ImmunoPET/fluorescence imaging and radioimmunotherapy ...

UNIVERSITY OF CALIFORNIA

Los Angeles

ImmunoPET/fluorescence imaging and radioimmunotherapy

of PSCA-positive prostate cancer

A dissertation submitted in partial satisfaction

of the requirements for the degree

Doctor of Philosophy in Molecular and Medical Pharmacology

by

Wen-Ting Katie Tsai

2018

Copyright by

Wen-Ting Katie Tsai

2018

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ABSTRACT OF THE DISSERTATION

ImmunoPET/fluorescence imaging and radioimmunotherapy

of PSCA-positive prostate cancer

by

Wen-Ting Katie Tsai

Doctor of Philosophy in Molecular and Medical Pharmacology

University of California, Los Angeles, 2018

Professor Anna Wu Work, Chair

Prostate cancer diagnosis and treatment options need to be improved as over- and under-

treatment, as well as disease recurrence and resistance to current therapies, continue to be

challenges. Prostate stem cell antigen (PSCA) is overexpressed in the majority of prostate

cancers and correlates with grade, stage, and metastatic potential. Antibodies, which are

highly specific for their target, can be labeled with radionuclides and fluorophores for

molecular imaging and therapy. This dissertation describes the use of humanized anti-PSCA

antibody fragments for dual-modality immuno-positron emission tomography

(immunoPET)/fluorescence imaging and for radioimmunotherapy of prostate cancer in

preclinical models.

Prostate cancer visualization could be improved by using immunoPET for preoperative non-

invasive disease detection, and fluorescence imaging for intraoperative guidance of tumor

resection; the power of both imaging modalities can be combined on a single agent. In this

work, the dual-labeled humanized anti-PSCA A11 cys-minibody (A11 cMb) was evaluated

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for successive immunoPET/fluorescence imaging in subcutaneous and orthotopic prostate

cancer models. A11 cMb was site-specifically conjugated with the near-infrared fluorophore

Cy5.5 and radiolabeled with 124I or 89Zr. 124I- and 89Zr-A11 cMb-Cy5.5 were used to detect

subcutaneous and intraprostatic PSCA-positive tumors. High contrast

immunoPET/fluorescence imaging with 124I-A11 cMb-Cy5.5 identified both high PSCA-

expression and moderate PSCA-expression subcutaneous tumors. 89Zr-A11 cMb-Cy5.5

immunoPET showed uptake in the prostate without interfering signal in the bladder, and ex

vivo fluorescence imaging clearly showed signal in the tumor and not the surrounding

seminal vesicles. These studies support the potential for dual-modality A11 cMb to be

translated for preoperative whole-body disease detection and real-time surgical guidance in

prostate cancer patients.

Prostate cancer that metastasizes often becomes resistant to current therapies and

eventually progresses, and alternative therapy options include radioimmunotherapy which

delivers a high radiation dose to the tumor with the aim of minimizing dose to normal organs.

Anti-PSCA antibody fragments radiolabeled with a cytotoxic radionuclide, such as the beta-

emitter 131I or 177Lu, may be effective for radioimmunotherapy with lower toxicity compared to

intact antibodies. 124I- and 89Zr-A11 minibody (A11 Mb) immunoPET was used to guide

dosimetry studies, and 131I- and 177Lu-A11 Mb was administered to 22Rv1-PSCA s.c. tumor-

bearing nude mice to complete dosimetry estimation. 131I-A11 Mb had a higher projected

therapeutic index, or tumor-to-radiosensitive tissue dose, and was used in subsequent

therapy studies. 131I-A11 Mb inhibited PSCA-positive tumor growth in a dose-dependent

manner and extended survival with minimal off-target toxicity. Additionally, preliminary

biodistribution studies in knock-in transgenic mice that express human-PSCA were similar to

that of nude mice. Therefore, radioimmunotherapy will likely not be toxic to organs with

normal PSCA expression (stomach, bladder), and A11 Mb may be suitable for human

translation.

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The results of this work demonstrate the potential of anti-PSCA minibodies as theranostic

agents for disease diagnosis, surgical guidance, and treatment. Additionally, the success of

anti-PSCA dual-modality imaging and radioimmunotherapy in preclinical models support

further evaluation in the clinic.

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The dissertation of Wen-Ting Katie Tsai is approved.

Samson A. Chow

Robert E. Reiter

Lily Wu

Anna Wu Work, Committee Chair

University of California, Los Angeles

2018

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I dedicate this dissertation to my mom, Yu-Yen, who passed away from lung cancer and is

part of my inspiration for working in cancer research. I also dedicate this work to my Dad,

Chih-Ling, who has a big heart and shows perseverance and passion in all aspects of his

life, as well as my sister Wen-Lin, my stepmom Ching-Ju, and my grandparents who have all

showed me endless love, kindness, and support.

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Table of Contents

1 Introduction ................................................................................................................. 1

1.1 Overview of prostate cancer ...................................................................................... 1

1.1.1 Prostate cancer epidemiology ...................................................................... 1

1.1.2 Prostate cancer biology ................................................................................ 2

1.1.3 Prostate cancer diagnosis ............................................................................ 3

1.1.4 Prostate cancer staging and stratification ..................................................... 3

1.1.5 Prostate cancer treatment ............................................................................ 5

1.2 Imaging of prostate cancer ........................................................................................ 6

1.2.1 Molecular imaging ........................................................................................ 6

1.2.2 SPECT imaging of prostate cancer .............................................................. 6

1.2.3 Overview of PET imaging of prostate cancer ............................................... 7

1.2.4 Lipid metabolism-based PET imaging .......................................................... 9

1.2.5 Amino acid transport-based PET imaging .................................................. 10

1.2.6 Androgen receptor-based PET imaging ..................................................... 10

1.2.7 18F-sodium fluoride ..................................................................................... 11

1.2.8 Overview of antigen-specific PET imaging of prostate cancer .................... 11

1.2.9 PSMA antibody-based PET imaging .......................................................... 11

1.2.10 PSMA small molecule-based PET imaging .............................................. 12

1.2.11 Other targets for prostate cancer PET imaging ........................................ 15

1.2.12 Near-infrared fluorescence imaging for intraoperative guidance ............... 16

1.2.13 Fluorescent imaging of prostate cancer .................................................... 18

1.2.14 Dual-modality imaging of prostate cancer ................................................ 18

1.3 Therapy of prostate cancer ...................................................................................... 19

1.4 Antibodies and antibody fragments .......................................................................... 26

1.5 Conclusion .............................................................................................................. 29

2 Development of the anti-PSCA A11 cys-minibody .................................................. 31

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2.1 Introduction ............................................................................................................. 31

2.1.1 PSCA-targeting antibodies ......................................................................... 31

2.1.2 Site-specific labeling and the development of A11 cMb. ............................. 32

2.2 Methods .................................................................................................................. 35

2.2.1 Cloning....................................................................................................... 35

2.2.2 Protein production and purification ............................................................. 35

2.2.3 Protein characterization ............................................................................. 35

2.2.4 Conjugation ................................................................................................ 36

2.2.5 A11 cMb-Cy5.5 characterization ................................................................ 37

2.2.6 Affinity of A11 cMb and conjugated derivatives .......................................... 37

2.3 Results .................................................................................................................... 38

2.3.1 Production/purification ................................................................................ 38

2.3.2 Biochemical characterization of A11 cMb ................................................... 38

2.3.3 Biochemical characterization of A11 cMb conjugated derivatives ............... 39

2.3.4 Affinity studies ............................................................................................ 40

2.4 Discussion ............................................................................................................... 41

3 Dual-modality imaging .............................................................................................. 43

3.1 Introduction ............................................................................................................. 43

3.1.1 Dual-imaging .............................................................................................. 43

3.1.2 ImmunoPET radionuclides ......................................................................... 44

3.1.3 Fluorophores .............................................................................................. 44

3.1.4 Evaluation of dual-labeled A11 cMb ........................................................... 44

3.2 Methods .................................................................................................................. 45

3.2.1 Cell lines and xenografts ............................................................................ 45

3.2.2 Quantitation of PSCA cell surface expression ............................................ 46

3.2.3 Dual-labeling of A11 cMb ........................................................................... 46

3.2.4 Labeling efficiency, purification, immunoreactivity ...................................... 47

3.2.5 Dual immunoPET/fluorescence imaging ..................................................... 48

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3.2.6 Biodistribution ............................................................................................ 48

3.2.7 Image analysis ........................................................................................... 48

3.2.8 Histology and immunohistochemistry ......................................................... 49

3.3 Results .................................................................................................................... 49

3.3.1 Radiolabeling ............................................................................................. 49

3.3.2 ImmunoPET/fluorescence imaging in PSCA-high s.c. model ..................... 50

3.3.3 ImmunoPET/fluorescence imaging in PSCA-moderate s.c. model ............. 53

3.3.4 ImmunoPET fluorescence imaging in intraprostatic orthotopic model ......... 55

3.4 Discussion ............................................................................................................... 58

4 Radioimmunotherapy of PSCA-positive prostate cancer ....................................... 63

4.1 Introduction ............................................................................................................. 63

4.2 Methods .................................................................................................................. 66

4.2.1 Cell Lines and Tumor Models ..................................................................... 66

4.2.2 Conjugations .............................................................................................. 67

4.2.3 Radiolabeling ............................................................................................. 67

4.2.4 Cell cytotoxicity assay ................................................................................ 69

4.2.5 PET/CT imaging ......................................................................................... 69

4.2.6 Biodistribution ............................................................................................ 69

4.2.7 Dose estimation ......................................................................................... 70

4.2.8 Radioimmunotherapy ................................................................................. 70

4.2.9 Tissue Analysis .......................................................................................... 71

4.2.10 Statistics .................................................................................................. 71

4.3 Results .................................................................................................................... 72

4.3.1 124I-A11 Mb and 89Zr-A11 Mb serial immunoPET/CT quantification ............. 72

4.3.2 131I- and 177Lu-A11 Mb radiolabeling ........................................................... 74

4.3.3 Dose estimation based on 131I- and 177Lu-A11 Mb ex vivo biodistributions .. 76

4.3.4 131I-A11 Mb in vitro cytotoxicity ................................................................... 79

4.3.5 131I-A11 Mb RIT therapeutic effect .............................................................. 80

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4.3.6 131I-A11 Mb RIT toxicity .............................................................................. 81

4.3.7 131I-A11 Mb biodistributions in hPSCA mice ................................................ 83

4.4 Discussion ............................................................................................................... 86

4.5 Conclusion .............................................................................................................. 89

5 Conclusion ................................................................................................................. 90

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List of Figures and Tables

Figure 1.1 Incidence of prostate cancer ................................................................................ 2

Figure 1.2: Gleason grading and scoring system .................................................................. 4

Figure 1.3: Molecular imaging strategies currently applied for prostate cancer ...................... 9

Figure 1.4: Targeted fluorescent imaging ............................................................................ 17

Figure 1.5: Prostate cancer pathways and corresponding therapies ................................... 22

Figure 1.6: Schematic representation of therapeutic radionuclides...................................... 24

Figure 1.7: Intact antibody and engineered antibody fragments .......................................... 28

Figure 1.8: Anti-PSCA immunoPET .................................................................................... 29

Figure 2.1: A11 cys-minibody .............................................................................................. 34

Figure 2.2: Schematic of A11 cMb site-specific conjugation ................................................ 36

Figure 2.3: Biochemical characterization of A11 cMb .......................................................... 39

Figure 2.4: Biochemical characterization of A11 cMb-Cy5.5 ................................................ 40

Figure 2.5: Binding and affinity of A11 cMb-Cy5.5 ............................................................... 41

Figure 3.1: Concept of dual-modality imaging with A11 cMb ............................................... 45

Figure 3.2: Schematic of the dual-labeled A11 cMb ............................................................ 47

Figure 3.3: 124

I-A11 cMb-Cy5.5 immunoPET/fluorescence of 22Rv1-PSCA s.c tumor-bearing

mice.................................................................................................................. 51

Figure 3.4: 124

I-A11 cMb-Cy5.5 immunoPET/fluorescence of PC3-PSCA s.c tumor-bearing

mice.................................................................................................................. 54

Figure 3.7: 89Zr-A11 cMb-Cy5.5 immunoPET/fluorescence of mice bearing 22Rv1-PSCA

intraprostatic tumors ......................................................................................... 57

Figure 4.1: Schematic of RIT radionuclides ......................................................................... 64

Figure 4.2: Schematic of RIT using radiolabeled A11 Mb .................................................... 65

Figure 4.3: Schematic of DTPA conjugation and 177Lu radiolabeling. .................................. 67

Figure 4.4: Schematic of 131I-A11 Mb radiolabeling. ............................................................ 68

Figure 4.5: Serial immunoPET/CT of non-tumor-bearing mice ............................................ 73

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Figure 4.6: Quantification of 124I- and 89Zr-A11 Mb immunoPET/CT of non-tumor-bearing

mice.................................................................................................................. 73

Figure 4.7: Biodistribution time activity curves used for RIT dose estimation ....................... 76

Figure 4.8: 131I-A11 Mb RIT induces cell death .................................................................... 79

Figure 4.9: 131I-A11 Mb RIT inhibits tumor growth and extends survival .............................. 80

Figure 4.10: Final tumor weights and IHC ........................................................................... 81

Figure 4.11: Mouse body weights over the course of therapy .............................................. 82

Figure 4.12: Blood and serum biochemical analysis ............................................................ 83

Figure 4.13: 131I-A11 Mb biodistribution of male non-tumor-bearing hPSCA mice ............... 86

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List of Tables

Table 1.1: Pathologic Grading System of the International Society of Urological Pathology .. 4

Table 1.2: European Association of Urology (EAU) risk groups and recommendations for

biochemical recurrence of localized and locally advanced prostate cancer ......... 5

Table 1.3: Radiopharmaceuticals for molecular imaging of prostate cancer .......................... 8

Table 1.4: Prostate cancer stages and corresponding standard treatments ........................ 20

Table 1.5: Clinical and preclinical targets and targeting molecules in prostate cancer RIT .. 25

Table 2.1: Theoretical and affinity properties of A11 Mb and A11 cMb ................................ 42

Table 3.1: Radiolabeling and immunoreactivity for 124I-A11 cMb, 124I-A11 cMb-Cy5.5, and

89Zr-A11 cMb-Cy5.5 .......................................................................................... 50

Table 3.2: Ex vivo biodistribution of 124I-A11 cMb and 124I-A11 cMb-Cy5.5 in 22Rv1-PSCA

tumor-bearing mice ........................................................................................... 52

Table 3.3: Ex vivo biodistribution of 124I-A11 cMb-Cy5.5 in PC3-PSCA tumor-bearing mice 55

Table 4.1: 131I-A11 Mb radiolabeling optimization ................................................................ 74

Table 4.2: 177Lu-A11 Mb radiolabeling optimization ............................................................. 75

Table 4.3: 131I- and 177Lu-A11 Mb radiolabeling and immunoreactivity from biodistribution and

therapy studies. ................................................................................................ 75

Table 4.4: 131I-A11 Mb biodistributions in 22Rv1-PSCA s.c. tumor-bearing nude mice ........ 77

Table 4.5: 177Lu-A11 Mb biodistributions in 22Rv1-PSCA s.c. tumor-bearing nude mice ..... 78

Table 4.6: Comparison of radiation dose to tumor and organs from administering maximum

tolerated activity of 177Lu-A11 Mb and 131I-A11 Mb. ........................................... 79

Table 4.7: 131I-A11 Mb biodistributions in non-tumor-bearing hPSCA transgenic mice ........ 85

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Acknowledgments

First, I would like to thank my mentor Dr. Anna Wu. Anna’s patience has been invaluable

during my graduate school experience. She graciously accepted me into her lab despite my

lack of experience with immunoPET imaging, therapy, or antibodies. Although working in

Anna’s lab has helped me acquire many new skills in these areas, more importantly, it has

helped shaped the way I think, write, and talk about science at a different level than before. I

am grateful Anna has always been willing to take the time to go through numerous

manuscript and presentation drafts, and she has always set aside time to provide guidance

and support. I would also like to thank my committee members Dr. Lily Wu, Dr. Robert

Reiter, and Dr. Sam Chow, for asking tough yet fair questions during committee meetings,

providing helpful suggestions that helped shape my projects, and showing a genuine interest

in my happiness as a PhD student and in my future after graduation.

I would also like to thank my lab family for their scientific and emotional support, and I am

very grateful they are not only great co-workers but also wonderful friends. I am lucky to

have two mentors who were postdocs when I first joined, Dr. Kirstin Zettlitz and Dr. Richard

Tavaré. I am so grateful they were willing to stick with me through all my mistakes and guide

me through my projects, including the ones that failed and did not make it into this thesis.

More importantly, they also set an example for the kind of scientist I hope to become. I am

grateful that Richard, who can always find ways to improve my work, has continued to offer

support even after his move to New York. I would also like to thank Kirstin, who has a

special intuition for both conducting and communicating research that inspires me to be a

better scientist. I am also lucky to have found a soul sister to be my partner porker and help

me survive, maybe even enjoy, half marathons, Whitney, and more adventures and

accomplishments to come.

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I also could not have made it through my PhD without Felix Bergara, the dad of the lab who

also has a heart of a child and one of the kindest people I have ever met. Besides providing

technical support in my projects, he always made sure Kirstin and I crossed the street safely,

got crumbs out of our hair, and he quickly become the person we ran to when something

was broken or lost, or we just needed a hug. I am also lucky to have met many other great

scientists and students who have come through the lab, including Dr. Amanda Freise who

provided a lot of support when I first started in the lab, Dr. Scott Knowles whose thesis on

using the anti-PSCA A11 minibody for immunoPET imaging was invaluable during my PhD,

Dr. Keyu Li, Arely Perez, and Kelly Huynh for their advice, support, and contributions to this

work. I also thank Dr. Chris Waldmann for our collaboration on a linker project, and for being

a great friend with very cute animals to pet-sit. Lastly, I would like to thank my colleague and

friend Dr. Tove Olafsen, the staff members of the Crump Institute Preclinical Imaging core,

Dr. Arion Chatziioannou, Dr. Naoko Kobayashi, and other Reiter lab members, who have all

helped me with my projects, and Dr. Mike van Dam and Dr. Peter Clark for their support.

I would also like to thank the UCLA service workers, the Pharmacology department staff

members Emily Fitch, Erika Corrin, and Elizabeth Arce, the administrators at CNSI, and Dr.

Greg Payne and the Graduate Program of Biosciences for their hard work and effort to keep

everything running smoothly. I am also thankful to have worked with a number of dedicated

and amazing women graduate students in Advancing Women in Science and Engineering.

I am extremely grateful to my friends from Davis, Cal, UCLA, and my boyfriend Costa for

their continued love and support, endless jokes, and spirit for new adventures traveling and

hiking. I am happy many of them introduced me to and joined me in rock climbing, which

was a great way to destress during my PhD and has become a huge joy in my life.

A portion of the introduction appeared as a review article in the Journal of Labelled

Compounds and Radiopharmaceuticals: Tsai, W.K. & Wu, A.M. Aligning physics and

physiology: Engineering antibodies for radionuclide delivery. J. Labelled Comp. and

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Radiopharm. 61, 693-714 (2018). I would like to acknowledge the co-author Dr. Anna Wu for

manuscript writing, reviewing, and editing. The work in Chapters 2 and 3 is published in

Theranostics: Tsai, W.K. et al. Dual-modality immunoPET/fluorescence imaging of prostate

cancer with an anti-PSCA cys-minibody. Theranostics. 8, 5903-5914 (2018). I’d like to

acknowledge the co-authors Dr. Kirstin Zettlitz and Dr. Richard Tavaré for study design,

supervision and help with experiments and data analysis, manuscript review and editing, Dr.

Naoko Kobayashi for performing immunohistochemistry experiments, Dr. Robert Reiter for

help with study design, and Anna Wu for help with study design, and manuscript review and

editing. Additionally, I would like to thank everyone who contributed technical support and

advice, including Felix Bergara, Kelly Huynh, Chong Hyun Chang, Waldemar Ladno,

Theresa Falls, Olga Sergeeva, Charles Zamilpa, Dr. Arion Chatziioannou, Roger Moore, and

Jeff Calimlim.

The work in Chapter 4 is in preparation for submission. I would like to acknowledge the co-

authors Dr. Kirstin Zettlitz for help with study design, data acquisition, and manuscript review

and editing, Dr. Magnus Dahlbom for help with the dosimetry calculations, Dr. Robert Reiter

for advice with study design, and Dr. Anna Wu for study design, manuscript review and

editing. I thank Dr. Paul Yazaki for providing the A11 minibody, Dr. Erik Larsson for providing

the S-factors, and Felix Bergara, Dr. Arion Chatziioannou, Dr. Sophie Poty, Dr. Xiaoxi Ling,

Dr. Roger Slavik, Dr. Jason Lee, Dr. Naoko Kobayashi, and Johnny Guan for technical

support and advice.

Funding that has supported me during this work include NIH grants R01 CA174294, P30

CA016042, Department of Defense W81 XWH-15-1-0725, and the Dr. Ursula Mandel

fellowship. Imaging, flow cytometry, pathology core services were supported by Jonsson

Comprehensive Cancer Center (P30 CA016042).

xvii

Vita

Education

Dec 2018 PhD (expected), Molecular and Medical Pharmacology, UCLA May 2012 BA, Integrative Biology, UC Berkeley Awards and fellowships

2018 Women in Molecular Imaging Network Scholar Award, WMIC 2018 2018 Student travel stipend, WMIC 2017 Best poster: Pharmacology Departmental Retreat, UCLA 2017 Student travel stipend, WMIC 2017 AbbVie Scholar-in-Training, AACR 2017 Student scholarship, Antibody Engineering & Therapeutics 2016 Best oral presentation: Pharmacology Departmental Retreat, UCLA 2015-17 Dr. Ursula Mandel Fellowship (UCLA) 2015-16 Isabel & Harvey Kibel Fellowship (UCLA) 2015 Student scholarship, Antibody Engineering & Therapeutics 2015 Travel award, Pharmacology Department Peer-reviewed publications

Tsai WK, Zettlitz KA, Tavaré R, Reiter RE, and Wu AM. Dual-modality immunoPET/fluorescence imaging of prostate cancer with an anti-PSCA cys-minibody. Manuscript accepted for Theranostics (Sept 2018). Tsai WK and Wu AM. Aligning physics and physiology: Engineering antibody fragments for radionuclide delivery. Journal of Labelled Compounds and Radiopharmaceuticals. 2018;61:693-714. Zettlitz KA, Tsai WK, Reiter RE, and Wu AM. Dual-modality immunoPET and near-infrared fluorescence (NIRF) imaging of pancreatic cancer using an anti-prostate stem cell antigen (PSCA) cys-diabody. Journal of Nuclear Medicine. 2018;59:1398-1405. Zettlitz KA, Yamada RE, Tavaré R, Tsai WK, Collins J, Ha NS, van Dam RM, Timmerman JM, Wu AM. 18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B-cell lymphoma in human CD20 transgenic mice. Manuscript accepted for European Journal of Nuclear Medicine and Molecular Imaging (Nov 2018). Freise AC, Zettlitz KA, Salazar FB, Tavaré R, Tsai WK, Hadjioannou A, Rozengurt N, Braun J, and Wu AM. ImmunoPET in inflammatory bowel disease: Imaging CD4+ T cells in a murine model of colitis. Journal of Nuclear Medicine. 2018;59:980-985. Zhang M, Kobayashi N, Zettlitz KA, Kono E, Yamashiro J, Tran C, Tsai WK, Jiang ZK, Wu AM, Reiter RE. Near-infrared-dye labeled anti-Prostate Stem Cell Antigen (PSCA) A11 minibody enables intraoperative fluorescence imaging and targeted surgery in translational mouse models of prostate cancer. Manuscript accepted for Clinical Cancer Research (Sept 2018).

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Oral Presentations

Tsai WK, Zettlitz KA, Dahlbom M, Reiter RE, and Wu AM. 131I- and 177Lu-A11 minibody for radioimmunotherapy of PSCA-expressing prostate cancer. Oral presentation accepted for: World Molecular Imaging Congress: 2018 Sept 13. Seattle, WA. Tsai WK, Zettlitz KA, Tavaré R, Reiter RE, and Wu AM. Dual-modality immunoPET/fluorescence imaging of prostate cancer using anti-PSCA A11 cys-minibody. Oral presentation at: UCLA Department of Molecular and Medical Pharmacology Retreat (2016). Huntington Beach, CA.

Poster Presentations

Tsai WK, Dahlbom M, Zettlitz KA, Reiter RE, and Wu AM. 131I- and 177Lu-A11 minibody for radioimmunotherapy of prostate stem cell antigen-expressing prostate cancer. Poster presented at: Antibody Engineering & Therapeutics (2017). San Diego, CA. Tsai WK, Zettlitz KA, Tavaré R, Reiter RE, and Wu AM. ImmunoPET/Fluorescence imaging of PSCA-positive prostate cancer using A11 cys-minibody. World Molecular Imaging Congress (2017). Philadelphia, Pennsylvania. Tsai WK, Zettlitz KA, Tavaré R, Salazar FB, Reiter RE, and Wu AM. Dual-modality immunoPET/fluorescence imaging of prostate cancer utilizing 89Zr- or 124I-anti-PSCA cys-minibody. American Association for Cancer Research Annual Meeting (2017). Washington, DC. Tsai WK, Tavaré R, Zettlitz KA, Salazar FB, Reiter RE, and Wu AM. Dual-modality immunoPET/fluorescence imaging of prostate cancer using anti-PSCA cys-minibody. UCSF Antibody Technology Research Symposium (2016). San Francisco, CA. Tsai WK, Tavaré R, Zettlitz KA, Salazar FB, Reiter RE, and Wu AM. Dual-modality immunoPET/fluorescence imaging of prostate cancer using anti-PSCA A11 cys-minibody. Poster presented at: Antibody Engineering and Therapeutics (2015). San Diego, CA. Tsai WK, Tavaré R, Zettlitz KA, Salazar FB, Knowles SK, Reiter RE, and Wu AM. Dual-modality immunoPET and fluorescence imaging of prostate cancer. Poster presented at: World Molecular Imaging Conference (2015). Honolulu, HI.

Chapter 1: Introduction

1

1 Introduction

1.1 Overview of prostate cancer

1.1.1 Prostate cancer epidemiology

Prostate cancer is the second most diagnosed solid cancer worldwide (1). The most

significant risk factors are age, race, and family. For example, the incidence in men ages 65

and older is much higher than ages 20-64, although the advent of prostate specific antigen

(PSA) screening led to a decline in incidence in the older age groups (Figure 1.1).

Additionally, incidence in African American men is 60% higher than in white men due to

aggressiveness and disparities in care (1). Although men with low-risk disease are more

likely to die with rather than from prostate cancer, progression to metastatic castration

resistant prostate cancer (mCRPC) is lethal.


2

Figure 1.1 Incidence of prostate cancer. The incidence initially increased after PSA screening was adopted in the late 1980s due to over-diagnosis. The implementation of active surveillance in the early 1990s led to a decline in incidence, most prominently in men ages 65+. Reprinted with permission from Springer Nature (1).

1.1.2 Prostate cancer biology

Prostate cancer typically develops in the peripheral zone, in contrast to benign prostatic

hyperplasia which originates from the transitional zone. Prostatic intraepithelial neoplasia

(PIN) can develop into prostate adenocarcinoma when the androgen receptor (AR), which

normally binds the androgens testosterone and 5α-dihydrotestosterone (DHT), becomes

constitutively active. Although androgen deprivation therapy can inhibit or slow tumor growth,

it is not curative and resistance can occur. Resistance mechanisms include AR gene

amplification which increases the sensitivity of tumor cells to androgens, AR mutation so that

it can be activated by other ligands, or splicing variation that allows AR to be constitutively

produced (2).


3

1.1.3 Prostate cancer diagnosis

Prostate cancer is typically first suspected after digital rectal exam (DRE) or high levels of

prostate-specific antigen (PSA). However, patients with benign prostatic hyperplasia and

other non-cancerous conditions may also have raised PSA levels, and therefore patients

with PSA levels above 4 ng/mL may receive an accompanying test such as a biopsy.

Biopsies are performed by taking 10-12 cores with transrectal ultrasound (TRUS) guidance,

and samples are analyzed using histology. Additionally, the use of multiparametic magnetic

resonance imaging (mpMRI) overlaid with real-time US allows for targeted biospies of

suspicious lesions, which was shown to improve diagnosis in two trials (1).

1.1.4 Prostate cancer staging and stratification

The histological appearance of the biopsies is graded using the Gleason scoring system,

which describes the likelihood of the disease to spread (Figure 1.2). The score (range 2-10)

is represented as a sum of two most common patterns (ie 3+4 =7) from the biopsy with the

highest pattern (3). In 2014, an updated grading system to describe Gleason scores 6-10

was established by the International Society of Urological Pathology, indicated in Table 1.1

(4). The clinical stage is based on the tumor, node, metastasis (TNM) system, which refers

to the extent of disease spread: confined to the primary tumor, spread to regional lymph

nodes, or metastasized to the rest of the body. Typically, the metastasis pattern begins with

regional pelvic lymph nodes, spreads next to distant lymph nodes and bone, and finally to

visceral organs such as liver in advanced disease.


4

Figure 1.2: Gleason grading and scoring system. Reprinted with permission from Springer Nature (1).

Table 1.1: Pathologic Grading System of the International Society of Urological Pathology. Table based from information in (4).

Grade Gleason Characteristic

1 6 (3+3) Well-formed glands 2 7 (3+4) Predominantly well-formed glands, some poorly formed 3 7 (4+3) Predominantly poorly formed glands 4 8 (4+4, 3+5, 5+3) Only poorly formed, fused, or cribiform glands 5 9, 10 (4+5, 5+4, 5+5) Lacks gland formation


5

Patients are stratified by risk (low, intermediate, and high), which is based on blood levels of

PSA, Gleason score, and TNM (Table 1.2). Risk stratification can be used to guide staging

and management, such as the recommendations to use CT/MRI and bone scan imaging for

intermediate and high-risk patients, or active surveillance for low-risk patients.

Table 1.2: European Association of Urology (EAU) risk groups and recommendations for biochemical recurrence of localized and locally advanced prostate cancer. Table adapted with permission from (3).

1.1.5 Prostate cancer treatment

The primary treatment options for men with localized disease are expectant management

(watchful waiting and active surveillance), surgery (radical prostatectomy), and radiation

therapy (external-beam radiation therapy, and brachytherapy). After treatment, biochemical

recurrence (BCR) may occur, which is characterized by rising PSA after therapy, even

without the presentation of symptoms. If the disease advances, bone scans and imaging

tests may be conducted to identify the site of BCR and inform the next course of treatment.

Any risk Low-risk Intermediate-risk High-risk Locally advanced Localized Localized Localized

PSA < 10 ng/mL and GS < 7 and cT1-2a

PSA 10–20 ng/mL or GS 7 or cT2b

PSA > 20 ng/mL or GS >7 or cT2c

any PSA any GS cT3–4 or cN+

Do not use CT and TRUS for local staging

Do not use additional imaging for staging

For metastatic screening, include at least cross-sectional abdominopelvic imaging (s.a. CT/MRI) and a bone scan for staging purposes. For local staging, use prostate mpMRI (in predominantly Gleason pattern 4).

Use prostate mpMRI for local staging. Perform metastatic screening including at least cross-sectional abdominopelvic imaging and a bone-scan.

GS = Gleason score; PSA = prostate-specific antigen; cT = clinical T, based on a physical exam or imaging (in contrast to pT, based on pathology of removed prostate). CT = computed tomography; mpMRI = multiparametric magnetic resonance imaging; MRI = magnetic resonance imaging; PCa = prostate cancer; TRUS = transrectal ultrasound.


6

Metastatic disease is primarily first treated with androgen deprivation therapy (ADT), and

those with more advanced disease may be treated with both radiation and ADT. Failure to

respond to ADT results in mCRPC, and many drugs have been evaluated for survival

extension. For example, the anti-androgens abiraterone or enzalutamide may be given as a

first-line treatment to mCRPC patients and have been shown to extended survival by a

median of 18 months, and the chemotherapeutic docetaxel could be offered as second-line

treatment. These therapies are discussed more in depth in section 1.3.

1.2 Imaging of prostate cancer

1.2.1 Molecular imaging

Despite recent advances prostate cancer care, there is a critical demand for improved

disease detection, staging and stratification. Noninvasive imaging methods, including

ultrasound, magnetic resonance imaging (MRI), positron emission tomography (PET), and

single-photon emission computed tomography (SPECT), have been adapted to aid prostate

cancer diagnosis, and the current FDA-approved molecular imaging probes (indicated in

Table 1.4) include 99mTc-methylene diphosphonate bone scintigraphy (99mTc-MDP), 111In-

anti-PSMA 7E11 antibody (ProstaScintTM), 18F-sodium fluoride, and 11C-choline. However,

each of these probes and modalities has its limitations. For example, MRI is a highly

sensitive and reliable strategy for local staging and identification of soft tissue lesions, but

may not be sensitive enough to detect lymph node metastases or differentiate from

inflammation (5). Therefore, the use of more than one imaging probe or modality may

provide more information on the disease extent to guide treatment decisions.

1.2.2 SPECT imaging of prostate cancer

99mTc-MDP SPECT is one of the most commonly used methods to detect bone metastases

through the process of chemisorption, or accumulation at the bone surface. However, 99mTc-


7

MDP is limited in the ability to detect micrometastases, and it is not specific for prostate

cancer (6). Prostate-specific antibodies include anti-PSMA antibody 7E11 (ProstaScint),

which binds to the intracellular domain of PSMA. Therefore, although approved by the FDA,

ProstaScint is limited in the ability to image prostate cancer cells with intact membranes.

Other PSMA-targeting antibodies that bind to the extracellular domain have been developed

for SPECT imaging, including J591 radiolabeled with 99mTc and with 111In. 111In-J591 SPECT

showed 94% of the bone lesions detected by conventional CT and bone scans in patients

with mCRPC (7), and J591 radiolabeled with positron-emitters was also used for PET

imaging studies. However, as the optimal imaging time point is 5-7 days, J591 derived

fragments were also evaluated. 99mTc-J591 cys-diabody was used for same-day SPECT

imaging of mice bearing subcutaneous PSMA-positive prostate carcinoma tumors, and

specificity was confirmed through blocking studies (8). PSMA-targeted radioligands have

also been developed as SPECT imaging agents. For example, 99mTc-PSMA I&S was

recently evaluated as a tool for radioguided surgery to intraoperatively confirm lesions

detected by presurgical PET imaging, as well as exclude additional lesions (9).

1.2.3 Overview of PET imaging of prostate cancer

Molecular imaging of prostate cancer by PET has been successful in identifying primary and

metastatic disease, and it is a powerful tool to guide therapy selection, stratify patients, and

monitor response to treatment. The most commonly used PET tracer, 2-deoxy-2-[18F]fluoro-

D-glucose (18F-FDG), had limited success in differentiating primary prostate carcinoma from

hyperplasia due to the low metabolism of most prostate cancer, and therefore other PET

tracers have been developed and evaluated in preclinical and clinical studies (Table 1.3,

Figure 1.3). 11C-choline, 18F-fluorocholine, and 11C-acetate are used to image lipogenesis in

prostate cancer, while the recently approved 18F-fluociclovine (FACBC) targets amino acid

transport systems (10).


8

Biochemical target/mechanism

Radiopharmaceutical Imaging technique

Bone matrix 99mTc-MDPa 99mTc-HDPa

SPECT

[18F]Sodium fluoridea PET

Glucose metabolism [18F]Fluorodeoxyglucose (FDG)a PET

Lipid metabolism [11C]Choline (CH)a [18F]Fluorocholine (FCH) [11C]Acetate [18F]Fluoroacetate

PET

Amino acid transport [11C]Methionine PET

[18F]FACBC PET

Androgen receptor [18F]FDHT PET

Gastrin-releasing peptide receptor (GRPR)

68Ga-BAY86–7548 64Cu-CB-TE2A-AR06

PET

Prostate-specific membrane antigen (PSMA)-antibody binding

111In-capromab pendetide (ProstaScint™)a 111In-DOTA-J591 mAb 177Lu-DOTA-J591 mAb

SPECT

89Zr-DFO-J591 mAb 89Zr-DF-IAB2M (J591 minibody)

PET

Small-molecule PSMA inhibitors

99mTc-MIP-1404 123I-MIP-1095

Planar/SPECT

68Ga-PSMA 68Ga-MIP-1588 18F-DCFBC 18F-DCFPyL

PET

aFDA approved

Table 1.3: Radiopharmaceuticals for molecular imaging of prostate cancer. Table reprinted with permission from Springer Nature (11).


9

Figure 1.3: Molecular imaging strategies currently applied for prostate cancer. Figure reprinted with permission from (6).

1.2.4 Lipid metabolism-based PET imaging

11C- and 18F-fluorocholine

11C-choline and 18F-fluorocholine PET are based on the increased use of choline by cancer

cells due to increased cell proliferation, likely related to the synthesis of phosphotidylcholine

which is used in cell membrane synthesis. 11C-choline PET was able to differentiate between

benign and malignant prostate cancer in patients (12), but due to the fast half-life of 11C (20

minutes), it can only be performed where an on-site cyclotron is available. Choline labeled

with 18F (t ½ = 110 min) allows for logistical flexibility, and 11C- and 18F-choline have a pooled

sensitivity and specificity of 86 and 93% for detection of metastases (13). 11C-choline was

approved by the US Food and Drug Administration (FDA) in 2012 for use in patients with

suspected recurrence, and the EAU recommends 11C-choline PET/CT for patients with

biochemical recurrence after radiotherapy (3).


10

11C- and 18F-acetate

Acetate is typically used in cells as acetyl-coA, and uptake of 11C- and 18F-acetate in

prostate cancer is likely due to the upregulation of fatty acid synthetase (6). Although 11C-

acetate was found to be highly specific, it also has low sensitivity (68%) and similarity in

uptake between prostate cancer and benign prostate hyperplasia (BPH) (6).

1.2.5 Amino acid transport-based PET imaging

18F-fluociclovine

18F-fluociclovine (FACBC), an analog of the amino acid L-leucine which is transported at a

higher rate in prostate cancer, was approved by the FDA and European Medicines Agency

(EMA) in 2016 for detection of recurrent prostate cancer. Compared to 11C-choline, FACBC

had higher sensitivity and specificity in patients who relapsed after surgery (14). Although

FACBC uptake can overlap with BPH nodules, one study showed that combination of

FACBC and MRI reduced the number of false positives and improved positive predictive

value compared to either modality alone (15). However, another study concluded FACBC

PET/MRI had low sensitivity for lymph node metastases, and therefore it cannot replace the

standard staging method (extended pelvic lymph node dissection) (16).

1.2.6 Androgen receptor-based PET imaging

18F-fluoro-dihydrotestosterone

The androgen receptor is the main driver of prostate cancer development and progression,

and 18F-fluoro-dihydrotestosterone (18F-FDHT) PET has been used to assess AR occupancy,

but it may not be a reliable imaging agent due to the reduction of binding in patients treated

with antiandrogens. However, 18F-FDHT may potentially be used to optimize doses of

antiandrogens (11).


11

1.2.7 18F-sodium fluoride

18F-sodium fluoride (18F-NaF) is an FDA-approved PET imaging agent for bone metastases.

Similar to diphosphonates, 18F-NaF is taken up by the bone through chemisorption. Although

it has been largely replaced by 99mTc-MDP, NaF clears more rapidly and results in higher

contrast (6). A review study reported high sensitivity (89%) and specificity (91%) based on

pooled data (17), and a prospective study demonstrated 18F-NaF PET was used to detect

significantly more metastases than PSMA-targeted 18F-DCFBC (see 1.2.10) in patients with

early and metastatic castrate-sensitive disease (18).

1.2.8 Overview of antigen-specific PET imaging of prostate cancer

While cell metabolism imaging has been successfully used to detect new lesions in patients

with biochemical recurrence, the lipid synthesis and amino acid transport processes

upregulated in prostate cancer can also occur in benign tissues. Therefore, antibodies,

antibody fragments, and peptides based on targeting prostate cancer biomarkers such as

prostate-specific membrane antigen (PSMA) (19,20) and gastrin-releasing peptide receptor

(GRPR) (6,21), have also been developed for PET imaging as well as therapy.

1.2.9 PSMA antibody-based PET imaging

Prostate specific membrane antigen (PSMA) is overexpressed in local prostate cancer,

lymph node and bone metastases, and correlates with disease progression and recurrence.

Antibodies and peptides targeting PSMA have been developed for immunoPET and

fluorescence imaging (22-24), as well as therapy. Example anti-PSMA antibodies that have

been evaluated in the clinic include 89Zr-radiolabeled J591 intact antibody and the IAB2M

minibody.


12

89Zr-J591

In a Phase I/II study, 50 patients with mCRPC were injected with 89Zr-J591. For detection of

bone lesions, 89Zr-J591 immunoPET was superior (95% accuracy) to conventional bone

scintigraphy, CT, and FDG, although detection of soft-tissue lesions (60% accuracy) was

similar to individual conventional imaging methods. However, due to the longer circulation

time of intact antibodies, the optimal imaging time point was 6-8 days. Therefore, J591

derivative antibody fragments were assessed in preclinical models, including IAB2M.

89Zr-IAB2M

In a preclinical comparison study of 89Zr-anti-PSMA J591 antibody fragments, the minibody

IAB2M resulted in higher tumor uptake than the diabody and faster clearance than the intact

antibody (25), and therefore it was developed further in the clinic. In a Phase I first-in human

study, 89Zr-IAB2M PET detected bone and soft tissues lesions in 17 of 18 patients with

metastatic prostate cancer by 48 hours, compared to baseline 99mTc-MDP (9 patients) or CT

(6 patients) to detect bone lesions, or MRI (14 patients) or 18F-FDG PET (10 patients) to

detect nodes or soft tissue lesions (Figure 4b) (19). Compared to PSMA-targeting peptides

(see below), little to no uptake was observed in lacrimal and salivary glands, and liver uptake

was attributed to minibody clearance and residualizing radiometal. The sensitivity and

specificity of 89Zr-IAB2M will be further assessed in a Phase II trial (NCT03675451), which

includes comparison to mpMRI and 68Ga-PSMA-11.

1.2.10 PSMA small molecule-based PET imaging

Small molecule PSMA ligands radiolabeled with 68Ga or 18F have been widely used for PET

imaging, although they have yet to be approved by the FDA or EMA. These peptide

mimetics clear rapidly, and those based on the urea backbone have advanced the farthest in

the clinic. The binding motif is composed of a Glu-urea-Lys motif which targets the catalytic

domain of PSMA, and linked to a chelator for radiometal labeling (ie 68Ga), or a prosthetic


13

group for 18F-labeling. Although the first PSMA ligands developed for PET were labeled with

68Ga, 18F has longer half-life which allows for ease of distribution. Additionally, 18F has a

lower positron energy and therefore leads to better spatial resolution, which may allow for

improved sensitivity for detection of small tumors. 68Ga-PSMA-11 (HBED-CC), 68Ga-PSMA-

617, 68Ga-PSMA-I&T, 18F-DCFBC, 18F-DCFPyL, and 18F-PSMA 1007 are among the most

commonly used ligands for PET, typically in patients with biochemical recurrence (26,27). In

these patients, diagnosis and staging is crucial to treatment planning, and studies have

shown that PSMA ligand PET informed a change in the therapy plan of 42-75% of patients

(27).

68Ga-PSMA-11

PSMA-11 includes a chelator HBED-CC, which can complex with 68Ga. 68Ga-PSMA-11 is

commonly used in the clinic for imaging patients with biochemical reoccurrence, especially in

Europe, and several studies have shown it has superior detection rates compared to choline

(26). Additionally, in patients who are candidates for salvage therapy and have PSA levels

below 0.5 ng/mL, 68Ga-PSMA-11 has been shown to detect disease at a higher rate than

other imaging modalities such as 18F-choline (28). However, limitations include the excretion

of 68Ga-PSMA-11 through the bladder, which may obscure sites of recurrence, and diuretics

can be used to help visualize the prostate (28).

68Ga-PSMA-617

PSMA-617 was developed with the binding properties of PSMA-11 and the chelator DOTA in

order to allow for radiolabeling with trivalent radionuclides 177Lu, 90Y, and 225Ac. PSMA-617

has been used for PET imaging (68Ga-PSMA-617) as well as radiotherapy (177Lu-PSMA-617)

(26).

68Ga-PSMA-I&T


14

PSMA-I&T is a theranostic agent that has been radiolabeled with 68Ga for PET imaging, 111In

for SPECT and radioguided surgery, and 177Lu for radioimmunotherapy (26). In first-in-

human studies, 68Ga-PSMA-I&T detected metastatic lesions with lower activity in

background organs compared to 68Ga-PSMA-11, although uptake in salivary glands was still

high (29). In a retrospective study, 68Ga-PSMA-I&T was shown to be effective at guiding

pretreatment staging in high-grade disease; however, its effectiveness may be limited for

detecting metastases in patients with Gleason score 7 or less (30).

18F-DCFBC and 18F-DCFPyL

In metastatic hormone-naïve and mCRPC patients, 18F-DCFBC was shown to be superior in

lesion detection compared to conventional imaging methods, although liver metastases and

some bone metastases had low contrast (31). However, 18F-DCFBC results in high

persistent activity in the blood, which led to the development of the second generation tracer

18F-DCFPyL for more rapid excretion and superior contrast (28).

18F-PSMA 1007

PSMA 1007 was designed as a companion diagnostic to 177Lu-PSMA-617 therapy,

consisting of a scaffold based on PSMA-617 and a prosthetic group for 18F-radiolabeling

(32). Unlike other PSMA-ligands, 18F-PSMA 1007 clears through the hepatobiliary route with

minimal urinary excretion, which may be advantageous for visualizing the pelvic region. In a

retrospective study, 18F-PSMA 1007 detected lesions at a higher rate (62%) than 68Ga-

PSMA-11 (46-58%) in patients with low levels of PSA (0.2-0.5 ng/mL) after radical

prostatectomy (33).

General imitations to PSMA imaging

Off-target accumulation of small molecule PSMA ligands occurs in the salivary glands, liver,

spleen, kidneys, and intestines, as well as neovasculature which may express PSMA (27).

The location of sacral and celiac ganglia near areas of potential lymph node metastases


15

could lead to false positives and misdiagnosis, and in one study, 68Ga-PSMA-11 uptake was

found in at least one ganglion in 89% of patients evaluated (34). Other cases of false

positives include benign diseases such as sarcoidosis and Paget’s disease, which have

been shown to have PSMA-radiotracer uptake (34). The spatial resolution of the clinical PET

scanner (5 mm) may lead to false negative diagnosis of lymph node metastases, and the

lack of PSMA overexpression on neuroendocrine differentiated prostate cancer could also

lead to misdiagnosis (34). The development of tracers targeting other antigens, as well as

multi-modality imaging, could help address these limitations and increase specificity and

sensitivity.

1.2.11 Other targets for prostate cancer PET imaging

GRPR

The gastrin-releasing peptide receptor (GRPR) is overexpressed on cancers such as

prostate cancer, and at low levels of normal prostate. However, although one study showed

positive correlation between GRPR expression and Gleason score, conflicting results from

another study showed an inverse correlation between GRPR and Gleason, PSA, and tumor

size (35). Agonists such as AMBA have been radiolabeled with 68Ga for PET in preclinical

and clinical studies, and 177Lu for RIT in several prostate cancer models. Antagonists can

target GRPR without activation and internalization of the receptor and have been

hypothesized to provide superior tumor-to-organ contrast; several antagonists have bene

evaluated for PET imaging in preclinical models (35). Lastly, a dual-targeting heterodimer

bombesin-RGD that binds both GRPR and the integrin αvβ3 was radiolabeled with 68Ga and

evaluated in a Phase I trial. 68Ga-BBN-RGD PET detected more lesions compared to 68Ga-

BBN, although 68Ga-RDG was not compared (36). However, it is not yet clear when to best

use 68Ga-BBN-RGD to guide therapy selection or identify disease heterogeneity (37).


16

PSCA

Prostate Stem Cell Antigen (PSCA) is a cell-surface marker upregulated in the majority of

prostate cancers and metastases, as well as pancreatic, bladder, and stomach cancer (38).

Increased expression of PSCA correlates with more severe tumor stage, Gleason score, and

progression towards androgen independence (39,40). As a cell-surface marker, PSCA is a

promising target for prostate cancer imaging due to overexpression in primary prostate

cancer (88-94%), bone metastases (87-100%), as well as lymph nodes and liver metastases

(67%).

Anti-PSCA pre-clinical molecular imaging has previously been used to detect PSCA-positive

prostate (41,42) and pancreatic cancer (43). The anti-PSCA antibody fragment, A11

minibody (A11 Mb) was radiolabeled with 124I and 89Zr and administered to mice bearing

22Rv1-PSCA prostate adenocarcinoma s.c. xenografts (41). 124I-A11 Mb PET imaging was

superior due to higher tumor-to-soft tissue contrast and therefore used in subsequent

preclinical and clinical studies. 124I-A11 Mb successfully detected PSCA-positive intratibial

xenografts with higher sensitivity and specificity than 18F-Fluoride bone scans (42). In mice

treated with the anti-androgen MDV-3100, uptake of 124I-A11 Mb also decreased in

correlation with PSCA downregulation (42), and therefore anti-PSCA imaging may be useful

to monitor response to therapy.

1.2.12 Near-infrared fluorescence imaging for intraoperative guidance

Although molecular imaging methods can identify the whole-body extent of disease,

surgeons currently rely on visual cues (color, morphology, elasticity) and experience to

identify tumorous tissue. Difficulty visualizing positive margins, including extracapsular

extensions and lymph node metastases, can increase the probability of incomplete resection

and therefore tumor recurrence (44,45). Wide-resection strategies can damage surrounding

tissues such as rectum, urinary sphincter, and erectile nerves, which can lead to urinary


17

incontinence and impotence (46). The transition to robot-assisted radical prostatectomy has

improved oncological, continence, and potency outcomes (47), and this surgical

advancement can be complemented with disease-specific optical imaging agents for further

improvement in patient outcome.

Figure 1.4: Targeted fluorescent imaging. A, Examples of targeted fluorophores. B, Near-infrared fluorescence allows for deeper tissue penetration (mm) to visualize the targeted tumor. Figure adapted from (48) and reprinted with permission from Springer Nature.

Near-infrared fluorescence (NIRF) image-guided surgery has emerged as a tool to visualize

tumor margins for improved resection (49,50). NIR fluorophores (traditional window 700-900

nm, recently extended to 1,700 nm (51)) allow for light penetration at a greater depth

(millimeters) than fluorophores in the visible light range (micrometers) (50), which is

accompanied by decreased background fluorescence and scattering and therefore

increased signal-to-noise (Figure xx). Non-targeted fluorescence-guided surgery using

indocyanine green (ICG) and methylene blue (MB) have been approved for use in clinical

trials. ICG in particular has been used for sentinel lymph node mapping in a variety of

cancers, hepatobiliary tumor imaging, and other solid tumors by passive accumulation (52).

However, active targeting can improve tumor retention and therefore tumor-to-background

contrast. NIR fluorophores used to conjugate to target-specific probes include IRDye800CW

Excitation light source and detection instrumentation

Excitation light

Fluorescence signal

Tumor

Targeted fluorophore

Mill

imete

rs

Antibodies Peptides

Small molecules Multimodality fluorophores

Activatablefluorophores

A B


18

(available clinical grade), ICG, and the cyanines Cy5, Cy5.5, or Cy7, which are more easily

detected in a preclinical setting (50,53).

1.2.13 Fluorescent imaging of prostate cancer

Fluorescence imaging probes targeting antigens such as PSMA and PSCA have been

developed for intraoperative guidance. The anti-PSMA antibody J591-ICG was used to

successfully detect PSMA-positive tumors in vivo (22). PSMA-targeted small molecules have

been used for fluorescent imaging in preclinical studies (54,55), and the anti-PSMA antibody

MDX1201-A488 is currently being evaluated in a Phase I trial to guide robotic assisted

radical prostatectomy (NCT02048150). PSCA-targeted antibody fragments have also been

evaluated for fluorescence guided surgery of prostate cancer. In one study, Cy5-labeled anti-

PSCA antibody fragment (A2 cDb) detected tumors implanted intramuscularly to mimic

invasive growth (56). Another anti-PSCA antibody fragment (A11 Mb) labeled with

IRDye800CW identified primary orthotopic prostate tumors and metastatic lesions in mice

expressing background human PSCA (57). Both probes were used for image-guided surgery

to facilitate tumor resection, and overall survival was improved compared to groups receiving

only white light surgery.

1.2.14 Dual-modality imaging of prostate cancer

Dual-modality probes are labeled with a radioisotope for PET or SPECT imaging in addition

to a fluorophore for fluorescent imaging. Dual-modality probes can provide preoperative

information on the location and extent of the disease, as well as intraoperative surgical

guidance. Combining two labels on a single agent allows for correspondence between the

two signals.

In one study, mice with intraperitoneal prostate cancer lesions were administered 111In-anti-

PSMA antibody-IRDye800CW. By SPECT, all lesions were visualized, while only the


19

superficial lesions were identified by fluorescence, supporting the use of a dual-modality

agent to localize tumors preoperatively to inform the intraoperative surgical plan (58).

However, intact antibodies have a long circulation half-life which lead to long imaging time

points post-injection. The small molecule PSMA-11 dual-labeled with 68Ga and IRCye800CW

was used for PET/fluorescence imaging of xenografts in mice, as well as fluorescence-

guided prostatectomy in pigs (59). 68Ga-gastrin-releasing peptide receptor (GRPR)

antagonist-IRDye 650 was used for PET/ fluorescence imaging of mice with prostate cancer

xenografts, although the additional of IRDye 650 resulted in increased kidney and blood

retention (60). Although small molecules can be successfully used for dual-modality imaging,

they are also more prone to biodistribution changes due to conjugation to chelators or

fluorophores. An antibody fragment allows for shorter imaging times compared to intact

antibodies, and can be labeled with dye-to-protein ratios with minimal pharmacokinetic

changes. Dual-labeled anti-PSCA A11 Mb (Chapter 3) (61) and A2 cDb (62) were

successfully used to detect PSCA-positive lesions by immunoPET/fluorescence, and these

proof-of-principal studies support the potential for clinical development.

1.3 Therapy of prostate cancer

1.3.1 Standard therapy for patients with local disease

Expectant management

Expectant management (watchful waiting and active surveillance) may be recommended for

men with slow-growing localized prostate cancer, especially at an older age, because the

benefit of more intensive therapies such as radiation may not outweigh the side effects.

Watchful waiting involves monitoring the patient and only treating symptoms when they

arise. Active surveillance typically includes DRE and PSA tests every 6 months, as well as

biopsies as needed. Studies have shown that despite ultimately needing treatment, patients

with low-risk disease have a better quality of life if they initially receive active surveillance

instead of surgery or radiation (4).


20

Surgery

Patients who have localized disease (Stage I/II) may undergo radical prostatectomy (RP) to

remove the prostate. Robotic-assisted RP and laparoscopic RP methods allow for minimal

invasiveness and sparing of surrounding nerves compared to standard open retropubic RP,

although studies have also shown incidence in positive margins and functional outcomes are

similar (4). Patients with intermediate and high-risk disease may also receive prostate lymph

node dissection (PLND) or extended PLND.

Table 1.4: Prostate cancer stages and corresponding standard treatments. Table based on (63) from the National Cancer Institute.

Radiation Therapy

Patients who have disease localized to the prostate and/or surrounding tissues but are not

good candidates for radical prostatectomy may receive radiation therapy. External beam

Stage (TNM) Standard Treatment

I

Watchful waiting or active surveillance Radical prostatectomy External-beam radiation therapy (EBRT) Interstitial implantation of radioisotopes

II

Watchful waiting or active surveillance Radical prostatectomy EBRT ± hormone therapy Interstitial implantation of radioisotopes

III

EBRT ± hormone therapy Hormone therapy ± radiation therapy Radical prostatectomy ± EBRT Watchful waiting or active surveillance

IV

Hormone therapy Bisphosphonates EBRT ± hormone therapy Palliative radiation therapy Palliative surgery with transurethral resection of prostate Watchful waiting or active surveillance

Recurrent

Hormone therapy Chemotherapy for hormone-resistant prostate cancer Immunotherapy Radiopharmaceutical therapy/alpha emitter radiation


21

radiation therapy (EBRT) involves the delivery of ionizing x-rays to the prostate and may be

planned using MRI or CT. Hypofractionation allows for the delivery of a larger dose per

fractionation (2.5-3.4 Gy x 19-28 fractions) and a lower overall dose (64). Stereotactic body

radiotherapy (SBRT) is form of hypofractionation (6-10 Gy x 3-7 fractions) that relies on

imaging to determine the 3D coordinates for more precise delivery than EBRT, although

acute urogenital toxicity may be higher (64). In contrast to externally delivered radiation,

brachytherapy involves the implantation of low-dose rate or high-dose rate radioactive

sources (64). For low-dose rate brachytherapy, 50-125 seeds containing 125I (145 Gy) or

103Pd (120 Gy) are directly implanted into the prostate. For high-dose rate brachytherapy,

catheters delivering 192Ir (12 Gy/h) are temporarily placed in the prostate, and this therapy is

often administered in the combination with EBRT.

1.3.2 Standard therapy for patients with metastatic disease

Androgen-targeted therapy

Androgen deprivation therapy (ADT) is the primary first-line treatment for patients with

metastatic prostate cancer, and the goal is to reduce the testosterone level to <20 ng/dL

(65). ADT options include using lutenizing hormone-releasing hormone (LHRH) receptor

(also known as gonadotropin receptor hormone, GnRH) agonists, AR antagonists (ie

enzalutamide), and testosterone production inhibitors (ie abieraterone, blocks the enzyme

CYP17) (Figure 1.5). Abiraterone is given in addition to prednisone and ongoing ADT.

Enzalutamide also inhibits testosterone but through a separate mechanism of binding to AR.


22

Figure 1.5: Prostate cancer pathways and corresponding therapies. Reprinted from (66) with permission from Springer Nature.

Radium-223

Patients with bone metastases may be administered radium-223 dichloride (223RaCl2), which

mimics calcium and binds to bone stroma that is newly formed. 223Ra is an alpha-emitter with

a short tissue range of 85 µm (maximum) and causes double-stranded DNA breaks with

limited cytotoxicity to healthy bone marrow. In a Phase III study, 223RaCl2 extended median

survival (14.9 mo) compared to placebo (11.3 mo) with limited hematological toxicity (67),

and it is offered as a potential first-line treatment for patients with mCRPC (68).

Chemotherapy

The standard chemotherapy for patients with mCRPC is a combination of docetaxel and

prednisone. If patients received abiraterone or enzalutamide as first-line treatment,


23

docetaxel is the second-line of treatment (68). For men with metastases at first presentation,

data from several clinical trials support the use of ADT and docetaxel as the new standard of

care (68).

1.3.3 Radioimmunotherapy overview

Radioimmunotherapy (RIT) enables specific delivery of the therapeutic radionuclide to the

site of disease, although the long biological half-life of intact antibodies may result in dose to

normal tissue (69). Using an antibody fragment (discussed in section 1.4) may be

advantageous in limiting dosage to normal organs, especially radiosensitive tissues such as

bone marrow; however, the kidney absorbed dose must be considered for fragment sizes

that are excreted via the renal route. Radionuclides for RIT emit beta, alpha, or Auger

electrons and cause cytotoxic DNA damage by numerous mechanisms including via reactive

oxygen species, single and double stranded breaks, and inhibition of DNA damage repair

mechanisms (70,71).

1.3.3 Radionuclides for radioimmunotherapy

Beta-emitters

Beta-emitting radionuclides for RIT have a low LET (0.2 keV/mm) and a relatively long range

in tissue (one to several mm), and they can therefore damage tissue throughout the tumor,

as well as adjacent normal tissue, due to cross-fire and radiation field effect across several

millimeters (Figure 1.6). Common beta emitters include 131I, which is readily available and

has long been used for thyroid cancer treatment. Both 131I and 177Lu co-emit a gamma

photon that can be detected by SPECT imaging that can complement RIT. Conjugated

antibodies radiolabeled with 177Lu and 90Y are catabolized to yield charged radiometal

chelate metabolites that residualize in tumor cells for increased tumor retention. A

disadvantage of 90Y is it emits almost exclusively beta particles and cannot be imaged by

conventional methods; however, 90Y produces bremsstrahlung photons and Cerenkov


24

radiation which can be detected by SPECT and by optical Cerenkov luminescence imaging,

respectively (72).

Alpha-emitters

Alpha emitters have a much shorter range (a few cell diameters) and a high LET (80-100

keV/µm), and they can be effective for smaller lesions and metastases (Figure 1.6). Alpha

emitters commonly used in RIT studies include 213Bi, 223Ra, the radiohalogen 211At, and the

radiometal 225Ac (73). 225Ac decays into four alpha-emitting daughter particles, two of which

emit an imageable gamma ray, and 225Ac-radiolabeled full length antibodies have been

efficacious in tumor-cell killing in in vitro and in vivo preclinical studies (74,75), as well as

clinical studies (76,77).

Figure 1.6: Schematic representation of therapeutic radionuclides. Beta-emitters have a low linear energy transfer (LET) of 0.2 keV/mm and a path length of 1-10 mm, which may reach throughout the tumor. Alpha-emitters have a high LET (80-100 keV/µm) and a path length of 50-90 µm, which corresponds to a few cell diameters. Auger-emitters have low energy but high LET (4-26 keV/µm) and a short path length of 2-500 nm. Intracellular deposit near the nucleus is necessary for therapeutic effect. Figure reprinted from (78) with permission from John Wiley and Sons.

Beta-emitter1-10 mm

Alpha-emitter50-90 µm

Tumor

Auger-emitter2-500 nm


25

1.3.3 Radioimmunotherapy for prostate cancer

Prostate cancer metastases typically localize to bone marrow and lymph nodes, and the

prostate s also a nonessential organ; therefore, prostate cancer can easily be targeted for

RIT (79). RIT targeting a variety of antigens have been evaluated in several preclinical and

clinical studies, and anti-PSMA RIT has been developed the furthest in the clinic (Table 1.5).

Table 1.5: Clinical and preclinical targets and targeting molecules in prostate cancer RIT. Table adapted from (79) and reprinted with permission from Elsevier.

PSMA-targeted RIT in the clinic

Although only two radioimmunotherapy agents have been approved by the FDA,

ibritumomab tiuxetan (Zevalin®) and tositumomab (Bexxar®), RIT agents for prostate cancer

are currently under clinical investigation. Several clinical trials have assessed PSMA-

targeted RIT in patients with mCRPC using 177Lu-radiolabeled J591 antibody, PSMA-617

ligand, and PSMA-I&T ligand. In Phase II trial, a single dose of 177Lu-J591 was administered

to patients with mCRPC and the maximum tolerated dose resulted in over 30% PSA decline

Target Constitution and Function of

Target Targeting molecule References

PSMA Integral type II membrane protein

J591 mAb, PSMA I&T peptides

(29,80,81)

hK2 Secretory protein (kallikrein-related peptidase family)

Murine 11B6 mAb, h11B6

(82)

Her2 Receptor, transmembrane protein

mAbs, ZHER2:V2Affibody

(83,84)

PSCA GPI-anchored cell surface antigen

A11 minibody unpublished

VE cadherin CD 144, involved in forming intercellular junctions

E4G10 mAb (85)

MUC-1 Glycoprotein upregulated in androgen-independent PCa cells

m170 mAbs, anti-MUC-1 scFvs

(86,87)

GRP-R Membrane protein, G-protein- coupled receptor

AMBA bombesin (peptide)

(88,89)

Lewis Y Carbohydrate antigen

Gangliosides hu3S193 mAb (90)


26

and survival benefit (21.8 mo vs 11.9 mo) (91). Although the hematological toxicity was

reversible, another clinical trial evaluated the benefit of dose fractionation, which allowed for

the delivery of a higher cumulative MTD and similar toxicity (92). As using an alpha-emitter

may address toxicity issues, 225Ac-J591 is currently being evaluated in a Phase I trial in

patients with mCRPC (NCT03276572).

A systematic review concluded that 177Lu-PSMA-671 and 177Lu-PSMA I&T resulted in a

better therapeutic effect and less hematologic toxicity than 177Lu-J591, although PSMA-PET

imaging that allows for selection of patients was not yet available during the J591-based

studies (93). In a German multicenter retrospective study, 177Lu-PSMA-617 beta-RIT (2-8

GBq, 1-4 cycles) was given as a third-line therapy to mCRPC patients. 40% of patients

responded after a single cycle (94), and 177Lu-PSMA-617 is currently being evaluated in a

Phase III trial (NCT03511664). However, 40% of patients from the retrospective study did

not have a PSA response (>50% PSA reduction from baseline), and therefore, 225Ac-PSMA-

617 alpha-RIT was explored as an alternative and evaluated in a small number of mCRPC

patients (95). 100 kBq/kg repeated every 2 months resulted in a therapeutic effect with

xerostomia as the dose-limiting toxicity. Due to the small patient sample, comparison cannot

be made with 177Lu-PSMA-617 RIT, although surprisingly the toxicity for salivary glands was

similar and may be due to small-molecule transporters rather than PSMA-expression.

Clinical trials evaluating combination therapy with RIT are also underway, including 177Lu-

PSMA-617 and 177Lu-J591 (NCT03545165), and 177Lu-PSMA-617 and Pembrolizumab

immunotherapy (NCT03658447).

1.4 Antibodies and antibody fragments

1.4.1 Antibody fragments

The specificity of antibodies and antibody fragments renders them excellent agents for

targeted delivery of radionuclides for molecular imaging and radioimmunotherapy of cell-


27

surface targets in oncology and immunology. The intact IgG antibody (~150 kDa) is

composed of antigen-binding (Fab) domains and a constant region (Fc) that interacts with

cell-surface receptors, such as Fcγ receptors (FcγR) on immune effector cells (Figure 1.7A)

and the neonatal Fc receptor (FcRn) involved in recycling. Advantages of using a

radiolabeled intact antibody for RIT include the ability to deliver a significant dose to the

target tumors, but this strategy may also lead to high radiation exposure to normal tissues.

Therefore, protein engineering can be applied to optimize affinity, improve pharmacokinetics,

enable uniform conjugation/radiolabeling, and guide excretion towards hepatic or renal

routes, in order to match the needs of the final clinical application.

Antibodies can be reformatted into a variety of fragments, including smaller monovalent

fragments such as single domain antibodies and scaffolds (Figure 1.7B), and monospecific

and bivalent fragments such as minibodies and diabodies (Figure 1.7C). Engineered

antibody fragments that lack the Fc clear more quickly compared to full-length antibodies for

high tumor-to-background images at shorter imaging times (next day for the minibody) (96).

The antibody size contributes to the route of clearance, as fragments above the renal

threshold (60 kDa) typically clear to the liver, and smaller fragments clear via the renal route.

The shorter circulation time compared to intact antibodies also translates to lower blood

absorbed dose that may be advantageous for radioimmunotherapy. Additionally, antibody

fragments can tolerate additional modifications, including fluorophore conjugation for dual-

modality immunoPET/fluorescence imaging, without significant changes in the

pharmacokinetics.


28

Figure 1.7: Intact antibody and engineered antibody fragments. Compared to A, intact IgG antibody, engineered antibody fragments range in size, specificity, avidity, and therefore pharmacokinetic properties. Several fragments and scaffolds for radionuclide delivery have been explored in preclinical and clinical studies, including B, monovalent antibody fragments and C, bivalent monospecific fragments. Figure from (78) and reprinted with permission from John Wiley and Sons.

1.4.2 Anti-PSCA antibody fragments

Anti-PSCA intact antibody, minibody, and diabody have been developed for immunoPET

imaging of prostate cancer. Figure 1.8 illustrates how 124I-radiolabeled intact antibody and

fragments immunoPET results in high contrast tumor-to-background images, with a much

earlier optimal imaging time point for the fragments. The A11 minibody (A11 Mb) is a

bivalent scFv-CH3 dimer (~80 kDa) with a human gamma 1 hinge. A11 Mb was generated by

affinity maturation of the humanized anti-PSCA antibody fragment (hu1G8) using yeast scFv

display (97). Compared to the parental hu1G8, A11 Mb has a serine to glycine substitution in

the VL CDR3, and four other substitutions. The serum half-life (t1/2) of the minibody (5-6 h in

mice) pairs well with longer-lived radionuclides, including positron-emitting iodine-124 (124I,

t1/2 = 4.2 days) and zirconium-89 (89Zr, t1/2 = 3.3 days), both of which also have been

successfully used in the clinic (96). Tumor uptake of 124I and 89Zr-minibodies peaks around 8


29

h post injection (p.i.), and unbound tracer is sufficiently cleared by 20 h p.i. to enable imaging

in mice with high tumor-to-blood ratios. Therefore, both 124I and 89Zr-A11 Mb immunoPET

were compared in prostate cancer preclinical models. Due to the successful detection of

PSCA-positive xenografts as discussed in 1.2.3, A11 Mb was purified (98) for first-in-human

immunoPET clinical studies (NCT02092948).

Figure 1.8: Anti-PSCA immunoPET. The intact antibody and fragments with corresponding Peak tumor-to-blood contrast for the intact antibody is obtained at a longer time p.i. (168 h) compared to the minibody (21 h) and diabody (8h). Figure adapted with permission from the American Society of Clinical Oncology (96), and Zettlitz, K., unpublished.

1.5 Conclusion

Prostate cancer can be resected more easily with a dual-modality imaging agent that can

preoperatively locate the disease and intraoperatively guide the surgery. As PSCA correlates

124I Intact 168 h

124I Minibody 21 h

124I Diabody 8 h


30

with disease stage, grade, and metastatic potential, it is a promising target for molecular

imaging based on the specificity of antibodies. Additionally, patients with metastatic

castration-resistant disease who no longer respond to currently available therapies could

benefit from alternative options. Radioimmunotherapy harness the power of antibodies and

cytotoxic radionuclides, and immunoPET can also inform dosimetry and patient selection.

In this dissertation, I evaluate the use of anti-PSCA A11 cys-minibody and A11 minibody for

dual-modality immunoPET/fluorescence imaging and radioimmunotherapy of prostate

cancer, respectively. In Chapter 2, I characterize the A11 cys-minibody to confirm binding

and similar affinity to the parental A11 minibody, and I test site-specific conjugation to the

near-infrared fluorophore Cy5.5 and characterize A11 cys-minibody-Cy5.5 in vitro. In

Chapter 3, I evaluate 124I-A11 cys-minibody-Cy5.5 dual-imaging in two subcutaneous

prostate cancer models and 89Zr-A11 cys-minibody-Cy5.5 in an orthotopic model. In Chapter

4, I compare the dosimetry of 131I- and 177Lu-A11 minibody to determine which beta-emitter

results in a better therapeutic index, and I evaluate the chosen radiolabeled A11 minibody

for radioimmunotherapy of a PSCA-positive subcutaneous prostate cancer model.

Chapter 2: Development of the anti-PSCA A11 cys-minibody

31

2 Development of the anti-PSCA A11 cys-minibody

2.1 Introduction

2.1.1 PSCA-targeting antibodies

Prostate stem cell antigen (PSCA) is a glycoprotein (123 amino acids) attached to the cell

membrane by a GPI anchor. The mRNA is expressed in the basal cells of the prostate, while

the protein is expressed in the basal, secretory, and neuroendocrine cells. In normal tissues,

PSCA is expressed in the bladder and neuroendocrine cells of the stomach and colon as

detected by immunohistochemistry.

Hybridoma screening was used to generate murine anti-PSCA antibodies including 1G8,

which recognizes an epitope located in the middle of PSCA (amino acids 46-85) (40).

Humanization was achieved by grafting the complementarity-determining regions (CDRs) to

the human Fv framework of anti-p185HER2 4D5v8 (trastuzumab). Yeast scFv display was

used to generate affinity matured minibody variants A2, A11, and C5. A11 ranked second in

affinity and was selected as the best tracer for immunoPET imaging (97).


32

The A11 minibody was previously developed for immunoPET imaging of prostate cancer.

124I-A11 Mb and 89Zr-A11 Mb immunoPET were compared in preclinical models, and 124I-A11

Mb resulted in superior tumor-to-soft tissue contrast in mice bearing 22Rv1-PSCA human

prostate carcinoma xenografts (41). The longer positron ranges of 124I and 89Zr influence

resolution, and therefore partial-volume correction was applied to improve ex vivo

quantification. 124I-A11 Mb PET was also evaluated in an intratibial tumor model representing

bone metastasis and successfully detected intratibial xenografts with higher sensitivity and

specificity than 18F-NaF bone scans (42). Additionally, decreased tumor uptake of 124I-A11

Mb in response to enzalutamide treatment corresponded with PSCA downregulation, and

was detectable before the tumor volume changed. Therefore, immunoPET of PSCA may be

explored for clinical translation to monitor response to androgen deprivation therapy. The

success of A11 Mb as an immunoPET probe naturally led to the consideration of using the

minibody for dual-modality immunoPET/fluorescence imaging.

2.1.2 Site-specific labeling and the development of A11 cMb.

Antibodies can be conjugated to moieties such as fluorophores or radiometal chelators

typically by N-hydroxysuccinimide (NHS) or isothiocyanate (SCN) chemistry, which react

with the ε–amino group of lysines on the antibody and require alkaline conditions (pH 7.2-9).

However, residues such as lysines are distributed randomly throughout the antibody, and

therefore conjugation location and degree of labeling cannot be controlled. In contrast,

site-specific labeling, such as modification based on cysteine residues, glycans, peptide

tags, and unnatural amino acids, allows for more precise control of the conjugate-to-protein

ratio (99). Site-specific labeling is also advantageous to improve the homogeneity of the

product, and to reduce changes in affinity by directing conjugation away from the binding

sites.


33

In previous studies, 124I radiolabeling (randomly labels to tyrosines) did not affect PSCA-

binding of A11 Mb. Therefore, for immunoPET/fluorescence imaging, the anti-PSCA

fragment was strategized to be dual-labeled by random radiolabeling with 124I or 89Zr

(chelated by SCN-DFO, which randomly labels to lysines), and site-specifically conjugating

with the fluorophore. The fluorophore can be conjugated to solvent-exposed cysteines using

thiol-labeling strategies such as maleimide chemistry, which can occur at pH 7, favorable for

antibodies. In the CH3 domain, a C-terminal cysteine was engineered to produce the A11

cys-minibody (A11 cMb). The sequence of A11 cMb and structural model of the variable

regions are shown in Figure 2.1. Biochemical characterization of the A11 cMb and A11 cMb

conjugated with maleimide-Cy5.5 (A11 cMb-Cy5.5) are presented in this chapter.


34

Figure 2.1: A11 cys-minibody. A, Annotated A11 cMb sequence, with the complementarity

determining regions (CDRs) determined by enhanced Chothia numbering scheme. The Igκ leader is cleaved during protein secretion. The sections underlined in pink and red respectively correspond to the CDRs in the light chain and heavy chain. B, 3D model of the A11 cMb variable domains VL (light) and VH (heavy) as computed by SWISS-MODEL homology modeling software and visualized in Pymol.

METDTLLLWVLLLWVPGSTGDIQLTQSPSTLSASMGDRVTITCS

ASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSG

TDFTLTISSLQPEDFATYYCQQWGSSPFTFGQGTKVEIKGSTSG

GGSGGGSGGGGSSEVQLVEYGGGLVQPGGSLRLSCAASGFNI

KDYYIHWVRQAPGKGLEWVAWIDPENGDTEFVPKFQGRATMS

ADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSSEP

KSCDKTHTCPPCGGGSSGGGSGGQPREPQVYTLPPSRDELTK

NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

KAAAGGGGSGGC

IgKappa Leader VL

VL

VL

18 aa Linker VH

VH

VH

CH325 aa Hinge

CH3

Cysteine

CH3

VL VH

Linker

CDRs (H)

CDRs (L)

A

B


35

2.2 Methods

2.2.1 Cloning

A11 cMb was engineered by Dr. Richard Tavaré using site-directed mutagenesis to add a

cysteine residue (GGGGSGGC) to the C-terminus of the parental anti-PSCA A11 Mb (41).

Mr. Felix Bergara subcloned A11 Mb from the vector pEE12 to the vector pSecTag2 A

(Thermo Fisher).

2.2.2 Protein production and purification

Dr. Richard Tavaré used the vector pSecTag2 A to transfect human embryonic kidney cells

(HEK 293-F, Life Technologies) by lipofection (Lipofectamine 2000, Thermo Fisher). Zeocin

selection was used to generate a stable cell line, and cells were grown in triple flasks and

cultured in serum-free media (Opti-MEM, Thermo Fisher). The collected supernatant (2-4 L)

was concentrated using tangential flow filtration to 50 mL and loaded onto a HiTrap Protein L

column (GE Healthcare) in PBS using an ÄKTA Purifier (GE Healthcare). A11 cMb protein

was eluted using an increasing amount of elution buffer (0.1 M glycine, pH 2.0) using in a

three-step process (30%, 60%, 100%) for the first three purifications, and using a two-step

process (80%, 100%) in later purifications. The protein was neutralized in 1 M Tris, pH 8.2,

and dialyzed three times in a dialysis cassette (Slide-a-Lyzer, Thermo Fisher) added to 4 L

of PBS. The dialyzed protein was concentrated (Vivaspin 2, Sartorius) to ~1-2 mg/mL, filter-

sterilized, and stored at 4ºC.

2.2.3 Protein characterization

The purity, assembly, and size of A11 cMb was tested using SDS-PAGE (Coomassie stain)

analysis and size exclusion chromatography (SEC). For SEC, a Superdex 200 10/30 GL

column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the

mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) was recorded. The following


36

protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and

carbonic anhydrase (29 kDa) (Sigma). Dynamic light scattering of A11 cMb (7.5 nm) was

accomplished on a particle size analyzer (Brookhaven ZetaPALS) and compared with

Bovine Serum Albumin (66 kDa, 6.2 nm) and another minibody fragment (GA101Mb ~80

kDa, 7.2 nm), and the size distribution by number was analyzed on BIC Particle Sizing

Software.

2.2.4 Conjugation

A11 cMb was site-specifically labeled with maleimide-Cy5.5 (mal-Cy5.5) by selective

reduction of and conjugation to C-terminal cysteines (Figure 2.2). In a typical reaction, 200

µg of protein at 1 mg/mL in phosphate-buffered saline (PBS) was reduced using a 2-fold

molar excess of tris(2-carboxyethyl)phosphine (TCEP, Pierce) for 30 min at room

temperature. Equimolar mal-Cy5.5 (Amersham, GE Healthcare) was added to the reduced

A11 cMb for 2 h at room temperature. Excess mal-Cy5.5 was removed using a Micro Bio-

Spin™ Size Exclusion Columns (Bio-Rad) pre-equilibrated with PBS. Dye-to-protein ratio

(D:P) was determined by measuring the protein (280 nm, A11 cMb extinction coefficient =

133950 Mol-1 cm-1) and Cy5.5 absorbance (675 nm, preset by manufacturer) with a

spectrophotometer (NanoDrop 2000) and using the formula ratio=dye (µM)/protein (µM).

Figure 2.2: Schematic of A11 cMb site-specific conjugation. To generate A11 cMb-Cy5.5, C-terminal cysteines are reduced and react with thiol-directed maleimide-Cy5.5.

A11 cMb

S-S Cy5.5

TCEP reduction

S S

Site-specific conjugation


37

2.2.5 A11 cMb-Cy5.5 characterization

Successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC).

SEC was performed similar to analysis of A11 cMb (2.2.3). Absorbance at 280 nm (protein)

and 675 nm (Cy5.5) was recorded.

Mass spectrometry of A11 cMb and A11 cMb-Cy5.5 was completed at City of Hope.

Samples were reduced and alkylated using iodoacetamide, then digested using trypsin and

GluC. Unmodified cysteines in the peptide digests are carbamidomethylated, a post-

translational modification with a mass shift of +57. Modified cysteines, such as the C-

terminal cysteine in A11 cMb Cy5.5, show a mass shift of +1038 from the mal-Cy5.5

conjugation.

2.2.6 Affinity of A11 cMb and conjugated derivatives

The 22Rv1-PSCA cell line was previously generated by transducing 22Rv1 human prostate

cell line (ATCC CRL-2505) with retrovirus to express PSCA (100). The PC3 human prostate

cell line (ATCC CRL-1435) was previously transfected to express PSCA (100) and FLuc to

produce the PC3-PSCA-FLuc cell line. These cells were used to determine the apparent

affinity of A11 cMb by flow cytometry. A11 cMb and A11 cMb-Cy5.5 were each incubated

with 100,000 cells at concentrations varying from 0-1 µM (2 hours on ice, triplicate). Samples

were washed three times with PBS + 1% FBS + 0.02% sodium azide. Goat anti-human IgG-

Dylight 649 (Jackson ImmunoResearch Laboratory, Inc.) was the secondary antibody used

to detect bound A11 cMb as previously described (41). Samples were run on the BD-

LSRFortessa X-20 analyzer and the results were analyzed using FlowJo (v9.3.2) to

determine mean fluorescence intensity of the cells. GraphPad Prism 7.0a was used to fit the

data using the one site saturation binding model.


38

2.3 Results

2.3.1 Production/purification

Due to low protein yield from a pooled transfection (0.4 mg/L), single colony selection was

used to obtain a higher producing stable cell line (0.6-0.7 mg/L). (make comment on failed

attempts to get better producer and could be helpful to re-engineer sequence?)

2.3.2 Biochemical characterization of A11 cMb

Purity and correct assembly of the A11 cMb (scFv-CH3 dimer) was confirmed by SDS-PAGE

analysis and SEC. A11 cMb migrated as a single band approximately at the molecular

weight calculated from the sequence (80.02 kDa) (Figure 2.3A). The size of the A11 cMb

was estimated to be 70 kDa from interpolation of the SEC elution profile with known SEC

standards (Figure 2.3B). A mean hydrodynamic diameter of 7.5 nm for A11 cMb was

determined by dynamic light scattering (Figure 2.3C).


39

Figure 2.3: Biochemical characterization of A11 cMb. A, Purity and correctly assembly of A11 cMb was confirmed by SDS-PAGE. Under non-reducing conditions (NR), A11 cMb migrated as a covalently bound scFv-CH3 homodimer approximately at 80 kDa as expected for a minibody. Under reducing conditions (R), A11 cMb migrated as a scFv-CH3 monomer. C, The size of A11 cMb was estimated to be 70 kDa from interpolation with the SEC standards of 200, 66, and 29 kDa. D, The mean hydrodynamic diameter of A11 cMb was estimated to be 7.5 nm as measured by dynamic light scattering, with a size distribution ranging from 6.1-11.6 nanometers (nm).

2.3.3 Biochemical characterization of A11 cMb conjugated derivatives

Purity and correct assembly of the A11 cMb site-specifically conjugated with the near-

infrared dye mal-Cy5.5 was confirmed by SDS-PAGE analysis and size exclusion

chromatography (SEC) (Figure 2.4). A11 cMb-Cy5.5 migrated as a single band

approximately at the molecular weight as unconjugated A11 cMb (80 kDa) (Figure 2.4A).

A11 cMb-Cy5.5 and dual-labeled DFO-A11 cMb-Cy5.5 (Section 3.2.6) eluted as single

peaks (27.07 min and 27.01 min, respectively) similar to the unconjugated A11 cMb,

confirming that dimeric minibody conformation was not impaired (Figure 2.4B). Similar

A B

R NR

160110

80

60

5040

30

20

260

10

3.5

kDa

C

25 30 351.0

1.5

2.0

2.5

A11 cMb=69.8 kDa

Elution Time (min)

Lo

g m

ole

cula

r siz

e (k

Da

)

6.1 6.9 7.9 8.9 10.2 11

.60

100

Diameter (nm)

Num

ber

A11 cMb = 7.5 nm

Mean size = 7.5 nm


40

elution profiles of the absorbance at 280 nm (protein) and 675 nm (Cy5.5) demonstrated the

fluorophore was associated with the protein (Figure 2.4B). Mass spectrometry confirmed

modification with Cy5.5 was specific to the C-terminal cysteine leaving the hinge unmodified

(Figure 2.4C).

Figure 2.4: Biochemical characterization of A11 cMb-Cy5.5. A, The Cy5.5 label visualized by unstained SDS-PAGE corresponds to the A11 cMb-Cy5.5 protein band visualized by Coomassie. B, Size exclusion chromatography (SEC) elution profiles show A11 cMb, A11 cMb-Cy5.5, and DFO-A11 cMb-Cy5.5 elute in a single peak (27.00 min, 27.07 min, and 27.01 min, respectively), demonstrating the conjugations did not disrupt the minibody dimeric conformation (protein at 280 nm, Cy5.5 at 675 nm). Absorption at 675 nm (Cy5.5) is higher for the conjugated A11 cMb samples. C, Mass spectrometry of peptides resulting from trypsin and GluC digests covered all cysteine residues. Only the peptide (boxed in red) with the C-terminal cysteine showed a mass shift of +1038, which was observed in the A11 cMb-Cy5.5 sample and not the A11 cMb sample.

2.3.4 Affinity studies

Flow cytometry analysis showed strong binding of A11 cMb-Cy5.5 to PSCA-expressing cells

(22Rv1-PSCA). The selectivity for PSCA was confirmed using PSCA-negative cells (22Rv1)

A B

CoomassieWhite Light

kDa

160110

80

60

5040

30

20

260

10

3.5

CA11 cMb54 exclusive unique peptides, 99 exclusive unique spectra, 1713 total spectra, 376/378 amino acids (99% coverage)

15 4027.00

0

10

0

10

15 4027.07

0

10

0

10

15 4027.01

0

10

0

10

ab

s 67

5 n

m

ab

s 2

80

nm

A11 cMb

Elution Time (min)

A11 cMb-Cy5.5 DFO-A11 cMb-Cy5.5


41

(Figure 2.5A). The apparent affinity (KD value) calculated from saturation binding curves for

A11 cMb-Cy5.5 (16.7 ± 3.4 nM, n=4, Figure 2.5B) was comparable to that of the parental

A11 Mb (13.7 ± 1.4 nM, (42)). This was also similar to the apparent affinities of A11 cMb-

Cy5.5 (22.7 ± 2.7 nM, n=7) and dual-labeled DFO-A11 cMb-Cy5.5 (26.8 ± 2.3 nM, n=3) to

PC3-PSCA cells (data not shown) (P>0.05 for all).

Figure 2.5: Binding and affinity of A11 cMb-Cy5.5. A, Flow cytometry analysis shows A11 cMb-Cy5.5 binding specifically to 22Rv1-PSCA cells. No binding to control 22Rv1 cells was detected. B, Saturation binding study of A11 cMb-Cy5.5 (22Rv1-PSCA and 22Rv1 cells) was used to calculate half-maximal binding KD using a one-site specific binding model (n=3, GraphPad).

2.4 Discussion

A11 cMb was successfully produced, and biochemical characterization (SDS-PAGE, SEC)

was similar to parental A11 Mb as expected. The theoretical properties of both A11 Mb and

A11 cMb are shown in Table 2.1.

Studies have shown that binding affinity can be affected by conjugation of large hydrophobic

moieties, such as near-infrared fluorophores (101). Therefore, the site-specific Cy5.5

modification was directed to engineered C-terminal cysteine away from the antigen binding

APC-A: Cy5.5

22Rv1

22Rv1-PSCAA11 cMb-Cy5.5

% of Max

0.01 0.1 1 10 100 10000

100

22Rv1

22Rv1-PSCA

A11 cMb-Cy5.5 (nM)

% of Max

KD = 13 nMR2 = 0.99

A B


42

sites. Cy5.5 conjugation conditions were established (mild TCEP reduction) that resulted in

A11 cMb-Cy5.5 with a dye-to-protein ratio of 0.6-0.8, and mass spectrometry confirmed the

fluorophore was conjugated only to the C-terminal cysteine. A11 cMb-Cy5.5 retained low

nanomolar affinity binding to PSCA-positive cells and was used in following dual-modality

imaging studies (Chapter 3).

Molecular

weight

Isoelectric

point

Extinction

coefficient

Affinity to

22Rv1-PSCA

A11 Mb 78.53 kDa 7.21 133825 Mol-1 cm-1 13.7 ± 1.4 nM

A11 cMb 80.02 7.11 133950 Mol-1 cm-1 16.7 ± 3.4 nM

Table 2.1: Theoretical and affinity properties of A11 Mb and A11 cMb. Molecular weight, isoelectric point, and extinction coefficients were calculated using the ExPASy Bioinformatics Resource Portal.

Chapter 3: Dual-modality imaging

43

3 Dual-modality imaging

3.1 Introduction

3.1.1 Dual-imaging

A dual-labeled molecule ensures that the radioactive and optical signals are carried on the

same molecule, and therefore targeting and imaging will be consistent. Dual-labeled

antibodies and peptides for SPECT/fluorescence and PET/fluorescence have been

evaluated in several preclinical models (102) including PSMA-positive prostate cancer

(58,103-105). Indium-111-DOTA-girentuximab-IRDye800CW was successfully used in a

recent clinical trial for preoperative and intraoperative guidance in renal cell carcinoma

patients (106). We propose that engineered antibody fragments are highly suited for dual-

labeling without perturbation of their kinetics and targeting, and offer next-day high contrast

immunoPET and fluorescence images. A recent study successfully used a dual-labeled

antibody fragment, 124I-A2 cDb-IRDye800CW, for same-day immunoPET/fluorescence to

detect PSCA-positive patient-derived pancreatic cancer xenografts (62). Therefore, dual

immunoPET/fluorescence imaging with an anti-PSCA antibody fragment could be used to

detect PSCA-positive prostate cancer. This chapter is adapted from text and figures

originally published in (61).


44

3.1.2 ImmunoPET radionuclides

For immunoPET using larger antibody fragments such as the minibody, longer-lived

radionuclides such as 89Zr and 124I can pair well for optimal imaging contrast 24-48 hours

post-injection. 89Zr-chelate metabolites cannot diffuse through cell membranes, which results

in increased uptake at target tumor tissues but also in normal tissues. In contrast, probes

radioiodinated via standard methods metabolize to free radioiodide or iodotyrosine, which

readily efflux from the cell and are rapidly cleared from systemic circulation, resulting in

reduced background although overall activity at tumor tissues may decrease over time.

3.1.3 Fluorophores

Near-infrared fluorophores allow for greater depth penetration compared to fluorophores in

the visible light range. While IRDye800CW has been approved for use in the clinic, cyanines

such as Cy5.5 are more easily detected in a preclinical setting. Therefore, Cy5.5 was used in

the following dual-modality studies.

3.1.4 Evaluation of dual-labeled A11 cMb

The cysteine-modified humanized anti-PSCA A11 Mb (A11 cMb) was developed and dually

labeled for successive immunoPET/fluorescence imaging (Figure 3.1). The efficacy was

demonstrated in two subcutaneous models and one orthotopic prostate cancer model, and

dual-labeled A11 cMb was successfully used to detect PSCA-positive tumors with both

imaging modalities.


45

Figure 3.1: Concept of dual-modality imaging with A11 cMb. Dual-labeled A11 cMb showed specific targeting to PSCA-positive prostate tumors, thereby enabling preoperative immunoPET and successive intraoperative fluorescence image guidance. Figure originally from (61).

3.2 Methods

3.2.1 Cell lines and xenografts

Male nu/nu mice (Foxn1nu, Jackson Laboratories) were used for all tumor models. The

22Rv1 human prostate cell line (ATCC) was previously transduced with retrovirus to express

PSCA (22Rv1-PSCA) (100). To establish tumors, 0.5-1x106 22Rv1 or 22Rv1-PSCA cells in

1:1 PBS:Matrigel (BD Bioscience) were implanted s.c. in the shoulder of 8- to 10-week-old

male nu/nu mice (25-30 g) and allowed to grow for 3-4 weeks. Mice were fed alfalfa-free

food to reduce autofluorescence in the stomach contents, although they still emitted

fluorescent signal.

22Rv1-PSCA was transfected with Firefly Luciferase (FLuc)-IRES-GFP (22Rv1-PSCA-FLuc)

as previously described (42). For the orthotopic intraprostatic model, 5x103 22Rv1-PSCA-

FLuc cells in 5 µl Hank’s Buffered Saline Solution (Sigma) were surgically implanted into the

anterior lobe of the prostate (107). Mice were monitored weekly by bioluminescence, and

tumors were allowed to grow for 6 weeks post-implantation.

The PC3 human prostate cell line (ATCC) was previously transfected to express PSCA (100)

and FLuc to produce the PC3-PSCA-FLuc cell line. 0.5-1x106 PC3 and PC3-PSCA-FLuc

Cy5.5

124I

Preoperative whole-bodyImmunoPET

Intraoperative near-infrared fluorescenceA11 cMb


46

cells in 1:1 PBS:Matrigel were implanted in the shoulder of male nu/nu mice and allowed to

grow for 3-4 weeks.

3.2.2 Quantitation of PSCA cell surface expression

22Rv1-PSCA antigen binding capacity (ABC) (Quantum Simply Cellular, Bangs

Laboratories), representative of the PSCA cell surface density, was quantified by flow

cytometry as 2.1x106 ± 2.5x105 (n=2) similar to previous published results (41). PC3-PSCA-

FLuc was quantified to have an ABC of 5.2 x105 ± 2.6x105 (n=3).

3.2.3 Dual-labeling of A11 cMb

The schematic of dual-labeling is shown in Figure 3.2. A11 cMb-Cy5.5 conjugation was

completed as described in chapter 2. Preliminary imaging studies (show these images?) with

a higher D:P of 2:1 showed altered pharmacokinetics and biodistribution with higher uptake

in the liver. Therefore, the optimized dye-to-protein (D:P) molar ratio of 0.6-0.8:1 was used to

minimize impacting the biodistribution.

124I iodination of A11 cMb and A11 cMb-Cy5.5

124I-A11 cMb, and 124I-A11 cMb-Cy5.5 were prepared using pre-coated Iodogen tubes

according to the manufacturer’s instructions (Pierce). In a typical reaction, 100 µg of 1 mg/ml

A11 cMb or A11 cMb-Cy5.5 was incubated with 0.5 mCi (18.5 MBq) Na-124I (3D Imaging

LLC) in 0.1 M Tris, pH 8.0 for 10 min at room temperature.

SCN-DFO and 89Zr radiolabeling of A11 cMb

A11 cMb was conjugated with the metal chelator p-isothiocyanatobenzyl-deferoxamine (p-

SCN-Bn-DFO, B-705, Macrocyclics) for 89Zr-radiolabeling (108). In a typical reaction, 3-fold

molar excess p-SCN-Bn-DFO was added to A11 cMb or A11 cMb-Cy5.5, and the reaction

was immediately adjusted to pH 9 using 0.1 M sodium bicarbonate (1/10 volume). The


47

reaction was allowed to proceed for 30 min at 37ºC, and excess SCN-DFO was removed by

size exclusion spin column.

89Zr-oxalate (3D Imaging LLC) in 1 M oxalic acid was buffered in 2 M Na2CO3 (0.4x volume

of 89Zr) and 1 M HEPES pH 7.2-7.5 (2.5x volume of 89Zr) (108). In a typical reaction, 89Zr-

A11 cMb-Cy5.5 was prepared by incubating 100 µg of 1 mg/ml DFO-A11 cMb-Cy5.5 with

0.5 mCi (18.5 MBq) 89Zr at pH 7 for 1 h at room temperature.

Figure 3.2: Schematic of the dual-labeled A11 cMb. Site-specific labeling to the A11 cMb C-terminal cysteines (reduced with TCEP) by maleimide-thiol chemistry. For radiolabeling, 124I and SCN-DFO (chelates 89Zr) are randomly labeled to surface exposed tyrosine or lysine residues, respectively. Figure originally from (61).

3.2.4 Labeling efficiency, purification, immunoreactivity

Radiolabeled proteins were purified by size exclusion spin columns pre-equilibrated with 1%

fetal bovine serum (FBS)/PBS. Radiolabeling efficiency and radiochemical purity were

assessed by Instant Thin Layer Chromatography (ITLC strips for radiolabeled antibodies,

Biodex Medical System) with 20 mM citrate pH 5.0 as the solvent for 89Zr-labeled proteins

and saline (0.9% sodium chloride) as the solvent for radioiodinated proteins. The strips were

analyzed by gamma counting (Wizard 3” 1480 Automatic Gamma Counter, Perkin-Elmer).

The immunoreactive fraction was determined by the Lindmo method (1x106 to 50x106 cells)

(109). Radiolabeled A11 cMb (0.9-1.8 ng) was incubated with excess antigen using 22Rv1-

PSCA (50x106 cells) or PC3-PSCA (50x106 cells) in 0.5 mL 1% FBS/PBS in triplicate for 1 h

at room temperature. 22Rv1 or PC3 cells were used as a negative control. Cells were

Random labeling 124I or 89Zr (-DFO-SCN)

Site-specific labelingmal-Cy5.5 or mal-DFO

S-S Cy5.5 Cy5.5


48

centrifuged and washed twice in 1% FBS/PBS, and the cell pellet and supernatant were

assessed by gamma counter.

3.2.5 Dual immunoPET/fluorescence imaging

Mice were administered Lugol’s potassium iodide solution (Sigma-Aldrich) and potassium

perchlorate (Sigma-Aldrich) to block nonspecific thyroid and stomach uptake, respectively

(110,111). 124I-A11 cMb (n=3, bilateral 22Rv1 and 22Rv1-PSCA s.c.), 124I-A11 cMb-Cy5.5

(n=3, bilateral 22Rv1 and 22Rv1-PSCA s.c.; n=5, bilateral PC3 and PC3-PSCA s.c.), or 89Zr

A11 cMb-Cy5.5 (n=5, orthotopic 22R1-PSCA) (20-90 µCi, 0.74-3.33 MBq, 10-20 µg) in 0.1

mL saline was injected via tail vein. Immediately after injection or at 22 h p.i., mice were

anesthetized with 1.5% isoflurane and 10-min static scans were acquired using an Inveon

microPET scanner (Siemens), followed by 1-min microCT scans (CrumpCAT (112)).

Following microPET/CT scans, post-mortem NIRF imaging was completed with the skin

removed in the IVIS Lumina II (Perkin Elmer) using the following settings: emission = Cy5.5,

excitation = 675 nm, 1 s exposure. The tumors and organs were excised and imaged ex vivo

to compare relative fluorescence signals without obstruction by other organs.

3.2.6 Biodistribution

Following the final imaging time point, blood and organs were collected, weighed, and

gamma-counted for ex vivo biodistribution. Uptake was calculated as % injected dose per

gram of tissue (%ID/g) based on a standard containing 1% of the ID.

3.2.7 Image analysis

MicroPET images were reconstructed by ordered subset expectation maximization-

maximum a posteriori (OSEM-MAP, Inveon Acquisition Workplace). Images were analyzed

and displayed using AMIDE (113). MicroPET/CT overlays are displayed as maximum


49

intensity projections (MIP) of the whole-body or 0.2 mm transverse slices. Fluorescence

images were analyzed using the Living Image software (Perkin Elmer). Biodistribution

graphs are depicted as box-and-whiskers (min to max), using GraphPad Prism (version 7.0a

for Mac OS X, GraphPad Software). Statistical analysis was performed using multiple

Student t tests, the significance threshold was set at 0.05, and p-values were corrected

using the Holm-Šídák method. All values in the tables are reported as mean ± SD.

3.2.8 Histology and immunohistochemistry

Freshly isolated tumors and organs of interest were fixed in 10% formalin/PBS. Samples

were embedded in paraffin (UCLA Translational Pathology Core Laboratory) and sectioned

into 4 µm sections, which were then stained with H&E or anti-PSCA antibody (ab56338,

Abcam).

3.3 Results

3.3.1 Radiolabeling

124I-A11 cMb and 124I-A11 cMb-Cy5.5 had specific activities of 1.0-3.9 µCi/µg (37-144

KBq/µg) and 0.6-5.9 µCi/µg (22-219 KBq/µg), respectively, and both probes retained high

immunoreactivity to PSCA-positive cells (>70%) (Table 3.1).

89Zr-A11 cMb-Cy5.5 had specific activities of (insert range, average 5.8 µCi/µg) and retained

high immunoreactivity to PSCA-positive cells (>70%) (Table 3.1).

124I-A11 cMb 124I-A11 cMb-Cy5.5 89Zr-A11 cMb-Cy5.5

Radiolabeling Efficiency

81 ± 21% n=3

69 ± 23% n=6

99 ± 1% n=3

Radiochemical Purity

99.1 ± 0.6% n=3

99.1 ± 0.90% n=6

99.7 ± 0.1% n=2

Specific Activity 2.5 ± 1.5 µCi/µg n=3

3.3 ± 2.1 µCi/µg n=6

5.8 ± 0.7 µCi/µg n=3


50

Immunoreactive fraction (r) for 22Rv1-PSCA

>0.76 n=3

>0.76 n=4

0.77 n=1

Immunoreactive fraction (r) for PC3-PSCA

n/a 0.82 n=1

0.80 n=1

Values are reported as mean ± standard deviation (SD).

Table 3.1: Radiolabeling and immunoreactivity for 124I-A11 cMb, 124I-A11 cMb-Cy5.5, and 89Zr-A11 cMb-Cy5.5. Table originally from (61).

3.3.2 ImmunoPET/fluorescence imaging in PSCA-high s.c. model

For the 22Rv1-PSCA s.c. model, 20 µg of 124I-A11 cMb or 124I-A11 cMb-Cy5.5 with a final

D:P of 0.7:1 was injected into nude mice. High contrast immunoPET images acquired 22 h

p.i. showed specific uptake of 124I-A11 cMb or 124I-A11 cMb-Cy5.5 in the 22Rv1-PSCA

tumors compared to minimal uptake in the antigen-negative 22Rv1 tumors, blood, and

background tissues (Figure 3.3A, B).


51

Figure 3.3: 124

I-A11 cMb-Cy5.5 immunoPET/fluorescence of 22Rv1-PSCA s.c tumor-bearing

mice. A, 124

I-A11 cMb and B 124

I-A11 cMb-Cy5.5 PET/CT scans show antigen specific uptake in 22Rv1-PSCA tumors (+, left shoulder) and minimal nonspecific uptake in 22Rv1 (-, right shoulder) tumors at 22 h post-injection (nude mice, n=3 per group). Images are represented as whole body maximum intensity projections (MIPs). C, Ex vivo biodistribution (22 hours p.i.) confirms high uptake in 22Rv1-PSCA tumors and low activity in all other tissues. The addition of Cy5.5 at a low dye-to-protein ratio did not alter biodistribution. D, E, Post-mortem Cy5.5 fluorescent images show specific signal in PSCA-positive tumors. The signal from the stomach (St) is due to autofluorescence. Scale units R.E.= Radiance Efficiency. Figure originally from (61).

0 5 10 15 20

Kidney

Liver

Blood

22Rv1 (-)

22Rv1-PSCA (+)

%ID/g

− 124I-A11 cMb

− 124I-A11 cMb-Cy5.5

124I-A11 cMb, 22 h p.i.

10

0.5

%ID

/g

+ - +- +

-

+ - -+

A

B

D

E

C

(+) (-)

8.9

4.5

R.E. x 108

11

4.2

+ -

R.E. x 108

10

0.5

124I-A11 cMb-Cy5.5, 22 h p.i.

+-

10

0.5

%ID

/g+ +- -

St


52

124I-A11 cMb 124I-A11 cMb-Cy5.5

%ID/g ± SD %ID/g ± SD

22Rv1-PSCA 12 ± 4.2 12 ± 1.3

22Rv1 1.7 ± 1.0 1.2 ± 0.6

Blood 3.3 ± 0.1 1.9 ± 0.5

Heart 1.2 ± 0.1 0.7 ± 0.3

Lung 1.7 ± 0.1 1.2 ± 0.1

Liver 0.6 ± 0.01 0.9 ± 0.1

Kidney 1.1 ± 0.04 1.0 ± 0.1

Spleen 0.7 ± 0.1 0.5 ± 0.1

Stomach 2.1 ± 0.3 1.3 ± 0.04

Intestine 0.4 ± 0.02 0.2 ± 0.1

Muscle 0.2 ± 0.01 0.1 ± 0.01

Pos:Neg Tumor 8.1 ± 2.8 13 ± 7.7

Pos Tum:Blood 3.7 ± 1.2 6.6 ± 1.9

Neg Tum:Blood 0.5 ± 0.3 0.6 ± 0.2

Pos Tum:Muscle 54 ± 20 125 ± 39

n=3 n=3

%ID/g: % injected dose per gram; Tum = Tumor Values are reported as mean ± SD. P=n.s. for all tissues

Table 3.2: Ex vivo biodistribution of 124I-A11 cMb and 124I-A11 cMb-Cy5.5 in 22Rv1-PSCA tumor-bearing mice. Table originally from (61).

Specific uptake in the PSCA-positive tumors was confirmed by ex vivo biodistribution at 22 h

p.i., with similar values for both 124I-A11 cMb-Cy5.5 (12 ± 1.3 %ID/g) and 124I-A11 cMb (12 ±

4.2 %ID/g, P>0.05) (Figure 3.3C, Table 3.2). Antigen specific uptake in 22Rv1-PSCA tumors

was significantly higher than nonspecific uptake in 22Rv1 tumors, resulting in positive-to-

negative tumor ratios of 13:1 and 8:1, for 124I-A11 cMb-Cy5.5 and 124I-A11 cMb, respectively

(Table 2). Importantly, no significant difference in uptake, clearance, and biodistribution

between the single (124I-A11 cMb) and the dual-modality (124I-A11 cMb-Cy5.5) tracer was

observed (Figure 3.3C, , Table 3.2).

Post-mortem fluorescent imaging was completed with the skin removed in order to mimic an

intraoperative setting. In mice injected with 124I-A11 cMb-Cy5.5, strong fluorescent signal


53

was detected in the 22Rv1-PSCA tumors with minimal background signal in 22Rv1 tumors

and surrounding muscle (Figure 3.3D). Ex vivo optical imaging of the resected tumors

allowed comparison of relative fluorescence signals without obstruction by other organs.

Consistent with the in situ results, high fluorescent signal was seen in the 22Rv1-PSCA

tumors compared to 22Rv1 tumors (Figure 3.3E).

3.3.3 ImmunoPET/fluorescence imaging in PSCA-moderate s.c. model

A subcutaneous PC3-PSCA tumor model was used to test the feasibility of imaging

moderate levels of PSCA (5.2 ± 2.6x105 antigens/cell). MicroPET/CT of nude mice (n=5)

bearing PC3-PSCA and control PC3 tumors showed high uptake of 124I-A11 cMb-Cy5.5 in

positive tumors at 22 h p.i. Nonspecific uptake in the stomach and thyroid is visible in images

set at a scale of 0.2-2 %ID/g (Figure 3.4A), as these organs scavenge free iodine and were

likely incompletely blocked. The ex vivo biodistribution confirms the imaging results with

significantly higher uptake in PC3-PSCA tumors (2.9 ± 0.6 %ID/g) compared to PC3 tumors

(0.7 ± 0.4 %ID/g) (P<0.01), resulting in a positive-to-negative tumor ratio of 5.6:1, and PC3-

PSCA tumor-to-muscle ratio of 54:1 (Figure 3.4B, Table 3.3).

Ex vivo optical imaging allowed comparison between the PC3-PSCA tumors, which were

detected by Cy5.5 fluorescence, and PC3 control tumors, which had little to no fluorescence

signal (Figure 3.4C). Ex vivo analysis showed expected autofluorescence in (stomach,

intestines) and additional signal in liver, kidneys, and bladder due to tracer clearance (

Figure 3.5).


54

Figure 3.4: 124

I-A11 cMb-Cy5.5 immunoPET/fluorescence of PC3-PSCA s.c tumor-bearing

mice. A, 124

I-A11 cMb-Cy5.5 PET/CT at 22 h p.i. (n=5) show specific uptake in subcutaneous PC3-PSCA (+) tumors and no nonspecific uptake in PC3 tumors (-). Low uptake is seen in thyroid (Th) and stomach (St) at this scale due to incomplete blocking, and in bladder (Bl) due to clearance. Images are represented as whole body MIPs. B, Ex vivo biodistribution (22 h p.i.) confirms higher %ID/g uptake in PC3-PSCA tumors than PC3 tumors. C, Ex vivo fluorescence (Cy5.5) imaging revealed strong fluorescent signal in PC3-PSCA tumors and excellent contrast to PC3 control tumors. R.E.= Radiance Efficiency. Figure originally from (61).

0.5

2

0.2

%ID

/g

–+

–+

– +– +– +

St

Th

R.E. x 108

2.3

1.5

(-) (+)

0 1 2 3 4

Stomach

Kidney

Liver

Blood

PC3 (-)

PC3-PSCA (+)

%ID/g

124I-A11 cMb-Cy5.5, 22 h p.i. A

B C


55

89Zr-A11 cMb-Cy5.5

%ID/g ± SD

Prostate Tumor 3.1 ± 0.5 Blood 0.9 ± 0.1

Heart 2.6 ± 0.7 Lung 2.4 ± 1.6

Liver 18 ± 2.7

Kidney 12 ± 1.2

Spleen 5.1 ± 2.1 Stomach 1.1 ± 0.2

Intestine 2.6 ± 0.3 Bone 4.4 ± 2.3

Muscle 0.4 ± 0.1

Testes 2.9 ± 1.4

Pros Tum:Blood 3.4 ± 0.2 Pros Tum:Muscle 7.7 ± 1.0

n=4, except blood n=3

Table 3.3: Ex vivo biodistribution of 124I-A11 cMb-Cy5.5 in PC3-PSCA tumor-bearing mice. Table originally from (61).

Figure 3.5: Ex vivo biodistribution of 124I-A11 cMb-Cy5.5 fluorescence signal compared with autofluorescence. A,. Fluorescent signal is specific to the PC3-PSCA tumors, and signal is also present in the organs of clearance (liver, kidney, and bladder). High autofluorescence is present in the stomach and intestines due to the food. B, A non-tumor-bearing mouse biodistribution confirmed autofluorescence in the stomach, intestine, and testes. R.E.= Radiance Efficiency. Figure originally from (61).

3.3.4 ImmunoPET fluorescence imaging in intraprostatic orthotopic model

In order to test dual-imaging at the natural site of prostate disease, an orthotopic model was

assessed. These studies were conducted using 89Zr because signal from 124I clearance to

R.E.x109

6.5

1.4

Heart Lungs

Bone

PC3

PC3-PSCA

Sem ves, prostate, bladder

Testes

Muscle

Tumors

Stomach

Intestines

Liver

Kidneys

Spleen

HeartLungs

Liver Kidneys

Spleen

StomachIntestine

Testes Sem ves, prostate, bladder

Stomach contents

BoneMuscle

R.E.x107

280

9.9

A B


56

the bladder interfered with visualization of activity accumulated in the prostate (data not

shown). Mice were intraprostatically implanted with 22Rv1-PSCA-Fluc-GFP cells, and

tumors grew extensively with significant bioluminescent signal (n=4) or low signal (n=1),

which was designated “limited disease” (Figure 3.6).

Figure 3.6: Comparison of extensive and limited intraprostatic tumors. A, 22Rv1-PSCA-GFP-Fluc intraprostatic tumors were imaged weekly by bioluminescence to determine the time point for PET/fluorescence imaging. A representative prostate tumor in a mouse with extensive disease was quantified in comparison to the bioluminescent signal from a tumor with limited disease, resulting in a 2 log difference of radiance signal. Rad= radiance. B, C,

The bioluminescence signal from extensive disease correlates with the corresponding 89Zr-A11 cMb-Cy5.5 PET and fluorescence images of the resected prostate tumor. R.E.= Radiance Efficiency. Figure originally from (61).

89Zr-A11 cMb-Cy5.5 immunoPET at 22 h p.i. showed specific uptake in the prostate tumors

with extensive disease compared to minimal prostate signal in the mouse with limited

disease (Figure 3.7A). Ex vivo biodistribution at (22 h p.i.) confirmed specific uptake in the

22Rv1-PSCA tumors (3.1 ± 0.5 %ID/g), and the tumor-to-blood ratio was quantified to be

3:1. Due to the residualizing nature of the radiometal, activity in organs of clearance is

5

0.5

%ID

/g

8

1

Rad x 106

8

1

Rad x 108

R.E.x108

3.1

1.0

A

B

C

Limited diseaseExtensive disease

R.E.x108

3.1

1.0

5

0.5

%ID

/g


57

retained at high levels compared with the iodinated A11 cMb (liver: 18 ± 2.7 %ID/g, kidneys:

12 ± 1.2 %ID/g, spleen: 5.1 ± 2.1 %ID/g) (Figure 3.7A-B, Table 4 need to add). Nonspecific

uptake in the bone is due to accumulation of free 89Zr. Importantly, the prostate tumor was

clearly distinguished by post-mortem fluorescence imaging compared to adjacent seminal

vesicles and bladder, which supports the feasibility of fluorescence guidance for prostate

cancer surgery (Figure 3.7C). PSCA-positive tumor growth in the prostate and absence of

neoplastic tissue in the surrounding seminal vesicles was confirmed by H&E and anti-PSCA

immunohistochemistry (IHC) (Figure 3.7D).

Figure 3.7: 89Zr-A11 cMb-Cy5.5 immunoPET/fluorescence of mice bearing 22Rv1-PSCA intraprostatic tumors. A, 89Zr-A11 cMb-Cy5.5 PET/CT (22 h p.i.) of nude mice (n=4) with intraprostatic orthotopic tumors (white dotted circle), compared to a mouse (n=1) with limited

A

C

D

Prostate tumor Sem ves

H&

EP

SC

A

B

89Zr-A11 cMb-Cy5.5, 22 h p.i.: Extensive Disease Limited disease

SV

Prostate tumor

B

Testes

Bone

Muscle

4.0

0.0

R.E. x 103

B

5.0

0.5%

ID/g

L

K

B

200 µm

0 5 10 15 20 25

Bone

Spleen

Kidney

Liver

Blood

22Rv1-PSCA

%ID/g


58

disease. Top: coronal whole body MIPs, bottom: 0.2 mm transverse sections, which do not include the bladder, black dotted circle). B, Ex vivo biodistribution (22 h p.i.) confirms higher %ID/g uptake in 22Rv1-PSCA prostate tumors compared to blood, and high clearance to the liver and kidney. C, Cy5.5 fluorescence signal is specific to the resected prostate with little to no signal in surrounding seminal vesicles, bladder, or background tissues (testes, bone, and muscle). R.E.= Radiance Efficiency. D, H&E confirms tumor growth in the prostate, which stained positively for PSCA while surrounding seminal vesicles were negative for PSCA. B– Bladder, K– Kidney, L– Liver, SV– Seminal Vesicle. Figure originally from (61).

3.4 Discussion

Primary prostate cancer and regional lymph node metastases can be treated by surgical

resection. However, patients often suffer from over and under-treatment due to inaccurate

diagnosis or inability to visualize positive margins during surgery. Dual-modality

PET/fluorescence imaging could provide non-invasive whole-body disease detection and

intraoperative fluorescence guidance, and therefore improve prostate cancer treatment.

In this study, the anti-PSCA antibody fragment A11 cys-minibody (A11 cMb) was used to

generate a novel dual-modality imaging probe. A11 cMb was conjugated with the near-

infrared dye Cy5.5 and radiolabeled with either 124I or 89Zr (124I-A11 cMb-Cy5.5 and 89Zr-A11

cMb-Cy5.5). Both tracers showed specific targeting to PSCA-expressing prostate cancer in

vivo resulting in whole body immunoPET scans visualizing the tumors and fluorescence

imaging distinguishing PSCA-positive cancer from surrounding healthy tissue.

Dual-labeled 124I-A11 cMb-Cy5.5 and 89Zr-A11 cMb-Cy5.5 retained high immunoreactivity for

PSCA-positive cells similar to 124I-A11 cMb (Table 1), demonstrating that dual-labeling did

not impair binding. Fluorophore conjugation can also affect the pharmacokinetics and

biodistribution of antibodies and small ligands (50,58), typically in the form of enhanced liver

uptake and faster blood clearance. 124I-A11 cMb-Cy5.5 with a dye-to-protein ratio (D:P) of

0.7-0.8:1 demonstrated similar tumor uptake and minimal changes in biodistribution

compared with 124I-A11 cMb.


59

Fluorescence-image guided surgery has been shown to improve tumor resection in

preclinical studies and several agents have entered clinical trials (48). Furthermore, dual-

modality imaging is increasingly studied in both preclinical and clinical studies (62,102,106).

Imaging with a dual-labeled antibody ensures similar biodistribution in both modalities and

facilitates clinical translation by avoiding separate testing of distinct imaging tracers. While

immunoPET provides whole-body, quantitative information with unlimited depth, it suffers

from low resolution and time constraints due to radioactive decay. NIRF fluorophores, on the

other hand, show low depth of penetration (114) but excellent cell-level resolution. Unlike the

radiolabel, the fluorophore does not have a physical half-life (cellular half-life = 3.9 days

(101)) and therefore allows longer detection of the signal for surgical guidance.

PSCA is expressed in virtually all prostate cancers (83-100%) as well as in prostate cancer

metastases to bone and lymph nodes (38,40,115,116). However, a wide range of PSCA

expression levels, correlating with progression and prognosis, was found by quantitative

reverse transcriptase-PCR analysis of clinical prostate metastases specimens (115).

Because increased PSCA corresponds with increased Gleason score and clinical stage, we

therefore evaluated the dual-modality A11 cMb in two s.c. models with different levels of

PSCA expression: 22Rv1-PSCA (high-PSCA) and PC3-PSCA (moderate PSCA).

Importantly, 124I-A11 cMb-Cy5.5 immunoPET/fluorescence imaging resulted in high contrast

PET and fluorescent images in both high- and moderate PSCA-positive prostate cancer

models. Although antigen expression does not necessarily correlate with tumor uptake, as

other factors such as tumor vasculature can affect accessibility, the dual-modality A11 cMb

could facilitate detection in different stages of prostate cancer.

The longer-lived radionuclides 124I and 89Zr are most commonly used for labeling antibodies,

and in this study, we compared specific tumor uptake and contrast to normal tissue of both

124I- and 89Zr-labeled A11 cMb-Cy5.5 in preclinical prostate cancer models. 124I and 89Zr differ


60

regarding their physical properties, protein conjugation chemistry, and biological metabolism

which can lead to significant differences in uptake and retention of the radioactive signal. 124I

is a nonresidualizing radiolabel, and upon internalization and degradation of radioiodinated

proteins, metabolites including iodotyrosine freely diffuse from the cell, leading to low

background and thereby high contrast. These characteristics are advantageous when

imaging targets retained on the cell surface, like PSCA that has previously been shown to

internalize slowly (41). Concordantly, immunoPET studies using 124I-labeled parental A11 Mb

(41), 124I-A11 cMb and 124I-A11 cMb-Cy5.5 achieved high-contrast images in the

subcutaneous models (22Rv1-PSCA, PC3-PSCA). A previous study using 124I-A11 Mb to

image intratibial xenografts supports the hypothesis that the very low background of 124I-A11

cMb-Cy5.5 will be advantageous for targeting bone metastases (42). In contrast, free 89Zr

accumulates in bone and may result in false positives (117).

A major benefit of imaging with a residualizing radiometal (89Zr) is better signal retention, as

89Zr-radiometabolites get trapped in the cell upon internalization, and higher spatial

resolution due to a shorter mean positron range (1.1 mm for 89Zr vs 3.0 mm for 124I). These

factors are crucial when imaging small structures like the prostate, hence 89Zr-A11 cMb-

Cy5.5 was used to image orthotopic prostate cancer. A further advantage compared with 124I

is that 89Zr is not excreted through urine, assuring minimal interference from activity in the

bladder.

89Zr-A11 cMb-Cy5.5 immunoPET/fluorescence imaging of the orthotopic model

demonstrated successful visualization and delineation of 22Rv1-PSCA tumors growing at

the site of natural disease, which is an important finding as tumor localization and

accessibility are known to affect tumor targeting and tracer uptake, and may account for the

difference in tumor uptake between the orthotopic and s.c. models. These promising results

and the fact that the dual-modality tracer is based on a humanized antibody fragment

suggest that 89Zr- and 124I-labeled A11 cMb-Cy5.5 could be clinically translated for imaging


61

and surgical guidance in prostate cancer. The results from an ongoing Phase I trial

evaluating 124I-parental A11 Mb for immunoPET (NCT02092948) will inform on the

pharmacokinetics of radiolabeled minibody in patients.

Future studies should include surgical studies to mimic the clinical setting (radical

prostatectomy) and confirm a survival benefit of resecting tumors guided by real-time

fluorescence imaging. Furthermore, more clinically relevant metastatic prostate cancer

models should be imaged to ensure that positive pelvic lymph nodes are detectable both

preoperatively using 89Zr-A11 cMb-Cy5.5 immunoPET and intraoperatively using

fluorescence, as the current clinical standard using frozen section histology to confirm which

lymph nodes to resect is time-intensive (118).

Dual-modality imaging can also be applied to antibodies against other prostate cancer

targets. For example, GRPR has been successfully targeted by 68Ga-HZ220-IRDye 650 in

mice bearing PC3 xenografts for PET/NIRF imaging (104), as well as by 68Ga-IRDye800CW-

BBN in a first-in-human study in patients with glioblastoma (119). Antibodies and peptides

targeting PSMA have been extensively explored for PET (120), and more recently validated

for optical imaging (50) and dual SPECT/fluorescence imaging (58,102,103). Dual-labeled

111In-PSMA-targeting-urea-IRDye800CW (103) and 111In-anti-PSMA antibody D2B-

IRDye800CW (58) successfully detected prostate cancer lesions in mice by SPECT/NIRF,

and the anti-PSMA antibody MDX1201-A488 is currently being evaluated for FIGS in

patients receiving robotic assisted laparoscopic prostatectomy (NCT02048150). However,

PSMA is also expressed in normal tissues, such as the ganglia close to the location of

typical lymph node metastases, which could result in false positives, and PSMA

overexpression is not present in 10% of patients (27). Therefore, it would be useful to also

explore PSCA as an additional target for prostate cancer diagnosis and therapy.


62

In summary, specific targeting to human PSCA-positive prostate cancer was achieved by in

vivo administration of 124I-A11 cMb-Cy5.5 and 89Zr-A11 cMb-Cy5.5 for immunoPET and

fluorescence imaging. Dual-modality imaging was successful in s.c. models with high and

moderate PSCA expression, demonstrating the ability of the A11 cMb to target a range of

antigen expression. In the intraprostatic model, fluorescence clearly distinguished the

prostate tumor from surrounding relevant tissues. This work has translational potential for

noninvasive preoperative whole-body imaging and additional real-time intraoperative

guidance in PSCA-positive prostate cancer.

Chapter 4: Radioimmunotherapy of PSCA-positive prostate cancer

63

4 Radioimmunotherapy of PSCA-positive prostate cancer

4.1 Introduction

Patients with metastatic castration resistant prostate cancer (mCRPC) are often treated with

anti-androgens, but the disease eventually progresses. Current second generation androgen

deprivation therapies such as abiraterone or enzalutamide may extend survival by 1-2 years;

however, 20-40% of patients do not respond, and almost all responders develop escape

mechanisms and secondary resistance (121). Therefore, new therapy options must be

explored. Radioimmunotherapy (RIT) uses an antibody to deliver cytotoxic radionuclides with

beta, alpha, or Auger-emissions which cause double-stranded breaks, reactive oxygen

species, and ultimately cell death. While beta-emitters have a longer range in tissue (up to

few mm) and can induce damage via cross-fire, alpha-emitters have a shorter range (few

cell diameters) and are typically more effective for smaller lesions (Figure 4.1). RIT is most

widely used to treat lymphoma and leukemia, but recently it has been explored for treating

solid tumors such as prostate cancer (79).


64

Figure 4.1: Schematic of RIT radionuclides. Figure reprinted from (78) with permission from John Wiley and Sons.

Several targets have been used in preclinical and clinical RIT studies for prostate cancer,

such as prostate specific membrane antigen (PSMA) (91,122), human kallikrein-related

peptidase 2 (82), and prostate stem cell antigen (PSCA) (123). PSMA-targeting agents

radiolabeled with the beta-emitter 177Lu have advanced the furthest in the clinic, including the

small molecules 177Lu-PSMA-617 and 177Lu-PSMA I&T (93), as well as 177Lu-J591 intact

antibody. However, 177Lu-PSMA-617 treatment resulted in xerostomia due to uptake in the

salivary glands (94). In contrast, the J591 intact antibody does not go to the salivary glands,

and the toxicity observed in patients who received a single dose of 177Lu-J591 was instead

myelosuppression that was reversible (91). J591 antibody could be a viable alternative, as

well as fragments such as the minibody (89Zr-IAB2M has been tested in patients as a PET

agent (19)). Alternative targets including PSCA should be explored, which may improve the

number of responders and even allow for combination RIT of more than one target. Prostate

stem cell antigen PSCA is a cell-surface target with overexpression on the majority of

primary and metastatic prostate cancer, low expression on normal prostate, stomach, and

bladder, and little to no expression in radiosensitive tissues (bone marrow, kidney). Upon

Beta-emitter1-10 mm

Alpha-emitter50-90 µm

Tumor

Auger-emitter2-500 nm


65

binding, PSCA internalizes slowly and is not shed, and therefore it may be a promising target

for RIT.

The goal of RIT is to deliver a high radiation dose to the tumor and minimize dose to normal

tissues (therapeutic index), especially radiosensitive organs such as the kidney and bone

marrow. The therapeutic index depends on the size and affinity of the antibody, radionuclide

properties, antibody-radionuclide metabolism, and target expression. Using an antibody

fragment, such as the minibody, may be advantageous for maximizing the therapeutic index

due to rapid serum clearance compared to intact antibody. 124I- and 89Zr-anti-PSCA A11

minibody (A11 Mb) immunoPET was previously used to detect prostate cancer in s.c. and

intratibial bone metastasis models, and 124I-A11 Mb was used to monitor response to anti-

androgen therapy (41,42). Therefore, we hypothesize that the A11 Mb could be radiolabeled

with a therapeutic radionuclide for RIT of prostate cancer (Figure 4.2).

Figure 4.2: Schematic of RIT using radiolabeled A11 Mb. A, A11 Mb targets PSCA-positive prostate cancer cells, and the beta-emission from 131I or 177Lu reaches throughout the tumor and causes cytotoxic effects, including DNA and ROS damage. B, A11 Mb is radiolabeled with 124I for PET imaging and 131I for RIT. A11 Mb is conjugated to SCN-DFO or p-SCN-Bn-CHX-A”-DTPA for 89Zr and 177Lu radiolabeling, respectively.

Beta-emitters have several characteristics that may be beneficial for RIT of solid tumors

including the ability to kill neighboring tumor cells by bystander effect. The beta-emitters

iodine-131 (131I, t1/2 = 8.0 d) and lutetium-177 (177Lu, t1/2 = 6.7 d) offer different properties that

PSCA+ Tumor Cell

B

– Lys –DTPAor DFO

124I � PET131I � RIT

89Zr � PET177Lu � RIT

– Tyr

A11 Mb

A


66

can affect the therapeutic index. The metabolites from conventionally radioiodinated probes

diffuse from cells radiometal chelate metabolites retained in tumor cells, which results in

lower off-target dose. On the other hand, radiometal chelate metabolites are retained in the

cells of not only the tumor, but also healthy tissues. Therefore, comparison of the dosimetry

of A11 Mb radiolabeled with either beta-emitter can help optimize therapeutic efficacy.

In this study, we evaluated anti-PSCA A11 minibody radiolabeled with 131I or 177Lu for

radioimmunotherapy of prostate cancer. 124I- and 89Zr-A11 Mb immunoPET imaging and

quantification was used to profile the pharmacokinetics of 131I- and 177Lu-A11 Mb,

respectively. 131I- and 177Lu-A11 Mb ex vivo biodistributions were used to calculate dosimetry

and select which radionuclide to proceed with in the therapy studies. 131I-A11 Mb RIT was

evaluated using mice bearing PSCA-positive s.c. tumors. Lastly, human-PSCA transgenic

mice were used to evaluate 131I-A11 Mb uptake in normal tissue.

4.2 Methods

4.2.1 Cell Lines and Tumor Models

To establish s.c. tumors, 0.5-1x106 22Rv1-PSCA cells in 1:1 Hank’s Buffered Saline Solution

(Sigma):Matrigel (BD Bioscience) were implanted in the shoulder of 8- to 10-week-old male

nu/nu mice (Foxn1nu, Jackson Laboratories), and tumors were allowed to grow for 10-14

days. Mice were administered 131I-A11 Mb treatment when tumors were 100-200 mm3.

Human PSCA knock-in (hPSCA-KI) C57BL/6J mice (18- to 27-week-old male and female)

were bred at UCLA (57). Protocols for all animal studies were approved by the UCLA Animal

Research Committee.


67

4.2.2 Conjugations

A11 Mb was conjugated with p-isothiocyanatobenzyl-deferoxamine (p-SCN-Bn-DFO, B-705,

Macrocyclics) to produce A11 Mb-DFO for 89Zr radiolabeling (108) as previously described

(41).

A11 Mb (97,98) was conjugated with the metal chelator 2-(p-isothiocyanatophenyl)-

cyclohexl-diethylenetriaminepentaacetic acid (p-SCN-Bn-CHX-A”-DTPA, B-355,

Macrocyclics) to produce A11 Mb-DTPA for 177Lu-radiolabeling. In a typical reaction under

metal-free conditions, A11 Mb (50 µl at 4 mg/ml) was incubated with 3-fold molar excess p-

SCN-Bn-CHX-A”-DTPA, and the reaction was immediately adjusted to pH 9 using 0.1 M

sodium bicarbonate (1/10 volume). The reaction was allowed to proceed for 1 hour at room

temperature, and excess p-SCN-Bn-CHX-A”-DTPA was removed using Micro Bio-Spin™

Size Exclusion Columns (Bio-Rad). A11 Mb-DTPA was dialyzed in 0.25 M ammonium

acetate (NH4OAc) pH 5.5 and 5 g/L Chelex 100 (Bio-Rad).

4.2.3 Radiolabeling

Radiolabeling with 89Zr and 124I for imaging studies was conducted as previously described

(41). Briefly, in a typical reaction, 100 µg of A11 Mb-DFO was incubated with 500 µCi (18.5

MBq) 89Zr (3D Imaging LLC), or A11 Mb with 124I (3D Imaging LLC) using conventional

iodination methods in Iodogen tubes (Pierce).

Figure 4.3: Schematic of DTPA conjugation and 177Lu radiolabeling.

+ 177Lu in 0.25 M NH4OAcpH 5.5, 1 h, 37C

177Lu177Lu

3x p-SCN-Bn-CHX-A”-DTPA1 h, RT

– Lysine –DTPA


68

177Lu-A11 Mb was prepared similar to previous protocols (124). 177LuCl3 was supplied by the

Isotope Program within the Office of Nuclear Physics in the Department of Energy’s Office of

Science. The 177LuCl3 was buffered in 250 nM NH4OAc pH 5 (2x volume of 177LuCl3). In a

typical reaction, 50 µg A11 Mb-DTPA was incubated with 0.5 mCi (18.5 MBq) 177Lu for 1 h at

37ºC (Figure 4.3). The reaction was quenched with 0.25 M NH4OAc/acetic acid pH 5.5/50

mM EDTA (0.1x volume of A11 Mb-DTPA + 177Lu) for 10 minutes.

Figure 4.4: Schematic of 131I-A11 Mb radiolabeling.

131I-A11 Mb was prepared using pre-coated Iodogen tubes according to the manufacturer’s

instructions (Pierce). In a typical reaction, 200 µg of A11 Mb was incubated with 4-10 mCi

(148-370 MBq) Na-131I (3D Imaging LLC) in 0.1 M Tris, pH 8.0 for 15 min at room

temperature (Figure 4.4). For the cell cytotoxicity assay, 60 µg 131I-isotype control minibody

(anti-CD20 GA101 cMb (125)) was radiolabeled with 600 µCi (22.2 MBq) Na-131I using the

same conditions.

Radiolabeled proteins were purified by size exclusion spin columns as previously described

(61). Radiolabeling efficiency and radiochemical purity were assessed by Instant Thin Layer

Chromatography (ITLC strips for radiolabeled antibodies, Biodex Medical System) with 20

mM citrate pH 5.0 (89Zr-A11 Mb), 0.25 M NH4OAc/acetic acid pH 5.5/50 mM EDTA (177Lu-

A11 Mb), or saline (radioiodinated proteins) as solvent. The strips were analyzed by gamma

counting (Wizard 3” 1480 Automatic Gamma Counter, Perkin-Elmer).

The stability of 131I- and 177Lu-A11 Mb was tested by incubation in mouse serum (Sigma) at

37ºC, and assessing the radiochemical purity at 4-24 hours by ITLC. Immunoreactivity was

131I

131I– Tyrosine

+ 131I in IODOGEN-coated tube15 min, RT


69

completed as previously described (61) using 22Rv1-PSCA and 22Rv1 control cells

immediately after radiolabeling, and at 24 and 72-96 hours.

4.2.4 Cell cytotoxicity assay

22Rv1-PSCA and 22Rv1 cells (1x104 cells) per well were plated in 96-well plates in 200 µl

RPMI and incubated for 24 hours. 5 µCi (0.19 MBq) (1 µg protein) of 131I-A11 Mb, 131I-GA101

cMb, 177Lu-A11 Mb, and unlabeled A11 Mb were added in triplicate with wells staggered to

minimize potential cross-fire (maximum radioisotope path length ~ 2 mm). At 48 h, 72 h, and

192 h, cells were assessed by crystal violet assay (OD at 550 nm). Percent cytotoxicity was

calculated by (ODcontrol-ODsample)/ ODcontrol x 100%, ODcontrol = wells without treatment.

4.2.5 PET/CT imaging

In order to block nonspecific radioiodine uptake in thyroid and stomach, mice were given

Lugol potassium iodide solution (Sigma-Aldrich) and potassium perchlorate (Sigma-Aldrich).

124I or 89Zr radiolabeled A11 Mb (40-50 µCi, 1.5-1.9 MBq, 20 µg) in 0.1 mL saline was

injected via tail vein (n=3). Immediately after injection, one mouse was anesthetized with

1.5% isoflurane and a 2 h dynamic scan (1 frame x 60s interval, 3 x 180s, 4 x 300s, 9 x

600s) was acquired using the G8 microPET scanner (Perkin Elmer), followed by a 1 min

microCT scan (CrumpCAT). 10-min static scans were acquired at 4 h, 8 h, 24 h, 48 h, and

72 h (n=3). 3D PET reconstruction was based on the Maximum Likelihood Estimation

Method (MLEM) algorithm and AMIDE (113) was used for ROI analysis and image display.

4.2.6 Biodistribution

131I- or 177Lu-A11 Mb (10-18 µCi, 0.37-0.67 MBq, 1 µg + 9 µg cold A11 Mb) in 0.1 mL saline

was injected via tail vein into 22Rv1-PSCA tumor-bearing male nude mice, or non-tumor-

bearing male and female hPSCA mice. At various time points (30 min, 2 h, 4 h, 8 h, 24 h, 48


70

h, 72 h, and 144 h; n=4 per time point), mice were terminated and blood and organs were

collected, weighed, and gamma-counted for ex vivo biodistributions. Bone marrow was

collected by flushing the femur with 200 µl PBS, and the sample was spun at 10,000 g for 5

min. The total activity in the organ represented as percentage of injected dose per organ

(%ID), and activity concentration represented as %ID per gram of tissue (%ID/g), were

calculated based on a standard of 1% ID.

4.2.7 Dose estimation

The non-linear two phase decay equation y=m1*e-m2*x+m3*e-m4*x was used to fit the

biodistribution data and determine the values for m1= kslow, m2=tauslow, m3=kfast, and

m4=taufast, where x=time, k=rate constant, tau=time constant. Calculations were performed

using GraphPad Prism (version 7.0a for Mac OS X, GraphPad Software). For each organ,

the area under the curve (AUC, %ID*h) was calculated from the equation AUC =

m1/(m2+λ)+m4/(m3+λ), using the decay constant λ=ln(2)/t1/2. The time-integrated activity

coefficient (TIAC, h) also referred to in the literature as residence time) was calculated from

TIAC=AUC*100%. The TIAC and mouse S-values (126) were used to determine the dose

rate (Gy/MBq) for each organ. The dose-limiting organs and maximum administered activity

for each radionuclide was determined by the maximum tolerated dose (Gy) for key organs

based on literature (77), with the assumption the values are similar between human and

mice. This assumption was made for a more conservative dose estimation, as mice have

been shown to tolerate higher dose to key organs, including kidney, compared to humans

(127). The dose to tumor was calculated using the sphere modeling program in

OLINDA/EXM (128).

4.2.8 Radioimmunotherapy

Mice bearing 22Rv1-PSCA s.c. tumors (100-200 mm3 on day 0) were administered 1 mCi

(37 MBq) (n=9), 300 µCi (11.1 MBq) (n=9), or 100 µCi (3.7 MBq) (n=9) 131I-A11 Mb (20 µg


71

protein dose), or saline (n=5). Mouse weight and tumor size (measured bi-directionally) were

recorded every other day in the control and low administrated activity groups, and every day

in the 1 mCi groups. Tumor size (mm3) was calculated as (length x width x width)/2. Mice

were sacrificed if body weight decreased more than 15-20% of the original weight. For the

tumor growth endpoint, mice were sacrificed when tumors reached ≥1000 mm3, and tumors

were weighed at the end of the study.

4.2.9 Tissue Analysis

Blood samples (n=3 per group) were collected at termination for whole blood count analysis,

which was performed by the UCLA Division of Laboratory Animals Medicine. Serum samples

for 1 mCi 131I-A11 Mb group (n=3) were prepared at termination for biochemical analysis to

assess potential acute toxicity to the liver (alkaline phosphatase, ALP) and kidney (blood

urea nitrogen, BUN, and creatine, CRE). BUN levels correlate with the degree of radiation-

induced toxicity as seen by histopathology and is also predictive of eventual renal failure,

and CRE is a marker of glomerular filtration rate, the preferred assay for renal injury in

humans (127); therefore, both are good markers for radioimmunotherapy toxicity to the

kidneys.

Immunohistochemistry (IHC) was performed by the UCLA Translational Pathology Core

Laboratory (TPCL) to stain samples for gamma H2AX (Phospho-Histone H2A.X Antibody,

Thermo Fisher), as well as PSCA (EK5 antibody, UCLA TPCL).

4.2.10 Statistics

To estimate the interaction between radionuclide (131I, 177Lu) and biodistribution by time

(%ID/organ for blood, tumor, kidney, liver), repeated measures ANOVA with Geisser-

Greenhouse correction and a significance threshold set to 0.05 was performed using

Graphpad Prism (version 8). For statistical analysis of biodistribution area under the curve,


72

as well as cell cytotoxicity, multiple Student’s t-tests in Prism were performed with the

significance threshold was set at 0.05, and P-values were corrected using the Holm–Šidák

method.

A linear mixed model with Type III tests of fixed effects was used to estimate the interaction

of tumor growth and therapy group (100 µCi, 300 µCi, 1 mCi, saline) (IBM SPSS Version 24,

2016). This test was completed with the help from the UCLA Statistical Consulting Group.

Survival curves were displayed using the survival table format in Prism, median survival was

calculated using the program’s built in survival curve analysis, and the log-rank (Mantel-Cox)

test was used for survival curve comparison. Values in the tables are reported as mean +/-

standard deviation.

4.3 Results

4.3.1 124I-A11 Mb and 89Zr-A11 Mb serial immunoPET/CT quantification

Serial immunoPET imaging was used to evaluate the kinetics and biodistribution of A11 Mb.

Therefore, the positron emitters 124I-A11 Mb and 89Zr-A11 Mb were used as surrogates for

the therapeutic radionuclides 131I and 177Lu, respectively. Figure 4.5 displays representative

time series of non-tumor-bearing mice (0-72 h post-injection, p.i.) injected with 124I-A11 Mb

(Figure 4.5A) and 89Zr-A11 Mb (Figure 4.5B). As expected, while 124I-A11 Mb (non-

residualizing) cleared from blood, liver, and kidney by 24-48 h p.i., 89Zr-A11 Mb (residualizing

radiometal) resulted in retention of activity in the kidneys (radiosensitive) and liver at 72 h.

The 72 h ex vivo biodistribution confirmed high activity in organs of clearance, and an

additional time point (144 h p.i.) was added for the subsequent biodistribution study.

These results were confirmed by ROI quantification of the heart (representative of blood),

liver, and kidney (Figure 4.6A). In mice administered 89Zr-A11 Mb, the kidney %ID/g

calculated from ROI quantification differed from biodistribution results at 72 h, likely due to

partial volume effect (Figure 4.6B).


73

Figure 4.5: Serial immunoPET/CT of non-tumor-bearing mice. A, 124I-A11 Mb (n=3) PET/CT images show whole body and bladder activity clears by 24-48 h (little to no activity seen at 72 h, B, 89Zr-A11 Mb PET/CT images (n=3) show activity clears from blood pool by 24 h, and clearance to kidney (K) and liver (L) results in high activity even at 72 h.

Figure 4.6: Quantification of 124I- and 89Zr-A11 Mb immunoPET/CT of non-tumor-bearing mice. A, 124I-A11 Mb ROI analysis confirms activity clears from the blood and organs by 24 hours. For the 72 h data points, %ID/g calculated from ex vivo biodistribution for heart, liver, and kidney, was plotted due to activity clearance and inability to quantify from ROIs. B, 89Zr-A11 Mb ROI analysis show high activity in the organs of clearance, liver and kidneys, at 72 hours. At 72 h, the kidney %ID/g from ex vivo biodistribution is higher than calculated from ROI, likely due to partial volume effect. ROI analysis was done by averaging %ID/g from 3 spheres placed in the heart and each kidney, and 6 spheres in the liver.

124I-A11 Mb

89Zr-A11 Mb

4 h 8 h 24 h4 min 48 h10

0.5

3

0.5

1

0.5

%ID

/g

%ID

/g

%ID

/g

4 min10

0.5

4 h 8 h 24 h 48 h 72 h

%ID

/g

A

B

K

L


74

4.3.2 131I- and 177Lu-A11 Mb radiolabeling

Radiolabeling with 131I and 177Lu was optimized in order to achieve high specific activity and

maintain immunoreactivity (Table 4.1, Table 4.2). 131I-A11 Mb with a specific activity (SA) of

4 µCi/µg (0.15 MBq/µg), typical for imaging studies, was used as a baseline comparison.

131I-A11 Mb with SA of 17 µCi/µg (0.63 MBq/µg) had high radiolabeling efficiency (Table

4.3), maintained radiolabeling stability (94%) at 24 h, and retained immunoreactivity (IR)

(52%) similar to 131I-A11 Mb with SA of 4 µCi/µg (0.15 MBq/µg) at 72 h. In contrast, 131I-A11

Mb with SA of 48 µCi/µg (1.8 MBq/µg) had slightly impaired IR (42%). Therefore, a target SA

of 15-20 µCi/µg (0.56-0.74 MBq/µg) was used for low administered activity therapy groups,

and a target SA of 50 µCi/µg (1.9 MBq/µg) was used for the 1 mCi (37 MBq) groups.

The highest specific activity achieved for 177Lu-A11 Mb from testing radiolabeling ratios of 5,

10, 20, and 50 µCi/µg (0.19, 0.37, 0.74, 1.85 MBq/µg) was 13 µCi/µg (0.48 MBq/µg). 177Lu-

A11 Mb with specific activity of 9.8 µCi/µg (0.36 MBq/µg) retained stability at 96 hours

(93%), and immunoreactivity at 4 h (52%) (Table 4.2 need to add). Therefore, a radiolabeling

ratio of 10 µCi/µg (0.37 MBq/µg) was used in the 177Lu-A11 Mb biodistribution study.

A B C

Starting Activity 5 µCi/µg 20 µCi/µg 50 µCi/µg

Specific Activity 4 µCi/µg 17 µCi/µg 48 µCi/µg

Radiolabeling Efficiency 93% 92% 91%

Bound Activity 4 h 92% 95% 94%

Bound Activity 24 h 91% 94% 93%

Immunoreactivity 4 h 66% 68% 61%



Table 4.1: 131I-A11 Mb radiolabeling optimization. Comparison of samples A-C with increasing starting activities showed high radiolabeling efficiency and high retention of bound


75

activity at 24 h for all samples. Sample C showed a slight decrease in immunoreactivity (30 mi 22Rv1-PSCA) at 24 and 72 h. Therefore, the ideal target specific activity of 131I-A11 Mb is 20 µCi/µg to maximize the amount of activity delivered without compromising immunoreactivity.

A B C D

Starting Activity 5 µCi/µg 10 µCi/µg 20 µCi/µg 50 µCi/µg

Specific Activity 4.9 µCi/µg 9.8 µCi/µg 12 µCi/µg 13 µCi/µg

Radiolabeling Efficiency 98% 98% 60% 27%

Radiochemical Purity - - 98% 86%

Bound Activity 24 h 94% 93% 97% 88%

Bound Activity 96 h 93% 93% 96% 88%

Immunoreactivity 4 h 48% 52% 57% -

Table 4.2: 177Lu-A11 Mb radiolabeling optimization. Comparison of samples A-D with increasing starting activities showed high retention of incorporated activity at 96 h, and similar immunoreactivity (30 mi 22Rv1-PSCA) at 4 h. However, samples C and D had low radiolabeling efficiencies. Therefore, ~10 µCi/µg was the target specific activity of 177Lu-A11 Mb used in following studies to retain bound activity and immunoreactivity. Future optimization of chelator conjugation ratios may allow for increased specific activity.

131I-A11 Mb 177Lu-A11 Mb

Specific Activity 17 ± 3.5 µCi/µg n=6

49 ± 2.7 µCi/µg n=5

10 ± 1.6 µCi/µg n=3

Radiolabeling Efficiency

95 ± 2.5% n=6

96 ± 2.6% n=5

82 ± 20% n=3

Radiochemical Purity

>96% n=2

99 ± 0.7% n=2

>98% n=2

Immunoreactivity (30 mi 22Rv1-PSCA)

58 ± 6.0% n=5

53 ± 1.4% n=2

55 ± 5.0% n=3

Table 4.3: 131I- and 177Lu-A11 Mb radiolabeling and immunoreactivity from biodistribution and therapy studies.


76

4.3.3 Dose estimation based on 131I- and 177Lu-A11 Mb ex vivo biodistributions

Dosimetry calculations for 131I- and 177Lu-A11 Mb were used to determine the optimal tumor-

to-normal tissue dose (therapeutic index). The ex vivo biodistribution data (Table 4.4, Table

4.5) from 22Rv1-PSCA s.c. tumor-bearing nude mice was used to plot decay-corrected %ID

time activity curves (Figure 4.7). The calculated tumor AUC was similar for 131I- and 177Lu-

A11 Mb. In contrast, the kidney AUC was greater for 177Lu-A11 Mb, and the maximum

administered activity was calculated to be 200 µCi (7.4 MBq) based on previously published

dose estimates (77) (tumor:kidney therapeutic index = 0.5, Table 4.6). For 131I-A11 Mb, the

bone marrow was the dose-limiting tissue and the calculated maximum tolerated activity was

1 mCi (37 MBq) (tumor:bone marrow therapeutic index = 22, Table 4.6). The dose delivered

to 0.1 g tumor from administering 1 mCi 131I-A11 Mb or 200 µCi 177Lu-A11 Mb was estimated

to be 35 Gy and 11 Gy, respectively. As 30-50 Gy is typically needed to effectively treat a

radiosensitive tumor (77), 131I-A11 Mb was chosen for the subsequent in vivo RIT studies.

Figure 4.7: Biodistribution time activity curves used for RIT dose estimation. A, 131I-A11 Mb and B, 177Lu-A11 Mb biodistributions in 22Rv1-PSCA s.c. tumor-bearing mice (n=4 per time point) show peak tumor uptake at 8 hours. As expected, high activity is retained in the liver and kidneys at 72 hours in mice injected with 177Lu-A11 Mb. There was a significant interaction between time and the radionuclide used on total organ activity for blood (F(7,42)=21.1, P<0.0001)), kidney (F(7,42)=27.9, P<0.0001)), and liver (F(7,42)=23.6, P<0.0001)), but no significant interaction for the tumor (F(7,41)=2.1, P=0.07)). The area under the curve for %ID/organ was used to calculate residence time and estimate dosimetry.


77

Table 4.4: 131I-A11 Mb biodistributions in 22Rv1-PSCA s.c. tumor-bearing nude mice (n = 4 per time point). Values are presented as total activity per organ (%ID ± standard deviation). Tumor:BM were not calculated for 48, 72, and 144 because %ID/BM was <10-3.

0.5 h 2 h 4 h 8 h 24 h 48 h 72 h 144 h AUC

(%ID*h)

Tumor 1.1 ± 0.3 1.1 ± 0.7 1.9 ± 1.3 2.5 ± 1.2 1.5 ± 1.0 0.9 ± 0.3 0.3 ± 0.2 0.0 ± 0.0 90

Blood 12 ± 2.1 7.4 ± 0.9 4.7 ± 2.0 5.2 ± 0.1 0.4 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 100

Kidney 2.2 ± 0.3 1.6 ± 0.2 0.9 ± 0.3 0.7 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 17

Liver 3.8 ± 0.4 2.8 ± 0.6 1.8 ± 0.5 1.4 ± 0.1 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 31

Stomach 1.3 ± 0.1 1.7 ± 0.2 1.3 ± 0.4 1.2 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 23

Intestine 0.2 ± 0.0 0.4 ± 0.0 0.5 ± 0.2 0.4 ± 0.0 0.3 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 14

Spleen 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.2

Heart 0.5 ± 0.1 0.5 ± 0.1 0.3 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 4.1

Lung 0.8 ± 0.0 0.6 ± 0.1 0.4 ± 0.0 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 8.1

Muscle 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8

Bone 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8

Bone marrow 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1

Ratios:

Tumor:Blood 0.1 ± 0.04 0.2 ± 0.1 0.6 ± 0.6 0.5 ± 0.2 4.4 ± 13.7 18 ± 0.8 20 ± 10 32 ± 32 0.9

Tumor:Kidney 0.6 ± 0.3 0.8 ± 0.6 2.3 ± 1.6 3.2 ± 1.6 13 ± 8.0 25 ± 9.6 21 ± 12 8.6 ± 6.7 5.2

Tumor:BM 120 ± 90 130 ± 90 330 ± 240 380 ± 180 1400 ±1100 710


78

0.5 h 2 h 4 h 8 h 24 h 48 h 72 h 144 h

AUC

[%ID*h]

Tumor 0.2 ± 0.1 1.0 ± 0.9 2.1 ± 0.4 1.1 ± 0.5 2.7 ± 1.7 0.8 ± 0.5 1.0 ± 0.7 0.5 ± 0.5 140

Blood 22 ± 2.5 16 ± 2.3 9.2 ± 1.4 6.5 ± 1.2 1.0 ± 0.2 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 150

Kidney 6.0 ± 1.1 7.7 ± 0.9 8.3 ± 1.3 9.9 ± 0.6 9.3 ± 0.6 7.2 ± 0.5 5.1 ± 0.5 2.5 ± 0.3 750

Liver 5.7 ± 0.6 6.7 ± 0.8 8.3 ± 1.1 8.3 ± 0.6 7.3 ± 0.7 6.9 ± 0.4 5.4 ± 0.5 3.8 ± 0.5 850

Stomach 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 20

Intestine 1.9 ± 0.4 2.7 ± 0.2 3.2 ± 0.4 3.2 ± 0.5 2.4 ± 0.1 1.5 ± 0.1 1.1 ± 0.0 0.5 ± 0.0 180

Spleen 0.3 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 40

Heart 0.9 ± 0.2 0.7 ± 0.1 0.6 ± 0.1 0.4 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.1 ± .0 0.0 ± 0.0 30

Lung 1.1 ± 0.1 1.0 ± 0.2 0.8 ± 0.2 0.6 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 30

Muscle 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.1 0.0 ± 0.2 0.0 ± 0.3 0.0 ± 0.4 0.0 ± 0.5 0.0 ± 0.6 4.5

Bone 0.0 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 9.8

Bone Marrow 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.3

Ratios:

Tumor:Blood 0.0 ± 0.0 0.1 ± 0.05 0.2 ± 0.04 0.1 ± 0.09 3.8 ± 1.3 5.6 ± 4.8 21 ± 15 95 ± 60 0.9

Tumor:Kidney 0.0 ± 0.00 0.1 ± 0.1 0.3 ± 0.1 0.1 ± 0.07 0.4 ± 0.1 0.1 ± 0.09 0.2 ± 0.1 0.3 ± 0.2 0.2

Tumor:BM 170 ± 120 270 ± 280 1300 ± 880 120 ± 110 340 ± 240 160 ± 140 68 ± 47 70 ± 58 60

Table 4.5: 177Lu-A11 Mb biodistributions in 22Rv1-PSCA s.c. tumor-bearing nude mice (n = 4 per time point). Values are presented as total activity per organ (%ID ± standard deviation). 131I-A11 Mb (1 mCi) 177Lu- A11 Mb (200 µCi)

Dose Tumor:Organ Dose Tumor:Organ


79

Tumor (0.1 g) 35 Gy - 11 Gy - Bone marrow 1.6 Gy 22 0.5 Gy 23 Kidney 3.3 Gy 10 20 Gy 0.5 Liver 0.6 Gy 58 2 Gy 5 Lung 3.0 Gy 11 2 Gy 5 Table 4.6: Comparison of radiation dose to tumor and organs from administering maximum tolerated activity of 177Lu-A11 Mb and 131I-A11 Mb.

4.3.4 131I-A11 Mb in vitro cytotoxicity

The ability of 131I-A11 Mb to kill tumor cells in an antigen-specific manner was determined by

cell cytotoxicity assay (Figure 4.8). Therefore, 22Rv1-PSCA and 22Rv1 control cells were

treated with 131I-A11 Mb, 177Lu-A11 Mb, or with the non-specific control 131I-anti-CD20 cMb

and non-radioactive A11 Mb. Treatment with 131I- and 177Lu-A11 Mb resulted in higher cell

death in PSCA+ cells (22Rv1-PSCA) compared to PSCA-negative cells and control

antibodies at all time points (P<0.05). At 168 h, both treatments specifically reduced cell

viability to 24%, compared with 80-100% viability for 131I-anti-CD20 cMb (81%), unlabeled

A11 Mb (103%), and 131I-A11 Mb added to 22Rv1 control cells (82%).

Figure 4.8: 131I-A11 Mb RIT induces cell death. In an in vitro cell cytotoxicity assay, 5 µCi 131I-A11 Mb treatments result in 24% viability of PSCA-positive cells at 168 h, compared to little to no inhibition of tumor cell growth in 131I-nonspecific Mb (P<0.001) and unlabeled A11 Mb control groups (P<0.001), as well as 131I-A11 Mb added to PSCA-negative cells (P<0.001). 177Lu-A11 Mb treatment had similar results.

0 48 72 168 h0

100

82

24

% viability

A11 Mb

131I-nonspecific Mb

131I-A11 Mb

131I-A11 Mb (control cells)

177Lu-A11 Mb


80

4.3.5 131I-A11 Mb RIT therapeutic effect

131I-A11 Mb was evaluated in 22Rv1-PSCA s.c. tumor-bearing mice for effects on tumor

growth and survival. In a dose escalation study, low (100 µCi and 300 µCi) and maximum

calculated maximum administered activities (1 mCi) were tested. 131I-A11 Mb inhibited

tumor-growth in a dose-dependent manner, with significant effects in the 1 mCi group

(Figure 4.9A). 131I-A11 Mb treatment also corresponded with extended median survival in the

300 µCi (18 d) and 1 mCi (24 d) groups compared to control mice receiving PBS (12 d)

(Figure 4.9B). The average tumor weight at time of sacrifice was similar for 100 µCi, 300

µCi, and control groups (1069 ± 274 mg), and was not higher for the 1 mCi group (831 ± 165

mg) (Figure 4.9C). The tumor from a mouse treated with 1 mCi and collected 48 h p.i. for

IHC stained positively for γH2AX, a marker for DNA double-strand breaks, demonstrating

RIT induces DNA damage at early time points (Figure 4.9D).

Figure 4.9: 131I-A11 Mb RIT inhibits tumor growth and extends survival. A, A single dose of 131I-A11 Mb delays tumor growth in a dose-dependent manner in 1 mCi (n=6), 300 µCi (n=8),

A B

– 1 mCi– 300 µCi– 100 µCi– Saline

Days Post-TherapyDays Post-Therapy

Tumor Size(mm3) % Survival

– 1 mCi A11 Mb– 1 mCi control cMb– A11 Mb– Saline

C D

0 12 16 24190

50

100

x x x x

0 1912 16 240

1000

2000

Days Post-Therapy

Tumor Size(mm3)

0 5014 250

1000

2000

Days Post-Therapy

0 14 250

50

100

x x

% Survival


81

and 100 µCi (n=7) therapy groups compared to saline control (n=5). B, Survival curves of mice administered 1mCi (median=24 d) and 300 µCi 131I-A11 Mb (median=19) have significantly survival curves (P<0.001 for both) compared to saline (median=12 d) while nonsignificant effect is observed in 100 µCi group (P=0.07). C, Spider plots of mice show a trend that unlabeled A11 Mb (n=6) does not inhibit tumor growth compared to saline (n=2), and 1 mCi A11 Mb (n=4) may inhibit tumor growth more than 1 mCi control cMb (n=4). However, the sample size is too small to draw conclusions. (Note: starting tumor size in A,B are larger than in C, D and therefore the data cannot be compared). D, The survival curve of mice that received 1 mCi control cMb (median=25 d) was not significantly different than mice that received unlabeled A11 Mb (median=14 d) (P=0.09). Survival was not calculated in the saline or 1 mCi groups due to small sample size and widespread data, respectively.

Figure 4.10: Final tumor weights and IHC. A, The average tumor weight at the time of sacrifice was 830 mg (1 mCi), 1050 mg (300 µCi), 1060 mg (100 µCi), and 940 mg (saline). This demonstrates the 1 mCi group did not have artificially extended survival due to sacrificing mice with tumors larger than other groups. B, Tumors excised from mice in the 1 mCi group stained positively for gamma H2AX, a marker of DNA damage, as well as PSCA.

4.3.6 131I-A11 Mb RIT toxicity

131I-A11 Mb off-target toxicity was monitored by assessing mouse body weight throughout

the therapy study, and assessing blood counts and biochemical levels representative of liver

and kidney damage at time of sacrifice. Despite an initial drop in weight among all therapy

and control groups in day 0-2, the majority of mice recovered by day 10 (Figure 4.11).

However, one mouse in the 1 mCi group was terminated at day 10 due to continued weight

loss and decrease in body conditioning score.

A B

5 µm

! H2AX PSCA HE

1 mCi 300 µCi 100 µCi Saline0

1000

2000

Tumor (mg)


82

Figure 4.11: Mouse body weights over the course of therapy. Mice in all therapy groups experienced weight loss at day 0-2, and the majority recovered by day 10. However, one mouse in the 1 mCi group was sacrificed due to a decrease in body conditioning score and weight loss of 20% of the original body weight.

Blood counts were not significantly different between control and therapy groups (P>0.05)

(Figure 4.12A). Biochemicals representative of damage to the liver (alkaline phosphatase,

alanine aminotransferase) and kidney (blood urea nitrogen, creatine) were not higher in mice

receiving 1 mCi 131I-A11 Mb (n=3) compared to mice that did not receive treatment (Figure

4.12B).

Days Post-Therapy

Ms weight (g)

– 1 mCi– 300 µCi– 100 µCi– A11 Mb– Saline

0 3020

35

0 3020

35

0 3020

35

0 3020

35

0 3020

35


83

Figure 4.12: Blood and serum biochemical analysis. A, Blood counts (white blood cells, WBC; neutrophils, NEU; lymphocytes, LYM; red blood cells, RBC) were not significantly different between the therapy groups at time of sacrifice (P>0.05). However, a single mouse from the 1 mCi group that was sacrificed due to decreased weight and body conditioning score had significantly higher lymphocytes (reasons unknown and therefore data not shown). B, Serum analysis of mice treated with 1 mCi 131I-A11 Mb (n=3) did not show increase in biochemicals representative of liver (alanine transferase, ALT) and kidney damage (blood urea nitrogen, BUN; creatine, CRE) compared with reported values for nude mice from Charles River.

4.3.7 131I-A11 Mb biodistributions in hPSCA mice

131I-A11 Mb biodistributions were completed in hPSCA transgenic mice in order to evaluate if

the expression of hPSCA in background tissues, including stomach, prostate, and bladder,

would alter the dosimetry calculations or lead to toxicity. Comparison of biodistributions of

male hPSCA mice at 2 h, 4 h, 8 h, 24 h, and 48 h and of female hPSCA mice at 8 h (Table

4.7) with the time activity curves from nude mice (Figure 4.13) suggests normal PSCA

expression would not alter the dosimetry estimation of 131I-A11 Mb RIT. Comparing male

hPSCA mice (n=4) and male nude mice, a significant interaction was observed between

mouse type and time on stomach %ID (Table 4.4), but the %ID in hPSCA mice was actually

lower, likely due to more complete radioiodine blockage. Therefore, radioimmunotherapy in

the more relevant hPSCA model likely will not result in increased dose to stomach or

bladder.

WBC NEU LYM RBC0

20# of cells

Saline100 µCi

1 mCi300 µCi

0.0

0.510

80

ALT(U/L)

BUN(mg/dL)

CRE(mg/dL)

Charles River nu/nu

131I-A11 MbA B


84

Dosimetry estimation models differ between human males and females, as females typically

have smaller organs and toxicity to the ovaries is a potential concern due to proximity to

source organs such as kidney and bladder (129). Therefore, one group of female hPSCA

mice were also administered 131I-A11 Mb and biodistribution was analyzed at 8 h. No

significant difference was observed between the biodistribution of male and female hPSCA

mice, and therefore radioimmunotherapy in other PSCA cancer models (pancreatic, bladder)

will likely not result in increased dose to organs in female mice. However, future studies will

need to include a more complete biodistribution in order to calculate dosimetry based on the

correct mouse weight and use a model that includes the ovaries.


85

2 h 4 h 8 h 8 h female 24 h 48 h

n = 4 n = 4 n = 4 n = 3 n = 4 n = 3

Blood 5.9 ± 0.7 4.7 ± 0.8 3.5 ± 0.3 2.5 ± 0.3 0.6 ± 0.1 0.1 ± 0.04 Stomach 0.4 ± 0.1 0.4 ± 0.02 0.6 ± 0.1 0.4 ± 0.03 0.3 ± 0.1 0.1 ± 0.02 Liver 2.1 ± 0.2 1.9 ± 0.1 1.4 ± 0.2 0.8 ± 0.1 0.3 ± 0.02 0.1 ± 0.02 Large intestine 0.7 ± 0.4 0.7 ± 0.2 0.7 ± 0.1 0.5 ± 0.1 0.2 ± 0.02 0.03 ± 0.0 Small intestine 1.6 ± 0.4 1.7 ± 0.2 1.2 ± 0.02 1.0 ± 0.1 0.3 ± 0.02 0.04 ± 0.02 Kidney 1.2 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.5 ± 0.03 0.1 ± 0.05 0.03 ± 0.01 Spleen 0.2 ± 0.02 0.14 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 0.02 ± 0.0 0.0 ± 0.0 Pancreas 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.1 ± 0.01 0.02 ± 0.01 0.0 ± 0.0 Heart 0.6 ± 0.1 0.5 ± 0.2 0.3 ± 0.1 0.2 ± 0.1 0.06 ± 0.01 0.0 ± 0.0 Lung 0.1 ± 0.01 0.6 ± 0.1 0.5 ± 0.1 0.3 ± 0.1 0.1 ± 0.01 0.01 ± 0.0 Prostate/ovaries 0.1 ± 0.02 0.2 ± 0.1 0.2 ± 0.1 0.03 ± 0.01 0.04 ± 0.01 0.0 ± 0.0 Seminal vesicle 0.2 ± 0.1 0.3 ± 0.2 0.1 ± 0.02 0.0 ± 0.0 0.04 ± 0.01 0.0 ± 0.0 Bladder 0.1 ± 0.01 0.1 ± 0.02 0.1 ± 0.05 0.1 ± 0.04 0.03 ± 0.01 0.0 ± 0.0 Muscle 0.04 ± 0.01 0.1 ± 0.02 0.1 ± 0.0 0.04 ± 0.01 0.01 ± 0.0 0.0 ± 0.0 Bone 1.5 ± 0.5 0.04 ± 0.02 0.03 ± 0.01 0.02 ± 0.01 0.01 ± 0.0 0.0 ± 0.0 Bone marrow 0.002 ± 0.0 0.001 ± 0.0 0.002 ± 0.0 0.001 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Table 4.7: 131I-A11 Mb biodistributions in non-tumor-bearing hPSCA transgenic mice. A significant interaction (P<0.0001) was observed between time and mouse type (male hPSCA and male nude, Table 4.4) on %ID of stomach (PSCA-positive in hPSCA mice), while there was not a significant interaction (P>0.05) for blood, liver, and kidney. Additionally, there was not a significant difference between male and female biodistributions at 8 h. Values are presented as %ID ± standard deviation.


86

Figure 4.13: 131I-A11 Mb biodistribution of male non-tumor-bearing hPSCA mice. Ex vivo biodistributions of hPSCA mice at 2, 4, 8, 24, and 48 h (squares and solid lines) p.i. did not show an increase in %ID of PSCA-positive stomach at any time point (see Table 4.7), nor blood, kidney, or liver (P>0.05) compared nude mice (circles and dotted lines). Therefore, the dose estimation for 131I-A11 Mb RIT is likely similar to that of nude mice without increased dose or toxicity to the stomach.

4.4 Discussion

Prostate cancer treatments such as androgen deprivation therapy can extend the life of

patients with mCRPC, yet the disease ultimately progresses and becomes resistant.

Radioimmunotherapy is a promising alternative (79), and the goal is to use antibodies to

deliver cytotoxic radiation to the tumor and minimize normal tissue toxicity. However, a high

radiation dose is required to treat solid tumors, and dosimetry must be performed to predict

the corresponding dose to radiosensitive normal tissues including bone marrow and kidney

(77). This balance depends on the agent’s clearance, binding affinity, metabolism, and the

choice of radionuclide.

0 482 4 8 240

5

10

15

Time (h)

%ID

- Blood- Kidney- Liver- Stomach

nudehPSCA


87

The anti-PSCA A11 minibody radiolabeled with 124I was previously used for immunoPET of

prostate tumor models and successfully detected intratibial bone metastases, as well as

response to anti-androgen therapy (42). Therefore, we propose beta-emitter-radiolabeled

A11 Mb could be an effective radioimmunotherapy agent. In this study, 124I- and 89Zr-A11 Mb

immunoPET were useful surrogates to profile the pharmacokinetics of radiolabeled A11 Mb.

131I- and 177Lu-A11 Mb biodistributions were used in dosimetry calculations that predicted

131I-A11 Mb would have a higher therapeutic index. Additionally, the maximum tolerated

activity of 131I-A11 Mb of 1 mCi (37 MBq) was estimated to deliver a tumoricidal dose (35

Gy), with bone marrow as the dose-limiting organ. 131I-A11 Mb dose escalation was

performed in mice bearing PSCA-positive xenografts, and tumor growth was inhibited in a

dose-dependent manner that translated to extended median survival.

Compared to 131I (nonresidualizing when labeled by conventional methods), the beta-emitter

177Lu (residualizing) has progressed further in the clinic as a successful RIT radiolabel for

prostate cancer, likely because it can deliver a higher dose to the tumor. However, both 131I-

and 177Lu-A11 Mb had similar cell-killing effects in vitro. When iodistribution dosimetry

calculations were performed, the 177Lu-A11 Mb time activity curves showed unexpected

clearance to the kidneys, and the corresponding therapeutic profile was not as promising as

131I-A11 Mb. Although 131I-A11 Mb showed tumor inhibitory effects in vivo, 177Lu may be

considered in future studies for increased dose to the tumor.

Future studies include re-engineering the minibody, modifying the therapy regimen, and

testing more relevant models. As the A11 Mb did not have the anticipated profile, it could be

re-engineered to a larger fragment to shifts clearance to the liver, yet retains similarly fast

pharmacokinetics compared to the intact antibody. Additionally, dose fractionation can allow

for the delivery of a higher cumulative dose, and a Phase I study currently evaluating the

efficacy of fractionated 177Lu-PSMA-617 (NCT03042468) may inform future studies using


88

anti-PSCA antibody fragments. Lastly, more relevant tumor models should be explored. A11

Mb-IRDye800CW was successfully used to detect metastases in the hPSCA transgenic

mouse model with normal expression of PSCA (57), and therefore this model would also be

useful for RIT evaluation. In preliminary dosimetry studies, the normal expression of PSCA

in hPSCA mice did not significantly affect the ex vivo biodistribution; therefore, 1 mCi 131I-

A11 Mb could also be tested in this mouse model without increased dose to stomach and

bladder.

Although other radioimmunotherapy agents have been evaluated for prostate cancer in

several preclinical and clinical studies (79), none are curative. In a systematic review of

clinical trials evaluating 177Lu-anti-PSMA antibodies and ligands, only 37% of patients

responded with PSA decline (93). Additionally, using radiolabeled intact antibodies (long

circulating half-life) can lead to higher bone marrow dose. Hematological toxicity was

observed in all patients administered a single dose of 177Lu-J591 (intact antibody) (91). In

contrast, small molecules (short circulating half-life) for RIT, such as 177Lu-PSMA-617 ligand,

result in less hematological toxicity. However, other adverse events including xerostomia

were observed in patients who received 177Lu-PSMA-617 (94), and some patients

discontinued treatment due to such toxicity. Using an anti-PSCA minibody may address both

toxicity issues, as it has a shorter half-life than intact antibodies and typically clears to the

liver (radioresistant). Additionally, PSCA is not expressed on the salivary glands and is

expressed at low levels on other radiosensitive organs (bone marrow, kidney), and it is a

promising target for radioimmunotherapy.

In addition to beta-radioimmunotherapy, there has been a recent surge in interest in alpha-

radioimmunotherapy; the majority of studies have been focused on peptides, but the

strategy can also be applied to the minibody. Radiolabeling A11 Mb with an alpha-emitter,

which has a short path length and high linear energy transfer, may increase therapeutic

efficacy especially if the disease has metastasized (130). In one study, over 63% of mCRPC


89

patients responded to 225Ac-PSMA-617 salvage therapy, which was greater than the

response rate in patients given 177Lu-PSMA-617 (76). However, 225Ac-PSMA-617-induced

salivary gland toxicity was surprisingly similar to 177Lu-PSMA-617, potentially due to small-

molecule transporters rather than PSMA-expression (95). Using antibodies or antibody

fragments may reduce off-target toxicity, and 225Ac-anti-PSMA antibody J591 is currently

being evaluated in mCRPC patients in a Phase I trial (NCT03276572). 211At-A11 Mb

administered in a fractionated regimen was shown to reduce the size of both subcutaneous

(200 mm3) and intratibial tumors (100-200 µm) (manuscript in preparation, courtesy of Dr.

Tom Bäck), and the results can inform future studies comparing the dosimetry and

therapeutic effect with beta-RIT. Additional combination therapies include using

chemotherapy to radiosensitize the tumors before administering RIT, which has been shown

to enhance the therapeutic effect in preclinical models (131).

4.5 Conclusion

Beta-emitter labeled A11 Mb was evaluated for radioimmunotherapy of PSCA-positive

prostate cancer. 131I- and 177Lu dosimetry calculations showed 131I-A11 Mb could deliver a

higher therapeutic dose while minimizing off-target effects. 131I-A11 Mb radioimmunotherapy

successfully inhibited tumor growth and extended survival of mice with PSCA-positive s.c.

xenografts with minimal toxicity. These results show anti-PSCA antibody fragments may be

effective agents for prostate cancer radioimmunotherapy in the clinic. This work supports the

potential of radiolabeled A11 Mb as a theranostic agent, where A11 Mb-based immunoPET

could help stratify patients, inform dosimetry for personalized treatment, and monitor

therapeutic efficacy, in addition to treating the patient potentially in combination with other

therapies.

Chapter 5: Conclusion

90

5 Conclusion

In this dissertation, I used radiolabeled antibody fragments for dual-modality imaging and

radioimmunotherapy of PSCA-positive prostate cancer. I dual-labeled the anti-PSCA A11

cys-minibody with a positron emitter (124I or 89Zr) and near-infrared fluorophore (Cy5.5) and

imaged PSCA-positive tumors by sequential immunoPET and fluorescence. 124I-A11 cMb-

Cy5.5 immunoPET was used to detect 22Rv1-PSCA (high expression) and PC3-PSCA

(moderate expression) subcutaneous tumors with high contrast, and fluorescence signal

was specific to PSCA-positive tumors. In addition, 89Zr-A11 cMb-Cy5.5 identified

intraprostatic 22Rv1-PSCA tumors by immunoPET, and ex vivo fluorescence imaging

delineated between the prostate and surrounding seminal vesicles. I also compared A11

minibody radiolabeled with the beta-emitters 131I and 177Lu and determined 131I-A11 Mb had a

higher therapeutic index. 131I-A11 Mb RIT caused dose-dependent tumor growth inhibition

and extended survival of mice bearing 22Rv1-PSCA subcutaneous tumors.

Positive prostate tumor margins are difficult to visualize during surgical resection, and over-

resection can damage surrounding healthy tissue and nerves while under-resection leads to

disease recurrence. Combining the advantages of immunoPET and fluorescence onto a

single agent can provide a preoperative whole-body image to confirm if the disease is

localized to the prostate or has spread to regional lymph nodes, as well as a corresponding


91

fluorescent signal for intraoperative guidance to improve resection accuracy. Anti-PSMA

agents have been evaluated for dual-modality imaging, yet PSMA expression in normal

ganglia may confound lymph node staging, and PSMA is not expressed in 10% of patients.

PSCA expression correlates with tumor grade and stage, and we showed dual-labeled A11

cMb could detect both high PSCA and moderate PSCA subcutaneous tumors, indicating the

potential utility of A11 cMb in different stages of prostate cancer. Dual-modality A11 cMb

was also able to visualize intraprostatic tumors, demonstrating accessibility to the site of

natural disease and supporting the potential for clinical translation. We believe dual-modality

immunoPET/fluorescence can be used for noninvasive whole-body imaging and real-time

intraoperative guidance in patients with PSCA-positive prostate cancer.

The A11 minibody has the potential to be used as a theranostic agent to provide an image

that confirms PSCA-overexpression and guides dosimetry estimation, and deliver cytotoxic

radioactivity to treat the lesions by radioimmunotherapy. The radionuclide emission type

(beta-emitter for larger lesions and alpha-emitter for small metastases) and pharmacokinetic

properties (residualizing and non-residualizing) contributes to the therapeutic index. I found

131I- and 177Lu-A11 Mb were both cytotoxic to PSCA-positive cells, but dosimetry estimation

guided by immunoPET and calculated from biodistributions showed 131I-A11 Mb would have

a higher tumor dose-to-radiosensitive organ dose ratio. As 131I-A11 Mb successfully inhibited

growth of PSCA-positive tumors and extended survival, A11 Mb may be similarly effective

when paired with an alpha-emitter to treat metastatic disease. Additionally, therapeutic

effects may be enhanced by combining radioimmunotherapy with other therapies (ie

immunotherapy, chemotherapy, alpha and beta emitters). Therefore, A11 Mb or another

anti-PSCA fragment modified to clear to the liver may be a useful theranostic for prostate

cancer patients.

In conclusion, anti-PSCA minibody-based imaging prostate cancer can guide surgery, and

radioimmunotherapy can be an effective treatment option to inhibit tumor growth and extend


92

survival. Dual-modality imaging may also be used to stratify patients before pursuing

surgical resection, while radioimmunotherapy could have an enhanced effect in combination

with other therapies. This theranostic approach could also be evaluated in other PSCA-

positive disease settings such as pancreatic cancer. These studies support the continuation

of immunoPET/fluorescence and radioimmunotherapy studies in the clinic.

Chapter 6: Appendix

93

6 Appendix

The following appendix describes the dosimetry calculations for the radioimmunotherapy

studies presented in Chapter 4. These calculations were completed in collaboration with Dr.

Magnus Dahlbom at UCLA. More information on fundamental concepts for calculating

radiation dose, as well as case studies, can be found in the book Fundamentals of Nuclear

Medicine Dosimetry by Dr. Michael G. Stabin (132).

6.1 Using biodistribution data to calculate TIAC

This section will describe how to enter biodistribution data into Prism and plot time activity

curves, which represents the total activity in the organ over time. The resulting values m1-

m4 (explained below) are used to calculate the area under the curve (AUC), which

represents cumulative activity, and subsequently the time integrated activity coefficient

(TIAC, previously known as residence time). The radionuclide decay constant (λ) is included

in the AUC and TIAC calculations, which removes effect of physical decay and isolates the

biological decay.

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6.1.1 Time activity curve calculations

1) In Prism, create XY table, format for replicates. Enter time (h) into X. Each organ is in

separate column group (Group A, B, C). Enter %ID/organ.

2) Analyze > Nonlinear Regression

a. Model: Create new equation > Equation Type: Explicit Equation > Definition:

Y=m1*exp(-m2*x)+m3(exp(-m4*x)

i. m1 = amplitude or time constant (fast)

ii. m2 = slope or uptake constant, (fast)

iii. m3 = time constant (slow)

iv. m4 = uptake constant (slow, <m2)

v. x = independent variable, time

b. Initial values: (nonlinear regression is iterative and must start with estimated

values for each variable within factor of five of actual value)

i. Blood fits exponential decay (two-phase model captures fast and slow

decay): m1 = 5, m2 = 0.1, m3 = 10, m4 = 0.01

ii. Uptake in tumor before washout: m1 = 5, m2 = 0.01, m3 = -10, m4 =

0.1

Chapter 6: Appendix

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3) Results Table: Best-fit values, std error, 95% CI, and goodness of fit for m1-m4

Chapter 6: Appendix

96

6.1.2 Time integrated activity coefficient (TIAC) calculations

1) In excel, enter m1-m4 values for all organs from Prism

2) AUC (%ID*h) = m1/m2+m3/m4

3) AUC with decay constant = m1/(m2+λ)+m4/(m3+λ), λ = ln(2)/t1/2, t1/2 in hours

a. λ for 131I = ln(2)/192.5 = 0.0036

b. λ for 177Lu= ln(2)/161.5 = 0.0043

4) TIAC = AUC (%ID*h) / (100%)

5) TIAC-error = sqrt((sd1/m2)^2 + (sd2*m1/m2^2)^2 + (sd3/m4)^2 + (sd4*m3/m4^2)^2 )

/ (100%)

6.2 Using S-values to calculate dose to organs

This section will describe how to use S-values to calculate dose to organs using 131I-A11 Mb

and the TIAC for bone marrow as an example. The 131I and 177Lu mouse S-values were

kindly provided by Dr. Erik Larsson, calculated using the MOBY mouse phantom (126). For

the full dataset please contact him at [email protected] and provide mouse weight and

radionuclide. OLINDA version 1.1 was used for the dose to tumor calculations presented in

chapter 4.

Chapter 6: Appendix

97

6.2.1 Dose to organ calculations

1) Assumptions may need to be made for organs not collected in the biodistribution. For

example, organs such as brain were omitted from the following calculations. The

same residence time was used for intestine and intestine contents, as well as left and

right lung, and left and right kidney. Additionally, the TIAC for femur bone was

extrapolated to ribs, spine, and skull.

2) Enter decay-corrected TIAC

3) Multiply S-value x TIAC = Dose to target organ

4) Sum up all doses = Total dose for organ, convert to Gy/µCi.

5) Use literature to obtain maximum tolerated dose for the organ (77). Maximum

tolerated activity = maximum dose/total dose. Based on this value, determine

administered activities to test in the therapy study, and calculate the corresponding

dose to organ.

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98

6.2.2 Using OLINDA to calculate the tumor dose

1) Input radionuclide, and enter TIAC of the s.c. tumor in spheres model input form.

2) The results file will show the total dose (mGy/MBq) for a range of sphere masses.

Use the total dose for the sphere mass of interest (i.e. 0.1 g) and calculate dose to

tumor for corresponding administered activity groups by multiplying total dose and

activity.

3) To control the source and target organs used for the tumor dose calculation, tumor

S-values can also be obtained from Dr. Erik Larsson. These values correspond to a

MOBY phantom with four subcutaneous tumor placement options (data not shown).

6.3 Dose calculation results from Chapter 4

6.3.1 131I-A11 Mb dose to organ calculations

Across a row, the summation of the values equal the total dose for the target organ.

Maximum tolerated dose from the literature (77) was used to identify the dose-limiting organ.

For 131I-A11 Mb , the bone marrow is the dose-limiting organ, as the maximum tolerated

dose of 1.5 Gy corresponds to a maximum tolerated activity of 955 µCi, while the maximum

tolerated activity for liver, lung and kidney is higher.

Chapter 6: Appendix

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Chapter 6: Appendix

100

6.3.2 177Lu-A11 Mb dose to organs calculations

For 177Lu-A11 Mb, the kidney is the dose-limiting organ, and the maximum tolerated dose of 20 Gy corresponds to a maximum tolerated activity

of 180 µCi.

Chapter 6: Appendix

101

6.3.3 Radionuclide comparison

The total dose to a 0.1 g was calculated in OLINDA. Administering the maximum tolerated

activity for 131I-A11 Mb (1000 µCi) and 177Lu-A11 Mb (200 µCi) results in a 35 Gy and 11Gy

dose delivered to a 0.1 g tumor, respectively. Therefore, 131I-A11 Mb was chosen for the

subsequent in vivo therapy studies.

Chapter 6: Appendix

102

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