Development of Novel Bioconjugation Strategies for Creating Cancer-Targeting Nanomaterials

Development of novel bioconjugation strategies for creating cancer-targeting nanomaterials

Andri Wardiana Bachelor of Science (Chemistry) Master of Biotechnology

https://orcid.org/0000-0003-1335-1320

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2020

Australian Institute for Bioengineering and Nanotechnology (AIBN)

Abstract

Nanomaterials used in therapy and in vivo imaging diagnostics have the potential to be highly specific through the use of monoclonal antibody targeting, and there are several promising antibody- targeted nanomaterial candidates being tested in clinical trials. Numerous bioconjugation approaches for the attachment of antibody and antibody fragments to the surface of nanoparticles have been investigated. The challenge is to create stable and reliable bio-nanomaterials, as the conjugation approaches are complex, require optimization, and have low conjugation efficiency. Indeed, the development of an optimal and efficient method of bioconjugation is crucial for routinely producing hybrid bio-nanomaterials suitable for therapeutic and in vivo diagnostic applications. In this study, we focused on the development of bio-nanomaterial conjugates targeting prostate cancer as a model disease. For receptor targeting, we focused on prostate specific membrane antigen (PSMA), since this transmembrane protein is over-expressed in all prostate cancer stages. As such, PSMA is an ideal target in prostate cancer diagnosis and therapy. Therefore, in this investigation, we describe an active-targeting ligand for prostate cancer diagnosis and therapy, based on the anti-PSMA J591 antibody. We have developed two novel methodologies for bioconjugation of the anti-PSMA antibody fragment to nanomaterials to create novel targeting bio-nanomaterials. Both methods were applied in novel applications and provide a simplistic and efficient way to create targeted nanomaterials for in vivo imaging and therapy. In the first method, we developed a bispecific antibody (BsAb) consisting of two antibody single-chain variable fragments (scFvs) linked together in a tandem scFv format by a simple glycine- serine peptide linker. One scFv is specific for PSMA (J591 anti-PSMA antibody) and the other scFv is specific for methoxy polyethylene glycol (mPEG) epitopes, which are present on the surface of many PEG based nanomaterials, including the hyperbranched PEG polymer (HBP) used in this study. The BsAb was expressed in CHO cells, purified and characterized as a stable monomeric species based on chromatography and dynamic light scattering size analysis methods. The BsAb could specifically bind to recombinant PSMA and mPEG HBP targets as determined by ELISA, as well as to native PSMA expressed in cancer cell lines as determined by immunoblot assays. The simple, one- step mixing of BsAb with HBP resulted in the non-covalent formation of bio-nano conjugates through BsAb-mPEG binding, evidenced by a size shift of the mPEG complex as determined by dynamic light scattering. This study highlighted the simple and rapid BsAb conjugation strategy, demonstrating formation of BsAb-HBP conjugates which could target cancer cells effectively in in vitro assays. Furthermore, the BsAb-HBP complex, loaded with cytotoxic drugs showed an enhanced in vitro accumulation and cytotoxic effect compared to non-targeted HBP with drug attached. i

However, development of this hybrid bionanomaterial proved to be complicated, as it exhibited different biological behaviors between in vitro and in vivo assays. The enhanced active-targeting of BsAb-HBP complexes vs untargeted HBP to tumors in xenograft cancer models was not as pronounced as in vitro assays. The BsAb-HBP also demonstrated increased localization within reticuloendothelial system (RES) organs, such as spleen, lungs and liver, compared to the unconjugated HBP. Therefore, it would suggest the need for further optimizations of ligand density in this antibody nanomaterial hybrid. The second method utilized chemoselective conjugation via the introduction of an azide- bearing unnatural amino acid (UAA) into an anti-PSMA J591 scFv, expressed in a periplasmic E. coli production system. We have evaluated the incorporation of azide-bearing UAA into J591 scFv and investigated the ability to bind to dibenzocyclo-octyne (DBCO) containing fluorescent dye or polymer through copper-free click chemistry reactions. In vitro analyses showed that this azide- modified scFv can specifically bind and internalize into PSMA-overexpressing PC3-PIP prostate cancer cells both independently and while bound to DBCO containing fluorescent dye or polymer. In vivo studies demonstrated specific binding of azide-modified J591 scFv, conjugated to fluorescently labelled DBCO, in PSMA positive xenograft tumor with rapid renal clearance and shorter circulation time, which is more ideal for the application of imaging tracers. The novel bioconjugation methodologies described here have contributed to the bio- nanomaterial research field, and have facilitated the creation of versatile antibody-nanoparticle conjugates, for the active targeting of bionanomaterials, as well as complex imaging contrast agents for in vivo diagnostic purposes. Furthermore, the utilizing of BsAbs presents a generic methodology for targeting drug-loaded nanomaterials to potentially any tumor-associated antigen. Currently, our group are utilizing this BsAbs for targeting HBP to other prostate cancer cell biomarkers, such as glypican-1 (GPC-1) antigen. Our group is also demonstrating the potential benefit of incorporating azide-bearing UAA into anti-PSMA J591 scFv for bioconjugation into larger structures such as a DBCO-containing thermo-responsive PEG-based polymer in micellar systems. The conjugation strategies developed here show much promise for simplifying the production of antibody nanomaterial hybrids, for use in targeted in vivo cancer diagnosis and for therapeutic applications, advancing the field of precision nanomedicine.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis.

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Publications included in this thesis

- Review Sivaram, A. J., Wardiana, A., Howard, C. B., Mahler, S. M., Thurecht, K. J. (2018). Recent Advances in the Generation of Antibody–Nanomaterial Conjugates. Adv. Healthcare Mater. 2018, 7, 1700607. https://doi.org/10.1002/adhm.201700607. Incorporated into Chapter 1 and 2.

Contributor Statement of contribution Amal J. Sivaram Conception and design (40%) Analysis and interpretation (35%) Drafting and production (40%) Andri Wardiana Conception and design (30%) Analysis and interpretation (30%) Drafting and production (40%) Christopher B. Howard Conception and design (5%) Analysis and interpretation (5%) Drafting and production (0%) Stephen M. Mahler Conception and design (0%) Analysis and interpretation (5%) Drafting and production (5%) Kristofer J. Thurecth Conception and design (25%) Analysis and interpretation (25%) Drafting and production (15%)

Submitted manuscripts included in this thesis

No manuscripts submitted for publication

Other publications during candidature

- Editorial Mahler, S.M., Wardiana, A., Jones, M.L., de, Bakker, C.J., Graham, G.G. and Howard, C.B. (2016), Biosimilars approved for treatment of inflammatory rheumatological diseases. Int J Rheum Dis, 19: 1043-1048. doi:10.1111/1756-185X.13037

- Conference papers Wardiana, A., Jones, M. L., Thurecth, K. J., Mahler, S. M. and Howard, C. B. Bioconjugation of an antibody fragment against prostate-specific membrane antigen with nanomaterials as a theranostic application for prostate cancer. Bioprocessing Network, Melbourne, Victoria, Australia. 11-13 October 2016. Poster presentation.

Wardiana, A., Howard, C. B. Jones, M. L. and Mahler, S. M (2016). Targeting nanomaterials to prostate cancer biomarkers for theranostic application. The International Conference on BioNano Innovation, Brisbane, Australia. 24-27 September 2017. Oral presentation.

Wardiana, A., Jones, M. L, Mahler, S. M., Thurecth, K. J. and Howard, C. B. Targeting nanomaterial to prostate specific membrane antigen using a bispecific antibody for prostate cancer theranostic applications. Protein and Antibody Engineering Summit (PEGS) Europe, Lisbon, Portugal 12-16 November 2018. Poster presentation.

Wardiana, A., Jones, M. L., Mahler, S. M. and Howard, C. B. Incorporation of non-canonical amino acid into an anti-PSMA scFv to generate a stable antibody drug conjugate for prostate cancer therapy. Antibody Engineering & Therapeutics Asia. Tokyo, Japan. 26-28 February 2019. Poster presentation.

Wardiana, A., Jones, M. L., Mahler, S. M., Bell, C. A., Thurecth, K. J. and Howard, C. B. Targeting nanomaterial to prostate specific membrane antigen using a bispecific antibody for prostate cancer theranostic applications. Protein Australia, Brisbane, Australia, 16-18 July 2019. Oral presentation.

Contributions by others to the thesis

This thesis was drafted and written by the Candidate under the supervision of Dr. Christopher Howard, Prof. Stephen Mahler and Dr. Martina Jones.

The nanoparticles and animal imaging used in this project are provided by A/Prof. Kristofer Thurecht group (AIBN, UQ). The HBP mPEG polymer was synthesized at the group by Dr. Craig Bell, and DEGMA-co-MAPFP polymer by Amal J Sivaram (PhD student). Mouse xenograft models were prepared and imaged by Dr. Nick Fletcher and Dr. Dewan Akhter. All animal imaging analyses were conducted at Central Advanced Imaging, UQ.

Elements of this research utilized the facilities, and the scientific and technical assistance of the National Biologic Facility (NBF), University of Queensland. NBF is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. This work was also performed in part at the Queensland nodes of the Australian National Fabrication Facility (ANFF), a company established under the NCRIS to provide nano-micro-fabrication facilities for Australia’s researchers.

Statement of parts of the thesis submitted to qualify for the award of another degree

None

Research Involving Human or Animal Subjects

Animal study included in Chapter 5 was conducted in compliance with the Australian National Health and Medical Research Council guidelines for the care and use of laboratory animals and with the approval of the Animal Ethics Unit of the University of Queensland. A copy of animal ethics certificate is included in Appendix B.

Acknowledgements

All the praise is due to Allah, the Lord of the Worlds, the Merciful, the Beneficent.

First of all, I would like to express my gratitude to my principal advisor, Dr. Christopher B Howard for always being available to help with the experiments in the lab since the first month of my PhD journey. His valuable guidance and encouragement have helped me to face all the challenging phases during my PhD research. I would like also to sincere my gratitude to Professor Stephen M Mahler as my co-advisor, for allowing me to join in his research group as a PhD student. I really appreciate for his insightful comments and advices for my research, especially in the beginning of my research and during thesis writing. I also want to express the high appreciation to Dr. Martina L Jones for her advices and editing the manuscripts for my candidature milestones.

I addition, my appreciation extends to all Mahler group, National Biologic Facility (NBF) and Centre for Biopharmaceutical Innovation (CBI) members at AIBN UQ for their advices and helped me during my PhD research. I specially thanks to Christopher de Baker, Michael Yeh, Aiden Bell (former NBF staffs) for helping me in the beginning of my research, and Dr. Christian Fletcher and Dr. Pie Huda for helping me in numerous of occasions in the last year of my PhD; Mohamed, Lyndon, Mathew, Ari, Irene, Elizabeth (Mahler’s students), Dinora, Nyssa and all CBI students. I also thanks also to our collaborator, Associate Professor Kristofer Thurecht and his members in Prostate Cancer targeting group, due to without them there would not have been this PhD project. My special thanks to Amal J Sivaram for our “symbiosis mutualism” to finish our PhD study including the papers, Joshua Simpson for teaching me in confocal microscopy, Dr. Craig Bell for providing the polymers and Dr. Nicholas Fletcher and Dr. Dewan Akhter for helping me in animal study and imaging.

My sincere thanks also for all my friends in Brisbane, Angklungers UQ-Indonesia Student Association, Ristekers in Brisbane, LPDP awardees in Brisbane, Indonesia Islamic Society in Brisbane (IISB) community, and brothers and sisters in Muslim Student Association-UQ, especially for Sudarjanto’s family, Yasar Atas, Krisna Suparka, Tezar, Fikry and Asep for their selfless support and encouragement.

I would not have achieved this far without the endless support and prayers from my family. My highly gratitude for my parents, Eman and Imas Setyawati, who, although no longer with us, I believe this journey is not possible without your motivation, inspiration and prayers. May Allah grants you the highest level of Paradise. For my dearest wife Gilang Hanita Mayasari, and my lovely son Athar Muhammad Wardiana, thank you for helping me survive, cheering me up and not letting me give up during the hard time. Thank you for your continuous support and love, making my life is so wonderful

vii and exciting. I would like also to say thank you for my step mother, Nur Samirah, my parents in law, Neneng Gustini and Jimmy Kardian, my siblings Uus, Wahyudi, Ai, Weni, Dini, and my brothers/sisters in laws, Iis, Nenden, Edang, Cecep, Lingga, Andri, Andry, and for all my nephews and nieces.

Finally, I would like to express highly appreciation to Indonesia Endowment Fund for Education (LPDP) for financially supporting my PhD study in Australia. Special thanks to Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), especially for Dr. Adi Santoso and Dr. Ratih Asmana Ningrum due to their faith in me to continue my study at UQ.

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Financial support

Biologics based experiments were supported by IC160100027: ARC Industrial Transformation Centre, “Centre for Biopharmaceutical Innovation”. Andri Wardiana is sponsored by the government of Indonesia through Indonesia Endowment Fund for Education (LPDP) to pursue postgraduate research study in the field of bioengineering and nanotechnology.

Keywords nanoparticle, antibody, bioconjugation, scFv, bispecific antibody, unnatural amino acid, click chemistry, prostate specific membrane antigen, targeted in vivo imaging diagnostics and therapy.

Australian and New Zealand Standard Research Classifications (ANZSRC)

100403, Medical Molecular Engineering of Nucleic Acids and Proteins, 50% 100703, Nanobiotechnology, 50%

Fields of Research (FoR) Classification

1007, Nanotechnology, 30% 1004, Medical Biotechnology, 40% 0601, Biochemistry and Cell Biology, 30%

Table of Contents

Abstract ...... i Declaration by author ...... iii Publications included in this thesis ...... iv Submitted manuscripts included in this thesis ...... iv Other publications during candidature ...... v Contributions by others to the thesis...... vi Statement of parts of the thesis submitted to qualify for the award of another degree ...... vi Research Involving Human or Animal Subjects ...... vi Acknowledgements...... vii Financial support ...... ix Keywords ...... ix Australian and New Zealand Standard Research Classifications (ANZSRC) ...... ix Fields of Research (FoR) Classification ...... ix Table of Contents ...... x List of Figures ...... xiii List of Tables ...... xv List of Abbreviation ...... xvi Chapter 1 Introduction ...... 1 1.1 Antibody-nanomaterials conjugate...... 1 1.2 Research aim and strategies ...... 3 Chapter 2 Literature Review ...... 6 2.1 Targeting NPs ...... 6 2.2 Conjugation strategies for targeting of mAbs into Nps ...... 9 2.3 Cancer biomarkers as a receptor target for cancer therapy ...... 14 2.3.1 Prostate specific membrane antigen (PSMA) ...... 16 Chapter 3 Non-covalent conjugation of an anti-PSMA antibody to a hyperbranched PEG polymer using bispecific antibodies as precision nanomedicine for prostate cancer ...... 17 3.1 Introduction ...... 17 3.2 Materials and methods ...... 19 3.2.1 Hyperbranched polymer ...... 19 3.2.2 Cell lines ...... 20 3.2.3 Cloning and protein expression of anti PSMA J591-mPEG BsAbs ...... 20 3.2.4 Protein purification ...... 21 3.2.5 Protein Characterization ...... 21

3.2.6 Target binding ELISA ...... 22 3.2.7 Western Blot ...... 23 3.2.8 Binding analysis of BsAb on prostate cancer cell lines by flow cytometry...... 23 3.2.9 Bioconjugation of BsAbs-Cy5 labelled hyperbranched polymer ...... 24 3.2.10 Optimal ratio of BsAb to mPEG-HBP ...... 24 3.2.11 Internalization and co-localization of BsAb-HBP bioconjugate into PSMA positive prostate cancer cells by confocal microscopy ...... 25 3.2.12 In vitro cell uptake study of BsAb-conjugated Doxorubicin loaded HBP into PSMA positive prostate cancer cells by confocal microscopy ...... 25 3.2.13 In vitro cytotoxicity study of BsAb-conjugated Doxorubicin loaded HBP ...... 26 3.3 Results...... 26 3.3.1 Protein production, purification and characterization ...... 26 3.3.2 Immuno-assay using ELISA and Western blot...... 28 3.3.3 Targeting efficiency of anti-PSMA J591-mPEG BsAb using flow cytometry analysis ...... 29 3.3.4 In vitro analysis of BsAb-Cy5 labelled HBP targeting capability ...... 30 3.3.5 Optimal ratio of J591-mPEG BsAb to HBP using flow cytometry assay ...... 33 3.3.6 In vitro cellular uptake and cytotoxicity studies of BsAb-conjugated Dox loaded HBP ...... 33 3.4 Discussion ...... 36 3.5 Conclusion ...... 40 Chapter 4 Incorporation of an azide-bearing unnatural amino acid into PSMA targeted scFv for molecular diagnostic applications ...... 41 4.1 Introduction ...... 41 4.2 Materials and methods ...... 42 4.2.1 Cell lines ...... 42 4.2.2 Polymers ...... 42 4.2.3 Cloning and protein expression anti-PSMA J591 scFv incorporated pAzF ...... 43 4.2.4 Protein purification ...... 44 4.2.5 Flow cytometry analysis of J591-scFv-pAzF ...... 45 4.2.6 Click chemistry reaction of J591 scFv-pAzF toward Cy5 labelled DBCO ...... 45 4.2.7 Binding and internalization studies of Cy5 labelled DBCO conjugated J591-scFv- pAzF into prostate cancer cells ...... 45 4.2.8 Click chemistry reaction of DBCO-PEG linker as a polymer model into J591 scFv- pAzF ...... 46 4.2.9 Verification of J591 scFv-pAzF-DPD conjugation by Mass Spectrometry ...... 46 4.2.10 Click chemistry reaction of DEGMA-co-MAPFP polymer with DBCO raft (DDM polymer) with J591 scFv azide...... 46

4.3 Results...... 47 4.3.1 Modified scFv production and bioactivity assay...... 47 4.3.2 Verification of azide-modified unnatural amino acid incorporation by click chemistry reaction toward DBCO containing imaging-dye...... 47 4.3.3 In vitro internalization study of scFv-dye conjugate uptake into prostate cancer cells ...... 48 4.3.4 Conjugation of azide-modified J591 scFv into DBCO PEG linker ...... 50 4.3.5 Conjugation of azide-modified J591 scFv into DEGMA-co-MAPFP polymer ...... 53 4.4 Discussion ...... 54 4.5 Conclusion ...... 56 Chapter 5 In vivo evaluation of biodistribution and tumor accumulation of antibody fragment-polymer/imaging dye conjugates in PSMA-positive tumor xenograft ...... 58 5.1 Introduction ...... 58 5.2 Materials and methods ...... 59 5.2.1 Animals ...... 59 5.2.2 Antibody fragment- polymer/imaging dye conjugates ...... 59 5.2.3 In vivo biodistribution, and tumor accumulation of BsAb-HBP bioconjugate in tumor bearing mice...... 60 5.2.4 In vivo biodistribution and selective tumor uptake of antibody fragment-dye conjugate using J591 scFv pAzF-Cy7 labelled DBCO in tumor bearing mice in comparison with BsAb-Cy5 labelled HBP ...... 60 5.3 Results...... 61 5.3.1 In vivo biodistribution and tumor uptake of BsAb-HBP conjugate in tumor bearing mice models ...... 61 5.3.2 Comparison of J591 scFv-pAzF conjugated Cy7 labelled DBCO and J591-mPEG BsAb conjugated Cy5 labelled PEG HBP for imaging purposes ...... 63 5.4 Discussion ...... 66 5.5 Conclusion ...... 67 Chapter 6 Concluding remarks ...... 69 References ...... 71 APPENDICES ...... 83

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List of Figures

Figure 1.1: Characteristic features of antibodies, NPs and antibody-NPs conjugates. Figure 1.2: Series of synthetic schemes showing various strategies for the developing antibody conjugated Nps. Figure 2.1: Schematic passive and active targeting NPs in solid tumor. Figure 2.2: The physicochemical properties of the targeting ligands and NPs which can affect the blood circulation, biodistribution, and cellular uptake and internalization by tumor cells. Figure 3.1: Detection, identification and characterization of J591-mPEG BsAb expression in CHO cells. Figure 3.2: Immuno assays and binding affinity of J591-mPEG BsAb into PSMA expressing prostate cancer cells. Figure 3.3: BsAb-Cy5 labelled HBP and its characterizations. Figure 3.4: The optimal ratio of BsAb:HBP. Figure 3.5: In vitro cellular uptake and cytotoxicity of BsAb-conjugated Dox loaded HBP. Figure 4.1: Design, cloning and expression of anti-PSMA J591 scFv incorporated pAzF in periplasmic space of E. coli system. Figure 4.2: Purification and affinity binding assay of J591 scFv-pAzF. Figure 4.3: Evaluation of UAA pAzF attachment into a single-chain fragment variable (scFv) of J591 antibody and its binding analysis. Figure 4.4: In vitro binding specificity and internalization studies of azide-modified J591-scFv conjugated into Cy5-labelled DBCO. Figure 4.5: Copper-free click chemistry reaction of J591 scFv-pAzF and DBCO-PEG linker under physiological condition. Figure 4.6: Verification of conjugation of DBCO-PEG4-DBCO with J591 scFv-pAzF. Figure 4.7: Analysis of J591-scFv- DDM polymer conjugates. Figure 5.1: In vivo biodistribution and tumor accumulation of free Cy5 labelled HBP and J591- mPEG BsAb conjugated to Cy5 labelled HBP in xenograft tumor models of PSMA positive prostate cancer, PC3-PIP (left flank) and PSMA negative prostate cancer, PC3 (right flank). Figure 5.2: Comparison of in vivo profile of J591 scFv-pAzF and J591-mPEG BsAb-HBP conjugate in xenograft tumor mice models bearing PSMA positive PC3-PIP (left flank) and PSMA negative PC3 (right flank).

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Figure 5.3: Ex vivo fluorescence of excised organs and tumors at 1, 4, and 24 hours post injection. Figure A.1: The BsAb J591-mPEG design. Figure A.2: SEC profile of protein standard (BioRad) with elution buffer 1xPBS in 20% ethanol. Figure A.3: Protein Identification and analysis tools using the ExPASy server. Figure A.4: The prediction of disorder protein sequence using algorithm by PONDR® protein disorder predictor.

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List of Tables

Table 2.1: Different strategies for the synthesis of antibody-NP conjugates: advantages and disadvantages. Table 2.2: Characteristic of membrane-associated proteins as cancer antigen. Table 5.1: Region of interest (ROI) ratio of PSMA positive tumor (PC3-PIP) to PSMA negative tumor (PC3) at 5 days after intravenous (i.v) injection of Cy5 labelled HBP alone and Cy5 labelled HBP conjugated to BsAb J591-mPEG.

List of Abbreviation aaRS Amino Acyl-tRNA Synthetase ADC Antibody Drug Conjugate ADCC Antibody Dependent Cellular Cytotoxicity BiTE Bispecific T Cell Engager BsAb Bispecific Antibody CDC Complement Dependent Cytotoxicity CHO Chinese Hamster Ovary CuAAC Cu(I) Catalyzed Alkyne–Azide Cycloaddition Cy5 Cyanine 5 Cy7 Cyanine 7 DART Dual-Affinity Retargeting Molecule DBCO Dibenzocyclo-Octyne DDM DEGMA-co-MAPFP Polymer with DBCO RAFT DEGMA-co-MAPFP Diethylene Glycol Methacrylate-co-Pentafluoro Phenyl Methacrylate DLS Dynamic Light Scattering Dox Doxorubicin DPD DBCO-PEG4-DBCO EDV Engeneic Delivery Vehicle EGFR Epidermal Growth Factor Receptor ELISA Enzyme Linked Immunosorbent Assay EMA European Medicine Agency EMEA European Medicine Evaluation Agency EpCAM Epithelial Cell Adhesion Molecule EphA2 Ephrin Type-A Receptor 2 EPR Enhanced Permeability and Retention Fab Antigen-Binding Fragment Fc Fragment Crystallizable FcγR Fragment Crystallizable-Gamma-Receptor FOLH1 Folate Hydrolase 1 GPCR G-Protein Coupled Receptor GPC-1 Glypican-1 GPI Glycosylphosphatidyl Inositol GS4 Glycine-4 x Serine

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HBP Hyperbranched Polymer hpi Hour Post Injection i.v Intravenous KD Binding Affinity kDa Kilo Dalton LPETG Leucine-Proline-Glutamic Acid-Threonine-Glycine LPS Lipopolysaccharide mAb Monoclonal Antibody MMAE Monomethyl Auristatin E mPEG Methoxy Polyethylene Glycol MW Molecular Weight MWCO Molecular Weight Cut Off NCI National Cancer Institute NHS N-Hydroxysuccinimide NK Natural Killer nm Nano meter nM Nano Molar NP Nanoparticle pAzF p-Azido-L-Phenylalanine PBS Phosphate Buffer Saline PBST Phosphate Buffer Saline Tween PEG Polyethylene Glycol PSMA Prostate Specific Membrane Antigen RES Reticuloendothelial System rHu Recombinant Human scFv Single-Chain Variable Fragment SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SEC Size Exclusion Chromatography SPAAC Strain Promoted Azide-Alkyne Click TKR Tyrosine Kinase Receptor TMB Tetramethylbenzidine UAA Unnatural Amino Acid US FDA United States Food Drug Administration VEGF Vascular Endothelial Growth Factor VH Variable Heavy xvii

VL Variable L

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Chapter 1 Introduction

Nanotechnology has been intensively researched as a powerful tool to investigate novel strategies for addressing the unmet clinical challenges in the treatment of various diseases, principally cancer, and also in the advancement of diagnostic applications. Nanoparticles (NPs) as drug delivery systems have had a significant impact in improving therapeutic efficacy for cancer treatment in comparison to systemic drug delivery, through specific targeting and subsequently lowering overall drug dosage, resulting in reduced cytotoxicity effects [1]. Moreover, increasing cellular uptake can potentially be achieved by active cellular targeting, through cell-specific ligand conjugation to NPs. Numerous ligands, such as proteins (antibodies, antibody fragments, transferrin, and growth factors), peptides, aptamers, carbohydrates, nucleic acids, and small molecules have been widely explored for NP-mediated drug delivery [2, 3]. Among these ligands, monoclonal antibodies (mAbs) are widely utilized for targeting NPs, due to their highly specificity. Currently, there are around 80 mAbs approved and marketed globally [4]. Even so, there are no actively-targeted NPs approved for clinical use in the diagnosis and treatment of cancer; however there are currently some promising candidates in clinical development [5]. The research reported in this thesis is focused on the development of novel targeted nanomedicines using novel bioconjugation strategies to create antibody-nanomaterial conjugates, for the potential diagnosis and treatment of cancer.

1.1 Antibody-nanomaterials conjugate

Numerous ligands have been investigated for the active targeting of NPs for tumor therapy. The high selectivity of therapeutic mAbs makes them an ideal ligand for actively targeting nanomedicines. Furthermore there are many mAbs under clinical development, creating an even greater number of potential targets for cancer treatments than exists at present [4]. As therapeutic agents, mAbs have a variety of mechanism of actions for cancer treatment, including directly attacking cancer cells through various mechanisms, blocking regulators of the immune system, triggering cell membrane destruction and inhibiting cell growth. However, the use of antibodies has several limitations, including the potential for immunogenicity, down-regulation of target receptors [6], and antibody drug resistance [7]. On the other hand, although typically lacking cellular specificity, several features of NPs such as stable drug encapsulation, multiple drug loading, controlled drug release and longer systemic circulation, offer advantages as next-generation nanomedicines. The conjugation of NPs with antibodies can generate a product combining the properties of the NPs themselves with the ability of the antibodies to specifically recognize the target

1 antigen. Moreover, there are two main benefits of using antibody-nanomaterial conjugates, including the improvement in the cellular uptake and also intracellular stability [8] (Figure 1.1).

Figure 1.1: Characteristic features of antibodies, NPs and antibody-NPs conjugates. The conjugation of antibody into NPs can generate a product combining both of properties of the NPs and the specificity of mAbs, improving cellular uptake as well as resulting intracellular stability. Redrawn from Sivaram et al., 2017 [7].

It is understandable that the properties of targeting antibodies are modified once conjugated to NPs, including antigen-binding specificity and affinity which can impact the pharmacokinetic and pharmacodynamic profiles and the potential off-targeting of the antibody. However, the specific affinity (strength of the physico-chemical interaction with antigen) of an antibody may be not be as critical, since the multiplicity of epitopes on the surface on the NP creates an avidity effect, resulting in an overall increased apparent affinity. In term of site specific conjugation and control of orientation of mAbs on the surface of NPs, several techniques have been developed, such as introduction of reactive groups at specific amino acid residue sites in the mAb, to enable attachment to the NP surface [9]. The various strategies involved in the synthesis of this hybrid material are shown in Figure 1.2.

Figure 1.2: Series of synthetic schemes showing various strategies for the developing antibody conjugated Nps. Adapted from Sivaram et al., 2017. Copyright Advanced Healthcare Materials [7].

1.2 Research aim and strategies

Antibody targeted NPs have shown great promise in cancer therapy applications, with several potential candidates having been tested in clinical trials [10-13]. Creating an efficient and stable antibody-nanomaterial conjugate is important for the generation of a homogeneous product with defined function(s). Several methods have been developed for site-specific conjugation to obtain homogeneity of the final product as well as enhancing receptor-binding activity [14, 15]. Many of these methods such as traditional chemical conjugation approaches have many challenges associated with 3 the generation of antibody-nanomaterial conjugates. The methods are complex, inefficient, conjugation may occur at sites involved in binding activity and multiple reagents are required. The primary overarching aim of this study is to develop simple methodologies to get around challenges faced using other conjugation strategies, to facilitate stable bioconjugation of an antibody fragment to a NP, to create an actively targeted nanomedicine, for imaging and therapeutic applications. In this study, we chose prostate cancer as a model disease. For receptor targeting, we utilize prostate specific membrane antigen (PSMA), since its expression is upregulated many fold in almost all prostate cancer stages, and can be related to tumor aggressiveness, metastatic disease and disease recurrence [16, 17], making this antigen an excellent target in prostate cancer detection and therapy. To target this receptor, we use J591 anti-PSMA antibody. Currently, this antibody is widely researched for prostate cancer treatment. A humanized version of J591 has been shown to be effective in imaging and delivering radionuclides, resulting in therapeutic responses in prostate cancer clinical trials [18-20]. The objective of this research is to develop novel bioconjugation strategies to rapidly and simply create hybrid antibody-nanomaterial formulations for application in diagnostics and as drug delivery tools. In this study two bioconjugation strategies were developed including: 1. A novel bispecific antibody (BsAb) platform technology. The BsAb exhibits dual specificity for methoxy-PEG (mPEG) epitopes on HBP nanomaterials and prostate cancer target PSMA receptor on cancer sites. The BsAb is mixed with fluorescence imaging dye- labelled hyperbranched methoxy-PEG polymer (HBP) or Doxorubicin (Dox) loaded HBP to obtain a homogenous bioconjugate. 2. A single chain antibody fragment targeting PSMA engineered with an azide group at a specific protein site using unnatural amino acid (UAA) incorporation. The azide UAA enables site specific and precise click chemistry conjugation of the antibody with cycloalkyne-containing fluorescent dyes or with polymer activated cycloalkyne- containing fluorescent dyes [21, 22]. To evaluate the suitability of these formulations as targeted nanocarriers, testing was required to be carried out, including protein characterization and confirmation of bioconjugation, as well as various in vitro and in vivo bioassays, namely: a. tumor cell uptake studies by flow cytometry and confocal microscopy, b. in vitro cytotoxicity study in PSMA expressing prostate cancer cells, c. biodistribution studies using animal imaging and tumor accumulation in xenograft mouse models to verify in vivo localization of these conjugate formulations.

4 By generating two different conjugation methods of antibody-nanomaterial, we can determine the feasibility of using these formulations for efficient actively targeted nanomedicines for potential application in cancer diagnosis and therapy.

Chapter 2 Literature Review

2.1 Targeting NPs

Over the past few decades, nanotechnology has been an emerging area, and has been applied to medicine for the treatment of diseases, especially in cancer medicine. The characteristic dimensions of the NP, which is in the size range 1-100 nm, can influence the physicochemical properties of the nanomaterials/nanomedicines. These properties can be employed for numerous practical applications, including diagnostics, imaging and drug delivery systems. Several nanocarriers have been extensively investigated for targeted delivery to increase therapeutic index in cancer treatment. Compared to traditional chemotherapy, targeted therapy utilizing encapsulated drugs in NPs is a promising method to enhance therapeutic efficacy while avoiding undesirable side effects for patients due to cytotoxic nature of anti-cancer agents [23]. NP encapsulation technologies are able to protect poorly soluble drugs [24, 25], and therapeutic molecules [26], resulting in stable formulations and favorable drug bioavailability. Nanotechnology-based formulations for chemotherapy drug delivery have been extensively employed and show superior pharmacokinetic properties and tumor site-specific delivery compared to those of conventional formulations. This nano-encapsulated drug circulates through the bloodstream and it is driven to the target tumor site for simultaneously in vivo drug release in a controlled manner. For some NPs, the size is big enough to be rapidly cleared in the kidney (<10 nm), but smaller than the size that can be recognized by the reticuloendothelial system (RES) organs (>200 nm) [27]. In addition, multifunctional NPs, which have diagnosis/imaging and therapy capabilities in one single entity, (i.e.theranostics) are being developed to diagnose and treat cancer [28, 29]. This system offers the opportunity to delineate disease boundaries, monitor therapy in a real time and offer prognostic options [30-32]. The first therapeutic NP used in clinical application was PEGylated liposomal Doxorubicin (Doxil®, Janssen Biotech, Inc. PA, USA) which received US Food Drug Administration (FDA) approval in 1995 for treating certain types of cancer (breast cancer, ovarian cancer, HIV associated Kaposi’s sarcoma and other solid tumors). Following this, several therapeutic NP compositions based on liposomal formulation were approved by US FDA to treat various cancer, including Daunorubicin liposomal (DaunoXome® (Galen Limited, Craigavon, UK), Vincristine liposomal (Marqibo®, Spectrum Pharmaceuticals, NV, USA) and Irinotecan Liposomal (Onivyde®, Ipsen Biopharmaceuticals, NJ, USA). A non-liposomal NP was also approved by US FDA for clinical use, an albumin-bound Paclitaxel formulation Abraxane® (Abraxis BioScience, CA, USA for treating advanced non-small-cell lung cancer, metastatic pancreatic cancer, and metastatic breast cancer [33, 34]. These therapeutic NPs utilize a passive targeting mechanism, taking advantage of leaky vasculature in tumor cells. The formation of new, irregular blood vessels in solid tumor, known as 6 angiogenesis, creates fenestrated capillaries, with gaps ranging from 200 to 2000 nm, depending on the tumor types as well as its environment and localization. In addition, the function of lymphatic drainage of tumors is also ineffective, allowing molecules smaller than 4 nm to diffuse back into the blood circulation, while larger molecules or NPs are retained and accumulate in the tumor. This process is known as the enhanced permeability and retention (EPR) effect which has been extensively investigated in the systemic administration of cancer nanomedicines [35]. However, according to recent meta-analysis studies, less than 1% of administered NPs are accumulated in solid tumors. This could be due to multiple physiological barriers involved in extravasation of NP through the tumor vasculature [36]. Other studies report that a major proportion of NPs are potentially cleared by the mononuclear phagocytic system (MPS), and some are accumulated in the liver or taken up by hepatocytes and Kupffer cells [37, 38]. Understanding tumor biology is fundamental to overcoming this limitation. The tumor environment is very complex and heterogeneous, contributing the irregular tumor vasculature and congested tumor lymphatic that may restrict the entry and transport NPs. For example, the degree of leakiness of tumor vasculature will depend on many factors, such as type of tumor and its stage, different xenograft models or the different location of grafted tumor. Moreover, since the first discovery of EPR effect by Matsumara and Maeda in 1986, investigations are mainly carried out in animal models, but are less well documented in humans, possibly due to the challenges in obtaining meaningful biodistribution data in human [35]. As a result, clinical outcomes of therapeutic NPs is highly heterogeneous with a high percentage failure rate, meaning that NP-mediated drug delivery will not always present the therapeutic benefits as anticipated in preclinical experiments [39]. Therefore, several studies have explored the strategies of tumor targeting utilizing the EPR effect to enhance patient responses [40]. One of the strategies to overcome heterogeneity of EPR-based tumor targeting is utilizing ligand conjugated to the surface of NPs (Figure 2.1) [41]. Ligand-mediated targeting, also called active targeting, allows the NPs to bind specifically to cell surface receptors, facilitating internalization into the tumor cells via receptor-mediated endocytosis. This approach aims to enhance the accumulation of NPs and internalization of drugs into target tumor cells. Numerous targeting molecules have been used for functionalization of NPs including antibodies, affibodies, aptamers, peptides, sugars, and small molecule receptor ligands. Currently, it is proposed that active targeting of NPs complements the EPR effect, resulting in better therapeutic outcomes with fewer off-target effects [9, 35, 40].

Figure 2.1: Schematic passive and active targeting NPs in solid tumor. Adapted from Peer et al., 2017 [41]. Passive targeting is achieved by utilizing EPR effect, taking the advantage of leaky vasculature and ineffective lymphatic drainage in tumor cells. In active cellular targeting, functionalization of NPs promotes the recognition and specific binding into target cells, whereas, (i) the carrier may release the cargo in tumor vicinity; (ii) attach to membrane cells and release the payload in a sustained manner, or (iii) cellular internalization via receptor binding endocytosis.

The attachment of ligands onto NPs can influence the properties of both NPs and targeting ligands. Ligand molecules can lose the translational and rotational freedom, while the geometry or size of NPs can also be altered. Therefore, it is crucial to determine the physicochemical properties of those molecules for targeting efficiency. For example, the consideration of NP size and shape can affect their blood circulation, cellular uptake and tumor accumulation. In addition, surface charge and hydrophobicity might also possibly affect their interaction with the target cells. The density of ligand on NPs also influences their affinity and maximal cellular uptake into target cancer cells. Lastly, choosing the right ligand molecule is a critical aspect for targeting efficiency (Figure 2.2)[35]. There are a variety of mAbs that have been identified for the targeting of biomarkers identified or associated with cancer as well as being used as stand-alone therapeutics. The simple, stable and efficient conjugation of mAbs and antibody fragments to NPs is critical for optimal active targeting of cancer.

Figure 2.2: The physicochemical properties of the targeting ligands and NPs which can affect the blood circulation, biodistribution, and cellular uptake and internalization by tumor cells. Adapted from Bertrand et al., 2014 [35].

2.2 Conjugation strategies for targeting of mAbs into Nps

The stable conjugation of mAbs to NPs is a key for successful targeting. In general, the conjugation of mAbs to NPs can be achieved through three different approaches, including through physical adsorption, covalent conjugation, and adaptor biomolecules. Table 2.1 outlines currently used bioconjugation strategies, including the summary of benefits and drawbacks of each strategy. These strategies are discussed in more details within the following sections. I. Conjugation via physical adsorption Numerous physical adsorption methods have been proposed to couple mAbs with several types of NPs. This method is one of the simplest ways to synthesise bioconjugates, mainly based on the physical interactions, such as hydrophobic interactions, electrostatic adsorption, and hydrogen binding or van der Waals forces of attractions [42]. It is a direct conjugation method, combining two components without any chemical modification. Research reported this non-specific interaction can obtain good results in terms of the orientation of antibodies.

9 However, several disadvantages arise such as poor reproducibility and low stability in different pH conditions (pH dependent) [43]. II. Conjugation via covalent binding Chemical approaches via covalent attachment of mAbs to NPs is the most common method for synthesis of bio-nano conjugates and a good technique to overcome the drawbacks of physical conjugations. Numerous reactive groups on amino acids can facilitate conjugation reaction, such as lysine (amine group), glutamic and aspartic acid (carboxyl group), and cysteine (via reduction of cysteine), as well as the carbohydrate moiety of mAbs (via periodate oxidation of cis-diols to aldehydes). In addition, the reactive groups can also be introduced to NPs, either via adsorption, by covalent linkage, or due to inherent nature of the NPs, such as bifunctional thiol in gold NPs [9]. a. Amide bond formation The amide bond is typically a stable linkage, can be formed via NHS activated group on the nanomaterial with the amine group on the antibody [44]. Another strategy utilizes carbodiimide as a crosslinking agent resulting a stable amide bond between the amino group of protein and carboxyl group on the NP surface [45]. b. Disulfide/thioether formation Thiol groups on mAb or NP can react with a maleimide group or a pyridylthio group to form thioether or a disulfide bond, respectively. A thiol group introduced into mAbs either using heterobifunctional reagents or the reduction of disulphide bonds of intact mAb can be directly conjugated to the surface of gold NPs [46]. Another example links thiolated mAbs with maleimide functionalized lipid on lipidic NPs, with a conjugation efficiency up to 99% while only slightly reducing antibody binding affinity [47]. In general, thiol- coupling chemistries are versatile for bioconjugation methods under mild conditions; however, there are several investigations that report an unstable reaction using this coupling method. Numerous approaches have been investigated to overcome this issue, such as utilizing both thiol groups within a dithiobridge in the native antibody. This approach can preserve the protein structure, and result in a quick and efficient coupling of mAbs to NPs [7]. c. Sugar moieties Carbohydrate residues in the hinge region of mAbs can be utilized for site-specific conjugation to NPs, and offers an advantage, whereby the antibody is specifically orientated on the NP at a site distant from the antibody-binding site. This coupling reaction can be achieved through several ways. For example, oxidation of polysaccharide moieties of Fc region for bioconjugation into magnetic NPs [7]. Another oxidation reaction is 10 through the hydroxyl group of sialic acid or mannose which results in aldehyde residues that can be modified to form hydrazone. These modified mAbs can be directly linked with gold nanorods [9]. d. Click chemistry The click reaction is one of the most promising covalent conjugation strategies and involves the formation of a stable and inert triazole linkage, combining an azide and an alkyne molecule. This reaction is fast and simple to use, versatile, highly stereospecific, and provide high product yields. As a consequence, it is widely applied in variety of research areas, such as in polymer chemistry, material sciences, and pharmaceutical sciences [48]. The concept was originally introduced by Sharpless in 2001 [49], using the copper I (Cu(I)) catalyzed alkyne–azide cycloaddition (CuAAC) to synthesize 1,2,3- triazole derivatives under mild conditions, which is one of the most applied among other click reactions [50]. Finneti and colleagues developed an alkyne-functionalized polymer to coat gold NPs, and were subsequently conjugated with azido-modified anti-CD63 mouse antibody through CuAAC click reaction [51]. However, the Cu(I) catalyst is readily oxidized, and it can interact with biomolecules which is related to toxicity. Some strategies have been employed to minimise the side effects of Cu(I) catalyst in click chemistry reaction, including the use of reducing agents (e.g. sodium ascorbate, hydrazine and hydroxylamine) to prevent the oxidation [52], and complexing the Cu using a chelator ( e.g (tris[(4-carboxyl-1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine)) to minimize toxicity issues [53]. As an alternative, Cu-free strain promoted azide-alkyne click (SPAAC) chemistry reaction was developed in 2004, utilizing cyclooctynes and azide in the absence of catalyst, and can be done under mild conditions [54]. Dibenzocyclooctyne (DBCO) and bicycle [6.1.0] nonyne (BCN) are representative of cyclooctyne which can be attributed either into mAbs or polymer. Jeong et al. reported the cyclooctyne functionalized anti Her-2 IgG attached in Fc region was successfully conjugated with azide-functionalized- silica-encapsulated NPs showing orientation control of antibody with high conjugation efficiency. Furthermore, this strategy has strong binding capability of antibody conjugated NPs, which was 8 times higher compared to EDCI/NHS coupling method [55]. The third generation of click-chemistry reaction has been proposed using Diels–Alder cycloaddition between a trans-cyclooctene and tetrazines. This method provides a faster reaction rate compared to the other two previous methods [56]. III. Conjugation via adaptor biomolecules The various strategies of covalent conjugation described above are generally non-site specific, resulting in sub-optimal orientation of antibody on the NP surface which can alleviate binding 11 efficiency, and may lead to aggregate formation. Non-covalent binding reactions using adaptor biomolecules presents a strategy to overcome these limitations. One of the most robust and extremely rapid strategies is through biotin-binding protein. Antibody is typically modified with biotin, and subsequently conjugated to streptavidin-modified NPs [57]. This non- covalent strategy has high-affinity interaction (KD ~1015) with numerous studies showing the stability of antibody-NP conjugate. For example, biotin (AviTag labelling) introduced into scFv targeting endothelial cells of blood brain barrier (BBB), was successfully incorporated into streptavidin modified quantum dots. This conjugate was fully internalized into BBB endothelial cells [58]. This strategy, however, will depend on the amount of biotinylated mAbs which can limit the interaction with avidin-modified NPs. To overcome this deficiency, antibody-conjugated NPs can be achieved by utilizing Fc-binding proteins, which can interact with the Fc region, leaving the Fab region as antibody-binding site fully active. Several Fc- binding proteins, including Protein A, Protein G, Protein A/G, and FcγR that were incorporated into NPs, were successfully conjugated into immunoglobulin of interest [59]. The secondary antibodies were also used for antibody-NPs conjugate, utilizing anti-polymer antibodies, which were incorporated into antigen-target antibody through bispecific design. Research claimed that these strategies offer the highly specific and stable conjugation, yielding homogeneity product [60-62]. Another promising technique is using nucleic acid hybridization strategy, incorporating a complimentary single-strand sequence into both antibody and NP. The coupling reaction was then achieved via that complimentary pairing. This hybrid has potential for cellular transfection and gene knockdown; however, as such structure typically exhibits cell type selectivity [63]. Enzyme-based conjugation is also utilized to overcome the limitation of chemistry conjugation. Sortase A, a Staphylococcus aureus transpeptidase, is an enzyme which specifically recognizes the leucine-proline-glutamic acid-threonine-glycine (LPETG) peptide motif and cleaves between the threonine and glycine residues forming a new amide bond, and subsequently bind into N-terminal glycine of other biomolecules. Leung et al. used this strategy for targeted imaging approach, involved the fusion of sortase A into anti-platelet scFv that was successfully coupled into polymeric system containing azide functionalized-alkyne modified PEG and poly(N-vinyl pyrrolidone) through azide-modified triglycine-containing peptide. This bio-click chemistry reaction offer a means to produce bioconjugate with a controlled and defined orientation of antibody compared to CuAAC reaction [14].

Table 2.1 Different strategies for the synthesis of antibody-NP conjugates: advantages and disadvantages. Adapted from Sivaram, et al., 2017 [7].

Strategy Functional groups Advantages Disadvantages Ref. involved

Physical Adsorption Hydrophobic Hydrophobicity Simple coupling Change in 3D confirmation, [64-66] interactions mechanism resulting in Ab denaturation; lack of specificity ; poor reproducibility Electrostatic Charged groups Doesn’t require Depends highly on the [67, 68] adsorption activation of antibodies environment and the and NPs; quick availability of charged groups; interaction interactions are highly pH dependent; varies with respect to physical properties of NP Hydrogen bonding Groups with H- Facile, versatile, no need Reversible attachment; poor [69, 70] bond donor and to modify the antibody reproducibility acceptor Covalent binding EDC-NHS amide Primary amine on Native antibodies can be Involves multiple steps; [71-74] bond antibody; used; highly efficient chemical modification of NPs; Carboxylic and active sites of antibodies can epoxide groups on interact with NPs and cause NPs less chance of antigen binding; loss of the reactive moieties with other interactions

Disulfide Disulfide group; High stability; better Chemical modification of the [75-79] functional groups Thiol group; ligand positioning antibodies /maleimide thiol Sulfhydryl group coupling Sugar moieties Gold surface; Thiol Oriented binding; Better Antibodies needs to be [80-82] reactive group; Specificity; Highest modified to interact with NPs; Hydrazide group; retention of antigen sugar moieties won’t be Amine group; binding capacity available for conjugation Piridyl group Click chemistry Mostly oriented binding; Presence of copper (Cu) [83-87] (Cu assisted) Strong binding; Quick catalyst can results in Azide group; interaction; Better yield instability of protein; copper Alkyne group; even at room catalyst hinders the Tetrazine and temperature; Zero By- fluorescence of the dyes; Trans-cyclooctene products toxicity issues with copper component catalyst requires Cu chelators to minimize toxicity; require modification of the Ab with the linker group Mostly oriented binding; Click chemistry strong binding; quick Require modification of the [88-90] (Cu free) interaction; better yield Ab with linker group (Bioorthogonal Azide group even at room click chemistry DBCO group 13

Tetrazine and temperature; zero by- trans-cyclooctene products component

Adaptor biomolecules Biotin-binding Biotin; Streptavidin; Mostly oriented binding; Expensive; Difficult to control [91-94] proteins Avidin; NeutrAvidin Highly stable interaction; NP valency; difficult to Resistant to varying control the binding site; external factors. labelling can be heterogeneous Secondary Anti-PEG antibodies Easy to control NP Reversible binding; [60, 62] antibodies valency; Ab remains orientation highly depend on unaffected the secondary antibodies; Secondary antibody must be monoclonal and it is expensive Fc binding proteins Protein A: Protein G; Ab remains unaffected Difficult to control NP [95-98] Protein A/G valency; Fc binding proteins (A,G) are expensive: Reversible binding Nucleic acid Oligonucleotide Highly specific Orientation highly depend on [99-103] hybridization sequences complementary base the nucleic acids; difficult to pairing; ideal for control ligand orientation on biosensor application NP surface

Enzyme based bio- Sortase A enzyme Precise control over Coupling limited by the large [104] conjugation conjugation; mild size fusion protein; multiple reaction condition preparation steps

2.3 Cancer biomarkers as a receptor target for cancer therapy

Typically, cancer cell therapeutics and in particular mAbs, target various membrane- associated proteins that include CD19, CD20, EGFR, PDL1 and others. The differential expression of membrane proteins on cancer cells defines the phenotype, and over the past few decades, many membrane-associated proteins have been investigated as potential targets for cancer therapy. These proteins, spanning the cell membrane (transmembrane or integral membrane proteins), or on the surface of the membrane (peripheral membrane proteins), are involved in the regulation of cancer cell formation, and have been extensively studied as cancer prognosis/diagnosis biomarkers and applied to targeted imaging and therapeutic applications. [105-107]. In general, the expression of membrane proteins is upregulated in tumor and tumor associated cells, including in malignant and angiogenetic cells [108], which can be discriminated from normal, healthy cells of tissues. Based on their biological function, there are at least 5 classifications of membrane-bound protein, including receptors (tyrosine kinase receptor (TKR) and G-protein coupled receptor (GPCR)), cell adhesion or anchoring proteins, cell membrane-associated enzymes, transporter proteins, and glycosylphosphatidyl inositol (GPI) proteins [106]. Several membrane-associated proteins have been widely studied as cancer biomarkers and disease targets, and numerous clinical applications have been reported, with treatment of breast,

14 lung and prostate cancer being the most prevalent, and combined, make up the majority of cancer related mortality (Table. 2.2). The research reported in this thesis is associated with the targeting of antibody-nanomaterial conjugates to the prostate specific membrane antigen which is overexpressed in prostate cancer.

Table 2.2 Characteristic of membrane-associated proteins as cancer antigen.

Protein Role in Cancer Clinical applications Ref. Tyrosine kinase receptors Colorectal, Uncontrolled cell Cetuximab, Panitumumab, [109, 110] EGFR Non-small cell lung proliferation, and Necitumumab cancer metastasis (ErbB-1) HER-2 (ErbB-2) Breast, Gastric, Cellular proliferation Trastuzumab, Pertuzumab, [111, 112] Esophaegal, Ovarian and differentiation Neratinib, Lapatinib, Afatinib EphA2 Solid tumors Metastasis and poor Anti-EphA2(1C1)-auristatin drug [113-115] clinical prognosis (toxin) conjugate; Dasatinib, synthetic small molecule inhibitor, binds to EphA2 VEGFR Colorectal Angiogenesis Bevacizumab, Ramicrumab [116, 117] Lung Cellular proliferation, Rilotumumab, Onartuzumab, [118-120] cMET /HGFR metastasis, invasion, Ficlatuzumab angiogenesis, and cell motility IGF-1R Solid tumors Cellular proliferation, Cixutumumab, Figitumumab [121-123] invasion and angiogenesis GPCR Chemokine Multiple Myeloma Distant metastasis, 68Ga-Pentixafor [124, 125] receptor, CXCR-4 and Lymphoma angiogenesis, and therapeutic resistance Cell adhesion molecules CEA Colon Tumor invasion and Labetuzumab [126, 127] metastases EpCAM Colon Cell signaling, Adecatumumab, Catumaxomab [128-130] proliferation and differentiation αvβ3 integrin Endothelium Tumor invasion, Etaracizumab [131, 132] angiogenesis, and metastases Cell membrane associated enzymes PSMA Prostate unclear Capromab, J591 [133] CD13 Macrophage Intracellular signaling - [134] CAIX Colon Regulator of pH Girentuximab [135, 136] GPI-anchored proteins Mesothelin Mesothelioma Peritoneal Amatuximab [137] implantation, and metastasis PSCA Prostate, Pancreatic unclear AGS-1C4D4 [138] uPAR Colon cell adhesion, ATN-658 [139] migration and proliferation Folate receptor Ovary DNA synthesis, Farletuzumab [140] methylation, and repair

15 2.3.1 Prostate specific membrane antigen (PSMA)

The overriding rationale for choosing and utilizing a membrane-bound protein as a target for cancer imaging and therapy, is the significant over-expression of that particular membrane bound protein in neoplastic growth compared to that in normal tissue. In prostate cancer disease, for example, several antigens are overexpressed, including PSMA, the epithelial cell adhesion molecule (EpCAM), ephrin type-A receptor 2 (EphA2) and the vascular endothelial growth factor (VEGF), all having potential as prognostic biomarkers in prostate cancer [141-144]. Among these antigens, PSMA has been demonstrated to be an ideal prognostic marker since PSMA expression is upregulated many fold in almost all prostate cancer stages, including tumor aggressiveness, metastatic disease and disease recurrence, but has a low expression in normal healthy tissues [16, 17, 145-147]. PSMA, known as glutamate carboxypeptidase II, or folate hydrolase 1 (FOLH1), is a type II transmembrane glycoprotein containing 750 amino acids, with MW around 100 kDa. PSMA has enzymatic activity, serving as transport or binding proteins or have hydrolytic activity [148]. PSMA is not only expressed in prostate tissues, but is also found at lower levels in other tissues, such as kidney, liver, small intestine, and brain [133, 149]. The PSMA protein has a unique 3-part structure, including an N-terminal internal portion (19-amino-acid), a transmembrane portion (24-amino-acid), and an external portion (707-amino-acid) [146, 150]. This large external portion, known as the extracellular domain of PSMA contains proline- and lysine rich domains with unknown function, a large catalytic domain, and a C-terminal domain. This domain has nine N-linked glycosylation sites, which are required for secretion of the cleavable extracellular domain, membrane expression and enzymatic activity [151, 152]. To date, numerous ligands including small molecules, antibodies, and antibody fragments targeting PSMA have been generated and radiolabeled for therapeutic use. These PSMA ligands are currently being used and investigated in pre-clinical and clinical studies for prostate cancer imaging and therapy [153]. For example, 111In-capromab-pendetide has been approved by US FDA for imaging soft tissue metastatic prostate cancer. Other radiolabeled PSMA ligands are being studied in phase II and III clinical trials as theranostics (combining diagnostic and therapy), such as 68Ga-PSMA-PET, 177Lu-PSMA-617, and 177Lu-anti PSMA J591 [154]. In this study, two different bioconjugation methods including BsAb platform technology and unnatural amino acid azide labelling will be used to tether anti-PSMA antibody fragments to nanomaterials for application in diagnostics, in vivo imaging and potential therapeutic delivery to prostate cancer tumors.

16 Chapter 3 Non-covalent conjugation of an anti-PSMA antibody to a hyperbranched PEG polymer using bispecific antibodies as precision nanomedicine for prostate cancer

3.1 Introduction

Antibodies have the capability to bind with high specificity to specific antigens, which makes them ideal reagents for biomedical research applications. As tools, numerous applications of antibodies have been widely reported and utilized in biomedical research, as secondary antibodies conjugated to enzymes or fluorophores for the purpose of detection of specific molecular entities, such as in immunohistochemistry, Western blot, immunocytochemistry, flow cytometry and ELISA [155-157]. In addition, antibodies are highly effective as therapeutic and in vivo diagnostic agents, and are utilized extensively in cancer medicine. They can be used as naked antibodies for therapeutic applications, taking advantage of immune effector function such as Antibody Dependent Cellular Cytotoxicity (ADCC) and Compliment Dependent Cytotoxicity (CDC), or they can be coupled with other different molecules, such as radioisotopes, chemotherapeutic drugs, nanomaterials, and fluorescent agents, that impart targeted imaging and cytotoxic capability [158]. Bispecific antibodies (BsAbs), also called ‘dual targeting antibodies’ have recently come to the attention of the global scientific community for development of diagnostic and therapeutic applications in cancer treatment [159]. BsAbs were discovered in the 1960s when the two different antigen-binding fragments (Fabs) of polyclonal sera were conjugated to make bispecific F(ab’)2 molecule [160]. However, the first approved BsAb is Catumaxomab, a trifunctional antibody combining EpCAM antigen, CD3 antigen for T-cell and an Fc portion for the recruitment of immune cells and received regulatory approval by European Medicine Agency (EMA) in 2009. Catumaxomab has an orphan drug status from FDA for the treatment of malignant ascites. In 2014, another BsAb, blinatumomab, was approved by FDA. Blinatumomab is a bispecific T cell engager (BiTE), comprising two scFvs, whereby one arm binds to antigen CD19, while the other arm targets CD3, which redirects and potently activates T cells. This BsAb is prescribed for the treatment relapsed or refractory (R/R) precursor B‐cell acute lymphoblastic leukemia (ALL) in adults and children [161]. The introduction and approval of BsAbs in the global market place has generated much attention and research activity within academia and industry, and the bioengineering of BsAbs has led to many candidate BsAbs entering clinical trials globally [162, 163]. In general, recombinant BsAbs can be differentiated based on the format and composition, including the presence or absence of Fc domain, symmetric or asymmetric architecture, and the number of binding sites [164]. A number of methods for the generation of BsAbs have been developed, including via chemical conjugation and

17 covalent attachment of fragments, genetic (protein or cellular) engineering, and through somatic fusion of two hybridoma lines (quadroma) [165]. Clinical applications of BsAbs are mainly for cancer therapy. Redirecting immune effector cells, such as T cells to tumor cells is common function for various designs of BsAbs, and as mentioned includes the clinically approved BsAbs using triomab (catuxomab) and BiTes (blinatumomab). Another BsAb design for the purpose of retargeting T cells to tumor cells is the Dual-affinity retargeting molecule (DART). The DART technology improves the stability of BsAbs and may decrease immunogenicity. Aside from T cell engagement, other strategies for engaging immune cells have been investigated, including the use of BsAbs to engage macrophage, monocytes and NK for the killing of tumor cells [166]. With multiple targeting sites within one single therapeutic entity, BsAbs also have significant potential for the treatment of other disease indications, including allergic, infectious and inflammatory diseases. BsAbs also have significant potential to be included in diagnostic applications, and have already been developed for detection of infectious diseases caused by bacterial and viral infections, as well as different types of cancers [159, 166]. In a new, novel area of application, BsAbs have recently been utilized for the creation of antibody-targeted NPs [167, 168]. In another example, our group at AIBN has developed a novel methodology of BsAbs for targeting NPs to tumor cells through non- covalent conjugation. One arm of the BsAb binds the NP material, while the other targets membrane-bound, tumor associated antigens. One example is the targeting of a bacterially derived NP with a BsAb with the dual targeting capability for lipopolysaccharide (LPS) and anti-epidermal growth factor receptor (EGFR). In this system, the NP, termed the Engeneic Delivery Vehicle (EDVTM) is naturally coated with LPS. These nuclear NPs of approximately 400 nm in diameter, can be loaded with drugs such as doxorubicin, and injected systemically for the treatment of solid tumors. The EDVsTM can be actively targeted to tumor cells by mixing with a BsAb preparation that targets EGFR and LPS [61]. In this way the EDVTM is coated with BsAb and imparts EGFR functionality. A simple and rapid method of conjugating antibody to NP has been developed using one-step mixing of mPEG NPs with BsAb containing a fusion Fab fragment of humanized anti-mPEG into scFv with specificity to the EGFR/HER2 tumor antigens utilizing 15 amino acids flexible linker ((G4S)3). The methoxyl group in mPEG NPs participates in binding activity of anti-mPEG Fab fragment obtaining homogeneity product since no chemical conjugation step in this process. This simple and rapid method is also interchangeability, which is can be applied for other target receptors [62, 169]. Our group at AIBN has also developed the similar technique as a novel methodology for targeting PEGylated NPs to tumor cells. This non-chemical conjugation system has been reported utilizing an anti-EGFR and anti-mPEG scFvs joined with G4S flexible linker and PEGylated NPs. It

18 was also demonstrated that utilizing hyperbranched mPEG results in stronger binding of BsAbs compared to that of linear mPEG due to avidity effects [170]. Herein we established the utility of a BsAb (tandem scFv consisting of anti-PSMA and anti- mPEG scFvs), and PEGylated NPs made of hyperbranched mPEG polymer (HBP) as an actively targeted nanomedicine for the potential diagnosis and treatment prostate cancer. PSMA has been extensively investigated as a target for prostate cancer and has shown to be a promising target for tumor imaging and therapy. Until now, there is only one clinical agent targeting PSMA approved by the US FDA for identification of metastatic prostate cancer, utilizing antibody-radiolabeled, 7E11- C5 mAb-111In conjugate, called ProstaScint®. However, this conjugate has a limitation due to the mAb only binding the intracellular domain of PSMA. Thus, only cells with damaged membrane, such as necrotic cancer cells will bind 7E11-C5[171]. Several mAbs have been reported to bind extracellular epitopes of PSMA including J591, D2B, 3C6 and 3/A12 which are widely utilized in research associated with prostate cancer imaging [172-175]. However, to date, only the J591 mAb has progressed to clinical trials. J591 mAb targets an extracellular domain of PSMA and binds to the majority of the apical domain, which is highly exposed and accessible. As a result, this antibody binds with high affinity to PSMA receptor [176]. In addition, a humanized version of J591 has been shown to be effective in imaging and delivering radionuclides resulting in therapeutic responses in prostate cancer in early phase 2 clinical trials [19, 177, 178]. In this study, we utilize J591 mAb as a ligand for targeting nanomaterials to PSMA receptor in prostate cancer, in the continuing development of useful targeted nanomedicines. We produced a BsAb by creating a tandem scFv consisting of the J591 scFv fused with an anti mPEG scFv by a G4S linker. The BsAb was mixed with a PEGylated NP (made of mPEG HBP), labelled with dye for detection and loaded with the cytotoxic drug doxorubicin. This study evaluated the capability of this BsAb to actively target PEGylated NPs to PSMA. To investigate this capability, in vitro targeting efficiency, cellular uptake and in vitro cytotoxicity were studied, as well as the optimal molar ratio of BsAb:HBP.

3.2 Materials and methods

3.2.1 Hyperbranched polymer

The hyperbranched polymers (HBP) used in this study was kindly provided by Professor Kristofer Thurecht (AIBN, UQ). The HBP was synthesized using protocols as described in Howard et al. [60] for Cy5-labelled HBP and in Pearce et al. [179] for Doxorubicin-loaded HBP.

3.2.2 Cell lines

Human prostate cancer cell lines, PC3 (ATCC CRL-1435) which is PSMA negative cell; LNCaP (ATCC CRL-1740) and PC3-PIP (PC3 transfected with human PSMA) which are PSMA positive cells, were cultured in RPMI 1640 (Gibco) containing 10 % fetal bovine serum (Gibco). All cells were incubated at 37 °C in 5% CO2.

3.2.3 Cloning and protein expression of anti PSMA J591-mPEG BsAbs

The BsAb sequences were synthesized and codon optimized for mammalian expression system by Geneart. Anti mPEG scFv [180] was incorporated with an anti-human PSMA J591 scFv comprising heavy chain variable region (GenBank: CCA78124.1) and kappa light chain variable region (GenBank: CCA78125.1) with flexible linker (G4S)3. A 6x Histidine and c-myc tags were incorporated at the N- and C-terminus respectively to facilitate purification as well as detection (Appendix A, Figure A.1) as described in Taylor et al. [61]. The optimized codon synthetic gene was cloned into a mammalian expression vector (NBF-347, National Biologics Facility) using HindIII and NotI restriction sites. The positive recombinant plasmid was expressed in a mammalian expression system using Chinese Hamster Ovary (CHO) cells. The CHO- XL99 which are adapted suspension cells from the attached CHO-K1 cell line (ATCC) was cultured in CD CHO medium with TM 8 mM GlutaMAX (Gibco) at 37 °C, 7.5% CO2, with shaking at 125 rpm. On the day of transfection, the cells were prepared at 3x106 cells/ml using dilution with fresh medium. The transient transfection was performed as the following: plasmid DNA (2 µg/mL) was diluted in OptiPro serum free media (Gibco) (12.5% total volume). Concurrently, the PEIpro (Polypus-transfection) (8 µg/mL) were also diluted in OptiPro serum free media (Gibco) (12.5% total volume), and incubated for 30-60 sec. The two solutions then were mixed by pipetting and incubated for 15 min at room temperature without disruption. The DNA-PEI complex then were added into the culture in a shake-flask and gently swirled. The culture was incubated at 37 °C, 7.5% CO2, and shaking at 125 rpm for 4 to 6 h. Following this incubation, the culture was diluted in the fresh CD CHO medium with 8 mM GlutaMAXTM at ratio 1:2 (v/v) containing 7.5% Efficient Feed A and B (Gibco) (v/v), and 0.4% anti-clumping agent

(Gibco). The culture incubation was then continued at 32 °C, 7.5% CO2, and shaking at 130 rpm for 13 days. The culture was then harvested by centrifugation at 4750xg for 10 min. Following this, the BsAb expression in CHO cells was evaluated using western blot analysis. Briefly, the supernatant was loaded into precast polyacrylamide gels NuPAGE™ 4-12% Bis-Tris protein gels (Invitrogen). The protein sample was run in 1x MES buffer (Invitrogen) for 35 min, 200 V, then semi-dry transferred into PVDF membrane (Biorad). The membrane was blocked in blocking solution

20 containing 2% milk in 1xPBST (0.05% Tween-20 in 1X phosphate buffer saline (PBS)) for 30 min, and was then incubated with anti-cMyc-HRP antibody (Miltenyi Biotec) diluted 1/5000 in blocking solution for 1 h at 4° C. The membrane was washed 3 times in PBST solution, and then soaked with ECL substrate (Novex™). The protein band was detected using the Chemidoc imaging system (Biorad).

3.2.4 Protein purification

The supernatants were filtered through 0.22 µm PES membrane (Millipore) then applied onto HisTrap excel column (GE Healthcare). The column was equilibrated with phosphate buffer containing 20 mM sodium phosphate, 500 mM NaCl pH 7.4. The BsAb supernatants were then loaded to the column, followed by washing using the buffer and eluting with the buffer containing 500 mM imidazole. The His eluate fraction was then buffer exchanged into 20 mM sodium phosphate, 500 mM NaCl pH 7.4 to remove imidazole using HiPrep 26/10 column (GE Healthcare). The purified protein was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) using Bolt 4-12% Bis-Tris Plus gels (Invitogen) and protein purity evaluated by coomassie blue staining of proteins (InvitrogenTM NovexTM SimplyBlueTM SafeStain).

3.2.5 Protein Characterization

Size exclusion chromatography (SEC) was performed by applying 500 µL of sample into Superdex 200 10/300 GL (GE Healthcare) column equilibrated in 1x PBS with 20% Ethanol, and connected to AKTA purification system. The purification system was run using the flow rate of 0.5 ml/min. The size distribution profile of the purified BsAb was also evaluated using dynamic light scattering (DLS) technique (DynaPro®Plate Reader, Wyatt Technology). 30 µl of 1 mg/mL BsAb was used for analysis. In addition, the identification of BsAb was examined using peptide mass fingerprinting (Mass Spectrometry Facility, School of Chemistry and Molecular Bioscience (SCMB), UQ). Briefly, the trypsinized and zip tipped samples (standard protocol at Mass Spectrometry Facility, SCMB UQ, Dr Amanda Nouwens) were separated using reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nano-system. Using a flow rate of 30 µl/min, samples were desalted on a Thermo PepMap 100 C18 trap (0.3 x 5 mm, 5 µm) for 5 min, followed by separation on a Vydac Everest C18 column (150 mm x 75 µm, 5um) at a flow rate of 300 nL/min. A gradient of 5-60% buffer B over 35 min where buffer A = 1 % ACN / 0.1% FA and buffer B = 80% ACN / 0.1% FA was used to separate peptides. Eluted peptides were directly analyzed on an Orbitrap Elite mass spectrometer (Thermo) using an

21 NSI electrospray interface. Source parameters included a capillary temperature of 275° C; S-Lens RF level at 63%; source voltage of 2.4 kV and maximum injection times of 200 ms for MS and 150 ms for MS2. Instrument parameters included an FTMS scan across m/z range 350-1800 at 30,000 resolution followed by information dependent acquisition of the top 10 peptides in the ion trap. Dynamic ion exclusion was employed using a 15 second interval. Charge state screening was enabled with rejection of +1 charged ions and monoisotopic precursor selection enabled. The protein identification was then performed using ProteinPilot™ Software 5.0.1 (Sciex).

3.2.6 Target binding ELISA

The target binding of the J591-mPEG BsAb was evaluated by indirect ELISA (enzyme linked immunosorbent assay) methods using recombinant human (rHu) PSMA as target protein (RnD Biosystems), and Doxoves®-Liposomal Doxorubicin HCl (FormuMax) and hyper branched polymer (HBP) based mPEG immobilized on ELISA plates. Individual wells of a 96-well maxisorp plate (Nunc) were coated with 100 μL of 10 μg/mL of HBP or Doxoves, or 10 μg/mL of target recombinant receptor (rHuPSMA) in 1 X PBS (pH 7.4) for 16–20 h at 4 °C. Following coating, the solution was decanted and 200 μL of 2% skim milk in PBST (PBS + 0.05% Tween 20) was added to each well for 60 min to block nonspecific binding. The blocker was decanted and 100 μL of purified J591-mPEG BsAb was added into each well. The negative control anti-EGFR-mPEG BsAb [60] was used for rHuPSMA, and control PBS was used as a negative for the polymers. The experiment was tested in triplicate wells for statistical purposes. The BsAbs and PBS was incubated for 2 h and then decanted, followed by washing step using manually washed in PBST for five times. Then, 100 μL of HRP labeled anti-c-myc antibody (Miltenyi Biotech) diluted 1/5000 in blocker was added to each well and incubated for 30 min. The c-myc antibody was then decanted, followed by final washed for five times manually with PBST. The colorimetric reaction was performed by adding 100 μL of 3,3′,5,5′- tetramethylbenzidine (TMB; Sigma) into each well, then incubated for 15 min or until adequate color development was identified. The TMB colorimetric reaction was neutralized using 100 μL of 2 M sulfuric acid and were immediately analyzed at an absorbance of 450 nm using the Spectramax plate reader. Average absorbance and standard deviation for each sample was determined and presented as histograms using excel software. This ELISA method was also used to confirm the specificity of J591-mPEG BsAb into mPEG molecules. Individual wells of a 96-well maxisorp plate (Nunc) were coated with 100 μL of 10 μg/mL of different size of linear mPEG molecules (MW of 2000, 5000, 10000, 20000, and 40000); hydroxyl PEG (OHPEG) linear as negative PEG molecules (MW of 2000, and 5000); HBP; and PBS. All

22 polymers were derived from Sigma and Jomar Life Sciences. The following ELISA protocol was the same as mentioned above.

3.2.7 Western Blot

Binding specificity of the BsAb J591-mPEG was evaluated by western blot analysis using rHuPSMA and cell lysate PSMA expressing prostate cancer cells (PC3-PIP and LNCaP). PC3 (PSMA negative) cell lysate was used as a negative control. Cell monolayers of the cells were removed from culture flasks with enzyme-free cell dissociation buffer (Gibco), centrifuged at 500 rpm for 4 minutes and cell pellet was washed three times with ice-cold phosphate buffer saline (PBS). The cell pellet was solubilized in RIPA buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NonidetP- 40) (1 ml per 10 X 106 cells) and 1x protease inhibitor cocktail (Roche). Lysate were clarified by centrifugation at 5000xg at 4 °C for 10 minutes and supernatants were recovered. BCA assay was used to determine the concentration of protein using bovine serum albumin (BSA) as the standards. Proteins (50-60 µg) were mixed with reducing buffer then separated by SDS-PAGE gel (Bolt 4-12% Bis-Tris Plus gels, Invitrogen) and transferred onto PVDF membranes (Trans-blot Turbo Transfer System, Bio-rad) and incubated with 5% milk blocking solution in 1 % tween in PBS (PBST) for 60 minutes, then with J591-mPEG BsAb (1/2500) in PBST milk at 4 °C for overnight. Membranes were incubated with anti c-myc HRP secondary antibody (Invitrogen) (1/5000) in PBST milk for 60 minutes, then were washed three times with PBST before added developing solution (Novex ECL Chemiluminescent, Invitrogen). The membrane was imaged using gel documentation system (Bio- Rad) under chemiluminescent blots-high resolution method.

3.2.8 Binding analysis of BsAb on prostate cancer cell lines by flow cytometry

The cell monolayer of prostate cancer cell lines were removed with enzyme-free cell dissociation buffer (Gibco), centrifuged at 500 rpm for 4 minutes and resuspended with fresh 10% FBS-PBS to give concentration of cells 2 x106 cells/mL. The BsAb at concentrations of 1× 10-6 M were added to 100 μL of cells (2 x 106 cell/mL) and incubated for 60 minutes at 4 °C. Following incubation, the cells were centrifuged gently at 2000 rpm for 2 minutes, the supernatant was pipetted off and the cells were washed three times with 200 μL of 10%FBS-PBS. After the final wash, the supernatant was removed from the cells and the pellet was resuspended in 50 μL of FITC labelled anti-c-myc secondary antibody (Miltenyi Biotec GmbH) diluted 1/11 in 10%FBS-PBS, then incubated in the dark for 60 minutes at 4 °C. Following incubation, the wash steps were repeated and then the cell pellet was resuspended in 100 μL of PBS for analysis by flow cytometry. Cells were

23 analyzed on the Accuri C6 Flow Cytometer using optical filter settings FL1 (533/30 nm) to detect FITC labelled secondary antibody. Data were evaluated using FCS Express 4 Flow based software.

3.2.9 Bioconjugation of BsAbs-Cy5 labelled hyperbranched polymer

The bioconjugation was performed using a simple method, one-step mixing conjugation reaction. To evaluate the bioconjugation reaction, DLS analysis was performed to evaluate different particle size of bioconjugate compared to BsAb alone. 30 μL of BsAb at concentrations of 1 mg/mL were mixed with 4 μL of 2 mg/mL of Cy5 labelled methoxy-Poly-ethylene glycol hyperbranched polymer (m-PEG-HBP) (Kristofer Thurecht lab collection, AIBN). The confirmation of bioconjugation of BsAb into HBP and its binding ability into cell membrane expressing PSMA were also performed using the same in vitro targeting technique by flow cytometry experiment as protocol mentioned above without the additional of secondary antibody incubation step. Cy5 dye attached on the HBP was utilized to detect binding activity. Briefly, 100 μL of BsAb at concentrations of 1 × 10-6 M in 10% FBS-PBS were mixed with 100 μL of 1 × 10-6 M Cy5 labelled methoxy-Poly- ethylene glycol hyperbranched polymer (m-PEG-HBP) in PBS by vortexing, then incubated in the dark for about 60 minutes. The negative control was used using the same concentration of Cy5 labelled HBP which was mixed with PBS. The cells were then analyzed through FL4 (675/25 nm) on an Acuri C6 Flow Cytometer (BD Bioscience, San Jose, CA) to detect Cy5. Data were evaluated using FCS Express 4 Flow based software.

3.2.10 Optimal ratio of BsAb to mPEG-HBP

To determine the optimal molar ratio of the BsAb J591-mPEG to mPEG on the hyperbranched polymer (HBP), free unbound BsAb into mPEG-HBP on the bioconjugate were measured by flow cytometry analysis. Six different molar ratios of bioconjugate were prepared (0.1:1; 0.5:1; 1:1; 2:1; 5:1; and 10:1) using fixed amount of mPEG-HBP. The same flow cytometry procedure for binding analysis of BsAb-Cy5 labelled HBP conjugate were performed as mentioned above using PSMA positive prostate cancer cells, PC3-PIP. The cells were then analyzed through Cy5 channel. Data were evaluated using FCS Express 4 Flow based software.

3.2.11 Internalization and co-localization of BsAb-HBP bioconjugate into PSMA positive prostate cancer cells by confocal microscopy

Cells were seeded on the coverslips until 80% confluence following which BsAb conjugated Cy5 labelled HBP (previously incubated at room temperature for 1h at ratio 1:1 in molarity) were added in fresh medium and incubated at 37 °C, 7.5% CO2 for 4 h. Following incubation, cells were washed with 1x PBS for three times then fixed with 4% paraformaldehyde (PFA) solution 0.5 mL for 20 minutes at room temperature. The PFA were removed and the cells were washed with 1x PBS for three times. Finally, the cover slips were mounted on slides containing Vectashield ® with DAPI (Cole-Parmer). Slides were observed and imaged under a confocal microscope (Leica TCS SP8) at Australian National Fabrication Facility's Queensland Node (ANFF-Q). For co-localization study, the cell were settled down on the glass bottom dish (dish/glass: 35/12mm) (WillCo Wells B.V) until 80% confluence. The bioconjugate (previously conjugated at ratio 1:1) were added in the fresh medium and incubated 37 °C, 7.5% CO2. After 24h, the cells were incubated with 5 µM LysoTracker Green DND-26 (Invitrogen) for 1 h in order to visualize lysosome. The medium were removed and the cells were washed with 1X PBS for three times, then fixed with 4% PFA at room temperature for 10-20 minutes. Cells were gently washed with 1x PBS, and 1:2000 Hoechst 33342 (Invitrogen) were added, and incubated for 10 min in room temperature. The staining solution was aspirated then the cells were washed with 1X PBS three times. The dish were observed and imaged under the Leica TCS SP8 confocal microscopy at ANFF-Q.

3.2.12 In vitro cell uptake study of BsAb-conjugated Doxorubicin loaded HBP into PSMA positive prostate cancer cells by confocal microscopy

Cells were seeded on the glass bottom dish (dish/glass: 35/12mm) (WillCo Wells B.V) until 80% confluence. The cell media were discarded and replaced with fresh media containing either 4 µg of free Doxorubicin (Dox) or BsAb-conjugated Dox loaded HBP (previously were mixed and incubated at room temperature for 1h at ratio 1:1 in molarity, containing 4 µg of Dox), then incubated at 37 °C, 7.5% CO2 for 24 h. The medium were removed and the cells were washed with 1xPBS for three times, then fixed with 4% PFA at room temperature for 10-20 minutes. The dish were observed and imaged using Dox channel under the Leica TCS SP8 confocal microscopy at ANFF-Q.

25 3.2.13 In vitro cytotoxicity study of BsAb-conjugated Doxorubicin loaded HBP

The PSMA positive prostate cancer cells, PC3-PIP were seeded in 96 well plate at a density 3 3 x 10 cells per well in complete medium. The cells were incubated overnight at 37 °C, 7.5% CO2. The cells medium were replaced by 100 µL of fresh medium containing either free BsAb, HBP-DOX, BsAb-HBP-DOX, or free DOX at three different concentrations, each triplicate (the BsAb/HBP ratio on the bioconjugate were 2:1; the concentration of HBP-DOX and BsAb were following the DOX concentration). The cells only were also grown as control wells. The cells were then incubated for 48 h, based on the optimum DOX released from the hyperbranched polymer, as described in previous research [179]. The cell viability assay were examined using CellTiter 96 Aqueous One solution Reagents, containing a novel tetrazolium 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] (Promega). Briefly, the cells were washed with 200 µL of fresh medium before the addition of 100 µL complete medium containing 20% of MTS reagents (v/v). Plate were incubated at 37 °C, 7.5% CO2 for 1 h, and colorimetric reaction of each well were measured at the absorbance of 490 nm using Spectramax plate reader. The values of treated wells were normalized to control wells. Cell viabilities were statistically significance calculated using unpaired t-test with the significance P< 0.05.

3.3 Results

3.3.1 Protein production, purification and characterization

The J591-mPEG BsAb was created by fusing two scFvs with specificities for PSMA and mPEG, utilizing a glycine-serine (GS4) linker (Figure 3.1 A). A secretion signal peptide was incorporated at the N terminus of the gene construct to enable secretion of the BsAb from CHO cells during culture; also 6X His and c-myc tags were incorporated at the N- and C-termini respectively, for purification and detection purposes. The BsAb were successfully expressed into supernatant, detected by Western blot analysis using anti c-myc HRP secondary antibody (Figure 3.1 B). The expressed BsAb was purified from supernatant by a HisTrap column. The His eluate fraction was then desalted into HiPrep column to remove the imidazole for biological assay purposes. SDS-PAGE analysis showed the expected MW of ~ 56 kDa (Figure 3.1 C). Identification of protein was examined using peptide mass finger printing. Results showed that 98 % of J591-mPEG BsAb sequence are matched with 95% confidence interval (p<0.05) (Figure 3.1 D). The native structure of the purified J591-mPEG BsAb was then evaluated using several analytical methods, including size exclusion chromatography (SEC), Dynamic Light Scattering

(DLS) and mass spectrometry (MS). SEC was used to estimate the molecular weight of the native protein, and to check the quality and size of protein, evaluating aggregation and/or degradation. The BsAb was analyzed by SEC on a Superdex 200 column run in PBS containing 20% ethanol. The ethanol was incorporated to reduce non-specific interactions of the BsAb with the resin that were observed from preliminary studies. SEC results indicated no large aggregate peaks and two main peaks, a main peak at 14.4ml and a shoulder peak at 15.7ml retention volumes (Figure 3.1 E). A comparison of the BsAb SEC profile with gel-filtration standard (Bio-rad) (Appendix A, Figure A.2) indicates a predicted molecular mass of BsAb in SEC is >158 kDa, which is much larger than expected molecular mass based on the amino acid sequence (~56 kDa) (Appendix A, Figure A.3). This larger MW than the one expected might be due to either protein dimerization or the partially intrinsic disorder character of J591-mPEG BsAb as predicted using algorithms (PONDR®) (Appendix A, Figure A.4). Another hydrodynamic technique was performed using DLS, an advanced method to measure particle size of molecule as well as to check the mono/polydispersity of macromolecule samples. DLS data displayed intensity of particle size distribution as shown in Figure 3.1 F. Results indicated polydispersity of sample with a prominent peak intensity of particle at diameter=7.4 nm. Polydispersity molecules might be explained by partially intrinsic disorder character of the BsAb [181, 182].

A B 1 2 C 1 2

62 kDa 62 kDa

49 kDa 49 kDa

38 kDa 38 kDa

E F

14.4

15.7

Figure 3.1: Detection, identification and characterization of J591-mPEG BsAb expression in CHO cells. A) BsAb gene design and format tethering two scFvs, an anti PSMA (green) and an anti-PEG (red) joined with flexible G4S linker [61]. Secretion peptide (SP) is for BsAb secretion from CHO cells into medium. 6xHis and c-myc are for protein purification and detection. B) Western blot analysis of culture supernatant using anti c-myc antibody confirmed the expression of J591-mPEG BsAb with expected MW of ~56 kDa. C) Purified J591-mPEG BsAb showed the purity of protein. 1: protein marker; 2: BsAb. D) Peptide mass fingerprinting analysis showing peptides group and a sequence coverage map, with 97.9 % of J591-mPEG BsAb sequence was matched (p<0.05). Green indicates the peptide has been identified with at least 95% confidence, yellow indicates at least 50% confidence, and grey indicates no spectral evidence. E) Size exclusion chromatography profile run on Superdex 200 in 1x PBS containing 20% ethanol showed a main peak at 14.4 mL, and a shoulder peak at 15.7 mL retention volume. F) Particle size distribution of purified BsAb using DLS analysis showing heterogeneity molecules. Particle size distribution represents light intensity versus estimated particle diameter with the main particle has 7.4 nm in diameter.

3.3.2 Immuno-assay using ELISA and Western blot

To evaluate the affinity of the anti-J591-mPEG BsAb for respective antigens, an ELISA was employed utilising immobilized antigens, including PSMA and mPEG-containing polymers, allowing the BsAb to bind with specific antigens. The results demonstrated that the BsAb binds specifically to PSMA target, and also binds with mPEG-containing polymers in liposomal doxorubicin and HBP based methoxyl PEG. No signal was detected when the BsAb negative control, an anti-EGFR/mPEG BsAb, was added into immobilized rHuPSMA wells. Similarly, the control PBS did not show any binding to polymers (Figure 3.2 A). The binding of the BsAb was also screened against different size linear mPEG molecules and OHPEG molecules. It was evident that the BsAb bound specifically to linear mPEG with optimal binding to linear chains with Mw of 2000, 5000 and to a lesser degree 10,000 compared to no binding to OHPEG linear chains of 2000 and 5000. There was limited binding to 20K or 40K linear mPEG (Figure 3.2 B). In agreement with ELISA data, Western blot analysis of J591-mPEG BsAb showed the detection of ~100 kDa band in rHuPSMA as well as in the PSMA-expressing prostate cancer cell, LNCaP and PC3-PIP cell extracts; PC3 cell extract (negative for PSMA) showed no protein band in Western blot (Figure 3.2 C). 28 3.3.3 Targeting efficiency of anti-PSMA J591-mPEG BsAb using flow cytometry analysis

To evaluate the binding of J591-mPEG BsAb to membrane-expressed PSMA, we examined the BsAb using two prostate cancer cell lines expressing PSMA receptor, PC3-PIP and LNCaP cells, and PC3 prostate cancer cell (PSMA negative) as a negative control. Cells were incubated with purified BsAb followed by incubation with FITC-conjugated anti c-myc antibody, then analyzed by flow cytometry. The results showed that J591-mPEG BsAb only binds to PSMA positive, PC3-PIP and LNCaP cells, but not to PSMA negative, PC3 cells (Figure 3.2 D), indicating that J591-mPEG BsAb binds native PSMA receptor in prostate cancer cell lines.

C D PC3 PC3-PIP LNCaP

Marker PC3 - LNCaP PC3-PIP - rHu PSMA

98 kDa

525/40- FITC 525/40- FITC 525/40- FITC

Figure 3.2: Immuno assays and binding affinity of J591-mPEG BsAb into PSMA expressing prostate cancer cells. A) ELISA binding data for J591-mPEG BsAb (red) demonstrated specificity for recombinant human PSMA and polymers

29 containing methoxyl PEG in Doxoves®-Liposomal Doxorubicin HCl and mPEG Hyperbranced Polymer (HBP). Negative controls were used using anti-EGFR-mPEG BsAb (orange) and phosphate buffer saline (PBS) (blue), showing no binding into the receptor and polymer targets. B) The BsAb has a specific binding into methoxyl PEG, including mPEG HBP and linear mPEG with MW of 2K, 5K and 10K compared to Hydroxyl PEG linear chains of 2K and 5K; but limited binding to 20K or 40K linear mPEG. C) Western blot analysis of BsAb using rHu PSMA and the cell lysate of PSMA positive human prostate cancer cells, LnCAP and PC3-PIP, as well as PSMA negative human prostate cancer cell lysate, PC3 as a negative control. D) BsAb targeting prostate cancer using flow cytometry demonstrated specificity binding into PSMA positive, PC3-PIP and LNCaP cells, and no bind in PSMA negative, PC3 cells. Light blue: cells with PBS only (control); green: cells with J59-mPEG BsAb.

3.3.4 In vitro analysis of BsAb-Cy5 labelled HBP targeting capability

A previous study has shown that BsAb-Cy5 labelled HBP complexes can be formed in a simple and rapid reaction using one-step mixing of BsAb that exhibits dual specificity for mPEG and the target receptor [60]. In this study, the bioconjugation protocol is shown in Figure 3.3 A. BsAb was mixed with increased amounts of HBP, incubated briefly and analyzed by DLS. The amounts of HBP added to the bioconjugation reaction were too low for accurate size determination by DLS of HBP in the reaction. HBP alone were 5nm diameter (size confirmed by Thurecht group) was provided for experiments. The confirmation of bioconjugate was performed using DLS analysis. Results showed that the diameter particle size distribution of bioconjugate was 13 nm and larger compared to BsAb alone, which was 7.4 nm (Figure 3.3 B). Imaging of cells by flow cytometry or confocal microscopy requires a detectable fluorescent probe. We utilized a far-red-fluorescent dye, Cy5, attached to the polymers. Flow cytometry analysis of BsAb-Cy5 labelled HBP conjugate through the Cy5 filter channel showed enhanced uptake of the conjugate into PSMA expressing cancer cells in comparison to HBP alone (Figure 3.3 C). Confocal microscopy images confirmed that significant uptake and internalization of the conjugates can be observed in PSMA positive, PC3-PIP cells, with fluorescence detected through the Cy5 channel. In PSMA negative, PC3 cells, no fluorescence signal in Cy5 channel was detected (Figure 3.3 D). In vitro co-localization studies were performed utilizing lysosome tracker, and showed that the conjugate was co-localized in the lysosome (Figure 3.4 E). These results demonstrated that J591-mPEG BsAb-Cy5 labelled HBP conjugates can effectively bind and internalize into cells expressing PSMA, making this conjugate an ideal candidate for further imaging development.

30 A

B BsAb

BsAb+HBP

31 C PC3 PC3-PIP LNCaP

660/20- Cy5 660/20- Cy5 660/20- Cy5 D

Hoechst Cy5 LysoTracker Merged Green DND-26

Figure 3.3: BsAb-Cy5 labelled HBP and its characterizations. A) Schematic representation of one step mixing antibody-NP conjugation of BsAb anti-PSMA-anti PEG and PEGylated hyperbranched polymer [7]. B) Particle size distribution of purified BsAb compared to BsAb-HBP conjugate using DLS analysis showing the increased diameter particle size of bioconjugate C) Flow cytometry of J591-mPEG BsAb-Cy5 labelled-HBP showing the bioconjugate has a binding activity into PSMA positive prostate cancer cell, PC3-PIP. Light blue: cells with PBS (control); orange: cells with HBP only; red: cells with BsAb-HBP conjugate. D) Confocal images showing uptake of Cy5-labelled HBP (red) following 1 h incubation of J591-mPEG BsAb-Cy5 labelled-HBP with PSMA positive LNCaP PC3-PIP cells and no cell uptake in PSMA negative PC3 cells. The cell nuclei were stained with DAPI. E) Immunofluorescent staining showing the co-localization of BsAb-HBP conjugate in cellular organelle (lysosome) in PSMA positive PC3-PIP cells (shown in white arrow in the merged image).

3.3.5 Optimal ratio of J591-mPEG BsAb to HBP using flow cytometry assay

To determine the optimal BsAb:HBP ratio in the one step mixing and formulation, formulations were prepared by mixing and vortexing the BsAb and HBP at six different molar ratios (0.1:1; 0.5:1; 1:1; 2:1; 5:1; and 10:1, with a fixed amount of mPEG-HBP) for 1 h at room temperature, before formulations were added to the PSMA positive PC3-PIP cells. As shown in Figure 3.4, the optimal BsAb:HBP molar ratio was around 5:1. The fluorescence intensity significantly increased with an increasing BsAb:HBP molar ratio (i.e. 0.1:1 to 5:1); however, the intensity was not significantly different between ratios of 5:1 and 10:1.

3.3.6 In vitro cellular uptake and cytotoxicity studies of BsAb-conjugated Dox loaded HBP

To determine cell uptake and accumulation, as well as the ability of BsAb-Doxil conjugates to deliver Dox into cells, PC3-PIP cells were treated with BsAb-Dox loaded HBP and incubated for 4h and 24 h. The free Dox was used as comparison to BsAb-Dox loaded HBP. These two formulas indeed demonstrated different cell uptake and accumulation behaviors. The free Dox was accumulated into the nucleus in 4 and 24 h. In contrast, the BsAb-Dox loaded HBP bioconjugate was retained in the outer nucleus membrane at 4h and at 24 h, the Dox was not released from the hyperbranched polymer. This suggest the in vitro stability of cellular uptake of Dox in NP formulation, without any undergoing degradation in the media (Figure 3.5 A). The ability of BsAb-HBP conjugate to release the Dox into the cells was further characterized by a cell viability assay using the MTS assay. Cells were treated with the following formulations: free BsAb, HBP-Dox, BsAb-HBP-Dox and free Dox with varying concentrations of Dox, and incubated for 48 h. As shown in Figure 3.5 B, the HBP-Dox, BsAb-HBP-Dox and free Dox at 1μg/ml Dox concentration resulted in cell viabilities of 80%, 60%, and 30% respectively, whereas for the BsAb alone 90% cell viability was maintained. The results suggest that BsAb-conjugated HBP-Dox showed 20% enhancement in cytotoxicity compared to that of HBP-Dox. Similar trends were observed at 0.5μg/ml and 0.1μg/ml concentration of Dox when comparing HBP-Dox vs the BsAb targeted HBP-Dox. BsAb targeted HBP-Dox had lower cell viabilities compared to HBP-Dox but cell killing effect was not as potent as 1μg/ml.

Figure 3.4: The optimal ratio of BsAb:HBP. Six different BsAb:HBP molar ratios (0.1:1; 0.5:1; 1:1; 2:1; 5:1; and 10:1, with fixed amount of mPEG-HBP) was assessed by flow cytometry analysis using PSMA positive PC3-PIP cells. Histogram (top) displayed as logarithmic scale (grey: cells with PBS (control), blue: cells with HBP alone, red: cells with BsAb-Cy5 labelled HBP conjugate). Bar charts (bottom) displayed linear scale, measuring median fluorescent intensity.

Figure 3.5: In vitro cellular uptake and cytotoxicity of BsAb conjugated Dox loaded HBP. A) Confocal fluorescence images PSMA expressing, PC3-PIP cells showing difference in uptake pattern in the cells treated with free Dox and Dox loaded BsAb-HBP conjugate. Images were taken after 4 and 24 h incubation time. B) The cytotoxicity of the BsAb, HBP- DOX, BsAb-HBP-Dox and free Dox at concentration of Dox 0.1, 0.5 and 1 µg/ml on PSMA expressing, PC3-PIP cells at 48 has quantified by an MTS assay. The bar charts represent mean and ± standard deviation, n=3. *) statistical significance calculated using unpaired t-tests (Significance P<0.05).

3.4 Discussion

Active targeting of drug-loaded NPs to tumor cells offers the potential advantage of enhancing tumor cell uptake of cytotoxic drugs at the tumor site, compared to passive targeting. Although both active and passive targeting take advantage of the EPR effect, actively targeted drug delivery allows further potential for effective cytotoxic drug delivery to tumor cells via specific tumor receptors and can result in enhanced tumor regression compared to that of passive targeting. Furthermore, it shows significant advantages over free drug therapy, with overall lower drug dosage and less adverse reactions [183]. Lower drug concentration and dosing reduces the horrible side-effects experienced by patients receiving chemotherapy and also lowers financial burden. There are a range of conjugation methods for attachment of antibodies, proteins to nanomaterials for application in diagnostics, drug targeting and in vivo imaging. Many of these traditional chemical conjugation methods are inefficient, require rigorous optimization. We engineered a novel bispecific format to facilitate antibody-NP conjugate as a versatile platform for precision nanomedicine. In this study, we developed a BsAb that binds the PSMA receptor in prostate cancer cells and also binds a m-PEG-HBP. When the BsAb is mixed with drug-loaded HBP NPs at an optimized ratio, the BsAb-HBP bio-nanomaterial can be utilized as an actively targeted drug delivery system for the potential treatment of prostate cancer. This BsAb design allows conjugation between antibody and nanomaterials via non-covalent binding resulting in a highly stable and homogenous product. Several techniques have been used to generate BsAbs including cell fusion and hybridoma technology (e.g Catumaxomab) or recombinant DNA technology, combining two antibodies, or antibody fragments (e.g. Blinatumomab) [184]. In this research, we used two scFv antibody fragments, an anti-PSMA scFv J591 and an anti-mPEG scFv, fused with a glycine 4-serine flexible linker, to produce a BsAb in tandem scFv format. The flexible linker allows the two scFvs to retain binding activity for their respective antigens when they are assembled as a BsAb [185]. It has been reported that this format has the optimal design in terms of expression yield and with respect to binding into nanocells NPs, for drug delivery of drug-loaded NPs to tumor cells site [61]. Furthermore, this tandem scFv format is utilized, for example, in the bispecific T cell engager (BiTE®) technology, which has been used to produce Blinatumomab, a T-cell engager approved for treatment of Non-Hodgkins Lymphoma, and other BsAbs configured in the BiTE format which are currently in clinical trial [186, 187]. It has been reported that scFv fragment offer several advantages compared to full-length IgG. An scFv fragment contains the smallest functional antigen-binding domain of antibodies, without the Fc region. Although the Fc domains are responsible for the function in antibodies (e.g. recruits antibodies effector functions and provides longer half-lives), there are situations where Fc- mediated

36 effects are undesirable. For instance, inappropriate activation of Fc receptor expressing cells may induce toxicity via cytokine release [188]. Therefore, the scFv format may display less immunogenicity than that of full length IgG antibody molecules, due to lack of the Fc domain, resulting in a lower immune response and avoiding the fast Fc clearance mechanism, while retaining almost affinity and specificity found in whole IgG [189]. In term of antibody-NPs conjugate, scFvs are able to couple in a more oriented manner to the surface of NPs [190, 191]. In addition, scFvs promote better tissue penetration compared to whole mAbs. However, due to their smaller size compared to full length IgG molecules, BsAbs such as Blinatumomab, display rapid renal clearance, resulting in a short half-life [192, 193]. Therefore, in this study, the tandem scFv design for BsAb was chosen, whereby one arm of the BsAb binds to methoxyl PEG containing polymer, and the other arm binds the tumor target PSMA. PEGylated NPs is common strategy to improve systemic circulation time. Due to the small size (around 55kDa), fast renal clearance of any BsAb that detaches from the NP would be expected. In terms of immunogenicity, the BsAb may display inherent immunogenicity, but would need to be tested in clinical trial. Conjugating of hyperbranched PEG polymer with the BsAb will impart multi valency and avidity of NPs, providing multiple binding sites into cancer receptor target [60]. Although the PEGylated NP possesses stealth properties, the non-covalent attachment of the BsAb to the exterior the PEGylated NP, forming a protein corona, may have an effect on these properties [194]. The hydrodynamic analytical methods by SEC and DLS were utilized to investigate the mass and size distribution profile of the BsAb respectively. The results indicated the BsAb had an approximate molecular mass of 150 kDa by SEC, which is 2-3 times that predicted MW. This suggests that the migration of the BsAb in the SEC matrix may be influenced by the protein structure or folding where this BsAb exists in different structural forms. As predicted by PONDR® protein disorder predictor, this BsAb with tandem scFv format of anti-PSMA/anti-mPEG scFvs might have partial intrinsically disordered protein (IDP) (Figure A.3) [182]. DLS is an in-solution method for determination of protein and particle sizes and unlike SEC is not influenced by the presence of a matrix. The DLS data indicates a 7-8nm diameter structure which correlates with approximately 50kDa protein. The DLS profile displayed a polydisperse particle distribution, which might be due to the IDP or attributed to the presence of multimer and monomer species, which can also be linked to instability of BsAbs in storage buffer [61]. The binding specificity of the BsAb was evaluated using several methods, including ELISA, Western blot and flow cytometry. ELISA data confirmed that the J591-mPEG BsAb binds to PSMA antigen, rHu-PSMA, as well as mPEG containing polymers. In Western blot and flow cytometry analyses, the data demonstrated the BsAb binding specificity to PSMA antigen expressed on LnCAP and PC3-PIP cells (PSMA positive cell lines), with no cross reactivity with the PC3 cells (PSMA negative) [195]. 37

It has been reported that J591 scFv binds the same PSMA epitope as the J591 complete IgG mAb, and so binding specificity is conserved with the J591 scFv format [196]. In this study we showed that our J591-mPEG BsAb binds to PSMA positive cell lines LNCaP and PC3-PIP cells, demonstrating that specificity for PSMA was conserved in the conversion of the J591 scFv to a tandem scFv BsAb format. Monovalent scFvs generally have lower overall affinity for their antigen compared to the affinity of bivalent IgG antibody molecules due to the avidity effect [197]. In the case of the tandem scFvs in this BsAb format, the BsAb forms a corona around the PEGylated HBP. The anti-mPEG arm of the BsAb binds strongly to the HBP due to the abundance (multiplicity) of m-PEG epitopes on the HBP. The exterior of the NP is covered with the anti-tumor target arm of the BsAb, and so imparts avidity in binding to the tumor receptor. HBP offers targeting efficiency compared to other nanomaterials due to its smaller size owing a greater potential in tumor penetration [198]. In addition, HBP provides multiple “arms” which allows for functionalizing with imaging and therapeutic agents. Importantly, the conjugation of BsAb with PEGylated HBP obtains a stable antibody-nanomaterial conjugate and improve the accumulation in tumor cells [60]. The bioconjugation of J591-mPEG BsAb and Cy5 labelled-HBP was carried out in a simple one step procedure, mixing the BsAb and HBP at an optimal molar ratio at room temperature to obtaining stable conjugation and homogeneous product [60]. The conjugation of the BsAb with HBP was confirmed by flow cytometry analysis, which also showed that the BsAb-HBP bionanomaterial binds to PSMA positive prostate cancer cells, LNCaP and PC3-PIP cells. Furthermore, the molar ratio of BsAb to HBP was optimized based on the fluorescence signal detected in flow cytometry analysis at six different BsAb:HBP molar ratios. As can be seen in Figure 3.6, the higher concentration of BsAb gave higher fluorescence intensity. However, the fluorescence intensity at ratio 5: 1 and ratio 10:1 was not significantly different, suggesting that the optimal molar ratio of the BsAb to HBP should be around 5 to 10:1. We also investigated the internalization of BsAb-HBP conjugates into relevant cells using confocal microscopy. Previous studies showed that J591 mAb alone readily binds the plasma membrane in 5 minutes and internalized into the cell within 20 minutes [199]. It has been reported that J591 mAb-dendrimer conjugates are internalized into PSMA positive LNCaP cells in 1 h [200]. In this study, we confirmed that 1 h incubation of BsAb-HBP conjugates within PSMA positive LNCaP and PC3-PIP cells resulted complete internalization. In addition, the co-localization study using organelle cell tracker (lysosome tracker) confirmed that BsAb-HBP conjugate was co-localized in the lysosome, suggesting that the internalization of BsAb-HBP conjugate in prostate cancer cells via an endosome-lysosome pathway. Therefore, this design is promising as an ideal vehicle for imaging and therapeutic drugs delivery.

In this study, we utilized two different PSMA positive prostate cancer cells, including LNCaP and PC3-PIP for cell uptake and internalization studies using flow cytometry and confocal fluorescence images. Results showed a marked difference in cell uptake behavior and internalization in these PSMA expressing prostate cancer cells. The uptake of conjugate by LNCaP cells is consistently lower than that by PC3-PIP cells in flow cytometry data. In addition, difference staining profile are clearly seen between PC3-PIP and LNCaP cells for internalization of BsAb-HBP conjugate through immune fluorescent staining. These phenomenon might be influenced by the over expression of PSMA in PC3-PIP cells, as PC3-PIP cells were created by transfecting PSMA encoding cDNA tr into PSMA negative PC3 cells [201], while LNCaP cells naturally express PSMA [202]. Importantly, we have demonstrated that the BsAb-HBP conjugate design could be utilized as a generic drug delivery system for the potential treatment of tumors with antibody-targeted, drug- loaded NPs. The cytotoxic drug Dox, loaded into the BsAb-HBP conjugate, exhibited excellent stability in the medium, as shown in the confocal microscopy images. The Dox in the HBP is able to be released at low pH, such as in the endosome, through hydrolysis of the hydrazone bond after internalization into the cells [179]. Molecular Dox is known to accumulate in the nucleus with minimal localization in vesicles. From confocal microscopy images, the Dox loaded BsAb-HBP was located in the outer nucleus membrane after 4 h incubation, and less than half staining the nucleus at 24 h, showing that the Dox was released in controlled manner. However, after 48 h, the Dox was successfully released from the BsAb-HBP-Dox conjugate and able to induce toxicity. The Dox in unconjugated HBP-Dox was also released into the cells with a lower cytotoxic effect compared to that of the Dox in conjugated BsAb-HBP-Dox. This suggests that attaching a ligand to the polymer which can specifically target PSMA positive prostate cancer cells, enhances the drug accumulation and cytotoxicity effects. In this in vitro cytotoxicity studies, the Dox loaded BsAb-HBP has a lower killing effect on cells as opposed to free Dox. This expected behavior, as the Dox in conjugated BsAb- HBP-Dox are released in controlled manner, while free Dox can bind into DNA and are able to accumulate within nucleus in a shorter time. However, the previous study showed that in in vivo tumor regression experiments, the Dox loaded ligand-conjugated HBP has more therapeutic effect than the free Dox does, with the volume of tumor decreased by 90% compared to 30% from original volume over the 28 day period, respectively. It is suggested that the HBP did not lead the Dox release during circulation, and therefore not imparting non-specific toxicity in animals. In contrast, the free Dox has a rapid clearance from blood stream following intravenous injection, decreasing efficiency in tumor accumulation and has a detrimental effect in the overall health of animals. Therefore, BsAb- HBP has an ability as targeted drug delivery carrier to increase therapeutic index and can reduce the amount of drug needed [179].

3.5 Conclusion

In summary, we have succeeded in generating BsAb using the tandem scFv format, targeting PSMA receptor on prostate cancer cells and mPEG containing HBP, for actively targeting NPs with antibodies. This BsAb format is non-covalently bound into mPEG containing HBP using one step mixing method, offering simple, rapid and efficient method to target mPEG NPs. This conjugation method overcomes instability of antibody conjugated to NPs resulted from chemical conjugation, leading to homogenous orientation of ligand which allows specific targeting of mPEG NPs to PSMA expressing prostate cancer cells. Due to multiple branches, the HBP used in this study can be functionalized by various cargos, including attachment of imaging dye and conjugation with cytotoxic drug. Therefore, the BsAb-HBP design has a potential to be used as an actively targeted nanocarrier for cancer diagnosis and treatment. The mixing of an anti-PSMA J591-mPEG BsAb with HBP resulted a targeted nanomaterial, which specifically binds into PSMA expressing cells and also improve the cellular uptake of this nanomaterials in the PSMA positive prostate cancer cells. Furthermore, in vitro studies confirm that attaching BsAb into drug loaded HBP facilitates enhanced drug accumulation and cytotoxicity effect compared to that unconjugated HBP.

Chapter 4 Incorporation of an azide-bearing unnatural amino acid into PSMA targeted scFv for molecular diagnostic applications

4.1 Introduction

Chemical modification strategies are extensively used to conjugate molecular diagnostic and/or therapeutic entities to antibodies or other proteins. There are myriad of coupling reactions currently available for creating protein bioconjugates. The classical methods for these bioconjugation techniques utilize modification of cysteine and lysine residues. Other approaches are targeting the N- and C-terminus of proteins, and the modification of native disulphide bonds via the bis-alkylation mechanism [203]. However, there have been numerous reports in the literature of the limitations of bioconjugation techniques, that can result in heterogeneous mixtures of protein conjugates. There have been recent reports of the incorporation of unnatural amino acids (UAA) into proteins, which can facilitate the conjugation chemistry of proteins for bioconjugation purposes [204]. UAA incorporation into proteins within living cells was first reported by the lab of Peter Schultz [205], to address the issues and problems that can arise through covalent modification of natural amino acid side chains, such as the generation of non-desirable products. UAA incorporation into proteins addresses this problem, rendering the highly specific coupling reaction with rapid and efficient under psychological conditions which can preserve the protein structure [206]. In addition, the site-specific incorporation of UAA into proteins by uniquely engineered tRNA/aminoacyl tRNA synthetase (tRNA/aaRS) pairs has gained interest as a promising modification strategy due to the feasibility of UAA addition at any site within any protein, irrespective of its size and solubility [207]. Currently, more than 200 UAA have been incorporated into proteins, and expressed in several living cell systems including bacterial, yeast, and mammalian [208]. In general, incorporating UAA into protein is achieved by introducing a non-sense codon, i.e. an amber codon (TAG) into the gene of interest, followed by co-transformation of additional plasmids encoding amino acyl-tRNA synthetase (aaRS)/ the suppressor tRNACUA pair. The certain UAA is added into cell culture media. The aaRS will catalyze the acylation of the suppressor tRNACUA with the UAA. When the mRNA containing the amber codon is being translated in the ribosome, the amber codon is recognized by the tRNACUA charged with the UAA resulting the addition of this unnatural amino acid into the growing polypeptide chain [209]. The most popular methodology of incorporating UAA into protein to facilitate selective bioconjugation via a covalent chemical bond formation is utilizing click chemistry reaction, including copper catalyzed azide alkyne cycloaddition, copper-free strain-promoted azide alkyne cycloaddition, photoclick cycloaddition, and inverse electron-demand Diels-Alder cycloaddition [210]. As an

41 example, this method has been utilized by Moatsou et al. to incorporate azide bearing protein with the alkyne containing poly [(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymer. They incorporated para-azido-phenylalanine (pAzF) UAA into protein at different positions to find the optimal optimum position that facilitates bioconjugation, while conserving protein structure and functionality. The incorporation of UAA into protein creates versatile protein mutant that are amenable to chemical conjugation, and which can be functionalized for a wide range of applications such as drug delivery, cellular imaging and molecular sensing [211]. Herein, we describe the expression in Escherichia coli of anti-PSMA J591 antibody single chain variable fragments (scFvs), with incorporated pAzF to facilitate the generation of a stable conjugate of anti-PSMA- pAzF with imaging dye or polymer, for targeting prostate cancer. The pAzF was genetically encoded into scFv by an engineered tRNA/aaRS pair derived from M. jannaschii that could incorporate pAzF in response to the amber codon TAG situated upstream at the N-terminus of the scFv gene coding sequences. Subsequently, the antibody fragment scFv-imaging dye/polymer conjugates were generated by the formation of a strong triazole linkage following strain-promoted, copper-free click chemistry reaction between the azide functional group in the pAzF incorporated scFvs and cycloalkyne functional group in the dibenzocyclooctyl (DBCO) containing fluorescent dyes or polymer. In this study, the characterization and verification of pAzF incorporation into J591 scFv were investigated, as well as the successful biorthogonal reactions with DBCO containing dye and DBCO containing polymer.

4.2 Materials and methods

4.2.1 Cell lines

Human prostate cancer cell lines, PC3 (ATCC CRL-1435) which is PSMA negative cell; and PC3-PIP (PC3 transfected with human PSMA) which is PSMA positive cells, were cultured in RPMI 1640 (Gibco) containing 10 % fetal bovine serum (Gibco). All cell lines were incubated at 37 °C in 5% CO2.

4.2.2 Polymers

DBCO-polymer model, DBCO-PEG4-DBCO (Jena Bioscience) with MW around 0.9 kDa was used as proved of concept for click chemistry reaction between azide-modified J591 scFv and polymer containing DBCO group. Diethyleneglycol methacrylate (DEGMA)-co-Pentafluorophenyl methacrylate (MAPFP) polymer with Dibenzocyclooctyne (DBCO) raft (DDM polymer) containing

Cy5 dye around 17.7 kDa in MW (unpublished) was kindly provided by Kris Thurecht group, AIBN UQ

4.2.3 Cloning and protein expression anti-PSMA J591 scFv incorporated pAzF

The anti-human PSMA J591 scFv sequence as mentioned above was synthesized and codon optimized for E. coli expression system by Geneart. Amber codon (TAG) was incorporated at the N- terminus by PCR followed by the strep tag coding sequence and a 6x His tag coding sequence was incorporated at the C-terminus for purification and detection purposes. The amplified gene was cloned into pET26b for periplasmic protein expression to produce soluble protein. Recombinant pET26b containing J591 scFv with amber codon was co-transformed into BL21 (DE3) competent cells with pEVOL-pAzF (Addgene, Schultz lab collection, kindly provided by Dr Angus Johnson) encoding a tRNA synthetase/tRNA pair for the in vivo incorporation of p-azido-l-phenylalanine (pAzF) in response to the amber codon (Figure 4.1). Colonies were grown on LB medium with kanamycin (30 µg/mL) and chloramphenicol (34 µg/mL). A single colony was grown in LB liquid until OD600 reached 0.5-0.8, followed by the addition of 1 mM of IPTG (Astral Scientific), 0.05% of L-arabinose (Sigma ) and 3mM of pAzF (Iris Biotech, GmbH), then incubated at 20 °C; 200 rpm for 16-20 hours. The cultures were harvested by centrifugation at 4000xg for 10 minutes. The soluble protein was extracted from the periplasm by resuspending cells thoroughly in 5mL of ice-cold sucrose solution per 1 gram cell pellet containing 30 mM Tris-HCl pH 8.0, 20 % (w/v) sucrose and 1 mM EDTA. The sample was incubated on ice for 30 minutes then centrifuged at 13.000 x g, 4 °C for 20 minutes. The sucrose fraction was kept and saved on ice. After that, the cell pellet was dissolved in 5 mL of ice-cold 5 mM MgSO4 per 1 gram cell pellet and 1x protease inhibitor cocktail (Roche), then lysozyme (Sigma) was added at final concentration 100 µg/mL. The sample was incubated on ice for 10 minutes then centrifuged at 13.000 x g, 4 °C for 20 minutes. The supernatant was kept on ice, then combined with sucrose fraction followed by dialysis against 1X D-PBS overnight at 4 °C, then purified by Strep-tag affinity chromatography.

Figure 4.1: Design, cloning and expression of anti-PSMA J591 scFv incorporated pAzF in periplasmic space of E. coli system. A) Plasmid design, pelB; promoter for periplasmic expression; TAG: amber stop codon for unnatural amino acid attachment site; StrepTag: strep tag coding sequence for purification purposes; VH: variable heavy chain sequence of J591 antibody; linker: flexible linker (G4S)3; VL: variable light chain of J591 antibody; His6: Histidine 6 X tag for purification and detection purposes; Stop: stop codon. B) A schematic of unnatural amino acid (UAA) incorporation method in periplasmic E. coli expression system. Plasmid encoding J591 scFv were co-transformed with plasmid encoding the suppressor tRNA and amino acyl-tRNA synthetase (aaRS). The UUA pAzF were added into growth medium for subsequent tRNA acylation and its incorporation into J591 scFv.

4.2.4 Protein purification

The protein was purified using StrepTrapTM HP 5 ml column (GE Healthcare), connected to AKTA purifier system, following provided manufacturer’s methods. Briefly, the column was equilibrated with binding buffer, containing 1 X PBS (20 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride) pH 7.4. Then, the filtered sample was applied into the column, and the flow through was collected. The column was washed with binding buffer (washed fraction was collected), followed by elution step using buffer containing 2.5 mM desthiobiotin in binding buffer and the elution fractions were saved. The purified protein was also characterized using size exclusion chromatography (SEC) (Superdex 75 10/300, GE Healthcare), connected to AKTA purifier system using 1 X PBS as the binding and elution buffers.

4.2.5 Flow cytometry analysis of J591-scFv-pAzF

Cell monolayer of grown PC3 (PSMA negative) and PC3-PIP (PSMA positive) cells were removed with enzyme-free cell dissociation buffer (Gibco), centrifuged at 500 rpm for 4 minutes and resuspended with fresh 10% FBS-PBS to give concentration of cells 2 x106 cells/ml. The scFv at concentration of 1× 10-6 M were added to 100 μL of cells (2 x 106 cell/mL) and treated as protocol described previously. FITC labelled anti-His secondary antibody (Miltenyi Biotec GmbH) was used as the secondary antibody. Then cells were analyzed on the Accuri C6 Flow Cytometer using optical filter settings FL1 (533/30 nm) to detect FITC labelled secondary antibody. Data were evaluated using FCS Express 4 Flow based software.

4.2.6 Click chemistry reaction of J591 scFv-pAzF toward Cy5 labelled DBCO

The incorporation of pAzF into J591 scFv was evaluated by copper-free click chemistry reaction using Cy5 labelled-DBCO (Sigma-Aldrich). 10µg of purified protein was mixed with 1µl of Cy5 labelled-DBCO, then incubated at 4 °C overnight. The reaction was evaluated by SDS-PAGE and detected through Cy5 channel protein imaging (ChemiDoc Imaging system, Bio-Rad).

4.2.7 Binding and internalization studies of Cy5 labelled DBCO conjugated J591-scFv-pAzF into prostate cancer cells

Target binding efficiency of conjugate was performed using flow cytometry analysis. The conjugates were incubated in PC3 and PC3-PIP cells and treated as protocol described above without the additional of secondary antibody incubation step. The cells were then analyzed on CytoFlex Flow Cytometer Platform (Beckman Coulter) through APC channel (690/50) to detect Cy5. Data were evaluated using FCS Express 4 Flow based software. For internalization study, the Cy5 labelled DBCO conjugated J591-scFv-pAzF were incubated in the grown cells (PC3 and PC3-PIP cells) with around 80% confluence at 37 °C, 7.5%

CO2 for 4 h. Following incubation, cells were washed with 1x PBS for three times then fixed with 4% paraformaldehyde (PFA) solution 0.5 mL for 20 minutes at room temperature. The PFA were removed and the cells were washed with 1x PBS for three times. Finally, the cover slips were mounted on slides containing Vectashield ® with DAPI (Cole-Parmer). Slides were observed and imaged under a confocal microscope (Leica TCS SP8) at Australian National Fabrication Facility’s Queensland Node (ANFF-Q).

4.2.8 Click chemistry reaction of DBCO-PEG linker as a polymer model into J591 scFv-pAzF

20 µl of 10 µM J591-pAzF was mixed with 2 µl of 10 mM DPD, then incubated at 37 °C, overnight with shaking at 300 rpm. The unconjugated DPD were removed through the 0.5 mL zeba spin desalting column with 7 kDa MWCO (Thermosfisher), following provided manufacturer’s protocol. The reaction were incubated again for another overnight in the same reaction conditions. The conjugated and unconjugated scFv’s were then analyzed using SDS PAGE.

4.2.9 Verification of J591 scFv-pAzF-DPD conjugation by Mass Spectrometry

Protein (~1 µ g) was analyzed using an Orbitrap Elite (Thermo) mass spectrometer and Dionex Ultimate 3000 nano LC system (Thermo). Sample was first desalted on a C4 PepMap300 pre-column (300 um x 5 mm, 5um 300A) using buffer A (30 ul / min) for 5 min and separated on an C4 Acclaim PepMap 300 (75 um x 150 mm) at a flow rate of 300 nL / min. A gradient of 10-70% buffer B over 10 min, followed by 70-98% buffer B over 0.5 min was used, where buffer A was 0.1% formic acid in water and buffer B was 80% acetonitrile / 0.1% formic acid. Eluted protein was directly analyzed on an Orbitrap Elite (Thermo) mass spectrometer interfaced with a NanoFlex source. MS was operated in positive ion mode using the ion trap analyzer. Source parameters included an ion spray voltage of 2.4 kV, temperature at 275° C, SID = 30V, S-lens = 70V, summed microscans = 3, FT vacuum = 0.1. MS analysis was performed across 800 – 2000 m/z. Data was deconvoluted using Thermo Protein Deconvolution ™ software across mass 1100-1800 m/z with minimum of 6-10 adjacent charges, target mass of 30 or 60 kDa as needed, and charge state range 10-100. Deconvoluted data is reported as average mass.

4.2.10 Click chemistry reaction of DEGMA-co-MAPFP polymer with DBCO raft (DDM polymer) with J591 scFv azide

Before the conjugation reaction, the DDM polymer was dissolved in water and kept stirring at 4 °C. Then, J591 scFv were added to the DDM polymer solution and the mixture was allowed to react overnight at 4°C. For a typical preparation, 5mg of DDM polymer (0.72 µmol) and 1 mg of scFv (0.036 µmol) were used (scFv to polymer ratio of ~1:20). After conjugation, samples were kept at 60°C for 10 minutes which will make the polymer insoluble. Upon centrifugation (1.4 rcf for 10 minutes), polymer scFv conjugate forms pellet and free scFv remains in the supernatant. The pellet was freeze dried and redissolved for further characterizations.

The conjugation reaction were analyzed using SDS PAGE. The bioactivity of conjugate was also confirmed by flow cytometry assay using PSMA expressing prostate cancer cells PC3-PIP with the protocol mentioned previously. The polymer alone was used as a negative control.

4.3 Results

4.3.1 Modified scFv production and bioactivity assay

Incorporation of azide-containing unnatural amino acid, pAzF, into J591 scFv was carried out using a periplasmic E. coli expression system, by co-transforming the pEVOL-pAzf plasmid encoding the aminoacyl-tRNA synthetase/suppressor tRNA pair, and an appropriate plasmid containing TAG amber codon at the N-terminus, into competent cells BL21 (DE3). A positive clone was selected and expressed in LB medium with appropriate inducers and amount of pAzF. The cells were harvested, followed by periplasmic extraction using the combination of EDTA-lysozyme and cold osmotic shock. The overall yield of J591 scFv-pAzF in periplasmic E. coli was up to 5-6 mg/L. The J591 scFv pAzF extracts were purified using Strep-tagged protein purification resulting in a protein preparation of high purity (Figure 4.2 A). The purified scFv was characterized by size exclusion chromatography confirming that the purified scFv preparation was predominantly monomeric protein, with a small percentage of dimers and protein aggregates (Figure 4.2 B). To evaluate targeting efficiency into membrane-expressed PSMA, the binding specificity of J591-scFv was verified using flow cytometry, utilizing PSMA-expressing prostate cancer cells (PC3-PIP) and PSMA negative prostate cancer cells (PC3), as the negative control. Flow cytometry results showed that scFv has binding specificity for PSMA receptor, as shown in Figure 4.2 C.

4.3.2 Verification of azide-modified unnatural amino acid incorporation by click chemistry reaction toward DBCO containing imaging-dye

To confirm the site-specific incorporation of UAA pAzF into anti PSMA J591 scFv, the click chemistry reaction of dibenzocyclo-octyne (DBCO) derivative toward mutant scFv was examined. DBCO-labelled Cyanine 5 dye was mixed with scFv; wild-type J591 scFv was used as a negative control. Coomassie blue staining clearly showed the bands of both mutant and wild type scFvs. However, fluorescent imaging of mutant scFv revealed a clear band, while the wild type scFv did not show any fluorescent labelling (Figure 4.3). Therefore, the results confirmed that the azide containing UAA has been successful incorporated into J591 scFv.

4.3.3 In vitro internalization study of scFv-dye conjugate uptake into prostate cancer cells

The internalization of J591 scFv-pAzF - DBCO Cy5 conjugate evaluated by immunofluorescence read out flow cytometry analysis through Cy5 channel (Figure 4.4 A) indicated that the conjugates specifically bind to PSMA expressing prostate cancer cells. In addition, immunofluorescence staining imaged using confocal microscopy showed that the conjugate was fully internalized into PSMA positive PC3-PIP cells, while no binding and internalization into PSMA negative PC3 cells was evident (Figure 4.4 B).

A B

49 kDa

38 kDa

28 kDa

C PC3 PC3-PIP

Events Events

525/40- FITC 525/40- FITC

Figure 4.2: Purification and affinity binding assay of J591 scFv-pAzF. A) SDS PAGE of purified J591 scFv-pAzF showing ~28 kDa in MW. B) Size exclusion chromatography shows that the scFv expressed in periplasmic space contains mainly monomeric protein with low protein aggregates and protein dimers. C) Flow cytometry analyses indicated that J591 scFv-pAzF can specifically bind into PSMA positive human prostate cancer cell line (PC3-PIP), but not into negative cell line (PC3). Histogram, grey: cells with PBS (control); red: cells with J591 scFv pAzF.

B M WT UAA M WT UAA

Coomassie blue Cy5 Channel

Figure 4.3: Evaluation of UAA pAzF attachment into a single-chain fragment variable (scFv) of J591 antibody and its binding analysis. A) Copper-free click chemistry reaction of Cy5 labelled DBCO with J591 scFv-pAzF. B) Comparison between J591 scFv wild type and J591 scFv-pAzF in coomassie blue and chemiluminescence Cy5 channel; M: protein marker; 1: J591 scFv wild type; 2: J591 scFv-pAzF.

Figure 4.4: In vitro binding specificity and internalization studies of azide-modified J591-scFv conjugated into Cy5-labelled DBCO. A) Flow cytometry assays showed that the scFv dye conjugates specifically binds into PSMA positive prostate cancer cells (PC3-PIP) and not into PSMA negative prostate cancer cells (PC3). B) Immunofluorescence analyses also showed the specificity of scFv dye conjugate in PSMA expressing prostate cancer cells (PC3-PIP).

4.3.4 Conjugation of azide-modified J591 scFv into DBCO PEG linker

To verify whether J591 scFv-pAzF can be used to attach other molecules, a DBCO PEG linker with MW around 1 kDa was chosen for copper-free click chemistry reaction, as a testbed for larger polymer. The linker has two DBCO molecules in the opposite arms which can be coupled into azide-bearing scFv (Figure 4.5). To 50 evaluate the conjugation, SDS PAGE analysis was performed. Results showed that more than one bands appear on the gel for conjugated sample as can be seen in Lane 3 in Figure 4.6 A. In addition, the less intense band of degraded protein of unconjugated scFv was also detected, as can be seen on the gel with lower MW at ~19 kDa (Lane 2). Further analysis was then performed using intact mass spectrometry to verify the MW of each band. In the unconjugated scFv, the intact mass was shown to be around 27-28 kDa. However, lower MW species for degraded protein was not detected (Figure 4.6 B). In the conjugated scFv, the intact mass results shown the same average mass with unconjugated scFv, i.e. around 27-28 kDa, and one higher species with average mass of 47 kDa (Figure 4.6 C). These results confirmed there were excess unconjugated scFvs in the mixture of product, and some scFvs were successfully conjugated with 1 kDa DBCO PEG linker in both sides. However, it is presumed that one side of DBCO reacted with 27 kDa scFv and another side of DBCO reacted with lower MW of degraded scFv which has around 19 kDa based on SDS PAGE gel result.

DBCO-PEG4 -DBCO Azide-modified J591 scFv

J591 scFv-DBCO-PEG4 –DBCO-J591 scFv

Figure 4.5: Copper-free click chemistry reaction of J591 scFv-pAzF and DBCO-PEG linker under physiological condition.

51 A B Unconjugated J591 scFV-pAzF (lane 2)

Degraded protein

C Conjugated J591 scFv-pAzF (lane 3)

Figure 4.6: Verification of conjugation of DBCO-PEG4-DBCO with J591 scFv-pAzF. A) SDS PAGE show the unconjugated and conjugated scFvs. Lane 1: Marker; lane 2: unconjugated scFv; lane 3: conjugated scFv shows several bands containing excess scFv (unconjugated) and scFv-polymer conjugates. B) Deconvoluted masses for unconjugated scFv as reported by Protein Deconvolution software. C) Deconvoluted masses for conjugated of scFv-DPD as reported by Protein Deconvolution software, measuring two MW species including excess scFv (unconjugated), and a higher MW species of ~47 kDa. 52 4.3.5 Conjugation of azide-modified J591 scFv into DEGMA-co-MAPFP polymer

For the conjugation of J591 scFv-pAzF with a higher MW polymer, an alkyne group formed as DBCO was incorporated in RAFT block co-polymer consists of DEGMA block and MAPFP prepared using RAFT agent at (~17.7 kDa). Cy5 fluorophore agent was incorporated within the polymer chain through the reaction with MAPFP for detection purposes. The conjugation of DBCO- functional group polymer with azide-bearing proteins was carried using copper free-click chemistry reaction. To evaluate the conjugation, we performed SDS PAGE and imaged the conjugation trough Cy5 filter. It was proposed that covalent attachment of protein via azide UAA to DBCO-polymer may influence the migration of Cy5-polymer. Results confirmed that the protein was successfully conjugated into polymer as can be seen under Cy5 channel. There was no signal in unreacted scFv, while smeared bands were shown for polymer alone and the conjugate. The conjugation reactions were also imaged after coomassie blue staining. The conjugates appeared on the gel as a smeared band with higher MW, compared to a distinct unconjugated scFv band (Figure 4.7 B). Such smears are commonly seen in protein-polymer conjugates, as well as in polymers alone due to the variation in polymer MW [212, 213]. Following protein-polymer bioconjugation, we then carried out flow cytometry analysis to confirm the protein-polymer bioconjugation as well as to evaluate binding ability of bioconjugation into cells expressing PSMA receptor. Results showed that the J591 scFv- pAzF - DBCO RAFT polymer conjugate binds into PSMA positive PC3-PIP cells. Flow cytometry histogram showing a shift in protein-polymer conjugates through Cy5 filter compared to control PBS and polymer alone (Figure 4.7 C).

B C PC3-PIP M 1 2 3 M 1 2 3

Events

Cy5 Channel Coomassie blue

660/20- Cy5

Figure 4.7: Analysis of J591-scFv- DDM polymer conjugates. A) Structure of DEGMA-co- MAPFP with DBCO RAFT (DDM) polymer (right); J591 scFv incorporated pAzF in N-terminus (left). B) SDS PAGE of the protein upon conjugation with polymer detected with Cy5 channel (right) and coomassie blue (left): M, marker; lane 1, J591 scFv- pAzf alone; lane 2, DBCO RAFT polymer alone; lane 3, J591-scFv- DBCO RAFT polymer conjugates. Red arrow shows the bioconjugates. C) Flow cytometry plots show Cy5 fluorescence in PSMA positive PC3-PIP cells incubated with J591- scFv-pAzF DBCO RAFT polymer conjugate: grey, control PBS; red, polymer alone; blue, J591-scFv- pAzF DBCO RAFT polymer conjugate.

4.4 Discussion

The incorporation of UAA into proteins and subsequent production in various cellular expression systems, has proven to be a novel method for protein engineering. Through residue- specific incorporation, the engineering of both physical and chemical properties of proteins is 54 possible, while incorporation at specific sites minimizes structural changes, thus conserving functionality. In this study, we used pAzF providing azide functionality, which was incorporated at the N-terminus of J591 anti-PSMA scFv. The incorporation of azide-bearing UAA facilitates biorthogonal click chemistry reactions via azide-alkyne cycloaddition. Since the TAG amber codon is a stop codon, the position of the TAG codon at the upstream of the gene encoding scFv ensures the generation of only homogeneous products, that is only anti-PSMA J591 scFv incorporated with pAzF. In addition, the incorporation of azide at N-terminal of scFv is favorable, as this position is solvent exposed and generally accessible for further chemical modifications [214]. For production a mutant J591 scFv with UAA incorporation, we expressed the protein in a periplasmic E. coli production system to obtain soluble protein at high yield, avoiding protein aggregation and suppressing the formation of dimers. In agreement with a previous study, soluble J591 scFv-pAzF expressed well in the periplasmic space of E. coli, with a low amount of dimer and the absence of aggregates. However, our results showed that the overall yield of soluble periplasmic J591 scFv-pAzF was about half of the yield of soluble periplasmic wild type scFv previously reported by Frigerio et al., 2013 [215]. This study showed that in general, expressing protein with UAA incorporation using amber codon has a limitation in term of protein yield [216]. The main reason for the low yield of UUA incorporated protein in comparison to the wild type is presumably due to inefficient translation caused by an incompatibility of orthogonal synthetase and its suppressor tRNA pairs with host cell organisms [208]. In addition, when standard periplasmic E. coli expression systems are utilized, our mutant scFv product is mostly expressed as inclusion body in the cytoplasmic space (data not shown). The tendency for protein accumulation as insoluble protein is probably due to the lack of processing signal peptidase, resulting the J591 scFv-pAzF failed to translocate into the periplasmic space of E. coli. However, this study suggests that expression of J591 scFv-pAzF in the periplasmic expression system is still superior compared to that in the cytoplasmic expression systems [217]. The disadvantages of expressing in the cytoplasmic space is the need for resolubilization from inclusion bodies and refolding of the protein which is an inefficient process, and can also produce protein aggregates and dimers. However, the most important reason for the unsuitability of cytoplasmic expression is the instability of the azide group in the presence of reducing agent, such as dithiothreitol (DTT), which is an essential agent in inclusion body solubilization and recombinant protein refolding. DTT, used for the reduction of disulphide bonds in this mutant scFv, can reduce the azide in pAzF to a corresponding amine group, resulting non-functional azide and prohibiting chemical conjugation reactions [218, 219]. Therefore, we found that the efficient production of azide- bearing UAA incorporated into J591 scFv is in periplasmic E. coli expression system. To evaluate the biological activity of the mutant scFv, a binding specificity assay using flow cytometry analysis was performed. The purified mutant scFv showed binding activity to PSMA- 55 expressing prostate cancer cells PC3-PIP, and showed no binding to PSMA-negative, PC3 prostate cancer cells. This binding study proved that the incorporation of azide-bearing unnatural amino acids at the N-terminus preserves the specific binding activity of the J591 anti-PSMA scFv. Following this, a biorthogonal reaction was performed to confirm the incorporation of azide UAA into J591 scFv using copper-free cycloaddition by employing cycloalkyne from DBCO, which is highly reactive towards the azide group. This catalyst-free click reaction avoided the use of the cytotoxic Cu(I) catalyst, which has been widely used in alkyne-azide cycloaddition. The DBCO containing cyanine dye (Cy5) was used for the copper-free click chemistry reaction and mixed with either wild- type J591 scFv or J591 scFv-pAzF. The detection under Cy5 channel confirmed that only mutant scFv containing azide group had been clicked with the DBCO group (Figure 4.3 B). Furthermore, in vitro binding studies were performed to evaluate the J591 scFv-pAzF conjugated Cy5 labelled DBCO using flow cytometry and confocal microscopy. Results showed that the scFv labelled with Cy5 dye, confirming the successful conjugation and has a specific binding and internalization into PSMA positive prostate cancer cells, PC3-PIP. The accessibility of the azide group incorporated at the N-terminus of scFv for coupling reaction was further evaluated through copper-free cycloaddition click chemistry reaction with DBCO containing polymers. Firstly, the reaction was demonstrated by reacting J591 scFv-pAzF with ~1 kDa PEG4 linker containing two DBCOs in the opposite arms. SDS-PAGE and intact mass analyses showed that the scFv was successfully conjugated to DBCO-PEG linker in the both sides. However, since degradation of protein was detected (Figure 4.6 A), the degraded scFv were conjugated into DBCO at one side of PEG linker. This might be because one side of DBCO in the linker is more sterically hindered compared to the other side, so that the less sterically hindered DBCO site was more amenable to react with degraded azide-scFv of lower MW. Secondly, the evaluation of azide accessibility for biorthogonal reaction was performed by reacting the Cy5 labelled DBCO containing DEGMA-co-MAPFP polymer (MW; ~17.7 kDa) with J591 scFv-pAzF. Results showed that the scFv was successfully conjugated to the DBCO polymer, confirmed by the SDS-PAGE analysis under Cy5 channel. Furthermore, flow cytometry analysis proved that the conjugate had binding specificity to PSMA-expressing prostate cancer cells.

4.5 Conclusion

There are a myriad of covalent conjugation methods that have been widely researched to obtain a stable conjugate for successful targeting of mAb to NPs. For conjugation, a suitable functional group has to be incorporated into the mAb or the surface of NP. In this research, we have successfully in vivo incorporated an UAA in E. coli expression system to functionalize the antibody 56 fragment anti-PSMA J591 scFv with azide group at the N-terminus providing bioorthogonality and a higher degree of specificity. Additionally, we have improved the efficient production of this modified scFv by expressing the protein in periplasmic space to obtain highly monomeric protein yields. In this study, we have successfully conjugated the azide-modified J591 scFv with DBCO containing imaging dye or polymers through copper-free cycloaddition click chemistry reaction, which proves a highly accessible of azide moiety in this mutant scFv for conjugation reaction. This covalent binding conjugation demonstrate a stable linkage, which exhibits the potential of various bioconjugations, such as for targeted imaging and therapeutic applications. In particular, we have demonstrated that the J591 scFv-pAzF- imaging dye/polymer conjugates are able to specifically bind into PSMA expressing prostate cancer cells. Further development of antibody-NPs conjugate via copper click chemistry reaction by controlling the density of ligand in polymeric micellar system will identify the interaction of this designed nanomaterial into target cells. Finally, this technology has the potential to be applied for other targeted nanomedicines, such as through covalently coupling to nanomaterial which has theranostic capabilities, or directly conjugating with cytotoxicity drug/toxin as antibody drug conjugate.

Chapter 5 In vivo evaluation of biodistribution and tumor accumulation of antibody fragment- polymer/imaging dye conjugates in PSMA-positive tumor xenograft

5.1 Introduction

The creation of bionanomaterials through attaching a targeting ligand such as an antibody fragment to a nanomaterial, for the active targeting of nanomedicines, has shown some potential in animal studies, which are essential investigations prior to clinical testing. Nanomedicine formulations (i.e. drug-loaded nanomaterials), have shown clinical benefits through improvement of pharmacokinetic profiles and reduced toxicity, compared with systemic delivery of chemotherapeutic drugs [220, 221]. Furthermore, actively targeted nanomedicine has a major advantage in a selective delivery of NPs to the specific tumor targets, remaining in the site for an extended period of time, thereby increasing the local accumulation of the NPs specifically to tumor sites [3, 222-224]. Antibodies and antibody fragments are attractive ligands for the active targeting of nanomaterials, due to their binding specificity for tumor targets. Several antibodies or antibody fragments conjugated to radiolabels, have been approved by FDA for cancer imaging, such as Capromab pendetite (ProstascintTM) for prostate cancer imaging, Arcitumomab (CEA-Scan™) for diagnostic imaging of colorectal cancer, and Nofetumomab merpentan (Verluma™) for small cell lung cancer imaging [225]. There are many studies that report the use of antibodies or antibody fragments for in vivo imaging for biodistribution studies and tumor accumulation in xenograft tumor models [226, 227]. Numerous research have also described in vivo biodistribution studies of NPs relative to NP size, shape, charge and surface chemistry for different biomedical applications [27]. However, only few reports are found on in vivo biodistribution and tumor accumulation studies with antibody functionalized NPs. NPs naturally accumulate into tumor sites through EPR effect, which makes them as an excellent tool for imaging and therapeutic applications [228]. One drawback of passive targeting of NPs via EPR is the lack of specificity to tumor sites. However there is potential to improve specificity, cellular uptake and tumor accumulation of NPs using an active targeting approach by the decoration of NPs with targeting ligands such as antibodies. It has been demonstrated that antibodies or antibody fragments conjugated to various NPs have more effect on tumor accumulation compared to unconjugated NP in xenograft mice models [229-231], confirming that the specific targeting of functionalized NPs is enhances accumulation and promote cellular uptake. This active targeting NPs has promising findings in some preclinical and early clinical studies. However, some studies reported the addition of targeting ligands may contribute to protein corona formation on NPs which can inhibit the biological functions of this bioconjugate and increased the elimination of NPs through mononuclear phagocyte system (MPS) [232]. This highlights the pros and cons of actively targeted 58

NPs in an in vivo and clinical setting and the importance for clever design and development of actively targeting NPs. In vivo kinetic biodistribution and tumor accumulation may differ between different types antibody formats, and antibody-NP conjugates, which influence the utilization in imaging and therapeutic applications. For diagnostic applications, targeting tumor deposition combined with rapid blood clearance are required, and minimal accumulation in healthy tissues to increase imaging contrast. While, in therapeutic applications, longer circulation times are desirable while minimizing accumulation and toxicity in healthy tissues [226]. Here we evaluated in vivo imaging properties, biodistribution, and tumor accumulation of two antibody targeted formats that have been developed, including J591-mPEG BsAb-conjugated imaging dye labelled PEGylated HBP and J591 scFv-pAzF-conjugated imaging dye labelled DBCO in xenograft mice models. We also demonstrated ex vivo images to support in vivo data and evaluated the xenograft tumor uptake as well as the distribution into functional organs, such as kidneys, lungs, spleen and hearth.

5.2 Materials and methods

5.2.1 Animals

For all animal models, eight-week-old Balb/c nude mice were acquired from the University of Queensland's Biological Resources and were allowed access to food and water ad libitum throughout the experiment. All the studies were approved by the Animal Ethics Committee of the University of Queensland, and Australian Code for the Care and Use of Animals for Scientific Purposes (see Appendix B).

5.2.2 Antibody fragment- polymer/imaging dye conjugates

All the antibody fragments and the conjugation methods were prepared as mentioned in chapter 3 and 4, including J591-mPEG BsAb which was non-covalently conjugated to Cy5 labelled HBP, and J591 scFv-pAzF which was covalently conjugated with Cy7 labelled DBCO through a click chemistry reaction.

5.2.3 In vivo biodistribution, and tumor accumulation of BsAb-HBP bioconjugate in tumor bearing mice

6 male Balb/c nu/nu mice (eight weeks old) were anaesthetized using 2% isofluorane and injected with 2x 106 PC3 cells in the right flank and 2x 106 PC3-PIP cells in the left flank to induce subcutaneous tumors. The tumors were grown for 20 days. The mice were split into 2 groups for experiments, Cy5 labelled HBP alone (non-targeted control) (n=3), and targeted BsAb-Cy5 labelled HBP conjugate (targeted) (n=3). 1 control mice (untreated) was prepared as a control mouse. The BsAb and Cy5 labelled HBP was mixed (1:1 molar ratio) in phosphate buffer saline and incubated at room temperature 1 h prior injection. The final concentration of Cy5 labelled HBP in injection formulation was 3 mg/mL. The mice were anaesthetized and injected via the tail vein with 200 µl injection volume. The fluorescence imaging was performed at 4, 24, 48 h and 5 days post-injection. Mice were anaesthetized during all procedures and imaged on an IVIS Lumina X5 imaging system (PerkinElmer Inc., Waltham, MA, USA) and analyzed using the Living Image software (PerkinElmer Inc.) at excitation 620 nm and emission filter of 670 nm. After 5 days, all the mice were euthanized and ex vivo images were performed using the same imaging protocols. All the fluorescence intensity data of the tumors and organs of treated mice were normalized into data of control (untreated) mouse.

5.2.4 In vivo biodistribution and selective tumor uptake of antibody fragment-dye conjugate using J591 scFv pAzF-Cy7 labelled DBCO in tumor bearing mice in comparison with BsAb- Cy5 labelled HBP

All the xenograft mice models were prepared as protocol above. BsAb-Cy5 labelled HBP as formula above, and J591 scFv pAzF at the concentration 10 µM were conjugated into Cy7 labelled DBCO via copper free click chemistry reaction in 1:1 molar ratio at 4 °C overnight. Following this, 6 xenograft mice with PC3 and PC3-PIP tumors were anaesthetized using 2% isofluorane and were co-injected with BsAb-Cy5 labelled HBP and J591 scFv pAzF-Cy7 labelled DBCO formula via the tail vein with 200 µl of total injection volume. 1 control mice (untreated) was prepared as a control mouse. The co-injected mice were split into 3 groups based on observation time, including 1-hour post injection (hpi), 4 hpi, and 24 hpi, with two mice for each group. Mice were anaesthetized during all time points and imaged on an IVIS Lumina X5 imaging system (PerkinElmer Inc., Waltham, MA, USA), followed by data analysis using the Living Image software (PerkinElmer Inc.). The 620Ex/670Em and 720 Ex/790Em were used to analyze Cy5 fluorescence at Bsab-Cy5 labelled HBP and Cy7 fluorescence at J591 scFv-pAzF-Cy7 labelled DBCO respectively. Following the fluorescence live imaging, the mice were euthanized for ex vivo imaging in each group. The ex vivo 60 images were performed using the same imaging protocols. All the fluorescence intensity data of the tumors and organs of treated mice were normalized into data of control (untreated) mouse.

5.3 Results

5.3.1 In vivo biodistribution and tumor uptake of BsAb-HBP conjugate in tumor bearing mice models

To investigate in vivo biodistribution and tumor uptake of BsAb-HBP conjugate, the images of mice were captured at 4, 24, 48 h and 5 days after administration with 200 µl of BsAb-HBP conjugate (1:1 in molar ratio), and HBP alone as a control at concentration 3mg/mL. As shown in Figure 5.1 A, the fluorescent intensity was primarily found in PC3-PIP (PSMA positive) cells; however, both BsAb- HBP conjugate and HBP alone gave the similar behavior in tumor uptake. It is presumably because of EPR effect. It was also observed from the 6 mice that the highest tumor targeting occurred in the BsAb-HBP targeted mouse 3. The high level of targeting in this mouse was also maintained over time. It was also observed that there was a slight improvement over time in tumor targeting in the BsAb-HBP cohort. The difference between BsAb conjugated to HBP and unconjugated HBP was in the blood circulation, whereas the HBP alone has more intense fluorescence, meaning it has longer circulation in the body. In addition, ex vivo images after 5 days administration, shown in Figure 5.1 B showed that fluorescence intensity of conjugated HBP was found higher in the clearance organs, such as kidney, liver and spleen compare to that for unconjugated HBP. For ex vivo tumors uptake, BsAb conjugated HBP has no significantly higher accumulation in PC3-PIP tumor compared to HBP alone. In contrast, the mean value is slightly higher for HBP comparing with BsAb conjugated HBP, seemingly due to EPR effect. However, based on the ROI ratio of PSMA positive to PSMA negative tumors, the BsAb conjugated HBP has the higher ratio with 2.4 compared to 1.6 for HBP alone (Table 5.1). Region of interest (ROI) ratio is measuring the fluorescence intensity in positive xenograft tumor compared to negative xenograft tumor. The higher ROI ratio value indicated more accumulation in positive xenograft tumor. Therefore, it is suggested that the attachment of BsAb facilitated enhanced BsAb-HBP accumulation in PSMA positive tumor.

Figure 5.1: In vivo biodistribution and tumor accumulation of free Cy5 labelled HBP and J591-mPEG BsAb conjugated to Cy5 labelled HBP in xenograft tumor models of PSMA positive prostate cancer, PC3-PIP (left flank) and PSMA negative prostate cancer, PC3 (right flank). A) Mice were imaged at 4, 24, 48 h, and 5 days post injection with an in vivo imaging system (IVIS) spectrum after being intravenously administered with J591-mPEG BsAb conjugated to Cy5 labelled HBP (1:1 in molar ratio) and Free Cy5 labelled HBP as a control. B) Ex vivo fluorescence of excised organs and tumors at 5 days post injections. Data represent mean (n=3) with standard deviation. The signal intensity of the tumors organs was measured by IVIS software and have been subtracted with the signal intensity of control mouse (untreated).

Table 5.1 Region of interest (ROI) ratio of PSMA positive tumor (PC3-PIP) to PSMA negative tumor (PC3) at 5 days after intravenous (i.v) injection of Cy5 labelled HBP alone and Cy5 labelled HBP conjugated to BsAb J591- mPEG.

i.v injection ROI ratio of PC3-PIP (PSMA +) to PC3 (PSMA -) tumors Cy5 labelled HBP 1.6 BsAb-Cy5 labelled HBP conjugate 2.4 62 5.3.2 Comparison of J591 scFv-pAzF conjugated Cy7 labelled DBCO and J591-mPEG BsAb conjugated Cy5 labelled PEG HBP for imaging purposes

In this study, we also compared the PEGylated antibody fragment format BsAb-HBP conjugate with the non-PEGylated format scFv-pAzF-DBCO conjugate for in vivo imaging properties and their distribution to organs and tumor uptake in xenograft models. Different cyanine dyes labelling was utilized to distinguish these two different formats, including a far-red fluorescent Cy5 attached on polymer for BsAb-PEG HBP and a near infra-red Cy7 attached on DBCO for scFv-pAzF- DBCO. The two formulas were co-injected into the same mouse, and the biodistribution profile and tumor uptake were evaluated at 1 h, 4 h, and 24 h post injection under Cy5 and Cy7 channel at the same time. The Cy5 channel corresponds to BsAb-HBP, while Cy7 channel corresponds to scFv- pAzF-DBCO. In general, in vivo data showed that both BsAb-HBP and scFv-pAzF DBCO specifically targeted PSMA positive xenograft. However, as shown in previous data, the accumulation of BsAb- HBP were also detected in PSMA negative tumors especially at 24 hpi. The scFv pAzF- DBCO showed high clearance through the kidney as early as 1 hpi. By 24 h, the signal in kidney was more intense, and the fluorescence started clearing in blood circulation as well as in xenograft tumor. In contrast, the kidney clearance was barely seen in BsAb-HBP conjugate, even after 24 h, and the fluorescence was still in the blood circulation with an increased accumulation into PSMA positive xenograft (Figure 5.2) Data were supported by ex vivo images results. The scFv-pAzF-DBCO targeted PSMA positive xenograft with the higher fluorescence intensity were detected at 4 hpi and the signal started clearing after 24 hpi. For the negative PSMA xenograft, the signal was lower than in positive xenograft and relatively in the same intensity. The fluorescence was mostly detected in kidneys, and continually increased until 24 hpi. In contrast, the fluorescence continually decreased in blood, and other organs including heart, lung, liver and spleen after 24 hpi. Interestingly, the fluorescence intensity in liver was also relatively higher compared to other organs, and tumors (Figure 5.3 A). The BsAb-HBP format also targeted PSMA positive xenograft, with higher fluorescence intensity compared to negative xenograft and increased after 4 and 24 hpi. In agreement with in vivo results, after 24 h, the fluorescence intensity of the BsAb also started rising in PSMA negative xenograft. For the organ’s accumulation, the higher fluorescence were detected in blood, followed by lungs and kidneys (Figure 5.3 B).

Figure 5.2: Comparison of in vivo profile of J591 scFv-pAzF and J591-mPEG BsAb-HBP conjugate in xenograft tumor mice models bearing PSMA positive PC3-PIP (left flank) and PSMA negative PC3 (right flank). Mice were co-injected with J591 scFv-pAzF-Cy7 labelled DBCO conjugate and J591-mPEG BsAb-Cy5 labelled HBP conjugate. Images were taken at 1, 4, and 24 hours post-injection of. Images under Cy7 channel (720Ex/790Em) (top) and Cy5 channel (620Ex/670Em) (bottom) represent in vivo biodistribution of J591 scFv-pAzF and J591-mPEG BsAb-HBP conjugate respectively.

64 A

Figure 5.3: Ex vivo fluorescence of excised organs and tumors at 1, 4, and 24 hours post injection. A) Fluorescence quantification of the organs and tumors through Cy7 channel (ex=720 nm; em= 790 nm), detecting J591-pAzF-DBCO conjugate (n=2, with standard deviation). B) Fluorescence quantification of the organs and tumors through Cy5 channel (ex= 620 nm; em= 670 nm), detecting J591-mPEG BsAb-HBP conjugate (n=2, with standard deviation). The signal intensity of the tumors organs was measured by IVIS software and have been subtracted with the signal intensity of control mouse (untreated).

5.4 Discussion

We evaluated the in vivo biodistribution and tumor accumulation of bioconjugates in xenograft mice models, including PEGylated format in BsAb-HBP and non-PEGylated format in scFv-DBCO. For the BsAb-HBP format, in vivo tumor uptake study demonstrates that after 5 days post injection, J591-mPEG BsAb-HBP conjugate was primarily accumulated and retained in PSMA positive xenograft tumor. In comparison with HBP alone, biodistribution of BsAb-HBP bioconjugate demonstrates an increased localization in clearance organs, such as liver, kidney and spleen. One of the undesirable effects of attaching active targeting ligand into nanomaterials is non-specific binding and increased uptake within reticuloendothelial system (RES) organs, such as spleen, lungs and liver, as well as kidneys [233]. In agreement with that, the attachment of BsAb into HBP as active targeting nanocarrier may display non-specific binding and increase uptake into clearance organs, resulting in shorter circulation time. Consequently, the cellular internalization and uptake into tumor cells will be not optimal. On the other hand, the longer blood circulation of unconjugated HBP increase the opportunity for tumor extravasation [234], resulting the enhanced accumulation through the utilization of EPR effect. Although, the BsAb conjugated HBP does not lead to a significant improvement in tumor accumulation in vivo, compared to HBP alone, after 5 days post injections, the fluorescent intensity (ROI ratio) of BsAb-HBP conjugate in PSMA positive xenograft tumor (PC3-PIP) was 2.4- fold greater than in PSMA negative xenograft tumor (PC3), compared to 1.6-fold greater for HBP alone (Table 5.1). These data may indicate the targeting selectivity of BsAb-HBP conjugate, thereby facilitating enhanced tumor internalization and accumulation via receptor-mediated endocytosis pathway [62]. Based on in vitro cellular uptake by flow cytometry (Figure 2.5), the increased BsAb amount conjugated into HBP results in increased cell targeting, with optimum molar ratio of BsAb:HBP was about 5:1. In this in vivo study, 1:1 of molar ratio was used since the highly concentrated of BsAb forms protein aggregates. Therefore, we could not prepare the injection formulation with high molar ratio of BsAb:HBP (5:1). The optimum density of BsAb in BsAb-HBP conjugate is anticipated to increase the avidity of antibody into receptor target in tumor which can aid rapid internalization and enhanced tumor accumulation. However, Cui et.al. reported that BsAb functionalization PEG particles results in different behavior in vitro and in vivo. The large amount of BsAb influenced the stealth properties of BsAb-PEG conjugate, resulting in off-target accumulation, whereas the increased accumulation of BsAb-PEG conjugate was found in spleen, liver and lungs rather than in positive tumor xenograft [194]. Co-injection of BsAb-HBP and scFv-pAzF DBCO into the same mouse were performed to evaluate the difference in in vivo biodistribution, and tumor accumulation of these two formats in xenograft mice models. This is also the first time that an antibody conjugated to Cy7 dye site

66 specifically via azide bearing unnatural amino acid has been trialed in vivo. In general, both designs have target specificity into PSMA expressing xenograft. The differences were observed in blood circulation time and organs biodistribution, especially in clearance organs. The rapid clearance and shorter circulation time was expected in non-PEGylated scFv format, as frequently reported in many studies due to the small MW [188, 226, 235]. However, the rapid renal clearance of this format is ideal for tumor diagnostic imaging and radiotherapy applications. The high ratio of tumor-to-background attained at early time points is more desired as imaging tracers [236]. The extremely high fluorescence intensity in the kidneys was observed presumably due to the excess of unconjugated small molecule DBCO-Cy7 in the solution which has a fast clearance through the kidneys. The higher accumulation of scFv-pAzF DBCO was also found in the liver at early time point, even though, the scFv generally do not accumulate in the liver [237-239]. The excess unconjugated imaging dye might be the reason for higher intensity as the fluorescence dye uptake might be retained longer in the liver. In contrast, the BsAb-HBP conjugate which is a PEGylated format has stealth properties avoiding rapid renal clearance and has a prolong circulation time [240]. Moreover, the multiple attachment sites of the BsAb-HBP formula may increase the avidity to target receptors, resulting in longer interaction on tumor sites and improved tumor accumulation. When this formula is used as targeted nanocarrier for drug delivery, a longer blood circulation may facilitate a longer time for the drugs to interact on the tumor sites. Therefore, this formula could be more valuable as theranostics applications, combining tumor diagnostic imaging and targeted drug delivery for therapy.

5.5 Conclusion

In this study, we examined in vivo imaging profiles, including the xenograft accumulation, biodistribution and clearance properties of two antibody conjugate formats, including J591-mPEG BsAb conjugated Cy5 labelled HBP, and J591 scFv-pAzF conjugated to Cy7 labelled DBCO. In general, we confirmed that both formats have shown the successful targeting into PSMA positive xenograft mice models. However, we observed the differences between PEGylated and non- PEGylated, and between single and multiple attachment site of antibody fragment for tumor accumulation, and biodistribution in other organs, including clearance organ (kidney), as well as RES organs (liver, spleen). Single attachment of scFv without PEGylation in J591-scFv-pAzF conjugated to Cy7 labelled DBCO showed a shorter circulation time which is expected due to the small size of this scFv-imaging dye conjugate (~28 kDa). Such design, however, is ideal to apply as imaging tracers. On the other hand, PEGylated format with multiple attachment sites of antibody fragment in BsAb-Cy5 labelled HBP conjugate demonstrated a longer half-life which provide enough time to accumulate in target tumor site. This benefit is essential for therapeutic application. However, this 67 study suggested that enhanced tumor accumulation is influenced by combination of longer half-lives and EPR effect, since it is also achieved by non-targeted agent, unconjugated HBP. In addition, BsAb functionalization PEGylated HBP increase in liver and spleen accumulation, resulting in reduction of tumor accumulation.

Chapter 6 Concluding remarks

NPs have potential as the next generation of nanomedicine, offering a number of applications to overcome the challenges in diagnosis, monitoring, prevention, and treatment of various diseases, particularly in cancer medicine. The potential to apply actively targeted therapeutics through conjugation of NPs with antibody targeting specific disease antigens opens up research possibilities exploring novel and simple conjugation methods, where a stable antibody-NP conjugate is crucial for successful targeting. During this project, we have successfully engineered two novel bioconjugation strategies, involving non-covalent and covalent conjugation methods to obtain a stable conjugate. Both these methods provide a simpler way to attach antibody fragments to NPs compared to current chemical and enzymatic based conjugation strategies which require rigorous optimization, validation and the generation of multiple reagents. In addition, this is the first time that unnatural amino acids incorporated into an antibody fragment have been applied in an in vivo imaging experiment and also the first time where two novel methods have been compared head to head in an in vivo cancer model. In this study we developed two bioconjugation formats. The first is a BsAb format, consisting of two scFvs, one scFv targets PSMA receptor and the other scFv targets methoxy-PEG (mPEG) nanomaterials. The BsAb, produced in mammalian cells, demonstrated binding specificity to PSMA expressing prostate cancer cells and non-covalent conjugation to m-PEG based HBP through simple mixing of BsAb with HBP which resulted in homogenous bioconjugates and overcomes potential instability of antibody-NP conjugate resulting from chemical conjugation. Furthermore, the HBP used in this study has versatile design which can be functionalized with various cargos, such as imaging agents and therapeutic drugs. Several in vitro analyses exhibited that the bioconjugate specifically targets and internalizes into PSMA positive prostate cancer cells and showed enhanced cellular uptake, demonstrated by confocal microscopy using PSMA positive prostate cancer cells. Moreover, in vitro cytotoxicity study showed that the BsAb-conjugated to Dox loaded HBP can enhance drug accumulation and cytotoxic effect compared to that unconjugated HBP, meaning this BsAb-HBP conjugate has major benefits as an actively targeted drug delivery system. The BsAb- HBP conjugate demonstrated a slight improvement in tumor targeting in xenograft mice models compared to HBP alone, while also increased liver and spleen accumulation. This result shows that functionalization of BsAb into PEGylated HBP can exhibit different biological behaviors between in vitro and in vivo. Therefore, further investigation of the BsAb structure and stability, and optimization of BsAb or mPEG HBP density in the bioconjugate will identify the optimal formulation of this hybrid material for targeted diagnosis and therapy. The second bioconjugation format focuses on a covalent conjugation that has been achieved via copper click chemistry reaction between azide-modified anti-PSMA J591 scFv and DBCO

69 functionalized NPs/imaging dye. In vitro incorporation of UAA to obtain azide-modified J591 scFv, provide selective labelling in the intracellular environment, resulting in a stable functionalized protein. We confirmed that a copper-free click chemistry reaction for antibody-NPs/imaging dye conjugate results in a stable conjugate under mild condition. Furthermore, in vitro studies confirmed the specific targeting of this antibody fragment-imaging dye conjugate into PSMA expressing prostate cancer cells. In vivo and ex vivo studies of azide-modified J591 scFv-imaging dye DBCO conjugate demonstrated high binding affinity into xenograft tumor target with rapid renal clearance, which may be more ideal for application as imaging tracers. We also demonstrated the ability to attach azide-modified J591 scFv to larger structures such as a DBCO containing thermo-responsive PEG based polymer in micellar system. This is very important for improvement of antibody half-life (via PEGylation) and binding affinity. Longer half-life would enhance time for target binding, and likely provide a more homogenous distribution into tumor sites. One major design consideration for targeted NPs is avoiding immune recognition. NPs injection via intravenous are often taken up by monocytes in the bloodstream or in the tumor microenvironment, and subsequently eliminated through the RES system, including lung, liver and spleen. By using click chemistry in combination with larger PEG polymer systems, there is potential to customize NPs with targeting ligands via site specific conjugation of scFv-azide to DBCO groups. This enables a systematic approach to balance and control targeting ligand density, placement and coverage on a PEG NP to maintain PEG stealth properties and avoid clearance and potentiate tumor targeting. This concept using DBCO- PEG polymer in combination with the UAA scFvs developed in this project is the basis of a paper that is currently under review by ACS Nano, “Controlling the biological fate of micellar nanoparticles: Balancing stealth and targeting” The potential benefit of copper-click chemistry reaction has led to the utilization of azide- modified scFv via UAA incorporation for other bioconjugation methods. Our group are currently developing the bioconjugation of azide-modified scFvs into DBCO containing toxin for targeting glioma for antibody drug conjugate application. For further investigation, we are determining the efficiency of conjugation for optimal bioconjugate design. In summary, this study has contributed to the bio-nanomaterial research field by developing novel bioconjugation strategies for the simple generation of antibody-nanomaterial conjugates for application as actively targeted drug delivery tools, as well as complex imaging contrast agent for diagnosis purposes. These advances will contribute in the generation of an era targeted cancer diagnosis and therapy as a precision nanomedicine.

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APPENDICES

Appendix A: Supplementary data for Chapter 3

MGWSCIILFLVATATGVHSHHHHHHEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHW VKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC AAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKA SQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLAITNVQSEDLAD YFCQQYNSYPLTFGAGTKLEIKRGGGGSEVKLEESGGGLVQPGGSMKLSCVAEESGGGLV QPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVTEIRSKSNNYATHYAESVKGRFTI SRDDSKGSVYLQMNNLRAEDTGIYYCSNRYYWGQGTLVTVSAGGGGSGGGGSGGGGSDI VMTQSHKFMSTSVRDRVTITCKASQDVNTSVAWYQQKPGQSPKLVIYWASTRHTGVPDR FTGSGSGTDFTLTISNVQSEDLADYFCLQYINYPYTFGGGTKLEIKEQKLISEEDLN*

Figure A.1: The BsAb J591-mPEG design. Signal Peptide -His6x- J59 Vh -(G4S)3- J591 Vk -G4S- anti-methoxyl-PEG

Vh -(G4S)3- anti-methoxyl-PEG Vk -cMyc-Tag*

SEC of protein standar 15.00 1

10.00

2 5.00 3 0.00 4 5 0.00 10.00 20.00 30.00 40.00 215 nm

Absorbance (mAU) Absorbance -5.00 280 nm

-10.00

-15.00 Volume (mL)

Peak Protein (Mw kDa) Retention volume (mL) 1 Thyroglobulin 13.41 (bovine) (669) 2 Aldolase (rabbit) 15.88 (158) 3 Ovalbumin 18.97 (chicken) (43) 4 Ribonuclease A 20.05 (14)

5 Vitamin B12 (1.35) 20.90

Figure A.2: SEC profile of protein standard (BioRad) with elution buffer 1xPBS in 20% ethanol.

Figure A.3: Protein Identification and analysis tools using the ExPASy server.

Figure A.4: The prediction of disorder protein sequence using algorithm by PONDR® protein disorder predictor [241, 242]. About 22.5 % of the BsAb protein sequences are predicted to be intrinsically disordered regions (IDRs).

APPENDIX B: Ethic approval certificate for conducting animal research