (81%)] with

regard to the shielding and patient discharge. These characteristics make 177Lu an attractive

option for therapy of cancers. However, the low abundance of the gamma emissions from 177Lu

limit its usefulness for imaging, and often a more abundant gamma emitter such as 111In is used

as a surrogate for imaging the biodistribution of the radiolabeled antibodies.

50

Table 1-9. Ongoing clinical trials for cancer treatment using 177Lu labeled RICs and

radiopeptides.

Disease Treatment Mechanism Phase

Kidney Cancer, Head and

Neck Cancer, Breast Cancer,

Non-small Cell Lung Cancer

(NSCLC), Colorectal Cancer,

Pancreatic Cancer, Ovarian

Cancer, Esophageal Cancer,

Gliomas

177Lu-J591 - J591: Anti-PSMA mAb Not

applicable

Prostate Cancer 177Lu-J591

Ketoconazole

Hydrocortisone 111In-J591

- Ketoconazole: inhibition of the

activity of enzymes necessary for the

conversion of cholesterol to steroid

hormones such as testosterone and

cortisol (229, 230)

- Hydrocortisone: to compensate for

cortisol insufficiency

Phase II

Prostate Cancer 177Lu-J591

Docetaxel

Prednisone

- Docetaxel: Inhibition of mitotic cell

division by preventing physiological

microtubule

depolymerisation/disassembly in the

absence of GTP (231, 232)

- Prednisone: Downregulation of

glucocorticoids synthesis

Phase I

Small Cell Lung Cancer

Non-small Cell Lung Cancer

(NSCLC)

TF2 mAb pre-

targeting 177Lu-IMP-288 111In-IMP-288

- TF2: anti-CEA/anti-HSG bispecific

mAb

- IMP-288 hapten HSG peptide

Phase I

Phase II

Pancreatic Carcinoma MVT-1075 with

blocking dose of

MVT-5873

- 5B1: anti CA19-9 mAb

- MVT-5873: Unlabelled 5B1 mAb

- MVT-1075: 177Lu- MVT-5873

Phase I

Small Cell Lung Cancer

Nivolumab 177Lu-DOTA-

TATE

- Nivolumab: Anti PD-1 mAb

- DOTA-TATE: Targeting

somatostatin receptor

Phase I

Phase II

Metastatic Renal Cell

Carcinoma

111In-DOTA-

cG250 177Lu-DOTA-

cG250

- cG250: Anti G250/CAIX/MN mAb Phase I

Phase II

Diffuse Large B-cell

Lymphoma

Betalutin (177Lu-

lilotomab

satetraxetan)

Betalutin: Anti-CD37 mAb Phase I

PSMA: prostate specific membrane antigen CEA: carcinoembryonic antigen

HSG: histamine-succinyl-glycine Dotatate: DOTA0-Tyr3-Octreotate

PD-1: Programmed cell death-1

51

Recently a randomized Phase 3 clinical trial of 177Lu-DOTA0-Tyr3-Octreotate (177Lu-

DOTATATE; Figure 1-9) in 229 patients with inoperable, progressive, somatostatin receptor

positive midgut carcinoid tumours (NETTER-1) showed markedly longer progression-free

survival and a significantly higher response rate than high-dose octreotide long-acting repeatable

(LAR) among patients with advanced midgut neuroendocrine tumours (233). 177Lu has

application for radiolabeling peptides (e.g. 177Lu-DOTATATE) as well as mAbs for

radiotherapeutic purposes (Table 1-9).

52

Figure 1-9. The chemical structure of 177Lu-DOTATATE

53

In summary, due to their potentially greater potency and lower non-specific toxicity to non-

targeted normal tissues including the hematopoietic stem cells in the bone marrow, α-particle and

Auger electron-emitting radionuclides are highly attractive for RIT of malignancies (Figure 1-

10A). However, their short range makes these forms of radiation most suitable for eradicating

single cells and small volume disease (<1 cm diameter) whereas the longer range of β-particles

emitted by 177Lu or 90Y makes this form of radiation more feasible for treating larger tumour

masses (>1 cm) (Figure 1-10B).

54

Figure 1-10. A) The cross-fire effect of the 2–10 mm range β-particles emitted by circulating

radiolabeled mAbs perfusing the bone marrow (BM) decreases the viability of hematopoietic

stem cells resulting in myelosuppression. In contrast, α-particles have a range of only 28–100 μm

and Auger electrons have a range <0.5 μm which greatly reduces or eliminates the BM toxicity

of radioimmunotherapy (RIT). B) Illustration of the track of α-particles, β-particles or Auger

electrons emitted by radiolabeled mAbs targeted to cancer cells. The short track length of α-

particles (28–100 μm) and Auger electrons (<0.5 μm) results in high linear energy transfer (LET)

values of 50–230 keV/μm and 4 to 26 keV/μm, respectively. β-particles have a track length of 2–

10 mm resulting in LET of 0.1–1.0 keV/μm. The high LET of α-particles (50–100 μm) and

Auger electrons makes these forms of radiation more powerful for killing cancer cells than β-

particles. Reprinted with permission from Aghevlian S et al. (191).

55

1.6.2 Clinical trials with RICs

Since the 1980s, clinical trials have tested numerous RICs for cancer but the field continues to

develop with the introduction of new humanized and fully human antibodies and radionuclides

(55). The results from clinical trials on the role of RIT as a neoadjuvant treatment to shrink a

tumour to a resectable state is also controversial and conflicting (55, 234). 131I labeled anti-CEA

mAb, Kab201, were the first RICs that were advanced to a phase I/II clinical trial for treatment

of 18 patients with locally advanced or metastatic PnCa (235). The overall response rate was 6

%. Several subsequent clinical trials have been performed with other RICs and will be discussed

in this section.

1.6.2.1 90Y-clivatuzumab tetraxetan

Humanized clivatuzumab (hPAM4) mAb specifically targets the mucin

glycoprotein MUC1 expressed on most PnCa. 90Y-clivatuzumab tetraxetan (trade name hPAM4-

Cide) is a radiopharmaceutical designed for the treatment of PnCa. In a Phase I clinical trial

thirty-eight previously untreated patients received gemcitabine 200 mg/m2 weekly for 4 weeks

with 90Y-hPAM4 given weekly in weeks 2, 3, and 4 (cycle 1), and the same cycle was repeated

in 13 patients (cycles 2–4) (Figure 1-11).

56

Figure 1-11. The protocol schema of RIT indicates fractionated RIT; 111In-hPAM4/90Y-hPAM4

indium-111 or yttrium-90 labeled humanized clivatuzumab tetraxetan; Gem: gemcitabine; PK,

pharmacokinetics.

57

The maximum tolerated dose of 90Y-hPAM4 was determined to be 12.0 mCi/m2 (444

MBq/m2) weekly for 3 weeks for cycle 1, with ≤9.0 mCi/m2 (333 MBq/m2) weekly for 3 weeks

for subsequent cycles. Six patients (16%) had partial responses and 16 patients (42%) had

stabilization as their best response (58% disease control). The median overall survival was 7.7

months for all 38 patients, including 11.8 months for those who received repeated cycles (46% [6

of 13 patients] ≥1 year), with improved efficacy at the higher RIT doses (236).

In March 2016 the phase III PANCRIT-1 trial in metastatic PnCa was terminated early due to

lack of improvement of overall survival compared to gemcitabine alone (237). However, as

mentioned, these patients had metastatic PnCa that had progressed on prior treatment regimens,

and there remains the potential to impact patient outcome in PnCa if treatment was commenced

earlier, especially in patients with surgically resectable tumours.

1.6.2.2 131Iodine-Tenatumomab

Tenatumomab is a murine mAb developed by Sigma-tau directed towards Tenascin-C expressed

on malignant pancreas, breast, colorectal, lung, ovarian or B and T cell Non-Hodgkin Limphoma

tissue sections. The use of the 131I-labeled tenatumomab for anti-cancer RIT was tested in a

phase I clinical trial, however due to negligible drug uptake into the tumour lesion this study was

terminated (238).

1.6.2.3 ²¹²Pb-TCMC-Trastuzumab

Alpha particle RIT using lead-212 (²¹²Pb), targeted to HER2 expressing cells by the trastuzumab

antibody was studied, as a potential treatment for patients with metastatic HER-2 expressing

tumours (e.g., ovarian, pancreatic, colon, gastric, endometrial, or breast). In this phase I trial 3

patients with HER-2 positive intraperitoneal cancers who had failed standard therapies received

i.p injection of 212Pb-TCMC-trastuzumab after 4 mg/kg of trastuzumab. Pharmacokinetics and

imaging after 0.2 mCi/m2 (7.4 MBq/m2) i.p. 212Pb-TCMC-trastuzumab in patients with HER-2-

expressing malignancy showed minimal distribution outside the peritoneal cavity, ≤6% urinary

excretion, and good tolerance (239). There is no information available on the effectiveness of

treatment.

58

1.6.2.4 90Y human mAb MN-14 (labetuzumab)

CEA has been recognized as an overexpressed cell surface antigen in many cancers, mainly in

gastrointestinal tract malignancy. 90Y labeled humanized anti-CEA antibody MN14 was

evaluated in phase I and II clinical trials to determine the safety of different dose levels in the

treatment of PnCa (https://clinicaltrials.gov/ct2/show/NCT00041639). No information is

available regarding the result of this trial.

1.6.2.5 177Lu human mAb 5B1 (MVT-1075) in combination with a blocking dose of unlabeled

5B1 (MVT-5873)

MVT-1075, is a human antibody-based RICs developed by 177Lu labelling of 5B1 mAb directed

towards CA19-9. Carbohydrate antigen 19-9, also called cancer antigen 19-9 or sialylated Lewis

(a) antigen) is a tumour marker that used primarily in the management of PnCa. MVT-1075 is

currently being evaluated in a phase I clinical study for the treatment of pancreatic, colon and

lung cancer in combination a blocking dose of unlabeled 5B1 (MVT-5873)

(https://clinicaltrials.gov/ct2/show/NCT03118349).

1.7 Increasing the specific activity (SA)

Increasing the specific activity of RICs may improve their potency for killing cancer cells and

their effectiveness of cancer treatment. Specific activity (SA) is the amount of radioactivity

carried by a RIC. Chelator complexes with radiometal in a 1:1 reaction, therefore the more

chelator present on an antibody the more radioactivity will be delivered to a cancer cell. Studies

have shown that amplification of radioactivity on a molecule of antibody using metal chelating

polymers (MCPs) or dendrimers can not only eradicate cancer cells more efficiently (240, 241)

but also overcome resistance to radiation in some cases (211). Furthermore, conjugation of

antibodies to MCPs or dendrimers provides reproducible labelling without interfering trace

metals. The concentration of trace metal contaminants increases with aging (decay) of the

radioisotope stock solution. Competition of contaminants with the radioisotope to complex the

chelator, can significantly reduce the labeling yield, while the chance of incorporating radiometal

is higher with a large number of chelators on the antibody.

59

1.7.1 Dendrimer

Dendrimers are homogeneous nano-sized, radially symmetric molecules. Their monodisperse

hyperbranched structure has a symmetric core, an inner shell, and an outer shell allowing for

versatile functionalization (242) (Figure 1-12). Dendritic macromolecules tend to linearly

increase in diameter and adopt a more globular shape with increasing dendrimer generation.

Figure 1-12. Three main parts of a dendrimer: the core, end-groups, and subunits linking the two

molecules.

60

The biological properties of dendrimers such as polyvalency, self-assembling, electrostatic

interactions, chemical stability, low cytotoxicity, and solubility renders them a promising

candidate in cancer therapy and imaging fields. Polyvalency provides for versatile

functionalization. The end-groups reaching the outer periphery of dendrimers can be conjugated

to chelating agents, targeting moieties, contrast agents for magnetic resonance imaging (MRI)

and drugs (243) (Figure 1-13).

Figure 1-13. Dendrimer with multiple surface functional groups (e.g. drugs, radioisotope, and

antibodies) can be directed to cancer cells by tumour-targeting entities such as antibodies

specific for tumour-associated antigens.

61

Dendrimers could be substituted with multiple metal chelators for radiolabeling to a high SA,

and conjugated to a mAb for tumour targeting (244, 245). The presence of large numbers of

atoms on a dendrimer molecule enables delivery of sufficient numbers of complexed metals as

MRI contrast agents, diagnostic or therapeutic radioisotopes to cancer cells. For neutron capture

therapy (NCT) of gliomas, Wu et al. developed boron-10 (10B) radiolabeled dendrimers

conjugated to anti-EGFR mAb cetuximab (IMC-C225) for targeted delivery of sufficient 10B to

brain tumours in rats (1100 atoms of 10B per molecule of dendrimer) (244). Polyamidoamine

dendrimers were modified with either 10 DOTA or 10 DTPA and were coupled to mAb 2E4.

Both the DTPA- and DOTA-dendrimer-antibody constructs were then labeled

with 90Y, 111In, 212Bi or stable Gd (III). Wu et al reported that conjugation of dendrimer to mAb

2E4 increased the labeling of 90Y to about a 47 fold higher SA compared to 90Y-labeled mAb

2E4 derivatized with one chelator per mAb 2E4 (1.74 MBq/μg vs. 0.037 MBq/μg) (246). In

another study dendrimers were conjugated to 43 derivatives of DTPA, 2-(p-

isothiocyanatobenzyl)-6-methyl-diethylene triamine penta-acetic acid (1B4M) for radiolabeling

with 111In. Kobayashi et al. demonstrated that 111In-G4-1B4M dendrimer conjugated to a murine

mAb IgG1 OST7 (470 MBq/μg) was labeled to a 54-fold higher SA than 111In-1B4M-OST7 (8.7

MBq/μg) (247). In our research group, dendrimers were reacted with 30 DTPA units then

conjugated through a thiol to maleimide-derivatized trastuzumab for labeling with 111In (128).

DTPA-denrimer-trastuzumab was further derivatized with NLS peptides for nuclear importation.

The substitution level of dendrimer was 1 or 2 dendrimers per trastuzumab. 111In labeling of

DTPA-dendrimer-trastuzumab yielded a 47 fold higher SA than 111In-DTPA-NLS-trastuzumab

(23.6 MBq/μg vs. 0.5 MBq/μg). Clonogenic assays showed that high SA 111In-DTPA-denrimer

RICs (5.5 MBq/μg) were 2-4 fold and 9 fold more cytotoxic to HER2 overexpressing SKBR-3

cells (1.3×106 receptors/cell) and low-HER2 density MDA-MB-231 cells (5×104 receptors/cell)

than low SA 111In-DTPA-NLS-trastuzumab (0.5 MBq/μg) (241). High SA of 111In-dendrimer-

trastuzumab overcomes the previously shown resistance of MDA-MB-231 cells to 111In-DTPA-

NLS-trastuzumab (211).

These studies show the great potential of chelate-functionalized dendrimers to greatly

increase the SA of mAb when labeled with radiometals compared to its direct conjugation to

metal chelators. High SA labeled mAbs can be more cytotoxic to cancer cells with high and low

expression of a particular target receptor such as HER2 (240).

62

1.7.2 Metal Chelating Polymer (MCP)

Polymers like dendrimers can be modified with metal chelators to serve the purpose of

increasing the specific activity for radiometal labeling of antibodies and thus, amplification of

radioactivity delivered to cancer or other diseased cells. Slinkin et al. conjugated Fab fragments

of RD110 mAb raised against cardiac myosin with Polylysine (PL) chelating polymers carrying

40 DTPA chelators for labeling 111In (248). Unlike normal myocardial cells, necrotic

cardiomyocytes cannot prevent binding of anti-myosin antibodies to intracellular myosin

allowing for imaging of necrotic cardiomyocytes by radiolabeled antimyosin mAbs. PL-DTPA-

Fab was labeled with 111In to a 50 fold higher SA (3.7 MBq/μg) than DTPA-Fab derivatized with

DTPA only. In vivo studies in New Zealand rabbits with experimental myocardium infarction

with 111In-PL-DTPA-Fab showed good accumulation of the labeled conjugate in infracted areas

with a target to normal ratio as high as 25:1 (248).

More recently, Dr. Mitch Winnik’s research group (Department of Chemistry, University of

Toronto) has been synthesizing MCPs for simultaneous detection of multiple cell specific

biomarkers on individual cells by high throughout Inductively Coupled Plasma-Mass

Spectrometry (ICP-MS) (249-251). In addition to carrying DOTA chelators for complexing

lanthanides and increasing the sensitivity of the method, this water soluble MCP also has a

functional maleimide end group allowing covalent binding to a mAb. Selective reduction of the

disulfide bonds in the hinge region of the mAb creates thiols which reacts with the maleimide

end group (132). ICP-MS studies showed that mAbs conjugated to lanthanides-labeled MCPs

determined the relative expression of CD33, CD34, CD38, CD45 and CD54 on two leukemic

cells lines (KG1a; THP-1) (249). Besides, multiple mAbs conjugated to MCPs but labeled with

different lanthanides could be used in a single assay without signal interference. Collaboration

with Dr. Winnik’s research group led to the development and synthesis of analogous MCPs to

complex radiometals studied in this thesis (Figure 1-14). Previously, another type of MCP with

polyglutamide (PGlu) backbone and 24-29 DTPA chelators (PGlu (DTPA) 24-29) was synthesised

by Dr. Winnik’s research group. Oxidation of glycans in the Fc domain of antibodies with

sodium-meta periodate (NaIO4) forms aldehyde groups which will further react with the

hydrazide end group on the MCP to form a hydrazone bond between the antibody and MCPs

(240, 252). This method is called site-directed coupling and preserves the antigen binding sites

63

from any chemical modification. Our group showed that the maximum achievable SA for 111In

labeling of trastuzumab-MCP was 90-fold higher compared to trastuzumab directly modified

with two DTPA, and this enhanced the cytotoxic potency in vitro against HER2-positive breast

cancer cells through the emission of Auger electrons by 111In (240).

Figure 1-14. Metal chelating polymer consists of a backbone harbouring multiple pendant

chelators for radiometal labeling to high specific activity as well as a functional end group for

conjugation to a mAb.

64

Dr. Winnik’s research group also synthesised an array of MCPs with varied backbones including

polyacrylamide (PAm), polyaspartamide (PAsp), or polyglutamide (PGlu) and multiple DTPA

units for labeling to the mAb to high SA with 111In. The shared feature of these MCPs was a

biotin end group for reaction to streptavidin derivatized trastuzumab Fab fragments (SAv-Fab)

(253-255). Bi-PAm(DTPA)40 had a net negative charge while Bi-PAsp(DTPA)33 and Bi-

PGlu(DTPA)28 were zwitterionic with a net neutral charge. Biodistribution studies of the MCPs

conjugated to trastuzamab Fab fragments in Balb/c mice showed that zwitterionic MCPs have

the most desirable properties with lower liver and spleen uptake (255). This study also showed

that saturation of DTPA chelators on Bi-PAm(DTPA)40 with stable 115In in order to reduce the

negative charge on the polymers also reduced the spleen and liver uptake. Polyethylene glycol

(PEG) modification may also improve the biodistribution profile of MCP-conjugated mAbs since

PEG can shield against opsonization by plasma proteins which promotes uptake by macrophages

and increases liver and spleen accumulation (256). Therefore a new generation of metal chelating

polymer (MCP) reagents were developed in this thesis that carry multiple polyethylene glycol

(PEG) pendant groups to provide stealth to MCP-based radioimmunoconjugates (RICs). A

hydrazinonicotinamide (HyNic) group was installed on the initiating end of this new generation

of MCP (HyNic-MCP). The conjugation of this functional end group and an aromatic aldehyde

introduced on ε-NH2 groups of lysines in antibodies forms a bis-aromatic hydrazone bond with a

measurable absorbance at 350 nm (257). Boyle et al. reported that F(ab)2 of the anti-EGFR

mAb, panitumumab conjugated to an MCP with a PGlu backbone incorporating 2-8 PEG (2

kDa) chains and 13-15 DOTA for complexing 64Cu, exhibited low liver and kidney uptake and

good tumour uptake [5-8 percent injected dose/g (%ID/g)] in human PnCa xenografts in NOD-

scid mice, allowing tumour imaging by PET (258).

In my thesis research project, a MCP with a PGlu backbone incorporating 10 PEG moieties

and presenting on average 13 DOTA that complex 111In or 177Lu conjugated to panitumumab via

hydrazine nicontinamide (HyNic) chemistry was studied in comparison to DOTA conjugated

panitumumab. More details are provided in Chapters 2 and 3. The overall aim of the research

was to develop 111In or 177Lu labeled MCP-panitumumab theranostics, and compare their in vitro

and in vivo properties with their DOTA-conjugated panitumumab counterparts under the same

conditions. The feasibility of providing reproducible labeling and high SA, may improve the

therapeutic outcome with these MCP radioimmunoconjugates.

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1.8 Hypothesis

The hypothesis of this thesis is that anti EGFR mAb panitumumab labeled with the Auger

electron emitter, 111In or the β-particle emitter, 177Lu will be useful agents for molecular imaging

and RIT of PnCa xenografts in mice. Modification of panitumumab with HyNIC-MCP that

harbours 13 pendant DOTA chelators and 10 PEG moieties to reduce liver and spleen uptake

could allow for dual labeling with 111In and 177Lu, more reproducible radiolabeling and higher

SA and potentially higher potency for killing cancer cells.

1.9 Objective and Aims

The overall objective of this research was to develop novel RICs for molecular imaging and RIT

of PnCa. The specific aims were:

I) To synthesis and characterize 111In- and/or 177Lu-labeled panitumumab-MCP and

panitumumab-DOTA conjugates in terms of specific activity (SA), stability, EGFR

binding affinity, biodistribution profile and to evaluate the imaging capability of 111In-

labeled panitumumab conjugates to visualize s.c. PANC-1 human PnCa xenografts in

NOD/SCID mice using microSPECT/CT imaging.

II) To evaluate in vitro toxicity and cellular dosimetry of panitumumab-MCP-177Lu

toward PANC-1 cells as well as their normal tissue toxicity, tumour growth inhibitory

effects and absorbed doses in tumour and normal tissues in NRG mice engrafted s.c.

with PANC-1 xenografts.

III) To evaluate and compare in vitro toxicity and cellular dosimetry of panitumumab-

DOTA-177Lu, panitumumab-DOTA-111In and panitumumab-MCP-111In towards

PANC-1 cells as well as their normal tissue toxicity, tumour growth inhibitory effects

and absorbed doses in tumour and normal tissues in NRG mice and NOD/SCID mice

engrafted s.c. with PANC-1 xenografts.

The methodology, results and discussion of the research addressing these three aims are

described in chapter 2, 3 and 4 of the thesis. The conclusion of the thesis and a discussion of

the future research directions are provided in chapter 5.

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Chapter 2: Panitumumab Modified with Metal-Chelating

Polymers (MCP) Complexed to 111In and 177Lu – An EGFR-

Targeted Theranostic for Pancreatic Cancer

67

This chapter represents a reprint of “Aghevlian S, Lu Y, Winnik MA, Hedley DW, Reilly RM.

Panitumumab Modified with Metal-Chelating Polymers (MCP) Complexed to 111In and 177Lu –

An EGFR-Targeted Theranostic for Pancreatic Cancer. Mol Pharm. 2018 Mar 5;15(3):1150-

1159” . Reprinted with permission.

All experiments were designed and carried out by Sadaf Aghevlian. MicroSPECT/CT was

performed with technical assistance from Deborah A Scollard and Teesha Kamal. The synthesis

and characterization of metal chelating polymer were performed by Dr. Yijie Lu at the

Department of Chemistry at the University of Toronto.

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2. Abstract

A metal-chelating polymer (MCP) with a polyglutamide (PGlu) backbone presenting on average

13 DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for complexing 111In or

177Lu and 10 polyethylene glycol (PEG) chains to minimize liver and spleen uptake was

conjugated to antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb),

panitumumab. Because panitumumab-MCP may be dual-labeled with 111In and 177Lu for SPECT,

or radioimmunotherapy (RIT) exploiting the Auger electrons or β-particle emissions,

respectively, we propose that panitumumab-MCP could be a useful theranostic agent for EGFR-

positive PnCa. Bioconjugation was achieved by reaction of a hydrazine nicotinamide (HyNIC)

group on the MCP with an aryl aromatic aldehyde introduced into panitumumab by reaction with

succinimidyl-4-formylbenzamide (S-4FB). The conjugation reaction was monitored by

measurement of the chromophoric bis-aryl hydrazone bond formed (ε350 nm = 24 500 M−1

cm−1 ) to achieve two MCPs/ panitumumab. Labeling of panitumumab-MCP with 111In or 177Lu

demonstrated that masses as small as 0.1 μg were labeled to >90% labeling efficiency (L.E.) and

a specific activity (SA) of >70 MBq/μg. Panitumumab-DOTA incorporating two DOTA per

mAb was labeled with 111In or 177Lu to a maximum SA of 65 MBq/μg and 46 MBq/μg,

respectively. Panitumumab-MCP-177Lu exhibited saturable binding to EGFR-overexpressing

MDA-MB-468 human breast cancer cells. The Kd for binding of panitumumabMCP-177Lu to

EGFR (2.2 ± 0.6 nmol/L) was not significantly different than panitumumab-DOTA-177Lu (1.0 ±

0.4 nmol/L). 111In and 177Lu were stably complexed to panitumumab-MCP. Panitumumab-MCP-

111In exhibited similar whole body retention (55−60%) as panitumumab-DOTA-111In in NOD-

scid mice up to 72 h postinjection (p.i.) and equivalent excretion of radioactivity into the urine

and feces. The uptake of panitumumab-MCP-111In in most normal tissues in NOD-scid mice with

EGFR-positive PANC-1 human pancreatic cancer (PnCa) xenografts at 72 h p.i. was not

significantly different than panitumumab-DOTA-111In, except for the liver which was 3-fold

greater for panitumumab-MCP-111In. Tumour uptake of panitumumab-MCP-111In (6.9 ± 1.3%

ID/g) was not significantly different than panitumumab-DOTA-111In (6.6 ± 3.3%ID/g). Tumour

uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In were reduced by

preadministration of excess panitumumab, suggesting EGFR-mediated uptake. Tumour uptake of

nonspecific IgG-MCP (5.4 ± 0.3%ID/g) was unexpectedly similar to panitumumab-MCP-111In.

69

An increased hydrodynamic radius of IgG when conjugated to an MCP may encourage tumour

uptake via the enhanced permeability and retention (EPR) effect. Tumour uptake of

panitumumab-DOTA-111In was 3.5-fold significantly higher than IgG-DOTA-111In. PANC-1

tumours were imaged by microSPECT/CT at 72 h p.i. of panitumumab-MCP-111In or

panitumumab-DOTA-111In. Tumours were not visualized with preadministration of excess

panitumumab to block EGFR, or with nonspecific IgG radioimmunoconjugates. We conclude

that linking panitumumab to an MCP enabled higher SA labeling with 111In and 177Lu than

DOTA-conjugated panitumumab, with preserved EGFR binding in vitro and comparable tumour

localization in vivo in mice with s.c. PANC-1 human PnCa xenografts. Normal tissue

distribution was similar except for the liver which was higher for the polymer

radioimmunoconjugates.

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Graphical abstract

71

2.1 Introduction

Radioimmunotherapy (RIT) employs monoclonal antibodies (mAbs) recognizing tumour-

associated receptors/antigens complexed to radionuclides that emit α-particles, β-particles or

Auger electrons for systemic radiation treatment of cancer (191, 259). RIT has been proven in

principle in a broad diversity of cancers in mouse tumour xenograft models and has been

effective for treatment of non-Hodgkin’s B-cell lymphoma in cancer patients (260). RIT of solid

tumours has been more challenging because of limited delivery and penetration of radiolabeled

mAbs into these tumours combined with dose-limiting hematopoietic system toxicity (261). The

barriers to delivery of radiolabeled mAbs to solid tumours include poor extravasation, high

tumour interstitial pressure (262), aberrant tumour vascularization (263), stromal barriers (264),

and a binding site barrier, which restricts deep tumour penetration due to binding to tumour cells

near blood vessels (265). Low and heterogeneous receptor/antigen expression have also been

reported to diminish the effectiveness of antibody-based therapies (266) and may play a role in

decreasing the effectiveness of RIT.

One strategy to overcome these delivery barriers may be to enhance the amount of radioactivity

targeted to tumour cells per receptor/antigen binding event through increasing the proportion of

antibody molecules carrying a radionuclide (i.e., specific radioactivity (SA)). Radiolabeling is

most commonly performed by conjugation of chelators to the mAbs such as DTPA

(diethylenetriaminepentaacetic acid) or DOTA (tetraazacyclododecane-1,4,7,10-tetraacetic acid)

that form a complex with radiometals, but to minimize the effect of chelator substitution on

binding affinity, usually only one to two DTPA or DOTA are conjugated per mAb molecule

(267). This low chelator substitution level limits the SA that is achievable, such that there may be

as few as 1 in 50 antibody molecules that are radiolabeled (240). To address this challenge, we

previously reported the synthesis of a metal-chelating polymer (MCP) composed of a

polyglutamide (PGlu) backbone presenting 24 or 29 DTPA per MCP for complexing 111In (268).

This MCP was conjugated to the Fc-domain of antihuman epidermal growth factor receptor-2

(HER2) mAb, trastuzumab (Herceptin, Roche) with good preservation of HER2 binding (Kd = 1

× 10–8 mols/L) (240). The SA achieved for labeling trastuzumab-MCP with 111In was increased

up to 90-fold compared with trastuzumab directly modified with two DTPA, and this increased

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the cytotoxic potency in vitro against HER2-positive breast cancer cells through the emission of

Auger electrons by 111In.

Nonetheless, an important consideration in the application of MCPs for high SA radiolabeling of

mAbs is their effect on tumour and normal tissue uptake. We previously examined the effect of

conjugation of MCPs with different backbones [polyacrylamide (PAm), polyaspartamide (PAsp),

or polyglutamide (PGlu)] that presented 28–40 DTPA for complexing 111In on the tumour and

normal tissue distribution of trastuzumab Fab in mice with s.c. HER2-positive SK-OV-3 human

ovarian cancer xenografts (269). Liver uptake was 5–13-fold higher for Fab conjugated to the

PAm MCP compared with a MCP with a PAsp or PGlu backbone. However, polymer charge

was also important, because saturation of uncomplexed DTPA on PAm MCP with stable indium

(115In) to eliminate residual anionic charges, decreased liver uptake by 1.5-fold. The highest

tumour uptake was observed with PAsp MCP-conjugated Fab. We then compared a PAm MCP

with 36 DTPA linked via a diethylenetriamine (DET) group to an MCP with 40 DTPA linked via

an ethylenediamine (EDA) group (270). The DET-functionalized polymer was zwitterionic when

DTPA was complexed to 111In or saturated with stable indium, whereas the EDA polymer was

anionic. In mice with s.c. SK-OV-3 tumours, zwitterionic polymer conjugates exhibited

decreased liver and kidney uptake, enhanced retention in the blood, and improved tumour uptake

compared with those conjugated to the anionic EDA MCP. Polyethylene glycol (PEG)

modification may also improve the biodistribution profile of MCP-conjugated mAbs since PEG

can shield against opsonization by plasma proteins which promotes uptake by macrophages and

increases liver and spleen uptake (256). We reported that F(ab′)2 of the antiepidermal growth

factor receptor (EGFR) mAb, panitumumab (Vectibix, Amgen) conjugated to an MCP with a

PGlu backbone incorporating 2–8 PEG (2 kDa) chains and 13–15 DOTA for complexing 64Cu,

exhibited low liver and kidney uptake and relatively good tumour uptake [5–8% injected dose/g

(%ID/g)] in human pancreatic cancer (PnCa) xenografts in NOD-scid mice, allowing tumour

imaging by positron-emission tomography (PET) (271).

In the current study, we describe for the first time a MCP with a PGlu backbone that incorporates

on average 10 PEG chains and 13 DOTA to complex 111In or 177Lu conjugated to panitumumab

via hydrazine nicotinamide (HyNic) chemistry (272). 111In [t1/2 = 2.8 days] emits 15 low energy

(<25 keV) Auger electrons that have a subcellular range ideal for killing single tumour cells or

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small clusters of tumour cells (e.g., micrometastases). In addition, 111In emits two high

abundance γ-photons [Eγ = 171 keV (90%) and Eγ = 245 keV (94%)] that enable single photon

emission computed tomography (SPECT) imaging. 177Lu [t1/2 = 6.7 days] emits moderate energy

β-particles [Eβ(max)=498 keV (78.6%), Eβ(max)=385 keV (9.1%) and Eβ(max)=176 keV

(12.2%)] that have a longer 2 mm range suitable for RIT of larger tumours. 177Lu also emits two

low abundance γ-photons [Eγ = 113 keV (6.4%) and Eγ = 208 keV (11%)] that can be imaged by

SPECT. Since the MCP provides multiple DOTA chelators for complexing 111In and 177Lu, this

provides an opportunity for dual-labeling of panitumumab to allow imaging as well as RIT of

different sized tumour deposits (“theranostic” approach). The higher abundance γ-photons

emitted by 111In would also increase the sensitivity of SPECT compared with imaging using only

the low abundance γ-photons emitted by 177Lu. Our aim in this study was to conjugate

panitumumab to these MCPs and compare the properties of the immunoconjugates to

panitumumab directly modified with DOTA. These properties included complexation of 111In

and 177Lu to high SA, EGFR immunoreactivity, stability and tumour and normal tissue

distribution including SPECT imaging in NOD-scid mice with s.c. PANC-1 human PnCa

xenografts. Panitumumab binds with high affinity to the EGFR (Kd = 5 × 10–11 M) (273) which

is overexpressed on >90% of PnCa tumours (274).

2.2 Materials and Methods

2.2.1 Cell Culture and Tumour Xenografts

PANC-1 human pancreatic adenocarcinoma cells (4 × 105 EGFR/cell) (143) and MDA-MB-468

human breast cancer cells (1.3 × 106 EGFR/cell) (275) were obtained from the American Type

Culture Collection (ATCC). Both cell lines were cultured in Dulbecco’s Modified Eagle’s

Medium (DMEM) with high glucose supplemented with 1% penicillin and streptomycin and

10% fetal bovine serum (Gibco-Invitrogen). Tumour xenografts (6–7 mm diameter) were

established in female nonobese diabetic severe combined immunodeficiency (NOD-scid) mice

(Charles River) by subcutaneous (s.c.) inoculation of 4 × 106 PANC-1 cells in serum free

DMEM (100 μL) into the left abdomen. All animal studies were conducted in compliance with

Canadian Council on Animal Care (CCAC) guidelines and were carried out under a protocol

approved by the Animal Care Committee at the University Health Network (AUP 2843.3).

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2.2.2 Radioimmunoconjugates (RICs)

Hydrazino nicotinamide (HyNic) end-capped metal-chelating polymers (MCP; Figure 2-1) were

synthesized and characterized as reported by Lu et al (271) Panitumumab (Vectibix; Amgen)

was reacted with succinimidyl-4-formylbenzamide (S-4FB; synthesized in-house) for 2 h in

phosphate-buffered saline (PBS), pH 8.0 to install aldehyde groups for reaction with HyNic-

MCP. The number of S-4FB conjugated to panitumumab was quantified colorimetrically by

reaction with 2-hydrazinopyridine (Sigma-Aldrich) which forms a chromophoric bis-aryl

hydrazone that absorbs at 350 nm (ε350 nm = 24 500 M–1 cm–1). S-4FB-modified

panitumumab was purified using an Amicon 30 kDa molecular weight cutoff (MWCO) spin

filter and reacted with 5 equivalent of HyNic-MCP to form a bis-aryl hydrazone chromophore

which absorbs at 354 nm (ε354 nm = 29000 M–1 cm–1). The desired number of MCP per

panitumumab was obtained by measuring the absorbance at 354 nm of the reaction mixture every

few minutes until the absorbance corresponded on an average of two MCPs linked to

panitumumab. Unconjugated polymers were removed from MCP-panitumumab by diluting first

in 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer pH 7.4 to block any unreacted

aldehydes and then in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer

pH 5.0 for subsequent radiolabeling, and passing through an Amicon spin filter (0.5 mL; 100

kDa MWCO; Millipore).

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Figure 2-1. Chemical structure of the hydrazino nicotinamide metal chelating polymer (HyNic-

MCP; Mr ∼ 33 kDa)a; (B,C) Synthesis of panitumumab-HyNic-MCP b; and (D)

immunoconjugates are labeled with 111InCl3 or 177LuCl3 in 0.1 M HEPES Buffer, pH 5.5.

a The polymer incorporates a polyglutamide backbone harboring on average 13 pendant DOTA

groups as well as 10 PEG chains. The chemically reactive group, HyNic, is indicated by the

blocked area.

b Following derivatization of ε-NH2 groups on lysine residues in panitumumab with S-4FB, the

hydrazine group on the MCP interacts with installed aldehyde groups on panitumumab to form a

bis-aryl hydrazone bond that absorbs at 354 nm. This reaction allows the measurement of the

number of MCP conjugated to panitumumab as the reaction proceeds.

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MCP-panitumumab was characterized by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) on a 4–20% Tris-HCl gel (Bio-Rad) stained with Coomassie blue.

Molecular weight standards (10–260 kDa) were used to calibrate the gel and determine the

molecular size of the immunoconjugates. For comparison, panitumumab was directly modified

with DOTA by reaction with the N-hydroxysuccinimide ester (DOTA-NHS; Macrocyclics).

Briefly, panitumumab (2 mg) was exchanged into metal-free NaHCO3 buffer pH 8.2 (100 μL),

then reacted with 10 equivalents of DOTA-NHS for 2 h at 25 °C. The immunoconjugates were

concentrated and purified using an Amicon spin filter (0.5 mL; 30 kDa MWCO; Millipore) in 0.1

M HEPES, pH 5.5 for subsequent radiolabeling. Similarly nonspecific human IgG (Sigma-

Aldrich) was modified with DOTA or MCP for radiolabeling with 111In or 177Lu. DOTA

conjugation efficiency was determined by trace-labeling a sample of the unpurified

immunoconjugates (10 μL) with 111InCl3 (Nordion) or 177LuCl3 (PerkinElmer) at 42 °C for 3 h

and measuring the proportion of 111In/177Lu-DOTA-panitumumab (Rf = 0.0) and unconjugated

111In/177Lu-DOTA (Rf = 1.0) by instant thin layer silica-gel chromatography (ITLC-SG; Pall)

developed in 0.1 M sodium citrate, pH 5.5. The conjugation efficiency was multiplied by the 10-

fold equivalent excess of DOTA in the reaction to calculate the number of DOTA/panitumumab.

The maximum specific activity (SA) achievable for 111In or 177Lu labeling of the

immunoconjugates was studied by labeling decreasing masses of panitumumab-MCP or

panitumumab-DOTA (100, 50, 10, 1, and 0.1 μg) with a constant amount (9.6 MBq) of 111InCl3

or/and 177LuCl3 and measuring the percent labeling efficiency (L.E.) by ITLC-SG. The feasibility

of dual labeling of the immunoconjugates with 111In and 177Lu was studied by addition of

111InCl3 (7.8 MBq) and 177LuCl3 (5.7 MBq) to 1 μg of panitumumab-MCP and panitumumab-

DOTA. Dual-isotope incorporation was measured by γ-counting with detection of the 177Lu X-

ray peak at 60 keV and 111In sum peak at 428 keV. Corrections were applied to eliminate

crossover from 111In to 177Lu and vice versa.

2.2.3 Measurement of Hydrodynamic Radius

To assess the effect of MCP conjugation on the molecular size of panitumumab, the

hydrodynamic radius of the immunoconjugates was measured by size-exclusion chromatography

(SEC, Viscotek) on tandem ViscoGEL G4000PWXL and G2500PWXL columns with

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polymethacrylate beads maintained at 30 °C on a high performance liquid chromatography

(HPLC) system equipped with a VE3210 UV/vis detector and VE3580 refractive index detector.

The eluent used was 0.2 M KNO3, in 25 mM sodium phosphate buffer pH 8.5 containing 200

ppm of NaN3 and the flow rate was 1.0 mL/min delivered using a VE1122 Solvent Delivery

System and VE7510 GPC Degasser (Viscotek). D-Glucose was used as an internal standard. A

calibration curve was constructed by plotting the elution time of polyethylene glycols (PEG,

0.54–23.8 kDa, Polymer Laboratories) as well as protein standards (12.4–440 kDa, Sigma-

Aldrich) vs their corresponding hydrodynamic radius (Rh). The scaling relationship

(hydrodynamic radius vs molecular weight) of PEG and protein standards were obtained from

the literature (276-280). On the basis of this relationship, the resulting hydrodynamic radius vs

retention time calibration curve was plotted.

2.2.4 EGFR Immunoreactivity

The EGFR binding affinity of 177Lu-labeled panitumumab-MCP was measured in saturation

binding assays using MDA-MB-468 human breast cancer cells (1.3 × 106 EGFR/cell) and

compared to panitumumab-DOTA-177Lu. Briefly, increasing concentrations (0 to 65 nmol/L) of

177Lu-labeled immunoconjugates (50 μL) were incubated with 1 × 106 MDA-MB-468 cells (100

μL) in 1.5 mL Eppendorf tubes at 4 °C for 3.5 h with gentle shaking. PBS (50 μL) was then

added to the cell suspensions and the tubes were centrifuged on an Eppendorf microcentrifuge

Model 5418 at 2,410 × g for 5 min to separate the unbound radioactivity in the supernatant from

the cell pellet. The total cell-bound radioactivity (TB) in the pellets was measured in a γ-counter

(PerkinElmer Wizard 3). The assay was repeated in the presence of a 50-fold molar excess of

unlabeled panitumumab (0.003–3.2 μM) to measure nonspecific binding (NSB). Specific binding

(SB) was calculated by subtracting NSB from TB and was plotted vs the concentration of 177Lu-

immunoconjugates added. The resulting curve was fitted by nonlinear regression to a one-site

receptor-binding model by Prism Ver. 4.0 software (GraphPad). The dissociation constant (Kd)

and maximum number of receptors per cell (Bmax) were calculated and compared for

panitumumab-MCP-177Lu and panitumumab-DOTA-177Lu.

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2.2.5 Stability of the RICs

The stability of 177Lu- or 111In-labeled panitumumab-MCP or panitumumab-DOTA RICs against

transchelation to ethylenediaminetetraacetic acid (EDTA) was evaluated. The RICs (100 μg; 1

μg/μL) were incubated with a 500-fold molar excess of EDTA (3.3 mM; pH = 7.4; Sigma-

Aldrich) for 1 h at room temperature. The proportion of 111In or 177Lu transchelated to EDTA or

remaining bound to panitumumab-MCP or panitumumab-DOTA was measured by ITLC-SG (Rf

= 1.0 and 0.0, respectively) and compared to RICs incubated in PBS, pH 7.0. In a separate

experiment 177Lu- or 111In-labeled panitumumab-MCP or panitumumab-DOTA (0.6 MBq/μg)

were incubated with human plasma (Sigma-Aldrich; P9523) in 0.5 mL Eppendorf tubes at a

concentration of 0.037 MBq/μL for up to 7 days at 37 °C. Samples were analyzed in duplicate by

size-exclusion HPLC (SE-HPLC) on a BioSep silica-based SEC-S2000 column (exclusion limit,

1000–300 000 kDa; Phenomenex) eluted with 100 mM NaH2PO4 buffer, pH 7.0, at a flow rate

of 0.8 mL/min. The SE-HPLC system (PerkinElmer) was interfaced to a flow scintillation

analyzer (FSA) radioactivity detector (PerkinElmer). Because HyNic-MCP interacts with the

silica matrix of the HPLC column, the stability of 177Lu- or 111In-labeled panitumumab-MCP was

alternatively studied by ultrafiltration through a Millipore centrifugal filter device (MWCO =

100 kDa; Amicon) and measuring the percentage of radioactivity filtered vs retained through the

device in a γ-counter.

The stability of the RICs in vivo was assessed by measuring the whole body retention in mice

and determining the proportion of radioactivity eliminated in the urine or feces. Groups of 4–5

female NOD-scid mice engrafted s.c. with PANC-1 cells were injected intravenously (i.v.) in the

tail vein with 37 MBq (10 μg) of panitumumab-MCP-111In or panitumumab-DOTA-111In. The

radioactivity retained in the whole body was measured in a radioisotope calibrator (Model CRC-

12; Capintec) immediately after injection and at 1, 4, 24, 48, and 72 h post injection (p.i.). Mice

were housed in metabolic cages and the cumulative percentage of the injected dose eliminated in

the urine and feces was collected and measured in the dose calibrator at the corresponding time

points.

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2.2.6 Imaging and Biodistribution Studies

Imaging and biodistribution studies were performed in groups of 5 NOD-scid mice bearing s.c.

PANC-1 tumours injected i.v. with 3.7 MBq (10 μg) of panitumumab-MCP-111In or

panitumumab-DOTA-111In. To determine the specificity of tumour uptake, other groups of mice

were injected with nonspecific human IgG labeled with 111In (IgG-DOTA-111In or IgG-MCP-

111In). The specificity of tumour uptake was also assessed by EGFR blocking performed by i.v.

administration of 1 mg of unlabeled panitumumab 1 day prior to injection of panitumumab-

MCP-111In or panitumumab-DOTA-111In. At 72 h p.i. one or two representative mice were

selected from each group and imaged on a NanoSPECT/CT tomograph (Bioscan) equipped with

4 NaI detectors and fitted with 1.4 mm multipinhole collimators [full width at half-maximum

height (fwhm) = 1.2 mm]. For imaging, mice were anaesthetized by inhalation of 2% isoflurane

in O2, and body temperature was maintained at 37 °C using a Minerve bed (Bioscan). A total of

24 projections were acquired in a 256 × 256 acquisition matrix with a minimum of 80,000 counts

per projection. Images were reconstructed using an ordered-subset expectation maximization

(OSEM) algorithm (9 iterations). Cone-beam CT images were acquired (180 projections, 1

s/projection, 45 kVp) and coregistration of micro-SPECT and CT images was performed using

InvivoScope software. For some mice, CT was performed on an eXplore Locus Ultra Preclinical

CT scanner (GE Healthcare) with routine acquisition parameters (80 kV, 50 mA-Voxel size of

154 × 154 × 154 μm) and coregistration of microSPECT and CT images performed using

Siemens Inveon Research Workplace software. Mice were sacrificed by cervical dislocation

under anesthesia, and the tumour and samples of normal tissues including blood were collected,

weighed, and counted in a γ-counter. Radioactivity uptake was expressed as % ID/g.

2.3 Statistical Analysis

Results were expressed as mean ± SEM and tested for significance (P < 0.05) using a two-sided

Student’s t test using Prism Ver. 4.0 software (GraphPad).

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2.4 Results

2.4.1 Radioimmunoconjugates

Panitumumab was first reacted with S-4FB to install aldehyde groups for conjugation to HyNic-

MCP (Figure 2-1). There were 4.1 ± 0.7 S-4FB groups on panitumumab based on the absorbance

at 350 nm of the bis-aryl hydrazone chromophore produced after reaction with 2-

hydrazinopyridine (281). S-4FB modified panitumumab was then reacted with an 5-fold excess

of HyNic-MCP for a sufficient length of time (75 min) to achieve substitution of approximately

two polymers per panitumumab molecule based on measurement of the bis-aryl hydrazone

chromophore at 354 nm formed in the reaction (Figure 2-2A,B). Panitumumab-MCP was

transferred to 0.1 M Tris pH 7.4 to block unreacted aldehydes and then to 0.1 M HEPES pH 5.0

to remove unconjugated MCP by ultrafiltration through an Amicon device. Reaction of

panitumumab with a 10-fold excess of DOTA-NHS resulted in substitution of 2.2 ± 0.5 DOTA

per antibody molecule. Analysis of panitumumab-MCP immunoconjugates by SDS-PAGE under

reducing and non-reducing conditions showed a broad upward band shift from 150 kDa toward

higher MW bands compared with panitumumab or panitumumab-DOTA indicating that there

was some variability in the number of MCP conjugated to panitumumab. Two major bands were

observed under reducing condition, at 50 and 25 kDa representing the heavy and light chains of

panitumumab (Figure 2-2C).

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Figure 2-2. (A) Reaction of panitumumab-S-4FB with HyNic-MCP was monitored by measuring

the absorbance at 354 nm due to formation of the bis-aryl hydrazone chromophore. (B) The

reaction was stopped at 75 min when the average number of MCP per panitumumab was 2,

which corresponded to an absorbance of 0.79. (C) SDS-PAGE analysis of panitumumab and

MCP- or DOTA-immunoconjugates on a 4–20% Tris-HCl gel stained with Coomassie Brilliant

Blue under nonreducing and reducing (2-mercaptoethanol) conditions. Lanes: MW: MW

markers (kDa), 2/5: panitumumab, 3/6: panitumumab-DOTA, 4/7: panitumumab-MCP. Under

nonreducing condition, the upward band shift in lane 4 associated with an increase in molecular

weight of panitumumab indicated the attachment of MCPs. Under reducing condition, there are

two major bands at 25 kDa and 50 kDa for panitumumab, panitumumab-DOTA and

panitumumab-MCP, which are the light and heavy chains of panitumumab, and an additional

diffuse higher MW band for panitumumab-MCP.

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Panitumumab-MCP and panitumumab-DOTA were labeled with 111In or 177Lu at high L.E. and

at increasing SA by incubation of decreasing masses with a constant amount (7.5–8.5 MBq) of

radioactivity (Table 2-1). A 3 h incubation period at 42 °C was employed to ensure high L.E.,

but shorter incubation periods may also be feasible (282). The maximum SA achievable with

111In based on a L.E. > 90% was 70.7 ± 3.2 MBq/μg and 65.3 ± 3.1 MBq/μg for panitumumab-

MCP and panitumumab-DOTA, respectively. The maximum SA for labeling with 177Lu was 71.5

± 9.2 MBq/μg and 46.2 ± 5.3 MBq/μg for panitumumab-MCP and panitumumab-DOTA,

respectively. Because there were approximately two MCPs incorporating 26 DOTA or two

DOTA directly conjugated to panitumumab, the maximum theoretical SA of panitumumab-MCP

immunoconjugates labeled with 111In or 177Lu is 9.1-fold higher than that of panitumumab-

DOTA, taking into account the higher MW of the polymer immunoconjugates. Thus, it may be

possible to achieve even higher SA by labeling smaller masses of panitumumab-MCP (<0.1 μg)

with 111In or 177Lu. The difference between the SA for labeling of the two immunoconjugates

was revealed further when 1 μg was incubated with both 111In (7.8 MBq) and 177Lu (5.7 MBq).

The SA of 1 μg of panitumumab-MCP labeled with 7.8 MBq of 111In and 5.7 MBq of 177Lu was

55.9 ± 4.8 MBq/μg and 20.5 ± 4.2 MBq/μg, respectively, while that of panitumumab-DOTA was

12.7-fold and 9.3-fold lower (4.4 ± 0.3 MBq/μg and 2.2 ± 0.3 MBq/μg, respectively). The 3.9-

fold higher theoretical SA of carrier-free 111InCl3 compared with 177LuCl3 (1.6 × 104 MBq/μg vs

4.0 × 103 MBq/μg, respectively) and the higher amount of 111In than 177Lu used for dual-labeling

(7.8 vs 5.7 MBq) contributed to the higher SA for 111In labeling.

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Table 2-1. Labelling Efficiency (L.E.) and Specific Activity (SA) of Panitumumab-MCP and

Panitumumab-DOTA with 111In or 177Lua.

Immunoconjugates Mass

(µg)

111In 177Lu

L.E. (%) SA

(MBq/µg) L.E. (%) SA (MBq/µg)

Panitumumab-

MCP

100 99.5±0.2 0.1± 0.0 99.6±0.4 0.1± 0.0

10 99±1 0.8± 0.0 99.3±0.4 0.9± 0.0

1 94±2 6.5± 0.4 95±2 9.0± 0.8

0.1 95±2 70.7 ± 3.2 90±3 71.5 ± 9.2

Panitumumab-

DOTA 100 95±3 0.1±0.01 99.4±0.4 0.1±0.01

10 96±2 0.91±0.0 99±0.8 0.9±0.01

1 92±3 6.6±0.6 95±2 5.5 ± 0.6

0.1 92±2 65.3 ± 3.1 74±2 46.2 ± 5.3

a Samples were labeled with 7.5–8.5 MBq of 111InCl3 or 177LuCl3 in 0.1 M HEPES buffer pH 5.0

for 3 h at 42 °C. Values reported are mean ± SEM (n = 6). L.E. was determined by ITLC-SG.

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2.4.2 Hydrodynamic Radius

The hydrodynamic radius of panitumumab-MCP and panitumumab-DOTA were characterized

by SEC-HPLC. A calibration curve was constructed by plotting the elution time of polyethylene

glycols (PEG, 0.54–23.8 kDa, Polymer Laboratories) and protein standards (12.4–660 kDa,

Sigma-Aldrich) against their corresponding hydrodynamic radius (Rh) (Table 2-2) and then

plotting “retention time” vs “Rh”. The scaling relationship (Rh vs MW) of PEG (Figure 2-3) and

protein standards were obtained from the literature (22-26). On the basis of this relationship, the

resulting Rh vs retention time calibration curve was plotted (Figure 2-3). The unconjugated MCP

and panitumumab-DOTA exhibited a peak Rh of 4.0 and 5.5 nm, respectively. Panitumumab-

MCP showed a main peak corresponding to an Rh of 5.5 nm which was attributed to

panitumumab and a shoulder peak corresponding to an Rh of 7.3 nm representing the MCP-

conjugate. On the basis of the integration of the area under the main peak and shoulder peak, it

was estimated that approximately 50% of panitumumab molecules were conjugated to MCP by

assuming the refractive index increment (dn/dc) of both MCP and panitumumab are identical.

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Figure 2-3. (A). The relationship of hydrodynamic radius (Rh) vs. molecular weight (MW) for

PEG standards measured by intrinsic viscosity using a capillary viscometer (black squares) and

by analytical ultracentrifugation (red diamonds), re-plotted from the literature. (B) Calibration

curve of Rh vs. retention time for PEG standards by size exclusion chromatography (open

squares: Rh obtained from the scaling relationship in Figure 2-3A and protein standards (blue

triangles: Rh obtained from the literature S1-S5). The number shown near each data point is the

MW (kDa) of the corresponding sample. (C) SEC profiles of panitumumab-DOTA,

panitumumab-MCP and unconjugated MCP at 30 °C in water containing 0.2 M KNO3, 25 mM

NaH2PO4 (pH 8.5), and 200 ppm NaN3. The corresponding Rh for the peak attributed to

panitumumab-DTPA, MCP and the shoulder peak from panitumumab-MCP were obtained from

panel (B).

86

Table 2-2. Hydrodynamic radii and retention times for PEG and protein standards by size

exclusion chromatography

MW (PEG) / kDa Rh (nm)a Retention time (min)b

24 5.1 13.84

22 5.0 13.79

10 3.2 14.44

8.0 2.8 14.59

5.7 2.3 14.83

3.8 1.8 15.51

2.0 1.3 16.32

1.4 1.0 16.65

0.95 0.8 17.18

0.54 0.6 17.94

Protein (MW / kDa)

Thyroglobulin from bovine thyroid (660) 8.6 12.75

Apoferritin (440) 6.7 13.12

β-Amylase (200) 5.1 13.62

Alcohol Dehydrogenase (150) 4.5 13.81

Albumin, bovine serum (66) 3.7 14.06

Carbonic Anhydrase (29) 2.5 14.71

Cytochrome c (12.4) 1.9 15.49

a: Hydrodynamic radii of PEG and protein standards were obtained from the literature (276-280).

b: Retention times were obtained by size-exclusion chromatography (SEC, Viscotek) on tandem

ViscoGEL G4000PWXL and G2500PWXL columns maintained at 30 oC on a high performance

liquid chromatography (HPLC) system equipped with a VE3210 UV/VIS detector and VE3580

refractive index detector. The eluent used was 0.2 M KNO3, in 25 mM sodium phosphate buffer

pH 8.5 containing 200 ppm NaN3 and the flow rate was 1.0 mL/min delivered using a VE1122

Solvent Delivery System and a VE7510 GPC Degasser (Viscotek). D-glucose was used as an

internal standard.

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2.4.3 EGFR Immunoreactivity

Both panitumumab-MCP-177Lu and panitumumab-DOTA-177Lu exhibited saturable binding to

EGFR-positive MDA-MB-468 human breast cancer cells and binding was significantly reduced

in the presence of a 50-fold excess of unlabeled panitumumab (Figure 2-4). Since MDA-MB-468

cells express a high density of EGFR (1.3 × 106) these cells were used to provide direct

(saturation) binding curves that were more readily fitted to a 1-site binding model for comparison

of the EGFR-binding properties of the RICs than PANC-1 PnCa cells which exhibit a 3.3-fold

lower EGFR density. The dissociation constant (Kd) and maximum number of binding sites/cell

(Bmax) for binding of panitumumab-MCP-177Lu to MDA-MB-468 cells were 2.2 ± 0.6 nmol/L

and 0.6 ± 0.07 × 106 receptors/cell, respectively. The Kd and Bmax for binding of panitumumab-

DOTA-177Lu to MDA-MB-468 cells were 1.0 ± 0.4 nmol/L and 0.5 ± 0.07 × 106 sites/cell. There

were no significant differences between the Kd values (P = 0.06) for panitumumab-MCP-177Lu

and panitumumab-DOTA-177Lu, indicating that both RICs exhibited high affinity binding to

EGFR.

88

Figure 2-4. Binding of (A) panitumumab-DOTA-177Lu or (B) panitumumab-MCP-177Lu to

EGFR positive MDA-468 human breast cancer cells in the absence (total binding; TB) or

presence (nonspecific binding; NSB) of a 50-fold excess of unlabeled panitumumab. Specific

binding (SB) was calculated by subtraction of NSB from TB. Values shown are the mean ± SEM

(n = 3). Specifically bound cpm at saturation were converted to nmoles for calculation of the

Bmax.

89

2.4.4 Radioimmunoconjugate Stability

Incubation of 177Lu- or 111In-labeled panitumumab-MCP or panitumumab-DOTA with a 500-fold

excess of EDTA revealed no appreciable release of radioactivity from either of these RICs

(Figure 2-5A), revaling high stability. SE-HPLC analysis of 177Lu- or 111In-labeled

panitumumab-DOTA incubated in human plasma at 37 °C showed a single peak with retention

time (tR) of 9.3 min up to 7 days, and the tR of this peak did not change over time (figure 2-5

B,C). SE-HPLC analysis of 111In- or 177Lu-labeled panitumumab-MCP was not feasible because

the MCP interacts with the column matrix, resulting in poor elution characteristics. However,

ultrafiltration of 177Lu- or 111In-labeled panitumumab-MCP (MW = 216 kDa) using a 100 kDa

MWCO filter and measuring the percentage of radioactivity filtered vs retained showed no

release of low MW radioactivity into the filtrate indicating stability of the RICs in human plasma

under these same conditions.

90

Figure 2-5. (A) Radioimmunoconjugates (RICs) were challenged with a 500-fold excess of

EDTA for 1 h at 25 ᵒC and analyzed by ITLC-SG. No transchelation of radiometal from the

radioimmunoconjugates to EDTA was observed as compared to the phosphate buffered saline

(PBS) control samples. Values shown are mean SEM (n=3). SEM is not visible due to very

small values. (B) SE-HPLC analysis of panitumumab-DOTA-111In and (C) panitumumab-MCP-

111In in human plasma at 37°C on a BioSep-S-2000 column eluted with 0.1M NaH2PO4 buffer

(pH=7.0) at a flow rate of 0.8mL/min. There was no change in the chromatographic properties

over an incubation time of 1 week (different colored lines).

91

The in vivo stability of panitumumab-MCP-111In and panitumumab-DOTA-111In was examined

by measuring the percent of injected radioactivity retained in mice over time after i.v. injection.

Both RICs were eliminated slowly from the body with 55.0 ± 0.9% and 59.7 ± 12.7% of the

injected radioactivity eliminated over 72 h (P > 0.05). The only significant difference in the

elimination of the RICs was at 4 h p.i. (P < 0.01; Figure 2-6A). Urinary and fecal excretion of

radioactivity was also measured up to 72 h p.i. (Figure 2-6B). Most of the radioactivity

eliminated from the body was excreted into the urine (34.1 ± 7.2% and 36.0 ± 1.6% for

panitumumab-MCP-111In and panitumumab-DOTA-111In, respectively; P > 0.05), whereas fecal

excretion was 4.9 ± 1.4% and 5.2 ± 2.0% for panitumumab-MCP-111In and panitumumab-

DOTA-111In, respectively (P > 0.05).

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Figure 2-6. (A) Percent of radioactivity retained in the whole body after i.v. (tail vein) injection

of panitumumab-MCP-111In or panitumumab-DOTA-111In up to 72 h p.i. in NOD-scid mice

engrafted s.c. with PANC-1 tumours. (B) Cumulative percent of radioactivity excreted in the

urine or feces up to 72 h p.i. in NOD-scid mice with PANC-1 tumours injected i.v. with

panitumumab-MCP-111In or panitumumab-DOTA-111In. * Statistically significant difference

between groups (P < 0.01). Data shown are the mean ± SEM (n = 4).

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2.4.5 Biodistribution and Imaging Studies

A biodistribution study comparing panitumumab-MCP-111In and panitumumab-DOTA-111In at

72 h p.i in NOD-scid mice bearing s.c. PANC-1 xenografts revealed an almost identical profile,

except for the liver (Figure 2-7). The liver uptake of panitumumab-MCP-111In was 2.9-fold

significantly greater than that of panitumumab-DOTA-111In (11.2 ± 1.0%ID/g vs 3.8 ±

1.3%ID/g; P = 0.001). There were no significant differences in tumour uptake between

panitumumab-MCP-111In and panitumumab-DOTA-111In (6.9 ± 1.3%ID/g vs 6.6 ± 3.3%ID/g,

respectively; P > 0.05). Preinjection of 100-fold excess (1 mg) of unlabeled panitumumab to

block EGFR significantly decreased the tumour uptake of panitumumab-MCP-111In and

panitumumab-DOTA-111In (0.02 ± 0.00%ID/g and 0.06 ± 0.02%ID/g, respectively). The tumour

and normal tissue distribution of nonspecific IgG-MCP-111In or IgG-DOTA-111In were studied as

additional controls to assess the specificity of tumour uptake of panitumumab RICs. The tumour

uptake of IgG-DOTA-111In (1.9 ± 0.3%ID/g) was 3.5-fold significantly lower than that of

panitumumab-DOTA-111In (P < 0.01). Unexpectedly, there was no significant difference

between the tumour uptake of IgG-MCP-111In (5.4 ± 0.3%ID/g) and panitumumab-MCP-111In

(6.9 ± 1.3%ID/g) (P > 0.05) in this study. There was also 2-fold significantly higher spleen

uptake of IgG-DOTA-111In (18.5 ± 1.7% ID/g) and IgG-MCP-111In (20.6 ± 3.5% ID/g) compared

with the corresponding panitumumab RICs (8.5 ± 3.5%ID/g vs 9.5 ± 0.6% ID/g). There was 3.1-

fold significantly greater liver uptake of IgG-DOTA-111In compared with panitumumab-DOTA-

111In (12.0 ± 1.7%ID/g vs 3.8 ± 1.3%ID/g, respectively; P < 0.001).

94

Figure 2-7. Tumour and normal tissue uptake [percent injected dose per gram (%ID/g)]

with/without preinjection of an excess of unlabeled panitumumab in NOD-scid mice bearing s.c.

PANC-1 xenografts at 72 h p.i. of (A) Panitumumab-DOTA-111In or (B) Panitumumab-MCP-

111In. Also shown in each panel is the tumour and normal tissue uptake of nonspecific IgG-

DOTA-111In or IgG-MCP-111In, respectively. * Statistically significant differences (P < 0.05).

Data shown are mean ± SEM (n = 5).

95

The ability of the RICs to image s.c. PANC-1 tumours in NOD-scid mice was evaluated by

performing SPECT/CT imaging studies (Figure 2-8). The images were in agreement with the

results of biodistribution studies. SPECT/CT images showed tumour uptake as well as liver and

spleen accumulation for both panitumumab-DOTA-111In and panitumumab-MCP-111In (Figure 2-

8A,D). Tumours were not visualized with nonspecific IgG-DOTA-111In or IgG-MCP-111In

(Figure 2-8C,F). There was decreased tumour uptake of the panitumumab RICs with

preadministration of an 100-fold excess of unlabeled panitumumab to block EGFR compared

with the images obtained without blocking (Figure 2-8B,E). Taken together, these images

indicated EGFR-mediated tumour uptake of both panitumumab RICs.

96

Figure 2-8. Anterior (left) and lateral (right) whole-body SPECT/CT images of NOD-scid mice

with s.c. PANC-1 human pancreatic xenografts (white circle) at 72 h p.i. of 37 MBq (10 μg) of

panitumumab-DOTA-111In or panitumumab-MCP-111In without (A and D, respectively) or with

(B and E, respectively) preinjection of an excess (1 mg) of unlabeled panitumumab 24 h prior to

injection of the 111In-labeled RICs. SPECT/CT images of NOD-scid mice at 72 h p.i. of 25 MBq

of nonspecific IgG-MCP-111In or IgG-DOTA-111In are shown in panels C and F, respectively.

Also visualized on all image panels are the liver (long arrow) and spleen (short arrow). Images

were adjusted to the same intensity. The units on the intensity bar are kBq.

97

2.5 Discussion

We describe for the first time in this study a novel MCP composed of a PGlu backbone that

presents on average 13 pendant DOTA for complexing 111In or 177Lu and 10 PEG chains to

minimize liver and spleen accumulation. The MCP was conjugated through a terminal HyNic

functional group to panitumumab modified with S-4FB by reaction with lysine residues on the

mAb. This bioconjugation strategy was first described by Schwartz et al. and patented in

2011.(17) The approach relies on formation of a stable bis-aryl hydrazone bond between the

aromatic aldehyde on SF-4B and the terminal hydrazine on the polymer (Figure 2-1). Because

formation of the bis-aryl hydrazone creates a chromophore that absorbs at 354 nm (Figure 2-2A),

the bioconjugation reaction can be monitored spectrophotometrically in “real-time” and the

reaction terminated when the desired number of polymers is conjugated to the mAb. In our case,

a reaction time of 75 min resulted in conjugation of approximately two MCPs to panitumumab

(Figure 2-2B). Polymer conjugation was confirmed by an upward shift in the band for

panitumumab on SDS-PAGE (Figure 2-2C).

The ability of panitumumab-MCP to complex 111In or 177Lu to high SA was examined by

determining the L.E. for decreasing masses (100 to 0.1 μg) incubated with a constant amount of

radioactivity (7.5–8.5 MBq). Masses of panitumumab-MCP as small as 0.1 μg were labeled to

>90% L.E. with 111In or 177Lu yielding a SA > 70 MBq/μg (Table 2-1). In contrast, while 0.1 μg

of panitumumab-DOTA was labeled with 111In to a L.E. > 90%, the L.E. for 177Lu was 74%. A

minimum of 1 μg of panitumumab-DOTA was needed to achieve a L.E. > 90% with 177Lu (Table

2-1). Thus, assuming a L.E. > 90%, the maximum SA practically achievable for panitumumab-

DOTA labeled with 111In or 177Lu was 65.3 ± 3.1 MBq/μg and 46.2 ± 5.3 MBq/μg, respectively.

Various factors affect the complexation of radiometals by metal chelators, including competing

metal ions (283) and stable daughter products that result from decay of the parent

radionuclide.(30) 111In is cyclotron-produced by the 111Cd(p,n)111In reaction and decays by

electron capture to stable 111Cd. 111In solutions contain Fe, Al, and Zn ions which along with the

111Cd decay product compete for binding to DOTA and decrease L.E. (283) 177Lu is reactor-

produced by neutron irradiation of 176Lu and decays by β-particle emission to 177Hf. It has been

reported that 177Hf does not interfere with 177Lu labeling of DOTA-conjugated peptides (284).

However, 176Lu would be complexed by DOTA and the impact of this competition with 177Lu

98

depends on the SA of 177Lu. In fact, it has been stated that there may be more than 3 times the

number of atoms of 176Lu in 177Lu solutions (285). Our results showed that small masses of

panitumumab-DOTA (0.1 μg) were more susceptible to competing metal interference in labeling

with 177Lu than 111In (Table 2-1). Moreover, panitumumab-MCP, due to the 13 DOTA available

for complexing radiometals is less effected by competing metal ions than panitumumab-DOTA

which incorporated only two DOTA, resulting in higher SA. We have similarly found that

conjugation of trastuzumab with an MCP incorporating 29 DTPA permitted labeling with 111In to

a much higher SA than trastuzumab modified with only two DTPA per mAb (240).

An important consideration with MCP conjugation of mAbs is the effect of the polymer on

receptor/antigen binding. The EGFR-binding properties of panitumumab-MCP-177Lu and

panitumumab-DOTA-177Lu were compared using MDA-MB-468 human breast cancer cells that

overexpress EGFR (1.3 × 106 receptors/cell). Both panitumumab-MCP-177Lu and panitumumab-

DOTA-177Lu exhibited saturable binding to MDA-MB-468 cells that was displaced by an excess

of unlabeled panitumumab, demonstrating specific EGFR-mediated binding (Figure 2-4). There

were no significant differences in the EGFR binding affinity of both RICs (Kd = 2.2 ± 0.6

nmol/L and 1.0 ± 0.4 nmol/L, respectively). These Kd values were similar to those reported by

our group for 123I-panitumumab (Kd = 0.7 ± 0.4 nmol/L) (286). We did not measure the EGFR

binding of 111In-labeled panitumumab-MCP or panitumumab-DOTA, but it is not expected that

changing the radiometal complexed by DOTA would affect these properties.

111In- or 177Lu-labeled panitumumab-MCP or panitumumab-DOTA were stable to loss of

radiometal when challenged in vitro with a 500-fold excess of EDTA (Figure 2-5A) or when

incubated in human plasma for up to 1 week (Figure 2-5BC). However, to investigate any

potential instability that may occur in vivo due to proteolytic degradation of the PGlu backbone

of the MCP or cleavage of the linkage between the MCP and panitumumab, the elimination of

radioactivity from the body in mice and excretion into the urine and feces were compared up to

72 h p.i. of panitumumab-MCP-111In or panitumumab-DOTA-111In. There was relatively high

whole body retention of radioactivity (55–60%) for both RICs (Figure 2-6A). Most radioactivity

was eliminated in the urine with only a small amount in the feces, and there were no significant

differences between panitumumab-MCP-111In and panitumumab-DOTA-111In (Figure 2-6B).

99

These results suggest that there was no instability of the RICs introduced by use of the MCP for

labeling with 111In compared to DOTA.

The tumour and normal tissue localization of panitumumab-MCP-111In and panitumumab-

DOTA-111In were compared at 72 h p.i. in NOD-scid mice with s.c. PANC-1 tumours (Figure 2-

7). There were no significant differences in normal tissue localization between the two RICs,

except for the liver which was 3-fold higher for panitumumab-MCP-111In. We previously

observed higher liver uptake of trastuzumab Fab conjugated to MCP with a PAm backbone that

harbored residual negative charges after labeling with 111In (269). The MCP used in this study

was designed with a PGlu backbone that exhibits lower liver uptake and the polymer was further

modified with PEG chains to shield the residual negative charges on uncomplexed DOTA after

labeling with 111In or 177Lu. It was expected that these design features would limit opsonization

by plasma proteins that lead to liver and spleen uptake (256). However, further optimization of

the MCP with additional PEG chains may be required to reduce the liver uptake of

panitumumab-MCP-111In (271). Liver uptake of the RICs is not due to EGFR binding since

panitumumab does not bind to mouse EGFR.(33) Liver uptake of panitumumab-DOTA-111In in

NOD-scid mice at 72 h p.i. was more than 4-fold lower than reported by Ogawa et al. for

panitumumab-DTPA-111In at 6 days p.i. in tumour-bearing nude mice (∼40% ID/g) (287).

Importantly, the tumour uptake of panitumumab-MCP-111In was not significantly different than

panitumumab-DOTA-111In (6.9 ± 1.3 vs 6.6 ± 3.3%ID/g, respectively; Figure 2-7; P > 0.05).

Furthermore, tumour uptake was EGFR-mediated because preadministration of a 100-fold excess

of unlabeled panitumumab 24 h prior to injection of the RICs greatly reduced the tumour uptake

of panitumumab-MCP-111In and panitumumab-DOTA-111In to 0.02 ± 0.00%ID/g and 0.06 ±

0.02%ID/g, respectively. The relatively low tumour uptake (<10% ID/g) of the RICs may be due

to a moderate EGFR expression level on PANC-1 cells (4 × 105 EGFR/cell), despite

overexpression compared with normal pancreas cells. In a previous study, we found that the

tumour uptake of 111In-labeled anti-EGFR mAb528 at 72 h p.i. in mice with MCF-7 human

breast cancer xenografts (1.5 × 104 EGFR/cell) was 8.4 ± 1.6% ID/g at 72 h p.i., while uptake in

MDA-MB-468 tumours (1.3 × 106 EGFR/cell) was 15.3 ± 2.5% ID/g (288). Other factors such as

tumour vascularity may affect the uptake of the RICs in PANC-1 tumours since one study

100

reported a discordance between EGFR expression level in tumour xenografts in mice and the

uptake of 89Zr-labeled anti-EGFR, cetuximab (289).

Interestingly, there was a major decrease in radioactivity in the blood and most normal tissues for

these RICs associated with preinjection of an excess of panitumumab. The reasons are not

known. However, Michel et al. reported that scid mice have very low levels of circulating IgG2a

which causes rapid blood clearance of injected IgG2a due to binding to neonatal Fc receptors

(FcRn) particularly in the spleen (290). Lower uptake of injected IgG2a compared with IgG1 in

scid mice was found for liver, kidneys, lungs and muscle. Since panitumumab is a human IgG2a,

it may similarly exhibit a rapid elimination from the blood and normal tissues in NOD-scid mice,

which have scid background. FcRn rescues immunoglobulins from degradation in lysosomes,

and Bleeker et al. showed that administration of an excess of IgG in C57BL/6 mice promoted

elimination of an IgG1 mAb from the blood by more than 40% at 72 h p.i. (291). This may

explain the greater elimination of radioactivity from the blood and normal tissues in NOD-scid

mice preadministered an excess of panitumumab (1 mg) compared with mice receiving only

small amounts (10 μg) of the RICs alone. Swiercz et al. reported that FcRn were present only at

very low or undetectable levels on tumour cell lines (although PANC-1 was not examined)

(292). Thus, we interpret the decreased uptake of radioactivity in PANC-1 tumours for the RICs

with preadministration of an excess of panitumumab as related to blocking EGFR and not FcRn

on these tumours.

To further examine the EGFR-mediated tumour uptake of panitumumab RICs, we compared

their biodistribution to nonspecific IgG modified with MCP or DOTA and labeled with 111In

(Figure 2-7). The tumour uptake of IgG-DOTA-111In (1.9 ± 0.3%ID/g) was 3.5-fold significantly

lower than panitumumab-DOTA-111In as expected. Unexpectedly, there was no significant

difference between the tumour uptake of IgG-MCP-111In (5.4 ± 0.3% ID/g) and panitumumab-

MCP-111In (6.9 ± 1.3%ID/g; P > 0.05). This may be due to a larger hydrodynamic radius for

IgG-MCP-111In compared with IgG-DOTA-111In, analogous to that found for panitumumab-MCP

vs DOTA-panitumumab (7.1 nm vs 5.1 nm; respectively; Figure 2-3). This increase in molecular

size may encourage nonspecific uptake of IgG-MCP-111In into tumours via the enhanced

permeability and retention (EPR) effect (293). We previously found that uptake of 111In-labeled

nonspecific IgG into human breast cancer xenografts via the EPR effect in some cases was

101

sufficient for tumour imaging, but the effect varied dependent on the tumour xenograft model

(294). Nonetheless, EGFR-binding likely contributes to the tumour uptake of panitumumab-

MCP-111In because saturation receptor-binding assays (Figure 2-4) revealed no significant

differences in EGFR-binding affinity between panitumumab-MCP-177Lu and panitumumab-

DOTA-177Lu. We did not study the tumour and normal tissue uptake of panitumumab-MCP-

177Lu or panitumumab-DOTA-177Lu, but 111In- and 177Lu-labeled mAbs exhibit similar

biodistribution properties (295, 296). Finally, microSPECT/CT at 72 h p.i. of panitumumab-

MCP-111In and panitumumab-DOTA-111In visualized s.c. PANC-1 human PnCa xenografts in

NOD-scid mice (Figure 2-8). Tumours were not visualized in mice preadministered an excess of

unlabeled panitumumab to block EGFR, or in mice injected with nonspecific IgG RICs. The

higher liver uptake of the polymer RICs was also apparent on the images.

2.6 Conclusions

Panitumumab was modified with a novel MCP that presents multiple DOTA chelators enabling

stable and efficient complexation of 111In or 177Lu. The EGFR binding affinity of panitumumab

was preserved following MCP conjugation. Panitumumab-MCP-111In exhibited EGFR-mediated

tumour uptake in mice with s.c. PANC-1 human PnCa xenografts that was not significantly

different than panitumumab-DOTA-111In. Normal tissue distribution was similar except for the

liver which was higher for the polymer radioimmunoconjugates. The ability to dual-label

panitumumab-MCP with 111In and 177Lu allows SPECT imaging as well as RIT exploiting the

Auger electrons emissions of 111In and β-particles emitted by 177Lu, thus enabling a theranostic

approach.

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Chapter 3: Radioimmunotherapy of PANC-1 Human Pancreatic

Cancer Xenografts in NRG Mice with Panitumumab Modified

with Metal-Chelating Polymers (MCP) Complexed to 177Lu

103

This chapter represents a reprint of Radioimmunotherapy of PANC-1 Human Pancreatic Cancer

Xenografts in NRG Mice with Panitumumab Modified with Metal-Chelating Polymers (MCP)

Complexed to 177Lu. All experiments were designed and carried out by Sadaf Aghevlian.

Dosimetry was performed with assistance from Dr. Zhongli Cai. The synthesis and

characterization of metal chelating polymer were performed by Dr. Yijie Lu at the chemistry

department, at University of Toronto.

104

3. Abstract

Our aim was to evaluate the effectiveness and normal tissue toxicity of radioimmunotherapy

(RIT) of s.c. PANC-1 human pancreatic cancer (PnCa) xenografts in NRG mice using anti-

EGFR panitumumab linked to metal-chelating polymers (MCP) that present 13 DOTA chelators

to complex the -emitter, 177Lu. The clonogenic survival (CS) of PANC-1 cells treated in vitro

with panitumumab-MCP-177Lu (0.3-1.2 MBq) and DNA double-strand breaks (DSBs) in the

nucleus of these cells were measured by confocal immunofluorescence microscopy for -H2AX.

Subcellular distribution of radioactivity for panitumumab-MCP-177Lu was measured and

absorbed doses to the cell nucleus calculated. Normal tissue toxicity was assessed in non-tumour

bearing NRG mice by monitoring body weight, complete blood cell counts (CBC) and serum

alanine aminotransferase (ALT) and creatinine (Cr) after i.v. injection of 6 MBq (10 g) of

panitumumab-MCP-177Lu. RIT was performed in NRG mice with s.c. PANC-1 tumours injected

i.v. with 6 MBq (10 g) of panitumumab-MCP-177Lu. Control mice received non-specific human

IgG-MCP-177Lu (6 MBq; 10 g), unlabeled panitumumab (10 g) or normal saline. The tumour

growth index (TGI) was compared. Tumour and normal organ doses were estimated based on

biodistribution studies. Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by

7.7-fold at the highest amount tested (1.2 MBq). Unlabeled panitumumab had no effect on the

CS of PANC-1 cells. γ-H2AX foci were increased by 3.8-fold by panitumumab-MCP-177Lu.

Panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. Administration of

panitumumab-MCP-177Lu (6 MBq; 10 g) to NRG mice caused no change in body weight, CBC

or ALT and only a slight increase in Cr compared to NRG mice treated with normal saline.

Panitumumab-MCP-177Lu strongly inhibited tumour growth in NRG mice (TGI = 2.3 ± 0.2)

compared to normal saline treated mice (TGI = 5.8 ± 0.5; P<0.01). Unlabeled panitumumab had

no effect on tumour growth (TGI = 6.0 ± 1.6; P>0.05). The absorbed dose of PANC-1 tumours

was 12.3 Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and

kidneys. We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to

overcome resistance to panitumumab in KRAS mutant PANC-1 tumours in NRG mice and may

be a promising approach to treatment of PnCa in humans.

105

Graphical abstract

106

3.1 Introduction

Pancreatic cancer (PnCa) is a rapidly fatal malignancy due to its often advanced stage at

diagnosis combined with the limited treatment options (297). Surgery remains the only hope for

long-term survival, but only 10-15% of patients are candidates for surgical resection due to local

invasion or distant metastasis (298). Even in patients who undergo surgical resection with

curative intent followed by chemotherapy, the median survival is 17-23 months. For patients

who are not candidates for surgery due to locally advanced PnCa, the median survival is 8-14

months and is 4-6 months for patients with metastatic PnCa (299). New therapeutic strategies are

needed for patients with PnCa. Stereotactic body radiation therapy (SBRT) has proven effective

for controlling local progression of PnCa providing freedom from local progression (FFLP) for

as long as a year in 80% of patients with locally advanced PnCa, but patients ultimately die of

metastatic disease (300). Radioimmunotherapy (RIT) which offers an opportunity to selectively

treat disseminated PnCa at any location in the body with radiation, may improve patient outcome

in patients with metastatic disease. RIT with the humanized monoclonal antibody (mAb),

clivatuzumab (Immunomedics, Morris Plains, NJ) targeting the cell surface mucin MUC1 (301)

labeled with the -emitter, 90Y [Emax=2.2 MeV (100%); t1/2=2.7 d] showed promising results

(partial remission or stable disease) in a Phase I/II trial combined with gemcitabine for treatment

of metastatic PnCa (302). Improved survival was found in patients with metastatic PnCa in a

subsequent Phase 1 trial that combined 90Y-clivatuzumab with gemcitabine chemotherapy

compared to RIT alone (7.8 vs. 3.4 months) (303). However, interim analysis of a Phase 3 trial

(PANCRIT-1) that randomized patients to RIT + gemcitabine and best supportive care (BSC) or

gemcitabine and BSC alone did not demonstrate improved survival in the RIT arm

(https://clinicaltrials.gov/ct2/show/NCT01956812) and the trial was discontinued. However,

patients in the PANCRIT-1 trial had metastatic PnCa that had progressed after at least two prior

chemotherapy regimens. Thus, there remains the potential that earlier treatment of PnCa in

patients with lower tumour burden may improve outcome. In addition, there is an emerging

interest in the use of RIT to eradicate small tumours and minimal residual disease to prevent

disease progression after other forms of cancer therapy (304). The shorter range (2 mm) -

particles emitted by 177Lu [t1/2 = 6.7 d; Emax = 0.5 MeV (78.6%); Emax = 0.38 MeV (9.1%);

Emax = 0.18 MeV (78.6%)] may be more appropriate for RIT of small tumours, while the higher

107

energy [(Emax = 2.2 MeV (100%)] and longer range (12 mm) -particles emitted by 90Y are

more useful for treatment of larger tumours. Modeling has revealed that the optimal tumour

diameter for treatment with 177Lu is ~2 mm, while for 90Y, the ideal tumour diameter for RIT is

34 mm (305). Indeed, de Jong et al. reported that co-treatment with a mixture of 177Lu- and 90Y-

labeled DOTATATE radiopeptides was more effective than either radiopeptide alone in

prolonging the survival of rats with both large and small somatostatin receptor-positive CA20948

s.c. pancreatic neuroendocrine tumours (306).

Panitumumab (Vectibix; Amgen, Thousand Oaks, CA) is a human IgG2 mAb that binds the

epidermal growth factor receptor (EGFR) (307). EGFR are overexpressed on PnCa (>90% of

cases) (308). Naked anti-EGFR mAbs have not proven effective for treatment of PnCa (309,

310) since there is often downstream KRAS mutation (311), but this is not limiting for RIT,

since the mAbs are used only to target radioactivity to tumours. We recently reported novel

radioimmunoconjugates (RICs) for RIT of PnCa consisting of panitumumab linked to metal-

chelating polymers (MCP) that complex 177Lu or 111In (312). The MCP are composed of a

polyglutamide backbone with 10 pendant polyethylene glycol (PEG) chains with the aim to

inhibit liver and spleen uptake of the RICs, and 13 DOTA (1,4,7,10-tetraazacyclododecane-

1,4,7,10-tetraacetic acid) chelators that strongly complex 177Lu or 111In (312). These polymer

immunoconjugates have theranostic capability since they may be labeled simultaneously to high

specific activity (SA) with 177Lu and 111In. The -particle emissions of 177Lu and Auger electrons

emitted by 111In enable RIT, while the -emissions of 111In [Eγ = 171 keV (90%) and Eγ = 245

keV (94%)] allow single photon emission computed tomography (SPECT).

In the current study, we examine the cytotoxicity of panitumumab-MCP-177Lu in vitro on EGFR-

positive PANC-1 human PnCa cells and its ability to inflict lethal DNA double-strand breaks

(DSBs) in the nucleus of these cells and estimate the absorbed doses in the cell nucleus. We

further report for the first time the effectiveness and normal tissue toxicity of RIT of PANC-1

tumours in NOD-Rag1−/−IL2RgammaC-null (NRG) mice with panitumumab-MCP-177Lu, and

estimate the absorbed doses in tumours and normal organs. We hypothesized that RIT with

panitumumab-MCP-177Lu would be effective for treatment of PANC-1 tumours at administered

amounts that are non-toxic to normal tissues.

108

3.2 Materials and Methods

3.2.1 Cell culture and tumour xenografts

PANC-1 human pancreatic adenocarcinoma cells (4.0 × 105 EGFR/ cell) (313) were purchased

from the American Type Culture Collection (ATCC, Manassas, VA, USA). PANC-1 cells were

cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (Gibco Life

Technologies; Burlington, ON, Canada) supplemented with 1% penicillin and streptomycin and

10% fetal bovine serum (Gibco-Invitrogen, Thermo Fisher Scientific, Waltham, MA). Tumour

xenografts (8-10 mm diameter) were established in female NOD-Rag1−/−IL2RgammaC-

null (NRG) mice (University Health Network, Toronto, ON) by subcutaneous (s.c.) inoculation

of 4 × 106 PANC-1 cells in serum free DMEM (100 μL) into the left flank.

3.2.2 Radioimmunoconjugates

The synthesis of hydrazino nicotinamide (HyNic) end-capped HyNic-MCP presenting 13 DOTA

chelators and 10 PEG chains to minimize liver and spleen uptake was previously reported (271).

Panitumumab-MCP immunoconjugates were constructed by cross-linking panitumumab IgG to

two HyNic-MCPs through an N-succinimidyl-4-formylbenzamide (S-4FB) moiety introduced

into panitumumab and stored in 0.1 M HEPES buffer, pH 5.5. Immunoconjugates (5-70 µg; 4-12

L) were radiolabeled by incubation with 177LuCl3 (0.75-49 MBq; 37 MBq/μL; PerkinElmer,

Waltham, MA) at 42 °C for 2 h (271). The final radiochemical purity of the RICs was >95%

determined by instant thin-layer silica gel chromatography (ITLC-SG; Pall) developed in 0.1 M

sodium citrate, pH 5.5. We previously reported that the KD for binding of panitumumab-MCP

complexed to 111In to EGFR-positive MDA-MB-468 human breast cancer cells was 2.2 ± 0.6

nmol/L (312). Non-specific human IgG (hIgG; Sigma-Aldrich, St. Louis, MO, Product No.

14506) was similarly modified with MCP and labeled with 177Lu. The RICs were shown to be

stable to loss of radiometal by challenge with an excess of ethylenediaminetetraacetic acid

(EDTA) and in plasma at 37 oC (312).

3.2.3 In vitro cytotoxicity studies

The clonogenic survival (CS) of PANC-1 cells treated in vitro with panitumumab-MCP-177Lu

was studied. Briefly, 2 × 105 cells were treated with 2.5 nmol/L (50-fold molar excess) of

109

panitumumab-MCP labeled with 0.3, 0.6 or 1.2 MBq of 177Lu [SA = 46.7, 93.5, 187.0

MBq/nmole] in 500 μL of growth medium in 24 well plates for 16 h. Controls consisted of cells

exposed to unlabeled panitumumab-MCP or to growth medium alone. Approximately 700 cells

were then seeded into 6 well plates and cultured for 12 d. Surviving colonies were stained with

methylene blue and colonies with >50 cells were counted. The plating efficiency (PE) was

determined by dividing the number of colonies formed by the number of cells seeded. The CS

was calculated by dividing the PE for treated cells by that for untreated cells (314).

Induction of DNA double-strand breaks (DSBs) in PANC-1 cells caused by panitumumab-MCP-

177Lu was assessed by immunofluorescence confocal microscopy for phosphorylated histone-2A

(γ-H2AX) which accumulates at sites of DSBs in the cell nucleus (315). Briefly, 2 × 105 PANC-

1 cells were seeded onto glass coverslips (Ted Pella, Redding, CA) in 24-well plates and cultured

overnight at 37 °C. The cells were then treated with panitumumab-MCP-177Lu as described

earlier. Controls consisted of cells exposed to unlabeled panitumumab-MCP or to growth

medium alone. Immunostaining for γ-H2AX and analysis of confocal microscopy images for the

density of γ-H2AX foci per nucleus area followed previously reported procedures (316).

Normalization of γ-H2AX foci per nucleus area rather than the number of foci in the nucleus

minimizes differences in cell nucleus size between cells.

3.2.4 Subcellular distribution and microdosimetry

The subcellular distribution of panitumumab-MCP-177Lu in PANC-1 cells was measured to

estimate the absorbed doses in the cell nucleus (microdosimetry). Briefly, 2 × 105 cells were

cultured overnight in wells in a 6-well plate. A 50-fold molar excess of panitumumab-MCP-

177Lu (1.2 MBq; 2.5 nmol/L) were added to the wells and the plates were incubated for 1, 4, 8, or

24 h at 37 °C in the absence or presence of excess of unlabeled panitumumab to block EGFR to

confirm the EGFR specificity of binding of panitumumab-MCP-177Lu to PANC-1 cells. Cell

fractionation studies were also performed for cells incubated with non-specific hIgG-MCP-177Lu

(1.2 MBq; 2.5 nmol/L). The medium was removed and the cells which were exposed to

panitumumab-MCP-177Lu were fractionated to determine the dose to the cell nucleus from the

cell surface, cytoplasm and nucleus source compartments. The cells were first rinsed with

phosphate-buffered saline (PBS), pH 7.5, then the proportion of internalized cell-bound

radioactivity was determined by displacing the cell-surface 177Lu with 1 mL of 200 mM sodium

110

acetate/500 mM NaCl, pH 2.5 at room temperature (RT). This procedure was repeated twice and

the cell membrane fractions were combined and measured in a γ-counter. The cell membrane

was then lysed twice in 500 μL of Nuclei EZ Lysis buffer (Sigma-Aldrich) on ice for 60 min. A

cell scraper was used to gently detach cells from each well and the cells were then transferred to

1.5 mL Eppendorf tubes. The tubes were centrifuged at 3,000 × g for 5 min to separate nuclear

and cytoplasmic radioactivity (supernatant). The radioactivity in the nucleus, membrane and

cytoplasm fractions was measured in a γ-counter and the percentage of radioactivity in each

fraction (relative to the total cell-bound 177Lu) was calculated. This cell fractionation procedure

has been previously validated by our group to yield pure cytoplasmic and nuclear fractions (317).

The absorbed dose in the nucleus (D) of a PANC-1 cell after exposure to 1.2 MBq of

panitumumab-MCP-177Lu (2.5 nmol/L) was estimated based on the cumulative amount of

radioactivity (As) on the cell surface, in the cytoplasm and in the cell nucleus as well as non cell-

bound radioactivity in the surrounding culture medium. As was calculated from the area under

the curve from 0 to 24 h (AUC0−24h; Bq × sec) using Prism Ver. 4.0 software (GraphPad). The

dose to the nucleus was estimated as D = As × S, and the S-values (Gy/Bq × sec) were calculated

using MCNP Monte Carlo code Ver. 5 (Los Alamos National Laboratory, Los Alamos, NM). A

mean cell radius of 17.1 μm and nucleus radius of 7.0 μm were used. These were measured by

fluorescence microscopy of PANC-1 cells and Image J software (National Institutes of Health,

Bethesda, MD) after staining of the nucleus with 4′,6-diamidino-2-phenylindole (DAPI) (221).

The absorbed doses were also calculated for PANC-1 cells exposed to panitumumab-MCP-177Lu

co-incubated with a 50-fold molar excess of unlabeled panitumumab to block EGFR, or non-

specific hIgG-MCP-177Lu (1.2 MBq; 2.5 nmol/L).

3.2.5 Evaluation of normal tissue toxicity

To identify a non-toxic dose for subsequent RIT studies, groups of 5 healthy, non-tumour

bearing female NRG mice were injected i.v. (tail vein) with 6.0 MBq (10 μg) of panitumumab-

MCP-177Lu or non-specific hIgG-MCP-177Lu in 100 μL of normal saline. These doses were

selected based on a previous study that reported that 8.0 MBq of 177Lu-labeled RICs were

administered safely without major toxicity to NRG mice (no loss in body weight) (318). NRG

mice were selected for these studies and for RIT studies, since unlike NOD/SCID mice, NRG

mice do not harbor a germ line defect in DNA repair (319) which renders NOD/SCID mice

111

unusually sensitive to radiation. Control mice received injections of normal saline or

panitumumab. Body weight was monitored every 2-4 d for 14 d. The mice were then sacrificed

and samples of blood were collected into EDTA-coated microtubes and centrifuged to separate

serum for biochemistry [serum alanine aminotransferase (ALT) and creatinine (Cr)] analyses.

ALT and Cr were measured using Infinity Creatinine or ALT clinical chemistry kits (Fisher

Diagnostics, Middletown, VA). Complete blood cell (CBC) counts, hematocrit (HCT) and

hemoglobin (Hb) were measured on a HemaVet 950FS instrument (Drew Scientific, Miami

Lakes, FL). The 14 d period for assessing normal tissue toxicity was selected based on the

requirements of Health Canada for acute toxicity testing of new drugs (320).

3.2.6 Radioimmunotherapy studies

The tumour-growth inhibitory effects of panitumumab-MCP-177Lu were studied in groups of

NRG mice (n=7) with s.c. PANC-1 xenografts. The number of mice in each group (7) was

sufficient to detect a minimum difference of 0.8 in the mean tumour growth index (TGI) with a

standard deviation of 0.5, an -value = 0.05 and power, -value = 0.80

(https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html). Mice received a single i.v. injection (tail

vein) of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu in 100 µL of normal saline. Control

groups of NRG mice were injected with unlabeled panitumumab (10 µg) or normal saline.

Tumour length (mm) and width (mm) were measured using calipers every 2-3 days until the

humane endpoint of the animal care protocol was reached (tumour diameter >15 mm). The

tumour volume (V; mm3) was calculated as V =length × width2 × 0.5 (321). The TGI was

calculated by dividing the tumour volume at each time point by the tumour volume at the

initiation of treatment. Similarly, the body weight index (BWI) was estimated by dividing the

body weight at each time point by the pre-treatment body weight.

3.2.7 Tumour and normal organ dosimetry

The absorbed doses in the tumour and normal organs in NRG mice with s.c. PANC-1 xenografts

after i.v. injection of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu were estimated. Groups of 5

mice were injected with RICs, then at selected time points ranging from 24 to 168 h p.i., the mice

were sacrificed by cervical dislocation under anesthesia, and the tumour and samples of normal

tissues including blood were collected, weighed, and counted in a γ-counter. Tumour and normal

112

organ uptake were expressed as MBq/g, then converted to MBq/organ by multiplying by

previously determined standard weights for mouse organs (322). The cumulative radioactivity in

the tumour and normal source organs from 0-168 h (Ã0-168h) was estimated using Prism Ver. 4.0

software (GraphPad, San Diego, CA) from the AUC0-168h (MBq × h) determined in

biodistribution studies. The cumulative radioactivity from 168 h to infinity (Ã168h-∞) was

calculated by dividing the radioactivity at the final measured time point by the elimination rate

constant for 177Lu (0.004345 h-1), thus assuming that further elimination occurs only by

radioactive decay without additional biological elimination. We have previously applied this

assumption to estimate the absorbed doses for other radiolabeled mAbs (322). The Ã0-∞ values of

each source organ (MBq h) were converted to Bq x sec by multiplying by (3.6 103

secs/h)(106 Bq/MBq). Then, these Ã0-∞ values were multiplied by the respective S-value (Gy/Bq

× sec) of the source to target organs for 177Lu using published mouse S-values (323), then

summing up all the absorbed doses to the target organ from all source organs. The dose delivered

to the tumour was estimated based on S-values of the sphere model in OLINDA/EXM, using the

average measured tumour volume at the time of treatment (324).

3.3 Statistical analysis

Results were expressed as mean SEM. Statistical significance was tested using an unpaired

two-tailed Student’s t-test (P<0.05) or in multiple comparisons by ANOVA (F-test; P<0.05)

using Prism Ver. 4.0 software (GraphPad).

3.4 Results

3.4.1 In vitro cytotoxicity studies

There was no effect of unlabeled panitumumab-MCP on the CS of PANC-1 cells (Figure 3-1a).

CS was decreased with exposure to increasing activity of panitumumab-MCP-177Lu. At the

highest radioactivity of panitumumab-MCP-177Lu studied (1.2 MBq), the CS of PANC-1 cells

was decreased by 7.4-fold compared to untreated cells. Exposure of PANC-1 cells to 0.6 MBq of

panitumumab-MCP-177Lu reduced the CS by 9.0-fold, but this was not significantly different

than cells treated with 1.2 MBq. The CS of cells exposed to 0.3 MBq of panitumumab-MCP-

177Lu was reduced by 1.9-fold, and this was significantly lower than cells exposed to 0.6 or 0.9

113

MBq. There were no significant differences in the density of γ-H2AX foci in the nucleus of

PANC-1 cells exposed to unlabeled panitumumab-MCP compared to untreated cells (Figure 3-

1b). Similarly, no differences in γ-H2AX foci were found in cells exposed to 0.3 MBq of

panitumumab-MCP-177Lu compared to cells exposed to these control treatments. However,

PANC-1 cells exposed to 0.6 MBq or 1.2 MBq of panitumumab-MCP-177Lu exhibited a

significantly greater density of γ-H2AX foci. At the highest amount tested (1.2 MBq), γ-H2AX

foci were increased by 3.8-fold by panitumumab-MCP-177Lu. Confocal immunofluorescence

microscopy for γ-H2AX (Figure 3-1c) showed more γ-H2AX foci in the nucleus of cells treated

with panitumumab-MCP-177Lu than for cells exposed to unlabeled immunoconjugates or

untreated cells.

114

Figure 3-1. (A) Clonogenic survival of PANC-1 human pancreatic cancer cells exposed to

increasing radioactivity of panitumumab-MCP-177Lu (0.3-1.2 MBq, 2.5 nmol/L) compared to

untreated cells (0.0 MBq). (B) Density of γ-H2AX foci in the nucleus of PANC-1 cells

representing sites of DNA double-strand breaks (DSBs) following exposure to increasing

amounts of panitumumab-MCP-177Lu or to unlabeled radioimmunoconjugates (RICs) or in

untreated cells. (C) Immunofluorescence confocal microscopy images of PANC-1 cells

exposed to these treatments. Bright foci in the nucleus of the cells (counterstained blue with

DAPI) are -H2AX foci, which are more prevalent in cells treated with panitumumab-MCP-

177Lu compared to untreated cells or cells exposed to unlabeled RICs.

Untr

eate

d

Unla

beled

RIC

s

177 Lu (0

.3M

Bq)

177 Lu (0

.6M

Bq)

177 Lu (1

.2M

Bq)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

g-H

2A

X f

oci

(In

teg

rate

d d

en

sit

y/n

ucle

us a

rea)

*

*

*

*

0.0 0.3 0.6 1.20

20

40

60

80

100

120

Radioactivity (MBq)

Clo

no

ge

nic

Su

rviv

al (%

)

*

*

*

*

Untreated UnlabeledRICs

177Lu (0.3 MBq)

177Lu (0.6 MBq)

177Lu (1.2 MBq)

A B

C

115

3.4.2 Subcellular localization and microdosimetry

There was a time-dependent increase in the percentage of radioactivity bound to PANC-1 cells

after incubation with panitumumab-MCP-177Lu for 1, 4, 8 or 24 h at 37 oC (Figure 3-2a). The

percentage of cell-bound radioactivity for non-specific hIgG-MCP-177Lu or panitumumab-MCP-

177Lu in the presence of excess of unlabeled panitumumab at 24 h was significantly lower than

for panitumumab-MCP-177Lu (1.21 ± 0.18% or 0.35 ± 0.01% vs. 4.16 ± 0.17%; P<0.001

respectively) indicating the EGFR-specificity of binding of panitumumab-MCP-177Lu RICs. The

percentage of the total radioactivity bound to the cells that was localized on the cell membrane,

in the cytoplasm or in the nucleus was measured by subcellular fractionation of cells treated with

panitumumab-MCP-177Lu (Figure 3-2b). Cell-membrane radioactivity decreased over 24 h,

representing internalization of the RICs, while cytoplasmic and nuclear radioactivity increased.

116

0

20

40

60

80

100Cell Membrane

Cytoplasm

Nucleus

1

1

1 4 8 24

4 8 24

4 8 24

Ce

ll B

ou

nd

(%

)

1 4 8 24 1 4 8 24 1 4 8 240.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0 Panitumumab-MCP-177Lu

Panitumumab-MCP-177Lu+ panitumumab

hIgG-MCP-177Lu

Time (h)

Cell

Bo

un

d (

%)

A B

Figure 3-2. (A) Percentage of radioactivity bound to PANC-1 cells at selected times after

incubation with panitumumab-MCP-177Lu in the absence or presence of excess of unlabeled

panitumumab or with non-specific hIgG-MCP-177Lu. (B) Percentage of total cell-bound

radioactivity which was localized on the cell membrane, in the cytoplasm or in the nucleus of

PANC-1 cells at selected times after incubation with panitumumab-MCP-177Lu.

117

The cumulative radioactivity in the cell surface, cytoplasm and nucleus source compartments

(Ãs) as well as non-cell bound radioactivity in the culture medium was used to estimate the

absorbed dose in the nucleus of a PANC-1 cell exposed to the maximum radioactivity (1.2 MBq)

of panitumumab-MCP-177Lu (Table 3-1). A total of 3.87 Gy of radiation was deposited in the

nucleus of PANC-1 cells when exposed to panitumumab-MCP-177Lu (1.2 MBq). The absorbed

dose was deposited mainly by 177Lu present on the cell surface, followed by cytoplasmic 177Lu,

then 177Lu in the nucleus. An additional 25% of the total absorbed dose in the nucleus of PANC-

1 cells was due to non-cell bound 177Lu in the culture medium through a cross-fire effect, during

the 24 h incubation period prior to recovering and seeding the cells for clonogenic assay. The

absorbed dose deposited in the nucleus of PANC-1 cells exposed to panitumumab-MCP-177Lu

was 2.7-fold higher than for panitumumab-MCP-177Lu co-incubated with a 50-fold molar excess

of unlabeled panitumumab to block EGFR (1.46 Gy), or non-specific hIgG-MCP-177Lu (2.06

Gy) (Table 3-1). A higher proportion of the dose deposited by these control treatments was due

to radioactivity present in the growth medium during the 16 h incubation period than for

panitumumab-MCP-177Lu.

118

Table 3-1. Absorbed doses in the nucleus of PANC-1 cells in vitro by panitumumab-MCP-177Lu a

Source

compartment

Ãs b

(Bq sec)

S-Factor

(Gy/Bq sec)

Dose to the cell nucleus (Gy)

Panitumumab-

MCP-177Lu

Panitumumab-

MCP-177Lu +

excess

panitumumab c

Non-specific

hIgG-MCP-

177Lu

Panitumumab-

MCP-177Lu

Panitumumab-

MCP-177Lu +

excess

panitumumab

Non-specific

hIgG-MCP-

177Lu

Cell surface 3989 367 544 3.55 10-4 1.42 0.13 0.19

Cytoplasm 2154 331 1040 4.02 10-4 0.86 0.13 0.42

Nucleus 663 101 468 6.93 10-4 0.46 0.07 0.32

Medium 9.8 1010 9.8 1010 9.8 1010 1.15 10-11 1.13 1.13 1.13

Total: 3.87 1.46 2.06

a Cells were incubated with 1.2 MBq (2.5 nmol/L) of radioimmunoconjugates for 24 h at 37 oC.

b Ãs: Cumulative radioactivity in the source compartment.

c Cells were co-incubated with 1.2 MBq (2.5 nmol/L) of panitumumab-MCP-177Lu and a 50-fold molar excess of unlabeled panitumumab to block

EGFR.

119

3.4.3 Evaluation of normal tissue toxicity

Administration of 6.0 MBq (10g) of panitumumab-MCP-177Lu to NRG mice caused no

significant decrease in red blood cell (RBC), white blood cell (WBC) or platelet (PLT) counts

(Figure 3-3a-c), Hb (Figure 3-3d) or HCT (Figure 3-3e) at 14 d post-injection compared to mice

receiving normal saline. These results indicated no hematologic toxicity. In addition, there were

no increases in serum ALT and only a slight increase in Cr in mice receiving RICs compared to

normal saline treated mice (Figure 3-3f-g), indicating no liver or kidney toxicity.

120

Figure 3-3. Hematology and serum biochemistry analyses of NRG mice administered 6.0

MBq (10g) of panitumumab-MCP-177Lu or normal saline. (A) red blood cells [RBC], (B)

white blood cells [WBC], (C) platelets [PLT], (D) hemoglobin [Hb], (E) hematocrit

[HCT], (F) serum alanine aminotransferase [ALT], (G) serum creatinine [Cr]. Individual

values are shown as well as the mean value (horizontal bars).

0

2

4

6

8

10R

BC

(´

10

6/m

L)

Panitumumab-MCP-177Lu

Normal saline

0

10

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HC

T (

%)

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C (

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03/m

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T (

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)

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T (

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(mm

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)

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)

A B C

D E F G

121

In addition, administration of panitumumab-MCP-177Lu or non-specific hIgG-MCP-177Lu caused

no change in body weight over 14 d compared to mice treated with unlabeled panitumumab or

normal saline, demonstrating no generalized normal tissue toxicity from these RICs administered

at these amounts (Figure 3-4a).

122

0 10 20 30 400.0

0.2

0.4

0.6

0.8

1.0

1.2

Panitumumab

Panitumumab-MCP-177Lu

Normal saline

hIgG-MCP-177Lu

A

Time (d)

Bo

dy

We

igh

t In

de

x (

BW

I)

0 10 20 30 400

2

4

6

8

10Panitumumab

Panitumumab-MCP-177Lu

Normal saline

*

hIgG-MCP-177Lu

*

B

*

Time (d)

Tu

mo

r g

row

th in

dex (

TG

I)

Figure 3-4. (A) Body weight index (BWI) for tumour bearing NRG mice at different times post

i.v. injection of 6.0 MBq (10 μg) of panitumumab-MCP-177Lu or non-specific hIgG-MCP-177Lu,

unlabeled panitumumab (10 μg) or normal saline. Values shown are the mean ± SEM (n = 7)

BWI. (B) Tumour growth index (TGI) in NRG mice with PANC-1 xenografts treated with a 6.0

MBq (10 g) of panitumumab-MCP-177Lu or hIgG-MCP-177Lu or in mice receiving unlabeled

panitumumab (10 g) or normal saline. Values are the mean ± SEM (n = 7) TGI. Significant

differences (P<0.05) between TGI at 33 days between treatment groups are indicated by

asterisks.

123

3.4.4 Radioimmunotherapy studies

Administration of 6.0 MBq (10 g) of panitumumab-MCP-177Lu to NRG mice significantly

inhibited the growth of PANC-1 xenografts (Figure 3-4b). The mean SEM TGI at 33 days in

surviving NRG mice treated with panitumumab-MCP-177Lu was 2.5 ± 0.3 (n=7) compared to 5.8

± 0.5 for mice receiving normal saline (n=3; P<0.001). Treatment of NRG mice with non-

specific hIgG-MCP-177Lu (n=4) slightly but non-significantly inhibited tumour growth (TGI =

4.0 ± 0.7) compared to normal saline treated mice (P=0.11) but these RICs were less effective

than panitumumab-MCP-177Lu (P=0.04). Treatment of NRG mice with unlabeled panitumumab

(10 g; n=3) did not slow tumour growth compared to normal saline treated control mice (TGI =

6.1 ± 1.1; P=0.81).

3.4.5 Tumour and normal organ dosimetry

The absorbed doses in PANC-1 tumours and in normal organ doses in NRG mice were

estimated based on the cumulative radioactivity (Ãs) in the tumour or source organs (Table 3-2)

without correction for radioactive decay determined in biodistribution studies (Figure 3-5 or 3-6)

and using mouse organ S-values (323). Absorbed doses in NRG mice with s.c. PANC-1 tumours

after i.v. injection of 6.0 MBq (10 g) of panitumumab-MCP-177Lu are shown in Table 3-3. The

tumour dose for panitumumab-MCP-177Lu was 12.3 ± 0.9 Gy while the highest normal organ

doses were received by the pancreas (19.3 ± 6.5 Gy), kidneys (15.7 ± 3.7 Gy) and liver (7.5 ± 1.6

Gy). The whole body dose was 1.4 ± 0.7 Gy.

124

0 24 48 72 96 120 144 168 1920.0

0.2

0.4

0.6

0.8

1.0

Heart

Lungs

Liver

Spleen

Pancreas

Stomach

Intestine

Kidney

Tumor

Time post injection (h)

177L

u (

MB

q)

Figure 3-5. Radioactivity in selected source organs at 24, 72, 120 and 168 h post i.v. injection of

6.0 MBq (10 µg) of panitumumab-MCP-177Lu in NRG mice. The cumulative radioactivity in

each source organ was calculated from the area-under-the-curve (AUC0-168h; MBq h). The

AUC168- was calculated by dividing the radioactivity at the last measured time point by the

decay constant for 177Lu (0.004345 h-1), assuming further elimination only by radioactive decay.

125

24 h

72 h

120

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168

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Time post injection (h)

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Time post injection (h)

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120

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168

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Time post injection (h)

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120

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Time post injection (h)

MB

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24 h

72 h

120

h

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Time post injection (h)

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72 h

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Time post injection (h)

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q/g

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120

h

168

h

0.0

0.2

0.4

0.6

0.8

1.0Tumor

Time post injection (h)

MB

q/g

Figure 3-6. Tumour and normal organ biodistribution of panitumumab-MCP-177Lu (MBq/g) in NRG

mice with s.c. PANC-1 human pancreatic cancer xenografts at selected times up to 168 h post-injection.

There was no correction for radioactive decay as these values were used for estimating the cumulative

radioactivity in these source organs (Ãs) for radiation dosimetry calculations.

126

Table 3-2. Cumulative radioactivity (ÃS) in source organs for panitumumab-MCP-177Lu injected

i.v. in NRG mice with s.c. PANC-1 tumour xenografts.

Source organ ÃS from 0-168 h

p.i.

(MBq h) a

ÃS from 168 to

(MBq h) b

Total ÃS from 0 to

(MBq h) c

Heart 7.39 3.72 11.11

Lungs 3.98 2.56 6.54

Liver 95.01 72.96 167.97

Spleen 15.59 25.13 40.72

Pancreas 8.14 13.90 22.04

Stomach 0.81 4.16 4.97

Intestine 8.56 16.98 25.54

Kidneys 14.85 3.15 18.00

Tumour 23.89 29.58 53.47

a Calculated by integrating the radioactivity vs. time curves for source organs shown in Figure 3-5.

b Calculated by dividing the radioactivity at 168 h p.i. of panitumumab-MCP-177Lu for source organs by

the decay constant for 177Lu (0.004345 h-1), assuming further elimination only by radioactive decay.

c These values were converted to Bq × sec for absorbed dose calculations (Table 3-3) by multiplying by

(3.6 × 103 secs/h)(106 Bq/MBq).

127

Table 3-3. Estimated radiation absorbed doses in the tumour and normal organs in NRG mice

with s.c. PANC-1 xenografts injected with panitumumab-MCP-177Lu

Target organ Dose (Gy) a Major source organ

contributor

Heart 3.37 ± 0.39 Heart (94.9%)

Lungs 4.31 ± 0.54 Lungs (90.6%)

Liver 7.53 ± 1.55 Liver (98.8%)

Spleen 14.80 ± 2.76 Spleen (97.8%)

Pancreas 19.31 ± 6.47 Pancreas (96.7%)

Stomach 1.87 ± 0.38 Stomach (56.9%)

Intestine 3.22 ± 1.58 Intestine (87.1%)

Kidneys 15.66 ± 3.70 Kidneys (97.3%)

Whole Body 1.40 ± 0.69 Liver (10.6%)

Tumour b 12.33 ± 0.86 Tumour (100%)

a Dose (D) was estimated for i.v. injection of 6 MBq (10 g) of panitumumab-MCP-177Lu and

represents the sum of all source organtarget organ pairs, calculated for each pair as was

calculated as D = Ãs S, where Ãs is the cumulative radioactivity in source organs (Table 3-2)

and S are the Snyder values for mice (323).

b Estimated using the sphere model in OLINDA/EXM (324) based on the measured tumour

diameter.

128

3.5 Discussion

Polymers that present multiple chelators for complexing metal ions (MCP) conjugated to mAbs

offer advantages compared to direct conjugation of chelators for radiometal labeling. For

example, MCP-conjugated mAbs may be labeled to higher specific activity (SA). We previously

reported that panitumumab modified with the same MCP described here presenting 13 DOTA

chelators was labeled to a SA up to 72 MBq/g with 177Lu, while achieving >90% labeling

efficiency (LE), almost two-fold higher than panitumumab conjugated directly to two DOTA (46

MBq/g) (312). Even greater differences (>10-fold) in SA were found between panitumumab-

MCP and panitumumab-DOTA dual-labeled with 177Lu and 111In (257, 312). Higher SA provides

greater potency in vitro for killing cancer cells, especially for cells that display low to moderate

receptor expression (325). However, there are challenges in studying high SA RICs for RIT in

vivo, since this requires administration of either a smaller mass or a larger radioactivity injected

dose at the same mass. A small mass may cause high liver uptake (326), decreasing tumour

uptake, while a larger radioactivity injected dose may exceed the maximum tolerated injected

dose in mice. Therefore, in the current study, we administered a mass of RICs (10 g)

complexed to 6 MBq of 177Lu that we previously found localized well in s.c. PANC-1 tumours in

NOD/SCID mice with only moderate liver uptake (312). Nonetheless, there remain practical

advantages to MCP conjugation of mAbs, including the availability of multiple chelators to

complex radiometals which minimizes interfering effects of contaminating trace metals or stable

decay products often found in commercially available radiometal solutions (327). In our

experience, MCP-conjugated mAbs provide very robust (LE>99%) and highly reliable (no

failures encounterd) radiometal complexation. These properties of MCP immunoconjugates

would provide an advantage in formulating a kit for labeling a RIT agent, as high LE (>95%) and

reliability are needed to minimize normal organ toxicities that may occur with unbound

radionuclides present even at small amounts. Finally, a mAb may be modified at a single site

with a MCP that presents multiple chelators, thus potentially avoiding decreased

immunoreactivity due to derivatization of the mAb with multiple chelators at many different

sites (328). Our studies described here provide to our knowledge the first report of RIT of

tumours in mice with panitumumab conjugated to a MCP for complexing the -emitter, 177Lu.

These studies extend the theranostic application from our previous report (312) in which we

129

demonstrated that panitumumab-MCP-111In imaged s.c. PANC-1 tumours in non-obese diabetic

severe combined immunodeficiency (NOD/SCID) mice by microSPECT/CT.

Panitumumab was linked to two MCP by reaction of HyNIC-modified polymers with an S-4FB

moiety introduced into the mAb (312). Panitumumab-MCP labeled with 111In exhibited high

affinity EGFR binding (KD = 2.2 ± 0.6 nmol/L) and was stable in vitro to loss of radiometal after

challenge with EDTA or in human plasma (312). In the current study, we determined the

cytotoxicity in vitro of panitumumab-MCP-177Lu on PANC-1 cells by clonogenic survival (CS)

assays and assessed lethal DNA DSBs inflicted by the emission of -particles by 177Lu probing

for -H2AX (315). Panitumumab-MCP-177Lu decreased the CS of PANC-1 cells by 9.0-fold at

the highest amount tested (1.2 MBq; Figure 3-1a) which was associated an increase in DNA

DSBs (Figure 3-1b,c). The lowest activity of panitumumab-177Lu tested (0.3 MBq) decreased the

CS of PANC-1 cells by 1.4-fold (Figure 3-1a), but there was no increase in DNA DSBs at this

amount compared to cells exposed to unlabeled panitumumab-MCP or untreated cells (Figure 3-

1b,c). Nuclear DNA is believed to be the principal target for radiation induced lethal damage, but

the cell membrane has also been shown to be an important target (213). Moreover, DNA DSBs

are repairable, and it is possible that these were inflicted in PANC-1 cells but were repaired prior

to probing for -H2AX at 16 h after exposure to this low amount of panitumumab-MCP-177Lu.

At higher amounts, the DNA repair processes are likely insufficient to repair all DNA DSBs.

Unlabeled panitumumab-MCP exhibited no toxicity on PANC-1 cells, indicating that the

polymer was not cytotoxic. Unlabeled panitumumab is not expected to be cytotoxic to PANC-1

cells, since these cells have a KRAS mutation that results in constitutive downstream EGFR

activation not blocked by panitumumab (329).

Binding of panitumumab-MCP-177Lu to PANC-1 cells was reduced by 11.8-fold in the presence

of an excess of unlabeled panitumumab and the binding of non-specific hIgG-MCP-177Lu to

PANC-1 cells was 3.5-fold lower than panitumumab-MCP-177Lu (Figure 3-2a). These results

indicate EGFR-specific cell binding. The absorbed dose in the nucleus of PANC-1 cells by

panitumumab-MCP-177Lu was estimated by measuring the cumulative radioactivity (Ãs) on the

cell surface, in the cytoplasm or in the nucleus source compartments (Figure 3-2b and Table 3-1)

and then applying microdosimetry models. A total of almost 4 Gy was deposited in the nucleus

of PANC-1 cells at the highest amount of radioactivity studied (1.2 MBq; Table 3-1). The S-

130

value for cell-bound 177Lu in the cytoplasm (4.02 10-4 Gy/Bq sec) was slightly greater than

for 177Lu on the cell surface (3.55 10-4 Gy/Bq sec) but the S-value was highest for 177Lu in

the nucleus (6.93 10-4 Gy/Bq sec). 177Lu emits moderate energy -particles (Emax = 0.18-0.5

MeV) that have a maximum range of ~2 mm, which accounts for the comparable S-values for

177Lu on the cell surface or in the cytoplasm. However, there is a distribution of energies with

many -particles exhibiting lower energy than the Emax (330). Low energy -particles have a

shorter range and would be more efficient for depositing dose in the nucleus when 177Lu is in the

nucleus, than when it decays occur on the cell surface or in the cytoplasm, explaining the higher

S-values for nuclear 177Lu. Approximately 25% of the absorbed dose deposited in the nucleus of

PANC-1 cells was due to unbound panitumumab-MCP-177Lu in the culture medium during the

16 h incubation period. This cross-fire effect is expected due to the 2 mm range -particles

emitted by 177Lu.

For RIT studies, we confirmed that an administered amount of panitumumab-MCP-177Lu (6.0

MBq; 10 g) previously found to cause no major normal tissue toxicity for RIT using other

177Lu-labeled mAbs in NRG mice (318), would be safe to administer to NRG mice for RIT of

PANC-1 tumours. NRG mice were selected for RIT because PANC-1 tumours efficiently engraft

into these mice, but unlike NOD/SCID mice, these mice do not harbor a germ-line defect in

DNA repair that renders NOD/SCID mice unusually radiation sensitive, including to RIT (319,

331). No decreases in CBC or increases in serum ALT or Cr compared to mice receiving normal

saline were noted (Figure 3-3) indicating no hematopoietic, liver or kidney toxicity at these

amounts. There was also no decrease in body weight in mice treated with panitumumab or with

non-specific IgG RICs (Figure 3-4a), indicating no generalized normal tissue toxicity. These

results are similar to previous studies by our group in which 3.7-11.1 MBq of 177Lu-labeled RICs

bispecific for HER2 and EGFR were safely administered to non-tumour bearing Balb/c mice

without normal tissue toxicity (332). Dose-limiting bone marrow toxicity has been reported at

higher administered amounts in humans for 177Lu-labeled mAbs (333). In one study, the

maximum tolerated dose (MTD) of 177Lu-labeled J591 for RIT of prostate cancer patients was

2,590 MBq/m2, but two or three fractionated doses of 1,110-2220 MBq/m2 were safely

administered over 4-6 months (334). Assuming a body surface area of 0.009 m2 for a mouse

(334), the amount of panitumumab-MCP-177Lu administered to NRG mice in our study was 667

131

MBq/m2, which is lower than these amounts administered to humans. However, a challenge in

assessing the toxicity of panitumumab-MCP-177Lu in mice is that panitumumab does not bind the

murine EGFR homologue, thus the absence of normal tissue toxicity from panitumumab-MCP-

177Lu in NRG mice may not preclude EGFR-mediated toxicity in humans. EGFR are present at

low levels on most epithelial tissues but are moderately expressed by the liver and kidneys (335,

336). Importantly however is that <3% of hematopoietic stem cells in the bone marrow, which is

one of the most radiation sensitive tissues are EGFR-positive (337). A Phase II trial of RIT with

anti-EGFR mAb 425 labeled with the low energy, subcellular range Auger electron emitter, 125I

improved survival in patients with glioblastoma multiforme without major (Grade 3 or 4)

toxicities including to EGFR-positive tissues (e.g. bowel or skin) after administration of 3

weekly doses of 1,800 MBq (~26 MBq/kg) (338). Nonetheless, it remains possible that the more

energetic and longer range -particles emitted by 177Lu may cause toxicity, which would need to

be evaluated in a Phase I clinical trial.

RIT with a single administered dose of panitumumab-MCP-177Lu (6 MBq; 10 g) strongly

inhibited the growth of PANC-1 tumours in NRG mice for up to 26 days before tumour growth

resumed (Figure 3-4b). In contrast, tumours in mice treated with unlabeled panitumumab or

normal saline showed rapid exponential growth at all times. As mentioned, PANC-1 cells have a

KRAS mutation which prevents panitumumab from inhibiting the growth of these tumours

(329). hIgG-MCP-177Lu also slowed the growth of PANC-1 tumours, but these non-specific

RICs were significantly less effective than panitumumab-MCP-177Lu. We did not study repeated

treatment with panitumumab-MCP-177Lu, but it is possible that tumour regrowth could be further

inhibited by administering a second dose. In addition, it may be feasible to increase the

administered dose to the maximum tolerated dose (MTD) to improve tumour response rather

than the No Observable Adverse Effect Level (NOAEL) dose used in this study. Nonetheless,

our results reveal that RIT with panitumumab-MCP-177Lu was able to overcome KRAS mutation

in PANC-1 tumours which caused resistance to panitumumab, suggesting that RIT could be a

promising approach to treatment of PnCa in humans (308). Furthermore, treatment of small

volume residual disease, including disseminated tumours to prevent metastatic progression after

surgical resection of the primary tumour could be the future of pancreatic cancer treatment using

RIT. We plan to conduct studies in an orthotopic patient derived xenograft (PDX) model of

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pancreatic cancer that metastasizes to the liver to investigate the role of RIT in this context (339).

We previously used this PDX model to study positron emission tomography (PET) of pancreatic

cancer with 64Cu-DOTA-panitumumab F(ab)2 fragments (143).

Finally, we estimated the absorbed doses in PANC-1 tumours and normal organs in mice using

biodistribution data (Figure 3-5) from administration of panitumumab-MCP-177Lu (Table 3-3).

The tumour dose was high (12.3 Gy) in agreement with the strong tumour growth inhibition

caused by panitumumab-MCP-177Lu. The highest normal doses were received by the kidneys

(15.7 Gy), pancreas (19.3 Gy), spleen (14.8 Gy) and liver (7.5 Gy). The MCP conjugated to

panitumumab were PEGylated but liver and spleen uptake remains a challenge for polymer

RICs. In other studies, we found that zwitterionic MCP conjugated to panitumumab provided

lower liver and spleen uptake than polyanionic MCP used in the current study (270).

Nonetheless, the absence of hepatic and renal toxicity (Figure 3-3) is consistent with the doses

deposited in the liver and kidneys and the tolerance of these tissues to radiation, with doses >30

Gy most likely to cause with liver toxicity and >23-25 Gy to cause renal toxicity in humans

(340). No bone marrow toxicity was found for panitumumab-MCP-177Lu (Figure 3-3) despite a

whole body absorbed dose of 1.4 Gy (Table 3-3). Whole body dose has been used as a predictor

of bone marrow toxicity from RIT with doses >2-3 Gy causing dose-limiting hematopoietic

toxicity in humans (333, 341). Mouse S-values were used to estimate the normal organ doses in

mice from panitumumab-MCP-177Lu (323). Due to the close proximity of organs in the mouse

and the maximum 2 mm range of the -particles emitted by 177Lu, the doses to normal organs in

NRG mice may overestimate the human doses, due to a significant cross-fire effect. Bitar et al.

point out that for energies >0.5 MeV, -particles cannot be considered as non-penetrating

radiation in mice, i.e. they have a significant cross-fire effect (323). This cross-fire effect is noted

by the main contributing source organ for the dose to the whole body being the liver (Table 3-3).

3.6 Conclusion

We conclude that panitumumab-MCP-177Lu inflicted DNA DSBs in PANC-1 human PnCa cells

in vitro that decreased the CS of these cells. Administration of 6 MBq (10 g) of panitumumab-

MCP-177Lu caused no normal tissue toxicity in NRG mice but strongly inhibited the growth of

s.c. PANC-1 tumours for up to 26 days. Unlabeled panitumumab was not effective for treating

133

PANC-1 tumours due to KRAS mutation. Our results demonstrate that RIT with panitumumab-

MCP-177Lu was able to overcome resistance to panitumumab caused by KRAS mutation in

PANC-1 tumours, suggesting that RIT may be a promising approach to the treatment of PnCa in

humans.

3.7 Acknowledgements

This work was supported by a grant from the Canadian Cancer Society and a grant from the

Cancer Research Society. S. Aghevlian received scholarships from the STARS21 strategic

training program in radiation research supported by the Terry Fox Foundation and the Centre for

Pharmaceutical Oncology (CPO) at the University of Toronto. S. Aghevlian is supported by the

Natural Sciences and Engineering Research Council of Canada (NSERC) through the Polymer

Nanoparticles for Drug Delivery (POND) Training Program. The authors would like to thank

Deborah Scollard at the Spatiotemporal Targeting and Amplification of Radiation Response

(STTARR) Innovation Centre for technical support.

134

Chapter 4: Comparison of Auger Electron-Emitting 111In- or -

Particle-Emitting 177Lu Complexed to Panitumumab for

Radioimmunotherapy of EGFR-Positive PANC-1 Human

Pancreatic Cancer Xenografts in NOD/SCID and NRG Mice

135

This chapter will be submitted to Molecular Pharmaceutics. All experiments were designed and

carried out by Sadaf Aghevlian. Dosimetry was performed with assistance from Dr. Zhongli Cai.

The synthesis and characterization of metal chelating polymer were performed by Dr. Yijie Lu at

the Department of Chemistry at the University of Toronto.

136

4. Abstract

Our hypothesis was that panitumumab modified with two DOTA chelators or with metal-

chelating polymers (MCP) that present 13 DOTA chelators to complex the -particle emitter,

177Lu or Auger electron (AE)-emitter, 111In, would be effective for radioimmunotherapy (RIT) of

subcutaneous (s.c.) EGFR-positive PANC-1 human PnCa xenografts in mice at administered

amounts that are non-toxic to normal tissues. The clonogenic survival (CS) of PANC-1 cells

exposed in vitro to panitumumab-DOTA-177Lu, panitumumab-DOTA-111In or panitumumab-

MCP-111In was determined. DNA double-strand breaks (DSBs) were assessed by

immunofluorescence for -H2AX. Absorbed doses to the cell nucleus was calculated based on

subcellular distribution of the radioimmunoconjugates (RICs). Normal tissue toxicity in vivo was

assessed by monitoring body weight, complete blood cell (CBC) counts and serum alanine

aminotransferase (ALT) and creatinine (Cr) in NOD/SCID mice injected i.v. with 10 MBq (10

g) of panitumumab-MCP-111In or panitumumab-DOTA-111In, or in NRG mice administered 6

MBq (10 g) of panitumumab-DOTA-177Lu. For RIT, NOD/SCID mice or NRG mice with s.c.

PANC-1 tumours were administered three amounts (10 MBq; 10 g) of panitumumab-MCP-

111In or panitumumab-DOTA-111In separated by 3 weeks or a single amount (6 MBq; 10 g) of

panitumumab-DOTA-177Lu, respectively. Control mice were treated with normal saline or

unlabeled panitumumab. Tumour growth was assessed by a tumour growth index (TGI).

Biodistribution studies were performed to calculate the cumulative radioactivity (Ãs) in normal

organs and tumour. The absorbed doses were estimated using D= Ãs×S equation and mouse S-

values. The tumour dose was estimated using OLINDA/EXM and the sphere model.

Panitumumab-DOTA-177Lu was 5-fold and 6.4-fold more cytotoxic than panitumumab-MCP-

111In or panitumumab-DOTA-111In, respectively, against PANC-1 cells which was associated

with more DNA DSBs and higher absorbed doses to the cell nucleus. There were no differences

in the cytotoxicity in vitro of panitumumab-MCP-111In and panitumumab-DOTA-111In. None of

the RICs caused any significant changes in body weight, decreased CBC or increases in ALT or

Cr compared to normal saline treated mice. Panitumumab-MCP-111In or panitumumab-DOTA-

111In inhibited PANC-1 tumour growth in NOD/SCID mice (TGI at 43 days = 3.9 ± 0.3 and 3.0 ±

0.4, respectively; P> 0.05) compared to normal saline and panitumumab treated mice (TGI = 9.8

± 1.6, 9.9 ± 1.4, respectively; P<0.001). Similarly, panitumumab-DOTA-177Lu inhibited tumour

137

growth in NRG mice (TGI = 2.9 ± 0.4). Absorbed doses in PANC-1 tumours were 11.5 ± 4.9 Gy

for panitumumab-DOTA-177Lu and 2.8 ± 0.7 Gy and 6.0 ± 2.2 Gy for panitumumab-MCP-111In

and panitumumab-DOTA-111In; respectively (P>0.05). The spleen and liver uptake were higher

for the MCP conjugates which makes these less attractive for RIT with 111In than the DOTA

conjugates. Generally, lower absorbed doses in normal organs for 111In than 177Lu may suggest

further escalation of the administered amount of 111In, while the whole body dose for 177Lu

doesn’t allow further escalation in the administered amount because of potential bone marrow

toxicity.

138

4.1 Introduction

In recent years there has been an increasing interest in targeting cancers overexpressing

epidermal growth factor receptor (EGFR) using the fully humanized anti-EGFR monoclonal

antibody (mAb), panitumumab (Vectibix, Amgen) (143, 286, 314, 342). Although pancreatic

cancer (PnCa) (274) overexpresses EGFR, it has the highest incidence of KRAS mutation among

all the EGFR positive cancers (309, 310) and cannot benefit from immunotherapy with

panitumumab due to constitutive KRAS-mediated downstream signal activation (311). In this

case, radioimmunotherapy (RIT) which exploits panitumumab only as a carrier for delivery of

radioisotopes to tumours could be the best treatment approach. We recently proposed a novel

metal chelating polymer (MCP) composed of a polyglutamide backbone and 13 DOTA

(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators for radiolabeling

panitumumab at high specific activity (SA) and presenting 10 pendant polyethylene glycol

(PEG) groups in order to reduce spleen and liver uptake of the radioimmunoconjugates (RICs)

(257). We showed that single intravenous (i.v.) injection (6 MBq; 10 μg) of panitumumab-MCP

labeled with 177Lu [β particles; Eβ(max)=498 keV (78.6%), Eβ(max)=385 keV (9.1%) and

Eβ(max)=176 keV (12.2%); t1/2 = 6.7 days], delayed the tumour growth of subcutaneous (s.c.)

PANC-1 human PnCa xenografts in NODRag1−/−IL2RgammaC-null (NRG) mice by about 2.6-

fold compared to control mice treated with normal saline or unlabeled panitumumab (343). In a

separate study, we showed that panitumumab-MCP and panitumumab-DOTA labeled with the γ-

emitter 111In [Eγ = 171 keV (90%) and Eγ = 245 keV (94%); (t1/2 = 2.8 d)] were able to image

s.c. PANC-1 xenografts in nonobese diabetic severe combined immunodeficiency (NOD-SCID)

mice at 72 h post injection (p.i.) by single photon emission computed tomography (SPECT) with

a tumour uptake of 6.9 ± 1.3 % injected dose per gram (%ID/g) and 6.6 ± 3.3 %ID/g;

respectively (257). 111In also emits 15 low energy (<25 keV) and nanometer-micrometer range

Auger electrons (AEs) that are well suited for eradicating micrometastases since the radiation

dose is deposited within the target volume of a single cell (200). The subcellular range of these

electrons yields high linear energy transfer (LET = 4-26 keV) that causes lethal DNA double-

strand breaks (DSBs), particularly if these electrons are emitted near the cell nucleus (191). RIT

with 111In-labeled mAbs has proven effective for treatment of tumour xenografts for a range of

cancers in mice (332, 344, 345). The potency of AEs can be enhanced by modification of the

mAbs with nuclear translocation sequences (NLS) to promote nuclear uptake after receptor-

139

mediated internalization (346, 347). However, since the EGFR harbours a putative NLS in the

transmembrane domain (348) that transports EGF (349) and anti-EGFR mAbs (350) to the

nucleus of cancer cells following receptor mediated internalization, there is no need to modify

panitumumab with NLS peptides to enhance the effectiveness of the AEs emitted by 111In (286,

343).

In the current study, we compared the 2 mm range β-particles emitted by 177Lu and the

nanometer range AEs emitted by 111In for killing PANC-1 pancreatic cancer cells in vitro, and

for treatment of PANC-1 xenografts in vivo in NRG mice and NOD/SCID mice. We also now

report the therapeutic efficacy of panitumumab-MCP-111In for comparison with our previously

reported results using panitumumab-MCP-177Lu (343). Our aim was to understand whether or

not the β-particles emitted by 177Lu or AEs emitted by 111In would be the most effective for RIT

of PANC-1 tumours in mice at administered amounts that were non-toxic to normal tissues. We

compared the normal tissue toxicity of panitumumab-DOTA-111In, panitumumab-DOTA-177Lu

and panitumumab-MCP-111In.

4.2 Materials and methods

4.2.1 Cell culture and tumour xenografts

EGFR positive PANC-1 human pancreatic adenocarcinoma cells (4.0 × 105 EGFR/ cell) (313)

were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and

cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (Gibco Life

Technologies; Burlington, ON, Canada) supplemented with 1% penicillin and streptomycin and

10% fetal bovine serum (Gibco-Invitrogen). Tumour xenografts (7-9 mm diameter) were

established in female NOD/SCID mice (Charles River) or in NRG mice (University Health

Network) by s.c. inoculation of 3-4 × 106 PANC-1 cells in serum free DMEM (100 μL) into the

left flank. All animal studies were conducted in compliance with Canadian Council on Animal

Care (CCAC) guidelines and were carried out under a protocol approved by the Animal Care

Committee at the University Health Network (AUP 2843.3).

140

4.2.2 Radioimmunoconjugates (RICs)

The synthesis of hydrazino nicotinamide (HyNic) end-capped MCPs (HyNic-MCP) presenting

13 DOTA chelators and 10 PEG chains was previously reported (271). Panitumumab-MCP

immunoconjugates were constructed by cross-linking panitumumab IgG to on average 2 HyNic-

MCPs through an N-succinimidyl-4-formylbenzamide (S-4FB) moiety introduced into

panitumumab (257). Panitumumab or non-specific IgG (hIgG; Sigma-Aldrich, St. Louis, MO,

Product No. 14506) was also modified with on average 2 N-hydroxysuccinimide ester (DOTA-

NHS; Macrocyclics) to construct panitumumab-DOTA conjugates (257). Unconjugated MCPs or

DOTA-NHS were removed from panitumumab-MCP or panitumumab-DOTA and buffer-

exchanged for subsequent radiolabeling by diluting in 0.1 M 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) buffer pH 5.0, and passing through an Amicon spin filter

(0.5 mL; 30 kDa MWCO; Millipore). Purified immunoconjugates (5−70 μg; 4−12 μL) were then

labeled with (0.75−49 MBq) 111InCl3 (Nordion) or 177LuCl3 (PerkinElmer) at 42 °C for 2 h (257).

The final radiochemical purity of RICs was >95% evaluated by instant thin-layer silica gel

chromatography (ITLC-SG; Pall) developed in 0.1M sodium citrate, pH 5.5. The Kd for binding

of panitumumab-MCP-177Lu and panitumumab-DOTA-177Lu to EGFR was 2.2 ± 0.6 nmol/L and

1.0 ± 0.4 nmol/L; respectively (257). The Kd values of the 111In-labeled RICs was not measured

as it was assumed that these would not be changed by complexation of a different radiometal.

4.2.3 Clonogenic survival (CS)

The cytotoxicity of panitumumab-DOTA-177Lu, panitumumab-DOTA-111In and panitumumab-

MCP-111In on PANC-1 cells were compared in clonogenic survival (CS) assays. Briefly, 2 × 105

cells were treated with growth medium containing 2.5 nmoles/L (50-fold molar excess) of RICs:

0.3, 0.6 or 1.2 MBq [specific activity (SA) = 46.7, 93.5, 187.0 MBq/nmole] in 24 well plates for

16 h. Controls consisted of cells exposed to unlabeled panitumumab-DOTA or growth medium

alone. Approximately 700 cells were then seeded into 6 well plates and cultured for 12 d.

Surviving colonies were stained with methylene blue and colonies of more than 50 cells were

counted. The plating efficiency (PE) was determined by dividing the number of colonies formed

by the number of cells seeded. The CS was calculated by dividing the PE for treated cells by that

for untreated cells.

141

4.2.4 Assessment of DNA damage

The accumulation of phosphorylated histone-2A (γ-H2AX) in the nucleus of PANC-1 cells due

to DNA double-stranded breaks (DSBs) caused by panitumumab-DOTA-177Lu, panitumumab-

DOTA-111In and panitumumab-MCP-111In was assessed by immunofluorescence confocal

microscopy (315). Briefly, 2× 105 cells were seeded onto glass coverslips (Ted Pella) in 24-well

plates and cultured overnight at 37 °C. The cells were then incubated with the RICs or unlabeled

panitumumab-DOTA or growth medium as described earlier. Immunostaining for γ-H2AX and

analysis of the images for the density of foci per nucleus area was conducted according to

previously reported procedures (316).

4.2.5 Subcellular distribution and cellular dosimetry

The subcellular distribution of panitumumab-DOTA-177Lu in PANC-1 cells was measured as

previously described for panitumumab-MCP-177Lu and non-specific hIgG-MCP-177Lu (343).

Briefly, 2 × 105 cells were cultured overnight in wells in a 6-well plate. Cells were then

incubated with a 50-fold molar excess of panitumumab-DOTA-177Lu (1.2 MBq; 2.5 nmoles/L) in

the absence or presence of excess unlabeled panitumumab (EGFR blocking; control group) or

with non-specific IgG-DOTA-177Lu (control group) for different times (1, 4, 8, and 24 h) at 37

°C. The medium was removed and the cells rinsed with phosphate-buffered saline (PBS), pH 7.5.

Cell-surface radioactivity was displaced with 1 mL of 200 mM sodium acetate/500 mM NaCl,

pH 2.5 at 22 ᵒC. This procedure was repeated twice and the combined cell membrane fractions

measured in a γ-counter. In order to determine the proportion of cell-bound radioactivity

internalized in the nucleus or remaining in cytoplasm, the cell membrane was lysed twice in 500

μL of Nuclei EZ Lysis buffer (Sigma-Aldrich) on ice for 60 min. The cells were gently detached

from each well using a cell scraper and were transferred to 1.5 mL Eppendorf tubes. The tubes

were centrifuged at 3,000 × g for 5 min to separate nuclear and cytoplasmic radioactivity

(supernatant). The fractions of nuclear and cytoplasmic radioactivity were measured separately

in a γ-counter and the percentage of radioactivity in each fraction (relative to total cell-bound

177Lu) was calculated.

142

The absorbed dose in the nucleus of a PANC-1 cell was determined as previously described for

panitumumab-MCP-177Lu and non-specific hIgG-MCP-177Lu (343). Briefly, the cumulative

amount of radioactivity (As) in the nucleus (D) after exposure to 1.2 MBq of panitumumab-

DOTA-177Lu (2.5 nmole/L) was estimated by plotting the amount of radioactivity in the nucleus

vs. time and determining the area under the curve (AUC0−24h; Bq × sec) using Prism Ver. 4.0

software (GraphPad). The absorbed dose in the nucleus was then estimated as D = As × S, and

the S-values (Gy/Bq × sec) were calculated using MCNP Monte Carlo code Ver. 5 (Los Alamos

National Laboratory). Following staining of the nucleus with 4′,6-diamidino-2-phenylindole

(DAPI), the mean cell radius and nucleus radius of PANC-1 cells were determined (17.1 μm and

7.0 μm; respectively) by fluorescence microscopy of cells and ImageJ software (National

Institutes of Health, Bethesda, MD) (221). The absorbed doses in the nucleus were also

calculated for the control groups. The absorbed doses in the nucleus from the corresponding

panitumumab-DOTA-111In and hIgG-DOTA-111In as well as panitumumab-MCP-111In and hIgG-

MCP-111In were estimated assuming that these would exhibit similar subcellular distribution as

panitumumab-DOTA-177Lu and panitumumab-MCP-177Lu (343). The t1/2 of 111In (2.8 d) was

employed to estimate As, and the S-factors for 111In were used to calculate these radiation doses

(323).

4.2.6 Evaluation of normal tissue toxicity

To evaluate normal tissue toxicity, groups of 5 healthy female NOD/SCID or NRG mice were

injected i.v. with an amount of panitumumab-DOTA-111In or panitumumab-MCP-111In (10.0

MBq; 10 μg) previously reported to be safe in other preclinical RIT studies (351). Similarly, an

amount of panitumumab-DOTA-177Lu (6.0 MBq; 10 μg) previously found to be safe was studied

(318, 343). NOD/SCID mice harbor a germ line mutation in DNA repair (319) which renders

these mice unusually sensitive to radiation. Thus the toxicity of panitumumab-DOTA-177Lu was

examined in NRG mice which do not have this mutation since it was anticipated that the β-

particles emitted by 177Lu may be more toxic to normal tissues than the AEs emitted by 111In, due

to an associated cross-fire effect (191). Control mice received injections of normal saline or

unlabeled panitumumab (10 μg). Body weight was monitored every 2-4 d for 14 d. The mice

were then sacrificed and samples of blood collected into EDTA-coated microtubes for

biochemistry [serum alanine aminotransferase (ALT) and creatinine (Cr)] and hematology

143

analyses. A complete blood cell (CBC) count, hematocrit (HCT) and hemoglobin (Hb) were

measured on a HemaVet 950FS (Drew Scientific) instrument.

4.2.7 Radioimmunotherapy (RIT) studies

The tumour-growth inhibitory effects of the RICs were studied in mice engrafted s.c. with

PANC-1 xenografts. NOD/SCID mice received three i.v. injections of 10 MBq of panitumumab-

DOTA-111In, panitumumab-MCP-111In or hIgG-MCP-111In (10μg/100 µL) every three weeks

whereas due to the longer half-life of 177Lu NRG mice received a single injection of 6 MBq of

panitumumab-DOTA-177Lu (10μg/100µL). Tumour length (mm) and width (mm) were measured

using calipers every 2-3 days until a humane endpoint of the animal care protocol was reached.

The tumour volume (V=mm3) was calculated as V=length×width2×0.5 (321). The tumour growth

index (TGI) was calculated by dividing the tumour volume at each time point by the tumour

volume at the start of treatment. Similarly, the body weight index (BWI) was estimated by

dividing the body weight at each time point by the pre-treatment body weight. The results were

compared to the control groups received saline (100µL) or unlabeled panitumumab

(10µg/100µL).

4.2.8 Tumour and normal organ dosimetry

The absorbed doses in the tumour and normal organs in NOD/SCID or NRG mice with s.c.

PANC-1 xenografts after injection of three amounts of 111In-labeled panitumumab-DOTA or

panitumumab-MCP (10 MBq; 10 μg) or one amount of panitumumab-DOTA-177Lu (6 MBq; 10

μg) were estimated as described previously (343). Briefly, groups of 5 NOD/SCID or NRG mice

were injected i.v. with one amount of these RICs and at selected time points from 24 to 168 h

p.i., the mice were sacrificed and the tumour and samples of normal organs collected and the

radioactivity measured in a -counter. The cumulative radioactivity in the tumour and these

normal source organs from 0-168 h (Ã0-168h) was estimated from the area under the curve [AUC0-

168h; Bq ×sec] using Prism Ver. 4.0 software (GraphPad). The cumulative radioactivity from 168

h to infinity (Ã168h-∞) was calculated by assuming elimination by physical decay without further

biological clearance. By multiplying the combined Ã0-∞ values by the S-value (Gy/Bq × sec) for

111In or 177Lu using published S-values for a mouse (323), the absorbed dose (Gy) for each organ

144

was estimated. The dose delivered to the tumour was estimated based on the S-values using the

sphere model in OLINDA/EXM radiation dose software, and the measured tumour size (324).

The absorbed dose for 111In-labeled RICs were estimated for three injections.

4.3 Statistical analysis

Results were expressed as mean ± SEM. Statistical significance was tested using an unpaired

two-tailed Student’s t-test or one-way ANOVA (p<0.05).

4.4 Results

4.4.1 Clonogenic survival (CS)

There was no effect of unlabeled panitumumab-DOTA on the CS of PANC-1 cells (Figure. 4-1).

There were no significant differences in the CS of PANC-1 cells exposed to panitumumab-MCP-

111In or panitumumab-DOTA-111In at 0.3 MBq (67.92 ± 4.35 vs 83.01 ± 5.44; P>0.5), 0.6 MBq

(64.91 ± 5.75 vs 71.48 ± 4.01) or 1.2 MBq (53.85 ± 6.85 vs 68.86 ± 3.34; P>0.5). However, at all

radioactivity amounts, 177Lu was more cytotoxic than 111In. At the highest amount of

radioactivity tested (1.2 MBq) panitumumab-DOTA-177Lu (10.69 ± 2.45) was 5-fold and 6.4-

fold more potent at reducing the CS of PANC-1 cells than panitumumab-MCP-111In or

panitumumab-DOTA-111In, respectively.

145

0.0 0.3 0.6 1.20

20

40

60

80

100

120

Panitumumab-MCP-111In

Panitumumab-DOTA-111In

Panitumumab-DOTA-177Lu

**

******

*** ***

***

Radioactivity (MBq)

Clo

no

ge

nic

Su

rviv

al (%

)

Figure 4-1. Clonogenic survival of PANC-1 cells exposed to increasing radioactivity (0.3 MBq,

0.6 MBq or 1.2 MBq) of panitumumab-DOTA-177Lu, panitumumab-MCP-111In or panitumumab-

DOTA-111In (2.5 nmoles/L) or to unlabeled panitumumab-DOTA (2.5 nmoles/L; 0.0 MBq) for

16 h then cultured for 12 d. Data shown are the mean ± SEM (n = 3–4). Significant differences

(P<0.05) between 111In- and 177Lu- labeled RICs are indicated by the asterisks. There are no

significant differences between panitumumab-MCP-111In and panitumumab-DOTA-111In.

146

4.4.2 Assessment of DNA damage

There were no differences in the integrated density of γ-H2AX foci per nucleus area in the

nucleus of cells exposed to growth medium and unlabeled panitumumab-DOTA (0.69 ± 0.05%

vs 0.76 ± 0.10% ; P > 0.05) (Figure 4-2A,B). At all radioactivity amounts (0.3, 0.6 and 1.2

MBq; 2.5 nmoles/L), panitumumab-DOTA-177Lu caused more DNA DSBs than panitumumab-

MCP-111In or panitumumab-DOTA-111In. At 1.2 MBq, panitumumab-DOTA-177Lu caused 2.7-

fold and 1.6-fold higher γ-H2AX foci than 111In-labeled panitumumab-DOTA or panitumumab-

MCP (3.1 ± 0.3% vs 1.1 ± 0.2% and 2.0 ± 0.2%). Exposure of cells to increasing amounts of

panitumumab-DOTA-177Lu increased γ-H2AX foci. Panitumumab-DOTA-111In at 1.2 MBq

caused more DNA DSBs than at 0.3 MBq but not at 0.6 MBq (Figure 4-2B).

147

Figure 4-2. (A) Representative confocal immunofluorescence microscopy images assessing γ-

H2AX foci (green foci) representing sites of unrepaired DNA DSBs in PANC-1 cells exposed

for 16 h to 0.3 MBq, 0.6 MBq or 1.2 MBq of 177Lu- labeled panitumumab-DOTA or 111In-

labeled panitumumab-DOTA or panitumumab-MCP. Cell nuclei were counterstained with DAPI

(blue). In the bottom set of panels are images of PANC-1 cells exposed to growth medium alone,

or unlabeled panitumumab-DOTA. (B) γ-H2AX foci were quantified as the integrated

density/nucleus area and are shown for untreated PANC-1 cells, cells treated with unlabeled

panitumumab-DOTA, panitumumab-MCP (343) or increasing amounts of RICs. Values shown

represent the mean ± SEM (n=3-4). Significant differences (P<0.05) between the RICs are

indicated by the asterisks.

148

4.4.3 Subcellular distribution and cellular dosimetry

The percentage of radioactivity bound to PANC-1 cells increased over time for all RICs. The

percentage of cell-bound radioactivity for panitumumab-DOTA-177Lu (11.0 ± 0.2%) was

significantly higher at 24 h after incubation compared to that for non-specific hIgG-DOTA-177Lu

(0.50 ± 0.32%; P<0.001) or panitumumab-DOTA-177Lu in the presence of 50-fold molar excess

unlabeled panitumumab (0.80 ± 0.05%; P< 0.001) indicating the EGFR cell-binding specificity

of the panitumumab-DOTA conjugates (Figure 4-3A). There were no significant changes with

time in the proportion of cell-bound radioactivity on the membrane, in the cytoplasm or nucleus

for panitumumab-DOTA-177Lu (Figure 4-3B).

149

1 4 8 24 1 4 8 24 1 4 8 24

0

1

2

3

4

5

6

7

8

9

10

11

12 Panitumumab-DOTA-177Lu

Panitumumab-DOTA-177Lu+ panitumumab

hIgG-DOTA-177Lu

Time (h)

Cell

Bo

un

d (

%)

0

20

40

60

80

100Cell Membrane

Cytoplasm

Nucleus

11

1 4 8 24

4 8 244 8 24

Ce

ll B

ou

nd

(%

)

A B

Figure 4-3. (A) Percentage of cell-bound radioactivity at selected time points (1, 4, 8 or 24 h)

after incubation of PANC-1 cells with panitumumab-DOTA-177Lu with or without excess

unlabeled panitumumab or with hIgG-DOTA-177Lu. (B) Percentage of radioactivity on the

membrane, in the cytoplasm or nucleus of PANC-1 cells at selected times after incubation with

panitumumab-DOTA-177Lu.

150

The subcellular fractionation data for all the time points were used to estimate absorbed doses in

the nucleus of PANC-1 cell from 111In- or 177Lu-labeled panitumumab-DOTA assuming that

substitution of 111In for 177Lu would not change cell binding or subcellular localization (Table 4-

1 and 4-2). Similarly, the absorbed dose in the nucleus from panitumumab-MCP-111In was

calculated based on the subcellular distribution of panitumumab-MCP-177Lu which has been

published in our previous study (343). The dose deposited in the nucleus was 3.5-fold and 6.9–

fold higher for panitumumab-DOTA-177Lu than panitumumab-DOTA-111In and panitumumab-

MCP-111In, respectively (Table 4-1 and 4-2). For the cells incubated with radiolabeled hIgG-

DOTA or hIgG-MCP or co-incubated with excess unlabeled panitumumab the absorbed doses to

the nucleus were significantly lower and radioactivity in the surrounding growth medium was the

major source contributor to the dose (Table 4-1 and 4-2).

151

Table 4-1. Cumulative radioactivity in the source component (Ãs (0-∞)) based on subcellular distribution of radioactivity after

incubation of PANC-1 cells with 1.2 MBq of 111In-labeled RICs (2.5 nmoles/L) in the absence or presence of 50-fold excess unlabeled

panitumumab for 24 h at 37 ᵒC.

Dose to the cell nucleus (Gy)

Source

compartment

S (Gy/Bq×sec)

111In

Panitumumab-

DOTA

Panitumumab-

DOTA+ excess

panitumumab

hIgG-

DOTA

Panitumumab-

MCP*

Panitumumab-

MCP + excess

panitumumab

hIgG-

MCP

Cell surface 7.35 10-5 1.01 0.06 0.02 0.29 0.02 0.03

Cytoplasm 1.03 10-4 0.3 0.01 0.007 0.22 0.03 0.1

Nucleus 5.06 10-4 0.64 0.04 0.06 0.34 0.05 0.23

Medium 3.39 10-12 0.33 0.33 0.33 0.32 0.33 0.33

Total

2.28 0.44 0.42 1.17 0.43 0.69

Major source

contributor (%)

Membrane

(44.3%)

Medium

(75.0%)

Medium

(79%)

Nucleus

(29%)

Medium

(76.7%)

Medium

(47.8%)

*The subcellular distribution of 177Lu–labeled RICs was previously reported and used to estimate the dose from the corresponding 111In-labeled

RICs.

152

Table 4-2. Cumulative radioactivity in the source component (Ãs (0-∞)) based on subcellular distribution of radioactivity after

incubation of PANC-1 cells with 1.2 MBq of panitumumab-DOTA-177Lu (2.5 nmoles/L) in the absence or presence of 50-fold excess

unlabeled panitumumab for 24 h at 37 ᵒC.

Dose to the cell nucleus (Gy)

Source compartment S (Gy/Bq×sec)

Lu-177 Panitumumab-DOTA

Panitumumab-DOTA+ excess

panitumumab hIgG-DOTA

Cell surface 3.55 10-4 4.86 0.3 0.12

Cytoplasm 4.02 10-4 1.18 0.05 0.03

Nucleus 6.93 10-4 0.87 0.05 0.09

Medium 1.15 10-11 1.13 1.13 1.13

Total

8.06 1.53 1.36

Major source contributor (%) Membrane (60.3%) Medium (73.9%) Medium (83.1%)

153

4.4.4 Evaluation of normal tissue toxicity

Administration of 10.0 MBq (10g) of panitumumab-DOTA-111In and panitumumab-MCP-111In

to non-engrafted NOD/SCID mice or 6.0 MBq (10g) of panitumumab-MCP-177Lu to NRG

mice caused no decrease in white blood cell (WBC), red blood cell (RBC) counts or hemoglobin

(Hb) (Figure 4-4A-C) or in hematocrit (HCT) (Figure 4D) or platelet (PLT) counts (Figure 4-

4E) at 14 d compared to NOD/SCID mice receiving normal saline. In addition, there were no

increases in serum ALT or Cr in mice receiving panitumumab-DOTA-111In compared to normal

saline treated mice (Figure 4-4F,G). Similarly, there was no significant difference in the blood

analysis of the NRG mice administered 6.0 MBq (10 µg) of panitumumab-DOTA-177Lu

compared to the control NRG mice receiving normal saline injection which was reported in our

previous study (343). There were some mouse strain differences noted between NOD/SCID mice

or NRG mice in serum ALT or Cr in normal saline controls (343). No significant differences

were observed in the body weight between the control group and treatment groups.

154

0

2

4

6

8

10 Normal saline (NOD/SCID)

Panitumumab-DOTA-111In (10 MBq)

Panitumumab-MCP-111In (10 MBq)

Panitumumab-DOTA-177Lu (6 MBq)

A

Normal saline (NRG)

WB

C (

10

3/

L)

0

2

4

6

8

10B

RB

C (

10

6/

L)

0

5

10

15

20C

Hb

(g

/dL

)

0

10

20

30

40

50D

HC

T (%

)

0

500

1000

1500

2000EP

LT

(

10

3/

L)

0

20

40

60

80

100

120F

Cr

(M

)

0

100

200

300G

AL

T (

U/L

)

Figure 4-4. Hematology and serum biochemistry analyses of NOD/SCID mice at 14 d after i.v.

injection of 10.0 MBq (10 μg) of panitumumab-DOTA-111In, panitumumab-MCP-111In or normal

saline. Also shown is the data for NRG mice administered 6.0 MBq (10 µg) of panitumumab-

DOTA-177Lu or normal saline (taken from the previous study (343). (A) white blood cells

[WBC], (B) Red blood cells [RBC], (C) hemoglobin [Hb], (D) hematocrit [HCT], (E) platelets

[PLT], (F) serum creatinine [Cr], (G) serum alanine aminotransferase [ALT], Individual values

are shown as well as the mean (horizontal bars). No significant difference was observed between

normal saline control and treatment groups.

155

4.4.5 Radioimmunotherapy (RIT) studies

Administration of three single amounts (10.0 MBq; 10 g) of panitumumab-DOTA-111In or

panitumumab-MCP-111In separated by 3 weeks to NOD/SCID mice strongly inhibited PANC-1

tumour growth (Figure 4-5A). There was no significant difference in the mean TGI at 43 d (time

point selected to include >3 surviving mice per group) for mice treated with panitumumab-

DOTA-111In or panitumumab-MCP-111In (3.97 ± 0.3 vs. 3.0 ± 0.4, respectively; P>0.05) but the

TGI for treatment with these 111In-labeled RICs was 2.5-3.0 fold significantly lower than for

control mice receiving normal saline or unlabeled panitumumab (TGI = 9.8 ± 1.6 and 9.9 ± 1.4,

respectively; P<0.001). Administration of a single amount (6.0 MBq; 10 g) of panitumumab-

DOTA-177Lu to NRG mice also significantly inhibited PANC-1 tumour growth compared to

control groups (TGI = 2.9 ± 0.4, P<0.001) (Fig. 4-5A). The tumour growth rate of PANC-1

xenografts in NOD/SCID administered normal saline or unlabeled panitumumab (TGI at 33 days

= 6.8 ± 1.2 and 5.3 ± 0.6; respectively) in the current study was not significantly different from

that previously studied in NRG mice (TGI at 33 days = 5.8 ± 0.5 and 6.1 ± 1.2; respectively)

(343).

156

0 10 20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

1.2

Normal saline

Panitumumab

Panitumumab-DOTA-111

In

Panitumumab-MCP-111

In

Panitumumab-DOTA-177

Lu

B

Time (d)

Bo

dy W

eig

ht

Ind

ex (

BW

I)

0 10 20 30 40 50 60 700

5

10

15

20

Normal saline

Panitumumab

Panitumumab-DOTA-111

In

Panitumumab-MCP-111

In

Panitumumab-DOTA-177

Lu

A

Time (d)

Tu

mo

r G

row

th In

de

x (

TG

I)

Figure 4-5. (A) Tumour growth index (TGI) in NOD/SCID mice with s.c. PANC-1 xenografts

receiving three amounts (10.0 MBq; 10 µg) of panitumumab-DOTA-111In, panitumumab-MCP-

111In, unlabeled panitumumab (10 µg) or normal saline separated by 3 weeks or in NRG mice

with s.c. PANC-1 human PnCa xenografts receiving a single injection of panitumumab-DOTA-

177Lu (6.0 MBq; 10 µg). (B) Body weight index (BWI) in the same treatment and control groups.

Values are mean ± SEM (n = 7-11). TGI at 43 d was significantly different (P<0.05) for 111In-

and 177Lu-labeled panitumumab RICs compared to normal saline or unlabeled panitumumab

controls.

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4.4.6 Tumour and normal organ dosimetry

Absorbed doses in the tumour and normal organs in NOD/SCID mice with s.c. PANC-1 tumours

after i.v. injection of 10 MBq (10 μg) of panitumumab-DOTA-111In, panitumumab-MCP-111In or

in NRG mice administered with 6 MBq (10 μg) of panitumumab-DOTA-177Lu are shown in

Table 4-2. The normal organ doses were estimated based on the cumulative radioactivity in

source organs determined in biodistribution studies (Figure 4-6) and using mouse organ S-values

(323). The tumour doses were calculated using the sphere model in OLINDA/EXM (352) and

the measured tumour size. There was no significant difference between the tumour dose for

panitumumab-MCP-111In and panitumumab-DOTA-111In (2.8 ± 0.7 Gy vs 6.0 ± 2.2 Gy,

respectively; P>0.05). However, for a single injection of 177Lu-labeled panitumumab-DOTA the

absorbed doses in the tumour (11.5 ± 4.9 Gy) and normal organs were significantly higher

compared to three injections of panitumumab-MCP-111In or panitumumab-DOTA-111In. The

absorbed dose in the whole body from panitumumab-DOTA-177Lu was 4.6-fold and 5.6–fold

higher for panitumumab-MCP-111In and panitumumab-DOTA-111In, respectively. The

accumulated radioactivity over time in the tumour and normal organs was lower for the 111In-

labeled RICs compared to 177Lu-labeled panitumumab-DOTA (Figure 4-6) leading to lower

absorbed doses in the tumour and normal organs. The highest normal organ absorbed doses from

all the RICs were deposited in the kidneys and spleen followed by the pancreas and liver (Table

4-2). Due to the higher liver and spleen uptake, the absorbed doses in these organs from

panitumumab-MCP-111In was 2.3–fold and 1.9-fold significantly higher compared to

panitumumab-DOTA-111In (10.3 ± 0.6 Gy vs 4.6± 0.3 Gy; P<0.01 and 19.5 ± 3.8 Gy vs 10.3 ±

3.6 Gy; P<0.05 respectively).

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0 24 48 72 96 120 144 168 1920.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0 HeartLungsLiver

Spleen

PancreasStomach

IntestineKidneysTumour

Panitumumab-DOTA-177LuA

Time post injection (h)

177L

u (

MB

q)

0 24 48 72 96 120 144 168 1920.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

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Heart

Liver

StomachPancreas

SpleenIntestine

Kidneys

Tumor

Lungs

Panitumumab-DOTA-111InB

Time post injection (h)

11

1In

(M

Bq

)

Panitumumab-MCP-111In

0 24 48 72 96 120

144

168

192

0.00

0.05

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1.401.451.501.551.60

Intestine

Heart

Lungs

Liver

Stomach

Pancreas

Spleen

Kidney

Tumor

C

11

1In

(M

Bq

)

Figure 4-6. Radioactivity (not corrected for decay) in selected organs at 24, 72, 120 and 168 h

post i.v. injection of a single amount (6 MBq; 10 μg) of panitumumab-DOTA-177Lu in NRG

mice or panitumumab-DOTA-111In (10 MBq ; 10 μg) or panitumumab-MCP-111In (10 MBq ; 10

μg) in NOD/SCID mice. The cumulative radioactivity up to 168 h (AUC0-168h; Bq × sec) was

obtained from the area under the curve integrated using GraphPad Prism Ver. 4.0. By assuming

that there was no further biological elimination from the organ, the radioactivity at the last time

point was divided by the decay constant of 177Lu (0.004345 h-1) or 111In (0.0107 h-1) to calculate

the AUC168h-∞. The total AUC0h-∞ was used to estimate the radiation absorbed doses.

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Table 4-3. Estimated absorbed doses in the tumour and normal organs in mice with s.c. PANC-1

xenografts injected with 111In- or 177Lu-RICs based on the AUC0h-∞ determined in biodistribution

studies at different time points (24, 72, 120 and 168 h p.i.; Figure 4-6).

Absorbed dose (Gy)a

Organ

177Lu (Single injection × 6 MBq) 111In (Three injections × 10 MBq)

Panitumumab-DOTA Panitumumab-MCP Panitumumab-DOTA

Heart 3.5 ± 0.3 2.3±0.2 2.4±0.3

Lungs 4.4 ± 1.0 2.3±0.2 2.7±1.0

Liver 6.9 ± 1.6 10.3±0.6 4.56±0.3

Spleen 10.2 ± 2.3 19.5±3.8 10.3±3.6

Pancreas 9.2 ± 5.2 9.6±3.0 5.2±1.3

Stomach 1.0 ± 0.2 2.8±0.2 1.5±0.3

Intestine 1.9 ± 0.3 2.3±0.2 2.5±0.0

Kidneys 19.3 ± 3.1 8.4±1.6 11.1±1.7

Whole Body 2.0 ± 0.1 0.4±0.0 0.4±0.0

Tumour b 11.5 ± 4.9 2.8±0.7 6.0±2.2

a Estimated using D= Ãs×S equation and mouse S-values (323).

b Estimated using the sphere model in OLINDA/EXM, and the measured tumour diameter.

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4.5 Discussion

We report here a promising new RIT approach for PnCa that exploits the overexpression of

EGFR on almost all (>90%) tumours (308) and a clinically used fully human anti-EGFR mAb,

panitumumab (Vectibix, Amgen). Panitumumab was linked to either two MCPs that present 13

DOTA chelators or to two DOTA chelators to complex the -emitter, 177Lu or the Auger electron

emitter, 111In. The MCP with multiple DOTA chelators can provide labeling at high SA (257)

and may overcome limited delivery of the RICs in PnCa due to stromal barriers (353). We

previously showed the therapeutic efficacy of 177Lu-labeled panitumumab-MCP on PANC-1

cells and on s.c. PANC-1 xenografts in NRG mice. In the current study we now evaluated the

RIT properties of 111In-labeled panitumumab-MCP and compared these with 111In- or 177Lu-

labeled panitumumab-DOTA. At all amount of radioactivity, panitumumab-DOTA-177Lu was

more potent in vitro for killing PANC-1 cells than 111In-labeled RICs (Figure 4-1) and its toxicity

was not different from the previously studied panitumumab-MCP-177Lu conjugates under the

same conditions (343). This greater cytotoxicity of 177Lu vs. 111In was associated with formation

of more DNA DSBs in the cell nucleus assessed by immunofluorescence for -H2AX (Figure 4-

2), as well as higher absorbed doses in the nucleus of PANC-1 cells for 177Lu than 111In (Table 4-

1 and 4-2). EGFR-specific cell binding of panitumumab-DOTA-177Lu conjugates was confirmed

by the lower cell-associated radioactivity compared to non-specific hIgG-DOTA-177Lu or

panitumumab-DOTA-177Lu in presence of excess unlabeled panitumumab (21.9–fold or 13.7–

fold lower; respectively) (Figure 4-3). The dose deposited in the nucleus of PANC-1 cells was

significantly lower for non-specific hIgG-DOTA-177Lu or panitumumab-DOTA-177Lu co-

incubated with excess panitumumab to block EGFR than panitumumab-DOTA-111In,

panitumumab-MCP-111In or panitumumab-DOTA-177Lu (Table 4-1). In contrast to

panitumumab-MCP-177Lu conjugates, subcellular fractionation study showed no changes with

time in the proportion of cell-bound radioactivity on the membrane, in the cytoplasm or the

nucleus for panitumumab-DOTA-177Lu, suggesting more efficient internalization of

panitumumab-MCP-177Lu into PANC-1 cells than panitumumab-DOTA-177Lu (343). However,

the lower cellular binding of panitumumab-MCP-177Lu compared to panitumumab-DOTA-177Lu

resulted in a 2–fold lower dose deposited in the nucleus of PANC-1. The limited cellular binding

of panitumumab-MCP-177Lu may be due to the steric hindrance in EGFR binding between the

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molecules of panitumumab-MCP-177Lu (50-fold molar excess) since these have larger

hydrodynamic radius compared to panitumumab-DOTA-177Lu (7.1 nm vs. 5.1 nm; respectively)

(257). It should be mentioned that saturation binding assays that were performed at lower

concentrations of the RICs (0.1 up to 12.5-fold molar excess) revealed similar binding affinity

for radiolabeled panitumumab-MCP and panitumumab-DOTA RICs (257).

The normal tissue toxicity of panitumumab-DOTA-111In, panitumumab-DOTA-177Lu and

panitumumab-MCP-111In was assessed to confirm that the amounts selected for RIT studies

would be safe to administer. Analysis of blood samples obtained from non-tumour bearing

NOD/SCID mice 14 days after administration of panitumumab-MCP-111In and panitumumab-

DOTA-111In (10 MBq; 10 μg) showed no hematopoietic, liver, or kidney toxicity compared to

control NOD/SCID mice receiving saline as measured by CBC, ALT and Cr (Figure 4-4).

Similarly, the normal tissue toxicity was assessed in non-tumour bearing NRG mice 14 days

after administration of panitumumab-DOTA-177Lu (6.0 MBq; 10 μg) or panitumumab-MCP-

177Lu (6.0 MBq; 10 μg) or saline. Hematology and serum biochemistry analysis for NRG mice

administered panitumumab-MCP-177Lu (6.0 MBq; 10 μg) and control NRG mice administered

normal saline were published in our previous study (343). Here we confirmed the safety of

panitumumab-DOTA-177Lu (6.0 MBq; 10 μg) in NRG mice, which caused no significant

decrease in CBC, or increase ALT and Cr (Figure 4-4compared to previously reported values for

panitumumab-MCP-177Lu (6.0 MBq; 10 μg) or normal saline injected NRG mice (343).

Administration of a single amount (6.0 MBq; 10 µg) of panitumumab-DOTA-177Lu or three

amounts (10.0 MBq; 10 µg) of panitumumab-MCP-111In or panitumumab-DOTA-111In separated

by 3 weeks significantly delayed tumour growth in NOD/SCID or NRG mice with s.c. PANC-1

tumours (TGI at 43 days = 2.9 ± 0.4, 3.0 ± 0.4 and 3.9 ± 0.3, respectively; P>0.05) compared to

saline and unlabeled panitumumab (TGI = 9.8 ± 1.6, 9.9 ± 1.4, respectively) (Figure 4-5).

Dosimetry (Table 4-3) estimated based on the biodistribution of the RICs (Figure 4-6) revealed

that at three injections of 111In-RICs (total 30 MBq) or one injection of 177Lu-RICs (6 MBq), the

absorbed dose in the tumour for panitumumab-DOTA-177Lu was 11.5 ± 4.9 Gy but these RICs

caused the same growth inhibitory effect on PANC-1 tumours as 6.0 ± 2.2 Gy deposited by

panitumumab-DOTA-111In and 2.8 ± 0.7 Gy deposited by panitumumab-MCP-111In. We

previously studied the tumour growth inhibition caused by a single administration of (6 MBq; 10

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μg) panitumumab-MCP-177Lu compared to normal saline or unlabeled panitumumab in NRG

mice engrafted s.c. with PANC-1 xenografts and the results were reported as the mean TGI ±

SEM at 33 days (343). The mean TGI at 33 days calculated for panitumumab-DOTA-177Lu in

this study is similar to that previously reported for panitumumab-MCP-177Lu (TGI = 1.8 ± 0.3 vs.

2.5 ± 0.3, respectively; P> 0.05) which can be explained by the similar absorbed doses in the

tumour (11.5 ± 4.9 Gy vs. 12.3 ± 0.9 Gy; P>0.05). Panitumumab-DOTA-177Lu and

panitumumab-MCP-177Lu significantly delayed tumour growth in NRG mice compared to

normal saline and unlabeled panitumumab (TGI = 5.8 ± 0.5 and TGI = 6.6 ± 1.1; P<0.05).

Panitumumab neither decreased the CS of PANC-1 cells in vitro (Figure 4-1) nor delayed

tumour growth of PANC-1 xenografts in vivo (Figure 4-5), likely due to a downstream mutation

in KRAS (329) that results in constitutive EGFR activation.

The equivalent tumour growth-inhibitory effects of 111In and 177Lu in vivo despite 2-4 fold higher

absorbed doses in PANC-1 tumours deposited by panitumumab-DOTA-177Lu than

panitumumab-DOTA-111In or panitumumab-MCP-111In; respectively (Table 4-3), could be

explained by tumour hypoxia. The DNA damaging effects of β-particles rely on reactive oxygen

species (ROS) while AEs are a form of high LET radiation that is cytotoxic under both normoxic

and hypoxic conditions (191). PANC-1 tumours in SCID mice have been reported to be hypoxic

which caused resistance to γ-irradiation (354) and may similarly cause resistance to β-particles.

In contrast, in the well-oxygenated cell culture system used in vitro, there is no hypoxia that

would restrict the effectiveness of the β-particles emitted by 177Lu. In addition, the cross-fire

effect from the 2 mm range β-particles contributes to absorbed dose in PANC-1 cells from

panitumumab-DOTA-177Lu in the medium in vitro, whereas there is a more limited cross-fire

effect from 111In, mainly from the low ionizing but penetrating γ-photon emissions (Table 4-1).

AEs have a higher relative biological effectiveness (RBE) than β-particles due to their high LET,

which means that at the same radiation dose, AEs are more damaging (191). Moreover, the

tumour absorbed doses were estimated from the cumulative radioactivity in the tumour (Ãs) and

OLINDA/EXM macrodosimetry (352). Thus, these macrodoses do not reflect the subcellular

doses deposited by 111In, especially in the cell nucleus which would be more important for

predicting the effectiveness of panitumumab-DOTA-111In.

It is challenging to compare β-particle or AE-emitters for RIT and it has been suggested that

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these may be best compared using administered amounts of radioactivity that cause equivalent

normal tissue toxicity (355). In our study we were most interested to compare 177Lu- and 111In-

labeled RICs at administered amounts that caused no normal tissue toxicity. Nonetheless, it

should be noted that similar antitumour effects were obtained after administration of three non-

toxic amounts of 111In-labeled panitumumab-DOTA and panitumumab-MCP (total = 30 MBq;

30 µg) but only one administration of 177Lu-labeled panitumumab-DOTA (6 MBq; 10 µg)

(Figure 4-5). In a previous study, 177Lu-labeled RICs bispecific for HER2 and EGFR were more

effective than 111In-labeled RICs, but both inhibited the growth of human breast cancer

xenografts co-expressing these receptors in mice (356).

Targeting overexpression of EGFR for RIT of PnCa may cause some normal tissue toxicity since

EGFR are displayed at low levels on most epithelial tissues and moderately by the liver and

kidneys (51, 336). Importantly, <3% of hematopoietic stem cells are EGFR-positive (357) which

should minimize hematopoietic toxicity, particularly for 111In-labeled RICs that require binding

and internalization for the AEs to be damaging to DNA (10). The 2 mm range β-particles emitted

by 177Lu may also cause non-specific bone marrow toxicity from a cross-fire effect, but our

results revealed no hematopoietic, liver or kidney toxicity in mice administered 111In- or 177Lu-

labeled RICs at the amounts studied (Figure 4-4). Nonetheless, it should be noted that

panitumumab does not bind to mouse EGFR (358), and EGFR-specific normal tissue toxicities

would need to be evaluated in a Phase 1 clinical trial. Previously, we found that 111In-EGF

caused no hematopoietic, liver or kidney toxicity at administered amounts up to 44.4 MBq in

mice or 85.1 MBq in rabbits (359) and up to 2,290 MBq in humans in a Phase I trial (360). Our

results suggest that panitumumab-DOTA-111In and panitumumab-MCP-111In as well as

panitumumab-DOTA-177Lu are promising agents for RIT of EGFR positive cancers such as

PnCa. However the MCP conjugates might have limitations over the DOTA conjugates due to

their higher liver and spleen uptake, therefore DOTA conjugates might be more promising for

RIT with 111In. There was no significant difference in the size of tumours treated with 111In and

177Lu but nanometer range AEs emitted by 111In might be more potent for treatment of small

volume disease, while 177Lu could be used for larger tumours (361). Although all the

aforementioned RICs provided strong tumour growth inhibition in mice without normal tissue

toxicity, the whole body dose, which is a predictor of bone marrow toxicity was much higher for

177Lu than 111In. This would limit the ability to further escalate the administered amount of 177Lu,

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but the amount of 111In could be further increased without exceeding a whole body dose of 2 Gy

(limit).

4.6 Conclusion

We found some differences in the relative potency of 111In vs. 177Lu comparing in vitro and in

vivo studies that may be due to hypoxia. On a per dose basis 111In was much more effective than

177Lu in vivo. In addition, 111In deposits lower doses in normal organs than 177Lu and this may

allow further escalation of the administered amount of 111In, while the whole body dose for 177Lu

doesn’t allow further escalation in the administered amount because of potential bone marrow

toxicity. Also, the higher liver and spleen uptake of the MCP conjugates makes these less

attractive for RIT with 111In than the DOTA conjugates.

4.7 Acknowledgments

Supported by grants from the Canadian Cancer Society Research Institute with funds from the

Canadian Cancer Society and from the Cancer Research Society. S. Aghevlian received

scholarships from the Strategic Training in Transdisciplinary Radiation Science for the 21st

Century (STARS21) strategic training program at the University Health Network supported by

the Terry Fox Foundation and from the Centre for Pharmaceutical Oncology (CPO) at the

University of Toronto.

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Chapter 5: Summary and Future Directions

166

5 Thesis Conclusion and Summary of Findings

The overall aim of this thesis research was to develop and characterize novel theranostics for

imaging and systemic RIT of PnCa using anti EGFR mAb panitumumab labeled with the Auger

electron emitter, 111In or β-particle emitter, 177Lu. Panitumumab was modified with either DOTA

chelators or with HyNIC-MCP that harbours 13 pendant DOTA chelators and 10 PEG moieties

to reduce liver and spleen uptake. The availability of multiple chelators on these polymers to

complex radiometals allow for dual labeling with 111In and 177Lu, as well as more reproducible

labeling efficiency and also achieving a higher SA which could provide higher toxicity towards

cancer cells.

The overall conclusions of the research described in this thesis are:

I) 111In- or 177Lu- labeled panitumumab-DOTA and panitumumab-MCP RICs were

constructed at high labeling yield and at high SA while preserving immunoreactivity

toward EGFR. MCP increases the SA of 111In and 177Lu dual labeled panitumumab up

to 12.5-fold compared to DOTA conjugation. EGFR-mediated tumour uptake of

panitumumab-DOTA and panitumumab-MCP as well as the EPR effect associated

with the larger hydrodynamic radius of MCP conjugates than DOTA conjugates (7.3

nm vs 5.5 nm; respectively) allowed visualization of EGFR overexpressing PANC-1

tumours in mice using microSPECT/CT imaging.

II) Panitumumab-MCP-177Lu reduced the CS of PANC-1 cells in vitro by 7.7-fold at 1.2

MBq (2.5 nmol/L) compared to unlabeled panitumumab due to a high absorbed dose

in the nucleus (3.84 Gy) causing 3.8-fold higher γ-H2AX foci in PANC-1 cells.

Intravenous (i.v.) injection of 6 MBq of panitumumab-MCP-177Lu (10 μg) delivered a

dose of 12.5 Gy to PANC-1 tumours and significantly inhibited tumour growth in

NRG mice compared to the control groups including saline, unlabeled panitumumab

and hIgG-MCP-177Lu (TGI at 33 days = 2.3 ± 0.2 vs. 5.8 ± 0.5, 6.0 ± 1.6 and 4.0 ± 0.7

respectively) without any observable normal tissue toxicity.

III) Panitumumab-DOTA-177Lu reduced the CS of PANC-1 cells in vitro by 6.4-fold and

5-fold at 1.2 MB (2.5 nmol/L) compared to panitumumab-DOTA-111In and

panitumumab-MCP-111In due to a higher absorbed dose in the nucleus (8.06 Gy v.s 2.2

Gy and 1.17 Gy; respectively) causing 2.7-fold higher γ-H2AX foci in PANC-1 cells.

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Intravenous (i.v.) injection of 6 MBq of panitumumab-DOTA-177Lu (10 μg) delivered

2-4 fold higher absorbed dose to PANC-1 tumours compared to 3 amounts of 10 MBq

of 111In-labeled RICs (10 μg), however as measured by TGI at 43 days p.i. of

panitumumab-DOTA-177Lu, panitumumab-DOTA-111In and panitumumab-MCP-111In

tumour growth was similarly delayed in mice with s.c. PANC-1 xenografts compared

to control mice treated with saline and unlabeled panitumumab (2.9 ± 0.43,3.03 ± 0.45

and 3.87 ± 0.35; respectively vs. 9.83 ± 1.56 and 9.87 ± 1.38; respectively) without

any observable normal tissue toxicity.

5.1.1 Chapter 2

This chapter characterized the in vitro and in vivo properties of 111In- or 177Lu- labeled

panitumumab-DOTA and panitumumab-MCP conjugates and their ability to visualize PANC-1

tumours in NOD/scid mice implanted s.c. with PANC-1 xenografts using microSPECT/CT

imaging. Panitumumab was modified with S-4FB through lysine groups in order to install

aldehyde groups for derivatization with HyNIC-MCP (two MCPs/panitumumab). For

comparison, using similar linkage chemistry, DOTA-NHS ester (succinimidyl ester) was selected

for reaction with lysines on panitumumab for synthesis of panitumumab-DOTA conjugates (2

DOTA/panitumumab). When 1 µg the RICs were dual-labeled with 111InCl3 (7.8 MBq) and 177Lu

Cl3 (5.7 MBq), the SA of panitumumab-MCP conjugates was 12.7-fold and 9.3-fold higher

compared to panitumumab-DOTA conjugates. Panitumumab-MCP-177Lu and panitumumab-

DOTA-177Lu exhibited saturable binding to EGFR overexpressing MDA-MB-468 cells (1.3 ×

106 receptors/cell) that was displaced by an excess of unlabeled panitumumab, demonstrating

specific EGFR-mediated binding. There were no significant differences in the EGFR binding

affinity of Panitumumab-MCP-177Lu and panitumumab-DOTA-177Lu (Kd = 2.2 ± 0.6 nmol/L

and 1.0 ± 0.4 nmol/L, respectively). 111In- or 177Lu-labeled panitumumab-MCP or panitumumab-

DOTA were stable to loss of radiometal when challenged in vitro with a 500-fold excess of

EDTA or when incubated in human plasma at 37ᵒC for up to 1 week. Comparison of the

elimination of radioactivity from the body in NOD/SCID mice engrafted s.c PANC-1 xenografts

and excretion into the urine and feces up to 72 h p.i. of panitumumab-MCP-111In or

panitumumab-DOTA-111In showed both RICs were slowly eliminated from the body with 40-

45% of the injected dose eliminated over 72h. Most of the radioactivity eliminated from the body

168

thorough the urinary system. These results suggest that there was no instability of the RICs

introduced by use of the MCP for labeling with 111In compared to DOTA. Biodistribution study

in NOD/SCID mice engrafted s.c PANC-1 xenografts at 72h p.i. showed no significant

difference in the tumour uptake of panitumumab-MCP-111In and panitumumab-DOTA-111In (6.9

± 1.3 vs 6.6 ± 3.3%ID/g, respectively). No significant differences were observed in normal tissue

localization between the two RICs, except for the liver which was 3-fold higher for

panitumumab-MCP-111In. EGFR-mediated tumour uptake of panitumumab-DOTA-111In was

confirmed by 3.5-fold reduced uptake of IgG-DOTA-111In, however similar tumour uptake of

nonspecific hIgG-MCP (5.4 ± 0.3%ID/g) to panitumumab-MCP-111In suggests EPR-mediated

tumour uptake due to the larger hydrodynamic radius of panitumumab-MCP than panitumumab-

DOTA (7.3 nm vs 5.5 nm; respectively). PANC-1 tumours were imaged by microSPECT/CT at

72 h p.i. of panitumumab-DOTA-111In, panitumumab-MCP-111In or hIgG-MCP-111In. It was

concluded that 111In- or 177Lu- labeled DOTA or MCP conjugated to panitumumab are stable

RICs with similar biodistribution profiles and could be useful theranostic agents for EGFR-

positive PnCa.

5.1.2 Chapter 3

This chapter evaluated the safety and efficacy of systemic RIT of s.c. PANC-1 human PnCa

xenografts in NRG mice using anti-EGFR panitumumab linked to MCP that presents 13 DOTA

chelators to complex the -emitter, 177Lu. Confocal immunofluorescence microscopy for -

H2AX showed that panitumumab-MCP-177Lu caused more DNA DSBs in the nucleus of PANC-

1 cells as the SA increased (0.3, 0.6, 1.2 MBq; SA = 46.7, 93.5, 187.0 MBq/nmole). Despite the

ineffectiveness of unlabeled panitumumab for killing PANC-1 cells, panitumumab-MCP-177Lu

reduced the CS of PANC-1 cells by 7.7-fold at 1.2 MBq, partly due to deposition of 3.84 Gy in

the nucleus of PANC-1 cells as measured by subcellular distribution of panitumumab-MCP-

177Lu at different time points from 1 to 24 h following incubation. Prior to RIT, toxicity study

showed that administration of panitumumab-MCP-177Lu (6 MBq; 10 g) to NRG mice caused

no significant change in body weight, CBC or ALT and Cr compared to NRG mice treated with

normal saline. RIT was performed in NRG mice with s.c. PANC-1 tumours injected i.v. with 6

MBq (10 g) of panitumumab-MCP-177Lu, hIgG-MCP-177Lu (6 MBq; 10 g), unlabeled

panitumumab (10 g) or normal saline (100 L). The tumour growth index (TGI) was compared.

169

Panitumumab-MCP-177Lu strongly inhibited tumour growth in NRG mice (TGI = 2.3 ± 0.2)

compared to normal saline treated mice (TGI = 5.8 ± 0.5; P<0.01). As expected due to KRAS

mutation, unlabeled panitumumab had no effect on tumour growth (TGI = 6.0 ± 1.6; P>0.05).

IgG-MCP-177Lu delayed tumour growth but it wasn’t as effective as Panitumumab-MCP-177Lu

(TGI = 4.0 ± 0.7; P>0.05). Tumour and normal organ doses were estimated based on

biodistribution studies at 1, 3, 5 and 7 days p.i. The absorbed dose of PANC-1 tumours was 12.3

Gy. The highest normal organ doses were absorbed by the pancreas, liver, spleen, and kidneys.

Due to presence of multiple DOTA chelators on the MCP, panitumumab-MCP conjugates can be

labeled at very high SA, however injecting a large radioactivity will exceed the maximum

tolerated injected dose in mice. Therefore, in this study, the injected amount limit was 6 MBq.

We conclude that EGFR-targeted RIT with panitumumab-MCP-177Lu was able to overcome

resistance to panitumumab in KRAS mutant PANC-1 tumours in NRG mice and may be a

promising approach to treatment of PnCa in humans.

5.1.3 Chapter 4

Chapter 4 focused on comparing the therapeutic efficacy of the β-particle emitter, 177Lu and

Auger electron-emitter, 111In targeted to EGFR positive PANC-1 cells in vitro or to s.c. PANC-1

xenografts in mice by exploiting DOTA-conjugated or MCP-conjugated panitumumab. The

clonogenic survival of PANC-1 cells was determined following exposure to increasing

radioactivity of (0.3-1.2 MBq; 2.5 nmole/L) panitumumab-DOTA-177Lu, panitumumab-DOTA-

111In or panitumumab-DOTA-111In (2.5 nmole/L) for 16 h then culturing for 12 days. At all

amounts of radioactivity panitumumab-DOTA-177Lu was more cytotoxic than 111In-labeled RICs.

In a separate study, PANC-1 cells were incubated with the RICs under the same condition and

only for 16 h to assess the intensity of γ-H2AX representing unrepaired DNA DSBs by

immunofluorescence. Panitumumab-DOTA-177Lu induced more DNA DSBs in the nucleus of

PANC-1 cells compared to 111In-labeled RICs. At 1.2 MBq, panitumumab-DOTA-177Lu induced

2.7-fold and 1.56-fold higher γ-H2AX foci than panitumumab-DOTA-111In and panitumumab-

MCP-111In; respectively, which is attributed to the higher absorbed dose in the nucleus by

panitumumab-DOTA-177Lu (8.06 Gy) compared to panitumumab-DOTA-111In and

panitumumab-MCP-111In (2.28 Gy and 1.17 Gy; respectively) as measured by subcellular

distribution of conjugates in PANC-1 cells. In the previous chapter we showed that

170

panitumumab-MCP-177Lu deposited 3.84 Gy in the nucleus of PANC-1 cells. It’s not clear why

the cellular binding of panitumumab-MCP is lower than panitumumab-DOTA however it’s

assumed that the larger hydrodynamic size of panitumumab-MCP-177Lu compared to

panitumumab-DOTA-177Lu might play a role. Stronger steric hindrance between the molecules

of panitumumab-MCP-177Lu (50-fold molar excess) might lower the cellular binding of

panitumumab-MCP-177Lu to PANC-1 cells resulting in a lower absorbed dose in the nucleus.

Administration to NRG mice of panitumumab-DOTA-177Lu (6 MBq; 10 µg) or to NOD/SCID

mice of panitumumab-DOTA-111In or panitumumab-MCP-111In (10 MBq; 10 µg) caused no

significant change in body weight, decreased CBC or increases in ALT or Cr compared to

normal saline treated NOD/SCID or NRG mice after 14 days. For RIT, NOD/SCID mice with

s.c. PANC-1 tumours received three amounts (10 MBq; 10 µg) of 111In-labeled RICs separated

by 3 weeks, whereas tumour bearing NRG mice received a single amount (6 MBq; 10 µg) of

panitumumab-DOTA-177Lu. Control NOD/SCID mice were treated with normal saline or

unlabeled panitumumab (10 µg). TGI at 43 day was calculated to include more than 3 surviving

mice in each group. This value for panitumumab-DOTA-111In or panitumumab-MCP-111In was

not significantly different from that for panitumumab-DOTA-177Lu (TGI = 3.02 ± 0.45, 3.87 ±

0.35 and 2.9 ± 0.43; respectively, P>0.5), whereas TGI at 43 days for both RICs were

significantly lower than that for normal saline and unlabeled panitumumab (TGI = 9.83 ± 1.56;

and TGI = 9.87 ± 1.38; respectively P<0.001). Tumour and normal organ doses were also

estimated based on biodistribution studies in NRG mice and NOD/SCID mice at different time

points. Absorbed doses in PANC-1 tumours were 11.52 ± 4.93 for panitumumab-DOTA-177Lu

and 2.79 ± 0.75 and 6.03 ± 2.22 for Panitumumab-MCP-111In and panitumumab-DOTA-111In;

respectively. The highest normal organ doses were received by the kidneys, spleen and pancreas.

Although studied under the same conditions, 111In deposited lower doses in the tumour and

normal organs, but the 111In-labeled RICs were still more effective than the 177Lu-RICs, possibly

because Auger electrons have higher RBE, thus at lower radiation doses they can be as effective

or more effective than β-particles (355).

In this study the mean TGI at 33 days for panitumumab-DOTA-177Lu was not significantly

different from that for panitumumab-MCP-177Lu (TGI = 1.83 ± 0.25 vs. TGI = 2.5 ± 0.3; P>

0.05) which can be explained by their equal absorbed dose in the tumour (11.52 ± 4.93 Gy vs.

12.33 ± 0.86 Gy; P>0.05). Panitumumab-DOTA-177Lu and panitumumab-MCP-177Lu

171

significantly delayed tumour growth in NRG mice compared to saline and unlabeled

panitumumab control mice (TGI = 5.8 ± 0.5 and TGI = 6.6 ± 1.1; P<0.05). In chapter 4 it was

shown that panitumumab-DOTA or panitumumab-MCP conjugates labeled with 177Lu or 111In

are promising RIT agents for treatment of PnCa in humans.

5.1.4 Future directions

In the past decade the rate of PnCa diagnosis has been increasing steadily and despite research.

there remain high mortality rates from this disease (362). The safety and efficacy identified in

animal studies is not generally translated to human trials because many of the animal models fail

to faithfully recapitulate the dynamic of human cancer. Studies have reported that compared with

subcutaneous xenograft models, orthotopic xenograft models in which tumour xenografts are

implanted into the natural organ, better simulate clinical cancer in terms of metastasis and

invasion, which may be attributed to the difference in microenvironment and lymphatic vessel

densities (363, 364). In this project the immunohistochemical studies showed that the

subcutaneous PANC-1 xenografts have a simple structure with homogeneous expression of

EGFR throughout the tumour (Appendix B). As opposed to subcutaneous xenograft models, the

tumour microenvironments of orthotopic xenograft models are more comparable to those in

humans and therefore rendering these models more relevant in predicting clinical outcomes in

humans (362). Furthermore, frequent metastases are observed in orthotopic xenograft models.

Studies have shown that up to 60% of orthotopic models of PnCa can disseminate to other organ

sites (365). Derosier et al. suggested in PnCa research the necessary second step is to employ

orthotopic xenograft mouse models to further validate results established in subcutaneous

xenograft mouse models (366). In the future, orthotopic tumour xenograft mouse models

for PnCa is recommended to further study the SPECT theranostics.

5.1.4.1 Bioluminescence orthotopic model of PANC-1

In PnCa, tumour cells with the ability to enter the blood circulation, i.e. circulating tumour cells

(CTCs) are responsible for development of metastases in distant organs particularly the liver,

lung and skeletal system (367). Recent studies have shown increasing interest in the application

of the luciferase-luciferin reaction for metastasis detection. Our lab has recently shown that tail

vein injection of luciferase-labeled MDA-MB-231 human breast cancer cells into NOD-SCID

mice and CD1 nude mice leads to metastases to the brain, lungs, liver, spleen and kidneys which

172

are visualized by injection of luciferin prior to bioluminescence imaging. Similarly, PANC-1

human PnCa cells can be tagged with luciferase (368-370) and administered i.v. into mice to

establish detectable metastases. The therapeutic efficacy of RICs particularly panitumumab-

DOTA-111In and panitumumab-MCP-111In in controlling metastases could be evaluated by

monitoring metastases using bioluminescence imaging. The high LET (4-26 keV/μm) of Auger

electrons emitted by 111In which travel only a short distance (nm to μm) in tissues makes 111In

suitable for eradicating micrometastases and CTCs. Since the role of imaging clinically would be

to detect metastases in order to better stage patients, the SPECT imaging properties of the RICs

can be studies in these metastatic as well as orthotopic models.

5.1.4.2 Evaluation of RICs in combination with other anti-cancer agents

According to the challenges of PnCa treatment mostly because of hypoxia (124, 371-373) and

low drug delivery [hypovascularity (71, 124, 371-373), stromal barrier (36, 297, 374, 375)]

combination of hypoxia-targeting drugs like evofosfamide (formerly known as TH-302) to

increase the sensitivity to radiation (376, 377) might be useful to improve the outcome of RIT.

RIT of PANC-1 tumours in mice with panitumumab-MCP and panitumumab-DOTA conjugates

labeled with111In or 177Lu significantly delayed tumour growth in mice compared to unlabeled

panitumumab, but didn’t result in tumour growth arrest. The therapeutic efficacy of these RICs

alone or in combination with drugs like nab-paclitaxel and evofosfamide could be studied and

compared with FOLFIRINOX or gemcitabine as the first line and second line treatment of PnCa

(378).

The SPECT theranostic RICs developed here may play an important role in future clinical

practice. Their ability to combine diagnostic and therapeutic capabilities may offer personalized

RIT for patients with EGFR-positive tumours. Beta-particles emitted by 177Lu could be useful for

RIT of millimetre-sized tumours whereas nanometer range Auger electrons emitted by 111In

could be more potent for treatment of micrometastases. Furthermore low absorbed doses in the

normal organs for 111In allow for dose escalation and more fractions. MCP provide an

opportunity for labeling panitumumab at high specific activity. While these high specific activity

probes have theoretical advantages in increasing the potency of the RICs, the benefit that MCP

conjugates may provide patients may need to be balanced against their higher accumulation in

the liver and spleen and the risk for low mass amounts to further promote uptake in these organs.

173

It would be necessary to test panitumumab-MCP conjugates at high specific activity side by side

with panitumumab-DOTA conjugates in a first-in human Phase I clinical trial in patients with

EGFR-positive tumours.

174

6 Appendices

175

6.1 Appendix A: Therapeutic efficacy of the 111In- and 177Lu-labeled

RICs side by side

For a better comparison, this section display all the results for 111In-labeled RICs and 177Lu-

labeled RICs side by side.

176

6.1.1 Appendix A1. Clonogenic survival

0.0 0.3 0.6 1.20

20

40

60

80

100

120

Panitumumab-MCP-111In

Panitumumab-DOTA-111In

Panitumumab-MCP-177Lu

Panitumumab-DOTA-177Lu

****** ***

Radioactivity (MBq)

Clo

no

ge

nic

Su

rviv

al (%

)

Figure S 1 Clonogenic survival of PANC-1 cells exposed to increasing radioactivity of 177Lu- or

111In- labeled panitumumab-MCP (2.5 nmol/L) or panitumumab-DOTA (2.5 nmol/L) or to

unlabeled immunoconjugates (2.5 nmol/L) for 16 h then cultured for 12 d. Data shown are the

mean ± SEM (n = 3–4). Significant differences (P<0.05) between 111In- or 177Lu- labeled RICs

are indicated by the asterisks. No significant deference was observed between panitumumab-

MCP-111In and panitumumab-DOTA-111In or between panitumumab-MCP-177Lu and

panitumumab-DOTA-177Lu.

177

6.1.2 Appendix A2: DNA damage assessment using γ-H2AX assay

Figure S 2. (A) Representative confocal immunofluorescence microscopy images assessing γ-

H2AX foci (green) representing sites of unrepaired DNA DSBs in PANC-1 cells exposed for 16

h to 0.3, 0.6 or 1.2 MBq of 177Lu- or 111In-labeled panitumumab-DOTA or panitumumab-MCP.

Cell nuclei were counterstained with DAPI (blue). In the bottom set of panels are images of

PANC-1 cells exposed to growth medium alone, or unlabeled panitumumab-DOTA or

panitumumab-MCP. (B) γ-H2AX foci were quantified as the integrated density/nucleus area and

are shown for untreated PANC-1 cells, cells treated with unlabeled radioimmunoconjugates

(RICs) or increasing amounts of 177Lu- or 111In-labeled RICs. Values shown represent the mean

± SEM (n= 3-4). Significant differences (P<0.05) are indicated by the asterisks.

178

6.1.3 Appendix A3: Absorbed dose in the nucleus of PANC-1 cells in vitro for 111In- or 177Lu-RICs

Table S 1. Absorbed dose in the nucleus were estimated following incubation of cells with 1.2 MBq (2.5 nmol/L) of 177Lu-RICs in the

absence or presence of 50-fold molar excess of unlabeled panitumumab to block EGFR for 24 h at 37 oC.

Dose to the cell nucleus (Gy)

Source

compartment

S

(Gy/Bq×sec)

Lu-177

Panitumumab-

DOTA

Panitumumab-

DOTA+ excess

panitumumab

hIgG-DOTA Panitumumab-MCP Panitumumab-

MCP + excess

panitumumab

hIgG-MCP

Cell surface 3.55 10-4 4.86 0.3 0.12 1.42 0.13 0.19

Cytoplasm 4.02 10-4 1.18 0.05 0.03 0.86 0.13 0.42

Nucleus 6.93 10-4 0.87 0.05 0.09 0.46 0.07 0.32

Medium 1.15 10-11 1.13 1.13 1.13 1.13 1.13 1.13

Total 8.06 1.53 1.36 3.87 1.46 2.06

Table S 2. Absorbed dose in the nucleus were estimated following incubation of cells with 1.2 MBq (2.5 nmol/L) of 111In-RICs in the

absence or presence of 50-fold molar excess of unlabeled panitumumab to block EGFR for 24 h at 37 oC.

Dose to the cell nucleus (Gy)

Source

compartment

S

(Gy/Bq×sec)

In-111

Panitumumab-

DOTA

Panitumumab-

DOTA+ excess

panitumumab

hIgG-DOTA Panitumumab-MCP Panitumumab-

MCP + excess

panitumumab

hIgG-MCP

Cell surface 7.35 10-5 1.01 0.06 0.02 0.29 0.02 0.03

Cytoplasm 1.03 10-4 0.3 0.01 0.007 0.22 0.03 0.1

Nucleus 5.06 10-4 0.64 0.04 0.06 0.33 0.05 0.23

Medium 3.39 10-12 0.33 0.33 0.33 0.33 0.33 0.33

Total 2.28 0.44 0.43 1.17 0.43 0.69

179

6.1.4 Appendix A4: Toxicity study

0

2

4

6

8

10 NOD/SCID - Normal saline

Panitumumab-DOTA-111In (10 MBq)

Panitumumab-MCP-111In (10 MBq)

Normal saline

Panitumumab-MCP-177Lu (6 MBq)

Panitumumab-DOTA-177Lu (6 MBq)

AW

BC

(

10

3/

L)

0

2

4

6

8

10B

RB

C (

10

6/

L)

0

5

10

15

20C

Hb

(g

/dL

)

0

10

20

30

40

50D

HC

T (

%)

0

500

1000

1500

2000EP

LT

(

10

3/

L)

0

20

40

60

80

100

120F

Cr

(M

)

0

100

200

300G

AL

T (

U/L

)

Figure S 3. Hematology and serum biochemistry analyses of NOD/SCID mice at 14 d after i.v.

injection of 10.0 MBq (10 μg) of panitumumab-MCP-111In, panitumumab-DOTA-111In or normal

saline and NRG mice administered 6.0 MBq (10 µg) of panitumumab-MCP-177Lu,

panitumumab-DOTA-177Lu or normal saline. (A) white blood cells [WBC], (B) Red blood cells

[RBC], (C) hemoglobin [Hb], (D) hematocrit [HCT], (E) platelets [PLT], (F) serum creatinine

[Cr], (G) serum alanine aminotransferase [ALT], Individual values are shown as well as the

mean (horizontal bars). No significant difference was observed between control and treatment

groups.

180

6.1.5 Appendix A5: RIT study with 177Lu-RICs

0 10 20 30 400.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline

panitumumab

panitumumab-MCP-177

Lu

panitumumab-DOTA-177

Lu

hIgG-MCP-177

Lu

A

Time (d)

Bo

dy W

eig

ht

Ind

ex (

BW

I)

0 10 20 30 400.0

2.5

5.0

7.5

10.0 Panitumumab

Panitumumab-MCP-177Lu

Panitumumab-DOTA-177Lu

Saline

hIgG-MCP-177

Lu

**

B

Time (d)T

um

or

gro

wth

ind

ex (

TG

I)

Figure S 4. Body weight index (BWI) in (A) NRG mice with s.c. PANC-1 human pancreatic

cancer (PnCa) xenografts receiving single amount (6.0 MBq; 10 g) of panitumumab-MCP-

177Lu, panitumumab-DOTA-177Lu or non-specific hIgG-MCP-177Lu or in mice receiving

unlabeled panitumumab (10 g) or normal saline. (B) Tumour growth index (TGI) in the

aforementioned groups. Values are mean ± SEM. Significant differences (P<0.05) between TGI

at 33 days after starting treatment between treatment groups are indicated by the asterisks. No

significant difference was observed in the TGI between panitumumab-MCP-177Lu and

panitumumab-DOTA-177Lu (2.3 ± 0.2 vs. 1.8 ± 0.25).

181

6.1.6 Appendix A6: RIT study with 111In-RICs

0 10 20 30 40 50 60 700.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline

Panitumumab

Panitumumab-DOTA-111

In

Panitumumab-MCP-111

In

hIgG-MCP-111

In

A

Time (d)

Bo

dy W

eig

ht

Ind

ex (

BW

I)

0 10 20 30 40 50 60 700

5

10

15

20

Saline

Panitumumab

Panitumumab-DOTA-111

In

***

Panitumumab-MCP-111

In

hIgG-MCP-111

In

B

Time (d)

Tu

mo

r G

row

th In

de

x (

TG

I)

Figure S 5. Body weight index (BWI) in (A) NOD/SCID mice with s.c. PANC-1 human

pancreatic cancer (PnCa) xenografts receiving radioimmunotherapy (RIT) with three amounts

(10.0 MBq; 10 g) of panitumumab-MCP-111In, panitumumab-DOTA-111In or non-specific

hIgG-MCP-111In separated by 3 weeks or in mice receiving unlabeled panitumumab (10 g) or

normal saline. The mice injected with started losing weight significantly since day 10 p.i.

suggesting toxicity of hIgG-MCP-111In (B). Tumour growth index (TGI) in the aforementioned

groups. Values are mean ± SEM. Significant differences (P<0.05) between TGI at 43 days after

starting treatment between treatment groups are indicated by the asterisks. No significant

difference was observed in the TGI between panitumumab-MCP-111In, panitumumab-DOTA-

111In (3.03 ± 0.45 vs. 3.87 ± 0.35).

182

6.1.7 Appendix A7: Tumour and normal organs dosimetry

Table S 3. Estimated absorbed doses in the tumour and normal organs in mice with s.c. PANC-1 xenografts injected with 111In- or

177Lu-RICs based on biodistribution study at different time points (1, 3, 5 and 7 days p.i.). Dose to infinity was calculated by dividing

the radioactivity at 168 h p.i. of RICs for source organs by the decay constant for 177Lu (0.004345 h-1) or 111In (0.0107 h-1), assuming

further elimination only by radioactive decay.

Absorbed dose (Gy)

Organ

177Lu (Single injection × 6 MBq) 111In (Three injections × 10 MBq)

Panitumumab-MCP Panitumumab-DOTA Panitumumab-MCP Panitumumab-DOTA

Heart 3.4 ± 0.4 3.5 ± 0.3 2.3 ± 0.2 2.4 ± 0.3

Lungs 4.3 ± 0.5 4.4 ± 1.0 2.3 ± 0.2 2.7 ± 1.0

Liver 7.5 ± 1.5 6.9 ± 1.6 10.3 ± 0.6* 4.6 ± 0.3

Spleen 14.8 ± 2.8 10.2 ± 2.3 19.5 ± 3.8 10.3 ± 3.6

Pancreas 19.3 ± 6.5 9.2 ± 5.2 9.6 ± 3.0 5.2 ± 1.3

Stomach 1.9 ± 0.4 1.0 ± 0.2 2.8 ± 0.2 1.5 ± 0.3

Intestine 3.2 ± 1.6 1.9 ± 0.3 2.3 ± 0.2 2.5 ± 0.0

Kidneys 15.7 ± 3.7 19.3 ± 3.1 8.4 ± 1.6 11.1 ± 1.7

Whole Body 1.40 ± 0.7 2.0 ± 0.1 0.4 ± 0.0 0.4 ± 0.0

Tumour 12.3 ± 0.9 11.52 ± 4.9 2.8 ± 0.7⸶ 6.0 ± 2.2

*The absorbed dose in the liver for panitumumab-MCP-111In is higher than that for panitumumab-DOTA-111In due to the higher liver

uptake of panitumumab-MCP-111In.

⸶No significant difference in the absorbed dose in tumour between 111In labeled panitumumab-DOTA and panitumumab-MCP.

183

6.2 Appendix B. Immunohistochemical staining of PANC-1 xenografts

obtained from NOD/SCID mice and NRG mice for characterization

of the tumour microenvironment

Methods: To compare the tumour microenvironment of human PANC-1 xenografts (n=5)

developed in NRG mice with that developed in NOD/SCID mice, the density of PANC-1 cells

(EGFR+), vascular endothelial cells (CD31+) and possible presence of fibroblasts (alpha-smooth

muscle actin (α-SMA+)) was determined by immunohistochemical staining (IHC). The tissues

were removed and snap-frozen in FSC 22 clear frozen section media (Leica Biosystems,

Germany) cooled in liquid nitrogen. 5 m frozen sections were obtained and air dried at room

temperature. Sections were then fixed with acetone for 10 minutes. Endogenous peroxidase and

biotin activities were blocked respectively using 0.3% hydrogen peroxide (Vector Labs). The

Abs, dilutions and detection kits used in this study are shown in Table S 3. After following kit

instructions, color development was performed with freshly prepared DAB (DAKO). Finally,

nuclei were counterstained lightly with Mayer’s Hematoxylin and coverslipped with Permount

mounting medium (Fisher). Reaction products and non-reactive nuclei were visualized in brown

(DAB) and blue respectively.

Table S 4. Staining of various antigens in PANC-1 tumour sections.

Antibody Source Clone Dilution (Incubation) Detection kit

CD 31 rat monoclonal BD Pharmingen MEC13.3 1/500 1hr Anti-Rat IgG

SMA rabbit polyclonal Abcam N/A 1/4000 1hr Anti-Rabbit IgG

184

EGFR

CD31

α-SMA

Results: Similar findings were observed in NOD/SCID mice and NRG mice. Both mouse strains

developed hypovascular tumours with low population of α-SMA+ cells. Although α-SMA is

widely used as a cancer associated fibroblast (CAF) marker, pericytes wrapping around the

endothelial cells also express this marker(379) as it can be seen in Figure S 6.

NOD/SCID NRG

Figure S 6. The expression of EGFR, CD31 and α-SMA on PANC-1 xenografts obtained from

NOD/SCID mice (on the left-hand side) and NRG mice (on the right-hand side). The brown

color indicates the reaction site where antibody detects the marker. No significant deference was

observed in the tumour microenvironment between the two mouse strains. Homogenous EGFR

expression and hypovascularity was shown by EGFR and CD31 Staining. It seems that many α-

SMA positive cells are perivascular cells.

185

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